The purpose of this project is to further our understanding of the mechanisms of cell-extracellular matrix interactions which regulate cell behavior during tumor progression and angiogenesis. In particular we are interested in the regulation of cell invasion and cell growth. These processes require remodeling of the extracellular matrix that occurs in a spatially and temporally controlled fashion. This involves both proteases and protease inhibitor activities that are tightly regulated. The tissue inhibitor of metalloproteinase (TIMP) family consists of four members: TIMP-1, TIMP-2, TIMP-3 and TIMP-4. All TIMP family members are secreted into the extracellular milieu and have a similar size core protein of 182-194 amino acids with 12 cysteine residues that form six intramolecular disulfides. Correct folding of the peptide backbone and formation of these disulfide bonds are required for functional MMP inhibitor activity. MMP inhibitor activity is further localized to the three amino-terminal disulfide loops and requires a free amino terminal cysteine residue. Studies have shown that the transcription of individual TIMPs are regulated independently of one another. TIMPs inhibit cell invasion (both tumor and endothelial cells) through reconstituted basement membranes in vitro, principally via inhibition of metalloproteinase activity. Both TIMP-1 and TIMP-2 demonstrate cell growth regulating and erythroid potentiating activities (EPA) that appear to be functionally distinct from inhibition of proteases.
The specific aims of these studies are: 1) to understand the relationship of protease inhibitor activity to the growth regulating activity of TIMPs; 2) to define the mechanisms through which individual members of the TIMP family regulate cell growth. To accomplish these goals we have developed a series of modified TIMP proteins and expression vectors, as well as both in vitro and in vivo models to examine the effects of TIMPs on cell growth. Using these reagents and model systems we have demonstrated that modulation of endothelial and tumor cell growth by TIMPs in vitro and in vivo is independent of the protease inhibitor activity of these proteins. In addition, direct cell surface binding of TIMPs has been demonstrated suggesting the presence of putative cell surface receptors for these extracellular matrix protease inhibitors. Signal transduction studies suggest that TIMP binding activates adenylate cyclase, protein kinase A and the protein tyrosine phosphatase SHP-1. Further experiments are directed at: 1) identification and characterization of TIMP receptors; 2) identification of specific peptide sequences responsible for the cell growth modulating properties of TIMPs; 3) proteomic and cDNA microarray characterization of cellular responses to TIMPs both in vitro and in vivo. Our findings confirm the antiangiogenic activity of TIMPs, as well as a unique growth inhibitory activity of TIMP-2 on human microvascular endothelial cells. Identification and characterization of TIMP-2-derived peptide sequences, and/or TIMP receptors may lead to development of novel anticancer therapies.
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