The role of IGFBP-3 in the regulation of IGFBP-4 proteolysis.
The biological effects of insulin-like growth factors (IGFs) are mediated by cell surface receptors but their bioavailability is regulated by IGF binding proteins (IGFBPs) which bind IGF with higher affinity than the receptor. Proteolytic cleavage of the binding proteins reduces their affinity for IGF making the IGF more available to the cell receptor. In the current study we have examined the regulation of IGFBP-4 protease produced by cultured human dermal fibroblasts. IGF-I and the analogs of IGF-I (LR3 and Des[1-3]) induced a dose dependent increase in both proliferation and IGFBP-3 production. Low concentrations of IGF-I induced a marked loss of IGFBP-4 by Western ligand blotting (WLB). This effect was confirmed by the ability of media collected from cells exposed to increasing concentrations of IGF-I to fragment recombinant IGFBP-4, an effect blocked by EDTA. IGFBP-4 proteolysis was observed when cells were exposed to Des[1-3] (albeit at higher concentrations) but not with LR3. Both analogs bind to the IGF receptor but do not bind to IGFBP-4 and have reduced (Des[1-3]) or no (LR3) affinity for IGFBP-3. This demonstrated that neither receptor activation nor ligand binding directly to IGFBP-4 was necessary for IGF induced proteolysis. Protease activity correlated with affinity for IGFBP-3 suggesting a role for IGFBP-3 in the regulation of IGFBP-4 proteolysis. This was confirmed by the ability of excess recombinant IGFBP-3 to inhibit the IGF-I and Des[1-3] induced proteolysis of IGFBP-4. Addition of IGF-I to media from cells unexposed to IGF induced IGFBP-4 proteolysis but this was not seen with LR3 which does not bind to IGFBP-3. Fragmentation occured at higher concentrations of Des[1-3] consistent with its reduced affinity for IGFBP-3. This data suggests that IGFBP-4 proteolysis is regulated in a novel manner by IGFBP-3 which is dependent on the relative proportions of the different binding proteins and the levels of IGFs.
Donnelly
M J
MJ
University Department of Surgery, Bristol Royal Infirmary, UK.
Holly
J M
JM
D000328:Adult; D015153:Blotting, Western; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D018972:Insulin-Like Growth Factor Binding Protein 3 / Q000502:physiology*; D018974:Insulin-Like Growth Factor Binding Protein 4 / Q000032:analysis / Q000378:metabolism*; D007334:Insulin-Like Growth Factor I / Q000031:analogs & derivatives / Q000494:pharmacology; D008666:Metalloendopeptidases / Q000378:metabolism*; D010446:Peptide Fragments / Q000494:pharmacology; D011266:Pregnancy-Associated Plasma Protein-A; D012867:Skin / Q000187:drug effects / Q000378:metabolism*
D018972:Insulin-Like Growth Factor Binding Protein 3; D018974:Insulin-Like Growth Factor Binding Protein 4; C084339:LR(3)IGF-I; D010446:Peptide Fragments; C058957:insulin-like growth factor 1, des-(1-3)-; D007334:Insulin-Like Growth Factor I; D008666:Metalloendopeptidases; D011266:Pregnancy-Associated Plasma Protein-A