No abstract

We report the cDNA cloning of SREBP-2, the second member of a family of basic-helix4oop-helix4leucine zipper (bHLH-Zip) transcription factors that recognize sterol regulatory element 1 (SRE-1). SRE-1, a conditional enhancer in the promoters for the low density lipoprotein receptor and 3-hydroxy-3-methylglutaryl-coenzyme A synthase genes, increases transcription in the absence of sterols and is inactivated when sterols accumulate. Human SREBP-2 contains 1141 amino acids and is 47% identical to human SREBP-la, the first recognized member ofthis family. The resemblance includes an acidic NH2 terminus, a highly conserved bHLH-Zip motif (71% identical), and an unusually long extension of 740 amino acids on the COOH-terminal side of the bHLH-Zip region. SREBP-2 possesses one feature lacking in SREBP-la-namely, a glutamine-rich region (27% glutamine over 121 residues). In vitro SREBP-2 bound SRE-1 with the same specificity as SREBP-la.In vivo it mimicked SREBP-la in activating transcription of reporter genes containing SRE-1. As with SREBP-la, activation by SREBP-2 occurred in the absence and presence of sterols, abolishing regulation. Cotransfection oflow amounts of pSREBP-la and pSREBP-2 into human embryonic kidney 293 cells stimulated transcription of promoters containing SRE-1 in an additive fashion. At high levels transcription reached a maximum, and the effects were no longer additive. The reason for the existence of two SREBPs and the mechanism by which they are regulated by sterols remain to be determined.A 10-base-pair (bp) element in the 5' flanking region confers sterol sensitivity upon the genes encoding the low density lipoprotein (LDL) receptor and 3-hydroxy-3-methylglutarylcoenzyme A (HMG-CoA) synthase. This element, sterol regulatory element 1 (SRE-1), enhances transcription when cells are depleted of sterols, and loses its activity when sterols accumulate (1, 2). This feedback mechanism allows cells to satisfy their cholesterol requirement from receptormediated uptake ofplasma lipoproteins and from endogenous synthesis, while preventing sterol overaccumulation during periods of reduced demand (3).In recent studies a group of SRE-1-binding proteins (SREBPs) were isolated by DNA affinity chromatography from nuclear extracts of human HeLa cells (2, 4). Upon SDS/ polyacrylamide gel electrophoresis, the proteins clustered in the range 59-68 kDa. The specificity of DNA binding was confirmed by the demonstration that binding correlated with transcriptional activity in a series of 16 point mutants in the SRE-1 and surrounding sequences. Each of the proteins bound to the 7 point mutants that were transcribed in vivo, but they failed to bind to 9 point mutants that were not transcribed (2, 4).The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.Sequences of six peptides were obtained from a mixed preparation of SREBPs, and a cDNA that encoded a protei...

There exist two distinct isozymes of prostaglandinendoperoxide synthase (PES). PES-2 mRNA is synergistically induced by lipopolysaccharide (LPS) and 12-Otetradecanoylphorbol-13-acetate (TPA) in bovine arterial endothelial cells. On the other hand, PES-1 mRNA is constitutively expressed under these conditions. Therefore, the promoter activities of the human genes for PES-1 and -2 in bovine arterial endothelial cells were examined. The 5-flanking region of the human PES-2 gene (nucleotides ؊327 to ؉59) showed promoter activity inducible by LPS and TPA using transient transfection analysis, whereas that of the PES-1 gene (nucleotides ؊1010 to ؉69) showed constitutive promoter activity. Destruction of both consensus sequences for the nuclear factor responsible for the interleukin-6 expression (NF-IL6) site (nucleotides ؊132 to ؊124) and the cyclic AMP response element (CRE) (nucleotides ؊59 to ؊53) of the human PES-2 gene markedly reduced the promoter activity (25%) of the PES-2 gene after combined treatment with LPS and TPA, although single destruction of the NF-IL6 site or the CRE slightly reduced the promoter activity (60 or 90%, respectively). Moreover, cotransfection experiments showed that a trans-acting factor, CCAAT enhancer binding protein ␦ (C/EBP␦), which binds to both the NF-IL6 site and the CRE, increased the promoter activity of the PES-2 gene mainly through the CRE. C/EBP␦ mRNA was rapidly induced by LPS. Collectively, these results suggest that transcription of the PES-2 gene in vascular endothelial cells is regulated through combination of the NF-IL6 site and the CRE and that C/EBP␦ functions as one of the trans-acting factors.

Dietary fish oil containing 3 highly unsaturated fatty acids has cardioprotective and anti-inflammatory effects. Prostaglandins (PGs) and thromboxanes are produced in vivo both from the 6 fatty acid arachidonic acid (AA) and the 3 fatty acid eicosapentaenoic acid (EPA). Certain beneficial effects of fish oil

Cell stimulation causes diacylglycerol kinase (DGK) to convert the second messenger diacylglycerol into phosphatidate, thus initiating the resynthesis of phosphatidylinositols and attenuating protein kinase C activity. Of the DGK isoforms so far reported, only porcine DGK from lymphocytes has been characterized in detail. Here we report the isolation and sequencing of complementary DNA clones that together cover the entire region encoding porcine DGK (relative molecular mass 80,000 (80K)). The deduced primary structure of this DGK contains the putative ATP-binding sites, two cysteine-rich zinc finger-like sequences similar to those found in protein kinase C, and two E-F hand motifs, typical of Ca2(+)-binding proteins like calmodulin. Indeed, we find that the activity of this DGK isoform is enhanced by micromolar concentrations of Ca2+ in the presence of deoxycholate or sphingosine. These properties of 80K DGK indicate that its action is probably linked with both of the second messengers diacylglycerol and inositol 1,4,5-trisphosphate.