Insulin-like growth factors (IGFs) are important regulators of cell growth and differentiation in many tissues. IGFs circulate in association with specific binding proteins (IGFBPs). Studies in this and other laboratories indicate that IGFBP-1 is a major short-term modulator of IGF bioavailability and that hepatic production of IGFBP-1 is rapidly regulated by glucocorticoids and insulin at the level of gene transcription. Changes in IGFBP-1 expression and IGF bioavailability may play an important role in the pathogenesis of complications of both hypo- and hyperinsulinemic states. Moreover, studies to date indicate that IGFBP-1 provides in important model system for understanding basic mechanisms by which insulin and glucocorticoids interact to regulate hepatic gene transcription. During an NIH FIRST Award, we identified and purified rat IGFBP-1, identified and utilized in vivo and cell culture model systems to examine the regulation of IGFBP-1, cloned the IGFBP-1 gene and its 5' promoter region and identified contiguous glucocorticoid (GRE) and insulin (IRS) response sequences in the proximal IGFBP-1 promoter. Competitive binding and supershift studies show that the major protein binding to this sequence is hepatocyte nuclear factor-3 (HNF-3) and that C/EBP proteins also interact with this sequence. To our knowledge, this represented the first demonstration that HNF-3 proteins may interact with an IRS and potentially contribute to effects of insulin on gene expression. Functional studies confirm that HNF-3 proteins enhance IGFBP-1 promoter activity at this site and mutation of this HNF-3 binding site also disrupts glucocorticoid effects and reduces the effect of insulin and glucocorticoids on promoter function. Gel shift studies indicate that C/EPB proteins in nuclear extracts also interact with this site and that still other factors may be required for insulin to exert its effects on basal IGFBP-1 promoter function. Based on these observations, we now will utilize fine mapping, site directed mutagenesis and co-transfection systems to examine the role of HNF-3 and/or C/EBP proteins in mediating interactions between glucocorticoids and insulin on IGFBP-1 promoter activity, and examine the role that interactions between and/or modifications on HNF-3, C/EBP and other factors may play in mediating effects of insulin on IGFBP-1 expression. In vivo footprinting will also be performed to determine whether insulin alters access of specific factors this site or the conformation of the IGFBP-1 promoter. The results of these studies should provide insight into specific mechanisms by which glucocorticoids and insulin interact to regulate hepatic gene expression and modulate biological effects of IGFs in disorders of metabolism.
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