Idiopathic pulmonary fibrosis (IPF) is a chronic interstitial lung disorder marked by persistent alveolar injury and dysregulated tissue repair, leading to progressive fibrosis and decline in pulmonary function [1, 2]. IPF predominantly affects middle-aged and elderly individuals, particularly men over 65 years of age, with its incidence steadily rising worldwide [3, 4]. The disease is associated with an unfavorable prognosis, with a median survival of approximately 3–5 years following diagnosis [5], severely impairing patients' quality of life and imposing a significant burden on healthcare systems[4]. While the etiology is multifactorial—involving environmental exposures, genetic predisposition, and immune dysregulation [6]—current treatments, such as pirfenidone and nintedanib, primarily aim to slow progression, though their efficacy varies and no curative options exist [7, 8]. Recently, the oral phosphodiesterase 4B (PDE4B) inhibitor nerandomilast has been approved in China, demonstrating antifibrotic and anti-inflammatory effects in Phase II trials [9], providing a novel therapeutic avenue. However, further elucidation of the underlying mechanisms and development of targeted therapies are essential to enhance long-term outcomes.

Multiple signaling cascades, such as TGF-β/Smad, Wnt/β-catenin, Notch, and inflammation-associated pathways, are disrupted in the pathogenesis of IPF [10]. These signaling cascades play vital roles in controlling cellular differentiation, proliferation, apoptosis, and the balance of extracellular matrix (ECM) production and degradation, with notable crosstalk such as TGF-β enhancing Wnt/β-catenin to promote ECM deposition and fibroblast activation, contributing to IPF progression [11]. Among these pathways, Wnt/β-catenin signaling is particularly notable for its pivotal role in promoting fibrotic progression. Wnt signaling stabilizes β-catenin, facilitating its nuclear accumulation and activation of downstream genes encoding ECM components and pro-fibrotic factors [12]. Upregulation of multiple Wnt/β-catenin pathway components has been observed in IPF lungs, correlating with disease severity [13]. Targeting Wnt/β-catenin shows therapeutic promise; for instance, inhibitors reduce collagen deposition by up to 50% and improve lung function in bleomycin-induced models [14]. Our recent work demonstrated that TRIOBP modulates miR-29b to influence the β-catenin pathway, underscoring its critical role in IPF pathogenesis [15]. These insights affirm β-catenin's centrality in IPF and position it as a viable therapeutic target.

CCAAT enhancer binding protein γ (C/EBPγ), a relatively understudied member of the C/EBP family, is the smallest and simplest, comprising only a leucine zipper and basic region without activation domains (AD) or negative regulatory domains (RD) [16]. This minimal structure has long been thought to constrain its functional potential. However, emerging evidence reveals C/EBPγ's involvement in various disease pathogeneses, highlighting its broader significance [17–20]. Its role in fibrotic disorders, however, remains poorly defined. In skin fibrosis, C/EBPγ is implicated in indirectly promoting fibroblast activation and matrix remodeling [21]. While direct evidence in pulmonary fibrosis is lacking, prior work shows that C/EBPγ is upregulated by IL-1β in LPS- or immunoglobulin-mediated lung inflammation models, conferring protection through decreased vascular permeability, restricted neutrophil infiltration, and reduced inflammatory mediator release [22]. Furthermore, C/EBPγ overexpression has been shown to attenuate inflammatory responses by antagonizing the transcriptional activity of other members such as C/EBPβ and C/EBPδ [22] and downregulating cytokines such as IL-6 [23]. Consequently, C/EBPγ is often viewed as an anti-inflammatory regulator, prompting speculation that it might mitigate fibrosis via immunomodulation. However, this notion has not been experimentally validated in pulmonary fibrosis, and critical gaps remain regarding its functional role in lung fibroblasts and its potential interactions with core fibrogenic signaling cascades. Importantly, fibrosis differs from acute inflammation; although inflammation can trigger remodeling, fibrotic progression entails sustained transcriptional changes, aberrant epithelial-mesenchymal transition, and extracellular matrix buildup. Thus, whether C/EBPγ acts protectively or paradoxically drives fibrosis in chronic injury settings remains a key unresolved question with clinical implications.

This study sought to investigate the potential involvement of C/EBPγ in pulmonary fibrosis, as its role in this process remains unknown. Through immunohistochemical and immunofluorescence analyses, we found that C/EBPγ expression was markedly elevated in lungs of IPF patients and similarly increased in the lungs of bleomycin-induced fibrotic mice. To clarify the function and molecular mechanisms of C/EBPγ in pulmonary fibrosis, we performed loss- and gain-of-function experiments in A549 and MRC-5 cells. C/EBPγ overexpression enhanced EMT in A549 cells, as well as fibroblast-to-myofibroblast transition (FMT) in MRC-5 cells. Mechanistically, C/EBPγ enhanced β-catenin signaling by stabilizing the protein and promoting its nuclear accumulation. Rather than directly regulating β-catenin transcription, C/EBPγ interacted with C/EBPα to repress Axin1 expression, thereby limiting β-catenin phosphorylation and subsequent degradation. In line with these observations, adeno-associated virus–driven C/EBPγ overexpression exacerbated bleomycin-induced pulmonary fibrosis in vivo, while its silencing mitigated the fibrotic response.

Human expression plasmids pcDNA3.1-C/EBPγ, pcDNA3.1-C/EBPα, and the truncation mutant pcDNA3.1-C/EBPγ ΔbZIP, as well as phage-Flag-tagged constructs for AXIN1 and β-catenin, and their corresponding empty vectors, were transfected into A549 and MRC-5 cells using Lipofectamine 3000 (Invitrogen, USA) according to the manufacturer’s instructions.

For gene silencing experiments, small interfering RNA (siRNA) targeting human C/EBPγ was purchased from RiboBio Co., Ltd. (Guangzhou, China). Transfections were carried out using the corresponding siRNA transfection reagent provided by the same manufacturer, following the recommended protocol.

Lung tissues and cultured cells were lysed in SDS buffer (P0013B, Beyotime, Shanghai, China) containing protease and phosphatase inhibitors. Protein levels in the supernatant were quantified with a BCA assay kit (Solarbio, Beijing, China). Equal protein samples were resolved on 8–12% SDS–PAGE gels and transferred to PVDF membranes (Millipore, Darmstadt, Germany). The membranes were blocked with 5% nonfat milk, then incubated with primary antibodies overnight at 4°C, followed by HRP-conjugated secondary antibodies at room temperature for 1 h. Immunoreactive bands were detected using the Odyssey Fc Imaging System (LI-COR Biosciences, Lincoln, NE, USA). The following antibodies were used: C/EBPγ (Thermo Fisher Scientific, #PA5-101128), β-actin (Affinity, #T0022), α-SMA (Cell Signaling Technology, #19245), COL1A1 (Cell Signaling Technology, #72026), E-cadherin (Cell Signaling Technology, 14472S), N-cadherin (Cell Signaling Technology, #13116), Vimentin (Proteintech, 10366-1-AP), Fibronectin (Cell Signaling Technology, #26836), β-catenin (Proteintech, #51067-2), phos-β-catenin (T41 + S45) (Abcam, #ab81305), C/EBPα(Proteintech, #29388-1).

For transcriptional analysis of tissue and cultured cells, total RNA was extracted using TRIzol reagent (Takara, Dalian, China) following the manufacturer’s protocol and previously published methods [24]. Complementary DNA (cDNA) was generated using the GoScript™ Reverse Transcription System (Promega, Madison, WI, USA). Quantitative real-time PCR (qRT–PCR) was carried out on a LightCycler 480 instrument (Roche, Basel, Switzerland) with SYBR Green Master Mix (Qiagen, Hilden, Germany) and cDNA as the template. Relative mRNA expression were determined using the 2^−ΔΔCt approach. Primer sequences applied in this study are provided in Table 1.

The contractile ability of MRC-5 fibroblasts was assessed using a collagen gel contraction model based on rat tail collagen type I (Corning, Corning Inc., NY, USA). MRC-5 cells were subjected to the corresponding transfection or pharmacological treatments prior to the assay.

Collagen mixtures were prepared on ice by combining rat tail Collagen I (3 mg/mL), 10× PBS, serum-free medium, and 0.1 M NaOH to adjust the pH to approximately 7.4. The concentration of collagen was finally adjusted to 1.5 mg/mL. Equal volumes of the collagen mixture and cell suspension (5 × 10⁵ cells/mL) were gently mixed and dispensed into 24-well plates (0.5 mL per well). Gels were allowed to polymerize at 37°C for 30–60 minutes, then covered with 0.5 mL of complete medium containing 10% FBS. After incubation for 24–48 hours, the gels were gently released from the well edges using a sterile pipette tip to permit contraction. Images were taken at 0 hour and again at 24 or 48 hours with an inverted microscope, and gel areas were measured using ImageJ (v1.52q). Contraction was quantified as the percentage reduction in area, calculated by (initial area − final area) / initial area × 100%, where a higher percentage reflected greater contractile strength.

MRC-5 cells were co-transfected with the appropriate plasmids and the TOPFlash-CP rapid-degradation luciferase reporter plasmid (Beyotime, Shanghai, China). After 48 hours of incubation, luciferase activity was determined using the Dual-Luciferase® Reporter Assay System (Promega, Madison, WI, USA) according to the manufacturer’s guidelines. In brief, cells underwent one wash with PBS before being lysed in 1× Passive Lysis Buffer (PLB, 100 µL per well in a 24-well plate) for 15 minutes at room temperature with gentle shaking. The lysates were then collected, centrifuged at 4°C to remove debris, and the collected supernatants were then transferred to 96-well plates for luminescence measurement.

Luciferase Assay Reagent II (LAR II) was prepared by reconstituting the lyophilized substrate in Luciferase Assay Buffer II and stored at − 80°C until use. Before measurement, both LAR II and cell lysates were equilibrated to room temperature. For each sample, 20 µL of cell lysate was mixed with 100 µL of LAR II solution, and luminescence was recorded immediately using a microplate luminometer. Relative light units (RLU) representing luciferase activity were adjusted according to total protein levels quantified via the BCA assay. Non-transfected controls (NTC) were included to adjust for background signals. All steps were carried out under low-light conditions to minimize nonspecific luminescence interference.

Lung samples were fixed in 4% paraformaldehyde, paraffin-embedded, and sliced into 4 µm sections. After deparaffinization and rehydration, the slices were incubated with an endogenous peroxidase blocking solution (Beyotime, Shanghai, China) for 10 min to quench peroxidase activity. Antigen retrieval was carried out in Tris-EDTA buffer (Beyotime) at 100°C for 10 minutes. After PBS rinsing, samples were blocked with blocking buffer at 37°C for 30 minutes, then incubated with primary antibodies against C/EBPγ at room temperature. Following additional PBS washes, biotin-conjugated secondary antibodies (Beyotime) were incubated at 37°C for 30 minutes. Staining was developed using a DAB substrate, followed by hematoxylin counterstaining. Images were obtained with a light microscope.

For immunocytochemistry, cells were seeded onto poly-L-lysine–coated coverslips, fixed with 4% paraformaldehyde for 30 min, and subsequently permeabilized using 0.03% Triton X-100 for 5 min. After three PBS rinses, they were blocked with 5% goat serum for 30 min and incubated with primary antibodies targeting β-catenin, HA, or C/EBPα overnight at 4°C. The following day, cells were exposed to Alexa Fluor 488– (green) or Alexa Fluor 594– (red) conjugated secondary antibodies at 37°C for 1 h. Nuclei were stained with DAPI, and fluorescence images were captured using a fluorescence or confocal microscope (LSM 700, Zeiss, Jena, Germany).

IPF constitutes a progressive interstitial lung disease of uncertain etiology, characterized by declining respiratory capacity and poor clinical outcomes [25]. The primary pathological characteristic of IPF is the abnormal activation of fibroblasts and the excessive accumulation of extracellular matrix (ECM) in the lung tissue [1, 26]. While antifibrotic agents like pirfenidone and nintedanib modestly delay disease progression, their therapeutic impact is limited, with median survival still confined to 3–5 years. Thus, new mechanisms and targeted therapies are urgently needed [27]. In recent years, the role of transcription factors in regulating the pathogenesis of IPF has garnered increasing attention [28–31]. This study found that C/EBPγ was significantly upregulated in both IPF patient lung tissues and in a mouse model of bleomycin-induced fibrosis. Functional experiments revealed that C/EBPγ knockdown suppressed the fibrotic phenotype, while its overexpression promoted EMT, fibroblast activation, and ECM deposition. Mechanistic investigations showed that C/EBPγ exerts a pro-fibrotic effect by downregulating AXIN1, thereby stabilizing β-catenin, promoting its nuclear translocation, and activating downstream signaling pathways. Additionally, C/EBPγ forms a heterodimer with C/EBPα, which reduces AXIN1 transcriptional activity and regulates the AXIN1–β-catenin axis. In the AAV-mediated mouse model, overexpression of C/EBPγ exacerbated pulmonary fibrosis, while its knockdown alleviated the disease. These results not only underscore the pro-fibrotic role of C/EBPγ in IPF but also shed light on the the regulatory network of transcription factor families.

Identified in 2003 as a major profibrotic signaling cascade in IPF, Wnt/β-catenin signaling serves as a pivotal mechanism underlying the progression of pulmonary fibrosis. Chilosi et al. first reported the abnormal nuclear accumulation of β-catenin in the lung tissues of IPF patients, demonstrating that the activation of this pathway is linked to the abnormal proliferation of lung epithelial cells and fibrosis [13]. Subsequent studies have confirmed that Wnt/β-catenin signaling is functionally upregulated in lung fibroblasts from IPF patients, promoting their proliferation and ECM deposition [32]. In experimental models, suppression of the β-catenin pathway markedly attenuated bleomycin-induced pulmonary fibrosis in mice, as evidenced by reduced inflammation and ECM accumulation [14, 33]. The Wnt co-receptor LRP5 has been identified as a key driver of IPF, and its deficiency has been shown to alleviate fibrosis in mice [34]. At the mechanistic level, β-catenin acts in synergy with TGF-β signaling, which promotes β-catenin activation and drives the differentiation of fibroblasts into myofibroblasts [35–38]. Moreover, downregulation of Wnt inhibitors such as DKK stabilizes β-catenin, thereby amplifying the fibrotic response [12, 39]. Additional signaling routes, such as Hedgehog, converge with Wnt/β-catenin to drive the mesenchymal stem cells differentiation into myofibroblasts [40, 41]. Collectively, these findings underscore that β-catenin acts not only as an “executor” of fibrosis but also as a key node integrating multiple signaling pathways. Existing studies agree that β-catenin remains continuously activated in the lungs of IPF patients, rather than being a transient stress response. Therefore, investigating the upstream factors or regulatory mechanisms of this pathway is crucial. In our study, we identified and characterized the transcription factor C/EBPγ as a key regulator of β-catenin stability. Specifically, C/EBPγ enhances β-catenin stability by downregulating AXIN1, thereby promoting its nuclear translocation and activates downstream fibrotic signaling. This mechanism aligns with the classical regulatory pattern of the Wnt/β-catenin pathway and also uncovers a potential new intervention point for the transcription factor family within this signaling axis. Notably, AXIN1, in addition to being a component of the β-catenin degradation complex, contains its own NES sequence, which functions as a molecular chaperone to promote the nuclear export of β-catenin [42, 43]. Thus, C/EBPγ-mediated downregulation of AXIN1 not only impairs the function of the degradation complex but also restricts the nuclear export of β-catenin, further enhancing its nuclear accumulation. This is consistent with the observation of β-catenin localization in the nucleus following C/EBPγ overexpression, as seen in immunofluorescence experiments. This mechanism supports the functional loop of the C/EBPγ-AXIN1-β-catenin axis at the cellular level, providing additional evidence for the enhanced β-catenin signaling and its fibrotic effects. This discovery not only broadens the regulatory network of Wnt/β-catenin in IPF but also offers a new perspective for understanding its pathogenesis and developing more targeted therapeutic strategies.

Members of the C/EBP family have been identified as critical regulators in fibrosis across different organs. For instance, in liver fibrosis, they regulate the expression of inflammatory factors and activation of hepatic stellate cells [44, 45]; in renal fibrosis, they influence EMT, FMT and ECM deposition; and in cardiac fibrosis, they are involved in cardiomyocyte reprogramming and collagen synthesis. Current research indicates that the involvement of this family in IPF is diverse and context dependent. Aside from C/EBPγ, other members have been widely studied: for example, C/EBPα primarily exerts a protective role in epithelial cells by inhibiting TGF-β signaling and upregulating cell cycle-related genes, thereby reducing EMT and fibroblast activation [46, 47]; in fibroblasts and macrophages, C/EBPβ activates the NF-κB pathway, promoting the expression of pro-inflammatory factors (such as IL-6, TNF-α) and ECM synthesis, thus exacerbating the fibrotic process [48, 49]; C/EBPδ plays a region- and cell type–specific dual role in pulmonary fibrosis—promoting fibrosis in alveolar epithelial cells and macrophages by inducing IL-6 and MCP-1 [50, 51], while suppressing it in Clara cells through upregulation of the protective proteins CCSP and SCGB3A2 [52–55]; C/EBPζ (CHOP) serves as an essential regulator of endoplasmic reticulum stress, driving type II alveolar epithelial cell apoptosis via an IRE1α/PERK-dependent pathway; its loss markedly alleviates fibrosis induced by hypoxia or repeated epithelial injury [56, 57]. These results underscore the multifaceted functions of the C/EBP family in various cell types involved in IPF—such as lung epithelial cells, fibroblasts, and immune cells—and their participation in multiple signaling pathways, including TGF-β, NF-κB, and HIF-1α. These insights are crucial for understanding the regulatory network of fibrosis. In our study, we found that elevated expression of C/EBPγ stimulated β-catenin signaling, which in turn promoted fibrotic progression. While some existing studies support the protective role of C/EBPγ in inflammation and aging, our experimental results offer a novel insight into the role of C/EBPγ in fibrosis. In this study, we employed several methods to investigate the impact of C/EBPγ on the β-catenin pathway. We observed that overexpression of C/EBPγ in both A549 and MRC-5 cells resulted in increased β-catenin protein levels and significant nuclear translocation, thereby verifying the β-catenin activation. The use of agonists and inhibitors further validated the dependence of this effect on the β-catenin pathway. Additionally, the regulation of AXIN1 by C/EBPα and the interaction between C/EBPγ and C/EBPα strengthened this evidence. We believe that these findings do not contradict existing studies but rather enhance our understanding of the diverse roles of C/EBP family members. For instance, C/EBPβ, a member of this family, is predominantly associated with pro-inflammatory and pro-fibrotic effects [48, 49], although some studies have also shown its inhibitory role in the β-catenin pathway [58]. Transcription factors typically regulate multiple target genes, forming cell type–dependent multidimensional regulatory networks. These networks can modulate both protective and pathogenic pathways under various physiological and pathological conditions, suggesting that their functions may be bidirectional. C/EBPγ’s structure and function distinguish it from other transcription factors in terms of its mode of transcriptional regulation. It interacts with other family members to fine-tune their transcriptional efficiency [17, 59]. In addition, C/EBPγ can interact with other bZIP-containing transcription factors, such as ATF4 [19]. This broadens the scope of C/EBPγ's role, potentially altering gene expression governed by both family-related and external transcription factors. Therefore, investigating C/EBPγ's diverse functions under various conditions not only broadens our understanding of the C/EBP family, but also deepens our knowledge of disease mechanisms and may help in developing more targeted therapeutic strategies.

Despite systematically demonstrating the pro-fibrotic role of C/EBPγ in IPF and its underlying mechanisms in both in vitro and in vivo models, several limitations should be noted. First, the study largely relied on the bleomycin-induced murine model and classical in vitro systems such as A549 and MRC-5 cells. While these models are widely used and experimentally tractable, they do not fully capture the heterogeneity, chronic progression, and multifactorial interactions characteristic of human IPF, including patient-specific variability. Second, our data reveal the C/EBPγ–C/EBPα heterodimer as a critical regulator of the AXIN1–β-catenin axis and underscore the central role of β-catenin signaling in C/EBPγ-mediated profibrotic effects. However, the broader effects of disrupting this heterodimer on pathways beyond β-catenin warrant further investigation, as well as its potential interactions with other family members (such as C/EBPδ) or additional coregulatory factors, including epigenetic modifiers. This inherent complexity may constrain a comprehensive understanding of the dynamic regulatory networks orchestrated by the C/EBP family. Third, although AAV-mediated in vivo modulation of C/EBPγ confirmed its functional impact, questions regarding long-term safety, dose optimization, and potential off-target effects remain unresolved. These limitations highlight important directions for future research, including validation in patient cohorts and integrative multi-omics profiling, which will be crucial for advancing our comprehension of the transcriptional regulatory landscape in IPF. Moreover, the development of strategies targeting specific heterodimer interfaces within defined pathological stages or cellular subpopulations may provide a promising avenue, aligning closely with the core principle of precision medicine: targeted interventions with maximal specificity.

This study uncovers a pro-fibrotic role of C/EBPγ in Bleomycin-induced pulmonary fibrosis: C/EBPγ forms a nonfunctional heterodimer with C/EBPα that suppresses AXIN1, leading to activation of the β-catenin pathway and promoting EMT, fibroblast activation, and ECM deposition. These findings not only contribute significantly to the understanding of the C/EBP family in fibrotic networks but also provide new insights into IPF pathogenesis. Looking forward, integrative multi-omics validation and strategies targeting specific dimerization interfaces may enable the development of novel precision therapies and accelerate translational progress in IPF management.