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. 2023 Nov 1:14:1167605.
doi: 10.3389/fimmu.2023.1167605. eCollection 2023.

Semaphorin 7a aggravates TGF-β1-induced airway EMT through the FAK/ERK1/2 signaling pathway in asthma

Haiying Peng  1 , Fei Sun  1 , Yunxiu Jiang  1 , Zihan Guo  1 , Xinyi Liu  1 , Anli Zuo  1 , Degan Lu  1
Affiliations

Semaphorin 7a aggravates TGF-β1-induced airway EMT through the FAK/ERK1/2 signaling pathway in asthma

Haiying Peng et al. Front Immunol. .

Abstract

Background: TGF-β1 can induce epithelial-mesenchymal transition (EMT) in primary airway epithelial cells (AECs). Semaphorin7A (Sema7a) plays a crucial role in regulating immune responses and initiating and maintaining transforming growth factor β1 TGF-β1-induced fibrosis.

Objective: To determine the expression of Sema7a, in serum isolated from asthmatics and non-asthmatics, the role of Sema7a in TGF-β1 induced proliferation, migration and airway EMT in human bronchial epithelial cells (HBECs) in vitro.

Methods: The concentrations of Sema7a in serum of asthmatic patients was detected by enzyme-linked immunosorbent assay (ELISA). The expressions of Sema7a and integrin-β1 were examined using conventional western blotting and real-time quantitative PCR (RT-PCR). Interaction between the Sema7a and Integrin-β1 was detected using the Integrin-β1 blocking antibody (GLPG0187). The changes in EMT indicators were performed by western blotting and immunofluorescence, as well as the expression levels of phosphorylated Focal-adhesion kinase (FAK) and Extracellular-signal-regulated kinase1/2 (ERK1/2) were analyzed by western blot and their mRNA expression was determined by RT-PCR.

Results: We described the first differentially expressed protein of sema7a, in patients with diagnosed bronchial asthma were significantly higher than those of healthy persons (P<0.05). Western blotting and RT-PCR showed that Sema7a and Integrin-β1 expression were significantly increased in lung tissue from the ovalbumin (OVA)-induced asthma model. GLPG0187 inhibited TGF-β1-mediated HBECs EMT, proliferation and migration, which was associated with Focal-adhesion kinase (FAK) and Extracellular-signal-regulated kinase1/2 (ERK1/2) phosphorylation.

Conclusion: Sema7a may play an important role in asthma airway remodeling by inducing EMT. Therefore, new therapeutic approaches for the treatment of chronic asthma, could be aided by the development of agents that target the Sema7a.

Keywords: TGF-β1; airway remodeling; asthma; epithelial-mesenchymal transition; semaphorin 7A.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Airway inflammation and AHR in an OVA-challenged mouse model. (A) The establishment of chronic asthma mouse model; (B) The level of Sema7a in serum of patients with asthma was higher than that in serum of healthy subjects. (C) The asthma group had higher IL-4 levels in the BALF than the control group and the anti-Sema7a group had lower IL-4 levels than the asthma group. (D) The asthma group had higher IL-5 levels in the BALF than the control group and the anti-Sema7a group had lower IL-4 levels than the asthma group. (E) The asthma group had higher IL-13 levels in the BALF than the control group and the anti-Sema7a group had lower IL-4 levels than the asthma group. (F) The asthma group had higher TGF-B 1 levels in the BALF than the control group and the anti-Sema7a group had lower IL-4 levels than the asthma group. (G) It does not reveal significant differences among the three groups of baseline airway responsiveness (at 0 mg/ml methacholine); Stimulated by acetylcholine, the C was significantly descended in asthma group compared with control group, while enhanced in anti-Sema7a group compared with asthma group. (H) Stimulated by acetylcholine, the R was significantly enhanced in asthma group compared with control group, while descended in anti-Sema7a group compared with asthma group. Data represent means ± SD. *P < 0.05, **P < 0.01.
Figure 2
Figure 2
Protein and mRNA expression in lung tissues of Sema7a and Integrin-β1 in each group. (A, B) The level of Sema7a protein in lung tissue of asthma group was higher than control group. (C) The level of Sema7a mRNA in lung tissue of asthma group was higher than control group. (D, E) The level of Integrin-β1 protein in lung tissue of asthma group was higher than control group. (F) The level of Integrin-β1 mRNA in lung tissue of asthma group was higher than control group. (G–I) Immunohistochemical analyses revealed that the expression of Sema7a in asthmatic epithelial cells was significantly higher than that in the control group. Data represent means ± SD.**P < 0.01.
Figure 3
Figure 3
Histological examination of lung tissue was performed 24 h after the final OVA challenge. Lung tissues were fixed, sectioned at 5 um thickness, and stained with hematoxylin-eosin (HE), periodic acid-Schiff (PAS), and Masson stain. (A, B) Quantified results of airway wall thickness (Wat) analyzed by Image-Pro® Plus 6.0 software. (C, D) Quantified results of airway wall thickness (Wat) analyzed by Image-Pro® Plus 6.0 software. (E, F) Quantitative analysis of collagen deposition by Image-Pro® Plus 6.0 software. Data represent means ± SD. *P < 0.05, **P < 0.01.
Figure 4
Figure 4
(A, B) 5ng/ml of TGF B 1 could induce obvious EMT changes in 16HBE cells. (C–E) TGF-β1 upregulated the expression of Integrin-β1, along with the upregulation of a-SMA and fibronectin, but downregulated E-cadherin compared with the control group; GLPG0187 significantly increased the expression of the Integrin-β1, E-cadherin and decreased that of the mesenchymal markers, fibronectin, a-SMA in a concentration-dependent manner. (F) GLPG0187 can reduce the 16HBE cells proliferation rate induced by TGF-β1. (G, H) TGF-B1 significantly upregulated the expression of the FAK, P-FAK, ERK and P-ERK, while the GLPG0187 can reduce the expression of the above signaling pathway indicators, **P < 0.01, #P>0.05.
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