The applicant has been a Fellow in Pediatric Endocrinology at Stanford for 3 1/2 years. His laboratory-based research has included characterization of insulin-like growth factor (IGF) peptide, receptor, and binding protein (IGFBP) expression in skin cells, leukemic lymphoblasts, and in sera of mice transgenic for IGF-II. IGF-I and IGF- II are important mitogenic factors that mediate most effects of growth hormone, and variable tissue and cellular IGFBP expression controls local delivery of IGFs to their cell-surface receptors. However, interaction of the IGFs and IGFBPs at the cellular level is poorly understood. This proposal builds upon the applicant's recent demonstration that in vivo levels of IGFBP-3 and -4 are regulated by IGF-I and IGF-II, but this regulatory control is not mediated by IGF receptors. Six related IGFBPs have been cloned and sequenced in the last few years. IGFBP-3 is the major serum IGF carrier protein and is expressed by many cells in culture, including human fibroblasts. In several fibroblast cell lines and an epidermal cell line (SCL-1), concentrations of IGFBP-3 in cell conditioned media are stimulated by exogenous IGF-I and IGF-II in a dose-dependent manner. In contrast, IGFBP-4 levels in fibroblasts are decreased by the IGFs, implying a different mechanism by which IGFs regulate each of these IGFBPs. Our preliminary studies utilizing receptor antibodies and synthetic IGF analogs indicate that both the IGFBP-3 and IGFBP-4 concentration changes are mediated independently of IGF binding to either the type 1 or type 2 IGF receptor. Although some evidence implicates direct IGF/IGFBP binding in modulation of IGFBP-3 levels, insulin (which does not bind IGFBPs) also regulates IGFBP-3 in some cell lines. changes in IGFBP-3 concentrations do not appear to be transcriptionally regulated. This proposal asks 1) how in vitro IGFBP-3 levels are increased by IGF-I and IGF-II via a receptor-independent, post-transcriptional signal; 2) whether the same cellular mechanism which increases IGFBP-3 could also cause decreased levels of IGFBP-4; 3) how insulin, which does not bind IGFBP-3 or -4, results in increases in IGFBP levels; and 4) why other monolayer cells fail to exhibit IGFBP-3 or IGFBP-4 changes in response to IGFs and insulin. These questions will be approached in human fibroblast, SCL-1, and keratinocyte cell lines and rat B104 neuroblastoma line by Northern analysis, ligand blot, affinity crosslinking, immunochemistry, and metabolic labeling, particularly examining partition of IGFBPs at the cell surface and potential signal transduction. Studies will make use of recently developed IGFBP-3 and IGFBP-4 antibodies, established receptor antibodies, and IGF-I and IGF-II analogs with altered affinity for IGFBPs and receptors, in order to elucidate details of IGF/IGFBP dynamics affecting growth regulation at the cellular level.
| Olney, R C; Anhalt, H; Neely, E K et al. (1995) A quantitative assay for IGF-I and IGF binding protein mRNAs: expression in malignant melanoma cells. Mol Cell Endocrinol 110:213-23 |
