Interleukin-17A promotes functional activation of systemic sclerosis patient-derived dermal vascular smooth muscle cells by extracellular-regulated protein kinases signalling pathway

Abstract

Introduction: Dermal vascular smooth muscle cells (DVSMCs) are important for vascular wall fibrosis in microangiopathy of systemic sclerosis (SSc). T helper 17 cell-associated cytokines, particularly interleukin-17A (IL-17A), have been demonstrated to play a role in the pathogenesis of SSc. However, the effect of IL-17A on the DVSMCs in microangiopathy of SSc has not been established. In the present study, we investigated the effect of IL-17A on the SSc patient-derived DVSMCs.

Methods: DVSMCs from patients with SSc and healthy subjects were incubated using IL-17A or serum derived from patients with SSc. Subsequently, the proliferation, collagen synthesis and secretion, and migration of DVSMCs were analysed using a cell counting kit-8 (CCK-8), dual-luciferase reporter assay, real-time reverse transcription-polymerase chain reaction (RT-PCR), Western blot, enzyme-linked immunosorbent assay (ELISA) and transwell assay. The protein phosphorylation of signalling pathways in the process of IL-17A-mediated DVSMC activation was investigated and validated by specific signalling pathway inhibitor.

Results: IL-17A and serum from patients with SSc could promote the proliferation, collagen synthesis and secretion, and migration of DVSMCs. IL-17A neutralising antibody could inhibit the IL-17A-induced activation of DVSMCs. Additionally, IL-17A induced the activation of extracellular-regulated protein kinases 1/2 (ERK1/2) in DVSMCs, and ERK1/2 inhibitor could block the IL-17A-elicited activation of DVSMCs.

Conclusions: Our results suggested that IL-17A derived from patients with SSc might induce the proliferation, collagen synthesis and secretion, and migration of DVSMCs via ERK1/2 signalling pathway, raising the likelihood that IL-17A and ERK1/2 might be promising therapeutic targets for the treatment of SSc-related vasculopathy.

Figure 1

IL-17A derived from SSc patient serum promotes the proliferation, collagen synthesis and secretion of SSc patient-derived DVSMCs. (A) DVSMCs were treated with different doses of IL-17A for 24 h, 48 h, 72 h, and cell proliferation was tested using cell counting kit-8. (B) The cells were treated with the serum of SSc patients for 24 h, 48 h, 72 h, and cell proliferation was tested using CCK8. (C) The activity of collagen1α1 or collagen3α1 proximal promoter was test using a dual luciferase reporter gene assay after being treated with different doses of IL-17A for 24 h. (D) The activity of collagen1α1 or collagen3α1 proximal promoter was also detected after being treated with the serum of SSc patients and healthy subject for 24 h. Data were represented as mean ratios of Firefly to Renilla luciferase activity. (E) The cells were cultured in the indicated doses of IL-17A for 24 h, the gene expression of collagen 1 and collagen 3 was measured using real-time RT-PCR analysis. (F) The cells were stimulated with the serum of SSc patients and healthy subjects for 24 h, and the gene expression of collagen 1 and collagen 3 was measured using real-time RT-PCR analysis. (G) The cells were treated with different doses of IL-17A for 24 h, the concentration of collagen 1, collagen 3 was detected using ELISA. (H) The cells were treated with the serum of SSc patients and healthy subjects for 24 h, and the concentration of collagen 1, collagen 3 was also detected by ELISA. (I, J) The cells were treated with different doses of IL-17A, serum of SSc patients or healthy individuals for 24 h, and the expressions of collagen 1, collagen 3, IL-17RA and α-SMA were measured using Western blot. GAPDH was used as a loading control. The experiment was repeated three times, and the data are presented as means ± standard deviation.

Figure 2

IL-17A induces ERK1/2 MAPK phosphorylation in SSc patient-derived DVSMCs. (A) SSc patient-derived DVSMCs were treated with 100 ng/ml IL-17A for 15, 30, 45 and 60 min, and the phosphorylation of ERK1/2, p38 MAPK and JNK were detected using Western blot. (B) The cells were exposed to the serum of SSc patients or healthy controls for 30 min, the phosphorylation of ERK1/2, p38 MAPK and JNK were detected by Western blot. (C) The cells were incubated with IL-17A (100 ng/ml) in the presence of PD98059 (10 μM/ml) for 30 min, the phosphorylation of ERK1/2 was detected using Western blot. (D) The cells were incubated with 5% SSc serum or healthy serum in the presence of PD98059 (10 μM/ml) for 30 min, the phosphorylation of ERK1/2 was detected using Western blot. GAPDH was used as a loading control. The experiment was repeated three times. DVSMCs, dermal vascular smooth muscle cells; ELISA, enzyme-linked immunosorbent assay; ERK, extracellular-regulated protein kinases; IL-17A, interleukin-17A; JNK, Jun N-terminal kinase; MAPK, mitogen-activated protein kinases; SSc, systemic sclerosis.

Figure 3

IL-17A induces proliferation, collagen synthesis and secretion of SSc patient-derived DVSMCs via ERK1/2 signing pathway. (A) DVSMCs were pre-treated with PD 98059 (10 μM/ml) for 2 h before incubation with IL-17A at 100 ng/ml for 24 h, 48 h, 72 h, and cell proliferation was tested using cell counting kit-8. (B) The cells were pre-treated with PD98059 for 2 h before incubation with the serum of SSc patients or healthy individuals for 24 h, 48 h, 72 h, and cell proliferation was tested using CCK8. (C) The cells were pre-treated with PD 98059 for 2 h before incubation with IL-17A at 100 ng/ml for 24 h. The activity of collagen1α1 or collagen3α1 proximal promoter was detected using a dual luciferase reporter gene assay. (D) After being pre-treated with PD98059 for 2h before incubation with the serum of SSc patients or healthy individuals for 24 h, the activity of collagen1α1 or collagen3α1 proximal promoter was also detected. Data were represented as mean ratios of Firefly to Renilla luciferase activity. (E, F) The cells were pre-treated with PD 98059 for 2 h before incubation with IL-17A or SSc serum for 24 h, the gene expression of collagen 1 and collagen 3 was measured using real-time RT-PCR analysis. (G, H) The cells were pre-treated with PD 98059 for 2 h before incubation with IL-17A or SSc serum for 24 h, the concentration of collagen 1, collagen 3 was detected using ELISA. (I, J) The cells were pre-treated with PD98059 for 2 h before incubation with IL-17A, serum from SSc patients and healthy individuals for 24 h, and the protein expressions of collagen 1, collagen 3, IL-17RA and α-SMA were measured using Western blot. GAPDH was used as a loading control. The experiment was repeated three times, and the data are presented as means ± standard deviation.

Figure 4

IL-17A promotes migration of SSc patient-derived DVSMCs by ERK signalling pathway. (A) SSc patient-derived DVSMCs were incubated with IL-17A (100 ng/ml), 5% healthy serum, 5% SSc serum with or without anti-IL-17A antibody (8 μg/ml), ERK inhibitor (PD98059, 10 μM/ml) or P38 inhibitor SB 203580 (10 μM/ml) for 24 h. Black arrows represent migrated cells in per high-power field (HPF) at 200 × magnification. Scale bar = 100 μm. (B) The number of migrated cells in per HPF. The experiment was repeated three times, and the data are presented as mean ± standard deviation. DVSMCs, dermal vascular smooth muscle cells; ELISA, enzyme-linked immunosorbent assay; ERK, extracellular-regulated protein kinases; IL-17A, interleukin-17A; SSc, systemic sclerosis.