CHARACTERIZATION AND HORMONAL-REGULATION OF A RAT OVARIAN INSULIN-LIKE-GROWTH-FACTOR BINDING PROTEIN-5 ENDOPEPTIDASE - AN FSH-INDUCIBLE GRANULOSA CELL-DERIVED METALLOPROTEASE

Previous studies established the existence of an FSH-inducible rat gra nulosa cell-derived insulin-like growth factor binding protein (IGFBP) -5 endopeptidase. It was the objective of this communication to charac terize this activity in some detail. Exposure of [I-125]rhlGFBP-5 subs trate to media conditioned by FSH-treated granulosa cells (a cell-free assay) produced two rhlGFBP-5 cleavage products (estimated size 19.5 and 17.5 kDa). The acquisition of IGFBP-5 endopeptidase activity in cu lture proved FSH (or PMSG) to be dose and time dependent. The addition of oFSH or rhFSH to the cell-free assay in turn, proved without effec t on IGFBP-5 endopeptidase activity, thereby arguing against the possi bility of an FSH receptor-independent phenomenon or of contaminating p ituitary-derived contribution. The ability of FSH to induce IGFBP-5 en dopeptidase activity proved relatively specific in that other granulos a cell agonists such as activin-A, IGF-I, GnRH, interleukin-1 beta, TN F alpha, TGF beta(1), EGF, or endothelin-1 failed to do so. However, t he concurrent provision of GnRH, TNF alpha, EGF, or endothelin-l prove d inhibitory to the IGFBP-5 endopeptidase-inducing property of FSH. Ac tivin-A and TGF beta(1), in turn further stimulated the FSH effect. Se nsitivity to EDTA, 1,10 phenanthroline, and high concentrations (great er than or equal to 0.1 mM) of Zn2+ suggested a Zn2+ metal loprotease. Insensitivity to TIMP-1 and TIMP-2 argued against a matrix metallopro tease (MMP). Relative insensitivity to PMSF, AMPSF, aprotinin, TPCK, a nd benzamidine argued against the possibility of a-serine protease. In sensitivity to pepstatin A and E64 argued against aspartic and cystein e proteases, respectively. Insensitivity to plasminogen activator inhi bitor-1 (PAI-1) and the presumed lack of free plasminogen in serum-fre e culture media argued against plasmin. Proteolysis was completely inh ibited over the acid pH range but proceeded unencumbered at neutral an d basic pH. Competition studies using unlabeled IGFBPs (1-6) as well a s cell-free proteolysis assays of [I-125]-labeled IGFBP-1, 2, 3, and 6 suggested a significant level of specificity for the FSH-induced/IGFB P-5-directed endopeptidase. Centricon-mediated fractionation of FSH-co nditioned media revealed the IGFBP-5 endopeptidase activity in the fra ction representing proteins of molecular weight >100K. Taken together, these observations document a secreted, granulosa cell-derived, high molecular weight, FSH-inducible, IGFBP-5-selective, neutral/basic pa-f avoring, non-MMP Zn2+ metalloprotease.