Sirolimus modifies cholesterol homeostasis in hepatic cells: a potential molecular mechanism for sirolimus-associated dyslipidemia.
ABSTRACT Sirolimus is a potent immunosuppressive agent, which is associated with dyslipidemia in clinical transplantation. The present study was undertaken to investigate the potential hepatocyte mechanisms by which sirolimus causes dyslipidemia.
Using both a quantitative assay of intracellular cholesterol and an [3H]-labeled cholesterol efflux assay, we studied the effect of sirolimus on cholesterol accumulation and cholesterol efflux in HepG2 cells in the absence or presence of inflammatory stress induced by interleukin-1beta. The gene and protein expression of molecules involved in cholesterol homeostasis were examined by real-time reverse-transcription polymerase chain reaction and Western blotting.
Sirolimus inhibited low-density lipoprotein (LDL) receptor (LDLr)-mediated cholesterol ester accumulation induced by interleukin-1beta in HepG2 cells. This inhibitory effect was mediated by down-regulation of sterol regulatory element-binding proteins (SREBP) cleavage activating protein (SCAP) and SREBP-2 mRNA expression. Using confocal microscopy, we demonstrated that sirolimus reduced translocation of SCAP-SREBP2 complex from endoplasmic reticulum to Golgi for activation, thereby inhibiting LDLr gene transcription. Reduction of LDLr in the liver may result in a delay of LDL-cholesterol clearance from circulation causing an increase of plasma cholesterol concentration. Furthermore, sirolimus increased cholesterol efflux mediated by adenosine triphosphate-binding cassette transporter A1 gene expression by increasing peroxisome proliferator-activated receptor-alpha and liver X receptor-alpha gene and protein expression. Increased cholesterol efflux from HepG2 cells may increase high-density lipoprotein cholesterol level and also contribute to apolipoprotein B lipoprotein formation by enhancing transfer of high-density lipoprotein cholesterol to apolipoprotein B lipoproteins.
This study demonstrates that the increase of LDL cholesterol by sirolimus is partly due to the reduction of LDLr on hepatocytes.
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ABSTRACT: The proteolytic cleavage of sterol regulatory element-binding proteins (SREBPs) is regulated by SREBP cleavage-activating protein (SCAP), which forms complexes with SREBPs in membranes of the endoplasmic reticulum (ER). In sterol-depleted cells, SCAP facilitates cleavage of SREBPs by Site-1 protease, thereby initiating release of active NH(2)-terminal fragments from the ER membrane so that they can enter the nucleus and activate gene expression. In sterol-overloaded cells, the activity of SCAP is blocked, SREBPs remain bound to membranes, and transcription of sterol-regulated genes declines. Here, we provide evidence that sterols act by inhibiting the cycling of SCAP between the ER and Golgi. We use glycosidases, glycosidase inhibitors, and a glycosylation-defective mutant cell line to demonstrate that the N-linked carbohydrates of SCAP are modified by Golgi enzymes in sterol-depleted cells. After modification, SCAP returns to the ER, as indicated by experiments that show that the Golgi-modified forms of SCAP cofractionate with ER membranes on density gradients. In sterol-overloaded cells, the Golgi modifications of SCAP do not occur, apparently because SCAP fails to leave the ER. Golgi modifications of SCAP are restored when sterol-overloaded cells are treated with brefeldin A, which causes Golgi enzymes to translocate to the ER. These studies suggest that sterols regulate the cleavage of SREBPs by modulating the ability of SCAP to transport SREBPs to a post-ER compartment that houses active Site-1 protease.Proceedings of the National Academy of Sciences 10/1999; 96(20):11235-40. · 9.74 Impact Factor
Article: ABCA1 and atherosclerosis.[show abstract] [hide abstract]
ABSTRACT: ATP binding cassette transporter A1 (ABCA1) mediates the cellular efflux of phospholipids and cholesterol to lipid-poor apolipoprotein A1 (apoA1) and plays a significant role in high density lipoprotein (HDL) metabolism. ABCA1's role in the causation of Tangier disease, characterized by absent HDL and premature atherosclerosis, has implicated this transporter and its regulators liver-X-receptoralpha (LXRalpha) and peroxisome proliferator activated receptorgamma (PPARgamma) as new candidates potentially influencing the progression of atherosclerosis. In addition to lipid regulation, these genes are involved in apoptosis and inflammation, processes thought to be central to atherosclerotic plaque progression. A Medline-based review of the literature was carried out. Tangier disease and human heterozygotes with ABCA1 mutations provide good evidence that ABCA1 is a major candidate influencing atherosclerosis. Animal and in vitro experiments suggest that ABCA1 not only mediates cholesterol and phospholipid efflux, but is also involved in the regulation of apoptosis and inflammation. The complex and beneficial interactions between apoA1 and ABCA1 seem to be pivotal for cholesterol efflux. The expression of the ABCA1 is tightly regulated. Furthermore the plaque microenvironment could potentially promote ABCA1 protein degradation thus compromising cholesterol efflux. PPAR-LXR-ABCA1 interactions are integral to cholesterol homeostasis and these nuclear receptors have proven anti-inflammatory and anti-matrix metalloproteinase activity. Therapeutic manipulation of the ABCA1 transporter is feasible using PPAR and LXR agonists. PPAR agonists like glitazones and ABCA1 protein stabilization could potentially modify the clinical progression of atherosclerotic lesions.Vascular Medicine 06/2005; 10(2):109-19. · 1.62 Impact Factor
- Nephrology Dialysis Transplantation 02/2003; 18(1):27-32. · 3.37 Impact Factor