Hepatic P-450 cholesterol 7 alpha-hydroxylase. Regulation in vivo at the protein and mRNA level in response to mevalonate, diurnal rhythm, and bile acid feedback.
Cholesterol 7 alpha-hydroxylase (P-450 Ch7 alpha) catalyzes the first and rate-limiting step in the hepatic conversion of cholesterol to bile acids. P-450 Ch7 alpha activity in rat liver is regulated at three independent levels: (a) feedback inhibition by bile acids (long term regulation); (b) midterm regulation through the diurnal cycle; (c) short term modulation by hormones and dietary factors. P-450 Ch7 alpha was purified to apparent homogeneity and in active form (turnover number = 10-15 min-1 P-450(-1)) from cholestyramine-fed female rats, and rabbit anti-P-450 Ch7 alpha polyclonal antibodies were then prepared. Liver microsomes were isolated from rats fed normal diet or diet containing the bile acid sequestrant cholestyramine and were then killed at either the apex (midnight) or nadir (noon) of the diurnal rhythm of P-450 Ch7 alpha activity. Direct comparison of microsomal P-450 Ch7 alpha enzyme activity levels with P-450 Ch7 alpha protein (Western blotting) and mRNA levels (Northern and slot blots) revealed that the 2.5-3-fold induction of P-450 Ch7 alpha activity with cholestyramine feeding can be fully accounted for by an increase in P-450 Ch7 alpha protein and mRNA. Turnover numbers of 7-9 nmol of 7 alpha-hydroxycholesterol/min/nmol of microsomal P-450 Ch7 alpha were observed for both induced and uninduced animals. Similarly, the postmidnight decrease in enzyme activity could be generally accounted for by a decrease in P-450 Ch7 alpha protein and mRNA, suggesting that these species have relatively short half-lives. The short term regulation of P-450 Ch7 alpha was examined following treatment with the cholesterol precursor mevalonic acid. A 2.5-fold increase in hepatic microsomal P-450 Ch7 alpha activity occurred within 150 min and was accompanied by a significant elevation of P-450 Ch7 alpha mRNA (up to 3-6-fold increase). These findings establish that hepatic cholesterol 7 alpha-hydroxylase activity is regulated in response to long term, midterm, and short term control factors primarily at a pretranslational level and that this regulation is of greater importance than proposed mechanisms based on allosteric effects of bile acids on P-450 Ch7 alpha protein, changes in cholesterol availability, or reversible phosphorylation of a putative P-450 Ch7 alpha phosphoprotein.
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
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Sundseth
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Waxman
D000818:Animals; D000906:Antibodies; D001483:Base Sequence; D001647:Bile Acids and Salts / Q000494:pharmacology*; D002790:Cholesterol 7-alpha-Hydroxylase / Q000235:genetics*; D002792:Cholestyramine Resin / Q000494:pharmacology*; D002940:Circadian Rhythm; D005246:Feedback; D005260:Female; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D008099:Liver / Q000187:drug effects / Q000201:enzymology*; D008297:Male; D008798:Mevalonic Acid / Q000494:pharmacology*; D008862:Microsomes, Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D012333:RNA, Messenger / Q000235:genetics*; D051381:Rats; D011919:Rats, Inbred Strains; D013250:Steroid Hydroxylases / Q000235:genetics*
D000906:Antibodies; D001647:Bile Acids and Salts; D015345:Oligonucleotide Probes; D012333:RNA, Messenger; D002792:Cholestyramine Resin; D013250:Steroid Hydroxylases; D002790:Cholesterol 7-alpha-Hydroxylase; D008798:Mevalonic Acid