In the studies reported herein, we show that two complementary experimental models: inbred strains of mice (i.e. C57BL/6 and C3H/HeJ), and a differentiated line of rat hepatoma cells (i.e. L35 cells), require the activation of cytokines by monocyte/macrophages to display bile acid negative feedback repression of cholesterol 7␣-hydroxylase (CYP7A1). Feeding a bile acid-containing atherogenic diet for 3 weeks to C57BL/6 mice led to a 70% reduction in the expression of hepatic CYP7A1 mRNA, whereas no reduction was observed in C3H/HeJ mice. The strain-specific response to repression of CYP7A1 paralleled the activation of hepatic cytokine expression. Studies using cultured THP-1 monocyte/ macrophages showed that the hydrophobic bile acid chenodeoxycholate, a well established potent repressor of CYP7A1, induced the expression of mRNAs encoding interleukin 1 (IL-1) and tumor necrosis factor ␣ (TNF␣). In contrast, the hydrophilic bile acid ursodeoxycholate, which does not repress CYP7A1, did not induce cytokine mRNA expression by THP-1 cells. Chenodeoxycholate activation of cytokines by THP-1 cells was blocked by the peroxisome proliferator-activated receptor ␥ agonist rosiglitazone. The expression of cytokines (e.g. IL-1 and TNF␣) by THP-1 cells paralleled with the ability of these cells to produce conditioned medium that when added to rat L35 hepatoma cells, repressed CYP7A1. Moreover, rosiglitazone, which blocks cytokine activation by macrophages, also blocked the repression of CYP7A1 normally exhibited by C57BL/6 mice fed the bile acid-containing atherogenic diet. The combined data indicate that the activation of cytokines may mediate CYP7A1 repression caused by feeding mice an atherogenic diet containing bile acids.Bile acids, the major metabolites produced from cholesterol, are amphipathic steroid detergents necessary for the digestion and absorption of fat soluble nutrients from the intestine (1-3). The conversion of cholesterol to bile acids is regulated by the expression of cholesterol 7␣-hydroxylase (CYP7A1), 1 a cytochrome P450 enzyme unique to the liver parenchymal cell (4 -6). Bile acid synthesis exhibits negative feedback regulation (7,8) by decreasing the enzymatic activity of CYP7A1 (9). It is generally accepted that bile acids can inhibit the transcription of the CYP7A1 gene (1-3).Many different experimental models have been used to examine bile acid negative feedback regulation of CYP7A1 and some have yielded conflicting results. Bile acid negative feedback repression of CYP7A1 has been experimentally demonstrated by infusing bile acids into the intestine of bile fistulae rats (10) and hamsters (11). The ability of different bile acids to repress CYP7A1 expression correlates with the hydrophobic index of the infused bile acid; CDCA is a potent repressor, whereas UDCA is not (12). The finding that infusing taurocholate into the portal vein of bile fistulae mice was unable to repress CYP7A1 led to the conclusion that a factor produced within the enterohepatic circulation may be required to repress CYP7A1...

Stable plasmid-driven expression of the liver-specific gene product cholesterol 7␣-hydroxylase (7␣-hydroxylase) was used to alter the cellular content of transcriptionally active sterol response element binding protein 1 (SREBP1). As a result of stable expression of 7␣-hydroxylase, individual single cell clones expressed varying amounts of mature SREBP1 protein. These single cell clones provided an opportunity to identify SREBP1-regulated genes that may influence the assembly and secretion of apoB-containing lipoproteins. Our results show that in McArdle rat hepatoma cells, which normally do not express 7␣-hydroxylase, plasmid-driven expression of 7␣-hydroxylase results in the following: 1) a linear relationship between (i) the cellular content of mature SREBP1 and 7␣-hydroxylase protein, (ii) the relative expression of 7␣-hydroxylase mRNA and the mRNA's encoding the enzymes regulating fatty acid, i.e. acetylCoA carboxylase and sterol synthesis, i.e. HMG-CoA reductase, (iii) the relative expression of 7␣-hydroxylase mRNA and microsomal triglyceride transfer protein mRNA, a gene product that is essential for the assembly and secretion of apoB-containing lipoproteins; 2) increased synthesis of all lipoprotein lipids (cholesterol, cholesterol esters, triglycerides, and phospholipids); and 3) increased secretion of apoB100 without any change in apoB mRNA. Cells expressing 7␣-hydroxylase contained significantly less cholesterol (both free and esterified). The increased cellular content of mature SREBP1 and increased secretion of apoB100 were concomitantly reversed by 25-hydroxycholesterol, suggesting that the content of mature SREBP1, known to be decreased by 25-hydroxycholesterol, mediates the changes in the lipoprotein assembly and secretion pathway that are caused by 7␣-hydroxylase. These data suggest that several steps in the assembly and secretion of apoB-containing lipoproteins by McArdle hepatoma cells may be coordinately linked through the cellular content of mature SREBP1.Apolipoprotein B100 (apoB) 1 is an unusually large (Ͼ500 kDa) amphipathic protein responsible for the assembly and secretion of plasma lipoproteins by the liver and intestine (reviewed in Refs. 1-3). Its concentration in plasma, as a component of LDL, is a major determinant of susceptibility to the development of atherosclerotic cardiovascular disease (4, 5). Hepatic derived apoB100-containing lipoproteins are the precursors of plasma LDL (6). Hepatic assembly and secretion of apoB-containing lipoproteins require an orchestration of many seemingly independent processes as follows: 1) the production of component lipids (cholesterol, cholesterol esters, triglycerides, and phospholipids); 2) the synthesis of apoB, a uniquely large polypeptide containing multiple amphipathic structural domains that irreversibly associate with phospholipids (7); 3) translocation across the endoplasmic reticulum that requires an intralumenal protein complex consisting of MTP and PDI (8); and 4) the assembly of VLDL particle within the endoplasmic reticulum (1, ...

Multiple AUUUA elements similar to those that regulate the degradation of several different mRNAs are conserved in the 3-untranslated region (3-UTR) of cholesterol-7␣-hydroxylase (CYP7A1) mRNAs from several species. We examined if stabilization of mRNA decay could account for the >20-fold increase in the expression of CYP7A1 mRNA without a detectable change in transcription following dexamethasone treatment of rat hepatoma cells (L35 cells). Following RNA polymerase II-dependent transcription block or protein synthesis block, the decay of CYP7A1 mRNA displayed a short half-life (ϳ30 min). Control experiments showed that in cells pre-treated with a RNA polymerase II inhibitor, dexamethasone had no detectable effect on CYP7A1 mRNA decay. Stable expression of luciferase reporter mRNAs in L35 cells showed that the CYP7A1 3-UTR was required to observe a dexamethasone induction. To examine the hypothesis that a labile protein is required for dexamethasone-induced mRNA stabilization, cells were stably transfected with a tetracycline-repressible promoter that drives the expression of a green fluorescent protein analogue (ECFP) with or without the 3-UTR of CYP7A1. Cells expressing ECFP with the 3-UTR of CYP7A1 displayed a 3-fold dexamethasone induction of ECFP mRNA, whereas cells expressing ECFP without the 3-UTR did not. Moreover, specific block of the transcription of ECFP containing the 3-UTR by adding the tetracycline analogue doxycycline clearly displayed dexamethasone-induced stabilization of mRNA decay. These data provide compelling evidence that a putative labile protein and the 3-UTR of CYP7A1 act together to decrease the rate of CYP7A1 mRNA degradation.

In this study, we explored how sterol metabolism altered by the expression of cholesterol-7␣-hydroxylase NADPH:oxygen oxidoreductase (7␣-hydroxylase) affects the ubiquitin-dependent proteasome degradation of translocation-arrested apoB53 in Chinese hamster ovary cells. Stable expression of two different plasmids that encode either rat or human 7␣-hydroxylase inhibited the ubiquitin conjugation of apoB and its subsequent degradation by the proteasome. Oxysterols (25-hydroxycholesterol and 7-ketocholesterol) reversed the inhibition of apoB degradation caused by 7␣-hydroxylase. The combined results suggest that the normally rapid proteasome degradation of translocation-arrested apoB can be regulated by a sterol-sensitive polyubiquitin conjugation step in the endoplasmic reticulum. Blocked ubiquitin-dependent proteasome degradation caused translocation-arrested apoB to become sequestered in segregated membrane domains. Our results described for the first time a novel mechanism through which the "quality control" proteasome endoplasmic reticulum degradative pathway of translocation-arrested apoB is linked to sterol metabolism. Sterol-sensitive blocked ubiquitin conjugation appears to selectively inhibit the proteasome degradation of apoB, but not 7␣-hydroxylase protein, with no impairment of cell vitality or function. Our findings may help to explain why the hepatic production of lipoproteins is increased when familial hypertriglyceridemic patients are treated with drugs that activate 7␣-hydroxylase (e.g. bile acid-binding resins).ApoB is the major structural protein responsible for the assembly of lipoproteins by the liver and intestine. Multiple forms of apoB, designated as the percentage of the N terminus of the largest secretory product apoB100 (4536 amino acids), are produced from a single gene transcript by mRNA editing and proteolytic cleavage (reviewed in Refs. 1-3). Overproduction of apoB-containing lipoproteins by the liver is responsible for familial combined hyperlipidemia (4). In addition, overproduction of triglyceride-rich lipoproteins is responsible for the human disease familial hypertriglyceridemia (5). In these patients, the secretion of triglyceride-rich lipoproteins varies in parallel with the rate of bile acid synthesis (6 -8). These findings suggest that the secretion of very low density lipoprotein triglyceride is linked to hepatic sterol metabolism via an as yet undefined mechanism that is dependent upon genes that contribute to hypertriglyceridemia.The rate of hepatic secretion of apoB is regulated post-transcriptionally. Only a portion of de novo synthesized apoB is secreted; the remaining portion is degraded intracellularly (9). Interruption of apoB translocation is one of several criteria that lead to increased intracellular degradation (reviewed in Ref. 10). Both translocation and lipid addition require the presence of microsomal triglyceride transfer protein (MTP) 1 in the ER (11-13). MTP exists in the ER lumen as a heterodimer with protein-disulfide isomerase (reviewed in Ref. 14). I...

It is known that hepatic levels of reduced glutathione correlate with the activity of the liver-specific enzyme cholesterol-7␣-hydroxylase. We examined the possibility that sulfhydryl reducing agents activate transcription of cholesterol 7␣-hydroxylase. Adding dithiothreitol (DTT, 1 mM) and dexamethasone to L35 hepatoma cells increased the content of 7␣-hydroxylase mRNA 3-fold above the levels observed with dexamethasone alone.

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