Characterization and hormonal regulation of a rat ovarian insulin-like growth factor binding protein-5 endopeptidase: An FSH-inducible granulosa cell-derived metalloprotease

Carol E. Resnick, Paul J. Fielder, Ronald (Ron) Rosenfeld, Eli Y. Adashi

Research output: Contribution to journalArticle

21 Citations (Scopus)

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-1β, TNFα, TGFβ, EGF, or endothelin-1 failed to do so. However, the concurrent provision of GnRH, TNFα, EGF, or endothelin-1 proved inhibitory to the IGFBP-5 endopeptidase-inducing property of FSH. Activin-A and TGFβ1 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.

Original languageEnglish (US)
Pages (from-to)1249-1257
Number of pages9
JournalEndocrinology
Volume139
Issue number3
DOIs
StatePublished - 1998

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Insulin-Like Growth Factor Binding Protein 5
Endopeptidases
Granulosa Cells
Metalloproteases
Insulin-Like Growth Factor Binding Protein 1
Endothelin-1
Conditioned Culture Medium
Epidermal Growth Factor
Gonadotropin-Releasing Hormone
Proteolysis
Tosylphenylalanyl Chloromethyl Ketone
Insulin-Like Growth Factor Binding Protein 6
Molecular Weight
FSH Receptors
Insulin-Like Growth Factor Binding Protein 2
Tissue Inhibitor of Metalloproteinase-2
Aprotinin
Tissue Inhibitor of Metalloproteinase-1
Cysteine Proteases
Plasminogen

ASJC Scopus subject areas

  • Endocrinology
  • Endocrinology, Diabetes and Metabolism

Cite this

Characterization and hormonal regulation of a rat ovarian insulin-like growth factor binding protein-5 endopeptidase : An FSH-inducible granulosa cell-derived metalloprotease. / Resnick, Carol E.; Fielder, Paul J.; Rosenfeld, Ronald (Ron); Adashi, Eli Y.

In: Endocrinology, Vol. 139, No. 3, 1998, p. 1249-1257.

Research output: Contribution to journalArticle

Resnick, Carol E. ; Fielder, Paul J. ; Rosenfeld, Ronald (Ron) ; Adashi, Eli Y. / Characterization and hormonal regulation of a rat ovarian insulin-like growth factor binding protein-5 endopeptidase : An FSH-inducible granulosa cell-derived metalloprotease. In: Endocrinology. 1998 ; Vol. 139, No. 3. pp. 1249-1257.
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N2 - 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-1β, TNFα, TGFβ, EGF, or endothelin-1 failed to do so. However, the concurrent provision of GnRH, TNFα, EGF, or endothelin-1 proved inhibitory to the IGFBP-5 endopeptidase-inducing property of FSH. Activin-A and TGFβ1 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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