TIMP-2 modulates VEGFR-2 phosphorylation and enhances phosphodiesterase activity in endothelial cells.
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.
Lee
Seo-Jin
SJ
Radiation Oncology Branch, Center for Cancer Research, National Cancer Institute, Bethesda, MD, USA.
Tsang
Patricia S
PS
Diaz
Tere M
TM
Wei
Bei-yang
BY
Stetler-Stevenson
William George
WG
D015056:1-Methyl-3-isobutylxanthine; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D002465:Cell Movement; D049109:Cell Proliferation; D006152:Cyclic GMP / Q000378:metabolism; D042783:Endothelial Cells / Q000378:metabolism*; D004789:Enzyme Activation; D005109:Extracellular Matrix / Q000378:metabolism; D006801:Humans; D009569:Nitric Oxide / Q000378:metabolism; D020030:Nitric Oxide Donors; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism; D051966:Phospholipase C gamma / Q000378:metabolism; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism*; D010766:Phosphorylation; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D019716:Tissue Inhibitor of Metalloproteinase-2 / Q000378:metabolism*; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism*; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000378:metabolism*
D020030:Nitric Oxide Donors; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D019716:Tissue Inhibitor of Metalloproteinase-2; D009569:Nitric Oxide; D052250:Nitric Oxide Synthase Type III; D040301:Vascular Endothelial Growth Factor Receptor-2; D051057:Proto-Oncogene Proteins c-akt; D010727:Phosphoric Diester Hydrolases; D051966:Phospholipase C gamma; D006152:Cyclic GMP; D002118:Calcium; D015056:1-Methyl-3-isobutylxanthine