Evidence for a novel insulin-like growth factor (IGF)-dependent protease regulating IGF-binding protein-4 in dermal fibroblasts.

Abstract

The mechanisms by which insulin-like growth factors (IGFs) reduce IGF-binding protein-4 (IGFBP-4) levels in cellular conditioned media are poorly understood. The effect of IGFs on IGFBP-4 levels in fibroblast conditioned media is not mediated via the type 1 or type 2 cellular IGF receptors, and the IGFs exert little or no effects on IGFBP-4 messenger RNA levels in human adult fibroblasts or in rat neuroblastoma cells. To determine whether the effects of IGFs on IGFBP-4 might be exerted through alterations in IGFBP-4 degradation, we incubated cell-free, fibroblast-conditioned media from either sheep or human dermal fibroblasts with or without IGF-I, IGF-II (each 1 microgram/ml), or insulin (10 micrograms/ml) for 72 h at 37 C. Samples were then analyzed by Western ligand blot using radiolabeled IGFs and by immunoblotting using a polyclonal antisera to human IGFBP-4. In the absence of IGFs, no apparent changes in the basal concentrations of the various IGFBPs were observed. In contrast, incubation of media with IGFs caused a 70-80% reduction in levels of both sheep and human IGFBP-4, whereas incubation with insulin was without effect. Similarly, incubation of cell-free conditioned media containing recombinant human IGFBP-4 with IGF-I caused a reduction in detectable levels of the 28K protein. The decrease in IGFBP-4 levels was accompanied by the appearance of an immunoreactive approximate 17-20K fragment that did not bind radiolabeled IGFs by ligand blot. The IGF-dependent decrease in IGFBP-4 was prevented by coincubation of the media with serine protease inhibitors, EDTA, or 1,10-phenanthrolene, suggesting that IGFs may activate an IGFBP-4 specific metallo-serine protease present in fibroblast conditioned media. Alternatively, binding of IGF-I or -II to IGFBP-4 may enhance the susceptibility of IGFBP-4 to proteolytic degradation. The demonstration that IGF-I and IGF-II can promote directly the proteolytic degradation of IGFBP-4 into fragments that do not bind IGFs provides a novel mechanism by which the IGFs may increase their own availability and/or activity in biological fluids.

• Authors: J Fowlkes, M Freemark
• Journal: Endocrinology (Endocrinology) Vol. 131 Issue 5 Pg. 2071-6 (Nov 1992) ISSN: 0013-7227 [Print] United States
• PMID: 1385096 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't, Research Support, U.S. Gov't, P.H.S.)

Chemical References

• Carrier Proteins
• Culture Media, Conditioned
• Insulin
• Insulin-Like Growth Factor Binding Protein 4
• Phenanthrolines
• RNA, Messenger
• Recombinant Proteins
• Somatomedins
• Insulin-Like Growth Factor I
• Insulin-Like Growth Factor II
• Edetic Acid
• Endopeptidases
• 1,10-phenanthroline

Topics

• Animals
• Blotting, Western
• Carrier Proteins (analysis, genetics, metabolism)
• Cells, Cultured
• Culture Media, Conditioned (chemistry, pharmacology)
• Edetic Acid (pharmacology)
• Endopeptidases (physiology)
• Fibroblasts (chemistry, metabolism)
• Humans
• Insulin (pharmacology)
• Insulin-Like Growth Factor Binding Protein 4
• Insulin-Like Growth Factor I (pharmacology)
• Insulin-Like Growth Factor II (pharmacology)
• Phenanthrolines (pharmacology)
• RNA, Messenger (analysis, genetics)
• Recombinant Proteins (analysis, genetics, metabolism)
• Sheep
• Skin (cytology)
• Somatomedins (pharmacology)