title: Tissue inhibitor of metalloproteinase-2 stimulates fibroblast proliferation via a cAMP-dependent mechanism.
abstract: In addition to inhibiting the proteolytic activity of the matrix metalloproteinases, 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 and normal dermal fibroblasts (Hs68) in a dose-dependent manner. This response is evident as early as 2 h and persists up to 48 h after treatment with recombinant TIMP-2 (rTIMP-2). The specificity of this response is demonstrated by the ability of affinity-purified polyclonal anti-TIMP-2 antibodies to ablate TIMP-2 mitogenesis and by the lack of response to TIMP-1. This response is also blocked by the presence of an adenylate 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 stimulates the production of cAMP in a time-dependent manner that differs for the two cell lines. Moreover, treatment of purified cell membranes with rTIMP-2 suppresses cholera toxin-mediated ADP-ribosylation of the GTP-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 cyclase and subsequent production of cAMP. Since cAMP binds to the regulatory subunit of cAMP-dependent protein kinase and activates kinase activity, we evaluated how treatment with rTIMP-2 affected both these parameters. We demonstrate in this report that the cAMP produced in response to treatment with rTIMP-2 binds to the type I regulatory subunit of cAMP-dependent protein kinase and stimulates kinase activity. These results are the first demonstration that TIMP-2 directly activates adenylate cyclase to produce cAMP, which increases cAMP-dependent protein kinase activity, resulting in stimulation of fibroblast mitogenesis.
authors.affiliation: Extracellular Matrix Pathology Section, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
authors.forename: M L
authors.initials: ML
authors.lastname: Corcoran
authors.forename: W G
authors.initials: WG
authors.lastname: Stetler-Stevenson
mesh_terms: D000067956:Adenylyl Cyclase Inhibitors; D002455:Cell Division / Q000187:drug effects*; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000096:biosynthesis / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D004789:Enzyme Activation; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects; D006801:Humans; D011506:Proteins / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000494:pharmacology; D013936:Thymidine / Q000378:metabolism; D019716:Tissue Inhibitor of Metalloproteinase-2
chemical_list: D000067956:Adenylyl Cyclase Inhibitors; D011506:Proteins; D011994:Recombinant Proteins; D019716:Tissue Inhibitor of Metalloproteinase-2; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D013936:Thymidine