Regulation of CCN2 mRNA expression and promoter activity in activated hepatic stellate cells

Abstract

The matricellular protein connective tissue growth factor (CCN2) is considered a faithful marker of fibroblast activation in wound healing and in fibrosis. CCN2 is induced during activation of hepatic stellate cells (HSC). Here, we investigate the molecular basis of CCN2 gene expression in HSC. Fluoroscence activated cell sorting was used to investigate CCN2 expression in HSC in vivo in mice treated with CCl₄. CCN2 and TGF-β mRNA expression were assessed by polymerase chain reaction as a function of culture-induced activation of HSC. CCN2 promoter/reporter constructs were used to map cis-acting elements required for basal and TGFβ-induced CCN2 promoter activity. Real-time polymerase chain reaction analysis was used to further clarify signaling pathways required for CCN2 expression in HSC. CCl₄ administration in vivo increased CCN2 production by HSC. In vitro, expression of CCN2 and TGF-β mRNA were concommitantly increased in mouse HSC between days 0 and 14 of culture. TGFβ-induced CCN2 promoter activity required the Smad and Ets-1 elements in the CCN2 promoter and was reduced by TGFβ type I receptor (ALK4/5/7) inhibition. CCN2 overexpression in activated HSC was ALK4/5/7-dependent. As CCN2 overexpression is a faithful marker of fibrogenesis, our data are consistent with the notion that signaling through TGFβ type I receptors such as ALK5 contributes to the activation of HSC and hence ALK4/5/7 inhibition would be expected to be an appropriate treatment for liver fibrosis.