Rat liver cholesterol 7 alpha-hydroxylase. Pretranslational regulation for circadian rhythm.

A complete cDNA encoding cholesterol 7 alpha-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. (1989) FEBS Lett. 257, 97-100) was totally sequenced. The cDNA contained a 1,509-base pair 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 base pairs. The predicted amino acid sequence displays less than 30% similarity to other sequenced cytochrome P-450s indicating that the 7 alpha-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 which were reported to be involved in rapidly degrading mRNA. Employing the specific antibodies and the cDNA as probes, a diurnal variation of the levels of the three factors, i.e. enzyme protein, mRNA, 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 10:00 p.m. and minimum at 10:00 a.m. No significant sexual difference was observed. Cholestyramine feeding increased all three factors at 10:00 a.m. close to the maximum levels of the normal rats, but did not show a significant increase at 10:00 p.m. On the contrary, starvation markedly decreased all three factors either at 10:00 a.m. or at 10:00 p.m., while maintaining still the diurnal variation. A good correlation of the levels of mRNA to the enzyme activities and the protein levels demonstrates that pretranslational regulation is most likely a mechanism for the circadian rhythm of 7 alpha-hydroxylase. The marked diurnal fluctuation of the amount of protein and the level of mRNA also indicates their rapid turnover. The short half-life of mRNA could be correlated with the structure of the 3'-untranslated region of the mRNA characteristic of rapidly degrading mRNA, 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.     

Rate-limiting, diurnal activity of hepatic microsomal cholesterol-7 alpha-hydroxylase in pigeons with high serum cholesterol

Efficiency of regulating serum cholesterol by cholesterol-7 alpha-hydroxylase was studied in pigeon strains hypo-(SR-39) and hypercholesterolemic (SR-37) with respect to dietary cholesterol. Diurnal hydroxylase activity in SR-37 was 10% of that in strain SR-39 adapted to a light-dark cycle and fed a non-cholesterol diet. Acrophase (6 p.m.) activity was 54-fold greater in SR-39 than in SR-37 pigeons. Dietary cholesterol elevated enzyme activity 2.8-fold in SR-37 pigeons. Dietary cholestyramine plus cholesterol increased hydroxylase activity 21-fold in SR-37 and 3-fold in SR-39 strain; yet, activity remained greater in SR-39. Cholestyramine feeding prevented elevated cholesterol levels in both groups. The circadian rhythms of hydroxylase and serum corticosterone were determined. The diurnal activity in SR-37 was 10% of that in SR-39 and acrophase activity was 34-fold greater in SR-39. Hormone levels were comparable. Programmed acrophase was asynchronous between strains. Hydroxylase activity was positively correlated with corticosterone levels and inversely correlated with serum cholesterol. A defect in the up-regulation of cholesterol-7 alpha-hydroxylase is proposed which limits the catabolism of cholesterol in strain SR-37.     

The role of glucocorticoids in the regulation of the diurnal rhythm of hepatic beta-hydroxy-beta-methylglutaryl-coenzyme A reductase and cholesterol 7 alpha-hydroxylase.

The microsomal activities of the hepatic enzymes hydroxymethylglutaryl-CoA reductase and cholesterol 7 alpha-hydroxylase exhibit a diurnal rhythm with maximum activities observed during the dark period and minimum activities around noon (12:00h). This diurnal rhythm was maintained for both enzymes after adrenalectomy, but the amplitude of variation for the activity of both enzymes was greatly decreased. A single injection of cortisol administered to adrenalectomized rats 3h before the expected maximum in enzyme activity resulted in a twofold increase in the activity of both enzymes 3h later, at values similar to those observed for control rats killed at the same time. This response appeared to require protein synthesis, since it was blocked by actinomycin D. However, the administration of cortisol to adrenalectomized rats 3 h before the expected minimum did not result in significant change in the activity of hydroxymethylglutaryl-CoA reductase and cholesterol 7 alpha-hydroxylase 3 h later. Kinetic studies of cholic acid metabolism in vivo demonstrated that adrenalectomy results in a significant decrease in the rate of synthesis of cholic acid and a considerable decrease in the pool size of cholic acid and its metabolic products. Treatment of adrenalectomized rats with cortisol increased the rate oonsistent with the effects of adrenalectomy and cortisol treatment on the activity of cholesterol 7alpha-hydroxylase.     

Increased cholesterol 7alpha-hydroxylase expression and size of the bile acid pool in the lactating rat

Maximal bile acid secretory rates and expression of bile acid transporters in liver and ileum are increased in lactation, possibly to facilitate increased enterohepatic recirculation of bile acids. We determined changes in the size and composition of the bile acid pool and key enzymes of the bile acid synthetic pathway [cholesterol 7alpha-hydroxylase (Cyp7a1), sterol 27-hydroxylase (Cyp27a1), and sterol 12alpha-hydroxylase (Cyp8b1)] in lactating rats relative to female virgin controls. The bile acid pool increased 1.9 to 2.5-fold [postpartum (PP) days 10, 14, and 19-23], compared with controls. A 1.5-fold increase in cholic acids and a 14 to 20% decrease in muricholic acids in lactation significantly increased the hydrophobicity index. In contrast, the hepatic concentration of bile acids and small heterodimer partner mRNA were unchanged in lactation. A 2.8-fold increase in Cyp7a1 mRNA expression at 16 h (10 h of light) demonstrated a shift in the diurnal rhythm at day 10 PP; Cyp7a1 protein expression and cholesterol 7alpha-hydroxylase activity were significantly increased at this time and remained elevated at day 14 PP but decreased to control levels by day 21 PP. There was an overall decrease in Cyp27a1 mRNA expression and a 20% decrease in Cyp27a1 protein expression, but there was no change in Cyp8b1 mRNA or protein expression at day 10 PP. The increase in Cyp7a1 expression PP provides a mechanism for the increase in the bile acid pool.     

Expression of cholesterol 7alpha-hydroxylase and delta(4)-3-ketosteroid 5beta-reductase genes in rat pancreatic hepatocyte-like cells.

Hepatocyte-like cells have been observed in the pancreas of the rat. We examined the bile acid biosynthetic function of these cells to determine whether they were real hepatocytes. This study investigated the existence of two liver-specific enzymes involved in bile acid biosynthesis (cholesterol 7alpha-hydroxylase and delta(4)-3-ketosteroid 5beta-reductase) in the hepatocyte-like cells. We could demonstrate cholesterol 7alpha-hydroxylase activity and its circadian rhythm in the hepatocyte-like cells. Northern blot analysis demonstrated the expression of messenger RNA for the 7alpha-hydroxylase and delta(4)-3-ketosteroid 5beta-reductase in the pancreatic hepatocyte-like cells. To measure the amount of the messenger RNA, we used the competitive polymerase chain reaction method for the 7alpha-hydroxylase. This quantitation revealed the existence of a circadian rhythm of cholesterol 7alpha-hydroxylase messenger RNA in the hepatocyte-like cells. These results indicated that bile acid biosynthesis was performed in the pancreatic hepatocyte-like cells as noted as in the liver parenchymal cells.     

Insulin is a dominant suppressor of sterol 12 alpha-hydroxylase P450 (CYP8B) expression in rat liver: possible role of insulin in circadian rhythm of CYP8B

Sterol 12 alpha-hydroxylase (CYP8B) is a key enzyme for regulating the cholic acid/chenodeoxycholic acid ratio in bile acid biosynthesis. The hepatic CYP8B level was elevated in streptozotocin-induced diabetic rats, and the elevated CYP8B was suppressed by insulin administration [Ishida, H. et al. (1999) J. Biochem. 126, 19-25]. The streptozotocin-induced elevation of hepatic CYP8B mRNA concomitantly responded to the decrement of the serum insulin level. The CYP8B mRNA level in the cultivated rat hepatoma H4TG cells was strongly suppressed by insulin, although it was affected by dibutyryl cAMP or thyroxine to lesser extents. These observations demonstrate that CYP8B expression is dominantly regulated by the direct action of insulin on hepatocytes. A marked circadian rhythm (maximum at 13:00-16:00 and minimum at 1:00) was observed both on the mRNA level and the activity of CYP8B. This rhythm was shifted from that of cholesterol 7 alpha-hydroxylase, a rate-limiting enzyme of bile acid biosynthesis, showing a maximum at 22:00 and a minimum at 10:00, and this shift might oscillate the cholic acid/chenodeoxycholic acid ratio, which is increased in the late afternoon and decreased at midnight. The rhythm of CYP8B was the inverse of the circadian variation of serum insulin level and was similar to the circadian rhythm of glucose 6-phosphatase. These facts and the potent suppressive effect of insulin on CYP8B indicate that the oscillation of the serum insulin may be a factor in producing the circadian rhythm of CYP8B.     

Regulation of cholesterol 7 alpha-hydroxylase in the liver. Cloning, sequencing, and regulation of cholesterol 7 alpha-hydroxylase mRNA.

Monospecific antibody against purified rat liver cholesterol 7 alpha-hydroxylase cytochrome P-450 was used to screen a lambda gt11 cDNA library constructed from immuno-enriched polysomal RNA of cholestyramine-treated female rat liver. Two types of cDNA clones differing in the length of the 3'-untranslated region were identified, and DNA sequences were determined. The full length clone contains 3561 base pairs plus a long poly(A) tail. The amino acid sequence deduced from the open reading frame revealed a unique P-450 protein containing 503 amino acid residues which belonged to a new gene family designated family VII or CYP7. Southern blot hybridization experiments indicated that the minimal size of P-450 VII gene was 11 kilobase pairs (kb), and there was probably only one gene in this new family. Northern blot hybridization using specific cDNA probes revealed at least two major mRNA species of about 4.0 kb and 2.1 kb, respectively. These two mRNA species may be derived from the use of different polyadenylation signals and reverse-transcribed to two types of cDNA clones. Cholesterol 7 alpha-hydroxylase mRNAs were induced 2- to 3-fold in rat liver by cholestyramine treatment. The mRNA level was rapidly reduced upon the removal of the inducer. Similarly, cholesterol feeding induced enzyme activity, protein, and mRNA levels in the rat by 2-fold, suggesting that cholesterol is an important regulator of cholesterol 7 alpha-hydroxylase in the liver. On the other hand, dexamethasone and pregnenolone-16 alpha-carbonitrile drastically reduced the activity, protein, and mRNA levels. These experiments suggest that the induction of cholesterol 7 alpha-hydroxylase activity by cholestyramine or cholesterol and inhibition of cholesterol 7 alpha-hydroxylase activity by bile acid feedback are results of the rapid turnover of cholesterol 7 alpha-hydroxylase enzyme and mRNA levels.     

Regulation of cholesterol 7 alpha-hydroxylase in the liver. Purification of cholesterol 7 alpha-hydroxylase and the immunochemical evidence for the induction of cholesterol 7 alpha-hydroxylase by cholestyramine and circadian rhythm

Two cholesterol 7 alpha-hydroxylase isozymes were purified from liver microsomes of cholestyramine-treated female rats by using anion exchange high performance liquid chromatography. These two cytochrome P-450 isozymes were similar in electrophoretic mobility, immunocross-reactivity, and Vmax but differed in Km for cholesterol, turnover number, and charges. Antibody against the major isozyme was raised in rabbit. This antibody specifically inhibited microsomal cholesterol 7 alpha-hydroxylase activity. Immunoblot of microsomal polypeptides indicated that microsomal cholesterol 7 alpha-hydroxylase enzyme levels were increased in parallel with cholesterol 7 alpha-hydroxylase activity upon the treatment of rats with diet supplemented with cholestyramine. Both cholesterol 7 alpha-hydroxylase activity and enzyme levels were drastically reduced immediately after the removal of cholestyramine from the diet. Cholesterol 7 alpha-hydroxylase activity was also detected in the microsomes of kidney, heart, and lung in about 7-27% of the level found in the liver. 3-Methylcholanthrene treatment induced cholesterol 7 alpha-hydroxylase activity and enzyme level. In contrast, pregnenolone-16 alpha-carbonitrile or dexamethasone treatment greatly depressed enzyme and activity in rats. Cholesterol 7 alpha-hydroxylase enzyme level was 2-3-fold higher in liver microsomes of rats maintained under the reversed light cycle than under the normal light cycle. In genetically obese Zucker rats, cholesterol 7 alpha-hydroxylase activity and enzyme level did not respond to the change in the light cycle, however, were induced to the same levels as in the lean rats by cholestyramine treatment. This study provided the first direct evidence that the bile acid feedback regulation and circadian rhythm of microsomal cholesterol 7 alpha-hydroxylase activity involved the induction of cholesterol 7 alpha-hydroxylase enzyme level.     

Assays for cholesterol 7 alpha-hydroxylase and 12 alpha-hydroxylase using high performance liquid chromatography

Rapid and accurate assay methods for cholesterol:NADPH oxidoreductase (EC 1.14.13.17, 7 alpha-hydroxylating) and 7 alpha-hydroxy-4-cholesten-3-one 12 alpha-hydroxylase (enzyme not yet registered) are described. 7 alpha-Hydroxylase utilizes the endogenous cholesterol of liver microsomes as substrate. The reaction products were separated by high performance liquid chromatography monitored at 214 nm. Much higher activity was obtained with the method compared to literature values, which were obtained using externally added radioactive cholesterol as the substrate. The 12 alpha-hydroxylase activity was measured using non-radioactive steroid as the substrate. The reaction products were separated by the chromatography and detected at 240 nm. Comparable activities were obtained by this method compared to those that were obtained using radioactive substrate.     

12 alpha-hydroxylase activity in human liver and its relation to cholesterol 7 alpha-hydroxylase activity.

Interruption of the enterohepatic circulation by cholestyramine causes a several-fold increase in bile acid synthesis, reflected in a stimulation of cholesterol 7 alpha-hydroxylase activity; the synthesis of cholic acid being stimulated to a greater extent than chenodeoxycholic acid. It is not known if this preferential increase in cholic acid is due to an increase of the 12 alpha-hydroxylase activity. The present study aimed at investigating the 12 alpha-hydroxylase activity and its relation to cholesterol 7 alpha-hydroxylase activity in liver microsomes of patients with different levels of cholesterol 7 alpha-hydroxylase activity. Liver biopsies were obtained from four gallstone-free patients, and seven untreated and two cholestyramine-treated gallstone patients undergoing cholecystectomy, and four patients with Crohn's disease undergoing intestinal resection. The combined group of cholestyramine-treated and ileum-resected patients had four times higher cholesterol 7 alpha-hydroxylase activity and two times higher 12 alpha-hydroxylase activity than the other patients. A positive correlation was obtained between cholesterol 7 alpha-hydroxylase activity and 12 alpha-hydroxylase activity (r = +0.69; n = 16). These results indicate that the increased ratio between the synthesis of cholic acid and chenodeoxycholic acid during cholestyramine treatment is due to a compensatory increase of the 12 alpha-hydroxylase activity.     

Structure of the rat gene encoding cholesterol 7 alpha-hydroxylase

Cholesterol 7 alpha-hydroxylase (7 alpha-hydroxylase) is a microsomal cytochrome P-450 that catalyzes the first and rate-limiting step in bile acid biosynthesis, the major catabolic pathway in cholesterol homeostasis. The gene encoding the rat 7 alpha-hydroxylase has been isolated and characterized. Southern blotting experiments demonstrated that the gene is present in a single copy in the rat genome. DNA sequence analysis showed that the 7 alpha-hydroxylase gene is unique among the characterized cytochrome P-450s in that it contains only six exons. Nuclease S1 and primer-extension mapping experiments positioned the 5'-ends of the 7 alpha-hydroxylase mRNA approximately 20-25 nucleotides downstream of a consensus TATAAA sequence. RNA blotting experiments demonstrated the presence of multiple 7 alpha-hydroxylase mRNAs that differ in the lengths of their 3'-untranslated regions.     

Purification of cholesterol 7 alpha-hydroxylase from human and rat liver and production of inhibiting polyclonal antibodies

Cholesterol 7 alpha-hydroxylase, the cytochrome P-450-dependent and rate-controlling enzyme of bile acid synthesis, was purified from rat and human liver microsomes. The purified fractions were assayed in a reconstituted system containing [4-14C]cholesterol, and cholesterol 7 alpha-hydroxylase activities in these fractions increased 500-600-fold relative to whole microsomes. Polyacrylamide gel electrophoresis of rat microsomes followed by immunoblotting with polyclonal rabbit antisera raised against purified cholesterol 7 alpha-hydroxylases revealed two peaks at molecular masses of 47,000 and 49,000 daltons for both rat and human fractions. Increasing amounts of rabbit anti-rat and anti-human antibodies progressively inhibited rat microsomal cholesterol 7 alpha-hydroxylase activity up to 80%. In contrast, monospecific antibodies raised against other purified cytochrome P-450 enzymes (P-450f, P-450g, and P-450j) did not inhibit rat or human cholesterol 7 alpha-hydroxylase activity. Immunoblots of rat microsomes with the rabbit anti-rat cholesterol 7 alpha-hydroxylase antibody demonstrated that the antibody reacted quantitatively with the rat microsomal enzyme. Microsomes from cholesterol-fed rats showed increased cholesterol 7 alpha-hydroxylase mass, whereas treatment with pravastatin, an inhibitor of hydroxy-methylglutaryl-coenzyme A reductase, reduced enzyme mass. Microsomes from starved rats contained slightly less cholesterol 7 alpha-hydroxylase protein than chow-fed control rats. These results indicate a similarity in molecular mass, structure, and antigenicity between rat and human cholesterol 7 alpha-hydroxylases; demonstrate the production of inhibiting anti-cholesterol 7 alpha-hydroxylase antibodies that can be used to measure the change in cholesterol 7 alpha-hydroxylase enzyme mass under various conditions; and emphasize the unique structure of cholesterol 7 alpha-hydroxylase with respect to other cytochrome P-450-dependent hydroxylases.     

Regulation of rat liver microsomal cholesterol 7 alpha-hydroxylase: presence of a cytosolic activator

Microsomal cholesterol 7 alpha-hydroxylase (EC 1.14.13.7) in rat liver was assayed by a single-isotope-incorporation method, and factors influencing its activity were studied. Crude cytosol contained a non-catalytic activator which was heat-stable and on-dialyzable. This activator enhanced cholesterol 7 alpha-hydroxylase catalytic activity. The stimulatory property of this cytosolic activator was not altered by cholestyramine feeding, and was retained after fractionation by ammonium sulfate of saturation up to 65%. 3-Hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) stimulated hydroxylase activity at concentrations up to 90 micro M. ATP at concentration greater than 1.2 mM inhibited hydroxylase activity. NaF was stimulatory at concentrations up to 50 mM with a maximum stimulatory effect at 10 mM, and was antagonistic in effect to ATP. HMG-CoA and ATP at the above inhibitory concentrations and higher abolished the activating effect of the cytosolic factor.     

HMGCoA reductase and cholesterol 7 alpha-hydroxylase in human liver

The activities of HMGCoA reductase and cholesterol 7 alpha-hydroxylase were assayed in liver biopsies of patients with or without cholestyramine treatment. The active dephosphorylated form of HMGCoA reductase and the activity of cholesterol 7 alpha-hydroxylase were under the detection limits in untreated subjects. After cholestyramine treatment activities of both enzymes were stimulated and the active form of HMGCoA reductase became detectable in four out of the five tested patients. In two subjects who received cholestyramine, the effect of exogenously added [4-14C] cholesterol on cholesterol 7 alpha-hydroxylase was tested. In the presence of Tween 80, the detergent by which [14C]cholesterol was suspended, the enzyme activity was profoundly inhibited and synthesis of 7 alpha-hydroxycholesterol was extremely low.