In nonhepatic cells, cholesterol 7alpha-hydroxylase induces the expression of genes regulating cholesterol biosynthesis, efflux, and homeostasis.

CHO cells expressing the liver-specific gene product cholesterol-7alpha-hydroxylase showed a 6-fold increase in the biosynthesis of [(14)C]cholesterol from [(14)C]acetate, as well as increased enzymatic activities of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase and squalene synthase. Cells expressing cholesterol-7alpha-hydroxylase contained less sterol response element-binding protein 1 (SREBP1) precursor, whereas the cellular content of mature SREBP1, as well as the mRNAs of cholesterol biosynthetic genes (HMG-CoA reductase and squalene synthase), were all increased approximately 3-fold. Cells expressing cholesterol-7alpha-hydroxylase displayed greater activities of luciferase reporters containing the SREBP-dependent promoter elements derived from HMG-CoA reductase and farnesyl diphosphate synthase, in spite of accumulating significantly more free and esterified cholesterol and 7alpha-hydroxycholesterol. While cells expressing cholesterol-7alpha-hydroxylase displayed increased SREBP-dependent transcription, sterol-mediated repression of SREBP-dependent transcription by LDL-cholesterol and exogenous oxysterols was similar in both cell types. Cells expressing cholesterol-7alpha-hydroxylase displayed greater rates of secretion of cholesterol as well as increased expression of the ABC1 cassette protein mRNA. Adding 25-hydroxycholesterol to the culture medium of both cell types increased the expression of ABC1 cassette protein mRNA. The combined data suggest that in nonhepatic CHO cells multiple regulatory processes sensitive to cellular sterols act independently to coordinately maintain cellular cholesterol homeostasis.

Chinese hamster ovary cells require the coexpression of microsomal triglyceride transfer protein and cholesterol 7alpha-hydroxylase for the assembly and secretion of apolipoprotein B-containing lipoproteins.

Due to the absence of microsomal triglyceride transfer protein (MTP), Chinese hamster ovary (CHO) cells lack the ability to translocate apoB into the lumen of the endoplasmic reticulum, causing apoB to be rapidly degraded by an N-acetyl-leucyl-leucyl-norleucinal-inhibitable process. The goal of this study was to examine if expression of MTP, whose genetic deletion is responsible for the human recessive disorder abetalipoproteinemia, would recapitulate the lipoprotein assembly pathway in CHO cells. Unexpectedly, expression of MTP mRNA and protein in CHO cells did not allow apoB-containing lipoproteins to be assembled and secreted by CHO cells expressing apoB53. Although expression of MTP in cells allowed apoB to completely enter the endoplasmic reticulum, it was degraded by a proteolytic process that was inhibited by dithiothreitol (1 mM) and chloroquine (100 microM), but resistant to N-acetyl-leucyl-leucyl-norleucinal. In marked contrast, coexpression of the liver-specific gene product cholesterol 7alpha-hydroxylase with MTP resulted in levels of MTP lipid transfer activity that were similar to those in mouse liver and allowed intact apoB53 to be secreted as a lipoprotein particle. These data suggest that, although MTP-facilitated lipid transport is not required for apoB translocation, it is required for the secretion of apoB-containing lipoproteins. We propose that, in CHO cells, MTP plays two roles in the assembly and secretion of apoB-containing lipoproteins: 1) it acts as a chaperone that facilitates apoB53 translocation, and 2) its lipid transfer activity allows apoB-containing lipoproteins to be assembled and secreted. Our results suggest that the phenotype of the cell (e.g. expression of cholesterol 7alpha-hydroxylase by the liver) may profoundly influence the metabolic relationships determining how apoB is processed into lipoproteins and/or degraded.

Translocation-arrested apolipoprotein B evades proteasome degradation via a sterol-sensitive block in ubiquitin conjugation.

In this study, we explored how sterol metabolism altered by the expression of cholesterol-7alpha-hydroxylase NADPH:oxygen oxidoreductase (7alpha-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 7alpha-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 7alpha-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 7alpha-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 7alpha-hydroxylase (e.g. bile acid-binding resins).