Background & aims: Inhibition of cholesterol 7alpha-hydroxylase (CYP7A1) by bile acids and inflammatory cytokines provides an important mechanism to protect hepatocytes from bile acid toxicity during cholestasis. Transforming growth factor beta1 (TGFbeta1) released by hepatic stellate cells during chronic liver injury plays a critical role in liver inflammation and fibrogenesis. The objective of this study is to investigate the role of TGFbeta1 in hepatic bile acid synthesis.
Methods: mRNA expressions in primary human hepatocytes and HepG2 cells were measured by quantitative real-time polymerase chain reaction. Reporter assay, glutathione-S-transferase pull-down assay, adenovirus-mediated gene transduction, and chromatin immunoprecipitation assay were used to study the mechanism of TGFbeta1 regulation of CYP7A1 gene transcription.
Results: TGFbeta1 inhibited the mRNA expression of CYP7A1 and bile acid synthesis in HepG2 cells and primary human hepatocytes. Mothers against decapentaplegic homolog (Smad3) inhibited both CYP7A1 promoter activity and mRNA expression by inhibiting DNA-binding activity of hepatocyte nuclear factor 4alpha (HNF4alpha). The histone deacetylase (HDAC) inhibitor Tricostatin A partially blocked the TGFbeta1 inhibition of CYP7A1 mRNA expression, whereas TGFbeta1 decreased histone 3 acetylation in the CYP7A1 chromatin. TGFbeta1 treatment and adenovirus Smad3 reduced HNF4alpha binding but increased the recruitment of Smad3, HDAC1, and a repressor mSin3A to the CYP7A1 chromatin.
Conclusions: This study provides the first evidence that TGFbeta1 represses CYP7A1 gene transcription in human hepatocytes by a mechanism involving Smad3-dependent inhibition of HNF4alpha and HDAC remodeling of CYP7A1 chromatin. The TGFbeta1/Smad3 signaling may reduce bile acid synthesis in the liver and prevent hepatocyte injury in cholestatic liver disease.