Extracellular matrix (ECM)-degrading proteinases of the matrix metalloproteinase (MMP) gene family have been implicated in the pathogenesis of chronic diseases, such as arthritis, diabetes, cirrhosis, and cancer progression. The MMPs also play a role in normal tissue development and remodeling. Endogenous protease inhibitors of several classes can regulate the ECM-degrading activities of the MMPs, but the tissue inhibitors of metalloproteinases (TIMPs) are the most specific and well studied. TIMPs can also modulate cell growth, differentiation and migration, as well as programmed cel death. These cellular effects may involve both indirect (via inhibition of MMPs) and direct (MMP-independent) mechanisms. Our laboratory examines at the level of molecular regulation how TIMPs alter cellular responses during tissue remodeling and differentiation. The hypothesis being tested is that, in addition to their ability to down-regulate MMP-dependent ECM remodeling, TIMPs selectively target cellular pathways involved in cell growth and differentiation, and together (growth regulation and MMP inhibition) these activities of the TIMPs facilitate the return of tissue homeostasis and/or differentiation.Recently we have demonstrated that TIMP-2 inhibits the mitogenic response of human microvascular endothelial cells to both basic fibroblast growth factor (FGF-2) and vascular endothelial growth factor (VEGF-A). This effect was demonstrated to be independent of MMP-inhibitory activity, as demonstrated by using an MMP null-inhibitor form of TIMP-2, Ala+TIMP-2. The mechanism involved requires TIMP-2 or Ala+TIMP-2 binding to the cell surface and is sensitive to orthovanadate a phosphotyrosine phosphatase inhibitor. We have characterized the integrin, alpha 3 beta 1, as the cell binding partner (receptor) for TIMP-2 and the SH2-domain containing protein tyrosine phosphatase, SHP-1, as essential for TIMP-2 inhibition of endothelial cell growth and angiogenesis (see Cell 114: 171-180, 2003). We have also shown that TIMP-1 promotes B lymphocyte differentiation and prevents induction of apoptosis in Burkitt's lymphoma cells in vitro and in vivo. This effect also appears to be SHP-1 dependent.Our general approach utilizes parallel in vitro cell culture and in vivo animal experiments to analyze the mechanisms of these effects. Human microvascular endothelial cells or B lymphocytes in the presence of exogenous wild type or mutant TIMPs, or with genetically altered TIMP levels will be studied in culture, as well as normal (wild type), transgenic and gene knock-out mice, and analyzed for growth, differentiation, angiogenesis and immune functions. The mechanisms by which TIMPs inhibit endothelial cell growth and angiogenesis will be compared and contrasted with the mechanisms by which TIMPs induce differentiation and inhibition apoptosis in B cells. the mechanisms that mediate these two distinct sets of processes and their molecular targets on the cell surface, cytoplasm and nucleus will be elucidated. The direct effects of TIMPs on the phenotype of endothelial cells and B cells will be studied to evaluate their roles in altering proliferation or inducing differentiation. These studies address an important aspect of the biology of chronic disease, including cancer, which is how do these disease states progress and how can we re-establish normal tissue homeostasis. The data obtained from these studies may form the basis of intervention and therapy in cancer, not only in the late stages of malignancy, but also in pre-malignant lesions.
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