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William G. Stetler-Stevenson, M.D., Ph.D.
Tissue Inhibitors of Metalloproteinases: Regulation of Cell Growth and Differentiation
The tissue inhibitors of metalloproteinases or TIMPs consist of a small family of four, homologous, low molecular weight proteins, TIMP-1, -2, -3 and -4. TIMP-2, the second member of this family discovered by our laboratory in 1989 is a unique member of this family. In 1993 we demonstrated that TIMP-2 selectively blocked human microvascular endothelial cell growth in vitro in response to pro-angiogenic factors such as FGF-2 or VEGF-A. Other distinguishing features of this gene is that it is the only member that is not nested within the gene structure of the synapsin gene family and the timp-2 gene also contains a large first intron (>60kB). In 1999, we described a novel form of TIMP-2 in which we appended a single Ala residue to the N-termianl cysteine residue, which we refer to as Ala+TIMP-2. The addition of this single alanine residue did not alter the tertiary structure of the protein but resulted in essentially complete loss of matrix metalloproteinase inhibitory activity. In 2001, we demonstrated that TIMP-2 could suppress receptor tyrosine kinase signaling independent of metalloproteinase inhibition, and that this activity was unique for TIMP-2, and not observed with other members of the TIMP family. In 2003, we demonstrated that the mechanism of this anti-proliferative effect was independent of the metalloproteinase inhibitory activity as demonstrated by an MMP-inhibitor null form known as Ala+TIMP-2, and was mediated by TIMP-2 binding to the alpha3 beta1 integrin on the surface of human microvascular endothelial cells. These findings suggest that further understanding of the anti-angiogenic activity of TIMP-2 might be exploited in human cancer therapy.
TIMP-2 cell surface binding and growth inhibitory activity is reduced by selective a3b1-blocking antibodies and is absent in b1-null fibroblasts. Furthermore, TIMP-2 pretreatment successfully blocks activation of the VEGFR-2 following VEGF-A stimulation or FGFR-1 activation following FGF-2 treatment. Investigations of the mechanism of these effects revealed that TIMP-2 interaction with the a3b1 receptor results in a decrease in the association of protein tyrosine phosphate activity with this integrin and an increase in PTP activity that is associated with VEGFR-2 and FGFR-1. Western blot analysis putatively identified the SH2-domain containing protein tyrosine phosphatase known as Shp-1 as increased in association with VEGFR-2 and FGFR-1 following TIMP-2 treatment.
In addition to these effects of TIMP-2 on endothelial cell growth in vitro, that both wild type human TIMP-2 and the Ala+TIMP-2 mutant inhibit angiogenesis in vivo. As demonstrated for the in vitro effects of TIMP-2 on endothelial cell growth, the in vivo anti-angiogenic effect of TIMP-2 can be reversed by the general protein tyrosine phosphatase inhibitor orthovanadate. Furthermore, experiments in mice deficient in Shp-1 activity demonstrate lack of TIMP-2-mediated antiangiogenic activity. This finding suggests that the principal mechanism of the anti-angiogenic effects of TIMP-2 in vivo is not related to the MMP inhibitory activity of this protein. The MMP inhibitory activity and in vivo anti-angiogenic effects of TIMP-2 are unrelated we have now shown that these activities reside in different but overlapping structural domains of the TIMP-2 protein.
Treatment of human microvascular endothelial cells with recombinant human TIMP-2 markedly alters the pattern of gene expression and migration in these cells in a pattern that is suggestive of endothelial differentiation. We have shown that TIMP-2 induced cell cycle arrest in G1 is mediated by de novo synthesis of p27. Interestingly, TIMP-2, itself an inhibitor of metalloproteinases, induced human microvascular endothelial cell expression of a novel, membrane bound protease inhibitor known as RECK. Enhanced expression of RECK results in a marked alteration of endothelial cell morphology and reduced endothelial cell migration in vitro. TIMP-2 induction of RECK expression is also independent of MMP inhibitory activity and is suppressed by the PTP inhibitor orthovanadate. Further examination of the mechanism of TIMP-2-induced RECK expression reveals that TIMP-2 alters the pattern of paxillin phosphorylation and increases the association of Crk with C3G, resulting in enhanced activationof the small G protein effector Rap-1. That this mechanism of reduced endothelial cell migration is specifically mediated by enhanced RECK expression is demonstrated by a loss of the TIMP-2-mediated effect in RECK-null cells.
Ongoing experiments are focused on detailed characterization of the effects of TIMP-2 to induce differentiaiton of alpha3 beta1 positive normal and tumor cells. We are using cell biological (in vitro) and genetic techniques to understand the mechanism of TIMP-2 induced cellular diifferentiation, with the aim of developing the concept that TIMP-2 induced differentiation may be an effective anti-angiogenic and anti-tumorigenic therapeutic. In addition, we are attempting to define the anti-mitogenic/differentiation -inducing domain(s) of the TIMP-2 protein with the specific aim of developing these peptides or small molecules analogs as novel cancer therapeutics.
This page was last updated on 6/7/2013.