MATRIX METALLOPROTEINASES DEGRADE INSULIN-LIKE GROWTH FACTOR-BINDING PROTEIN-3 IN DERMAL FIBROBLAST-CULTURES

Insulin-like growth factor binding protein-3 (IGFBP-3) is degraded by a Zn2+-dependent protease(s) produced by human dermal fibroblasts in v itro (Fowlkes, J. (1994) Endocrine J. 2, 63-68). Initial studies using IGFBP-3-substrate zymography identified several IGFBP-3-degrading pro teases with M(r) 52,000-72,000, which were inhibitable by EDTA and wer e shifted to lower M, species after treatment of conditioned medium wi th an organomercurial, suggesting that they might represent one or mor e of the matrix metalloproteinases (MMPs). Immunoblotting of condition ed medium demonstrated the presence of proMMP-1 (52 and 55 kDa), proMM P-3 (58 and 60 kDa), and proMMP-2 (72 kDa) whose molecular masses corr esponded identically to those of the IGFBP-3 degrading proteases. Degr adation of recombinant human (rh) IGFBP-3 by conditioned media was blo cked (>80% inhibition) by tissue inhibitor of metalloproteinases-l, a specific inhibitor of all MMPs, while removal of MMPs -1, -2, and -3 f rom conditioned medium by sequential immunoaffinity and gelatin-Sephar ose 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 li ttle or no homology with the other five cloned IGFBPs. These studies s uggest that MMPs, beyond their previously described functions as extra cellular degrading enzymes, may also exert effects on cellular growth and proliferation via degradation of IGFBP-3, thus enhancing IGF bioav ailability.