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.
Highlights
In addition to inhibiting the proteolytic activity of the matrix metalloproteinases, tissue inhibitors of metalloproteinases (TlMPs) promote the growth of cells in the absence of other exogenous growth factors
Due to a growing body of evidence for the growth-promoting activities of the TIMPs, we have investigated the signal transduction mechanisms involved in TIMP-2-mediated growth stimulation of normal dermal fibroblasts and fibrosarcoma cells
In the absence of serum or exogenous growth factors, rTIMP-2 mediates a mitogenic response in normal dermal fibroblasts and fibrosarcoma cells by stimulating adenylate cyclase to produce cAMP which, in turn, activates cAMP-dependent protein kinase (PKA)
Summary
Vol 270, No 22, Issue of June 2, pp. 13453-13459, 1995 Printed in U.S.A. Tissue Inhibitor of Metalloproteinase-2 Stimulates Fibroblast Proliferation via a cAMP-dependent Mechanism*. The TIMPs bind with high affinity in a 1:1 molar ratio to active matrix metalloproteinases, resulting in loss of proteolytic activity In addition to their function as matrix metalloproteinase inhibitors, a growing body of experimental evidence suggests that TIMPs behave as cytokines and stimulate cellular proliferation in the absence of other growth factors. In the absence of serum or exogenous growth factors, rTIMP-2 mediates a mitogenic response in normal dermal fibroblasts and fibrosarcoma cells by stimulating adenylate cyclase to produce cAMP which, in turn, activates cAMP-dependent protein kinase (PKA). This is the first report demonstrating that TIMP-2 stimulates growth by activation of PKA. :j:To whom correspondence should be addressed: The Extracellular Matrix Pathology Section, Laboratory of Pathology, NCI, NIH, Bldg. 10, Rm. 2A33, Bethesda, MD 20892
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