Proteolytic activation of SREBPs during adipocyte differentiation

A member of sterol regulatory element-binding protein (SREBP) family, SREBP-1, is a key regulator of adipocyte differentiation. Expression of the SREBP-1 gene is induced during adipocyte differentiation, but proteolytic activation of the synthesized precursor form of SREBP-1 has not been well analyzed. The proteolytic processing of SREBPs is severely suppressed in sterol loaded culture cells. Here we report that a splicing isoform, SREBP-1a, is predominantly expressed in 3T3-L1 preadipocytes and adipocytes, and that the nuclear active form of SREBP-1 protein increases in adipocyte differentiation. We further show that the amount of nuclear SREBP-2 protein also increases despite no increase in SREBP-2 mRNA, suggesting that proteolytic cleavage of SREBPs is induced in lipid loaded adipocytes. Northern blot analyses reveal that mRNA levels for SREBP cleavage-activating protein (SCAP), Site-1 protease (S1P), and Site-2 protease (S2P), which participate in the proteolytic processing of SREBPs, are relatively unaffected in adipogenesis. These results demonstrate that SREBP-2 appears to promote adipocyte differentiation as well as SREBP-1 and that the proteolytic activation of SREBPs may be induced by an as-yet unidentified mechanism in lipid loaded adipocytes.     

Piperine Induces Hepatic Low-Density Lipoprotein Receptor Expression through Proteolytic Activation of Sterol Regulatory Element-Binding Proteins

Elevated plasma low-density lipoprotein (LDL) cholesterol is considered as a risk factor for atherosclerosis. Because the hepatic LDL receptor (LDLR) uptakes plasma lipoproteins and lowers plasma LDL cholesterol, the activation of LDLR is a promising drug target for atherosclerosis. In the present study, we identified the naturally occurring alkaloid piperine, as an inducer of LDLR gene expression by screening the effectors of human LDLR promoter. The treatment of HepG2 cells with piperine increased LDLR expression at mRNA and protein levels and stimulated LDL uptake. Subsequent luciferase reporter gene assays revealed that the mutation of sterol regulatory element-binding protein (SREBP)-binding element abolished the piperine-mediated induction of LDLR promoter activity. Further, piperine treatments increased mRNA levels of several SREBP targets and mature forms of SREBPs. However, the piperine-mediated induction of the mature forms of SREBPs was not observed in SRD–15 cells, which lack insulin-induced gene–1 (Insig–1) and Insig–2. Finally, the knockdown of SREBPs completely abolished the piperine-meditated induction of LDLR gene expression in HepG2 cells, indicating that piperine stimulates the proteolytic activation of SREBP and subsequent induction of LDLR expression and activity.     

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.     

Sterol regulatory element-binding proteins induce an entire pathway of cholesterol synthesis

To evaluate the effects of sterol regulatory element-binding proteins (SREBPs) on the expression of the individual enzymes in the cholesterol synthetic pathway, we examined expression of these genes in the livers from wild-type and transgenic mice overexpressing nuclear SREBP-1a or -2. As estimated by a Northern blot analysis, overexpression of nuclear SREBP-1a or -2 caused marked increases in mRNA levels of the whole battery of cholesterogenic genes. This SREBP activation covers not only rate-limiting enzymes such as HMG CoA synthase and reductase that have been well established as SREBP targets, but also all the enzyme genes in the cholesterol synthetic pathway tested here. The activated genes include mevalonate kinase, mevalonate pyrophosphate decarboxylase, isopentenyl phosphate isomerase, geranylgeranyl pyrophosphate synthase, farnesyl pyrophosphate synthase, squalene synthase, squalene epoxidase, lanosterol synthase, lanosterol demethylase, and 7-dehydro-cholesterol reductase. These results demonstrate that SREBPs activate every step of cholesterol synthetic pathway, contributing to an efficient cholesterol synthesis.