花生四烯酸诱导肌球蛋白磷酸化被抑制的平滑肌细胞 迁移的信号传导途径

在应用肌球蛋白轻链激酶特异抑制剂ML-7抑制了肌球蛋白轻链磷酸化后,花生四烯酸(arachidonic acid,AA)仍可诱导兔血管平滑肌细胞（SM3）发生迁移.为了进一步阐明其信号传导途径,应用多种信号抑制剂,采用免疫印迹、Boyden小室和提取细胞膜蛋白等实验方法,对上述迁移作用的信号传导途径进行了深入的研究.结果显示,PTX（Gi蛋白抑制剂）、U73122（PLC抑制剂）、staurosporine (PKC抑制剂)、PD98059（ERK1/2抑制剂）和SB203580（p38抑制剂）分别可拮抗上述AA诱导的SM3细胞迁移作用,而SP600125（JNK抑制剂）的作用较弱.免疫印迹结果显示,AA可提高SM3细胞中PKC(ε)、ERK1/2、p38和JNK信号的磷酸化水平,呈时间依赖性, PTX或U73122可抑制上述作用;staurosporine可抑制由AA 引起的ERK1/2和JNK的磷酸化水平增强,但对p38的磷酸化水平无影响.还发现AA可促进PLCβ2的细胞膜移位, PTX可抑制其作用.上述结果表明,当肌球蛋白轻链的磷酸化被抑制后, AA可通过Gi蛋白的活化促进PLCβ2向细胞膜移位,进而通过激活PKC(ε)、ERK1/2、p38和JNK等信号转导途径而诱导SM3细胞发生迁移     

内皮素-1对大鼠血管平滑肌细胞产生MCP-1的影响及其机制

目的：观察内皮素-1（ET-1）对大鼠血管平滑肌细胞（VSMCs）产生单核细胞趋化蛋白-1（MCP-1）的影响及其机制。方法：培养大鼠血管平滑肌细胞（VSMCs）。细胞分为2组：ET-1刺激组：以不同浓度ET-1刺激VSMCs不同时间;阻断剂干预组：VSMCs分别与不同阻断剂[ET_AR、ET_BR阻断剂BQ123、BQ788,抗氧化剂N-乙酰半胱氨酸（NAC）,ERK、p38MAPK、JNK及NF-κB抑制剂PD98059、SB203580、SP600125及PDTC]预先孵育30 min,再加入ET-1刺激24 h。在预定时间,以酶联免疫吸附（ELISA）法、逆转录聚合酶链反应（RT-PCR）法分别测定不同因素下VSMCs MCP-1蛋白质及mRNA表达量。VSMCs分别与不同阻断剂（BQ123、BQ788、NAC、PD98059、SB203580及SP600125预先孵育20 min,再加入ET-1刺激5 min,免疫印迹（WB）法测定VSMCs胞浆中细胞外调节蛋白激酶（ERK）、p38丝裂原活化蛋白激酶（p38MAPK）、c-Jun氨基末端激酶（JNK）及其各自磷酸化蛋白质的水平。各项检测均重复3次。结果：ET-1能刺激VSMCs MCP-1蛋白质及mRNA表达,其表达量随ET-1浓度及刺激时间的增加呈升高趋势（P<0.05,P<0.01）;BQ123、NAC、PD98059、SB203580及PDTC能显著抑制ET-1诱导的大鼠VSMCs MCP-1蛋白质及mRNA表达（P<0.01）,而BQ788及SP600125对此作用无明显影响。BQ123、NAC与PD98059或SB203580能分别抑制ET-1刺激后VSMCs胞浆内ERK及p38MAPK的磷酸化（P<0.05,P<0.01）,而ET-1对JNK的磷酸化无明显激活作用。结论：ET-1通过ET_AR、ROS、ERK、p38MAPK及NF-κB诱导大鼠VSMCs产生MCP-1。     

CGRP抑制AngⅡ诱导HUVECs的损伤及其ERK1/2信号通路

目的:探索降钙素基因相关肽(CGRP)对经血管紧张素Ⅱ (AngⅡ)损伤的人脐静脉内皮细胞(HUVECs)的保护作用且CGRP与细胞外信号调节激酶(ERK1/2)的关系.方法:不同浓度的CGRP、AngⅡ处理体外培养的HUVECs,噻唑蓝比色法检测HUVECs活力;流式细胞仪分析HUVECs凋亡率及其增殖指数;显微镜观察HUVECs的形态学变化;Western blot检测p-ERK1/2的表达.结果:AngⅡ (0.1-100 nmol/L)浓度依赖性降低HUVECs的活力,而CGRP (0.1-1000 nmol/L)浓度依赖性增加HUVECs的活力;HUVECs增殖指数PI值受AngⅡ、CGRP及PD98059(ERK1/2抑制剂)影响;AngⅡ孵育HUVECs在第10min时ERK1/2磷酸化水平可达到最大;CGRP能抑制AngⅡ诱导的HUVECs内ERK1/2磷酸化水平;CGRP8-37(CGRP受体拮抗剂)可部分减弱CGRP抑制ERK1/2磷酸化水平作用;PD98059(ERK1/2抑制剂)作用下,ERK1/2磷酸化水平显著降低,但是对细胞内总ERK1/2水平表达无明显影响.结论:CGRP可抑制AngⅡ对HUVECs的损伤作用,可能与CGRP抑制信号通路ERK1/2有关.     

JNK、ERK信号通路在NaAsO2诱导骨髓间充质干细胞增殖中的作用

目的:研究c-ink氨基末端激酶(JNK)、细胞外信号调节激酶(ERK)在亚砷酸钠(NaAsO2)诱导骨髓间充质干细胞(BMSC)增殖中的作用.方法:体外培养骨髓间充质干细胞,四甲基偶氮唑盐比色法(MTT法)检测细胞增殖,Western-blot检测磷酸化JNK、ERK表达水平.结果:低浓度1、2μ mol/L NaAsO2对BMSC有明显的促进增殖作用;高浓度16、32μ mol/LNaAsO2则对细胞生长产生抑制作用,具有一定剂量-效应关系;2、4、8μ mol/LNaAsO2处理BMSC 24h后,JNK磷酸化表达水平明显增加,ERK磷酸化表达水平明显降低;JNK抑制剂SP600125可明显降低高浓度16、32μmol/LNaAsO2的生长抑制作用;ERK抑制剂PD98059可抑制低浓度1、2μ mol/LNaAsO2对BMSC的促增殖作用.结论:低浓度NaAsO2激活ERK信号通路,提高细胞增殖率,可被抑制剂PD98059阻断;高浓度NaAsO2激活JNK信号通路,提高细胞凋亡率,可被抑制剂SP600125阻断.NaAsO2致癌机制可能与JNK、ERK信号通路作用相关.     

柴胡提取物诱导人类白血病细胞HL-60的细胞凋亡及其机理研究

柴胡提取物诱导人类白血病细胞HL-60的细胞凋亡从而抑制其细胞生长.为了研究该过程的作用机理,我们研究了丝裂原活化蛋白激酶(MAPKs),包括胞外信号调节激酶(ERK1/2),c-jun氨基末端蛋白激酶(JNK)和p38丝裂原活化蛋白激酶(MAPK),在该过程中的磷酸化特征与动态变化.结果表明,柴胡提取物显著的增加了p38丝裂原活化蛋白激酶和胞外信号调节激酶(ERK1/2)的磷酸化作用,其增加值在测试范围内与测试剂量和作用时间成正相关,但在柴胡提取物诱导人类白血病细胞HL-60的细胞凋亡过程中,没有发现对氨基末端蛋白激酶(JNK)表现出磷酸化活性.柴胡提取物诱导白血病HL-60的细胞凋亡部分归结于对p38丝裂原活化蛋白激酶的上调节作用,这种上调节作用能够受到p38 MAPK特异性的抑制剂SB203580的部分逆转,而MEK的抑制剂U0126则对柴胡提取物诱导HL-60细胞凋亡过程中的胞外信号调节激酶(ERK1/2)的磷酸化具有显著的协同效应.这是首次报道柴胡提取物在诱导人白血病细胞HL-60细胞凋亡过程中参与p38丝裂原活化蛋白激酶的磷酸化,同时柴胡提取物作为胞外信号调节激酶(ERK1/2)抑制剂的协同作用物具有相应的药物学功能.     

JNK、ERK信号通路在NaAsO_2诱导骨髓间充质干细胞增殖中的作用

目的：研究c-jnk氨基末端激酶（JNK）、细胞外信号调节激酶（ERK）在亚砷酸钠（NaAs02）诱导骨髓间充质干细胞（BMSC）增殖中的作用。方法：体外培养骨髓间充质干细胞,四甲基偶氮唑盐比色法（MTT法）检测细胞增殖,Western-blot检测磷酸化JNK、ERK表达水平。结果：低浓度1、2μmol/LNaAs02对BMSC有明显的促进增殖作用;高浓度16、32μmol/LNaAs02则对细胞生长产生抑制作用,具有一定剂量-效应关系;2、4、8μmol/LNaAs02处理BMSC24h后,JNK磷酸化表达水平明显增加,ERK磷酸化表达水平明显降低;JNK抑制剂SP600125可明显降低高浓度16、32μmol/LNaAs02的生长抑制作用;ERK抑制剂PD98059可抑制低浓度1、2μmol/LNaAs02对BMSC的促增殖作用。结论：低浓度NaAs02激活ERK信号通路,提高细胞增殖率,可被抑制剂PD98059阻断;高浓度NaAs02激活JNK信号通路,提高细胞凋亡率,可被抑制剂SP600125阻断。NaAs02致癌机制可能与JNK、ERK信号通路作用相关。     

ERK激活对SDF-1引起的大鼠海马神经元GABA分泌抑制的影响

目的:观察细胞外调节激酶(ERK)信号通路激活对基质细胞衍生因子(SDF-1)引起离体培养的大鼠海马神经元γ-氨基丁酸(GABA)分泌的影响。方法:新生SD大鼠海马神经元离体培养,Western blot法观察ERK1/2信号通路的磷酸化水平;ELISA法和RT-PCR技术检测ERK1/2特异性阻断剂PD98059作用于离体培养的海马神经元后GABA分泌的改变;谷氨酸脱羧酶(GAD65/67)和γ-氨基丁酸转运体(GAT)的蛋白表达量及GAD65和GAT-1 mRNA表达水平。结果:SDF-1作用于海马神经元可引起ERK1/2磷酸化水平明显升高,同时加用CXCR4受体阻断剂AMD3100,可阻断SDF1引起的ERK1/2激活;SDF-1可明显抑制离体培养的海马神经元GABA的分泌,同时加用ERK1/2特异性抑制剂PD98059,可部分逆转SDF-1对GABA分泌的抑制作用;SDF-1作用于离体培养的海马神经元,可抑制谷氨酸脱羧酶GAD65和GABA转运体GAT-1 mRNA的生成;ERK抑制剂PD98059可有效翻转SDF-1的作用。Western blot结果发现SDF-1可抑制海马神经元GAT-1和GAD65/67蛋白的表达,加用ERK1/2抑制剂可部分恢复GAT-1和GAD65/67蛋白合成。结论:SDF1作用于离体培养的海马神经元CXCR4,通过激活ERK1/2信号通路进而抑制GAD蛋白表达,可能是其介导GABA分泌抑制的通路之一。     

细胞外信号调节激酶在小鼠神经干细胞增殖中的作用

本文旨在探讨细胞外信号调节激酶(extracellular signal-regulated kinase 1/2, ERK1/2)对小鼠神经干细胞增殖的影响.分离E14.5小鼠皮层神经干细胞,通过Western blot检测神经干细胞增殖过程中磷酸化ERK1/2的表达情况,以及不同浓度PD98059处理对神经干细胞ERK1/2磷酸化及神经球形成的影响,并用CCK-8法检测PD98059对神经干细胞增殖的影响.结果显示:ERK1/2在体外培养的神经下细胞增殖过程中被激活;PD98059显著抑制ERK1/2磷酸化及神经干细胞的成球率,且存在剂量效应依赖关系;加入PD98059后神经干细胞的生长被抑制.以上结果表明,ERK1/2在小鼠神经干细胞增殖中具有重要的作用,阻断ERK1/2信号通路后可抑制神经干细胞的增殖.     

ERK1/2通路参与15-羟二十碳四烯酸收缩缺氧大鼠肺动脉的过程

缺氧诱导的15-羟二十碳四烯酸（15-hydroxyeicosatetraenoic acid,15-HETE）是引起肺动脉收缩的重要介导因子。15-HETE引起肺动脉收缩的信号转导途径尚不清楚。本研究旨在确定细胞外信号调节激酶1／2（extracellular signal-regulated kinase-1／2,ERK1／2）信号转导通路是否参与15-HETE收缩缺氧火鼠肺动脉的过程。采用组织浴槽肺动脉环张力检测、蛋白质免疫印迹Western blot）和免疫细胞化学方法。制备缺氧大鼠动物模型,成年雄性Wistar大鼠在低氧环境下（吸入氧分数为0．12）正常喂养9d。显微分离直径1-1．5mm肺动脉,剪成长为3mm的动脉环,进行血管张力检测。用ERK1／2上游激酶（MEK）抑制剂PD98059抑制ERK1／2活性。结果显示,PD98059可明显抑制15-HETE对缺氧大鼠肺动脉环的收缩作用。在去除内皮的肺动脉环,PD98059仍叮明显降低15-HETE的缩血管作用。Western blot和免疫细胞化学结果都显示,15-HETE能促进ERK1／2磷酸化。由此表明ERK1／2信号转导通路参与15-HETE收缩缺氧大鼠肺动脉的过程。     

在fMLP刺激的HL-60细胞中PI3-K介导的肌动蛋白聚合

趋化肽fMLP能够诱导中性粒细胞粘附、游走和吞噬.为了澄清这种趋化反应的发生机理,将HL-60细胞诱导分化为中性粒细胞样细胞,然后利用激酶特异性抑制剂研究了在fMLP刺激下细胞内PI3-K、p38和ERK激酶在肌动蛋白聚合中的作用.结果0.1 μmol/L的PI3-K抑制物Wortmannin抑制fMLP诱导的肌动蛋白聚合;而50 μmol/L的p38激酶抑制物SB203580和50 μmol/L的ERK激酶抑制物PD098059对fMLP诱导的肌动蛋白聚合没有影响, 但p38和ERK在不同程度上受到PI3-K的调节.这说明PI3-K介导的肌动蛋白聚合信号传导途径不同于PI3-K介导的p38和ERK激活途径.     

Ferulic acid inhibits endothelial cell proliferation through NO down-regulating ERK1/2 pathway

The aim of this study was to determine the antiproliferative mechanism of ferulic acid (FA) on serum induced ECV304 cell, a human umbilical vein endothelial line. The results suggest that FA significantly suppressed ECV304 cells proliferation and blocked the cell cycle in G0/G1 phase. Treatment of the cells with FA increased nitric oxide (NO) production and inactivated the extracellular signal-regulated kinase (EERK1/2), and the NO donor, sodium nitroprusside, inhibited both ECV304 cells proliferation and phosphorylation of ERK1/2. However, the NO synthase inhibitor, Nomega-nitro-L-arginine methyl ester, caused ECV304 cells proliferation. PD 98059, the inhibitor of ERK1/2, had no effect on the NO production. These results indicate that NO suppressed ECV304 cells proliferation through down-regulating ERK1/2 pathway. Moreover, the inhibition of cell cycle progression was associated with the decrement of cyclin D1 expression and phosphorylation of retinoblastoma protein (pRb) by increment of p21 level. The findings not only present the first evidence that FA is a potent inhibitor on ECV304 cells proliferation, but also reveal the potential signaling molecules involved in its action.     

Upregulation of vascular endothelial growth factor by heat-killed Listeria monocytogenes in macrophages

We investigated the effect of heat-killed Listeria monocytogenes (HKLM) on the expression of vascular endothelial growth factor (VEGF) in RAW264.7 macrophage-like cells. The expression of VEGF was induced in RAW264.7 cells treated with HKLM. Pretreatment of cells with cycloheximide, a protein synthesis inhibitor, inhibited the induction of VEGF mRNA by HKLM. Induction of VEGF by HKLM was partially inhibited by treatment of cells with SB203580, a p38 mitogen-activated protein kinase (MAPK) inhibitor, or a neutralizing antibody against tumor necrosis factor-alpha (TNF-alpha). In addition, HKLM induced phosphorylation of p38 MAPK. These results suggest that p38 MAPK and TNF-alpha are involved in the VEGF expression induced by HKLM in RAW264.7 cells. We confirmed that increased VEGF expression is immunohistochemically detected in splenic macrophages of mice infected with L. monocytogenes (L. monocytogenes). VEGF is thought to be involved in inflammatory reactions induced by L. monocytogenes infection.     

Inhibition of the MEK/ERK signaling pathway by the novel antimetastatic agent NAMI-A down regulates c-myc gene expression and endothelial cell proliferation.

Imidazolium trans-imidazoledimethyl sulfoxide-tetrachlororuthenate (NAMI-A) is a novel ruthenium-containing experimental antimetastatic agent. Compelling evidence ascribes a pivotal role to endothelial cells in the orchestration of tumor angiogenesis and metastatic growth, suggesting antiangiogenic therapy as an attractive approach for anticancer treatment. In this context, activation of the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) signaling pathway has been found fundamental in transducing extracellular stimuli that modulate a number of cellular process including cell proliferation, migration and invasion. Here we show that exposure of the transformed endothelial cell line ECV304 to NAMI-A significantly inhibited DNA synthesis, as well as the expression of the proliferating cell nuclear antigene (PCNA). These responses were associated with a marked down-regulation of ERK phosphorylation in serum-cultured cells. In addition, NAMI-A markedly reduced serum stimulated- and completely suppressed phorbol 12-myristate 13-acetate (PMA)-triggered MAPK/ERK kinase activity. NAMI-A was also able to inhibit the phosphorylation of MEK, the upstream activator of ERK, and, similar to both the protein kinase C (PKC) inhibitor GF109203X and the MAPK/ERK (MEK) inhibitor PD98059, it completely counteracted PMA-induced ERK phosphorylation. Finally, NAMI-A and PD98059 down regulated c-myc gene expression to the same extent in serum-cultured cells and dose-dependently counteracted, and ultimately abolished, the increase in c-myc gene expression elicited by PMA in serum-free cells. These results suggest that inhibition of MEK/ERK signaling by NAMI-A may have an important role in modulating c-myc gene expression and ECV304 proliferation.     

PACAP-27 tyrosine phosphorylates mitogen activated protein kinase and increases VEGF mRNAs in human lung cancer cells

The effects of pituitary adenylate cyclase activating polypeptide (PACAP) on human lung cancer cell line NCI-1299 mitogen activated protein kinase (MAPK) tyrosine phosphorylation and vascular endothelial cell growth factor (VEGF) expression were investigated. PACAP-27 (100 nM) increased MAPK tyrosine phosphorylation 3-fold, 5 min after addition to NCI-H1299 cells. PACAP caused tyrosine phosphorylation in a concentration-dependent manner being half-maximal at 10 nM PACAP-27. PACAP-27 or PACAP-38 (100 nM) but not PACAP28-38 or VIP caused increased MAPK tyrosine phosphorylation using NCI-H1299 cells. Also, the increase in MAPK tyrosine phosphorylation caused by PACAP-27 was totally inhibited by 10 microM PACAP(6-38), a PAC(1) receptor antagonist or 10 microM PD98059, a MAPKK inhibitor. These results suggest that PAC(1) receptors regulate tyrosine phosphorylation of MAPK in a MAPKK-dependent manner. PACAP-27 (100 nM) caused increased VEGF mRNA in NCI-H1299 cells after 8 h. The increase in VEGF mRNA caused by PACAP-27 was partially inhibited by PACAP(6-38), PD98059 and H-89. Addition of VIP to NCI-H1299 cells caused increased VEGF mRNA, which was totally inhibited by H89, a PKA inhibitor. These results suggest that PAC(1) and VPAC(1) receptors regulate VEGF expression in lung cancer cells.     

MAPK pathway mediates the protective effects of onychin on oxidative stress-induced apoptosis in ECV304 endothelial cells

Our recent studies have shown that onychin could protect rabbit aortic rings from lysophosphatidylcholine-induced injury by preserving endothelium-dependent relaxation and alleviating acute endothelial damage mediated by oxidative stress. However, the effect of onychin on apoptosis of endothelial cells induced by oxidative stress was not evaluated. In the present study, we investigated the effect of onychin on Hydrogen Peroxide (H2O2) induced apoptosis of ECV304 endothelial cells. Cultured human umbilical vein endothelial cell line (ECV304) was pretreated with vehicle (DMSO), genistein, or different concentrations of onychin (0.1, 0.3, 1, 3, and 10 micromol/L) for 30 minutes and then exposed to 1 mmol/L H2O2 for 24 hours. Cell apoptosis was determined by TUNEL and flow cytometric analysis. Meanwhile, Western-blot was used to measure the expression of phospho-ERK1/2, phospho-p38 and caspase-3. Our data showed that onychin treatment exhibited a protective effect on ECV304 endothelial cells from H2O2-induced apoptosis in a concentration-dependent manner. Moreover, onychin attenuated H2O2-induced phosphorylation of p38MAPK and increased H2O2-induced phosphorylation of ERK1/2. Furthermore, onychin decreased the activation of caspase-3. The opposing effects of onychin on phosphorylation levels of p38MAPK and ERK1/2, and its caspase-3 inhibition might play a role in the beneficial effect of onychin on endothelial injury.     

Regulation of vascular endothelial growth factor expression by EMMPRIN via the PI3K-Akt signaling pathway

Extracellular matrix metalloproteinase (MMP) inducer (EMMPRIN) is a cell surface glycoprotein overexpressed in many solid tumors. In addition to its ability to stimulate stromal MMP expression, tumor-associated EMMPRIN also induces vascular endothelial growth factor (VEGF) expression. To explore the underlying signaling pathways used by EMMPRIN, we studied the involvement of phosphoinositide 3-kinase (PI3K)-Akt, mitogen-activated protein kinase (MAPK), JUN, and p38 kinases in EMMPRIN-mediated VEGF regulation. Overexpression of EMMPRIN in MDA-MB-231 breast cancer cells stimulated the phosphorylation of only Akt and MAPKs but not that of JUN and p38 kinases. Conversely, inhibition of EMMPRIN expression resulted in suppressed Akt and MAPK phosphorylation. Furthermore, the PI3K-specific inhibitor LY294002 inhibited VEGF production by EMMPRIN-overexpressing cells in a dose- and time-dependent manner. On the other hand, the MAPK inhibitor U0126 did not affect VEGF production. In vivo, EMMPRIN-overexpressing tumors with elevated VEGF expression had a high level of phosphorylation of Akt and MAPK. Finally, when fibroblast cells were treated with recombinant EMMPRIN, Akt kinase but not MAPK was phosphorylated concomitant with an increase in VEGF production. Both the activation of Akt kinase and the induction of VEGF were specifically inhibited with a neutralizing antibody to EMMPRIN. Our results show that in both tumor and fibroblast cells EMMPRIN regulates VEGF production via the PI3K-Akt pathway but not via the MAPK, JUN, or p38 kinase pathways.     

Sphingosine kinase activation regulates hepatocyte growth factor induced migration of endothelial cells

Hepatocyte growth factor (HGF)-induced migration of endothelial cells is critical for angiogenesis. Sphingosine kinase (SPK) is a key enzyme catalyzing the formation of sphingosine-1-phosphate (S1P), a lipid messenger that is implicated in the regulation of a wide variety of important cellular events through both intracellular and extracellular mechanisms. The aim of this study was to investigate whether activation of SPK is involved in the migration of endothelial cells induced by HGF. The biological functions of HGF are mediated through the activation of its high-affinity tyrosine kinase receptor, c-met protooncogene. In the present study, Treatment of ECV304 endothelial cells with HGF resulted in tyrosine phosphorylation of c-Met and activation of SPK in a concentration-dependent manner. Either Ly294002 or PD98059, specific inhibitor of the PI3K and ERK/MAPK pathways, respectively, blocked the HGF-induced activation of SPK. HGF stimulation significantly increased intracellular S1P level, but no detectable secretion of S1P into the cell culture medium was observed. Treatment of ECV304 cells with pertussis toxin (PTX) has no effect on the HGF-induced migration, indicating extracellular S1P is dispensable for this process. Overexpression of wild-type SPK gene in ECV 304 cells increased the intracellular S1P and enhanced the HGF-induced migration, whereas inhibition of cellular SPK activity by N,N-dimethylsphingosine (DMS), a potent inhibitor of SPK, or by expression of a dominant-negative SPK (DN-SK) blocked the HGF-induced migration of ECV 304 cells. It is suggested that PI3K and ERK/MAPK mediated the activation of SPK and would be involved in the HGF-induced migration of endothelial cells. These results elucidate a novel mechanism by which intracellularly generated S1P mediates signaling from HGF/c-Met to the endothelial cell migration.     

Endogenous production of nitric oxide by vascular endothelial growth factor down-regulates proliferation of choriocarcinoma cells

The trophoblast-like choriocarcinoma cell line BeWo expresses a receptor for vascular endothelial growth factor (VEGF) and proliferates in response to VEGF. Nitric oxide (NO) seems to play a key role in the VEGF-induced proliferation of endothelial cells but the NO mechanistic regulation of VEGF-stimulated trophoblast proliferation is presently unclear. We assessed the effect of exogenous VEGF on BeWo cell proliferation by [3H]thymidine incorporation. The VEGF-induced proliferation of BeWo cells was significantly increased by the NO synthase (NOS) inhibitor, N(omega)-nitro-l-arginine methyl ester (L-NAME), but was inhibited by the NO donor, sodium nitroprusside. Treatment of the cells with 10 ng/ml of VEGF increased not only eNOS expression but also NO production. The extracellular signal-regulated kinase (Erk) of the mitogen-activated protein kinase (MAPK) family was activated by VEGF as demonstrated by the phosphorylation of Erk in Western blots. The effects of VEGF on NO production and the expression of endothelial NOS (eNOS) were attenuated by treating BeWo cells with the selective inhibitor of MAPK kinase, PD98059. VEGF-stimulated proliferation of BeWo cells was inhibited by the tyrosine kinase inhibitor genistein but increased by PD98059. Other kinase inhibitors, including LY294002 (phosphoinositide 3-kinase inhibitor) and SB203580 (P38 MAPK inhibitor), had no effect on the proliferation of the cells and NO production. These results indicate that endogenous NO production down-regulates the VEGF-stimulated proliferation of BeWo cells and that the activation of Erk plays an important role in this mechanism.     

An involvement of SR-B1 mediated PI3K-Akt-eNOS signaling in HDL-induced cyclooxygenase 2 expression and prostacyclin production in endothelial cells

It is well-known that sphingosine-1-phosphate (S1P), the phospholipid content of HDL, binding to S1P receptors can raise COX-2 expression and PGI(2) release through p38MAPK/CREB pathway. In the present study we assess the action of SR-B1 initiated PI3K-Akt-eNOS signaling in the regulation of COX-2 expression and PGI(2) production in response to HDL. We found that apoA1 could increase PGI(2) release and COX-2 expression in ECV 304 endothelial cells. Furthermore, SR-B1 was found to be involved in HDL induced up-regulation of COX-2 and PGI(2). Over-expressed SR-B1 did not significantly increase the expression of COX-2 and the PGI(2) levels, but knock-down of SR-B1 by siRNA could significantly attenuate COX-2 expression and PGI(2) release together with p38MAPK and CREB phosphorylation. Consistently, the declines of p-p38MAPK, p-CREB, COX-2 and PGI(2) were also observed after incubation with LY294002 (25μmol/L; PI3K special inhibitor) or L-NAME (50μmol/L; eNOS special inhibitor). In addition, we demonstrated the increases of PGI(2) release, COX-2 expression and p38MAPK phosphorylation, when nitric oxide level was raised through the incubation of L-arginine (10 or 20nmol/L) in endothelial cells. Taking together, our data support that SR-B1 mediated PI3K-Akt-eNOS signaling was involved in HDL-induced COX-2 expression and PGI(2) release in endothelial cells.     

RNA-seq analysis provide new insights into mapk signaling of apolipoproteinciii-induced inflammation in porcine vascular endothelial cells

Apolipoprotein CIII (ApoCIII) has been shown to be associated with the inflammatory response, but the mechanism of its inflammatory effects remains unclear. Because vascular endothelial cells (VECs) play a key role in the development of inflammation, the present study was performed to investigate inflammatory mechanisms induced by ApoCIII in VECs. In this study, we screened differentially expressed genes (DEGs) using RNA-sequencing. The results identified 390 up-regulated genes and 257 down-regulated genes. We performed GO functional classification and KEGG pathway analysis for DEGs. Analysis of sequencing data showed that 21 genes were related to the MAPK pathway. Finally, we investigated whether ApoCIII regulates the expression of pro-inflammatory cytokines via MAPK signaling pathway. The results showed that ApoCIII increased the expression levels of IL-6, TNF-α, VCAM-1 and ICAM-1 in VECs. ApoCIII activated the phosphorylation of ERK1/2 and p38 MAPK. An inhibitor of ERK1/2 and p38 MAPK decreased the protein levels of IL-6 and TNF-α. Our findings demonstrate that ApoCIII induces pro-inflammatory cytokine production in VECs via activation of ERK1/2 and p38 MAPK phosphorylation.