Angiogenesis describes the development of new blood vessels from pre-existing vessels. The hijacking of this physiological process by tumours allows them to develop their own supplies of nutrients and oxygen, enabling their growth and metastasis. A large body of literature has accumulated over the last 20 years relating to angiogenesis, including signalling pathways involved in this process. One such pathway uses Slit-Roundabout proteins that are implicated in the development of cancers and tumour angiogenesis. The Roundabout family of receptors are large, single-pass transmembrane cell surface receptors involved in directing cell migration in response to their cognate Slit ligands. Although best known for their role in neuronal development, Slits and Roundabouts have now been implicated in myogenesis, leukocyte chemotaxis and tumour angiogenesis, confirming that the Robo signalling pathway functions across multiple cell types. We review here the evidence for a role for Slits and Roundabouts in cancer. In particular, we focus on the role of Robo1 and Robo4 in tumour angiogenesis and discuss the signalling pathways downstream of these proteins mediating endothelial cell migration.
This study aimed to further elucidate the function of Roundabout proteins in endothelium. We show that both Robo1 and Robo4 are present in human umbilical vein endothelial cells (HUVECs) and have knocked expression down using small interfering RNA (siRNA) technology. Roundabout knockout endothelial cells were then studied in a variety of in vitro assays. We also performed a yeast 2-hybrid analysis using the intracellular domain of Robo4 as bait to identify interacting proteins and downstream signaling. Both Robo1 and Robo4 siRNA knockdown and transfection of Robo4-green fluorescent protein inhibited endothelial cell movement and disrupted tube formation on Matrigel. Consistent with a role in regulating cell movement, yeast 2-hybrid and glutathione-S-transferase pulldown analyses show Robo4 binding to a Wiskott-Aldrich syndrome protein (WASP), neural Wiskott-Aldrich syndrome protein, and WASP-interacting protein actin-nucleating complex. We have further shown that Robo1 forms a heterodimeric complex with Robo4, and that transfection of Robo4GFP into HUVECs induces filopodia formation. We finally show using Robo1 knockdown cells that Robo1 is essential for Robo4-mediated filopodia induction. Our results favor a model whereby Slit2 binding to a Robo1/Robo4 heterodimer activates actin nucleation-promoting factors to promote endothelial cell migration.
The Wiskott-Aldrich syndrome protein (WASP) family activates the Arp2/3 complex leading to the formation of new actin filaments. Here, we study the involvement of Scar1, Scar2, N-WASP, and Arp2/3 complex in dorsal ruffle formation in mouse embryonic fibroblasts (MEFs). Using platelet-derived growth factor to stimulate circular dorsal ruffle assembly in primary E13 and immortalized E9 Scar1 ؉/؉ and Scar1 null MEFs, we establish that Scar1 loss does not impair the formation of dorsal ruffles. Reduction of Scar2 protein levels via small interfering RNA (siRNA) also did not affect dorsal ruffle production. In contrast, wiskostatin, a chemical inhibitor of N-WASP, potently suppressed dorsal ruffle formation in a dose-dependent manner. Furthermore, N-WASP and Arp2 siRNA treatment significantly decreased the formation of dorsal ruffles in MEFs. In addition, the expression of an N-WASP truncation mutant that cannot bind Arp2/3 complex blocked the formation of these structures. Finally, N-WASP ؊/؊ fibroblast-like cells generated aberrant dorsal ruffles. These ruffles were highly unstable, severely depleted of Arp2/3 complex, and diminished in size. We hypothesize that N-WASP and Arp2/3 complex are part of a multiprotein assembly important for the generation of dorsal ruffles and that Scar1 and Scar2 are dispensable for this process.
Objective-We aimed to characterize the expression and function of a novel transcript that bioinformatics analysis predicted to be endothelial specific, called endothelial-specific molecule-2 (ECSM2). Methods and Results-A full-length cDNA was isolated and predicted ECSM2 to be a putative 205-amino acid transmembrane protein that bears no homology to any known protein.Quantitative polymerase chain reaction analysis in vitro and in situ hybridization analysis in vivo confirmed ECSM2 expression to be exclusively endothelial, and localization to the plasma membrane was shown. Knockdown of ECSM2 expression in human umbilical vein endothelial cells using siRNA resulted in both reduced chemotaxis and impaired tube formation on matrigel, a solubilized basement membrane, both processes involved in angiogenesis. A yeast 2 hybrid analysis using the ECSM2 intracellular domain identified filamin A as an interacting protein. This interaction was confirmed by precipitation of filamin-A from endothelial cell lysates by a GST-tagged intracellular domain of ECSM2. Conclusion-This study is the first to characterize a novel cell surface protein ECSM2 that regulates endothelial chemotaxis and tube formation, and interacts with filamin A. These studies implicate a role for ECSM2 in angiogenesis via modulation of the actin cytoskeleton. Key Words: endothelial genes Ⅲ transmembrane proteins Ⅲ filamin Ⅲ cell signaling Ⅲ chemotaxis Ⅲ angiogenesis E ndothelial cells perform a wide variety of physiological functions and also play a central role in the pathogenesis of many diseases including atherosclerosis and cancer. As such, many laboratories have sought to identify novel endothelial genes using a variety of techniques. 1-8 We previously described a bioinformatics approach 9 to identify novel endothelial genes such as Robo4 10,11 and endothelial cell-specific molecule-2 (ECSM2) now described here. The ECSM2 EST encoded a conceptual protein of 205 amino acids that contained a putative transmembrane region but no other functional domains and had no homology to any known protein.Pelossi et al subsequently showed that ECSM2 is a marker of primary hemangioblasts and endothelial progenitors and not other hematopoietic progenitor cells, 12 but little else has been published on the protein.We now report the isolation of the ECSM2 cDNA, the identification of putative orthologs, and analysis of the ECSM2 protein. The bioinformatics prediction of endothelial specificity 9 was confirmed both in vitro and in vivo by ECSM2 expression analysis. Predicted plasma membrane expression was confirmed by cell surface expression of an ECSM2-GFP fusion protein in endothelial cells. To characterize the function of ECSM2, its expression was knocked down using siRNA. This resulted in reduced chemotaxis and impaired tube formation on matrigel, a solubilized basement membrane extract. These data suggest a role for ECSM2 in angiogenesis, a complex process involving the migration, proliferation, and lumen formation by endothelial cells. A yeast 2 hybrid screen was per...
We have applied search algorithms to expression databases to identify genes whose expression is restricted to the endothelial cell. Such genes frequently play a critical role in endothelial biology and angiogenesis. Two such genes are the roundabout receptor Robo4 and the ECSCR (endothelial-cell-specific chemotaxis regulator). Endothelial cells express both Robo1 and Robo4, which we have knocked down using siRNA (small interfering RNA) and then studied the effect in a variety of in vitro assays. Both Robo4 and Robo1 knockdown inhibited in vitro tube formation on Matrigel. Transfection of Robo4 into endothelial cells increased the number of filopodial extensions from the cell, but failed to do so in Robo1-knockdown cells. Separate immunoprecipitation studies showed that Robo1 and Robo4 heterodimerize. We conclude from this and other work that a heteroduplex of Robo1 and Robo4 signals through WASP (Wiskott-Aldrich syndrome protein) and other actin nucleation-promoting factors to increase the number of filopodia and cell migration. Knockdown of the transmembrane ECSCR protein in endothelial cells also reduced chemotaxis and impaired tube formation on Matrigel. Yeast two-hybrid analysis and immunoprecipitation studies showed that, in contrast with the roundabouts, ECSCR binds to the actin-modulatory filamin A. We conclude that all three of these genes are critical for effective endothelial cell migration and, in turn, angiogenesis.
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