Abstract
Genistein has been implicated for its anti-atherogenic effects. We investigated the molecular mechanisms behind the impact of genistein on expression of LDLR, the receptor for LDL-cholesterol, and related signaling pathways in HepG2 cells. Genistein increased mRNA and protein levels of LDLR in a time-dependent manner. In order to find out the effects of genistein on the transcriptional levels, a luciferase reporter construct containing LDLR promoter (pLDLR-luc) was constructed and examined for its response to genistein. Genistein increased the reporter activity but failed to increase transcriptional activity when sterol-regulatory element (SRE) in the LDLR promoter was deleted. Genistein increased nuclear translocation of SREBP-2 and DNA binding activity of SREBP-2 to LDLR promoter by chromatin immunoprecipitation assay (CHIP). Pre-treatment of 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF), serine protease inhibitor, prevented the effects of genistein while brefeldin A causing the fusion of the endoplasmic reticulum (ER) and the Golgi apparatus did not, suggesting that genistein may have an effect on SREBP-2 trafficking from the ER to the Golgi apparatus. Insig-1 protein levels were not changed by genistein. Among mitogen-activated protein kinases (MAPK), genistein phosphorylated JNK but not p38 and ERK signals. JNK inhibitor (SP600126) abolished genistein-stimulated levels of LDLR and nuclear SREBP-2. To minimize the effects of c-Jun, a transcription factor activated by JNK signals, a truncated LDLR luciferase construct that contained SRE but lacked the c-jun putative binding site was constructed. Genistein still was able to boost the transcriptional activity of the truncated LDLR construct. All the genistein effects were abolished by the addition of cholesterol. In conclusion, genistein has the anti-atherogenic effects by activating JNK signals and SREBP-2 processing, which is followed by up-regulation of LDLR. However, the beneficial effects of genistein could be affected by the amount of cellular cholesterols.
Highlights
Hypercholesterolemia is a strong risk factor for the development of atherosclerosis, a hallmark of cardiovascular disorders [1]
When Low Density Lipoprotein Receptor (LDLR) promoter activity was investigated using a luciferase construct containing the proximal region of the LDLR gene, genistein increased transcriptional activity of the construct (Figure 2A)
In a previous animal study, the anti-atherogenic effects of genistein appeared to mediate LDLR based on a lack of the effects in LDLR knock-out mice [19], but the exact molecular mechanisms behind the actions of genistein were not investigated
Summary
Hypercholesterolemia is a strong risk factor for the development of atherosclerosis, a hallmark of cardiovascular disorders [1]. Familial hypercholesterolemia suggests that hepatic expression of LDLR, a receptor for LDL-cholesterol, is crucial for the regulation of blood cholesterol levels [2,3]. Extensive studies indicate that statins improve lipid profiles by inhibiting the activity of HMGCR, a rate-limiting enzyme of cholesterol synthesis, thereby increasing expression of LDLR [4]. Statins only reduce cardiovascular risk by about 20 to 40% [5]. Diet alone successfully lowers blood LDL-cholesterol to a degree comparable to statin treatment [6]. Combination therapy using statins and a dietary regime may further reduce blood cholesterol levels [5]. The neutral antagonist treatment of type 1 cannabinoid receptor may improve blood lipid profile [7,8]
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