吗啡对μ阿片受体介导的ERK磷酸化的影响

目的：利用表达μ阿片受体的中国仓鼠卵巢细胞,研究吗啡急慢性处理下对细胞外信号调节激酶（ ERK）磷酸化的影响。方法免疫印迹检测磷酸化ERK水平,获取1 h急性处理的ERK磷酸化变化时程和36 h慢性处理以及纳洛酮催促下的ERK磷酸化变化。结果1μmol·L-1吗啡可以快速诱导ERK磷酸化水平短暂升高,5 min时达峰（ P〈0.01）,吗啡诱导的ERK磷酸化水平升高具有显著的浓度依赖性。10μmol·L-1吗啡慢性处理36 h后ERK磷酸化水平与对照组比较,差异无统计学意义,但纳洛酮急性催促5或10 min均导致ERK磷酸化显著下降（与对照比较,P〈0.01）。结论吗啡急慢性刺激下以及纳洛酮催促下ERK磷酸化表现出不同的变化形式,提示μ阿片受体介导下ERK相关的信号通路发生了代偿。     

异氟醚可能通过ERK磷酸化增强肾动脉平滑肌收缩

目的实验观察异氟醚对去内皮肾动脉血管平滑肌收缩增强的作用,并观察肾动脉血管平滑肌细胞在异氟醚的作用下细胞内MAPK通路的激活状态,以探讨异氟醚引发肾动脉血管平滑肌收缩增强的可能信号通路。方法①去内皮肾动脉条,3%皂苷处理使膜通透,咖啡因诱除内质网内贮钙,利用10-6钙离子浓度EGTA缓冲液平衡后,应用接近最大的钙离子浓度的EGTA缓冲液使肾动脉条压缩达平衡,加入不同浓度异氟醚EGTA缓冲溶液,观察动脉条张力变化。②1%、3%、5%异氟醚分别作用于培养的肾动脉血管平滑肌细胞,提取细胞内蛋白,Westenblot检测ERK磷酸化的变化。结果异氟醚能使压缩达平衡的肾动脉环张力进一步增强,且与异氟醚浓度呈剂量依赖性。随异氟醚浓度的增加,培养的肾动脉血管平滑肌细胞内ERK1/2(p44/42)磷酸化逐步增强,并随时间增强,在15min后,逐渐下降。结论异氟醚引起的肾动脉血管条收缩增强可能与异氟醚引起MAPK系统活化有关     

乙酰胆碱通过调节IL-1β/IL-1RA平衡改善炎症微环境的机制探讨（英文）

本研究旨在探讨乙酰胆碱的抗炎机制,为阿尔茨海默病(Alzheimer’s disease, AD)炎性病理机制的验证和治疗药物的筛选提供进一步依据。研究首先建立Aβ25-35诱导小胶质细胞系BV-2细胞炎性损伤的体外模型,观察氯化乙酰胆碱(ACh)抑制炎症反应的作用。于有或无α7nAChR的阻断剂(α-银环蛇毒)存在的情况下,采用ELISA方法评价ACh作用后IL-1β和IL-1RA的表达及二者比率,并分别利用Westernblot和EMSA方法测定MAPK(JNK,p38和ERK)及NF-κB通路相关分子的表达及磷酸化水平。结果显示,ACh能够显著降低IL-1β/IL-1RA比率,对抗Aβ25-35诱导的IL-1系统炎症活性变化,维持IL-1亚家族平衡。预先给予α7nAChR阻断剂能够阻断ACh对IL-1β和IL-1RA蛋白表达的抑制作用。同时,α7nAChR阻断剂能够阻断ACh对下游ERK1/2MAPK蛋白的磷酸化水平和NF-κB的活化的调节作用。总之,乙酰胆碱能够抑制Aβ诱导的BV-2细胞中IL-1亚家族的炎症活性、维持该亚家族的平衡稳定,该作用由α7nAChR介导,有ERK1/...     

抗氧化剂SS-31对异氟醚麻醉小鼠脑源性神经营养因子信号通路及认知功能的影响

目的 观察抗氧化剂SS-31对异氟醚麻醉小鼠脑源性神经营养因子(BDNF)信号通路及认知功能的影响。方法 老年雄性C57BL/6小鼠42只随机均分为3组(n=14)：氧气+生理盐水组(Con组)、异氟醚组+生理盐水组(Iso组)和异氟醚+SS-31组(SS-31组)。根据分组,于氧气或异氟醚吸入前30 min腹腔注射等容生理盐水或SS-31(5 mg/kg)。气体吸入2 h后各组取6只小鼠检测海马组织中活性氧自由基(ROS)及三磷酸腺苷(ATP)水平,以及BDNF、酪氨酸激酶B受体(TrkB)、磷酸化TrkB(p-TrkB)和磷酸化环磷酸腺苷反应元件结合蛋白(p-CREB)水平,24 h后各组余8只小鼠行旷场实验和条件恐惧性实验。结果 与Iso组比较,SS-31组ROS水平降低,ATP水平升高,BDNF、p-TrkB和p-CREB含量升高,24 h场景实验僵直时间增加(P<0.05)。结论 线粒体抗氧化肽SS-31可改善异氟醚麻醉所致小鼠认知功能障碍,其机制可能与清除线粒体内ROS,增加ATP的产生,调节BDNF信号通路有关。     

远志寡糖酯A对C6细胞MAPK/ERK信号通路的影响

目的探究远志寡糖酯A(tenuifoliside A)对大鼠胶质瘤细胞(C6细胞)的神经营养作用及可能机制。方法远志寡糖酯A 60,30,10,6,3和0.6μmol·L-1处理C6细胞24 h,MTT法检测检测细胞存活率;远志寡糖酯A 10μmol·L-1处理C6细胞0,5,10,15,30,45,60,120 min,Western blotting测定C6细胞中p-ERK,ERK,BDNF的表达水平;选取各有效剂量分别处理C6细胞24 h,蛋白免疫印记法测定C6细胞中BDNF的表达水平;用ERK1/2特异性拮抗剂U0126 10μmol·L-1预处理C6细胞30 min,再用远志寡糖酯A 10μmol·L-1处理15 min,Western blotting检测p-ERK,ERK,BDNF的表达水平。结果与正常组细胞相比,30,10,6和3μmol·L-1剂量能促进C6细胞增殖,60μmol·L-1剂量对C6细胞有抑制作用,远志寡糖酯A 0.6μmol·L-1剂量对细胞增殖作用不明显;远志寡糖酯A诱导C6细胞中ERK1/2磷酸化具有时效性,在5 min起效,15 min时ERK1/2磷酸化水平达到最高峰,1 h后回落;不同浓度的远志寡糖酯A促C6细胞内BD-NF表达呈剂量依赖性;和溶剂组相比,ERK1/2拮抗剂处理组的p-ERK,ERK,BDNF的表达水平显著降低。结论远志寡糖酯A对大鼠胶质瘤细胞有促增殖作用,其机制可能与激活MAPK/ERK通路,磷酸化ERK1/2,进而促进CREB磷酸化,最终促进BDNF表达有关。     

二苯乙烯苷调控MAPKs信号通路诱导大肠癌SW116细胞凋亡

目的研究二苯乙烯苷(THSG)对大肠癌SW116细胞凋亡的诱导作用及其可能的分子机制。方法体外常规培养SW116细胞,采用不同剂量(0、5、10、20、40、80、120 mmol/L)THSG处理SW116细胞,CCK8法分析不同剂量药物处理24、48、72 h后细胞的存活率;不同浓度(0、10、20、40 mmol/L)THSG干预SW116细胞24 h后,Annexin/PI双染后流式细胞仪检测SW116细胞凋亡情况,Western blot方法检测SW116细胞中凋亡相关蛋白PARP和Pro-caspase 3的表达及MAPKs信号通路p38、ERK、JNK的磷酸化水平;p38、ERK、JNK特异性抑制剂干预细胞后,Western blot方法检测抑制剂对p38、ERK、JNK磷酸化的抑制作用。结果 THSG呈剂量和时间依赖性地抑制大肠癌SW116细胞的增殖,诱导细胞凋亡;THSG干预SW116细胞后,Cleaved PARP表达显著升高、Pro-caspase 3表达显著降低,胞内p38和JNK磷酸化水平显著升高(P<0.05),然而ERK磷酸化水平显著降低(P<0.05)。结论特异性抑制剂阻断ERK激活,可以增强THSG诱导SW116细胞凋亡,特异性阻断p38、JNK活化,能够部分逆转THSG对p38和JNK磷酸化和细胞凋亡的诱导作用。THSG通过激活p38和JNK信号传导,抑制ERK信号途径,诱导大肠癌SW116细胞凋亡。     

丹参酮ⅡA磺酸钠对AngⅡ诱导的心肌细胞肥大反应中磷酸化细胞外信号调节激酶1/2的作用

目的从细胞外信号调节激酶1/2(ERK1/2)激活角度研究丹参酮ⅡA磺酸钠(sodium tanshinoneⅡ Asulfonate,STS)对血管紧张素Ⅱ(angiotensinⅡ,AngⅡ)诱导的心肌肥大反应中的作用。方法培养新生大鼠心肌细胞,考马斯亮蓝法测定心肌细胞蛋白含量、[3H]-亮氨酸参入法测定蛋白合成速率作为心肌肥大指标;用免疫荧光标记法和Western blot测定磷酸化ERK1/2蛋白(p-ERK1/2)表达。结果(1)AngⅡ(1μmol·L-1)处理24h,心肌细胞[3H]-亮氨酸参入率、蛋白含量明显增加,STS能明显抑制AngⅡ介导心肌细胞[3H]-亮氨酸参入率、蛋白含量的增加;(2)AngⅡ刺激心肌细胞可见胞核内出现磷酸化ERK1/2荧光染色,丹参酮ⅡA可阻断AngⅡ引起的ERK1/2活化、入核过程;(3)用AngⅡ(1μmol·L-1)处理心肌细胞5min,磷酸化ERK1/2蛋白(p-ERK1/2)表达即开始增加,10min左右时最明显。以AngⅡ(1μmol·L-1)处理心肌细胞10min,磷酸化ERK1/2蛋白(p-ERK1/2)表达为标准,预先以STS(2,10,50μmol·L-1)处理心肌细胞30min,发现STS可明显抑制AngⅡ诱导的心肌细胞磷酸化ERK1/2蛋白表达;(4)预先以不同浓度STS处理心肌细胞30min,发现STS对AngⅡ诱导的心肌细胞磷酸化ERK1/2蛋白表达的抑制作用存在剂量依赖性。结论STS可以抑制AngⅡ诱导的心肌肥厚,其机制与抑制磷酸化ERK1/2表达有关。     

氟伐他汀对糖基化终末产物诱导肾小管细胞转分化及ERK1/2信号通路的影响

目的观察氟伐他汀对糖基化终末产物(AGEs)诱导肾小管上皮细胞(HKC)转分化及ERK1/2信号通路的影响。方法将肾小管上皮细胞(HKC)分为对照组、AGEs刺激组、AGEs加氟伐他汀组和AGEs加ERK1/2阻断剂PD98059组,采用免疫细胞化学检测平滑肌肌动蛋白(α-SMA)的表达情况;Western blot检测α-SMA、E-钙黏着糖蛋白(E-cadher-in)、Ⅰ型胶原(collagenⅠ,ColⅠ)、细胞外信号调节酶1和2(ERK1/2)及其磷酸化蛋白(p-ERK1/2)的表达;酶联免疫吸附实验(ELISA)测定细胞上清液中转化生长因子-β1(TGF-β1)的分泌;逆转录-聚合酶链反应(RT-PCR)检测α-SMA和E-cadherin mRNA的表达。结果与对照组相比,AGEs组肾小管细胞α-SMA和Col I蛋白表达明显上调,细胞培养上清中TGF-β1含量增加,α-SMA mRNA的表达增加,而E-cadherin蛋白和mRNA表达下调;AGEs刺激细胞15minp-ERK1/2表达明显增强,1h达到高峰。PD98059和氟伐他汀能够抑制AGEs刺激引起的HKC细胞α-SMA和ColI的表达及ERK1/2的磷酸化;减少TGF-β1的含量;下调AGEs刺激引起的HKC细胞α-SMA mRNA的表达;同时能够逆转AGEs刺激引起的HKC细胞E-cadherin蛋白和mR-NA的下调表达。结论氟伐他汀抑制AGEs诱导肾小管上皮细胞转分化和胶原I的合成可能是通过抑制ERK1/2信号通路活化实现的。     

雌激素受体α在六氟双酚A诱导细胞外调节蛋白激酶激活中的作用研究

目的研究六氟双酚A(bisphenol AF,BPAF)诱导T47D乳腺癌细胞中细胞外调节蛋白激酶(extracellular regulated protein kinases,ERK)激活的分子机制,探讨雌激素受体α(estrogen receptorα,ERα)在BPAF诱导ERK激活过程中的作用。方法通过蛋白免疫印迹实验(Western Blot)检测ERK磷酸化水平,研究BPAF对ERK磷酸化的诱导作用。运用表达ERαshRNA的慢病毒感染细胞,下调表达ERα,研究ERα在BPAF激活ERK过程中的作用。结果Western Blot结果显示,BPAF在浓度为50 nmol/L时即可诱导ERK磷酸化水平显著升高(P0.05),并且随着剂量的升高呈现出剂量-效应关系。当BPAF浓度达到1μmol/L时ERK磷酸化水平达到最高(P0.05)。BPAF诱导ERK激活时间效应实验中,BPAF处理细胞5~60 min时,ERK磷酸化水平均显著升高(P0.05),在BPAF处理细胞15 min时,ERK磷酸化水平达到最高。在运用表达ERαshRNA的慢病毒感染细胞后,ERα的表达水平显著降低(P0.05)。与正常细胞相比,在下调表达ERα的细胞中,BPAF对ERK磷酸化的诱导作用显著降低(P0.05)。结论 BPAF能够诱导ERK磷酸化,并且呈现剂量-效应关系。ERα在BPAF诱导的ERK激活过程中起到重要作用。     

ERK1/2信号通路在角质细胞生长因子-2诱导角膜基质细胞增殖中的作用

目的观察角质细胞生长因子(keratinocyte growthfactor-2,KGF-2)对角膜基质细胞的增殖作用及ERK1/2信号在角膜基质细胞增殖中的调控作用,探讨KGF-2对角膜基质细胞增殖的可能机制。方法体外培养角膜基质细胞,MTT法检测细胞增殖,Western blot检测磷酸化ERK1/2表达水平。结果 KGF-2在1～100 mg·L-1对角膜基质细胞有明显的促进增殖作用且呈现剂量-效应关系;100 mg·L-1 KGF-2处理角膜基质细胞,5、15、30 min后ERK1/2磷酸化表达水平明显升高,60、90、120、180 min后ERK1/2磷酸化表达水平逐渐减弱;20μmol.L-1 ERK1/2抑制剂PD98059可抑制KGF-2对角膜基质细胞的促增殖作用。结论 KGF-2激活ERK1/2信号通路,提高细胞增殖率,可被抑制剂PD98059阻断;ERK1/2信号通路调控角膜基质细胞的增殖,在角膜基质细胞损伤中发挥作用。     

Relevance of mitogen activated protein kinase (MAPK) and phosphotidylinositol-3-kinase/protein kinase B (PI3K/PKB) pathways to induction of apoptosis by curcumin in breast cells

Following observations that curcumin inhibited proliferation (IC(50)=1-5 microM), invasiveness and progression through S/G2/M phases of the cell cycle in the non-tumourigenic HBL100 and tumourigenic MDA-MB-468 human breast cell lines, it was noted that apoptosis was much more pronounced in the tumour line. Therefore, the ability of curcumin to modulate signalling pathways which might contribute to cell survival was investigated. After pre-treatment of cells for 20 min, curcumin (40 microM) inhibited EGF-stimulated phosphorylation of the EGFR in MDA-MB-468 cells and phosphorylation of extracellular signal regulated kinases (ERKs) 1 and 2, as well as ERK activity and levels of nuclear c-fos in both cell lines. At a lower dose (10 microM), it also inhibited the ability of anisomycin to activate JNK, resulting in decreased c-jun phosphorylation, although it did not inhibit JNK activity directly. In contrast, the activation of p38 mitogen activated protein kinase (MAPK) by anisomycin was not inhibited. Curcumin inhibited basal phosphorylation of Akt/protein kinase B (PKB) in both cell lines, but more consistently and to a greater extent in the MDA-MB-468 cells. The MAPK kinase (MKK) inhibitor U0126 (10 microM), while preventing ERK phosphorylation in MDA-MB-468 cells, did not induce apoptosis. The PI3K inhibitor LY294002 (50 microM) inhibited PKB phosphorylation in both cells lines, but only induced apoptosis in the MDA-MB-468 line. These results suggest that while curcumin has several different molecular targets within the MAPK and PI3K/PKB signalling pathways that could contribute to inhibition of proliferation and induction of apoptosis, inhibition of basal activity of Akt/PKB, but not ERK, may facilitate apoptosis in the tumour cell line.     

Novel I1-imidazoline S43126 enhance insulin action in PC12 cells

The I(1)-imidazoline receptor is a novel target for drug development for hypertension and insulin resistance, major disorders associated with type 2 diabetes. In the present study, we examined the effects of a novel imidazoline agonist S43126, on phosphorylation of protein kinase B (PKB/Akt) and extracellular signal-regulated kinase (ERK1/2) in PC12 cells. We further examined the effects of S43126 on insulin stimulated PKB and ERK phosphorylation. PC12 cells were treated with varying doses of S43126 (10(-10) to 10(-6) M) or insulin (10(-10) to 10(-6) M) or combination treatment with insulin (10(-6) M) and varying doses of S43126 (10(-6) - 10(-11) M) for 10 min. Western blot analysis of treated samples showed that S43126 increased both ERK1/2 and PKB phosphorylation by 5 fold. Combination treatment with insulin (10(-6) M) and varying doses of S43126 (10(-6) - 10(-11) M) enhanced phosphorylation of PKB and ERK1/2 above the level of insulin alone, in a dose and time dependent manner. Treatment with siRNA against Nischarin (mouse homologue of I(1)-imidazoline receptor) reduced the phosphorylation of both ERK and PKB following combination treatments. These results indicate that S43126 has the potential to augment insulin action and should be further studied as a possible candidate drug for the treatment of insulin resistance states.     

肺癌组织ERK1/2磷酸化水平与化疗患者预后的关系评估

目的探讨细胞外信号调节激酶(Extracellular regulated kinase,ERK)的磷酸化水平与肺癌细胞生物学行为及化疗患者预后的关系。方法采用双染免疫组化检测50例非小细胞肺癌(NACLC)组织中ERK1/2和p-ERK1/2的表达,并分析二者与肺癌生物学行为、化疗疗效、患者生存期的关系。结果 50例肺癌标本ERK1/2阳性率62.0%,p-ERK1/2阳性率46.0%;ERK1/2和p-ERK1/2分化低、有淋巴结转移的肺癌表达明显升高,p-ERK1/2水平还与临床分期密切相关;ERK1/2和p-ERK1/2表达与肺癌化疗疗效有关,ERK1/2和p-ERK1/2阳性组肺癌治疗有效率明显低于相应的阴性组肺癌;Kaplan-Meier生存曲线及log-rank分析发现,p-ERK1/2表达水平与肺癌患者总生存期(OS)和无瘤生存期(DFS)明显相关,p-ERK1/2高表达组肺癌患者OS和DFS比阴性组肺癌缩短,而ERK1/2与患者OS和DFS无明显相关。结论肺癌组织存在ERK1/2信号蛋白激活现象,ERK1/2被激活后的磷酸化水平能够反映肺癌的生物学行为,并对患者的预后判断有一定帮助。     

5-HT(2C) receptor activation is a common mechanism on proerectile effects of apomorphine, oxytocin and melanotan-II in rats

This study was conducted to determine the phosphorylation state of N-methyl-d-aspartate (NMDA) NR1 subunit on serine residues 896 (Ser896) and 897 (Ser897), the extracellular signal-regulated kinase 1/2 (ERK1/2), and the cyclic AMP response element-binding protein (CREB) after repeated exposure to cocaine (20 mg/kg, once daily for 9 days) in the dorsal striatum of rats. The real-time changes of glutamate concentration evoked by repeated cocaine injections were examined using a glutamate biosensor in order to evaluate the correlation between glutamate concentration and the change in these phosphoproteins. The results of this study showed that the immunoreactivity of phosphorylated (p)NMDA NR1 subunit at Ser896 and Ser897 as well as pERK1/2, but not pCREB, in the dorsal striatum was increased at 30 min and then returned to basal levels 4 h after repeated cocaine injections. Similarly, glutamate responses evoked by repeated cocaine injections were also increased 30 min after repeated cocaine injections for 3 days and were prolonged by the 9th day of treatment. However, the glutamate responses were not detected at 4 h after repeated cocaine injections for 5 days. In addition, the elevated immunoreactivity of the phosphoproteins 2 h after repeated cocaine injections was attenuated by the blockade of dopamine D1 receptors and NMDA receptors with the SCH23390 or MK801 antagonists, respectively. These findings suggest that glutamate release and dopamine D1 and NMDA receptor stimulation after repeated exposure to cocaine are associated with NMDA NR1 subunit, ERK1/2 and CREB phosphorylation in the dorsal striatum.     

Anti-inflammatory treatment with the p38 mitogen-activated protein kinase inhibitor SB239063 is neuroprotective, decreases the number of activated microglia and facilitates neurogenesis in oxygen-glucose-deprived hippocampal slice cultures

We investigated the effect of the p38 mitogen-activated protein kinase inhibitor SB239063 on inflammation and neurogenesis after ischemia in organotypic hippocampal slice cultures. Our study shows that after oxygen-glucose deprivation, the p38 mitogen-activated protein kinase (MAPK) and the extracellular-signal-regulated kinase 1/2 (ERK1/2) are strongly activated. The p38 MAPK phosphorylation returned to basal levels within 1 h after oxygen-glucose deprivation, whereas the ERK1/2 phosphorylation reached the basal level only after 24 h. Treatment with 20 muM and 100 muM SB239063 strikingly reduced cell death after oxygen-glucose deprivation and significantly diminished microglia activation in the cornu ammonis (CA-region), but not in the area dentata. Levels of the pro-inflammatory cytokine IL-1beta were reduced by 84% after treatment with SB239063 whereas the cytokines IL-6 and TNF-alpha were not affected. After 6 days, neurogenesis was significantly increased in the posterior periventricle. Based on these findings, our study shows that anti-inflammatory treatment with SB239063 reduces cell death, inflammation and microglia activation and, at high concentrations, enhances the oxygen-glucose deprivation-induced neurogenesis in the posterior periventricle.     

Role of mechanical and redox stress in activation of mitogen-activated protein kinases in primary cultured rat hepatocytes

Mechanical stress is known to activate signaling cascades, including mitogen-activated protein kinase (MAPK) pathways. Although mechanical stress has been implicated in hepatic cirrhosis and liver regeneration following hepatectomy, the signaling pathway(s) that may be activated in hepatocytes in response to mechanical stress have not been determined. Using primary cultured rat hepatocytes to examine cellular signaling in response to mechanical stress associated with medium change, we observed that the phosphorylation status of extracellular signal-regulated kinase 1/2 (ERK1/2), Jun N-terminal kinase and p38 MAPK, but not Akt, was altered. MAPK activation, especially ERK1/2, was rapidly increased within 5 min, followed by a subsequent decrease to below basal levels between 30 min and 1 h following medium change. MAPK/ERK kinase (MEK1/2) phosphorylation followed the same pattern. The phosphorylation of Raf-1 in response to medium change was also consistent with Raf-1 serving as an upstream regulator of MEK1/2-ERK1/2 signaling. Phosphorylation of ERK1/2 was increased by mechanical stress alone, suggesting that mechanical stress may be primarily responsible for ERK1/2 activation in response to medium change. Medium change produced a marked decline in oxidized glutathione and malondialdehyde levels, and the antioxidant N-acetyl-l-cysteine decreased basal ERK1/2 phosphorylation, suggesting a role for oxidative stress in maintaining basal ERK1/2 phosphorylation in cultured hepatocytes. These data suggest that medium change results in immediate activation of the MAPK signaling pathway due to mechanical stress, followed by a subsequent inactivation of MAPK signaling due to a reduction in oxidative stress levels. These processes may be associated with alteration of hepatic hemodynamic circulation observed in hepatic diseases and in liver transplantation.     

Calcium and the replication of human vascular smooth muscle cells: studies on the activation and translocation of extracellular signal regulated kinase (ERK) and cyclin D1 expression

Since the precise role of sarcoplasmic reticular Ca2+ in mediating vascular smooth muscle cells (VSMC) proliferation is unknown, the effect of pre-incubation with thapsigargin on extracellular signal regulated kinase (ERK) activation, the translocation of activated of ERK 1/2 to the nucleus, cyclin D1 expression, the onset of S phase and cytosolic Ca2+ levels were studied. Human saphenous vein VSMCs (hVSMC) were incubated with 10 nM thapsigargin for 24 h followed by stimulation with fetal calf serum and the activation of ERK1/2 and cyclin D1 assessed by western blotting, the intracellular distribution of ERK1/2 using indirect immunofluorescence, the onset of S-phase with the incorporation of bromodeoxyuridine and sarcoplasmic reticular Ca2+ status using FURA-2. Thapsigargin had a marginal effect on ERK1/2 activation only at 5 min and 10 min after stimulation with fetal calf serum. In contrast, the rapid translocation of ERK1/2 to the nucleus was completely blocked by thapsigargin. S phase was delayed by 8 h by thapsigargin which co-incided with the recovery of cytosolic Ca2+ levels and cyclin D1 expression. It is concluded that the inhibitory effect of thapsigargin (depletion of Ca2+ pools) on hVSMC replication is mediated through the inhibition of translocation of activated ERK1/2 to the nucleus and not to the phosphorylation of ERK, per se, which in turn prevents cyclin D1 expression and thus progression of the cell cycle.     

Reciprocal regulation of extracellular signal regulated kinase 1/2 and mitogen activated protein kinase phosphatase-3

Mitogen activated protein kinase phosphatase-3 (MKP-3) is a putative tumor suppressor. When transiently overexpressed, MKP-3 dephosphorylates and inactivates extracellular signal regulated kinase (ERK) 1/2. Little is known about the roles of endogenous MKP-3, however. We previously showed that MKP-3 is upregulated in cell lines that express oncogenic Ras. Here we tested the roles of endogenous MKP-3 in modulating ERK1/2 under conditions of chronic stimulation of the Ras/Raf/MEK1/2/ERK1/2 pathway by expression of oncogenic Ras. We used two cell lines: H-ras MCF10A, breast epithelial cells engineered to express H-Ras, and DLD-1, colon cancer cells that express endogenous Ki-Ras. First, we found that MKP-3 acts in a negative feedback loop to suppress basal ERK1/2 when oncogenic Ras stimulates the Ras/Raf/MEK1/2/ERK1/2 cascade. ERK1/2 was required to maintain elevated MKP-3, indicative of a negative feedback loop. Accordingly, knockdown of MKP-3, via siRNA, increased ERK1/2 phosphorylation. Second, by using siRNA, we found that MKP-3 helps establish the sensitivity of ERK1/2 to extracellular activators by limiting the duration of ERK1/2 phosphorylation. Third, we found that the regulation of ERK1/2 by MKP-3 is countered by the complex regulation of MKP-3 by ERK1/2. Potent ERK1/2 activators stimulated the loss of MKP-3 within 30 min due to an ERK1/2-dependent decrease in MKP-3 protein stability. MKP-3 levels recovered within 120 min due to ERK1/2-dependent resynthesis. Preventing MKP-3 resynthesis, via siRNA, prolonged ERK1/2 phosphorylation. Altogether, these results suggest that under the pressure of oncogenic Ras expression, MKP-3 reins in ERK1/2 by serving in ERK1/2-dependent negative feedback pathways.     

Activation of mitogen-activated protein kinases by tributyltin in CCRF-CEM cells: role of intracellular Ca(2+)

Effects of tributyltin chloride (TBT) and other organotin compounds on mitogen-activated protein kinases (MAPKs) were examined in CCRF-CEM human T lymphoblastoid cells. In response to the incubation with 0.25-2 microM TBT for 1 h, the levels of the phosphorylated form of extracellular signal-regulated protein kinase (ERK), c-Jun NH(2)-terminal kinase (JNK), and p38 MAPK increased in a dose-dependent manner. The phosphorylation was observed after 15 min and lasted for 4 h following exposure to 1 microM TBT, while the cell viability was not lowered significantly within 6 h. On the other hand, no clear changes were found in the total protein levels of ERK, JNK, and p38 MAPK. The in vitro activities of MAPKs also increased in response to TBT exposure. The potentials of MAPKs phosphorylation and of cellular damage were TBT > dibutyltin dichloride (DBT) > monobutyltin trichloride (MBT). When compared to other triorganotin compounds such as trimethyltin chloride (TMT), triphenyltin chloride (TPT), and triethyltin bromide (TET), TBT exposure induced the most marked phosphorylation of MAPKs. Chelation of intracellular Ca(2+) suppressed TBT-induced MAPKs phosphorylation almost completely, but removal of external Ca(2+) did not. The present results showed that tributyltin is a potent activator of ERK, JNK, and p38 MAPK pathways, and Ca(2+) mobilized from intracellular stores plays an important role for the phosphorylation of MAPKs in this human T cell line.