Browsing by Author "Eleswarapu, Satyanarayana"

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    Growth hormone regulation of insulin-like growth factor-I gene expression may be mediated by multiple distal signal transducer and activator of transcription 5 binding sites
    Eleswarapu, Satyanarayana; Gu, Zhiliang; Jiang, Honglin (Endocrine Society, 2008-05)
    The transcription factor signal transducer and activator of transcription (STAT)-5 mediates GH stimulation of IGF-I gene expression in the liver. Previous studies suggested that STAT5 might exert this effect by binding to an IGF-I intron 2 region and a distal 5' -flanking region each containing two STAT5 binding sites. Here we report the identification of three additional chromosomal regions containing a total of five putative STAT5 binding sites that may mediate GH-induced STAT5 activation of IGF-I gene expression in the mouse liver. By comparing an 170-kb mouse genomic DNA containing the IGF-I gene with the corresponding human sequence, we identified 19 putative STAT5 binding sites that bear the consensus sequence of STAT5 binding site and are conserved across the two species. Chromatin immunoprecipitation assays indicated that five chromosomal regions containing a total of nine of the 19 putative STAT5 binding sites were bound by STAT5 in the mouse liver in response to GH administration and that these bindings preceded or coincided with GH-increased IGF-I gene transcription. Two of the five chromosomal regions correspond to those previously identified in other species, and the three new chromosomal regions that contain a total of five putative STAT5 binding sites are IGF-I intron 3 regions located at least 26 kb from the transcription start site. Gelshift assays confirmed the binding of the five new putative STAT5 binding sites as well as three of the four previously identified STAT5 binding sites to GH-activated STAT5 from the mouse liver. Cotransfection analyses indicated that, although each of the five chromosomal regions was able to mediate STAT5 activation of reporter gene expression, together they mediated greater STAT5 activation of reporter gene expression in response to GH. Overall, these results suggest that GH-induced STAT5 activation of IGF-I gene expression in the mouse liver might be collectively mediated by at least eight STAT5 binding sites located located in distal intronic and 5' -flanking regions of the IGF-I gene.
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    Growth Hormone Stimulates Transcription of the Fibroblast Growth Factor 21 Gene in the Liver through the Signal Transducer and Activator of Transcription 5
    Yu, Jie; Zhao, Lidan; Wang, Aihua; Eleswarapu, Satyanarayana; Ge, Xiaomei; Chen, Daiwen; Jiang, Honglin (Endocrine Society, 2012-02)
    Fibroblast growth factor 21 (FGF21) is a recently discovered metabolic regulator. Interestingly, FGF21 is also known to inhibit Janus kinase 2 (JAK2)-signal transducer and activator of transcription 5 (STAT5) signaling from the GH receptor in the liver, where FGF21 mRNA is predominantly expressed. In this study, we tested the hypothesis that FGF21 gene expression in the liver is controlled by GH through STAT5. We found that GH injection to cattle increased FGF21 mRNA expression in the liver. Mapped by a 5'-rapid amplification of cDNA ends assay, transcription of the FGF21 gene in the bovine liver was mainly initiated from a nucleotide 24 bp downstream of a TATA box. The bovine FGF21 promoter contains three putative STAT5-binding sites. EMSA confirmed the ability of them to bind to liver STAT5 protein from GH-injected cattle. Chromatin immunoprecipitation assays demonstrated that GH administration increased the binding of STAT5 to the FGF21 promoter in the liver. Cotransfection analyses showed that GH induced reporter gene expression from the FGF21 promoter in a STAT5-dependent manner. GH also stimulated FGF21 mRNA expression in cultured mouse hepatocytes. These data together indicate that GH directly stimulates FGF21 gene transcription in the liver, at least in part, through STAT5. This finding, together with the fact that FGF21 inhibits GH-induced JAK2-STAT5 signaling in the liver, suggests a novel negative feedback loop that prevents excessive JAK2-STAT5 signaling from the GH receptor in the liver. (Endocrinology 153: 750-758, 2012)
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    Growth Hormone-Activated STAT5 May Indirectly Stimulate IGF-I Gene Transcription through HNF-3 gamma
    Eleswarapu, Satyanarayana; Ge, Xiaomei; Wang, Ying; Yu, Jie; Jiang, Honglin (Endocrine Society, 2009-12)
    IGF-I is abundantly expressed in the liver under the stimulation of GH. We showed previously that expression of hepatocyte nuclear factor (HNF)-3 gamma, a liver-enriched transcription factor, was strongly stimulated by GH in bovine liver. In this study, we determined whether GH-increased HNF-3 gamma might contribute to GH stimulation of IGF-I gene expression in bovine liver and the underlying mechanism. A sequence analysis of the bovine IGF-I promoter revealed three putative HNF-3 binding sites, which all appear to be conserved in mammals. Chromatin immunoprecipitation assays showed that GH injection increased binding of HNF-3 gamma to the IGF-I promoter in bovine liver. Gel-shift assays indicated that one of the three putative HNF-3 binding sites, HNF-3 binding site 1, bound to the HNF-3 gamma protein from bovine liver with high affinity. Cotransfection analyses demonstrated that this HNF-3 binding site was essential for the transcriptional response of the IGF-I promoter to HNF-3 gamma in CHO cells and to GH in primary mouse hepatocytes. Using similar approaches, we found that GH increased binding of the signal transducer and activator of transcription 5 (STAT5) to the HNF-3 gamma promoter in bovine liver, that this binding occurred at a conserved STAT5 binding site, and that this STAT5 binding site was necessary for the HNF-3 gamma promoter to respond to GH. Taken together, these results suggest that in addition to direct action, GH-activated STAT5 may also indirectly stimulate IGF-I gene transcription in the liver by directly enhancing the expression of the HNF-3 gamma gene. (Molecular Endocrinology 23: 2026-2037, 2009)
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    Mechanisms of Growth Hormone Regulation of Insulin-Like Growth Factor-I Gene Expression in Liver
    Eleswarapu, Satyanarayana (Virginia Tech, 2009-02-02)
    The overall objective of this research was to understand the mechanims by which growth hormone (GH) regulates insulin-like growth factor-I (IGF-I) gene expression in liver. Previous studies have suggested that GH regulation of IGF-I gene expression in liver is mediated by binding of the transcription factor signal transducer and activator of transcription (STAT) 5 to four binding sites located distantly from the IGF-I promoter. The first specific objective of this research was to determine whether additional STAT5 binding sites were involved in GH stimulation of IGF-I gene expression in liver. Sequence analysis of 170 kb of mouse genomic DNA revealed nineteen consensus STAT5 binding sequences corresponding to fourteen ~200 bp chromosomal regions that were conserved in the corresponding human DNA sequence. Eight of these chromosomal regions were able to mediate STAT5 activation of reporter gene expression in cotransfection experiments. Two of these chromosomal regions corresponded to those previously identified. Gel-shift assays indicated that the eight new STAT5 binding sites and three of the four previously identified STAT5 binding sites could bind GH-activated STAT5 from mouse liver. Together, these results suggest that GH stimulation of IGF-I gene transcription in the mouse liver may be mediated by at least eleven STAT5 binding sites located distantly from the IGF-I promoter. In a previous study, I found that liver expression of liver-enriched transcription factor hepatocyte nuclear factor 3γ (HNF-3γ) was increased by GH in cattle. Therefore, the second specific objective of this research was to determine how GH stimulates HNF-3γ gene expression and whether the increased HNF-3γ mediates GH stimulation of IGF-I gene expression in bovine liver. Sequence analysis of the bovine HNF-3γ promoter revealed the presence of two putative binding sites for STAT5. The proximal putative STAT5 binding site appears to be conserved in other mammals. Chromatin immunoprecipitation (ChIP) assays demonstrated that GH increased the binding of STAT5 to the HNF-3γ promoter in bovine liver and that this binding was associated with increased HNF-3γ expression. Gel-shift assays demonstrated that the proximal STAT5 binding site in the HNF-3γ promoter could bind GH-activated STAT5 from bovine liver. Cotransfection analyses showed that the proximal STAT5 binding site was necessary for the HNF-3γ promoter to be activated by GH. The promoter of the bovine IGF-I gene contains three putative HNF-3 binding sites that seem to be evolutionarily conserved. ChIP assays indicated that GH stimulated the binding of HNF-3γ to the IGF-I promoter in bovine liver. Gel-shift assays showed that one of the putative HNF-3 binding sites could bind HNF-3γ protein from bovine liver. Co-transfection analyses demonstrated that this HNF-3 binding site was necessary for HNF-3γ activation of reporter gene expression from the IGF-I promoter. In summary, the results of this dissertation research suggest that GH-activated STAT5 directly stimulates IGF-I gene transcription in liver by binding to at least eleven distantly located STAT5 binding sites in the IGF-I locus and indirectly stimulates IGF-I gene transcription by enhancing HNF-3γ gene expression in the liver.