Regulation of the hamster cholesterol 7 alpha-hydroxylase gene (CYP7A): Prevalence of negative over positive transcriptional control
Biophysics; down-regulation; Biochemistry & Molecular Biology; hmg-coa reductase; messenger-rna levels; Bile acids; protein-kinase-c; cultures; primary; thyroid-hormone; rat hepatocytes; hormonal-regulation; hypophysectomized rats
Cholesterol 7 alpha-hydroxylase plays a crucial role in cholesterol homeostasis. We investigated the regulation of this enzyme in the hamster, a suitable animal model for studying cholesterol metabolism. DNase I hypersensitivity assay revealed the presence of a hypersensitive region in the proximal promoter. Both negative (bile acids, phorbol esters and insulin) and positive (glucocorticoid hormones) effects were mediated through sequences in the region 318 bp upstream of the ATG codon. All-trans-retinoic acid, cAMP, and LDL did not affect transcriptional activity. These findings show that the hamster cholesterol 7 alpha-hydroxylase gene undergoes a predominant negative regulation, as opposed to the rat CYP7A homologous gene. (C) 1996 Academic Press, Inc.
DeFabiani E; Crestani M; Marrapodi M; Pinelli A; Chiang J Y L; Galli G
Biochemical and Biophysical Research Communications
1996
1996-09
Journal Article or Conference Abstract Publication
<a href="http://doi.org/10.1006/bbrc.1996.1412" target="_blank" rel="noreferrer noopener">10.1006/bbrc.1996.1412</a>
Hormonal regulation of cholesterol 7 alpha-hydroxylase specific activity, mRNA levels, and transcriptional activity in vivo in the rat
bile-acid biosynthesis; Biochemistry & Molecular Biology; cholesterol; cholesterol 7 alpha-hydroxylase; cultures; gene; gene cyp7; glucocorticoid; hepatic cholesterol; hepatocyte cultures; liver; messenger-rna levels; monolayer-cultures; primary; protein-kinase-c; regulation; sterol 27-hydroxylase; thyroid; thyroid-hormone
In primary cultures of rat hepatocytes, transcription of the cholesterol 7 alpha-hydroxylase gene is induced synergistically by glucocorticoid and thyroid hormones. The objective of the present study was to evaluate the role of glucocorticoid and thyroid hormones in the maintenance of cholesterol 7 alpha-hydroxylase gene expression in vivo. Male Sprague-Dawley rats underwent adrenalectomy (A), thyroidectomy (T), adrenalectomy + thyroidectomy (A + T), hypophysectomy (H), or sham surgery (paired controls). Ten days post surgery, livers were harvested and cholesterol 7 alpha-hydroxylase specific activity, steady-state mRNA levels, and transcriptional activity were determined. Serum corticosterone levels were <2% of paired controls in A, A + T and H rats. Free thyroxine index was <32% of paired controls in rats with T and H. When compared to sham-operated controls, A + T and H led to decreases in cholesterol 7 alpha-hydroxylase specific activities of 44 +/- 8% and 57 +/- 3%, respectively (P < 0.03 and < 0.05). Similar changes were observed in cholesterol 7 alpha-hyroxylase steady-state mRNA levels, which decreased by 43 +/- 10% (P < 0.001) and 56 +/- 19% (P < 0.05), respectively. Cholesterol 7 alpha-hydroxylase transcriptional activity in A + T and H rats decreased by 34 +/- 11% (P < 0.01) and 61 +/- 4% (P < 0.001), respectively. The observed decreases were greater after H than after A + T, suggesting the possibility that another pituitary hormone plays a role in regulation of cholesterol 7 alpha-hydroxylase. Thyroidectomy alone led to a decrease in cholesterol 7 alpha-hydroxylase specific activity of 37 +/- 7% (P < 0.05) and a trend toward decreased steady-state mRNA levels (21 +/- 12%; P = ns). Adrenalectomy did not significantly decrease cholesterol 7 alpha-hydroxylase specific activity or mRNA levels. Neither thyroidectomy nor adrenalectomy alone affected transcriptional activity. We conclude that under physiologic circumstances, full expression of the cholesterol 7 alpha-hydroxylase gene requires synergistic action of glucocorticoids and thyroid hormone.
Pandak W M; Heuman D M; Redford K; Stravitz R T; Chiang J Y L; Hylemon P B; Vlahcevic Z R
Journal of Lipid Research
1997
1997-12
Journal Article
n/a
The stimulatory effect of LXR alpha is blocked by SHP despite the presence of a LXR alpha binding site in the rabbit CYP7A1 promoter
alpha-fetoprotein transcription; bile-acid biosynthesis; Biochemistry & Molecular Biology; cholesterol 7 alpha-hydroxylase; cholesterol 7-alpha-hydroxylase gene; dietary-cholesterol; dietary-cholesterol; factor; farnesoid X receptor; farnesoid X receptor; inhibition; liver X receptor; messenger-rna levels; nuclear receptor; orphan; rat; SHP; taurocholate; transcription
The transcription of the cholesterol 7 alpha-hydroxylase gene (CYP7A1) is greatly decreased in cholesterol-fed rabbits. To determine whether the molecular structure of the promoter is responsible for this downregulation, we cloned the rabbit CYP7A1 promoter, identified the binding sites for a-fetoprotein transcription factor (FTF) and liver X receptor (LXR alpha), and studied the effects of FTF, LXR alpha, and SHP on its transcription. Adding LXR alpha/retinoid X receptor together with their ligands (L/R) to the promoter/reporter construct transfected into HepG2 cells greatly increased its activity. FTF did not increase promoter activity, nor did it enhance the stimulatory effect of L/R. Mutating the FTF binding site abolished the promoter baseline activity. Increasing amounts of SHP abolished the effect of L/R, and FTF enhanced the ability of SHP to decrease promoter activity below baseline levels. Thus, downregulation of CYP7A1 in cholesterol-fed rabbits is attributable secondarily to the activation of farnesoid X receptor, which increases SHP expression to override the positive effects of LXR alpha. Although FFF is a competent factor for maintaining baseline activity, it does not further enhance and may suppress CYP7A1 transcription.
Shang Q; Pan L X; Saumoy M; Chiang J Y L; Tint G S; Salen G; Xu G R
Journal of Lipid Research
2006
2006-05
Journal Article
<a href="http://doi.org/10.1194/jlr.M500449-JLR200" target="_blank" rel="noreferrer noopener">10.1194/jlr.M500449-JLR200</a>
FAILURE OF INTRAVENOUS-INFUSION OF TAUROCHOLATE TO DOWN-REGULATE CHOLESTEROL 7-ALPHA-HYDROXYLASE IN RATS WITH BILIARY FISTULAS
3-hydroxy-3-methylglutaryl; bile-acid synthesis; cholesterol; coenzyme; coenzyme-a reductase; feedback-regulation; Gastroenterology & Hepatology; hepatic; hepatocytes; liquid-chromatography; liver; messenger-rna levels; performance; transcriptional regulation
Background/Aims: The decrease in cholesterol 7 alpha-hydroxylase induced by intraduodenal infusion of taurocholate in bile fistula vats may be indirect, i.e., mediated through release or absorption of an intestinal factor in response to the presence of bile salts in the intestine. The aim of this study was to determine if negative feedback regulation of cholesterol 7 alpha-hydroxylase can be shown when equimolar concentrations of taurocholate are administered intravenously, thus bypassing the intestine. Methods: After 96 hours of biliary diversion, taurocholate (36 mu mol.h(-1).100 g rat(-1)) was infused into the rats either intravenously or intraduodenally for the final 24 hours. Livers were then harvested for analysis of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase specific activity, cholesterol 7 alpha-hydroxylase specific activity, messenger RNA levels, and transcriptional activity. Results: Intra-duodenally administered taurocholate significantly decreased HMG-CoA reductase and cholesterol 7 alpha-hydroxyfase specific activity by more than 50% and cholesterol 7 alpha-hydroxylase steady-state messenger RNA levels and transcriptional activity by 50%-75%. In contrast, intravenous administration of taurocholate failed to down-regulate either cholesterol 7 alpha-hydroxylase or HMG-CoA reductase. Conclusions: Passage of taurocholate through the intestine strongly potentiates negative feedback regulation of cholesterol 7 alpha-hydroxylase. A putative intestinal factor, released or absorbed in the presence of bile acids in the intestinal lumen, may play a role in the regulation of bile acid synthesis.
Pandak W M; Heuman D M; Hylemon P B; Chiang J Y L; Vlahcevic Z R
Gastroenterology
1995
1995-02
Journal Article
<a href="http://doi.org/10.1016/0016-5085(95)90083-7" target="_blank" rel="noreferrer noopener">10.1016/0016-5085(95)90083-7</a>
Cholesterol 7 alpha-hydroxylase activities from human and rat liver are modulated in vitro posttranslationally by phosphorylation/dephosphorylation
bile-acid synthesis; cloning; dietary-cholesterol; enzyme; Escherichia coli; expression; Gastroenterology & Hepatology; hmg-coa reductase; messenger-rna levels; Phosphatase; purification
Purified cholesterol 7 alpha-hydroxylases (C7 alpha H) from human and rat liver microsomes, and from transformed Escherichia coli expression systems, were incubated with 0.3 mmol/L [gamma-P-32] adenosine triphosphate (ATP) in the presence and absence of bacterial alkaline phosphatase (AP) or rabbit muscle adenosine 3',5'-cyclic monophosphate (cAMP) dependent protein kinase. The amounts of P-32 incorporation after separation of human and rat C7 alpha H proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were related to C7 alpha H catalytic activities (determined by a radioisotope incorporation method) and enzyme protein mass (determined by Western blotting and laser densitometry). Both human and rat C7 alpha H activities significantly decreased after dephosphorylation by AP (-57%--72%) and increased up to twofold with phosphorylation by rabbit muscle cAMP-dependent protein kinase. The increases in C7 alpha H activities were proportional to the amounts of cAMP-dependent protein kinase used, and were coupled to P-32 incorporation into the purified enzymes, Both the activation of C7 alpha H and the amounts of P-32 incorporation were time-dependent and reached a maximum after 1 hour of incubation with 5 U of cAMP-dependent protein kinase. In a second set of experiments, purified human and rat Liver C7 alpha H were dephosphorylated by 30-minute incubation with AP, followed by inactivation of the phosphatase by the inhibitor NaF, and rephosphorylation of C7 alpha H by 30-minute incubation with rabbit muscle cAMP-dependent protein kinase or bovine heart cAMP-independent protein kinase. Rephosphorylation of the dephosphorylated C7 alpha H proteins by cAMP-dependent protein kinase increased C7 alpha H catalytic activities up to fourfold, and the stimulation in catalytic activities paralleled the increases in P-32 incorporation into the purified enzymes. Bovine heart protein kinase was as potent as rabbit muscle cAMP-dependent protein kinase in stimulating catalytic activity and P-32 incorporation into the human C7 alpha H protein. Because the protein mass of these purified enzymes did not change, the short-term regulation or catalytic efficiency of C7 alpha H (activity per protein mass unit) is modulated, in vitro, posttranslationally by a phosphorylation/ dephosphorylation mechanism in both the human and the rat enzymes.
Nguyen L B; Shefer S; Salen G; Chiang J Y L; Patel M
Hepatology
1996
1996-12
Journal Article
<a href="http://doi.org/10.1053/jhep.1996.v24.pm0008938182" target="_blank" rel="noreferrer noopener">10.1053/jhep.1996.v24.pm0008938182</a>