CREB1 and Smad3 mediate TGF-β3-induced Smad7 expression in rat hepatic stellate cells

Abstract

Transforming growth factor (TGF)-β3 has previously been reported to antagonize hepatic fibrosis in vivo and in vitro. The present study aimed to investigate the mechanism underlying the involvement of TGF-β3 in hepatic fibrosis. Short hairpin (sh)RNA-cAMP-responsive element binding protein (CREB) 1 and small interfering (si)RNA-Smad3 were utilized to silence the expression of CREB1 and Smad3 in hepatic stellate cells (HSCs), whereas the vector PRSV-CREB1 was used to induce CREB1 overexpression in HSCs. Cells were treated with or without exogenous TGF-β3 or TGF-β1, and mRNA and protein expression levels were assessed using reverse transcription-quantitative polymerase chain reaction and western blot analysis. Untreated cells served as the control group. Exogenous TGF-β3 increased Smad7 mRNA and protein expression levels in rat HSCs, and CREB1 and Smad3 appeared to be implicated in the mechanism of Smad7. CREB1 knockdown inhibited the TGF-β3-induced upregulation of Smad7, whereas its overexpression potentiated the Smad7 upregulation in HSCs; conversely, CREB1 manipulations had no effect on Smad7 expression under basal conditions. In addition, TGF-β3-induced Smad7 upregulation was blocked when the activity of p38, a kinase upstream of CREB1, was inhibited. Furthermore, silencing Smad3 resulted in decreased Smad7 expression under basal conditions and in TGF-β3-stimulated cells. Notably, Smad7 expression appeared to also be induced by exogenous TGF-β1, independent of CREB1. The present study demonstrated that TGF-β3 increased Smad7 expression in HSCs, whereas CREB1 and Smad3 appeared to participate in the mechanism of induction. Smad3 is the key regulator whereas CREB-1 acts as a co-regulator. These results suggested that this mechanism may underlie the antagonizing effects of TGF-β3 on hepatic fibrosis.