Brahma-related gene 1 regulates the proliferation of alveolar type II epithelial cells via the JAK1/2-PI3K/AKT signaling pathway

doi:10.21203/rs.3.rs-2215820/v1

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Brahma-related gene 1 regulates the proliferation of alveolar type II epithelial cells via the JAK1/2-PI3K/AKT signaling pathway

https://doi.org/10.21203/rs.3.rs-2215820/v1

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Background

Alveolar type II epithelial cells (AECIIs), a crucial part of the alveolar epithelium, can secrete surfactant-associated proteins and act as progenitor cells of the alveoli. The number of AECIIs in lung tissues is closely related to the pathogenesis and pathological process of numerous lung diseases. Brahma-related geng1 (Brg1), a catalytic subunit of ATPase, is a core component of the mammalian switch/sucrose non-fermentable (SWI/SNF) ATP-dependent chromatin-remodeling complex, which achieves chromosome recombination and further regulates gene expression in an ATP-dependent manner. Brgl plays a pivotal role in regulating cell differentiation, proliferation, and apoptosis. Unfortunately, almost no research exists on the relationship between Brg1 and AECIIs. Therefore, we aimed to investigate the effect of Brg1 on the quantity of AECIIs as well as the possible mechanisms.

Methods

C57BL/6 mice with the Brg1 gene specifically knocked out in AECII epithelial cells (Brg1^fl/fl mice) were constructed to analyze the effect of brg1 gene expression on the number of AECIIs in vivo. The number of ACEIIs was detected and compared in the Brg1^fl/fl group and wild-type (WT) group using immunohistochemistry, flow cytometry, and immunofluorescence. The Brg1 gene in immortalized mouse pulmonary alveolar type II (ImpacII) cells was knocked down using lentiviral vectors. The migration and invasion of ImpacII were observed using cell scratch assay and transwell migration assay. The proliferation of ImpacII was monitored using the cell clone assay, CCK-8 cell proliferation assay, and cell cycle assay. The proliferation-related proteins including Ki67, p-JAK1/2/JAK1/2, p-STAT6/STAT6, p-PI3K/PI3K, and p-AKT/AKT were detected using Western blot and immunofluorescence in ImpacII cells. To explore the specific molecular mechanism of Brg1 regulating ImpacII proliferation, the binding sequences of Brg1 in ImpacII cells were sought using chromatin immunoprecipitation-sequence (CHIP-seq) and further confirmed by chromatin immunoprecipitation-qPCR (CHIP-qPCR). The interactive relationship between JAK1/2 and PI3K was verified by co-immunoprecipitation (Co-IP) assay.

Results

Knocking out brg1 facilitated the proliferation of AECIIs in vivo. Knocking down brg1 induced the proliferation in association with the migration and invasion of ImpacII in vitro. Mechanistically, knocking down brg1 activated the JAK1/2-PI3K/AKT signaling pathway and induced the expression of proliferation-related protein Ki67. Furthermore, CHIP-seq and CHIP-qPCR results showed that Brg1 could bind to the JAK1/2 promoter region and regulate the activity of the JAK1/2-PI3K/AKT signaling pathway. Co-IP confirmed that JAK1/2 interacted with PI3K.

Conclusion

Knocking out Brg1 promoted the proliferation, migration, and invasion of AECIIs via the JAK1/2-PI3K/AKT signaling pathway. This represents a potential therapeutic target and a novel prognostic indicator in various pulmonary diseases.

Brg1

alveolar type II epithelial cells

immortalized mouse pulmonary alveolar type II cells

proliferation

JAK1/2-PI3K/AKT

Alveolar type II epithelial cells (AECIIs), together with alveolar type I epithelial cells (AECIs), form pulmonary alveoli. They occupy 2–5% of the alveolar surface area and play a crucial part in maintaining pulmonary homeostasis^[1–4]. AECIIs synthesize and secrete surfactant-associated proteins that can reduce the alveolar surface tension and regulate the pulmonary initial immunity^[5]. They also function as progenitor cells of the alveoli, displaying self-renewal capabilities and differentiating into AECIs to repair and maintain the integrity of the alveoli during lung injury^{[2, 6–9]}. Some studies have shown that Wnt signaling niches maintained the stemness of AECIIs and inhibited the differentiation of AECII progenitors^{[9, 10]}. Other studies have reported that Yap/Taz signaling promoted the differentiation of AECII progenitors into AECIs and regulated alveolar regeneration^{[11, 12]}. AECII dysfunction has been associated with the pathogenesis of numerous pulmonary diseases, including pulmonary fibrosis, pulmonary infection, acute respiratory distress syndrome (ARDS), bronchopulmonary dysplasia (BPD), and lung cancer^[12–17], but this remains incompletely understood. Therefore, exploring the function of AECIIs may guide the clinical treatment of pulmonary diseases. Surfactant-related protein C (SFTPC/SPC), a component of surfactant-associated protein, as well as the precursor protein of SFTPC (pro-SFTPC/pro-SPC), is only expressed in AECIIs and usually considered a marker of AECIIs^{[18, 19]}. Consequently, the expressions of SPC and pro-SPC partly reflect the quantity and function of AECIIs.

Brahma-related gene 1 (Brg1), also known as SMARCA4 in human cells, is the core ATPase catalytic subunit of the mammalian switch/sucrose non-fermentable (SWI/SNF) chromatin-remodeling complex, which repositions and recomposes the nucleosome in an ATP-dependent manner^[20–23]. As a key factor of the chromatin-remodeling complex, Brgl is involved in regulating DNA replication, DNA damage repair, and gene expression by altering the structure of chromatin and specific interactions with nuclear proteins to make genomic regions more accessible^{[24, 25]}. Numerous previous studies have confirmed that the Brgl gene plays a pivotal role in regulating cell differentiation, proliferation, and apoptosis^[26–28]. The latest research has reported that Brg1 interacted with hemogen in mammals to promote the proliferation and differentiation of hematopoietic cells as well as fetal erythroid maturation by enhancing the chromatin accessibility of erythroid gene promoters and enhancers^[29]. Tapan et al. found that Brg1 with normal bromodomain function affected skeletal myogenesis through binding to promoters of target genes and then regulating cell cycle exit when muscle differentiation is induced^[30]. Moreover, our previous studies have suggested that knockdown of Brg1 in 16HBE cells increased the proliferation and migration ability of 16HBE^[31]. Unfortunately, limited research exists on the effects of Brg1 on AECIIs. Therefore, this study investigated the association between the Brg1 gene and the function of AECIIs.

2.1. Animals

SPF female C57BL/6 mice, aged 4–6 weeks, were purchased from the Experimental Animal Center of Chongqing Medical University (Chongqing, China). SFTPC-rtTA/(TetO)7 transgenic mice (The Jackson Laboratory, CA, USA) were hybridized with Brg1^loxP/loxP mice (kindly provided by Prof. Yujun Shi, Medical College of Sichuan University). Seven days after birth, DNA was extracted from fingernails, and then genotypes were detected according to previous studies^[31]. The homozygotes, also known as alveolar type II epithelial cell–specific Brg1-knockout (SFTPC-Cre/Brg^fl/fl), were fed with breast milk for 3 weeks and then received 2% doxycycline in drinking water for 14 days to induce recombinant enzyme activity and further targeted cleavage of the Brg1 gene in AECIIs. We changed the doxycycline water every 2 days. Meanwhile, WT mice received sterile water. All experimental mice were housed under specific pathogen-free conditions, with the temperature controlled at 20–25℃, the humidity maintained at 50–60%, and a 12h/12h dark/light cycle. The study was approved by the Ethics Committee of Chongqing Medical University.

2.2. Immunohistochemical assessment of lung tissues

The left lung tissues were fixed in 4% formalin, dehydrated, embedded in paraffin, and sectioned into 4-µm-thick slices. The sections were dewaxed in xylene and rehydrated in ethanol. Then, 10mM sodium citrate buffer was used for antigen retrieval, as previously described^[32]. The tissue slices were subjected to immunohistochemical staining using the Rabbit two-step kit (Zhongshan Golden Bridge, Beijing, China) according to the manufacturer’s instructions. Rabbit antibody against SPC (1:200; Abcam, Cambridge, UK) was used for IHC staining. Tissues or cells that were stained brown were recorded as positive for SPC. The intensity of IHC staining was quantified using Image-Pro Plus 6.0.

2.3. Flow cytometry of lung tissues

The lung tissues were prepared into a single-cell suspension by digesting with 0.2% collagenase type I (1640 configuration) at 37℃ for 15 min, then repeatedly cut into pieces in a 200-mesh screen and washed with 1640 medium. The single-cell suspension was fixed with 4% paraformaldehyde, then the membrane was broken with 0.5% TritonX-100 and blocked with rat serum. Next, the single-cell suspension was stained with rabbit anti-pro-SPC (1:200, Abcam, Cambridge, UK) at room temperature for 1.5 h, then stained with PE-labeled goat anti-rabbit second antibody (1:500, Abcam, Cambridge, UK) at room temperature for 30 min, avoiding light, and washed twice with PBS. Finally, the single-cell suspension was resuspended in 200 µL of PBS after centrifugation on the machine, and the results were analyzed using FlowJo-V10 software.

2.4. Cell culture and treatments

Immortalized mouse pulmonary alveolar type II (ImpacII) cells were constructed successfully, as in a previous study,^[33] and maintained in Dulbecco’s phosphate-buffered saline medium (DMEM) containing 10% fetal bovine serum (FBS; Gibco, CA, USA) at 37°C and 5% CO₂. Short hairpin RNAs (shRNAs) against Brg1 (Brg1-shRNA) and negative control-shRNA (NC-shRNA), designed and cloned into pGMLV-SC5 RNAi backbone adenoviral vectors, were purchased from Genechem (Genechem, Shanghai, China). The ImpacII cells were infected with the adenovirus vectors following the manufacturer’s instructions and screened for the stably transfected strains with puromycin. In addition, to verify that Brg1 knockdown was successful in the ImpacII cells, the expression of Brg1 was detected with Western blot, q-PCR, and cell immunofluorescence. Cells were divided into the control group (ImpacII), negative control group (Brg1-sc), and knockdown group (Brg1-sh).

2.5. Cell migration, cell proliferation, and cell cycle

Cell migration was measured with transwell and cell scratch assay. The ImpacII cells were cultured with serum-free medium for 24 h in transwell and stained with crystal violet. The ImpacII cells, which were cultured in a cell-culture plate, were wounded with pipette tips, and photos were taken 24 h after the wound. Then, the wound width was analyzed by Image J. Cell proliferation was determined using cell cloning and the CCK-8 Kit (Dojindo Lab, Kumamoto, Japan) according to the manufacturer’s instructions. The cell cycle was tested using a cell cycle kit (KeyGen Biotech, Jiangsu, China) following the manufacturer’s protocols, and the results were analyzed by FlowJo-V10.

2.6. Immunofluorescence detection

The expression of pro-SPC in lung tissues and Ki67 in ImpacII cells was determined using immunofluorescence staining. The left lung tissues were made into paraffin sections. The sections were dewaxed and rehydrated, with antigen retrieval as previously described. The lung tissues were then blocked with 5% bovine serum albumin (BSA) and stained with rabbit anti-SPC (1:200, Abcam, UK) at 4℃ overnight. After washing three times with PBS, the lung tissues were stained at 37℃ for 1h with goat anti-rabbit Cy3 secondary antibodies (1:200; Beyotime, Shanghai, China). Finally, nuclei were stained with DAPI (4,6-diamidino-2-phenylindole; Beyotime, Shanghai, China) for 15 min at room temperature. The ImpacII cells were spread evenly on cell slides, and the cells were fixed with 4% paraformaldehyde, then the cytomembrane was broken with 0.5% TritonX-100, and the cells were blocked with 5% BSA. Next, the cells were stained with rabbit anti-Ki67 (1:50, Affinity Biosciences, Jiangshu, China) and goat anti-rabbit Cy3 (1:200, Beyotime, Shanghai, China). Nuclei were then stained with DAPI. Finally, the fluorescence of the samples was captured using a confocal laser scanning microscope (A1þR, Nikon, Tokyo, Japan), and the fluorescence intensity was quantified using Image-Pro Plus 6.0.

2.7. Western blot and co-immunoprecipitation (Co-IP) assay

The total protein from the lung tissue and ImpacII cells was isolated using a total protein extraction kit (KeyGenBiotech, Jiangsu, China) according to the manufacturer’s protocol. The protein concentration was detected using the NanoDrop One microUV-visible spectrophotometer (ThermoFisher Scientific, Shanghai, China). Protein samples were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes. Membranes were then blocked with 5% BSA for 2h at room temperature and incubated overnight at 4℃ with the following primary antibodies: rabbit anti-Brg1 (1:500, Zen-Biosciences, China), rabbit anti-Ki67 (1:500, Affinity Biosciences, Jiangshu, China), rabbit anti-phospho-JAK1/2 (1:1000; CST, USA), rabbit anti-JAK1/2 (1:500, Zen-Biosciences, China), rabbit anti-phospho-STAT6 (1:1000; CST, USA), rabbit anti-STAT6 (1:500, Zen-Biosciences, China), rabbit anti-phospho-PI3K (1:1000; CST, USA), rabbit anti-PI3K (1:1000, CST, USA), rabbit anti-phospho-AKT (1:1000; CST, USA), rabbit anti-AKT (1:1000, Proteintech, China), and rabbit anti-β-actin (1:1000; Proteintech, China). Subsequently, membranes were incubated with horseradish peroxidase-linked goat anti-rabbit IgG secondary antibody (1:5000; Proteintech, China). Finally, the immunoreactivity was visualized using an enhanced chemiluminescence (ECL) kit (Thermo Fisher, USA), and the results were analyzed by Image Lab.

The Co-IP was performed using Protein A/G Magnetic Beads (MedChemExpress, NJ, USA) following the manufacturer’s instructions. The rabbit anti-JAK1/2 (1:500, Zen-Biosciences, China) and rabbit anti-PI3K (1:1000, CST, USA) were used as the IP group antibody, and the rabbit anti-IgG (Abcam, Cambridge, UK) was used as a control antibody. The harvested samples were divided into input (protein pretreated with nothing), IgG (pre-treated with Protein A/G Magnetic Beads and rabbit anti-IgG), anti-JAK1/2 (pre-treated with Protein A/G Magnetic Beads and rabbit anti-JAK1/2), and anti-PI3K (pre-treated with Protein A/G Magnetic Beads and rabbit anti-PI3K). The results were finally analyzed by Western blot.

2.8. Chromatin immunoprecipitation

The chromatin immunoprecipitation (CHIP) was performed using a CHIP kit (Milipore, MA, USA) in accordance with the kit’s instructions. The chromatin in the ImpacII cells was cross-linked with protein using formaldehyde (1%), then cells were lysed fully to release chromatin. Subsequently, DNA was fragmented into 200-500bp pieces using ultrasonic disruption. DNA pieces were incubated overnight at 4℃ with the rabbit anti-Brg1 (1:20, ZEN-Biosciences, Chengdu, China) or rabbit anti-IgG and magnetic beads at room temperature for 2h. The DNA and protein complexes were de-crosslinked after the magnetic bead-antibody/chromatin complexes were washed four times. The DNA was purified and dissolved in 30 µL of Elution buffer. Finally, the DNA samples were subjected to CHIP-seq and CHIP-qPCR analysis. The primer sequences of JAK1 were forward 5-GGCCCTAGACCTTAGCACAA-3 and reverse 5-TGCCCTGGGTTGACATACTT-3. The primer sequences of JAK2 were forward 5-ACAAGGGCTGATCTCACACA-3 and reverse 5-CGTTTAGCATGGCCAGACTG-3.

2.9. Realtime RT-PCR

The total RNA from ImpacII cells and lung tissues was extracted using a Total RNA Extraction Kit (BioFlux, Beijing, China), then reverse-transcribed into cDNA using the PrimeScript RT Reagent Kit (Takara, Otsu, Japan). Realtime RT-PCR reactions were performed using Real MasterMix (SYBR Green, Tiangen, Beijing, China). The primer sequences of Brg1 were forward 5-GAGGAGGTCCGGCAGAAG AAATC-3 and reverse 5-TTCTTCTGCTTCTTGCTCTC-3. The primer sequences for GAPDH were forward 5-CAGCGACACCCACTCCTCCACCTT-3 and reverse 5-CATGAGGTCCACCACCCTGTTGCT-3.

2.10. Statistical analyses

Data are expressed as the mean ± standard error of the mean. GraphPad Prism 8.0 software was used for the statistical analyses. One-way analysis of variance or t-test analysis was used to determine statistically significant differences in the tested variables among the different groups. If an overall test was significant, Tukey’s test was used for specific comparisons between groups. Statistical significance was set at p < 0.05. All experiments were repeated at least three times, with consistent outcomes.

3.1. Knockout of Brg1 increased the number of AECIIs in the lung tissues

To investigate the effects of Brg1 on the number of AECIIs in the lung tissue, we generated AECII-specific Brg1-knockout (Brg1^fl/fl) mice using Cre/loxP technology as before^[31]. SPC and pro-SPC are specific markers of AECIIs. Thus, the number of AECIIs was determined by detecting SPC- or pro-SPC-positive cells. The number of SPC- or pro-SPC-positive cells in lung tissue was detected by immunohistochemistry (Fig. 1A-B), immunofluorescence (Fig. 1E-F), and flow cytometry (Fig. 1D). The immunohistochemistry (Fig. 1B), and immunofluorescence (Fig. 1E-F) results showed that more SPC-positive cells existed in the Brg1^fl/fl group mice compared to the other group mice. The flow cytometry (Fig. 1C-D) of lung tissues showed that the percentage of pro-SPC cells of the Brg1^fl/fl group was markedly higher than that of the WT group. These results suggest that the number of AECIIs increased in Brg1 knockout mice.

3.2. Knockdown of Brg1 promoted the proliferation and migration of immortalized mouse pulmonary alveolar type II cells

We successfully constructed immortalized mouse ImpacII cells^[33].To validate the effects of Brg1 on the ImpacII cells in vitro, the Brg1 gene in ImpacII cells was knocked down using adenovirus, and then the proliferation and migration of the ImpacII cells were detected by transwell migration assay, cell scratch assay, cell clone, CCK-8, and cell cycle. Transwell migration assay (Fig. 2A) showed that the cell migration rate in the Brg1 knockdown group (Brg1-sh) was higher than that in the virus empty control group (Brg1-sc). The cell scratch assay (Fig. 2B-C) results revealed that the repair of scratch wounds at 24 h after wounding in the Brg1-sh group increased significantly compared with that in the Brg1-sc group, whereas the Brg1-sc group showed no difference compared with the blank control group (ImpacII). This indicates that the migration of the Brg1-sh group increased compared to that of the Brg1-sc group. We also found that the clone formation rate of ImpacII cells increased when Brg1 was silenced from cell clone assay (Fig. 2D). CCK-8 (Fig. 2E) showed that cells in the Brg1-sh group proliferated more quickly than those in the other two groups. In addition, we found by cell cycle assay that the population of cells that were in the G2 phase in the Brg1-sh group was greater than that in the other two groups (Fig. 2F). Ki67 is a marker of cell proliferation state, closely related to mitosis and indispensable in cell proliferation. Therefore, cell immunofluorescence and Western blot of ImpacII cells were performed to detect the expression level of Ki67 proteins. The results of cell immunofluorescence showed that the Ki67 expression level in the Brg1-sh group was higher than that in the Brg1-sc group. This was confirmed by Western blot. The Brg1-sc group showed no difference compared with the ImpacII group.

Taken together, these data suggest that the proliferation and migration in association with the invasion of ImpacII cells were enhanced remarkably when Brg1 was knocked down.

3.3. Brg1 bound indirectly to the JAK1/2 promoter and then regulated the PI3K/AKT signaling pathway in vitro

Brg1, an important epigenetic factor, regulates the expression of various genes through chromosomal recombination. To further explore the specific molecular mechanism of Brg1 regulating the proliferation of ImpacII cells, CHIP-seq (Fig. 3A) was used to seek the binding sequence of Brg1 in ImpacII cells. The results revealed that Brg1 bound indirectly to the JAK1/2 promoter in ImpacII cells, which was confirmed by ChIP-qPCR (Fig. 3B). In addition, CHIP-seq (Fig. 3A) showed Brg1 and regulated the PI3K-AKT signaling pathway in ImpacII cells. Previous studies have confirmed that the PI3K-AKT signaling pathway, whose upstream signaling molecule is JAK1/2, plays a crucial role in regulating various cellular functions, including proliferation, migration, survival, transcription, and protein synthesis. To further verify the relationship between JAK1/2 and PI3K, Co-IP assay (Fig. 3C) was performed. The results showed JAK1/2 interaction with PI3K. Collectively, these data suggest that Brg1 affected the proliferation of ImpacII cells by binding to the JAK1/2 promoter, promoting JAK1/2 protein expression and then regulating the PI3K/AKT signaling pathway.

3.4. Knockdown of Brg1 activated the JAK/STAT6-PI3K/AKT signaling pathway in immortalized mouse pulmonary alveolar type II cells

To verify whether Brg1 promoted the proliferation and migration of ImpacII via the JAK-PI3K/AKT pathway, the Brg1 gene in ImpacII cells was knocked down. Subsequently, Western blot was used to detect the expression of p-JAK1/2, JAK1/2, p-STAT6, STAT6, p-PI3K, PI3K, p-AKT, and AKT proteins in ImpacII cells (Fig. 4A). The results showed that the ratios of p-PI3K/PI3K (Fig. 4B), p-STAT6/STAT6 (Fig. 4C), p-PI3K/PI3K (Fig. 4D), and p-AKT/AKT (Fig. 4E) of Brg1-sh were significantly increased compared with those of Brg1-sc, and no difference existed in the ratios of these proteins between the Brg1-sc and ImpacII groups. We concluded that knocking down Brg1 may promote the proliferation and migration of ImpacII by activating the JAK1/2-PI3K/AKT signaling pathway.

Epigenetics refers to heritable changes in gene expression without changes in the DNA sequence, mainly including changes in DNA methylation, histone acetylation, and chromatin remodeling^[34–36]. The core catalytic subunit of the chromatin-remodeling complex (Brg1) is a phosphorylated nuclear protein. Its main role is to regulate gene transcription by rearranging the nucleosome position and histone DNA interaction through ATP hydrolysis, thus promoting transcriptional activation or repression of target genes, which is a type of epigenetics^[37]. Brg1 has been reported to play an important role in promoting cell proliferation and regulating the cell cycle. In human non-small cell lung cancer (NSCLC) cells, Brg1 depletion was found to change the morphology of the cells and increase their tumorigenic potential^[38]. Brg1 recruitment to enhancers following B-cell activation was associated with increased chromatin accessibility and transcriptional activation of their coupled promoters, thereby regulating the expression of cell cycle–associated genes^[39]. Strobeck et al. found that Brg1 could inhibit the release of E2F transcription factor and downregulate the expression of cyclin E and cyclin A by binding phosphorylated retinoblastoma protein (pRB protein) to form a complex, thus interfering with the proliferation of fibroblasts^[40]. Brg1 may also control gene transcription by promoting the expression of genes responsible for cell cycle progression in studied breast cancer cells^[41]. All these studies suggest that Brg1 plays an important role in the regulation of cell proliferation. In this study, we verified that knockdown of the Brg1 gene in AECIIs could promote their proliferation and increase them in mice, in both in vivo and in vitro experiments. This study is the first to find the effect of Brg1 on AECIIs, which can provide a basis for the future study of lung diseases related to AECIIs.

A variety of signaling pathways are involved in the regulation of cell proliferation, among which the JAK/STAT6-PI3K/AKT signaling pathway is a ubiquitous classical signaling pathway in mammals. It plays an important role in mediating cell differentiation, proliferation, autophagy, and apoptosis^[42]. One study found that insulin-like growth factor-2 messenger RNA-binding protein 3 (IGF2BP3) facilitated bladder cancer cell proliferation by activating the JAK/STAT signaling pathway^[43]. In bladder cancer research, microRNA-294 could promote cellular proliferation and motility through the PI3K/AKT and JAK/STAT pathways by upregulation of NRAS gene expression^[44]. Another study indicated that isobavachalcone exerted its anti-rheumatoid arthritis effect by regulating cell proliferation and survival via the PI3K/AKT and JAK/STAT signaling pathways^[45]. In our study, we performed ChIP-seq of ImpacII cells and found that the Brg1 gene could be involved in the regulation of the PI3K/AKT signaling pathway. In hepatocellular carcinoma cells, downregulating Brg1 decreased lipid droplet deposition by upregulating glycosylated lysosomal membrane protein and altering the PI3K/AKT pathway^[46]. Brg1 could also regulate the phosphorylation of Akt at Ser473 to directly promote the expression of cyclin D1 and promote the growth of prostate cancer cells^[47]. These studies also validated our sequencing results, suggesting that Brg1 may affect the proliferation of AECIIs by regulating the PI3K/AKT signaling pathway. At the same time, combined with our CHIP-qPCR results, it has been found that Brg1 could bind to the JAK1/2 gene promoter region. Research has also identified Brg1 as a chromatin-remodeling factor that upregulates the transcriptome of B-cells during their activation to promote rapid cell proliferation and to mount an effective immune response^[48]. The SWI/SNF (switch/sucrose nonfermentable or BAF, Brg/Brahma-associated factors) complexes were regulated by Jak/Stat3 activity in embryonic stem cells. Knocking down Brm during early, mid, and late stages (days 3, 6, and 9 after initial iPSC induction) and knocking down Baf170 during late-stage (day 9) reprogramming improved the numbers of iPSC colonies formed^[49]. In this study, we found that knockdown of the Brg1 gene in AECII cells increased the activation of the JAK/STAT6 and PI3K/AKT pathways, and Co-IP assay found that the JAK1/2 protein bound to PI3K. The results of this study are consistent with previous reports, and we verified for the first time that the Brg1 gene may regulate downstream PI3K and JAK1/2 in AECIIs by binding to the JAK1/2 promoter region.

In conclusion, specific knockdown of Brg1 in type II alveolar epithelial cells of C57BL/6 mice can increase the number of AECⅡs in mouse lungs. Through in vitro experiments, we found that the increased number of AECⅡs was related to the increased proliferation ability of Brg1 gene knockdown cells. The increase in the number and function of AECIIs in lung tissue can enable the lung to repair the injury in time^[50]. The results of this study can provide new targets and directions for the study and treatment of respiratory diseases such as pulmonary fibrosis, asthma, and acute lung injury.

AECIIs

alveolar type II epithelial cells

Brg1: Brahma-related geng1;

SWI/SNF: switch/sucrose non-fermentable;

Brg1^fl/fl mice: C57BL/6 mice with Brg1 gene specifically knocked out in alveolar type II epithelial cells;

WT: wild-type C57BL/6 mice;

ImpacII: immortalized mouse pulmonary alveolar type II;

CHIP-seq: chromatin immunoprecipitation-sequence;

CHIP-qPCR: chromatin immunoprecipitation-realtime-PCR;

Co-IP: co-immunoprecipitation;

AECIs: alveolar type I epithelial cells;

ARDS: acute respiratory distress syndrome;

BPD: bronchopulmonary dysplasia;

SFTPC/SPC: surfactant-related protein C

pro-SFTPC/pro-SPC: precursor protein of SFTPC;

shRNAs: short hairpin RNAs;

NC-shRNA: negative control-shRNA;

Brg1-sc: immortalized mouse pulmonary alveolar type II cells stably expressing a Brg1-negative control vector;

Brg1-sh: immortalized mouse pulmonary alveolar type II cells stably expressing a Brg1 knockdown vector.

Acknowledgments

We thank Prof. Yujun Shi (West China Hospital of Sichuan University) for giving us the SFTPC-Cre-Brg1^fl/fl mice and Prof. Tongchuan He (The University of Chicago Medical Center) for providing us with ImpacII cells.

Funding

This research was supported by grants from the Postdoctoral Science Foundation of Chongqing Natural Science Foundation (No.cstc2020jcyj-bshX0075), he Postdoctoral Science Foundation of Chongqing Natural Science Foundation (No.CSTB2022NSCQ-BHX0639), the General Project of Basic Research of Ministry of Education Key Laboratory of Child Developmental Disease Research, the Children’s Hospital Affiliated to Chongqing Medical University (No.GBRP-202120), and the Program for Youth Innovation in Future Medical University (W0013).

Consent for publication

Not applicable.

Availability of data and material

All data and materials are contained and described within the manuscript.

Competing interests

The authors declare that they have no competing interests.

Ethics approval and consent to participate

The study was approved by the Ethics Committee of Chongqing Medical University.

Authors’ contributions

MZX and HJQ designed and performed the experiments and wrote the manuscript. LHZ analyzed and interpreted the data. LLY and LYR completed the experiment. WJZ and ZF contributed to the conception and design of the study, the revision of the manuscript, and the final approval of the version to be published.

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Brahma-related gene 1 regulates the proliferation of alveolar type II epithelial cells via the JAK1/2-PI3K/AKT signaling pathway

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Abstract

Background

Methods

Results

Conclusion

Figures

1. Introduction

2. Materials And Methods

2.1. Animals

2.2. Immunohistochemical assessment of lung tissues

2.3. Flow cytometry of lung tissues

2.4. Cell culture and treatments

2.5. Cell migration, cell proliferation, and cell cycle

2.6. Immunofluorescence detection

2.7. Western blot and co-immunoprecipitation (Co-IP) assay

2.8. Chromatin immunoprecipitation

2.9. Realtime RT-PCR

2.10. Statistical analyses

3. Results

3.1. Knockout of Brg1 increased the number of AECIIs in the lung tissues

4. Discussion

Abbreviations

Declarations

References

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