CHOLESTEROL HOMEOSTASIS: INVOLVEMENT OF HISTONE DEACETYLASES IN THE MOLECULAR REGULATORY PATHWAY OF CHOLESTEROL7ALPHA-HYDROXYLASE

Cholesterol Homeostasis: involvement of histone deacetylases in the molecular regulatory pathway of Cholesterol 7α-hydroxylase Cholesterol 7α-Hydroxylase (CYP7A1) is the major check-point of bile acid (BA) biosynthesis, quantitatively the most important route of cholesterol disposal in mammals. BA returning to the liver via enterohepatic circulation repress CYP7A1 expression. BA induce histone deacetylase 7 (HDAC7) translocation to nucleus and the sequential recruitment of HDAC7, 3, 1 and of the corepressor SMRTα creates a repressor complex on the nuclear receptor HNF-4 bound to CYP7A1 promoter in a human hepatoma cell line. Previous results showed that non-selective HDAC inhibitors increase CYP7A1 expression in vitro and in vivo by preventing the negative feedback exerted by BA and reduce serum cholesterol in mice. Based on these seminal findings, our aim was to define the role of specific HDACs and corepressors in the regulation of CYP7A1. To this end, we tested class selective HDAC inhibitors in vitro and in vivo. By using a reporter cell line containing CYP7A1 promoter upstream of luciferase gene, we demonstrated that the class I selective HDAC inhibitor MS275 totally prevented the repressive effect of BA on CYP7A1; class II selective inhibitor MC1568 only elicited a partial recovery of CYP7A1 promoter driven transcription. In addition, MS275 significantly increased liver Cyp7a1 expression in C57Bl/6J mice. A mild induction, albeit not statistically significant, was observed even after SAHA and MS275 treatment. HDAC1, 3 and 7 silencing in the same reporter cell line pointed out the importance of HDAC1 and 7 in the regulation of CYP7A1 transcription To unravel the role of specific HDACs and corepressors we cloned shRNA against Hdac1, 3, 4, 5, 7 and Smrt in adenovectors and we tested them in primary hepatocytes. Hdac1, 7 and Smrt silencing significantly increased Cyp7a1 transcription, highlighting their involvement in the regulation of this gene. Adenoviral infection of C57BL/6J mice confirmed Hdac7 and Smrt as crucial players in the regulation of BA metabolism in vivo. Generation of HDAC7 liver-specific KO mice fed western diet allowed to investigate the role of HDAC7 in vivo: we observed body weight and LDL-cholesterol reduction, increased liver BA consistent with increased Cyp7A1 expression, lower liver lipid accumulation and a major fraction of lipid poor pre-beta HDL profile compared to wild type mice. The results obtained with the investigations performed during my doctorate training show that specific HDACs affect CYP7A1 transcription highlighting their role in the regulation of cholesterol and lipid homeostasis and represent a step forward contributing to shed light on the mechanisms of CYP7A1 regulation and their impact in lipid homeostasis.