Summary: Extracellular matrix (ECM)-degrading proteinases of the matrix metalloproteinase (MMP) gene family have been implicated in the pathogenesis of several chronic diseases, such as arthritis, diabetes, cirrhosis, and cancer progression. The MMPs also play a role in normal tissue development and remodeling. Interestingly, tumorigenesis is akin to developmental processes in that both require remodeling of an established tissue microenvironment. MMPs have also been implicated in embryologic development, with several MMP-deficient mouse models showing developmental defects. 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. The TIMPs are a family of four small highly conserved proteins. The TIMPs have been identified in species ranging from drosophila, zebra fish and C. elegans to humans, suggesting that these proteins are ancient eukaryotic proteins. The demonstration that TIMP 1 and TIMP-2 inhibited tumor cell invasion and metastasis in mouse xenograft models during the early 1990s, spurred a significant effort on the part of the pharmaceutical industry to develop synthetic MMP inhibitors as potential new cancer therapeutics. Unfortunately, these synthetic MMP inhibitors failed in all subsequent human clinical trials. Although many hypotheses have been put forward to explain the failure of these drugs in human cancer patients, the true nature of this failure remains unknown. The mammalian TIMP family has four members, which share significant homology and structural identity at the protein level. TIMP-2 is unique as a member of the TIMP family in that in addition to inhibiting MMPs TIMP-2 selectively interacts with MT1-MMP to facilitate the cell-surface activation of pro-MMP-2. Thus, TIMP-2 functions both as an inhibitor of MMPs, and is required for the cellular mechanism of pro-MMP-2 activation. TIMP-2 also has a distinct gene structure compared with the other three members of the TIMP family. An interesting relationship exists between the TIMPs and the synapsin gene family in that three members of the TIMP family are nested within the synapsin genes. The synapsin 1 gene nests TIMP-1, synapsin 2 nests TIMP-4 and synapsin 3 nests TIMP-3. TIMP-2 is the only member of the TIMP family that is not nested within a gene of the synapsin family. The synapsin-TIMP gene nesting relationship began phylogenetically as far back as Drosophila. A recent report describes a nested gene within the very large (60 kb) first intron of the TIMP-2 gene, known as differential display clone 8 (DDC8), which at first was thought to encode a testis specific protein. Furthermore, it has been shown that the brain of the TIMP-2-deficient mouse generated by deletion of exon 1 contains TIMP-2 mRNA encoding exons 2-5 downstream of DDC8 sequence, suggesting alternative splicing between these two genes. TIMPs 1 through 3 have been shown to be anti-angiogenic. Initially, this activity was attributed solely to their MMP inhibitory activity. However, with the recent report that TIMP-4 does not inhibit angiogenesis in vivo, that TIMP-3 may act as a competitive inhibitor for vascular endothelial cell growth factor (VEGF-A) binding to the VEGF receptor-2 (VEGFR-2), and that TIMP-1 anti-angiogenic effects may be both MMP-dependent and MMP-independent investigators have begun to challenge this concept. Our laboratory was the first to demonstrate that TIMP-2 inhibits the mitogenic response of human microvascular endothelial cells to both basic fibroblast growth factor (FGF-2) and VEGF-A, and that these effects are completely independent of MMP-inhibitory activity. The mechanism was shown to be the first example of integrin-mediated heterologous receptor inactivation of receptor tyrosine kinases. Specifically, the mechanism involves TIMP-2 or Ala+TIMP-2 binding to the cell surface via the integrin, alpha 3 beta 1 receptor. TIMP-2 binding to the &#945;3&#946;1 receptor results in activation of a protein tyrosine phosphatase; known as Sh-2 domain phosphatase-1 (SHP-1) that prevents subsequent autophosphorylation of the homodimeric receptor tyrosine kinases (VEGFR-2 or FGF receptor-1) (see Cell 114: 171-180, 2003). We have continued to study additional downstream signaling events associated with TIMP-2 binding to the alpha3 beta1 integrin receptor. We have shown that this interaction results in G1 cell cycle arrest of endothelial cells in vitro, and this inhibition of cellular proliferation is mediated by de novo synthesis of the cyclin-dependent kinase (Cdk) inhibitor p27Kip1 resulting in reduction of both Cdk-2 andCdk-4 activity, and subsequent hypo-phosphorylation of pRb (see JBC 281:3711-3721, 2006). In collaboration with Dr. Cheng-Kui Qu in the Department of Pathology at the University of Maryland Medical School, we have shown that in Shp-1-deficient mice, known as the moth-eaten variable mouse model, TIMP-2 is unable to inhibit angiogenesis in vivo. Focusing on proximal events associated with TIMP-2 binding to the alpha3 beta1 integrin receptor we have shown that this interactions results in increased phosphorylation of Src at tyrosine-527 that is mediated by enhanced association with the c-terminal Src kinase (Csk), a negative regulator of Src activity. Subsequent to the negative regulation of Src we observed altered phosphorylation of the integrin-associated scaffolding protein paxillin at tyrosine residues 31 and 118. This reduced phosphorylation of paxillin at these sites results in disassembly of the adapter protein Crk interaction with the downstream guanidine exchange factor (GEF) known as DOCK-180. DOCK-180 is responsible for activation of the small G protein Rac1 that is known to promote cell migration. In contrast, TIMP-2 interaction with alpha3 beta1 integrin alters paxillin phosphorylation that instead results in the formation of a complex in which the GEF component is replaced with C3G, thus promoting formation of a paxillin/Crk/C3G. The formation of this complex results in activation of the small G-protein Rap-1, which is associated with reduced cell migration and reduced vascular permeability, possibly mediated by enhanced phosphorylation of vascular endothelial cadherin (see Oncogene 25:3230-4234, 2006). These findings are consistent with our prior demonstration that TIMP-2 interaction with the alpha3 beta1 integrin receptor reduces human microvascular cell migration in a time-dependent fashion that was mediated by Rap-1-dependent increase in the cell surface associated protease inhibitor known as reversion-enhancing-cysteine rich protein with Kazal motifs or simply RECK. RECK was originally identified in morphologic revertants of v-Ki-ras-transformed NIH3T3 cell line. This suggests that enhanced RECK expression is associated with a more differentiated, less transformed phenotype