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 1 beta (IL-1 beta) 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-1 beta 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-1 beta 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.
Chen
Ya-xi
YX
Centre for Lipid Research, Key Laboratory of Molecular Biology on Infectious Diseases, Ministry of Education, Chongqing 400010, China.
Ruan
Xiong-zhong
XZ
Huang
Ai-long
AL
Li
Qiu
Q
Moorhead
John F
JF
Varghese
Zac
Z
D045744:Cell Line, Tumor; D002784:Cholesterol / Q000032:analysis; D004721:Endoplasmic Reticulum / Q000378:metabolism; D025461:Feedback, Physiological; D006801:Humans; D053583:Interleukin-1beta / Q000494:pharmacology*; D047908:Intracellular Signaling Peptides and Proteins / Q000032:analysis / Q000235:genetics; D008565:Membrane Proteins / Q000032:analysis / Q000235:genetics; D021381:Protein Transport; D012333:RNA, Messenger / Q000032:analysis; D011973:Receptors, LDL / Q000032:analysis / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D051782:Sterol Regulatory Element Binding Protein 2 / Q000032:analysis / Q000235:genetics
D053583:Interleukin-1beta; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D012333:RNA, Messenger; D011973:Receptors, LDL; C497390:SREBF2 protein, human; C102196:SREBP cleavage-activating protein; D051782:Sterol Regulatory Element Binding Protein 2; D002784:Cholesterol