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 CCl 4 . 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. CCl 4 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.

Transforming Growth Factor-β (TGF-β)-mediated Connective Tissue Growth Factor (CTGF) Expression in Hepatic Stellate Cells Requires Stat3 Signaling Activation

“…The molecular details how TGF-␤ induces CTGF production in HSCs are still largely unsolved. Current available data suggest an ALK5-dependent mechanism as dominant for this induction (26). Other signaling pathways such as the TGF-␤ non-canonical MAPK, PI3K/Akt, and PKC pathways may also participate in this process; the underlying mechanisms, however, are yet to be clarified (26).…”

“…In addition to the canonical Smad pathway, TGF-␤ can also activate other signaling molecules such as Erk1/2, JNK, p38 MAPK, PKC, and PI3K/Akt, which is largely cell type-dependent (25). Previous studies have demonstrated that selective inhibition of those signaling pathways resulted in a potent reduction of CTGF mRNA expression in activated HSCs (26,27). The underlying mechanisms, however, have not been clarified yet.…”

Connective tissue growth factor hammerhead ribozyme attenuates human hepatic stellate cell function

“…We and others have shown that CCN2 mRNA and protein are expressed increasingly during progressive activation of cultured primary rat HSCs, or in response of the cells to stimulation by TGF-b, vascular endothelial growth factor, lipid peroxidation products, acetaldehyde or platelet-derived growth factor-BB [8,[13][14][15][16] . We have shown further that CCN2 promoter activity is enhanced in a Smad7-dependent fashion by TGF-b in primary rat HSCs transfected with the CCN2 promoter, luciferase reporter constr uct [10] , and that CCN2 production in the HSCs is stimulated by TGF-b1 [6] .…”

“…We have shown further that CCN2 promoter activity is enhanced in a Smad7-dependent fashion by TGF-b in primary rat HSCs transfected with the CCN2 promoter, luciferase reporter constr uct [10] , and that CCN2 production in the HSCs is stimulated by TGF-b1 [6] . We recently showed that TGF-b-induced CCN2 promoter activity in activated mouse HSCs requires Smad and Ets-1 elements in the CCN2 promoter [16] , as described for normal fibroblasts or mesangial cells [17][18][19][20] . However, CCN2 promoter activity in activated HSCs is uniquely antagonized by ALK4/5/7 inhibition [16] .…”

“…We recently showed that TGF-b-induced CCN2 promoter activity in activated mouse HSCs requires Smad and Ets-1 elements in the CCN2 promoter [16] , as described for normal fibroblasts or mesangial cells [17][18][19][20] . However, CCN2 promoter activity in activated HSCs is uniquely antagonized by ALK4/5/7 inhibition [16] . CCN2 gene regulation in activated HSCs is distinct from that of scleroderma, which is independent of Smad/ALK5-mediated TGF-b signaling, but is dependent on endothelin-1 [17,21,22] , or pancreatic cancer, which depends on activated ras/MEK/ERK rather than TGF-b or endothelin-1 [23] .…”