Properties of an insulin-like growth factor-binding protein-4 protease that is secreted by smooth muscle cells

Alexander Parker, A. Gockerman, W. H. Busby, D. R. Clemmons

Research output: Contribution to journalArticle

85 Citations (Scopus)

Abstract

Smooth muscle cells (SMC) secrete insulin-like growth factor (IGF)- binding protein-4 (IGFBP-4) and an IGFBP-4 protease. The purpose of this study was to determine the characteristics of this IGFBP-4 protease and to compare its inhibitor profile to those of IGFBP-5 and IGFBP-2 proteases, which are also present in SMC-conditioned medium. Cultured SMC were exposed to serum-free medium for periods of 24-72 h, and the amount of proteolytic activity in the conditioned medium was assessed by its capacity to degrade pure IGFBP-4. Minimal activity (e.g. <20% of IGFBP-4 degraded in 24 h at 37 C) was present in conditioned medium unless IGF-I or IGF-II was added. This resulted in more than 60% of the intact IGFBP-4 being degraded in 14 h. The activity was a calcium-dependent serine protease and was inhibited by EDTA or 3,4-dicloroisocoumarin. Calcium, but not zinc, could restore proteolytic activity. Heparin alone inhibited IGFBP-4 proteolysis by more than 60%. When heparin cofactor-II and antithrombin-III (AT-III) were added alone, they each had an effect. The combination of heparin plus AT-III was no more active than heparin alone, but the combination of heparin cofactor-II and heparin resulted in near complete inhibition. Peptides that contained the active sites of AT-III or α1-antichymotrypsin were potent inhibitors of the IGFBP- 4 protease. The medium also contained proteolytic activities for IGFBP-2 and IGFBP-5. Comparison of the inhibitor profiles for the IGFBP-4 and IGFBP-5 proteolytic activities revealed major differences, but the IGFBP-2 proteolytic activity was very similar to that of the IGFBP-4 protease. IGFBP- 4 zymography showed a band with a molecular mass estimate of 48 kilodaltons. In contrast, when IGFBP-2 was used as the substrate, a single band at 36 kilodaltons was visualized. These data taken together with the protease inhibitor results suggest that the IGFBP-2, IGFBP-4, and IGFBP-5 proteases are members of a similar family of calcium-dependent serine proteases, but they are distinct proteases. As IGFBP-4 is a potent inhibitor of IGF action, and the activity of this protease is regulated by IGF exposure, the protease represents a novel system for regulating the actions of IGF-I in this cell type.

Original languageEnglish (US)
Pages (from-to)2470-2476
Number of pages7
JournalEndocrinology
Volume136
Issue number6
StatePublished - 1995
Externally publishedYes

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Insulin-Like Growth Factor Binding Protein 4
Pregnancy-Associated Plasma Protein-A
Smooth Muscle Myocytes
Insulin-Like Growth Factor Binding Protein 5
Peptide Hydrolases
Insulin-Like Growth Factor Binding Protein 2
Antithrombin III
Heparin
Conditioned Culture Medium
Heparin Cofactor II
Serine Proteases
Somatomedins
Calcium
Insulin-Like Growth Factor I
Insulin-Like Growth Factor II
Serum-Free Culture Media
Protease Inhibitors
Edetic Acid
Proteolysis

ASJC Scopus subject areas

  • Endocrinology
  • Endocrinology, Diabetes and Metabolism

Cite this

Properties of an insulin-like growth factor-binding protein-4 protease that is secreted by smooth muscle cells. / Parker, Alexander; Gockerman, A.; Busby, W. H.; Clemmons, D. R.

In: Endocrinology, Vol. 136, No. 6, 1995, p. 2470-2476.

Research output: Contribution to journalArticle

Parker, Alexander ; Gockerman, A. ; Busby, W. H. ; Clemmons, D. R. / Properties of an insulin-like growth factor-binding protein-4 protease that is secreted by smooth muscle cells. In: Endocrinology. 1995 ; Vol. 136, No. 6. pp. 2470-2476.
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