Down-regulation of the rat hepatic sterol 27-hydroxylase gene by bile acids in transfected primary hepatocytes: possible role of hepatic nuclear factor 1 alpha

In vitro and in vivo studies have shown that the sterol 27-hydroxylase (CYP 27) gene is transcriptionally repressed by hydrophobic bile acids. The mole cular mechanism(s) of repression of CYP27 by bile acids is unknown. To iden tify the bile acid responsive element (BARE) and transcription factor(s) th at mediate the repression of CYP27 by bile acids, constructs of the CYP27 5 '-flanking DNA were linked to either the CAT or luciferase reporter gene an d transiently transfected into primary rat hepatocytes. Taurocholate (TCA), taurodeoxycholate (TDCA) and taurochenodeoxycholate (TCDCA) significantly reduced CAT activities of the -840/+23, -329/+23, and -195/+23 mCAT constru cts. A -76/+23 construct showed no regulation by bile acids. When a DNA fra gment (-110/-86) from this region was cloned in front of an SV 40 promoter it showed downregulation by TDCA. 'Super'-electrophoretic mobility shift as says (EMSA) indicated that both HNF1 alpha and C/EBP bind to the -110 to -8 6 bp DNA fragment. Recombinant rat HNF1 alpha and C/EBP alpha competitively bound to this DNA fragment. 'Super'-EMSA showed that TDCA addition to hepa tocytes in culture decreased HNF1 alpha, but not C/EBP, binding to the -110 /-86 bp DNA fragment. A four base pair substitution mutation (-103 to -99) in this sequence eliminated TCA and TDCA regulation of the (-840/ + 23) con struct. The substitution mutation also eliminated (>95%) HNF1 alpha, but no t C/EBP, binding to this DNA fragment. We conclude that bile acids repress CYP27 transcription through a putative BARE located between -110 and -86 bp of the CYP27 promoter. The data suggest that bile acids repress CYP27 tran scriptional activity by decreasing HNF1 alpha binding to the CYP27 promoter . (C) 1999 Elsevier Science Ltd. All rights reserved.