Effect of the BMPR-II-SMAD3/MRTF complex on proliferation and migration of ASMCs and the mechanism in asthma

Phenotype transformation of airway smooth muscle (ASM) is the key feature of airway remodelling in asthma. A variety of smooth muscle-specific genes and proteins, including SMAD3, BMPR-II, and MRTF, are involved in airway remodelling in asthma. Bone morphogenetic protein (BMP) signalling plays an important role in the physiological and pathological processes of asthma. As a receptor of BMP signalling, BMPR-II has important roles in variety of cellular processes. However, the understanding of the roles and underlying mechanism of BMPR-II in airway smooth muscle cells (ASMCs) in asthma remains incomplete. First, we observed significant increases in BMPR-II, SMAD3 and MRTF in asthmatic ASMCs at both the mRNA and protein levels. Second, we observed that silencing of siBMPR-II and siMRTF inhibits proliferation, migratory capacity and intracellular Ca2+ concentration in ASMCs. Furthermore, our results revealed these three factors, BMPR-II, SMAD3 and MRTF, form a complex that affects the bioactivities of ASMCs. Taken together, this study indicates that the BMPR-II-SMAD3/MRTF signalling pathway is involved in the process of ASM remodelling, providing novel avenues for the identification of new therapeutic modalities.


Introduction
Asthma is a common chronic respiratory disease that poses a serious potential 46 threat to patients' physical and mental health [1]. Meanwhile, it also causes significant 47 financial burdens to patients and their families [2]. Symptoms of asthma attacks 48 primarily include chest tightness, wheezing, short of breath, and coughing. These subepithelial fibrosis, goblet cell hyperplasia, and ASMC hyperplasia and 67 hypertrophy [6]. Recently, increasing research has found that the mass of the ASM 68 layer in asthmatic airway tissue is significantly thicker than that in non-asthmatics [7]. 69 Biopsies from patients with severe asthma reveal larger ASM areas and shorter 70 distance between the subepithelial basement membrane and airway smooth muscle. 71 Both ASM zone and SBM-ASM distance are closely related to FEV1 before and after 72 use of bronchodilators in asthma patients, indicating that ASM content may be closely 73 related to the severity of airway obstruction and may represent one of the critical   provided filtered water and pellet feed ad libitum. Twenty rats were randomly 123 assigned to either the control or asthma group with 10 rats per group.

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To create an asthma model, rats were intraperitoneally injected with 100 µg   The remaining tissue samples were used for isolation of primary ASMCs. applied to slides, which were then examined using a microscope (Olympus, Japan).

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Cell proliferation was measured using the Cell Counting Kit-8 assay (CCK8, 159 Junxin, Suzhou, China) according to the manufacturer's instructions. Cells were 160 plated into 96-well plates at a concentration of 0.2x10 5 cells/well. After their 161 respective treatments, 10 µL CCK8 agents were added into each well, and cells were 162 incubated for 2 hr followed by measurement of the absorbance 450 nm using a 163 microplate reader (Thermo).

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ASMCs were digested using 0.25% trypsin, 400 µL serum culture medium was added 167 to Transwell chambers of a 24-well plate, and cells at a concentration of 2 × 10 5 /ml in 168 serum-free medium were added to Transwell chamber inner chambers in 100 µL. antibodies at room temperature for 1 h. Then, membranes were exposed to ECL 203 reagent (Junxin, Suzhou, China). β-actin was measured as a loading control.

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Antibodies used in this study are listed below: anti-BMPR-II antibody (Abcam   were markedly thicker than in the control group (Fig. 1A). Smooth muscle α-actin 257 (α-SMA) immunofluorescence was performed to identify ASMCs in asthmatic rats.

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These results in the allergic asthma group are shown in Figure 1B. We observed that As shown in Figure 2A and 2B, the viability and migration ability of ASMCs 265 isolated from asthmatic rats were higher than ASMCs isolated from the control group.

BMPR-II, SMAD3, and MRTF are upregulated in ASMCs from asthmatic rats 269
To explore the role of BMPR-II, SMAD3, and MRTF in asthma, we detected were significantly up-regulated in ASMCs from asthmatic rats (Fig. 4D-F). These Ca 2+ concentration in ASMCs (Fig. 6A-C). Collectively, these results demonstrate 321 that the BMPR-II-SMAD3/MRTF complex affects cell viability, migration capacity, 322 and intracellular Ca2+ concentration in asthmatic rat ASMCs.  collection and analyses of data, decision to publish, or preparation of the article.