TISSUE INHIBITOR OF METALLOPROTEINASE-2 STIMULATES FIBROBLAST PROLIFERATION VIA A CAMP-DEPENDENT MECHANISM

In addition to inhibiting the proteolytic activity of the matrix metal loproteinases, tissue inhibitors of metalloproteinases (TIMPs) promote the growth of cells in the absence of other exogenous growth factors. TIMP-2 stimulates the proliferation of fibrosarcoma (HT-1080) cells a nd normal dermal fibroblasts (Hs68) in a dose-dependent manner. This r esponse is evident as early as 2 h and persists up to 48 h after treat ment with recombinant TIMP-2 (rTIMP-2). The specificity of this respon se is demonstrated by the ability of affinity-purified polyclonal anti -TIMP-2 antibodies to ablate TIMP-2 mitogenesis and by the lack of res ponse to TIMP-1. This response is also blocked by the presence of an a denylate cyclase inhibitor, 9-(tetrahydro-2-furyl) adenine (SQ22536). Although SQ22536 did not affect untreated fibroblasts or fibrosarcoma cells, this inhibitor completely abrogates the proliferative response induced by rTIMP-2. Treatment of these cells with rTIMP-2 also stimula tes the production of cAMP in a time-dependent manner that differs for the two cell lines. Moreover, treatment of purified cell membranes wi th rTIMP-2 suppresses cholera toxin-mediated ADP-ribosylation of the G TP-binding protein, Gs alpha subunit. These results indicate that the alpha beta gamma heterotrimer is dissociated by treatment with rTIMP-2 , which may facilitate the Gs alpha-mediated activation of adenylate c yclase and subsequent production of cAMP. Since cAMP binds to the regu latory subunit of cAMP-dependent protein kinase and activates kinase a ctivity, we evaluated how treatment with rTIMP-2 affected both these p arameters. We demonstrate in this report that the cAMP produced in res ponse to treatment with rTIMP-2 binds to the type I regulatory subunit of cAMP-dependent protein kinase and stimulates kinase activity. Thes e results are the first demonstration that TIMP-2 directly activates a denylate cyclase to produce cAMP, which increases cAMP-dependent prote in kinase activity, resulting in stimulation of fibroblast mitogenesis .