ABSTRACT
In this study, we examine the effects of tissue inhibitor of metalloproteinases-2 (TIMP-2) on the phosphorylation status of specific phosphotyrosine residues on the vascular endothelial cell growth factor receptor-2 (VEGFR-2) cytoplasmic tail and examine the effects on associated downstream signaling pathways. To focus on metalloproteinase-independent mechanisms, we used the TIMP-2 analog known as Ala+TIMP-2 that is deficient in matrix metalloproteinase-inhibitory activity. Our experiments are designed to compare the effects of VEGF-A stimulation with or without Ala+TIMP-2 pretreatment, as well as basal responses in human microvascular endothelial cells. Our results show that Ala+TIMP-2 selectively alters the phosphorylation pattern of VEGFR-2 after VEGF-A stimulation and disrupts the downstream activation of PLC-gamma, Ca(+2) flux, Akt, and eNOS, as well as decreasing cGMP levels. Moreover, we observed an Ala+TIMP-2-induced reduction in cGMP levels typically elevated by exogenous NO donors implicating Ala+TIMP-2 in the direct activation of an isobutylmethylxanthine (IBMX)-sensitive cGMP phosphodiesterase activity. TIMP-2 suppression of endothelial mitogenesis and angiogenesis involves at least two mechanisms, one mediated by protein tyrosine phosphatase inhibition of VEGFR-2 activation as well as downstream signaling and a second mechanism involving direct activation of an IBMX-sensitive phosphodiesterase activity.
Subject(s)
Endothelial Cells/metabolism , Phosphoric Diester Hydrolases/metabolism , Tissue Inhibitor of Metalloproteinase-2/metabolism , Vascular Endothelial Growth Factor A/metabolism , Vascular Endothelial Growth Factor Receptor-2/metabolism , 1-Methyl-3-isobutylxanthine , Calcium/metabolism , Cell Line , Cell Movement , Cell Proliferation , Cyclic GMP/metabolism , Enzyme Activation , Extracellular Matrix/metabolism , Humans , Nitric Oxide/metabolism , Nitric Oxide Donors , Nitric Oxide Synthase Type III/metabolism , Phospholipase C gamma/metabolism , Phosphorylation , Proto-Oncogene Proteins c-akt/metabolismABSTRACT
We have previously reported that tissue inhibitor of metalloproteinases-2 (TIMP-2), an endogenous inhibitor of matrix metalloproteinase, modulates angiogenic responses through the MMP inhibition-independent activity. In this study, we investigate the molecular mechanisms of TIMP-2-mediated growth inhibition in response to fibroblast growth factor-2 (FGF-2). Pre-treatment with a protein tyrosine phosphatase inhibitor orthovanadate or expression of a dominant negative Shp-1 mutant fails to induce TIMP-2 inactivation of FGF-2 signaling pathways in human microvascular endothelial cells. We also show that TIMP-2 inhibition of FGF-2-induced p42/44(MAPK) activation and cell proliferation is associated with TIMP-2 binding to integrin alpha3beta1 on endothelial cell surfaces, as demonstrated by use of anti-integrin alpha3 or beta1 blocking antibodies, or disruption of integrin alpha3 expression by siRNA. Collectively, our results indicate that TIMP-2 inhibits FGF-2 signaling pathways through association with integrin alpha3beta1 and Shp-1-dependent inhibition of p42/44(MAPK) signaling, which in turn, results in suppression of FGF-2-stimulated endothelial cell mitogenesis.
Subject(s)
Endothelial Cells/drug effects , Fibroblast Growth Factor 2/pharmacology , Signal Transduction/drug effects , Tissue Inhibitor of Metalloproteinase-2/pharmacology , Base Sequence , Cell Line, Tumor , Cell Proliferation/drug effects , Cells, Cultured , Endothelial Cells/cytology , Endothelial Cells/physiology , Fibroblast Growth Factor 2/physiology , Humans , Integrin alpha3beta1/antagonists & inhibitors , Integrin alpha3beta1/genetics , Integrin alpha3beta1/physiology , MAP Kinase Signaling System/drug effects , MAP Kinase Signaling System/physiology , Matrix Metalloproteinases/physiology , Mitogen-Activated Protein Kinase 1/antagonists & inhibitors , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/antagonists & inhibitors , Mitogen-Activated Protein Kinase 3/metabolism , Neovascularization, Physiologic , Protein Tyrosine Phosphatase, Non-Receptor Type 6/physiology , RNA, Small Interfering/genetics , Receptor, Fibroblast Growth Factor, Type 1/physiology , Recombinant Proteins/pharmacology , Signal Transduction/physiology , Tissue Inhibitor of Metalloproteinase-2/physiologyABSTRACT
Tissue inhibitors of metalloproteinases (TIMPs) suppress matrix metalloproteinase (MMP) activity critical for extracellular matrix turnover associated with both physiologic and pathologic tissue remodeling. We demonstrate here that TIMP-2 abrogates angiogenic factor-induced endothelial cell proliferation in vitro and angiogenesis in vivo independent of MMP inhibition. These effects require alpha 3 beta 1 integrin-mediated binding of TIMP-2 to endothelial cells. Further, TIMP-2 induces a decrease in total protein tyrosine phosphatase (PTP) activity associated with beta1 integrin subunits as well as dissociation of the phosphatase SHP-1 from beta1. TIMP-2 treatment also results in a concomitant increase in PTP activity associated with tyrosine kinase receptors FGFR-1 and KDR. Our findings establish an unexpected, MMP-independent mechanism for TIMP-2 inhibition of endothelial cell proliferation in vitro and reveal an important component of the antiangiogenic effect of TIMP2 in vivo.
