主动脉弓缩窄诱导大鼠心肌肥大Raf/MEK/ERK通路的变化

目的:通过观察心肌肥大大鼠加速纤维肉瘤/丝裂素活化蛋白激酶激酶/胞外信号调节蛋白激酶(Raf/MEK/ERK)通路关键因子的基因和蛋白表达及蛋白磷酸化修饰水平上的变化,了解Raf/MEK/ERK通路在心肌肥大调控中的作用。方法: 20只SD大鼠随机分为假手术组和模型组,通过主动脉弓缩窄(TAC)法建立心肌肥大模型,12周后颌下静脉取血分离血清,检测氨基末端脑钠肽前体(NT-proBNP)含量,之后进行超声心动图测定和麻醉下的血流动力学测定,收集心肌标本,观察心肌组织的病理学改变,检测心肌组织Raf/MEK/ERK通路的关键因子基因、蛋白表达水平及蛋白磷酸化水平的变化。结果:与假手术组比较,TAC模型组大鼠超声心动图的左室舒张末期室间隔厚度(IVSd)、左室收缩末期室间隔厚度(IVSs)、左室后壁舒张末期厚度(LVPWd)、左室后壁收缩末期厚度(LVPWs)显著增厚(P＜0.05,P＜0.01),左室收缩末期内径(LVIDs)显著减小(P＜0.01),左心室质量(LV Mass)、左心系数LW(LV Mass/Weight)比值显著增加(P＜0.05,P＜0.01);大鼠心率(HR)、左心室最大收缩速率(+dp/dtmax)、左心室最大舒张速率(-dp/dtmax) 均显著降低(P＜0.01),血清中NT-pro BNP含量显著增加(P＜ 0.01);心肌细胞排列杂乱,心肌细胞肥大、胞质明显增多,炎症细胞浸润,出现大量胶原纤维沉积,大面积心肌细胞呈现蓝色;大鼠心肌组织中c-Raf在Ser259和Ser338上的磷酸化蛋白phospho-c-Raf (Ser259)和phospho-c-Raf (Ser338) 表达水平显著升高(P＜0.01),其下游MEK1/2、ERK1/2的磷酸化蛋白phospho-MEK1/2(Ser217/Ser221)和phospho-ERK1/2 (Thr202/Tyr204)表达水平也显著增高(P＜0.01)。结论: Raf/MEK/ERK通路在心肌肥大中的调控作用,可能通过激活关键因子c-Raf、MEK1、MEK2、ERK1和ERK2特异性位点的磷酸化实现的。     

雌激素快速影响大鼠发育期海马NMDA受体活性的机制

为了探讨雌激素对发育期大鼠海马NMDA受体活性的快速影响,对出生后18d的雄性大鼠进行苯甲酸雌二醇皮下注射,1h后用WesternBlot检测海马NMDA受体NR1和NR2B亚基、雌激素β受体、ERK1/2蛋白的表达,以及NR2B和ERK1/2的磷酸化水平;并通过海马内给予雌激素受体拮抗剂ICI182,780和MEK1/2抑制剂U0126预处理,进一步分析苯甲酸雌二醇影响NR2B和ERK1/2磷酸化的作用机制。结果显示,苯甲酸雌二醇不影响NR1、NR2B、ERβ和ERK1/2的表达,但能快速增强NR2B和ERK1/2的磷酸化水平。雌激素受体拮抗剂ICI182,780和MEK1/2抑制剂U0126均能明显抑制苯甲酸雌二醇诱导的NR2B和ERK1/2磷酸化水平的增加。以上结果提示,雌激素可能通过雌激素受体的非基因组机制激活ERK/MAPK信号转导通路,快速诱导NMDA受体NR2B亚基磷酸化,激活NMDA受体。     

毒蝇碱型乙酰胆碱受体经Ras—ERK—1／2信号通路上调Bcl—2和磷酸化Bad表达

毒蝇碱型乙酰胆碱受体 (muscarinicacetylcholinereceptor,mAChR)和Bcl 2家族蛋白均具有调控神经细胞凋亡和生存的作用 ,然而mAChR和Bcl 2家族蛋白之间的内在联系即信号转导通路仍然不清楚。为此 ,对mAChR调控神经母细胞瘤SH SY5Y细胞生存蛋白Bcl 2和磷酸化Bad的信号转导通路进行了研究。结果显示 :(1)mAChR激动剂卡巴可 (carbachol)不仅活化SH SY5Y细胞的MEK/ERK 1/ 2 ,而且上调Bcl 2和磷酸化Bad的表达 ;(2 )mAChR拮抗剂阿托品、MEK抑制剂PD980 5 9、PKC抑制剂bisindolymaleimide I和Src抑制剂PP1均能完全阻断或显著减弱卡巴可的上述作用 ,但G蛋白脱偶联剂百日咳毒素和PI 3激酶抑制剂wortmannin对卡巴可的上述作用无明显影响 ;(3)显性负突变Ras和Raf均能阻断卡巴可上调转染至SH SY5Y细胞内的Bcl 2启动子的转录调控活性。结果表明 :mAChR通过Gq/ 11、PKC和Src依赖的Ras ERK 1/ 2信号转导通路上调SH SY5Y细胞Bcl 2和磷酸化Bad蛋白表达。这一研究将有助于揭示神经递质、神经营养因子和神经营养药物等抑制神经细胞凋亡、促进神经细胞生存的分子机制。     

cAMP反应元件结合蛋白介导磷酸化细胞外信号调节激酶在神经病理性痛形成中的作用

采用行为学、免疫组织化学和Western blot方法,观察鞘内注射细胞外信号调节激酶(extracellular signal-regulate kinase,ERK)信号转导通路阻滞剂对慢性压迫性损伤(chronic constriction injury,CCI)大鼠痛行为及脊髓背角内磷酸化cAMP反应元件结合蛋白(phosphorylated cAMP response-element binding protein,pCREB)和Fos表达变化的影响,探讨ERK／CREB转导通路在神经病理性疼痛中的作用。结果表明,CCI可明显增加双侧脊髓背角pCREB、损伤侧脊髓背角浅层Fos阳性神经元表达,以CCI后3与5d时尤为显著。鞘内沣射促分裂原活化蛋白激酶激酶(mitogen-activated protein kinase kinase,MEK)阻滞剂U0126及ERK反义寡核苷酸在减轻大鼠痛行为的同时,能明显抑制双侧脊髓背角内pCREB的表达,同时,Fos阳性神经元的表达也明显减少。大鼠痛行为及脊髓背角pCREB和Fos的表达在时相上一致。上述结果提示pCREB参与pERK介导的神经病理性疼痛。     

宽频噪音对大鼠AD模型不同脑区ERK、GDNF表达及ABR阈值的影响

目的 :观察大鼠AD模型颞叶和额叶在 98dB宽频噪音暴露 5min前后不同脑区ERK、GDNF表达及ABR阈值的影响。方法 :取体重 15 0～ 2 2 0 gSD大鼠 ,雌雄不拘 ,经行为训练筛选 ,剔除记忆差 (指“稍有记忆”和“无记忆”)大鼠后 ,随机分 3组 :双侧海马CA1区 (AP3.2～ 3.4 ,L2 .0～ 2 .4 ,H2 .8～ 3.0 )微量注射谷氨酸组 (n =8) ;双侧海马CA1区微量注射生理盐水组 (n =8) ;空白对照组 (n =10 )。采用WesternBlot及图像分析技术 ,结合ABR测定方法。结果 :①空白对照组大鼠额叶ERK表达明显多于其它两组鼠 ,且加噪音后有较显著的上调趋势 ;②各组动物颞区皮质神经元加噪音后ERK表达明显增强 ,且均强于额叶各组 ,也有较显著的上调趋势 ;③空白对照组大鼠额叶GDNF加噪音后表达多于加噪音前的同组鼠 ,有较明显的上调趋势 ;④谷氨酸组加噪音后颞区皮质神经元GDNF表达有较明显的下调趋势 ;⑤颞区空白对照组GDNF表达远弱于额叶。结论 :AD模型大鼠额叶ERK表达较少 ,不受噪音刺激影响 ,颞区则相反 ,且明显上调 ;宽频噪音抑制大鼠AD模型颞叶GDNF表达。     

大鼠内侧前额叶皮质内ERK1/2 活化参与脊髓损伤后抑郁情绪

目的:观察细胞外信号调节激酶1/2(ERK1/2)的活化在脊髓损伤引起抑郁中的作用。方法:应用Western blot和行为药理学方法,观察脊髓损伤后(SCI)大鼠内侧前额叶皮质内(mPFC)ERK1/2及磷酸化-ERK1/2(p-ERK1/2)的表达情况及ERK1/2磷酸化抑制剂U0126对抑郁样行为的影响。结果:脊髓损伤后的第2天到第8周,SCI模型大鼠的BBB评分均显著低于假手术组,差异具有统计学意义(p0.05)。脊髓损伤后8周-12周,SCI模型大鼠强迫游泳不动时间与假手术组相比明显缩短,mPFC内pERK1/2蛋白表达水平明显升高,总ERK 1/2的蛋白水平则未见组间差异,而给予U0126的大鼠的不动时间与给药之前相比明显延长增加,mPFC内pERK1/2蛋白表达水平较SCI模型大鼠明显降低,差异均具有统计学意义(P0.05)。结论:内侧前额叶皮质内ERK1/2的激活参与了脊髓损伤后引起的突触可塑性,在相关的抑郁样行为的产生中发挥了重要的作用。     

细胞MEK1/2蛋白及其相关通路在单纯疱疹病毒Ⅱ型（HSV-2）复制中的作用

基于细胞Raf/MEK/ERK信号通路与病毒复制的关系,应用Western印迹检测 p-ERK1/2蛋白的表达、用终点滴定法测定病毒增殖量（TCID_５０）,以及观察感染细胞的细胞病变效应（CPE）等,揭示单纯疱疹病毒Ⅱ型(HSV-2)复制与 ERK通路的关系. 结果表明,HSV-2的复制可引起细胞ERK通路的活化;用U0126预先抑制ERK通路的活化,或用特异性siRNA敲减MEK1/2基因的表达可显著地抑制病毒复制.提示ERK信号通路以及MEK1/2蛋白对HSV-2的复制具有重要的作用.该研究对进一步阐明细胞ERK通路各激酶蛋白在病毒复制中的作用机制、寻找抗病毒作用靶标等奠定了良好的基础.     

PBK/TOPK在乳腺癌细胞中介导MEK非依赖性ERK激活中作用机制分析

该文探讨了乳腺癌细胞中表皮生长因子(EGF)介导的MEK非依赖性ERK激活通路。Western blot检测EGF刺激下,siRNA抑制MEK1/2后的T47D细胞的p-ERK水平,以验证T47D细胞中存在EGF介导的MEK非依赖性ERK激活的通路。接着使用可能参与MEK非依赖性ERK激活的激酶的小分子抑制剂抑制相关激酶(AC、PKC、Src、PI3K、PDK1和Akt)活性后,检测T47D细胞EGF介导ERK的磷酸化水平。siRNA抑制MEK1/2表达后,T47D细胞在EGF刺激后的仍保留部分p-ERK,即在T47D细胞中,存在EGF介导的MEK非依赖性的ERK磷酸化通路。小分子抑制剂抑制AC、PKC、Src对MEK非依赖性ERK激活途径影响不大。而使用小分子抑制剂抑制PI3K、PDK1和Akt后,ERK的磷酸化水平显著降低,提示PI3K/Akt通路下游的激酶参与T47D中EGF介导的MEK非依赖性ERK激活途径。siRNA干扰PI3K/Akt通路下游PBK/TOPK后并使用U0126抑制MEK功能后,几乎检测不到p-ERK,提示PBK/TOPK参与T47D细胞中EGF介导的MEK非依赖性ERK激活途径。乳腺癌抗雌激素药物耐药株T47D细胞存在EGF介导的MEK非依赖性ERK激活途径,且该途径受PI3K/Akt下游的PBK/TOPK调控。     

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.     

福莫特罗对大鼠骨髓间充质干细胞向成骨细胞分化基因表达的影响

目的:研究β2肾上腺素能激动剂福莫特罗(Formoterol)对大鼠的体外骨髓间充质干细胞(MMSC)向成骨细胞分化的影响,进而探讨其作用机制。方法:取SD大鼠的骨髓间充质干细胞,用条件培养液诱导分化后分别加入不同浓度药物,在不同时间点采用RT-PCR法检测细胞分化中Runx2和Osterix的mRNA的表达,采用westernblot法检测细胞中MEK和ERK1/2的磷酸化。结果:在细胞分化早期,加入已知浓度10-7mol/L的Formoterol可抑制成骨样细胞细胞特异性转录因子Runx2mRNA表达;在细胞分化晚期,浓度10-7mol/L的Formoterol也可抑制成骨样细胞OsterixmRNA表达。在加入浓度10-7mol/的Formoterol作用30min后,MEK和ERK1/2的蛋白磷酸化表达均下降。结论:β2受体激动剂可抑制MMSC细胞体外向成骨样细胞的分化,并且可抑制MEK和ERK1/2磷酸化的表达。     

Increased Membrane/Nuclear Translocation and Phosphorylation of p90 KD Ribosomal S6 Kinase in the Brain of Hypoxic Preconditioned Mice

Our previous studies have demonstrated that hypoxic precondition (HPC) increased membrane translocation of protein kinase C isoforms and decreased phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) in the brain of mice. The goal of this study was to determine the involvement of p90 KD ribosomal S6 kinase (RSK) in cerebral HPC of mice. Using Western-blot analysis, we found that the levels of membrane/nuclear translocation, but not protein expression of RSK increased significantly in the frontal cortex and hippocampus of HPC mice. In addition, we found that the phosphorylation levels of RSK at the Ser227 site (a PDK1 phosphorylation site), but not at the Thr359/Ser363 sites (ERK1/2 phosphorylated sites) increased significantly in the brain of HPC mice. Similar results were confirmed by an immunostaining study of total RSK and phospho-Ser227 RSK. To further define the cellular populations to express phospho-Ser227 RSK, we found that the expression of phospho-Ser227 RSK co-localized with neurogranin, a neuron-specific marker, in cortex and hippocampus of HPC mice by using double-labeled immunofluorescent staining method. These results suggest that increased RSK membrane/nuclear translocation and PDK1 mediated neuron-specific phosphorylation of RSK at Ser227 might be involved in the development of cerebral HPC of mice.     

Association of upregulated Ras/Raf/ERK1/2 signaling with autism

Autism is a neurodevelopmental disorder characterized by impairments in social interaction, verbal communication and repetitive behaviors. BTBR mouse is currently used as a model for understanding mechanisms that may be responsible for the pathogenesis of autism. Growing evidence suggests that Ras/Raf/ERK1/2 signaling plays death-promoting apoptotic roles in neural cells. Recent studies showed a possible association between neural cell death and autism. In addition, two studies reported that a deletion of a locus on chromosome 16, which includes the MAPK3 gene that encodes ERK1, is associated with autism. We thus hypothesized that Ras/Raf/ERK1/2 signaling could be abnormally regulated in the brain of BTBR mice that models autism. In this study, we show that expression of Ras protein was significantly elevated in frontal cortex and cerebellum of BTBR mice as compared with B6 mice. The phosphorylations of A-Raf, B-Raf and C-Raf were all significantly increased in frontal cortex of BTBR mice. However, only C-Raf phosphorylation was increased in the cerebellum of BTBR mice. In addition, we further detected that the activities of both MEK1/2 and ERK1/2, which are the downstream kinases of Ras/Raf signaling, were significantly enhanced in the frontal cortex. We also detected that ERK1/2 is significantly over-expressed in frontal cortex of autistic subjects. Our results indicate that Ras/Raf/ERK1/2 signaling is upregulated in the frontal cortex of BTBR mice that model autism. These findings, together with the enhanced ERK1/2 expression in autistic frontal cortex, imply that Ras/Raf/ERK1/2 signaling activities could be increased in autistic brain and involved in the pathogenesis of autism.     

Angiotensin II promotes the phosphorylation of cyclic AMP‐responsive element binding protein (CREB) at Ser133 through an ERK1/2-dependent mechanism

In cells from the adrenal medulla, angiotensin II (AII) regulates both the activity and mRNA levels of catecholamine biosynthetic enzymes whose expression is thought to be under the control of cAMP-responsive element (CRE) binding protein (CREB). In this study, we evaluated the effect of AII stimulation on CREB phosphorylation at Ser133 (pCREB) in bovine adrenal chromaffin cells (BACC). We found that AII produces a rapid and AII type-1 receptor (AT1)-dependent increase in pCREB levels, which is blocked by the MEK1/2 inhibitor U0126 but not by H-89, SB203580 or KN-93, suggesting that it is mediated by the extracellular-regulated protein kinases 1 and 2 (ERK1/2) and not by cAMP-dependent protein kinase (PKA), p38 mitogen-activated protein kinase (p38MAPK) or Ca(2+)/calmodulin-dependent protein kinases (CaMKs) dependent pathways. Gel-shift experiments showed that the increase in pCREB levels is accompanied by an ERK1/2-dependent upregulation of CRE-binding activity. We also found that AII promotes a rapid and reversible increase in the activity of the non-receptor tyrosine kinase Src and that the inhibition of this enzyme completely blocks the AII-induced phosphorylation of ERK1/2, the CREB kinase (p90)RSK and CREB. Our data support the hypothesis that in BACC, AII upregulates CREB functionality through a mechanism that requires Src-mediated activation of ERK 1/2 and (p90)RSK.     

Depolarization-induced differentiation of PC12 cells is mediated by phospholipase D2 through the transcription factor CREB pathway

The present study examined the role of phospholipase D2 (PLD2) in the regulation of depolarization-induced neurite outgrowth and the expression of growth-associated protein-43 (GAP-43) and synapsin I in rat pheochromocytoma (PC12) cells. Depolarization of PC12 cells with 50 mmol/L KCl increased neurite outgrowth and elevated mRNA and protein expression of GAP-43 and synapsin I. These increases were suppressed by inhibition of Ca²⁺-calmodulin-dependent protein kinase II (CaMKII), PLD, or mitogen-activated protein kinase kinase (MEK). Knockdown of PLD2 by small interfering RNA (siRNA) suppressed the depolarization-induced neurite outgrowth, and the increase in GAP-43 and synapsin I expression. Depolarization evoked a Ca²⁺ rise that activated various signaling enzymes and the cAMP response element-binding protein (CREB). Silencing CaMKIIδ by siRNA blocked KCl-induced phosphorylation of proline-rich protein tyrosine kinase 2 (Pyk2), Src kinase, and extracellular signal-regulated kinase (ERK). Inhibition of Src or MEK abolished phosphorylation of ERK and CREB. Furthermore, phosphorylation of Pyk2, ERK, and CREB was suppressed by the PLD inhibitor, 1-butanol and transfection of PLD2 siRNA, whereas it was enhanced by over-expression of wild-type PLD2. Depolarization-induced PLD2 activation was suppressed by CaMKII and Src inhibitors, but not by MEK or protein kinase A inhibitors. These results suggest that the signaling pathway of depolarization-induced PLD2 activation was downstream of CaMKIIδ and Src, and upstream of Pyk2(Y881) and ERK/CREB, but independent of the protein kinase A. This is the first demonstration that PLD2 activation is involved in GAP-43 and synapsin I expression during depolarization-induced neuronal differentiation in PC12 cells.     

Leukotriene D4 mediates survival and proliferation via separate but parallel pathways in the human intestinal epithelial cell line Int 407

We demonstrated previously that leukotriene D4 (LTD4) regulates proliferation of intestinal epithelial cells through a CysLT receptor by protein kinase C (PKC)epsilon-dependent stimulation of the mitogen-activated protein kinase ERK1/2. Our current study provides the first evidence that LTD4 can activate 90-kDa ribosomal S6 kinase (p90RSK) and cAMP-responsive element-binding protein (CREB) via pertussis-toxin-sensitive Gi protein pathways. Transfection and inhibitor experiments revealed that activation of p90RSK, but not CREB, is a PKCepsilon/Raf-1/ERK1/2-dependent process. LTD4-mediated CREB activation was not affected by expression of kinase-dead p90RSK but was abolished by transfection with the regulatory domain of PKCalpha (a specific dominant-inhibitor of PKCalpha). Kinase-negative mutants of p90RSK and CREB (K-p90RSK and K-CREB) blocked the LTD4-induced increase in cell number and DNA synthesis (thymidine incorporation). Compatible with these results, flow cytometry showed that LTD4 caused transition from the G0/G1 to the S+G2/M cell cycle phase, indicating increased proliferation. Similar treatment of cells transfected with K-p90RSK resulted in cell cycle arrest in the G0/G1 phase, consistent with a role of p90RSK in LTD4-induced proliferation. On the other hand, expression of K-CREB caused a substantial buildup in the sub-G0/G1 phase, suggesting a role for CREB in mediating LTD4-mediated survival in intestinal epithelial cells. Our results show that LTD4 regulates proliferation and survival via distinct intracellular signaling pathways in intestinal epithelial cells.     

Activation of p90 ribosomal S6 kinase by ORF45 of Kaposi's sarcoma-associated herpesvirus and its role in viral lytic replication

The extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) pathway is essential for infection by a variety of viruses. The p90 ribosomal S6 kinases (RSKs) are direct substrates of ERK and functional mediators of ERK MAPK signaling, but their roles in viral infection have never been examined. We demonstrate that ORF45 of Kaposi's sarcoma-associated herpesvirus (KSHV) interacts with RSK1 and RSK2 and strongly stimulates their kinase activities. The activation of RSK by ORF45 is correlated with ERK activation but does not require MEK. We further demonstrate that RSK1/RSK2 is activated during KSHV primary infection and reactivation from latency; a subset of RSK1/RSK2 is present in the viral replication compartment in the nucleus. Depletion of RSK1/RSK2 by small interfering RNA or the specific inhibitor BI-D1870 suppresses KSHV lytic gene expression and progeny virion production, suggesting an essential role of RSK1/RSK2 in KSHV lytic replication.