ERK1/2信号通路介导同型半胱氨酸对PC12细胞的损伤作用

目的观察同型半胱氨酸(Homocysteine,Hcy)对PC12细胞的损伤作用,探讨胞外信号调节蛋白激酶1/2(extracellular signal-regulated protein kinases1and2,ERK1/2)通路在其中的作用。方法台盼蓝拒染法和MTT比色法检测细胞存活率,相差倒置显微镜观察细胞形态,Hoechst33258染色荧光照相术检测细胞凋亡,罗丹明123染色荧光照相术和流式细胞术检测细胞线粒体膜电位(mitochondrial membrane potential,MMP),Western blot检测ERK1/2磷酸化的水平。结果Hcy(2.5～20mmol/L)作用24h后,可浓度依赖性地抑制PC12细胞的存活率;10mmol/L Hcy处理24h后,PC12细胞出现核固缩等典型的凋亡特征;Hcy能明显地降低PC12细胞的MMP;Hcy能诱导ERK1/2磷酸化,特异性的ERK1/2阻断剂U0126可显著抑制Hcy对PC12细胞的损伤作用。结论Hcy可引起PC12细胞损伤,此作用与其抑制MMP及诱导ERK1/2磷酸化有关。     

STAT3抑制剂Stattic激活ERK通路诱导THP-1细胞IL-8的产生及细胞凋亡

目的观察信号转导子与转录激活子3(STAT3)抑制剂Stattic对THP-1细胞产生白细胞介素8(IL-8)和细胞凋亡的影响,并探讨其机制。方法采用(0、 1、 5、 10、 15、 20)μmol/L Stattic处理THP-1细胞0、 1、 3、 6、 12、 24h,实时荧光定量PCR检测细胞IL-8、 IL-6、 IL-1β、肿瘤坏死因子α(TNF-α)的mRNA水平, ELISA检测细胞培养上清液IL-8蛋白水平,流式细胞术检测THP-1细胞的凋亡, Western blot法检测细胞STAT3、细胞外信号调节激酶(ERK)的蛋白磷酸化水平;采用(0、 1、 5、 10)μmol/L的ERK通路选择性抑制剂U0126预处理THP-1细胞,反转录PCR检测U0126对Stattic诱导THP-1细胞表达IL-8 mRNA水平的影响。结果 (10～20)μmol/LStattic显著上调IL-8在THP-1细胞中的mRNA和蛋白表达,仅(15、 20)μmol/LStattic能诱导THP-1细胞凋亡; Stattic处理THP-1细胞1、 3、 6、 12、 24 h,均显著上调IL-8的mRNA水平,以3 h时最为明显,在6 h以后呈时间依赖性上调IL-8的蛋白水平并诱导THP-1细胞凋亡;Stattic呈浓度和时间依赖性抑制STAT3磷酸化,时间依赖性地诱导ERK磷酸化,在(1、 5、 10、 15、 20)μmol/L时均显著诱导ERK磷酸化。另外, U0126显著抑制Stattic诱导的IL-8 mRNA表达。结论 STAT3抑制剂Stattic通过激活ERK信号通路诱导THP-1细胞凋亡和IL-8产生。     

ERK1/2信号通路在IGF-1诱导新生小鼠海马神经干细胞增殖分化中的作用

目的:探讨细胞外信号调节激酶1/2(ERK1/2)信号通路在胰岛素样生长因子-1(insulin-like growth factor-1,IGF-1)诱导新生小鼠神经干细胞(neural stem cells,NSCs)增殖分化中的作用。方法:取新生C57BL/6J小鼠海马体外分离、培养NSCs,分别加入终浓度100 ng/ml的IGF-1和20μmol/L的抑制剂U0126。CCK-8试剂盒检测IGF-1对NSCs增殖的影响;免疫荧光细胞染色法检测IGF-1对细胞分化的影响;Western blot检测ERK1/2和p-ERK1/2蛋白的表达,并对各组进行比较。结果:CCK-8测定细胞增殖,第3 d U0126组相对OD值显著低于IGF-1组,第7 d时IGF-1组OD值则显著高于对照组和U0126组(P<0.05)。倒置荧光显微镜下可观察到IGF-1组βⅢTubulin和GFAP阳性分化率均明显高于对照组和U0126组(P<0.05)。Western blot结果显示:IGF-1组蛋白表达量显著高于对照组和U0126组(P<0.05),IGF-1促进ERK磷酸化,U0126则抑制ERK的活化。结论:IGF-1可显著诱导NSCs的增殖和分化,ERK1/2信号通路可能具有重要作用,同时IGF-1可能参与ERK1/2的活化。     

神经再生素激活PC12细胞ERK1/2的实验研究

本实验初步探讨了神经再生素 ( NRF)作用于 PC12细胞的信号传导途径。采用 MTT法检测 NRF对无血清培养的PC12细胞存活率的影响 ;用磷酸化的 Elk单克隆抗体 ,通过 Western blot法观察 NRF的不同浓度 ( 10、10 0、10 0 0 ng/ m l)及不同作用时间 ( 0 min、15 min、3 0 min、1h、2 h)对无血清培养的 PC12细胞 ERK1/ 2活性的影响 ;还运用 MEK1/ 2阻断剂 U0 12 6,观察NRF激活 ERK1/ 2是否需要其上游信号蛋白 MEK1/ 2的激活。结果表明 ,NRF作用于无血清培养的 PC12细胞 ,可以激活ERK1/ 2 ,存在明显的剂量效应关系和时间依赖作用 ,在 10 0 0 ng/ ml浓度下最大 ,作用 2 h时达峰值 ;U0 12 6作用后 ,可抑制 NRF作用于 PC12细胞引起的 ERK1/ 2的激活。由此可见 ,NRF对无血清培养的 PC12细胞具有保护作用 ,可能通过激活 ERK1/ 2磷酸化级联途径来介导这些作用。     

SHP-2通过ERK和JNK通路调节PC12细胞应对NGF的细胞生存和凋亡

目的：探讨蛋白酪氨酸磷酸酶SHP-2在神经生长因子(NGF)作用下大鼠嗜铬细胞瘤PC12细胞生存及NGF撤除后细胞凋亡的过程中的作用及机制。方法：SHP-2 抑制剂NSC87877作用于PC12细胞, MTT测定PC12细胞存活率;流式细胞仪检测细胞凋亡率。将pIRES-GFP空载体、pIRES-GFP-SHP-2野生型和pIRES-GFP-SHP-2C459S突变体通过脂质体方法转染PC12细胞;加入NGF作用１h和撤除NGF 5 h后分别用Western blotting方法检测细胞外信号调节蛋白激酶(ERK)、磷酸化ERK（p-ERK）、c-Jun氨基末端激酶(JNK)及磷酸化JNK (p-JNK)表达变化。结果：MTT和流式细胞仪检测表明SHP-2 可以促进PC12细胞生存,抑制细胞凋亡。Western blotting结果显示无SHP-2抑制剂组和转染pIRES-SHP-2野生型组p-ERK表达在加入NGF的过程中升高;撤除NGF后,各组p-ERK表达均降低,pIRES-GFP-SHP-2C459S突变体组和pIRES-GFP组p-ERK表达明显低于pIRES-GFP-SHP-2野生型组, NGF去除+ SHP-2抑制剂组的表达水平明显低于NGF去除对照组; NGF去除+SHP-2抑制剂组p-JNK表达高于NGF去除对照组;pIRES-GFP-SHP-2C459S突变体组高于pIRES-GFP空载体组,pIRES-GFP-SHP-2野生型组低于pIRES-GFP空载体组。结论：SHP-2可能通过对ERK的正向激活,抑制JNK的激活,增强NGF作用下PC12细胞生存及抑制NGF撤除后所致细胞凋亡,从而在NGF的信号转导中起到一个中心环节的作用。     

ERK通路激动剂EGF及抑制剂U0126对HepG2肝癌细胞迁移和侵袭能力的影响

目的研究丝裂原活化蛋白激酶(MAPK)亚族细胞外调节蛋白激酶(ERK)通路在HepG2肝癌细胞迁移和侵袭中的作用。方法体外培养人肝癌HepG2细胞,采用ERK通路激动剂和抑制剂:表皮生长因子(EGF)及U0126刺激48h,分别应用MTS法、划痕实验法及Transwell小室法检测EGF和U0126对HepG2细胞增殖能力、迁移能力和侵袭能力的影响,qPCR和Western blot法分别检测ERK mRNA和磷酸化ERK蛋白的表达变化。结果细胞增殖、迁移和侵袭实验结果均显示,激动剂EGF可显著提高HepG2肝癌细胞的增殖、迁移及侵袭能力(P0.01),而U0126均对以上指标有抑制效应(P0.05)。qPCR和Western blot结果显示,EGF可上调ERK mRNA和磷酸化蛋白的表达(P0.01),而U0126对ERK mRNA无明显影响(P0.05),但可显著下调ERK磷酸化蛋白的表达(P0.01)。结论 ERK通路参与HepG2细胞的增殖、迁移和侵袭过程,EGF和U0126对以上指标的影响可能与ERK mRNA的表达及蛋白磷酸化过程相关。     

Erk信号途径在B104 CM诱导神经干细胞向少突胶质细胞前体分化中的作用

目的探讨细胞外信号调节激酶(Erk)信号途径及下游转录因子cf-os、cj-un等在神经母细胞瘤B104细胞系来源的条件培养基(B104 CM)诱导神经干细胞(NSCs)向少突胶质细胞前体(OPCs)分化中的作用。方法从形态学上观察Erk1/2特异性抑制剂U0126阻断对B104 CM诱导NSCs向OPCs分化的影响;分别以Western blotting和RT-PCR法检测对照组、B104 CM诱导组和U0126预孵组NSCs中Erk的磷酸化和转录因子cf-os、cj-un、c-myc的表达情况。结果U0126预孵可阻断B104 CM诱导的NSCs向OPCs分化;B104 CM可引起NSCs中Erk1/2迅速磷酸化和cf-os、cj-unmRNA表达上调,该作用可被U0126阻断。结论B104 CM通过活化Erk信号途径及其随后上调转录因子cf-os、cj-un的表达诱导NSCs向OPCs分化。     

大鼠重型弥漫性脑创伤后ERK1/2信号途径的改变及意义

目的:探讨重型弥漫性脑创伤后细胞外信号调节激酶1/2(ERK1/2)信号的改变及意义。方法:SD大鼠分为正常对照组、创伤组、ERK1/2抑制剂U0126高、低剂量组。Marmarou等法制作弥漫性脑创伤模型。伤后30min、1、6、24、48、72h光镜和电镜下观察伤后脑组织形态变化;Western blotting法检测海马区磷酸化ERK1/2和c-Fos蛋白表达;TUNEL法检测海马区凋亡细胞;伤后3-7d水迷宫法测试动物学习记忆功能。结果:伤后,创伤组脑组织损伤严重,神经细胞出现变性坏死和凋亡改变、神经轴索变性断裂;磷酸化ERK1/2蛋白表达在伤后30min明显增高,6h达高峰,高表达状态持续至伤后24h;c-Fos在伤后30min明显增高,6h达高峰,24h接近正常水平;神经细胞凋亡数目伤后6h明显增多,72h达高峰;大鼠搜索安全岛时间延长。U0126干预后,脑组织形态损伤程度、磷酸化ERK1/2和c-Fos表达、神经细胞凋亡数目回降;大鼠搜索安全岛时间缩短;上述变化在U0126高剂量组中更为显著。结论:ERK1/2信号途径参与大鼠重型弥漫性脑创伤病理损伤过程,并在伤后神经细胞凋亡进程中发挥关键作用。     

Changes of extracellular signal regulated kinase and caspase-8 expression in arteriae cerebri of mice with cerebral vasospasm model

目的:研究细胞外信号调节激酶(ERK)、半胱氨酸天冬氨酸特异性蛋白酶-8(caspase-8)在脑血管痉挛(CVS)后早期脑损伤中的作用,并探讨ERK特异性抑制剂U0126通过此途径发挥作用的保护机制.方法:采用非开颅血管内穿线法制备小鼠蛛网膜下腔出血(SAH)并脑血管痉挛模型,给予U0126,术后行神经功能评分,分别在术后12、24、48 h3个时相点取右侧大脑动脉标本,应用免疫印迹检测各组磷酸化ERK1/2( p-ERK1/2)、caspase-8蛋白表达,TUNEL法检测大脑中动脉内皮细胞凋亡.结果:模型组小鼠神经功能评分降低,大脑中动脉出现严重脑血管痉挛,随脑血管痉挛时间延长,各组小鼠p-ERK1/2、caspase-8蛋白均有不同程度增强,凋亡细胞增多.与模型组比较,治疗组小鼠神经功能评分增加,各时相点3项指标的表达均不同程度下调.脑血管痉挛12～48 h p-ERK1/2与caspase-8呈正相关.结论:脑血管痉挛后,ERK表达增强可通过激活caspase-8信号途径诱导大脑动脉内皮细胞凋亡;U0126对脑血管痉挛具有一定的治疗作用,其机制之一可能是通过部分阻抑ERK通路活化,减少凋亡实现的.     

黄芩素和U0126 体外协同抗人膀胱癌细胞的作用及机制研究

目的：探讨黄芩素和U0126体外协同抗人膀胱癌T-24细胞的作用及机制研究。方法：用黄芩素、U0126及二者联合处理T-24细胞,流式细胞术检测细胞周期、细胞凋亡;显微镜下计数细胞数量变化;TUNEL法检测细胞凋亡指数;实时定量PCR和Western blot分别检测T-24 细胞胞外信号调节激酶1/2(ERK1/2) 、CyclinD1、GSK-3β和AKT ＲNA水平、蛋白水平,分析黄芩素与U0126对膀胱癌细胞凋亡和增殖的影响。结果：T-24细胞经不同浓度黄芩素处理后, 细胞凋亡率显著增加;T-24细胞经黄芩素处理24 h,G0/G1 期细胞比例明显增多,S 期细胞比例明显下降,细胞数减少,采用黄芩素联合U0126 处理T-24细胞24 h,S 期细胞比例显著降低;黄芩素或U0126单独处理T-24细胞引起明显细胞凋亡,二者联合处理凋亡更明显;利用两种药物单独或联合作用T-24细胞24 h,GSK-3β、ERK1/2、AKT磷酸化水平显著降低, ERK1/2、CyclinD1的 mRNA表达减少,联合处理作用更显著。结论：黄芩素和U0126 均可诱导T-24细胞凋亡,增加G0/G1 期细胞比例,降低S 期细胞比例,联合应用效果更明显。     

Dorsomorphin stimulates neurite outgrowth in PC12 cells via activation of a protein kinase A-dependent MEK-ERK1/2 signaling pathway

In this study, we investigated the effect of dorsomorphin, a selective inhibitor of bone morphogenetic protein (BMP) signaling, on rat PC12 pheochromocytoma cell differentiation. PC12 cells can be induced to differentiate into neuron-like cells possessing elongated neurites by nerve growth factor, BMP2, and other inducers. Cells were incubated with BMP2 and/or dorsomorphin, and the extent of neurite outgrowth was evaluated. Unexpectedly, BMP2-mediated neuritogenesis was not inhibited by co-treatment with dorsomorphin. We also found that treatment with dorsomorphin alone, but not another BMP signaling inhibitor, LDN-193189, induced neurite outgrowth in PC12 cells. To further understand the mechanism of action of dorsomorphin, the effects of this drug on intracellular signaling were investigated using the following signaling inhibitors: the ERK kinase (MEK) inhibitor U0126; the tropomyosin-related kinase A inhibitor GW441756; and the protein kinase A (PKA) inhibitor H89. Dorsomorphin induced rapid and sustained ERK1/2 activation; however, dorsomorphin-mediated ERK1/2 activation and neuritogenesis were robustly inhibited in the presence of U0126 or H89, but not GW441756. These findings suggest that dorsomorphin has the potential to induce neuritogenesis in PC12 cells, a response that requires the activation of PKA-dependent MEK-ERK1/2 signaling.     

Sargachromenol, a novel nerve growth factor-potentiating substance isolated from Sargassum macrocarpum, promotes neurite outgrowth and survival via distinct signaling pathways in PC12D cells

We previously found that the methanol extract of a marine brown alga, Sargassum macrocarpum showed marked nerve growth factor (NGF)-dependent neurite outgrowth promoting activity to PC12D cells. The active substance purified was elucidated to be sargachromenol. The median effective dose (ED50) was 9 microM against PC12D cells in the presence of 10 ng/ml NGF, although it showed no neurotrophic effect on its own. Pretreatment of cells with protein kinase A (PKA) inhibitor or U0126 substantially suppressed the sargachromenol-enhanced neurite outgrowth from PC12D cells, suggesting that the activation of cyclic AMP-mediated protein kinase and mitogen-activated protein (MAP) kinase 1/2 was apparently required for the action of sargachromenol. On the other hand, sargachromenol significantly promoted the survival of neuronal PC12D cells at 0-50 ng/ml NGF in serum-free medium. Neither PKA inhibitor nor U0126 could inhibit the survival supporting effect of sargachromenol, whereas wortmannin significantly blocked the sargachromenol-induced survival supporting effect on neuronal PC12D cells, suggesting that sargachromenol rescued neuronal PC12D cells by activating phosphatidylinositol-3 kinase. These results demonstrate that sargachromenol promotes neuronal differentiation of PC12D cells and supports the survival of neuronal PC12D cells via two distinct signaling pathways.     

Effect of scoparone on neurite outgrowth in PC12 cells

The neurite outgrowth-promoting effects of scoparone isolated from the stem bark of Liriodendron tulipifera were investigated in PC12 cells. At a concentration of 200 microM, scoparone markedly induced neurite outgrowth from PC12 cells. Scoparone at 200 microM also enhanced the outgrowth of neurites from cells in the presence of nerve growth factor (NGF, 2 ng/ml). The levels of intracellular cyclic AMP and concentration of Ca2+ were also increased by 200 microM scoparone. In addition, scoparone at 200 microM increased the activities of extracellular signal-regulated protein kinase (ERK), cyclic AMP-dependent protein kinase (PKA), protein kinase C (PKC) and Ca2+/calmodulin kinase II (CaMK II). However, scoparone-induced neurite outgrowth was blocked by a mitogen-activated protein kinase inhibitor (U0126), a PKA inhibitor (H89), a PKC inhibitor (GF109203X) and a CaMK II inhibitor (KN62). These kinase inhibitors also reduced the scoparone-induced neurite outgrowth associated with NGF. These results suggest that scoparone can induce neurite outgrowth by stimulating the upstream steps of ERK, PKA, PKC and CaMK II in PC12 cells.     

Inhibition of NGF-induced neurite outgrowth of rat pheochromocytoma cells (PC12) following administration of dioxyamphetamine

Amphetamine analogs are known to induce not only neurotoxicity at serotonergic axon terminals but also neocortical neuronal degeneration. However, a much less studied aspect involves the impact of amphetamine exposure on neuronal development. The present study investigated whether pretreatment of PC12 cells with dioxyamphetamine (DA) alters differentiation of PC12 cells by NGF and, if so, which components of the Ras/Raf/MEK/ERK pathway known to be involved in the differentiation response to NGF are particularly affected. Though exposure of PC12 cells to DA 1 h prior to NGF treatment resulted in apopotosis, several PC12 cells survived. However, neurite outgrowth of these NGF-responsive cells was repressed. Immunoblots of whole cell extracts revealed a strong induction rather than inhibition of ERK phosphorylation up to 48 h after DA/NGF treatment. Our results indicate that NGF-mediated neurite outgrowth was inhibited by pretreatment with DA, and this blockage of NGF-induced neuritogenesis was not due to an inhibition of ERK phosphorylation.     

Low concentrations of nitric oxide (NO) induced cell death in PC12 cells through activation of p38 mitogen-activated protein kinase (p38 MAPK) but not via extracellular signal-regulated kinases (ERK1/2) or c-Jun N-terminal protein kinase (JNK)

Nitric oxide (NO), a highly reactive gaseous molecule, has been previously reported to induce apoptosis-like cell death even at a low concentration in PC12 cells. In this study, we examined NO-induced activation of members of the mitogen-activated protein kinase (MAPK) family, i.e., p38 MAPK, extracellular signal-regulated kinases (ERK1/2), and c-Jun N-terminal protein kinase (JNK). Following the exposure of PC12 cells to an NO donor, (+)-(E)-4-ethyl-2-[hydroxyimino]-5-nitro-3-hexenamide (NOR3; 100 muM), the phosphorylation level of p38 MAPK increased time dependently from 2 to 6 h, but that of both ERK1/2 and JNK did not. Treatment with a p38 MAPK inhibitor SB203580 partially blocked the NOR3-induced cell death. Neither PD98059, U0126 (inhibitors of ERK1/2) nor SP600125 (a specific inhibitor of JNK) treatments had any significant effect on the NOR3-induced cell death. These findings suggest that the activation of a p38 MAPK pathway, but not that of ERK1/2 or JNK, plays an essential role in the apoptosis-like cell death induced by low concentrations of NO.     

The mitogen-activated/extracellular signal-regulated kinase kinase 1/2 inhibitor U0126 induces glial fibrillary acidic protein expression and reduces the proliferation and migration of C6 glioma cells

The extracellular signal-regulated kinase (ERK) signaling pathway has been implicated in diverse cellular functions. ERK and its activating kinase, mitogen-activated/extracellular signal-regulated kinase kinase (MEK), are downstream of cell surface receptors known to be up-regulated in many malignant gliomas. We sought to investigate the role of ERK in glioma cell migration, proliferation and differentiation using the rat-derived C6 glioma cell line and the MEK inhibitor, U0126. Treatment of C6 cells with U0126 caused a significant concentration-dependent reduction in cell proliferation and migration and also induced expression of glial fibrillary acidic protein, a marker of astrocytic differentiation. These results suggest that the ERK pathway regulates glioma cell proliferation, migration and differentiation.     

Involvement of BREK, a serine/threonine kinase enriched in brain, in NGF signalling

We identified AATYK2 (Apoptosis-Associated Tyrosine Kinase 2) through a database search as a kinase specifically expressed in the brain. After characterization, we renamed it BREK (Brain-Enriched Kinase). Mouse BREK mRNA is expressed predominantly in brain, especially in olfactory bulb, olfactory tubercle, hippocampus, striatum, cerebellum, and cerebral cortex. Levels of expression and phosphorylation of BREK were high at 0-2 weeks after birth, suggesting that BREK is involved in neural development and functions during the early postnatal period. Phosphoamino acid analysis following in vitro kinase reaction revealed that BREK is a catalytically active, serine/threonine kinase. In PC12 cells, BREK was phosphorylated rapidly upon stimulation with nerve growth factor (NGF) in a protein kinase C-dependent pathway. In differentiated PC12 cells, BREK was enriched in cell bodies and growth cones, and also present along neurites. Introduction of a kinase-defective mutant of BREK into PC12 cells enhanced both ERK phosphorylation and neurite outgrowth in response to NGF, suggesting that BREK is a negative regulator of NGF-induced neuronal differentiation. Thus, we conclude that BREK is a new member of the family of protein serine/threonine kinases and that it plays important roles in NGF-TrkA signalling.