Impaired growth in diabetes mellitus involves reduced generation and action of insulin-like growth factors (IGFs). The IGFs are mitogenic polypeptides, which circulate in predominant association with an IGF binding protein, IGFBP-3. IGFBP-3 prolongs the halflife of circulating IGFs, facilitates targeting of IGFs to specific tissue and cell types, and can both potentiate and inhibit IGF actions. Abundance of IGFBP-3 mRNA supports the liver as a major site of synthesis of circulating IGFBP-3, but the specific metabolic and hormonal factors that regulate IGFBP-3 in the liver are not known. IGFBP-3 is not released in conventional liver cell systems, limiting present studies largely to in vivo models, where separation of regulatory events is difficult. Preliminary studies by the P.I. have established that co-cultures of rat hepatocytes with intact or heat-killed nonparenchymal cells exhibit hormone-regulated IGFBP-3 expression. Strong correlations between IGFBP- 3 mRNA abundance and levels of protein in conditioned medium indicate pre-translational regulation. Thus, this proposal emphasizes mechanisms of molecular regulation, with the following specific aims: 1. To evaluate models for study of cellular regulation of IGFBP-3, we will quantify IGFBP-3 mRNA abundance in hepatocytes co-cultured with different subpopulations of nonparenchymal cells, and dissect the permissive contributions of soluble and insoluble factors produced by nonparenchymal cells. 2. To define the receptor-related mechanisms mediating stimulation of IGFBP-3 by insulin or IGF-1, we will assess IGFBP-3 expression in co-cultures incubated with added polyclonal antibodies to IGF-1 and/or to insulin receptors, and with addition of IGF-1 mutants with altered affinity for IGFBPs or the Type 1 IGF receptor. 3. To delineate the critical role of transcription, we will quantify transcription rate and mRNA stability in normal and diabetic animals, and in co-cultured hepatocytes with or without added insulin and/or dexamethasone -- conditions which lead to reproducible changes in IGFBP-3 expression, and represent major abnormalities seen in vivo. 4. to characterize the DNA regions which mediate regulation of IGFBP-3 transcription, DNase I hypersensitivity analysis will be used to identify areas likely involved in transcriptional control, transient transfection will be used to define hormone-sensitive promoter regions, and footprinting and gel mobility shift analysis will be used to characterize regions contacted by putative hormone-regulated transcription factors. If time permits, 5. to attempt isolation of metabolically regulated transcription factors, we will screen lambdaZap expression libraries via """"""""Southwestern blotting"""""""" with catenated probes reflecting critical DNA- binding sites, and clone and sequence the identified cDNAs. These studies should provide understanding of the molecular regulation of a critical IGF binding protein, and provide insight into the pathophysiology of impaired growth in diabetes mellitus.
| Phillips, L S; Pao, C I; Villafuerte, B C (1998) Molecular regulation of insulin-like growth factor-I and its principal binding protein, IGFBP-3. Prog Nucleic Acid Res Mol Biol 60:195-265 |
| Villafuerte, B C; Zhao, W; Herington, A C et al. (1997) Identification of an insulin-responsive element in the rat insulin-like growth factor-binding protein-3 gene. J Biol Chem 272:5024-30 |
| Villafuerte, B C; Zhang, W N; Phillips, L S (1996) Insulin and insulin-like growth factor-I regulate hepatic insulin-like growth factor binding protein-3 by different mechanisms. Mol Endocrinol 10:622-30 |
