Gli2-regulated activation of hepatic stellate cells and liver fibrosis by TGF-β signaling

Abstract

The Hedgehog (Hh) signaling pathway is correlated with hepatic stellate cells (HSCs) activation and liver fibrosis. Gli2 is a key transcription effector of Hh signaling. However, the role of Gli2 in HSC-mediated liver fibrosis progression is largely unknown. In the present study, we investigated the effect of Gli2 on liver fibrogenesis and its possible mechanism using conditional knockout (cKO) Gli2 mice and HSC models. Wild-type (WT) and GFAP-CreERT;Gli2^flox/flox male mice were exposed to CCl₄ for 1 mo to induce liver fibrosis. Primary HSCs were isolated from mice and the transition of HSCs into a myofibroblastic phenotype was evaluated. Livers from mice underwent histological, immunohistochemical, and immunofluorescence analyses. The expression levels of proteins and genes were evaluated by Western blot (WB) analysis and quantitative real-time polymerase chain reaction (qRT-PCR), respectively. RNA-seq was used to screen differentially expressed genes. Results showed that CCl₄ treatment induced liver fibrosis, promoted HSCs activation and proliferation, and upregulated Hh signaling activity. The cKO of Gli2 in GFAP-CreERT;Gli2^flox/flox mice decreased liver fibrosis as well as HSC activation and proliferation. In vitro studies showed that KO of Gli2 in HSCs blocked cell proliferation and activation by decrease of cyclin D1/D2 expression. The RNA-seq results revealed that the expression levels TGF-β1 ligands were downregulated in Gli2 KO HSCs. Furthermore, overexpression of Gli2 rescued proliferation and activation of HSCs by upregulation of TGF-β signaling activity. Our data demonstrated that Gli2 regulated HSC activation and liver fibrosis by TGF-β signaling, thus providing support for future Gli2-based investigations of liver fibrosis therapy.NEW & NOTEWORTHYGli2 is a key transcription effector of Hh signaling. We found that Hh/Gli2 signaling activity was upregulated in CCl₄-induced liver fibrosis. Conditional deletion of the Gli2 gene in HSCs ameliorated CCl₄-induced liver fibrosis and HSCs activation. Moreover, Gli2 promoted activation of HSCs through upregulation of cyclin expression and TGF-β signaling activity. Thus, our data provide strong support for future investigations on Gli2 inhibition to slow liver fibrosis progression in humans.

Keywords: Gli2; Hedgehog signaling; TGF-β signaling; hepatic stellate cells; liver fibrosis.