Transcriptional activities of nuclear SREBP-1a, -1c,and -2 to different target promoters of lipogenic and cholesterogenic genes

Recent studies on the in vivo roles of the sterol regulatory element binding protein (SREBP) family indicate that SREBP-2 is more specific to cholesterogenic gene expression whereas SREBP-1 targets lipogenic genes. To define the molecular mechanism involved in this differential regulation, luciferase-reporter gene assays were performed in HepG2 cells to compare the transactivities of nuclear SREBP-1a, -1c, and -2 on a battery of SREBP-target promoters containing sterol regulatory element (SRE), SRE-like, or E-box sequences. The results show first that cholesterogenic genes containing classic SREs in their promoters are strongly and efficiently activated by both SREBP-1a and SREBP-2, but not by SREBP-1c. Second, an E-box containing reporter gene is much less efficiently activated by SREBP-1a and -1c, and SREBP-2 was inactive in spite of its ability to bind to the E-box. Third, promoters of lipogenic enzymes containing variations of SRE (SRE-like sequences) are strongly activated by SREBP-1a, and only modestly and equally by both SREBP-1c and -2. Finally, substitution of the unique tyrosine residue within the basic helix-loop-helix (bHLH) portion of nuclear SREBPs with arginine, the conserved residue found in all other bHLH proteins, abolishes the transactivity of all SREBPs for SRE, and conversely results in markedly increased activity of SREBP-1 but not activity of SREBP-2 for E-boxes. These data demonstrate the different specificity and affinity of nuclear SREBP-1 and -2 for different target DNAs, explaining a part of the mechanism behind the differential in vivo regulation of cholesterogenic and lipogenic enzymes by SREBP-1 and -2, respectively.     

葡萄糖转运蛋白GLUT4表达的调节机制

胰岛素反应性的葡萄糖转运蛋白4（glucose transporter 4,GLUT4）在葡萄糖的摄取和代谢过程中发挥着重要作用。GLUT4蛋白表达水平直接影响机体葡萄糖的利用。肌细胞增强因子2（myocyte enhancer factor 2,MEF2）、过氧化物酶体增殖物激活受体（peroxisome proliferator activated receptors,PPARs）、CCAAT增强子结合蛋白α（CCAAT enhancer binding protein α,C/EBP-α）、固醇类反应元件结合蛋白1c(sterol response element binding protein 1c,SREBP-1c）等转录因子可以上调或下调Glut4基因转录。激素、代谢以及一些病理状态可以通过改变转录因子的量或活性影响Glut4。本文综述了在Glut4基因表达中发挥作用的转录因子,以及在特定的生理或病生理状态下Glut4基因表达调控的机制。     

Induction of LPL gene expression by sterols is mediated by a sterol regulatory element and is independent of the presence of multiple E boxes

Overexpression of the adipocyte differentiation and determination factor-1 (ADD-1) or sterol regulatory element binding protein-1 (SREBP-1) induces the expression of numerous genes involved in lipid metabolism, including lipoprotein lipase (LPL). Therefore, we investigated whether LPL gene expression is controlled by changes in cellular cholesterol concentration and determined the molecular pathways involved. Cholesterol depletion of culture medium resulted in a significant induction of LPL mRNA in the 3T3-L1 preadipocyte cell line, whereas addition of cholesterol reduced LPL mRNA expression to basal levels. Similar to the expression of the endogenous LPL gene, the activity of the human LPL gene promoter was enhanced by cholesterol depletion in transient transfection assays, whereas addition of cholesterol caused a reversal of its induction. The effect of cholesterol depletion upon the human LPL gene promoter was mimicked by cotransfection of expression constructs encoding the nuclear form of SREBP-1a, -1c (also called ADD-1) and SREBP-2. Bioinformatic analysis demonstrated the presence of 3 potential sterol regulatory elements (SRE) and 3 ADD-1 binding sequences (ABS), also known as E-box motifs. Using a combination of in vitro protein-DNA binding assays and transient transfection assays of reporter constructs containing mutations in each individual site, a sequence element, termed LPL-SRE2 (SRE2), was shown to be the principal site conferring sterol responsiveness upon the LPL promoter. These data furthermore underscore the importance of SRE sites relative to E-boxes in the regulation of LPL gene expression by sterols and demonstrate that sterols contribute to the control of triglyceride metabolism via binding of SREBP to the LPL regulatory sequences.     

The E-box like sterol regulatory element mediates the suppression of human Delta-6 desaturase gene by highly unsaturated fatty acids

Delta-6 Desaturase (D6D) catalyzes the first step of the synthesis of highly unsaturated fatty acids (HUFA) that play pivotal roles in many biological functions. The D6D expression is under feedback regulation by dietary HUFA. We co-transfected D6D promoter-reporter constructs to HepG2 cells with an expression vector of nuclear form sterol regulatory element binding protein-1c (SREBP-1c). A 90-bp region of the D6D promoter was required for the activation by SREBP-1c as well as for the suppression of the promoter activity by HUFA. The region contained two candidates of sterol regulatory element (SRE). Mutation analysis identified E-box like SRE (SRE-2) as essential for both SREBP-1c activation and HUFA suppression. SRE-2 has a core sequence of CAGCAG, and is also conserved in stearoyl CoA desatruases. Because HUFA are primarily incorporated into phospholipids (PL), our results suggest that the primary role of SREBP-1c in liver is the regulation of fatty acid supply for PL rather than for triglycerides.     

Clusterin: a protective mediator for ischemic cardiomyocytes?

We examined the relationship between clusterin and activated complement in human heart infarction and evaluated the effect of this protein on ischemic rat neonatal cardiomyoblasts (H9c2) and isolated adult ventricular rat cardiomyocytes as in vitro models of acute myocardial infarction. Clusterin protects cells by inhibiting complement and colocalizes with complement on jeopardized human cardiomyocytes after infarction. The distribution of clusterin and complement factor C3d was evaluated in the infarcted human heart. We also analyzed the protein expression of clusterin in ischemic H9c2 cells. The binding of endogenous and purified human clusterin on H9c2 cells was analyzed by flow cytometry. Furthermore, the effect of clusterin on the viability of ischemically challenged H9c2 cells and isolated adult ventricular rat cardiomyocytes was analyzed. In human myocardial infarcts, clusterin was found on scattered, morphologically viable cardiomyocytes within the infarcted area that were negative for complement. In H9c2 cells, clusterin was rapidly expressed after ischemia. Its expression was reduced after reperfusion. Clusterin bound to single annexin V-positive or annexin V and propidium iodide-positive H9c2 cells. Clusterin inhibited ischemia-induced death in H9c2 cells as well as in isolated adult ventricular rat cardiomyocytes in the absence of complement. We conclude that ischemia induces the upregulation of clusterin in ischemically challenged, but viable, cardiomyocytes. Our data suggest that clusterin protects cardiomyocytes against ischemic cell death via a complement-independent pathway.