Knockdown of Rap1b Enhances Apoptosis and Autophagy in Gastric Cancer Cells via the PI3K/Akt/mTOR Pathway.

Gastric cancer (GC) is the fourth most common malignancy and the second leading cause of cancer mortality around the world. However, the regulatory mechanisms of GC tumorigenesis and cancer cell motility are completely unknown. We investigated the role of a RAS-related protein (Rap1b) in the progression of GC. Our results showed that the expression of Rap1b is aberrantly upregulated in GC tissue samples and human GC cell lines, and the high expression of Rap1b indicated a positive correlation with poor prognosis in patients with GC. Inhibition of endogenous Rap1b dramatically reduced the cell cycle progression but strongly enhanced the apoptosis capacity of human GC cell lines MKN-28 and SGC-7901 cells compared with the control group. Western blotting assay showed that Rap1b inhibition resulted in a significant increase in the ratio of LC3-II to LC3-I, and the levels of p62 protein were decreased in both MKN-28 and SGC-7901 cells. Furthermore, PI3K/Akt/mTOR activation was found to be maintained in a low level in the normal gastric mucosal epithelial cells, while it was significantly upregulated in GC cells, which could be decreased by Rap1b inhibition. The PI3K inhibitor LY294002 was enhanced but activator insulin-like growth factor 1 (IGF-1) blocked the Rap1b silencing-induced enhancement of apoptosis and autophagy in MKN-28 and SGC-7901 cells. In conclusion, we demonstrate that Rap1b expression is aberrantly increased in GC, resulting in the inhibition of autophagy and apoptosis of GC cells by the PI3K/Akt/mTOR pathway. This might provide a new understanding and represent a novel therapeutic target for human GC.

Small GTPase Rap1 Is Essential for Mouse Development and Formation of Functional Vasculature.

BACKGROUND: Small GTPase Rap1 has been implicated in a number of basic cellular functions, including cell-cell and cell-matrix adhesion, proliferation and regulation of polarity. Evolutionarily conserved, Rap1 has been studied in model organisms: yeast, Drosophila and mice. Mouse in vivo studies implicate Rap1 in the control of multiple stem cell, leukocyte and vascular cell functions. In vitro, several Rap1 effectors and regulatory mechanisms have been proposed. In particular, Rap1 has been implicated in maintaining epithelial and endothelial cell junction integrity and linked with cerebral cavernous malformations. RATIONALE: How Rap1 signaling network controls mammalian development is not clear. As a first step in addressing this question, we present phenotypes of murine total and vascular-specific Rap1a, Rap1b and double Rap1a and Rap1b (Rap1) knockout (KO) mice. RESULTS AND CONCLUSIONS: The majority of total Rap1 KO mice die before E10.5, consistent with the critical role of Rap1 in epithelial morphogenesis. At that time point, about 50% of Tie2-double Rap1 KOs appear grossly normal and develop normal vasculature, while the remaining 50% suffer tissue degeneration and show vascular abnormalities, including hemorrhages and engorgement of perineural vessels, albeit with normal branchial arches. However, no Tie2-double Rap1 KO embryos are present at E15.5, with hemorrhages a likely cause of death. Therefore, at least one Rap1 allele is required for development prior to the formation of the vascular system; and in endothelium-for the life-supporting function of the vasculature.

The small GTPase Rap1b negatively regulates neutrophil chemotaxis and transcellular diapedesis by inhibiting Akt activation.

Neutrophils are the first line of cellular defense in response to infections and inflammatory injuries. However, neutrophil activation and accumulation into tissues trigger tissue damage due to release of a plethora of toxic oxidants and proteases, a cause of acute lung injury (ALI). Despite its clinical importance, the molecular regulation of neutrophil migration is poorly understood. The small GTPase Rap1b is generally viewed as a positive regulator of immune cell functions by controlling bidirectional integrin signaling. However, we found that Rap1b-deficient mice exhibited enhanced neutrophil recruitment to inflamed lungs and enhanced susceptibility to endotoxin shock. Unexpectedly, Rap1b deficiency promoted the transcellular route of diapedesis through endothelial cell. Increased transcellular migration of Rap1b-deficient neutrophils in vitro was selectively mediated by enhanced PI3K-Akt activation and invadopodia-like protrusions. Akt inhibition in vivo suppressed excessive Rap1b-deficient neutrophil migration and associated endotoxin shock. The inhibitory action of Rap1b on PI3K signaling may be mediated by activation of phosphatase SHP-1. Thus, this study reveals an unexpected role for Rap1b as a key suppressor of neutrophil migration and lung inflammation.

Rap1b in smooth muscle and endothelium is required for maintenance of vascular tone and normal blood pressure.

OBJECTIVE: Small GTPase Ras-related protein 1 (Rap1b) controls several basic cellular phenomena, and its deletion in mice leads to several cardiovascular defects, including impaired adhesion of blood cells and defective angiogenesis. We found that Rap1b(-/-) mice develop cardiac hypertrophy and hypertension. Therefore, we examined the function of Rap1b in regulation of blood pressure. APPROACH AND RESULTS: Rap1b(-/-) mice developed cardiac hypertrophy and elevated blood pressure, but maintained a normal heart rate. Correcting elevated blood pressure with losartan, an angiotensin II type 1 receptor antagonist, alleviated cardiac hypertrophy in Rap1b(-/-) mice, suggesting a possibility that cardiac hypertrophy develops secondary to hypertension. The indices of renal function and plasma renin activity were normal in Rap1b(-/-) mice. Ex vivo, we examined whether the effect of Rap1b deletion on smooth muscle-mediated vessel contraction and endothelium-dependent vessel dilation, 2 major mechanisms controlling basal vascular tone, was the basis for the hypertension. We found increased contractility on stimulation with a thromboxane analog or angiotensin II or phenylephrine along with increased inhibitory phosphorylation of myosin phosphatase under basal conditions consistent with elevated basal tone and the observed hypertension. Cyclic adenosine monophosphate-dependent relaxation in response to Rap1 activator, Epac, was decreased in vessels from Rap1b(-/-) mice. Defective endothelial release of dilatory nitric oxide in response to elevated blood flow leads to hypertension. We found that nitric oxide-dependent vasodilation was significantly inhibited in Rap1b-deficient vessels. CONCLUSIONS: This is the first report to indicate that Rap1b in both smooth muscle and endothelium plays a key role in maintaining blood pressure by controlling normal vascular tone.

Podocyte-specific RAP1GAP expression contributes to focal segmental glomerulosclerosis-associated glomerular injury.

Injury to the specialized epithelial cells of the glomerulus (podocytes) underlies the pathogenesis of all forms of proteinuric kidney disease; however, the specific genetic changes that mediate podocyte dysfunction after injury are not fully understood. Here, we performed a large-scale insertional mutagenic screen of injury-resistant podocytes isolated from mice and found that increased expression of the gene Rap1gap, encoding a RAP1 activation inhibitor, ameliorated podocyte injury resistance. Furthermore, injured podocytes in murine models of disease and kidney biopsies from glomerulosclerosis patients exhibited increased RAP1GAP, resulting in diminished glomerular RAP1 activation. In mouse models, podocyte-specific inactivation of Rap1a and Rap1b induced massive glomerulosclerosis and premature death. Podocyte-specific Rap1a and Rap1b haploinsufficiency also resulted in severe podocyte damage, including features of podocyte detachment. Over-expression of RAP1GAP in cultured podocytes induced loss of activated ß1 integrin, which was similarly observed in kidney biopsies from patients. Furthermore, preventing elevation of RAP1GAP levels in injured podocytes maintained ß1 integrin-mediated adhesion and prevented cellular detachment. Taken together, our findings suggest that increased podocyte expression of RAP1GAP contributes directly to podocyte dysfunction by a mechanism that involves loss of RAP1-mediated activation of ß1 integrin.

Rap1 and Rap2 antagonistically control endothelial barrier resistance.

Rap1 and Rap2 are closely related proteins of the Ras family of small G-proteins. Rap1 is well known to regulate cell-cell adhesion. Here, we have analysed the effect of Rap-mediated signalling on endothelial permeability using electrical impedance measurements of HUVEC monolayers and subsequent determination of the barrier resistance, which is a measure for the ease with which ions can pass cell junctions. In line with its well-established effect on cell-cell junctions, depletion of Rap1 decreases, whereas activation of Rap1 increases barrier resistance. Despite its high sequence homology with Rap1, depletion of Rap2 has an opposite, enhancing, effect on barrier resistance. This effect can be mimicked by depletion of the Rap2 specific activator RasGEF1C and the Rap2 effector MAP4K4, establishing Rap2 signalling as an independent pathway controlling barrier resistance. As simultaneous depletion or activation of both Rap1 and Rap2 results in a barrier resistance comparable to control cells, Rap1 and Rap2 control barrier resistance in a reciprocal manner. This Rap1-antagonizing effect of Rap2 is established independent of junctional actin formation. These data establish that endothelial barrier resistance is determined by the combined antagonistic actions of Rap1 and Rap2.

Rap1b facilitates NK cell functions via IQGAP1-mediated signalosomes.

Rap1 GTPases control immune synapse formation and signaling in lymphocytes. However, the precise molecular mechanism by which Rap1 regulates natural killer (NK) cell activation is not known. Using Rap1a or Rap1b knockout mice, we identify Rap1b as the major isoform in NK cells. Its absence significantly impaired LFA1 polarization, spreading, and microtubule organizing center (MTOC) formation in NK cells. Neither Rap1 isoform was essential for NK cytotoxicity. However, absence of Rap1b impaired NKG2D, Ly49D, and NCR1-mediated cytokine and chemokine production. Upon activation, Rap1b colocalized with the scaffolding protein IQGAP1. This interaction facilitated sequential phosphorylation of B-Raf, C-Raf, and ERK1/2 and helped IQGAP1 to form a large signalosome in the perinuclear region. These results reveal a previously unrecognized role for Rap1b in NK cell signaling and effector functions.

Rap1b is critical for glycoprotein VI-mediated but not ADP receptor-mediated alpha2beta1 activation.

BACKGROUND: The platelet alpha2beta1 integrin functions as both an adhesion and signaling receptor upon exposure to collagen. Recent studies have indicated that alpha2beta1 function can be activated via inside-out signaling, similar to the prototypical platelet integrin alphaIIbbeta3. However, signaling molecules that regulate alpha2beta1 activation in platelets are not well defined. A strong candidate molecule is the small GTPase Rap1b, the dominant platelet isoform of Rap1, which regulates alphaIIbbeta3 activation. OBJECTIVES: We hypothesized that Rap1b positively regulates alpha2beta1 during agonist-induced platelet activation. METHODS: To test whether Rap1b activates alpha2beta1 downstream of glycoprotein (GP)VI or other platelet receptors, we stimulated platelets purified from Rap1b-/- or wild-type mice with diverse agonists and measured alpha2beta1 activation using fluorescein isothiocyanate-labeled monomeric collagen. We also examined the role of Rap1b in outside-in signaling pathways by analyzing adhesion and spreading of Rap1b-/- or wild-type platelets on monomeric, immobilized collagen. Finally, we monitored the activation status of related Rap GTPases to detect changes in signaling pathways potentially associated with Rap1b-mediated events. RESULTS: Rap1b-/- platelets displayed comparable ADP-induced or thrombin-induced alpha2beta1 activation as wild-type platelets, but reduced convulxin-dependent alpha2beta1 activation. Rap1b-/- platelets exhibited increased spreading on immobilized collagen but similar adhesion to immobilized collagen compared to wild-type platelets. Rap1b-/- platelets also showed Rap1a and Rap2 activation upon agonist stimulation, possibly revealing functional compensation among Rap family members. CONCLUSIONS: Rap1b is required for maximal GPVI-induced but not ADP-induced activation of alpha2beta1 in murine platelets.

A critical role of Rap1b in B-cell trafficking and marginal zone B-cell development.

B-cell development is orchestrated by complex signaling networks. Rap1 is a member of the Ras superfamily of small GTP-binding proteins and has 2 isoforms, Rap1a and Rap1b. Although Rap1 has been suggested to have an important role in a variety of cellular processes, no direct evidence demonstrates a role for Rap1 in B-cell biology. In this study, we found that Rap1b was the dominant isoform of Rap1 in B cells. We discovered that Rap1b deficiency in mice barely affected early development of B cells but markedly reduced marginal zone (MZ) B cells in the spleen and mature B cells in peripheral and mucosal lymph nodes. Rap1b-deficient B cells displayed normal survival and proliferation in vivo and in vitro. However, Rap1b-deficient B cells had impaired adhesion and reduced chemotaxis in vitro, and lessened homing to lymph nodes in vivo. Furthermore, we found that Rap1b deficiency had no marked effect on LPS-, BCR-, or SDF-1-induced activation of mitogen-activated protein kinases and AKT but clearly impaired SDF-1-mediated activation of Pyk-2, a key regulator of SDF-1-mediated B-cell migration. Thus, we have discovered a critical and distinct role of Rap1b in mature B-cell trafficking and development of MZ B cells.

Enhanced cortico-amygdala efficacy and suppressed fear in absence of Rap1.

Auditory fear conditioning, a model for fear learning, is thought to be mediated by synaptic changes in the cortical and thalamic inputs to the lateral amygdala (LA); however, the specific roles of both pathways are still debated. Here, we report that a CaMKII-alpha-Cre-mediated knock-out (KO) of the rap1a and rap1b genes impaired synaptic plasticity and increased basal synaptic transmission in the cortical but not thalamic input to the LA via presynaptic changes: increases in glutamate release probability and the number of glutamate quanta released by a single action potential. Moreover, KO mice with alterations in the cortico-LA pathway had impaired fear learning, which could be rescued by training with a more aversive unconditional stimulus. These results suggest that Rap1-mediated suppression of synaptic transmission enables plasticity in the cortico-amygdala pathway, which is required for fear learning with a moderately aversive unconditional stimulus.

Defective angiogenesis, endothelial migration, proliferation, and MAPK signaling in Rap1b-deficient mice.

Angiogenesis is the main mechanism of vascular remodeling during late development and, after birth, in wound healing. Perturbations of angiogenesis occur in cancer, diabetes, ischemia, and inflammation. While much progress has been made in identifying factors that control angiogenesis, the understanding of the precise molecular mechanisms involved is incomplete. Here we identify a small GTPase, Rap1b, as a positive regulator of angiogenesis. Rap1b-deficient mice had a decreased level of Matrigel plug and neonatal retinal neovascularization, and aortas isolated from Rap1b-deficient animals had a reduced microvessel sprouting response to 2 major physiological regulators of angiogenesis: vascular endothelial growth factor (VEGF) and basic fibroblasts growth factor (bFGF), indicating an intrinsic defect in endothelial cells. Proliferation of retinal endothelial cells in situ and in vitro migration of lung endothelial cells isolated from Rap1b-deficient mice were inhibited. At the molecular level, activation of 2 MAP kinases, p38 MAPK and p42/44 ERK, important regulators of endothelial migration and proliferation, was decreased in Rap1b-deficient endothelial cells in response to VEGF stimulation. These studies provide evidence that Rap1b is required for normal angiogenesis and reveal a novel role of Rap1 in regulation of proangiogenic signaling in endothelial cells.