Cell differentiation-related signaling pathways: biological functions, diseases and therapeutic targets

Abstract

Cell differentiation, the process by which stem cells acquire specialized functions, underpins embryonic development, tissue homeostasis, and regeneration. This process is governed by evolutionarily conserved signaling pathways, including Wnt, Notch, Hedgehog, TGF-β, and Hippo, which integrate extracellular cues with intrinsic genetic programs to determine cell fate. Here, we provide a comprehensive overview of the molecular architecture of these pathways, spanning ligand–receptor interactions to downstream transcriptional regulation. We further highlight multilayered regulatory mechanisms such as epigenetic modifications, non-coding RNAs, feedback loops, and pathway crosstalk that fine-tune signaling outputs to ensure spatiotemporal precision in fate decisions. We also discuss how tissue-specific contexts and interactions among these pathways generate distinct differentiation outcomes in different organs and disease settings. Dysregulation of these networks contributes to diverse pathological states, driving uncontrolled proliferation in cancer, maladaptive remodeling in cardiovascular disease, neuronal loss in neurodegeneration, metabolic imbalance, digestive dysfunction, chronic inflammation in respiratory and autoimmune diseases, and skeletal disorders. Reflecting their clinical importance, we also summarize recent therapeutic advances, including small-molecule inhibitors, monoclonal antibodies, gene-based strategies, and pathway-modulating compounds, which target these pathways to restore normal cellular function. Particular attention is given to distinguishing preclinical findings from clinical applications and to the translational challenges of pathway-targeted therapies. Collectively, this review underscores the central role of differentiation-related signaling in health and disease and highlights emerging opportunities for therapeutic intervention.

Introduction

Cell differentiation is the process by which pluripotent or multipotent stem and progenitor cells acquire specialized molecular, structural, and functional identities. Pluripotent stem cells, represented by embryonic stem cells (ESCs), can generate nearly all somatic cell types, whereas multipotent stem cells are restricted to defined lineage compartments.^1–3 Differentiation is therefore not a simple loss of developmental potential, but a coordinated transition involving changes in transcriptional programs, chromatin accessibility, metabolism, cell morphology, and interactions with the surrounding microenvironment. This process is indispensable for embryonic development and organogenesis and remains active throughout adult life to sustain tissue renewal, repair, and physiological homeostasis.^4,5 Its precision is especially important because inappropriate maintenance of stemness, premature lineage commitment, or failure of terminal maturation can impair regeneration, accelerate aging, and promote disease.⁶ Thus, understanding how cells convert extracellular information into stable and context-appropriate identities is a central question in developmental biology, stem cell biology, and regenerative medicine.

Although common molecular principles are shared across tissues, differentiation is strongly shaped by lineage history and local niche signals. During hematopoiesis, hematopoietic stem cells generate diverse blood and immune lineages through sequential fate restriction controlled by the bone marrow microenvironment and pathways such as Wnt.⁷ In the central nervous system, neural stem and progenitor cells balance self-renewal with neuronal and glial differentiation under the influence of Notch and other developmental signals.⁸ Myogenesis similarly requires the coordinated activation of lineage-determining transcription factors, including MyoD, together with extracellular cues such as Wnt and bone morphogenetic proteins.^9,10 These examples illustrate that cell fate is not dictated by a single pathway or transcription factor. Instead, it emerges from the integration of intrinsic genetic programs with ligand availability, receptor expression, signal duration, mechanical inputs, metabolic state, and epigenetic competence. At the center of this regulatory system are five evolutionarily conserved pathways—Wnt, Notch, Hedgehog (Hh), transforming growth factor-β (TGF-β), and Hippo—which translate extracellular, cell-cell, and mechanical cues into transcriptional outputs that determine proliferation, quiescence, lineage commitment, and terminal differentiation.¹¹ Their effects are further modified by growth factors and cytokines, which regulate the balance between self-renewal and differentiation in a tissue- and stage-dependent manner.^12–14

Current research has moved beyond describing these pathways as independent linear cascades. Increasing evidence shows that their biological outputs are controlled by multilayered regulatory mechanisms, including DNA methylation, histone modification, non-coding RNAs, feedback loops, pathway crosstalk, and biomechanical regulation. The same pathway can therefore produce opposite outcomes depending on cell type, developmental stage, signal strength, and disease context. Wnt signaling may maintain stem-cell renewal in one tissue but promote differentiation or pathological proliferation in another; Notch can preserve progenitor populations or induce lineage commitment depending on ligand-receptor combinations and signaling duration; TGF-β can suppress early tumorigenesis yet drive fibrosis and immune evasion in advanced disease; and Hippo pathway activity can either constrain or permit YAP/TAZ-dependent growth and plasticity. Recent advances in single-cell and spatial omics, organoid systems, live-cell imaging, CRISPR-based perturbation, and biomaterial-guided culture models are now enabling these context-dependent responses to be examined at higher resolution. This systems-level perspective is particularly important because dysregulated differentiation signaling contributes to developmental abnormalities, malignant transformation, immune dysfunction, tissue fibrosis, metabolic imbalance, neurodegeneration, and impaired regeneration.¹⁵ Precise regulation of these pathways is therefore essential for maintaining stem-cell pool stability and normal tissue function.^16,17 At the same time, the therapeutic consequences of manipulating these pathways are inherently complex. A pathway that is pathogenic in one cellular compartment may remain indispensable for repair or homeostasis in another, and the direction of benefit may change with disease stage. This context dependence helps explain why promising preclinical interventions have not always translated into durable clinical responses and why tissue selectivity, treatment timing, and biomarker-guided patient selection have become major priorities.

This review focuses on the biological functions, disease relevance, and therapeutic potential of Wnt, Notch, Hh, TGF-β, and Hippo signaling in cell differentiation. We first outline the historical milestones that established the concept of inductive signaling and led to the discovery of these core pathways. We then compare their molecular architecture, including ligands, receptors, intracellular mediators, transcriptional effectors, and the epigenetic and feedback mechanisms that shape signaling output. Particular attention is given to pathway crosstalk, tissue-specific heterogeneity, and shared regulatory principles that convert dynamic signals into stable cell-fate decisions. We subsequently examine how disruption of these pathways contributes to cancer and tumorigenesis, cardiovascular, neurodegenerative, metabolic, digestive, respiratory, autoimmune, and skeletal diseases. Finally, we summarize pathway-targeted interventions, including small-molecule inhibitors, monoclonal antibodies, gene-based approaches, natural compounds, and cell- or biomaterial-based strategies, while distinguishing preclinical evidence from established or emerging clinical applications. By integrating basic mechanisms with disease-specific and translational evidence, this review aims to clarify when pathway activation or inhibition may be beneficial, identify the major obstacles to therapeutic translation, and highlight priorities for developing safer, more precise, and context-dependent differentiation-directed therapies.

Historical perspective and milestones in research on cell differentiation-related signaling pathways

Research on cell differentiation-related pathways has a long history (Fig. 1). The scientific investigation of cellular differentiation originated within the embryological domain, where pioneering observations demonstrated that developing cells acquire specialized functional identities during ontogeny. During the late 19th and early 20th centuries, investigators predominantly focused on elucidating the intrinsic genetic determinants governing developmental trajectories. Wilhelm Roux, through his seminal experiments on amphibian embryos, proposed the “mosaic” theory of development, which posited that ontogenesis proceeds via autonomous, predetermined cell lineages.¹⁸ This mechanistic view was subsequently challenged by Hans Driesch, whose experiments with echinoderm embryos demonstrated the regulative capacity of the early embryo.¹⁹ Although isolated embryonic blastomeres retain the capacity to develop into complete organisms, normal morphogenesis is achieved through precisely orchestrated interactions among embryonic constituents. This integrative perspective was further substantiated by Hans Spemann’s landmark contributions. Through innovative transplantation experiments using amphibian embryos, Spemann and Mangold demonstrated that specific cellular domains could generate inductive signals that direct the differentiation of adjacent tissues, a phenomenon subsequently termed embryonic induction.²⁰ This discovery represented a fundamental advancement in our understanding of intercellular communication mechanisms. These investigations collectively established that cellular differentiation is not exclusively regulated by cell-autonomous gene expression programs but is profoundly influenced by extracellular signaling cues and intercellular molecular dialogs.

Fig. 1.

Historical perspective and milestones in research on cell differentiation-related signaling pathways. This timeline charts the progression from late-19th-century theories of mosaic and self-regulating development to the discovery of embryonic induction in the early 20th century. From the mid-20th century onward, advances in molecular biology enabled the characterization of morphogens and signaling cascades and progressively led to landmark discoveries related to TGF-β, Notch, Wnt, Hedgehog, and Hippo signaling. Modern research now integrates these elements into gene regulatory networks to elucidate tissue maintenance and translate findings into therapies. The graphics were created with BioRender (https://www.biorender.com). TGF-β transforming growth factor-beta

Advances in biochemical and molecular biology techniques in the mid-20th century enabled the isolation of functional molecules from tissues and cells, paving the way for elucidation of signaling pathways. These investigative approaches revealed that extracellular communication, mediated via secreted morphogens or membrane-anchored ligands, is indispensable for orchestrating coordinated morphogenesis.²¹ Upon ligand-receptor engagement, conformational alterations or receptor oligomerization events initiate sophisticated intracellular signal transduction cascades. These molecular cascades typically encompass sequential enzyme activation, second messenger generation, and ultimately culminate in comprehensive transcriptional reprogramming.^22,23 Through these highly conserved molecular mechanisms, cells effectively interpret environmental stimuli and execute lineage-specific differentiation programs.^24,25 Throughout embryonic development, conserved signaling pathways are deployed repeatedly to regulate proliferation, migration, polarity, and differentiation.²⁶ These pathways operate through spatiotemporally precise mechanisms to orchestrate morphogenesis. Despite structural and mechanistic diversity, a unifying feature is the reliance on signal-dependent transcription factors that regulate cell-type-specific gene expression programs.

The identification of major signaling pathways including Wnt, Notch, Hh, Hippo, and TGF-β has been pivotal to understanding the molecular logic of differentiation. The TGF-β pathway was initially delineated within oncological contexts. In 1978, De Larco and Todaro identified secreted polypeptide growth factors produced by murine sarcoma virus-transformed fibroblasts that promoted anchorage-independent growth.²⁷ Later, Sporn and Roberts refined this characterization and demonstrated the presence of TGF in both neoplastic and physiologically normal cellular contexts. Among the identified isoforms, TGF-β exhibited ubiquitous expression in normal tissues and was demonstrated to be critical in both carcinogenesis and normal developmental morphogenesis.²⁸ Other key signaling cascades were also uncovered through classical genetic studies. The Notch mutant phenotype was first described by Dexter in Drosophila in 1914 and was subsequently characterized genetically by Morgan and colleagues.²⁹ Mammalian homologs of Notch (Notch1/2/3/4) were subsequently discovered.³⁰ The Wnt gene family emerged from the discovery of the mouse mammary oncogene int-1 in 1982³¹, followed by the demonstration in 1987 that the Drosophila segment-polarity gene wingless is homologous to int-1.³² In 1980, Nüsslein-Volhard and Wieschaus identified Hh as one of the Drosophila segment-polarity genes, named after the spiny phenotype of mutant embryos.³³ Three mammalian Hh homologs (Desert, Indian, and Sonic) were later cloned in mice.³⁴ The Hippo pathway was defined in 2003 through Drosophila genetic studies identifying the core kinase Hippo, whose loss caused excessive tissue growth by increasing proliferation and reducing apoptosis.^35,36 Together, these discoveries laid the conceptual foundation for understanding how extracellular cues are integrated into developmental programs. By linking cell-extrinsic signaling to gene regulatory networks, these pathways provide a unified model of how cells acquire specialized identities and maintain tissue architecture.

Alongside the discovery of these pathways, early investigations underscored the central role of growth factors and their receptors as key mediators of cell differentiation. Growth factors such as epidermal growth factor (EGF), fibroblast growth factor (FGF), and nerve growth factor (NGF) orchestrate complex developmental programs through precise receptor-mediated signaling. The epidermal growth factor receptor (EGFR) was the first transmembrane receptor tyrosine kinase to be cloned and sequenced. EGFR and its related family members bind to more than ten ligands within the EGF and neuregulin/regulin families.³⁷ These ligand-receptor systems are essential for the development and maintenance of various organs³⁸, and play an important role in the differentiation of epithelial cells.³⁹ The mammalian FGF family comprises 22 members that contribute to embryonic development, tissue repair, and a broad range of physiological and pathological processes. During differentiation, recombinant FGF proteins can direct lineage commitment by modulating intracellular signaling pathways. For example, FGF-2 enhances the proliferation and differentiation of neural stem cells (NSCs)⁴⁰, while FGF-6 promotes activation and myogenic differentiation of satellite cells.⁴¹ FGF-10 and other factors have been shown to guide pluripotent stem cells toward cardiomyocyte lineages.⁴² Similarly, FGF-2 and FGF-7 facilitate the hepatic differentiation of progenitor cells, aiding in liver regeneration and disease modeling.⁴³ NGF, the first neurotrophic factor to be identified, governs neuronal differentiation and also contributes to the development of myeloid and lymphoid cell lineages.⁴⁴ These growth factors exert their effects through major intracellular cascades, which converge on transcriptional regulators to reprogram gene expression. Although both EGF and NGF/FGF activate the mitogen-activated protein kinase (MAPK) cascade, EGF causes transient activation of the MAPK cascade, whereas NGF/FGF causes both transient and sustained activation.⁴⁵ This difference in signaling kinetics is believed to underlie distinct lineage outcomes. The spatial and temporal availability of growth factors, their receptor specificity, and downstream signaling profiles collectively ensure the precise control of cell differentiation. These foundational insights established that extracellular cues, mediated by growth factor pathways, are essential for guiding cell behavior during embryogenesis and tissue morphogenesis.

Composition and regulatory mechanisms of cell differentiation-related signaling pathways

Cell differentiation, a fundamental process in multicellular organisms, is precisely regulated by a complex network of signaling pathways that govern lineage specification and functional specialization. These pathways integrate extracellular stimuli with intracellular signaling machinery to activate transcriptional programs that drive cell fate transitions. At the core of differentiation-related signaling networks lie evolutionarily conserved molecular components, including ligands, transmembrane receptors, intracellular mediators, and transcription factors. Collectively, these elements translate environmental signals into epigenetic and transcriptional modifications, enabling cells to adopt tissue-specific identities.⁴⁶ Recent advances have revealed the dynamic interplay between canonical signaling pathways and their multi-layered regulatory mechanisms, highlighting the mechanisms by which cells maintain precision in both developmental timing and spatial organization.

The complexity of differentiation regulation extends beyond linear signal transduction. Epigenetic modifications, such as DNA methylation and histone acetylation, establish heritable chromatin states that commit cells into specific lineages.⁴⁷ Concurrently, non-coding RNAs (ncRNAs), including microRNAs (miRNAs) and long non-coding RNAs (lncRNAs), modulate pathway activity by targeting key components such as receptors or transcription factors. For instance, LINC-ROR promotes osteogenic differentiation of mesenchymal stem cells by acting as a competing endogenous RNA for miR-138 and miR-145, thereby derepressing ZEB2 and activating Wnt/β-catenin signaling.⁴⁸ Feedback loops further enhance robustness. For example, TGF-β signaling promotes differentiation while simultaneously activating inhibitors such as SMAD7 to prevent excessive activation.⁴⁹ Such multilayered regulation ensures adaptive responses to fluctuating microenvironments.

Notably, differentiation pathways rarely operate in isolation. Crosstalk between Wnt, Notch, and bone morphogenetic protein (BMP) signaling pathways exemplifies synergistic or antagonistic interactions during fate determination.⁵⁰ Tissue-specific adaptations further diversify pathway outputs: Notch signaling directs T-cell maturation in hematopoiesis but regulates epidermal stratification in skin.^51–53 This context-dependent modulation arises from combinatorial interactions with lineage-determining transcription factors and extracellular matrix (ECM) components, highlighting the need to explore signaling networks within their physiological contexts. Elucidating these principles is expected to not only expand our understanding of developmental biology but also inform therapeutic strategies for regenerative medicine and cancer treatment.

Core components of signaling pathways

This section details the core components and regulatory mechanisms of differentiation-related signaling networks, emphasizing recent advances in understanding their spatiotemporal dynamics and therapeutic implications (Fig. 2).

Fig. 2.

Composition of cell differentiation-related signaling pathways. The conserved TGF-β, Wnt, Notch, Hippo, and Hedgehog pathways orchestrate cellular differentiation through ligand–receptor interactions that activate intracellular effectors, including SMADs, β-catenin, NICD, YAP/TAZ, and GLI. These effectors regulate lineage-specific transcription through pathway-specific nuclear mechanisms and partners such as TCF/LEF, CSL, MAML, and TEAD; notably, YAP/TAZ accumulate in the nucleus when the Hippo kinase cascade is inactive. The graphics were created with BioRender (https://www.biorender.com). SMAD mothers against decapentaplegic, GSK-3β glycogen synthase kinase 3β, TCF/LEF T-cell factor/lymphoid enhancer factor, NICD Notch intracellular domain, CSL CBF1/Su(H)/Lag-1, MST1/2 mammalian sterile 20-like kinase 1/2, LATS1/2 large tumor suppressor kinase 1/2, YAP/TAZ yes-associated protein/transcriptional coactivator with PDZ-binding motif, TEAD TEA domain transcription factor, TGFβR transforming growth factor-β receptor, Hh hedgehog, PTCH patched, SMO smoothened, Gli glioma-associated oncogene homolog. CK1α casein kinase 1 alpha, MAML Mastermind-like transcriptional coactivator

Ligands: initiators of differentiation signals

As is well documented, signaling networks are the backbone of cellular differentiation, orchestrating intricate processes that guide cells toward specific lineages. These networks are initiated by ligands, which are secreted proteins or small molecules that bind to specific receptors on the cell surface to trigger a cascade of intracellular signaling events. Among key ligands that induce differentiation, Wnt proteins, TGF-β superfamily members, and Notch ligands represent critical regulators of cellular fate.

Wnt proteins are lipid-modified glycoproteins that play a pivotal role in embryonic patterning and stem cell maintenance. Wnt3a, a canonical Wnt ligand, activates β-catenin-dependent transcription, thereby promoting mesodermal differentiation through the regulation of target genes such as Brachyury and Tbx6.^54,55 This process is closely regulated by the Wnt/β-catenin pathway, wherein the stabilization and nuclear translocation of β-catenin are essential for the activation of Wnt target genes. Recent studies have established the role of non-canonical Wnt signaling, such as Wnt5a, which activates β-catenin-independent pathways to regulate planar cell polarity during epithelial differentiation.⁵⁶ This ligand-specific activation highlights the complexity of Wnt signaling, where subtle differences in ligand structure can lead to divergent downstream effects.

The TGF-β superfamily represents another critical group of ligands that mediate cellular differentiation. This superfamily includes BMPs, Activin, and TGF-β, each with distinct roles in regulating lineage commitment. BMP4 is a well-studied ligand that induces osteogenic differentiation by activating SMAD1/5/8 signaling, leading to the expression of osteoblast-specific genes such as Runx2 and Osterix.^57–59 Similarly, Activin A, another member of the TGF-β superfamily, plays a vital role in endodermal lineage specification. In human ESCs, Activin A signaling through SMAD2/3 up-regulates the expression of endodermal markers, guiding cells toward pancreatic, hepatic, or intestinal fates.^60,61 The specificity of TGF-β signaling is further modulated by the presence of inhibitory SMADs, which form negative feedback loops to regulate pathway activity. Recent studies have also reported the role of non-canonical TGF-β signaling, including the activation of PI3K/Protein kinase B (AKT) and MAPK pathways, which can influence cellular behaviors such as migration and survival.⁶²

Notch signaling, on the other hand, relies on membrane-bound ligands such as Jagged and Delta-like proteins to mediate cell-cell communication. This pathway is particularly important during neural development, where it regulates the diversification of neural progenitor cells (NPCs) through a process termed lateral inhibition. For example, Delta-like ligands expressed on the surface of neural progenitors activate Notch receptors on neighboring cells, suppressing their ability to adopt an identical fate and ensuring the generation of a diverse pool of neural cells⁶³. The Notch signaling cascade involves the cleavage and nuclear translocation of the Notch intracellular domain (NICD), which forms a complex with transcription factors to activate target genes, including Hes1 and Hey1. These genes, in turn, regulate the expression of lineage-specific markers and maintain the balance between progenitor self-renewal and differentiation. Recent advances have also implicated Notch signaling in the regulation of immune cell differentiation, where it influences T-cell specification and the maintenance of hematopoietic stem cells.

The interplay between these signaling networks is further shaped by the heterogeneity of ligands and receptors, which can lead to pathway specificity and context-dependent outcomes. For instance, while Wnt3a activates canonical β-catenin-dependent signaling to promote mesodermal differentiation, Wnt5a engages non-canonical pathways to regulate planar cell polarity during epithelial differentiation. In contrast, BMP4 signaling can also promote extraembryonic endoderm differentiation under specific culture conditions in human ESCs, demonstrating the context-dependent nature of TGF-β signaling.^64–66 Taken together, these findings highlight the importance of delineating the spatiotemporal dynamics and cellular context in which these signaling networks operate, given their significant implications for both basic research and therapeutic applications.

Receptors: gatekeepers of signal transduction

The spatiotemporal regulation of cell differentiation-related signaling pathways relies on an intricate molecular network mediated by receptors, and its functional characterization has emerged as a central focus in developmental and regenerative medicine. As the core hub of transmembrane signaling, the receptor family constitutes a molecular switching system that determines cell fate through ligand-specific recognition, conformational dynamic rearrangement, and differential recruitment of downstream signaling modules. Earlier studies have demonstrated that receptor activation thresholds are synergistically regulated by their oligomerization status, subcellular localization, and epigenetic modifications, and that these properties directly regulate the balance between the maintenance of stem cell pluripotency or directed differentiation.

Transmembrane receptors, encompassing Frizzled (FZD) receptors in the Wnt signaling pathway, transforming growth factor-β receptor type II (TGFβRII), and Notch receptors, are key components of these signaling cascades. Upon ligand binding, these receptors undergo conformational alterations that trigger downstream signaling events. For instance, in the Notch signaling pathway, ligand endocytosis in the signal-sending cell promotes proteolytic processing of the Notch receptor. γ-Secretase cleavage of the receptor releases NICD, which then translocates to the nucleus to regulate target-gene expression involved in cell differentiation and fate determination.⁶⁷ Similarly, TGFβRII, upon binding to TGF-β ligands, phosphorylates and activates transforming growth factor-β receptor type I (TGFβRI), which in turn phosphorylates SMAD proteins to regulate gene expression.⁶⁸ In addition to transmembrane receptors, intracellular receptors also play a decisive role in cell differentiation. Nuclear receptors, such as retinoic acid receptors, directly bind to lipophilic ligands such as retinoic acid to regulate neural differentiation. Retinoic acid, a derivative of vitamin A, serves as a morphogen in developing tissues, establishing concentration gradients that dictate cellular fate and patterning. The binding of retinoic acid to retinoic acid receptors triggers the activation of target genes involved in neurogenesis and the specification of NPCs.⁶⁹

Previous studies have identified receptor oligomerization as an emerging regulatory mechanism in cell signaling. For example, TGF-β receptors form heterotetrameric complexes that dictate the kinetics of SMAD activation. This oligomerization not only enhances the specificity of ligand binding but also modulates the intensity and duration of downstream signaling.^70,71 Likewise, receptor tyrosine kinases typically dimerize upon ligand binding, leading to autophosphorylation and the activation of downstream effectors such as the MAPK and PI3K pathways. These pathways are essential regulators of cell proliferation, survival, and differentiation.⁷² The regulation of these signaling pathways is further complicated by the involvement of various co-receptors, antagonists, and regulatory proteins. For instance, in the Wnt signaling pathway, FZD receptors are generally modulated by co-receptors such as low-density lipoprotein receptor-related protein 5/6 (LRP5/6), which enhance the stability and signaling capacity of the Wnt-FZD complex. These co-receptors facilitate the formation of a larger signaling complex that promotes β-catenin stabilization and subsequent transcriptional activation of Wnt target genes.⁷³ Conversely, antagonists such as Dickkopf (DKK) proteins bind to LRP5/6 to inhibit the formation of the Wnt-FZD-LRP complex, thereby suppressing Wnt signaling. This antagonistic mechanism is crucial for maintaining precise control of Wnt signaling activity during development and tissue homeostasis.⁷⁴

In addition to DKK proteins, other antagonists such as SFRPs can directly bind to Wnt ligands, thereby preventing their interaction with FZD receptors. This antagonistic mechanism not only modulates the intensity of Wnt signaling but also contributes to the establishment of Wnt gradients in developing tissues.⁷⁵ Furthermore, intracellular regulatory proteins such as β-catenin and its binding partners, including TCF/LEF transcription factors, are tightly controlled by a complex interplay of kinases, ubiquitin ligases, and phosphatases. For example, glycogen synthase kinase-3β (GSK-3β) phosphorylates β-catenin, targeting it for proteasomal degradation in the absence of Wnt signaling. This phosphorylation-dependent regulation ensures that β-catenin is maintained at low levels, thereby inhibiting inappropriate activation of Wnt target genes.⁷⁶ Moreover, studies have described the role of R-spondin proteins in modulating Wnt signaling. R-spondins bind to LGR4/5/6 and antagonize the transmembrane E3 ubiquitin ligases RNF43 and ZNRF3, thereby reducing Frizzled receptor ubiquitination and internalization. This increases the abundance of Wnt receptors at the cell surface and amplifies Wnt/β-catenin signaling.^77–79 The regulation of cell differentiation-related signaling pathways involves a complex interplay of receptors, co-receptors, antagonists, and intracellular regulatory proteins. These components coordinate to ensure that signaling pathways are activated with precise thresholds, durations, and spatial patterns, enabling cells to interpret and respond to developmental signals with high fidelity.

Intracellular signaling proteins: signal amplifiers and integrators

Central to processes governing cell differentiation are intracellular signaling proteins that act as both amplifiers and integrators of extracellular signals, ultimately determining transcriptional outputs that drive differentiation. These signaling molecules include GTPases, kinases, and second messengers, each playing distinct yet interconnected roles in the cellular differentiation machinery.

GTPases, particularly Rho family GTPases such as RhoA and Rac1, are critical regulators of cytoskeletal remodeling, a process integral to cellular migration and differentiation. For instance, during epithelial-mesenchymal transition (EMT), a key process in developmental and pathological events, Rho GTPases modulate the dynamic rearrangement of actin filaments, enabling the acquisition of mesenchymal traits in migrating neural crest cells.⁸⁰ This transition is not only a prerequisite for proper embryonic development but also a mechanism frequently hijacked in pathological processes such as cancer metastasis. Kinases, on the other hand, serve as central nodes in signal transduction cascades, integrating and amplifying signals from growth factors and other extracellular cues. MAPK cascades, including the ERK and p38 pathways, exemplify this role. Specifically, these kinases phosphorylate and activate downstream targets such as MyoD, a master regulator of myogenic differentiation, thereby modulating transcriptional programs required for muscle cell development.⁸¹ The specificity and intensity of these signaling events are tightly regulated by upstream signals, ensuring that differentiation proceeds in a context-dependent manner.

Second messengers, such as calcium ions, further add complexity to these signaling networks. Calcium oscillations, triggered by the activation of IP3 receptors, play a major role in coordinating cellular responses during differentiation. For example, during cardiomyocyte growth, calcium signals activate the calcineurin/NFAT pathway, which in turn regulates the expression of genes essential for cardiac lineage commitment.⁸² These oscillatory patterns of calcium signaling are not merely passive phenomena but rather highly regulated events that encode specific information for downstream effectors. Recent advances in single-cell RNA sequencing have offered unprecedented insights into the context-dependent nature of these signaling pathways. Indeed, studies have revealed that the same signaling molecules can exert different effects contingent upon cellular context. For instance, AKT1 and AKT2, two closely related isoforms of AKT kinase, exhibit distinct roles in differentiation. AKT phosphorylates SOX2 to stabilize the protein and support pluripotency, whereas AKT2 plays an essential role in adipocyte differentiation and maintenance.^83,84 Such isoform-specific functions highlight the complexity of signaling networks and the need for a more comprehensive understanding of their regulation. Signaling pathways that regulate cell differentiation are composed of an intricate interplay of GTPases, kinases, and second messengers, each contributing uniquely to cellular differentiation. These pathways are tightly regulated by upstream signals and exhibit context-dependent behaviors that ensure precise control over cellular identity and function.

Transcription factors: executors of differentiation programs

Transcription factors, epigenetic modifiers, and emerging mechanisms involving biomolecular condensates collectively ensure precise spatiotemporal control over the acquisition of cellular identity. Recent advances in single-cell genomics, CRISPR-based functional screens, and super-resolution imaging have revolutionized our understanding of the mechanisms by which these components interact dynamically during fate determination. Terminal differentiation requires the coordinated activity of specialized transcription factors that bind lineage-specific enhancers and promoters to activate differentiation-associated genes. Pioneer factors, such as FOXA2 in endoderm specification and FOXA1 in mammary epithelial differentiation, initiate cellular reprogramming by accessing condensed chromatin through intrinsic chromatin-opening domains.^85–87 These factors facilitate nucleosome displacement and recruit ATP-dependent chromatin remodelers such as SWI/SNF complexes to establish accessible chromatin domains. Subsequent lineage-determining transcription factors then bind these primed regulatory elements to execute differentiation programs: MyoD1 drives myogenesis through direct activation of muscle-specific genes like MyoG and Desmin, while PAX6 regulates neurogenesis by governing retinal progenitor cell fate through modulation of Notch signaling components.^88–92

The recruitment of epigenetic modifiers by transcription factors fosters a permissive chromatin landscape for differentiation. SWI/SNF complexes (BAF/PBAF variants) facilitate transcription factor access through nucleosome sliding, while histone acetyltransferases such as p300 establish H3K27ac marks at active enhancers.^93,94 CEBPA has also been shown to interact directly with DNMT3A and inhibit its DNA methyltransferase activity, illustrating a direct interface between lineage-determining transcription factors and epigenetic enzymes.⁹⁵ Emerging evidence highlights the role of transcription factor dimerization and liquid-liquid phase separation in forming transcriptional condensates that enhance target gene specificity.

Transcription-factor activation domains can form phase-separated condensates that concentrate components of the transcriptional machinery.⁹⁶ Coactivators such as MED1 and BRD4 form condensates at super-enhancers, whereas OCT4 phase separation contributes to topologically associating domain reorganization during cell-fate transitions.⁹⁷ The kinetics of condensed chromatin loops in OCT4 phase-separated condensates contribute to topologically associating domain reorganization. Disruption of OCT4 phase separation attenuates topologically associating domain reorganization and reprogramming, which can be rescued by the incorporation of intrinsically disordered domains into OCT4. Topologically associating domain reorganization plays a major role in the regulation of fate transitions during cell differentiation.⁹⁸ These findings unveil that phase separation not only amplifies transcriptional output but also serves as a regulatory node integrating signaling inputs with chromatin state.

Regulatory mechanisms in signaling pathways

Cell differentiation, a fundamental process in developmental biology and tissue homeostasis, is regulated by an intricate network of signaling pathways whose activity is tightly regulated through epigenetic modifications, ncRNAs, and feedback loops (Fig. 3). These regulatory mechanisms ensure precise spatiotemporal control over gene expression programs that drive lineage commitment while concomitantly suppressing pluripotency. Recent advances in single-cell epigenomics and CRISPR-based functional genomics have offered valuable insights into the molecular mechanisms underlying these processes, particularly in the context of DNA methylation, histone modifications, and ncRNA-mediated regulation.

Fig. 3.

Regulatory mechanisms in signaling pathways. Epigenetic DNA Modifications: DNMT3A/B methylate and silence pluripotency genes (OCT4, NANOG); TET-mediated demethylation supports cardiac gene activation; Histone Acetylation: TLX recruits HDAC3/5 to repress p21/pten; HDAC inhibition upregulates NeuroD/Ngn1 promoting neurogenesis; Non-coding RNA Action: lncMREF recruits Smarca5/p300 to activate MyoD for myogenesis; circHIPK3 knockdown promotes autophagy and osteogenesis through the HuR/ATG16L1 axis; Feedback Regulation: MST1/2 inhibit YAP and TGF-β/SMAD signaling, thereby limiting fibroblast differentiation and renal fibrosis; Wnt7 signaling triggers Jagged1-Notch feedback regulating neurogenesis timing. The graphics were created with BioRender (https://www.biorender.com). DNMT3A/3B DNA (cytosine-5)-methyltransferase 3A/3B, TET ten-eleven translocation, HDAC3/5 histone deacetylase 3/5, TLX T-cell leukemia homeobox, lncMREF long non-coding muscle regeneration factor, HuR Hu antigen R, ATG16L1 autophagy related 16 like 1

At the epigenetic level, DNA methylation dynamics mediated by DNA methyltransferases (DNMTs) and ten-eleven translocation (TET) enzymes constitute a critical regulatory axis.⁹⁹ During differentiation, DNMT3A and DNMT3B catalyze the methylation of CpG islands at pluripotency gene promoters, such as OCT4 and NANOG, effectively silencing these loci to enable lineage specification.^100,101 Conversely, TET enzymes oxidize 5-methylcytosine to 5-hydroxymethylcytosine, a process essential for activating neurogenic loci during neuronal differentiation. Notably, TET enzyme regulation of DNA demethylation kinetics is critical for human cardiomyocyte growth, given that triple TET inactivation in human ESCs disrupts cardiac mesodermal patterning and blocks cardiomyocyte generation. Genome-wide analysis has shown that TET loss leads to promoter hypermethylation of Wnt inhibitors, induces aberrant Wnt signaling through NKX2-5 silencing, and impairs cardiac progenitor cell specification. TET activity is required for the demethylation of cardiac structural genes at specific stages during progenitor cell maturation.¹⁰² Histone acetylation, regulated by histone acetyltransferases and histone deacetylases (HDACs), further modulates chromatin accessibility. Additionally, HDACs regulate NSC function through the transcriptional repressor TLX, a key regulator of NSCs. HDAC3 and HDAC5 are recruited to the promoters of TLX target genes, leading to transcriptional repression of genes such as p21 and pten, which are involved in cell proliferation. Disruption of the TLX-HDAC interaction up-regulates p21 and pten expression, thereby inhibiting NSC proliferation. Lastly, HDAC inhibitors promote neuronal differentiation by upregulating genes such as NeuroD, Ngn1, and Math1, as well as promoting neuronal production in both adult and embryonic NSCs.¹⁰³

ncRNAs have emerged as indispensable regulators of differentiation through post-transcriptional and epigenetic mechanisms. The expression level of miR-134 is low in mouse ESCs but up-regulated during neural differentiation and down-regulated during induced pluripotent stem cell (iPSC) induction from NPCs. Inhibition of miR-134 enhances iPSC reprogramming efficiency, whereas its overexpression represses iPSC induction.¹⁰⁴ lncRNAs exhibit more complex regulatory roles. For example, lncMREF is a conserved positive regulator of muscle regeneration in multiple species, promoting myogenic differentiation and regeneration by interacting with Smarca5, which facilitates chromatin accessibility and recruits p300/CBP/H3K27ac to upregulate MyoD and other myogenic regulators, thereby enhancing muscle cell differentiation.¹⁰⁵ Interestingly, silencing circHIPK3 promotes osteogenesis in human bone marrow mesenchymal stem cells, while silencing linear mHIPK3 exerts no significant effects. Besides, silencing circHIPK3 enhances autophagic flux and thereby promotes osteogenesis, whereas autophagy inhibition attenuates bone formation. In addition, circHIPK3 interacts with HuR and ATG16L1 to modulate ATG16L1 expression, thereby influencing autophagy and bone generation.¹⁰⁶ Overall, these findings highlight the multi-layered regulatory capacity of ncRNAs in modulating differentiation-related pathways.

Feedback regulation ensures dynamic control over signaling intensity and duration. Mammalian sterile 20-like kinases 1/2 (MST1/2) act as negative feedback regulators of Yes-associated protein (YAP) and TGF-β/SMAD signaling, suppressing fibroblast differentiation and limiting renal fibrosis. Experimental studies in mice with unilateral ureteral obstruction showed that MST1/2 deletion or inhibition promoted myofibroblast accumulation and fibrosis.¹⁰⁷ Similarly, Celsr3 and FZD3 gene mutations affect the generation of neurons and glial cells during cortical development. Defective neurons in these mutants fail to activate Notch signaling in NPCs due to impaired Wnt7 signaling, leading to delayed neurogenesis and reduced gliogenesis. Celsr3 and FZD3 enable immature neurons to respond to Wnt7, upregulate Jagged1, and establish a negative feedback mechanism that optimizes NPC differentiation timing via Notch activation.¹⁰⁸

Emerging technologies such as CUT&Tag and ATAC-seq have revealed that these regulatory layers are highly coordinated. Earlier studies have leveraged scATAC-seq and CUT&Tag technologies to explore epigenetic landscape changes during early retinal cell differentiation, as well as identify enhancers, transcription factor motifs, and interactions critical for cell state-specific gene expression. Key transcription factors, including Otx2, Atoh7, Pou4f2, and Isl1, collaboratively and competitively regulate gene expression to drive the differentiation of retinal ganglion cells from retinal progenitor cells, revealing a general mechanism for neural cell type specification.¹⁰⁹ As multi-omics datasets continue to expand, systems-level integration of these mechanisms will be critical for elucidating pathological differentiation states in cancer and regenerative medicine.

Crosstalk and integration among signaling pathways

Cell differentiation is governed by a dynamic interplay of signaling pathways that integrate extracellular cues into precise transcriptional programs. While individual pathways such as Wnt, Notch, BMP, and FGF have well-characterized roles in lineage specification, their functional crosstalk and synergistic integration are critical for fine-tuning differentiation outcomes, ensuring tissue homeostasis, and preventing pathological states such as cancer or developmental disorders (Fig. 4).

Fig. 4.

Signaling crosstalk and integration in cell differentiation. In Lgr5+ liver organoids, Wnt agonism with RSPO1 supports hepatocyte differentiation, whereas combined inhibition of Wnt, Notch, and MAPK directs enteroendocrine differentiation in intestinal organoids; TWIST1 knockdown in human adipose-derived stem cells enhances osteogenesis via synergistic BMP and FGF/ERK activation upregulating TAZ; microenvironmental context modulates effects, e.g., BMP-2/FGF-2 in calvarial vs. femoral sites; Cardiogenesis is regulated by non-autonomous crosstalk: endocardial YAP/TAZ induces Nrg1, activating cardiomyocyte ErbB4; cleaved ErbB4-YAP complex drives proliferation; Non-canonical and epigenetic integration: CSP-TTK21 promotes p300-mediated histone acetylation activating neurogenic genes (e.g., NeuroD1); non-canonical Wnt11/Ror2 signaling modulates PAR-1 to regulate neural crest polarity and specification. The graphics were created with BioRender (https://www.biorender.com). Lgr5 leucine-rich repeat-containing G-protein coupled receptor 5, RSPO1 R-spondin 1, Nrg1 neuregulin 1, CSP-TTK21 cell-penetrating peptide-conjugated TTK21, NeuroD1 neurogenic differentiation 1, Ror2 receptor tyrosine kinase-like orphan receptor 2, PAR-1 kinase partitioning defective 1 kinase

Recent advances in single-cell transcriptomics, CRISPR-based pathway modulation, and live-cell imaging have revealed intricate mechanisms by which these pathways converge to regulate cell fate decisions. For instance, the Wnt/β-catenin and Notch pathways play important roles in cell differentiation. Lgr5 marks Wnt-driven self-renewing stem cells in tissues such as the small intestine, colon, and hair follicles. A 3D culture system enables the clonal expansion of single Lgr5⁺ liver progenitor cells into organoids that preserve tissue architecture. These organoids, cultured with the Wnt agonist RSPO1, can differentiate into functional hepatocytes upon transplantation into Fah^-/- mice.¹¹⁰ Concurrently, Lgr5⁺ adult intestinal stem cells are maintained by Wnt signaling and can be expanded into 3D organoids to investigate differentiation processes. Inhibiting EGFR or MAPK signaling induces a quiescence state in Lgr5⁺ cells, which can be reactivated to undergo differentiation into hormone-producing enteroendocrine cells. Combined inhibition of Wnt, Notch, and MAPK pathways efficiently generates diverse enteroendocrine cell subtypes in vitro.¹¹¹

Similarly, the BMP and FGF pathways synergistically regulate osteogenic differentiation. TWIST1 knockdown in human adipose-derived stem cells enhances osteogenic differentiation by endogenously activating BMP and ERK/FGF signaling pathways, which drive the upregulation of the osteogenic modulator transcriptional coactivator with PDZ-binding motif (TAZ). Mechanistically, the pro-osteogenic effects of TWIST1 silencing critically rely on TAZ, considering that co-silencing both TWIST1 and TAZ abolishes the differentiation enhancement, validating the role of TAZ as the downstream mediator of this regulatory cascade. Importantly, pharmacological inhibition of either BMP or FGF/ERK signaling suppresses TAZ expression and reverses the osteogenic boost, highlighting the interconnected roles of these pathways in mediating human adipose-derived stem cell differentiation.¹¹² Of note, BMP-2 and FGF-2 exhibit contrasting osteogenic differentiation effects depending on the bone marrow microenvironment. The former induces robust bone formation in extramedullary (marrow-scarce) calvarial defects and triggers bone resorption in medullary (marrow-abundant) femoral sites, whereas the latter exerts the opposite effect, promoting osteoblast activity in the femur without significantly impairing hematopoietic function.¹¹³

Pathway integration generally involves shared downstream effectors or epigenetic modifiers. For example, Hippo-YAP signaling coordinates cardiogenesis via Nrg1/ErbB4 crosstalk. In endocardial cells, Hippo pathway effectors YAP/TAZ drive Nrg1 expression, activating ErbB2/4 receptors on cardiomyocytes to promote proliferation. ErbB4 cleavage releases an intracellular domain that binds to YAP, forming a TEA domain transcription factor (TEAD) transcriptional complex that activates pro-proliferative genes. This dual Hippo-ErbB interaction integrates cell-autonomous and non-autonomous YAP signaling, orchestrating cardiac development and regeneration.¹¹⁴ Moreover, YAP/TAZ govern endothelial differentiation by driving actin remodeling and Myc-mediated proliferation/metabolism, which are essential for sprouting angiogenesis and vascular integrity. In contrast, their loss disrupts tip cell formation, junction assembly, and barrier function, resulting in hemorrhage and pathological neovascularization defects.¹¹⁵ These findings position Hippo-YAP/TAZ signaling as a pivotal regulator of vascular differentiation and therapeutic targets. Additionally, epigenetic regulators such as histone acetyltransferase p300 serve as nodal hubs for pathway convergence. The brain-permeable activator CSP-TTK21 enhances p300/CBP-mediated lysine acetylation, elevating the expression of key neurogenesis genes (e.g., NeuroD1, Tubb3) to drive dendritic maturation and functional neuron formation in the dentate gyrus. This epigenetic activation promotes the differentiation of neuronal progenitors into branched, doublecortin-positive neurons, linking the epigenetic role of p300 to synaptic function and action potential dynamics.¹¹⁶ Mounting evidence highlights the role of non-canonical signaling in pathway crosstalk. For instance, noncanonical Wnt signaling regulates neural crest differentiation through Wnt11-like proteins and Ror2/Dishevelled signaling, independent of β-catenin, as demonstrated by functional studies in Xenopus. Wnt11R, expressed in adjacent neuroectoderm, drives neural crest specification by modulating PAR-1 kinase activity, which is essential for cell polarity and differentiation, with PAR-1 RNA rescuing neural crest markers following pathway inhibition.¹¹⁷ Likewise, coordinated Wnt and TGF-β signaling in pluripotent stem cells shapes mesendodermal patterning and lineage commitment.¹¹⁸ Such a dynamic regulatory framework has been exploited in in vitro differentiation protocols.

Tissue-specific heterogeneity of signaling pathways

Cell differentiation, a biological process regulated at multiple levels, is highly dependent on tissue-specific reprogramming of signaling pathways for its fate decision. Although evolutionarily conserved pathways such as Notch, Wnt, and TGF-β constitute the central framework of differentiation regulation, their signaling outputs exhibit significant microenvironmental dependence. Tissue-specific differences in receptor heterodimer expression profiles, co-regulator libraries, and epigenetic landscapes collectively shape the temporal and spatial dynamics of the signaling pathway responses. This heterogeneity not only determines the diversity of differentiation programs induced by identical signaling molecules across various tissues but also ensures that differentiation is precisely adapted to the local physiological demands by establishing tissue-specific feedback regulatory networks.

The Notch signaling pathway governs cell fate determination through a conserved mechanism involving ligand-receptor binding (e.g., Delta-like ligands or Jagged family members to Notch1-4 receptors), which triggers sequential proteolytic cleavage and nuclear translocation of the NICD.¹¹⁹ In the nucleus, NICD recruits the CSL transcription factor complex to activate target genes such as Hes1 and Hey1, which orchestrate lineage-specific differentiation programs.¹²⁰ Notably, tissue-specific regulation of Notch signaling arises from microenvironmental ligand availability, receptor isoform expression, and cross-talk with other pathways. Jagged2/Notch1 signaling drives sebocyte differentiation in skin, as antibody blockade in mice depletes mature cells and expands progenitors, with reversibility confirming dynamic control. Notch1 enforces terminal differentiation by suppressing progenitor proliferation, thereby maintaining sebaceous gland homeostasis. This axis acts as a molecular switch balancing stem cell renewal with lineage commitment.¹²¹ Conversely, Notch1-Jagged1 cis-inhibition blocks differentiation to preserve hematopoietic stem cells in embryonic aortic clusters, while spatial attenuation of Notch activity accompanies hematopoietic stem cell maturation. Forced Notch1 activation overrides this inhibitory mechanism, driving lineage commitment through hematopoietic programs. This self-limiting mechanism spatially orchestrates hematopoietic stem cell maintenance and differentiation within arterial hematopoietic niches.¹²² Stromal cells further fine-tune Notch outputs by spatially presenting membrane-bound ligands. For example, thymic epithelial cells express Delta-like ligand 4 (DLL4) to drive T-cell specification.¹²³ Such tissue-specific regulatory networks illustrate the mechanism by which Notch signaling balances proliferation and differentiation across diverse contexts, with dysregulation implicated in pathologies ranging from atopic dermatitis to T-cell acute lymphoblastic leukemia.

The Wnt signaling cascade bifurcates into two principal branches, namely the canonical β-catenin-mediated pathway and β-catenin-independent non-canonical routes, exhibiting remarkable tissue-specific functional specialization. Wnt signaling promotes osteogenic differentiation via stage-specific synergies and antagonisms: synergizing with estrogen signaling to amplify matrix mineralization while counterbalanced by Notch-mediated β-catenin suppression. Hh primes osteoprogenitor commitment through RUNX2 induction upstream of Wnt, whereas Wnt remains critical in Osx1⁺ precursors to sustain osteoblast maturation and prevent chondrogenic diversion. Temporal Wnt dependency is indispensable, with its inhibition at any differentiation stage blocking terminal maturation, contrasting Hh’s dispensability after lineage initiation.¹²⁴ The Wnt pathway is partially heterogeneous regarding the differentiation of intestinal epithelial cells. Wnt/β-catenin signaling preserves intestinal stem cell plasticity by suppressing terminal differentiation, given that its ablation triggers rapid crypt collapse and forces stem cells into differentiation, thus disrupting epithelial homeostasis. This dual role is essential for tissue maintenance but becomes pathologically hyperactivated in 90% of colorectal cancer (CRC). A stromal-supported intestinal culture system enables sustained epithelial proliferation and multilineage differentiation, critically relying on Wnt activation for stem cell expansion and Notch modulation for secretory lineage specification.¹²⁵ This system faithfully replicates the Wnt-driven niche dynamics essential for balancing intestinal stem cell maintenance and differentiation.

The TGF-β superfamily regulates cellular differentiation through context-dependent integration of canonical SMAD-dependent and non-canonical signaling cascades. In immune regulation, SMAD7 suppresses nuclear factor-κB (NF-κB)-driven inflammation via dual mechanisms: stabilizing inhibitor of κBα to block nuclear translocation and directly inhibiting the transcriptional activity of NF-κB/p65. TGF-β/SMAD7 signaling further intersects with Wnt pathways through context-dependent stabilization or degradation of β-catenin, where SMAD7 recruits GSK-3β to enhance β-catenin proteolysis during inflammatory homeostasis while paradoxically promoting its stabilization during apoptosis induction. This cross-regulation extends to cell adhesion dynamics, as SMAD7 upregulates E-cadherin and suppresses β-catenin activation in carcinomas, countering EMT. The pleiotropic roles of SMAD7 in immune modulation and Wnt/TGF-β crosstalk demonstrate its therapeutic duality, balancing anti-inflammatory effects against risks of disrupting tissue integrity or oncogenic signaling.¹²⁶ On the other hand, mesenchymal lineages exhibit diametrically opposed responses. MBD2 drives fibroblast-to-myofibroblast differentiation in pulmonary fibrosis via epigenetic regulation, as its deletion attenuates TGF-β/SMAD3-mediated global DNA hypermethylation and halts fibrogenesis.¹²⁷ Mechanistically, TGF-β1 establishes a feedforward loop with SMAD3 to upregulate MBD2, which represses Erdr1 by binding methylated CpG islands, amplifying profibrotic signaling and differentiation.

Unifying regulatory principles of signaling pathways

Despite their structural diversity, differentiation-related signaling pathways share several unifying mechanistic principles that underlie context-dependent cell fate decisions. Recognizing these shared regulatory logics enables a systems-level understanding of differentiation signaling and may facilitate the development of therapeutic strategies that account for dynamic, tissue-specific pathway behavior rather than targeting isolated molecular components.

The conversion of graded morphogen inputs into binary cell fate decisions is driven by nonlinear dynamics and feedback architecture. Thresholding is achieved through molecular mechanisms such as oligomer binding¹²⁸, multisite phosphorylation¹²⁹, and stoichiometric inhibition¹³⁰, which generate sigmoidal signal-response curves that filter out low-level noise. The stability and permanence of these decisions are governed by specific regulatory motifs: positive feedback loops can induce bistability and hysteresis, allowing a transient signal to lock a cell into a new state even after the stimulus is removed.¹²⁸

Cellular signal transduction is increasingly recognized not as a static “on-off” switch but as a sophisticated, nonlinear process where information is encoded into specific temporal dynamical patterns. A unifying principle across these pathways is the evolutionary optimization of signal shapes—such as steep, gradual, or overshooting—to balance energetic efficiency with transmissional reliability.¹³¹ This temporal regulation allows cells to execute complex survival strategies, such as the Nrf2-mediated activation to induce transient cell cycle arrest during early oxidative stress.¹³² Ultimately, the geometry of a signal’s time-course—governed by the relaxation scales of the decoding network—serves as a critical selective pressure in the design of biological communication.

Oscillatory activity is a widespread and essential feature of cell signaling, moving beyond the traditional view of constant signal inputs to a dynamic framework that enables cells to self-organize in space and time. These rhythms, often driven by delayed negative feedback loops, allow for multiplexing information where different dynamical regimes code for distinct biological outcomes like differentiation or self-renewal.¹²⁸ In the Notch pathway, oscillations of the DLL1 and the core component Hes1 are critical for maintaining the progenitor state in neural and muscular stem cells, as well as coordinating the vertebrate segmentation clock.^133,134 The modulation of phase shifts between Wnt and Notch signaling is fundamental to mesoderm segmentation.¹³⁵

Feedback control is a fundamental feature of biological signaling networks, providing the robust homeostasis and high-fidelity transduction necessary for complex tissue development and cellular decision-making. Negative feedback often involves the transcriptional upregulation of pathway inhibitors, creating loops that limit signal duration and intensity to prevent pathological overactivation.¹³⁶ The Hippo pathway similarly employs feedback to regulate organ size and stem cell niches, where downstream effectors like YAP/TAZ promote the expression of their own negative regulators to maintain homeostatic set points.¹³⁷ In a mathematical model of hematopoietic stem-cell-niche interactions, bidirectional regulation exhibited proportional-integral feedback properties and accounted for recovery of the HSC compartment after irradiation but apparent lack of recovery after direct HSC ablation.¹³⁸

Mechanical signals are now recognized as fundamental regulators of conserved signaling pathways, acting as a physical “checkpoint” for biochemical activity. The Hippo pathway is perhaps the most documented, where high mechanical tension—sensed via substrate stiffness or cell spreading—promotes the nuclear translocation of YAP/TAZ.¹³⁹ This is regulated by mechanosensitive proteins like TRIP6 and LIMD1, which sequester Hippo kinases under force to prevent YAP phosphorylation and cytoplasmic retention.¹⁴⁰ Similarly, the Notch pathway utilizes a direct mechanosensing mechanism where the binding of a ligand on a neighboring cell provides the necessary pulling force to unfold the Notch receptor, exposing a cryptic cleavage site required for signal activation.¹⁴¹ Wnt and TGF-β signaling are frequently modulated at the cell-matrix interface; for instance, focal adhesion kinase (FAK) activation and integrin-mediated force transmission can tune the availability of TGF-β by mechanically releasing it from the ECM, highlighting a broad spectrum of mechanical inputs that can orchestrate complex gene expression programs across these pathways.¹⁴²

The behavior of adult stem cells is fundamentally governed by the niche, a specialized microenvironment that integrates structural, mechanical, and biochemical signals to regulate the balance between quiescence, self-renewal, and differentiation.^138,143 The Wnt pathway is a central regulator, promoting self-renewal in the intestine, skin, and bone marrow, often requiring R-spondins to drive expansion.^144,145 Notch signaling tunes these outcomes through contact-dependent decisions; for example, lung basal cells use Jagged2-Notch to maintain secretory progeny¹⁴⁶, while intestinal Notch activity prevents premature secretory commitment.¹⁴⁷ Hh signaling serves as a critical feedback regulator, such as in the hair follicle where progeny-derived SHh sustains stem-cell activation.¹⁴⁸

Pathological roles of cell differentiation-related signaling pathways and therapeutic advances

Cancer and tumorigenesis

Disruptions in normal differentiation processes are closely linked to cancer initiation and progression, leading to uncontrolled cell growth and tumor formation. Signaling pathways governing cell differentiation play crucial roles in these pathological events.¹⁴⁹ Unraveling the mechanisms by which these pathways are altered in cancer may provide insights into tumor biology and inform the development of targeted therapies (Table 1, Fig. 5).

Table 1.

Therapeutic advances of cell differentiation-related signaling pathways in cancer and tumorigenesis

Signaling pathway	Agents	Mechanism	Disease	Study phase	Therapeutic advances	Reference
Wnt signaling	LGK974	PORCN inhibitor	Head and neck squamous cell carcinoma	Preclinical	Show high response rate in head and neck cancer cell lines with loss-of-function mutations in the Notch signaling pathway.	159
	LGK974	PORCN inhibitor	Epithelial ovarian cancer	Preclinical	Inhibit tumor growth, prevent ascites formation, and prolong survival in mouse models.	160
	ETC159	PORCN inhibitor	Colorectal cancer	Preclinical	Reduce the expression of genes associated with cell cycle, stem cells, and proliferation, and increase differentiation markers.	161
	ETC159	PORCN inhibitor	Pancreatic cancer	Preclinical	Suppress the growth of Wnt-driven pancreatic cancer synergistically and potently both in vitro and in vivo.	162
	Vantictumab	FZD1/2/5/7/8 inhibitor	Breast, pancreatic, colon and lung cancer	Preclinical	Inhibit the growth of various tumors, reduce the frequency of tumor-initiating cells, and demonstrate synergistic activity when combined with standard-of-care chemotherapeutic agents.	163
	Ipafricept	Wnt signaling antagonist	Sarcoma, basal cell cancer, hepatocellular carcinoma, ovarian cancer, metastatic pancreatic cancer	Preclinical	Reduce tumor growth and decrease CSC frequency, both as a single agent and in combination with other chemotherapeutic agents.	164
	XAV939	Tankyrase inhibitor	Head and neck squamous cell carcinoma and colon cancer	Preclinical	Attenuate CSC progression and eliminate CSC-mediated chemoresistance.	165
	IWR-1	Tankyrase inhibitor	Osteosarcoma	Preclinical	Demonstrate synergistic cytotoxicity when combined with doxorubicin, reverse drug resistance, and decrease tumor progression in animal models.	165
	Trifluoperazine	Phenothiazine-like antipsychotic drug	Lung cancer	Preclinical	Inhibit CSC tumor spheroid formation, down-regulate CSC markers, and inhibit Wnt/β-catenin signaling.	165
	ONC201	Multi-pathway inhibitor	Prostate cancer and glioblastoma	Preclinical	Downregulate stem cell pathways like Wnt signaling, modulate key self-renewal genes, and inhibit self-renewal and CSC markers in prostate cancer and glioblastoma cells.	166
	FZD4 cDNA	Wnt pathway restoration	Lung cancer	Preclinical	Restore endothelial WNT/β-catenin signaling, normalize tumor vasculature, and inhibit tumor growth and metastasis in vivo.	167
TGF-β signaling	Vactosertib	TGFβRI kinase inhibitor	Non-MSI-high metastatic colorectal or gastric cancer	Phase Ib/IIa (NCT03724851)	Show a manageable safety profile and clinical activity in metastatic colorectal cancer when combined with pembrolizumab, with no objective responses observed in metastatic gastric cancer.	175
	Vactosertib	TGFβRI kinase inhibitor	Relapsed/refractory multiple myeloma	Phase Ib (NCT03143985)	Inhibit the growth and viability of human and murine MM cells, and achieve a PFS-6 of 82% when combined with pomalidomide in a preliminary efficacy assessment.	176
	SRK181-mIgG1	TGF-β1 inhibitor	Solid cancers	Preclinical	Overcome primary resistance to checkpoint inhibitors and show efficacy and safety when used in combination with immune checkpoint blockade.	177
	Fresolimumab	Pan-TGF-β neutralizing antibody	Advanced malignant melanoma or renal cell carcinoma	Phase I (NCT00356460)	Demonstrate acceptable safety and preliminary antitumor activity, including a partial response, in patients with advanced malignant melanoma or renal cell carcinoma.	178
	AVID200	TGF-β1/3 ligand trap	Myelodysplastic syndrome-associated anemia	Preclinical	Sequester TGF-β1/3 and relieve TGF-β-mediated suppression of hematopoietic progenitor growth in myelodysplastic syndrome-associated anemia.	179
	Bintrafusp alfa	TGF-β/PD-L1 inhibitor	Advanced, pretreated human papillomavirus-associated cancers	Phase I (NCT02517398)	Achieve a confirmed objective response rate of 30.5%, including five complete responses, with a disease control rate of 44.1%.	180
	Trabedersen	TGF-β2-specific antisense oligodeoxynucleotide	High-grade glioma, advanced pancreatic carcinoma, metastatic melanoma, metastatic colorectal carcinoma	Phase III (NCT00761280) Phase I/II (NCT00844064)	Show antitumor activity and tolerability in preclinical studies and advance to phase I/II and phase III clinical evaluation.	181
	Vigil vaccine	TGF-β inhibitor	Advanced ovarian cancer	Phase II (NCT01309230)	Show treatment-associated immune responses and exploratory recurrence-free survival benefits.	182
Hedgehog signaling	5E1	SHh antibody	Gastric cancer	Preclinical	Inhibit gastric cancer cell self-renewal capacity and chemoresistance.	187
	Vismodegib	SMO inhibitor	Advanced basal cell carcinoma	Phase II (NCT00833417)	Achieve investigator-assessed objective response rates of 48.5% in mBCC and 60.3% in laBCC, with durable responses and a median overall survival of 33.4 months in the mBCC group.	189
	Sonidegib	SMO inhibitor	Advanced basal cell carcinoma	Phase II (NCT01327053)	Achieve objective response rates of 56.1% in laBCC and 7.7% in mBCC, with median response durations exceeding 24 months and 2-year overall survival rates of 93.2% and 69.3%, respectively.	190
	GANT61	GLI inhibitor	Lung cancer, acute myeloid leukemia, rhabdomyosarcoma, and neuroblastoma	Preclinical	Show antitumor activity across multiple cancer types, induce G1 cell-cycle arrest, promote cytotoxic activity, and attenuate EMT to slow cell migration.	193
Notch signaling	RO4929097	γ-secretase inhibitor	Advanced solid tumors	Phase I (NCT01145456)	Show preliminary antitumor activity, including one partial response in nasopharyngeal cancer and prolonged stable disease in three patients.	200
	RO4929097	γ-secretase inhibitor	Advanced sarcoma	Phase Ib/II (NCT01154452)	Show no objective responses.	201
	Brontictuzumab	Notch1 inhibitor	Selected solid tumors	Phase I (NCT01778439)	Show preliminary efficacy in patients with adenoid cystic carcinoma and Notch1 pathway activation, including two unconfirmed partial responses and four cases of prolonged disease stabilization.	202
	Enoticumab	DLL4 inhibitor	Advanced solid tumors	Phase I (NCT00871559)	Show preliminary antitumor activity, with two patients achieving a partial response and 16 others showing stable disease.	203
	Demcizumab	DLL4 inhibitor	Solid tumors	Phase I (NCT00744562)	Show preliminary activity, including one unconfirmed partial response and disease stabilization or response in 16 evaluable patients treated with 10 mg/kg every other week.	204
	ABT-165	DLL4/VEGF inhibitor	Cancer	Preclinical	Induce significant tumor growth inhibition and outperform single-target antibodies in preclinical models.	205
Hippo signaling	Verteporfin	YAP inhibitor	Retinoblastoma	Preclinical	Inhibit the growth, proliferation, and viability of retinoblastoma cells.	213
	Verteporfin	YAP inhibitor	Gastric cancer	Preclinical	Inhibit the growth, proliferation, and viability of gastric CSCs.	214
	Dasatinib	Src inhibitor	Colon cancer	Preclinical	Show greater efficacy against β-catenin-active cancer cells than against β-catenin-inactive cells.	217
	AZD0530	Src inhibitor	Colorectal cancer	Preclinical	Inhibit YAP expression and reduce nuclear YAP accumulation.	218
	PP2	Src inhibitor	Colorectal cancer	Preclinical	Reduce YAP1 protein expression and promote intestinal epithelial differentiation in HT29 and Caco-2/15 cell models, supporting its use as a mechanistic Src-YAP modulator rather than an established therapy.	220
	PF-573228	FAK inhibitor	Diffuse gastric cancer	Preclinical	Show sensitivity to FAK inhibition and combined YAP and PI3K pathway blockade in RHOAY42C murine models.	221
	Defactinib	FAK inhibitor	Diffuse gastric cancer	Preclinical	Show sensitivity to FAK inhibition and combined YAP and PI3K pathway blockade in RHOAY42C murine models.	221
	VT3989	TEAD palmitoylation inhibitor	Mesothelioma and solid tumors	Phase I/II (NCT04665206)	Disrupt YAP/TAZ-TEAD transcriptional activity through TEAD palmitoylation inhibition and demonstrate early clinical activity in YAP/TAZ-activated solid tumors.	223
	IK-930	TEAD palmitoylation inhibitor	Advanced solid tumors	Phase I (NCT05228015)	Inhibit TEAD autopalmitoylation and downstream transcription and advance to phase I evaluation.	224
	IAG933	YAP-TEAD protein-protein interaction inhibitor	Mesothelioma and solid tumors	Preclinical	Disrupt YAP-TEAD transcriptional activity and show antitumor activity in Hippo-dependent and RAS-MAPK-altered cancer models.	225

PORCN porcupine O-acyltransferase, FZD Frizzled, CSC cancer stem cell, cDNA complementary deoxyribonucleic acid, Wnt wingless-related integration site, TGF-β transforming growth factor-β, TGFβRI transforming growth factor-β receptor type I, MM multiple myeloma, PFS-6 6-month progression-free survival, AML acute myeloid leukemia, PD-L1 programmed death-ligand 1, GM-CSF granulocyte-macrophage colony-stimulating factor, shRNA short hairpin RNA, SHh Sonic Hedgehog, SMO Smoothened, mBCC metastatic basal cell carcinoma, laBCC locally advanced basal cell carcinoma, GLI glioma-associated oncogene homolog, EMT epithelial-mesenchymal transition, DLL4 delta-like ligand 4, VEGF vascular endothelial growth factor, Src proto-oncogene tyrosine-protein kinase Src, YAP Yes-associated protein, TAZ transcriptional coactivator with PDZ-binding motif, FAK focal adhesion kinase, PI3K phosphoinositide 3-kinase, RAS-MAPK RAS-mitogen-activated protein kinase, TEAD TEA domain transcription factor

Fig. 5.

Therapeutic advances of cell differentiation-related signaling pathways in cancer and tumorigenesis. a Wnt: Targeting the Wnt cascade occurs at multiple levels, including using porcupine inhibitors (e.g., LGK974) to block ligand secretion, and tankyrase inhibitors (e.g., XAV939) to stabilize the destruction complex, promoting β-catenin degradation. b TGF-β: The pathway is targeted by neutralizing antibodies such as Fresolimumab, TGFβRI/ALK5 kinase inhibitors such as Vactosertib and Galunisertib, and immunotherapeutic approaches such as the Vigil vaccine. c Hippo: Modulation of the kinase cascade involves agents like verteporfin to inhibit YAP/TAZ-TEAD transcriptional activity or Src inhibitors (e.g., Dasatinib) to target upstream regulators. d Notch: Targeting includes γ-secretase inhibitors, such as RO4929097, together with receptor- or ligand-directed biologics that suppress Notch signaling. e Hedgehog: Therapeutic strategies include SMO inhibitors (e.g., Sonidegib, Vismodegib) to block membrane signaling, and GLI inhibitors (e.g., GANT61) to inhibit nuclear GLI transcription. The graphics were created with BioRender (https://www.biorender.com). PORCN porcupine O-acyltransferase, Src proto-oncogene tyrosine-protein kinase Src

Wnt signaling pathway

Pathological roles

In neoplastic transformation, aberrant Wnt signaling has been strongly associated with malignant progression through its effects on uncontrolled proliferation, differentiation defects, and sustenance of cancer stem cell (CSC) populations.^150,151

In numerous types of cancer, hyperactivation of the canonical Wnt/β-catenin pathway contributes to the accumulation of nuclear β-catenin, which binds to TCF/LEF transcription factors to drive the expression of oncogenic targets such as c-Myc and Cyclin D1.¹⁵² This aberrant signaling not only sustains the self-renewal capacity of CSCs but also impedes their differentiation into mature, non-proliferative cells.¹⁵³ For instance, in CRC, mutations in APC or β-catenin stabilize the latter, resulting in constitutive Wnt activation that maintains CSCs in an undifferentiated state while simultaneously promoting uncontrolled proliferation. Similarly, in hepatocellular carcinomas (HCC) and gastric adenocarcinomas, stabilizing mutations in β-catenin or destruction complex components mimic this mechanism, underscoring the conserved role of Wnt dysregulation across malignancies.¹⁵⁴

Furthermore, a critical consequence of Wnt signaling in cancer differentiation is its role in EMT, a process enabling cancer cells to acquire migratory and invasive abilities. Wnt/β-catenin directly upregulates EMT-related transcription factors, encompassing Snail1 and Snail2, which repress epithelial markers such as E-cadherin.¹⁵⁵ Non-canonical Wnt pathways, including Wnt/Ca²⁺ and Wnt/PCP, further promote metastasis by modulating cytoskeletal dynamics and calcium-dependent signaling.¹⁵⁶ For example, Wnt5a and Wnt11, ligands associated with non-canonical pathways, are frequently overexpressed in aggressive tumors and correlate with poor clinical outcomes due to their ability to promote cell migration and tissue invasion. These pathways foster a permissive environment for circulating tumor cells to evade differentiation signals and colonize distant organs.

Therapeutic advances

Porcupine inhibitors targeting PORCN effectively inhibit the post-translational modification of Wnt ligands, particularly their acylation, thereby minimizing Wnt ligand secretion.¹⁵⁷ LGK974, a PORCN inhibitor, has demonstrated significant efficacy in MMTV-Wnt1 mammary tumor models and human head and neck squamous cell carcinoma (HNSCC) models.^158,159 Meanwhile, LGK974 reduces epithelial ovarian cancer cell viability in vitro and suppresses tumor growth in vivo.¹⁶⁰ ETC159, an oral PORCN inhibitor, comprehensively inhibits Wnt secretion and activity, demonstrating robust therapeutic potential in CRC patient-derived xenograft models with RSPO translocations.¹⁶¹ According to a prior investigation, the combination of ETC159 with the PI3K inhibitor GDC-0941 inhibited RNF43-mutant pancreatic cancer cell proliferation and xenograft growth.¹⁶²

OMP-18R5 (vantictumab), a monoclonal antibody targeting FZD1/2/5/7/8, suppressed tumor growth in breast, pancreatic, colon, lung, and HNSCC xenograft models.¹⁶³ OMP-54F28 (ipafricept) shares a similar mechanism. Ipafricept demonstrates marked anti-tumor effects in MMTV-Wnt1 cancer models. Combined with gemcitabine, it significantly reduces CSC frequency in pancreatic patient-derived xenograft models and outperforms monotherapy.¹⁶⁴ Preclinical evidence indicated that sequential administration of ipafricept or vantictumab before taxane treatment enhances antitumor activity by overcoming taxane resistance and promoting mitotic catastrophe.¹⁶⁴

Furthermore, Wnt/β-catenin inhibitors show promise in targeting CSCs. LGK974 (Wnt974) suppresses mammary CSC proliferation. Niclosamide selectively inhibits ovarian CSCs by downregulating LRP6/β-catenin.¹⁶⁵ ONC201 inhibits CSC-related genes in prostate and glioblastoma tumors.¹⁶⁶ XAV939 attenuates CSC-mediated chemoresistance in HNSCC and colon cancer by inhibiting β-catenin. IWR-1 suppresses osteosarcoma CSC self-renewal and enhances doxorubicin sensitivity. Trifluoperazine inhibits lung CSC spheroid formation by downregulating Wnt signaling.¹⁶⁵

In addition to small-molecule inhibitors and monoclonal antibodies, gene-based strategies targeting the WNT–FZD axis have emerged as a promising means of restoring differentiation control. Endothelial-specific loss of FOXF1 in tumor-associated endothelial cells promoted lung tumor growth and metastasis by reducing FZD transcription and suppressing WNT/β-catenin signaling. Notably, nanoparticle-mediated endothelial delivery of FZD4 cDNA restored WNT signaling, normalized tumor vasculature, and inhibited lung cancer progression in vivo. These findings highlight the translational potential of cell-type-specific gene therapy to re-establish lineage stability within the tumor microenvironment (TME).¹⁶⁷

TGF-β signaling pathway

Pathological roles

In early tumorigenesis, TGF-β exerts tumor-suppressive effects by promoting cellular differentiation and apoptosis. Through canonical SMAD-dependent pathways, it induces cell cycle arrest by upregulating Cyclin-dependent kinase inhibitors and suppressing oncogenes such as c-Myc.¹⁶⁸ Simultaneously, TGF-β promotes apoptosis by modulating Bcl-2 family proteins and activating caspases, particularly in epithelial and hematopoietic cells.¹⁶⁹ These mechanisms maintain tissue homeostasis by eliminating premalignant cells or directing them into differentiation programs, as evidenced by their role in suppressing colorectal and HCC initiation.¹⁷⁰

However, advanced cancers frequently develop resistance to TGF-β‘s differentiation signals, hijacking the pathway to drive dedifferentiation and metastasis. Tumor cells frequently harbor mutations in TGF-β receptors or SMAD4, enabling them to bypass growth-inhibitory signals while exploiting non-canonical pathways such as MAPK/ERK and PI3K/AKT.¹⁷¹ This shift triggers EMT, wherein TGF-β upregulates transcription factors to repress epithelial markers (E-cadherin) and enhance migratory capacity. In aggressive breast and lung cancers, TGF-β interacts with stromal components to remodel the ECM via MMP-2/9 secretion, facilitating invasion. Notably, the pathway also induces the immunosuppressive differentiation of regulatory T cells (Tregs) and cancer-associated fibroblasts, establishing a permissive microenvironment for undifferentiated tumor stem cells.¹⁷²

Therapeutic advances

Some small molecule inhibitors can suppress the kinase activity of TGF-β receptor by binding to its ATP-binding domain, thereby blocking downstream signaling, and demonstrate significant potential in the treatment of cancer by inhibiting the TGF-β signaling pathway.¹⁷³

Vactosertib (TEW-7197) is a highly selective oral TGFβRI/ALK-5 kinase inhibitor that has been shown to inhibit tumor growth and progression in multiple cancer models. Specifically, it acts on tumor cells and their microenvironment through various mechanisms, including inhibiting tumor stem cell properties, preventing EMT, and reducing metastatic potential.¹⁷⁴ In a phase Ib/IIa study of patients with non-MSI-high metastatic colorectal or gastric cancer, vactosertib plus pembrolizumab demonstrated a manageable safety profile and clinical activity in metastatic colorectal cancer, whereas no objective responses were observed in gastric cancer.¹⁷⁵ In relapsed/refractory multiple myeloma, vactosertib combined with pomalidomide was evaluated in a phase Ib trial and showed preliminary therapeutic activity.¹⁷⁶

Overall, TGF-β receptor kinase inhibitors show great promise in cancer treatment, especially when used in combination with other treatment strategies, such as chemotherapy and immunotherapy. However, their clinical application remains challenging, and future studies are warranted to further explore their mechanisms and develop more effective combination treatment options.

TGF-β-directed antibodies can interfere with the binding of TGF-β ligands to their corresponding receptors, as well as block potential TGF-β activation. These two steps are essential for TGF-β to exert its pro-tumor and immunosuppressive effects.

SRK181-mIgG1, a potent and highly selective inhibitor of TGF-β1, has been shown to overcome primary resistance to immune checkpoint inhibitors in preclinical models.¹⁷⁷ Fresolimumab, a pan–TGF-β neutralizing monoclonal antibody targeting TGF-β1, TGF-β2, and TGF-β3, showed a partial response in a phase I clinical trial in melanoma patients.¹⁷⁸

AVID200 is an engineered TGF-β-ligand trap, which is formed from the fusion of the extracellular domain of TGFβR and the human Fc domain. It possesses antibody-like properties and displays 1000-fold greater selectivity for TGF-β1 and TGF-β3 compared to TGF-β2.¹⁷⁹ Bintrafusp alfa is an innovative bifunctional fusion protein formed by the fusion of an anti-PD-L1 antibody with the extracellular domain of TGFβRII that can simultaneously block TGF-β signaling and PD-L1-mediated immunosuppression.¹⁸⁰

Antisense oligonucleotides can specifically target and inactivate genes associated with the TGF-β pathway involved in cancer progression, particularly those genes that are challenging to inhibit using small molecules or monoclonal antibodies. AP12009 (Trabedersen) is an antisense oligonucleotide that specifically targets TGF-β2 and has been investigated in glioma and other solid tumors. In a randomized, active-controlled phase IIb study involving patients with recurrent and/or refractory high-grade gliomas, AP12009 did not significantly improve the 6-month tumor-control rate overall; however, a prespecified analysis showed improved 14-month tumor control with 10 μM AP12009 in the anaplastic astrocytoma subgroup compared with standard chemotherapy. A subsequent phase III study was initiated but terminated early, whereas intravenous AP12009 was assessed in a phase I/II dose-escalation study in patients with advanced pancreatic carcinoma, metastatic melanoma, or metastatic colorectal carcinoma.¹⁸¹

The Vigil vaccine is derived from the patient’s tumor cells, which are transfected in vitro with a plasmid encoding the immune-stimulating cytokine GM-CSF and a bifunctional short hairpin ribonucleic acid designed to specifically reduce the expression of furin, a key converting enzyme responsible for producing TGF-β1 and TGF-β2. A phase II study evaluated Vigil as maintenance therapy in patients with advanced ovarian cancer and reported treatment-associated immune responses and exploratory recurrence-free survival benefits.¹⁸²

Hedgehog signaling pathway

Pathological roles

Aberrant activation of the Hh pathway, either through mutations in key components or ligand-dependent mechanisms, drives tumorigenesis by maintaining cancer cells in an undifferentiated, stem-like state. For instance, in basal cell carcinoma (BCC) and medulloblastoma, constitutive Hh activation via PTCH1 mutations sustains CSC self-renewal and resistance to differentiation signals, a mechanism validated in both human tumors and murine models.¹⁸³

The pathway’s impact on differentiation extends beyond cell-autonomous effects to reshape the TME. Hh ligands secreted by cancer cells engage stromal components such as cancer-associated fibroblasts and immune cells through paracrine signaling, creating an environment that perpetuates dedifferentiation. In triple-negative breast cancer, Hh activation promotes immunosuppressive Treg differentiation while suppressing pro-inflammatory Th17 cells, effectively maintaining an immunologically “cold” TME. This stromal reprogramming is mediated by metabolic remodeling, where Hh inhibition limits Treg bioenergetic capacity and shifts the balance toward Th17-mediated antitumor immunity. Similarly, in HCC, Hh signaling stabilizes PD-L1 expression via GLI1 activation, linking pathway activity to immune evasion and poor differentiation.^184,185

Therapeutic advances

Overexpression of SHh leads to hyperactivation of the Hh signaling pathway and is positively correlated with tumor burden in nearly all cancer types.¹⁸⁶ Therefore, targeting SHh presents an attractive therapeutic strategy. SHh-targeting inhibitors include the monoclonal antibody 5E1 and the Hh-interacting protein.

5E1 is a monoclonal anti-SHh antibody that blocks the binding of SHh to PTCH1. It suppresses the expression of Hh pathway target genes, including Cyclin D1, in CRC cell lines while restoring epithelial-like characteristics. Studies in gastric CSCs demonstrate that 5E1 significantly inhibits self-renewal capacity and chemoresistance.¹⁸⁷ Additionally, 5E1 has been demonstrated to enhance the efficacy of radiotherapy and cisplatin in cervical cancer.

Smoothened (SMO), a critical transmembrane signaling protein in the Hh pathway, is inhibited by PTCH1 in the absence of Hh ligands, preventing its translocation to primary cilia. This inhibition leads to the sequestration and degradation of GLI transcription factors by the suppressor of fused in the cytoplasm. Upon Hh ligand binding, PTCH1-mediated inhibition is relieved, allowing SMO to become activated within the primary cilia. Activated SMO promotes the release of GLI transcription factors, which then translocate to the nucleus and initiate transcription of target genes that promote cell proliferation, survival, and stemness. SMO inhibitors directly bind to SMO, block downstream signaling, and suppress GLI activation, thereby inhibiting tumor growth and metastasis, rendering them promising anticancer agents.¹⁸⁸

In cancer treatment, SMO inhibitors have demonstrated potential across various cancer types. Vismodegib, the first FDA-approved SMO inhibitor, achieved investigator-assessed objective response rates of 48.5% in metastatic BCC and 60.3% in locally advanced BCC.¹⁸⁹ Sonidegib, another SMO inhibitor, exhibited an objective response rate of 57% in locally advanced BCC.¹⁹⁰

Despite these promising outcomes, resistance remains a key challenge. In a small cohort of 28 patients with advanced BCC receiving continuous vismodegib, 6 patients (21%) developed at least one tumor regrowth during treatment.¹⁹¹ Overcoming acquired resistance remains crucial for expanding the clinical utility of SMO inhibitors.

GLI inhibitors target the terminal effectors of the Hh pathway—GLI transcription factors (GLI1, GLI2, and GLI3). In the canonical Hh pathway, GLI activation depends on upstream SMO signaling; however, in non-canonical pathways, GLI can be activated independently through crosstalk with other signaling cascades. GLI inhibitors disrupt Hh signaling through multiple mechanisms. For example, GANT61 binds to the zinc finger domain of GLI proteins, thereby blocking their DNA binding and transcriptional activity.¹⁹² These inhibitors suppress tumor cell proliferation, induce cell cycle arrest, activate apoptotic pathways, inhibit EMT, and downregulate stemness-associated genes (NANOG, SOX2, and OCT4), thereby reducing tumor aggressiveness and therapeutic resistance.¹⁹³

Notch signaling pathway

Pathological roles

The Notch signaling pathway plays a context-dependent role in cancer differentiation, functioning as either a tumor suppressor or an oncogene across different malignancies. In gastric cancer (GC), hyperactivation of Notch3, driven by epigenetic silencing of the tumor-suppressive miRNAs miR-491-5p and miR-875-5p, promotes dedifferentiation by upregulating PHLDB2 and sustaining AKT-mTOR signaling. This process maintains cancer stem-like properties and contributes to chemoresistance.¹⁹⁴ Similarly, in CRC, Notch3 overexpression correlates with tumor recurrence and promotes stemness by directly activating RNA-binding protein Musashi homolog 1, a marker of undifferentiated epithelial cells. This oncogenic role also extends to pancreatic ductal adenocarcinoma, where Notch2 interacts with mutant K-RAS to reprogram acinar cells into duct-like progenitors, thereby accelerating dysplastic progression.¹⁹⁵ Paradoxically, Notch signaling can act as a tumor suppressor. In prostate cancer, it inhibits androgen receptor activity through Hey1, potentially restraining differentiation-resistant tumor growth. In contrast, loss of Notch signaling in lung and skin cancers promotes malignant transformation.¹⁹⁶

Notch’s influence on differentiation is intricately linked to its modulation of the TME. For example, in non-small cell lung cancer (NSCLC), Notch signaling polarizes tumor-associated macrophages toward an antitumor M1 phenotype by suppressing suppressor of cytokine signaling 3 and the Wnt/β-catenin pathways, indirectly promoting differentiation. Moreover, the pathway regulates NK cell maturation: While DLL1-mediated Notch signaling enhances NK cytotoxicity by upregulating CD16 and KIRs, its downregulation in GC and patients with NSCLC compromises antitumor immunity, enabling the proliferation of undifferentiated cancer cells. These dual roles highlight Notch’s capacity to either enforce or subvert differentiation programs depending on cellular crosstalk within the TME.¹⁹⁷

Therapeutic advances

γ-secretase inhibitors (GSIs), initially developed for treating Alzheimer’s disease (AD), are now widely investigated as anticancer drugs due to their ability to inhibit Notch signaling activation.^198,199 In a phase I study, RO4929097 combined with gemcitabine showed acceptable tolerability and limited preliminary antitumor activity in patients with advanced solid tumors.²⁰⁰ A phase Ib/II trial evaluating the combination of RO4929097 and vismodegib in advanced sarcoma reported no objective responses.²⁰¹

Brontictuzumab (OMP-52M51), a monoclonal antibody targeting Notch1, effectively suppresses Notch activation. In a phase I trial, 48 patients with solid tumors tolerated brontictuzumab well at the maximum tolerated dose.²⁰² Enoticumab (REGN421), a human IgG1 antibody targeting DLL4, blocked Notch signaling and demonstrated tolerability and partial responses in phase I trials for solid tumors.²⁰³ Demcizumab (OMP-21M18), a humanized anti-DLL4 IgG2 mAb, indicated antitumor activity in pretreated solid tumors. In a phase I dose-escalation and expansion study, demcizumab showed manageable tolerability at doses ≤5 mg/kg weekly, whereas prolonged treatment at higher doses was associated with an increased risk of congestive heart failure. Preliminary antitumor activity was observed, mainly as disease stabilization and tumor shrinkage in patients with previously treated solid tumors.²⁰⁴ ABT-165, a dual-targeting DLL4/VEGF immunoglobulin, demonstrated preclinical efficacy, while navicixizumab demonstrated modest activity and adverse events in phase I solid tumor trials.²⁰⁵

Hippo signaling pathway

Pathological roles

The Hippo pathway, through its core kinase cascade (MST1/2-LATS1/2) and downstream effectors YAP/TAZ, plays multifaceted roles in tumorigenesis.^206,207 Dysregulation of Hippo signaling, particularly aberrant YAP activation, promotes tumor-cell proliferation and cell-cycle progression. For instance, Sekido et al. identified YAP activation in more than 70% of cases of malignant mesothelioma. They reported that this activation enhanced the transcription of positive cell cycle regulators, such as CCND1 and FOXM1, thereby driving the proliferation of malignant mesothelioma cells.^208,209

Besides, the Hippo signaling pathway can induce local invasion and distant metastasis, which enables cancer cells to spread into other tissues and contribute to cancer-related mortality. Previous studies have outlined that the dysregulation of the Hippo signaling pathway is a significant contributor to metastasis in various types of cancer. For example, Lamar et al. noted that in breast cancer and melanoma, constitutive YAP activation induced lung metastases in mice and upregulated transcription factors such as TEAD, which in turn promoted the expression of genes involved in cell movement and invasion.²¹⁰ Additionally, YAP activation has been linked to anoikis resistance, a significant feature for metastatic cells to survive detachment from the primary tumor site.²¹¹ Collectively, these findings highlight the crucial role of the Hippo pathway in cancer metastasis and suggest that targeting YAP could be a promising therapeutic method to inhibit metastatic progression.

Therapeutic advances

As a downstream effector of the Hippo signaling pathway, YAP forms the YAP-TEAD complex by binding with TEAD transcription factors to activate the transcription of genes involved in cell proliferation and metastasis. Disrupting the formation of this complex has emerged as an important strategy to inhibit Hippo signaling and offers a promising method for cancer treatment.²¹²

Verteporfin, a benzoporphyrin derivative originally employed as a photosensitizer in ophthalmic treatments, has been found to inhibit cancer cell proliferation and tumor growth by binding to YAP and changing its conformation, thereby blocking its interaction with TEAD.²¹³ In GC cell lines and xenotransplantation models, Verteporfin reduces the expression of YAP-TEAD downstream target genes, including CTGF and CYR61, and induces cell cycle arrest.²¹⁴

Bromodomain and extra-terminal domain protein (BET) inhibitors target BRD4 and other members of the BET protein family and represent a promising class of anti-YAP/TAZ therapeutics. Because YAP/TAZ can recruit BRD4 to chromatin, BET inhibitors may exert anticancer effects by disrupting this interaction and thereby inhibiting the Hippo pathway signaling. Studies have indicated that BET inhibitors can suppress YAP/TAZ-driven tumorigenesis in vivo. Several clinical trials of BET inhibitors have been initiated, although only a few have been completed. Due to limited and inconsistent data, no definitive conclusions can yet be drawn, highlighting the need for further clinical research to clarify the therapeutic potential of BET inhibitors.²¹⁵

YAP activity is influenced by various factors, including cell polarity, adhesive junctions, cytoskeleton, mechanical forces, G-protein-coupled receptor ligands, and certain stress signals. Moreover, cell adhesion to the ECM affects YAP activity. Therefore, inhibition of proteins associated with these regulatory factors may modulate YAP function. One potential method for inhibiting YAP is the use of Src inhibitors. Src is a proto-oncogene tyrosine kinase involved in cell adhesion and movement. Dasatinib, a multi-target kinase inhibitor approved by the FDA for the first-line treatment of chronic myeloid leukemia, has been shown to significantly upregulate YAP phosphorylation by inhibiting the Src-LATS pathway. This promotes YAP retention in the cytoplasm, reduces YAP-TEAD binding, and thereby weakens YAP’s transcriptional activity.^216,217 Additionally, selective Src-family kinase inhibitors have been used as preclinical tools to modulate YAP activity. AZD0530 suppresses Src-dependent YAP activation in the gp130–Src–YAP axis, whereas PP2 reduces YAP1 protein expression and promotes intestinal epithelial differentiation in HT29 and Caco-2/15 cell models.^218–220

FAK inhibitors can also inhibit YAP through a similar mechanism. FAK is a central component of focal adhesions and regulates cytoskeletal organization and associated signaling proteins. In GC, FAK activation contributes to cancer development and metastasis by promoting cell proliferation, migration, invasion, and adhesion. Zhang et al.‘s study demonstrated that FAK inhibitors PF-573228 and Defactinib could decrease YAP protein levels in the GC cell line SNU-719.²²¹ In an in vitro study on CRC by Tan et al., FAK inhibitors increased phosphorylated YAP levels and decreased mRNA and protein expression of CSC markers CD133, ALDH1, and Lgr5.²²² Moreover, the FAK inhibitor PF573228 can effectively prevent the nuclear accumulation of YAP in transitional amplification cells.²¹⁹

Recent advances have increasingly focused on TEAD-centered inhibitors targeting the palmitoylation pocket, which is essential for TEAD stability and YAP/TAZ binding. VT3989, a small-molecule TEAD palmitoylation inhibitor, disrupts YAP/TAZ–TEAD transcriptional activity and has advanced into phase I/II clinical trials in mesothelioma and other YAP/TAZ-activated solid tumors, demonstrating early signs of clinical activity.²²³ IK-930 is an orally bioavailable TEAD palmitoylation inhibitor that suppresses TEAD autopalmitoylation and downstream transcriptional programs. It was evaluated in a first-in-human phase I study in patients with advanced solid tumors.²²⁴ Distinct from palmitoylation blockade approaches, IAG933 directly disrupts the YAP-TEAD protein–protein interaction and has demonstrated potent antitumor activity in Hippo-dependent and RAS-MAPK-altered cancers.²²⁵ Structure-based covalent TEAD inhibitors further validate the palmitoylation pocket as a druggable vulnerability within the Hippo pathway.²²⁶ Emerging PROTAC-based degraders targeting YAP or TEAD represent an additional approach to achieve sustained suppression of Hippo-driven transcriptional programs.

Overall, targeting cell adhesion and cytoskeletal components, particularly through Src and FAK inhibitors, is a promising method for cancer treatment. These inhibitors reduce YAP transcriptional activity by promoting its phosphorylation and cytoplasmic retention, thereby inhibiting cancer cell proliferation and metastasis.

Cardiovascular diseases

Cardiovascular diseases (CVDs), including hypertension, heart failure (HF), atherosclerosis, stroke, and ischemic heart diseases, continue to be a significant cause of death worldwide. Cell differentiation plays a crucial role in CVD pathogenesis.^227,228 Dysregulation of normal differentiation pathways in endothelial cells, cardiomyocytes, fibroblasts, or smooth muscle cells can exacerbate disease progression.²²⁹ Consequently, therapeutic strategies aimed at modulating cell differentiation hold significant potential to promote regeneration and mitigate tissue damage in CVDs (Table 2, Fig. 6).

Table 2.

Therapeutic advances of cell differentiation-related signaling pathways in cardiovascular diseases

Signaling pathway	Agents	Mechanism	Disease	Study phase	Therapeutic advances	Reference
Wnt signaling	SFRP-2	Wnt signaling inhibitor	Myocardial disorders and ischemic heart injury	Preclinical	Bind Wnt3a and inhibit its ability to induce caspase and transcriptional activities.	233
	XAV939	Tankyrase inhibitor	Myocardial infarction	Preclinical	Reduce myocardial infarct size, improve cardiac function, suppress fibrosis and apoptosis, and promote angiogenesis.	234
	CHIR99021	GSK-3 inhibitor	Myocardial infarction	Preclinical	Generate MSC-like cells from hiPSCs with TGF-β induction, improve cardiac function, increase angiogenesis, and reduce scar size in an AMI rat model.	236
TGF-β signaling	Galunisertib	TGFβRI inhibitor	Myocardial infarction	Preclinical	Improve long-term cardiac function and reduce cardiac fibrosis by inhibiting TGF-β-dependent fibroblast activation.	238
Hedgehog signaling	Vismodegib	SMO inhibitor	Atherosclerosis	Preclinical	Ameliorate lipid deposition and oxidative stress in plaques without affecting circulating cholesterol levels.	242
Notch signaling	Jagged1 peptide mimic hydrogel	Notch agonist	Myocardial infarction	Preclinical	Improve cardiac function and angiogenesis, reduce fibrosis, and promote cardiomyocyte proliferative responses after intramyocardial delivery in a rat myocardial infarction model.	246

SFRP secreted frizzled-related protein, GSK-3 glycogen synthase kinase 3, MSC mesenchymal stem cell, hiPSC human induced pluripotent stem cell, AMI acute myocardial infarction, TGF-β transforming growth factor-β, TGFβRI transforming growth factor-β receptor type I, SMO Smoothened

Fig. 6.

Therapeutic advances of cell differentiation-related signaling pathways in cardiovascular diseases. a Wnt: Targeting employs inhibitors (e.g., XAV939) and modulators (e.g., SFRP-2/5, CHIR99021) to regulate β-catenin stability and its downstream transcriptional activity. b TGF-β: Interventions include antisense oligonucleotides targeting TGF-β expression and small-molecule TGFβRI inhibitors such as Galunisertib to modulate fibrotic responses and tissue repair. c Hippo: Preclinical genetic modulation of Hippo–YAP signaling has been explored to enhance cardiomyocyte proliferation and cardiac repair. d Notch: Therapeutic modulation includes localized Notch activation strategies, such as Jagged1 peptide mimic hydrogel, to support cardiac repair after myocardial infarction. e Hedgehog: Preclinical SMO inhibition with vismodegib suppresses downstream GLI-mediated signaling in cardiovascular disease models. The graphics were created with BioRender (https://www.biorender.com). SFRP secreted frizzled-related protein

Wnt signaling pathway

Pathological roles

Wnt signaling regulates critical cellular processes in cardiomyocytes, endothelial cells, and smooth muscle cells, which are essential for heart and vascular structure and function.²³⁰ In cardiac regeneration, the Wnt signaling pathway is essential for stem cell differentiation into cardiomyocytes. Muneer et al. demonstrated that inhibiting Wnt signaling promotes the differentiation of mesenchymal stem cells (MSCs) into cardiac progenitor cells. This process involves upregulation of cardiac-specific genes and proteins, resulting in the formation of functional cardiomyocytes. These pre-differentiated MSCs showed improved survival, homing, and integration into the infarcted myocardium, significantly improving cardiac function in a rat model of myocardial infarction.²³¹

Moreover, the Wnt signaling pathway is involved in the development of coronary artery atherosclerosis. Wnt3a, Wnt5a, and Wnt5b are upregulated in diseased coronary arteries and contribute to plaque development by promoting vascular smooth muscle cell (VSMC) proliferation, movement, and calcification. Specifically, Wnt3a activates the canonical pathway, driving VSMC proliferation, while Wnt5a and Wnt5b, which serve through the non-canonical pathway, induce inflammation and foam cell formation. These processes collectively contribute to the formation and destabilization of atherosclerotic plaques.²³²

Therapeutic advances

The Wnt/β-catenin signaling pathway has dual roles in cardiovascular development and disease, contributing to myocardial repair while also being involved in pathological processes, including myocardial fibrosis and atherosclerosis. Therapeutic methods targeting this pathway primarily modulate its activity by inhibiting or activating specific components. SFRP proteins, including SFRP-2 and SFRP-5, modulate Wnt signaling by binding Wnt ligands and attenuating downstream pathway activity. In particular, SFRP-2 has been reported to protect cardiac cells from apoptosis by blocking canonical Wnt3a signaling.²³³ XAV939 is a tankyrase inhibitor that improves the activity of the β-catenin-degrading complex by stabilizing the Axin protein and inhibiting classical Wnt signaling.²³⁴ In animal models, XAV939 has been indicated to alleviate myocardial hypertrophy and fibrosis, thereby reducing the risk of cardiac remodeling.²³⁵

CHIR99021, a GSK-3 inhibitor that activates Wnt/β-catenin signaling, has been used to support cardiovascular repair in preclinical models. In one study, combined CHIR99021 and TGF-β induction generated MSC-like cells from hiPSCs, and repeated intraperitoneal administration of these hiPSC-derived MSCs improved cardiac function, increased angiogenesis, and reduced scar size in a rat model of acute myocardial infarction.²³⁶

TGF-β signaling pathway

Pathological roles

The TGF-β signaling pathway plays a pivotal role in CVDs, influencing key processes, including fibrosis and atherosclerosis. TGF-β1, a central cytokine in fibrosis, stimulates fibroblast activation and their transformation into myofibroblasts, which produce ECM components. Reactive oxygen species and NOX enzymes, particularly NOX4, facilitate TGF-β-induced fibroblast activation. TGF-β signaling is also implicated in atherosclerosis by regulating endothelial and smooth muscle cell functions, such as proliferation, movement, and ECM production. Dysregulated TGF-β signaling can promote plaque formation and instability, thereby exacerbating atherosclerotic complications.²³⁷

Therapeutic advances

Current therapeutic methods focus on disrupting pathological TGF-β signaling, while preserving its essential homeostatic functions. Small-molecule inhibitors such as galunisertib (LY2157299), which selectively target TGFβRI kinase activity, have demonstrated efficacy in preclinical models of myocardial infarction by reducing fibrosis and improving cardiac function.²³⁸ Emerging methods include antisense oligonucleotides to suppress TGF-β1 mRNA expression as well as gene therapy vectors that deliver soluble TGF-β decoy receptors.²³⁹

Hedgehog signaling pathway

Pathological roles

The Hh signaling pathway plays a crucial role in various CVDs, including vascular calcification (VC) and ion channel regulation. VC is a common disorder in patients with chronic kidney disease and is associated with higher death rates. Huang et al. have indicated that the Hh signaling pathway is significantly involved in the pathogenesis of VC. Specifically, differentially expressed genes in VC are enriched in processes, such as ossification, osteoblast differentiation, and the Hh signaling pathway. In vitro studies have demonstrated that the SMO agonist markedly alleviates VSMC calcification, whereas the Hh signaling inhibitor cyclopamine significantly exacerbates VC.²⁴⁰

Moreover, the Hh signaling pathway is crucial for regulating ion channels, which are relevant to CVDs. In the study by Li et al., SHh was demonstrated to enhance calcium oscillations in hippocampal astrocytes, potentially influencing calcium channel regulation in cardiovascular contexts. The Hh signaling pathway has also been associated with the regulation of ion channels such as TRPV1, which play a significant role in atherosclerosis and the formation of macrophage-foam cells.²⁴¹

Therapeutic advances

Pharmacological inhibition of the Hh pathway has revealed paradoxical advantages in early atherosclerosis. The SMO inhibitor Vismodegib, originally developed for cancer treatment, was found to reduce plaque formation and lipid deposition in apoE-deficient mice by improving macrophage autophagy and cholesterol efflux. This mechanism appears to function independently of lipid-lowering effects, as Vismodegib treatment did not alter circulating cholesterol levels but did specifically promote autophagosome maturation in foam cells. The drug’s ability to accelerate lipid droplet clearance through autophagy-lysosomal pathways positions it as a promising candidate for treating lipid metabolism diseases in cardiovascular disorders.²⁴²

Notch signaling pathway

Pathological roles

During heart development, Notch ligands and receptors are expressed sequentially, which is crucial for myocardial patterning and cardiac valve formation. Specifically, Jagged1 and DLL4 are essential for EMT and valve development, while Notch1 and Notch4 are required to maintain endothelial function and stabilize blood vessels.^243,244

Moreover, mutations in Notch family genes are associated with a wide range of congenital heart defects. Guida et al. identified that Notch1 mutations are associated with bicuspid aortic valve and aortic valve disorder, whereas Jagged1 mutations are linked to Tetralogy of Fallot and Alagille syndrome. These mutations often lead to incomplete penetrance and variable expression, highlighting the complexity of Notch signaling in cardiovascular development.²⁴⁵

Therapeutic advances

The Notch signaling pathway plays a crucial role in cardiomyocyte survival, angiogenesis, and cardiac repair. Its aberrant activation or suppression can result in abnormal vascular remodeling or impaired myocardial regeneration. DLL4-Notch inhibition modulates angiogenesis, though chronic DLL4 blockade may cause cardiovascular toxicity including heart failure and pulmonary hypertension.

A preclinical study further suggested that localized activation of Notch signaling may support cardiac repair after myocardial infarction. Intramyocardial delivery of a Jagged1 peptide mimic hydrogel activated Notch1 signaling in the infarcted myocardium, improved cardiac function, increased neovascularization, reduced fibrosis, and promoted cardiomyocyte proliferative responses in a rat myocardial infarction model. This biomaterial-based strategy highlights the therapeutic potential of spatially controlled Notch activation, although its efficacy and safety remain to be validated in clinical studies.²⁴⁶

Hippo signaling pathway

Pathological roles

Wang et al. observed that YAP expression significantly decreases during the differentiation of cardiovascular progenitor cells into SMCs. Moreover, YAP overexpression in cardiovascular progenitor cells reduces the efficiency of their differentiation into SMCs. This effect may occur because YAP competes with Nkx2.5 for binding to Myocardin, thereby inhibiting the SMC differentiation process.²⁴⁷

Additionally, the Hippo signaling pathway participates in the differentiation of the epicardium into coronary arteries. Singh et al. indicated that inhibition of YAP and TAZ impairs EMT in the epicardium, reduces epicardial cell proliferation, and hinders their differentiation into coronary endothelial cells. YAP and TAZ may regulate Tbx18 and Wt1 expression to control epicardial cell behavior and influence coronary vascular development.²⁴⁸

Therapeutic advances

Existing drug development efforts focus on multi-level modulation of the Hippo pathway, such as small-molecule inhibitors of MST1/2 and gene therapies targeting YAP/TAZ transcriptional activity. Preclinical genetic studies have shown that attenuation of the Hippo kinase cascade can enhance cardiomyocyte proliferation and cardiac regeneration.^249,250 However, challenges remain in achieving tissue-specific targeting, prompting innovations in nanoparticle-based delivery systems to reduce off-target effects. Besides, the discovery of post-translational modifications regulating LATS1/2 kinase activity—including phosphorylation and ubiquitination—has opened novel avenues for precision therapeutics. As research advances, temporal regulation of Hippo–YAP signaling across acute injury and chronic remodeling is expected to guide translational methods, potentially revolutionizing the treatment of ischemic cardiomyopathy and other intractable cardiovascular conditions.^251,252

Neurodegenerative diseases

Neurodegenerative diseases (NDDs) are a group of diseases characterized by the progressive degeneration and death of neurons in the central nervous system, resulting in cognitive, motor, and sensory impairments. Common NDDs include AD, Parkinson’s disease (PD), Huntington’s disease, amyotrophic lateral sclerosis, and multiple sclerosis.^253,254 Cell differentiation plays a crucial role in the pathology of NDDs and provides potential for developing therapeutic strategies (Table 3, Fig. 7). By regulating the differentiation of NSCs to replace damaged neurons, it may be possible to alleviate symptoms of the disorder. However, significant challenges remain, including the management of inflammatory responses and precise directional control of differentiation, both of which are key areas of ongoing research.²⁵⁵

Table 3.

Therapeutic advances of cell differentiation-related signaling pathways in neurodegenerative diseases

Signaling pathway	Agents	Mechanism	Disease	Study phase	Therapeutic advances	Reference
Wnt signaling	Atrial natriuretic peptide	Wnt signaling agonist	Parkinson’s disease	Preclinical	Exert neuroprotective effects through Wnt pathway activation and protect neuronal cells from PD-like neurotoxic insults.	261
	DKK1-neutralizing antibodies	DKK1 inhibitor	Alzheimer’s disease	Preclinical	Suppress Aβ-induced synapse loss in mouse brain slices and counteract DKK1-associated synaptic impairment.	262
Notch signaling	Uscharin	Notch inhibitor	Alzheimer’s disease and Parkinson’s disease	Preclinical	Reduce Hes1 and Hes5 expression and promote neuronal differentiation by accelerating NICD degradation.	275
	Quinic acid	Notch pathway modulator	Neurodegenerative diseases	Preclinical	Promote NSC proliferation, maintenance, migration, and differentiation into mature neurons through modulation of Notch-associated signaling.	276

PD Parkinson’s disease, DKK1 Dickkopf-related protein 1, Aβ amyloid-β, Hes hairy and enhancer of split, NICD Notch intracellular domain, NSC neural stem cell

Fig. 7.

Therapeutic advances of cell differentiation-related signaling pathways in neurodegenerative diseases. a Wnt: Therapeutic strategies deploy DKK1-neutralizing antibodies, lithium chloride, SB216763, and atrial natriuretic peptide to fine-tune β-catenin stability. b TGF-β: Interventions aim to activate ALK5-dependent TGFβR signaling to support adult hippocampal neurogenesis and cognitive resilience. c Hippo: The PLIN4-Hippo signaling axis represents a potential therapeutic target for limiting sevoflurane-associated neuronal ferroptosis. d Notch: Quinic acid modulates Notch-associated signaling and promotes neurogenesis, whereas uscharin suppresses Notch activity by accelerating NICD degradation. e Hedgehog: The panel illustrates SMO inhibition by vismodegib as an experimental approach to suppress Hedgehog–GLI signaling; its relevance to neurodegenerative diseases remains preclinical. The graphics were created with BioRender (https://www.biorender.com)

Wnt signaling pathway

Pathological roles

Dysregulation of the Wnt pathway is implicated in the progression of multiple NDDs, such as PD and AD.²⁵⁶ In AD, the Wnt pathway is often downregulated, contributing to synaptic dysfunction and amyloid-β (Aβ) accumulation. In PD, disrupted Wnt signaling is associated with dopaminergic neuron loss and mitochondrial dysfunction²⁵⁷. Consequently, restoring Wnt signaling has been introduced as a promising strategy to promote neuronal survival and improve synaptic function.²⁵⁸

Some studies have demonstrated the role of the Wnt signaling pathway in regulating cell differentiation in NDDs. For example, Sahebdel et al. demonstrated that trabecular meshwork-derived mesenchymal stem cells exhibit high neural potency, as evidenced by increased expression of neural and dopaminergic markers, including MAP2 and Nurr1. Further analysis revealed that activation of the Wnt/β-catenin pathway significantly enhances dopaminergic differentiation in trabecular meshwork-derived mesenchymal stem cells, whereas inhibition of the pathway reduces it. This highlighted the potential of Wnt signaling to modulate trabecular meshwork-derived mesenchymal stem cell differentiation into dopaminergic neurons, which could be beneficial for treating NDDs such as PD.²⁵⁹ Oh et al. investigated the role of MSCs in enhancing neuronal differentiation through the Wnt/β-catenin signaling pathway in an AD model. They demonstrated that MSCs significantly upregulated neurogenic markers in Aβ-treated NPCs. Furthermore, MSC treatment upregulated Wnt/β-catenin signaling components, including β-catenin and neurogenin-1, leading to higher neurogenesis and neuronal differentiation in in vitro and in vivo AD models. These findings suggest that MSCs may promote endogenous neurogenesis by enhancing Wnt signaling, offering a potential therapeutic strategy for AD.²⁶⁰

Therapeutic advances

Colini Baldeschi et al. found that atrial natriuretic peptide influences Wnt/catenin signaling through FZD-mediated mechanisms. As a Wnt regulator, atrial natriuretic peptide can effectively reverse abnormal Wnt signaling in various pathological conditions by restoring the balance between Wnt off and Wnt on states. It may have broad therapeutic potential in NDDs associated with Wnt signaling dysregulation.²⁶¹

The DKK protein DKK1 is an antagonist of Wnt signaling that binds to LRP5/6. DKK1 promotes synaptic disintegration by disrupting the distribution of synaptic components; however, blocking DKK1 activity may help preserve synaptic integrity in AD. Furthermore, a direct link exists between DKK1-induced synaptic loss and increased Aβ production, suggesting a positive feedback mechanism. Specifically, Aβ can induce DKK1 expression, which in turn further promotes Aβ production by inhibiting Wnt signaling.²⁶² Therefore, DKK1 inhibitors targeting the Wnt signaling pathway may represent a viable therapeutic method for treating NDDs.

In the brain, GSK-3β regulates multiple critical cellular processes by modulating signaling pathways, including the Wnt pathway, and therefore may serve as a potential therapeutic target for NDDs. Zhang et al. reported that activation of the Wnt signaling pathway through GSK inhibitors, including lithium chloride and SB216763, exerted neuroprotective effects in PD cell models.²⁶³ Valproic acid, another GSK inhibitor, was indicated to reduce neuronal loss, improve hippocampal synaptic structure, and enhance cognitive performance in mouse models of AD.²⁶⁴

TGF-β signaling pathway

Pathological roles

The TGF-β signaling pathway plays a multifaceted role in NDDs. TGF-β1, a key member of the TGF-β superfamily, has been indicated to exert neuroprotective effects in experimental models of AD. In a Scopolamine-induced AD-like model, Kara et al. reported that TGF-β1 treatment significantly improved learning and memory performance, reduced neuronal damage, and decreased Aβ-40 accumulation. Mechanistically, TGF-β1 increased α-secretase expression while decreasing β and γ-secretase levels, thereby lowering Aβ-40 levels and caspase-3 immunoreactivity. These findings suggest that TGF-β1 may serve as a potential therapeutic agent for AD by enhancing cognitive function and preventing neuronal damage.²⁶⁵

Additionally, the TGF-β signaling pathway plays a crucial role in cognitive function and synaptic plasticity. In the hippocampus, TGF-β1 facilitates long-term potentiation, a key process for learning and memory. Disruptions in TGF-β signaling have been correlated with cognitive deficits in several neurological conditions, including AD and cerebrovascular disease. Research has demonstrated that targeting TGF-β signaling can improve cognitive function, highlighting its potential as a promising target for cognitive impairment.²⁶⁶

Besides, Kandasamy et al. employed a combination of histological analysis, gene expression profiling, and in vivo experiments to explore how TGF-β1 influences NPCs in the hippocampal dentate gyrus. They found that TGF-β1 signaling promotes stem cell quiescence and neuronal survival. In a transgenic mouse model, elevated TGF-β1 decreased NPC proliferation while increasing the survival of newly generated neurons. Gene expression analysis revealed that TGF-β1 regulates genes involved in cell cycle arrest, stem cell maintenance, neuronal differentiation, and survival. These studies suggest that TGF-β1 signaling may serve as a potential therapeutic target for modulating neuroregenerative processes in age-related conditions and NDDs.²⁶⁷

Therapeutic advances

Therapeutic strategies aim to restore deficient signaling by supplementing TGF-β1 or activating its receptors, as indicated in preclinical models to reduce Aβ-induced neurodegeneration and suppress pro-inflammatory cytokine production. Notably, ALK5-dependent TGF-β signaling regulates adult hippocampal neurogenesis, enhancing neuronal migration, dendritic arborization, and cognitive resilience upon activation. Genetic activation of ALK5 in the forebrain improves memory function, positioning ALK5 agonists as promising candidates for interventions targeting cognitive decline.²⁶⁸

Hedgehog signaling pathway

Pathological roles

Dysregulation of the Hh pathway contributes to the pathogenesis of AD and PD. In AD, impaired Hh signaling has been linked to reduced neurogenesis and diminished synaptic plasticity in the hippocampus, a region vital for memory formation. The SHh subfamily plays a key role in maintaining the integrity of the blood-brain barrier, thereby limiting the entry of neurotoxic substances such as β-amyloid from the peripheral circulation into the brain. Additionally, SHh signaling promotes the survival of NSCs in the subventricular zone and hippocampus, which are essential for adult neurogenesis and cognitive resilience. Experimental studies have indicated that pharmacological activation of SHh pathways can mitigate neuronal loss and inflammation, potentially by suppressing pro-inflammatory cytokines such as IL-1β and TNF-α.²⁶⁹

In PD, the Hh pathway is involved in mechanisms that support dopaminergic neuron survival. SHh signaling can protect midbrain dopaminergic neurons, whose degeneration is a hallmark of PD. In preclinical models, SHh delivery or pathway activation has been associated with reduced oxidative stress and inflammatory injury. These findings suggest that controlled activation of Hh signaling may have neuroprotective potential in PD, although the available evidence remains preclinical.²⁷⁰

Therapeutic advances

Chronic overactivation of Hh signaling exacerbates neuroinflammation by promoting the release of pro-inflammatory cytokines and inducing neuronal apoptosis. Inhibitors targeting SMO, such as cyclopamine and FDA-approved drug vismodegib, can alleviate this pathological inflammation by suppressing downstream GLI transcription factors, thereby reducing microglial activation and neuronal loss in models of neuroinflammatory injury. Beyond its role in inflammation, Hh signaling is essential for maintaining NSC populations in adult neurogenic niches such as the hippocampus. By promoting NSC self-renewal and preventing premature differentiation, activation of this pathway supports endogenous repair mechanisms. However, excessive stimulation may lead to aberrant proliferation. This balance is particularly critical in glioblastoma, where Hh inhibitors such as vismodegib have been clinically investigated to suppress tumorigenic GLI activity.²⁷¹

Notch signaling pathway

Pathological roles

The Notch signaling pathway plays a multifaceted role in NDDs by regulating neural development, synaptic plasticity, and neuroinflammatory responses. In AD, Notch signaling is intricately linked to γ-secretase. Mutations in PSEN1, the catalytic subunit of γ-secretase, not only increase amyloid-β production but also dysregulate NICD release, impairing NSCs’ self-renewal and promoting neuronal loss. In human iPSC-derived cortical spheroids, PSEN1 mutations such as L435F abnormally increase Notch target genes (Hes1 and Hey1), leading to excessive neural progenitor proliferation and reduced neuronal differentiation—a phenomenon linked to early AD pathogenesis. Notably, impaired long-term potentiation in Notch-deficient models suggests its crucial role in hippocampal synaptic plasticity and cognitive decline.^272,273

In PD, Notch signaling intersects with pathways that regulate dopaminergic neuron survival and oxidative stress. Activation of Notch1 in midbrain neurons mitigates mitochondrial dysfunction and apoptosis by suppressing ROS, whereas inhibition of Notch1 exacerbates α-synuclein-induced toxicity. Paradoxically, chronic overactivation of Notch in microglia amplifies neuroinflammation through NF-κB signaling, thereby accelerating dopaminergic neuron degeneration. This dual role complicates its therapeutic targeting. A similar pattern is observed in ischemic brain injury models: Early activation of Notch signaling promotes neurovascular repair through VEGF-mediated angiogenesis, whereas sustained activation triggers inflammatory cascades that worsen secondary neuronal damage.²⁷⁴

Therapeutic advances

The role of the Notch signaling pathway in NDDs is complex and diverse, and researchers have identified compounds that can modulate Notch signaling to treat NDDs.

Yoneyama et al. extracted a compound from Calotropis gigantea and found that it promotes the differentiation of NSCs. This compound appears to inhibit the Notch signaling pathway, thereby facilitating the differentiation of NSCs into neurons. Specifically, uscharin reduces the protein levels of Hes1 and Hes5 and suppresses Notch pathway activity by accelerating the degradation of the NICD. This study identifies uscharin as a potential candidate compound for developing new neuroregenerative therapies targeting the Notch signaling pathway.²⁷⁵

Niaz et al. reported that quinic acid promotes neurogenesis by modulating Notch-associated signaling. In primary hippocampal cell cultures, quinic acid supported the proliferation, maintenance, migration, and differentiation of NSCs into mature neurons, accompanied by changes in the expression of Notch-related genes and proteins. These findings suggest that quinic acid may serve as a potential Notch pathway modulator for neuroregenerative applications.²⁷⁶

Additionally, the Notch signaling pathway regulates neural cytoskeletal plasticity. Its activation leads to alterations in neuronal morphology and function, including neurite remodeling and reduced neurotransmitter release. These morphological changes can be reversed using Notch pathway inhibitors, which enhance cytoskeletal plasticity. Therefore, the Notch signaling pathway may serve as a pharmacological target to protect the neuronal cytoskeleton and prevent NDDs.²⁷⁷

Hippo signaling pathway

Pathological roles

The Hippo signaling pathway is significantly implicated in NDDs, particularly AD. In AD, this pathway plays a key role in regulating neuronal survival and apoptosis. Abnormal activation of the Hippo pathway in the entorhinal cortex, a crucial brain region for memory formation and consolidation, leads to neuronal loss and plaque formation. Dysregulation of the pathway’s primary effectors, YAP/TAZ, accelerates neuronal inflammation, aging, degeneration, and cell death, thereby contributing to cognitive decline.²⁷⁸

Moreover, components of the Hippo pathway, particularly YAP and MST1, are expressed in microglia and may be altered in AD. Studies have demonstrated that YAP activation can reduce the production of pro-inflammatory cytokines in microglia, whereas MST1 signaling contributes to microglial activation and neuronal apoptosis under ischemic conditions. Accordingly, the Hippo pathway may represent a potential therapeutic target for AD by modulating microglial-mediated neurodegeneration. However, further research is needed to clarify the specific roles of Hippo signaling in microglia and its impact on AD progression.²⁷⁹

Therapeutic advances

Zeng et al. found that sevoflurane treatment significantly increased PLIN4 expression in cultured mouse HT22 hippocampal neuronal cells, leading to ferroptosis. Silencing PLIN4 mitigated ferroptosis and partially reversed the sevoflurane-induced inhibition of the Hippo signaling pathway. Specifically, sevoflurane treatment increased PLIN4 mRNA levels by 2.9-fold, and PLIN4 upregulation exacerbated neuronal ferroptosis by suppressing the Hippo signaling pathway. These results suggest that targeting PLIN4 and Hippo signaling pathways may provide new, promising strategies for preventing sevoflurane-associated neurotoxicity and NDDs.²⁸⁰

Metabolic diseases

Metabolic diseases, such as obesity, diabetes, and dyslipidemia, are characterized by chronic metabolic imbalances that profoundly affect cell differentiation and function. In these conditions, cellular metabolism is often reprogramed, thereby influencing the behavior and fate of immune cells, stem cells, and tissue-specific cells.^281–283 Understanding how metabolic alterations regulate cell differentiation and the associated signaling pathways is crucial for developing targeted therapies that address the underlying pathophysiology of metabolic diseases (Table 4, Fig. 8).

Table 4.

Therapeutic advances of cell differentiation-related signaling pathways in metabolic diseases

Signaling pathway	Agents	Mechanism	Disease	Study phase	Therapeutic advances	Reference
Wnt signaling	Metformin	Wnt/β-catenin pathway activator	Diabetic osteoporosis	Preclinical	Promote osteogenic differentiation and alleviate osteoporosis under hyperglycemic conditions.	286
	Exendin-4	GLP-1 receptor agonist	T2D-associated cognitive impairment	Preclinical	Reduce tau hyperphosphorylation and alleviate cognitive impairment in T2D model mice and high-glucose-damaged HT-22 hippocampal neuronal cells by enhancing Wnt/β-catenin/NeuroD1-mediated brain insulin signaling.	287
TGF-β signaling	Green tea peptides	TGF-β/SMAD pathway inhibitor	Diabetic nephropathy	Preclinical	Reduce glomerular hypertrophy and tubulointerstitial injury.	291
	Imperatorin	TGF-β/SMAD pathway inhibitor	Diabetic nephropathy	Preclinical	Improve renal function, reduce serum creatinine and urea nitrogen levels, and alleviate pathological damage of kidney tissue in DN mice.	292
Notch signaling	Gliquidone	Notch signaling inhibitor	Diabetic nephropathy	Preclinical	Improve kidney function, reduce levels of urinary protein, blood urea nitrogen and serum creatinine, and suppress proliferation and fibrosis of renal tubular epithelial cells.	302
	DAPT	Notch inhibitor	Post-acute pancreatitis diabetes	Preclinical	Promote the differentiation of pancreatic Krt5+ cells into islet β cells and increase the number of newborn small islets, thereby improving glucose tolerance.	303
Hippo signaling	Quercetin	Hippo pathway reactivator	Diabetic nephropathy	Preclinical	Inhibit the proliferation of mesangial cells and improve kidney function.	305

GLP-1 glucagon-like peptide-1, T2D type 2 diabetes, HT-22 mouse hippocampal neuronal cell line, TGF-β transforming growth factor-β, DN diabetic nephropathy, NeuroD1 neurogenic differentiation 1, DAPT N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine tert-butyl ester, Krt5 keratin 5

Fig. 8.

Therapeutic advances of cell differentiation-related signaling pathways in metabolic diseases. a Wnt: Metformin activates Wnt/β-catenin signaling in diabetic osteoporosis, whereas exendin-4, a GLP-1 receptor agonist, activates the Wnt/β-catenin/NeuroD1 axis in T2D-associated cognitive impairment. b TGF-β: Interventions leverage natural compounds like green tea peptides and imperatorin to modulate SMAD2/3/4 signaling cascades. c Hippo: Quercetin modulates the Hippo network by targeting upstream regulators, thereby controlling YAP/TAZ nuclear translocation and TEAD-mediated transcription. d Notch: Gliquidone suppresses Notch/Snail1 signaling, whereas DAPT inhibits γ-secretase-mediated Notch cleavage, thereby modulating metabolic signaling and tissue repair. e Hedgehog: Vismodegib-mediated inhibition of SMO blocks Hedgehog signal transduction, suppressing GLI-dependent gene expression programs. The graphics were created with BioRender (https://www.biorender.com). GLP-1R glucagon-like peptide-1 receptor

Wnt signaling pathway

Pathological roles

Wnt signaling plays a crucial role in metabolic diseases, regulating insulin secretion, adipogenesis, and lipid metabolism.²⁸⁴ In obesity and type 2 diabetes mellitus (T2D), dysregulated canonical Wnt/β-catenin signaling disrupts adipose tissue homeostasis. Under physiological conditions, Wnt activation inhibits adipogenesis by suppressing PPARγ and redirecting MSCs toward osteogenic lineages. However, chronic suppression of this pathway in mature adipocytes promotes lipid accumulation and visceral fat expansion. Conversely, non-canonical Wnt pathways, such as Wnt5a signaling, contribute to vascular oxidative stress in obesity by activating NOX through USP17/RAC1, thereby exacerbating insulin resistance and endothelial dysfunction.²⁸⁴

Moreover, the pathway regulates pancreatic β-cell biology, with TCF7L2, a key transcriptional effector of Wnt signaling, emerging as a primary genetic risk factor for diabetes. Variants in TCF7L2 impair insulin secretion by disrupting β-cell proliferation, survival, and glucose sensing. Additionally, aberrant Wnt signaling exacerbates metabolic inflexibility by inhibiting AMPK activity. This metabolic reprogramming promotes enhanced glycolysis and glutaminolysis, partly through Wnt-mediated stabilization of β-catenin, which redirects metabolic fluxes to support cellular proliferation in insulin-resistant tissues.²⁸⁵

Therapeutic advances

Huang et al. reported that a hyperglycemic environment can suppress the proliferation and osteogenic differentiation of MC3T3-E1 cells, whereas metformin treatment can enhance their proliferation and osteogenic differentiation under hyperglycemic conditions. Moreover, it can elevate the alkaline phosphatase activity and the expression of Runx2 and osteocalcin proteins, thereby stimulating osteogenic differentiation and alleviating osteoporosis. Other studies have demonstrated that metformin can activate the Wnt/β-catenin signaling pathway, suggesting that it is a novel treatment for diabetic osteoporosis.²⁸⁶

Kang et al. investigated the potential of Exendin-4 to ameliorate cognitive impairment and tau hyperphosphorylation associated with T2D. They reported that Exendin-4 suppressed tau hyperphosphorylation in T2D model mice and high-glucose-damaged HT-22 hippocampal neuronal cells by activating the Wnt/β-catenin/NeuroD1 pathway. Specifically, Exendin-4 upregulated NeuroD1 expression, enhanced Ins2 transcription, increased brain insulin signaling, and reduced GSK-3β activity, thereby alleviating AD-like pathological changes rather than promoting AD development.²⁸⁷

TGF-β signaling pathway

Pathological roles

Dysregulation of TGF-β signaling contributes to obesity, T2D, non-alcoholic fatty liver disease (NAFLD), and metabolic syndrome. In adipose tissue, TGF-β inhibits adipogenesis by suppressing PPARγ and C/EBPs via SMAD3.²⁸⁵ Paradoxically, in obesity, chronic TGF-β1 overactivation drives pathological fibrosis through SMAD2/3-dependent collagen and PAI-1 induction, leading to insulin resistance. M2 macrophages exacerbate this by secreting TGF-β1, which triggers adipose progenitor senescence via mitochondrial ROS-mediated DNA damage, impairing adipose plasticity during aging.^239,288

In the liver, TGF-β signaling is a central driver of metabolic dysfunction-associated steatohepatitis (MASH) progression. Cholesterol enrichment in hepatocyte membranes alters the balance between canonical (SMAD2/3) and non-canonical (AKT) TGF-β signaling by modulating raft-dependent TGFβRI/TGFβRII receptor complex formation.²⁸⁹ Specifically, cholesterol enrichment suppresses TGF-β-induced AKT phosphorylation, favoring fibrogenic SMAD signaling that accelerates collagen deposition and hepatic stellate cell activation. The E3 ubiquitin ligase NEDD4 emerges as a critical regulator, as it ameliorates MASH by ubiquitinating and degrading decorin, a proteoglycan that typically enhances TGF-β/SMAD signaling to promote lipid accumulation and inflammation. Conversely, NEDD4 deficiency amplifies TGF-β-driven fibrogenesis, highlighting this axis as a therapeutic target.²⁹⁰ Moreover, in diabetic nephropathy (DN), hyperglycemia-induced oxidative stress activates renal TGF-β/SMAD signaling, driving mesangial cell hypertrophy, ECM accumulation, and glomerulosclerosis.²³⁹

Therapeutic advances

Green tea peptides ameliorate diabetic nephropathy in mice by reducing blood glucose, serum creatinine, urea nitrogen, and urinary albumin/creatinine ratio, attenuating glomerular hypertrophy and tubulointerstitial injury, downregulating p-SMAD2/3, α-SMA, ZO-1, and vimentin, and upregulating SMAD7, thereby inhibiting the TGF-β/SMAD signaling pathway.²⁹¹

A study found that treatment with imperatorin, a furanocoumarin derivative derived from natural plants, significantly improved renal function, reduced serum creatinine and urea nitrogen levels, and alleviated pathological damage of kidney tissue in mice with DN. Additionally, imperatorin inhibited the TGF-β/SMAD2/3 signaling axis in kidney tissue, downregulated the expression level of EMT-associated proteins, and suppressed the levels of inflammatory factors, including TNF-α and IL-1β. These results indicate that imperatorin can target the TGF-β signaling pathway to treat DN.²⁹²

Hedgehog signaling pathway

Pathological roles

The Hh signaling pathway influences cell proliferation, differentiation, and tissue homeostasis, all of which are critical in metabolic regulation. Dysregulation of the Hh pathway has been implicated in a range of metabolic disorders, such as obesity, diabetes, and NAFLD.²⁹³

In obesity, the Hh pathway plays a crucial role in adipogenesis and energy homeostasis. Studies have indicated that activation of the SHh pathway can inhibit white adipose tissue differentiation, thereby reducing fat storage and improving insulin sensitivity.²⁹⁴ Conversely, in diseases, including NAFLD and non-alcoholic steatohepatitis (NASH), the Hh pathway is often upregulated, leading to liver fibrosis and inflammation. SHh ligands produced by damaged hepatocytes activate hepatic stellate cells, leading to their transdifferentiation into myofibroblasts, which promote fibrogenesis.²⁹⁵

In diabetes, the Hh pathway is involved in pancreatic β-cell function and insulin production. Increased Hh signaling has been indicated to induce β-cell dedifferentiation, thereby reducing insulin production and contributing to hyperglycemia.²⁹⁶ Furthermore, the Hh pathway interacts with other signaling pathways, including Wnt/β-catenin and TGF-β, which also play crucial roles in metabolic regulation. These interactions can modulate insulin sensitivity and glucose tolerance, underscoring the complexity of Hh signaling in metabolic homeostasis.²⁹⁷

Therapeutic advances

In the liver, chronic Hh activation promotes the progression of MASH and fibrosis. Studies have indicated that Hh signaling regulates hepatic stellate cell metabolism. Quiescent hepatic stellate cells undergo glycolytic reprogramming during transdifferentiation into myofibroblasts, with lactate accumulation fueling fibrogenesis. Pharmacological inhibition of Hh signaling using SMO antagonists, including vismodegib, reverses this metabolic reprogramming. This intervention suppresses HIF1α-mediated glycolysis and promotes the reversion of hepatic stellate cells to a quiescent state, thereby ameliorating fibrosis.^298,299

Notch signaling pathway

Pathological roles

The Notch signaling pathway plays a significant role in metabolic disorders, particularly in metabolic bone diseases. It regulates the differentiation and function of osteoblasts and osteoclasts and influences key processes such as cartilage and bone formation and bone resorption. Mutations in genes involved in Notch signaling are closely associated with several metabolic bone diseases, including Alagille syndrome, Adams-Oliver syndrome, and spondylocostal dysostosis. These diseases are characterized by imbalances in bone metabolism, often leading to osteoporosis and other skeletal deformities.³⁰⁰

Beyond bone metabolism, the Notch signaling pathway influences other metabolic processes. For instance, it has been indicated to dynamically regulate adult β-cell proliferation and maturation, which is essential for maintaining glucose homeostasis. This suggests a potential role for Notch signaling in metabolic diseases such as diabetes, although further research is needed to fully elucidate its role in these conditions.³⁰¹

Therapeutic advances

Tian et al. investigated the mechanisms by which Gliquidone protects against DN. It was found to improve kidney function in diabetic mice, decrease urinary protein levels, blood urea nitrogen, and serum creatinine, while inhibiting the proliferation and fibrosis of renal tubular epithelial cells by blocking the Notch/Snail1 signaling pathway, thereby delaying DN progression. This indicates that gliquidone can effectively treat DN by targeting the Notch signaling pathway.³⁰²

Previously, Zhang et al. explored whether reducing Notch activity could improve diabetes associated with acute pancreatitis (AP). The results indicated that suppression of the Notch signaling pathway promoted the differentiation of pancreatic Krt5⁺ cells into islet β cells and increased the number of newborn small islets, thereby improving glucose tolerance. These findings suggest that targeting the Notch signaling pathway may stimulate the regeneration of islet β cells, providing a novel strategy for treating post-AP diabetes.³⁰³

Hippo signaling pathway

Pathological roles

In metabolic disorders such as obesity and diabetes, dysregulation of the Hippo pathway can affect adipogenesis and insulin sensitivity. YAP/TAZ, key effectors of this pathway, regulate adipocyte differentiation and lipid metabolism, both of which are central to these disorders.³⁰⁴

Therapeutic advances

Du et al. reported that mesangial cell proliferation is significantly increased under hyperglycemic conditions, accompanied by marked inactivation of the Hippo signaling pathway. Treatment with quercetin inhibited the hyperglycemia-induced cell proliferation by reactivating the Hippo signaling pathway. Specifically, quercetin enhanced the phosphorylation of MST1 and Lats1, while downregulating the YAP expression and nuclear translocation, thereby suppressing cell proliferation. In a db/db diabetic mouse model, quercetin treatment reduced mesangial cell proliferation and improved kidney function through activation of the Hippo signaling pathway. These findings suggest that quercetin may alleviate early DN by targeting the Hippo signaling pathway.³⁰⁵

Digestive diseases

Digestive diseases refer to those affecting the gastrointestinal tract, liver, pancreas, and gallbladder. These diseases can be acute or chronic and may manifest as impaired digestion, absorption, and nutrient utilization.^306,307 The development of digestive diseases involves diverse signaling pathways and cell differentiation (Table 5, Fig. 9).

Table 5.

Therapeutic advances of cell differentiation-related signaling pathways in digestive diseases

Signaling pathway	Agents	Mechanism	Disease	Study phase	Therapeutic advances	Reference
Wnt signaling	PRI-724	CBP/β-catenin interaction inhibitor	Cirrhosis	Phase I/IIa (NCT03620474)	Show general tolerability and exploratory anti-fibrotic signals, but do not demonstrate significant histological fibrosis regression.	314
	IL-38	Wnt pathway modulator	Intestinal inflammation	Preclinical	Promote intestinal stem cell differentiation and epithelial barrier repair by inducing WNT3A-dependent signaling and a beneficial IL-1β response.	315
	Berberine	Wnt/β-catenin pathway modulator	Ulcerative colitis	Preclinical	Restore the biological barrier by regulating intestinal flora and activate the Wnt pathway to promote the expression of tight junction proteins, thus repairing the integrity of the intestinal epithelial barrier.	316
TGF-β signaling	HH-F3	TGF-β inhibitor	Liver injury and fibrosis	Preclinical	Reduce the deposition of collagen, α-SMA, and other ECM components, and inhibit the proliferation and migration of hepatic stellate cells.	319
	Xanthohumol	TGF-β inhibitor	Intestinal fibrosis	Preclinical	Inhibit Smad2/3 phosphorylation and CAGA-dependent transcription, thereby reducing the expression of fibrosis markers such as α-SMA and collagen.	320
Hedgehog signaling	Oxy210	Hedgehog/TGF-β inhibitor	Metabolic dysfunction-associated steatohepatitis	Preclinical	Alleviate liver fibrosis, inflammation, and hypercholesterolemia in mice after 16 weeks of treatment.	324
Notch signaling	J2H-1702	11β-HSD1 inhibitor	Liver fibrosis	Preclinical	Block the differentiation of hepatic stellate cells into myofibroblasts and alleviate collagen deposition.	330
	Astragaloside	Notch inhibitor	Cholestatic hepatic fibrosis	Preclinical	Inhibit fibrosis progression, reduce the differentiation of hepatic progenitor cells into bile duct epithelial cells, and suppress abnormal bile duct proliferation.	331
Hippo signaling	ADMSCs	ADMSC-based modulation of Hippo signaling	Liver fibrosis	Preclinical	Improve liver function in rats with hepatic fibrosis and decrease collagen deposition and fibrotic marker expression.	336

CBP CREB-binding protein, IL-38 interleukin-38, TGF-β transforming growth factor-β, Hh Hedgehog, α-SMA α-smooth muscle actin, ECM extracellular matrix, CAGA SMAD-responsive CAGA element, 11β-HSD1 11β-hydroxysteroid dehydrogenase type 1, ADMSCs adipose-derived mesenchymal stem cells

Fig. 9.

Therapeutic advances of cell differentiation-related signaling pathways in digestive diseases. a Wnt: IL-38 and berberine promote Wnt-dependent intestinal repair, whereas PRI-724 inhibits CBP/β-catenin-mediated transcription. b TGF-β: Natural agents, including Graptopetalum paraguayense-derived preparations and xanthohumol, modulate the TGFβR–SMAD cascade and SMAD2/3 phosphorylation. c Hippo: Adipose-derived mesenchymal stem cells (ADMSCs) modulate Hippo signaling and reduce YAP/TAZ–TEAD transcriptional activity, thereby suppressing hepatic stellate cell activation and fibrotic remodeling. d Notch: J2H-1702 suppresses hepatic stellate-cell activation, whereas astragaloside attenuates ductular reactions; both reduce fibrosis through Notch-related mechanisms. e Hedgehog: Oxy210 inhibits both Hedgehog and TGF-β signaling, thereby attenuating hepatic stellate cell activation and hepatic fibrotic and inflammatory remodeling. The graphics were created with BioRender (https://www.biorender.com). IL-38 interleukin 38, ADMSCs adipose-derived mesenchymal stem cells

Wnt signaling pathway

Pathological roles

The Wnt/β-catenin pathway contributes to the pathogenesis of digestive diseases through tissue-specific mechanisms. In liver fibrosis and NAFLD, activation of this pathway promotes hepatic stellate cell proliferation and transdifferentiation into myofibroblasts, increasing ECM production, including collagen I and III.³⁰⁸ Inflammatory cytokines further regulate Wnt signaling in hepatic stellate cells; for example, TNF-α and IFN-γ may modulate this pathway through AKT activation or induction of Wnt antagonists such as DKK1.³⁰⁹

Beyond its effects on hepatic stellate cells, the Wnt/β-catenin pathway also modulates lipid metabolism and inflammation in NAFLD. Deregulated activation of this pathway stimulates hepatic steatosis and progression to NASH.³¹⁰ Studies have reported that some dietary components, such as docosahexaenoic acid, can modulate Wnt signaling. Docosahexaenoic acid upregulates the Wnt3a expression in macrophages, inducing an anti-inflammatory phenotype and alleviating liver steatosis in pediatric NAFLD.³¹¹

In inflammatory bowel disease (IBD), the Wnt/β-catenin pathway sustains the integrity of the intestinal epithelial barrier and disruption of this pathway may potentially damage the barrier function, leading to chronic inflammation and tissue damage in IBD.³¹² In addition, the Wnt signaling intersects with key inflammatory pathways, such as NF-κB and MAPK, collectively modulating the expression of pro-inflammatory cytokines and chemokines. This crosstalk regulates intestinal homeostasis and tissue repair during inflammation.³¹³

Therapeutic advances

PRI-724, a selective CBP/β-catenin interaction inhibitor, has been clinically evaluated as an anti-fibrotic therapy for hepatitis B virus- or hepatitis C virus-induced liver cirrhosis. In an investigator-initiated, open-label, non-randomized, multicenter phase I/IIa study, PRI-724 was generally tolerated and showed exploratory anti-fibrotic signals, including improvements in liver stiffness, MELD score, and serum albumin in some patients. However, statistically significant histological fibrosis regression was not demonstrated. Thus, PRI-724 should be regarded as an investigational CBP/β-catenin-targeted therapy for cirrhosis rather than an established anti-fibrotic treatment.³¹⁴

Dinarello et al. found that IL-38 maintains intestinal homeostasis by regulating the Wnt signaling pathway. IL-38 promoted WNT3A expression and induced a controlled, beneficial IL-1β response, thereby supporting intestinal stem cell differentiation and intestinal organoid growth. Inhibition of Wnt signaling attenuated the pro-repair effects of IL-38, indicating that the Wnt pathway is a key mediator of IL-38-dependent epithelial homeostasis and barrier repair.³¹⁵

Dong et al. demonstrated that berberine alleviates DSS-induced colitis via the Wnt/β-catenin pathway. Berberine restores the biological barrier by regulating intestinal flora, and activates the Wnt pathway thereby upregulating the levels of tight junction proteins (ZO-1 and Occludin), to repair the integrity of the intestinal epithelial barrier. Proteomics revealed significant activation of the Wnt pathway, whereas the inhibitor FH535 abolished the protective effect of berberine. In addition, berberine regulates immune homeostasis by inhibiting ILC1/Th17 pro-inflammatory cells and increasing the number of ILC3/Treg anti-inflammatory cells, and its mechanism is closely related to Wnt-mediated mucosal repair and immune balance.³¹⁶

TGF-β signaling pathway

Pathological roles

Genetic studies have identified several risk loci containing SMAD2, SMAD3, SMAD4, and SMAD7 in IBD patients, particularly those with ulcerative colitis. In IBD, the TGF-β signaling pathway affects the onset of inflammation and goblet cell differentiation.³¹⁷

In the context of NAFLD and NASH, the TGF-β signaling pathway plays a crucial role in regulating liver fibrosis and the progression of these diseases. Specifically, TGF-β1 contributes to the development of liver fibrosis associated with NAFLD and NASH. At a mechanistic level, it activates hepatic stellate cells, which are the primary cell type responsible for controlling collagen production and deposition within the liver. This activation predominantly occurs through the SMAD3-dependent signaling pathway, leading to increased ECM production and subsequent fibrosis.³¹⁸

Therapeutic advances

Hsu et al. explored the mechanism by which Graptopetalum paraguayense and its active component HH-F3 protected against liver fibrosis by inhibiting the TGF-β signaling pathway. The results indicated that the Graptopetalum paraguayense extracts and HH-F3 significantly inhibited TGF-β1-mediated SMAD2/3 phosphorylation and MAPK pathway activation, thereby reducing the deposition of collagen, α-SMA, and other ECM in hepatic stellate cells and DEN-induced rat hepatic fibrosis models. Moreover, it inhibited the proliferation and migration of hepatic stellate cells. TGF-β, as the core driver of liver fibrosis, can promote the fibrosis process by activating downstream SMAD-dependent and independent pathways, whereas Graptopetalum paraguayense/HH-F3 effectively alleviated liver injury and fibrosis by blocking TGF-β signaling.³¹⁹

Yun et al. found that Xanthohumol treatment inhibits intestinal fibrosis by blocking the TGF-β signaling pathway. In TGF-β1-stimulated human intestinal fibroblasts, Xanthohumol prevents the interaction between TGFβRI and SMAD3 by binding to the L45 loop of TGFβRI, reduces SMAD2/3 phosphorylation and CAGA-dependent transcription, thereby reducing the expression of fibrosis markers such as α-SMA and collagen. In addition, Xanthohumol inhibited the binding of NF-κB to SMAD3 in the α-SMA promoter region, reversing the fibroblast activation phenotype. It has been observed that the TGF-β signaling leads to abnormal ECM deposition in intestinal fibrosis by activating the SMAD pathway, whereas Xanthohumol exerts dual anti-fibrotic effects by targeting the TGF-β receptor complex.³²⁰

Hedgehog signaling pathway

Pathological roles

The Hh pathway plays a context-dependent role in liver repair. After acute injury, such as partial hepatectomy, its activation promotes hepatocyte proliferation and liver progenitor cell expansion, thereby supporting tissue regeneration. However, sustained Hh activation during chronic liver injury exacerbates fibrogenesis and cirrhosis, partly by promoting hepatic stellate cell transdifferentiation into myofibroblasts.³²¹

The Hh pathway is dysregulated and promotes disease progression in NAFLD. Specifically, this pathway is activated by lipotoxicity and hepatocyte injury, enhancing the progression of NASH and fibrosis. Excessive activation of the Hh signaling was reported to induce EMT in hepatocytes and cholangiocytes, leading to accumulation of progenitor cells and fibrogenic myofibroblasts.³²²

Therapeutic advances

Evidence from prior investigations indicates that the Hh signaling pathway contributes to the occurrence of IBD. It can modulate intestinal homeostasis via regulating the interaction between intestinal interstitial cells and epithelial cells. Inhibition of this pathway has been linked to severe IBD inflammation. In IBD, Hh alleviates intestinal damage by suppressing the NF-κB inflammatory pathway, regulating immune cells (such as Tregs), and stimulating the production of the Wnt ligand from interstitial cells.³²³

Hui et al. developed Oxy210, a novel inhibitor of the Hh signaling pathway, and explored its therapeutic effect on MASH. Hh signaling pathway synergizes with TGF-β pathway to promote hepatic stellate cell activation, thereby stimulating liver fibrosis. Treatment with Oxy210 downregulated the expression of pro-fibrotic genes in hepatic stellate cells by inhibiting Hh and TGF-β signaling, and significantly alleviated liver fibrosis, inflammation and hypercholesterolemia in mice after 16 weeks of treatment. These findings suggest that dual inhibition of Hh and TGF-β signaling may represent a potential therapeutic strategy for MASH.³²⁴

Notch signaling pathway

Pathological roles

In liver fibrosis, Notch activation in hepatic stellate cells fosters fibrosis through a positive feedback loop with the TGF-β/SMAD pathway, and modulates hepatic stellate cell phenotypic transformation and inflammatory responses.^325,326

Additionally, it has been implicated in the regulation of hepatocyte and biliary epithelial cell proliferation during liver regeneration, particularly after partial hepatectomy, where its activation promotes liver cell regeneration.³²⁷ Notch2 is a key regulator of biliary epithelial cell fate, playing a crucial role in bile duct development and regeneration. Mutations in Jagged1 or Notch2, identified in Alagille syndrome, can lead to biliary paucity as a result of impaired bile duct formation.³²⁸

The Notch signaling pathway plays an important role in regulating macrophage polarization and thereby influences the progression of liver diseases. Activation of Notch signaling generally favors pro-inflammatory M1-like macrophage polarization and enhances the expression of inflammatory mediators, whereas reduced Notch activity may favor M2-like phenotypes in several experimental contexts. Through this context-dependent regulation of macrophage states, dysregulated Notch signaling can alter the balance between inflammatory injury, fibrogenesis, and tissue repair in the liver.³²⁹

Therapeutic advances

Kim et al. designed a novel 11β-HSD1 inhibitor, J2H-1702, and investigated its antifibrotic mechanism in a mouse model of liver fibrosis induced by thioacetamide. Inhibition of 11β-HSD1 lowered the concentration of intrahepatic cortisol, leading to the inhibition of hepatic stellate cell activation, and Notch signaling pathway was significantly down-regulated following RNA sequencing. Inhibition of the Notch signaling pathway could directly prevent the differentiation of hepatic stellate cells into myofibroblasts and alleviate collagen deposition. In addition, the inhibitor promoted hepatic stellate cell clearance by elevating the activity of NK cells, further inhibiting fibrosis. Notably, the Notch pathway can promote hepatic stellate cell activation in liver fibrosis, indicating that targeting Notch may be a robust therapeutic strategy.³³⁰

Mu et al. investigated the protective effect of astragaloside on cholestatic hepatic fibrosis induced by bile duct ligation. Notably, the astragaloside treatment prevented fibrosis by inhibiting Notch signaling pathway, suppressing the differentiation of hepatic progenitor cells into bile duct epithelial cells and abnormal bile duct proliferation. Activation of the Notch pathway can induce the bile duct response and fibrosis in cholestasis, but treatment with astragaloside significantly downregulated the expression of bile duct markers (CK7/CK19/OV6/SOX9) and collagen deposition by blocking this pathway.³³¹

Hippo signaling pathway

Pathological roles

In gastrointestinal diseases including IBD, the Hippo pathway modulates intestinal epithelial regeneration, immune regulation, gut microbiota, and angiogenesis. YAP overexpression enhances intestinal epithelial proliferation and mucosal regeneration in DSS-induced colitis models³³², and YAP in macrophages regulates gut microbiota homeostasis.³³³ The Hippo pathway also interacts with Wnt and Notch to modulate intestinal homeostasis.³³⁴

It is also essential in liver development, homeostasis, and regeneration. The Hippo pathway regulates hepatocyte proliferation and differentiation during liver development and maintains liver size by controlling cell growth and apoptosis. Furthermore, this pathway is activated in response to liver injury to promote tissue repair and regeneration. For instance, YAP activation stimulates hepatocyte proliferation following partial hepatectomy or chemically induced liver injury.³³⁵

Therapeutic advances

Elsewhere, Liu et al. investigated the mechanism by which adipose-derived mesenchymal stem cells decrease the activity of hepatic stellate cells through the Hippo signaling pathway to alleviate liver fibrosis. Results indicated that adipose-derived mesenchymal stem cell grafts markedly improved liver function in rats with CCl₄-induced hepatic fibrosis, reduced collagen deposition and the expression of fibrotic markers (α-SMA, TGF-β1, and collagen I), and increased the inhibitory phosphorylation of YAP/TAZ. This reduced YAP/TAZ-dependent transcription and inhibited hepatic stellate cell proliferation and activation. In vitro experiments further confirmed that adipose-derived mesenchymal stem cells inhibited TGF-β1-induced hepatic stellate cell activation via the Hippo pathway, whereas inhibition of the upstream Hippo kinase cascade weakened these therapeutic effects. The Hippo signaling pathway can modulate the development and progression of diverse digestive diseases (such as liver fibrosis). Abnormal activation of its effector protein YAP/TAZ may stimulate the hepatic stellate cell activity and ECM deposition, while inhibition of YAP/TAZ can suppress fibrogenesis. The study showed that the Hippo pathway may be a potential therapeutic target for liver fibrosis.³³⁶

Respiratory diseases

Respiratory diseases refer to those influencing the respiratory system, such as asthma, chronic obstructive pulmonary disease (COPD), and other forms of pneumonia. They are generally characterized by impaired respiratory function and are often associated with chronic inflammation and tissue damage.³³⁷ Cell differentiation and signaling pathways contribute to the pathogenesis of respiratory diseases (Fig. 10).

Fig. 10.

Therapeutic advances of cell differentiation-related signaling pathways in respiratory diseases. a Wnt: PRI-724 is depicted as a CBP/β-catenin interaction inhibitor that suppresses downstream β-catenin-dependent transcription in preclinical pulmonary fibrosis models. b TGF-β: Antifibrotic agents, including pirfenidone, nintedanib, and Zangsiwei, modulate profibrotic TGF-β signaling. c Hippo: Strategies target the pathological activation of effectors using YAP inhibitors to modulate cellular proliferation and environmental sensing. d Notch: Restoration of Notch signaling represents a potential strategy in contexts where cigarette smoke disrupts Notch-mediated airway epithelial maintenance and repair. e Hedgehog: Therapeutic targeting utilizes pirfenidone to inhibit GLI-mediated transcription, thereby reducing the Hedgehog-driven fibrotic response in respiratory pathologies. The graphics were created with BioRender (https://www.biorender.com)

Wnt signaling pathway

Pathological roles

In the respiratory system, Wnt signaling plays essential roles in lung development and disease. Mice lacking Wnt2/2b or β-catenin fail to form lungs³³⁸, while overexpression of Wnt5a negatively influences epithelial branching.³³⁹

In chronic lung diseases, the Wnt pathway is implicated in fibrotic and obstructive conditions. In idiopathic pulmonary fibrosis (IPF), the canonical Wnt/β-catenin signaling is highly activated as confirmed by increased expression of Wnt target genes and nuclear β-catenin activity in various cell types, which leads to excessive ECM deposition and fibrosis.³⁴⁰ In contrast, patients with COPD present with inhibited canonical Wnt signaling. This may be linked to impaired alveolar epithelial cell proliferation and wound healing, inducing emphysema development.³⁴¹

Aging is a major factor driving the development and progression of chronic lung diseases. For instance, aging-related changes in Wnt signaling, such as a shift from canonical to non-canonical signaling in some contexts, have been reported. High levels of non-canonical ligands like Wnt5a were detected in aged lungs, which is linked to impaired lung repair and increased susceptibility to COPD.³⁴¹ This suggests that dysregulated Wnt signaling during aging may elevate the risk of chronic lung diseases by disrupting tissue homeostasis and repair mechanisms. Therefore, further research is needed to clarify the complex interplay between Wnt signaling, aging, and chronic lung disease pathogenesis, which lays a foundation for developing novel therapeutic strategies targeting this pathway.

Therapeutic advances

The Wnt signaling pathway is integral to lung development, epithelial repair, and the pathophysiology of respiratory diseases. Aberrant activation of the canonical Wnt/β-catenin signaling pathway has been implicated in IPF, where it facilitates fibroblast proliferation and myofibroblast differentiation, leading to excessive ECM deposition. Notably, increased nuclear β-catenin levels and elevated expression of Wnt ligands have been identified in fibrotic lung tissues.³⁴²

Pharmacological inhibition of Wnt signaling has shown therapeutic potential in preclinical models of pulmonary fibrosis. For instance, PRI-724 ameliorated bleomycin-induced pulmonary fibrosis in mice by inhibiting CBP/β-catenin signaling.³⁴³

TGF-β signaling pathway

Pathological roles

In chronic lung diseases, TGF-β signaling is dysregulated and contributes to pathogenesis. For instance, TGF-β can drive fibroblast activation and myofibroblast differentiation, leading to ECM deposition and tissue remodeling in pulmonary fibrosis. Studies have reported high expression of TGF-β in fibrotic lungs, and its inhibition can attenuate fibrosis progression. In COPD, TGF-β signaling is compartment- and cell-specific; TGF-β1 expression is increased in the small-airway epithelium and correlates with smoking exposure and small-airway obstruction.^344,345

In asthma, TGF-β can alter immune response and modulate airway remodeling. Specifically, it enhances airway smooth muscle proliferation, ECM deposition, and airway hyperresponsiveness. Furthermore, TGF-β participates in T cell homeostasis, promoting the differentiation of Tregs and Th17 cells while inhibiting Th1 and Th2 cells. This dual action contributes to the immune dysregulation observed in asthma. Previous studies have demonstrated that the expression level of TGF-β in bronchoalveolar lavage fluid and bronchial specimens from asthmatic patients is increased, highlighting its role in disease progression.^346,347

Therapeutic advances

The TGF-β signaling pathway plays a significant role in the treatment of respiratory diseases, including asthma, COPD, pulmonary fibrosis, and lung cancer. As a key mediator of fibrosis, TGF-β facilitates fibroblast activation, EMT, and ECM deposition through both SMAD-dependent and independent pathways. However, due to its immunosuppressive and anticancer properties, completely inhibiting TGF-β may pose safety risks. Currently marketed drugs, such as pirfenidone and nintedanib, slow the progression of pulmonary fibrosis by partially modulating TGF-β signaling. In contrast, innovative selective inhibitors, particularly those targeting integrin-mediated TGF-β activation, have demonstrated improved safety profiles.³⁴⁸

Wu et al. explored how the Tibetan drug Zangsiwei inhibits the TGF-β/SMAD pathway to mitigate lung injury caused by particulate matter, employing a multi-omics integration approach. They identified fifty-eight components through serum metabolomics and network pharmacology, which are predicted to regulate the fibrotic pathway via TGFβRI, SMAD2/3, and other targets. In vivo experiments demonstrated that ZANGSIWEI reduced the expression of TGFβRI, α-SMA, and collagen, while also inhibiting inflammatory infiltration in the alveoli and the formation of fibrous nodules in mice exposed to silica and PM2.5. Additionally, in vitro experiments confirmed that serum containing ZANGSIWEI blocked the activation of MRC-5 fibroblasts induced by TGF-β1 and downregulated the phosphorylation of SMAD2/3, indicating that ZANGSIWEI can effectively inhibit TGF-β/SMAD signaling through multiple components. This research offers a molecular foundation for developing traditional drug therapies for fibrotic diseases, including pneumoconiosis.³⁴⁹

Hedgehog signaling pathway

Pathological roles

In IPF, it was observed that the Hh pathway was reactivated in alveolar epithelial cells and fibroblasts, contributing to fibroblast activation, myofibroblast differentiation, and excessive ECM deposition, which are the important hallmarks of the disease. Dysregulation of the Hh signaling, particularly following the activation of transcription factors such as GLI1 and GLI2, promotes EMT and fibroblast proliferation, stimulating the progression of lung fibrosis.³⁵⁰

Genetic studies have identified SNPs near the Hh-interacting protein gene in COPD, which encodes a key inhibitor of the Hh pathway. These SNPs have been linked to a reduction in lung function, increased susceptibility to COPD, and enhanced emphysema progression. Furthermore, Hh-interacting protein expression is downregulated in COPD lungs, suggesting the loss of negative regulation of the Hh pathway, which regulates airway remodeling and fibrosis. In COPD, impaired Hh signaling can disrupt the normal balance in epithelial and mesenchymal interactions, leading to impaired lung repair and chronic inflammation.³⁵¹

Therapeutic advances

Zhang et al. investigated the impact of pirfenidone on early pulmonary fibrosis in a lipopolysaccharide (LPS)-induced acute respiratory distress syndrome (ARDS) model by inhibiting the Hh signaling pathway-mediated endothelial-to-mesenchymal transition (EndMT). The study revealed that the expression of GLI1, a key transcription factor within the Hh pathway, was significantly upregulated in the LPS-induced ARDS model, alongside an increase in EndMT-related markers (such as α-SMA and vimentin) and a decrease in endothelial markers (such as CD31 and VE-cadherin), which collectively promote pulmonary fibrosis. Pirfenidone was shown to downregulate the expression of GLI1 and fibrotic factors, thereby inhibiting the EndMT process, reducing the inflammatory response, decreasing collagen deposition, and improving lung injury. Additional experiments confirmed that the Hh pathway inhibitor GANT58 or GLI1 knockdown could replicate the antifibrotic effects of pirfenidone, suggesting that the Hh pathway may represent a critical therapeutic target for addressing EndMT and fibrosis in respiratory diseases, thereby offering a novel strategy for treating ARDS and associated pulmonary fibrosis.³⁵²

Notch signaling pathway

Pathological roles

In respiratory diseases, the Notch pathway modulates cell differentiation, proliferation, and tissue remodeling through crosstalk with autophagy. Impaired autophagy accelerates fibroblast-to-myofibroblast differentiation and epithelial cell senescence, promoting pulmonary fibrosis. Notch signaling downregulates PTEN via Hes1, activating PI3K/AKT/mTOR signaling and suppressing autophagy, while also regulating the NLRP3 inflammasome via Notch1 to drive fibrosis progression.³⁵³

The role of the Notch signaling in disease progression is mediated by two key mechanisms: myofibroblast activation and epithelial/endothelial cell transition. Notch1 signaling promotes the differentiation of myofibroblasts, increasing the production of ECM and fibrosis. This process is central to the development of IPF. Inhibiting the Notch signaling can reduce myofibroblast activation and ECM accumulation, implying that targeting the Notch may exert therapeutic benefits.³⁵⁴ In addition, the Notch signaling stimulates EMT and EndMT, both of which participate in IPF. Notch1 signaling upregulates the expression of transcription factors like Snail and Slug, which drive EMT and contribute to fibrosis.³⁵⁵

Therapeutic advances

Di Vincenzo et al. investigated the detrimental impact of cigarette smoke on the wound healing processes in airway epithelial cells. Their findings revealed that cigarette smoke disrupts repair mechanisms by interfering with EMT and the Notch-1 signaling pathways. The study provided evidence that exposure to cigarette smoke led to a decrease in the expression of several key components of the Notch-1 pathway, including Notch1, Jagged1, and Hes1. Additionally, levels of basal cell markers such as TP63 and SCGB1A1 were also reduced. In contrast, TGF-β1 was shown to activate Notch-1 and facilitate the repair process. Furthermore, the inhibition of Notch signaling resulted in delayed wound healing, underscoring the critical role of the Notch-1 pathway in normal repair processes.³⁵⁶

In addition, the Notch pathway is involved in maintaining airway epithelial balance by regulating the differentiation of basal cells into ciliated or secretory cells. Its dysregulation can lead to goblet metaplasia or basal cell hyperplasia. For example, inhibition of the Jagged1-Notch2 signaling pathway reduces IL-13-induced goblet cell proliferation, and restoring Notch activity can improve epithelial repair disorders in COPD. In addition, abnormalities affecting the Notch pathway impact lung adenocarcinoma stem cells, indicating that targeting Notch ligands (such as DLL3) or receptors (such as Notch3) could potentially treat lung cancer and other respiratory diseases.³⁵⁷

Hippo signaling pathway

Pathological roles

The Hippo signaling pathway regulates the pathogenesis of diverse respiratory diseases, including acute lung injury (ALI), IPF, pulmonary arterial hypertension (PAH), pneumonia, and asthma. In ALI, the Hippo pathway participates in the repair process by promoting the proliferation and differentiation of lung epithelial progenitor cells and stimulating the repair of pulmonary capillary endothelium. For instance, YAP, a major target of the Hippo pathway, enhances the self-renewal of AECIIs and their differentiation into AECIs in response to mechanical tension, which promotes alveolar regeneration.³⁵⁸ Additionally, YAP activates the sprouting and remodeling of neovessels through various angiogenesis factors, improving the repair of the pulmonary capillary endothelium.³⁵⁹

It has been shown that the YAP/TAZ signaling is aberrantly activated in alveolar epithelial cells and fibroblasts, contributing to excessive collagen deposition and fibrosis in IPF. Moreover, activation of the YAP/TAZ promotes the proliferation and activation of lung fibroblasts, increasing collagen deposition and IPF progression.³⁶⁰ In PAH, YAP activation promotes the proliferation of pulmonary arterial vascular smooth muscle cells via the PI3K/AKT pathway, inducing pulmonary vascular remodeling and worsening of PAH.³⁶¹

Therapeutic advances

The Hippo signaling pathway regulates cell proliferation, inflammatory response and fibrosis process via targeting the core components MST1/2, LATS1/2 and effector YAP/TAZ-TEAD complex. In ALI, YAP accelerates tissue repair by promoting alveolar type II epithelial cell regeneration and pulmonary capillary repair. Inhibition of the YAP activity can block abnormal proliferation of pulmonary artery smooth muscle mediated by PI3K/AKT pathway and improve vascular remodeling in PAH. For pneumonia, YAP inhibits NF-κB overactivation by stabilizing IkB and alleviating alveolar inflammation. In IPF, targeting YAP/TAZ inhibits mechanical stress-induced fibroblast activation and collagen deposition while delaying alveolar epithelial cell senescence. In addition, the Hippo pathway influences the occurrence of airway inflammation in asthma by regulating the Notch4-Treg cell axis and airway smooth muscle reactivity via the S1P signaling. These findings suggest that agents that regulate the Hippo pathway activity (such as YAP inhibitors or agonists) are likely to be potential strategies for treating respiratory diseases, especially in terms of intervention in lung tissue regeneration, anti-fibrosis and immune regulation.³⁵⁹

Autoimmune diseases

In autoimmune diseases, the immune system attacks the body’s own tissues, causing chronic inflammation and tissue damage. These diseases can influence various organs and systems, including the gastrointestinal tract and lungs.³⁶² Cell differentiation and signaling pathways participate in the development and progression of autoimmune diseases (Fig. 11).

Fig. 11.

Therapeutic advances of cell differentiation-related signaling pathways in autoimmune diseases. a Wnt: Therapeutic interest centers on vitamin D’s inhibition of Wnt/β-catenin signaling to modulate immune cell differentiation and inflammatory responses. b TGF-β: The panel illustrates TGFβR inhibition as a potential strategy to modulate SMAD-dependent immune responses in autoimmune disease. c Hippo: Ginsenosides modulate YAP-dependent immune regulation, influencing myeloid-derived suppressor cells and T-cell responses. d Notch: DAPT inhibits γ-secretase-mediated Notch cleavage, whereas DLL/Jagged-targeting antibodies and miR-146a modulate Notch signaling through distinct mechanisms. e Hedgehog: Preclinical si-S1A3-Chol-mediated SMO silencing suppresses GLI-dependent transcription in experimental autoimmune arthritis. The graphics were created with BioRender (https://www.biorender.com)

Wnt signaling pathway

Pathological roles

The Wnt signaling pathway modulates the occurrence of systemic and localized bone loss in RA, due to the imbalance in the osteoblast-osteoclast axis. Pro-inflammatory cytokines such as TNFα, IL-1β, and IL-6 activate the RANK/RANKL pathway, thereby increasing osteoclast activity and bone resorption. Meanwhile, these cytokines inhibit the Wnt signaling, which is crucial for bone formation and homeostasis, by increasing the levels of inhibitors like DKK1 and sclerostin. This dysregulation of Wnt signaling may induce the bone complications detected in RA, including erosions and systemic osteoporosis. Upregulated DKK1 expression in RA patients leads to more severe bone damage, suggesting its potential to be a biomarker and therapeutic target.^363,364

Besides bone metabolism, the Wnt signaling also regulates immune cell differentiation and proliferation. It modulates the proliferation, differentiation, and self-renewal of hematopoietic stem cells, which can differentiate into immune cells such as T cells, B cells, and macrophages. Impaired Wnt signaling has been linked to the development of autoimmune conditions or immune deficiency. For example, the expression of a stable form of β-catenin was observed to prolong the survival of Tregs, and its activation in inflammatory conditions stimulated the occurrence of autoimmune responses. Pro-inflammatory cytokines such as TNFα and IL-1β inhibit the Wnt signaling by inducing DKK1 and sclerostin, mediating the association of inflammation with Wnt pathway dysregulation in autoimmune diseases.^365,366

Therapeutic advances

In their study, Zou et al. investigated how vitamin D enhances the management of systemic lupus erythematosus (SLE) using a mouse model by inhibiting the Wnt/β-catenin signaling pathway. Their findings revealed that vitamin D significantly down-regulated the expression of Wnt ligands, specifically Wnt1 and Wnt3a. Additionally, it inhibited the nuclear translocation of β-catenin and the expression of its downstream target genes (c-Myc and Cyclin D1). This action contributed to the reduction of impaired apoptosis in Th1/Th2 immune cells and lowered the levels of autoantibodies (ANA, anti-DSDNA, anti-SNRNP). Notably, the overactivation of the Wnt signaling pathway enhances the stability of β-catenin in conjunction with GSK-3β, leading to worsened autoimmune reactions. Conversely, vitamin D treatment effectively obstructs this pathway, restores immune equilibrium, and alleviates SLE symptoms, thus offering a promising new approach for treating autoimmune diseases through targeting the Wnt pathway.³⁶⁷

TGF-β signaling pathway

Pathological roles

In autoimmune thyroid diseases, TGF-β plays dual roles, acting both as an immunosuppressive factor and as a facilitator of autoimmune responses, depending on the stage of the disease and the specific signaling pathways activated. In Graves’ disease, TGF-β deficiency caused a marked increase in expression of MHC class II antigens and T-cell activity, while treatment with the recombinant TGF-β abolished these effects.³⁶⁸ Similarly, in Hashimoto’s thyroiditis, TGF-β levels were found to be initially decreased, contributing to the disease onset by altering Th1/Th2 balance and inhibiting Tregs. However, it increases afterwards to promote fibrosis and thyroid cell depletion.³⁶⁹

The dual role of TGF-β has been documented in other autoimmune conditions, such as multiple sclerosis, where TGF-β signaling participates in the maintenance of the immune tolerance. In MSCs, TGF-β modulates their immunosuppressive potential, with MSC-derived TGF-β1 playing a role in the induction of Tregs and modulation of the Th1/Th2/Th17 balance, which is critical for controlling autoimmune responses.^370,371 Therefore, understanding the precise mechanisms and context-specific actions of TGF-β will pave the way for the development of targeted therapies to treat autoimmune diseases.

Therapeutic advances

Zhang et al. demonstrated that high sugar intake exacerbates autoimmune diseases by promoting Th17 cell differentiation. It also activates the latent state of TGF-β by increasing the level of ROS in T cells, particularly mitochondria-derived ROS. This, in turn, drives Th17 cell differentiation and worsens the pathological processes associated with mouse colitis and experimental autoimmune encephalomyelitis. Data from experimental studies indicate that TGF-β activation promotes proinflammation in a high-glucose environment: In vitro, high glucose activates TGF-β signaling in a ROS-dependent manner, enhancing the generation of RORγt-dependent Th17 cells; In vivo, TGFBR2-deficient T cells show a poor response to the autoimmune-enhancing effects induced by high glucose, confirming the significance of TGF-β signaling. It was also observed that TGF-β activation preferentially promoted Th17 rather than Treg differentiation in specific cytokine environments, such as in the presence of IL-6, providing new insights into the interaction between metabolic factors and cytokine networks in autoimmune diseases. This also implies that targeting the TGF-β pathway or ROS may be a therapeutic strategy for alleviating hyperglycemia-related autoimmune pathology.³⁷²

Hedgehog signaling pathway

Pathological roles

In RA, the Hh pathway is over-activated in synovial tissues, promoting the proliferation and invasive potential of fibroblast-like synoviocytes (FLS) and contributing to cartilage damage and joint inflammation.^373,374 Evidence shows that the Hh signaling regulates pancreatic β-cell function and insulin production in diabetes, and its dysregulation can enhance the development of insulin-dependent diabetes mellitus³⁷⁴. It also affects the polarization of macrophages and activation of NKT cells, which participate in the generation of immune responses and disease pathology.^375,376

Therapeutic advances

In RA, Hh pathway hyperactivation drives FLS to acquire an invasive, tumor-like phenotype which manifests with unregulated proliferation, migration, and secretion of destructive enzymes and cytokines. Researchers developed si-S1A3-Chol, a cholesterol-conjugated, chemically modified siRNA targeting SMO. When delivered via intra-articular injection in collagen-induced arthritis mice, si-S1A3-Chol inhibited SMO expression, blocked nuclear translocation of the transcription factor GLI1, and decreased the levels of downstream factors like Cyclin D1 and MMP-9. This improved the cell-cycle control (G0/G1 arrest), inhibited synovitis, and preserved joint function without systemic toxicity.³⁷⁷ Localized delivery can mitigate the risks associated with systemic Hh inhibition, such as gastrointestinal or developmental side effects seen following oral administration of SMO inhibitors such as vismodegib.

Clinically, repurposed SMO inhibitors and emerging agents like si-S1A3-Chol are poised to be crucial therapeutic strategies. However, there are challenges regarding how to balance its efficacy and off-tissue effects. Future studies should optimize delivery systems and develop combinatorial regimens with immune checkpoint modulators to exploit the Hh pathway plasticity to treat autoimmune diseases.

Notch signaling pathway

Pathological roles

The Notch signaling pathway influences the initiation and progression of autoimmune diseases, including RA and atherosclerosis. In RA, Notch signaling could regulate angiogenesis, which influences the progression of synovial inflammation and joint destruction. It has been reported that the Notch signaling, particularly via its interaction with HIF-1α, promotes angiogenesis and inflammation in RA-affected joints, which exacerbates disease severity and joint damage.^378,379 Moreover, the Notch signaling can alter the polarization of macrophages and differentiation of T cells, both of which participate in the regulation of immune responses associated with RA. For instance, the Notch signaling pathway can promote the differentiation of M1 macrophages, important pro-inflammatory factors, while inhibiting the formation of anti-inflammatory M2 macrophages. This imbalance promotes chronic inflammation which is a major feature of RA.³⁸⁰

In atherosclerosis, the Notch signaling can modulate immune cell function and inflammation. It also regulates the polarization of macrophages into the pro-inflammatory M1 subtype, enhancing the severity of plaque formation and progression. The Notch signaling also interferes with T-cell differentiation, thereby promoting the development of Th1 and Th17 cells leading to inflammation and atherosclerotic plaque instability. Conversely, it inhibits the differentiation of Tregs, which can protect against the development of atherosclerosis. This suggests that targeting Notch signaling may shift the immune balance towards a more anti-inflammatory state, thus reducing atherosclerosis progression.³⁸¹ Overall, these studies show that the Notch signaling pathway can regulate immune cell function and inflammation in autoimmune diseases, suggesting that it is a promising target for disease treatment.

Therapeutic advances

Pharmacological agents have been designed to modulate the Notch signaling in autoimmunity. They include GSIs such as DAPT, which inhibit proteolytic cleavage of Notch receptors to prevent release of the NICD and the transcription of downstream genes to ameliorate RA progression owing to suppression of FLS and MMP secretion while also reducing demyelination in experimental autoimmune encephalomyelitis models.³⁸² Moreover, ligand-specific antibodies that fine-tune immune responses have been reported. For instance, agents targeting Jagged1 can downregulate the Th2-mediated allergic inflammation, whereas anti-DLL4 antibodies can mitigate Th1-induced pathologies.³⁸³ Alternatively, miRNA-based modulators like miR-146a, a natural Notch1 inhibitor often downregulated in RA monocytes, was delivered as a synthetic mimic to myeloid cells, which resulted in the suppression of the Notch1 signaling, anti-inflammatory M2 macrophage polarization, and alleviation of bone erosion in collagen-induced arthritis models. Collectively, these approaches leverage molecular, antibody-directed, and epigenetic mechanisms to restore immune balance.³⁸⁴

Hippo signaling pathway

Pathological roles

In autoimmune diseases such as RA, IBD, and psoriasis, the Hippo pathway regulates immune cell differentiation, proliferation, and inflammatory responses. For instance, in RA, inactivation of the Hippo pathway can increase YAP/TAZ activity, which promotes the abnormal growth and migration of FLS, thus contributing to joint destruction.³⁸⁵ Similarly, in IBD, YAP/TAZ regulate intestinal epithelial cell proliferation and mucosal regeneration, and dysregulation of these proteins contributes to chronic inflammation. The Hippo pathway interacts with other pathways such as TGF-β and TLRs, to alter immune responses and autoimmune disease progression.³⁸⁶

Therapeutic advances

The Hippo pathway regulates the function of macrophages, T cells, and dendritic cells through core molecules such as MST1/2, YAP/TAZ, and interacts with immune-related pathways such as NF-κB and TGF-β to influence autoimmune diseases. In RA, YAP promotes synovial fibroblast proliferation and angiogenesis, while MST1 deficiency exacerbates inflammation and bone erosion. YAP/TAZ were reported to activate the intestinal epithelial regeneration by stimulating the Wnt/β-catenin signaling in IBD, and its loss caused intestinal barrier breakdown. In psoriasis, YAP stimulates pathological progression by regulating keratinocyte proliferation and angiogenesis. The Hippo pathway can influence immune homeostasis by balancing Th17/Treg differentiation. A study showed that inhibition of MST1 enhanced Th17 response and reduced Treg expression. These mechanisms suggest that targeting the Hippo pathway can alter immune cell differentiation and the generation of inflammatory responses, making it a potential strategy for treating autoimmune diseases.³⁸⁶

Zhang et al. showed that ginsenoside treatment ameliorated experimental autoimmune hepatitis (AIH) by modulating the Hippo-YAP/TAZ signaling pathway. Furthermore, ginsenosides can stimulate the proliferation and function of myeloid-derived suppressor cells (MDSCs), especially the monocytic subset, by activating glucocorticoid receptor and YAP, a core effector of the Hippo pathway, increasing their potential to secrete immunosuppressive molecules (such as iNOS, Arg-1 and NO). It also inhibits the production of ROS. In addition, ginsenosides can remodel the immune microenvironment via targeting the YAP signaling cascade, promoting the differentiation of Tregs and preventing overactivation of Th17 cells to alleviate concanavalin A-induced hepatic inflammatory damage. Moreover, the Hippo-YAP pathway can connect the innate immunity (MDSCs) and adaptive immunity (T-cell balance), providing a new target for AIH therapy.³⁸⁷

Skeletal diseases

Skeletal diseases comprise a wide range of disorders that impact bone and cartilage, including osteoporosis, osteoarthritis (OA), fracture nonunion, heterotopic ossification (HO), and rare genetic skeletal dysplasias. These conditions are intricately associated with abnormal stem and progenitor cell fate decisions, impaired differentiation of osteoblasts and chondrocytes, excessive osteoclast activity, and age-related changes in bone marrow mesenchymal stromal cells toward adipogenesis and senescence. Emerging evidence suggests that the Wnt, TGF-β/BMP, Hh, Notch, and Hippo signaling pathways play crucial roles in regulating skeletal homeostasis, and their dysregulation contributes to both degenerative and developmental skeletal pathologies.³⁸⁸ Consequently, therapeutic strategies aimed at targeting these pathways represent a promising avenue for restoring lineage balance, enhancing bone regeneration, and mitigating pathological ossification (Fig. 12).

Fig. 12.

Therapeutic advances of cell differentiation-related signaling pathways in skeletal diseases. a Wnt: Therapeutics promote bone formation by neutralizing endogenous Wnt inhibitors, using DKK1 inhibitors or the anti-sclerostin antibody romosozumab to enhance β-catenin-mediated transcription. b TGF-β: Modulation employs rhBMP-2 to stimulate SMAD1/5/8, while TGFβRI inhibitors manage SMAD2/3-dependent fibrotic or regulatory responses. c Hippo: Therapeutics utilize Verteporfin to inhibit YAP/TAZ-TEAD interaction, modulating environmental sensing and proliferation in skeletal cells. d Notch: DAPT inhibits γ-secretase-mediated NICD release, whereas anti-NOTCH2 antibodies selectively block NOTCH2 receptor signaling. e Hedgehog: In preclinical models, Hedgehog agonists enhance bone repair, whereas GANT58 suppresses pathological Hedgehog activation and ectopic ossification. The graphics were created with BioRender (https://www.biorender.com)

Wnt signaling pathway

Pathological roles

The Wnt/β-catenin pathway plays a crucial role in maintaining skeletal homeostasis by regulating the differentiation and function of bone marrow mesenchymal stem cells, osteoblasts, osteoclasts, and osteocytes. A decrease in Wnt activity negatively impacts osteoblastogenesis, promotes marrow adipogenesis, and is linked to age-related bone loss and osteoporosis. Furthermore, genetic and experimental studies have demonstrated that abnormalities in components of the Wnt pathway, including LRP5/6 and antagonists such as sclerostin and DKK1, are significantly associated with lowbone-mass phenotypes, impaired bone formation, and an increased risk of fractures.³⁸⁸

Therapeutic advances

Therapeutically, Wnt activation has emerged as one of the most advanced pathway-based approaches in skeletal medicine. The anti-sclerostin monoclonal antibody romosozumab enhances Wnt signaling and is approved for the treatment of osteoporosis, promoting increased bone formation and mass while reducing fracture risk.³⁸⁹ Furthermore, dual inhibition of sclerostin and DKK1 has demonstrated synergistic anabolic effects in preclinical models, resulting in enhanced bone mass accrual and more effective fracture repair compared to the blockade of either antagonist alone.³⁹⁰ These findings bolster the idea that modulating the Wnt pathway presents a logical approach for addressing osteoporosis and other skeletal disorders associated with compromised osteogenesis.

TGF-β signaling pathway

Pathological roles

The TGF-β/BMP superfamily pathways regulate the fate and function of bone marrow mesenchymal stem/stromal cells, osteoblasts, osteocytes, chondrocytes, and osteoclasts in a stage-specific and context-dependent manner. BMP signaling promotes osteogenic and chondrogenic differentiation, supporting bone formation and repair, while TGF-β signaling has dynamic effects, including progenitor cell recruitment, coupling bone resorption to formation, regulating osteocyte function, and maintaining the bone matrix microenvironment. Disruption of the balance between TGF-β and BMP signaling can lead to skeletal pathologies.³⁹¹

In metabolic bone disease, excessive or mislocalized TGF-β activity contributes to uncoupled bone remodeling and impaired bone quality, while inadequate BMP activity can weaken osteoblast differentiation and bone formation. This imbalance is particularly relevant to osteoporosis and age-related bone loss, where reduced osteogenesis, increased marrow adipogenesis, and defective regenerative responses are common pathological features. TGF-β/BMP dysregulation also affects the differentiation potential and senescence-associated behavior of skeletal stem/progenitor cells, linking this pathway to the age-related deterioration of bone homeostasis. The pathway is also strongly implicated in degenerative joint disease. In OA, abnormal activation of TGF-β signaling in subchondral bone has been shown to disturb the osteochondral unit, promote aberrant bone remodeling, and accelerate cartilage degeneration. Concurrently, altered BMP signaling can influence chondrocyte hypertrophy, matrix turnover, and osteophyte formation, further contributing to disease progression. Thus, TGF-β/BMP signaling is not only important for skeletal development but also for maintaining the adult joint microenvironment.³⁹¹

Inherited and acquired disorders related to ectopic or abnormal ossification exemplify the pathogenic role of TGF-β/BMP signaling. Camurati-Engelmann disease results from activating mutations in TGFB1, which lead to heightened TGF-β activity, disrupted bone remodeling, and progressive skeletal sclerosis. In contrast, fibrodysplasia ossificans progressiva (FOP) is caused by activating mutations in ACVR1/ALK2, a BMP type I receptor, resulting in inappropriate osteochondrogenic differentiation and progressive HO in soft tissues. A critical mechanistic aspect of FOP is the abnormal responsiveness of the mutant ACVR1 to activin A, which erroneously activates downstream osteogenic signaling pathways and facilitates the formation of bone in non-skeletal tissues. Together, these findings suggest that dysregulation of TGF-β/BMP signaling is implicated in a wide array of skeletal disorders, including osteoporosis and OA, as well as rare but insightful genetic disorders.³⁹²

Therapeutic advances

Among the various pathway-based skeletal therapies, recombinant BMPs have already found their place in clinical applications. Recombinant human BMP-2 has been extensively utilized in spinal fusion and certain trauma cases, with evidence indicating that fusion rates are either comparable to or even exceed those achieved with autologous bone grafts in suitable scenarios. These results underscore the osteoinductive capabilities of BMP-based therapies in facilitating bone regeneration and enhancing structural repair. Nevertheless, the clinical deployment of recombinant human BMP-2 necessitates meticulous consideration of dosage and specific indications, as excessive BMP activity can elevate the risk of inflammation, ectopic ossification, and other local complications.³⁹³

In parallel, more selective suppression of excessive TGF-β signaling is emerging as a precision strategy for skeletal dysplasia. Bone-targeted delivery of a TGFβRI inhibitor restored bone turnover and improved the skeletal phenotype in preclinical models of Camurati–Engelmann disease. This finding suggests that, in skeletal disorders driven by pathological TGF-β activation, inhibiting this pathway may help rebalance bone remodeling and alleviate abnormal bone accumulation. Additionally, activin A blockade has shown lesion-suppressive effects in FOP, further highlighting the translational potential of targeting the broader TGF-β/BMP axis in skeletal disease. Together, these studies indicate that therapeutic modulation of this pathway is highly context-dependent, with BMP enhancement being more suitable for bone repair, while inhibition of excessive TGF-β-related signaling may be more effective in sclerosing bone disorders and pathological ossification.³⁹⁴

Hedgehog signaling pathway

Pathological roles

Hh signaling, specifically through IHh and SHh, is critical for skeletal development, chondrocyte maturation, osteoblast differentiation, and fracture repair. Dysregulation of Hh activation has been associated with OA-related cartilage degeneration and pathological ectopic ossification, including trauma-induced tendon ossification and HO. Conversely, inadequate Hh activity may hinder reparative osteogenesis and contribute to impaired fracture healing, particularly in aged or metabolically compromised environments.

Therapeutic advances

Therapeutic modulation of Hh signaling is highly context-dependent. On the regenerative front, pharmacological activation of SMO through Hh agonists has demonstrated an ability to enhance fracture healing in aged mice and improve bone repair in preclinical models. Conversely, in conditions characterized by pathological ossification, inhibiting Hh signaling may offer therapeutic benefits. For instance, the use of GANT58 to suppress Hh signaling resulted in a reduction of osteogenic and chondrogenic differentiation, as well as a decrease in trauma-induced tendon ossification in vivo. These findings indicate that both Hh agonists and antagonists can possess therapeutic potential in skeletal diseases, contingent upon the clinical objective—whether it is to facilitate bone repair or to inhibit ectopic bone formation.^395,396

Notch signaling pathway

Pathological roles

Notch signaling regulates the fate of skeletal stem and progenitor cells, orchestrating osteoblast differentiation, osteoclastogenesis, cartilage homeostasis, and fracture repair. Gain-of-function Notch2 mutations drive osteoporosis, acroosteolysis, and fractures in Hajdu-Cheney syndrome.^397,398 In aging bone, elevated Notch activity diminishes osteolineage commitment, increases marrow adiposity, and compromises repair. In articular cartilage, transient Notch activation facilitates repair, while prolonged activation promotes OA-like degeneration.

Therapeutic advances

Preclinical studies suggest that selectively modulating Notch signaling could offer therapeutic benefits for skeletal diseases. In a mouse model of Hajdu–Cheney syndrome, the administration of an anti-Notch2 antibody successfully reversed the osteopenic phenotype. This finding underscores the potential of receptor-selective inhibition as a treatment strategy for gain-of-function skeletal disorders.³⁹⁸ In parallel, γ-secretase inhibition with DAPT suppressed proliferation and osteogenic/chondrogenic differentiation of tissue-resident mesenchymal progenitors and reduced ectopic cartilage and bone formation in traumatic HO models.³⁹⁹ These findings suggest that Notch-targeted interventions may be beneficial in both rare osteolytic syndromes and acquired pathological ossification, although careful temporal and tissue-specific control will be essential.

Hippo signaling pathway

Pathological roles

The Hippo pathway, via its downstream effectors YAP and TAZ, regulates mesenchymal lineage allocation, osteoblast differentiation, osteocyte function, cartilage maintenance, and overall bone quality. Dysregulated Hippo signaling has been linked to osteoporosis, OA, bone tumors, and impaired skeletal regeneration. In bone marrow stromal cells, YAP/TAZ influence the balance between osteogenesis and adipogenesis, while in chondrocytes and joint tissues they modulate cartilage integrity and degenerative remodeling.⁴⁰⁰ These observations position Hippo signaling at the core of skeletal mechanotransduction and age-related skeletal degeneration.

Therapeutic advances

Hippo-targeted skeletal therapies are primarily in the preclinical stage, yet they are progressing swiftly. Research utilizing OA models has demonstrated that the localized administration of the YAP inhibitor verteporfin can reduce cartilage degeneration. This finding suggests that excessive YAP activity can be effectively managed in cases of degenerative joint disease. Concurrently, additional studies indicate that YAP/TAZ activity plays a beneficial role in osteogenesis and fracture repair when situated in the correct cellular environments. This implies that future therapies based on the Hippo pathway will likely necessitate a nuanced approach, requiring modulation that is both tissue-specific and stage-specific, rather than merely implementing broad inhibition or activation.^401,402 Thus, Hippo signaling represents a promising but mechanistically nuanced target for osteoporosis, OA, and bone repair.

Translational challenges for cell differentiation pathway-targeted therapies

Despite encouraging therapeutic advances, translating differentiation-related signaling pathways into durable clinical benefits remains challenging because these pathways also play essential roles in normal tissue homeostasis. On-target toxicities therefore represent a significant barrier to clinical development. For example, Wnt inhibitors have been associated with gastrointestinal and bone-related adverse effects, reflecting the physiological importance of Wnt signaling in tissue renewal and skeletal maintenance. Likewise, systemic TGF-β blockade has raised concerns regarding cardiovascular safety, impaired tissue repair, and structural toxicity, underscoring the need for careful dose optimization and intermittent scheduling strategies during clinical development.^403,404 Another major challenge is identifying patients most likely to benefit from pathway-targeted interventions. Because pathway activity is highly context-dependent, biomarker-driven stratification will be essential for improving efficacy and limiting unnecessary toxicity. Potential approaches include using pathway activation signatures, mutational status, and tissue-specific transcriptional or protein profiles to identify biologically responsive patient subsets. In this context, predictive biomarker development should move beyond single-gene alterations and increasingly incorporate multi-omic and lineage-context information, particularly for pathways such as Wnt, TGF-β, Notch, and Hippo, which exhibit strong tissue-specific outputs.⁴⁰⁵

Adaptive resistance often significantly limits the long-term efficacy of pathway-directed therapies. In numerous contexts, the inhibition of a specific signaling axis may initiate compensatory rewiring through parallel or interacting pathways, including PI3K–AKT, MAPK, Wnt, Hh, and inflammatory signaling networks. This feedback adaptation can sustain cancer stemness, survival, or mesenchymal plasticity despite initial target suppression. Furthermore, extensive pathway crosstalk complicates therapeutic responses, as Notch, Wnt, Hh, TGF-β, and Hippo signaling frequently operate within interconnected regulatory circuits rather than in isolation.^406,407

These considerations provide a compelling rationale for the implementation of combination-based treatment strategies. Mechanistically informed combinations may enhance efficacy by concurrently suppressing compensatory signaling, reducing microenvironmental support, or promoting differentiation restoration. Particularly promising avenues include the combination of pathway inhibitors with immunotherapy, anti-fibrotic agents, or other targeted therapies, especially in tumors or chronic diseases characterized by stromal remodeling, immune evasion, or lineage plasticity. More broadly, future advancements will hinge on balancing efficacy with safety, integrating biomarker-guided patient selection, and designing context-specific combination regimens that take into account tissue biology and pathway interdependence. A deeper understanding of differentiation context dependency will therefore be essential for the safe translation of pathway-targeted interventions into sustainable therapeutic outcomes.

Conclusions and perspectives

Over the past several decades, research on cell differentiation has progressed from descriptive observations of embryonic development to a mechanistic framework centered on conserved signaling networks. Wnt, Notch, Hedgehog, TGF-β, and Hippo signaling collectively control the balance among stem-cell maintenance, lineage commitment, maturation, and tissue-specific function. Although these pathways differ in their ligands, receptors, intracellular mediators, and transcriptional effectors, they share several organizational principles. They convert extracellular, cell–cell, and mechanical information into gene-regulatory responses; use positive and negative feedback to define signaling thresholds and duration; and interact with chromatin regulators, non-coding RNAs, metabolic programs, and the ECM. Their outputs are therefore determined not only by whether a pathway is activated, but also by where, when, for how long, and in which cellular state activation occurs. This context-dependent view provides a more accurate explanation of cell-fate control than treating each pathway as an isolated linear cascade.

A major conclusion of this review is that pathway crosstalk and tissue-specific heterogeneity are central to both normal differentiation and disease. Wnt and Notch cooperate or oppose one another during intestinal, neural, and hematopoietic differentiation; TGF-β/BMP signaling intersects with Wnt, MAPK, and Hippo effectors during mesenchymal and epithelial fate decisions; and YAP/TAZ integrate biochemical signaling with mechanical tension and matrix stiffness. The same molecular component can consequently exert distinct or even opposite effects across organs and disease stages. For example, transient pathway activation may support regeneration after acute injury, whereas persistent activation may sustain fibrosis, inflammation, stemness, or malignant progression. Likewise, broad pathway inhibition may suppress a pathological cell population while impairing physiological renewal in neighboring tissues. These observations emphasize that differentiation-related signaling should be understood as a dynamic network whose function depends on lineage identity, microenvironmental inputs, temporal patterning, and compensatory interactions.

Dysregulation of these networks contributes to a wide spectrum of human diseases, including cancer and tumorigenesis, cardiovascular, neurodegenerative, metabolic, digestive, respiratory, autoimmune, and skeletal diseases (Fig. 13). In cancer, altered Wnt, Notch, Hh, TGF-β, and Hippo signaling can maintain cancer stem-cell properties, promote dedifferentiation and epithelial–mesenchymal transition, remodel the tumor microenvironment, and support therapeutic resistance. In cardiovascular, digestive, respiratory, and skeletal disorders, abnormal pathway activity can redirect progenitor-cell fate, activate fibroblasts or myofibroblasts, disturb tissue repair, and drive pathological remodeling. In neurodegenerative and metabolic diseases, defective differentiation signaling affects neuronal survival, adult neurogenesis, adipogenesis, insulin-related processes, and organ-specific regeneration. Autoimmune diseases further illustrate the importance of context, because the same pathway may regulate both tissue-resident cells and immune-cell differentiation. The therapeutic approaches reviewed here—including small-molecule inhibitors, monoclonal antibodies, ligand traps, antisense oligonucleotides, gene-based strategies, natural compounds, cell therapies, and biomaterial-assisted delivery—demonstrate that these pathways are therapeutically tractable. However, the level of evidence varies substantially, and many interventions remain preclinical or exploratory rather than established treatments.

Fig. 13.

Overview of human diseases associated with dysregulated cell differentiation-related signaling pathways. Dysregulation of core cell differentiation-related signaling pathways is associated with a broad spectrum of pathological conditions across multiple organ systems, including cancer and tumorigenesis, cardiovascular diseases, neurodegenerative diseases, metabolic diseases, digestive diseases, respiratory diseases, autoimmune disorders, and skeletal diseases. The central diagram illustrates the process of cell differentiation, whereby multipotent cells give rise to specialized cell types, whose aberrant regulation contributes to the pathogenesis of the listed disorders. The graphics were created with BioRender (https://www.biorender.com)

Several limitations continue to restrict clinical translation. First, pleiotropy creates a narrow therapeutic window: pathway components that drive disease are often required for normal stem-cell maintenance, immune regulation, vascular integrity, or wound repair. Second, pathway activity is highly heterogeneous between patients, tissues, and cellular compartments, making a single molecular marker insufficient for treatment selection. Third, compensatory rewiring can restore downstream transcription through parallel pathways such as PI3K–AKT, MAPK, inflammatory signaling, or alternative members of the same ligand–receptor family. Fourth, conventional two-dimensional cultures and some animal models do not adequately reproduce human tissue architecture, aging, immune–stromal interactions, or chronic disease evolution. Fifth, many studies infer pathway activity from the abundance of one component rather than measuring spatial localization, phosphorylation state, target-gene output, or signaling dynamics. These limitations complicate comparisons across studies and may contribute to the gap between promising mechanistic findings and inconsistent clinical responses.

Addressing these challenges will require strategies that prioritize precision over indiscriminate pathway activation or inhibition. Tissue-restricted and cell-type-specific delivery systems—including nanoparticles, engineered antibodies, local hydrogels, viral vectors, and targeted oligonucleotides—may reduce systemic toxicity while preserving physiological signaling elsewhere. Temporal control is equally important; intermittent dosing, stage-specific intervention, and reversible modulation may be preferable to continuous blockade of pathways involved in tissue renewal. Biomarker development should integrate mutations with pathway activation signatures, phosphoproteomic profiles, cell-state composition, and spatial information rather than relying on single genes. Mechanistically informed combination therapies may prevent compensatory signaling, but combinations should be selected on the basis of demonstrated network dependencies rather than empirical escalation. Human organoids, patient-derived cells, organ-on-chip systems, and co-culture models incorporating immune, stromal, vascular, and epithelial compartments should be used alongside animal models to improve prediction of efficacy and toxicity. Standardized reporting of disease context, cell type, treatment timing, dose, and pathway readouts would also strengthen reproducibility and facilitate comparison across studies.

Future research should focus on resolving signaling dynamics at single-cell and tissue scales and translating that information into clinically actionable decisions. Single-cell multi-omics and spatial transcriptomics can identify rare pathogenic cell states and determine which cells respond to treatment, while live-cell imaging and lineage tracing can reveal how signal duration and oscillation influence fate transitions. CRISPR-based perturbation screens can map causal pathway interactions, and artificial intelligence may assist in integrating genomic, epigenomic, proteomic, imaging, and clinical data to predict therapeutic vulnerabilities. These technologies should be coupled with longitudinal patient sampling to distinguish drivers of disease initiation from adaptive responses that emerge during progression or treatment. Prospective validation will be essential before computationally defined pathway states can guide therapy. Future trials should therefore incorporate pharmacodynamic measurements, lineage-resolved biomarkers, and long-term surveillance for effects on regeneration, immunity, and secondary tissue remodeling. Particular care will be required when interventions are applied to children, older adults, or patients with chronic inflammatory and fibrotic conditions, in whom baseline regenerative capacity and pathway activity may differ substantially. Ultimately, the goal is not simply to switch a pathway on or off, but to restore an appropriate differentiation trajectory in the correct cells at the correct time. Achieving this objective will require close integration of developmental biology, systems biology, pharmacology, biomaterials, and clinical investigation. Such an approach may convert the broad biological importance of differentiation-related signaling pathways into safer, more durable, and more personalized therapeutic benefits.

Author contributions

Xiao Xu, Xuyong Wei, and Zhe Yang conceived the review. Ze Xiang, Jiarui Li, Yunyang Xu, Chenhao Xu and Shuhui Li performed the literature search and drafted the manuscript. Xiao Xu, Xuyong Wei, Zhe Yang and Shusen Zheng critically revised the manuscript. All authors have read and approved the review article.

Competing interests

The authors declare no competing interests.