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.     

Circadian rhythms of sterol 12alpha-hydroxylase, cholesterol 7alpha-hydroxylase and DBP involved in rat cholesterol catabolism

Circadian rhythms of important enzymes involved in the conversion of cholesterol to bile acids [sterol 12alpha-hydroxylase (12alpha-hydroxylase) and cholesterol 7alpha-hydroxylase (7alpha-hydroxylase)] and an albumin site D-binding protein (DBP) were examined in rats. When the animals were fed freely, they usually ate in the dark and the circadian rhythms of activities of 12alpha-hydroxylase and 7alpha-hydroxylase showed the same peaks (at 10 p.m.) and lows (at 2 p.m.). Their mRNA levels were determined at four timepoints: 3 a.m., 10 a.m., 3 p.m. and 10 p.m. A maximum of the rhythm of 12alpha-hydroxylase was observed at 3 p.m. and the minimum at 3 a.m. These results are distinct from those of 7alpha-hydroxylase, whose maximum point was at 10 p.m. and minimum at 3 p.m. When the rats were fed only in the day-time (from 9 a.m. to 5 p.m.), a marked shift of the activity and mRNA rhythms was observed with both enzymes. The circadian rhythms of the activities of both enzymes showed the same peaks (at 3 p.m.), but the mRNA levels of 12alpha-hydroxylase were distinct from those of 7alpha-hydroxylase, whose maximum point was at 3 a.m. and minimum at 10 p.m. Differences between the maximum and the minimum points of each enzyme mRNA level were statistically significant (P < 0.01 for 12alpha-hydroxylase and 0.05 for 7alpha-hydroxylase). Moreover, circadian rhythms of DBP were also markedly shifted with the change of feeding period. The maximum mRNA level was observed at 10 p.m. instead of 10 a.m. and the minimum was at 10 a.m. instead of 10 p.m.     

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.     

On the possible use of the serum level of 7 alpha-hydroxycholesterol as a marker for increased activity of the cholesterol 7 alpha-hydroxylase in humans

The possibility was investigated that the serum level of 7 alpha-hydroxycholesterol can be used as a marker for cholesterol 7 alpha-hydroxylase activity. Six patients with gallstone disease were found to have a mean level of 7 alpha-hydroxycholesterol in serum of 30 +/- 4 ng/ml (mean +/- SEM) as measured by isotope dilution-mass spectrometry, using deuterated 7 alpha-hydroxycholesterol as internal standard. After treatment with cholestyramine in a dose of 8 g twice daily for 2-3 weeks preoperatively, the serum level increased to 128 +/- 20 ng/ml (P less than 0.001). Eight other patients with gallstone disease had a mean level of 7 alpha-hydroxycholesterol in serum of 29 +/- 7 ng/ml. Treatment with chenodeoxycholic acid, 15 mg per kg body weight per day for 3-4 weeks before surgery, decreased the mean level to 20 +/- 7 ng/ml (P greater than 0.05). The activity of the cholesterol 7 alpha-hydroxylase in liver biopsies taken during operation was found to be 38 +/- 5 pmol/min per mg of protein in the group of patients treated with cholestyramine and 1.3 +/- 0.5 pmol/min per mg in the group of patients treated with chenodeoxycholic acid. Liver biopsies from a group of untreated patients (n = 13) had a mean cholesterol 7 alpha-hydroxylase activity of 7.6 +/- 1.5 pmol/min per mg.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ellagic acid affects mRNA expression levels of genes that regulate cholesterol metabolism in HepG2 cells

Ellagic acid has been shown to improve cholesterol metabolism in animal studies, but the molecular mechanisms underlying this function have not been fully understood. We performed DNA microarray analysis to elucidate the effects of ellagic acid on cholesterol metabolism in HepG2 hepatocytes. This revealed that the expression levels of several genes related to cholesterol metabolism, including the low-density lipoprotein receptor (LDLR), were changed by ellagic acid treatment. Using a real-time PCR and immunoblot we confirmed that ellagic acid treatment up-regulated mRNA and protein expression level of the LDLR. Moreover, In the presence of 25 μM ellagic acid, extracellular apoB protein and MTP mRNA levels were significantly decreased. These findings indicate that ellagic acid improves cholesterol metabolism through the up-regulation of LDLR, down-regulation of MTP mRNA and reduces extracellular apoB levels. The ellagic acid-induced up-regulation of LDLR occurred via the extracellular signal-regulated kinase (ERK) signaling pathway in HepG2 hepatocytes.

Abbreviations: LDLR: low-density lipoprotein receptor; apoB: apolipoprotein B; PKC: diacylglycerol-protein kinase C; MAPK: mitogen-activated protein kinase; ERK: p42/44 extracellular signal-regulated kinase; JNK: c-Jun N-terminal kinase; VLDLR: very low density lipoprotein receptor; PPARδ: peroxisome proliferator-activated receptor δ; SREBPs: sterol regulatory element-binding proteins; MTP: microsomal triacylglycerol transfer protein; LPDS: lipoprotein-deficient serum     

Half-life of cholesterol 7alpha-hydroxylase activity and enzyme mass differ in animals and humans when determined by a monoclonal antibody against human cholesterol 7alpha-hydroxylase

A monoclonal antibody against human cholesterol 7alpha-hydroxylase was produced, and the half-life of the enzyme was studied. Both the activity and protein mass of the enzyme were measured at timed intervals during microsomal incubation. The enzyme activity dropped rapidly; the half-life was 1, 1.7 and 3h in humans, rats and rabbits, respectively. In contrast, the protein mass, measured by immunoblotting, declined slowly; its half-life was 5h in the human and 9h in the rat and rabbit enzymes. This suggests that there may be vulnerable sites responsible for the enzyme activity. Addition of dithiothreitol (DTT) restored the decreased activity considerably, indicating that at least one sulfhydryl group is involved in the vulnerability. These results show considerable decrement in cholesterol 7alpha-hydroxylase activity due to sulfhydryl groups.     

Determination of hepatic cholesterol 7alpha-hydroxylase activity in man

Methods were developed to determine the activity of the microsomal enzyme cholesterol 7alpha-hydroxylase in human liver. The enzyme assay could be performed with as little as 20 mg of fresh liver tissue, thus making the procedure applicable to specimens obtained by percutaneous liver biopsy. Optimal assay conditions were determined and the identity and radioactive purity of the reaction product, cholest-5-ene-3beta,7alpha-diol (7alpha-hydroxycholesterol) were established. Specific enzyme activity was measured in a number of patients with disorders of lipid metabolism.     

Expression of cholesterol 7alpha-hydroxylase restores bile acid synthesis in McArdle RH7777 cells

Bile acid synthesis involves several enzymes and occurs only in liver cells. The first and rate-determining step is catalyzed by cholesterol 7alpha-hydroxylase (cyp7a). McArdle RH7777 hepatoma cells do not synthesize bile acids and do not express the cyp7a gene. A synthetic cyp7a gene was stably expressed in this cell line to determine if restoration of cyp7a activity is sufficient to reconstitute the bile acid synthetic pathway. The transfected cells contained the recombinant cyp7a mRNA and the corresponding protein. Microsomes from recombinant cells converted cholesterol into 7alpha-hydroxycholesterol, indicating that the recombinant enzyme was active. Radiolabeled bile acids, originated from exogenously supplied radiolabeled cholesterol, were detected in the culture medium of recombinant cells. Thus, expression of cyp7a is sufficient in restoring bile acid synthesis in McArdle RH7777 cells. The results also show that the additional complement of enzymatic activities required to convert cholesterol into bile acids has remained active in this cell line.     

Effects of internal biliary bypass on the regulation of hepatic cholesterol 7alpha-hydroxylase activity in rats.

In order to re-evaluate the importance of the enterohepatic circulation of bile acid in the regulation of the activities of hepatic cholesterol 7alpha-hydroxylase, bile acid metabolism was examined in internal biliary bypass models of rats. A polyethylene tube was inserted into the common bile duct and another side of the tube was placed in the duodenum (DD), lower jejunum (JD), cecum (CeD), or transverse colon (CoD) as internal biliary bypass models and in the urinary bladder as an external biliary drainage (ED). After bile diversion for 7 days in each group, hepatic cholesterol 7alpha-hydroxylase activities, bile acid concentrations in bile, serum, and portal vein, biliary bile acid compositions, and intestinal absorption rates of infused labeled taurocholic acid were analyzed. Hepatic cholesterol 7alpha-hydroxylase activity was similar in the JD group compared with the DD group, however, it was significantly up-regulated in the CeD (227% of the DD group), CoD (312%), and ED groups (316%). Biliary, serum, and portal bile acid concentrations were not significantly changed in the DD, JD, and CeD groups but those were significantly lower in the CoD and ED groups compared with the DD group. The proportion of the secondary bile acids was significantly increased in the CeD group and was decreased in the CoD and ED groups. The absorption rate of taurocholic acid was almost 100%, 56%, and 23% in the JD, CeD, and the CoD group, respectively. As the cholesterol 7alpha-hydroxylase activity was not significantly changed in the JD group and the predominance of secondary bile acids did not suppress the enzyme activity in the CeD group, the luminal factor, which is absorbed in the presence of bile acids, and the bile acid metabolites are not likely the regulatory factor. The cholesterol 7alpha-hydroxylase activity seems to be primarily regulated by the intestinal absorption of bile acids and partly by the intestinal mucosal factor which is linked to the intestinal bile acid absorption.     

On the saturation of the cholesterol 7 alpha-hydroxylase in human liver microsomes

The relationships between cholesterol 7 alpha-hydroxylase activity, pool of free microsomal cholesterol, and degree of substrate saturation of the enzyme were studied in untreated (n = 5), cholesterol-fed (n = 4), and cholestyramine-treated (n = 6) gallstone patients undergoing cholecystectomy. Highly accurate methods based on isotope dilution-mass spectrometry were used for assay of the cholesterol 7 alpha-hydroxylase activity and for determination of the concentration of free cholesterol in the microsomes. The cholesterol-enriched diet increased the cholesterol 7 alpha-hydroxylase activity about twofold. Cholestyramine treatment was associated with a five- to sixfold increase of the cholesterol 7 alpha-hydroxylase activity. The concentration of free microsomal cholesterol remained essentially unchanged. The apparent degree of saturation of the enzyme was calculated to be 85% in the untreated patients, 86% in the cholesterol-fed patients, and 67% in those treated with cholestyramine. A significant negative correlation was obtained between enzyme activity and apparent substrate saturation. It is concluded that the apparent substrate saturation of the cholesterol 7 alpha-hydroxylase in human liver microsomes is high but that availability of cholesterol may limit the enzyme activity to some extent a high bile acid synthesis rates.     

Changes in mRNA expression of 3-hydroxy-3-methylglutaryl coenzyme A reductase and cholesterol 7 alpha-hydroxylase in chickens

3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) and cholesterol 7 alpha-hydroxylase (CYP7A1), essential enzymes of cholesterol synthesis and excretion, respectively, were isolated from a chicken liver cDNA library. When their recombinant proteins were overexpressed in HNK293 cells, corresponding enzyme activities were observed. The complete open reading frames of MHGR and CYP7A1 contained (i) 2625 base pairs (bp), predicting a protein of 875 amino acids, and (ii) 1539 bp, predicting a protein of 513 amino acids, respectively. By Northern blot analysis, chicken HMGR mRNA expression was detected in most tissues examined, however, the highest levels were found in liver, brain and ileum. CYP7A1 mRNA was detected only in the liver. Changes in chicken HMGR and CYP7A1 mRNA expression with nutritional state were examined and were shown to respond to certain nutritional treatments, i.e. fast refeeding and cholesterol supplementation. HMGR and CYP7A1 mRNA levels were significantly increased with maturation (i.e. egg producing), when compared to immature chickens. However, these stimulations were not associated with estrogen, although this does enhance triacylglycerol and very low density lipoprotein secretion by the chicken liver. The present study is the first to report the molecular characterization of HMGR and CYP7A1, key enzymes of cholesterol metabolism in avian species.     

Stimulation of the activity and mRNA level of hepatic triacylglycerol lipase by triiodothyronine in HepG2 cells.

We have studied the effects of triiodothyronine (T3) on the production of hepatic triacylglycerol lipase (HTGL) in the human hepatocellular carcinoma cell line, HepG2, by measuring its activity and mRNA levels. The HTGL activity released into the medium by heparin, increased after the addition of T3 in a both time- (27% increase after 24 and 75% increase after 48 h) and dose-dependent manner (maximum activity with over 0.2 micrograms/ml of T3 in the medium). Messenger RNA levels of HTGL in cells incubated with T3 for 24 and 48 h were increased by 33% and 98% compared to those of the control. These results suggest that the production of HTGL may be regulated by thyroid hormone at the level of gene expression.