Characterization and Hormonal Regulation of a Rat Ovarian Insulin-Like Growth Factor Binding Protein-5 Endopeptidase: An FSH-Inducible Granulosa Cell-Derived Metalloprotease

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Abstract
Previous studies established the existence of an FSH-inducible rat granulosa cell-derived insulin-like growth factor binding protein (IG- FBP)-5 endopeptidase. It was the objective of this communication to characterize this activity in some detail. Exposure of (125I) rhIGFBP-5 substrate to media conditioned by FSH-treated granulosa cells (a cell-free assay) produced two rhIGFBP-5 cleavage products (estimat- ed size 19.5 and 17.5 kDa). The acquisition of IGFBP-5 endopeptidase activity in culture proved FSH (or PMSG) to be dose and time de- pendent. The addition of oFSH or rhFSH to the cell-free assay in turn, proved without effect on IGFBP-5 endopeptidase activity, thereby arguing against the possibility of an FSH receptor-independent phe- nomenon or of contaminating pituitary-derived contribution. The ability of FSH to induce IGFBP-5 endopeptidase activity proved rel- atively specific in that other granulosa cell agonists such as activin-A, IGF-I, GnRH, interleukin-1b, TNFa, TGFb1, EGF, or endothelin-1 failed to do so. However, the concurrent provision of GnRH, TNFa, EGF, or endothelin-1 proved inhibitory to the IGFBP-5 endopepti- dase-inducing property of FSH. Activin-A and TGFb1 in turn further stimulated the FSH effect. Sensitivity to EDTA, 1,10 phenanthroline, and high concentrations ($0.1 mM )o f Zn 21 suggested a Zn21 metal- loprotease. Insensitivity to TIMP-1 and TIMP-2 argued against a matrix metalloprotease (MMP). Relative insensitivity to PMSF, AMPSF, aprotinin, TPCK, and benzamidine argued against the pos- sibility of a serine protease. Insensitivity to pepstatin A and E64 argued against aspartic and cysteine proteases, respectively. Insen- sitivity to plasminogen activator inhibitor-1 (PAI-1) and the pre- sumed lack of free plasminogen in serum-free culture media argued against plasmin. Proteolysis was completely inhibited over the acid pH range but proceeded unencumbered at neutral and basic pH. Competition studies using unlabeled IGFBPs (1- 6) as well as cell-free proteolysis assays of (125I)-labeled IGFBP-1, 2, 3, and 6 suggested a significant level of specificity for the FSH-induced/IGFBP-5-directed endopeptidase. Centricon-mediated fractionation of FSH-conditioned media revealed the IGFBP-5 endopeptidase activity in the fraction 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 pH-favoring, non-MMP Zn21 metalloprotease. (Endocrinology 139: 1249 -1257, 1998)

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