Activation of the silent endogenous cholesterol-7-alpha-hydroxylase gene in rat hepatoma cells: a new complementation group having resistance to 25-hydroxycholesterol.

The oxysterol 25-hydroxycholesterol acts both as a regulatory sterol determining the expression of genes governed by sterol regulatory elements and as a substrate for 7-alpha-hydroxylase, the first and rate-limiting enzyme in the bile acid synthetic pathway. Most wild-type nonhepatic cells are killed by the cytotoxic action of 25-hydroxycholesterol. In contrast, liver cells, which express 7-alpha-hydroxylase activity, are resistant to killing by 25-hydroxycholesterol. We examined the possibility that selection for resistance to 25-hydroxycholesterol might lead to the derivation of a cell line expressing 7-alpha-hydroxylase. A rat hepatoma cell line (7-alpha-hydroxylase minus) was transfected with human DNA and screened for resistance to 25-hydroxycholesterol. Although parental hepatoma cells were all killed within a week, a 25-hydroxycholesterol-resistant cell line (L35 cells) which showed stable expression of 7-alpha-hydroxylase activity and mRNA was obtained. These cells exhibited normal inhibition of cholesterol biosynthesis by 25-hydroxycholesterol. Blocking 7-alpha-hydroxylase activity with ketoconazole also blocked the resistance of L35 cells to 25-hydroxycholesterol. Isolation of microsomes from these cells showed levels of 7-alpha-hydroxylase activity (22.9 pmol/min/mg of protein) that were comparable to the activity (33.2 pmol/min/mg) of microsomes isolated from the livers of rats killed during the high point of the diurnal cycle. Parental cells had no detectable activity. These data show a new complementation group for 25-hydroxycholesterol resistance: expression of 7-alpha-hydroxylase. Dexamethasone increased both the activity and the cellular content of mRNA coding for 7-alpha-hydroxylase. Since dactinomycin blocked the ability of dexamethasone to induce mRNA, active transcription is required. Southern analysis of genomic DNA showed that L35 cells contain the rat (endogenous) gene but not the human gene. Furthermore, the RNA expressed by L35 cells is similar in size to rat RNA and is distinct from the human form of 7-alpha-hydroxylase. The combined data indicate that L35 cells are resistant to 25-hydroxycholesterol because they express 7-alpha-hydroxylase. The mechanism responsible involves activation of the endogenous (silent) gene of the parental rat hepatoma cell.

The effect of idebenone, a coenzyme Q analogue, on hydrophobic bile acid toxicity to isolated rat hepatocytes and hepatic mitochondria.

Oxidant stress induced by hydrophobic bile acids has been implicated in the pathogenesis of liver injury in cholestatic liver disorders. We evaluated the effect of idebenone, a coenzyme Q analogue, on taurochenodeoxycholic acid (TCDC)-induced cell injury and oxidant stress in isolated rat hepatocytes and on glycochenodeoxycholic acid (GCDC)-induced generation of hydroperoxides in fresh hepatic mitochondria. Isolated rat hepatocytes in suspension under 9% oxygen atmosphere were preincubated with 0, 50, and 100 micromol/l idebenone for 30 min and then exposed to 1000 micromol/l TCDC for 4 h. LDH release (cell injury) and thiobarbituric acid reactive substances (measure of lipid peroxidation) increased after TCDC exposure but were markedly suppressed by idebenone pretreatment. In a second set of experiments, the addition of 100 micromol/l idebenone up to 3 h after hepatocytes were exposed to 1000 micromol/l TCDC resulted in abrogation of subsequent cell injury and markedly reduced oxidant damage to hepatocytes. Chenodeoxycholic acid concentrations increased to 5.15 nmol/10(6) cells after 2 h and to 7.05 after 4 h of incubation of hepatocytes with 1000 micromol/l TCDC, and did not differ in the presence of idebenone. In freshly isolated rat hepatic mitochondria, when respiration was stimulated by succinate, 10 micromol/l idebenone abrogated the generation of hydroperoxides during a 90-minute exposure to 400 micromol/l GCDC. These data demonstrate that idebenone functions as a potent protective hepatocyte antioxidant during hydrophobic bile acid toxicity, perhaps by reducing generation of oxygen free radicals in mitochondria.

Post-transcriptional regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase by mevalonate.

We have examined the mechanisms of sterol-independent regulation of the expression of 3-hydroxy-3-methylglutaryl coenzyme-A (HMG-CoA) reductase by mevalonate in Chinese hamster ovary (CHO) cells. Serum lipoproteins, 25-hydroxycholesterol, or mevalonate each repress HMG-CoA reductase activity by fivefold or more, and mevalonate lowers the rate of reductase synthesis by twofold. However, while the expression of the HMG-CoA reductase promoter construct, T42 delta CAT, in stable transfectants is also repressed by serum lipoproteins and 25-hydroxycholesterol, mevalonate is without effect. In addition, while 25-hydroxycholesterol reduces the steady-state level of endogenous HMG-CoA reductase mRNA by more than threefold, mevalonate again has no effect. Mevalonate does partially regulate the expression of both the artificial promoter construct pTK-Kx3-CAT, containing three copies of the sterol regulatory element, SRE-1, and the full-length LDL receptor promoter construct, pLDLRCAT-6500 as well as the expression of functional LDL receptors. This transcriptional regulation appears to be mediated by sterol end products generated from added mevalonate. In CHO cells starved for mevalonate due to a mutation in the biosynthetic pathway, addition of 20 mM mevalonate accelerates the rate of degradation of HMG-CoA reductase by threefold whether new sterol biosynthesis is blocked or not. In such cells, addition of 25-hydroxycholesterol, by itself, also decreases the half-life of reductase from 11.6 to 2.3 h. In contrast, in cells acutely treated with a reductase inhibitor, sterol-accelerated degradation of reductase is only observed in the presence of submillimolar level of mevalonate. We conclude that large concentrations of exogenous mevalonate fail to generate a transcriptional regulator of HMG-CoA reductase in CHO cells but do lead to the formation of translational regulator(s) of reductase synthesis. In contrast, sterol regulators derived from exogenous mevalonate appear to be capable of downregulating the LDL receptor promoter. We further conclude that in the absence of pretreatment with a reductase inhibitor, the regulatory signals generated by sterols and nonsterols for accelerated degradation of HMG-CoA reductase are mutually independent. However, the enzyme synthesized in the presence of reductase inhibitors appears to exhibit an obligatory corequirement for low-dose mevalonate for sterol-accelerated degradation.

Substrate stimulation of 7 alpha-hydroxylase, an enzyme located in the cholesterol-poor endoplasmic reticulum.

We examined the role of cholesterol in altering the activity of the microsomal cytochrome P-450 enzyme, cholesterol-NADPH:oxygen oxidoreductase (cholesterol 7 alpha-hydroxylase). Liposomes were used to deliver cholesterol to hepatic microsomes. Formation of 7 alpha-hydroxycholesterol was quantitated by isotope dilution/gas chromatography-mass spectrometry. As the liposomal cholesterol/phospholipid molar ratio increased, 7 alpha-hydroxylase activity increased, whereas the activity of another microsomal cytochrome P-450 enzyme, ethylmorphine N-demethylase, decreased. To determine if the degree of stimulation was affected by the endogenous activity (without liposomes), microsomes, from rats fed chow alone or chow containing cholestyramine, taurocholate, or cholesterol were challenged with cholesterol-enriched liposomes. The degree of stimulation was dependent upon the endogenous activity: cholestyramine-fed much greater than cholesterol = chow control greater than taurocholate-fed. To determine if cholesterol stimulates 7 alpha-hydroxylase by increasing membrane viscosity, microsomes were incubated with liposomes having the same cholesterol/phospholipid molar ratio as microsomes, but different viscosities. Dipalmitoylphosphatidylcholine (high viscosity) liposomes increased microsomal viscosity and decreased 7 alpha-hydroxylase activity. In contrast, dioleoylphosphatidylcholine (low viscosity) liposomes decreased microsomal viscosity and increased enzyme activity. Since greater viscosity inhibits 7 alpha-hydroxylase, cholesterol cannot stimulate the enzyme by increasing membrane viscosity. The data suggest that cholesterol stimulates production of 7 alpha-hydroxycholesterol by providing substrate.

Role of oxidant stress in the permeability transition induced in rat hepatic mitochondria by hydrophobic bile acids.

Hydrophobic bile acids may cause hepatocellular necrosis and apoptosis during cholestatic liver diseases. The mechanism for this injury may involve mitochondrial dysfunction and the generation of oxidant stress. The purpose of this study was to determine the relationship of oxidant stress and the mitochondrial membrane permeability transition (MMPT) in hepatocyte necrosis induced by bile acids. The MMPT was measured spectrophotometrically and morphologically in rat liver mitochondria exposed to glycochenodeoxycholic acid (GCDC). Freshly isolated rat hepatocytes were exposed to GCDC and hepatocellular necrosis was assessed by lactate dehydrogenase release, hydroperoxide generation by dichlorofluorescein fluorescence, and the MMPT in cells by JC1 and tetramethylrhodamine methylester fluorescence on flow cytometry. GCDC induced the MMPT in a dose- and Ca(2+)-dependent manner. Antioxidants significantly inhibited the GCDC-induced MMPT and the generation of hydroperoxides in isolated mitochondria. Other detergents failed to induce the MMPT and a calpain-like protease inhibitor had no effect on the GCDC-induced MMPT. In isolated rat hepatocytes, GCDC induced the MMPT, which was inhibited by antioxidants. Blocking the MMPT in hepatocytes reduced hepatocyte necrosis and oxidant stress caused by GCDC. Oxidant stress, and not detergent effects or the stimulation of calpain-like proteases, mediates the GCDC-induced MMPT in hepatocytes. We propose that reducing mitochondrial generation of reactive oxygen species or preventing increases in mitochondrial Ca(2+) may protect the hepatocyte against bile acid-induced necrosis.