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

Endocrinology. 1992 Nov;131(5):2071-6. doi: 10.1210/endo.131.5.1385096.

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Blotting, Western
  • Carrier Proteins / analysis
  • Carrier Proteins / genetics
  • Carrier Proteins / metabolism*
  • Cells, Cultured
  • Culture Media, Conditioned / chemistry
  • Culture Media, Conditioned / pharmacology
  • Edetic Acid / pharmacology
  • Endopeptidases / physiology*
  • Fibroblasts / chemistry*
  • Fibroblasts / 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
  • RNA, Messenger / genetics
  • Recombinant Proteins / analysis
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Sheep
  • Skin / cytology*
  • Somatomedins / pharmacology*

Substances

  • 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