Novel role of p66Shc in ROS-dependent VEGF signaling and angiogenesis in endothelial cells.
ABSTRACT p66Shc, a longevity adaptor protein, is demonstrated as a key regulator of reactive oxygen species (ROS) metabolism involved in aging and cardiovascular diseases. Vascular endothelial growth factor (VEGF) stimulates endothelial cell (EC) migration and proliferation primarily through the VEGF receptor-2 (VEGFR2). We have shown that ROS derived from Rac1-dependent NADPH oxidase are involved in VEGFR2 autophosphorylation and angiogenic-related responses in ECs. However, a role of p66Shc in VEGF signaling and physiological responses in ECs is unknown. Here we show that VEGF promotes p66Shc phosphorylation at Ser36 through the JNK/ERK or PKC pathway as well as Rac1 binding to a nonphosphorylated form of p66Shc in ECs. Depletion of endogenous p66Shc with short interfering RNA inhibits VEGF-induced Rac1 activity and ROS production. Fractionation of caveolin-enriched lipid raft demonstrates that p66Shc plays a critical role in VEGFR2 phosphorylation in caveolae/lipid rafts as well as downstream p38MAP kinase activation. This in turn stimulates VEGF-induced EC migration, proliferation, and capillary-like tube formation. These studies uncover a novel role of p66Shc as a positive regulator for ROS-dependent VEGFR2 signaling linked to angiogenesis in ECs and suggest p66Shc as a potential therapeutic target for various angiogenesis-dependent diseases.
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ABSTRACT: Angiogenesis is a key process involved in normal development and wound repair, as well as ischemic heart and limb diseases, and atherosclerosis. Vascular endothelial growth factor (VEGF), a potent angiogenesis factor, stimulates proliferation, migration, and tube formation of endothelial cells (ECs), primarily through the VEGF receptor type2 (VEGFR2). Reactive oxygen species (ROS) function as signaling molecules to mediate biological responses. In ECs, NADPH oxidase is one of the major sources of ROS and consists of catalytic subunits (Nox1, Nox2, and Nox4), p22phox, p47phox, p67phox, and the small GTPase Rac1. VEGF stimulates ROS production via activation of gp91phox (Nox2)-based NADPH oxidase, and ROS are involved in VEGFR2-mediated signaling linked to EC migration and proliferation. Moreover, ROS derived from NADPH oxidase are involved in postnatal angiogenesis. Localizing NADPH oxidase and its regulators at the specific subcellular compartment is an important mechanism for activating specific redox signaling events. This review focuses on a role of NADPH oxidase-derived ROS in angiogenesis and critical regulators involved in generation of spatially and temporally restricted ROS-dependent VEGF signaling at leading edge, focal adhesions/complexes, caveolae/lipid rafts, and cell-cell junctions in ECs. Understanding these mechanisms should facilitate the development of new therapeutic strategies to modulate new blood vessel formation.Antioxidants and Redox Signaling 07/2007; 9(6):731-9. · 7.19 Impact Factor
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ABSTRACT: Endothelial cell (EC) proliferation and migration are important for reendothelialization and angiogenesis. We have demonstrated that reactive oxygen species (ROS) derived from the small GTPase Rac1-dependent NAD(P)H oxidase are involved in vascular endothelial growth factor (VEGF)-mediated endothelial responses mainly through the VEGF type2 receptor (VEGFR2). Little is known about the underlying molecular mechanisms. IQGAP1 is a scaffolding protein that controls cellular motility and morphogenesis by interacting directly with cytoskeletal, cell adhesion, and small G proteins, including Rac1. In this study, we show that IQGAP1 is robustly expressed in ECs and binds to the VEGFR2. A pulldown assay using purified proteins demonstrates that IQGAP1 directly interacts with active VEGFR2. In cultured ECs, VEGF stimulation rapidly promotes recruitment of Rac1 to IQGAP1, which inducibly binds to VEGFR2 and which, in turn, is associated with tyrosine phosphorylation of IQGAP1. Endogenous IQGAP1 knockdown by siRNA shows that IQGAP1 is involved in VEGF-stimulated ROS production, Akt phosphorylation, endothelial migration, and proliferation. Wound assays reveal that IQGAP1 and phosphorylated VEGFR2 accumulate and colocalize at the leading edge in actively migrating ECs. Moreover, we found that IQGAP1 expression is dramatically increased in the VEGFR2-positive regenerating EC layer in balloon-injured rat carotid artery. These results suggest that IQGAP1 functions as a VEGFR2-associated scaffold protein to organize ROS-dependent VEGF signaling, thereby promoting EC migration and proliferation, which may contribute to repair and maintenance of the functional integrity of established blood vessels.Circulation Research 09/2004; 95(3):276-83. · 11.86 Impact Factor
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ABSTRACT: A decrease in reactive oxygen species (ROS) production has been associated with extended life span in animal models of longevity. Mice deficient in the p66Shc gene are long-lived, and their cells are both resistant to oxidative stress and produce less ROS. Our microarray analysis of p66Shc(-/-) mouse tissues showed alterations in transcripts involved in heme and superoxide production and insulin signaling. Thus, we carried out analysis of ROS production by NADPH oxidase (PHOX) in macrophages of control and p66Shc knock-out mice. p66Shc(-/-) mice had a 40% reduction in PHOX-dependent superoxide production. To confirm whether the defect in superoxide production was a direct consequence of p66Shc deficiency, p66Shc was knocked down with siRNA in the macrophage cell line RAW264, and a 30% defect in superoxide generation was observed. The pathway of PHOX-dependent superoxide generation was investigated. PHOX protein levels were not decreased in mutant macrophages; however, the rate and extent of phosphorylation of p47phox was decreased in mutants, as was membrane translocation of the complex. Consistently, phosphorylation of protein kinase Cdelta, Akt, and ERK (the kinases responsible for phosphorylation of p47phox) was decreased. Thus, p66Shc deficiency causes a defect in activation of the PHOX complex that results in decreased superoxide production. p66Shc-deficient mice have recently been observed to be resistant to atherosclerosis and to oxidant injury in kidney and brain. Because phagocyte-derived superoxide is often a component of oxidant injury and inflammation, we suggest that the decreased superoxide production by PHOX in p66Shc-deficient mice could contribute significantly to their relative protection from oxidant injury and consequent longevity.Journal of Biological Chemistry 11/2009; 285(2):1153-65. · 4.65 Impact Factor