PROTEOLYSIS OF INSULIN-LIKE GROWTH FACTOR-BINDING PROTEIN-3 BY HUMAN SKIN KERATINOCYTES IN CULTURE IN COMPARISON TO THAT IN SKIN INTERSTITIAL FLUID - THE ROLE AND REGULATION OF COMPONENTS OF THE PLASMIN SYSTEM

Proteolysis of insulin-like growth factor (IGF)-binding protein-3 (IGF BP-3) is an important determinant of IGF action on cells. We have inve stigated this in a human skin keratinocyte cell line HaCaT. Although t hese cells did not normally produce an active IGFBP-3 protease, additi on of plasminogen resulted in a dose-dependent proteolysis of endogeno us and exogenous IGFBP-3, producing fragments similar to those cleaved by skin interstitial fluid, but different from those generated by pla smin. Protease inhibitor profiles suggested the enzyme in the conditio ned medium to be a calcium-dependent serine protease. Exogenous IGFBP- 3 either inhibited or slightly stimulated IGF-I-induced cell prolifera tion when it was coincubated or preincubated with the cells, respectiv ely. Both effects were attenuated in the presence of plasminogen. Prei ncubation of cells with IGF-I or long R-3 IGF-I divergently changed pl asminogen activator inhibitor-1 and -2 secretion, but only IGF-I block ed IGFBP-3 proteolysis. Such inhibition was also observed in a cell-fr ee protease assay. IGF-I, however, had no effect on plasmin-induced IG FBP-3 degradation. Together, these data indicate that an IGFBP-3 prote ase similar to that in skin interstitial fluid is generated in plasmin ogen-treated HaCaT cells, and it attenuates the effects of IGFBP-3 on IGF action. IGF-I, probably by coupling with IGFBP-3, can protect it f rom the action of this protease.