Matrix metalloproteinases degrade insulin-like growth factor-binding protein-3 in dermal fibroblast cultures

TY - JOUR

T1 - Matrix metalloproteinases degrade insulin-like growth factor-binding protein-3 in dermal fibroblast cultures

AU - Fowlkes, J L

AU - Enghild, J J

AU - Suzuki, K

AU - Nagase, H

PY - 1994

Y1 - 1994

N2 - Insulin-like growth factor binding protein-3 (IG-FBP-3) is degraded by a Zn(2+)-dependent protease(s) produced by human dermal fibroblasts in vitro (Fowlkes, J. (1994) Endocrine J. 2, 63-68). Initial studies using IG-FBP-3-substrate zymography identified several IGFBP-3-degrading proteases with M(r) 52,000-72,000, which were inhibitable by EDTA and were shifted to lower M(r) species after treatment of conditioned medium with an organomercurial, suggesting that they might represent one or more of the matrix metalloproteinases (MMPs). Immunoblotting of conditioned medium demonstrated the presence of proMMP-1 (52 and 55 kDa), proMMP-3 (58 and 60 kDa), and proMMP-2 (72 kDa) whose molecular masses corresponded identically to those of the IGFBP-3-degrading proteases. Degradation of recombinant human (rh) IGFBP-3 by conditioned media was blocked (> 80% inhibition) by tissue inhibitor of metallo-proteinases-1, a specific inhibitor of all MMPs, while removal of MMPs -1, -2, and -3 from conditioned medium by sequential immunoaffinity and gelatin-Sepharose chromatography resulted in the complete loss of IGFBP-3-degrading proteinase activity. Furthermore, human MMP-1, MMP-3, and to a lesser extent MMP-2 degraded rhIGFBP-3 in vitro. Sequence analysis of rhIGFBP-3 cleavage sites produced by MMP-1, -2, or -3 demonstrated that each cleaved within the mid-region of the binding protein, a domain with little or no homology with the other five cloned IGFBPs. These studies suggest that MMPs, beyond their previously described functions as extracellular degrading enzymes, may also exert effects on cellular growth and proliferation via degradation of IGFBP-3, thus enhancing IGF bioavailability.

AB - Insulin-like growth factor binding protein-3 (IG-FBP-3) is degraded by a Zn(2+)-dependent protease(s) produced by human dermal fibroblasts in vitro (Fowlkes, J. (1994) Endocrine J. 2, 63-68). Initial studies using IG-FBP-3-substrate zymography identified several IGFBP-3-degrading proteases with M(r) 52,000-72,000, which were inhibitable by EDTA and were shifted to lower M(r) species after treatment of conditioned medium with an organomercurial, suggesting that they might represent one or more of the matrix metalloproteinases (MMPs). Immunoblotting of conditioned medium demonstrated the presence of proMMP-1 (52 and 55 kDa), proMMP-3 (58 and 60 kDa), and proMMP-2 (72 kDa) whose molecular masses corresponded identically to those of the IGFBP-3-degrading proteases. Degradation of recombinant human (rh) IGFBP-3 by conditioned media was blocked (> 80% inhibition) by tissue inhibitor of metallo-proteinases-1, a specific inhibitor of all MMPs, while removal of MMPs -1, -2, and -3 from conditioned medium by sequential immunoaffinity and gelatin-Sepharose chromatography resulted in the complete loss of IGFBP-3-degrading proteinase activity. Furthermore, human MMP-1, MMP-3, and to a lesser extent MMP-2 degraded rhIGFBP-3 in vitro. Sequence analysis of rhIGFBP-3 cleavage sites produced by MMP-1, -2, or -3 demonstrated that each cleaved within the mid-region of the binding protein, a domain with little or no homology with the other five cloned IGFBPs. These studies suggest that MMPs, beyond their previously described functions as extracellular degrading enzymes, may also exert effects on cellular growth and proliferation via degradation of IGFBP-3, thus enhancing IGF bioavailability.

KW - Amino Acid Sequence

KW - Carrier Proteins

KW - Cells, Cultured

KW - Collagenases

KW - Culture Media, Conditioned

KW - Extracellular Matrix

KW - Fibroblasts

KW - Gelatin

KW - Gelatinases

KW - Glycoproteins

KW - Humans

KW - Insulin-Like Growth Factor Binding Proteins

KW - Matrix Metalloproteinase 1

KW - Matrix Metalloproteinase 2

KW - Matrix Metalloproteinase 3

KW - Metalloendopeptidases

KW - Molecular Sequence Data

KW - Recombinant Proteins

KW - Sequence Analysis

KW - Skin

KW - Substrate Specificity

KW - Tissue Inhibitor of Metalloproteinases

M3 - Journal article

C2 - 7523391

SN - 0021-9258

VL - 269

SP - 25742

EP - 25746

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

IS - 41

ER -