Tissue Specificity Of Feedback Regulation Of Cholesterol Uptake And Biosynthesis Under Inflammatory Stress: A Potential New Mechanism Of Foam Cells Formation

Objectives Cholesterol is derived from two pathways, one is the uptake of plasma via LDL receptor-mediated endocytosis, the other is from cellular synthesis. In the pathway of cholesterol synthesis, HMG-CoA reductase is the important rate-limiting enzyme. Either HMG-CoA reductase or LDLr activity is under the control via a feedback system that is dependent on intracellular cholesterol concentration. We have demonstrated that inflammatory cytokines promote foam cell formation by increasing cholesterol uptake via disrupting cholesterol-mediated LDL receptor feedback regulation, inhibiting cholesterol efflux via reducing ABCA1 gene expression in peripheral cells, such as kidney mesangial cells (HMCs) and vascular smooth muscle cells (VSMC). The aim of this study was to investigate (1) if there are differences between HMCs and human hepatic cell line HepG2 regarding regulation of cholesterol exogenous uptake via the LDL receptor under physiological and inflammatory conditions; (2) if inflammatory cytokines affect in HMGCoA reductase mediated cholesterol synthesis in HMCs and HepG2 and cause statins (competitive inhibitors of HMGCoA reductase) resistance ; (3) the molecular mechanisms by which inflammatory cytokines override the normal cholesterol-mediated suppression of the LDLr/HMGCoA reductase induced by a high concentration of LDL through examining the mRNA expression of the sterol regulatory element binding protein (SREBP-2), as well as SREBP cleavage-activating protein (SCAP) intracellular translocation between the ER and Golgi under the influence of inflammation.Material and Methods HMCs and HepG2 were used in all experiments. Intracellular total cholesterol (TC), free cholesterol (FC) and cholesterol ester (CE) were measured by enzymic assay. Oil Red O staining was used to examine cell morphology. [14C] acetic acid sodium was added to cells medium and incubation was continued for 1 hr to estimate cellular sterol synthesis. Total cellular RNA was isolated from above cells for detecting LDL receptor, HMGCoA reductase, SREBP-2 and SCAP mRNA level with real-time quantitative PCR. LDL receptor and HMGCoA reductase protein expression was measured by Western blotting. HMGcoA reductase activity was measured by Thin Layer Chromatography detecting the amount of mevalonate-3-14C that is formed from 3-14C HMGcoA. Confocal microscopy was used to investigate the translocation of SCAP-SREBP complex from the ER to Golgi after dual staining with anti-human SCAP antibody and anti-Golgin antibody.Results We demonstrated that LDL loading increased intracellular cholesterol level dose-dependently. The accumulation of intracellular cholesterol ester in HMCs is more than in HepG2 cells under IL-1βstress. Increased intracellular cholesterol reduced LDL receptor mRNA and protein expression in both HepG2 and HMCS cells, but to a greater degree in HMCs. Compactin, a HMGCoA reductase activity inhibitor, increased LDL receptor mRNA and protein expression in both cell types by decreasing intracellular cholesterol level, thereby regulating cholesterol exogenous uptake via the LDL receptor. HepG2 cell was more sensitive than HMCs regarding LDL receptor upregulation. However, IL-1βenhanced LDL receptor mRNA and protein expression, and overrode the suppression of LDL receptor induced by high concentrations of LDL cholesterol in both HepG2 and HMCs.In addition, we demonstrated that LDL loading also reduced HMGCoA reductase mRNA, protein expression and enzymatic activity in both cell types. The inhibition in HMCs was more sensitive than in HepG2 which prevent lipid accumulation in peripheral cell HMCs. However, IL-1βincreased cholesterol level in both cells in the presence of LDL by increasing HMGCoA reductase mRNA, protein expression and activity in HMCs, but only increasing activity, not mRNA and protein expression in HepG2 cells. Compactin increased HMGCoA reductase mRNA level and inhibited HMGCoA reductase activity in both cell types, but the effect of compactin was weakened in presence of IL-1β.LDL also reduced SREBP-2 and SCAP mRNA level, inhibited its translocation from the endoplasmic reticulum to the Golgi. Interestingly, there was more SCAP accumulation in the Golgi even in the presence of high concentration of LDL in HepG2 cells rather than HMCs. However, IL-1βdisrupts normal distribution of SCAP in HMCs by enhancing translocation of SCAP from the ER to the Golgi even in the presence of high concentration of LDL.Conclusion LDL receptor and HMGCoA reductase are normally regulated via a feedback system that is dependent on intracellular cholesterol level in the both cells, but to more sensitive in HMCs which prevent lipid accumulation in peripheral cell HMCs. However, IL-1βdisrupts cholesterol-mediated LDL receptor/HMGCoA reductase feedback regulation in peripheral cells, permitting intracellular accumulation of cholesterol and causing foam cell formation. HMCs is more sensitive to inflammation than HepG2 cells which could be one reason why HMCs easy to become foam cells under inflammatory stress. Inflammation also causes statin resistance by disrupting HMGCoA reductase transcriptional and activity, therefore a high concentration of statin may be required to achieve the same biological effect. SCAP location is an important factor for the sensitivity of LDL receptor and HMGCoA reductase regulation in the both cell types.