Transcriptional regulation of human sterol 27-hydroxylase gene (CYP27A1)

Mitochondrial sterol 27-hydroxylase (CYP27A1) catalyses the side-chain oxidation of the classical pathway in the liver and initiates the alternative pathway of bile acid synthesis. It is well established that mutation of this gene could cause cerebrotendinous xanthomatosis (CTX), a cholesterol storage disease with premature atherosclerosis. The sterol 27-hydroxylase is stimulated by glucocorticoids and inhibited by bile acids. Little is known about the regulation of the sterol 27-hydroxylase gene. In this work, I cloned and sequenced the 4.3 kb 5'-flanking region of human CYP27A1. I carried out a detailed study of the transcriptional regulation of the human CYP27A1 using transient transfection assays in human hepatocellular carcinoma (HepG2), human embryonic kidney (HEK293) and Chinese hamster ovary (CHO) cells. I mapped the response elements of the physiological regulators in the 5'-upstream region of the gene. Glucocorticoid receptor (GR) and dexamethasone (Dex) strongly stimulated the reporter activity in HepG2 cells, but not in HEK293 or CHO cells. Thyroid hormone receptor alpha (TRalpha) and TRbeta increased the reporter activity in the presence of thyroxine (T4) in HEK 293 and CHO cells, but not in HepG2 cells. Co-transfection with cAMP response element binding protein (CREB) stimulated the promoter activity in HepG2 and CHO, but not in HEK293 cells. Cotransfection with CREB and CREB binding protein (CBP) further stimulated promoter activity in HepG2 cells, but not in CHO cells. Bile acids strongly suppressed the CYP27A1/Luc reporter activity in HepG2 cells when cotransfected with farnesoid X receptor (FXR). All the taurine-conjugated bile acids strongly suppressed the CYP27A1/Luc reporter activity in HEK293 cells when cotransfected with Na+ taurocholate cotransporter protein (NTCP). Bile acids and FXR strongly reduced CYP27A1 mRNA expression levels in HepG2 cells. The bile acid response element was mapped to a region 147 bp upstream of the translation start codon. Hepatocyte nuclear factor 4alpha (HNF4alpha) strongly stimulated CYP27A1 transcription and the small heterodimer partner (SHP) inhibited HNF4alpha transactivation of the CYP27A1. Electrophoretic mobility shift assay (EMSA) with in vitro synthesized HNF4alpha and antibody super-shift assay showed that HNF4alpha bound to a sequence in the proximal promoter. Mutation of the HNF4 binding site reduced the basal activity of CYP27A1 and the response to bile acid. My results suggest that HNF4alpha is important not only in transactivation of the CYP27A1 gene but also in the bile acid inhibition of CYP27A1 transcription. Human CYP27A1 is highly regulated by physiological regulators in a tissue-specific manner. The study of CYP27A1 gene regulation is important in understanding the mechanism of cholesterol homeostasis and the pathology of lipid storage disease such as atherosclerosis and CTX.