Netrin-1 is critical for axonal pathfinding which shares similarities with formation of vascular network. Here we report that netrin-1 induction of angiogenesis is mediated by an increase in endothelial nitric oxide (NO ⅐ ) production, which occurs via a DCC-dependent, ERK1͞2-eNOS feed-forward mechanism. Exposure of mature aortic endothelial cells to netrin-1 resulted in a potent, dose-dependent increase in NO ⅐ production, detected by electron spin resonance. Scavenging NO ⅐ with 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO) abolished netrin-1 stimulated angiogenesis. Netrin-1-stimulated NO ⅐ production or angiogenesis was inhibited by DCC antibody, DCC small interfering RNA (siRNA), specific inhibitors (PD98059, U0126), or siRNAs for MEK1͞2. PTIO attenuated ERK1͞2 phosphorylation, indicating a feed-forward mechanism. Netrin-1 induced a time-dependent phosphorylation of eNOS s1179, s116 and a rapid dephosphorylation of eNOSt497. Only eNOS s1179 was sensitive to U0126 or PTIO. These data characterized a mechanism whereby netrin-1 promotes angiogenesis, which may broadly relate to cardiovascular, neuronal and cancer physiology.nitric oxide N etrin-1 is one of the first discovered axon-guiding molecules that are critical for neuronal development (1, 2). It is a secreted protein that is released to circulation after being produced by a variety of cells (3). Upon activation of its attraction-selective receptor deleted in colorectal cancer (DCC) or neogenin, netrin-1 induces axonal outgrowth and crossover through the midline (2, 4); this is at least partially mediated by activation of the mitogen activated protein kinase ERK1͞2 (5).Formation of vascular network shares many similarities with neuronal pathfinding (6). A number of mitogens including VEGF and EGF have been shown to regulate endothelial cell growth and proliferation. Interestingly, netrin-1 is structurally homologous to the endothelial mitogens. It has an N-terminal type IV laminin repeat, followed by three cystein-rich EGF modules and a positively charged C-terminal domain. A recent study demonstrated that netrin-1 stimulates growth of umbilical vein endothelial cells and vascular smooth muscle (7). However, in the presence of the repulsive receptor UNC5B in developing capillaries, netrin-1 induces endothelial filopodial retraction (8). Our current study uniquely studied proliferation and migration of adult mature aortic endothelial cells, and endothelial outgrowth from adult mouse aortic discs to examine the angiogenic effects of netrin-1. Compared to umbilical vein or developing vessels, aorta shares the closest physiology with large adult coronary arteries where therapeutic angiogenesis is beneficial for patients with ischemic coronary artery diseases (9, 10). More importantly, the signaling mechanisms underlying netrin-1 modulation of angiogenesis were identified.Using the highly specific and sensitive electron spin resonance technology for direct and characteristic measurement of nitric oxide gas radical (NO ⅐ ), we examined a potent...

BackgroundMany serious diseases have a genetic basis which, from an evolutionary point of view, should have been selected against, resulting in very low frequencies. The remarkable sustained prevalence of a number of disease-associated alleles is therefore surprising. We believe that antagonistic pleiotropy, when multiple effects of a gene have opposing effects on fitness (e.g., sickle cell disease), may be more widespread than typically considered. We hypothesize that, rather than being an exception to the rule of genetic disorders, antagonistic pleiotropy may be common.MethodsWe surveyed the medical literature in order to determine whether sufficient evidence exists to reassess the nature of antagonistic pleiotropy; from being considered an unusual scenario to one that is anticipated. We also used a simple population genetic model to examine the feasibility of antagonistic pleiotropy to act as a mechanism to maintain polymorphism for serious genetic disorders even if the benefits are subtle.ResultsWe identified a number of examples of antagonistic pleiotropy where the deleterious effect, the beneficial effect, and the exact molecular cause have been demonstrated. We also identified putative cases in which there is circumstantial evidence or a strong reason to expect antagonistic pleiotropy in a genetic disorder. The population genetic model demonstrates that alleles with severe deleterious health effects can be maintained at medically relevant frequencies with only minor beneficial pleiotropic effects.ConclusionWe believe that our identification of several cases of antagonistic pleiotropy, despite the lack of research on this question and the varied natures of the types of these disorders, speaks to both the underappreciated nature of this phenomenon and its potentially fundamental importance. If antagonistic pleiotropy is as common as our research suggests, this may explain why so many serious diseases, even apparently environmentally caused ones, have a genetic component. Furthermore, acceptance of a genome full of antagonistically pleiotropic genetic interactions poses important implications for clinical treatment and disease prevention research, especially genetically based therapies.

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We have identified two vesicular proteins, designated evectin (evt)-1 and -2. These proteins are Ϸ Ϸ25 kDa in molecular mass, lack a cleaved N-terminal signal sequence, and appear to be inserted into membranes through a Cterminal hydrophobic anchor. They also carry a pleckstrin homology domain at their N termini, which potentially couples them to signal transduction pathways that result in the production of lipid second messengers. evt-1 is specific to the nervous system, where it is expressed in photoreceptors and myelinating glia, polarized cell types in which plasma membrane biosynthesis is prodigious and regulated; in contrast, evt-2 is widely expressed in both neural and nonneural tissues. In photoreceptors, evt-1 localizes to rhodopsin-bearing membranes of the post-Golgi, an important transport compartment for which specific molecular markers have heretofore been lacking. The structure and subcellular distribution of evt-1 strongly implicate this protein as a mediator of postGolgi trafficking in cells that produce large membrane-rich organelles. Its restricted cellular distribution and genetic locus make it a candidate gene for the inherited human retinopathy autosomal dominant familial exudative vitreoretinopathy and suggest that it also may be a susceptibility gene for multiple sclerosis.

Germ line CQ transcripts can be induced by 1L-4 in the human B cell line, BL-2. Utilizing a IFN-y activation sitelike DNA sequence element located upstream of the L exon, we demonstrated by gel mobility shift assays that IL-4 induced a binding activity in the cytosol and nucleus of BL-2 cells. This factor was designated 1L4 NAF (IL-4-induced nuclear-activating factors) and was identified as a tyrosine phosphoprotein, which translocates from the cytosol to the nucleus upon 11-4 treatment. Because these are the characteristics of a signal transducer and activator of transcription (Stat) protein, we determined whether antibodies to Stat proteins will interfere with gel mobility shift and found that antibodies to 11-4 Stat, also known as Stat6, but not antibodies to other Stat proteins, interfere with the formation of the IL-4 NAF complex. Congruous with the involvement of a Stat protein, 1L-4 induced robust Janus kinase 3 (JAK3) activity in BL-2 cells. Cotransfection of JAK3 with 11-4 Stat into COS-7 cells produced an intracellular activity which bound the same IFN-y activation site-like sequence and comigrated with 1L-4 NAF in electrophoretic mobility shift assay. These results show that 1L-4 NAF is 11-4 Stat, which is activated by JAK3 in response to 1L-4 receptor engagement. (J. Clin. Invest. 1995. 96:907-914.)

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Engagement of the B cell receptor for antigen (BCR) leads to immune responses through a cascade of intracellular signaling events. Most studies to date have focused on the BCR and protein tyrosine phosphorylation. Because spleen tyrosine kinase, Syk, is an upstream kinase in multiple BCR-regulated signaling pathways, it also affects many downstream events that are modulated through the phosphorylation of proteins on serine and threonine residues. Here, we report a novel phosphopeptide enrichment strategy and its application to a comprehensive quantitative phosphoproteomics analysis of Syk-dependent downstream signaling events in B cells, focusing on serine and threonine phosphorylation. Using a combination of the Syk inhibitor piceatannol, SILAC quantification, peptide fractionation, and complementary PolyMAC-Ti and PolyMAC-Zr enrichment techniques, we analyzed changes in BCR-stimulated protein phosphorylation that were dependent on the activity of Syk. We identified and quantified over 13 000 unique phosphopeptides, with a large percentage dependent on Syk activity in BCR-stimulated B cells. Our results not only confirmed many known functions of Syk, but more importantly, suggested many novel roles, including in the ubiquitin proteasome pathway, that warrant further exploration.

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