TIMP-2 is a member of the tissue inhibitor of metalloproteinase family (TIMP family). This family currently consist of four members: TIMP-1, TIMP-2, TIMP-3 and TIMP-4. All TIMP family members share a similar size core protein of 182-194 amino acids and contain 12 cysteine residues that form six intramolecular disulfides. Correct folding and formation of these disulfide is required for MMP inhibitor activity. MMP inhibitor activity is further localized to the amino-terminal three disulfide loops and requires a free amino terminal cystine residue Our studies have shown that TIMP-2 transcription is regulated independently of both TIMP-1 and TIMP-3.TIMP-2 inhibits tumor cell invasion through reconstituted basement membranes in vitro, and this inhibitor demonstrates erythroid potentiating activity (EPA). TIMP-2 inhibits proteolytic opening of the blood brain barrier in hemorrhagic stroke models. In addition to their function as MMP inhibitors, a growing body of experimental evidence suggests that TIMPs can stimulate cellular proliferation in the absence of other growth factors. In the presence of growth factors, however, TIMP-2 antagonizes the growth of cells. In order to understand the disparate effects of TIMP-2 on growth, the signal transduction mechanisms utilized by TIMP-2 were evaluated. The growth promoting effects are presumably mediated by putative TIMP receptors. Recent studies have demonstrated selective cell surface binding of TIMP-2 to HT-1080 cells that is not competed by TIMP-1. 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). The increase in cAMP which may involve activation of a G-protein is required for proliferation. This is the first demonstration that TIMP-2 stimulates growth by activation of PKA. This activity is not ablated by reductive alkylation of recombinant TIMP-2. Growth modulating activity is also retained by Ala-N-terminal TIMP-2 mutants that are completely devoid of MMP inhibitor activity. TIMP-2 peptides have been generated by Asp-N protease digestion of reduced and alkylated TIMP-2. this has led to the identification of specific peptide sequences responsible for the cell growth modulating properties of TIMP-2. The utility of these peptides for isolation of the putative TIMP-2 receptor, as well as modulation of the angiogenic response in vivo, are being explored. Recent studies have developed TIMP-2 mutants that are completely devoid of MMP inhibitor activity. These TIMP-2 mutants are being studied in in vitro and in vivo models to examine the link between MMP inhibitor activity and the previously characterized growth modulating activities of TIMP-2.TIMP-2 is anti-angiogenic and the mechanism for this effect is two fold: through inhibition of endothelial cell proliferation and blocking endothelial cell-mediated matrix proteolysis. We have used the Ala-N-terminal TIMP-2 mutant and TIMP-2 peptides described above to examine in detail the differential effects of TIMPs on stimulated growth of human microvascular endothelial cells, endothelial cell migration and attachment. Our findings confirm that the antiangiogenic activity of TIMP-2 is multifactorial and that the use of TIMP-2-derived peptide sequences can complement the antiangiogenic activity of MMP inhibitors.
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