Deletion of SMARCA4 impairs alveolar epithelial type II cells proliferation and aggravates pulmonary fibrosis in mice

Alveolar epithelial cells (AECs) injury and failed reconstitution of the AECs barrier are both integral to alveolar flooding and subsequent pulmonary fibrosis (PF). Nevertheless, the exact mechanisms regulating the regeneration of AECs post-injury still remain unclear. SMARCA4 is a part of the large ATP-dependent chromatin remodelling complex SWI/SNF, which is essential for kidney and heart fibrosis. We investigates SMARCA4 function in lung fibrosis by establishing PF mice model with bleomycin firstly and found that the expression of SMARCA4 was mainly enhanced in alveolar type II (ATII) cells. Moreover, we established an alveolar epithelium-specific SMARCA4-deleted SP-C-rtTA/(tetO)7-Cre/SMARCA4f/f mice (SOSM4Δ/Δ) model, as well as a new SMARCA4-deleted alveolar type II (ATII)-like mle-12 cell line. We found that the bleomycin-induced PF was more aggressive in SOSM4Δ/Δ mice. Also, the proliferation of ATII cells was decreased with the loss of SMARCA4 in vivo and in vitro. In addition, we observed increased proliferation of ATII cells accompanied by abnormally high expression of SMARCA4 in human PF lung sections. These data uncovered the indispensable role of SMARCA4 in the proliferation of ATII cells, which might affect the progression of PF.


Introduction
Pulmonary fibrosis (PF) is a fatal disease with unclear pathogenic mechanism. Most PF patients show a progressive decline in pulmonary function, which eventually leads to respiratory failure and death. 1 Conventional therapy consisting of glucocorticoids or immunosuppressive drugs has been affirmed ineffective. Currently, lung transplantation is considered to be an effective treatment approach, but only in limited number of patients, with a 5 year post-transplantation survival rate of about 50%. Recently, two novel disease-modifying therapies, nintedanib and pirfenidone, have shown promising results, nonetheless both these drugs are based on pleiotropic mechanisms of action. 2,3 The limited treatment approaches are caused by the elusive pathogenic mechanisms. Therefore, further explorations of the PF pathogenesis are exigent.
As a major component of interstitial lung disease, PF is characterised by repeated damage of alveolar type I (ATI) and type II (ATII) cells. Under normal conditions, when these cells die, the pulmonary stem cells proliferate and differentiate into the functional cells, consequently repairing the injury. Otherwise, they are replaced by abundant fibroblasts 2,4 and extracellular matrix proteins, 5 resulting in destruction of normal pulmonary tissue, thickening of the walls and honeycombing of the lungs. 6 In general, insufficient proliferation and differentiation of pulmonary stem cells into the functional cells following lung injury constitute a predisposition to PF. As the pulmonary stem cell populations, ATII can secrete surfactant and serve as a progenitor for ATI cells in response to injurious stimuli. 7 Therefore, exploring the influences of ATII cell proliferation may be helpful for illustrating the pathogenesis of PF.
In recent years, SMARCA4, a catalytic subunit of the yeast switch in mating type (SWI)/sucrose nonfermentation (SNF) chromatin remodelling complex, 8 was reported to be involved in the progression of fibrosis in many different organ systems. 9e13 In hearts following pathological stress, SMARCA4 and forkhead box M1 (FoxM1) transcription factor complexes regulate the transcription of angiotensin through conversion of enzyme (Ace) and Ace2 by binding to their promoter regions in the coronary endothelial cells. These in turn trigger angiotensin I-to-II conversion, followed by cardiac hypertrophy and fibrosis. 12 Furthermore, SMARCA4 could also be activated by cardiac stresses and could form a complex with histone deacetylase (HDAC) and poly (ADP ribose) polymerase (PARP) to induce a pathological a-myosin heavy chain (Myh6) to b-myosin heavy chain (Myh7) shift in cardiomyocytes, and to promote myocyte proliferation and accelerate cardiac fibrosis. 11 Beside myocardial fibrosis, SMARCA4 has been shown to be abnormally elevated in renal fibrosis following ischaemiareperfusion injury by binding to the promoters of proinflammatory or profibrotic genes to accentuate their transcription. 13 Moreover, SWI/SNF complex has also been reported to influence the regeneration following liver injury and ear wound, 14 and to influence the proliferation of various cell types. 8,15e17 Nevertheless, very little is known about the role of SMARCA4 in pulmonary fibrosis.
In this study, we established the PF model with bleomycin (BLM) and found elevation of SMARCA4 expression mainly in ATII cells of BLM-induced PF mice. After constructing lung epithelium-specific SMARCA4 depleted mice and SMARCA4-depledted ATII cell line, we found that the proliferation of ATII cells was decreased with the loss of SMARCA4 in vivo and in vitro. Moreover, the human lung sections from PF patients also confirmed the increased proliferation of ATII cells accompanied by abnormally high expression of SMARCA4, further uncovering the critical role of SMARCA4 in PF pathogenesis.

Bleomycineinduced lung fibrosis
All the protocols were performed in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals (Library of Congress Control Number: 2010940400, revised 2011) guidelines and relevant regulations. A total of 2.5 or 5.0 mg/kg of bleomycin (Sigma, USA) was intratracheally (i.t.) injected in wild-type or in transgenic 8e10 weeks old mice. All mouse experiments were approved by the Ethics Committee of Chongqing Medical University.

Primary ATII cells isolation
Primary ATII cells were isolated from adult mice as previously described. 22 Magnetic sorting was used for CD45negtive and epithelial-cell adhesion molecule (EpCAM)positive selection. Every ATII cell sample was collected from three different mice sharing the same genotype and administration.

Human lung tissue samples
Human lung tissues were obtained from patients undergoing surgical lung biopsy for idiopathic pulmonary fibrosis (IPF) and from surgical specimens showing normal lung parenchyma distant from the tumour nodules (control). The serial adjacent slides were collected according to previously described method 23e25 and were used for histological staining. All patients read and signed the informed consents. This study was approved by the Research Ethics Committee of Chongqing Medical University and undertaken in accordance with the principles of the revised Declaration of Helsinki.

Statistical analysis
All values are expressed as means AE SEM. Data were analysed using two-way analysis of variance. A student's t test analysis was used for the single-parameter comparisons. Statistical differences were analysed using SPSS software (version 16). p < 0.05 was considered statistically significant. KaplaneMeier survival analysis was performed using Prism 5 (GraphPad Software, La Jolla, CA) and statistical significance was determined by log-rank test.
(Additional data on materials and methods are provided in the "Electronic Supplementary Material")).

ATII cells in the lung tissues of PF mice showed increased SMARCA4 expression
To investigate the function of SMARCA4 in PF, we remodelled the PF procession in mice. Briefly, PF mice models were established by bleomycin intratracheal injection, and were consequently sacrificed 21 days post-bleomycin administration. Lung fibrosis was assessed by H&E, Masson's trichrome staining and the content of acid-soluble lung collagen. Histological results showed thickened alveolar walls, damaged lung structure, formation of fibrous masses and collagen deposition in bleomycin-treated mice. The Ashcroft score was significantly higher in PF models. Furthermore, higher acid-soluble lung collagen was observed in PF models compared to saline-treated mice (Suppl. Fig. S1AeC). Moreover, the survival rate of PF models was lower than that of control group (Suppl. Fig. S1D). To sum up, these results suggested successful establishment of PF models.
Accounting for SMARCA4-positive cells, we found positive cells to be significantly increased in the lung tissues of PF models (Fig. 1A, C); these data were further confirmed by immunoblotting approaches (Fig. 1B, D). Given that many different cell types, i.e. myofibroblasts (marked by smooth muscle-specific a-actin, a-SMA), macrophages (marked by CD11b) and ATII cells (marked by surfactant protein C, SPC), participated in the pathogenesis of PF, 5,23,26,27 we further investigated the SMARCA4 expression using immunohistochemistry of lung serial sections. 23e25 Interestingly, the enhanced SMARCA4 expression was observed in the lung epithelial cells (Supple Fig. S2A, B), while the myofibroblasts and macrophages with aberrance expression of SMARCA4 were rare in lungs of PF models (Suppl. Fig. S2C-F). For more accurate detection, the ATII cells were isolated (Suppl. Fig. S3 and Fig. 1E) by immunomagnetic bead cell sorting and the increase of SMARCA4 in those cells were confirmed by immunoblotting and flow cytometry approach ( Fig. 1G and H). Furthermore, higher number of ATII cells recovered from PF mice 21 days postbleomycin treatment was observed compared to control mice (Fig. 1F). In addition, compared to control group, higher proliferation of ATII cells in PF mice was detected following BrdU incorporation (Fig. 1I), indicating that the elevated SMARCA4 might enhance the ATII cells proliferative capacity during PF procession.

Mice with epithelial SMARCA4 deficiency were viable and healthy
To further explore the function of the elevated SMARCA4 in ATII cells during PF development, ATII cells specific SMARCA4 knock-down mice were bred by crossing SPC-rtTA/(tetO) 7 -Cre mice with SMARCA4 f/f mice (Suppl. Fig. S4A). After genotyping (Suppl. Fig. S4BeD), we fed the mice with doxycycline for one week. Comparing with the WT mice (SPC-rtTA/(tetO) 7 -Cre/SMARCA4 þ/þ ), only about 40% of SMARCA4 expression was detected in the isolated ATII cells in SOSM4 D/D and SOSM4 D/þ mice, while the expression of SPC was not impacted (Fig. 2A). The knockdown efficiency was further confirmed by flow cytometry (Fig. 2B). The residual SMARCA4 expression in the homozygotes might probably occurred due to incomplete excision by SPC-Cre. 7 Moreover, the similarity of SMARCA4 expression between the SOSM4 D/D and SOSM4 D/þ was possibly caused by the same reason. Also, SOSM4 D/D and SOSM4 D/þ mice were healthy and did not show any signs of polypnea or emaciation until seven months postdoxycycline administration. Furthermore, the histology of the lung tissue of SOSM4 D/D and SOSM4 D/þ mice was normal comparing with their littermates (WT) (Fig. 2C and D). To conclude, the obtained data indicated that the SMARCA4 knock-down in ATII cells did not compromise the respiratory function in mice.

Epithelial SMARCA4 deficiency aggravates bleomycin-induced PF
Given that knock-down of SMARCA4 in ATII cells was insufficient to cause PF independently, to further elucidate the function of enhanced SMARCA4 in PF model, we constructed the PF model in SOSM4 D/D mice and their littermates (WT) following feeding with Dox for one week. As high dose of bleomycin (5 mg/kg) would induce severe pulmonary fibrosis and lead to death rapidly in both of them, we reduced the dosage to 2.5 mg/kg. Then, the different responses of SOSM4 D/D and WT mice to bleomycin were distinguishable.
After bleomycin administration, all the mice showed PF in different levels. Also, 60% reduction of SMARCA4 protein in isolated ATII cells lysates were observed in SOSM4 D/D mice compared to their littermates (WT) (Fig. 3A), which was further confirmed by flow cytometry (Fig. 3B and C). Interestingly, we found that SOSM4 D/D mice tend to die earlier than their littermates following bleomycin exposing (Fig. 3D). Moreover, the lung tissues of SOSM4 D/D mice showed augmented fibrosis with histological examination compared with their littermates (Fig. 3F and G). Also, the acid-soluble lung collagen in response to bleomycin was significantly higher in SOSM4 D/D mice compared to WT mice  . 3E). Ultimately, these data suggested that the deletion of SMARCA4 in ATII cells could exacerbate PF induced by bleomycin in mice.

Loss of SMARCA4 reduced the proliferation of ATII cells
To future unveil the potential mechanism behind this phenotype, we explored the endoplasmic reticulum (ER) stress marked by X-box binding protein 1 (XBP1) mRNA splicing and heavy-chain Ig binding protein (BiP) accumulation, which in turn led to reactive oxygen species production from ATII cells and therefore contributed to aberrant lung remodelling followed by enhanced susceptibility of PF. 28e31 Our data showed that BiP mRNA and XBP1 splicing in whole-lung tissue samples and isolated ATII cells were similar between SOSM4 D/D mice and their littermates (Suppl. Fig. S5). Furthermore, without bleomycin stimulation, reduction of SMARCA4 in ATII cells did not affect the  Figs. 2A and 3A) that potentially underwent transcriptional inhibition 32 and was involved in ER stress. 31 Hence, there could be other mechanisms to explain the influence of SMARCA4 on the progression of PF.
It has been shown that SMARCA4 influences cell proliferation in many different diseases. 15,11,33 Considering that proliferation of ATII cells is vital for the progression of PF, 4,34,35 we further investigated the ATII proliferation ability following SMARCA4 knock-down. Notably, lower number of ATII cells was observed in injured lung sections of SOSM4 D/D mice compared to WT mice (Fig. 4A, F upper  panels, 4B, E). While, the accumulation of macrophages and myofibroblasts in impaired lung tissues were not influenced by lung epithelium-specific SMARCA4 deletion (Fig. 4A, F middle and lower panels, 4C, D). To further look insight to the affected cell functions, we detected the apoptosis and proliferation in the lung tissues by TUNEL staining and BrdU incorporation. Finally, no differences in apoptosis were observed between SOSM4 D/D and WT mice (Suppl Fig. S7A). But, interestingly, the proliferative ability of ATII cells was stronger in SOSM4 D/D mice compared to WT mice (Fig. 4G), which was re-confirmed by cell proliferation marker, PCNA, as well (Suppl Fig. S6B).
To examine the effect of SMARCA4 on the proliferation of ATII cells, we knocked down SMARCA4 in immortalised ATII cell line mle-12 using small interfering RNA (Suppl. Fig. S8, Fig. 5A). Our data indicated that transient SMARCA4 knock-down slowed the growth of mle-12 (Fig. 5B), possibly caused by the reduced cell proliferation according to BrdU incorporation assay and PCNA protein detection (Fig. 5A, C, D). Additionally, the cells were arrested at G0/G1 following SMARCA4 deletion (Fig. 5E). Nevertheless, no difference in mle-12 cell apoptosis was observed (Suppl. Fig. S7). In general, SMARCA4 was indispensable for the cell proliferation of ATII cells. Considering the protective effect of ATII cells on PF progression, our results indicated that ATII cells specific SMARCA4 deletion might exacerbate PF by resisting ATII cells proliferation.

The ATII cells of human IPF lung tissues showed enhancement of SMARCA4 expression
To determine whether SMARCA4 in PF mouse model is associated with human PF, we detected the expression of SMARCA4 in ATII cells from lung biopsy specimens. Consequently, we observed that SMARCA4 expression was markedly increased in ATII cells from lung sections of the individuals with PF compared to control lung tissues (Fig. 6). These observations were similar to those verified in our mouse models (Suppl. Fig. S2A, B), suggesting that the hyperplastic ATII cells were accompanied with the enhancement of SMARCA4 in lung tissues of IPF patients.

Discussion
Extensive loss of AECs from the alveolar wall leads to proliferation and activation of fibroblasts and excessive collagen deposition, which in turn induce fibrosis. 36 ATII cells, which are progenitor cells of the pulmonary epithelium, have been shown to patch the loss of AECs following lung injury. 7 Therefore investigating the mechanisms underlying the proliferation of ATII cells appears to be the key for understanding the pathogenesis of PF. In this study, we uncovered the pivotal role of epithelial SMARCA4 in PF pathogenesis. Postnatal knock-down of SMARCA4 in ATII cells resulted in exacerbated lung scarring and collagen accumulation post-injury. Furthermore, the impaired lungs of epithelial SMARCA4-knockdown mice exhibited less cell count and decreased proliferation of ATII cells. The suppression of SMARCA4 deletion on cell proliferation was confirmed on mle-12 cell line. We observed that hyperplastic ATII cells were accompanied with enhancement of SMARCA4 in human IPF lungs. In conclusion, our data indicated that epithelial SMARCA4 was essential for ATII cell proliferation following lung injury, which illustrated a novel pathogenic mechanism of PF.
To date, the exact pathophysiology of PF remains unclear. Different studies have reported the implication of epithelial and mesenchymal cells with PF. Myofibroblasts, the activated form of fibroblasts, are believed to be important in wound healing and fibrosis. 37 The extracellular matrix proteins they produce, such as collagen, have been shown to accumulate in tissues, which in turn replace normal structure required for proper organ function. 5 Macrophages, the major elevated inflammatory cells in PF, are also critical in the pathogenesis of fibrosis. 23 The effects of macrophages on PF depend on different subsets. M1 cells produce proinflammatory cytokine in order to exacerbate fibrosis, while M2 cells can ameliorate fibrotic diseases. 38 Repetitively impaired ATII cells lead to uncontrolled recruitment and activation of mesenchymal cells. 36 Furthermore, misfolding of surfactant protein A (SPA) and SPC leads to ER stress and reactive oxygen species production from ATII cells, which contributing to aberrant lung remodelling. 28e31 In the present study, we detected an aberrant expression of SMARCA4 in the lungs of PF mice and assessed the expression of the same among the three distinct cell types. Interestingly, our data indicated enhanced expression of SMARCA4 mainly in ATII cells. However, after SMARCA4 knockdown, the ER stress of ATII cells was not affected. Although the high expression of SMARCA4 in myofibroblasts was independent of PF, depletion of SMARCA4 promoted the differentiation of fibroblasts to myofibroblasts (data not shown).
SMARCA4, an essential catalytic subunit of SWI/SNF chromatin remodelling complex, is involved in many fibrotic diseases. In human hypertrophic hearts, SMARCA4 is highly activated and strongly correlated with disease severity. Mice with endothelial or myocardial SMARCA4 deletion have shown resistance to stress-induced cardiac hypertrophy and fibrosis. 11,12 Renal ischaemia-reperfusion injury has also shown to cause progressive increase and binding of SMARCA4 to proinflammatory/profibrotic genes in renal cortex. 13 Furthermore, SWI/SNF complex participates in liver regeneration and affects the pathogenesis of hepatic fibrosis. 14 However, our results suggested that SMARCA4 could serve as a protective factor in PF.
The mechanism of SMARCA4 on the progression of fibrosis are distinct in different organs. In hearts following pathological stressed, SMARCA4 chromatin remodeler and forkhead box M1 (FoxM1) transcription factor cooperate within coronary endothelial cells to trigger the Ace2-to-Ace enzyme switch, angiotensin I-to-II conversion, and cardiac hypertrophy and fibrosis. 12 Moreover, in response to angiotensin II stimulation, SMARCA4 and H3K4 methylation complex (Ash2/Wdr5) are recruited to the endothelin (ET-1) promoter region in endothelial cells to enhance the transcription of ET-1, and consequently induce cardiac hypertrophy and fibrosis. 10 Focusing on the cardiomyocytes, SMARCA4 is activated by cardiac stresses and forms a complex with histone deacetylase (HDAC) and poly (ADP ribose) polymerase (PARP) which in turn induces a pathological Myh6 to Myh7 shift, suppress cardiac differentiation and promote myocyte proliferation. 11 In renal ischaemiareperfusion injury, SMARCA4 binds to the promoters of proinflammatory/profibrotic genes to accentuate the transcription of these genes. 13 In our study, deletion of SMARCA4 impairs ATII cells proliferation and aggravates PF induced by bleomycin, demonstrating a protective role of SMARCA4 in lung fibrosis.
SMARCA4 is essential for cell proliferation. For example, SMARCA4 is indispensable for the proliferation of neuroblastoma cells, 17 while it decreases the proliferation ability of the lung adenocarcinoma NCI-H522 cells. 33 Regarding non-neoplastic disease, it promotes myocyte proliferation and suppresses cardiac differentiation simultaneously. 11 Our research revealed that SMARCA4 was essential for the proliferation ATII cells, which were considered putative alveolar stem cells. 35 Contrary, Chapman et al. have shown that in the severe lung injury caused by bleomycin, integrin a6b4-positive Sftpc-negative cells generated ATII cells are not associated with the proliferation of pre-existing ATII cells. 39 Nevertheless, many different studies have supported the theory that Sftpc-positive ATII underwent clonal proliferation and generated multiple ATI and ATII cells following lung injury. 7,40 The probable mechanisms of that SMARCA4 deletion could affect the cell proliferation were different in disparate cell types. In SMARCA4-null myocardium, the Bmp10 expression was nearly abolished, while p57 kip2 , a cyclin-dependent kinase inhibitor which is suppressed by Bmp10, appeared ectopically and finally caused proliferation reduction. 11 In SMARCA4-deleted neuroblastoma cells, the expression of several key genes related to cell growth and proliferation, such as PI3KCA and BCL2, were impacted. 17 Nonethless, the molecular mechanisms of SMARCA4 deletion in ATII cells that might explain the phenotype of the transgenic mice were not involved in this article and need to be further explored.
In the present study, hyperplastic ATII cells were found in the lung tissues of IPF patients and mice models, as other reports. 36 The hyperplastic ATII cells is often observed in PF