STAT5 activators modulate acyl CoA oxidase (AOX) expression in adipocytes and STAT5A binds to the AOX promoter in vitro

Growth hormone (GH) diminishes adipose tissue mass in vivo and prolactin (PRL) can also modulate adipocyte metabolism. Both GH and PRL are potent activators of STAT5 and exert a variety of effects on adipocyte gene expression. In this study, we have demonstrated that GH and PRL increase the mRNA of acyl CoA oxidase in 3T3-L1 adipocytes. We also identified seven putative STAT elements in the murine AOX promoter. We observed that GH modulates protein binding to the majority of these promoter elements. However, GH induced very potent binding to -1841 to -1825 of the murine AOX promoter. EMSA supershift analysis revealed that this site was specifically bound by STAT5A, but not by STAT1 or STAT3. Taken together, these data strongly suggest that GH directly induces the expression of AOX in adipocytes through STAT5A binding to the -1841 to -1825 site within the AOX promoter. Our observations are consistent with other studies that demonstrate that STAT5 activators modulate fatty acid oxidation.     

Enhancer role of STAT5 in CD2 activation of IFN-gamma gene expression

IFN-gamma is an important immunoregulatory protein with tightly controlled expression in activated T and NK cells. Three potential STAT binding regions have been recognized within the IFN-gamma promoter: 1) an IL-12-mediated STAT4 binding site at -236 bp; 2) a newly identified IL-2-induced STAT5 binding element at -3.6 kb; and 3) CD2-mediated STAT1 and STAT4 binding to an intronic element in mucosal T cells. However, functional activation of these sites remains unclear. In this study we demonstrate CD2-mediated activation of the newly characterized -3.6-kb IFN-gamma STAT5 binding region. CD2 signaling of human PBMC results in activation of the -3.6-kb IFN-gamma promoter, whereas mutation of the -3.6-kb STAT5 site attenuates promoter activity. Functional activation is accompanied by STAT5A but little STAT5B nucleoprotein binding to the IFN-gamma STAT5 site, as determined by competition and supershift assays. STAT5 activation via CD2 occurs independent of IL-2. Western and FACS analysis shows increased phospho-STAT5 following CD2 signaling. AG490, a tyrosine kinase inhibitor affecting Jak proteins, inhibits CD2-mediated IFN-gamma mRNA expression, secretion, and nucleoprotein binding to the IFN-gamma STAT5 site in a dose-dependent fashion. This report is the first to describe CD2-mediated activation of STAT5 and supports STAT5 involvement in regulation of IFN-gamma expression.     

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.     

IL-4 modulates transcriptional control of the mannose receptor in mouse FSDC dendritic cells

The mannose receptor is a 175 kDa protein found on the surface of macrophages and dendritic cells whose functions include clearance of extracellular hydrolases, internalization of pathogens, and antigen capture. Receptor expression is closely linked to the functional state of these cells and is regulated by cytokines. Previous work has shown that treatment of macrophages and dendritic cells with interleukin-4 leads to increased mannose receptor expression. We have examined the mechanism of this IL-4-mediated up-regulation in the murine dendritic cell line FSDC. IL-4 increased mannose receptor activity, protein, and mRNA. The mannose receptor promoter was functional in FSDCs using transient transfection assays, and IL-4 treatment increased promoter activity 2.6-fold. The responsive region was localized to the proximal 228 bp. Electrophoretic mobility shift assays detected an IL-4-inducible protein that bound to the mannose receptor promoter at a site spanning the region between -147 and -108 bp. The sequence TTAC(N)4CACC (-135 and -124 bp) is similar to the IL-4 response region in the Fc receptor II. Mutation of the flanking TT and CC in this motif blocked IL-4 responsiveness and binding of the IL-4-induced mannose receptor binding protein. This protein does not appear to be STAT6 since neither an anti-STAT6 antibody nor a STAT6 consensus oligonucleotide altered factor binding.