Angiogenesis, the formation of new blood vessels from existing vasculature, is regulated primarily by endothelial cell activity. We show herein that the Ras family GTPase Rap1 has a key role in the regulation of angiogenesis by modulating endothelial cell functions. Blood vessel growth into fibroblast growth factor 2 (FGF2)-containing Matrigel plugs was absent from rap1a ؊/؊ mice, and aortic rings derived from rap1a ؊/؊ mice failed to sprout primitive tubes in response to FGF2, when the tissue was embedded in Matrigel. Knocking down either rap1a or rap1b, two closely related rap1 family members, in human microvascular endothelial cells (HMVECs) by utilizing siRNA confirmed that Rap1 plays key roles in endothelial cell function. The rap1a or rap1b knockdown resulted in decreased adhesion to extracellular matrices and impaired cell migration. HMVEC monolayers lacking Rap1 had increased permeability, and Rap1-deficient endothelial cells failed to form three-dimensional tubular structures when they were plated on Matrigel in vitro. Finally, the activation levels of extracellular signal-regulated kinase (ERK), p38, and Rac, which are important signaling molecules in angiogenesis, were all reduced in response to FGF2 when either of the Rap1 proteins was depleted. These observations place Rap1 centrally in the human angiogenic process and suggest that both the Rap1a and Rap1b proteins are required for angiogenesis and that Rap1 is a critical mediator of FGF-induced ERK activation.Angiogenesis, the formation of new capillaries from preexisting vasculature, is an essential process, both during development (46) and throughout life (5). Maladaptive angiogenesis contributes to numerous cardiovascular diseases such as atherosclerosis, brain ischemia, hypertension, and stroke (5). The process of angiogenesis is usually initiated by increased endothelial cell permeability and proliferation, followed by the proteolysis of basement membrane components. These cells then sprout and migrate toward a site of needed blood supply where they reestablish junctions, form tube structures, and subsequently stabilize into mature capillaries (37). However, the mechanisms underlying this process are only poorly understood.Rap1 is a Ras family small GTPase that serves as a molecular switch. It couples extracellular stimuli to intracellular effectors and their resulting biological responses by cycling between inactive GDP-and active GTP-bound states. In response to ligand binding to multiple cell surface receptors, Rap1 is activated by guanine nucleotide exchange factors (GEFs) and is subsequently converted back to its inactive GDP-bound state by GTPase-activating proteins (GAPs) (3, 4, 44). There exist two closely related Rap1 family members, Rap1a and Rap1b, which are encoded by separate genes but share 95% amino acid identity and may have complementary as well as distinct biological functions.Rap1 was first reported to antagonize Ras by binding to but not activating the c-Raf-1 kinase (11,27). However, in cell types that express B-Raf, Rap1 ...
The Ras-related GTPases Rap1a and 1b have been implicated in multiple biological events including cell adhesion, free radical production, and cancer. To gain a better understanding of Rap1 function in mammalian physiology, we deleted the Rap1a gene. Although loss of Rap1a expression did not initially affect mouse size or viability, upon backcross into C57BL/6J mice some Rap1a−/− embryos died in utero. T cell, B cell, or myeloid cell development was not disrupted in Rap1a −/− mice. However, macrophages from Rap1a null mice exhibited increased haptotaxis on fibronectin and vitronectin matrices that correlated with decreased adhesion. Chemotaxis of lymphoid and myeloid cells in response to CXCL12 or CCL21 was significantly reduced. In contrast, an increase in FcR-mediated phagocytosis was observed. Because Rap1a was previously copurified with the human neutrophil NADPH oxidase, we addressed whether GTPase loss affected superoxide production. Neutrophils from Rap1a−/− mice had reduced fMLP-stimulated superoxide production as well as a weaker initial response to phorbol ester. These results suggest that, despite 95% amino acid sequence identity, similar intracellular distribution, and broad tissue distribution, Rap1a and 1b are not functionally redundant but rather differentially regulate certain cellular events.
E-cadherin based adherens junctions are finely regulated by multiple cellular signaling events. Here we show that the Ras-related Rap1 GTPase is enriched in regions of nascent cell-cell contacts and strengthens E-cadherin junctions: constitutively active Rap1 expressing MDCK cells exhibit increased junctional contact and resisted calcium depletion-induced cell-cell junction disruption. E-cadherin disengagement activated Rap1 and this correlated with E-cadherin association with the Rap GEFs, C3G and PDZ-GEF I. PDZ-GEF I associated with E-cadherin and β-catenin whereas C3G interaction with E-cadherin did not involve β-catenin. Knockdown of PDZ-GEF I in MDCK cells decreased Rap1 activity following E-cadherin junction disruption. We hereby show that Rap1 plays a role in the maintenance and repair of E-cadherin junctions and is activated via an "outside-in" signaling pathway initiated by E-cadherin and mediated at least in part by PDZ-GEF I. KeywordsRap1; PDZ-GEF; Guanine nucleotide exchange factor; adherens junction; E-cadherin; C3G Epithelial cells establish structurally and functionally distinct cell-cell adhesions with their neighbors. Three types of cell junctions, tight junctions (TJs), adherens junctions (AJs) and desmosomes contribute to cell-cell adhesion and specialized proteins mediate each of these interactions. Adherens junctions are the first cell-cell contacts to form and their establishment is necessary for the subsequent formation of tight junctions and desmosomes [Adams et al., 1996;Gumbiner et al., 1988;Pokutta and Weis, 2002]. Transmembrane Ecadherin is the major component of epithelial cell adherens junctions. The extracellular domains of E-cadherin form calcium dependent homophilic interactions with cadherin molecules on an adjacent cell [Pokutta et al., 1994] while its cytoplasmic tail acts as a docking site for β-catenin [Chen et al., 1999] [Pokutta andWeis, 2002], α-catenin and the actin cytoskeleton [Adams et al., 1996;Vasioukhin et al., 2000].In addition to serving a structural role, adherens junctions also regulate the growth, proliferation and migration of epithelial cells [Perez-Moreno et al., 2003]. They are essential during both normal growth and developmental processes as well as in pathological situations such as carcinoma growth and metastasis. The formation and maintenance of epithelial cell junctions are dynamic processes and undergo active regulation by both the extracellular milieu and intracellular signals. The Rho family GTPases, Rac and Cdc42 play a critical role in the regulation of E-cadherin based AJs 1999]. E-cadherin in turn activates Rac and Cdc42 during the initiation of cell-cell contacts to drive the process of junction formation and maintenance of the mature contact [Arthur et al., 2002;Braga, 2000;Braga et al., 1999;Braga et al., 1997;Nakagawa et al., 2001;Noren et al., 2001]. Here, we have analyzed junctional regulation by another small GTPase, Rap1.Rap1 is a member of the Ras-family of GTP binding proteins [Pizon et al., 1988] that cycles between active...
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