Mechanisms of dysregulation of low-density lipoprotein receptor expression in HepG2 cells induced by inflammatory cytokines

Background Low-density lipoprotein （LDL） receptor is normally regulated via a feedback system that is dependent on intracellular cholesterol levels. We have demonstrated that cytokines disrupt cholesterol-mediated LDL receptor feedback regulation causing intracellular accumulation of unmodified LDL in peripheral cells. Liver is the central organ for lipid homeostasis. The aim of this study was to investigate the regulation of cholesterol exogenous uptake via LDL receptor and its underlying mechanisms in human hepatic cell line （HepG2） cells under physiological and inflammatory conditions. Methods Intracellular total cholesterol （TC）, free cholesterol （FC） and cholesterol ester （CE） were measured by an enzymic assay. Oil Red O staining was used to visualize lipid droplet accumulation in cells. Total cellular RNA was isolated from cells for detecting LDL receptor, sterol regulatory element binding protein （SREBP）-2 and SREBP cleavage-activating protein （SCAP） mRNA levels using real-time quantitative PCR. LDL receptor and SREBP-2 protein expression were examined by Western blotting. Confocal microscopy was used to investigate the translocation of SCAP-SREBP complex from the endoplasmic reticulum （ER） to the Golgi by dual staining with anti-human SCAP and anti-Golgin antibodies. Results LDL loading increased intracellular cholesterol level, thereby reduced LDL receptor mRNA and protein expression in HepG2 cells under physiological conditions. However, interleukin 1β （IL-1β） further increased intracellular cholesterol level in the presence of LDL by increasing both LDL receptor mRNA and protein expression in HepG2. LDL also reduced the SREBP and SCAP mRNA level under physiological conditions. Exposure to IL-1β caused over-expression of SREBP-2 and also disrupted normal distribution of SCAP-SREBP complex in HepG2 by enhancing translocation of SCAP-SREBP from the ER to the Golgi despite a high concentration of LDL in the culture medium. Conclusions IL-1β disrupts cholesterol-mediated LDL receptor feedback regulation by enhancing SCAP-SREBP complex translocation from the ER to the Golgi, thereby increasing SREBP-2 mediated LDL receptor expression even in the presence of high concentration of LDL. This results in LDL cholesterol accumulation in hepatic cells via LDL receptor pathway under inflammatory stress.

Endoplasmic reticulum stress is involved in acetylated low-density lipoprotein induced apoptosis in THP-1 differentiated macrophages

Background Cardiovascular disease is a major cause of mortality and morbidity in patients with chronic kidney disease Macrophage death in advanced atherosclerosJs promotes necrosis and plaque destabilization. In vitro data from peritoneal macrophages show apoptosis triggered through endoplasmic reticulum （ER） stress caused by free cholesterol accumulation plays an important role. Here we used THP-1 cells differentiated by 100 ng/ml of phorbol 12-myristate 13-acetate （PMA） for five days as an in vitro model, to investigate if acetylated low-density lipoprotein （AcLDL） loading could also induce apoptosis and its underlying mechanisms. Methods Oil red O staining was used to examine the lipid droplets. Confocal microscopy was used to visualize the uptake of AcLDL. Hoechst 33258 stain and the caspase 3,7 assay were used to detect apoptosis. High performance liquid chromatography was used in the intracellular free cholesterol （FC） and cholesterol ester （CE） assay. Western blotting was used to demonstrate the protein level. Real-time PCR was used to detect the changes of mRNAs. ER free cholesterol was also assayed. Results Confocal microscopy showed THP-1 cells differentiated by 100 ng/ml of PMA for five days uptake more AcLDL than differentiated for two days. Hoechst 33258 stain showed AcLDL could induce apoptosis in THP-1 macrophages in a time and dose dependent manner. Exposure of THP-1 macrophages to 100 ug/ml of AcLDL for 24 hours resulted in a significant increase in caspase 3,7 activity, a significant increase in FC and CE mass of 1.5 and 2.4-fold, meanwhile, a significant increase in transcription factor C/EBP homologous protein and a decrease in Bcl-2 both in protein and mRNA levels were observed with an 8-fold rise of free cholesterol in the ER. Conclusion ER stress is involved in AcLDL induced apoptosJs in THP-1 macrophages with free cholesterol accumulation in the ER.

The molecular pathogenic role of inflammatory stress in dysregulation of lipid homeostasis and hepatic steatosis

Non-alcoholic Fatty Liver Disease(NAFLD)is becoming the leading cause of chronic liver injury in developed countries and China.Chronic systemic inflammation plays a decisive role and is fundamental in the progression of NAFLD from simple steatosis(SS)toward higher risk nonalcoholic steatohepatitis(NASH)states.However,the exact mechanisms by which inflammation leading to NASH are incompletely understood.In this review,we focus the role of the cross talk between inflammation and lipid homeostasis on the progression of NAFLD.