CREPT is required for pulmonary fibrosis induced by bleomycin

CREPT is required for pulmonary fibrosis induced by bleomycin | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article CREPT is required for pulmonary fibrosis induced by bleomycin Jiayu Wang, Jian Sheng, Sihan Liu, Jianghua Li, Jun Chu, Minghan Wang, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4805438/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Pulmonary fibrosis is a chronic and progressive disease that originates from interstitial lung diseases and ultimately exhibits respiratory failure in patients. The disease is characterized by focal accumulation and excessive production of extracellular matrix (ECM) from over-activated fibroblasts in the lung. Although many extrinsic factors have been identified to boost fibroblast proliferation and activation, it remains unclear how fibrosis is regulated by intrinsic factors. Methods Pulmonary fibrosis mouse model was induced by intratracheal injection of bleomycin (BLM) into CREPT WT and CREPT KO mice. In vitro study, the proliferation of mouse lung fibroblasts (MLFs) was assessed using CCK-8 assays and expression of fibrotic protein was examined following transforming growth factor (TGF)-β stimulation in MLFs. Results In this study, we found that deletion of CREPT alleviated BLM induced pulmonary fibrosis. Deletion of CREPT resulted in attenuated murine lung fibroblast proliferation, TGF-β-induced fibroblast-to-myofibroblast activation, and ECM deposition. Consistently, deletion of CREPT decreased the expression of fibrotic marker genes such as a-SMA , Col1a1 , and FN1 but had no influence on the inflammation response upon the BLM challenge. Conclusions In summary, we report that CREPT is required for BLM induced pulmonary fibrosis in mice. Our study unravels an intrinsic molecular mechanism for the development of pulmonary fibrosis and provides a new target for the therapy of the interstitial lung disease. Pulmonary fibrosis fibroblast-to myofibroblast activation CREPT Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Background Interstitial lung disease (ILD) comprises many chronic lung diseases characterized by varying degrees of inflammation and fibrosis [ 1 ]. The interstitial disorder of the lung likely develops from a multifaceted interaction of genetic and environmental risk factors, aging-related events, and epigenetic profibrotic reprogramming. However, the mechanism of the progression of fibrosis remains poorly understood [ 2 ]. Pulmonary fibrosis is a clinical phenotype that reflects the end stage of chronic interstitial lung diseases characterized by progressive accumulation of extracellular matrix (ECM) in the peripheral lung, accompanied by destruction of functional alveolar gas exchange units. The most severe form of pulmonary fibrosis is idiopathic pulmonary fibrosis, a relentlessly progressive disorder that causes respiratory failure and death eventually [ 3 ]. The lung contains more than 40 different cell types [ 4 ], despite the diverse cellular composition, most of the increased ECM deposited in idiopathic pulmonary fibrosis is ascribed to the activated myofibroblasts in fibroblast foci [ 5 ]. The ECM accumulation leads to lesions that do not arise in healthy lungs but are a common feature of idiopathic pulmonary fibrosis. In clinics, the lesion numbers correlate with the survival of the patients. Many studies revealed that the lung lesions, in particular the epithelial cell injury, initiates the inflammation responses, which are categorized into different phases. During the initial injury phase, alveolar epithelial cells are activated and recruit inflammatory cells such as macrophages [ 6 ] and neutrophils [ 7 ]. Both the activated epithelial cells and immune cells release potent fibrogenic growth factors, including TGF-β, PDFG, IL-6, TNF-α, and Wnt [ 8 ], to exacerbate the pathologic process. In the next phase, these growth factors, particularly TGF-β, induce apoptosis of alveolar epithelial cells and boost activation, invasion, or apoptosis resistance of fibroblasts and myofibroblasts [ 9 ]. With the progress of the disease into the third phase, fibroblast foci, which form three-dimensional networks in lungs with usual interstitial pneumonia [ 10 ], re-dominantly occur in subepithelial regions of apoptotic or hyperplastic alveolar epithelial cells. In summary, fibrosis is characterized by an initial injury and development of subsequent remodeling of the alveolar epithelial-mesenchymal unit, which produces elevated numbers of myofibroblasts. In the past decade, substantial progress has been made in identification of potential cellular causes of the increased myofibroblasts that occur specifically in fibroblast foci in idiopathic pulmonary fibrosis [ 11 ]. To date, several factors have been reported to be involved in the initiation and progression of pulmonary fibrosis. However, these inducers are mainly extrinsic factors such as virus infection (COVID19 [ 12 ]), aging and environmental factors, including smoking, dust inhalation, and asbestos exposure [ 13 ]. Under the challenge of these inducers, the alveolar epithelial cells are subjected to repeated micro-injury that leads to an aberrant repair response and abnormal secretion of inflammatory cytokines. These cytokines promote activation and expansion of fibroblasts/myofibroblasts, which lead to excessive ECM production and deposition [ 14 ]. As a consequence, fibroblastic foci form, with a feature of accumulated fibroblasts and myofibroblasts. To mimic this pathological process, bleomycin (BLM) injury has been widely used in the mouse model to represent all the hallmarks of human lung fibrosis. BLM induces lung damage and a transient fibrotic response with increased numbers of α-SMA + myofibroblasts in the alveolar region [ 15 ]. The BLM induced lung injury progresses through acute injury and inflammatory phase (1–7 days post- endotracheal injection of BLM), transition phase from inflammation to active fibrosis (7–14 days post of BLM), and chronic fibrosis phase (14–21 days post of BLM) [ 16 ]. During the chronic fibrosis phase, the myofibroblast population is expanded and the deposition of ECM, such as collagens, is increased [ 17 ]. The increased deposition of lung collagen usually peaks at about 28 days post-BLM treatment [ 18 ]. Overall, BLM-induced injury boosts pathological alterations of the pulmonary fibrosis with inflammation response and activation of myofibroblasts. CREPT, also named RPRD1B, was identified as a tumor-related protein due to its upregulation in tumors [ 19 ]. Previous studies demonstrated that deletion of CREPT impeded tumorigenesis but overexpression of CREPT promoted tumor formation. CREPT was demonstrated to upregulate cyclin D1 and cyclin B1 expression to accelerate cell cycle at both G1 and G2 phases[ 19 , 20 ]. Recently, we reported that CREPT is required for the maintenance of murine intestinal stem cells as deletion of CREPT in the intestinal epithelium of mice (Vil-CREPT KO ) resulted in lower body weight and slow migration of epithelial cells in the intestine [ 21 ]. We have provided evidence that CREPT participates in the regulation of intestine regeneration after irradiation and chemical-induced damage. On the other hand, we demonstrated that CREPT enhanced STAT3 transcriptional activity[ 22 ], a major regulator related to cell proliferation and inflammation responses[ 23 ]. Here, we report that CREPT, as an intrinsic factor, is required for BLM-induced pulmonary fibrosis. Deletion of CREPT attenuates BLM-induced lung fibrosis and reduces the expression of genes encoding fibrosis markers (e.g., α-SMA, FN1, COL1A1). We show that CREPT enhances fibroblast activation, proliferation, and excessive ECM production induced by TGF-β. Material and Methods Mice All mice used in this study were on a C57BL/6 background. To generate inducible systemic CREPT knockout mice, ERT2-Cre mice with mice homozygous for a floxed allele of CREPT (CREPT fl/fl ). ERT2-Cre +/− CREPT fl/fl mice were then maintained by breeding to CREPT fl/fl mice. ERT2-Cre +/− CREPT fl/fl mice were treated with intraperitoneal injections of 100 mg/kg of Tamoxifen (Sigma) (diluted in 100 µL of sunflower seed oil with 10% ethanol) or an equal volume of oil once a day for five consecutive days, then wait for a week to get CREPT-knockout mice or wild-type mice. All mice, including WT, were bred and maintained in a specific pathogen-free animal facility at Tsinghua University. The mice were maintained on a 12/12-h light/ dark cycle at 22 to 26°C with sterile pellet food and water ad libitum. All animal procedures were under the approval of the Institutional Animal Care and Use Committee at Tsinghua University. Model of BLM induced pulmonary fibrosis (BIPF) Age-matched male mice between 6 and 8 weeks of age (22 to 25 g) were used for induction of BIPF. Mice were divided randomly into two groups: (A) saline-only (PBS), or (B) BLM (Bleo). In brief, mice were anesthetized with 2.5% avertin (100 uL/10g) ( Sigma-Aldrich T48402 ) via intraperitoneal injection. Lung injury and pulmonary fibrosis were induced by single-dose administration of BLM hydrochloride (Sigma Aldrich, Germany), which was dissolved in sterile PBS and given at 2.5 mg/kg (intratracheal instillation) bodyweight. The control group was treated with sterile PBS only. Mice were sacrificed at designated time points (days 7 and 14), after instillation. Treated animals were continuously under strict observation with respect to phenotypic changes, abnormal behavior and signs of body weight loss. Antibodies The anti-beta Tubulin antibody (AT0003, Engibody, China) was from Sigma. beta Actin (A5441, Thermo Fisher, USA). Antibody against alpha smooth muscle Actin (ab5694) were purchase from abcam. An anti-CREPT antibody (3E10) was raised in our lab. Immunofluorescence staining The cells were fixed using 4% PFA after washing with 1× PBS. The cells were permeabilized using 0.3% Triton X-100 followed by incubation in 1% bovine serum albumin to avoid non‐specific binding of antibodies. The cells were then incubated with primary (α-SMA with a final concentration of 1:100, CREPT with a final concentration of1:50) and secondary (with a final concentration of 1:100; goat anti‐mouse TRITC (ZF-0313, ZSGB-Bio, China), and goat anti-rabbit FITC (ZF-0311, ZSGB-Bio, China) antibodies followed by PBS washing for three times at intervals of 5 min each. The cells were then incubated with 0.2 µg/mL DAPI (4′,6‐diamidino‐2‐phenylindole, dilactate), a nucleic acid stain, for 10 min. The slides were imaged using a confocal microscope (FV3000, Olympus, Japan). Isolation of primary murine lung fibroblast Primary murine lung fibroblasts were isolated from mice and purified using a modification of published methods[ 24 ]. Briefly, mouse lungs were cut into small pieces, minced, and digested enzymatically by DNase I in DMEM with 5% FBS for 90 min. After filtration cells were centrifuged, washed, and cultured in 6-cm dishes in DMEM medium containing 10% FBS. Bronchoalveolar lavage. CREPT WT and CREPT KO mice were subjected to bronchoalveolar lavage thrice with 0.5 ml of 0.9% NaCl. The BALF samples were collected and centrifuged at 500 g for 5 min, and the supernatant of the lavage fluid was used to measure total protein concentration using BCA Protein Assay Kit (Thermo Fisher Scientific, MA, USA). More than three hundred cells were counted in BLM-treated group and about one hundred cells were counted in sham group under 400-fold magnification, and the absolute number of each cell type were calculated. Cell proliferation assay Cell proliferative ability was evaluated by Cell Count Kit-8 (CCK-8) assay. For CCK-8 assay, cells were plated onto a 96-well plate (Corning, USA), at a density of 1 × 10 3 cells/well and cultured for the indicated time. 10 µL of CCK-8 reagent (Dojindo Molecular Technologies, Japan) and 100 µL of medium were added to the plates, which were then incubated with 5% CO 2 at 37°C for 2 h. After the incubation, the optical density (OD450) value was assessed by a microplate reader (BioTek, USA) for the evaluation of cell proliferation ability. Statistical analysis The mean ± standard deviation was used to present the data. Independent sample t-test was employed to compare differences between two groups, while one-way analysis of variance (ANOVA) followed by Tukey’s test was utilized for comparisons among multiple groups. Statistical significance of group effects was determined at P < 0.05 level. Results Deletion of CREPT attenuates BLM-induced lung fibrosis To evaluate the role of CREPT in the pulmonary fibrotic process, we crossed the CREPT fl/fl mouse with the ERT2-Cre +/− mouse to generate CREPT deletion mice controlled by tamoxifen induction (Fig. 1A). A western blot showed no CREPT expression in lung tissues of ERT2-Cre +/− ,CREPT fl/fl mice after the injection of tamoxifen for 5 days, indicating that CREPT was effectively deleted (Fig. 1B). Then, we challenged the mice with BLM via intra-tracheal instillation to build up a mouse model of lung fibrosis (Fig. 1C). CREPT WT (ERT2-Cre +/− ) mice were used as a control to compare with CREPT KO (ERT2-Cre +/− ,CREPT fl/fl ) mice under BLM challenge (BLM) or PBS treatment. All the mice were sacrificed and lung tissues were harvested 14 days after BLM administration (Fig. 1C). We determined to examine the lung coefficient (Lung [g]/body weight [kg]), which reflects the lung injury severity [ 25 ]. The result showed that the lung coefficient was increased in CREPT WT mice by BLM, suggesting that serious lung injury occurred under the BLM challenge (Fig. 1D). However, we observed that the increase of the lung coefficient was retarded in CREPT KO mice (Fig. 1D, right columns). Both H&E and Masson’s trichrome staining experiments showed that BLM led to extensive lung fibrosis severely in CREPT WT mice but mildly in CREPT KO mice at 14 days, suggesting that deletion of CREPT preserves the lung structure and attenuates the fibrotic changes (Fig. 1E). These results suggested that global deletion of CREPT markedly reduced the pulmonary fibrosis derived from the BLM treatment. Deletion of CREPT reduces fibrogenic gene expression during BLM-induced pulmonary fibrosis To reveal the role of CREPT on the gene expression during fibrosis, we determined to examine the level of α-smooth muscle actin (α-SMA), a marker of activated myofibroblasts in the alveolar region during pulmonary fibrosis [ 26 ]. A Western blot showed that the level of α-SMA was dramatically elevated upon BLM challenge in the lungs of wild type mice but remained no significant change in the lungs of CREPT KO mice (Fig. 2A). Of note, the levels of α-SMA were markedly lower in CREPT KO mice compared to CREPT WT mice in response to BLM challenge (Fig. 2A, WT vs KO). Furthermore, we examined the mRNA levels of α-SMA and other marker genes including Col1a1 and FN1 , which feature for lung myofibroblast differentiation. The results showed that deletion of CREPT significantly repressed the expression of a-SMA , Col1a1 , and FN1 under BLM challenge compared with control mice. (Fig. 2B). In addition, we performed an immunohistochemical (IHC) staining experiment to demonstrate the levels of α-SMA and COL1A1 in the lung. The result showed that α-SMA + myofibroblasts dominated in the lung, forming significant fibrotic foci with disappeared alveolar structures but thickened alveolar walls in the wild type mouse under the BLM challenge (Fig. 2C, upper panels). Notably, we observed that under normal conditions, the lung from CREPT KO mice remained in typical structures similar to the lung from the wild type mice, although the α-SMA + myofibroblasts appeared to be stronger in the BLM-challenged lungs than the control lungs in the CREPT KO mice (Fig. 2C, compare right two upper panels). We observed similar alterations of COL1A1 as well. (Fig. 2C, bottom panels). Taken together, all the results suggest that deletion of CREPT reduces the occurrence of pulmonary fibrosis by repressing the activation of myofibroblasts. CREPT deletion affects no inflammatory response to BLM. As the BLM challenge induces damage to epithelial cells, which boosts inflammatory responses to further activate myofibroblasts for mediating pulmonary fibrosis, we questioned whether CREPT deletion influences the pulmonary inflammation. To this end, we examined the inflammation alterations in the lungs of CREPT WT and CREPT KO mice after BLM challenge for 7 days (Fig. 3A). We collected the bronchoalveolar lavage fluid (BALF) and measured the total protein concentration, which represents the alveolar infiltration of inflammatory cells including macrophages, neutrophils, and lymphocytes [ 27 ]. The result showed the total protein concentrations in the BALF were significantly increased in both CREPT WT and CREPT KO mice after BLM challenge (Fig. 3B, BLM vs PBS, P = 0.0004). However, we observed no difference in the protein concentrations between CREPT WT and CREPT KO mice (Fig. 3B, WT vs. KO, right columns). This result suggests that the deletion of CREPT has no effect on the overall inflammation response during BLM challenge. In the meanwhile, we examined the levels of inflammatory cytokines including TNF-α, IL-6, and IL-1β in the BALF. Interestingly, we observed that the level of TNF-α was dramatically increased upon BLM challenge (Fig. 3C, BLM vs PBS, P = 0.0045). However, we observed no significant differences of TNF-α (Fig. 3C, WT vs KO), IL-6 (Fig. 3D) and IL-1β (Fig. 3E) in the BALFs between CREPT WT and CREPT KO mice with or without BLM challenge. These results suggest that deletion of CREPT has no effect on the development of pulmonary inflammation in response to BLM treatment. CREPT promotes the proliferation and activation of murine lung fibroblasts We next addressed how deletion of CREPT reduced pulmonary fibrosis. Our aforementioned results indicated that deletion of CREPT reduced the activation of myofibroblasts. We reasoned that CREPT might directly regulate myofibroblast proliferation from alveolar fibroblasts. Since these cells in CREPT WT and CREPT KO mice received similar inflammation stimulation after BLM challenge, we speculated that CREPT might function as an intrinsic factor to regulate myofibroblast proliferation. This echoes our previous studies in cancer cells where CREPT was found to promote cancer cell proliferation via accelerating cell cycle progression [ 19 ]. To examine this hypothesis, we isolated the mouse lung fibroblasts (MLFs) from the CREPT KO and CREPT WT mice (Fig. 4A). A Western blot analysis showed that CREPT was completely deleted in the MLFs after the addition of tamoxifen in CREPT KO mice (Fig. 4B). Then we performed a cell proliferation experiment using MLFs. The result showed that MLFs from the CREPT KO mice grew with a decreased rate compared with those from wild type mice (Fig. 4C, red vs blue curves, P = 0.0004). This result suggests that CREPT is critical for the proliferation of MLFs in the in vitro culture condition. To determine if CREPT regulates the differentiation of lung fibroblasts into myofibroblasts in response to cytokine stimulation, we examined the level of αSMA in the MLFs. In an assay using TGF-β stimulation, which was widely used as a cytokine to activate fibroblasts[ 28 ], we observed that CREPT deletion rendered the response of αSMA in the MLFs from CREPT KO mice in comparison with that from CREPT WT mice (Fig. 4D). These results suggest that deletion of CREPT reduced the expression of α-SMA in the MLFs, implying that CREPT regulates the differentiation of fibroblasts into myofibroblasts. Consistent with the Western blot analyses, an immunostaining experiment further demonstrated that α-SMA + cells were reduced when CREPT was deleted under the in vitro cultural condition (Fig. 4E). All these results collectively suggest that CREPT is required for lung fibroblast activation upon inflammatory cytokine stimulations. Discussion Pulmonary fibrosis is a major public health problem with limited therapeutic options. Treatment options are restricted to two clinically approved drugs, Nintedanib (Ofev)[ 29 ] and pirfenidone (Esbriet)[ 30 ], both of which slow progression but do not halt or reverse the fibrosis. Many studies have been carried out to understand the pathogenesis of pulmonary fibrosis. Most studies focused on the factors in the regulation of fibroblast differentiation into myofibroblasts under inflammatory challenges. In this study, we determined to identify intrinsic factors that control the activation of the fibroblasts during fibrotic responses. We observed that CREPT deficiency mice developed mildly pulmonary fibrosis upon BLM challenge. Using different assays, we demonstrated that deletion of CREPT indeed decreased the activation of fibroblasts, while overexpression of CREPT promoted the proliferation of fibroblasts (data not shown). In particular, we observed that a-SMA, an important marker for fibroblast activation, as well as other markers such as Col1a1 and FN1, was dramatically decreased when CREPT was deleted both in vivo and in vitro . Therefore, we speculate that CREPT plays an important role during the pulmonary fibrosis (Fig. 5). Pulmonary fibrosis is often observed in the lung biopsy samples from patients with usual interstitial pneumonia resulting from chronic hypersensitivity pneumonitis, suggesting an inflammatory process preceding the development of fibrosis [ 31 ]. Many studies suggest that various inflammatory responses may lead to a pro-fibrotic environment and cytokine milieu (including TGF-β[ 32 ], IL-6, TNF-α, PDGF[ 33 ], and WNT[ 34 ]). Shared downstream pathways may activate and sustain a complex interplay leading to fibroblast activation and differentiation into myofibroblasts, further orchestrate fibrogenesis [ 35 ]. However, the clinical trials using anti-inflammatory agents failed to improve outcome of idiopathic pulmonary fibrosis patients [ 36 ]. These clinical results suggest that the activation of fibroblasts could be maintained during the chronic inflammation. In this study, we revealed that CREPT contributes to the activation of the fibroblasts under BLM-induced chronic inflammation. Interestingly, we observed that deletion of CREPT had no influence on the chronic inflammation per se, as the cytokine production (TNF-α, IL-6, and IL-1β) in CREPT KO mice were maintained at the similar levels to the wildtype mice. In this context, we reasoned that CREPT may help fibroblast proliferate or activate without affecting the inflammation initiation from epithelial cells and inflammation exacerbation from immune cells such as macrophages and neutrophils. Indeed, in our IHC experiments, we observed no alteration of CREPT expression in epithelium in the wildtype mice under BLM challenge (data not shown), although the mice produced significantly high amounts of cytokines. The unchanged expression of CREPT in the epithelial cells during the BLM challenge might explain the reason why the inflammation responses were not altered upon CREPT deletion. Our results suggest that CREPT regulates the activation of fibroblasts as deleting CREPT repressed fibroblast proliferation and differentiation. However, we observed no elevated expression of CREPT in the fibroblasts under inflammation challenges (data not shown). Considering the significant phenotype from the deletion experiments, we speculate that the minimal amount of endogenous CREPT is enough to maintain the fibroblast activation. Since elevated CREPT was frequently observed in varieties of tumors, we considered that the basal CREPT is maintained in fibroblasts to avoid their over-proliferation. Nevertheless, the role of CREPT on fibroblast activation without affecting inflammation is particularly of interest for the development of drugs against fibrosis. In this study, we found that deletion of CREPT attenuated the expression of fibrotic genes, including α-SMA , Col1a1 , and FN . We have employed qRCR, Western blot, and IHC experiments to consistently demonstrate the levels of these fibrotic markers. All these results suggest that CREPT participates in the regulation of these gene expression during the activation of fibroblasts. However, we did not elucidate how CREPT regulates the gene expression under inflammation. Previous studies have demonstrated that CREPT is a positive regulator of transcriptional factors such as STAT3 and β-catenin/TCF4 [ 37 ] and promotes the activity of RNAPII at the regions of promoters and terminators in the CCND1 gene [ 38 ]. In cancer cells, CREPT has been attributed to promoting the proliferation of cells by accelerating the cell cycle progression. In this study, we observed that CREPT functioned in fibroblast proliferation and differentiation stimulated by TGF-β or TNF-α in an in vitro culture condition. We speculate that CREPT may act as a coactivator to facilitate gene expression via binding to the promoter region of fibrogenic genes. As our current results from the whole body CREPT deletion mouse provide evidence for the fibroblast activation during pulmonary fibrosis, a fibroblast-specific conditional mouse line will be a super to demonstrate the role of CREPT in this tough disease. Conclusion In summary, we demonstrate that CREPT plays a crucial role in the pathogenesis of pulmonary fibrosis. CREPT deficiency ameliorates the BLM induced lung fibrosis in mice. These results revealed a critical yet previously unrecognized role of CREPT in the regulation of lung fibrosis. Our study opens up opportunities for drug discovery, precision targets and therapeutic interventions in pulmonary fibrosis. Declarations Author information J.W. and J. S. made equal contributions to this work. All the authors read and approved the submitted manuscript. Author details 1 State Key Laboratory of Membrane Biology, School of Medicine, Tsinghua University, Beijing 100084, China. 2 Department of Surgery, The Second Affiliated Hospital of Jiaxing University, No. 397, Huangcheng North Road, Jiaxing 314000. 3 SXMU-Tsinghua Collaborative Innovation Center for Frontier Medicine, Shanxi Medical University, Taiyuan, Shanxi Province 030001, China. Co-corresponding authors Zhijie Chang, Tsinghua University. Tel: +86 10 62785076, Fax: +86 10 62773624, E-mail: [email protected] . Xiaoguang Wang, The Second Affiliated Hospital of Jiaxing University. Tel: +86 573 82053235, E-mail: [email protected] . Chenxi Cao, The Second Affiliated Hospital of Jiaxing University. Tel: +86 573 82050295, E-mail: [email protected] . Ethics approval and consent to participate All experimental procedures involving animals were conducted in strict accordance with the Basel Declaration and followed the protocol approved by the Institutional Animal Care and Use Committee of Tsinghua University School of Medicine. All animal handling procedures adhered to the guidelines for the Care and Use of Laboratory Animals published by the National Institute of Health. Consent for publication Not applicable. Competing interests The authors declare no competing interests. Funding This project was supported by funding from the National Natural Science Foundation of China (grant. no. 81830092) and the Public Welfare Science and Technology Program of Jiaxing City (grant. no. 2024AZ30004; Jiaxing China). Author Contribution J.W. and J. S. made equal contributions to this work. J.W. , J. S., S.L, J.L., and J.C. collected data and assisted with data analyses. J.W. wrote the original draft. Z.C.,X.W., C.C., Y.W., M.W. and F.R. assisted in preparing the manuscript and critically reviewed it. All the authors read and approved the submitted manuscript. Acknowledgements Not applicable. Data availability No datasets were generated during the current study. References Richeldi, L., H.R. Collard, and M.G. Jones, Idiopathic pulmonary fibrosis. The Lancet, 2017. 389 (10082): p. 1941-1952. Flaherty, K.R., et al., Fibroblastic foci in usual interstitial pneumonia: idiopathic versus collagen vascular disease. Am J Respir Crit Care Med, 2003. 167 (10): p. 1410-5. 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Conte, E., et al., Effect of pirfenidone on proliferation, TGF-β-induced myofibroblast differentiation and fibrogenic activity of primary human lung fibroblasts. Eur J Pharm Sci, 2014. 58 : p. 13-9. Klinkhammer, B.M., J. Floege, and P. Boor, PDGF in organ fibrosis. Mol Aspects Med, 2018. 62 : p. 44-62. Dou, C., et al., P300 Acetyltransferase Mediates Stiffness-Induced Activation of Hepatic Stellate Cells Into Tumor-Promoting Myofibroblasts. Gastroenterology, 2018. 154 (8): p. 2209-2221.e14. Homer, R.J., et al., Modern concepts on the role of inflammation in pulmonary fibrosis. Arch Pathol Lab Med, 2011. 135 (6): p. 780-8. Torrisi, S.E., et al., Evolution and treatment of idiopathic pulmonary fibrosis. Presse Med, 2020. 49 (2): p. 104025. Zhang, Y., et al., CREPT/RPRD1B, a recently identified novel protein highly expressed in tumors, enhances the β-catenin·TCF4 transcriptional activity in response to Wnt signaling. J Biol Chem, 2014. 289 (33): p. 22589-22599. Zhang, Y., et al., CREPT facilitates colorectal cancer growth through inducing Wnt/β-catenin pathway by enhancing p300-mediated β-catenin acetylation. Oncogene, 2018. 37 (26): p. 3485-3500. Additional Declarations No competing interests reported. Supplementary Files SupplementaryfileoriginalBlotimages.pdf Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4805438","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":340206322,"identity":"9f4d2dc1-df6a-427d-b902-20fc26f6d17d","order_by":0,"name":"Jiayu Wang","email":"","orcid":"","institution":"Tsinghua University","correspondingAuthor":false,"prefix":"","firstName":"Jiayu","middleName":"","lastName":"Wang","suffix":""},{"id":340206323,"identity":"66b47ab8-7498-48ab-841f-e184d2d8b306","order_by":1,"name":"Jian Sheng","email":"","orcid":"","institution":"The Second Affiliated Hospital of Jiaxing University","correspondingAuthor":false,"prefix":"","firstName":"Jian","middleName":"","lastName":"Sheng","suffix":""},{"id":340206324,"identity":"491192c3-297c-401f-b911-b0b926d7dd14","order_by":2,"name":"Sihan Liu","email":"","orcid":"","institution":"Tsinghua University","correspondingAuthor":false,"prefix":"","firstName":"Sihan","middleName":"","lastName":"Liu","suffix":""},{"id":340206325,"identity":"03c9c5b1-a3ef-4a3b-a474-e436958d887d","order_by":3,"name":"Jianghua Li","email":"","orcid":"","institution":"Tsinghua University","correspondingAuthor":false,"prefix":"","firstName":"Jianghua","middleName":"","lastName":"Li","suffix":""},{"id":340206326,"identity":"20a8b98a-a8a0-4b0b-a8bc-676e05efa684","order_by":4,"name":"Jun Chu","email":"","orcid":"","institution":"Tsinghua University","correspondingAuthor":false,"prefix":"","firstName":"Jun","middleName":"","lastName":"Chu","suffix":""},{"id":340206327,"identity":"d8a5e505-bc4b-4b05-a58d-bd008882ccae","order_by":5,"name":"Minghan Wang","email":"","orcid":"","institution":"Tsinghua University","correspondingAuthor":false,"prefix":"","firstName":"Minghan","middleName":"","lastName":"Wang","suffix":""},{"id":340206328,"identity":"3414db4e-6e9a-448f-b570-9a0238a2cab9","order_by":6,"name":"Yinyin Wang","email":"","orcid":"","institution":"Tsinghua University","correspondingAuthor":false,"prefix":"","firstName":"Yinyin","middleName":"","lastName":"Wang","suffix":""},{"id":340206329,"identity":"25712f7d-1232-40df-a056-2f7296ec72bf","order_by":7,"name":"Fangli Ren","email":"","orcid":"","institution":"Tsinghua University","correspondingAuthor":false,"prefix":"","firstName":"Fangli","middleName":"","lastName":"Ren","suffix":""},{"id":340206330,"identity":"dfc352c4-891d-4b5b-8647-b3f9b75c3171","order_by":8,"name":"Chenxi Cao","email":"","orcid":"","institution":"The Second Affiliated Hospital of Jiaxing University","correspondingAuthor":false,"prefix":"","firstName":"Chenxi","middleName":"","lastName":"Cao","suffix":""},{"id":340206331,"identity":"f7289bfc-62c1-4c32-be32-fe21cd669fdd","order_by":9,"name":"Xiaoguang Wang","email":"","orcid":"","institution":"The Second Affiliated Hospital of Jiaxing University","correspondingAuthor":false,"prefix":"","firstName":"Xiaoguang","middleName":"","lastName":"Wang","suffix":""},{"id":340206332,"identity":"32337cf6-e524-471a-ade3-556d47ed9ec3","order_by":10,"name":"Zhijie Chang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA/klEQVRIiWNgGAWjYDACCcYGgwQGBjkGBsYGEAIBA6K0GPOQoAVCJfaASKK0yM9ubih4UGGXvp//cNvDrzsOyzOwN2+TYKi5g1ML45yDQIedSc7tkUhsN5Y9c9iwgedYmQTDsWc4tTBLJDYYJLYdAGphbJOWbDvM2CCRYwb04GGcWtjAWv4dSOfhPwjWYt8g/wa/Fh6wloYDCTwMiW2SH9sOJzZI8ODXIgHSknAs2bDnRmKbNGNbenIbT1qxRcIx3FrkZ6Q/M/xRYyfP3n/8meTPNmvbfvbDG298qMGtBeQdeDQw84C4IFYCPg1AhQ9gLMYf+FWOglEwCkbBCAUAmbVUDlGEGXwAAAAASUVORK5CYII=","orcid":"","institution":"Tsinghua University","correspondingAuthor":true,"prefix":"","firstName":"Zhijie","middleName":"","lastName":"Chang","suffix":""}],"badges":[],"createdAt":"2024-07-26 05:14:42","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4805438/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4805438/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":63831216,"identity":"9ebcf73a-20fb-4b43-ac3a-52d1569fc29d","added_by":"auto","created_at":"2024-09-02 19:05:53","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1071641,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"Figuremanuscript1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4805438/v1/87d27a59ba4549ddd7fc6850.jpg"},{"id":63830375,"identity":"d192f5f0-1338-4610-af8a-f1e201d3240d","added_by":"auto","created_at":"2024-09-02 18:57:53","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1200712,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"Figuremanuscript2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4805438/v1/6a1edda827c8fd50a3613233.jpg"},{"id":63830378,"identity":"9fc831d0-d37e-4925-bf3a-9d0c65d81eb0","added_by":"auto","created_at":"2024-09-02 18:57:53","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":517136,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"Figuremanuscript3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4805438/v1/594fc71e1173b69f50002b76.jpg"},{"id":63830374,"identity":"8594cc1a-7878-4d9a-b878-c4d5668bce76","added_by":"auto","created_at":"2024-09-02 18:57:53","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":619111,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"Figuremanuscript4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4805438/v1/f891653c6c382339a8ed4d19.jpg"},{"id":63830377,"identity":"419335b4-4bb5-401b-b446-a5e7987c7142","added_by":"auto","created_at":"2024-09-02 18:57:53","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":408369,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"Figuremanuscript5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4805438/v1/a5d683d29a15546663c7e5bb.jpg"},{"id":86849877,"identity":"6db54629-e35d-4055-b381-46c01323cda2","added_by":"auto","created_at":"2025-07-16 09:32:03","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4649234,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4805438/v1/08e3187c-0255-4728-b6ba-7510e4a2f7b0.pdf"},{"id":63830380,"identity":"d2971b5c-55f9-4372-bc6e-5c47ff15c10f","added_by":"auto","created_at":"2024-09-02 18:57:53","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1466385,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryfileoriginalBlotimages.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4805438/v1/1d3e53311c8ab0e3c65a972b.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"CREPT is required for pulmonary fibrosis induced by bleomycin","fulltext":[{"header":"Background","content":"\u003cp\u003eInterstitial lung disease (ILD) comprises many chronic lung diseases characterized by varying degrees of inflammation and fibrosis [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The interstitial disorder of the lung likely develops from a multifaceted interaction of genetic and environmental risk factors, aging-related events, and epigenetic profibrotic reprogramming. However, the mechanism of the progression of fibrosis remains poorly understood [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Pulmonary fibrosis is a clinical phenotype that reflects the end stage of chronic interstitial lung diseases characterized by progressive accumulation of extracellular matrix (ECM) in the peripheral lung, accompanied by destruction of functional alveolar gas exchange units. The most severe form of pulmonary fibrosis is idiopathic pulmonary fibrosis, a relentlessly progressive disorder that causes respiratory failure and death eventually [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe lung contains more than 40 different cell types [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], despite the diverse cellular composition, most of the increased ECM deposited in idiopathic pulmonary fibrosis is ascribed to the activated myofibroblasts in fibroblast foci [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. The ECM accumulation leads to lesions that do not arise in healthy lungs but are a common feature of idiopathic pulmonary fibrosis. In clinics, the lesion numbers correlate with the survival of the patients. Many studies revealed that the lung lesions, in particular the epithelial cell injury, initiates the inflammation responses, which are categorized into different phases. During the initial injury phase, alveolar epithelial cells are activated and recruit inflammatory cells such as macrophages [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e] and neutrophils [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Both the activated epithelial cells and immune cells release potent fibrogenic growth factors, including TGF-β, PDFG, IL-6, TNF-α, and Wnt [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], to exacerbate the pathologic process. In the next phase, these growth factors, particularly TGF-β, induce apoptosis of alveolar epithelial cells and boost activation, invasion, or apoptosis resistance of fibroblasts and myofibroblasts [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. With the progress of the disease into the third phase, fibroblast foci, which form three-dimensional networks in lungs with usual interstitial pneumonia [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], re-dominantly occur in subepithelial regions of apoptotic or hyperplastic alveolar epithelial cells. In summary, fibrosis is characterized by an initial injury and development of subsequent remodeling of the alveolar epithelial-mesenchymal unit, which produces elevated numbers of myofibroblasts.\u003c/p\u003e \u003cp\u003eIn the past decade, substantial progress has been made in identification of potential cellular causes of the increased myofibroblasts that occur specifically in fibroblast foci in idiopathic pulmonary fibrosis [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. To date, several factors have been reported to be involved in the initiation and progression of pulmonary fibrosis. However, these inducers are mainly extrinsic factors such as virus infection (COVID19 [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]), aging and environmental factors, including smoking, dust inhalation, and asbestos exposure [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Under the challenge of these inducers, the alveolar epithelial cells are subjected to repeated micro-injury that leads to an aberrant repair response and abnormal secretion of inflammatory cytokines. These cytokines promote activation and expansion of fibroblasts/myofibroblasts, which lead to excessive ECM production and deposition [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. As a consequence, fibroblastic foci form, with a feature of accumulated fibroblasts and myofibroblasts. To mimic this pathological process, bleomycin (BLM) injury has been widely used in the mouse model to represent all the hallmarks of human lung fibrosis. BLM induces lung damage and a transient fibrotic response with increased numbers of α-SMA\u003csup\u003e+\u003c/sup\u003e myofibroblasts in the alveolar region [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. The BLM induced lung injury progresses through acute injury and inflammatory phase (1\u0026ndash;7 days post- endotracheal injection of BLM), transition phase from inflammation to active fibrosis (7\u0026ndash;14 days post of BLM), and chronic fibrosis phase (14\u0026ndash;21 days post of BLM) [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. During the chronic fibrosis phase, the myofibroblast population is expanded and the deposition of ECM, such as collagens, is increased [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The increased deposition of lung collagen usually peaks at about 28 days post-BLM treatment [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Overall, BLM-induced injury boosts pathological alterations of the pulmonary fibrosis with inflammation response and activation of myofibroblasts.\u003c/p\u003e \u003cp\u003eCREPT, also named RPRD1B, was identified as a tumor-related protein due to its upregulation in tumors [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Previous studies demonstrated that deletion of CREPT impeded tumorigenesis but overexpression of CREPT promoted tumor formation. CREPT was demonstrated to upregulate cyclin D1 and cyclin B1 expression to accelerate cell cycle at both G1 and G2 phases[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Recently, we reported that CREPT is required for the maintenance of murine intestinal stem cells as deletion of CREPT in the intestinal epithelium of mice (Vil-CREPT\u003csup\u003eKO\u003c/sup\u003e) resulted in lower body weight and slow migration of epithelial cells in the intestine [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. We have provided evidence that CREPT participates in the regulation of intestine regeneration after irradiation and chemical-induced damage. On the other hand, we demonstrated that CREPT enhanced STAT3 transcriptional activity[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], a major regulator related to cell proliferation and inflammation responses[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHere, we report that CREPT, as an intrinsic factor, is required for BLM-induced pulmonary fibrosis. Deletion of CREPT attenuates BLM-induced lung fibrosis and reduces the expression of genes encoding fibrosis markers (e.g., α-SMA, FN1, COL1A1). We show that CREPT enhances fibroblast activation, proliferation, and excessive ECM production induced by TGF-β.\u003c/p\u003e"},{"header":"Material and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMice\u003c/h2\u003e \u003cp\u003eAll mice used in this study were on a C57BL/6 background. To generate inducible systemic CREPT knockout mice, ERT2-Cre mice with mice homozygous for a floxed allele of CREPT (CREPT\u003csup\u003efl/fl\u003c/sup\u003e). ERT2-Cre\u003csup\u003e+/\u0026minus;\u003c/sup\u003eCREPT\u003csup\u003efl/fl\u003c/sup\u003e mice were then maintained by breeding to CREPT\u003csup\u003efl/fl\u003c/sup\u003e mice. ERT2-Cre\u003csup\u003e+/\u0026minus;\u003c/sup\u003eCREPT\u003csup\u003efl/fl\u003c/sup\u003e mice were treated with intraperitoneal injections of 100 mg/kg of Tamoxifen (Sigma) (diluted in 100 \u0026micro;L of sunflower seed oil with 10% ethanol) or an equal volume of oil once a day for five consecutive days, then wait for a week to get CREPT-knockout mice or wild-type mice. All mice, including WT, were bred and maintained in a specific pathogen-free animal facility at Tsinghua University. The mice were maintained on a 12/12-h light/ dark cycle at 22 to 26\u0026deg;C with sterile pellet food and water ad libitum. All animal procedures were under the approval of the Institutional Animal Care and Use Committee at Tsinghua University.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eModel of BLM induced pulmonary fibrosis (BIPF)\u003c/h2\u003e \u003cp\u003eAge-matched male mice between 6 and 8 weeks of age (22 to 25 g) were used for induction of BIPF. Mice were divided randomly into two groups: (A) saline-only (PBS), or (B) BLM (Bleo). In brief, mice were anesthetized with 2.5% avertin (100 uL/10g) \u003cb\u003e(\u003c/b\u003eSigma-Aldrich T48402\u003cb\u003e)\u003c/b\u003e via intraperitoneal injection. Lung injury and pulmonary fibrosis were induced by single-dose administration of BLM hydrochloride (Sigma Aldrich, Germany), which was dissolved in sterile PBS and given at 2.5 mg/kg (intratracheal instillation) bodyweight. The control group was treated with sterile PBS only. Mice were sacrificed at designated time points (days 7 and 14), after instillation. Treated animals were continuously under strict observation with respect to phenotypic changes, abnormal behavior and signs of body weight loss.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eAntibodies\u003c/h2\u003e \u003cp\u003eThe anti-beta Tubulin antibody (AT0003, Engibody, China) was from Sigma. beta Actin (A5441, Thermo Fisher, USA). Antibody against alpha smooth muscle Actin (ab5694) were purchase from abcam. An anti-CREPT antibody (3E10) was raised in our lab.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eImmunofluorescence staining\u003c/h2\u003e \u003cp\u003eThe cells were fixed using 4% PFA after washing with 1\u0026times; PBS. The cells were permeabilized using 0.3% Triton X-100 followed by incubation in 1% bovine serum albumin to avoid non‐specific binding of antibodies. The cells were then incubated with primary (α-SMA with a final concentration of 1:100, CREPT with a final concentration of1:50) and secondary (with a final concentration of 1:100; goat anti‐mouse TRITC (ZF-0313, ZSGB-Bio, China), and goat anti-rabbit FITC (ZF-0311, ZSGB-Bio, China) antibodies followed by PBS washing for three times at intervals of 5 min each. The cells were then incubated with 0.2 \u0026micro;g/mL DAPI (4\u0026prime;,6‐diamidino‐2‐phenylindole, dilactate), a nucleic acid stain, for 10 min. The slides were imaged using a confocal microscope (FV3000, Olympus, Japan).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eIsolation of primary murine lung fibroblast\u003c/h2\u003e \u003cp\u003ePrimary murine lung fibroblasts were isolated from mice and purified using a modification of published methods[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Briefly, mouse lungs were cut into small pieces, minced, and digested enzymatically by DNase I in DMEM with 5% FBS for 90 min. After filtration cells were centrifuged, washed, and cultured in 6-cm dishes in DMEM medium containing 10% FBS.\u003c/p\u003e \u003cp\u003e \u003cb\u003eBronchoalveolar lavage.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eCREPT\u003csup\u003eWT\u003c/sup\u003e and CREPT\u003csup\u003eKO\u003c/sup\u003e mice were subjected to bronchoalveolar lavage thrice with 0.5 ml of 0.9% NaCl. The BALF samples were collected and centrifuged at 500 g for 5 min, and the supernatant of the lavage fluid was used to measure total protein concentration using BCA Protein Assay Kit (Thermo Fisher Scientific, MA, USA). More than three hundred cells were counted in BLM-treated group and about one hundred cells were counted in sham group under 400-fold magnification, and the absolute number of each cell type were calculated.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eCell proliferation assay\u003c/h2\u003e \u003cp\u003eCell proliferative ability was evaluated by Cell Count Kit-8 (CCK-8) assay. For CCK-8 assay, cells were plated onto a 96-well plate (Corning, USA), at a density of 1 \u0026times; 10\u003csup\u003e3\u003c/sup\u003ecells/well and cultured for the indicated time. 10 \u0026micro;L of CCK-8 reagent (Dojindo Molecular Technologies, Japan) and 100 \u0026micro;L of medium were added to the plates, which were then incubated with 5% CO\u003csub\u003e2\u003c/sub\u003e at 37\u0026deg;C for 2 h. After the incubation, the optical density (OD450) value was assessed by a microplate reader (BioTek, USA) for the evaluation of cell proliferation ability.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation was used to present the data. Independent sample t-test was employed to compare differences between two groups, while one-way analysis of variance (ANOVA) followed by Tukey\u0026rsquo;s test was utilized for comparisons among multiple groups. Statistical significance of group effects was determined at P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 level.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eDeletion of CREPT attenuates BLM-induced lung fibrosis\u003c/h2\u003e \u003cp\u003eTo evaluate the role of CREPT in the pulmonary fibrotic process, we crossed the CREPT\u003csup\u003efl/fl\u003c/sup\u003e mouse with the ERT2-Cre\u003csup\u003e+/\u0026minus;\u003c/sup\u003e mouse to generate CREPT deletion mice controlled by tamoxifen induction (Fig.\u0026nbsp;1A). A western blot showed no CREPT expression in lung tissues of ERT2-Cre\u003csup\u003e+/\u0026minus;\u003c/sup\u003e,CREPT\u003csup\u003efl/fl\u003c/sup\u003e mice after the injection of tamoxifen for 5 days, indicating that CREPT was effectively deleted (Fig.\u0026nbsp;1B). Then, we challenged the mice with BLM via intra-tracheal instillation to build up a mouse model of lung fibrosis (Fig.\u0026nbsp;1C). CREPT\u003csup\u003eWT\u003c/sup\u003e (ERT2-Cre\u003csup\u003e+/\u0026minus;\u003c/sup\u003e) mice were used as a control to compare with CREPT\u003csup\u003eKO\u003c/sup\u003e (ERT2-Cre\u003csup\u003e+/\u0026minus;\u003c/sup\u003e,CREPT\u003csup\u003efl/fl\u003c/sup\u003e) mice under BLM challenge (BLM) or PBS treatment. All the mice were sacrificed and lung tissues were harvested 14 days after BLM administration (Fig.\u0026nbsp;1C). We determined to examine the lung coefficient (Lung [g]/body weight [kg]), which reflects the lung injury severity [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. The result showed that the lung coefficient was increased in CREPT\u003csup\u003eWT\u003c/sup\u003e mice by BLM, suggesting that serious lung injury occurred under the BLM challenge (Fig.\u0026nbsp;1D). However, we observed that the increase of the lung coefficient was retarded in CREPT\u003csup\u003eKO\u003c/sup\u003e mice (Fig.\u0026nbsp;1D, right columns). Both H\u0026amp;E and Masson\u0026rsquo;s trichrome staining experiments showed that BLM led to extensive lung fibrosis severely in CREPT\u003csup\u003eWT\u003c/sup\u003e mice but mildly in CREPT\u003csup\u003eKO\u003c/sup\u003e mice at 14 days, suggesting that deletion of CREPT preserves the lung structure and attenuates the fibrotic changes (Fig.\u0026nbsp;1E). These results suggested that global deletion of CREPT markedly reduced the pulmonary fibrosis derived from the BLM treatment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eDeletion of CREPT reduces fibrogenic gene expression during BLM-induced pulmonary fibrosis\u003c/h2\u003e \u003cp\u003eTo reveal the role of CREPT on the gene expression during fibrosis, we determined to examine the level of α-smooth muscle actin (α-SMA), a marker of activated myofibroblasts in the alveolar region during pulmonary fibrosis [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. A Western blot showed that the level of α-SMA was dramatically elevated upon BLM challenge in the lungs of wild type mice but remained no significant change in the lungs of CREPT\u003csup\u003eKO\u003c/sup\u003e mice (Fig.\u0026nbsp;2A). Of note, the levels of α-SMA were markedly lower in CREPT\u003csup\u003eKO\u003c/sup\u003e mice compared to CREPT\u003csup\u003eWT\u003c/sup\u003e mice in response to BLM challenge (Fig.\u0026nbsp;2A, WT vs KO). Furthermore, we examined the mRNA levels of \u003cem\u003eα-SMA\u003c/em\u003e and other marker genes including \u003cem\u003eCol1a1\u003c/em\u003e and \u003cem\u003eFN1\u003c/em\u003e, which feature for lung myofibroblast differentiation. The results showed that deletion of CREPT significantly repressed the expression of \u003cem\u003ea-SMA\u003c/em\u003e, \u003cem\u003eCol1a1\u003c/em\u003e, and \u003cem\u003eFN1\u003c/em\u003e under BLM challenge compared with control mice. (Fig.\u0026nbsp;2B). In addition, we performed an immunohistochemical (IHC) staining experiment to demonstrate the levels of α-SMA and COL1A1 in the lung. The result showed that α-SMA\u003csup\u003e+\u003c/sup\u003e myofibroblasts dominated in the lung, forming significant fibrotic foci with disappeared alveolar structures but thickened alveolar walls in the wild type mouse under the BLM challenge (Fig.\u0026nbsp;2C, upper panels). Notably, we observed that under normal conditions, the lung from CREPT\u003csup\u003eKO\u003c/sup\u003e mice remained in typical structures similar to the lung from the wild type mice, although the α-SMA\u003csup\u003e+\u003c/sup\u003e myofibroblasts appeared to be stronger in the BLM-challenged lungs than the control lungs in the CREPT\u003csup\u003eKO\u003c/sup\u003e mice (Fig.\u0026nbsp;2C, compare right two upper panels). We observed similar alterations of COL1A1 as well. (Fig.\u0026nbsp;2C, bottom panels). Taken together, all the results suggest that deletion of CREPT reduces the occurrence of pulmonary fibrosis by repressing the activation of myofibroblasts.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCREPT deletion affects no inflammatory response to BLM.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAs the BLM challenge induces damage to epithelial cells, which boosts inflammatory responses to further activate myofibroblasts for mediating pulmonary fibrosis, we questioned whether CREPT deletion influences the pulmonary inflammation. To this end, we examined the inflammation alterations in the lungs of CREPT\u003csup\u003eWT\u003c/sup\u003e and CREPT\u003csup\u003eKO\u003c/sup\u003e mice after BLM challenge for 7 days (Fig.\u0026nbsp;3A). We collected the bronchoalveolar lavage fluid (BALF) and measured the total protein concentration, which represents the alveolar infiltration of inflammatory cells including macrophages, neutrophils, and lymphocytes [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The result showed the total protein concentrations in the BALF were significantly increased in both CREPT\u003csup\u003eWT\u003c/sup\u003e and CREPT\u003csup\u003eKO\u003c/sup\u003e mice after BLM challenge (Fig.\u0026nbsp;3B, BLM vs PBS, P\u0026thinsp;=\u0026thinsp;0.0004). However, we observed no difference in the protein concentrations between CREPT\u003csup\u003eWT\u003c/sup\u003e and CREPT\u003csup\u003eKO\u003c/sup\u003e mice (Fig.\u0026nbsp;3B, WT vs. KO, right columns). This result suggests that the deletion of CREPT has no effect on the overall inflammation response during BLM challenge. In the meanwhile, we examined the levels of inflammatory cytokines including TNF-α, IL-6, and IL-1β in the BALF. Interestingly, we observed that the level of TNF-α was dramatically increased upon BLM challenge (Fig.\u0026nbsp;3C, BLM vs PBS, P\u0026thinsp;=\u0026thinsp;0.0045). However, we observed no significant differences of TNF-α (Fig.\u0026nbsp;3C, WT vs KO), IL-6 (Fig.\u0026nbsp;3D) and IL-1β (Fig.\u0026nbsp;3E) in the BALFs between CREPT\u003csup\u003eWT\u003c/sup\u003e and CREPT\u003csup\u003eKO\u003c/sup\u003e mice with or without BLM challenge. These results suggest that deletion of CREPT has no effect on the development of pulmonary inflammation in response to BLM treatment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eCREPT promotes the proliferation and activation of murine lung fibroblasts\u003c/h2\u003e \u003cp\u003eWe next addressed how deletion of CREPT reduced pulmonary fibrosis. Our aforementioned results indicated that deletion of CREPT reduced the activation of myofibroblasts. We reasoned that CREPT might directly regulate myofibroblast proliferation from alveolar fibroblasts. Since these cells in CREPT\u003csup\u003eWT\u003c/sup\u003e and CREPT\u003csup\u003eKO\u003c/sup\u003e mice received similar inflammation stimulation after BLM challenge, we speculated that CREPT might function as an intrinsic factor to regulate myofibroblast proliferation. This echoes our previous studies in cancer cells where CREPT was found to promote cancer cell proliferation via accelerating cell cycle progression [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. To examine this hypothesis, we isolated the mouse lung fibroblasts (MLFs) from the CREPT\u003csup\u003eKO\u003c/sup\u003e and CREPT\u003csup\u003eWT\u003c/sup\u003e mice (Fig.\u0026nbsp;4A). A Western blot analysis showed that CREPT was completely deleted in the MLFs after the addition of tamoxifen in CREPT\u003csup\u003eKO\u003c/sup\u003e mice (Fig.\u0026nbsp;4B). Then we performed a cell proliferation experiment using MLFs. The result showed that MLFs from the CREPT\u003csup\u003eKO\u003c/sup\u003e mice grew with a decreased rate compared with those from wild type mice (Fig.\u0026nbsp;4C, red vs blue curves, P\u0026thinsp;=\u0026thinsp;0.0004). This result suggests that CREPT is critical for the proliferation of MLFs in the \u003cem\u003ein vitro\u003c/em\u003e culture condition.\u003c/p\u003e \u003cp\u003eTo determine if CREPT regulates the differentiation of lung fibroblasts into myofibroblasts in response to cytokine stimulation, we examined the level of αSMA in the MLFs. In an assay using TGF-β stimulation, which was widely used as a cytokine to activate fibroblasts[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], we observed that CREPT deletion rendered the response of αSMA in the MLFs from CREPT\u003csup\u003eKO\u003c/sup\u003e mice in comparison with that from CREPT\u003csup\u003eWT\u003c/sup\u003e mice (Fig.\u0026nbsp;4D). These results suggest that deletion of CREPT reduced the expression of α-SMA in the MLFs, implying that CREPT regulates the differentiation of fibroblasts into myofibroblasts. Consistent with the Western blot analyses, an immunostaining experiment further demonstrated that α-SMA\u003csup\u003e+\u003c/sup\u003e cells were reduced when CREPT was deleted under the \u003cem\u003ein vitro\u003c/em\u003e cultural condition (Fig.\u0026nbsp;4E). All these results collectively suggest that CREPT is required for lung fibroblast activation upon inflammatory cytokine stimulations.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003ePulmonary fibrosis is a major public health problem with limited therapeutic options. Treatment options are restricted to two clinically approved drugs, Nintedanib (Ofev)[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] and pirfenidone (Esbriet)[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], both of which slow progression but do not halt or reverse the fibrosis. Many studies have been carried out to understand the pathogenesis of pulmonary fibrosis. Most studies focused on the factors in the regulation of fibroblast differentiation into myofibroblasts under inflammatory challenges. In this study, we determined to identify intrinsic factors that control the activation of the fibroblasts during fibrotic responses. We observed that CREPT deficiency mice developed mildly pulmonary fibrosis upon BLM challenge. Using different assays, we demonstrated that deletion of CREPT indeed decreased the activation of fibroblasts, while overexpression of CREPT promoted the proliferation of fibroblasts (data not shown). In particular, we observed that a-SMA, an important marker for fibroblast activation, as well as other markers such as Col1a1 and FN1, was dramatically decreased when CREPT was deleted both \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e. Therefore, we speculate that CREPT plays an important role during the pulmonary fibrosis (Fig.\u0026nbsp;5).\u003c/p\u003e \u003cp\u003ePulmonary fibrosis is often observed in the lung biopsy samples from patients with usual interstitial pneumonia resulting from chronic hypersensitivity pneumonitis, suggesting an inflammatory process preceding the development of fibrosis [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Many studies suggest that various inflammatory responses may lead to a pro-fibrotic environment and cytokine milieu (including TGF-β[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], IL-6, TNF-α, PDGF[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], and WNT[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]). Shared downstream pathways may activate and sustain a complex interplay leading to fibroblast activation and differentiation into myofibroblasts, further orchestrate fibrogenesis [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. However, the clinical trials using anti-inflammatory agents failed to improve outcome of idiopathic pulmonary fibrosis patients [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. These clinical results suggest that the activation of fibroblasts could be maintained during the chronic inflammation. In this study, we revealed that CREPT contributes to the activation of the fibroblasts under BLM-induced chronic inflammation. Interestingly, we observed that deletion of CREPT had no influence on the chronic inflammation per se, as the cytokine production (TNF-α, IL-6, and IL-1β) in CREPT KO mice were maintained at the similar levels to the wildtype mice. In this context, we reasoned that CREPT may help fibroblast proliferate or activate without affecting the inflammation initiation from epithelial cells and inflammation exacerbation from immune cells such as macrophages and neutrophils. Indeed, in our IHC experiments, we observed no alteration of CREPT expression in epithelium in the wildtype mice under BLM challenge (data not shown), although the mice produced significantly high amounts of cytokines. The unchanged expression of CREPT in the epithelial cells during the BLM challenge might explain the reason why the inflammation responses were not altered upon CREPT deletion. Our results suggest that CREPT regulates the activation of fibroblasts as deleting CREPT repressed fibroblast proliferation and differentiation. However, we observed no elevated expression of CREPT in the fibroblasts under inflammation challenges (data not shown). Considering the significant phenotype from the deletion experiments, we speculate that the minimal amount of endogenous CREPT is enough to maintain the fibroblast activation. Since elevated CREPT was frequently observed in varieties of tumors, we considered that the basal CREPT is maintained in fibroblasts to avoid their over-proliferation. Nevertheless, the role of CREPT on fibroblast activation without affecting inflammation is particularly of interest for the development of drugs against fibrosis.\u003c/p\u003e \u003cp\u003eIn this study, we found that deletion of CREPT attenuated the expression of fibrotic genes, including \u003cem\u003eα-SMA\u003c/em\u003e, \u003cem\u003eCol1a1\u003c/em\u003e, and \u003cem\u003eFN\u003c/em\u003e. We have employed qRCR, Western blot, and IHC experiments to consistently demonstrate the levels of these fibrotic markers. All these results suggest that CREPT participates in the regulation of these gene expression during the activation of fibroblasts. However, we did not elucidate how CREPT regulates the gene expression under inflammation. Previous studies have demonstrated that CREPT is a positive regulator of transcriptional factors such as STAT3 and β-catenin/TCF4 [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e] and promotes the activity of RNAPII at the regions of promoters and terminators in the \u003cem\u003eCCND1\u003c/em\u003e gene [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. In cancer cells, CREPT has been attributed to promoting the proliferation of cells by accelerating the cell cycle progression. In this study, we observed that CREPT functioned in fibroblast proliferation and differentiation stimulated by TGF-β or TNF-α in an \u003cem\u003ein vitro\u003c/em\u003e culture condition. We speculate that CREPT may act as a coactivator to facilitate gene expression via binding to the promoter region of fibrogenic genes. As our current results from the whole body CREPT deletion mouse provide evidence for the fibroblast activation during pulmonary fibrosis, a fibroblast-specific conditional mouse line will be a super to demonstrate the role of CREPT in this tough disease.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn summary, we demonstrate that CREPT plays a crucial role in the pathogenesis of pulmonary fibrosis. CREPT deficiency ameliorates the BLM induced lung fibrosis in mice. These results revealed a critical yet previously unrecognized role of CREPT in the regulation of lung fibrosis. Our study opens up opportunities for drug discovery, precision targets and therapeutic interventions in pulmonary fibrosis.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eAuthor information\u003c/h2\u003e \u003cp\u003eJ.W. and J. S. made equal contributions to this work. All the authors read and approved the submitted manuscript.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eAuthor details\u003c/strong\u003e \u003cp\u003e \u003csup\u003e1\u003c/sup\u003eState Key Laboratory of Membrane Biology, School of Medicine, Tsinghua University, Beijing 100084, China.\u003c/p\u003e \u003cp\u003e \u003csup\u003e2\u003c/sup\u003eDepartment of Surgery, The Second Affiliated Hospital of Jiaxing University, No. 397, Huangcheng North Road, Jiaxing 314000.\u003c/p\u003e \u003cp\u003e \u003csup\u003e3\u003c/sup\u003eSXMU-Tsinghua Collaborative Innovation Center for Frontier Medicine, Shanxi Medical University, Taiyuan, Shanxi Province 030001, China.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eCo-corresponding authors\u003c/strong\u003e \u003cp\u003eZhijie Chang, Tsinghua University. Tel: +86 10 62785076, Fax: +86 10 62773624, E-mail: [email protected].\u003c/p\u003e \u003cp\u003eXiaoguang Wang, The Second Affiliated Hospital of Jiaxing University. Tel: +86 573 82053235, E-mail: [email protected].\u003c/p\u003e \u003cp\u003eChenxi Cao, The Second Affiliated Hospital of Jiaxing University. Tel: +86 573 82050295, E-mail: [email protected].\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e \u003cp\u003e All experimental procedures involving animals were conducted in strict accordance with the Basel Declaration and followed the protocol approved by the Institutional Animal Care and Use Committee of Tsinghua University School of Medicine. All animal handling procedures adhered to the guidelines for the Care and Use of Laboratory Animals published by the National Institute of Health.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent for publication\u003c/strong\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eCompeting interests\u003c/strong\u003e \u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis project was supported by funding from the National Natural Science Foundation of China (grant. no. 81830092) and the Public Welfare Science and Technology Program of Jiaxing City (grant. no. 2024AZ30004; Jiaxing China).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eJ.W. and J. S. made equal contributions to this work. J.W. , J. S., S.L, J.L., and J.C. collected data and assisted with data analyses. J.W. wrote the original draft. Z.C.,X.W., C.C., Y.W., M.W. and F.R. assisted in preparing the manuscript and critically reviewed it. All the authors read and approved the submitted manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eNot applicable.\u003c/p\u003e\u003ch2\u003eData availability\u003c/h2\u003e \u003cp\u003eNo datasets were generated during the current study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eRicheldi, L., H.R. Collard, and M.G. 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Exp. Med, 2006. \u003cstrong\u003e203\u003c/strong\u003e: p. 1021\u0026ndash;1031.\u003c/li\u003e\n\u003cli\u003eAkamatsu, T., et al., \u003cem\u003eDirect isolation of myofibroblasts and fibroblasts from bleomycin-injured lungs reveals their functional similarities and differences.\u003c/em\u003e Fibrogenesis \u0026amp; Tissue Repair, 2013. \u003cstrong\u003e6\u003c/strong\u003e(1): p. 15.\u003c/li\u003e\n\u003cli\u003eGattinoni, L., E. Carlesso, and P. Caironi, \u003cem\u003eStress and strain within the lung.\u003c/em\u003e Curr Opin Crit Care, 2012. \u003cstrong\u003e18\u003c/strong\u003e(1): p. 42-7.\u003c/li\u003e\n\u003cli\u003eDing, H., et al., \u003cem\u003eTGF-\u0026beta;-induced \u0026alpha;-SMA expression is mediated by C/EBP\u0026beta; acetylation in human alveolar epithelial cells.\u003c/em\u003e Mol Med, 2021. \u003cstrong\u003e27\u003c/strong\u003e(1): p. 22.\u003c/li\u003e\n\u003cli\u003eVasakova, M., et al., \u003cem\u003eCytokine gene polymorphisms and BALF cytokine levels in interstitial lung diseases.\u003c/em\u003e Respir Med, 2009. \u003cstrong\u003e103\u003c/strong\u003e(5): p. 773-9.\u003c/li\u003e\n\u003cli\u003eKim, K.K., D. Sheppard, and H.A. Chapman, \u003cem\u003eTGF-\u0026beta;1 Signaling and Tissue Fibrosis.\u003c/em\u003e Cold Spring Harb Perspect Biol, 2018. \u003cstrong\u003e10\u003c/strong\u003e(4).\u003c/li\u003e\n\u003cli\u003eRicheldi, L., et al., \u003cem\u003eEfficacy and safety of nintedanib in idiopathic pulmonary fibrosis.\u003c/em\u003e N Engl J Med, 2014. \u003cstrong\u003e370\u003c/strong\u003e(22): p. 2071-82.\u003c/li\u003e\n\u003cli\u003eNoble, P.W., et al., \u003cem\u003ePirfenidone in patients with idiopathic pulmonary fibrosis (CAPACITY): two randomised trials.\u003c/em\u003e Lancet, 2011. \u003cstrong\u003e377\u003c/strong\u003e(9779): p. 1760-9.\u003c/li\u003e\n\u003cli\u003eSgalla, G., et al., \u003cem\u003eIdiopathic pulmonary fibrosis: pathogenesis and management.\u003c/em\u003e Respir Res, 2018. \u003cstrong\u003e19\u003c/strong\u003e(1): p. 32.\u003c/li\u003e\n\u003cli\u003eConte, E., et al., \u003cem\u003eEffect of pirfenidone on proliferation, TGF-\u0026beta;-induced myofibroblast differentiation and fibrogenic activity of primary human lung fibroblasts.\u003c/em\u003e Eur J Pharm Sci, 2014. \u003cstrong\u003e58\u003c/strong\u003e: p. 13-9.\u003c/li\u003e\n\u003cli\u003eKlinkhammer, B.M., J. 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Boor, \u003cem\u003ePDGF in organ fibrosis.\u003c/em\u003e Mol Aspects Med, 2018. \u003cstrong\u003e62\u003c/strong\u003e: p. 44-62.\u003c/li\u003e\n\u003cli\u003eDou, C., et al., \u003cem\u003eP300 Acetyltransferase Mediates Stiffness-Induced Activation of Hepatic Stellate Cells Into Tumor-Promoting Myofibroblasts.\u003c/em\u003e Gastroenterology, 2018. \u003cstrong\u003e154\u003c/strong\u003e(8): p. 2209-2221.e14.\u003c/li\u003e\n\u003cli\u003eHomer, R.J., et al., \u003cem\u003eModern concepts on the role of inflammation in pulmonary fibrosis.\u003c/em\u003e Arch Pathol Lab Med, 2011. \u003cstrong\u003e135\u003c/strong\u003e(6): p. 780-8.\u003c/li\u003e\n\u003cli\u003eTorrisi, S.E., et al., \u003cem\u003eEvolution and treatment of idiopathic pulmonary fibrosis.\u003c/em\u003e Presse Med, 2020. \u003cstrong\u003e49\u003c/strong\u003e(2): p. 104025.\u003c/li\u003e\n\u003cli\u003eZhang, Y., et al., \u003cem\u003eCREPT/RPRD1B, a recently identified novel protein highly expressed in tumors, enhances the \u0026beta;-catenin\u0026middot;TCF4 transcriptional activity in response to Wnt signaling.\u003c/em\u003e J Biol Chem, 2014. \u003cstrong\u003e289\u003c/strong\u003e(33): p. 22589-22599.\u003c/li\u003e\n\u003cli\u003eZhang, Y., et al., \u003cem\u003eCREPT facilitates colorectal cancer growth through inducing Wnt/\u0026beta;-catenin pathway by enhancing p300-mediated \u0026beta;-catenin acetylation.\u003c/em\u003e Oncogene, 2018. \u003cstrong\u003e37\u003c/strong\u003e(26): p. 3485-3500.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Pulmonary fibrosis, fibroblast-to myofibroblast activation, CREPT","lastPublishedDoi":"10.21203/rs.3.rs-4805438/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4805438/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003ePulmonary fibrosis is a chronic and progressive disease that originates from interstitial lung diseases and ultimately exhibits respiratory failure in patients. The disease is characterized by focal accumulation and excessive production of extracellular matrix (ECM) from over-activated fibroblasts in the lung. Although many extrinsic factors have been identified to boost fibroblast proliferation and activation, it remains unclear how fibrosis is regulated by intrinsic factors.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003ePulmonary fibrosis mouse model was induced by intratracheal injection of bleomycin (BLM) into CREPT\u003csup\u003eWT\u003c/sup\u003e and CREPT\u003csup\u003eKO\u003c/sup\u003e mice. In vitro study, the proliferation of mouse lung fibroblasts (MLFs) was assessed using CCK-8 assays and expression of fibrotic protein was examined following transforming growth factor (TGF)-β stimulation in MLFs.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eIn this study, we found that deletion of CREPT alleviated BLM induced pulmonary fibrosis. Deletion of CREPT resulted in attenuated murine lung fibroblast proliferation, TGF-β-induced fibroblast-to-myofibroblast activation, and ECM deposition. Consistently, deletion of CREPT decreased the expression of fibrotic marker genes such as \u003cem\u003ea-SMA\u003c/em\u003e, \u003cem\u003eCol1a1\u003c/em\u003e, and \u003cem\u003eFN1\u003c/em\u003e but had no influence on the inflammation response upon the BLM challenge.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eIn summary, we report that CREPT is required for BLM induced pulmonary fibrosis in mice. Our study unravels an intrinsic molecular mechanism for the development of pulmonary fibrosis and provides a new target for the therapy of the interstitial lung disease.\u003c/p\u003e","manuscriptTitle":"CREPT is required for pulmonary fibrosis induced by bleomycin","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-09-02 18:57:48","doi":"10.21203/rs.3.rs-4805438/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"0c0b1ffd-4183-41c2-8ddd-d85bf0901f4b","owner":[],"postedDate":"September 2nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-07-16T09:23:54+00:00","versionOfRecord":[],"versionCreatedAt":"2024-09-02 18:57:48","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4805438","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4805438","identity":"rs-4805438","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}