The cloning of cholesterol 7 α‐hydroxylase: Will molecular techniques help us understand the physiology of bile acid synthesis?

The rate-limiting step in bile acid biosynthesis is catalyzed by the microsomal cytochrome P-450 cholesterol 7 α-hydroxylase. The expression of this enzyme is subject to feedback regulation by sterols and is thought to be coordinately regulated with enzymes in the cholesterol supply pathways, including the low-density lipoprotein receptor and 3-hydroxy-3-methylglutarylcoenzyme A reductase and synthase. Here we report the purification of rat microsomal P-450 cholesterol 7 α-hydroxylase and the determination of a partial amino acid sequence. Oligonucleotides derived from peptide sequence were used to clone a full-length cDNA encoding microsomal P-450 cholesterol 7 α-hydroxylase. DNA sequence analysis of the cDNA revealed a microsomal P-450 cholesterol 7 α-hydroxylase protein of 503 amino acids with a predicted molecular weight of 56,890, which represents a novel family of cytochrome P-450 enzymes. Transfection of a microsomal P-450 cholesterol 7 α-hydroxylase cDNA into simian COS cells resulted in the synthesis of a functional enzyme whose activity was stimulated in vitro by the addition of rat microsomal cytochrome P-450 reductase protein. RNA blot hybridization experiments indicated that the messenger RNA for microsomal P-450 cholesterol 7 α-hydroxylase is found only in the liver. The levels of this messenger RNA increased when bile acids were depleted by dietary cholestyramine and decreased when bile acids were consumed. Dietary cholesterol led to an increase in microsomal P-450 cholesterol 7 α-hydroxylase messenger RNA levels. The enzymatic activity of microsomal P-450 cholesterol 7 α-hydroxylase paralleled the observed changes in messenger RNA levels. These results suggest that bile acids and sterols are able to alter the transcription of the microsomal P-450 cholesterol 7 α-hydroxylase gene and that this control explains the previously observed feedback regulation of bile acid synthesis. A complete cDNA encoding cholesterol 7 α-monooxygenase (EC 1.14 13.17), which had been isolated from rat liver cDNA libraries by using specific antibodies to the enzyme (Noshiro M, Nishimoto M, and Okuda K. FEBS Lett 1989;257:97–100) was totally sequenced. The cDNA contained a 1,509 bb open-reading frame encoding 503 amino acid residues (Mr = 56,880) and an unusually long 3′-untranslated region rich in AT sequence in the total length of 3,545 bp. The predicted amino acid sequence displays less than 30% similarity to other sequenced cytochrome P-450s, indicating that the 7 α-hydroxylase constitutes a novel family of cytochrome P-450. The AT-rich region often contained ATTTA motifs, 5′-AAT-3′ or 5′-TAA-3′ trinucleotides reported to be involved in rapidly degrading messenger RNA. Using the specific antibodies and the cDNA as probes, a diurnal variation of the levels of the three factors (i.e., enzyme protein, messenger RNA and enzyme activity) was studied on rat livers prepared at various times of the day. In normal animals, all three factors exhibited maximum level at 10 PM and minimum at 10 AM. No significant sexual difference was observed. Cholestyramine feeding increased all three factors at 10 AM close to the maximum levels of the normal rats but did not show a significant increase at 10 PM. On the contrary, starvation markedly decreased all three factors either at 10 AM or at 10 PM, while still maintaining the diurnal variation. A good correlation of the levels of messenger RNA to the enzyme activities and the protein levels demonstrates that pretranslational regulation is most likely a mechanism for the circadian rhythm of 7 α-hydroxylase. The marked diurnal fluctuation of the amount of protein and the level of messenger RNA also indicates their rapid turnover. The short half-life of messenger RNA could be correlated with the structure of the 3′-untranslated region of the messenger RNA characteristic of rapidly degrading messenger RNA (i.e., abundance of motif, AUUUA and existence of 5′-AAU-3′ or 5′-UAA-3′ trinucleotides in single-stranded regions of the secondary structure).