Mechanisms of Growth Hormone Regulation of Insulin-Like Growth Factor-I Gene Expression in Liver
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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 3Î³ (HNF-3Î³) was increased by GH in cattle. Therefore, the second specific objective of this research was to determine how GH stimulates HNF-3Î³ gene expression and whether the increased HNF-3Î³ mediates GH stimulation of IGF-I gene expression in bovine liver. Sequence analysis of the bovine HNF-3Î³ 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-3Î³ promoter in bovine liver and that this binding was associated with increased HNF-3Î³ expression. Gel-shift assays demonstrated that the proximal STAT5 binding site in the HNF-3Î³ promoter could bind GH-activated STAT5 from bovine liver. Cotransfection analyses showed that the proximal STAT5 binding site was necessary for the HNF-3Î³ 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-3Î³ to the IGF-I promoter in bovine liver. Gel-shift assays showed that one of the putative HNF-3 binding sites could bind HNF-3Î³ protein from bovine liver. Co-transfection analyses demonstrated that this HNF-3 binding site was necessary for HNF-3Î³ 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-3Î³ gene expression in the liver.
- Doctoral Dissertations