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Characterization and Hormonal Regulation of a Rat Ovarian Insulin-Like Growth Factor Binding Protein-5 Endopeptidase: An FSH-Inducible Granulosa Cell-Derived Metalloprotease¹

Division of Reproductive Endocrinology, Department of Obstetrics and Gynecology (C.E.R., E.Y.A.), University of Maryland School of Medicine, Baltimore, Maryland 21201; Stanford University, Department of Pediatrics (P.J.F.), Palo Alto, California 94305; and Department of Pediatrics (R.G.R.), Oregon Health Sciences University, Portland, Oregon 97201

Address all correspondence and requests for reprints to: Dr. Eli Y. Adashi, Division of Reproductive Sciences, Department of Obstetrics and Gynecology, University of Utah Health Sciences Center, 546 Chipeta Way, Mailbox No. 20, Salt Lake City, Utah 84108. E-mail: eadashi{at}hsc.utah.edu

Previous studies established the existence of an FSH-inducible rat granulosa cell-derived insulin-like growth factor binding protein (IGFBP)-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 (estimated 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 dependent. 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 phenomenon or of contaminating pituitary-derived contribution. The ability of FSH to induce IGFBP-5 endopeptidase activity proved relatively specific in that other granulosa cell agonists such as activin-A, IGF-I, GnRH, interleukin-1ß, TNF, TGFß₁, EGF, or endothelin-1 failed to do so. However, the concurrent provision of GnRH, TNF, EGF, or endothelin-1 proved inhibitory to the IGFBP-5 endopeptidase-inducing property of FSH. Activin-A and TGFß₁ in turn further stimulated the FSH effect. Sensitivity to EDTA, 1,10 phenanthroline, and high concentrations (0.1 mM) of Zn²⁺ suggested a Zn²⁺ metalloprotease. 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 possibility of a serine protease. Insensitivity to pepstatin A and E64 argued against aspartic and cysteine proteases, respectively. Insensitivity to plasminogen activator inhibitor-1 (PAI-1) and the presumed 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 Zn²⁺ metalloprotease.

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