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ScientificWorldJournal
2019 Jan 01;2019:4639165. doi: 10.1155/2019/4639165.
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RASGRP2 Suppresses Apoptosis via Inhibition of ROS Production in Vascular Endothelial Cells.
Sato T
,
Takino JI
,
Nagamine K
,
Nishio K
,
Hori T
.
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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.
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