3.8 | Cyclopamine suppresses WISP-1-induced fibrogenesis in vitro
Subsequently, we performed an in vitro assay to confirm the role of the Hedgehog signaling in the pro-fibrotic effect of WISP-1 on LF fibrosis. Firstly, we established the WISP-1 overexpression cell lines. As shown in Figures 8A, WISP-1 expression increased after plasmid transfection and the levels of Hedgehog signaling-related proteins such as Gli1 and Smo were significantly reduced by Hedgehog signaling inhibitor cyclopamine.
Previously, our in vitro experiment suggested that WISP-1 increased the collagen expression in human LF fibroblasts (Sun et al., 2017). Next, we sought to explore the intrinsic mechanisms by which WISP-1 regulated collagen expression. Having observed that the WISP-1 activated Hedgehog-Gli1 signaling in LF cells in vitro and Gli1 over-expression abundantly enhanced the expression of collagen in the present study. We predicted that WISP-1 might increase collagen expression via the Hedgehog-Gli1 pathway. To conform this, we detected the expression of collagen with or without WISP-1 over-expression in the presence or absence of Hedgehog inhibitor (cyclopamine) in human LF cells. The protein expression detected by western blot demonstrated that Hedgehog-Gli1 signaling was a crucial adaptor for WISP-1 induced collagen expression, as the upregulation of collagen expression induced by WISP-1 was significantly attenuated by cyclopamine (Fig. 8B).
Having demonstrated that WISP-1 enhanced LF fibroblast viability, we next sought to explore the possible mechanisms. According to the results of the EdU assay, LF cell proliferation was significantly enhanced by WISP-1 over-expression. However, cyclopamine inhibited WISP-1-induced cell proliferation, suggesting that WISP-1 promoted LF cell proliferation via the Hedgehog signaling (Fig. 8C). Furthermore, flow cytometry demonstrated that the percentage of G0/G1 phase LF cells with WISP-1 over-expression was significantly lower as compared with the control. However, this response was suppressed by incubation with cyclopamine (Fig. 8D).
To confirm whether the positive effect of WISP-1 on LF cell viability is associated with apoptosis inhibition, we first examined the percentage of apoptotic cells by flow cytometry. This percentage comprised the sum of early and late apoptotic cells and was significantly reduced with WISP-1 overexpression in LF cells. However, the percentage of the apoptotic cells was significantly increased after following incubation with cyclopamine (Fig. 8E). Take together, above data suggested that WISP-1 promoted collagen expression and cell proliferation, and inhibited cell apoptosis through the Hedgehog pathway.
3.9 | WISP-1 promotesa-SMA expression through Hedgehog signaling
During fibrosis, the trans-differentiation of fibroblasts into myofibroblasts influences the production of ECM and secretes fibrosis related factors, which is a key cellular event that drives the fibrosis response in various tissues and organs. a-SMA is regarded as a marker of activated myofibroblasts (Mack & Yanagita, 2015; Shu & Lovicu, 2017; Rosenkranz, 2004). We found that the fluorescence intensity of a-SMA in LF tissues from LSCS group was significantly higher than that of LDH group (Fig. 9A), indicating that the transition of fibroblasts into myofibroblasts was also the important mechanism of LF fibrosis. It has been reported that CCN protein induces a-SMA expression in the fibrosis of many organs (Chen et al., 2019; Morales et al., 2011). Therefore, we suspected that WISP-1 might also induce a-SMA expression in LF fibroblast during fibrogenesis. In order to confirm this, we conducted a gain of function experiment in which we overexpressed WISP-1 by plasmid transfection. Similar to the WISP-1 and Gli1 expression results, we found that WISP-1 overexpression significantly increase a-SMA expression. In addition, the crucial role of WISP-1 in a-SMA expression in LF cells was further demonstrated by the loss-of-function in which we knocked down WISP-1. The results showed that WISP-1 knockdown significantly reduced a-SMA expression (Fig. 9B and 9C). Meanwhile, further evidence of the role of WISP-1 in a-SMA expression was generated from our experiments with immunofluorescence staining measuring fluorescence intensity (Fig. 9D).
As we had shown that Hedgehog-Gli1 signaling is required for WISP-1-induced fibrogenesis, we predicted that WISP-1 might also increase a-SMA expression via the Hedgehog-Gli1 pathway. To confirm this, we detected the level of a-SMA with or without WISP-1 overexpression in the presence or absence of cyclopamine in human LF fibroblasts. The protein expression of a-SMA in the LF cells detected by western blot was significantly reduced by cyclopamine (Fig. 9E). Next, the immunofluorescence staining was also perform to examine the expression level of a-SMA. As shown in Fig. 9F, the fluorescence intensity of a-SMA after WISP-1 overopression was higher in LF cells than that of control group. Meanwhile, WISP-1 knockdown in human LF cells substantially reduced the the fluorescence intensity of a-SMA. Furthermore, the increased fluorescence intensity of a-SMA induced by WISP-1 overopression was significantly suppressed by cyclopamine. These results indicated that WISP-1 induced a-SMA expression in human LF fibroblasts by targeting Hedgehog signaling.