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.