Endothelial cell (EC) activation and directional migration is an initial step in angiogenesis that is involved in the pathogenesis of tumor growth and diabetic retinopathy. We previously identified NgBR as a new receptor that is essential for Nogo-B-stimulated EC migration in vitro. Recently, we demonstrated that NgBR is essential for in vivo angiogenesis in zebrafish. In Preliminary Results, we present new data showing that NgBR binds farnesylated Ras (F-Ras) and in so doing, activate Ras-PI3K-Akt dependent pathways to induce EC migration. Although Ras, an oncogene, is well studied in cancer, its role in endothelial cell biology remains relatively unclear. In fact, VEGF can remarkably increase Ras activation in EC. Constitutively activated Ras can stimulate the phosphorylation of Akt, a key player for EC migration. Our preliminary data show that Ras knockdown decreases VEGF-induced EC migration by about 50% and completely abolishes Nogo-B-induced EC migration. It suggests that the role of Ras in regulating EC migration deserves further investigation. Further, considering the fact that NgBR knockdown decreases VEGF-induced EC migration by about 60% and completely abolishes Nogo-B-induced EC migration, NgBR-mediated Ras-PI3K-Akt signaling likely represents a much more prominent pathway than previously considered. In this application, we will determine the mechanisms by which NgBR interacts with F-Ras to initiate Ras-dependent EC migration. The hypothesis of our application is: "NgBR recruits F-Ras via specific sites in its cytoplasmic domain, which is an essential step for Nogo-B- and VEGF-stimulated endothelial cell migration." To test this hypothesis, we will: 1. identify which regions in the cytoplasmic domain of NgBR are required for binding F-Ras. Furthermore, we will use NgBR loss of function mutants to determine if and the extent to which EC migration and angiogenesis are dependent on NgBR-mediated Ras translocation;2. determine the mechanism by which NgBR regulates Nogo-B/VEGF- stimulated Ras activation;3. determine the roles of NgBR in coordinating with VEGFR2 to induce Ras- dependent EC migration and angiogenesis. This application is innovative because it is the first to define the role of NgBR as a novel scaffold protein in modulating Ras translocation and in activating Ras-PI3K-akt pathway. Successful findings from this application have a "huge" impact on vascular biology because it will have uncovered or revealed an entirely new pathway for stimulating EC migration and angiogenesis. Although Ras is a well-studied oncogene in cancer, the exact mechanisms by which Ras translocates to plasma membrane to influence EC function are unknown. Our proposed studies will be the first to define the mechanisms by which NgBR mediates Ras activation to influence EC migration. Findings from these studies will allow us to develop new and novel anti-angiogenic therapies to protect against diabetic retinopathy and tumor angiogenesis.
This research is relevant to public health since endothelial cell proliferation and migration is a major contribution to pathological angiogenesis, which is a hallmark of cancer as well as various ischemic and inflammatory diseases. Our research has demonstrated NgBR as a new Ras modulator to activate a novel signaling pathway to promote blood vessel formation. Research supported by this grant may help identify new therapeutic targets to reduce pathological blood vessel formation so as to improve the quality of life of people suffering with tumor growth and other vascular malformation diseases.
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|Kilari, Sreenivasulu; Remadevi, Indulekha; Zhao, Baofeng et al. (2013) Endothelial cell-specific chemotaxis receptor (ECSCR) enhances vascular endothelial growth factor (VEGF) receptor-2/kinase insert domain receptor (KDR) activation and promotes proteolysis of internalized KDR. J Biol Chem 288:10265-74|