8-bromoadenosine cyclic 3',5'-phosphate rapidly increases 3-hydroxy-3-methylglutaryl coenzyme A reductase mRNA in human granulosa cells: role of cellular sterol balance in controlling the response to tropic stimulation

Exposure of cultured human granulosa cells to 8-bromoadenosine cyclic 3',5'-phosphate (8-bromo-cAMP) resulted in a rapid increase in the content of the mRNA for 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, a rate-limiting enzyme in the de novo synthesis of cholesterol. HMG-CoA reductase mRNA levels increased within 2 h of stimulation and remained elevated for at least 6 h. Treatment of granulosa cells with 25-hydroxycholesterol, a soluble cholesterol analogue, in combination with aminoglutethimide to block conversion of cellular sterols to pregnenolone, resulted in suppression of HMG-CoA reductase mRNA. When cells were stimulated with 8-bromo-cAMP in the presence of 25-hydroxycholesterol and aminoglutethimide, the increase in HMG-CoA reductase mRNA provoked by the tropic agent was markedly attenuated. This indicates that 8-bromo-cAMP raises HMG-CoA reductase mRNA levels indirectly by accelerating steroidogenesis and depleting cellular sterol pools, thus relieving sterol-mediated negative feedback of HMG-CoA reductase gene expression. 25-Hydroxycholesterol in the presence of aminoglutethimide suppressed low-density lipoprotein (LDL) receptor mRNA, but 8-bromo-cAMP effected a significant stimulation of LDL receptor mRNA levels when added with hydroxysterol and aminoglutethimide. These findings reveal differential regulation of HMG-CoA reductase and LDL receptor mRNAs in the presence of sterol negative feedback.     

Blockade of cholesterol absorption by ezetimibe reveals a complex homeostatic network in enterocytes

Enterocyte cholesterol homeostasis reflects aggregated rates of sterol synthesis, efflux, and uptake from plasma and gut lumen. Cholesterol synthesis and LDL uptake are coordinately regulated by sterol regulatory element-binding proteins (SREBP), whereas sterol efflux is regulated by liver X receptors (LXR). How these processes are coordinately regulated in enterocytes, the site of cholesterol absorption, is not well understood. Here, we treat mice with ezetimibe to investigate the effect of blocking cholesterol absorption on intestinal SREBPs, LXRs, and their effectors. Ezetimibe increased nuclear SREBP-2 8-fold. HMG-CoA reductase (HMGR) and LDL receptor (LDLR) mRNA levels increased less than 3-fold, whereas their protein levels increased 30- and 10-fold, respectively. Expression of inducible degrader of LDLR (IDOL), an LXR-regulated gene that degrades LDLRs, was reduced 50% by ezetimibe. Coadministration of ezetimibe with the LXR agonist T0901317 abolished the reduction in IDOL and prevented the increase in LDLR protein. Ezetimibe-stimulated LDLR expression was independent of proprotein convertase subtilisin/kexin type 9 (PSCK9), a protein that degrades LDLRs. To maintain cholesterol homeostasis in the face of ezetimibe, enterocytes boost LDL uptake by increasing LDLR number, and they boost sterol synthesis by increasing HMGR and other cholesterologenic genes. These studies reveal a hitherto undescribed homeostatic network in enterocytes triggered by blockade of cholesterol absorption.     

Effect of cutaneous permeability barrier disruption on HMG-CoA reductase, LDL receptor, and apolipoprotein E mRNA levels in the epidermis of hairless mice.

Disruption of the permeability barrier results in an increase in cholesterol synthesis in the epidermis. Inhibition of cholesterol synthesis impairs the repair and maintenance of barrier function. The increase in epidermal cholesterol synthesis after barrier disruption is due to an increase in the activity of epidermal HMG-CoA (3-hydroxy-3-methylglutaryl CoA) reductase. To determine the mechanism for this increase in enzyme activity, in the present study we have shown by Western blot analysis that there is a 1.5-fold increase in the mass of HMG-CoA reductase after acute disruption of the barrier with acetone. In a chronic model of barrier disruption, essential fatty acid deficiency, there is a 3-fold increase in the mass of HMG-CoA reductase. Northern blot analysis demonstrated that after acute barrier disruption with acetone or tape-stripping, epidermal HMG-CoA reductase mRNA levels are increased. In essential fatty acid deficiency, epidermal HMG-CoA reductase mRNA levels are increased 3-fold. Thus, both acute and chronic barrier disruption result in increases in epidermal HMG-CoA reductase mRNA levels which could account for the increase in HMG-CoA reductase mass and activity. Additionally, both acute and chronic barrier disruption increase the number of low density lipoprotein (LDL) receptors and LDL receptor mRNA levels in the epidermis. Moreover, epidermal apolipoprotein E mRNA levels are increased by both acute and chronic perturbations in the barrier. Increases in these proteins in response to barrier disruption may allow for increased lipid synthesis and transport between cells and facilitate barrier repair.     

Dissociated regulation of macrophage LDL receptor and apolipoprotein E gene expression by sterol

The control of apoE gene expression by sterols and the relationship between regulation of the apoE and low density lipoprotein (LDL) receptor genes were investigated in a human macrophage line. Incubation of THP1 cells in either LDL or acetylated LDL increased apoE mRNA levels 4- to 15-fold. In addition, the cellular abundance of these two mRNA species (apoE and LDL receptor) was inversely regulated by cellular cholesterol content over an identical dose-response relationship. Regulation of the LDL receptor and apoE genes could, however, be temporally dissociated in response to the accumulation or removal of lipoprotein-derived (exogenous) cholesterol and in response to perturbation of endogenous cellular cholesterol biosynthesis. In addition, we observed that the apoE gene responded more promptly to 25-hydroxycholesterol than to exogenous cholesterol. These data support the concept that the apoE gene be considered among the family of genes sensitively regulated by cellular sterol balance but suggest that the molecular mechanism accounting for the modulation of the LDL receptor and apoE genes are distinct, with the relationship between cell sterol balance and apoE gene regulation being more complex.     

Increased cholesterol biosynthesis and hypercholesterolemia in mice overexpressing squalene synthase in the liver

Squalene synthase (SS) is the first committed enzyme for cholesterol biosynthesis, located at a branch point in the mevalonate pathway. To examine the role of SS in the overall cholesterol metabolism, we transiently overexpressed mouse SS in the livers of mice using adenovirus-mediated gene transfer. Overexpression of SS increased de novo cholesterol biosynthesis with increased 3-hydroxy-3-methyglutaryl-CoA (HMG-CoA) reductase activity, in spite of the downregulation of its own mRNA expression. Furthermore, overexpression of SS increased plasma concentrations of LDL, irrespective of the presence of functional LDL receptor (LDLR). Thus, the hypercholesterolemia is primarily caused by increased hepatic production of cholesterol-rich VLDL, as demonstrated by the increases in plasma cholesterol levels after intravenous injection of Triton WR1339. mRNA expression of LDLR was decreased, suggesting that defective LDL clearance contributed to the development of hypercholesterolemia. Curiously, the liver was enlarged, with a larger number of Ki-67-positive cells. These results demonstrate that transient upregulation of SS stimulates cholesterol biosynthesis as well as lipoprotein production, providing the first in vivo evidence that SS plays a regulatory role in cholesterol metabolism through modulation of HMG-CoA reductase activity and cholesterol biosynthesis.