Nuclear accumulated μ-calpain in AT2 cell participates in pulmonary fibrosis via inactivating FoxO3a

Nuclear accumulated μ-calpain in AT2 cell participates in pulmonary fibrosis via inactivating FoxO3a | 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 Nuclear accumulated μ-calpain in AT2 cell participates in pulmonary fibrosis via inactivating FoxO3a Qiao LI, Yu-Tong YE, Yi-Liang ZHU, Yu TIAN, Miao-Feng WANG, Yuan FANG, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3787538/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 µ-calpain is implicated in pulmonary fibrosis, however its role in the aberrant differentiation of alveolar epithelial type II cells (AT2), a hallmark of pulmonary fibrosis remains unclear, and its targeted transcription factor has not been addressed. Here, examination of the specimen of fibrosis patients revealed excessive proliferation of AT2 cells. In parallel, AT2 cells exhibited substantial calpain 1 (CAPN1), a catalytic subunit of µ-calpain, and phosphorylated FoxO3a (p-FoxO3a), an important transcription factor in lung tissue. Of note, targeted knockdown of CAPN1 in AT2 cells blocked the progression of bleomycin-induced pulmonary fibrosis, manifested as reduced poorly aerated regions in chest CT image, and decreased content of hydroxyproline and α-SMA. Analysis of nuclear fraction displayed an accumulation of CAPN1 and loss of FoxO3a, which was accompanied with activation of Akt. Knockdown of CAPN1 in A549 cells with siRNA antagonized the process of epithelial-mesenchymal transition and blunted FoxO3a phosphorylation and Akt activation. Conversely, overexpression of CAPN1 accelerated mesenchymal transition, enhanced its nuclear accumulation and the translocation of p-FoxO3a out of nucleus. Finally, inhibition of Akt decreased calpain-elicited FoxO3a phosphorylation, meanwhile, transfection of FoxO3a mutant carrying Thr32A and Ser253A mitigated the calpain-stimulated mesenchymal transition. Collectively, we conclude that nuclear accumulation of µ-calpain in AT2 cells is a critical step to aggravate pulmonary fibrosis. we also identify that inactivation of FoxO3a in a Akt-dependent phosphorylation manner confers to calpain-elicited the aberrant differentiation of AT2 cells. pulmonary fibrosis calpain alveolar epithelial Type II cells FoxO3a epithelial to mesenchymal transition Akt Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Idiopathic pulmonary fibrosis (IPF) is the most common type of pulmonary fibrosis, which has only a medium survival time of only 2–4 years from diagnosis [ 1 ]. IPF predominantly presents as usual interstitial pneumonia (UIP) with radiological images, and was characterized by alveolar architecture disruption and extracellular collagen deposition under histological observation, which ultimately leads to respiratory failure and death [ 2 ]. Although Nintedanib and Pirfenidone are currently approved to delay the rate of lung function decline of IPF patients [ 3 , 4 ], the problem that both drugs fail to halt the progression of pulmonary fibrosis prompted us to gain further insight into its pathophysiological mechanisms. Mesenchymal transition is an important initiating event in IPF [ 5 , 6 ]. Till now, alveolar epithelial Type II cells (AT2), endothelial cells, resident fibroblasts have been reported to act as progenitor cells in animal models[ 7 , 8 ]. Upon the profibrotic cytokine transforming growth factor-β (TGFβ) stimulation, the progenitor cells differentiates into the so-called myofibrolasts, which produce substantial collagen-rich extracellular matrix[ 9 ]. The key evidence for the involvement of AT2 cells in the progression of epithelial-mesenchymal transition (EMT) is that AT2 cells express mesenchymal markers α-smooth muscle action (α-SMA) and vimentin during their differentiation[ 10 , 11 ]. Recently, cell fate-map studies corroborate that the aberrant differentiation of AT2 cells is a hallmark and a major contributor to pulmonary fibrosis [ 5 , 12 ]. Thus, it is necessary to address the molecular mechanisms behind the role of AT2 cells in pulmonary fibrosis. µ-calpain is a superfamily of Ca 2+ -dependent nonlysosomal cysteine protease, which consist of a large catalytic subunit, namely calpain 1 (CAPN1) and a small regulatory subunit CAPN4 [ 13 ]. We and others have demonstrated that CAPNs participate in a variety of pathophysiological processes, i.e., plasma membrane disruption, mitochondrial damage, transform of vascular permeability and cell motility [ 14 – 17 ]. Of note, µ-calpain is implicated in the progression of IPF patients [ 18 , 19 ]. Conditional genetic ablation of CAPN1 in pleural mesothelial cell and macrophage has been documented to blunt bleomycin-induced pulmonary fibrosis [ 19 , 20 ]. Although a reciprocal interaction between µ-calpain and TGFβ, and activation of Smad2/3, Akt, and the extracellular signal-regulated kinase explain the harmful effects [ 18 , 21 ], very little is known on the transcription factors involved, and the role of µ-calpain in the fate of AT2 cells remains unclear. FoxO3a belongs to the forkhead box protein class O subfamily. It is a transcription factor involved in cell proliferation, differentiation, apoptosis, and metabolism [ 22 ]. The transcriptional activity of FoxO3a depends on its phosphorylation status. Upon phosphorylation, FoxO3a translocates from the nucleus to cytosol and becomes inactivated [ 23 , 24 ]. FoxO3a stimulates the expression of p27, a negative regulator of the G1-S transition in cell cycle progression, which impedes the proliferation of fibroblast and protects against pulmonary fibrosis [ 24 ]. FoxO3a decline/suppression has been implicated in coronavirus disease 2019 (COVID)-pulmonary fibrosis, and bleomycin-induced pulmonary fibrosis [ 25 , 26 ]. Thus, we hypothesized that FoxO3a is a targeted transcription factor of µ-calpain, which dictates the fate of AT2 cells. In this study, we firstly examined the expression of CAPN1 and FoxO3a in AT2 cells in the specimen of IPF patients. Secondly, we targeted knockdown of CAPN1 in AT2 cells and evaluated the outcome of bleomycin-induced pulmonary fibrosis. Finally, we determined whether FoxO3a is a targeted transcription factor of CAPN1 in the EMT process, and investigated the role of phosphorylation modification via Akt in this scenario. Our data for the first demonstrate that nuclear accumulation of µ-calpain is a critical step in the progression of IPF, which inactivates FoxO3a via Akt-dependent phosphorylation, and accelerates the transition of AT2 cells to mesenchymal population. Materials and methods Materials Bleomycin was purchased from Nippon (Kayaku, Japan). PD150606 (Cat#HY-100529) and TGFβ (Cat#HY-P70543) were obtained from MedChem Express (Monmouth Junction, NJ, USA). Antibodies against Calpain 1 (Cat#C5736), STFPC (Cat#PA5-71680), p-FoxO3a (Ser253), FoxO3a (Cat#12819), E-cadherin (Cat#3195), α-SMA (Cat#19245), COL1A1 (Cat#72026), p-Akt (Ser473, Cat#4060), Akt (Cat#4685S), and GAPDH (Cat#2118) was obtained from Sigma-Aldrich(Merck, USA), Invitrogen (Eugene, OR, USA), Abcam (Cambridge, MA, USA) and Cell Signaling Technology (Danvers, MA, USA) respectively. Tetramethylrhodamine-conjugated Goat anti-Rabbit IgG (Cat#T2769) and LipofectamineTM 3000 (Cat#L3000015) were purchased from Thermo Fisher (Illinois, IL, USA). DyLight488 AffiniPure Goat Anti-Mouse IgG (Cat#E032210) was purchased from EarthOx (SanFrancisco, CA, USA). The primers and siRNAs were synthesized by GenePharma (Shanghai, China), and pcDNA3.1(+)-Flag-CAPN1 and pcDNA3.1(+)-GFP-FoxO3a-mutant were purchased from Sangon Biotech (Shanghai, China). BCA Protein Assay Kit was acquired from Thermo Fisher (Waltham, MA, USA). Animals Pathogen-free C57BL/6 mice aged at 8 to 10 weeks were provided by the Laboratory Animal Centre of the Air Force Medical University. sftpc-CreERT2 mice were purchased from Cyagen Com. (Suzhou, China). The animals were housed in constant temperature and humidity with a 12 h light cycle. Animals were allowed food and water ad libitum. Male mice were used for all experiments, and all animal experiments were conducted in strict accordance with the Guidelines of Health and guidelines for use, and were permitted by the Animal Welfare and Ethics Institution of Air Force Military Medical University (IACUC-20230099). Human tissues Ethical approval for this study (No. 20202052) was approved by the Medical Ethics Committee of the Xijing Hospital of Air Force Medical University. The study was conducted in accordance with the World Medical Association's Code of Ethics (Declaration of Helsinki). Informed consent was obtained from each subject for the study protocol. Fours lung tissue biopsies were obtained with the help of bronchoscopic or ultrasound-guidance in Xijing Hospital. IPF patients were diagnosed with the histopathologic examination and/or high resolution computed tomography. Four normal lung biopsies from resection of cancer were used as controls. Preparation of pulmonary fibrosis model Wild type C57/BL6 mice were anesthetized with an intraperitoneal injection of 0.3% pentobarbital sodium and randomly divided into: (1) control group (CON), (2) bleomycin group (BLM). To study the role of µ-calpain in AT2 cells, sftpc-CreERT2 mice were randomly divided into (1) scrambled RNA group (Sc), (2) CAPN1-shRNA group (Sh), (3) scrambled RNA with bleomycin group (Sc + B), and (4) CAPN1-shRNA with bleomycin group (Sh + B). Bleomycin was dissolved in saline, and a volume of 50 µl containing 2.5 mg/kg bleomycin was intratracheally given using a 22 G plastic cannula. Same amount of saline was delivered to the mice as a control. At day 21, mice were euthanized, and the lung tissue was harvested[ 27 ]. AAV-FLEX-shRNA-CAPN1 tagged with GFP and its delivery to tomato-tagged sftpc-CreERT2 mice A GFP-tagged adenovirus associated virus vector (AAV2/6) containing either mir30-based shRNA against CAPN1 or scramble RNA, with the reading frames inverted in a flip-excision (FLEX) cassette were prepared by HANBio Com. (Shanghai, China) [ 28 ]. The interfering sequence to CAPN1 was CUGGGCUGCUCCAUUAAUA. After receiving 1 mg tamoxifen intraperitoneally injection for continuous 7 days to induce Cre expression in AT2 cells, adult sftpc-CreERT2 mice was administered via trachea a volume of 35 µl AAV2/6 virus suspensions with a titer of 1.5×10 12 vg/ml. Three weeks later, pulmonary fibrosis was induced by bleomycin as described above. Mice were euthanized on day 21 after bleomycin injection, and lung tissue was harvested In vivo micro-computed tomography (micro-CT) analysis Briefly, a non-invasive micro-CT scanner (IvIS Lumina K series III, PerkinElmer) with computer software (Living Image v. 4.3.1, PerkinElmer) was employed to analyze lung structure. The X-ray system used a micro-focus tube with a spot size of 5 mm. The images were acquired at 90 kV, 160 mA and the wide field of view scanning at 40 mm. Lung regions were classified into 4 categories by CT attenuation densities: hyper-inflated, density range from − 861 to -1000 Hounsfield units (HU); normally-aerated, range from − 435 to -860 HU; poorly-aerated, density range from − 121 to -434 HU; non-aerated, range from − 120 to + 120 HU. And then a total mask was established to cover the density range of -1000 to + 121 HU. 8–10 axial CT images that could be recognized in the lung were painted. The poorly aerated regions were calculated to reflect pulmonary fibrosis, as described previously [ 29 , 30 ]. Hematoxylin-eosin and Masson's trichrome staining, and hydroxyproline measurement At the end of the experiment, the left lung tissues in mice were fixed in 4% paraformaldehyde solution for 24 h, and then embedded in paraffin. A mid-sagittal tissue section was prepared with 4 µm thickness. The sections were then stained with hematoxylin-eosin to evaluate alveolar structure [ 31 ]. Masson's trichrome staining was performed to determine collagen deposition. Ashcroft score ranged from 0 to 8 was evaluated by an examiner, who was blind to the mice allocation, to determine the severity of pulmonary fibrosis, as described previously [ 32 ]. To determine hydroxyproline content, the lung tissues were homogenated and centrifuged. The supernatant was used to measure the content of hydroxyproline using the Hydroxyproline assay kit according to the manufacturer's protocol [ 18 ]. Cell transfection of constructed-expression vectors and siRNA Full-length human FoxO3a (NM_001455.4) cDNA was synthesized and cloned into the HindIII and NotI sites of pcDNA3.1 which contains GFP. CAPN1 (NM_005186.4) was tagged with Flag and cloned into BamHI and XhoI sites of pcDNA3.1. To study the role of FoxO3a phosphorylation in the process of mesenchymal transition, the phosphorylation sites including Thr32 and Ser253 were mutated to alanine. The sequence alignment for the cDNAs was carried out using DNAMAN software (version 6.0). CAPN1-siRNA was employed to determine the role of µ-calpain in the process of mesenchymal transition, the sequences were as follows: CAPN1-siRNA: 5′-GGUUACCGAGUGGUACGAGUU-3′. Scrambled siRNA: 5′-UUCUCCGAACGUGUCACGUTT-3′. The human type II A549 alveolar epithelial cells were cultured with Dulbecco’s Modified Eagle Medium containing 10% fetal bovine serum. When the density of the cells reached 60% confluence, the cells were transfected with CAPN1-siRNA or plasmid vector containing CAPN1 or FoxO3a mutant, which was dissolved in a mixture of Lipofectamine 3000 reagent and Opti-MEM. Scramble RNA and empty vector were used negative controls. Six hour later, the medium was replaced with DMEM containing10% FBS. The transfection efficiency was evaluated with Western blot [ 33 ]. At 24 h after transfection, the cells were incubated with 5 or 10 ng/ml TGFβ for another 24 h, and then harvested to evaluate mesenchymal transition. Immunofluorescence The slice was blocked with phosphate buffer containing 5% donkey serum in at room temperature for 30 min, and permeabilization with 0.3% Triton-X 100 for 15 min. After that, the slice was incubated with antibodies against SFTPC, CAPN1, E-cadherin, α-SMA, p-FoxO3a, Flag and FoxO3a at 4°C overnight. All the antibodies were used at a dilution of 1:100. After the slice were incubated with tetramethylrhodamine-conjugated secondary antibodies against rabbit IgG and/or DyLight488-conjugated secondary antibodies against mouse IgG for 1 hour at room temperature, the nuclei were counterstained with DAPI (20 µg/ml). Finally, the slice was examined with a laser confocal microscope equipped with an FV-10-ASW system (Olympus FV1000, Tokyo, Japan), as described previously [ 31 ] Western blotting Total protein was extracted with a lysis buffer containing (in mM) Tris (pH = 7.4) 50, NaCl 150, 1% NP-40, 0.5% sodiumdeoxycholate, 0.1% SDS. After centrifugation at 12,000 g for 15 min, the supernatants were collected. To extract nuclear proteins, the tissue or cell was incubated with a buffer containing (in mM) Tris (pH = 8.0) 10, KCl 10, MgCl2 1.5, DTT 1, EDTA 0.2, 5% glycerol, and protease inhibitors. The sample was then snap-frozen in liquid nitrogen, and homogenated. After centrifugation at 12,000 g for 10 min, the sediments were lysed with a buffer containing (in mM) Tris (pH = 8.0) 20, NaCl 420, MgCl2 1.5, DTT 0.5, EDTA 0.2, 25% glycerol, and protease inhibitors. The lysate was centrifuged at 12,000 g for 10 min, and the supernatants were collected as the nuclear fraction. Protein concentration was determined with BCA method. The samples were separated with 6–10% SDS-polyacrylamide gels and transferred to the PVDF membranes. After blocking with 5% bovine serum albumin in Tris buffer, the membranes were incubated with antibodies against CAPN1, collagen I, α-SMA, E-cadherin, FoxO3a, p-FoxO3a, Akt, p-Akt, GAPDH, Lamin B at 4°C overnight (1:1000 for all), followed by incubation with horseradish peroxidase-conjugated secondary antibodies (1:5000) at room temperature for 1 h. Proteins were visualized using an ultrasensitive ECL chemiluminescence substrate. GAPDH and Lamin B were used as loading controls. The signal intensity was quantified with the aid of Image J software [ 15 , 31 ]. Statistical analysis All data are presented as the mean ± standard error of the mean, which were performed in at least three independent biological experiments. GraphPad Prism was used to perform statistical analysis. Student’s t-test was employed for comparisons within two groups. ANOVA followed by Tukey’s post hoc test was performed for multiple group comparisons. P value less than 0.05 was considered statistically significant. Results 1. An increase of both µ-calpain and p-FoxO3a in AT2 cells represents a hallmark in IPF patients Compared with the control group, IPF patients exhibited excessive proliferation of AT2 cells, manifested as positive staining of SFTPC protein (Fig. 1 A & B). These cells were clustered, and the percentage of positive cells reached 45.43±1.6 per field, which was in contrast to 10.73±0.9 per field in the control group (Fig. 1 A-C). These results suggest that the impediment of AT2 to AT1 cells differentiation is a critical pathological characteristic of pulmonary fibrosis. Immunofluorescence double staining revealed that substantial CAPN1 and p-FoxO3a were located in AT2 cells (Fig. 1 A & B). The percentage of CAPN1- and p-FoxO3a-positive cells rose from 7.3±0.9, and 8.72 ±1.4 in the control group to 53.22±4.4 and 47.0±2.8 per field respectively (Fig. 1 C). These results raise the possibility that both molecules are involved in pulmonary fibrosis. 2. Targeted knockdown of CAPN1 in AT2 cells ameliorates bleomycin-induced pulmonary fibrosis Compared with the ones treated with saline (control group), mice in bleomycin-treated group displayed disruption of the architecture of alveolar, an increase of the interalveolar septal thickness and collagen deposition (Fig. 2 A & B), manifested as HE and Masson’s trichrome staining. Furthermore, bleomycin resulted in the excessive proliferation of AT2 cells, which express substantial CAPN1 and phosphorylated FoxO3a (Fig. 2 C-E). These results corroborate that µ-calpain and FoxO3a are involved in the aberrant differentiation of AT2 cells. To determine the role of µ-calpain in this scenario, FLEx switch (Cre-On) adeno-associated viral vector carrying GFP and shRNA against CAPN1 was delivered via trachea to Tomato-tagged sftpc-CreERT2 mice, which specifically knockdown CAPN1 in AT2 cells. As expected, bleomyocin caused excessive proliferation of AT2 cells, manifested as the red fluorescence emitted by tagged protein tomato. Furthermore, AT2 cells in bleomyocin-treated mice also exhibited green fluorescence emitted by GFP, indicating the successful delivery of AAV vectors carrying shRNA against CAPN1 to AT2 cells. Of particular interest, shRNA-CAPN1 did not antagonize bleomycin-induced an increase of AT2 cell number (Fig. 3 A & B). These results indicate that µ-calpain is not involved in AT2 cells proliferation in the process of pulmonary fibrosis. Chest CT scanning showed that shRNA-CAPN1 reduced the nonaerated regions and the content of hydroxyproline in bleomycin-treated mice (Fig. 3 C-E). Biochemical data and Western blotting revealed that CAPN1-shRNA antagonized bleomycin-induced alterations of α-SMA, Collagen-1, and E-cadherin content (Fig. 3 F). These results suggest that µ-calpain is an instigator to aggravate mesenchymal transition. Last but not the least, both scramble-RNA and shRNA had no appreciable effects in normal mice. 3. Nuclear accumulation of µ-calpain and loss of FoxO3a are detected in fibrosis tissue As transcriptional factor is a major determinant in mesenchymal transition, nuclear fraction was isolated. Compared with that in the scramble group, the mice in bleomycin-treated group showed a decline of FoxO3a in the nucleus, but an increase of p-FoxO3a in whole cell lysates (Fig. 4 A). µ-calpain, and its downstream molecule Akt, were also examined. To our surprise, bleomycin resulted in an accumulation of CAPN1 and Akt activation in the nuclear fraction (Fig. 4 B & C), along with an increase of CAPN1 and p-Akt in whole cell lysates (Fig. 4 B & C). Moreover, all these deleterious effects were blocked by targeting knockdown of CAPN1 in AT2 cells with shRNA (Fig. 4 ). These results suggest that nuclear accumulation of CAPN1 stimulated FoxO3a phosphorylation via Akt and accelerated the translocation of phosphorylated FoxO3a out of nucleus. 4. Nuclear accumulation of µ-calpain precedes FoxO3a phosphorylation and the mesenchymal transition After 10 ng/ml TGFβ stimulation for 48 h, normal polygonal shape epithelial cells exhibit a spindle -like appearance (Fig. 5 A). Confocal images and Western blotting showed that these cells presented more mesenchymal marker α-SMA, and less alveolar epithelial marker E-cadherin (Fig. 5 B & C), indicating the differentiation of epithelial cells into myofibroblasts. Furthermore, stimulated an increase of total CAPN1 and nuclear accumulation of CAPN1 (Fig. 5 D). In contrast, TGFβ resulted in a decrease of FoxO3a in the nucleus, and an increase in FoxO3a phosphorylation in the cytoplasm (Fig. 5 E-F). Of particular interest, all these alterations were blocked by CAPN1-siRNA. These results suggest that nuclear accumulation of µ-calpain precedes FoxO3a phosphorylation and the mesenchymal transition. To establish the critical role of µ-calpain, pcDNA3.1-CAPN1-Flag overexpression plasmid was constructed. As expected, the cells overexpressing CAPN1 were more sensitive to TGFβ stimulation than that in the control group. TGFβ at 5 ng/ml, was inclined to induce mesenchymal transition, and presented less alveolar epithelial marker E-cadherin, and more mesenchymal marker α-SMA (Fig. 6 A). Immunofluorescence images and Western blotting showed that the cells overexpressing CAPN1 exhibited strong-positive staining of CAPN1 and Flag in the nucleus (Fig. 6 B & C). Furthermore, an increased CAPN1 caused less FoxO3a in the nuclei fraction, and more p-FoxO3a in the cytoplasm upon TGFβ stimulation (Fig. 6 D & E). In addition, the overexpression of CAPN1 increased the level of both total and nuclear p-Akt after TGFβ stimulation (Fig. 6 F). These results suggest that Akt is involved in µ-calpain-elicited the phosphorylation of FoxO3a and mesenchymal transition. 5. Calpain inactivates FoxO3a in an Akt-dependent phosphorylation manner Compared with the control group, TGFβ enhanced Akt phosphorylation at residue Ser473, and FoxO3a phosphorylation at residue Ser253 (Fig. 7 A). Moreover, 20 µM LY294002, an inhibitor of Akt, not only blocked Akt phosphorylation, but also the phosphorylation of FoxO3a, which were accompanied with the impediment of mesenchymal transition (Fig. 7 A). These results validate that Akt is involved in regulating FoxO3a activity via phosphorylation modification. To determine whether the transcription activity of FoxO3a depends on its phosphorylation status, the phosphorylation sites of both Thr32 and Ser253 were mutated to alanine, and pcDNA3.1-GFP-FoxO3a mutant were constructed. Compared with the control, the cells transfected with FoxO3a mutant were more resistant to TGFβ (Fig. 7 B), manifested as less α-SMA. Western blotting data showed that FoxO3a mutant reduced the phosphorylation of endogenous FoxO3a and α-SMA, but increased E-cadherin (Fig. 7 C). These results support that FoxO3a is involved in maintaining the epithelial character. Furthermore, taking into consideration the finding that calpain activated Akt (Fig. 4 B, & Fig. 6 F), these data suggest that FoxO3a is regulated by µ-calpain via Akt-dependent manner. Discussion Previous studies have demonstrated that calpain is upregulated in IPF patients [ 18 , 19 ]. Thus, it is meaningful to identify the cells source in this clinical setting. Here, we observed that (1) IPF patients exhibited numerous AT2 cells, which expressed substantial µ-calpain subunit CAPN1 and p-FoxO3a, (2) Targeted knockdown of µ-calpain subunit CAPN1 in AT2 cells reversed the bleomycin-induced pulmonary fibrosis, (3) CAPN1 was accumulated in the nucleus, whereas p-FoxO3a translocated out of nucleus, (4) Inhibition of Akt alleviated the FoxO3a phosphorylation, and mutation of the phosphorylation residues of FoxO3a antagonized calpain-elicited mesenchymal transition. Our data provide evidence for the first that nuclear accumulation of µ-calpain in AT2 cells is a critical step to aggravate pulmonary fibrosis, and identify FoxO3a as a transcription factor conferring to µ-calpain-elicited mesenchymal transition. Our data suggest that blockade of the aberrant differentiation of AT2 cells via inhibiting µ-calpain is a new strategy to mitigate the progression of IPF. Here, specimens from IPF patients exhibited numerous SFTPC-positive cells. These results are consistent with previous findings that a hyperplasia of AT2 cells is a prominent feature of IPF [ 5 ]. Mir30-base AAV-FLEX system is a new technique to specifically knockdown the targeted molecule in specific cell [ 34 , 35 ]. Using this method, we observed that CAPN1-shRNA decreased the CAPN1 content (Fig. 4 ) and pulmonary fibrosis. These results for the first demonstrate that targeted regulation of CAPN1 in AT2 cells is a potential strategy to treat IPF. Our data are in contrast to previous findings showing that µ-calpain in pleural mesothelial cells and macrophages were involved in the process of IPF [ 19 , 20 ]. Therefore, the contribution of µ-calpain in different type of cells to the progression of IPF needs to be further evaluated. One interesting observation in this study was CAPN1-shRNA did not alter the number of AT2 cells. These results strongly suggest that µ-calpain participates in mesenchymal transition. In addition, AT2 cells have been recently identified to differentiate to a specialized cell population marked with keratin-8/claudin-4 positive, a regenerative intermediate cell state which exhibited senescence and failed to differentiate to AT1 cells [ 9 , 36 ]. Thus, the role of µ-calpain in this scenario also warranted further studied. µ-calpain is a cytosolic and substrate-specific cysteine protease [ 37 ]. Here, we observed an accumulation of the catalytic subunit CAPN1 in the nucleus after bleomycin treatment. These data are consistent with previous findings showing nuclear translocation of µ-calpain in mammary gland involution [ 38 ]. Furthermore, these results corroborate the point that modification of transcription factor activity is an important step to trigger mesenchymal transition. However, the results are in contrast to our previous findings showing an accumulation of µ-calpain in the plasma membrane and mitochondria after stressful stimulation [ 14 , 15 ]. Thus, the unidentified regulator, which is in charge of the trajectory of µ-calpain, warrants to be elucidated. It has been reported that Smad-dependent pathway and Akt-dependent pathway were involved in calpain-elicited pulmonary fibrosis [ 21 , 39 ]. However, the transcription factors located downstream of calpain remains elusive. Here, we demonstrated that nuclear accumulation of calpain was accompanied with the translocation of p-FoxO3a out of nucleus, and that blockade of FoxO3a phosphorylation antagonized calpain-elicited mesenchymal transition. These results for the first provide evidence that FoxO3a protects against the aberrant differentiation of AT2 cells, and that FoxO3a is a new targeted transcription factor of calpain. In addition to FoxO3a, Twist1, β-catenin, lymphoid enhancer factor and snail are also been demonstrated to participate in pulmonary fibrosis [ 40 – 42 ]. Whether calpain acts as a nexus to regulate these transcription factors in the process of pulmonary fibrosis merits further investigation. Till now, the function of FoxO3a in fibroblast in the lung tissue is well documented [ 43 ]. Knockdown of FoxO3a stimulated the proliferation of fibroblast, thus exacerbating pulmonary fibrosis [ 25 ]. Here, we demonstrated that AT2 cells in IPF patients did exhibit substantial p-FoxO3a. These results for the first provide evidence that FoxO3a participates in the aberrant differentiation of AT2 cells in pulmonary fibrosis. Furthermore, transfection of FoxO3a mutant carrying Thr32 and Ser253A mitigated the process of mesenchymal transition, these data support the point that FoxO3a is involved in maintaining the epithelial character. In this study, knockdown of CAPN1 reduced the level of p-FoxO3a and Akt activation, and the Akt inhibitor alleviated endogenous FoxO3a phosphorylation. This is consistent with previous findings that FoxO3a was regulated by Akt [ 23 , 24 ]. Particularly, these data demonstrate that µ-calpain inactivates FoxO3a in Akt-dependent manner [ 44 ], and that blockade of p-FoxO3a activity by µ-calpain is a critical step in instigating the process of the aberrant differentiation of AT2 cells, which accelerates the progression of IPF. Taken together, our data for the first time demonstrate that nuclear accumulation of µ-calpain in AT2 cells is a critical step to aggravate the prognosis of pulmonary fibrosis, which involves inactivation of FoxO3a in Akt-dependent manner, instigating the differentiation of AT2 cells to mesenchymal population. We propose that interfering mesenchymal transition by AT2 cell-specific weakening µ-calpain activity is a new approach to improve the prognosis of IPF. Further comprehensive understanding the biological behavior of µ-calpain in AT2 cells differentiation will help exploiting better strategies to limit IPF. Declarations Funding The work was supported by grants from the National Natural Science Foundation of China (no: 81570054 & 81772070). Competing Interest The authors declare that they have no relevant financial interests to disclose. Consent to participate Not appliciable Consent for publication The participant has consented to the submission of the case report to the journal. Availability of data and material The data that support the findings of this study are available on request from the corresponding author, Feng ZHAO, Xijing Hospital. Email address: [email protected] . Acknowledgments The authors thanks Ms. Li-Xia HAO for preparing tissue sections, and Mr. Hai-Feng ZHANG for taking confocal images. Ethics approval All animal experiments were conducted in strict accordance with the Guidelines of Health and guidelines for use, and were permitted by the Animal Welfare and Ethics Institution of Air Force Military Medical University (IACUC-20230099). References Raghu G, et al. Idiopathic pulmonary fibrosis (an Update) and progressive pulmonary fibrosis in adults: An official ATS/ERS/JRS/ALAT clinical practice guideline. Am J Respir Crit Care Med. 2022;205(9):e18–e47. Mei Q, et al. Idiopathic pulmonary fibrosis: An update on pathogenesis. Front Pharmacol. 2021;12:797292. de Andrade JA, et al. Effect of antifibrotic therapy on survival in patients with idiopathic pulmonary fibrosis. Clin Ther. 2023;45(4):306–15. Lancaster L, et al. Safety and survival data in patients with idiopathic pulmonary fibrosis treated with nintedanib: pooled data from six clinical trials. BMJ Open Respir Res. 2019;6(1):e000397. Katzen J, Beers MF. Contributions of alveolar epithelial cell quality control to pulmonary fibrosis. J Clin Invest. 2020;130(10):5088–99. Nieto MA, et al. EMT: 2016. Cell. 2016;166(1):21–45. Chapman HA. Epithelial-mesenchymal interactions in pulmonary fibrosis. Annu Rev Physiol. 2011;73:413–35. Hewlett JC, Kropski JA, Blackwell TS. Idiopathic pulmonary fibrosis: Epithelial-mesenchymal interactions and emerging therapeutic targets . Matrix Biol, 2018. 71–2: p. 112–127. Kobayashi Y, et al. Persistence of a regeneration-associated, transitional alveolar epithelial cell state in pulmonary fibrosis. Nat Cell Biol. 2020;22(8):934–46. Yamada M, et al. Dual-immunohistochemistry provides little evidence for epithelial-mesenchymal transition in pulmonary fibrosis. Histochem Cell Biol. 2008;129(4):453–62. Selman M, Pardo A. The leading role of epithelial cells in the pathogenesis of idiopathic pulmonary fibrosis. Cell Signal. 2020;66:109482. Nureki SI, et al. Expression of mutant Sftpc in murine alveolar epithelia drives spontaneous lung fibrosis. J Clin Invest. 2018;128(9):4008–24. Nian H, Ma B. Calpain-calpastatin system and cancer progression. Biol Rev Camb Philos Soc. 2021;96(3):961–75. Zhang RR, et al. Severe burn-induced mitochondrial recruitment of calpain causes aberrant mitochondrial dynamics and heart dysfunction. Shock. 2023;60(2):255–61. Lu HT, et al. CaMKII/calpain interaction mediates ischemia/reperfusion injury in isolated rat hearts. Cell Death Dis. 2020;11(5):388. Friedrich EE, et al. Endothelial cell Piezo1 mediates pressure-induced lung vascular hyperpermeability via disruption of adherens junctions. Proc Natl Acad Sci U S A. 2019;116(26):12980–5. Garcia-Trevijano ER, et al. Calpains, the proteases of two faces controlling the epithelial homeostasis in mammary gland. Front Cell Dev Biol. 2023;11:1249317. Zou M, et al. Inhibition of the ERK1/2-ubiquitous calpains pathway attenuates experimental pulmonary fibrosis in vivo and in vitro. Exp Cell Res. 2020;391(1):111886. Zhou LL, et al. Pleural mesothelial cell migration into lung parenchyma by calpain contributes to idiopathic pulmonary fibrosis. J Cell Physiol. 2022;237(1):566–79. Zhang L, et al. Myeloid cell-specific deletion of Capns1 prevents macrophage polarization toward the M1 phenotype and reduces interstitial lung disease in the bleomycin model of systemic sclerosis. Arthritis Res Ther. 2022;24(1):148. Li FZ, et al. Crosstalk between calpain activation and TGF-beta1 augments collagen-I synthesis in pulmonary fibrosis. Biochim Biophys Acta. 2015;1852(9):1796–804. Liu Y, et al. Critical role of FOXO3a in carcinogenesis. Mol Cancer. 2018;17(1):104. Qian W, et al. Astragaloside IV modulates TGF-b1-dependent epithelial-mesenchymal transition in bleomycin-induced pulmonary fibrosis. J Cell Mol Med. 2018;22(9):4354–65. Nho RS, et al. Pathological alteration of FoxO3a activity promotes idiopathic pulmonary fibrosis fibroblast proliferation on type i collagen matrix. Am J Pathol. 2011;179(5):2420–30. Al-Tamari HM, et al. FoxO3 an important player in fibrogenesis and therapeutic target for idiopathic pulmonary fibrosis. EMBO Mol Med. 2018;10(2):276–93. Wang S, et al. A single-cell transcriptomic landscape of the lungs of patients with COVID-19. Nat Cell Biol. 2021;23(12):1314–28. Gul A, et al. Pulmonary fibrosis model of mice induced by different administration methods of bleomycin. BMC Pulm Med. 2023;23(1):91. Xu B, et al. Gasdermin D plays a key role as a pyroptosis executor of non-alcoholic steatohepatitis in humans and mice. J Hepatol. 2018;68(4):773–82. Song S et al. Intracellular hydroxyproline imprinting following resolution of bleomycin-induced pulmonary fibrosis . Eur Respir J, 2022. 59(5). Xue Z, et al. Combination therapy of tanshinone IIA and puerarin for pulmonary fibrosis via targeting IL6-JAK2-STAT3/STAT1 signaling pathways. Phytother Res. 2021;35(10):5883–98. Du PR, et al. Calpain inhibition ameliorates scald burn-induced acute lung injury in rats. Burns Trauma. 2018;6:28. Kasamatsu H, et al. A cysteine proteinase inhibitor ALLN alleviates bleomycin-induced skin and lung fibrosis. Arthritis Res Ther. 2023;25(1):156. Li Y, et al. Calpain activation contributes to hyperglycaemia-induced apoptosis in cardiomyocytes. Cardiovasc Res. 2009;84(1):100–10. Liu XY, et al. Lentiviral miR30-based RNA interference against heparanase suppresses melanoma metastasis with lower liver and lung toxicity. Int J Biol Sci. 2013;9(6):564–77. Mattugini N, et al. Inducing Different Neuronal Subtypes from Astrocytes in the Injured Mouse Cerebral Cortex. Neuron. 2019;103(6):1086–95. e5. Strunz M, et al. Alveolar regeneration through a Krt8 + transitional stem cell state that persists in human lung fibrosis. Nat Commun. 2020;11(1):3559. Ono Y, Saido TC, Sorimachi H. Calpain research for drug discovery: challenges and potential. Nat Rev Drug Discov. 2016;15(12):854–76. Arnandis T, et al. Differential functions of calpain 1 during epithelial cell death and adipocyte differentiation in mammary gland involution. Biochem J. 2014;459(2):355–68. Tan WJ, et al. Calpain 1 regulates TGF-beta1-induced epithelial-mesenchymal transition in human lung epithelial cells via PI3K/Akt signaling pathway. Am J Transl Res. 2017;9(3):1402–9. Valenzi E et al. Single-nucleus chromatin accessibility identifies a critical role for TWIST1 in idiopathic pulmonary fibrosis myofibroblast activity . Eur Respir J, 2023. 62(1). Wettstein G, et al. Inhibition of HSP27 blocks fibrosis development and EMT features by promoting Snail degradation. FASEB J. 2013;27(4):1549–60. Bayati P, et al. Induced pluripotent stem cells modulate the Wnt pathway in the bleomycin-induced model of idiopathic pulmonary fibrosis. Stem Cell Res Ther. 2023;14(1):343. Wang B, Pan J, Liu Z. Unraveling FOXO3a and USP18 Functions in Idiopathic Pulmonary Fibrosis through Single-Cell RNA Sequencing of Mouse and Human Lungs. Glob Med Genet. 2023;10(4):301–10. Habrowska-Gorczynska DE, et al. FOXO3a and its regulators in prostate cancer. Int J Mol Sci. 2021;22(22):12530. Additional Declarations No competing interests reported. Supplementary Files 7.WB.docx 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. 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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-3787538","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":263370221,"identity":"aaff12a6-ed8b-41b1-befe-9ad1a901573e","order_by":0,"name":"Qiao LI","email":"","orcid":"","institution":"Xijing Hospital","correspondingAuthor":false,"prefix":"","firstName":"Qiao","middleName":"","lastName":"LI","suffix":""},{"id":263370224,"identity":"1a73c220-4cce-4a83-b9d9-0f4c4b4b2097","order_by":1,"name":"Yu-Tong YE","email":"","orcid":"","institution":"Xi'an Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yu-Tong","middleName":"","lastName":"YE","suffix":""},{"id":263370228,"identity":"67c95485-547f-4f4f-b6e3-7f8691e83f05","order_by":2,"name":"Yi-Liang ZHU","email":"","orcid":"","institution":"Tianshui First-People's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yi-Liang","middleName":"","lastName":"ZHU","suffix":""},{"id":263370229,"identity":"f98163d0-b323-4410-adca-bb1fb2f289a1","order_by":3,"name":"Yu TIAN","email":"","orcid":"","institution":"Xijing Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yu","middleName":"","lastName":"TIAN","suffix":""},{"id":263370231,"identity":"0884a27f-8083-42df-9007-02c7564a2b6d","order_by":4,"name":"Miao-Feng WANG","email":"","orcid":"","institution":"Xijing Hospital","correspondingAuthor":false,"prefix":"","firstName":"Miao-Feng","middleName":"","lastName":"WANG","suffix":""},{"id":263370233,"identity":"0aade639-903e-40f1-b9e6-158b2b46ff86","order_by":5,"name":"Yuan FANG","email":"","orcid":"","institution":"Xijing Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yuan","middleName":"","lastName":"FANG","suffix":""},{"id":263370234,"identity":"b2142047-f581-40eb-b31b-1f2a210261d3","order_by":6,"name":"Lu-Yao HAN","email":"","orcid":"","institution":"Xijing Hospital","correspondingAuthor":false,"prefix":"","firstName":"Lu-Yao","middleName":"","lastName":"HAN","suffix":""},{"id":263370235,"identity":"c4c6061e-4db1-4de6-b68f-4d838e061ea1","order_by":7,"name":"Ran-Ran ZHANG","email":"","orcid":"","institution":"Fourth Military Medical University","correspondingAuthor":false,"prefix":"","firstName":"Ran-Ran","middleName":"","lastName":"ZHANG","suffix":""},{"id":263370237,"identity":"2665db3d-6526-477c-8f94-e070c4741ee5","order_by":8,"name":"Xiao HUANG","email":"","orcid":"","institution":"Xijing Hospital","correspondingAuthor":false,"prefix":"","firstName":"Xiao","middleName":"","lastName":"HUANG","suffix":""},{"id":263370238,"identity":"0e7dda73-633c-4152-99fa-09189e2e23f6","order_by":9,"name":"Jing-Jun ZHOU","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAwElEQVRIiWNgGAWjYBACxoYDbEDKhh/CZSNeS5pkA9FaoMoOk6CFufH4swcf285L6E47Y8DwoewwA//sBoIOSzec2XZbwux2jgHjjHOHGSTuHCCo5Zg0b9vtOpAWZt62wwwGEgmEtBxsA2o5B7aF+S9xWg6zAbUcgGhhJE7LMTbJGeeSgVrSCg72nEvnkbhBQIvhjOPPJD6U2QG1JG988KPMWo5/BkEtBxAcEJMHv3ogkOdvIKhmFIyCUTAKRjoAAFDmRZguQPXGAAAAAElFTkSuQmCC","orcid":"","institution":"Fourth Military Medical University","correspondingAuthor":true,"prefix":"","firstName":"Jing-Jun","middleName":"","lastName":"ZHOU","suffix":""},{"id":263370241,"identity":"3d55890b-1273-4206-937b-457bba3ba2b2","order_by":10,"name":"Feng ZHAO","email":"","orcid":"","institution":"Xijing Hospital","correspondingAuthor":false,"prefix":"","firstName":"Feng","middleName":"","lastName":"ZHAO","suffix":""}],"badges":[],"createdAt":"2023-12-21 14:44:22","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3787538/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3787538/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":49073066,"identity":"2de363e7-6534-4074-83c6-86b5672c35bd","added_by":"auto","created_at":"2024-01-02 17:24:03","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":712611,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative double-label immunofluorescence images and group results of the percentage positive-cells in IPF patients. \u003cstrong\u003eA.\u003c/strong\u003e Double-label staining with antibodies against SFTPC and CAPN1. \u003cstrong\u003eB.\u003c/strong\u003eDouble-label staining with antibodies against SFTPC and p-FoxO3a. Scale bar, 50 mm. \u003cstrong\u003eC.\u003c/strong\u003e Group results on the percentage of SFTPC-, CAPN1-, and p-FoxO3a-positive cells respectively. The data are expressed mean±SEM, **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, vs. the CON group. CAPN1, calpain 1, CON, control; IPF, idiopathic pulmonary fibrosis.\u003c/p\u003e","description":"","filename":"Fig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3787538/v1/dbbc1359621b6064a068742d.jpg"},{"id":49073067,"identity":"ef20841e-c178-46db-ac76-968755422aaa","added_by":"auto","created_at":"2024-01-02 17:24:03","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1082934,"visible":true,"origin":"","legend":"\u003cp\u003eBleomycin\u003cstrong\u003e \u003c/strong\u003estimulates the proliferation of AT2 cells, which expresses substantial CAPN1 and p-FoxO3. \u003cstrong\u003eA \u0026amp; B.\u003c/strong\u003e HE and Masson staining images showing the pulmonary fibrosis caused by bleomycin. The mice were sacrificed on the day of 21 after bleomycin treatment. \u003cstrong\u003eC \u0026amp; D.\u003c/strong\u003e Representative confocal images showing the co-localization of CPAN1 or p-FoxO3a with SFTPC. Scale bar, 50 mm. \u003cstrong\u003eE.\u003c/strong\u003e Group results of the percentage of SFTPC-positive, CAPN1-positive and p-FoxO3a-positive cells. The data are expressed mean±SEM, **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, vs. the CON group. CAPN1, calpain 1, CON, control; BLM, bleomycin.\u003c/p\u003e","description":"","filename":"Fig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3787538/v1/492dff95c91f2722708fcd35.jpg"},{"id":49073063,"identity":"885538fe-49e2-4301-a5fa-a0db3a6694ae","added_by":"auto","created_at":"2024-01-02 17:24:03","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":892021,"visible":true,"origin":"","legend":"\u003cp\u003eAT2 cell-conditioned knockdown of CAPN1 ameliorates bleomycin-induced pulmonary fibrosis. \u003cstrong\u003eA.\u003c/strong\u003e Representative confocal images showing the amount of AT2 cells, indicated by red fluorescence emitted by tomato, and the delivery of shRNA-CAPN1, indicated by green fluorescence emitted by GFP. The mice were sacrificed on the day of 21 after bleomycin treatment. \u003cstrong\u003eB.\u003c/strong\u003e Group results on the percentage of AT2 cell, and the GFP-positive cells. \u003cstrong\u003eC.\u003c/strong\u003e Representative axial and coronal images of lung with micro-CT scanning. \u003cstrong\u003eD.\u003c/strong\u003e Grouped results of poor aeration volume on chest CT.\u003cstrong\u003e E\u003c/strong\u003e. Grouped results of the hydroxyproline content.\u003cstrong\u003e F.\u003c/strong\u003e Representative blotting and densitometric analysis of epithelial and fibrotic protein markers. The chest CT were captured on the day of 21 after bleomycin treatment. After that, the mice were euthanized, and the lung tissues were harvested. The data are expressed mean±SEM, **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, vs. the Sc group,\u003csup\u003e #\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01 vs. the Sc + B group. Sc, scramble RNA; Sh, CAPN1-shRNA; B, bleomycin, COL1A1, alpha-1 type 1 collagen, a-SMA, alpha smooth muscle actin, E-CAD, epithelial type-cadherin.\u003c/p\u003e","description":"","filename":"Fig3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3787538/v1/eb42aafc304be9d850bd5613.jpg"},{"id":49073068,"identity":"d41e0992-4fdf-4be2-a35b-24369d7ecd74","added_by":"auto","created_at":"2024-01-02 17:24:03","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":456671,"visible":true,"origin":"","legend":"\u003cp\u003eBleomycin treatment results in the loss of FoxO3a, the accumulation of CAPN1, and Akt activation in nuclear fraction. \u003cstrong\u003eA.\u003c/strong\u003e Representative Western blotting and grouped densitometric analysis of nuclear and total protein level of FoxO3a and p-FoxO3a. \u003cstrong\u003eB.\u003c/strong\u003e Representative Western blotting and grouped densitometric analysis of nuclear and total protein level of CAPN1. \u003cstrong\u003eC.\u003c/strong\u003e Representative Western blotting and grouped densitometric analysis of nuclear and total protein level of p-Akt. Western blots were performed in six independent biological experiments and for three technique replicates per sample. The data are expressed mean±SEM, **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01 vs. the Sc group,\u003csup\u003e #\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01 vs. the Sc+B group. B, bleomycin; Sc, scrambled RNA; Sh, CAPN1-shRNA.\u003c/p\u003e","description":"","filename":"Fig4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3787538/v1/bc2f9036d826af226c8ee625.jpg"},{"id":49073064,"identity":"88e15540-18b3-4243-9848-fef9c32b9ce5","added_by":"auto","created_at":"2024-01-02 17:24:03","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2127462,"visible":true,"origin":"","legend":"\u003cp\u003eKnockdown of CAPN1 blunted FoxO3a phosphorylation and mesenchymal transition in response to TGFb. \u003cstrong\u003eA.\u003c/strong\u003e Representative images of the living cell morphology. \u003cstrong\u003eB.\u003c/strong\u003e Immunostaining of the epithelial cell marker E-cadherin (E-CAD) and fibrotic protein marker a-SMA. \u003cstrong\u003eC.\u003c/strong\u003e Representative Western blotting and grouped densitometric analysis of E-CAD, and a-SMA.\u003cstrong\u003e D-E\u003c/strong\u003e. Representative Western blotting and grouped results of densitometric analysis of CAPN1, FoxO3a and p-FoxO3a in the nuclear fraction and whole cell lysates. \u003cstrong\u003eF\u003c/strong\u003e. Confocal images showing the intracellular distribution of CAPN1 and p-FoxO3a. Scale bar, 20 mm. The data are expressed mean±SEM, **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01 vs. CON; \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01 vs. scRNA+TGFb group. scRNA, scrambled RNA.\u003c/p\u003e","description":"","filename":"Fig5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3787538/v1/66003c9028e96425e5e0a0f1.jpg"},{"id":49073299,"identity":"5573bd6e-9bf7-4056-a7d8-c8454a2e9698","added_by":"auto","created_at":"2024-01-02 17:32:03","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":671825,"visible":true,"origin":"","legend":"\u003cp\u003eKnockdown of CAPN1 blunted the mesenchymal transition response to TGFb.\u003cstrong\u003eA.\u003c/strong\u003e Representative Western blotting and grouped densitometric analysis of E-CAD and a-SMA. \u003cstrong\u003eB.\u003c/strong\u003e Confocal images showing an accumulation of CAPN1 and Flag in the nucleus upon TGFb stimulation. \u003cstrong\u003eC.\u003c/strong\u003eRepresentative western blotting and grouped densitometric analysis of CAPN1 and Flag in nucleus. \u003cstrong\u003eD.\u003c/strong\u003e Representative images showing nuclear loss of FoxO3a in cells overexpressing CAPN1. \u003cstrong\u003eE.\u003c/strong\u003e Representative Western blotting and grouped densitometric analysis of p-FoxO3a in whole cell lysates, and FoxO3a in nuclear extracts. \u003cstrong\u003eF.\u003c/strong\u003e Representative western blotting and grouped densitometric analysis of Akt in whole cell lysates, and p-Akt nuclear extracts. Scale bar, 20 mm. The data are expressed mean±SEM. *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01 vs. V+TGFb group. V, plasmid vector; O, plasmid overexpressing CAPN1.\u003c/p\u003e","description":"","filename":"Fig6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3787538/v1/64873c9dfe063176a361c7bb.jpg"},{"id":49073300,"identity":"d18165e8-2005-484b-a576-da751bc6cdac","added_by":"auto","created_at":"2024-01-02 17:32:03","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":655886,"visible":true,"origin":"","legend":"\u003cp\u003eFoxO3a was phosphorylated by Akt and FoxO3a mutant blunted the process of mesenchymal transition. \u003cstrong\u003eA.\u003c/strong\u003e Akt inhibitor LY294002 blocked the phosphorylation of Akt at Ser473 and FoxO3a at Ser 253, and antagonized the mesenchymal transition process caused by 10 ng/ml TGFb. \u003cstrong\u003eB.\u003c/strong\u003e Confocal images showing a decrease of a-SMA in cells overexpressed FoxO3a mutant after TGFb stimulation. Scale bar, 20 mm. \u003cstrong\u003eC. \u003c/strong\u003eRepresentative Western blotting and grouped densitometric analysis of FoxO3a, p-FoxO3a, and the mesenchymal transition protein markers a-SMA and E-cadherin (E-CAD) in cells transfected with FoxO3a mutant. The data are expressed mean±SEM. **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01 vs. control (CON); \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01 vs. the corresponding group without Akt inhibitor or mutant. CON, Control; Vector, plasmid vector; Mutant, FoxO3a mutant overexpression.\u003c/p\u003e","description":"","filename":"Fig7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3787538/v1/934af2a7bc96c230f329d9b4.jpg"},{"id":49074162,"identity":"deda5105-02d0-4009-979f-8c708bacf177","added_by":"auto","created_at":"2024-01-02 17:48:05","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1730892,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3787538/v1/60eacdfb-d184-465c-9494-59924c570652.pdf"},{"id":49073071,"identity":"94199262-6dad-45d2-a66d-32eef8f2b64a","added_by":"auto","created_at":"2024-01-02 17:24:05","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":34035425,"visible":true,"origin":"","legend":"","description":"","filename":"7.WB.docx","url":"https://assets-eu.researchsquare.com/files/rs-3787538/v1/95c40fc2080fc1c9586b142e.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Nuclear accumulated μ-calpain in AT2 cell participates in pulmonary fibrosis via inactivating FoxO3a","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIdiopathic pulmonary fibrosis (IPF) is the most common type of pulmonary fibrosis, which has only a medium survival time of only 2\u0026ndash;4 years from diagnosis [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. IPF predominantly presents as usual interstitial pneumonia (UIP) with radiological images, and was characterized by alveolar architecture disruption and extracellular collagen deposition under histological observation, which ultimately leads to respiratory failure and death [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Although Nintedanib and Pirfenidone are currently approved to delay the rate of lung function decline of IPF patients [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], the problem that both drugs fail to halt the progression of pulmonary fibrosis prompted us to gain further insight into its pathophysiological mechanisms.\u003c/p\u003e \u003cp\u003eMesenchymal transition is an important initiating event in IPF [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Till now, alveolar epithelial Type II cells (AT2), endothelial cells, resident fibroblasts have been reported to act as progenitor cells in animal models[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Upon the profibrotic cytokine transforming growth factor-β (TGFβ) stimulation, the progenitor cells differentiates into the so-called myofibrolasts, which produce substantial collagen-rich extracellular matrix[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. The key evidence for the involvement of AT2 cells in the progression of epithelial-mesenchymal transition (EMT) is that AT2 cells express mesenchymal markers α-smooth muscle action (α-SMA) and \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003evimentin\u003c/span\u003e during their differentiation[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Recently, cell fate-map studies corroborate that the aberrant differentiation of AT2 cells is a hallmark and a major contributor to pulmonary fibrosis [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Thus, it is necessary to address the molecular mechanisms behind the role of AT2 cells in pulmonary fibrosis.\u003c/p\u003e \u003cp\u003e\u0026micro;-calpain is a superfamily of Ca\u003csup\u003e2+\u003c/sup\u003e-dependent nonlysosomal cysteine protease, which consist of a large catalytic subunit, namely calpain 1 (CAPN1) and a small regulatory subunit CAPN4 [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. We and others have demonstrated that CAPNs participate in a variety of pathophysiological processes, i.e., plasma membrane disruption, mitochondrial damage, transform of vascular permeability and cell motility [\u003cspan additionalcitationids=\"CR15 CR16\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Of note, \u0026micro;-calpain is implicated in the progression of IPF patients [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Conditional genetic ablation of CAPN1 in pleural mesothelial cell and macrophage has been documented to blunt bleomycin-induced pulmonary fibrosis [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Although a reciprocal interaction between \u0026micro;-calpain and TGFβ, and activation of Smad2/3, Akt, and the extracellular signal-regulated kinase explain the harmful effects [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], very little is known on the transcription factors involved, and the role of \u0026micro;-calpain in the fate of AT2 cells remains unclear.\u003c/p\u003e \u003cp\u003eFoxO3a belongs to the forkhead box protein class O subfamily. It is a transcription factor involved in cell proliferation, differentiation, apoptosis, and metabolism [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. The transcriptional activity of FoxO3a depends on its phosphorylation status. Upon phosphorylation, FoxO3a translocates from the nucleus to cytosol and becomes inactivated [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. FoxO3a stimulates the expression of p27, a negative regulator of the G1-S transition in cell cycle progression, which impedes the proliferation of fibroblast and protects against pulmonary fibrosis [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. FoxO3a decline/suppression has been implicated in coronavirus disease 2019 (COVID)-pulmonary fibrosis, and bleomycin-induced pulmonary fibrosis [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Thus, we hypothesized that FoxO3a is a targeted transcription factor of \u0026micro;-calpain, which dictates the fate of AT2 cells.\u003c/p\u003e \u003cp\u003eIn this study, we firstly examined the expression of CAPN1 and FoxO3a in AT2 cells in the specimen of IPF patients. Secondly, we targeted knockdown of CAPN1 in AT2 cells and evaluated the outcome of bleomycin-induced pulmonary fibrosis. Finally, we determined whether FoxO3a is a targeted transcription factor of CAPN1 in the EMT process, and investigated the role of phosphorylation modification via Akt in this scenario. Our data for the first demonstrate that nuclear accumulation of \u0026micro;-calpain is a critical step in the progression of IPF, which inactivates FoxO3a via Akt-dependent phosphorylation, and accelerates the transition of AT2 cells to mesenchymal population.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMaterials\u003c/h2\u003e \u003cp\u003eBleomycin was purchased from Nippon (Kayaku, Japan). PD150606 (Cat#HY-100529) and TGFβ (Cat#HY-P70543) were obtained from MedChem Express (Monmouth Junction, NJ, USA). Antibodies against Calpain 1 (Cat#C5736), STFPC (Cat#PA5-71680), p-FoxO3a (Ser253), FoxO3a (Cat#12819), E-cadherin (Cat#3195), α-SMA (Cat#19245), COL1A1 (Cat#72026), p-Akt (Ser473, Cat#4060), Akt (Cat#4685S), and GAPDH (Cat#2118) was obtained from Sigma-Aldrich(Merck, USA), Invitrogen (Eugene, OR, USA), Abcam (Cambridge, MA, USA) and Cell Signaling Technology (Danvers, MA, USA) respectively. Tetramethylrhodamine-conjugated Goat anti-Rabbit IgG (Cat#T2769) and LipofectamineTM 3000 (Cat#L3000015) were purchased from Thermo Fisher (Illinois, IL, USA). DyLight488 AffiniPure Goat Anti-Mouse IgG (Cat#E032210) was purchased from EarthOx (SanFrancisco, CA, USA). The primers and siRNAs were synthesized by GenePharma (Shanghai, China), and pcDNA3.1(+)-Flag-CAPN1 and pcDNA3.1(+)-GFP-FoxO3a-mutant were purchased from Sangon Biotech (Shanghai, China). BCA Protein Assay Kit was acquired from Thermo Fisher (Waltham, MA, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003ePathogen-free C57BL/6 mice aged at 8 to 10 weeks were provided by the Laboratory Animal Centre of the Air Force Medical University. sftpc-CreERT2 mice were purchased from Cyagen Com. (Suzhou, China). The animals were housed in constant temperature and humidity with a 12 h light cycle. Animals were allowed food and water ad libitum. Male mice were used for all experiments, and all animal experiments were conducted in strict accordance with the Guidelines of Health and guidelines for use, and were permitted by the Animal Welfare and Ethics Institution of Air Force Military Medical University (IACUC-20230099).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eHuman tissues\u003c/h2\u003e \u003cp\u003eEthical approval for this study (No. 20202052) was approved by the Medical Ethics Committee of the Xijing Hospital of Air Force Medical University. The study was conducted in accordance with the World Medical Association's Code of Ethics (Declaration of Helsinki). Informed consent was obtained from each subject for the study protocol. Fours lung tissue biopsies were obtained with the help of bronchoscopic or ultrasound-guidance in Xijing Hospital. IPF patients were diagnosed with the histopathologic examination and/or high resolution computed tomography. Four normal lung biopsies from resection of cancer were used as controls.\u003c/p\u003e \u003c/p\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003ePreparation of pulmonary fibrosis model\u003c/h2\u003e \u003cp\u003eWild type C57/BL6 mice were anesthetized with an intraperitoneal injection of 0.3% pentobarbital sodium and randomly divided into: (1) control group (CON), (2) bleomycin group (BLM). To study the role of \u0026micro;-calpain in AT2 cells, sftpc-CreERT2 mice were randomly divided into (1) scrambled RNA group (Sc), (2) CAPN1-shRNA group (Sh), (3) scrambled RNA with bleomycin group (Sc\u0026thinsp;+\u0026thinsp;B), and (4) CAPN1-shRNA with bleomycin group (Sh\u0026thinsp;+\u0026thinsp;B). Bleomycin was dissolved in saline, and a volume of 50 \u0026micro;l containing 2.5 mg/kg bleomycin was intratracheally given using a 22 G plastic cannula. Same amount of saline was delivered to the mice as a control. At day 21, mice were euthanized, and the lung tissue was harvested[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eAAV-FLEX-shRNA-CAPN1 tagged with GFP and its delivery to tomato-tagged sftpc-CreERT2 mice\u003c/h2\u003e \u003cp\u003eA GFP-tagged adenovirus associated virus vector (AAV2/6) containing either mir30-based shRNA against CAPN1 or scramble RNA, with the reading frames inverted in a flip-excision (FLEX) cassette were prepared by HANBio Com. (Shanghai, China) [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. The interfering sequence to CAPN1 was CUGGGCUGCUCCAUUAAUA.\u003c/p\u003e \u003cp\u003eAfter receiving 1 mg tamoxifen intraperitoneally injection for continuous 7 days to induce Cre expression in AT2 cells, adult sftpc-CreERT2 mice was administered via trachea a volume of 35 \u0026micro;l AAV2/6 virus suspensions with a titer of 1.5\u0026times;10\u003csup\u003e12\u003c/sup\u003evg/ml. Three weeks later, pulmonary fibrosis was induced by bleomycin as described above. Mice were euthanized on day 21 after bleomycin injection, and lung tissue was harvested\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eIn vivo micro-computed tomography (micro-CT) analysis\u003c/h2\u003e \u003cp\u003eBriefly, a non-invasive micro-CT scanner (IvIS Lumina K series III, PerkinElmer) with computer software (Living Image v. 4.3.1, PerkinElmer) was employed to analyze lung structure. The X-ray system used a micro-focus tube with a spot size of 5 mm. The images were acquired at 90 kV, 160 mA and the wide field of view scanning at 40 mm. Lung regions were classified into 4 categories by CT attenuation densities: hyper-inflated, density range from \u0026minus;\u0026thinsp;861 to -1000 Hounsfield units (HU); normally-aerated, range from \u0026minus;\u0026thinsp;435 to -860 HU; poorly-aerated, density range from \u0026minus;\u0026thinsp;121 to -434 HU; non-aerated, range from \u0026minus;\u0026thinsp;120 to +\u0026thinsp;120 HU. And then a total mask was established to cover the density range of -1000 to +\u0026thinsp;121 HU. 8\u0026ndash;10 axial CT images that could be recognized in the lung were painted. The poorly aerated regions were calculated to reflect pulmonary fibrosis, as described previously [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eHematoxylin-eosin and Masson's trichrome staining, and hydroxyproline measurement\u003c/h2\u003e \u003cp\u003eAt the end of the experiment, the left lung tissues in mice were fixed in 4% paraformaldehyde solution for 24 h, and then embedded in paraffin. A mid-sagittal tissue section was prepared with 4 \u0026micro;m thickness. The sections were then stained with hematoxylin-eosin to evaluate alveolar structure [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Masson's trichrome staining was performed to determine collagen deposition. Ashcroft score ranged from 0 to 8 was evaluated by an examiner, who was blind to the mice allocation, to determine the severity of pulmonary fibrosis, as described previously [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. To determine hydroxyproline content, the lung tissues were homogenated and centrifuged. The supernatant was used to measure the content of hydroxyproline using the Hydroxyproline assay kit according to the manufacturer's protocol [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eCell transfection of constructed-expression vectors and siRNA\u003c/h2\u003e \u003cp\u003eFull-length human FoxO3a (NM_001455.4) cDNA was synthesized and cloned into the HindIII and NotI sites of pcDNA3.1 which contains GFP. CAPN1 (NM_005186.4) was tagged with Flag and cloned into BamHI and XhoI sites of pcDNA3.1. To study the role of FoxO3a phosphorylation in the process of mesenchymal transition, the phosphorylation sites including Thr32 and Ser253 were mutated to alanine. The sequence alignment for the cDNAs was carried out using DNAMAN software (version 6.0). CAPN1-siRNA was employed to determine the role of \u0026micro;-calpain in the process of mesenchymal transition, the sequences were as follows:\u003c/p\u003e \u003cp\u003eCAPN1-siRNA: 5\u0026prime;-GGUUACCGAGUGGUACGAGUU-3\u0026prime;.\u003c/p\u003e \u003cp\u003eScrambled siRNA: 5\u0026prime;-UUCUCCGAACGUGUCACGUTT-3\u0026prime;.\u003c/p\u003e \u003cp\u003eThe human type II A549 alveolar epithelial cells were cultured with Dulbecco\u0026rsquo;s Modified Eagle Medium containing 10% fetal bovine serum. When the density of the cells reached 60% confluence, the cells were transfected with CAPN1-siRNA or plasmid vector containing CAPN1 or FoxO3a mutant, which was dissolved in a mixture of Lipofectamine 3000 reagent and Opti-MEM. Scramble RNA and empty vector were used negative controls. Six hour later, the medium was replaced with DMEM containing10% FBS. The transfection efficiency was evaluated with Western blot [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. At 24 h after transfection, the cells were incubated with 5 or 10 ng/ml TGFβ for another 24 h, and then harvested to evaluate mesenchymal transition.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eImmunofluorescence\u003c/h2\u003e \u003cp\u003eThe slice was blocked with phosphate buffer containing 5% donkey serum in at room temperature for 30 min, and permeabilization with 0.3% Triton-X 100 for 15 min. After that, the slice was incubated with antibodies against SFTPC, CAPN1, E-cadherin, α-SMA, p-FoxO3a, Flag and FoxO3a at 4\u0026deg;C overnight. All the antibodies were used at a dilution of 1:100. After the slice were incubated with tetramethylrhodamine-conjugated secondary antibodies against rabbit IgG and/or DyLight488-conjugated secondary antibodies against mouse IgG for 1 hour at room temperature, the nuclei were counterstained with DAPI (20 \u0026micro;g/ml). Finally, the slice was examined with a laser confocal microscope equipped with an FV-10-ASW system (Olympus FV1000, Tokyo, Japan), as described previously [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eWestern blotting\u003c/h2\u003e \u003cp\u003eTotal protein was extracted with a lysis buffer containing (in mM) Tris (pH\u0026thinsp;=\u0026thinsp;7.4) 50, NaCl 150, 1% NP-40, 0.5% sodiumdeoxycholate, 0.1% SDS. After centrifugation at 12,000 g for 15 min, the supernatants were collected. To extract nuclear proteins, the tissue or cell was incubated with a buffer containing (in mM) Tris (pH\u0026thinsp;=\u0026thinsp;8.0) 10, KCl 10, MgCl2 1.5, DTT 1, EDTA 0.2, 5% glycerol, and protease inhibitors. The sample was then snap-frozen in liquid nitrogen, and homogenated. After centrifugation at 12,000 g for 10 min, the sediments were lysed with a buffer containing (in mM) Tris (pH\u0026thinsp;=\u0026thinsp;8.0) 20, NaCl 420, MgCl2 1.5, DTT 0.5, EDTA 0.2, 25% glycerol, and protease inhibitors. The lysate was centrifuged at 12,000 g for 10 min, and the supernatants were collected as the nuclear fraction. Protein concentration was determined with BCA method. The samples were separated with 6\u0026ndash;10% SDS-polyacrylamide gels and transferred to the PVDF membranes. After blocking with 5% bovine serum albumin in Tris buffer, the membranes were incubated with antibodies against CAPN1, collagen I, α-SMA, E-cadherin, FoxO3a, p-FoxO3a, Akt, p-Akt, GAPDH, Lamin B at 4\u0026deg;C overnight (1:1000 for all), followed by incubation with horseradish peroxidase-conjugated secondary antibodies (1:5000) at room temperature for 1 h. Proteins were visualized using an ultrasensitive ECL chemiluminescence substrate. GAPDH and Lamin B were used as loading controls. The signal intensity was quantified with the aid of Image J software [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll data are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean, which were performed in at least three independent biological experiments. GraphPad Prism was used to perform statistical analysis. Student\u0026rsquo;s t-test was employed for comparisons within two groups. ANOVA followed by Tukey\u0026rsquo;s post hoc test was performed for multiple group comparisons. \u003cem\u003eP\u003c/em\u003e value less than 0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003e1. An increase of both \u0026micro;-calpain and p-FoxO3a in AT2 cells represents a hallmark in IPF patients\u003c/b\u003e \u003c/p\u003e \u003cp\u003eCompared with the control group, IPF patients exhibited excessive proliferation of AT2 cells, manifested as positive staining of SFTPC protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA \u0026amp; B). These cells were clustered, and the percentage of positive cells reached 45.43\u0026plusmn;1.6 per field, which was in contrast to 10.73\u0026plusmn;0.9 per field in the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-C). These results suggest that the impediment of AT2 to AT1 cells differentiation is a critical pathological characteristic of pulmonary fibrosis. Immunofluorescence double staining revealed that substantial CAPN1 and p-FoxO3a were located in AT2 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA \u0026amp; B). The percentage of CAPN1- and p-FoxO3a-positive cells rose from 7.3\u0026plusmn;0.9, and 8.72 \u0026plusmn;1.4 in the control group to 53.22\u0026plusmn;4.4 and 47.0\u0026plusmn;2.8 per field respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). These results raise the possibility that both molecules are involved in pulmonary fibrosis.\u003c/p\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e2. Targeted knockdown of CAPN1 in AT2 cells ameliorates bleomycin-induced pulmonary fibrosis\u003c/h2\u003e \u003cp\u003eCompared with the ones treated with saline (control group), mice in bleomycin-treated group displayed disruption of the architecture of alveolar, an increase of the interalveolar septal thickness and collagen deposition (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA \u0026amp; B), manifested as HE and Masson\u0026rsquo;s trichrome staining. Furthermore, bleomycin resulted in the excessive proliferation of AT2 cells, which express substantial CAPN1 and phosphorylated FoxO3a (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC-E). These results corroborate that \u0026micro;-calpain and FoxO3a are involved in the aberrant differentiation of AT2 cells.\u003c/p\u003e\u003cp\u003eTo determine the role of \u0026micro;-calpain in this scenario, FLEx switch (Cre-On) adeno-associated viral vector carrying GFP and shRNA against CAPN1 was delivered via trachea to Tomato-tagged sftpc-CreERT2 mice, which specifically knockdown CAPN1 in AT2 cells. As expected, bleomyocin caused excessive proliferation of AT2 cells, manifested as the red fluorescence emitted by tagged protein tomato. Furthermore, AT2 cells in bleomyocin-treated mice also exhibited green fluorescence emitted by GFP, indicating the successful delivery of AAV vectors carrying shRNA against CAPN1 to AT2 cells. Of particular interest, shRNA-CAPN1 did not antagonize bleomycin-induced an increase of AT2 cell number (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA \u0026amp; B). These results indicate that \u0026micro;-calpain is not involved in AT2 cells proliferation in the process of pulmonary fibrosis.\u003c/p\u003e \u003cp\u003eChest CT scanning showed that shRNA-CAPN1 reduced the nonaerated regions and the content of hydroxyproline in bleomycin-treated mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC-E). Biochemical data and Western blotting revealed that CAPN1-shRNA antagonized bleomycin-induced alterations of α-SMA, Collagen-1, and E-cadherin content (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). These results suggest that \u0026micro;-calpain is an instigator to aggravate mesenchymal transition. Last but not the least, both scramble-RNA and shRNA had no appreciable effects in normal mice.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3. Nuclear accumulation of \u0026micro;-calpain and loss of FoxO3a are detected in fibrosis tissue\u003c/h2\u003e \u003cp\u003eAs transcriptional factor is a major determinant in mesenchymal transition, nuclear fraction was isolated. Compared with that in the scramble group, the mice in bleomycin-treated group showed a decline of FoxO3a in the nucleus, but an increase of p-FoxO3a in whole cell lysates (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). \u0026micro;-calpain, and its downstream molecule Akt, were also examined. To our surprise, bleomycin resulted in an accumulation of CAPN1 and Akt activation in the nuclear fraction (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB \u0026amp; C), along with an increase of CAPN1 and p-Akt in whole cell lysates (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB \u0026amp; C). Moreover, all these deleterious effects were blocked by targeting knockdown of CAPN1 in AT2 cells with shRNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). These results suggest that nuclear accumulation of CAPN1 stimulated FoxO3a phosphorylation via Akt and accelerated the translocation of phosphorylated FoxO3a out of nucleus.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e4. Nuclear accumulation of \u0026micro;-calpain precedes FoxO3a phosphorylation and the mesenchymal transition\u003c/h2\u003e \u003cp\u003eAfter 10 ng/ml TGFβ stimulation for 48 h, normal polygonal shape epithelial cells exhibit a spindle -like appearance (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Confocal images and Western blotting showed that these cells presented more mesenchymal marker α-SMA, and less alveolar epithelial marker E-cadherin (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB \u0026amp; C), indicating the differentiation of epithelial cells into myofibroblasts. Furthermore, stimulated an increase of total CAPN1 and nuclear accumulation of CAPN1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). In contrast, TGFβ resulted in a decrease of FoxO3a in the nucleus, and an increase in FoxO3a phosphorylation in the cytoplasm (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE-F). Of particular interest, all these alterations were blocked by CAPN1-siRNA. These results suggest that nuclear accumulation of \u0026micro;-calpain precedes FoxO3a phosphorylation and the mesenchymal transition.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo establish the critical role of \u0026micro;-calpain, pcDNA3.1-CAPN1-Flag overexpression plasmid was constructed. As expected, the cells overexpressing CAPN1 were more sensitive to TGFβ stimulation than that in the control group. TGFβ at 5 ng/ml, was inclined to induce mesenchymal transition, and presented less alveolar epithelial marker E-cadherin, and more mesenchymal marker α-SMA (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Immunofluorescence images and Western blotting showed that the cells overexpressing CAPN1 exhibited strong-positive staining of CAPN1 and Flag in the nucleus (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB \u0026amp; C). Furthermore, an increased CAPN1 caused less FoxO3a in the nuclei fraction, and more p-FoxO3a in the cytoplasm upon TGFβ stimulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD \u0026amp; E).\u003c/p\u003e \u003cp\u003eIn addition, the overexpression of CAPN1 increased the level of both total and nuclear p-Akt after TGFβ stimulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF). These results suggest that Akt is involved in \u0026micro;-calpain-elicited the phosphorylation of FoxO3a and mesenchymal transition.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e5. Calpain inactivates FoxO3a in an Akt-dependent phosphorylation manner\u003c/h2\u003e \u003cp\u003eCompared with the control group, TGFβ enhanced Akt phosphorylation at residue Ser473, and FoxO3a phosphorylation at residue Ser253 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). Moreover, 20 \u0026micro;M LY294002, an inhibitor of Akt, not only blocked Akt phosphorylation, but also the phosphorylation of FoxO3a, which were accompanied with the impediment of mesenchymal transition (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). These results validate that Akt is involved in regulating FoxO3a activity via phosphorylation modification.\u003c/p\u003e \u003cp\u003eTo determine whether the transcription activity of FoxO3a depends on its phosphorylation status, the phosphorylation sites of both Thr32 and Ser253 were mutated to alanine, and pcDNA3.1-GFP-FoxO3a mutant were constructed. Compared with the control, the cells transfected with FoxO3a mutant were more resistant to TGFβ (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB), manifested as less α-SMA. Western blotting data showed that FoxO3a mutant reduced the phosphorylation of endogenous FoxO3a and α-SMA, but increased E-cadherin (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). These results support that FoxO3a is involved in maintaining the epithelial character. Furthermore, taking into consideration the finding that calpain activated Akt (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, \u0026amp; Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF), these data suggest that FoxO3a is regulated by \u0026micro;-calpain via Akt-dependent manner.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003ePrevious studies have demonstrated that calpain is upregulated in IPF patients [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Thus, it is meaningful to identify the cells source in this clinical setting. Here, we observed that (1) IPF patients exhibited numerous AT2 cells, which expressed substantial \u0026micro;-calpain subunit CAPN1 and p-FoxO3a, (2) Targeted knockdown of \u0026micro;-calpain subunit CAPN1 in AT2 cells reversed the bleomycin-induced pulmonary fibrosis, (3) CAPN1 was accumulated in the nucleus, whereas p-FoxO3a translocated out of nucleus, (4) Inhibition of Akt alleviated the FoxO3a phosphorylation, and mutation of the phosphorylation residues of FoxO3a antagonized calpain-elicited mesenchymal transition. Our data provide evidence for the first that nuclear accumulation of \u0026micro;-calpain in AT2 cells is a critical step to aggravate pulmonary fibrosis, and identify FoxO3a as a transcription factor conferring to \u0026micro;-calpain-elicited mesenchymal transition. Our data suggest that blockade of the aberrant differentiation of AT2 cells via inhibiting \u0026micro;-calpain is a new strategy to mitigate the progression of IPF.\u003c/p\u003e \u003cp\u003eHere, specimens from IPF patients exhibited numerous SFTPC-positive cells. These results are consistent with previous findings that a hyperplasia of AT2 cells is a prominent feature of IPF [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Mir30-base AAV-FLEX system is a new technique to specifically knockdown the targeted molecule in specific cell [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Using this method, we observed that CAPN1-shRNA decreased the CAPN1 content (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e) and pulmonary fibrosis. These results for the first demonstrate that targeted regulation of CAPN1 in AT2 cells is a potential strategy to treat IPF. Our data are in contrast to previous findings showing that \u0026micro;-calpain in pleural mesothelial cells and macrophages were involved in the process of IPF [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Therefore, the contribution of \u0026micro;-calpain in different type of cells to the progression of IPF needs to be further evaluated. One interesting observation in this study was CAPN1-shRNA did not alter the number of AT2 cells. These results strongly suggest that \u0026micro;-calpain participates in mesenchymal transition. In addition, AT2 cells have been recently identified to differentiate to a specialized cell population marked with keratin-8/claudin-4 positive, a regenerative intermediate cell state which exhibited senescence and failed to differentiate to AT1 cells [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Thus, the role of \u0026micro;-calpain in this scenario also warranted further studied.\u003c/p\u003e \u003cp\u003e\u0026micro;-calpain is a cytosolic and substrate-specific cysteine protease [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Here, we observed an accumulation of the catalytic subunit CAPN1 in the nucleus after bleomycin treatment. These data are consistent with previous findings showing nuclear translocation of \u0026micro;-calpain in mammary gland involution [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Furthermore, these results corroborate the point that modification of transcription factor activity is an important step to trigger mesenchymal transition. However, the results are in contrast to our previous findings showing an accumulation of \u0026micro;-calpain in the plasma membrane and mitochondria after stressful stimulation [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Thus, the unidentified regulator, which is in charge of the trajectory of \u0026micro;-calpain, warrants to be elucidated.\u003c/p\u003e \u003cp\u003eIt has been reported that Smad-dependent pathway and Akt-dependent pathway were involved in calpain-elicited pulmonary fibrosis [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. However, the transcription factors located downstream of calpain remains elusive. Here, we demonstrated that nuclear accumulation of calpain was accompanied with the translocation of p-FoxO3a out of nucleus, and that blockade of FoxO3a phosphorylation antagonized calpain-elicited mesenchymal transition. These results for the first provide evidence that FoxO3a protects against the aberrant differentiation of AT2 cells, and that FoxO3a is a new targeted transcription factor of calpain. In addition to FoxO3a, Twist1, β-catenin, lymphoid enhancer factor and snail are also been demonstrated to participate in pulmonary fibrosis [\u003cspan additionalcitationids=\"CR41\" citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Whether calpain acts as a nexus to regulate these transcription factors in the process of pulmonary fibrosis merits further investigation.\u003c/p\u003e \u003cp\u003eTill now, the function of FoxO3a in fibroblast in the lung tissue is well documented [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Knockdown of FoxO3a stimulated the proliferation of fibroblast, thus exacerbating pulmonary fibrosis [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Here, we demonstrated that AT2 cells in IPF patients did exhibit substantial p-FoxO3a. These results for the first provide evidence that FoxO3a participates in the aberrant differentiation of AT2 cells in pulmonary fibrosis. Furthermore, transfection of FoxO3a mutant carrying Thr32 and Ser253A mitigated the process of mesenchymal transition, these data support the point that FoxO3a is involved in maintaining the epithelial character. In this study, knockdown of CAPN1 reduced the level of p-FoxO3a and Akt activation, and the Akt inhibitor alleviated endogenous FoxO3a phosphorylation. This is consistent with previous findings that FoxO3a was regulated by Akt [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Particularly, these data demonstrate that \u0026micro;-calpain inactivates FoxO3a in Akt-dependent manner [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e], and that blockade of p-FoxO3a activity by \u0026micro;-calpain is a critical step in instigating the process of the aberrant differentiation of AT2 cells, which accelerates the progression of IPF.\u003c/p\u003e \u003cp\u003eTaken together, our data for the first time demonstrate that nuclear accumulation of \u0026micro;-calpain in AT2 cells is a critical step to aggravate the prognosis of pulmonary fibrosis, which involves inactivation of FoxO3a in Akt-dependent manner, instigating the differentiation of AT2 cells to mesenchymal population. We propose that interfering mesenchymal transition by AT2 cell-specific weakening \u0026micro;-calpain activity is a new approach to improve the prognosis of IPF. Further comprehensive understanding the biological behavior of \u0026micro;-calpain in AT2 cells differentiation will help exploiting better strategies to limit IPF.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe work was supported by grants from the National Natural Science Foundation of China (no: 81570054 \u0026amp; 81772070).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no relevant financial interests to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot appliciable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe participant has consented to the submission of the case report to the journal.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are available on request from the corresponding author, Feng ZHAO, Xijing Hospital. Email address: [email protected].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thanks Ms. Li-Xia HAO for preparing tissue sections, and Mr. Hai-Feng ZHANG for taking confocal images.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal experiments were conducted in strict accordance with the Guidelines of Health and guidelines for use, and were permitted by the Animal Welfare and Ethics Institution of Air Force Military Medical University (IACUC-20230099).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eRaghu G, et al. Idiopathic pulmonary fibrosis (an Update) and progressive pulmonary fibrosis in adults: An official ATS/ERS/JRS/ALAT clinical practice guideline. Am J Respir Crit Care Med. 2022;205(9):e18\u0026ndash;e47.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMei Q, et al. Idiopathic pulmonary fibrosis: An update on pathogenesis. Front Pharmacol. 2021;12:797292.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ede Andrade JA, et al. Effect of antifibrotic therapy on survival in patients with idiopathic pulmonary fibrosis. Clin Ther. 2023;45(4):306\u0026ndash;15.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLancaster L, et al. Safety and survival data in patients with idiopathic pulmonary fibrosis treated with nintedanib: pooled data from six clinical trials. BMJ Open Respir Res. 2019;6(1):e000397.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKatzen J, Beers MF. Contributions of alveolar epithelial cell quality control to pulmonary fibrosis. J Clin Invest. 2020;130(10):5088\u0026ndash;99.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNieto MA, et al. EMT: 2016. Cell. 2016;166(1):21\u0026ndash;45.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChapman HA. Epithelial-mesenchymal interactions in pulmonary fibrosis. Annu Rev Physiol. 2011;73:413\u0026ndash;35.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHewlett JC, Kropski JA, Blackwell TS. \u003cem\u003eIdiopathic pulmonary fibrosis: Epithelial-mesenchymal interactions and emerging therapeutic targets\u003c/em\u003e. Matrix Biol, 2018. 71\u0026ndash;2: p.\u0026nbsp;112\u0026ndash;127.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKobayashi Y, et al. Persistence of a regeneration-associated, transitional alveolar epithelial cell state in pulmonary fibrosis. Nat Cell Biol. 2020;22(8):934\u0026ndash;46.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYamada M, et al. Dual-immunohistochemistry provides little evidence for epithelial-mesenchymal transition in pulmonary fibrosis. Histochem Cell Biol. 2008;129(4):453\u0026ndash;62.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSelman M, Pardo A. The leading role of epithelial cells in the pathogenesis of idiopathic pulmonary fibrosis. Cell Signal. 2020;66:109482.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNureki SI, et al. Expression of mutant Sftpc in murine alveolar epithelia drives spontaneous lung fibrosis. J Clin Invest. 2018;128(9):4008\u0026ndash;24.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNian H, Ma B. Calpain-calpastatin system and cancer progression. Biol Rev Camb Philos Soc. 2021;96(3):961\u0026ndash;75.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang RR, et al. Severe burn-induced mitochondrial recruitment of calpain causes aberrant mitochondrial dynamics and heart dysfunction. Shock. 2023;60(2):255\u0026ndash;61.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLu HT, et al. CaMKII/calpain interaction mediates ischemia/reperfusion injury in isolated rat hearts. Cell Death Dis. 2020;11(5):388.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFriedrich EE, et al. Endothelial cell Piezo1 mediates pressure-induced lung vascular hyperpermeability via disruption of adherens junctions. Proc Natl Acad Sci U S A. 2019;116(26):12980\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGarcia-Trevijano ER, et al. Calpains, the proteases of two faces controlling the epithelial homeostasis in mammary gland. Front Cell Dev Biol. 2023;11:1249317.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZou M, et al. Inhibition of the ERK1/2-ubiquitous calpains pathway attenuates experimental pulmonary fibrosis in vivo and in vitro. Exp Cell Res. 2020;391(1):111886.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhou LL, et al. Pleural mesothelial cell migration into lung parenchyma by calpain contributes to idiopathic pulmonary fibrosis. J Cell Physiol. 2022;237(1):566\u0026ndash;79.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang L, et al. Myeloid cell-specific deletion of Capns1 prevents macrophage polarization toward the M1 phenotype and reduces interstitial lung disease in the bleomycin model of systemic sclerosis. Arthritis Res Ther. 2022;24(1):148.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi FZ, et al. Crosstalk between calpain activation and TGF-beta1 augments collagen-I synthesis in pulmonary fibrosis. Biochim Biophys Acta. 2015;1852(9):1796\u0026ndash;804.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu Y, et al. Critical role of FOXO3a in carcinogenesis. Mol Cancer. 2018;17(1):104.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eQian W, et al. Astragaloside IV modulates TGF-b1-dependent epithelial-mesenchymal transition in bleomycin-induced pulmonary fibrosis. J Cell Mol Med. 2018;22(9):4354\u0026ndash;65.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNho RS, et al. Pathological alteration of FoxO3a activity promotes idiopathic pulmonary fibrosis fibroblast proliferation on type i collagen matrix. Am J Pathol. 2011;179(5):2420\u0026ndash;30.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAl-Tamari HM, et al. FoxO3 an important player in fibrogenesis and therapeutic target for idiopathic pulmonary fibrosis. EMBO Mol Med. 2018;10(2):276\u0026ndash;93.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang S, et al. A single-cell transcriptomic landscape of the lungs of patients with COVID-19. Nat Cell Biol. 2021;23(12):1314\u0026ndash;28.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGul A, et al. Pulmonary fibrosis model of mice induced by different administration methods of bleomycin. BMC Pulm Med. 2023;23(1):91.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXu B, et al. Gasdermin D plays a key role as a pyroptosis executor of non-alcoholic steatohepatitis in humans and mice. J Hepatol. 2018;68(4):773\u0026ndash;82.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSong S et al. \u003cem\u003eIntracellular hydroxyproline imprinting following resolution of bleomycin-induced pulmonary fibrosis\u003c/em\u003e. Eur Respir J, 2022. 59(5).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXue Z, et al. Combination therapy of tanshinone IIA and puerarin for pulmonary fibrosis via targeting IL6-JAK2-STAT3/STAT1 signaling pathways. Phytother Res. 2021;35(10):5883\u0026ndash;98.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDu PR, et al. Calpain inhibition ameliorates scald burn-induced acute lung injury in rats. Burns Trauma. 2018;6:28.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKasamatsu H, et al. A cysteine proteinase inhibitor ALLN alleviates bleomycin-induced skin and lung fibrosis. Arthritis Res Ther. 2023;25(1):156.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi Y, et al. Calpain activation contributes to hyperglycaemia-induced apoptosis in cardiomyocytes. Cardiovasc Res. 2009;84(1):100\u0026ndash;10.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu XY, et al. Lentiviral miR30-based RNA interference against heparanase suppresses melanoma metastasis with lower liver and lung toxicity. Int J Biol Sci. 2013;9(6):564\u0026ndash;77.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMattugini N, et al. Inducing Different Neuronal Subtypes from Astrocytes in the Injured Mouse Cerebral Cortex. Neuron. 2019;103(6):1086\u0026ndash;95. e5.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStrunz M, et al. Alveolar regeneration through a Krt8\u0026thinsp;+\u0026thinsp;transitional stem cell state that persists in human lung fibrosis. Nat Commun. 2020;11(1):3559.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOno Y, Saido TC, Sorimachi H. Calpain research for drug discovery: challenges and potential. Nat Rev Drug Discov. 2016;15(12):854\u0026ndash;76.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eArnandis T, et al. Differential functions of calpain 1 during epithelial cell death and adipocyte differentiation in mammary gland involution. Biochem J. 2014;459(2):355\u0026ndash;68.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTan WJ, et al. Calpain 1 regulates TGF-beta1-induced epithelial-mesenchymal transition in human lung epithelial cells via PI3K/Akt signaling pathway. Am J Transl Res. 2017;9(3):1402\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eValenzi E et al. \u003cem\u003eSingle-nucleus chromatin accessibility identifies a critical role for TWIST1 in idiopathic pulmonary fibrosis myofibroblast activity\u003c/em\u003e. Eur Respir J, 2023. 62(1).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWettstein G, et al. Inhibition of HSP27 blocks fibrosis development and EMT features by promoting Snail degradation. FASEB J. 2013;27(4):1549\u0026ndash;60.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBayati P, et al. Induced pluripotent stem cells modulate the Wnt pathway in the bleomycin-induced model of idiopathic pulmonary fibrosis. Stem Cell Res Ther. 2023;14(1):343.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang B, Pan J, Liu Z. Unraveling FOXO3a and USP18 Functions in Idiopathic Pulmonary Fibrosis through Single-Cell RNA Sequencing of Mouse and Human Lungs. Glob Med Genet. 2023;10(4):301\u0026ndash;10.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHabrowska-Gorczynska DE, et al. FOXO3a and its regulators in prostate cancer. Int J Mol Sci. 2021;22(22):12530.\u003c/span\u003e\u003c/li\u003e\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, calpain, alveolar epithelial Type II cells, FoxO3a, epithelial to mesenchymal transition, Akt","lastPublishedDoi":"10.21203/rs.3.rs-3787538/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3787538/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u0026micro;-calpain is implicated in pulmonary fibrosis, however its role in the aberrant differentiation of alveolar epithelial type II cells (AT2), a hallmark of pulmonary fibrosis remains unclear, and its targeted transcription factor has not been addressed. Here, examination of the specimen of fibrosis patients revealed excessive proliferation of AT2 cells. In parallel, AT2 cells exhibited substantial calpain 1 (CAPN1), a catalytic subunit of \u0026micro;-calpain, and phosphorylated FoxO3a (p-FoxO3a), an important transcription factor in lung tissue. Of note, targeted knockdown of CAPN1 in AT2 cells blocked the progression of bleomycin-induced pulmonary fibrosis, manifested as reduced poorly aerated regions in chest CT image, and decreased content of hydroxyproline and α-SMA. Analysis of nuclear fraction displayed an accumulation of CAPN1 and loss of FoxO3a, which was accompanied with activation of Akt. Knockdown of CAPN1 in A549 cells with siRNA antagonized the process of epithelial-mesenchymal transition and blunted FoxO3a phosphorylation and Akt activation. Conversely, overexpression of CAPN1 accelerated mesenchymal transition, enhanced its nuclear accumulation and the translocation of p-FoxO3a out of nucleus. Finally, inhibition of Akt decreased calpain-elicited FoxO3a phosphorylation, meanwhile, transfection of FoxO3a mutant carrying Thr32A and Ser253A mitigated the calpain-stimulated mesenchymal transition. Collectively, we conclude that nuclear accumulation of \u0026micro;-calpain in AT2 cells is a critical step to aggravate pulmonary fibrosis. we also identify that inactivation of FoxO3a in a Akt-dependent phosphorylation manner confers to calpain-elicited the aberrant differentiation of AT2 cells.\u003c/p\u003e","manuscriptTitle":"Nuclear accumulated μ-calpain in AT2 cell participates in pulmonary fibrosis via inactivating FoxO3a","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-02 17:23:58","doi":"10.21203/rs.3.rs-3787538/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":"50d0f28b-0bca-4aa3-a9f5-2605f491c0d7","owner":[],"postedDate":"January 2nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-01-09T10:29:16+00:00","versionOfRecord":[],"versionCreatedAt":"2024-01-02 17:23:58","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3787538","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3787538","identity":"rs-3787538","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}