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.     

STAT1 pathway is involved in activation of caprine arthritis-encephalitis virus long terminal repeat in monocytes.

The caprine arthritis-encephalitis virus (CAEV) long terminal repeat (LTR) is activated by gamma interferon (IFN-gamma) in promonocytic cells. We have previously shown that a 70-bp element is necessary and sufficient for the response of the CAEV LTR to this cytokine. At the 5' end, this 70-bp IFN-gamma response element contains sequence similarity to the gamma activated site (GAS). Here we demonstrate that the putative GAS element in the CAEV LTR binds specifically to a cellular factor induced by IFN-gamma in promonocytic cells. Substitution mutations in this consensus sequence eliminate binding of the inducible factor. The GAS element from the 70-bp motif is sufficient to confer responsiveness to IFN-gamma using a heterologous minimal promoter. Consistent with the binding data, the same mutations in the GAS element eliminate responsiveness to IFN-gamma in the context of both a functional CAEV LTR and a heterologous promoter. The cellular factor that binds to the GAS element is present from 5 min to 14 h after stimulation with IFN-gamma. Binding of the nuclear factor to the GAS element in the CAEV LTR is inhibited by antibody directed against STAT1 (p91/84). Thus, the GAS sequence in the CAEV LTR is essential for the response to IFN-gamma and a STAT1-like factor binds to this site. The STAT-1 signaling pathway provides at least one mechanism for activation of the CAEV LTR by IFN-gamma in monocytes. These data are the first demonstration of a role for a STAT family member in the regulation of a viral promoter.     

Sequence determinants for the tandem recognition of UGU and CUG rich RNA elements by the two N--terminal RRMs of CELF1

CUGBP, Elav-like family member 1 (CELF1) is an RNA binding protein with important roles in the regulation of splicing, mRNA decay and translation. CELF1 contains three RNA recognition motifs (RRMs). We used gel retardation, gel filtration, isothermal titration calorimetry and NMR titration studies to investigate the recognition of RNA by the first two RRMs of CELF1. NMR shows that RRM1 is promiscuous in binding to both UGU and CUG repeat sequences with comparable chemical shift perturbations. In contrast, RRM2 shows greater selectivity for UGUU rather than CUG motifs. A construct (T187) containing both binding domains (RRM1 and RRM2) was systematically studied for interaction with tandem UGU RNA binding sites with different length linker sequences UGU(U)_xUGU where x = 1–7. A single U spacer results in interactions only with RRM1, demonstrating both steric constraints in accommodating both RRMs simultaneously at adjacent sites, and also subtle differences in binding affinities between RRMs. However, high affinity co-operative binding (K_d ~ 0.4 µM) is evident for RNA sequences with x = 2–4, but longer spacers (x ≥ 5) lead to a 10-fold reduction in affinity. Our analysis rationalizes the high affinity interaction of T187 with the 11mer GRE consensus regulatory sequence UGUUUGUUUGU and has significant consequences for the prediction of CELF1 binding sites.     

The reductase NCB5OR is responsive to the redox status in beta-cells and is not involved in the ER stress response

The novel reductase NCB5OR (NADPH cytochrome b5 oxidoreductase) resides in the ER (endoplasmic reticulum) and may protect cells against ER stress. Levels of BiP (immunoglobulin heavy-chain-binding protein), CHOP (CCAAT/enhancer-binding protein homologous protein) and XBP-1 (X-box-binding protein-1) did not differ in WT (wild-type) and KO (Ncb5or-null) tissues or MEFs (mouse embryonic fibroblasts), and XBP-1 remained unspliced. MEFs treated with inducers of ER stress demonstrated no change in Ncb5or expression and expression of ER-stress-induced genes was not enhanced. Induction of ER stress in beta-cell lines did not change Ncb5or expression or promoter activity. Transfection with Ncb5or-specific siRNA (small interfering RNA) yielded similar results. Microarray analysis of mRNA from islets and liver of WT and KO animals revealed no significant changes in ER-stress-response genes. Induction of oxidative stress in betaTC3 cells did not alter Ncb5or mRNA levels or promoter activity. However, KO islets were more sensitive to streptozotocin when compared with WT islets. MEFs incubated with nitric oxide donors showed no difference in cell viability or levels of nitrite produced. No significant differences in mRNA expression of antioxidant enzymes were observed when comparing WT and KO tissues; however, microarray analysis of islets indicated slightly enhanced expression of some antioxidant enzymes in the KO islets. Short-term tBHQ (t-butylhydroquinone) treatment increased Ncb5or promoter activity, although longer incubation times yielded a dose-dependent decrease in activity. This response appears to be due to a consensus ARE (antioxidant-response element) present in the Ncb5or promoter. In summary, NCB5OR does not appear to be involved in ER stress, although it may be involved in maintaining or regulating the redox status in beta-cells.