PI3K信号通路介导apelin-13促进单核细胞-血管内皮细胞黏附的研究

本室以前已经报道G蛋白偶联受体APJ的内源性配体多肽apelin-13促进单核细胞-血管内皮细胞黏附,本文研究PI3K信号途径是否参与apelin-13促进单核细胞-血管内皮细胞黏附,探讨apelin/APJ系统的细胞信号转导机制.MPO方法检测细胞黏附;Western blot方法检测PI3K、VCAM-1的表达.Western blot方法结果显示,apelin-13(0、0.5、1、2、4μmol/L)浓度依赖性刺激血管内皮细胞PI3K磷酸化,以1μmol/L最为明显;1μmol/L apelin-13时间依赖性促进血管内皮细胞PI3K磷酸化,在30 min增加最为显著;PI3K抑制剂LY294002明显抑制apelin-13诱导的VCAM-1表达和单核细胞-血管内皮细胞黏附.上述结果表明,PI3K信号途径介导apelin-13促进单核细胞-血管内皮细胞黏附.     

ERK1/2参与自发性高血压大鼠离体血管环对apelin-13舒张反应性降低作用

研究自发性高血压大鼠(spontanously hypertensive rat,SHR)离体血管环对G蛋白偶联受体APJ的内源性配体apelin-13的血管收缩与舒张反应及其与一氧化氮(NO)和ERK1/2通路关系．采用离体血管环体外灌流方法用Power-Lab生物信息采集仪检测血管环的张力．实验分组如下：新福林(Phenylephrine,PE)组,乙酰胆碱(acetylcholine,Ach)组,apelin-13组,apelin-13 + PE组,apelin-13 + Ach组,PD98059(ERK1/2抑制剂) + PE组,PD98059 + Ach组,LNNA(L-nitro-arginine,硝基左旋精氨酸,一氧化氮合酶抑制剂) + PE组,LNNA+Ach组,apelin-13(预孵育) + PD98059 + PE组,apelin-13(预孵育)+PD98059+ Ach组,apelin-13(预孵育) + LNNA + PE组和apelin-13(预孵育) + LNNA + Ach组,以WKY大鼠血管环为对照组．培养大鼠血管平滑肌细胞,Western blot检测ERK1/2蛋白表达．结果显示：a．apelin-13对于有内皮的血管表现出浓度依赖性舒张作用,血管舒张百分比SHR < WKY大鼠,而对于去除内皮血管,apelin-13则表现出收缩血管的作用,且收缩张力SHR>WKY大鼠,apelin-13预孵育,能减少SHR和WKY大鼠血管对新福林的缩血管反应性,增加对乙酰胆碱的舒张反应性;b．NOS抑制剂LNNA阻断NO形成后,血管环对apelin-13的舒张反应明显抑制,且SHR组较WKY组对apelin的舒张反应减少更明显,提示apelin-13的舒血管效应至少部分依赖NO通路,而SHR高血压大鼠NO通路障碍减弱了apelin对血管的舒张作用;c．ERK1/2抑制剂PD98059预孵育后血管环对apelin-13表现出浓度依赖性的收缩,与去除内皮后apelin-13的收缩血管效应趋势一致,血管收缩张力SHR＞WKY大鼠,PD98059逆转了apelin-13引起的血管舒张效应;d．Apelin-13促大鼠VSMCs ERK1/2磷酸化增加并呈剂量依赖性和时间依赖性,ERK1/2抑制剂PD98059可以减少apelin-13诱导ERK1/2的磷酸化．结果表明,自发性高血压大鼠离体血管环对apelin-13舒张反应性降低, NO通路和ERK1/2通路介导了apelin-13的舒张血管作用．     

转化生长因子-β1及Sirt1/2参与高血压诱导的血管平滑肌细胞缝隙连接蛋白-43的表达及细胞增殖

为探讨转化生长因子-β1及组蛋白去乙酰化酶Sirt1和Sirt2在高血压诱导的血管平滑肌细胞缝隙连接蛋白-43表达及细胞增殖中的作用,研究了腹主动脉窄缩诱导高血压大鼠和正常大鼠胸主动脉转化生长因子-β1、缝隙连接蛋白-43、Sirt1和Sirt2,以及细胞增殖标志蛋白增殖细胞核抗原表达的变化;观察Sirt1和Sirt2在转化生长因子-β1刺激大鼠VSMCs缝隙连接蛋白-43的表达及细胞增殖中的作用。结果显示,高血压大鼠胸主动脉的转化生长因子-β1、缝隙连接蛋白-43、TGF-β1、Sirt1和Sirt2的表达及细胞增殖较正常大鼠均明显升高;转化生长因子-β1促进了大鼠血管平滑肌细胞缝隙连接蛋白-43的表达和增殖,Sirt1与Sirt2的抑制剂Salermide有效抑制了转化生长因子-β1诱导的血管平滑肌细胞缝隙连接蛋白-43的表达与细胞增殖。结果表明,高血压通过上调转化生长因子-β1来诱导VSMCs缝隙连接蛋白-43的表达和细胞增殖,而Sirt1和Sirt2可能在其中起调控作用。     

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。     

血管紧张素-(1-7)对大鼠血管平滑肌细胞PKC和ERK1/2的抑制作用

采用Westernblot、氚 胸腺嘧啶 ( 3H TdR)和氚 亮氨酸 ( 3H Leu )掺入等技术和方法 ,用血管紧张素Ⅱ(AngⅡ )和血管紧张素 ( 1 7) [Ang ( 1 7) ]刺激大鼠血管平滑肌细胞 (VSMCs) ,观察和分析Ang ( 1 7)对VSMCs增殖及蛋白激酶C (PKC)和胞外调节蛋白激酶 (ERK)表达的影响。Ang ( 1 7)能明显抑制基础和AngⅡ刺激下的VSMCsPKC ζ和ERK1/ 2蛋白表达 (P <0 0 1或P <0 0 5 ) ,减少3H TdR和3H Leu掺入量 (P <0 0 1或P <0 0 5 )。结果提示 ,Ang ( 1 7)对VSMCs增殖有抑制作用 ,这可能与影响PKC ζ和ERK1/ 2蛋白表达有关。     

酸化环境对血管平滑肌细胞(VSMC)增殖的影响及姜黄素对此的作用

探讨姜黄素对缺血缺氧引起细胞外环境酸化下血管平滑肌增殖的影响及可能机制。在pH 6.9的环境下体外培养大鼠胸主动脉血管平滑肌细胞(VSMC),分别以姜黄素、PCTX-1等处理细胞,并设置空白对照组;采用Western-Blot法测定ASICS 1a蛋白及磷酸化ERK1/2蛋白表达,CCK-8法测定各处理组对VSMC增殖影响。结果显示pH 6.9组显著刺激VSMC的ASICS 1a和ERK1/2磷酸化表达,并促使VSMC增殖;而姜黄素显著抑制大鼠胸主动脉血管平滑肌细胞上ASICS 1a及细胞内磷酸化ERK1/2蛋白表达,从而抑制VSMC增殖。在缺血缺氧所致酸化环境下,大鼠胸主动脉血管平滑肌上的ASIC 1a表达增多,引起细胞内ERK1/2磷酸化,从而对血管平滑肌的增殖进行调控,而姜黄素则表现出抑制作用。     

ERK活化对哮喘大鼠气道重塑及CyclinD1表达的影响

目的探讨细胞外信号调节蛋白激酶（ERK）对哮喘大鼠气道重塑及CyclinD1表达的作用。方法原代培养大鼠的平滑肌细胞（ASMCs）,给予ERK激动剂表皮生长因子EGF和抑制剂PD98059干预ASMCs生长,依处理方式不同分为5组：（1）正常对照组（2）哮喘对照组;（3）E组：EGF20 ng/mL;（4）P＋E组,PD98059 10μmol/L1 h后添加EGF 20 ng/mL;（5）PD组,PD98059 10μmol/L。采用四甲基偶氮唑盐（MTT）法检测气道平滑肌细胞（ASMCs）增殖能力,流式细胞术（FCM）测定细胞周期和cyclinD1的蛋白含量,RT-PCR方法检测cyclinD1mRNA表达水平。结果（1）与哮喘对照组比较,E组ASMCs S＋G2/M期比例、吸光度A值、cyclinD1蛋白阳性表达率和cyclinD1 mRNA的A值均显著升高,PD组均显著降低（P〈0.05）。P＋E组与哮喘对照在此4项指标上比较无明显差异。（2）哮喘（对照组、E组、PD组和P＋E组）组与正常对照组,其S＋G2/M期比例、吸光度A值、cyclinD1蛋白和cyclinD1 mRNA的表达均显著增高（P〈0.05）。结论ERK活性促进哮喘大鼠ASMCs的增殖,增加cyclinD1在哮喘平滑肌细胞中的表达,导致气道重塑的形成,提示ERK可能对CyclinD1的表达具有调节作用。     

血管平滑肌细胞中ERK通路与TGF-β_1/Smad通路的相互作用

目的:探讨血管平滑肌细胞(VSMC)中TGF-β/Smad与ERK信号转导通路是否存在相互调节关系。方法:原代培养的大鼠胸主动脉平滑肌细胞,分四组:①对照组,②TGF-β1组,③ERK阻断剂(PD98059)组和④TGF-β1+ERK阻断剂(PD98059)组。分别用Western blot法检测VSMC内Smad2/3、ERK1/2蛋白表达及磷酸化Smad2/3、磷酸化ERK1/2蛋白含量,RT-PCR方法测VSMC中Smad2、Smad3mRNA的表达。结果:①与对照组相比,TGF-β1组P-Smad2/3、P-ERK1/2蛋白含量增多(P0.05),ERK阻断剂组P-Smad2/3、P-ERK1/2蛋白含量减少(P0.05),TGF-β1+ERK阻断剂组P-Smad2/3、P-ERK1/2蛋白含量无差异;与TGF-β1组相比,TGF-β1+ERK阻断剂组P-Smad2/3、P-ERK1/2蛋白含量减少(P0.05)。各组间Smad2/3、ERK1/2蛋白表达无差异。②各组的Smad2、Smad3mRNA表达无差异。结论:TGF-β1诱导的Smad2/3蛋白磷酸化依赖ERK通路激活,但ERK通路对Smad2/3蛋白和mRNA表达水平无影响。     

E1A激活基因阻遏子过表达诱导体外培养大鼠平滑肌细胞分化

为探讨E1A激活基因阻遏子蛋白(CREG)对血管平滑肌细胞表型转换的调控机制,应用pRC/CMV-hCREG真核表达载体转染体外培养的大鼠血管平滑肌细胞(VSMCs),观察了转染前后细胞的表型改变.结果发现,pRC/CMV-hCREG质粒转染后,大鼠平滑肌细胞增殖受抑,细胞分化标志蛋白SM α-actin表达显著增加.免疫共沉淀分析发现,CREG与血清反应因子(SRF)发生相互作用形成复合体,在CREG基因过表达时,与CREG结合的SRF蛋白增加.凝胶迁移阻滞分析(GSMA)和抗体凝胶迁移阻滞分析(supershift)显示,过表达的CREG蛋白与SRF蛋白共同结合到 SM α-actin基因启动子区CArG位点上,可能参与 SM α-actin表达的调控. 构建SM α-actin promoter- pCAT^® 3报告基因载体并与pRC/CMV-hCREG质粒共转染VSMCs也证实,CREG蛋白过表达可增强SM α-actin基因表达.上述研究提示,CREG蛋白可能是调控VSMCs表型转换的重要分子.     

电磁辐射对大鼠海马Raf／MEK／ERK信号通路的影响

目的：研究电磁辐射后大鼠海马Raf／MEK／ERK通路相关信号分子的表达变化规律。探讨辐射损伤机制。方法：分别采用X波段高功率微波（X-HPM）、S波段高功率微波（S-HPM）及电磁脉冲（EMP）模拟源辐射大鼠,建立电磁辐射动物模型。通过Western blot检测海马Raf-1、磷酸化Raf-1和磷酸化ERK的表达。结果：三种电磁辐射后6h-14d,Raf-1表达均下调,以7d最为显著,至28d基本恢复,辐射组间未见明显差异。辐射后6h和7d,磷酸化Raf-1和磷酸化ERK表达均上调,6h较为明显,磷酸化ERK的变化以两微波组更为显著。S-HPM辐射后6h～14d,磷酸化Raf-1表达持续上调,磷酸化ERK的变化呈波浪状,以6h和3d为高峰。结论：Raf／MEK／ERK信号通路参与了电磁辐射所致海马损伤;ERK通路过度活化导致神经元凋亡与坏死可能是电磁辐射致认知功能障碍的重要机制。     

The C-terminus of Raf-1 acts as a 14-3-3-dependent activation switch

The Raf-1 protein kinase is a major activator of the ERK MAPK pathway, which links signaling by a variety of cell surface receptors to the regulation of cell proliferation, survival, differentiation and migration. Signaling by Raf-1 is regulated by a complex and poorly understood interplay between phosphorylation events and protein–protein interactions. One important mode of Raf-1 regulation involves the phosphorylation-dependent binding of 14-3-3 proteins. Here, we have examined the mechanism whereby the C-terminal 14-3-3 binding site of Raf-1, S621, controls the activation of MEK-ERK signaling. We show that phosphorylation of S621 turns over rapidly and is enriched in the activated pool of endogenous Raf-1. The phosphorylation on this site can be mediated by Raf-1 itself but also by other kinase(s). Mutations that prevent the binding of 14-3-3 proteins to S621 render Raf-1 inactive by specifically disrupting its capacity to bind to ATP, and not by gross conformational alteration as indicated by intact MEK binding. Phosphorylation of S621 correlates with the inhibition of Raf-1 catalytic activity in vitro, but 14-3-3 proteins can completely reverse this inhibition. Our findings suggest that 14-3-3 proteins function as critical cofactors in Raf-1 activation, which induce and maintain the protein in a state that is competent for both ATP binding and MEK phosphorylation.     

Raf-1 activation disrupts its binding to keratins during cell stress

Keratins 8 and 18 (K8/18) heteropolymers may regulate cell signaling via the known K18 association with 14-3-3 proteins and 14-3-3 association with Raf-1 kinase. We characterized Raf-keratin-14-3-3 associations and show that Raf associates directly with K8, independent of Raf kinase activity or Ras-Raf interaction, and that K18 is a Raf physiologic substrate. Raf activation during oxidative and toxin exposure in cultured cells and animals disrupt keratin-Raf association in a phosphorylation-dependent manner. Mutational analysis showed that 14-3-3 residues that are essential for Raf binding also regulate 14-3-3-keratin association. Similarly, Raf phosphorylation sites that are important for binding to 14-3-3 are also essential for Raf binding to K8/18. Therefore, keratins may modulate some aspects of Raf signaling under basal conditions via sequestration by K8, akin to Raf-14-3-3 binding. Keratin-bound Raf kinase is released upon Raf hyperphosphorylation and activation during oxidative and other stresses.     

Structure and Sites of Phosphorylation of 14-3-3 Protein: Role in Coordinating Signal Transduction Pathways

The 14-3-3 family are homo- and heterodimeric proteins whose biological role has been unclear for some time, although they are now gaining acceptance as a novel type of adaptor protein that modulates interactions between components of signal transduction pathways, rather than by direct activation or inhibition. It is becoming apparent that phosphorylation of the binding partner and possibly also the 14-3-3 proteins may regulate these interactions. 14-3-3 isoforms interact with a novel phosphoserine (Sp) motif on many proteins, RSX_1,2SpXP. The two isoforms that interact with Raf-1 are phosphorylated in vivo on Ser185 in a consensus sequence motif for proline-directed kinases. The crystal structure of 14-3-3 indicates that this phosphorylation could regulate interaction of 14-3-3 with its target proteins. We have now identified a number of additional phosphorylation sites on distinct mammalian and yeast isoforms.     

Ischemia activates JNK/c-Jun/AP-1 pathway to up-regulate 14-3-3γ in astrocyte

Ischemia occurs in the brain as the result of stroke and other related injuries and few therapies are effective. If more is understood then potential treatments could be investigated. It was previously reported that 14-3-3γ could be up-regulated by ischemia in astrocyte to protect cells from ischemia-induced apoptosis. In this study, we attempted to uncover the mechanism responsible for this 14-3-3γ up-regulation in primary culture of astrocytes under ischemic-like conditions. It was found that in vitro ischemia may activate PI3K/Akt and MAPK signaling pathways. Astrocyte cultures were treated with LY294002 (PI3K inhibitor), U0126 (ERK inhibitor), SB203580 (p38 inhibitor) and SP600125 (JNK inhibitor). Only SP600125 could inhibit the ischemia-induced 14-3-3γ up-regulation in astrocytes. At the same time, we observed an ischemia-induced nuclear translocation of p-c-Jun, a major downstream component of JNK. Inhibition of AP-1 with curcumin also inhibited 14-3-3γ up-regulation indicating that ischemia-induced up-regulation of 14-3-3γ in astrocyte involves activation of the JNK/p-c-Jun/AP-1 pathway.     

14-3-3 Facilitates Ras-Dependent Raf-1 Activation In Vitro and In Vivo

14-3-3 proteins complex with many signaling molecules, including the Raf-1 kinase. However, the role of 14-3-3 in regulating Raf-1 activity is unclear. We show here that 14-3-3 is bound to Raf-1 in the cytosol but is totally displaced when Raf-1 is recruited to the plasma membrane by oncogenic mutant Ras, in vitro and in vivo. 14-3-3 is also displaced when Raf-1 is targeted to the plasma membrane. When serum-starved cells are stimulated with epidermal growth factor, some recruitment of 14-3-3 to the plasma membrane is evident, but 14-3-3 recruitment correlates with Raf-1 dissociation and inactivation, not with Raf-1 recruitment. In vivo, overexpression of 14-3-3 potentiates the specific activity of membrane-recruited Raf-1 without stably associating with the plasma membrane. In vitro, Raf-1 must be complexed with 14-3-3 for efficient recruitment and activation by oncogenic Ras. Recombinant 14-3-3 facilitates Raf-1 activation by membranes containing oncogenic Ras but reduces the amount of Raf-1 that associates with the membranes. These data demonstrate that the interaction of 14-3-3 with Raf-1 is permissive for recruitment and activation by Ras, that 14-3-3 is displaced upon membrane recruitment, and that 14-3-3 may recycle Raf-1 to the cytosol. A model that rationalizes many of the apparently discrepant observations on the role of 14-3-3 in Raf-1 activation is proposed.     

14-3-3 proteins as potential therapeutic targets

The 14-3-3 family of phosphoserine/phosphothreonine-binding proteins dynamically regulates the activity of client proteins in various signaling pathways that control diverse physiological and pathological processes. In response to environmental cues, 14-3-3 proteins orchestrate the highly regulated flow of signals through complex networks of molecular interactions to achieve well-controlled physiological outputs, such as cell proliferation or differentiation. Accumulating evidence now supports the concept that either an abnormal state of 14-3-3 protein expression, or dysregulation of 14-3-3/client protein interactions, contributes to the development of a large number of human diseases. In particular, clinical investigations in the field of oncology have demonstrated a correlation between upregulated 14-3-3 levels and poor survival of cancer patients. These studies highlight the rapid emergence of 14-3-3 proteins as a novel class of molecular target for potential therapeutic intervention. The current status of 14-3-3 modulator discovery is discussed.     

α3β1 integrin promotes cell survival via multiple interactions between 14-3-3 isoforms and proapoptotic proteins

Laminin-5 and α3β1 integrin promote keratinocyte survival; however, the downstream signaling pathways for laminin-5/α3β1 integrin-mediated cell survival had not been fully established. We report the unexpected finding of multiple interactions between 14-3-3 isoforms and proapoptotic proteins in the survival signaling pathway. Ln5-P4 motif within human laminin-5 α3 chain promotes cell survival and anti-apoptosis by inactivating Bad and YAP. This effect is achieved through the formation of 14-3-3ζ/p-Bad and 14-3-3σ/p-YAP complexes, which is initiated by α3β1 integrin and FAK/PI3K/Akt signaling. These complexes result in cytoplasmic sequestration of Bad and YAP and their subsequent inactivation. An increase in Akt1 activity in cells induces 14-3-3ζ and σ, p-Bad, and p-YAP, promoting cell survival, whereas decreasing Akt activity suppresses the same proteins and inhibits cell survival. Suppression of 14-3-3ζ with RNA-interference inhibits cell viability and promotes apoptosis. These results reveal a new mechanism of cell survival whereby the formation of 14-3-3ζ/p-Bad and 14-3-3σ/p-YAP complexes is initiated by laminin-5 stimulation via the α3β1 integrin and FAK/PI3K/Akt signaling pathways, thereby resulting in cell survival and anti-apoptosis.