Vascular Endothelial Growth Factor Receptor-2 Activates ADP-ribosylation Factor 1 to Promote Endothelial Nitric-oxide Synthase Activation and Nitric Oxide Release from Endothelial Cells

Vascular endothelial growth factor (VEGF) induces angiogenesis and regulates endothelial function via production and release of nitric oxide (NO), an important signaling molecule. The molecular basis leading to NO production involves phosphatidylinositiol-3 kinase (PI3K), Akt, and endothelial nitric-oxide synthase (eNOS) activation. In this study, we have examined whether small GTP-binding proteins of the ADP-ribosylation factor (ARF) family act as molecular switches to regulate signaling cascades activated by VEGF in endothelial cells. Our results show that this growth factor can promote the rapid and transient activation of ARF1. In endothelial cells, this GTPase is present on dynamic plasma membrane ruffles. Inhibition of ARF1 expression, using RNA interference, markedly impaired VEGF-dependent eNOS phosphorylation and NO production by preventing the activation of the PI3K/Akt signaling axis. Furthermore, our data indicate that phosphorylation of Tyr⁸⁰¹, on VEGF receptor 2, is essential for activating Src- and ARF1-dependent signaling events leading to NO release from endothelial cells. Lastly, this mediator is known to regulate a broad variety of endothelial cell functions. Depletion of ARF1 markedly inhibits VEGF-dependent increase of vascular permeability as well as capillary tubule formation, a process important for angiogenesis. Taken together, our data indicate that ARF1 is a novel modulator of VEGF-stimulated NO release and signaling in endothelial cells.     

Identification of New Interactions between Endolysosomal Tethering Factors

Proper functioning of the precisely controlled endolysosomal system is essential for maintaining the homeostasis of the entire cell. Tethering factors play pivotal roles in mediating the fusion of different transport vesicles, such as endosomes or autophagosomes with each other or with lysosomes. In this work, we uncover several new interactions between the endolysosomal tethering factors Rabenosyn-5 (Rbsn) and the HOPS and CORVET complexes. We find that Rbsn binds to the HOPS/CORVET complexes mainly via their shared subunit Vps18 and we mapped this interaction to the 773–854 region of Vps18. Based on genetic rescue experiments, the binding between Rbsn and Vps18 is required for endosomal transport and is dispensable for autophagy. Moreover, Vps18 seems to be important for β1 integrin recycling by binding to Rbsn and its known partner Vps45.     

A microarray-based approach identifies ADP ribosylation factor-like protein 2 as a target of microRNA-16

microRNAs (miRNAs) are generally thought to negatively regulate the expression of their target genes by mRNA degradation or by translation repression. Here we show an efficient way to identify miRNA target genes by screening alterations in global mRNA levels following changes in miRNA levels. In this study, we used mRNA microarrays to measure global mRNA expression in three cell lines with increased or decreased levels of miR-16 and performed bioinformatics analysis based on multiple target prediction algorithms. For further investigation among the predicted miR-16 target genes, we selected genes that show an expression pattern opposite to that of miR-16. One of the candidate target genes that may interact with miR-16, ADP-ribosylation factor-like protein 2 (ARL2), was further investigated. First, ARL2 was deduced to be an ideal miR-16 target by computational predictions. Second, ARL2 mRNA and protein levels were significantly abolished by treatment with miR-16 precursors, whereas a miR-16 inhibitor increased ARL2 mRNA and protein levels. Third, a luciferase reporter assay confirmed that miR-16 directly recognizes the 3'-untranslated region (3'-UTR) of ARL2. Finally, we showed that miR-16 could regulate proliferation and induce a significant G0/G1 cell cycle arrest, which was due at least in part, to the down-regulation of ARL2. In summary, the present study suggests that integrating global mRNA profiling and bioinformatics tools may provide the basis for further investigation of the potential targets of a given miRNA. These results also illustrate a novel function of miR-16 targeting ARL2 in modulating proliferation and cell cycle progression.     

ADP-Ribosylation: Activation,Recognition, and Removal

ADP-ribosylation is a type of posttranslational modification catalyzed by members of the poly(ADP-ribose) (PAR) polymerase superfamily. ADP-ribosylation is initiated by PARPs, recognized by PAR binding proteins, and removed by PARG and other ADP-ribose hydrolases. These three groups of proteins work together to regulate the cellular and molecular response of PAR signaling, which is critical for a wide range of cellular and physiological functions.     

Oxidatively stressed lymphocytes remain in Go/Gla on mitogenic stimulation

Thiol modifiers and oxidants inhibit lymphocyte activation. To investigate which of the many cell functions sensitive to oxidation are critical in this inhibition, mouse splenic lymphocytes were treated with oxidants prior to exposure to mitogen, and progression into the cell cycle was assayed. Different treatments were used to chemically dissect different potential targets within the cell: copper phenanthroline (CuP), to oxidize surface sulfhydryls; N-ethyl maleimide (NEM), to alkylate extra- and intracellular thiols; and hydrogen peroxide, which generates the highly reactive hydroxyl radical within the cell. Progression into the cell cycle was assayed with acridine orange (AO) and assays of interleukin-2 (IL-2) production and IL-2 receptor (IL-2R) expression. The contribution of ADP-ribosylation to inhibition of mitogenesis was assessed using 3-aminobenzamide (3AB) to inhibit adenosine 5′-diphosphate (ADP)-ribose transferases. The results indicate that the CuP and NEM treatments both produce two independent inhibitory effects, that is, a failure in the production of and response to IL-2. Cells treated with these compounds were able to progress only through G1a upon mitogenic stimulation. H₂O₂ had more complex effects. Both ADP-ribosylation and modulations of cytosolic Ca²⁺ were involved in the inhibitory effects. With lower inhibitory doses of H₂O₂, lymphocytes were completely unresponsive to mitogen and failed to exit Go upon mitogenic stimulation. If intra- and extracellular Ca²⁺ were buffered before treatment with H²O², higher concentrations were required, and under these conditions cells were able to enter G1a but could not progress into G1b. Under neither of these conditions could cells produce IL-2 or express IL-2R.