Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
ScientificWorldJournal
2019 Jan 01;2019:4639165. doi: 10.1155/2019/4639165.
Show Gene links
Show Anatomy links
RASGRP2 Suppresses Apoptosis via Inhibition of ROS Production in Vascular Endothelial Cells.
Sato T
,
Takino JI
,
Nagamine K
,
Nishio K
,
Hori T
.
???displayArticle.abstract???
We have identified ras guanyl releasing protein 2 (rasgrp2) as a blood vessel related gene from Xenopus embryo. In addition, we reported that RASGRP2 is also expressed in human umbilical vein endothelial cells (HUVEC). It is known that RASGRP2 activates Ras-related protein 1 (Rap1). However, the function of RASGRP2 in human vascular endothelium remains unknown. Therefore, we performed functional analysis of RASGRP2 using immortalized HUVEC (TERT HUVEC). We established a stable RASGRP2 overexpressing cell line (TERT HUVEC R) and mock cell line (mock). Furthermore, we compared the activity of Rap1 and the generation of intracellular reactive oxygen species (ROS), which is related to cell death, in both cell lines. Significant increase in Rap1 activity was observed in the TERT HUVEC R compared to the mock. Furthermore, apoptosis by tumor necrosis factor-α (TNF-α) stimulation was significantly more reduced in the TERT HUVEC R than in the mock. In the mock, apoptosis induced by TNF-α stimulation was decreased by pretreatment with diphenyleneiodonium (DPI), which is an inhibitor of NADPH oxidase (NOX). However, in the TERT HUVEC R, apoptosis induced by TNF-α stimulation was not reduced after pretreatment of DPI. Furthermore, there was no reduction in ROS production in the TERT HUVEC R after DPI pretreatment. In addition, the difference in the degree of apoptosis induced by TNF-α stimulation in both cell lines was consistent with the difference in ROS production in the cell lines. From these results, it was suggested that RASGRP2 activates Rap1 and the activated Rap1 suppresses apoptosis via NOX inhibition.
Figure 1. Effect of RASGRP2 overexpression on Rap1 activation and NOX4 expression. (a) Expression of endogenous RASGRP2 protein with overexposure using X-ray film. 1: HUVEC, 2: TERT HUVEC. (b) Activated Rap1 in TERT HUVEC was recovered using RalGDS-RBD Agarose Beads and validated by western blot. M: mock cells; R: TERT HUVEC R cells. 1 and 2: each of two single cell clones obtained by establishment. (c) Expression of NOX4 protein in TERT HUVEC.
Figure 2. Cell viability by TNF-α stimulation. Cells were treated with 20 ng/mL TNF-α for either 24 h or 48 h. Cell viability at 0 h was taken as 100%. Blue circle: Mock untreated; red cross: Mock TNF-α; green asterisk: TERT HUVEC R untreated; purple square: TERT HUVEC R TNF-α; n=3; a = P<0.01; Mock untreated versus Mock TNF-α; b = P<0.01, TERT HUVEC R untreated versus TERT HUVEC R TNF-α; c = P<0.01, Mock TNF-α versus TERT HUVEC R TNF-α.
Figure 3. Effect of ROS inhibitors on TNF-α stimulation. Cells were treated with 20 ng/mL TNF-α for either 24 h or 48 h. Cell viability at 0 h was taken as 100%. (a) and (b) represent the cell viability of the mock cells and TERT HUVEC R cells pretreated with 5 mM NAC, respectively. Blue circle: untreated; red cross: TNF-α; green asterisk: NAC; purple square: TNF-α + NAC. (c) and (d) represent the cell viability of mock cells and TERT HUVEC R cells pretreated with 20 μM DPI, respectively. Blue circle: untreated; red cross: TNF-α; green asterisk: DPI; purple square: TNF-α + DPI. (e) and (f) represent the cell viability of mock cells and TERT HUVEC R cells pretreated with 30 μM apocynin, respectively. Blue circle: untreated; red cross: TNF-α; green asterisk: apocynin; purple square: TNF-α + apocynin. n=3; A = P<0.01, untreated versus TNF-α; B = P<0.01, TNF-α versus TNF-α + NAC; C = P<0.01, TNF-α versus TNF-α + DPI; D = P<0.01, TNF-α versus TNF-α + apocynin.
Figure 4. Apoptosis by TNF-α stimulation. Each cell line was treated with 20 ng/mL TNF-α for 24 h and stained using NucView 488. (a) Cells were photographed with fluorescence microscopy (40× magnification). (b) A fluorescent image was taken and digitized using ImageJ. M: mock cells, R: TERT HUVEC R cells. n=3; ∗∗p < 0.01.
Figure 5. Change in intracellular ROS amount by TNF-α stimulation. Each cell line was treated with 20 ng/mL TNF-α for 4 h and stained using CellROX® Green. (a) Cells were photographed with fluorescence microscopy (40× magnification). (b) A fluorescent images were taken and digitized using ImageJ. M: mock cells; R: TERT HUVEC R cells. n=3; ∗∗p < 0.01.
Figure 6. Effect of Rap1 siRNA on TNF-α stimulation. (a) Activated Rap1 in TERT HUVEC under Rap1 siRNA conditions was recovered using RalGDS-RBD agarose beads and validated by western blot. M: mock cells; R: TERT HUVEC R cells. (b) and (c) represent the cell viability of mock cells and TERT HUVEC R cells in TNF-α stimulation under condition Rap1 knocked down. Blue circle: control siRNA; red cross: control siRNA + TNF-α; green asterisk: Rap1 siRNA; purple square: Rap1 siRNA + TNF-α; n=3; A = P<0.01, Rap1 siRNA versus Rap1 siRNA + TNF-α; B = P<0.01, control siRNA + TNF-α versus Rap1 siRNA + TNF-α.
Figure 7. Proposed model of signaling pathway involving RASGRP2 in vascular endothelial cells.
Ago,
Nox4 as the major catalytic component of an endothelial NAD(P)H oxidase.
2004, Pubmed
Ago,
Nox4 as the major catalytic component of an endothelial NAD(P)H oxidase.
2004,
Pubmed
Basuroy,
Nox4 NADPH oxidase mediates oxidative stress and apoptosis caused by TNF-alpha in cerebral vascular endothelial cells.
2009,
Pubmed
Birukova,
Rap-afadin axis in control of Rho signaling and endothelial barrier recovery.
2013,
Pubmed
Caloca,
F-actin-dependent translocation of the Rap1 GDP/GTP exchange factor RasGRP2.
2004,
Pubmed
Canault,
Human CalDAG-GEFI gene (RASGRP2) mutation affects platelet function and causes severe bleeding.
2014,
Pubmed
Carbo,
Integrin-independent role of CalDAG-GEFI in neutrophil chemotaxis.
2010,
Pubmed
Cullere,
Regulation of vascular endothelial barrier function by Epac, a cAMP-activated exchange factor for Rap GTPase.
2005,
Pubmed
Datla,
Important role of Nox4 type NADPH oxidase in angiogenic responses in human microvascular endothelial cells in vitro.
2007,
Pubmed
Deng,
Inhibition of protein kinase C β(2) prevents tumor necrosis factor-α-induced apoptosis and oxidative stress in endothelial cells: the role of NADPH oxidase subunits.
2012,
Pubmed
Dupuy,
Activation of the Rap1 guanine nucleotide exchange gene, CalDAG-GEF I, in BXH-2 murine myeloid leukemia.
2001,
Pubmed
Englaro,
Tumor necrosis factor alpha-mediated inhibition of melanogenesis is dependent on nuclear factor kappa B activation.
1999,
Pubmed
Hanahan,
Accessories to the crime: functions of cells recruited to the tumor microenvironment.
2012,
Pubmed
Katagiri,
Rap1-mediated lymphocyte function-associated antigen-1 activation by the T cell antigen receptor is dependent on phospholipase C-gamma1.
2004,
Pubmed
Liu,
Dengue virus infection differentially regulates endothelial barrier function over time through type I interferon effects.
2009,
Pubmed
Miyazaki,
Establishment of a method for evaluating endothelial cell injury by TNF-α in vitro for clarifying the pathophysiology of virus-associated acute encephalopathy.
2017,
Pubmed
Nagamine,
Blood cell and vessel formation following transplantation of activin-treated explants in Xenopus.
2007,
Pubmed
,
Xenbase
Nagamine,
XRASGRP2 expression during early development of Xenopus embryos.
2008,
Pubmed
,
Xenbase
Nagamine,
Identification of the gene regulatory region in human rasgrp2 gene in vascular endothelial cells.
2010,
Pubmed
,
Xenbase
Riches,
Trisomy 12 chronic lymphocytic leukemia cells exhibit upregulation of integrin signaling that is modulated by NOTCH1 mutations.
2014,
Pubmed
Stadtmann,
Rap1a activation by CalDAG-GEFI and p38 MAPK is involved in E-selectin-dependent slow leukocyte rolling.
2011,
Pubmed
Stone,
Regulation and Function of the RasGRP Family of Ras Activators in Blood Cells.
2011,
Pubmed
Stork,
Multiple roles of Rap1 in hematopoietic cells: complementary versus antagonistic functions.
2005,
Pubmed
Subramanian,
Phosphorylation of CalDAG-GEFI by protein kinase A prevents Rap1b activation.
2013,
Pubmed
Takino,
Ras guanyl nucleotide releasing protein 2 affects cell viability and cell-matrix adhesion in ECV304 endothelial cells.
2013,
Pubmed
,
Xenbase
Xia,
Luteolin protects HUVECs from TNF-α-induced oxidative stress and inflammation via its effects on the Nox4/ROS-NF-κB and MAPK pathways.
2014,
Pubmed
Yan,
Nox4 and redox signaling mediate TGF-β-induced endothelial cell apoptosis and phenotypic switch.
2014,
Pubmed
Zhang,
The role of inflammatory cytokines in endothelial dysfunction.
2008,
Pubmed