Transcriptional factor KLF9 in pulmonary fibrosis: from molecular mechanisms to precision intervention—a narrative review

Abstract

Background and Objective

Pulmonary fibrosis (PF) is a devastating and progressive respiratory disease characterized by extracellular matrix (ECM) remodelling and functional decline, yet current therapeutic options remain limited. Recent studies have highlighted the critical, albeit complex, role of Krüppel-like factor 9 (KLF9) in the pathogenesis of PF. This review aims to comprehensively integrate the existing fragmented mechanistic evidence, delineate the differential regulatory networks of KLF9, and outline forward-looking strategies to promote the transition of KLF9 from a mechanistic candidate molecule to a clinically translatable target.

Methods

A comprehensive literature search was conducted on databases including PubMed, Web of Science, and Embase for articles published up to March 14, 2026. The search focused on keywords such as (“pulmonary fibrosis” OR “idiopathic pulmonary fibrosis” OR “lung fibrosis”) AND (“Krüppel-like factor 9” OR “KLF9” OR “BTEB1”).

Key Content and Findings

Evidence indicates that KLF9 exhibits differential regulatory networks in alveolar epithelial cells, fibroblasts, and macrophages. Based on its functional paradox across diverse cell types, conventional systemic interventions targeting KLF9 face inherent limitations, including the “undruggable” nature of transcription factors and physical barriers in fibrotic lungs. To overcome these obstacles, advanced targeted strategies are required.

Conclusions

While KLF9 plays a pivotal role in the fibrotic microenvironment, realizing its therapeutic potential necessitates a paradigm shift in intervention methods. Forward-looking strategies, including single-cell/spatial multi-omics analysis, ligand-modified inhaled nano-delivery, and stage-dependent dynamic dosing systems [such as proteolysis-targeting chimera (PROTAC) technology], represent promising avenues for precise KLF9 targeting in PF.

Introduction

Background

Pulmonary fibrosis (PF) is a group of chronic interstitial lung diseases characterised by progressive scarring of the lung parenchyma and irreversible decline in gas exchange function. Among them, idiopathic PF (IPF) is the most common subtype with the worst prognosis (1-3). Epidemiological data show that the global incidence of IPF is significantly increasing with population ageing; the median survival after diagnosis is only 3–5 years, and the 5-year survival rate is lower than that of many common malignancies (4-6). Although the anti-fibrotic drugs pirfenidone and nintedanib have been approved for clinical use, they only slow the annual decline in forced vital capacity by approximately 50% and cannot reverse established fibrotic lesions, and they are often accompanied by adverse reactions such as gastrointestinal discomfort and photosensitive rash (7-9). Therefore, in-depth elucidation of the molecular pathological mechanisms of PF and identification of novel molecular targets with high selectivity and intervenability remain core issues that need to be addressed in current clinical and basic research (10-12).

The pathogenesis of PF involves repeated micro-injury of alveolar epithelial cells, aberrant repair responses, transdifferentiation of fibroblasts into myofibroblasts, excessive deposition of extracellular matrix (ECM), and persistent immune microenvironment dysregulation (13-15). In recent years, dysregulation of transcriptional networks has been recognised as a key upstream event driving the above pathological steps. Krüppel-like factors (KLFs) are a family of transcription factors characterised by highly conserved C2H2-type zinc finger structures, and they are widely involved in fundamental life processes such as cell proliferation, differentiation, inflammation and tissue remodelling (16-18). The family members KLF4 and KLF6 have been shown to play pivotal roles in vascular inflammation, macrophage polarisation and liver/intestinal fibrosis, respectively (19-21), suggesting that this family represents a reservoir of targets with potential interventional value in tissue fibrosis.

Rationale and knowledge gap

KLF9 [formerly basic transcription element-binding protein 1 (BTEB1)], an important member of this family, was previously studied mainly for its transcriptional repressor functions in tumours (22,23). However, recent evidence has revealed multiple roles of KLF9 in oxidative stress, metabolic reprogramming and inflammatory regulation, making it an emerging candidate molecule that is rapidly gaining attention in PF research. Currently, there are differing views on the role of KLF9 in PF: the mainstream view holds that KLF9 is a pro-fibrotic “oxidative stress amplifier”, and its genetic deletion exhibits clear lung-protective effects in bleomycin-induced PF animal models (24-26). At the same time, recent cross-evidence from cardiovascular injury repair models shows that in the context of macrophage-mediated tissue injury repair, KLF9 can participate in regulating reparative polarisation, and its deletion instead leads to impaired healing, poor matrix deposition, and increased risk of organ rupture (27). This cell-type specificity and phase dependency mean that KLF9 cannot be simply classified as either pro-fibrotic or anti-fibrotic, but rather presents a “spatiotemporal functional paradox” with translational challenges (28,29).

Current clinically used pirfenidone and nintedanib mainly exert their effects by inhibiting fibroblast proliferation and ECM synthesis downstream of transforming growth factor-β (TGF-β) signalling, but their targets are relatively limited, making it difficult to simultaneously block multiple pathological links such as epithelial injury, macrophage-mediated inflammation amplification, and metabolic reprogramming. Moreover, both drugs require long-term continuous oral administration, offer limited flexibility for dynamic adjustment based on disease phases, and fibrosis often rapidly recurs after drug withdrawal. Consequently, there is a lack of precise targeted countermeasures for the interstitial inflammatory burst during acute exacerbation of IPF (AE-IPF). As an upstream transcriptional regulatory node integrating oxidative stress, metabolic disturbance, and immune polarisation, KLF9 can theoretically intervene in the fibrotic process at an earlier stage. Furthermore, although the functional antagonism of KLF9 in different cells poses difficulties for systemic administration, it also provides a unique opportunity to design a “cell-selective + phase-dynamic” precision intervention—that is, to achieve controllable modulation of this target through targeted delivery and stage-dependent dosing.

Objective

This review aims to comprehensively integrate the existing fragmented mechanistic evidence, delineate the differential regulatory networks of KLF9 in alveolar epithelial cells, fibroblasts and macrophages, and based on its functional paradox, discuss forward-looking strategies to overcome the limitations of conventional systemic intervention, including single-cell/spatial multi-omics analysis, ligand-modified inhaled nano-delivery and stage-dependent dynamic dosing systems, in order to promote the transition of KLF9 from a mechanistic candidate molecule to a clinically translatable target. We present this article in accordance with the Narrative Review reporting checklist (available at https://jtd.amegroups.com/article/view/10.21037/jtd-2026-1767/rc).

Methods

To map the molecular mechanisms and translational strategies of KLF9 in PF, a comprehensive literature search was performed. Databases including PubMed, Web of Science, and Embase were queried for relevant peer-reviewed articles. The detailed search strategy is summarized in Table 1. Please note that a narrative review is less methodologically demanding than a systematic review, as it does not require a search of all literature in a field. Therefore, the search strategy summary is mainly used for more transparent reporting.

Table 1. The search strategy summary.

Items	Specification
Date of search	March 15, 2026
Databases searched	PubMed, Web of Science, and Embase
Search terms used	(“Pulmonary fibrosis” OR “idiopathic pulmonary fibrosis” OR “lung fibrosis”) AND (“Krüppel-like factor 9” OR “KLF9” OR “BTEB1”)
Timeframe	Inception to March 14, 2026
Inclusion and exclusion criteria	Inclusion: English language, peer-reviewed articles, original experimental studies, and reviews relevant to the topic
	Exclusion: non-English literature, conference abstracts without full text
Selection process	The literature search and initial screening based on titles and abstracts were conducted independently by A.K. The final selection of full-text articles was discussed and validated by both authors (A.K. and Y.L.) to reach a consensus
Any additional considerations	Reference lists of the included articles were manually screened to identify any further relevant studies

BTEB1, basic transcription element-binding protein 1; KLF9, Krüppel-like factor 9.

Molecular biological characteristics of KLF9

This section summarises the molecular features of KLF9 that underpin its regulatory roles. It first describes the gene structure and functional domains, particularly the C-terminal zinc finger DNA-binding domain and the N-terminal Sin3a interaction domain (SID) that mediates transcriptional repression. It then examines the expression patterns and multi-level regulatory mechanisms controlling KLF9 activity, including transcriptional control by nuclear factor erythroid 2-related factor 2 (Nrf2) and hormonal signals, as well as post-transcriptional and post-translational modifications. A summary table (Table 2) is provided at the end to compare the differential targets and functional outcomes of KLF9 across various cell types and disease contexts.

Table 2. Summary of differential targets and functional phenotypes of KLF9 in multiple organs and cellular microenvironments.

Tissue/cell type	Disease model	Pathological phase/signal	Key downstream targets/mechanisms	Cellular phenotype and outcome	References
Alveolar epithelial cells	Lung injury/fibrosis	Chronic stress (Nrf2)	Transcriptional suppression of Txnrd2, Prdx6	Amplified oxidative injury, pro-apoptosis and senescence	(24,25,30)
Macrophages	Tissue injury repair	Acute injury phase	Upregulation of GSDMD, activation of NF-κB	Drives inflammatory storm and M1 polarization	(30-32)
	Myocardial infarction repair	Repair phase (myocardium)	Regulation of inflammatory attenuation network	Limits excessive inflammation, promotes matrix homeostasis remodelling	(27)
Mesenchymal cell populations	Lung/kidney interstitial pathology	TGF-β, lipotoxicity	Transcriptional suppression of Ppargc1a (encoding PGC-1α), activation of FABP4	Energy and lipid metabolic reprogramming, excessive ECM deposition	(18,24,33)
Tumor epithelial cells	Gastrointestinal cancer	Epigenetic silencing/methylation	Suppression of MMP9, MMP28	Restricts invasion and metastatic potential	(31,34,35)

The repair-phase role of KLF9 in macrophages is primarily based on cross-organ evidence from cardiovascular injury models; direct validation in PF remains to be established. Summary of differential targets and functional phenotypes of KLF9 in multiple organs and cellular microenvironments. This table compares the pathological phases, key downstream targets/mechanisms, cellular outcomes, and references for KLF9 across alveolar epithelial cells, macrophages (acute injury phase and myocardial repair phase), mesenchymal cell populations, and tumour epithelial cells. The repair-phase role of KLF9 in macrophages is primarily based on cross-organ evidence from cardiovascular injury models; direct validation in pulmonary fibrosis remains to be established. ECM, extracellular matrix; FABP4, fatty acid-binding protein 4; GSDMD, gasdermin D; KLF9, Krüppel-like factor 9; MMP, matrix metallopeptidase; NF-κB, nuclear factor-κB; Nrf2, nuclear factor erythroid 2-related factor 2; PF, pulmonary fibrosis; PGC-1α, peroxisome proliferator-activated receptor gamma coactivator 1-alpha; Ppargc1a, peroxisome proliferator-activated receptor gamma coactivator 1-alpha; Prdx6, peroxiredoxin 6; TGF-β, transforming growth factor-β; Txnrd2, thioredoxin reductase 2.

Gene structure and functional domains

The KLF9 gene is located on the long arm of human chromosome 9, region 1, band 3 (9q13), and encodes a nuclear protein of 244 amino acids (36,37). The molecular basis for its transcriptional regulatory function lies in its unique dual-module structure. The carboxy (C)-terminal region contains three tandem C2H2-type zinc finger structures, a typical hallmark of the KLF family, which enables high-affinity recognition and binding to GC boxes and CACCC boxes in the promoter regions of target genes, thereby precisely anchoring to regulatory regions of antioxidant enzymes, matrix metalloproteinases and metabolism-related genes (38,39). The amino (N)-terminal region contains a conserved SID, which can directly recruit the SIN3A/histone deacetylase (HDAC) co-repressor complex (40,41). By catalysing histone deacetylation and compacting local chromatin structure, KLF9 achieves transcriptional repression of target genes. This SID-mediated repression mechanism is also present in the family member KLF4 (42) and constitutes the molecular basis for KLF9-mediated changes in the oxidative microenvironment and metabolic remodelling (16,43).

Expression patterns and activity regulation

KLF9 expression exhibits marked tissue and pathological stage specificity. In normal adult lung tissue, its basal expression level is low; however, it can be rapidly upregulated under various pathological stimuli. For example, in a lipopolysaccharide (LPS)-induced acute lung injury model, increased KLF9 expression promotes alveolar macrophage pyroptosis by upregulating gasdermin D (GSDMD) (30); in a bleomycin-induced PF model, KLF9 expression increases in a time-dependent manner as fibrosis progresses (24,44,45); in lung tissue from patients with end-stage α1-antitrypsin deficiency-associated chronic obstructive pulmonary disease (COPD), KLF9 expression can be increased by more than 30-fold (45). Furthermore, KLF9 also exhibits high pathological sensitivity in multiple systemic scenarios such as optic nerve regeneration, melanoma, and osteoarthritis (46-48), suggesting that it is a regulatory factor that broadly responds to microenvironmental stress. Notably, the ability of KLF9 to inhibit epithelial-mesenchymal transition (EMT) and cell invasion in gastrointestinal tumours (22,34,49) provides cross-field circumstantial evidence for understanding its potential regulatory role in fibroblast activation and ECM remodelling—the mechanisms of EMT in tumour cells and fibroblast activation share pathological similarities.

Transcriptional regulation

Oxidative stress is an important inducer of KLF9. Upon accumulation of reactive oxygen species (ROS), Nrf2 is activated and translocates to the nucleus, directly binding to the antioxidant response element (ARE) in the KLF9 promoter and driving its transcription (24,50). In addition, the glucocorticoid receptor (GR), triiodothyronine (T3), and its nuclear receptor thyroid hormone receptor (TR)/retinoid X receptor (RXR) heterodimer can also activate KLF9 expression (50-52).

Post-transcriptional and post-translational regulation

Multiple microRNAs (miRNAs; e.g., miR-889-3p, miR-130a-3p, miR-625-3p, etc.) can bind to the 3'-untranslated region (3'-UTR) of KLF9 mRNA to inhibit its translation or promote its degradation (53-55); long non-coding RNAs and circular RNAs can act as competitive endogenous RNAs (ceRNAs) to sponge the above miRNAs, thereby relieving the inhibition of KLF9 (35,56,57). At the protein level, mitogen-activated protein kinase (MAPK) pathway-mediated phosphorylation can enhance the binding affinity of KLF9 to target gene promoters (31,49); whereas HDAC1/2/6-catalysed deacetylation exposes its lysine residues, promoting recognition by E3 ubiquitin ligases and subsequent proteasomal degradation, forming a “deacetylation-ubiquitination” degradation axis (31,34). This multi-level regulation endows KLF9 with the ability to rapidly respond to microenvironmental changes and also provides a molecular basis for its complex roles in PF.

KLF9 in the core regulatory network of PF

This section systematically delineates the functional heterogeneity of KLF9 across different cell types and pathological phases in PF. It first introduces the overall expression pattern and paradoxical roles of KLF9. It then examines its interactions with key pro-fibrotic signalling pathways, including TGF-β/Smad, Wnt/β-catenin, and oxidative stress/Nrf2, as well as epigenetic and miRNA dysregulation. Finally, it details the KLF9-driven phenotypic changes in alveolar epithelial cells, fibroblasts/myofibroblasts, and macrophages, highlighting the stage-dependent duality that underlies the “spatiotemporal functional paradox”. The core regulatory network is illustrated in Figure 1.

Figure 1.

KLF9 integrates upstream signals and drives cell-type-specific fates in PF. KLF9 serves as a central hub receiving multiple upstream pro-fibrotic signals, including Wnt, TGF-β, and Nrf2-driven oxidative stress (ROS). In alveolar epithelial cells, KLF9 suppresses the antioxidant genes Txnrd2 and Prdx6, amplifying oxidative stress and promoting apoptosis and senescence. In fibroblasts, KLF9 drives activation and excessive ECM deposition. In macrophages, KLF9 exhibits a stage-dependent biphasic function: it promotes M1 pro-inflammatory polarization during the acute phase, while contributing to M2-like tissue remodelling during the repair phase. Solid lines indicate direct or validated regulations; dashed lines indicate indirect or context-dependent regulations. The black downward arrow indicates the KLF9-mediated repression of Txnrd2 and Prdx6, leading to ROS accumulation. Created in BioRender. Kelimujiang A. [2026]. Available online: https://BioRender.com/ikcbadz. ECM, extracellular matrix; KLF9, Krüppel-like factor 9; Nrf2, nuclear factor erythroid 2-related factor 2; PF, pulmonary fibrosis; Prdx6, peroxiredoxin 6; ROS, reactive oxygen species; TGF-β, transforming growth factor-β; Txnrd2, thioredoxin reductase 2.

The Nrf2-KLF9-ROS positive feedback loop: a threshold shift from protection to injury

Oxidative stress is a critical driver of the initiation and progression of PF (58-60). Under physiological conditions, the transcription factor Nrf2 is a master regulator maintaining intracellular redox homeostasis, exerting cytoprotective effects by inducing multiple antioxidant genes such as Nqo1, Gclc, and Gpx2 (61,62). However, in the PF microenvironment, long-term sustained high-intensity oxidative stress can lead to a pathological consequence. Zucker et al. (24) first discovered that although massive nuclear entry of Nrf2 activates KLF9 transcription, KLF9 does not cooperate in antioxidant defence but instead directly binds to and represses several key antioxidant effector genes, including mitochondrial thioredoxin reductase 2 (Txnrd2), peroxisome proliferator-activated receptor gamma coactivator 1-alpha (Ppargc1a, encoding PGC-1α), and peroxiredoxin 6 (Prdx6). This constitutes a vicious “Nrf2-KLF9-ROS” positive feedback loop: Nrf2 upregulates KLF9, KLF9 represses antioxidant genes leading to elevated ROS, and ROS further activate Nrf2, thus constituting a self-amplifying cycle.

The mechanistic basis for the operation of this feedback lies in the threshold effect of target repression. Among the numerous antioxidant genes, KLF9-mediated repression of Prdx6 is particularly important. Prdx6 is not only a key enzyme for clearing intracellular peroxides but also possesses independent phospholipase A2 (iPLA2) activity, participating in the repair of oxidatively damaged cell membranes and mitochondrial membrane lipids (25,63). When both Txnrd2 and Prdx6 (which combine ROS scavenging and damaged membrane lipid repair functions) are simultaneously suppressed, the accumulation of intracellular ROS irreversibly breaches the safety threshold of the endogenous buffering system, leading to uncontrolled lipid peroxidation chain reactions and impaired mitochondrial function, thereby converting the originally adaptive Nrf2 system into a pro-injury pathway. It should be noted that this mechanism currently derives mainly from in vitro stress models and some bleomycin mouse studies; its in situ validation in different cell populations in IPF patient lung tissue remains insufficient, but it provides a referable molecular framework for understanding the epithelial injury effects of KLF9.

Alveolar epithelial cells: oxidative damage, senescence and a possible ferroptosis link

Repeated injury and impaired regeneration of alveolar epithelial cells, especially type II alveolar epithelial cells (AEC2s), are considered important initiating events in the progression of PF (64-66). KLF9 in this cell population mainly exerts pro-injury effects. First, by repressing Txnrd2 and Prdx6, KLF9 impairs the ability of epithelial cells to clear ROS, leading to accumulation of oxidative DNA damage and inducing apoptosis and the release of senescence-associated secretory phenotype (SASP) (24,67,68). Senescent AEC2s further alter the local microenvironment and promote fibroblast activation by secreting factors such as interleukin (IL)-6, IL-8, and TGF-β.

In recent years, ferroptosis, an iron-dependent, lipid peroxidation-characterised regulated cell death pathway, has been identified as an important route of epithelial injury in IPF (69,70). In cardiovascular ischaemia-reperfusion injury models, KLF9 has been shown to transcriptionally activate high mobility group box 2 (HMGB2), thereby driving cardiomyocyte apoptosis, inflammation and ferroptosis (71). Although direct in situ validation of KLF9-regulated ferroptosis in lung tissue is currently lacking, this cross-organ molecular linkage (used here as a mechanistic reference from other organs) suggests that KLF9 may be involved in coupling epithelial oxidative injury to ferroptosis. Particularly given the aforementioned direct repression of Prdx6 (a key anti-lipid peroxidation enzyme) by KLF9, this provides a highly compatible biochemical basis for KLF9-driven ferroptosis. This hypothesis points to a direction for future research: detecting lipid peroxidation markers [malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE)] and glutathione peroxidase 4 (GPX4) expression in alveolar epithelial cell-specific KLF9 knockout/knock-in models may clarify the role of the KLF9-HMGB2 axis in ferroptosis in PF.

Fibroblasts/myofibroblasts: metabolic reprogramming and lipotoxicity

Excessive ECM deposition is the terminal manifestation of the fibrotic process, and the transdifferentiation of fibroblasts into myofibroblasts is the core cellular event achieving this process (72). Compared with epithelial cells, direct functional evidence for KLF9 in lung fibroblasts is relatively limited, but clues from other mesenchymal lineages provide suggestive indications.

Energy metabolic reprogramming. KLF9 represses the expression of PGC-1α, a master regulator of mitochondrial biogenesis (24), thereby affecting cellular energy metabolic homeostasis. Upon TGF-β stimulation, fibroblasts need to shift from quiescent oxidative phosphorylation to aerobic glycolysis (the Warburg effect) to meet the demands for adenosine triphosphate (ATP) and biosynthetic precursors for rapid proliferation and ECM synthesis (72). KLF9-mediated PGC-1α repression may synergise with TGF-β signalling to relieve mitochondrial constraints on metabolic flux, thereby promoting myofibroblast activation.

Lipid metabolism disturbance and lipotoxicity. A recent study in a renal tubulointerstitial fibrosis model demonstrated that KLF9 directly transcriptionally activates fatty acid-binding protein 4 (FABP4) (31). High FABP4 expression leads to enhanced intracellular fatty acid uptake and esterification, causing abnormal lipid droplet accumulation and lipotoxicity, which in turn aggravates endoplasmic reticulum stress, mitochondrial dysfunction, and interstitial fibrosis. Single-cell sequencing studies in recent years have confirmed that lung fibroblasts from IPF patients exhibit significant lipid droplet accumulation and lipid metabolic reprogramming features (73,74). Therefore, the KLF9-FABP4 axis may represent a conserved cross-organ pro-fibrotic mechanism. Future studies should validate the direct transcriptional regulation of FABP4 by KLF9 in IPF patient-derived lung fibroblasts and assess whether small-molecule FABP4 inhibitors can block KLF9 overexpression-driven collagen synthesis.

Macrophages: stage-dependent inflammation amplification and repair modulation

Lung macrophage populations exhibit high phenotypic heterogeneity at different stages of fibrosis development, maintenance, and resolution (75,76). KLF9 in this cell population displays a biphasic character with translational significance.

Acute injury phase (pro-inflammation and pyroptosis)

In LPS-induced acute lung injury models, KLF9 activates the nuclear factor-κB (NF-κB) pathway by directly binding to the TLR2 promoter, driving macrophages to polarise towards the classical M1 pro-inflammatory phenotype and releasing inflammatory factors such as tumour necrosis factor-α (TNF-α), IL-6, and IL-1β (77). At the same time, KLF9 upregulates the expression of the pyroptosis execution protein GSDMD, promoting alveolar macrophage pyroptosis and releasing damage-associated molecular patterns (DAMPs) that further amplify the local inflammatory response (30). Inhibition of KLF9 at this stage exerts organ-protective effects.

Tissue repair phase (inflammation resolution and pro-healing)

It should be noted that the direct experimental evidence currently revealing KLF9-mediated tissue repair comes mainly from acute ischaemia models such as myocardial infarction (27), and its exact role in chronically progressive PF awaits validation using cell-specific genetic models. In that myocardial repair study, although macrophage-specific Klf9 deletion alleviated early inflammatory injury, it subsequently hindered the transition to a reparative M2-like phenotype, leading to poor collagen scaffold construction in the infarct area, sparse neovascularisation, and increased risk of ventricular rupture (27). Mechanistically, KLF9 does not directly activate M2 marker genes but acts as an upstream regulatory element maintaining the temporal attenuation of interferon-γ (IFN-γ)/signal transducer and activator of transcription 1 (STAT1) pro-inflammatory signals, thereby creating conditions for macrophages to clear necrotic debris and secrete pro-remodelling factors [such as TGF-β, vascular endothelial growth factor (VEGF), and IL-10] (27,78).

Although reparative fibrosis (e.g., post-infarct scar formation) and pathological progressive fibrosis (e.g., IPF) have distinctly different end-stage phenotypes, the underlying molecular logic of macrophages handling necrotic debris and temporally attenuating STAT1 signals to regulate matrix remodelling is highly conserved in evolution. Therefore, indiscriminate sustained inhibition of KLF9 during the repair phase of PF may interfere with this physiological attenuation network, affecting matrix remodelling and alveolar epithelial regeneration. This “phase paradox” is a major obstacle to conventional systemic intervention strategies.

KLF9’s cell-specific functional paradox and controversies in PF

Although KLF9 has been identified as a critical transcriptional regulator in PF, its specific roles are highly context-dependent, presenting a spatiotemporal functional paradox across different cellular microenvironments. Existing studies reveal seemingly contradictory conclusions regarding its function. In alveolar epithelial cells and fibroblasts, KLF9 predominantly acts as a pro-fibrotic driver. By amplifying oxidative stress and suppressing antioxidant defense mechanisms (such as repressing the Nrf2 pathway and its targets like Txnrd2 and Prdx6), KLF9 exacerbates epithelial apoptosis and promotes fibroblast activation and excessive ECM deposition (24,25).

Conversely, emerging evidence suggests that KLF9 may mediate distinct immunomodulatory functions in macrophages, particularly by regulating macrophage polarization during specific injury and resolution phases (27). This cellular heterogeneity explains why conventional systemic blockade of KLF9—which indiscriminately inhibits the factor across all tissues—may yield ineffective outcomes by disrupting essential macrophage-mediated tissue repair. Recognizing this functional paradox fundamentally highlights the limitations of systemic interventions and underscores the urgent need for cell-selective, precision-targeted strategies.

Translational targeting: limitations, advanced strategies and future directions

This section discusses the obstacles to translating KLF9-targeted interventions into clinical practice and proposes potential solutions. It first analyses the inherent limitations of conventional systemic inhibition, including the functional paradox across cell types, the “undruggable” nature of transcription factors, and physical barriers in fibrotic lungs. It then introduces advanced strategies to overcome these challenges: spatial multi-omics to map the spatiotemporal expression landscape of KLF9, inhaled nano-delivery systems for cell-selective targeting, and proteolysis-targeting chimera (PROTAC) technology combined with stage-dependent dynamic dosing. A framework integrating these approaches is presented in Figure 2.

Figure 2.

Framework of precision targeted strategies to overcome the “spatiotemporal functional paradox” of KLF9 in PF. The left panel illustrates the biological barriers for drug delivery in fibrotic lungs, including the mucus layer and dense ECM. The upper right panel depicts engineered nanodelivery systems (e.g., LNPs, exosomes, and ligand-modified particles) designed to penetrate these barriers. By incorporating targeting ligands, these systems enable gene silencing and KLF9 knockdown specifically in activated fibroblasts. This strategy can be combined with standard anti-fibrotic drugs for synergistic therapy. The lower panel displays a stage-dependent decision flow for dynamic administration: during the acute/inflammatory phase, KLF9 is inhibited to limit tissue injury; during the repair/resolution phase, KLF9 inhibition is paused to preserve its physiological functions in tissue repair. Created in BioRender. Kelimujiang A. [2026]. Available online: https://BioRender.com/tybb01b. ECM, extracellular matrix; KLF9, Krüppel-like factor 9; LNP, lipid nanoparticle; mRNA, messenger RNA; PF, pulmonary fibrosis; siRNA, small interfering RNA.

Inherent limitations of conventional systemic intervention

If a conventional small-molecule kinase inhibitor approach (such as an analogue of nintedanib) is used to systemically inhibit KLF9, multiple difficulties will be encountered. First, KLF9 exerts pro-fibrotic effects in alveolar epithelial cells and fibroblasts, but participates in inflammation resolution and tissue healing during the macrophage repair phase. This target functional paradox means that systemic inhibition may interfere with repair programs while reducing fibrosis, potentially offsetting the net clinical benefit (79). Second, as a nuclear transcription factor, KLF9 lacks a typical small-molecule binding pocket, making it conventionally ‘undruggable’ and difficult to obtain highly selective small-molecule inhibitors. Third, the dense ECM deposition and destruction of the alveolar capillary bed in fibrotic lung tissue form physical barriers that reduce the efficiency of drug penetration across the blood-air barrier to reach target cells (80,81). Therefore, even if an ideal KLF9 inhibitor were obtained, clinical translation would still face difficulties if cell selectivity and delivery efficiency are not addressed. The key downstream targets of KLF9 across different cell types are summarized in Table 3.

Table 3. Summary of KLF9 downstream targets and cell-specific functional phenotypes in PF.

Cell type	Key downstream targets/pathways	Regulatory direction by KLF9	Cellular phenotype and functional effect	References
Alveolar epithelial cells	Nrf2, Txnrd2, Prdx6	Inhibition/downregulation	Increased ROS accumulation, amplified oxidative stress, accelerated cellular apoptosis and senescence	(24)
Fibroblasts	TGF-β signalling, ECM-related genes, FABP4	Activation/upregulation	Enhanced myofibroblast differentiation, excessive collagen and ECM deposition, lipotoxicity	(24,31)
Macrophages	Macrophage polarization markers (e.g., M1/M2)	Context-dependent modulation	Regulation of immunomodulation, macrophage polarization, and tissue repair during resolution phases	(27)

ECM, extracellular matrix; FABP4, fatty acid-binding protein 4; KLF9, Krüppel-like factor 9; Nrf2, nuclear factor erythroid 2-related factor 2; PF, pulmonary fibrosis; Prdx6, peroxiredoxin 6; ROS, reactive oxygen species; TGF-β, transforming growth factor-β; Txnrd2, thioredoxin reductase 2.

Spatial multi-omics: mapping the spatiotemporal dynamic landscape of KLF9 in the lung

The first step in resolving the “spatiotemporal paradox” is to precisely map the expression landscape of KLF9 in fibrotic lung tissue. Conventional bulk RNA sequencing mixes and averages signals from different cell subsets, which may obscure specific changes in KLF9 in particular pathological regions or rare cell subsets. Recent advances in single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics provide tools to address this issue (82,83). scRNA-seq can identify specific cell subsets with abnormally elevated KLF9 in IPF lung tissue, such as a high-oxidative-stress AEC2 subset in a transitional state or a platelet-derived growth factor receptor β (PDGFRβ)⁺ fibroblast subset. Spatial transcriptomics can map gene expression information back to the tissue in situ, localising regions of KLF9 enrichment in fibroblastic foci (84,85). Combined with pseudo-time analysis, the dynamic trajectory of KLF9 expression as the disease evolves from the inflammatory phase to the fibrotic phase and then to the repair phase can also be constructed. This information will provide a basis for identifying target cell populations and time windows for subsequent targeted interventions.

To practically translate this concept, a specific research design combining spatial transcriptomics (e.g., 10× Genomics Visium) with scRNA-seq is highly recommended. For example, recent pioneer studies have successfully utilized spatial transcriptomics to dissect the complex architecture of the “fibroblastic niche” in IPF lungs (86). Building upon this methodological framework, future studies could map intact IPF tissue sections, focusing specifically on actively remodelling fibroblastic foci compared to adjacent normal alveolar margins.

By integrating scRNA-seq data, this design allows for the precise deconvolution of cell types within spatial niches. Consequently, researchers can dynamically trace the expression trajectory of KLF9, identifying whether its peak expression co-localizes with senescent epithelial cells at the injury edge or with activated myofibroblasts in the core fibrotic matrix. Such a spatially resolved multi-omics approach will bridge current knowledge gaps, providing a high-resolution roadmap of KLF9’s epigenetic networks across different disease stages.

Inhaled nano-delivery: a technological route to cell-selective targeting

For local pulmonary lesions and the cell-specificity paradox of KLF9, inhaled nano-delivery systems represent a translationally promising solution (87). Compared with intravenous injection, nebulised inhalation can directly deliver drugs to the alveolar surface, avoiding hepatic first-pass metabolism and reducing off-target toxicity from systemic exposure. Current core design strategies include: encapsulating small interfering RNA (siRNA) or messenger RNA (mRNA) inhibitors in lipid nanoparticles (LNPs) or polymeric nanoparticles with a particle size controlled below 100 nm to facilitate penetration of mucus and ECM barriers; modifying the nanoparticle surface with targeting ligands [such as anti-PDGFRβ antibodies, anti-fibroblast activation protein (FAP) antibodies or arginylglycylaspartic acid (RGD) peptides] to actively recognise activated lung fibroblasts or injured epithelial cells (88-90); and using PEGylated surface coatings to reduce non-specific uptake by alveolar macrophages (91,92). In animal experiments, inhaled siRNA-LNPs have already demonstrated inhibitory effects on bleomycin-induced PF (93,94).

To overcome the biological barriers in fibrotic lungs, such as the dense mucus layer and excessive ECM deposition, inhaled nano-delivery systems have emerged as a highly feasible strategy. Recent advancements have successfully demonstrated the potential of this approach in PF. For instance, inhalable biomimetic liposome-based nanocarriers have been utilized to locally deliver anti-fibrotic agents directly to the lungs, significantly reducing systemic toxicity while enhancing local drug accumulation (95). Similarly, targeted LNPs modified with specific surface ligands have been developed to deliver siRNA via inhalation, achieving highly precise gene silencing in the pulmonary microenvironment (96).

Applying this advanced technology to KLF9 presents a promising therapeutic avenue. By formulating KLF9-targeted siRNA within ligand-modified LNPs (e.g., functionalized with peptides specific to activated fibroblasts or injured epithelial cells), we can achieve cell-specific silencing of KLF9. This localized modulation via inhalation maximizes the therapeutic index and effectively circumvents the spatiotemporal paradox of KLF9 by sparing its functional integrity in macrophages.

In terms of mechanistic trade-offs in targeting strategies, it should be noted that macrophages also rely on KLF9 to drive M1 inflammation during the acute phase, so inhibiting KLF9 in macrophages during the acute phase could theoretically also confer anti-inflammatory benefits. However, given that fibroblasts are the direct executors of excessive ECM deposition and lung structure destruction, and that macrophages require KLF9 for inflammation resolution during the repair phase, our proposed strategy prioritises the targeting of activated lung fibroblasts through ligand modification, while reducing macrophage uptake via PEGylation to preserve their later repair functions. For acute-phase macrophage-mediated excessive inflammation, short-term combination with already-marketed anti-inflammatory drugs (such as low-dose glucocorticoids or the inherent anti-inflammatory effects of nintedanib itself) could be considered as bridging therapy in the early disease phase. Looking further ahead, microenvironment-responsive smart nanoparticles (e.g., ROS-hyper-responsive release or reversible ligand shielding technology) could be designed to achieve dynamic targeting switching, temporarily inhibiting macrophage KLF9 during the inflammatory phase and automatically releasing the inhibition during the repair phase.

PROTAC technology and dynamic stage-dependent dosing: regulation in the time dimension

In addition to spatial selectivity, targeting KLF9 requires overcoming its “undruggable” nature as a transcription factor. Novel modalities such as inhaled siRNA (acting at the mRNA level, as discussed above) and PROTAC (acting at the protein level) offer unprecedented opportunities (94). Compared with conventional small-molecule inhibitors, PROTAC utilizes the ubiquitin-proteasome system to catalytically degrade target proteins, offering higher efficiency and overcoming the lack of distinct binding pockets on transcription factors. Regardless of the therapeutic modality (siRNA or PROTAC), based on the phase paradox of KLF9, the treatment should not adopt a long-term continuous inhibition model. A rational dynamic dosing strategy is “hit-and-run”: administering short-term KLF9-targeted formulations during the acute exacerbation phase [guided by surging biomarkers like lactate dehydrogenase (LDH), C-reactive protein (CRP), or Krebs von den Lungen-6 (KL-6)] to quickly curb the “KLF9-ROS” inflammatory and fibrotic cascades, followed by prompt drug washout. Such formulations, ideally with a controllable half-life, provide pharmacokinetic convenience, allowing the lung microenvironment to transition smoothly into the “repair observation period”, thereby preserving the indispensable physiological functions of KLF9 in tissue healing (95).

Combinatorial strategies: integrating standard care with KLF9 precision intervention

Currently, the clinical management of PF heavily relies on broad-spectrum anti-fibrotic drugs, primarily Nintedanib and Pirfenidone. While these agents effectively slow disease progression by globally inhibiting tyrosine kinases or modulating TGF-β pathways, they lack cell-specific targeting and fail to completely halt or reverse underlying cellular dysfunctions. Notably, recent pharmacological studies indicate that these standard-of-care drugs broadly intersect with oxidative stress pathways and antioxidant mechanisms in the fibrotic microenvironment (97).

To maximize clinical translational value, we propose a novel combinatorial intervention strategy. This approach involves utilizing existing systemic anti-fibrotic drugs as a foundational therapy to stabilize the overall fibrotic milieu, concurrently coupled with KLF9-targeted precision nanomedicines (e.g., inhaled PROTACs or siRNA-LNPs). While the standard care mitigates broad fibrogenic signalling, the localized KLF9-targeted intervention can precisely clear senescent epithelial cells or deactivate specific fibroblast sub-populations. Such a synergistic paradigm, aimed at achieving supra-additive therapeutic efficacy, capitalizes on the broad stabilization provided by current drugs while overcoming their lack of specificity through the precise spatiotemporal modulation of KLF9.

Future research priorities

Based on the above analysis, future work should focus on the following priorities. First, at the basic mechanism level, generate alveolar epithelial cell-specific, fibroblast-specific, and macrophage-specific conditional KLF9 knockout mice. This will help clarify the differential contributions of KLF9 in each cell population to fibrotic phenotype, redox status, and repair outcome in bleomycin-induced models. Second, at the technological method level, integrate scRNA-seq and spatial transcriptomics to map the three-dimensional spatiotemporal atlas of KLF9 expression in human IPF lungs, and identify upstream signals driving KLF9 upregulation in AE-IPF (such as Nrf2, GR, and hypoxia). Finally, at the translational validation level, test the efficacy and cell selectivity of inhaled KLF9-siRNA nanoparticles in IPF patient-derived precision-cut lung slices (PCLS), and evaluate their synergistic effects with nintedanib or pirfenidone, providing proof-of-concept data for entry into clinical trials.

Strengths and limitations of this review

The strength of this review lies in its comprehensive integration of the seemingly contradictory roles of KLF9 across different cell types—including alveolar epithelial cells, fibroblasts, and macrophages—in PF. By conceptualising KLF9 as a “spatiotemporal functional paradox” rather than a simple pro- or anti-fibrotic factor, this article provides a novel and nuanced perspective on a complex molecular target. Furthermore, we do not merely summarise mechanistic pathways; rather, we propose forward-looking, actionable translational strategies, such as single-cell multi-omics mapping, ligand-modified nano-delivery, and stage-dependent dynamic dosing, thereby bridging the gap between basic mechanistic research and future clinical applications.

However, several limitations must be acknowledged. First, as a narrative review, the literature search and selection processes may be subject to inherent selection bias compared to the strict, exhaustive methodology of a systematic review. Second, the current evidence regarding KLF9 in PF is predominantly derived from preclinical animal models and in vitro cellular assays. Direct clinical trial data targeting KLF9 are currently lacking, which limits the immediate clinical generalisability of the proposed therapeutic strategies. Finally, because the downstream targets of KLF9 are highly dependent on the microenvironment and epigenetic context, some of the regulatory networks mapped in this review may require further validation in human pathological samples to confirm their full translational value.

Conclusions

In summary, KLF9 emerges as a critical, highly dynamic transcriptional regulator in the pathogenesis of PF. Rather than acting strictly as a unilateral driver or suppressor of fibrosis, KLF9 exhibits distinct, context-dependent functions: it amplifies oxidative stress and promotes fibrogenesis in epithelial cells and fibroblasts, while potentially mediating essential reparative processes in macrophages during specific injury phases. This “spatiotemporal paradox” renders conventional systemic blockade strategies potentially ineffective or even detrimental. Overcoming this translational hurdle requires a paradigm shift towards precision medicine. Future research must prioritise decrypting the cell-specific epigenetic networks of KLF9 using spatial transcriptomics and developing targeted inhaled nano-delivery systems to achieve cell-selective and phase-dynamic modulation. Only through such precise spatiotemporal targeting can the mechanistic potential of KLF9 be successfully translated into safe and effective therapeutic countermeasures for patients with PF.

Supplementary

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Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.