Mesenchymal stem cells alleviate epithelial-to-mesenchymal transition by modulating the IRE1α branch of the endoplasmic reticulum stress response

doi:10.21203/rs.3.rs-1533966/v1

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Research Article

Mesenchymal stem cells alleviate epithelial-to-mesenchymal transition by modulating the IRE1α branch of the endoplasmic reticulum stress response

https://doi.org/10.21203/rs.3.rs-1533966/v1

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posted 13 Apr, 2022

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Background: Idiopathic pulmonary fibrosis (IPF) is the most common idiopathic interstitial lung disease, and it carries a poor prognosis due to the lack of efficient diagnosis and treatments. Epithelial-mesenchymal transition (EMT) plays a key role in the pathogenesis of IPF. Endoplasmic reticulum (ER) stress contributes to fibrosis via EMT-mediated pathways. Mesenchymal stem cells (MSCs) are a promising treatment for pulmonary fibrosis and ameliorate lung fibrosis via paracrine effects in animal models. However, the specific mechanisms are not known. We previously reported that MSCs attenuated endothelial injury by modulating ER stress and endothelial-to-mesenchymal transition (EndMT). The present investigated whether modulation of ER stress and EMT-related pathways played essential roles in MSC-mediated alleviation of IPF.

Methods and results: We constructed a transforming growth factor-β1 (TGF-β1)-induced fibrosis model in A549 cells. TGF-β1 induced EMT in A549 cells, and MSC coculture ameliorated EMT, which was characterized by increased E-cadherin and decreased vimentin levels. ER stress participated in TGF-β1-induced EMT in A549 cells, and MSCs inhibited the expression of XBP-1s XBP-1u and BiP, which were upregulated by TGF-β1. Inhibition of ER stress contributed to MSC-mediated amelioration of EMT in A549 cells, in which modulation of the IRE1α-XBP1 branch of the ER stress pathway may play an important role. MSC transplantation alleviated bleomycin-induced pulmonary fibrosis in mice. MSC treatment decreased the expression of ER stress- and EMT-related genes and proteins, and inhibition of the IRE1α/XBP1 pathway was the most obvious effect.

Conclusions: The present study demonstrated that MSCs alleviated EMT by modulating ER stress and blockade of the IRE1α-XBP1 pathway may play a critical role. The current study provides a novel vision for the application of MSCs for IPF treatment and elucidates the mechanism of the preventive effects of MSCs against pulmonary fibrosis.

mesenchymal stem cells

epithelial-to-mesenchymal transition

endoplasmic reticulum stress

Idiopathic pulmonary fibrosis (IPF) is the most common idiopathic interstitial lung disease, which is characterized by chronic, progressive worsening of pulmonary function. IPF has a poor prognosis with a median survival of 2–3 years due to the lack of efficient diagnosis and treatments[1]. IPF also places a heavy financial burden on patients and society because treatment costs approximately 25,000 USD/person-year[2]. The triggers for IPF are not clear, and a multitude of factors likely play a role, including smoking, environmental exposures, male sex, and aging[3].

Considerable studies demonstrated that uninterrupted alveolar injuries and abnormal repairs contributed to subsequent fibrosis[4]. During lung injury repair, the continuous epithelial-mesenchymal transition (EMT) contributes to extracellular matrix accumulation and tissue remodeling[5]. EMT is the process by which epithelial cells lose tight junctions and apical-basal polarity and alter their shape from an epithelial-like to a mesenchymal-like phenotype under certain conditions[6]. EMT leads to fibrosis in a number of organs, including the kidney[7], liver[8], intestine[9] and lung. The role of EMT in the pathogenesis of IPF was widely reported and confirmed[10], and suppression of EMT improves IPF in animal models[11]. Many factors cause EMT, and activation of the unfolded protein response (UPR) and subsequent endoplasmic reticulum (ER) stress may contribute to fibrosis via EMT-mediated pathways[6]. Continuous ER stress is a causative factor of EMT in alveolar epithelial cells subjected to ER stress inducers[12]. However, the precise underlying mechanisms between EMT, ER stress and IPF are not clear.

Mesenchymal stem cells (MSCs) have become a hotspot in cell therapy due to their self-renewal and multilineage differentiation properties. MSCs have therapeutic potential for various diseases due to their immunomodulatory and paracrine properties[13]. Our previous studies found that MSCs showed promising outcomes for the treatment of diabetes mellitus[14], nonalcoholic fatty liver disease (NAFLD)[15], hyperuricemic nephropathy[16] and diabetic nephropathy[17] in animal models. For IPF, some studies suggested that MSCs ameliorated bleomycin-induced lung fibrosis via paracrine effects in mice[18, 19]. MSCs also reduced hypoxia-induced alveolar EMT in vitro by inhibiting the expression of transforming growth factor-β1 (TGF-β1)[20]. Stanniocalcin 1 secreted by MSCs reduces oxidative and ER stress and the level of profibrotic factors in AECs[21], but the specific molecular mechanism of these beneficial effects are not clear. Therefore, we investigated links between ER stress and EMT and hypothesized that modulation of ER stress and EMT-related pathways would play important roles in MSC-mediated alleviation of IPF. The present study also investigated the regulatory effects of MSCs on ER stress and EMT in bleomycin-induced lung fibrosis in mice and the molecular mechanisms involved.

2.1 Cell Culture

The human alveolar epithelial cell line (A549 cells) was kindly provided by Sichuan University and cultured in DMEM/F-12 medium (G4613, Servicebio, Wuhan, China) supplemented with 10% fetal bovine serum (FBS, Gibco, Waltham, MA, USA) at 37°C. Human umbilical cord-derived mesenchymal stem cells (huMSCs) were purchased from Zhongqiao Xinzhou Co., Ltd. (DF-GMP-ZB09BA, Shanghai, China). MSCs were cultured in MSC Complete Medium (ZQ-1320, Zhongqiao Xinzhou) supplemented with 5% serum substitute (ZQ-1320S, Zhongqiao Xinzhou) and antibiotics (S110JV, BasalMedia, Shanghai, China) at 37°C in 5% CO₂. For coculture experiments, huMSCs were seeded in a Transwell insert (Corning, New York, NY, USA) and cultured in HUMSC Complete Medium overnight before coculture with A549 cells at a ratio of 2:5 in DMEM/F-12 medium.

2.2 Cell Treatment

The control groups were untreated cells (Cont). A549 cells were treated with TGF-β1 (100 − 21, PeproTech, Rocky Hill, NJ, USA) for 72 h with or without pretreatment with the ER stress inhibitor TUDCA (abs816166; Absin, Shanghai, China) or IRE1α-specific inhibitor 4µ8c (T6363, Topscience, Shanghai, China) for 1 h. Tunicamycin (TM, M4798, AbMole, Houston, TX, USA) was used to induce ER stress in A549 cells.

2.3 Cell Viability

Cell viability was measured using Cell Counting Kit 8 (CCK8, CK04, Dojindo, Japan) according to the manufacturer’s instructions. Briefly, the supernatant was removed, and CCK8 solution was added to each well at a 1:10 dilution with cell culture medium. The absorbance at 450 nm was measured using a microplate reader (Thermo Scientific, San Jose, CA, USA) after 2–4 h.

2.4 Transmission Electron Microscopy (TEM)

A549 cells were prefixed and embedded. After the indicated treatments, the sections were observed under a transmission electron microscope (JEM-1400, Japan).

2.5 RNA Isolation and Quantitative Real-Time PCR

Total RNA from A549 cells and lung tissues was extracted using TRIzol reagent (15596026, Thermo Scientific, USA) and reverse-transcribed into cDNA using a Reverse Transcription Kit (R312, Vazyme, Nanjing, China). Quantitative real-time PCR (Q-PCR) was performed using SYBR Green qPCR Master Mix (B21203, Bimake, Houston, Texas, USA) in a CFX96 Real-Time PCR System (BioRad, Hercules, CA, USA). The primer information is listed in the Supplemental Table, and the primer for the reference gene (β-actin) was obtained from Sangon Biotech (Shanghai, China). The 2^−△△CT method was used to quantify relative gene expression.

2.6 Western Blotting

Total protein from A549 cells and lung tissues was extracted using radioimmunoprecipitation assay (RIPA) buffer (Beyotime, Shanghai, China). After SDS-PAGE, the membranes were blocked with Protein Free Rapid Blocking Buffer (PS108, EpiZyme) and incubated with primary antibodies against CHOP (A5462, Bimake), BiP (11587-1-AP; Proteintech, Wuhan, China), ATF6 (D262665, Sangon, China), ATF4 (A5514, Bimake), XBP-1s (24868-1-AP, Proteintech), XBP-1u (25997-1-AP, Proteintech), IRE1α (A00683-1, Boster, Wuhan, China), phospho-IRE1α (S724, human) (ab124945, Abcam, Cambridge, UK), phospho-IRE1α (S724, mouse) (530878, ZEN-BIO, Chengdu, China), Vimentin (ET1610-39, HuaBio, Hangzhou, China), and E-cadherin (340341, ZEN-BIO) overnight. GAPDH (bs-0755R, Bioss, Beijing, China) was used as the internal reference protein. After incubation with HRP-conjugated goat anti-rabbit IgG (H + L) (AS014, ABclonal, Wuhan, China) and HRP-conjugated goat anti-mouse IgG (H + L) (AS003, ABclonal), the immunoblots were visualized using a ChemiDoc™ Imaging System (Bio-Rad) and quantified using NIH ImageJ software (http://rsb.info.nih.gov).

2.7 Immunofluorescence

Immunofluorescence experiments were performed as previously described[22]. Lung tissue slices were incubated with anti-BiP (11587-1-AP, Proteintech) and anti-vimentin antibodies (BF8006, Affinity Biosciences, Changzhou, China). The cell climbing slices were incubated with anti-vimentin (BF8006, Affinity Biosciences) and anti-E-cadherin antibodies (3195T, CST, MA, USA). After staining with secondary antibodies and DAPI, the slices were observed under a fluorescence microscope (Olympus, Japan).

2.8 Animal Experiments

C57BL/6 mice were housed in the animal center of Guizhou University of Traditional Chinese Medicine in accordance with the Guide for the Care and Use of Laboratory Animals. Male wild-type C57BL/6 mice (8 weeks old, n = 15) were purchased from China Three Gorges University [SCXK (E) 2017-0012, Hubei, China]. After a 1-week adaptive period, mice were randomly assigned to the sham (Cont, n = 5), model (BLM, n = 5), and MSC treatment groups (BLM + MSCs, n = 5). Mice were anesthetized and intratracheally injected with 3.5 mg/kg bleomycin (B107423, Aladdin, Shanghai, China) to induce pulmonary fibrosis. The control mice received the same volume of saline. The animals were placed in a vertical position with rotation to evenly distribute the drug. One day after surgery, mice in the BLM + MSCs group received mouse bone marrow MSCs (passages 2 to 4, 5×10⁵ cells/mouse suspended in 200 µL of PBS) every other week twice via tail vein injection. Mouse MSCs were purchased from Procell Life Science & Technology Co., Ltd. (CP-M131, Wuhan, China). Mice in the Cont and BLM groups received the same volume of PBS. Twenty-eight days after surgery, the mice were euthanized, and lung tissues and serum were harvested. The Institutional Animal Care and Use Committee of Guizhou University of Traditional Chinese Medicine approved all experimental procedures.

2.9 Hydroxyproline Assay

The hydroxyproline content in mouse lungs was determined using a hydroxyproline assay kit (A030-2-1, NNJCBIO, Nanjing, China) according to the manufacturer’s instructions. The hydroxyproline content is expressed in µg/mg left lung (wet weight).

2.10 Histology

Mouse pulmonary tissues were fixed and embedded in paraffin. The tissues were sliced for hematoxylin and eosin (HE) staining (D006, NJJCBIO) and Masson's staining (D026, NJJCBIO). The slices were observed under a light microscope (Leica, Germany). Collagen area, alveolar space and numbers were quantified using ImageJ software.

2.11 Enzyme-Linked Immunosorbent Assay

The concentrations of IL-1β (70-EK201B, MultiSciences, Hangzhou, China), IL-6 (RK00008, ABclonal), TNF-α (SEKM-0034, Solarbio, Beijing, China) and TGF-β1 (SEKM-0035, Solarbio) in serum were measured using ELISA kits according to the manufacturers’ instructions.

2.12 Statistical Analysis

All experiments were performed at least three times. The data were analyzed using one-way analysis of variance (ANOVA) and Duncan’s multiple-range test, Student’s t test or Bonferroni post hoc test. Data are presented as the means ± SEM, and differences between experimental groups were considered statistically significant at P < 0.05.

3.1 TGF-β1 induced EMT in A549 cells

TGF-β1 is commonly used as an inducer in models of EMT in A549 cells[5]. We first determined the cytotoxicity of TGF-β1 on A549 cells. A549 cells were exposed to different concentrations (5, 10, and 20 ng/ml) of TGF-β1 for 24, 48 and 72 h. CCK8 measurements showed that TGF-β1 treatment did not significantly affect cell viability (Fig. 1A). We treated A549 cells with 10 ng/ml TGF-β1 for 72 h, and the cell morphology of A549 cells changed from an epithelial shape to a fibroblast-like shape (Fig. 1B). The protein levels of the fibroblast marker vimentin were markedly increased, and the epithelial marker E-cadherin decreased, which is consistent with the time course (Fig. 1C). Immunofluorescence staining showed that vimentin was increased and E-cadherin was decreased after TGF-β1 treatment in A549 cells (Fig. 1D). Collectively, these results indicated that TGF-β1 induced EMT in A549 cells.

3.2 MSCs ameliorated EMT in A549 cells

We examined whether MSCs ameliorated EMT in A549 cells. Phase-contrast images revealed that MSCs coculture maintained the epithelial shape of A549 cells after TGF-β1 treatment (Fig. 2A). Q-PCR showed that MSCs restored the gene expression of E-cadherin and inhibited the expression of vimentin compared to the TGF-β1 group (Fig. 2B). Western blot analysis also showed that MSCs restored the protein expression of E-cadherin and suppressed the expression of vimentin (Fig. 2C). The coculture of A549 cells with MSCs without TGF-β1 did not influence the protein expression of EMT-related markers (Fig. 2D). Consistently, immunofluorescence staining showed a significant loss of E-cadherin and enhancement of vimentin protein expression in A549 cells after TGF-β1 treatment, and MSC coculture reversed these alterations (Fig. 2E). These findings suggested that MSCs ameliorated TGF-β1-induced EMT in A549 cells.

3.3 ER stress is involved in TGF-β1-induced EMT in A549 cells

Growing evidence indicates that ER stress drives EMT in different cellular systems[6, 12]. Therefore, we tested whether TGF-β1 induced ER stress in A549 cells. We found that the protein levels of some ER stress markers, including ATF6, ATF4, XBP-1s and BiP, were increased after 10 ng/ml TGF-β1 treatment for different times (Fig. 3A). Notably, TGF-β1 slightly upregulated CHOP, which indicates that ER stress did not induce apoptosis in A549 cells after TGF-β1 treatment for 72 h. Next we investigated whether ER stress induced EMT in A549 cells. A549 cells were cultured with different concentrations of a specific ER stress activator, TM (1, 2 and 5 µM) for 48 h, and cell viability was decreased 50% compared to the Cont group (Fig. 3B). Then A549 cells were treated with TM (0.50, 1 and 2 µM) for 48 h, and TM upregulated the protein levels of BiP, ATF4, ATF6, XBP-1s and CHOP (Fig. 3C). As expected, TM increased the expression of vimentin and decreased the expression of E-cadherin in A549 cells, which indicated the occurrence of EMT (Fig. 3D). Collectively, these results demonstrated that ER stress was involved in TGF-β1-induced EMT in A549 cells.

3.4 MSCs ameliorated ER stress in A549 cells

Our previous study showed that MSCs ameliorated PA-induced ER stress in human umbilical vein endothelial cells[22]. Therefore, we investigated whether MSCs alleviated ER stress in A549 cells. The results showed that MSCs significantly decreased the protein levels of XBP-1s XBP-1u and BiP, which were upregulated by TGF-β1, but MSCs did not obviously inhibit ATF4 or ATF6 expression in A549 cells (Fig. 4A). Q-PCR also revealed that MSCs significantly decreased the expression of many ER stress-related genes, including ATF6, XBP-1s, and BiP (Fig. 4B). Coculture of MSCs with A549 cells without TGF-β1 did not influence the protein levels of ER stress-related genes (Fig. 4C). To obtain more accurate results, TEM was performed. We found that the structure of the ER was ordered and tubular in A549 cells of the Cont group. However, the ER was irregular and dilated after TGF-β1 treatment, which was appreciably improved by MSC coculture (Fig. 4D). These results suggested that MSCs alleviated TGF-β1-induced ER stress in A549 cells.

3.5 Suppression of ER stress contributed to MSC-mediated amelioration of EMT in A549 cells

To determine whether MSCs inhibit EMT by suppressing ER stress, we used a specific inhibitor of ER stress, TUDCA. TUDCA alone did not influence the expression of ER stress-related proteins (BiP, ATF6, ATF4, XBP-1u or XBP-1s) (Fig. 5A) but significantly inhibited TGF-β1-induced ER stress in A549 cells (Fig. 5B). TUDCA also restored the protein levels of E-cadherin and suppressed the expression of vimentin compared to the TGF-β1 group, which suggests that attenuation of ER stress helped ameliorate EMT (Fig. 5C, D). These results demonstrated that attenuation of ER stress contributed to MSC-mediated amelioration of EMT in A549 cells.

3.6 MSCs attenuated EMT via the IRE1α/XBP1 pathway

ER stress responses are comprised of three branches of signaling pathways: ATF6, PERK, and IRE1α. Figure 1 shows that MSCs particularly inhibited XBP-1s protein levels in A549 cells. Therefore, we hypothesized that the IRE1α/XBP1 pathway played an important role in MSC-mediated amelioration of EMT in A549 cells. To test this hypothesis, we first examined an upstream signaling molecule of XBP1, IRE1α. Western blot analysis showed that TGF-β1 prominently up-regulated IRE1α and phosphorylated (p)-IRE1α (S724), and MSC coculture suppressed IRE1α and p-IRE1α protein levels (Fig. 6A). Similar results were obtained using Q-PCR (Fig. 6B). To verify whether MSCs ameliorated EMT by inhibiting the IRE1α-XBP1 pathway in A549 cells, we introduced a specific inhibitor of IRE1α activity, 4µ8c (Fig. 6C). The results showed that 4µ8c treatment attenuated the TGF-β1-induced EMT-related protein alterations (Fig. 6D). Overall, our study indicated that the IRE1α-XBP1 pathway is at least partially associated with MSC-mediated amelioration of EMT in A549 cells.

3.7 MSCs decreased bleomycin-induced pulmonary fibrosis in mice

The aforementioned results showed that MSCs attenuated EMT by regulating ER stress in vitro. We performed follow-up in vivo experiments to confirm the effects of MSCs (Fig. 7A). Twenty-eight days after the initial surgery, bleomycin (BLM) treatment decreased body weight, and MSC transplantation reversed it (Fig. 7B). However, no morphological differences were observed in the lung tissues of all groups of mice (Fig. 7C). Compared to the BLM group, the serum levels of TNF-α, IL-1β, and IL-6 were lower in BLM + MSC mice, which indicated that MSCs reduced inflammation in lung fibrosis mice (Fig. 7D-F). HE staining revealed that the pulmonary alveolar space and numbers were obviously decreased, the connective tissue was markedly increased, and the alveolar wall was thickened in mice receiving bleomycin. The alveolar space and number were significantly increased in the BLM + MSCs group, and the connective tissue was significantly lower compared to the BLM group (Fig. 7G-I).

Next, pulmonary fibrosis was assessed. Hydroxyproline (HYP) assays were used to assess the collagen content in the lung. The HYP content was significantly increased in the BLM group compared to the Cont group, and MSCs decreased the HYP levels (Fig. 8B). Masson trichrome staining showed that collagen was increased in the alveolar and interstitial regions compared to the control, but a remarkable decrease in collagen deposition was observed in the MSC treatment group (Fig. 8B, C). Upon the development of lung fibrosis, the serum level of TGF-β1 was significantly increased in BLM-treated mice and was decreased by MSC transplantation (Fig. 8D). These results indicated that MSCs decreased bleomycin-induced pulmonary fibrosis in mice.

3.8 MSCs ameliorated ER stress and EMT in the lungs of lung fibrosis mice

We first assessed whether MSC transplantation decreased EMT in lung tissues. We analyzed EMT using E-cadherin and vimentin levels. Q-PCR and Western blotting showed that E-cadherin was decreased, and vimentin was increased in BLM-treated mice, which were reversed by MSC treatment (Fig. 9A, B). ER stress markers were evaluated. MSC treatment markedly decreased the expression of several ER stress-related genes (Atf4, Ire1α, Xbp-1s, Bip and Chop) in lung tissues (Fig. 9C). However, the protein expression levels of ATF6, IRE1α, p-IRE1α (S724), XBP-1s, XBP-1u, BiP and CHOP were upregulated in lung tissues of BLM-treated mice and decreased after MSC transplantation (Fig. 9D). Notably, the inhibition of the IRE1α/XBP1 pathway by MSCs was the most obvious effect. To more intuitively observe ER stress and EMT in lung tissues, dual immunofluorescence staining of BiP and vimentin was performed. As shown in Fig. 9E, a significant increase in BiP and vimentin was observed in lung tissues in the BLM group, which was attenuated in the BLM + MSC group. Taken together, these results indicated that MSCs attenuated ER stress and EMT in lungs from lung fibrosis mice, in which suppression of the IRE1α/XBP1 pathway may play a critical role.

The present research examined the effects and underlying molecular mechanisms of MSCs in in vitro and in vivo models of IPF. We found that MSCs alleviated TGFβ1-induced EMT by inhibiting ER stress in A549 cells. We showed that the IRE1α/XBP-1 pathway may play an important role in MSC-mediated alleviation of EMT. We also confirmed the results in a bleomycin-induced IPF mouse model (Fig. 10).

The pathogenesis of IPF is not clearly understood, and several factors likely play a role. Chronic alveolar epithelial cell (AEC) injuries deserve attention. There are two types of AECs: type I (AT-I) and type II (AT-II). AT-I cells execute gas exchange, and AT-II cells serve as progenitor cells and differentiate into AT-I cells after injury[23]. The current paradigm considers that AECs play a central role in the pathogenesis of IPF due to their abnormal regenerative function. Chronic AEC injuries may occur due to various continuous stimuli, such as inflammation, stress, pollutants and infection[24]. Aberrant alveolar repair and the imbalance between profibrotic and antifibrotic mediators lead to IPF[25]. A recent study showed that AT-II activated local fibroblasts and accelerated profibrotic microenvironment formation via paracrine signaling, and fibrosis developed with excessive extracellular matrix accumulation[26]. Growing evidence showed that EMT contributed to promotion of the profibrotic microenvironment by dysregulating paracrine signaling in AT-II cells[27]. Therefore, we focused our studies on EMT. TGFβ is one of the most powerful cytokines driving fibrosis, and it is widely used to induce EMT in AECs[28]. The present study treated A549 cells with TGF-β1 for 72 h and successfully constructed an EMT model in vitro. Subsequent experiments showed that MSCs ameliorated EMT in A549 cells. Notably, many studies reported that MSCs promoted EMT and increased migration and invasion in A549 cells[29]. While other studies showed that MSCs prevented EMT in TGF-β-treated A549 cells[30]. The contradictions in these studies are likely attributable to the different sources of MSCs, the different coculture systems (e.g., the cell number and proportion) and the variability in the method of cell isolation. Beane OS reported that BM-MSCs from aging donors functioned abnormally compared to MSCs derived from younger donors[31]. Therefore, establishing a clear and intact evaluation system for MSCs is critical.

The ER is an important organelle in eukaryotic cells, and it is responsible for the synthesis, folding, and maturation of most proteins in cells. Under normal conditions, protein homeostasis is maintained by the balance between protein synthesis and degradation[32]. However, the UPR is activated when unfolded and misfolded proteins accumulate in the ER. The UPR is a cytoprotective response that attempts to restore protein homeostasis, but excessive or prolonged UPR results in ER stress[33]. In recent decades, ER stress was demonstrated as an important pathological mechanism in various diseases, such as diabetes, NAFLD, atherosclerosis, IPF and cancer[34, 35]. Increased ER stress is a causative factor of EMT in AECs subjected to ER stress inducers[12]. Our previous study showed that ER stress inducers (TM and thapsigargin) induced EndMT in HUVECs[22]. Therefore, we evaluated ER stress in TGF-β1-treated A549 cells. ER stress includes three signaling pathways, the PERK/ATF4, ATF6 and IRE1α/XBP1 pathways. Therefore, we detected the protein expression of ATF4, ATF6, XBP-1s, BiP and CHOP. TGF-β1 upregulated the protein expression of ATF4, ATF6, XBP-1s and BiP, which indicated that ER stress occurred in A549 cells. Notably, the expression of CHOP, which mediates ER stress-induced apoptosis, was not increased (P > 0.05). In contrast, we noticed that palmitic acid robustly increased the expression of CHOP and other ER stress-related proteins in HUVECs, and cell viability was significantly decreased after 24 h in our previous study[22]. TGF-β1 did not decrease cell viability after 72 h. Therefore, we speculate that TGF-β1-induced EMT is a comparatively gentle process that does not induce ER stress-induced apoptosis.

ER stress was reported to induce EMT in AECs. To confirm this effect, we treated A549 cells with the ER stress inducer TM. TM upregulated the expression of ATF4, ATF6, XBP-1s and CHOP. TM also increased the protein levels of vimentin and decreased the expression of E-cadherin in A549 cells, which indicated the occurrence of EMT. Therefore, we conclude that ER stress is involved in TGF-β1-induced EMT. However, multiple signaling pathways are involved in TGF-β1-induced EMT in A549 cells, including classic pathways such as the Smad, PI3K/AKT, and RhoA/ROCK pathways[36]. We paid more attention to ER stress because of the potent anti-ER stress capacity of MSCs, as we previously reported[37], and we presumed that ER stress was a key target in MSC-mediated amelioration of EMT. It was a pleasant surprise that MSCs prominently suppressed the protein expression of XBP-1s and XBP-1u, which prompted us to detect the expression of the upstream molecules of XBP1 signaling, IRE1α/p-IRE1α (S724). MSC coculture obviously suppressed IRE1α and p-IRE1α mRNA and protein levels. Inhibition of IRE1α activity using the IRE1 RNase inhibitor 4µ8c produced similar results, which indicates that modulation of the IRE1/XBP1 pathway may be an important mechanism in MSC protection against TGF-β1-induced EMT. However, MSCs exert their beneficial effects in numerous ways. Therefore, it is difficult to conclude that modulation of the IRE1/XBP1 pathway is the most important mechanism for all of the protective effects of MSCs. We observed that 4µ8c demonstrated less protection than MSCs against EMT in A549 cells. Therefore, we speculate that the pathways involved in the protection of MSCs are multifaceted, and further studies are needed.

To further verify the in vitro results, we constructed a BLM-induced IPF mouse model. We found that MSCs decreased collagen deposition, reversed histological alterations and reduced serum inflammatory factors and TGF-β1 levels in mice compared to the BLM group. Several recent studies reported the protective effects of MSCs on IPF animal models. Kuo-An Chu et al. reported that human umbilical mesenchymal stem cell transplantation effectively reversed IPF in rats. MSCs inhibited inflammation, prevented collagen synthesis, activated macrophages to synthesize MMP-9 and promoted TLR-4 expression in AECs[38]. Kun Huang[39] and Shirong Ni[40] showed that MSCs attenuated BLM-induced fibrosis by inhibiting oxidative stress and the Nrf2 pathway. However, few studies focused on the effects of MSCs on ER stress and EMT in BLM-treated mice. To the best of our knowledge, the current study is the first report to provide a comprehensive assessment of the three UPR signaling branches and found that MSCs particularly inhibited the IRE1α/XBP1 pathway in an IPF mouse model. These results are consistent with the in vitro results. Therefore, suppression of the IRE1α/XBP1 pathway is likely key for MSC-mediated amelioration of EMT. We also found that CHOP protein levels were increased in the BLM group and reduced by MSC transplantation, which indicated more serious ER stress activation than in vitro experiments. This result may be due to the differences in experimental conditions between in vitro and in vivo, and the in vivo physiological environment is much more complex than the in vitro physiological environments.

In conclusion, we demonstrated that MSCs ameliorated TGFβ1-induced EMT by modulating ER stress in A549 cells, and blockade of the IRE1α-XBP1 pathway was associated with these effects. MSCs attenuated ER stress and EMT in the lungs in vivo, which contributed to the amelioration of lung fibrosis in BLM-treated mice. The current study provides a novel vision for the application of MSCs for IPF treatment and elucidates the mechanism of the preventive effects of MSCs against pulmonary fibrosis.

IPF: Idiopathic pulmonary fibrosis; EMT: Epithelial-mesenchymal transition; EndMT: Endothelial-to-mesenchymal transition; MSCs: Mesenchymal stem cells; ER: Endoplasmic reticulum; TGF-β1: Transforming growth factor-β1; UPR: Unfolded protein response; NAFLD: Nonalcoholic fatty liver disease; huMSCs: Human umbilical cord-derived mesenchymal stem cells; TM: Tunicamycin; CCK8: Cell Counting Kit 8; Q-PCR: Quantitative real-time PCR; HE: Hematoxylin and eosin; BLM: Bleomycin; HYP: Hydroxyproline; AEC: Alveolar epithelial cell.

Acknowledgements

We thank Dr. Min Zhu for providing A549 cells.

Authors' contributions

The study was conceived by Ruixi Luo, and Weiyi Tian. Ruixi Luo performed the experiments and data collection with help from Yaqiong Wei, Peng Chen and La Wang. The paper was written by Ruixi Luo and edited by Wenjia Wang, Ping Wang and Weiyi Tian.

Funding

This study was supported by the Science and Technology Foundation of Guizhou Province (QKHJC-ZK[2021] YIBAN345), the Science and Technology Foundation of Guizhou Health Commission ([2021]92), Program of National Natural Science Foundation of China (81760790 and 81960796), and Guizhou Science and Technology Innovation Talent Team (Guizhou Science and Technology Cooperation Platform Talent [2020]5010).

Availability of data and materials

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Ethics approval and consent to participate

This study was approved by the Institutional Animal Care and Use Committee of Guizhou University of Traditional Chinese Medicine (20210041).

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

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Mesenchymal stem cells alleviate epithelial-to-mesenchymal transition by modulating the IRE1α branch of the endoplasmic reticulum stress response

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Abstract

Figures

1. Introduction

2. Materials And Methods

2.1 Cell Culture

2.2 Cell Treatment

2.3 Cell Viability

2.4 Transmission Electron Microscopy (TEM)

2.5 RNA Isolation and Quantitative Real-Time PCR

2.6 Western Blotting

2.7 Immunofluorescence

2.8 Animal Experiments

2.9 Hydroxyproline Assay

2.10 Histology

2.11 Enzyme-Linked Immunosorbent Assay

2.12 Statistical Analysis

3. Results

3.1 TGF-β1 induced EMT in A549 cells

3.2 MSCs ameliorated EMT in A549 cells

3.3 ER stress is involved in TGF-β1-induced EMT in A549 cells

3.4 MSCs ameliorated ER stress in A549 cells

3.5 Suppression of ER stress contributed to MSC-mediated amelioration of EMT in A549 cells

3.6 MSCs attenuated EMT via the IRE1α/XBP1 pathway

3.7 MSCs decreased bleomycin-induced pulmonary fibrosis in mice

3.8 MSCs ameliorated ER stress and EMT in the lungs of lung fibrosis mice

4. Discussion

Abbreviations

Declarations

References

Supplementary Files

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