A potential new approach for treating systemic sclerosis: Dedifferentiation of SSc fibroblasts and change in the microenvironment by blocking store-operated Ca2+ entry
Fig 6
The differentiation of fibroblasts induced by exogenous factors was TGF-β1 independent.
(A) Human dermal fibroblasts (F) from healthy people were treated with the conditioned medium of each group, and the alteration in cell morphology was then observed. The conditioned medium was obtained from each group of SSc fibroblasts after a one-day incubation and used to treat the fibroblasts (group 1 conditioned medium, G1CM; group 2 conditioned medium, G2CM; group 3 conditioned medium, G3CM). (B) Treatment with the conditioned medium from each group of SSc fibroblasts promoted the TG-induced Ca2+ influx. Right panel: the mean area under the intracellular Ca2+ response curves after TG was applied (n >120 cells, mean ± SD, *, p<0.05; ***, p<0.001). (C) Analysis of the expression levels of TGF-β1, α-SMA, and GAPDH after treatment with the conditioned medium for 24 h. (D) The conditioned media blocked the cell migration. (E) Three extracellular molecules, gelatin-1 (Gel-1), FAM20A, and human albumin (Alb), were screened by analysis with the IPA system, which identified the common components in the media from the three groups. Analysis of the expression of gelatin-1, FAM20A, and human albumin in the three groups by LC/MS (mean ± SD, *, p<0.05; **, p<0.01; ***, p<0.001). Treatment with gelatin-1 (0.25 ng/ml), FAM20A (1 μg/ml), and human albumin (2 mg/ml) in fibroblasts for 14 days and the subsequent effects on (F) cell morphology, (G) SOCE activity and (H) α-SMA expression. Right panel: the mean area under the intracellular Ca2+ response curves after the addition of extracellular CaCl2 (n >120 cells, mean ± SD, *, p<0.05; **, p<0.01). (I) The quantification of (H) protein expression is shown (data were normalized to the protein expression of the internal control, GAPDH; mean ± SD, **, p<0.01; ***, p<0.001).