title: Characterization and hormonal regulation of a rat ovarian insulin-like growth factor binding protein-5 endopeptidase: an FSH-inducible granulosa cell-derived metalloprotease.
abstract: 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-1beta, TNF alpha, TGF beta1, EGF, or endothelin-1 failed to do so. However, the concurrent provision of GnRH, TNF alpha, EGF, or endothelin-1 proved inhibitory to the IGFBP-5 endopeptidase-inducing property of FSH. Activin-A and TGF beta1 in turn further stimulated the FSH effect. Sensitivity to EDTA, 1,10 phenanthroline, and high concentrations (> or = 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.
authors.affiliation: Department of Obstetrics and Gynecology, University of Maryland School of Medicine, Baltimore 21201, USA.
authors.forename: C E
authors.initials: CE
authors.lastname: Resnick
authors.forename: P J
authors.initials: PJ
authors.lastname: Fielder
authors.forename: R G
authors.initials: RG
authors.lastname: Rosenfeld
authors.forename: E Y
authors.initials: EY
authors.lastname: Adashi
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D005260:Female; D005640:Follicle Stimulating Hormone / Q000494:pharmacology*; D006107:Granulosa Cells / Q000201:enzymology*; D018975:Insulin-Like Growth Factor Binding Protein 5 / Q000378:metabolism*; D008666:Metalloendopeptidases / Q000378:metabolism*; D008969:Molecular Sequence Data; D009928:Organ Specificity; D051381:Rats; D017207:Rats, Sprague-Dawley; D011962:Receptors, FSH / Q000502:physiology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
chemical_list: D018975:Insulin-Like Growth Factor Binding Protein 5; D011962:Receptors, FSH; D005640:Follicle Stimulating Hormone; D008666:Metalloendopeptidases; D013755:Tetradecanoylphorbol Acetate