Gene expression regulation by Ca²⁺ signaling: an updated systematic review

Abstract

All life processes depend on the precise spatiotemporal expression of genes, which involves orderly processes including transcription, posttranscriptional processing, translation, and posttranslational modification. Accumulating evidence demonstrates that Ca²⁺ is the most critical second messenger that orchestrates nearly all fundamental biological processes vital for maintaining normal physiological functions. Ca²⁺ homeostasis/signaling is primarily maintained through Ca²⁺ influx, cytoplasmic Ca²⁺ release, Ca²⁺ store cycling, and binding and release of Ca²⁺ buffers. Their coordinated interactions ensure that Ca²⁺ concentrations remain within the physiologically appropriate range. Ca²⁺ signaling must be appropriately activated or suppressed during cellular signal transduction to support specific functions, and its dysregulation can trigger various pathological conditions. This review summarizes recent progress in Ca²⁺ signaling regulatory networks, including the roles of key regulatory elements/toolkits, the functional significance of Ca²⁺ signals in different microdomains, and the influence of Ca²⁺ signaling on gene expression, along with the underlying mechanisms at various stages of gene expression. The involvement of Ca²⁺, both independently and collaboratively, in the nucleus, cytoplasm, subcellular microdomains such as mitochondria, and the extracellular space, in the multi-level regulation of gene expression, has been extensively studied. This information is essential for understanding the mechanisms underlying gene expression and for advancing the diagnosis and treatment of diseases. Finally, we propose forward-looking recommendations to address current research gaps, aiming to provide valuable references for researchers in this field.

Graphical abstract

Supplementary Information

The online version contains supplementary material available at 10.1186/s11658-026-00872-5.

Introduction

In broad terms, gene expression involves multistep processes such as transcription, posttranscription, translation, and posttranslational processing and modification. A variety of biomacromolecules, including DNA, RNA, proteins, and signaling pathways precisely choreograph each process of gene expression in a critically coordinated manner. Abnormalities in gene expression with spatiotemporal dysregulation may contribute to the onset of diseases and even lead to cell death. Growing evidence shows that calcium ions (Ca²⁺) are involved in most physiological activities, including proliferation, development, cycle, differentiation, apoptosis, and excitability regulation [1–3]. Ca²⁺ signaling characteristics with dynamic coding ability, spatiotemporal specific decoding mechanism, multi-pathway regulatory network, and complex interaction with other signaling systems enable cells to respond precisely to physiological stimuli, such as cytokines, hormones, and small metabolites, and adapt to complex microenvironmental changes. The imbalance of cellular Ca²⁺ homeostasis affects gene expression at multiple steps, ultimately shaping the behaviors and fate of cells, which might be one of the pathological mechanisms of numerous diseases, such as cardiomyopathy, ischemia–reperfusion injury, Alzheimer’s disease, Angelman syndrome, infertility, and cancers [4–9]. The underlying mechanism is largely attributed to the disordered spatiotemporal expression of numerous genes related to Ca²⁺ signal regulation. Once Ca²⁺ homeostasis is disrupted, it forces cells to mount well-defined responses, either activation of Ca²⁺-dependent regulation systems that quickly bring intracellular Ca²⁺ back to baseline, or engagement of Ca²⁺-triggered death mechanisms, such as calpain activation, mitochondrial permeability transition, or caspase-mediated apoptosis [10, 11]. Therefore, an in-depth exploration of the regulation and mechanism of Ca²⁺ signaling on gene expression is important for understanding many physiological and pathological processes and the diagnosis and treatment of Ca²⁺ dyshomeostasis-causing diseases.

Ca²⁺ homeostasis and signaling are two aspects of Ca²⁺ management that play critical roles in cellular physiological and pathological processes. Ca²⁺ homeostasis mainly refers to the maintenance of physiological Ca²⁺ levels in different compartments, whereas Ca²⁺ signaling pertains to the process in which Ca²⁺ participates in regulating various cellular functions by transmitting specific information within the cells under stimulation. Together, they represent the dual role of Ca²⁺ in biological processes and cannot be viewed or discussed in isolation from one another. Both involve processes such as the binding, distribution, release, refilling, transport, and recycling of Ca²⁺. These processes jointly maintain Ca²⁺ homeostasis or achieve signal transduction through Ca²⁺ toolkits, including channels, pumps, exchangers, or transporters on plasma and organelle membranes and Ca²⁺ buffers in the intracellular microenvironment. In the resting state, free Ca²⁺ accounts for only approximately 1% of the total cellular Ca²⁺ in mammalian cells [12]. The Ca²⁺ concentration in the cytoplasm is approximately 100 nM, in Ca²⁺ stores such as the endoplasmic reticulum (ER) and mitochondria is around 100–1000 μM and 100–200 nM, respectively, whereas the extracellular Ca²⁺ concentration is as high as 1–2 mM [13, 14]. The balance between the free and bound forms of Ca²⁺ in the cell and homeostasis in different regions within the cell are precisely controlled by Ca²⁺ toolkits. Given that Ca²⁺ toolkits have been systematically summarized in previous reviews [10, 15], we present their composition and functions in Fig. 1. Besides mediating the flow of Ca²⁺, the toolkits might also be regulated by Ca²⁺ itself in a feedback-adjusted manner at the transcription level [16, 17] or by manipulating protein activities [18]. For instance, Ca²⁺ signaling can phosphorylate the AU-rich element RNA-binding factor (AUF1) protein and bind it to the 3'- untranslated region (UTR) of SERCA2a (Sarco/ER Ca²⁺ ATPase) mRNA, resulting in mRNA degradation [19].

Fig. 1.

The schematic diagram of Ca²⁺ homeostasis regulation. Ca²⁺ homeostasis is rigorously orchestrated across cellular compartments by the specialized Ca²⁺ toolkits. Extracellular Ca²⁺ enters the cytosol through Ca²⁺-permeable channels and exchangers, while cytosolic Ca²⁺ is extruded via pumps or sequestered within Ca²⁺ stores (e.g., endoplasmic reticulum (ER) by SERCAs, mitochondria (Mito) by MUC, and Golgi apparatus (Go) by SPCA) and other subcellular microdomains. This compartmentalization maintains the resting cytosolic Ca²⁺ concentration at approximately 100 nM, which can transiently rise to 1 μM-mM levels upon stimulation. Such spatiotemporal regulation is mediated by coordinated actions of Ca²⁺ toolkits, which orchestrate Ca²⁺ influx, efflux, storage, and recycling. Nuclear Ca²⁺ homeostasis is governed by nuclear pore complexes (NPCs) and nuclear Ca²⁺ toolkits such as envelope channels (e.g., IP3R) and Ca²⁺-binding proteins (e.g., SII and ChB). Store-operated Ca²⁺ entry (SOCE) through TRP/ORAI channels elevates both cytosolic and nuclear Ca²⁺, while NPCs enable passive diffusion to buffer transient Ca²⁺ fluctuations. Crosstalk between cytosolic and nuclear compartments further integrates Ca²⁺ signals. Lys lysosomes, TRP Transient receptor potential, PMCA Plasma membrane Ca²⁺ ATPase, SERCA Sarco/Endoplasmic Reticulum Ca²⁺ ATPase, SPCA Secretory pathway Ca²⁺-ATPase, MCU mitochondrial Ca²⁺ uniporter, STIM Stromal interaction molecule, IP3R Inositol 1,4,5-trisphosphate receptor, SII Secretogranin II, ChB Chromogranins B

This paper comprehensively reviews the intracellular Ca²⁺ homeostasis regulatory networks, the role of Ca²⁺ signals in different cellular compartments, and the underlying mechanisms of gene expression regulation in a broad sense. We focus on the latest research findings in this field and propose reasonable suggestions for addressing the current research gaps and deficiencies on this topic. Considering the similarity/association in content and mechanisms, the present review does not strictly distinguish the differences between the two terms of Ca²⁺ homeostasis and signaling and combines them for discussion.

Regulatory role of Ca²⁺ signaling/homeostasis on gene transcription

Ca²⁺ signaling/homeostasis in the nucleus

Gene transcription in mammalian cells is conducted using DNA as a template, and the nucleus is the core organelle where gene transcription regulation occurs. Therefore, precise regulation of nuclear Ca²⁺ signaling is indispensable for ensuring the fidelity of gene transcription and executing specialized cellular functions and cell fate specification through genetic and epigenetic mechanisms [20]. Although the cell nucleus is the second highest Ca²⁺ organelle with 10 mM free and bound Ca²⁺ together, after secretory granules [21], the free Ca²⁺ ions in the resting state are approximately 100 nM. Ca²⁺ channels, pumps, and exchangers on the nuclear envelope and Ca²⁺ stores, nuclear Ca²⁺-binding proteins, and the nuclear pore complex jointly maintain nuclear Ca²⁺ homeostasis [22]. In addition, studies have found that the nucleus contains a reticular Ca²⁺ store (nucleoplasmic reticulum), which is structurally integrated with the ER and nuclear envelope, providing a mechanism for the rapid local release or response of Ca²⁺ [23]. Some neurons lack this structure, and in such neurons, the function of Ca²⁺ stored in the nucleus may be taken over by the nuclear envelope [24]. In addition, many Ca²⁺-regulating proteins or channels previously reported to be located in the ER also have nuclear localization, mainly in the nucleoplasmic reticulum, including inositol 1,4,5-triphosphate receptor (IP3R), ryanodine receptor (RYR), and SERCA [25, 26]. Besides participating in the regulation of cytoplasmic Ca²⁺ homeostasis, they are crucial for the modulation of nuclear Ca²⁺ signals. The upregulation of IP3R1 expression in the nucleus of human atrial cardiomyocytes in patients with atrial fibrillation leads to nuclear Ca²⁺ overload. This may be caused by the downregulation of miR-26a, which relieves its translational inhibition of IP3R1 [27]. Besides regulating Ca²⁺ homeostasis in the ER by interacting with SERCA2, phospholamban, a key small, integral membrane protein primarily located in cardiac muscle, also regulates Ca²⁺ homeostasis in the nucleus through IP3R and RYR2 channels [28]. The large pore diameter and nonselective permeability of the nuclear pore complex (NPC) allow Ca²⁺ to diffuse freely in and out of the nucleus, providing a rapid Ca²⁺ buffering mechanism. In addition, recent evidence from atrial and ventricular cardiomyocytes suggests that Ca²⁺ influx controlled by store-operated Ca²⁺ entry (SOCE) also leads to an increase in nuclear Ca²⁺, in which transient receptor potential channels (TRPC) and ORAI Ca²⁺ channels play an important role, with approximately 33–67% reduction in SOCE activity if these two channels are inhibited. These results indicate that the maintenance of nuclear Ca²⁺ homeostasis requires the participation of other cytoplasmic Ca²⁺ stores and channels on the cell membrane [29]. In addition, various Ca²⁺-binding proteins in the nucleus are significant regulators of nuclear Ca²⁺ signaling and homeostasis. Secretogranin II colocalizes with Chromogranins B in IP3-sensitive nucleoplasmic Ca²⁺ store vesicles, which function in the storage and control of Ca²⁺ in the nucleus by IP3R-mediated Ca²⁺ release [30, 31]. S100A1 and A4 are largely localized in the cytosol, whereas S100A2 and A6 are found mainly in the nucleus [32]. These Ca²⁺ buffers are specifically expressed in the nucleus, reflecting the unique regulation of nuclear Ca²⁺ homeostasis.

The nucleus itself, as another distinct Ca²⁺ store, participates in the regulation of cellular functions, such as cell proliferation [33], development and differentiation [34], cell cycle, senescence, death, and chromosome assembly [35, 36]. For example, Ca²⁺ signaling in the nucleus can independently govern cell proliferation by regulating the expression of asparaginyl endopeptidase, legumain (LGMN). The expression of LGMN decreases significantly, and cell proliferation slows down when the Ca²⁺ concentration decreases in the nucleus [37]. Nuclear Ca²⁺ signaling in neurons regulates the recruitment and response of microglia after traumatic brain injury through the osteoprotegerin signaling pathway [38]. High concentrations of Ca²⁺ in the nucleus (sub-millimolar levels) can induce changes in DNA structure [39] and regulate chromosome condensation [40], which is important for ensuring genetic material stability. The elevation of Ca²⁺ in the nuclear and cytoplasmic compartments can enhance the activity of DNase X, resulting in DNA fragmentation and apoptosis of the nucleus, a process accompanied by changes in the expression of various Ca²⁺ transporters and regulatory factors, such as SERCA2, RyR1, IP3R1, cluster of differentiation 38 (CD38), and IP3 [41]. These changes in the expression of various genes triggered by fluctuations in nuclear Ca²⁺ levels indicate that nuclear Ca²⁺ subtly participates in the regulation of gene expression, which will be reviewed below.

Gene transcription regulation by the nuclear Ca²⁺

The direct regulation of gene expression by nuclear Ca²⁺ has garnered significant attention and has been extensively reviewed in previous studies [42, 43]. In this review, we focus on the key discoveries in this field and highlight recent advancements. Studies have demonstrated that the physiological roles of Ca²⁺ signals in the nucleus may differ from those in the cytoplasm of the cell. For instance, in glucose-stimulated insulinoma cells, activator protein-1, cAMP response element-binding protein (CREB), and serum response element (SRE)-mediated gene transcription are regulated by cytoplasmic Ca²⁺ signals, with no effect observed upon chelation of ER or nuclear Ca²⁺. Overexpression of the mitochondrial protein Fis-1 or inhibition of GTPase Drp-1 alters Ca²⁺ signal-mediated gene transcription following glucose stimulation [44]. Consequently, Ca²⁺ signaling regulates gene expression under various conditions and stimuli with distinct characteristics. CREB primarily responds to increases in nuclear Ca²⁺ signals, whereas the SRE is mainly regulated by cytoplasmic Ca²⁺ signals without requiring an increase in the nuclear Ca²⁺ concentration. This phenomenon has been verified across various research models, including hippocampal neurons and the anterior pituitary tumor 20 cell line [45, 46]. Indeed, CREB can be regulated either cooperatively or independently by cytoplasmic or nuclear Ca²⁺ signaling [47] depending on the stimuli or cell types. In hippocampal neurons, CREB can be activated by nuclear Ca²⁺ alone via the CaM kinase pathway and does not require the import of cytoplasmic proteins into the nucleus. However, an increase in nuclear Ca²⁺ is unnecessary for ERK-mediated CREB activation, which is mediated by cytosolic Ca²⁺ and refers to the cytoplasmic-nuclear shuttling of kinases and CREB in skeletal muscle [46, 47]. In addition, EGF-induced transcriptional activation of Elk-1, an ETS domain TF, depends on nuclear rather than cytoplasmic Ca²⁺ signaling [48]. These findings indicate that nuclear Ca²⁺ signaling plays a direct and independent role in the transcription of genes. However, given the interconnection between cytoplasmic and nuclear Ca²⁺ signals, in many cases, it is impossible to distinguish whether the result of gene expression regulation is directly influenced by Ca²⁺ in the nucleus or is produced after the cytoplasmic Ca²⁺ signal is transmitted to the nucleus, such as diffusion through NPC. This is a point that requires particular consideration when analyzing the contribution of nuclear Ca²⁺ signals.

Nuclear Ca²⁺ signaling regulates gene expression through multiple mechanisms, one of which involves CREB phosphorylation [49]. The translocation of Calmodulin (CaM) from the cytoplasm to the nucleus drives CREB phosphorylation [50]. Nuclear-localized CREB is also regulated by Ca²⁺ influx via the big-conductance Ca²⁺-activated K⁺ channels in the nuclear membrane [51]. Microinjection of BAPTA to chelate nuclear Ca²⁺ abolishes the reaction of the CRE element, indicating that nuclear Ca²⁺ is a direct factor in its response [52]; however, this solitary evidence requires further research to be confirmed. Moreover, some L-type voltage-gated Ca²⁺ channels (LTCC), such as CaV1.2, can effectively activate TFs, such as CREB and myocyte enhancer factor 2 (MEF-2) [53]. Ca²⁺-CaM binds directly to the isoleucine-glutamine (IQ) domain at the C-terminus of LTCCs. This binding event subsequently initiates Ca²⁺ influx into the nucleus, a process mediated by the mitogen-activated protein kinase (MAPK) signaling pathway [54]. Other Ca²⁺-regulated proteins also regulate the involvement of CREB in gene transcription. For example, the retention time of Jacob protein, also named Nsmf, a NMDA receptor synapto-nuclear signaling and neuronal migration factor, transduces signals from N-methyl-d-aspartate receptors (NMDARs)-associated signalosomes to the nucleus and regulates CREB-dependent gene expression via dynamic binding to LaminB1 and CREB, correlates significantly with increased nuclear Ca²⁺ concentration. A transient increase in Ca²⁺ causes Jacob to dissociate from LaminB1 in the nuclear lamina and bind to CREB to coregulate transcription [52]. Furthermore, in pathological conditions such as Alzheimer’s disease, amyloid β-oligomers inhibit neuronal activity-induced nuclear Ca²⁺ signaling, interfering with the normal expression of neuroprotective genes such as neuronal PAS domain protein 4 (Npas4), brain-derived neurotrophic factor (BDNF), RyR2, and NAD(P)H quinone dehydrogenase 1 (Nqo1) [55].

Various stimuli can elevate nuclear Ca²⁺ concentrations, regulating the transcription of genes. For example, far-infrared irradiation inhibits cell proliferation by promoting checkpoint kinase 2 protein phosphorylation at Thr68 by increasing intranuclear Ca²⁺ and nuclear translocation of CaM [56]. Upon stimulation by hepatocyte growth factor, c-Met protein on the cell membrane rapidly translocates into the nucleus in a manner dependent on importin β and adaptor protein Gab1, leading to increased intranuclear IP3 levels and subsequent activation of the IP3R receptor channel in the nucleus to release Ca²⁺. In contrast, vasopressin (AVP) activates plasma membrane receptors in SkHep1 cells. It triggers PLCβ to hydrolyze PIP2 into cytoplasmic IP3, which binds ER-localized IP3R to release Ca²⁺, elevating cytoplasmic Ca²⁺. This signal is abolished by cytoplasmic IP3 buffers but not by nuclear ones, verifying the dependence on cytoplasmic IP3 and ER release [57]. Moreover, various G-protein-coupled receptors (GPCRs) on the nuclear membrane may mediate the regulation of intranuclear Ca²⁺ homeostasis. Bitter taste receptors are one such receptor type, and their activation can lead to increased Ca²⁺ levels in the nucleus and mitochondria, inducing apoptosis [58]. After epidermal growth factor (EGF) activates its receptor epidermal growth factor receptor (EGFR), the nuclear EGFR signal is increased, activating PLCδ4, which is specifically expressed in the nucleus, to hydrolyze nuclear PI(4,5)P2, increasing IP3R-mediated nuclear Ca²⁺ signals and ultimately altering cell proliferation and cell cycle progression [59]. Although multiple PLC isoforms, such as PLCδ1 and PLCβ1, exist in the nucleus, they may be regulated by different receptor tyrosine kinases [60], suggesting that diverse regulatory mechanisms participate in the regulation of intranuclear Ca²⁺ signaling. Interestingly, PLCβ4 in the nucleus is also involved in the regulation of cytoplasmic Ca²⁺ homeostasis. Studies on neural cells have shown that under normal conditions, PLCβ4 located in the nucleus regulates nuclear Ca²⁺ homeostasis by affecting its activity through Sentrin-specific protease 2 (SENP2)-mediated SUMOylation. Meanwhile, the absence of SENP2 leads to the abnormal expression of various Ca²⁺ homeostasis regulatory proteins and a significant decrease in cytoplasmic Ca²⁺ levels [61].

Nuclear Ca²⁺ signaling is modulated by biomechanical stimuli, including cell spreading and osmotic stretching, through mechanisms involving cytoplasmic Ca²⁺ propagation and nuclear envelope transport. After cell adhesion and spreading, Ca²⁺ from the perinuclear space causes a significant increase in nuclear Ca²⁺ levels, which induces changes in gene expression. Studies using isolated nuclei have confirmed that Ca²⁺ levels in the perinuclear space undergo changes in response to mechanical stimulation, Ca²⁺ store inhibitor thapsigargin, and Ca²⁺ ionophores, and this responsiveness may be mediated by mechanically sensitive Ca²⁺ channels on the nuclear membrane [37]. In addition, electrical stimulation has been shown to modulate nuclear Ca²⁺ signaling. A study in neonatal mouse ventricular myocytes utilized both conventional (Fluo4-AM) and novel genetically encoded calcium indicators (GECIs) to monitor Ca²⁺ dynamics. Specifically, the green fluorescent G-GECO1 (targeting the cytoplasm) and the red fluorescent NLS-R-GECO (targeting the nucleus), both driven by the CMV promoter, were employed. The results indicated that electrical stimulation can trigger transient increases in nuclear Ca²⁺ concentration, and that these nuclear transients are slower than those in the cytoplasm [62]. Moreover, given the continuity between the cytoplasm and the nucleus, whereby changes in cytoplasmic Ca²⁺ lead to parallel changes in nuclear Ca²⁺, chelating cytoplasmic Ca²⁺ can eliminate nuclear Ca²⁺ transients, further suggesting that nuclear Ca²⁺ changes under electrical stimulation originate from the cytoplasm [62].

Gene transcription occurs in the nucleus and within mitochondria-semiautonomous organelles that house their own genome and transcribe 37 genes encoding essential respiratory chain components through polycistronic transcription. Mitochondrial Ca²⁺ homeostasis is mainly maintained by the mitochondrial Ca²⁺ uniporter (MCU) and Na⁺/Ca²⁺ exchanger, which affect cellular metabolism, energy production, and signal transduction. While direct evidence linking Ca²⁺ signaling to mitochondrial gene transcription remains limited, the mitochondrial transcription factor A (TFAM) is modulated by phosphorylation via multiple Ca²⁺-dependent kinases (e.g., PKA, CaMKII, PKC), which dynamically regulate TFAM–DNA interactions, potentially fine-tuning its capacity to initiate transcription and coordinate mitochondrial genome maintenance [63]. TFAM can directly bind to the promoter region of mitochondrial DNA to regulate the expression of mitochondrial genes or randomly bind to DNA to maintain the stability and copy number of mitochondrial DNA, indirectly managing gene transcription [64]. There is clear evidence that TFAM regulates nuclear gene expression via cytoplasmic Ca²⁺ signaling. Mitochondrial membrane depolarization hinders the uptake of Ca²⁺ from the cytoplasm into the mitochondria, resulting in cytosolic Ca²⁺ overload that subsequently activates the Calcineurin and CaMK pathways to regulate nuclear gene expression [65, 66]. For example, transcription of the nuclear-encoded SERCA2 gene is directly regulated by TFAM through promoter binding, and TFAM deficiency significantly downregulates SERCA2 expression, impairing Ca²⁺ homeostasis in cardiomyocytes [64]. In addition, inhibition of mitochondrial Ca²⁺ uptake through silencing of MCU leads to Ca²⁺ oscillations in the cytosol, consequently inducing the expression of their target genes in the nuclear genome by activating Ca²⁺-sensitive TFs, such as nuclear factors of activated T cells (NFATCs) and NF-κB [42]. These findings demonstrate that mitochondria and the nucleus can exchange information through Ca²⁺ signals to regulate gene transcription. However, the extensive cross-regulation of the mitochondrial and nuclear Ca²⁺ signaling axes and the underlying pathological significance and mechanisms require further investigation.

Gene transcription regulation by the cytoplasmic Ca²⁺

The regulatory role of cytoplasmic Ca²⁺ signaling in modulating cellular physiology through gene expression has been extensively reviewed [67–69]. These networks are activated by diverse physiological or pathological stimuli and serve as critical intermediaries in translating extracellular signals into transcriptional responses. The participation of cytoplasmic Ca²⁺ in transcription regulation is mainly indirectly realized by the Ca²⁺-related kinases and TFs after the activation of Ca²⁺ toolkits, since Ca²⁺ is spatially separated from the location where gene transcription occurs. Therefore, this section focuses on recent advances in cytoplasmic Ca²⁺-mediated transcriptional regulation, particularly highlighting the involvement of TFs as pivotal bridges connecting upstream pathways and downstream regulatory nodes. Figure 2 illustrates the well-characterized Ca²⁺-sensitive TF regulatory networks.

Fig. 2.

Ca²⁺ Signaling regulates gene transcription via transcription factors (TFs). A NFATCs family. The Ca²⁺-Calcineurin-NFATCs signaling axis transduces dynamic Ca²⁺ fluctuations into the miscellaneous transcription events. Dephosphorylated NFATC family members (NFATC1-4) undergo nuclear translocation to activate the responding genes with isoform-specific decoding of Ca²⁺ signals. NFATC2 responds to Ca²⁺ release-activated Ca²⁺ channel (CRAC)-mediated store-operated Ca²⁺ entry (SOCE), whereas NFATC3 activation requires synchronized nuclear Ca²⁺ pulses. T-cell receptor (TCR) activation, L-type Ca²⁺ channels (LTCCs), and mitochondrial Ca²⁺ uniporter (MCU) mediated Ca²⁺ flux differentially regulate NFATC isoforms to orchestrate immune surveillance, maintain the stem cell niche, and regulate pathogenic cascades in autoimmune diseases and cancers. B TFEB, DREAM, and CREB. The lysosomal Ca²⁺ messenger nicotinic acid adenine dinucleotide phosphate (NAADP) and IP3R-mediated Ca²⁺ signals converge to activate TFEB, a master regulator of autophagy-lysosomal genes. Transcription factor EB (TFEB) activation involves coordinated regulation by cluster of differentiation 38 (CD38) and leucine-rich repeat kinase 2 (LRRK2) at the plasma membrane and ER Ca²⁺ signaling interfaces. Upon dephosphorylation via protein phosphatase 2A (PP2A), TFEB translocates to the nucleus, driving autophagy initiation. Ca²⁺ dynamics govern transcriptional repression through the downstream regulatory element antagonist modulator (DREAM) complex by binding to DNA under low Ca²⁺ conditions. Elevated Ca²⁺ triggers DREAM dissociation, relieving transcription repression and enabling cyclic-AMP response binding protein (CREB) activation through liberated CREB binding protein (CBP) recruitment sites. CREB is phosphorylated by ERK, Ras/MAPK, CaMKII/IV and/or PKA cascades following Ca²⁺ influx through LTCCs, N-methyl-D-aspartic acid receptor (NMDARs), membrane depolarization, or SOCE. C Other TFs. Ca²⁺ influx or IP3R-mediated Ca²⁺ release signaling dynamically regulates the activity of RELB Proto-Oncogene, NF-κB Subunit (RELB), signal transducer and activator of transcription 3 (STAT3), and Trithorax-like (Trl). Specifically, RELB sustains colorectal cancer cell survival through noncanonical NF-κB signaling, transient receptor potential channels (TRPC) channel-mediated Ca²⁺ influx activates STAT3, and IP3R-dependent SOCE triggers Trl activation to drive SET-domain-containing 2 (Set2) expression. The Ca²⁺/Calmodulin complex inhibits vitamin D receptor activity, downregulating Cytochrome P450 family 24 subfamily A member 1 (CYP24A1) transcription and thereby controlling vitamin D metabolism. Heat stress-induced Ca²⁺ signaling activate Myeloblastosis transcription factors 3 (MYB3) and Microtubule-associated protein 2–4 (AP2-4), further regulating glycerol-3-phosphate acyltransferase (GPATs) expression. Calcineurin-responsive zinc finger 1 (Crz1) promotes glucan-chitin polymer synthesis by modulating Phosphate starvation response regulator 2 (Phr2) expression to maintain cell wall integrity. In addition, Na⁺/Ca²⁺ exchangers (NCX1)-mediated Ca²⁺ extrusion inhibits the activation of the GATA binding protein 4/heart and neural crest derivatives expressed 2 (gata4/hand2). HSPs Heat shock proteins, AKR1B1 Aldo–keto reductase family 1, Notch3 Neurogenic locus notch homolog protein 3, Sox2 SRY-box transcription factor 2, c-MYC Myelocytomatosis oncogene, FoxM1 Forkhead box M1, ANP Atrialnatriureticpeptide, BNP Brain natriuretic peptide, CTSD Cathepsin D, LAMP1 Lysosomal-associated membrane protein 1, BECN1 Beclin 1, PER1 period circadian regulator 1, GEM GTP-binding protein overexpressed in skeletal muscle, Hrk Harakiri, BCL2 interacting protein, GFAP Glial fibrillary acidic protein, Npas4 Neuronal PAS domain protein 4, BNDF Brain-derived neurotrophic factor, Nrg1 Neuregulin

NFATC family

NFATs or NFATCs are a family of five TFs, including NFATC1, NFATC2, NFATC3, NFATC4, and NFAT5, collectively known as NFATs. They are the most extensively studied Ca²⁺-dependent TFs, play important roles in excitable and nonexcitable cells, and are implicated in the regulation of multiple physiological and pathological processes. The Ca²⁺-Calcineurin–NFATC1–4 signaling pathway is involved in the development of autoimmune diseases, as summarized in a previous review [70]. In addition, inhibiting the SOCE–Calcineurin–NFATCs pathway can enhance the antitumor potential of chimeric antigen receptor T-cell (CRA-T) immunotherapy. Classic immunosuppressants targeting this pathway, such as cyclosporine A and tacrolimus, which specifically inhibit Calcineurin activity to block NFATCs activation, are clinically established to prevent graft rejection in solid organ or hematopoietic stem cell transplantation. However, their overuse may weaken the efficacy of CRA-T immunotherapy, requiring careful dosage adjustment to balance graft tolerance and antitumor activity [71]. Therefore, intervention at key nodes of the NFATCs pathway holds promise for the treatment of various autoimmune diseases and cancers. Furthermore, the manipulation of NFATCs signaling has also shown good application prospects in the treatment of many other diseases, such as Alzheimer’s disease, nonalcoholic steatohepatitis, and cardiac hypertrophy, which highlights the importance of Ca²⁺ signal-mediated NFATCs in regulating gene expression [72, 73]. Altering the phosphorylation level and subsequent nuclear translocation of NFATCs is the primary mechanism regulating gene transcription. Although all NFATCs are subject to Ca²⁺ regulation, distinct isoforms exhibit varying Ca²⁺ signal modulation mechanisms. For instance, Calcineurin-mediated dephosphorylation of NFATC2 necessitates repetitive or prolonged increases in localized Ca²⁺ signals driven by SOCE via CRAC channels. Specifically, when ER Ca²⁺ stores are depleted, the ER-resident Ca²⁺ sensor STIM1 aggregates at ER-plasma membrane (ER-PM) junctions, recruits the CRAC channel pore subunit ORAI1, and activates the CRAC channels. This spatial restriction of the STIM1–Orai1 interaction leads to a concentrated Ca²⁺ influx at ER–PM junctions, forming localized Ca²⁺ “hotspots.” This sustained local Ca²⁺ accumulation effectively activates Calcineurin, triggering the dephosphorylation of NFATC2 [70]. However, NFATC3 activation requires elevated nuclear Ca²⁺ levels. The activation and deactivation kinetics differ significantly between these isoforms, with NFATC3 undergoing rephosphorylation within the nucleus much more rapidly than NFATC2 [74]. The differences in Ca²⁺ sensitivity and nuclear translocation dynamics of the NFATCs subtypes constitute a hierarchical or redundant response system. NFATC2 response to Ca²⁺ microdomains near open Orai1 channels. NFATC3 stimulation requires both local Ca²⁺ entry and a nuclear Ca²⁺ increase [75]. NFATC3 can respond quickly to weak Ca²⁺ signals, making it suitable for rapid and dynamic signal coding. However, NFATC2 is suitable for homeostatic regulation, whereas NFATC1 drives persistent gene expression under pathological conditions [76, 77]. This dynamic response diversity enables cells with enhanced capabilities of temporal information processing to precisely coordinate the spatiotemporal dynamics of immune responses and malignant phenotypes; however, it also provides a basis for the targeted treatment of specific subtypes. Here, we summarize the latest progress on the regulation of gene expression by each subtype of NFATCs, particularly in Ca²⁺ signaling.

Alterations in Ca²⁺ signaling triggered by external stimuli or pathological conditions can lead to long-term changes in gene expression, affecting cellular function. GPCRs and Ca²⁺ channels located on the cell membrane play crucial roles in mediating signal transduction cascades in response to sensory stimuli. The activation of NFATC1 exemplifies this regulatory mechanism. For instance, acetic acid triggers Ca²⁺ influx via GPR43, leading to the activation of myocyte enhancer factor 2A (MEF2A), peroxisome proliferator-activated receptor γ coactivator-1α, and NFATC1 [78]. NFATC1 plays a Ca²⁺-dependent role in cancer cell proliferation and in the maintenance of stem cells. Formyl peptide receptor 3 functions as a tumor suppressor by reducing intracellular Ca²⁺ levels and nuclear translocation of NFATC1, leading to decreased Notch receptor 3 expression and glycolysis. In addition, NFATC1 interacts with the sex-determining region Y-box 2 (SOX2) promoter to regulate stemness [79]. Recent research indicates that the activation of the Calcineurin–NFATC1 pathway, driven by elevated cytoplasmic Ca²⁺, can influence the cell cycle and proliferation by modulating histone expression levels, which might be linked to changes in acetylation and chromatin structure [80].

A recent study demonstrated that NFATC2 enhances the expression of Pdia3 through nuclear translocation in a Ca²⁺-dependent manner during T-cell activation, augmenting interactions with signal transducer and activator of transcription 1 or pyruvate kinase M2 and promoting the expression of Th1 and Th17 lineage-related genes, respectively [81]. Researchers have used PD-1 antibodies combined with calcium carbonate nanoparticles conjugated with 12-myristate-13-acetate to elevate cytoplasmic Ca²⁺ concentrations in T cells, activating NFATC2 and NF-κB signaling to enhance antitumor efficacy [82]. Therefore, Ca²⁺-NFATC signaling in T cells may represent a conserved regulatory mechanism for the adaptive evolution of the immune system [83]. Mechanistically, NFATC2 participates in transcriptional regulation similar to NFATC1. Following endothelial cell activation, ephrin-A1/EphA2 influences vascular cell adhesion molecule-1 expression via Ca²⁺ signaling-mediated activation of NFATC2 rather than NF-κB [84]. Activation of transient receptor potential channel 1 induces Ca²⁺ influx and facilitates the nuclear translocation of the NFATC2–NFATC2IP complex, promoting muscle growth [85]. Ca²⁺–NFATC2 pathway plays a crucial role in stem cell function. In human limbal epithelial stem cells, WNT16b promotes proliferation and maintenance of stemness via the Ca²⁺–Calcineurin A–NFATC2 pathway. Increased cytoplasmic Ca²⁺ induces NFATC2 nuclear translocation, which coregulates the expression of target genes, such as myelocytomatosis viral oncogene homolog (c-Myc) and forkhead box protein M1 (FoxM1), with epigenetic factors, including histone deacetylase 3, general control of amino acid synthesis yeast homolog-like 2, and WD repeat-containing protein 5 [86]. In liver cancer stem cells, SOCE–Calcineurin promotes NFATC2 nuclear translocation to regulate the expression of stem cell maintenance genes, such as Nanog homeobox, Octamer-binding transcription factor 4 (OCT4), SOX2, and fibroblast growth factor 19 [87].

SOCE is a critical regulator of NFATC2 activation. Upon SOCE activation, the resultant elevation in cytoplasmic Ca²⁺ levels triggers a CaM-Calcineurin-mediated signaling cascade. Calcineurin subsequently mediates the dephosphorylation of NFATC2, enabling its nuclear translocation and transcriptional activation of target genes. This process is also exquisitely regulated by A-kinase anchoring protein 79, which interacts with the N-terminus of STIM1-gated Orai1 to anchor Calcineurin and NFATC2 near SOCE domains, further reinforcing the specificity of localized Ca²⁺ signals for NFATC2 activation [88]. In breast cancer cells, although STIM1, a component of SOCE, affects cell migration by regulating NFATC2, it does not depend on SOCE-mediated Ca²⁺ signals [89], indicating that NFATCs may be regulated in a cell-specific manner. In addition, the MCU serves as a central regulator of interorganellar Ca²⁺ transfer between the ER and mitochondria, which is implicated in modulating NFATC2 transcriptional activity, suggesting the involvement of mitochondrial Ca²⁺ signaling in this process [90].

NFATC3 is another Ca²⁺-regulated TF subtype. Studies on cardiomyocytes have shown that inhibiting bone marrow stromal cell antigen-1 decreases nuclear Ca²⁺ levels and inhibits NFATC3 nuclear entry [91], whereas FK506-binding protein 52 modulates MDM2 transcription by enhancing NFATC3 nuclear translocation and transcriptional activity in cancer cells [92]. The NFATC3 pathway is also regulated by light signals. The reactive oxygen species (ROS) produced by near-UV light leads to an increase in cytoplasmic Ca²⁺ and follows the nuclear import of NFATC3 after the activation of LTCC rather than ROS itself causing the nuclear import of NFATC3 [93]. Under pathological conditions, transient receptor potential vanilloid 4 (TRPV4) upregulates the expression of TRPC6 through the Ca²⁺-dependent Calcineurin–NFATC3 signaling pathway, leading to fibrosis of cardiomyocytes [94].

Research on NFATC4 is limited. NFATC4 translocates to the nucleus to regulate gene expression in a Ca²⁺-dependent manner, influenced by SOCC, during estrogen 17β-estradiol-induced embryonic stem cell proliferation [95]. Studies conducted in a mouse model of neuronal ceroid lipofuscinosis type 1 (CLN1 disease) have demonstrated that the nuclear translocation of NFATC4 is governed not only through S-palmitoylation modification catalyzed by palmitoyl acyltransferases ZDHHC4 and ZDHHC8, but also in a Ca²⁺-dependent manner, requiring Calcineurin activated by Ca²⁺–CaM to mediate NFATC4 dephosphorylation, a step essential for its nuclear translocation. Decreased S-palmitoylation levels of NFATC4 mediated by ZDHHC4 and ZDHHC8 result in the suppression of its target gene IP3R consequently disrupting lysosomal Ca²⁺ homeostasis [96].

NFAT5 is an atypical member of the NFAT family that does not directly rely on Ca²⁺ signaling for its activation. It was initially identified as an osmotic regulator that influences gene transcription under osmotic stress. NFAT5 is regulated by Calcineurin, indicating potential indirect Ca²⁺-mediated regulation in specific contexts. In T lymphocytes, the activation of the T-cell receptor (TCR) can indirectly enhance the protein accumulation of NFAT5 or affect the gene expression mode regulated by it through the Calcineurin pathway [97, 98]. Experimental evidence in cellular models indicates that the initiation of transcription of LTCC Cacna1c is activated by NFAT5 via binding to the consensus sequence TGGAAGCGTTC in the promoter of Cacna1c [99], which is crucial for cardiomyocyte development and maturation. Nfat5 knockdown reduced Cacna1c expression and L-type Ca²⁺ current in neonatal mouse ventricular myocytes, whereas morpholino-mediated Nfatc5 depletion in zebrafish caused noncontractile ventricles, and the phenotype was rescued by overexpressing Cacna1c or NFAT5, confirming that NFAT5 maintains LTCC-mediated Ca²⁺ influx for cardiac electrophysiological maturation. In non-transformed cells, such as T lymphocytes, NFAT5 is activated at 360–380 mOsm/kg [100]. This observation lays a critical foundation for discerning the activation threshold of NFAT5, as osmotic pressure emerges as a key modulator of Ca²⁺ signaling [101]. Future investigations are warranted to elucidate whether alterations in osmotic pressure-induced NFAT5 activation involve the modulation of intracellular Ca²⁺ signaling and subsequent gene expression.

TFEB (transcription factor EB)

TFEB acts as a Ca²⁺-dependent TF that exerts critical functions across diverse cell types, with its dysfunction being implicated in human diseases such as Parkinson’s disease, Alzheimer’s disease, and Pompe disease [102]. TFEB phosphorylated at distinct serine residues by various protein kinases, including mechanistic target of rapamycin complex 1, PKCβ, ERK2, and GSK-3β, binds to the cytosolic adaptor protein 14-3-3 and remains inactive in the cytosol and in an inactive state. In contrast, dephosphorylated TFEB translocates to the nucleus and becomes activated [103]. Its activity is mediated by TPCN2- or TRPML1-dependent lysosomal Ca²⁺ signaling. CD38 and Leucine-rich repeat kinase 2 (LRRK2) act as upstream activators that interact with each other at the plasma membrane, facilitating NAADP–TPCN2-mediated Ca²⁺ signaling and nuclear translocation of TFEB [104]. The lysosomal Ca²⁺ channel-like protein TRPML1 has a similar activation mechanism for TFEB [105]. TFEB can be activated by Calcineurin, and this activation depends on Ca²⁺ released by the lysosomal Ca²⁺ channel Mcoln1; Calcineurin promotes the nuclear translocation of TFEB through its dephosphorylation [106]. Besides its activation via Calcineurin, TFEB undergoes dephosphorylation by protein phosphatase 2A, which potentiates its nuclear translocation and drives the expression of genes central to autophagosome and lysosome biogenesis [107]. Beyond lysosomal Ca²⁺ regulation, ER Ca²⁺ signaling also triggers TFEB via Ca²⁺/Calcineurin-dependent nuclear translocation [107, 108]. Activation of Heme oxygenase triggers PERK-mediated Ca²⁺ release from the ER, leading to TFEB dephosphorylation, nuclear translocation, and subsequent modulation of downstream lysosomal biogenesis-related genes (Lamp-2a, Lamp2, M6PR, CTSD, CTSB, ATP6V1H) and autophagy-related genes (LC3, SQSTM1, RAB7A). Downregulation of these genes causes lysosomal dysfunction and autophagy flux inhibition, preventing bovine mammary epithelial cells from clearing intracellular Mycoplasma bovis and leading to increased bacterial proliferation [109]. In addition, certain pharmaceuticals, such as resveratrol, have been shown to trigger ER Ca²⁺ signaling, promoting TFEB activation. The study used cell models, such as HepG2 hepatocellular carcinoma cells and primary mouse hepatocytes, demonstrating a significant upregulation of TFEB expression following resveratrol treatment. This highlights the sophisticated regulatory interplay between Ca²⁺ signaling, protein phosphatase activity, and TFEB function. The interaction of TFEB with various TFs makes Ca²⁺-mediated TFEB regulation more diverse [110]. For example, TFEB can form complexes with β-catenin-TCF–LEF1 and participate in Wnt signaling. TFEB and NFAT are coinvolved in Ca²⁺ signal-mediated renal injury repair and the development of chronic kidney disease [111]. TFEB is activated via lysosomal and ER Ca²⁺ signaling, depending on phosphorylation or dephosphorylation, interacts with other TFs, and plays a core role in autophagosome/lysosome biogenesis and disease-related processes.

Downstream regulatory element antagonist modulator (DREAM)

DREAM, also known as KCNIP3 or KChIP3, is another TF that is directly regulated by Ca²⁺ signaling and interacts with voltage-dependent K⁺ channels of the Kv4 class to modulate their function. In the absence of Ca²⁺, DREAM binds to specific DNA sequences, inhibiting gene transcription. An increase in Ca²⁺ concentration prompts DREAM to dissociate from DNA, lifting this inhibition. This study identified DREAM as the first EF-hand protein known to bind DNA and directly regulate transcription in a Ca²⁺-dependent manner [112, 113]. Its interaction with Kv4 potassium channels is distinct from its transcriptional regulatory roles, whereas DREAM, as an auxiliary subunit of the Kv4 channel, binds to the α subunit to modulate its trafficking to the cell membrane and channel gating properties in a Ca²⁺-dependent manner [114, 115]. Mechanistically, in the absence of Ca²⁺, DREAM binds to the leucine-charged domain within the kinase-inducible domain of CREB, impeding the recruitment of CREB-binding protein (CBP, a CREB coactivator) by phosphorylated CREB, blocking CBP-mediated transcriptional activation at CRE sites in a Ca²⁺-dependent manner [116]. Research on mouse neural precursor cells has demonstrated that DREAM activates glial fibrillary acidic protein (GFAP) expression by binding to a specific site on the GFAP promoter, both before and after stimulation by the neuropeptide pituitary adenylate cyclase-activating polypeptide. This activation requires cAMP signaling to elevate intracellular Ca²⁺ levels and relies on an intact DREAM Ca²⁺-binding domain [117]. Similar activation mechanisms have been observed in hematopoietic progenitor cells, GT1-7 cells, and thyroid carcinoma cells for the activation of Hrk (harakiri, BCL2 interacting protein), Gonadotrophin-releasing hormone, Calcitonin, respectively [118–120]. Furthermore, interference with other TFs, such as thyroid transcription factor-1, is another important way for DREAM to exert gene transcription regulation [121].

CREB

As a critical signal transduction molecule, the activity of the Ca²⁺-dependent TF CREB is not only directly regulated by nuclear Ca²⁺-dependent phosphorylation but is also indirectly modulated by cytoplasmic Ca²⁺-activated signaling cascades. This dual regulation underpins its pivotal role in diverse physiological processes (e.g., synaptic plasticity, memory consolidation, and cell survival) and pathological states (e.g., neurodegeneration, cancer progression, and metabolic disorders) in mammalian cells. Ca²⁺ influx mediated by NMDARs (NMDA receptors), cell membrane depolarization, or other stimuli, and Ca²⁺ release from intracellular stores leading to cytoplasmic Ca²⁺ overload are key mechanisms for CREB phosphorylation and activation. Specifically, low concentrations of NMDA stimulation (e.g., 1–5 μM) can sustain CREB activation for up to 3 h because such low concentrations trigger NMDAR activation that further elicits LTCC-mediated Ca²⁺ propagation, a process required for sustained CREB activation, whereas excitotoxic high concentrations (e.g., 50 μM) induce only transient CREB phosphorylation levels decline by 15 min poststimulation and return to basal levels by 45 min, indicating that different NMDA concentrations result in distinct CREB activation patterns [122]. Only the Ca²⁺ signals mediated by LTCC that propagate to the soma, rather than dendrite-restricted Ca²⁺ signals generated by NMDARs, can activate CREB-dependent transcription [123], further emphasizing the uniqueness of CREB activation by distinct Ca²⁺ microdomains. In addition, membrane depolarization in neurons and muscle cells triggers Ca²⁺ influx, which activates CREB to regulate the expression of target genes [124, 125]. The functional coupling between Kv2.1 potassium channels and Cav1.2 Ca²⁺ channels regulates gene transcription via depolarization-induced Ca²⁺ signaling. Disruption of this coupling markedly attenuates Cav1.2-dependent phosphorylation of CREB and impairs the expression of its target gene c-fos [126]. Two autism-related mutations, S143F and G113S, in the β2 subunit of voltage-gated Ca²⁺ channels activate the Ras–ERK–CREB pathway to regulate gene transcription [127]. Both Ca_V1 and Ca_V2 channels activate CREB via Ca²⁺ signaling. Ca_V1 channels use local Ca²⁺ to trigger CaMKII-dependent signaling to the nucleus, whereas Ca_V2 channels elevate Ca²⁺ microns away to activate CaMKII near Ca_V1 channels, with the latter’s Ca²⁺ overload preferentially buffered by intracellular stores [128]. Hydrogen peroxide-induced ERK signaling can still activate CREB in a Ca²⁺-free medium and when RyR receptor-mediated Ca²⁺ store release is inhibited [47]. In addition, SOCE-mediated Ca²⁺ influx significantly activates CREB [129], further supporting the role of Ca²⁺ stores in CREB activation.

The alteration of phosphorylation modification is the most significant way in which cytoplasmic Ca²⁺ signals affect CREB transcriptional activity. Phosphorylation at multiple sites is involved in the activation of its transcriptional activity, especially at the serine 133 site, which is mediated by kinases such as MAPK, CaMKII, CaMKIV, and Ras/ERK-dependent kinases (such as RSK and SK) [130–132]. However, some studies suggest that phosphorylation at Ser142 and 143 is activated specifically by Ca²⁺ influx, whereas phosphorylation at Ser133 can be induced by various stimuli [133]. CREB protein also exists in mitochondria, and its phosphorylation modification at the Ser133 site is regulated by Ca²⁺ signals, but it is not clear whether mitochondrial CREB participates in the transcriptional regulation of downstream genes [134]. From a mechanistic perspective, cytoplasmic Ca²⁺ signaling primarily drives the activation of CREB through multiple protein kinases or interacting factors, although the specific pathways involved exhibit context-dependent variations. In addition, Ca²⁺ signal regulatory elements are also subject to feedback regulation by CREB or CREB-regulated proteins. For instance, CREB can directly bind to the promoter regions of genes encoding key Ca²⁺ signaling components, such as TRPC3 and SERCA2, to modulate their transcription [135, 136]. Following light-induced LTCCs activation, the increase in cytoplasmic Ca²⁺ stimulates CREB-promoted transcription of the G-protein GEM, which inhibits LTCC activity, demonstrating feedback mechanisms in gene transcription regulation to prevent channel over-activation [137]. In another study, the expression of MCU in cardiomyocytes was regulated by lncRNA-mediated Ca²⁺ signaling, and this process was CaMKII/CREB-dependent [138], indicating elaborate crosstalk between TFs and Ca²⁺ homeostatic managing elements.

Other Ca²⁺-dependent TFs

Numerous other TFs regulate gene transcription in a Ca²⁺-dependent manner. For example, Crz1, a novel Ca²⁺-regulated TF, facilitates the formation of a glucan-chitin polymer at the bud neck of yeast cells by modulating the expression of the phosphate starvation response regulator 2 (Phr2) [139]. The Ca²⁺/CaM pathway inhibits the vitamin D receptor, allowing membrane-localized tuberous sclerosis complex 2 to enter the nucleus and suppress the transcriptional activity of the receptor, reducing the transcription level of its target 25-Hydroxyvitamin D-24-hydroxylase (CYP24A1) [140]. In addition, heat stress-induced Ca²⁺ fluctuations may influence the expression of glycerol-3-phosphate acyltransferases by activating the TFs MYB3 and AP2-4 [141]. RELB (RELB Proto-Oncogene, NF-κB Subunit), signal transducer and activator of transcription 3 (STAT3), and trithorax-like (Trl) proteins are TFs influenced by IP3R3-mediated Ca²⁺ signaling. RELB supports colorectal cancer cell survival via nonclassical NF-κB signaling [142]. STAT3 is activated by TRPC-mediated Ca²⁺ influx, affecting astrocytic functions [143], and Trl is triggered by Ca²⁺ influx through Orai-mediated SOCE following IP3R activation, leading to the expression of genes such as the epigenetic modifier Set2, a histone methyltransferase for H3K36me3, which sustains SOCE [144]. This study establishes a bidirectional regulatory circuitry between Ca²⁺ signaling and epigenetic modifications, including histone methylation patterns (e.g., H3K27me3) and chromatin remodeling, which reciprocally modulate Ca²⁺-responsive gene expression networks.

Besides positively promoting gene expression, elevated Ca²⁺ levels can exert negative regulation of gene transcription, with the nonselective cation channel TRPV4 providing a paradigm for Ca²⁺-dependent gene suppression. TRPV4 is present in the nucleus, where it coregulates downstream gene expression with β-catenin under resting conditions. Upon activation, TRPV4 results in a concurrent increase in Ca²⁺ in the cytoplasm and nucleus and initiates Ca²⁺-dependent nuclear export of β-catenin, consequently inhibiting gene expression [145]. Ca²⁺ efflux mediated by the Ca²⁺ exchanger NCX1 may be involved in TFs activity, which is crucial for the formation of ventricular cardiomyocytes in zebrafish. A mutation in the 154th leucine residue of NCX1 leads to intracellular Ca²⁺ retention by inhibiting the activation of TFs GATA4 and HAND2, resulting in the abnormal formation of cardiomyocytes [146]. Some scholars have developed a method for real-time monitoring of cold Ca²⁺-dependent transcription for drug discovery on the basis of the principle that NCX1 mediates gene transcription through Ca²⁺ signaling [147]. Ca²⁺ signaling pathways converge on analogous mechanisms to regulate gene expression through TFs. These mechanisms consistently involve three core components: (i) activation or modulation of specific kinases (e.g., CaM kinases I/II, CaMKII); (ii) posttranslational modifications of TFs, particularly phosphorylation; and (iii) nuclear translocation of activated TFs to access the target genes. Given this fundamental conservation across pathways, a systematic review of each individual Ca²⁺-sensitive TFs is therefore omitted for conciseness. In this study, Supplementary Table S1 summarizes the roles of Ca²⁺-dependent TFs with clear evidence, along with the signal networks they mediate. Gene transcription governed by cytoplasmic Ca²⁺ overload or oscillation via Ca²⁺ influx or Ca²⁺ store mobilization involves numerous kinases or phosphatase-mediated protein phosphorylation processes; however, the specific TF responding to these pathways have not been fully elucidated. Therefore, the regulatory network of Ca²⁺-dependent TFs requires further investigation within specific cell types or in response to distinct environmental stimuli, as the functional regulatory complexes and pathways governing gene expression also depend on context-specific synergistic interactions with kinases, Ca²⁺-binding proteins, coactivators and corepressors.

Regulation of gene transcription by the extracellular Ca²⁺

The cellular survival milieu critically depends on extracellular Ca²⁺, which orchestrates transcriptional programs through Ca²⁺ channel-mediated influx and ion flow-independent mechanisms. Studies on stem and cancer cells have highlighted the significance of extracellular Ca²⁺ in gene transcription modulation, although the involvement of Ca²⁺ ion flow remains uncertain. Hematopoietic stem cells (HSCs) thrive in a low- Ca²⁺ environment, as evidenced by the Ca²⁺ concentration in bone marrow interstitial fluid being four times lower than that in blood. Similarly, HSCs cultured in vitro require lower Ca²⁺ levels than other somatic cells, indicating that reduced extracellular Ca²⁺ is crucial for maintaining stemness and preventing differentiation [148]. In the skin, Ca²⁺ levels are lower in the basal layer, increase in the intermediate spiny and granular layers, and decrease again in the stratum corneum, reflecting the proliferative potential of various cell types. Keratinized cells maintain their self-renewal capacity under low-Ca²⁺ conditions, whereas high-Ca²⁺ conditions induce differentiation. Correspondingly, pluripotent stem cells transition from a naive to a primed state with reduced intracellular Ca²⁺ levels [149]. Given that these processes rely on specific gene expression, extracellular Ca²⁺ may play a significant role in transcriptional regulation. For instance, changes in extracellular Ca²⁺ levels in pancreatic alveolar cells significantly affect early response gene expression, with c-fos levels increasing by 102% and 163% at Ca²⁺ concentrations of 1 mM and 10 mM, respectively [150]. Furthermore, this concentration-dependent effect implies that extracellular Ca²⁺ may directly influence the gene expression [150]. Evidence of the regulation of nuclear gene expression by Ca²⁺ signaling has been demonstrated through the Ca²⁺-sensing receptor (CaSR) on the cell membrane. In human parathyroid tumors, extracellular Ca²⁺ activates CaSR, triggering the Gα12–13 or Gαq–11 pathways. This activation results in the dephosphorylation and nuclear accumulation of the TF Yes-associated protein 1 (YAP1), which collaborates with transcriptional enhanced associate domain (TEAD) to regulate downstream targets such as cysteine-rich angiogenic inducer 61, connective tissue growth factor, and Wnt family member 5A [151]. Similarly, research on triple-negative breast cancer has revealed that high extracellular Ca²⁺ reduces CaSR sensitivity, leading to the expression of early response genes FOS/FOSB and subsequently activating MAGE family member C2 (MAGEC2). MAGEC2 has thus been identified as a high Ca²⁺-inducible gene that facilitates cancer cell growth [152]. Certain voltage-gated Ca²⁺ channels, such as Cav1.2, regulate stimulus-transcription coupling without requiring Ca²⁺ influx. This process relies on the binding of extracellular Ca²⁺ to the channel pore region, specifically the EEEE motif of Cav1.2, to trigger downstream signaling, akin to the mechanism of gene transcription that is independent of NMDA-mediated Ca²⁺ influx [153, 154]. The recognition of Ca²⁺ occupancy within the open ion pore and its contribution to the excitation coupling processes that precede the influx of Ca²⁺. Moreover, extracellular Ca²⁺ interacts with cholecystokinin-induced nuclear Ca²⁺ signaling, likely by first influxing into the cytoplasm through plasma membrane channels and then diffusing into the nucleus via permeable NPCs to regulate nuclear Ca²⁺ levels and crucially influence immediate-early gene expression [150]. This suggests that extracellular Ca²⁺ can modulate gene expression through synergistic interactions with other pathways, independent of significant changes in cytoplasmic Ca²⁺ concentrations.

Posttranscriptional regulation of gene expression by Ca²⁺ signaling

Once transcribed, the precursor mRNA must undergo further processing and maturation to serve as a functional template for protein translation. This maturation process encompasses several critical steps, including 5' capping, 3' polyadenylation, alternative splicing, and mRNA stability. Although direct evidence for the involvement of Ca²⁺ signals in the enzymatic processes of 5’ capping or 3’ polyadenylation is lacking, Ca²⁺-dependent modulation of key regulatory proteins, such as CaMKIV, orchestrates the expression of downstream genes that encode factors that regulate RNA processing events [155]. Overexpression of constitutively active CaMK IV specifically decreases stress axis regulated exon (STREX) inclusion in the Big Potassium channel mRNA and mediates the alternative splicing of ion channel pre-mRNAs. This establishes an indirect regulatory role for Ca²⁺ signals in the RNA processing pathway. Ca²⁺ signals are particularly well-documented in the precise regulation of alternative splicing, which represents one of the most extensively studied aspects of posttranscriptional modification. Ca²⁺ signals, initiated by the activation of LTCCs or NMDARs, lead to the activation of CaMKIV [155]. This regulates alternative splicing, either by directly phosphorylating splicing factors or by inducing the expression of splicing regulators, ultimately altering splice site usage and the abundance of mRNA/protein isoforms. These findings were systematically reviewed by scholars [156], and subsequent studies have provided further confirmation [157–160]. Ca²⁺ signaling has also been extensively reported to regulate the stability of mRNAs. For instance, phosphorylated RNA-binding protein CPEB1 by CaMKII or Aurora A recruits polyadenylation complexes (such as CPSF and PAP) to promote the elongation of polyadenylation tails of target mRNAs, enhancing mRNA stability; however, its dephosphorylation mediated by Calcineurin results in the opposite effect [161]. Ca²⁺ signaling can influence the stability of mRNA 3′-UTRs through transacting factors, such as AUF1 (which often promotes decay) and HuR (which often promotes stability). These factors modulate mRNA stability via changes in their phosphorylation status, which can be regulated by Ca²⁺-dependent kinases, including PKC [162]. In addition, Ca²⁺ signaling, mediated by the activity of proteins such as the Ca²⁺ pumps SERCA2a and SERCA2b, can regulate the stability of mRNAs. This occurs through cis-acting stability determinants within their 3'-UTR regions, which interact with transacting factors [163]. Furthermore, Ca²⁺ signaling can impact mRNA translation by regulating nonsense-mediated mRNA decay. Although the exact mechanism remains unclear, it may influence specific steps in the translation elongation process [164]. The well-documented regulatory roles of Ca²⁺ signaling in posttranscriptional regulation are shown in Fig. 3A.

Fig. 3.

Ca²⁺ Signaling regulates posttranscriptional processes. A Ca²⁺ signaling dynamically regulates precursor mRNA splicing and mature mRNA stability. Upon entering cells via n-methyl-d-aspartate receptors (NMDARs), or L-type Ca²⁺ channels (LTCCs), Ca²⁺ activate CaMKIV to phosphorylates splicing factors to regulate the frequency of selective usage of exons, thereby driving alternative splicing events. CaMKIV targets pre-mRNA element1/2 (CaRRE1/2), probably the UAGG motif to control the inclusion of the exons (e.g., ZERO, STREX and e22) in Big potassium channel mRNA. In parallel, CaMKII/Aurora A and Calcineurin modulate mRNA stability by adjusting the phosphorylation status of RNA-binding proteins (RBP) Cytoplasmic polyadenylation element binding protein 1 (CPEB1). The phosphorylation state of CPEB1 is dynamically regulated by CaMKII, Aurora A, and Calcineurin. In addition, PKC phosphorylates AU-rich element RNA-binding factor 1 (AUF1) to enhance mRNA degradation, while phosphorylated HuR to stabilize transcripts. Furthermore, Ca²⁺ signals influence mRNA stability and translation fidelity by modulating nonsense-mediated decay (NMD) and the different expression of Calmodulin (CaM) isoforms. B Ca²⁺ influx or release alters the activity of protein kinases, miRNA and translation factors, thereby impacting protein synthesis. Ca²⁺ derived from receptors or channels differentially regulate translation. Sustained Ca²⁺ influx through LTCCs (e.g., ApolipoproteinE4 (ApoE4) stimulus) prolonged inhibits global translation by eukaryotic elongation factor 2 (eEF2) phosphorylation, whereas transient Ca²⁺ transients via NMDA receptor activation (e.g., ApoE3 or 4 stimulus) induce reversible, but rapid translational pauses. The mammalian target of rapamycin complex 1 (mTORC1) pathway regulates translation initiation by multiple effectors such as 70 kDa ribosomal protein S6 kinase (p70S6K1), eukaryotic elongation factor 2 kinase (eEF2K), and ribosomal protein S6 (rpS6) in a Ca²⁺/CaM signaling dependent manner. Ca²⁺-dependent dissociation of Myo5a from actin-ribonucleoprotein complexes also adjusts mRNA transport efficiency and subsequent locally protein synthesis. Increased Ca²⁺ inhibits the translation of Calmodulin and Sorcin by binding the miR-1 within their 3'-UTR. In addition, Ca²⁺ released for endoplasm reticulum (ER) stimulated by thapsigargin or A23187 inhibit protein synthesis by eIF2α phosphorylation. ZERO–E22–STREX BK channel splice variants, SRSF1 Serine/Arginine-rich splicing factor 1, HuR Human resistance protein R, TSC Tuberous sclerosis complex, Rheb Ras homolog, mTORC1 binding, rpS6 ribosomal protein S6, MYO5A Myosin VA

In addition, the Calcineurin pathway, CaM, and many Ca²⁺-binding proteins significantly affect mRNA stability. Increasing Calcineurin activity enhances the ability of actin 3 'UTR to regulate mRNA levels. For example, in C2C12 muscle cells, simultaneous transfection of the Calcineurin gene with sustained activity and a LacZ reporter vector containing a full-length utrophin 3 'UTR increased LacZ mRNA expression levels by approximately two-fold, suggesting that Ca²⁺ signaling can regulate mRNA stability by activating a specific signaling pathway [165]. The CaM gene has three subtypes (CALM1, CALM2, and CALM3), with 76% nucleotide sequence homology. Despite the identical 149-amino-acid protein products, functional differences exist across subtypes, potentially arising from codon usage bias, tissue-specific expression levels, and mRNA structural dynamics, which affect mRNA stability and translation efficiency. The CALM3 gene might produce relatively stable mRNA, which is suitable for low transcription levels or temporary deletion, whereas CALM2 has a short mRNA half-life, which might provide better temporal control of CaM levels [166]. This finding implies that Ca²⁺ signals indirectly affect mRNA stability by regulating the expression of different CaM. In addition, annexin family Ca²⁺ regulatory proteins are involved in mRNA transport and translation regulation. Annexin A2 specifically binds to the 5'UTR of c-myc, whereas Annexin A13, Annexin A7, and Annexin A11 bind to the 3'UTR of c-myc, showing binding affinity in the nanomolar range [167].

Translation regulation of mRNA by Ca²⁺ signaling

Mature mRNA is transported to ribosomes on the rough ER to initiate protein translation, a process linked to Ca²⁺ signaling since 1987 [168, 169]. Despite the established role of Ca²⁺ signaling in gene transcription, few studies have explored its involvement at the translational level. Generally, Ca²⁺ influences mRNA translation by binding to or affecting kinases or translation initiation factors to regulate translation initiation, modulating translation-related protein activity via other signaling pathways, or directly affecting mRNA localization and transport (Fig. 3B).

Research on cellular responses to hypoxic conditions has revealed that Ca²⁺ influx after hypoxic stimulation specifically facilitates the selective translation of cPKC-α and mTOR-mediated HIF-1α and HIF-2α while suppressing overall protein synthesis [170]. Phosphorylation of eukaryotic elongation factor 2 (eEF2) can significantly affect the pace of protein synthesis, and investigations have indicated that Ca²⁺ signaling regulates its phosphorylation. Exposure of neurons to Apolipoprotein E3 induces a transient Ca²⁺ influx via NMDA receptors, triggering rapid but reversible suppression of protein synthesis that returns to baseline levels. Conversely, Apolipoprotein E4 stimulation induces a sustained elevation in Ca²⁺ levels via NMDA receptors and LTCCs, leading to prolonged inhibition of protein synthesis mediated by phosphorylated eEF2 [171]. Furthermore, alterations in Ca²⁺ signaling owing to changes in neuronal activity also influence spine density through CaMKI activation and subsequent phosphorylation of eukaryotic translation initiation factor 4G [172]. Research on CaM further underscores the role of Ca²⁺ in protein synthesis, which is vital for axonal growth in hippocampal neurons. Mechanistic studies have revealed that this process modulates rRNA synthesis in the nucleus through Ca²⁺-dependent interactions with DEAD-box RNA helicase 21 [173], a critical prestep for protein synthesis. Disruption of Calcineurin similarly impairs protein synthesis, particularly by affecting the formation of the mRNA cap-binding complex and elongation processes [174]. Furthermore, Ca²⁺ signaling, triggered by amino acids such as Arginine and Lysine, serves as a prestart factor driving de novo protein synthesis by mTORC1 activation [175].

Extensive pharmacological evidence supports the role of Ca²⁺ signaling in protein synthesis through multiple kinase pathways. Prolonged exposure of rat pancreatic beta cells to glibenclamide (a sulfonylurea oral hypoglycemic drug) activates protein translation factors, such as eIF2α, leading to a significant increase in insulin synthesis. This effect is fully blocked by verapamil, which is a blocker of LTCC; however, it only blocks some. Inhibition of mTOR by rapamycin, PKA by Rp-8-Br-cAMPs, and MEK by U0126 partially blocks glibenclamide-induced protein synthesis, indicating that Ca²⁺-dependent mTOR, PKA, and MEK signaling pathways mediate this effect [176]. Upon mTORC1, it phosphorylates p70S6K1, which in turn phosphorylates eIF4B to enhance its activity and promotes PDCD4 degradation via phosphorylation and subsequent ubiquitination, thereby relieving PDCD4-mediated inhibition of eIF4A and facilitating the assembly of eIF4A into the eIF4E–eIF4G complex to promote translation of specific mRNAs [177]. Furthermore, MNK1/2 phosphorylates eIF4E, enhancing its binding to the mRNA cap structure and promoting translation initiation [178]. Conversely, mTORC1 inhibition leads to the dephosphorylation of 4E-binding proteins, preventing their dissociation from eIF4E and inhibiting translation initiation [179]. Similar evidence from pancreatic acinar cells has demonstrated that both the Ca²⁺ ionophore A23187 and the SERCA inhibitor thapsigargin significantly suppress eIF2B activity and protein synthesis. This effect is likely owing to the phosphorylation of eIF2α triggered by the rapid and irreversible depletion of Ca²⁺ in the ER. Interestingly, protein synthesis can partially recover upon Ca²⁺ repletion [180]. This finding underscores the importance of extracellular Ca²⁺ flow and the maintenance of intracellular Ca²⁺ store homeostasis for protein synthesis. Interestingly, recent studies have revealed that Ca²⁺ signals regulate protein synthesis through distinct mechanisms depending on their origin or the receptors and channels involved. Taking Ca²⁺ signaling-mediated protein synthesis after stimulation of NMDAR and mGluRs as examples. NMDAR stimulation triggers a temporally resolved biphasic translation response, characterized by three sequential phases: increased translation inhibition, reduced translation inhibition, and enhanced translation activation. Ca²⁺ influx through LTCCs is crucial for alleviating translation inhibition and facilitating activation, whereas Ca²⁺ release from the ER and SOCE are indispensable for the sustained translation activation phase [181]. In contrast, mGluR stimulation induces a sustained reduction in translation inhibition, accompanied by a persistent increase in translation activation [181]. These findings underscore the multifaceted, spatiotemporally constrained, and context-sensitive regulation of Ca²⁺ signaling in protein synthesis control.

Another mechanism by which Ca²⁺ signaling regulates protein translation is the modulation of the structure and function of the mRNA translation complex. Studies have shown that Ca²⁺ affects the structural properties of ribonucleoprotein complexes. Upon intracellular Ca²⁺ signal initiation, Myo5a dissociates from ribonucleoprotein complexes on the actin cytoskeleton, underscoring its involvement in mRNA transport, indirectly modulating protein translation efficiency [182, 183]. In addition, Ca²⁺ signaling critically regulates miRNA expression, which indirectly influences mRNA translation efficiency [184]. For instance, in cardiomyocytes, miR-1 modulates cell growth and function by inhibiting the translation of CaM and Sorcin-coding mRNAs via highly conserved target sites within their 3′-UTR [185, 186]. In addition, in response to Ca²⁺ influx, cytoplasmic polyadenylation element-binding protein (CPEB) is phosphorylated by upstream kinases such as CaMKII and Aurora A, leading to its activation [161, 187]. Activated CPEB promotes polyadenylation of cytoplasmic target mRNAs and subsequent translation. This mechanism functions as a key translational switch in processes, including oocyte maturation and long-term synaptic plasticity [161]. Disruptions in protein translation can impact intracellular Ca²⁺ balance and cell fate [188], underscoring the complex interplay between protein translation and Ca²⁺ regulation. Although significant progress has been made, key aspects of Ca²⁺ signaling involvement in the control of protein translation require further elucidation. For instance, the precise mechanisms by which Ca²⁺ signals regulate kinases, such as mTOR, PKA, and MEK, the spatiotemporal dynamics underlying Ca²⁺-dependent modulation of specific protein translation, and the context-dependent regulatory roles of distinct Ca²⁺ microdomains under specific stimuli or developmental stages necessitate comprehensive investigation.

Posttranslational regulation of proteins by Ca²⁺ signaling

Although posttranslational regulation of proteins is not part of the core process of gene expression, it constitutes a critical extension and an indispensable step for the realization of encoded-gene biological functions. To understand the underlying mechanisms of the complete genetic information flow from DNA to functional proteins, we further provide a brief overview of the role of Ca²⁺ signaling in protein folding, assembly, localization, and posttranslational modifications (PTMs), processes that are crucial for protein function and cellular homeostasis. Fluctuations in Ca²⁺ levels directly influence the interaction between molecular chaperones and protein disulfide isomerase, facilitating proper protein folding. It inhibits the formation of complexes between ERp57 and Calnexin, affecting the efficiency of disulfide bond formation [189]. Calreticulin, a key Ca²⁺-binding chaperone within the ER, modulates folding efficiency and accuracy through interactions with Ca²⁺ handling molecules, substrates, and stress sensors [190, 191]. Ca²⁺ can stabilize protein folding by binding to specific amino acid residues and altering their conformation. For instance, the EF-hand domain of CaM undergoes conformational changes upon Ca²⁺ binding, activating downstream proteins such as CaMK and CaN [192]. Current evidence suggests that Ca²⁺ signaling regulates the assembly of proteins. CaM dynamically influences protein polymerization and depolymerization by binding to specific proteins. During cilia formation, the interaction between CaM and microtubule-associated proteins governs the dynamic restructuring of microtubules, potentially regulating protein assembly via phosphorylation [193]. Moreover, in protein self-assembly, diverse protein structure conformations can self-assemble by adjusting the Ca²⁺ levels [194]. Furthermore, Ca²⁺ signaling influences protein localization by modulating cytoskeletal reorganization. Elevated intracellular Ca²⁺ levels trigger the activation of CaM-dependent protease calpain, which remodels cell morphology and movement by cleaving and rearranging cytoskeletal proteins, impacting protein movement along the cytoskeleton [195]. It also regulates membrane protein localization through PTMs [196] or by modulating the reorganization of the cytoskeleton mediated by small GTPases such as Rac1. This Ca²⁺/CaM–Rac1 interplay is a key mechanism that affects directional cell migration by modulating protein aggregation and membrane translocation [197–199].

Ca²⁺ signaling orchestrates diverse PTMs, which is achieved mainly in two ways: direct modulation of target protein modification processes or indirect mediation via Ca²⁺-dependent proteins and Ca²⁺-associated signaling pathways. Here, we depict the major protein modifications that have been demonstrated to be regulated by Ca²⁺ signaling (Fig. 4). As discussed in the preceding section on gene transcription regulation, Ca²⁺ signaling plays a crucial role in modulating phosphorylation patterns via various kinases and TFs, influencing gene expression. In this section, we complementally delve into the impact of Ca²⁺ signaling on nontranscriptional pathways, such as muscle contraction, neurotransmitter release, neural plasticity, and regulation of enzyme activity and ion channels, bypass the need for long-term gene transcriptional responses. Instead, they depend on fast, transient Ca²⁺ signaling-mediated modulation of protein function. Ca²⁺ signaling rapidly affects the functions of specific proteins by directly binding to the regulatory domains of the kinases or phosphatases, or by indirectly triggering the signals via Ca²⁺ flux, fluctuation, or release. For instance, in cardiomyocytes, Ca²⁺-bound CaM activates CaMKII, which phosphorylates RyR2 at Ser2815 in humans (Ser2814 in mice) to enhance sarcoplasmic reticulum Ca²⁺ release and regulate cardiac contraction by increasing RyR2 open probability and Ca²⁺ sensitivity [200]. Phosphorylation of the Ser3 site of Cofilin by Ca²⁺ influx-activated LIM kinase inhibits its ability to cleave F-actin, thus stabilizing actin fibers [201]. Ca²⁺ influx-activated CaMKII phosphorylates the Ser9 site of the synaptic protein synapsin, releasing its anchoring effect on synaptic vesicles and promoting the fusion of vesicles with the presynaptic membrane [202]. Within the tumor microenvironment, lncRNA FOXC2-AS1 enhances ER Ca²⁺ release to activate proline-rich tyrosine kinase 2, leading to the phosphorylation of multiple tyrosine residues (Tyr397/576/861) on FAK and driving tumor invasion and chemoresistance [203]. In a counter-regulatory paradigm, flavonoids suppress IP3 receptor-mediated Ca²⁺ release in platelets, reducing ERK1/2 phosphorylation at Thr202/Tyr204, and inhibiting platelet aggregation [204].

Fig. 4.

Ca²⁺ signaling regulates posttranslational modifications (PTMs) of proteins. Ca²⁺ signaling modulates six major PTMs. Phosphorylation: Ca²⁺ initiate a signaling cascade by binding to Calmodulin (CaM), subsequently activating kinases such as CaMKII and AMPK. Activated kinases phosphorylate specific proteins including Ryanodine Receptor 2 (RyR2), synaptotagmin 7 (Syt7), and ATP-sensitive potassium channel (KATP) involved in muscle contraction and neurotransmission [218, 219]. Ca²⁺ oscillations directly activate CaMKII to phosphorylates Emi2/XErp1, while Calcineurin (CaN) to dephosphorylates XErp1/Cdc20, thus regulating the exit of meiosis and fertilization [220, 221]. Methylation: Ca²⁺ signaling might influence protein methylation by regulating methyltransferases or Ca²⁺-associated proteins. Calmodulin methylation at Lys-115 impacts its target interactions. CD28 activation enhances protein arginine methyltransferases (PRMT) activity, resulting in the arginine methylation of proteins such as Vav1 [222, 223]. Ca²⁺ signaling affects SOX2 expression by inhibiting H3K27me3 levels. Ca²⁺-dependent protein arginine deiminases (PADs) can catalyze histone citrullination to dynamically and antagonistically cooperates with methylation [20]. Acetylation: Ca²⁺ modulates protein acetylation. Ca²⁺ binding inhibits parvalbumin acetylation, and influences Calmodulin and Calreticulin acetylation. It can influence overall protein acetylation levels by modulating the activity of transacetylase (TAase) [224]. Calreticulin (CALR) is acetylated at lysine residues K359 and K363, which essential for the interaction between CALR and ATG9A and depends on the Ca²⁺-binding capacity of its C-terminal domain [225]. In addition, mitochondrial Ca²⁺ promote acetyl-CoA production to drove histone H3K27 acetylation at the tribbles homolog 3 locus [206]. Glycosylation: Ca²⁺ signaling modulates glycosylation by the glycosyltransferase or its cofactors. The glycosylation of Capsular polysaccharide phosphotransferase Y (CPsY), Growth arrest-specific protein 1 (Gas1p) and pH-response regulator 1 (Phr1) are regulated under a high Ca²⁺ condition [226, 227]. PLCβ-mediated intracellular Ca²⁺ stores release promotes scavenger receptor SR-A glycosylation. In addition, Ca²⁺ toolkits such as STIM1, Orai1, and Cav3.2 undergo glycosylation modification. Ubiquitination: Ca²⁺, CaM, and CaM-dependent kinases, along with their signaling pathways, influence protein ubiquitination. Elevated Ca²⁺ levels could enhance ubiquitin–proteasome system regulation. Increased cytosolic Ca²⁺ activates neural precursor cell expressed, developmentally down‑regulated 4 (Nedd4) [211], ring finger protein 167 (RNF167), ubiquitin-specific protease 6 (TRE17), and cullin 3 ubiquitinase to affect targets function or lead to degradation [228]. Furthermore, after being ubiquitinated, Ca²⁺ signaling-related proteins are degraded in lysosomes through endocytosis-mediated processes. SUMOylation: Ca²⁺ signaling modulates SUMOylation at global or individual protein levels. KCl enhances global SUMOs conjugation with proteins reliant on Ca²⁺ influx. FOXM1 and MEF2A SUMOylation were regulated by the cytoplasmic Ca²⁺ fluctuation. Conversely, Ca²⁺ can suppress global SUMOylation via calpain-mediated cleavage of SUMO-activating enzyme subunit 2 (SAE2) [229]. In addition, the SENP2–PLCβ4 signaling axis regulates Ca²⁺ release from the neuronal ER and maintains Ca²⁺ homeostasis by altering the SUMO status of PLCβ4 [61]. Emi2: FBXO43 F-Box protein 43, Plx1 Xenopus polo-like kinase, XErp1 Xenopus Emi1-related protein 1, Vav1 Vav guanine nucleotide exchange factor 1, HDACs Histone deacetylases, ATG9A Autophagy related 9A, TRIB3 Tribbles pseudokinase 3, Gdt1p Gcr1-dependent translation factor 1, Gas1p β-1,3-glucanosyltransferase 1, Phr1 pH-response regulator 1, P2Y6R Pyrimidinergic receptor P2Y6, SR-A Scavenger receptor class A, CUL3 Cullin-3, AMPAR α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor, MEF2A Myocyte enhancer factor 2A, FOXM1 Forkhead box protein M1, SENP2 Sentrin-specific protease 2

Ca²⁺ signaling also affects the methylation, acetylation, glycosylation, ubiquitination, and SUMOylation modification of proteins. Protein methylation is regulated through Ca²⁺-associated proteins or by regulating methyltransferase activity. For instance, trimethylation at Lys-115 of CaM directly influences its interaction with target proteins [205]. CaM kinase II (CaMKIIa) can modulate SOX2 expression by reducing H3K27me3 levels and depends on the presence of the histone methyltransferase EZH2 [206]. Alterations in Ca²⁺ concentration directly also influence protein acetylation such as parvalbumins, Calreticulin (CALR), and CAM. Specifically, Ca²⁺ binding can inhibit N-terminal acetylation of several parvalbumins, modulating their interactions with the target proteins [207]. Acetylation levels of CaM are notably elevated in the central nervous system, particularly in the hippocampus, and are contingent upon NMDA receptor activation. However, reductions in CaM acetylation levels in mice lead to decreased CaMKIα activity, resulting in memory impairment [208]. Further research has demonstrated that steroid receptor coactivator 3 (SRC3) directly mediates CaM acetylation, and that inhibition of SRC3 weakens the Ca²⁺-CaM-CaMKIIα pathway, attenuating fear memory [209]. In addition, Ca²⁺ acts as a direct cofactor or stabilizer for glycosyltransferases. Pathological Ca²⁺ signaling drives disease progression via glycosylation cascades. In atherosclerotic pathogenesis, P2Y6 receptor activation triggers PLCβ-mediated intracellular Ca²⁺ elevation, which may promote scavenger receptor SR-A glycosylation and upregulation, thereby accelerating pathological inflammation and lipid deposition [210]. Ubiquitination is crucial for protein degradation, and signal transduction with protein degradation being the most extensively studied and linked to Ca²⁺ signaling. Ca²⁺, CaM, and CaM-dependent kinases, along with their signaling pathways, influence protein ubiquitination by modulating ubiquitin ligases and deubiquitinating enzymes [211]. Ca²⁺ signaling is vital for the regulation of E3 ligase activity evidenced by the identification of Ca²⁺- or CaM-binding domains in a comprehensive analysis of E3 ubiquitin ligase sequences. Increased cytosolic Ca²⁺ concentration activates NEDD4 by disrupting the interaction between the C2 domain and the homologous to E6AP C-terminus domain [211, 212]. In neurons, LGCC opening activates the E3 ligases NEDD4 and RNF167, facilitating AMPA receptor endocytosis and degradation [213]. Furthermore, Ca²⁺ or CaM interactions with target proteins are crucial for regulating ubiquitination; elevated Ca²⁺ levels increase TRE17 ubiquitination, a process diminished in CaM-binding-deficient TRE17 mutants [214]. Ca²⁺ signaling also plays a crucial role in SUMOylation, affecting protein stability and function. Depolarization induced by KCl enhances global SUMO conjugation by enhancing protein binding to SUMOs, with this effect predominantly reliant on Ca²⁺ influx signaling [215]. Decreasing intracellular Ca²⁺ levels triggers the SUMOylation of FOXM1, leading to its accumulation at the inner nuclear membrane, expediting the G2/M transition of the cell cycle, and reducing apoptosis [216]. SUMOylation of the MEF2A lysine 403 site promotes dendritic claw differentiation, whereas Ca²⁺-dependent signaling pathways facilitate the switch from SUMOylation to acetylation at 403, impeding dendritic claw differentiation [217]. Nevertheless, research on Ca²⁺ signaling in protein processing and PTMs remains largely unexplored. Furthermore, studying how Ca²⁺ signaling influences chromatin-level events, including histone modifications and remodeling complexes, is essential to fully elucidate its role in regulating gene expression at posttranslational levels.

Ca²⁺-gene expression axis in pathology and intervention

Dysfunction of Ca²⁺ toolkit and its regulatory network participates in the development of various diseases, and a variety of drugs for disease treatment have been developed on the basis of the Ca²⁺ signaling-gene expression regulatory axis. Here, we summarize the four main strategies used to modulate Ca²⁺ homeostasis in the treatment of diseases. (1) Blocking or boosting the activity of Ca²⁺ channels or pumps: LTCC blockers (CCB), such as nimodipine and isradipine, show mood-stabilizing and neuroprotective effects in neuropsychiatry [230]. By targeting TRP and LTCC channels, CCB have been recognized for their therapeutic potential in cancer treatment, and some compounds, such as Mibefradil and Cannabinoids, are being evaluated in multiple clinical trials [231]. CDN1163 was used to promote SERCAs pump activity by modulating the protein conformational state that favors Ca²⁺ recycle from the cytoplasm to the ER [232]. In addition, altering the SUMOylation of Ca²⁺ regulatory proteins to control Ca²⁺ homeostasis is an alternative solution. SUMOylation of SERCA2 at K480 and K585 increases Ca²⁺ reuptake into the ER [233, 234]. (2) Targeting key Ca²⁺ signaling molecules, such as Calcineurin, CaMK, or CaM, to interfere with signal transmission, including TFs entering the nucleus. In immunological diseases, traditional immunosuppressants, such as cyclosporine A (CsA) and tacrolimus (FK506), which target the Calcineurin-NFATCs pathways, have become the cornerstone of organ transplantation and the treatment of autoimmune diseases. These drugs exert their effects mainly by inhibiting the activity of Calcineurin phosphatase, blocking the dephosphorylation and nuclear translocation of NTATCs, suppressing the expression of immune-related genes such as IL-2 [70]. The activation of CaMK depends on an increase in intracellular Ca²⁺ concentration. Inhibiting CaMK can reduce pathological Ca²⁺ influx and leakage, thereby alleviating Ca²⁺ overload-related pathologies [235]. Ca²⁺/CaM-competitive CaMK II inhibitors (KN-62, KN-93), peptide inhibitors (AC3-I, AIP), and ATP-competitive CaMK II inhibitors have been used in preclinical studies for the treatment of cardiovascular diseases [236]. CaM inhibitors, such as W-7 and CaM peptides, inhibit the interaction between CaM and target proteins, thus impairing CaM function [237, 238]. (3) Directly intervening in Ca²⁺-dependent TFs binding to coactivators to regulate transcription in the nucleus. Small molecules, such as KG-501, and peptides disrupt the CREB/CBP complex and attenuate target gene expression [239]. Along with NFATC1, coactivators p300 promote activation of the β myosin heavy chain promoter in a Calcineurin-dependent manner [240], and the p300 inhibitor A‑485, L002 might restrain NFATC1 function [233]. (4) Using Ca²⁺ buffers to chelate the overloaded Ca²⁺. Metal chelators, such as EDTA, EGTA, BAPTA, and DP-b99, and Ca²⁺ binding proteins, such as S100A1, parvalbumins, and calbindin-D28k, can chelate excess Ca²⁺ to reduce cytotoxicity [241–243], However, there are few successful clinical applications, and most are still in preclinical research. Nevertheless, to enhance the therapeutic effects, a combination of multiple strategies should be considered in future research and practical applications. Leveraging the role of Ca²⁺ signaling in gene expression could lead to the development of more specific and effective drug targets. Drugs designed to modulate the activity of Ca²⁺ channels, pumps, binding proteins, or related TFs, separately or simultaneously, could correct gene expression disorders and treat diseases. By regulating cell proliferation, differentiation, and gene expression through Ca²⁺ signaling, we can optimize cell culture and differentiation conditions to enhance stem cell treatment outcomes.

Conclusion and prospects

Ca²⁺ signaling orchestrates cellular homeostasis and intercellular communication through the coordinated action of Ca²⁺ toolkits. This signaling cascade exerts multilayered control over gene expression, modulating transcription initiation or repression via Ca²⁺-responsive TFs, regulating posttranscriptional RNA processing through Ca²⁺-dependent splicing regulators or mRNA stability modulation, controlling translational efficiency via eIF2α kinase or mRNA translation complex, and modifying protein functional modification through various PTMs. Crucially, these regulatory mechanisms exhibit distinct spatiotemporal patterns across cellular microdomains, enabling compartmentalized signal decoding. However, several challenges and opportunities warrant careful consideration: (1) elucidating the spatiotemporal dynamics of Ca²⁺ signaling within subcellular compartments. Leveraging subcellular and organelle-level imaging technologies or multicolor coimaging technology, real-time monitoring of the dynamics of Ca²⁺ signaling within Ca²⁺ microdomains and manipulation of key molecules involved in Ca²⁺ signaling networks by integrating gene editing techniques can be performed to elucidate their roles in spatiotemporal regulation. (2) Identifying novel Ca²⁺-sensitive TFs and their functional interactions with epigenetic mechanisms, such as noncoding RNA regulation, chromatin remodeling, genomic imprinting, X chromosome inactivation, and other signaling pathways, such as cAMP, MAPK, and PI3K–Akt. (3) Uncovering the interaction of Ca²⁺ signaling with metabolism, such as sugar, protein, and lipid metabolism, and their crosstalk with gene expression. (4) Characterization of cell-type-specific Ca²⁺ signaling networks in specific physiological and pathological contexts. (5) Exploring the therapeutic potential of modulating critical nodes in Ca²⁺ signaling pathways implicated in the dysregulation of gene expression. Addressing these research priorities will not only advance our mechanistic understanding of Ca²⁺-mediated gene regulation but also pave the way for innovative diagnostic and therapeutic strategies for a broad range of diseases.

Abbreviations

AMPA

α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid

AMPK

AMP-activated protein kinase

AVP

Vasopressin

BAPTA

1,2-Bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid

BDNF

Brain-derived neurotrophic factor

CaM

Calmodulin

CaMK

Calcium/calmodulin-dependent protein kinase I/II/IV

CaSR

Calcium-sensing receptor

CCB

Calcium channel blocker

CPEB

Cytoplasmic Polyadenylation Element-Binding

CPSF

Cleavage and polyadenylation specificity factor

CRAC

Ca²⁺ release-activated Ca²⁺ channel

CRE

CAMP response element

CREB

CAMP response element-binding

DREAM

Downstream regulatory element antagonist modulator

EGF

Epidermal growth factor

EGFR

Epidermal growth factor receptor

ER

Endoplasmic reticulum

ERK

Extracellular signal-regulated kinase

ETS

E26 transformation-specific

FAK

Focal adhesion kinase

GECI

Genetically Encoded Ca²⁺ Indicator

GFAP

Glial fibrillary acidic protein

HuR

Human antigen R

LacZ

β-Galactosidase gene

LGMN

Legumain

LIM

Lin-11, Isl-1, Mec-3 domain

LTCC

L-type voltage-gated Ca²⁺ channel

MAGE

Mitogen-activated protein kinase

MAPK

Mitogen-activated protein kinase

MCU

Mitochondrial Ca²⁺ uniporter

MEK

Mitogen-activated protein kinase kinase

MMP

Mitochondrial membrane potential

NFATC

Nuclear factors of activated T cells

NMDA

N-methyl-D-aspartate

NMDAR

N-methyl-d-aspartate receptors

NPC

Nuclear pore complex

PAP

Poly(A) polymerase

PKA

CAMP-dependent protein kinase

PTM

Posttranslational modification

RELB

RELB Proto-Oncogene, NF-κB Subunit

ROS

Reactive oxygen species

RSK

Ribosomal S6 kinase

RYR

Ryanodine receptor

SENP

Sentrin-specific protease

SERCA

Sarco/Endoplasmic Reticulum Ca²⁺ ATPase

SOCC

Store-operated Ca²⁺ channel

SOCE

Store-operated Ca²⁺ entry

SRE

Serum response element

STREX

Stress axis regulated exon

SUMO

Small ubiquitin-like modifier

TCR

T-cell receptor

TEAD

Transcriptional Enhanced Associate Domain

TF

Transcription factor

TFAM

Mitochondrial transcription factor A

TFEB

Transcription factor EB

TRPC

Transient receptor potential channels

UTR

Untranslated region

WD

WD repeat-containing domain

EZH2

Enhancer of zeste homolog 2

P2Y6

Purinergic receptor P2Y6

PLCβ

Phospholipase C beta

LGCC

Ligand-gated Ca²⁺ channel

FOXC2-AS1

FOXC2 antisense RNA 1

Author contributions

ZXN and ZXH conceived, and supervised the research. ZM and WHY created the figures. ZXN and ZM wrote the manuscript, and ZXH revised the manuscript. All authors read and approved the final manuscript.

Funding

This study was funded by the National Natural Science Foundation of China (82471646 and 32271167) and Large Instruments Open Foundation of Nantong University (KFJN2564).

Data availability

No datasets were generated or analyzed during the current study.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.