|
|
GH Inhibition of IGF-I STAT5b Expression« Back to Volume 23, Issue 3, November 2007 - Table of Contents Ligand binding of the growth hormone (GH) receptor activates, via the Jak2 tyrosine kinase, the Stat transcription factors and the MAP kinase and PI3 kinase/Akt pathways. As is well known to the readers of GGH, GH-stimulated transcription of the insulin-like growth factor (IGF)-I gene requires the Jak2/Stat5b mechanism. However, GH signaling also leads to transcriptional repression of a cohort of genes, including the IGF binding protein (IGFBP)-1. Ono et al sought to elucidate the mechanism of this facet of GH action. Hypophysectomized Sprague-Dawley rats were given a single systemic pulse of GH, and hepatic RNA was isolated 30, 60 or 120 minutes afterwards. By both microarray and RT-PCR methods, GH acutely increased the mRNA levels of IGF-I and Socs-2 while decreasing that of IGFBP-1. GH also acutely induced the nuclear accumulation of phosphorylated Stat5b. Adenoviral-mediated delivery of a constitutively active Stat5b construct to livers of GH-deficient rats similarly increased IGF-I and Socs-2 expression while decreasing IGFBP-1. To further examine the transcriptional regulation of IGFBP-1, Cos-7 cells were transiently transfected with a rat IGFBP-1 promoter-luciferase reporter construct as well as an expression vector for mouse GH receptor. Cotransfection with wild-type or constitutively activated FoxO1, a transcription factor important for IGFBP-1 expression, stimulated promoter activity. GH treatment altered neither IGFBP-1 promoter activity nor the abundance of the FoxO1 proteins. In contrast, when wild type Stat5b was also co-transfected, GH treatment led to a 35%-50% reduction of IGFBP-1 promoter activity with either type of FoxO1; GH stimulated posphorylation of the wild-type but not constitutively activated FoxO1, and abundance of the FoxO1 proteins again were not altered. Thus, GH-induced IGFBP-1 repression is mediated by Stat5b and not Akt (the constitutively activated FoxO1 is Akt resistant.) Because IGFBP-1 expression is also repressed by insulin, which acts via Akt inhibition of FoxO1, the authors sought to further examine the interactions between Akt, Stat5b and FoxO1. A tamoxifen-inducible Akt fusion protein, iAkt, repressed IGFBP-1 promoter activity in the presence of wild type, but not a constitutively activated, FoxO1; the former form of FoxO1 was phosphorylated by Akt while the latter cannot be. In contrast, a constitutively activated Stat5b did not phosphorylate FoxO1. Further experiments were performed to mechanistically examine Stat5b inhibition of FoxO1. Using a luciferase reporter construct driven by a minimal promoter containing 3 copies of IRSA (one of the tandem FoxO1 binding sequences found in the IGFBP-1 promoter), the FoxO1 binding site was shown sufficient for GH and Stat5b inhibition of FoxO1-stimulated gene transcription. To examine the possibility of reciprocal inhibition, a luciferase reporter construct driven by the Stat5b-dependent HS7 response element (found in the IGF-I gene) was examined. It increased activity in response to GH in the absence of FoxO1, and increased further still when wild type or constitutively activated FoxO1 were cotransfected, even though there were no FoxO1 binding sites in the HS7-promoter sequences. Thus, competition for transcriptional co-factors does not seem to be the mechanism of Stat5b’s inhibition of FoxO1 activity. A dominant-negative Stat5b was shown to lose the ability to mediate GH inhibition of IGFBP-1 promoter activity, in both co-transfected Cos-7 cells in vitro and in GH-treated hypophysectomized rats in vivo. Co-transfected Cos-7 cells further showed that GH induced nuclear accumulation of Stat5b, but neither nuclear levels of FoxO1 protein nor its DNA-binding ability were reduced by activated Stat5b. Direct protein-protein interactions between FoxO1 and Stat5b from Cos-7 nuclear extracts were not detected by co-immunoprecipitation assays or avidin-biotin complex DNA binding assay. Finally, the authors returned to their hepatic microarray results from GH-stimulated hypophysectomized rats. They compared the list of GH-repressed genes to genes repressed by adenovirally introduced constitutively activated Stat5b. Eighty-nine gene transcripts were similarly reduced by both mechanisms. In silico search for FoxO1 binding sites within phylogenetically conserved (rat and human) regions of these genes revealed 19 hits, or 21% of the repressed genes. Of 322 randomly selected genes 19% were not regulated by GH or Stat5b and were also found to contain Fox01 binding sites. Thus, FoxO1 inhibition accounts for only a subset of transcriptional repression by GH/Stat5b. Ono M, Chia DJ, Merino-Martinez R, Flores-Morales A, Unterman TG, Rotwein P. Signal transducer and activator of transcription (stat) 5b-mediated inhibition of insulin-like growth factor binding protein-1 gene transcription: a mechanism for repression of gene expression by growth hormone. Mol Endocrinol. 2007;21:1443-57. Editor’s CommentThrough a well constructed series of experiments, Ono et al. clearly showed that GH inhibits IGFBP-1 expression via activated Stat5b and FoxO1. However, the exact mechanism of FoxO1 inhibition by Stat5b remains elusive; FoxO1 protein degradation, nuclear exclusion and impaired DNA binding ability were all ruled out, as was direct protein-protein interaction between Stat5b and FoxO1. Nonetheless, this paper expands our thinking along two lines. First, GH, via activated Stat5b, not only induces gene expression (eg. IGF-I), but also represses transcription of other genes, such as IGFBP-1. Thus, the genetic response to GH/Stat5b signaling is a richer compilation of coordinated alterations than previously appreciated. Second, the mechanism whereby IGFBP-1 expression is repressed by GH is clearly distinct from that of insulin (activated Akt phosphorylating FoxO1, thereby sequestering it out of the nucleus and impairing its ability to transcribe IGFBP-11 ). Although we are used to thinking of GH as counter-regulatory to insulin, in certain circumstances, like IGFBP-1 expression as shown here, the two hormones can act synergistically because they effect the same molecular change through separate pathways. Adda Grimberg, MD Reference - (linked to
|
Volume 24 Number 1
May 2008
www.GGHjournal.com
ISSN 1932-9032
)