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 (IGFBP-3) is an important determinant of IGF action on cells. We have investigated this in a human skin keratinocyte cell line HaCaT. Although these cells did not normally produce an active IGFBP-3 protease, addition of plasminogen resulted in a dose-dependent proteolysis of endogenous and exogenous IGFBP-3, producing fragments similar to those cleaved by skin interstitial fluid, but different from those generated by plasmin. Protease inhibitor profiles suggested the enzyme in the conditioned medium to be a calcium-dependent serine protease. Exogenous IGFBP-3 either inhibited or slightly stimulated IGF-I-induced cell proliferation when it was coincubated or preincubated with the cells, respectively. Both effects were attenuated in the presence of plasminogen. Preincubation of cells with IGF-I or long R3 IGF-I divergently changed plasminogen activator inhibitor-1 and -2 secretion, but only IGF-I blocked IGFBP-3 proteolysis. Such inhibition was also observed in a cell-free protease assay. IGF-I, however, had no effect on plasmin-induced IGFBP-3 degradation. Together, these data indicate that an IGFBP-3 protease similar to that in skin interstitial fluid is generated in plasminogen-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 from the action of this protease.
Xu
S
S
Department of Surgery, University of Bristol, Bristol Royal Infirmary, United Kingdom.
Savage
P
P
Burton
J L
JL
Sansom
J
J
Holly
J M
JM
D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D005110:Extracellular Space / Q000378:metabolism*; D005341:Fibrinolysin / Q000502:physiology*; D006801:Humans; D018972:Insulin-Like Growth Factor Binding Protein 3 / Q000037:antagonists & inhibitors / Q000378:metabolism* / Q000494:pharmacology; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology; D015603:Keratinocytes / Q000187:drug effects / Q000378:metabolism*; D010447:Peptide Hydrolases / Q000378:metabolism*; D010958:Plasminogen / Q000494:pharmacology; D017395:Plasminogen Activator Inhibitor 1 / Q000378:metabolism; D017396:Plasminogen Activator Inhibitor 2 / Q000378:metabolism; D011480:Protease Inhibitors / Q000494:pharmacology; D012867:Skin / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*
D018972:Insulin-Like Growth Factor Binding Protein 3; D017395:Plasminogen Activator Inhibitor 1; D017396:Plasminogen Activator Inhibitor 2; D011480:Protease Inhibitors; D007334:Insulin-Like Growth Factor I; D010958:Plasminogen; D010447:Peptide Hydrolases; D005341:Fibrinolysin