微波辐射对PC12细胞Raf/MEK/ERK信号通路相关分子表达的影响

体外培养PC12细胞,将其诱导分化为神经元后,建立微波辐射细胞模型,采用免疫印迹技术和图像分析技术研究微波辐射后Raf/MEK/ERK信号通路相关分子的动态表达变化规律,进一步探讨微波辐射损伤的分子机制。结果发现,微波辐射后6h～3d,假辐射组和辐射组PC12细胞中Raf-1、ERK表达均呈先增加后减少趋势,两组差别不显著,但辐射组Raf-1、ERK和CREB的磷酸化水平均较假辐射组明显升高,表明Raf/MEK/ERK信号通路活化增强可能是微波辐射致神经细胞损伤的重要机制。     

HIF-1α/ERK通路活化调控微波长期辐射致大鼠海马神经元线粒体损伤

本文旨在探讨微波辐射致大鼠海马神经元线粒体损伤中HIF-1α和ERK通路分子表达的改变及意义,为深入研究微波辐射损伤机制和防治提供新靶标.2.5,5和10mW/cm2的微波辐射100只雄性Wistar大鼠,辐射时间为6min/次,5次/周,连续辐射1月,于辐射后6h,7d,14d,1周和2月,采用Real-timePCR,Westernblot和免疫组织化学检测海马中hif-1αmRNA,HIF-1α,ERK1/2和p-ERK1/2表达.结果发现,大鼠海马hif-1αmRNA和HIF-1α蛋白分别在2.5和5mW/cm2组于辐射后14d和1月明显增加,10mW/cm2组辐射后14d～2月降低.但海马ERK1/2未见明显改变.假辐射组p-ERK1/2于海马神经元胞浆中呈弱阳性,2.5mW/cm2组p-ERK1/2表达无明显变化,5和10mW/cm2辐射后7d～1月,p-ERK1/2于海马神经元胞浆和胞核中呈阳性或强阳性.2.5,5和10mW/cm2微波长期辐射后大鼠海马HIF-1α和p-ERK1/2表达的改变,表明HIF-1α和ERK通路活化参与微波辐射致海马线粒体损伤的过程,并可能发挥修复线粒体损伤的作用.     

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。     

去卵巢大鼠海马CA1／CA2区ERK1／2的基础活性和诱导活性降低

目的：观察去卵巢大鼠空间学习记忆能力的变化与海马中胞外信号调节激酶1／2（ERK1／2）通路的关系。方法：雌性sD大鼠随机分为假手术组和去卵巢组,饲养4个月后采用Morris水迷宫测试空间学习记忆能力,于测试前将各组组内又分为训练组和非训练组,训练组用于测定经学习记忆训练诱发的ERK1／2的诱导活性,非训练组用于测定未经学习记忆训练时的ERK1／2的基础活性,Western blot方法检测海马CA1／CA2区p-ERK1／2蛋白及Raf激酶抑制蛋白（RKIP）的变化。结果：①与假手术组比较,去卵巢组大鼠的空间学习记忆能力明显下降（P〈0．05）。②各组中的训练大鼠p-ERK1／2蛋白水平明显高于非训练大鼠（P〈0．05）。③去卵巢组训练及非训练大鼠的p-ERK1／2蛋白水平均相应低于假手术组训练及非训练大鼠（P〈0．05）。④去卵巢组RKIP蛋白表达水平明显高于假手术组（P〈0．05）。结论：雌激素缺乏大鼠的空间学习记忆能力下降与海马CA1／CA2区ERK1／2通路的基础和诱导活性降低以及该通路的抑制蛋白RKIP的表达增加有关。     

不同波段电磁辐射对大鼠Sertoli细胞雄激素受体和卵泡刺激素受体表达的影响

探讨不同波段电磁辐射对大鼠睾丸Sertoli细胞雄激素受体(androgen receptor,AR)和卵泡刺激素受体(follicle stimulating hormone receptor,FSHR)表达的影响。原代培养的Sertoli细胞分别经场强6×104 V/m的电磁脉冲(electromagnetic pulse,EMP)、平均功率密度为100 mW/cm2的S波段微波(S-band high power microwave,S-HPM)和X波段微波(X-bandhigh power microwave,X-HPM)辐射4 min。应用real-time RT-PCR和Western blot方法检测Sertoli细胞AR的表达,结果显示具有不同程度的降低(P<0.05,P<0.01);Wistar大鼠分别经S-HPM、X-HPM和EMP照射20 min,应用免疫组化方法检测睾丸组织AR的表达,发现辐射后7 d有显著降低(P<0.05,P<0.01)。三者比较,AR的表达总体呈EMP>X-HPM>S-HPM的趋势,而FSHR的表达无明显变化。Sertoli细胞AR表达的变化,可能参与了电磁辐射致...     

丙戊酸钠活化大鼠海马和额叶ERK-1/2信号传导通路

为探讨慢性服用丙戊酸钠对中枢神经系统细胞外调控激酶 (ERK) 1/ 2信号传导通路活性的影响 ,阐明丙戊酸钠治疗躁狂抑郁症作用的可能分子机制 ,将 4 0只雄性Wistar大鼠随机分为实验组和对照组 ,每组各 2 0只 .实验组大鼠用含丙戊酸钠的饲料喂养 ,对照组大鼠用常规饲料喂养 ,4周后取大鼠海马和额叶组织制备蛋白质样本 ,蛋白质印迹方法分析海马和额叶组织丝裂原活化的蛋白激酶激酶 (MEK)、ERK 1/ 2、MAPK活化的蛋白激酶 1(RSK1)、cAMP效应元件结合因子 (CREB)的磷酸化水平以及Bcl 2的表达水平 ,电泳迁移率变动分析(EMSA)方法分析海马和额叶组织激活蛋白 1(AP 1)的DNA结合活性 .与对照组比较 ,丙戊酸钠显著增强海马和额叶MEK、ERK 1/ 2、RSK1、CREB和AP1的活性 ,上调海马和额叶Bcl 2的表达 .结果表明 :慢性服用丙戊酸钠激活中枢神经系统ERK 1/ 2信号传导通路、上调中枢神经系统Bcl 2蛋白表达 ,这些作用可能与丙戊酸钠治疗躁狂抑郁症的作用有关     

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。     

14-3-3参与apelin-13促进大鼠血管平滑肌细胞增殖ERK1/2信号途径研究

本室以前已经报道了G蛋白偶联受体APJ的内源性配体多肽,apelin-13,通过激活ERK1/2促进大鼠血管平滑肌细胞增殖.本文研究14-3-3信号蛋白是否参与apelin-13促进大鼠血管平滑肌细胞增殖ERK1/2信号途径,探讨apelin/APJ系统的细胞信号转导机制.组织贴块法培养大鼠胸主动脉VSMCs;Western blotting方法检测14-3-3、pRaf-1、Raf-1、pERK1/2、ERK1/2、cyclinD1、cyclinE的表达;MTT方法观察14-3-3抑制剂Difopein对VSMCs的增殖作用;免疫共沉淀方法检测14-3-3和Raf-1蛋白复合物的形成.Western blotting方法结果显示,apelin-13(0、0.5、1、2、4μmol/L)浓度依赖性刺激大鼠VSMCs 14-3-3表达、Raf-1和ERK1/2磷酸化,以2μmol/L最为明显;2μmol/L apelin-13时间依赖性刺激大鼠VSMCs 14-3-3表达、Raf-1和ERK1/2磷酸化,在4 h增加最为显著;14-3-3蛋白抑制剂Difopein明显抑制apelin-13诱导的Raf-1磷酸化、ERK1/2磷酸化、cyclinD1及cyclinE表达;免疫共沉淀方法发现apelin-13诱导14-3-3与Raf-1结合增加,而Difopein明显抑制两者结合;MTT法显示Difopein明显抑制apelin-13诱导的血管平滑肌细胞增殖.上述结果表明,Apelin-13通过14-3-3/Raf-1复合物-ERK1/2信号转导通路促进大鼠血管平滑肌细胞增殖.     

不同波段电磁辐射致大鼠睾丸支持细胞的损伤效应

为探讨不同波段电磁辐射对大鼠睾丸支持细胞(Sertoli细胞)损伤效应的异同.将原代培养的Sertoli细胞经场强6×104V/m的电磁脉冲(electromagnetic pulse,EMP)、平均功率密度为100mW/cm2的S-波段高功率微波(S-band high power microwave,S-HPM)和...     

Cyclosporin A通过MEK／ERK1／2信号通路调节滋养细胞titin表达

探讨MEK／ERK1／2信号通路在CyclosporinA(CsA)诱导滋养细胞表达titin中的作用。应用RT—PCR、Western blot检测CsA诱导的滋养细胞titin的表达水平,Western blot检测CsA作用于滋养细胞后ERK1／2的活化程度,并观察MEK特异性抑制剂U0126对其mRNA转录的影响。发现CsA以时间和剂量依赖方式诱导titin表达,并刺激滋养细胞ERK1／2的活化,U0126以剂量依赖方式抑制CsA诱导的titin表达。结果表明CsA通过活化MEK／ERK1／2信号通路诱导滋养细胞titin的表达,改变其生物学行为,从而有利于胚胎着床及早期发育。     

Oligonol, an oligomerized lychee fruit-derived polyphenol, activates the Ras/Raf-1/MEK1/2 cascade independent of the IL-6 signaling pathway in rat primary adipocytes

Oligonol is a lychee fruit-derived low-molecular form of polyphenol. In this study, the effect of Oligonol on the mitogen activated-protein kinase (MAPK) signaling pathway in primary adipocytes was investigated to examine the mechanism underlying the enhanced levels of phosphorylated extracellular-signaling regulatory kinase1/2 (ERK1/2) that accompany an in vitro increase in lipolysis. Oligonol significantly elevated the levels of activated Ras and the phosphorylation of Raf-1 and MAPK/ERK kinase1/2 (MEK1/2) with no increase in pan-Raf-1 and -MEK1/2 proteins. The increase in phosphorylation of Raf-1 and MEK1/2 with Oligonol was inhibited completely by pretreatment with GW5074, a selective Raf-1 inhibitor, or PD98059, a selective MEK1/2 inhibitor. IL-6 also activated the MAPK signaling pathway in adipocytes through the association with its receptor. IL-6-induced phosphorylation of Raf-1 and MEK1/2 was significantly inhibited by pretreatment with the IL-6 receptor antibody. Under such a condition, however, the levels of phosphorylated Raf-1 and MEK1/2 with Oligonol still remained significantly higher, and there was a significant decrease in secretion of IL-6 from adipocytes, compared with untreated control cells. These results suggest that Oligonol activates the Ras/Raf-1/MEK1/2 signaling pathway, independent of the IL-6 signaling pathway, leading to activation of ERK1/2 proteins in primary adipocytes.     

Opposing roles of Ras/Raf oncogenes and the MEK1/ERK signaling module in regulation of expression and adhesive function of surface transglutaminase

Tissue transglutaminase (tTG) serves as a potent and ubiquitous integrin-associated adhesion co-receptor for fibronectin on the cell surface and affects several key integrin functions. Here we report that in fibroblasts, activated H-Ras and Raf-1 oncogenes decrease biosynthesis, association with beta1 integrins, and surface expression of tTG because of down-regulation of tTG mRNA. In turn, the reduction of surface tTG inhibits adhesion of H-Ras- and Raf-1-transformed cells on fibronectin and, in particular, on its tTG-binding fragment I(6)II(1,2)I(7-9), which does not interact directly with integrins. Analysis of Ras/Raf downstream signaling with specific pharmacological inhibitors reveals that the decrease in tTG expression is mediated by the p38 MAPK, c-Jun NH2-terminal kinase, and phosphatidylinositol 3-kinase pathways. In contrast, increased activation of the ERK pathway by constitutively active MEK1 stimulates tTG mRNA expression, biosynthesis, and surface expression of tTG, whereas MEK inhibitors or dominant negative MEK1 exert an opposite effect. This modulation of surface tTG by ERK signaling alters adhesion of cells on fibronectin and its fragment that binds tTG. Furthermore, transient stimulation of ERK signaling in untransformed fibroblasts by adhesion on fibronectin or growth factors elevates tTG biosynthesis, increases complex formation with beta1 integrins, and raises surface expression of tTG. Finally, ERK activation is required for growth factor-induced redistribution of tTG on the surface of adherent fibroblasts and co-clustering of beta1 integrins and tTG at cell-matrix adhesion contacts. Together, our data indicate that down-regulation of surface tTG by Ras and Raf oncogenes contributes to adhesive deficiency of transformed fibroblasts, whereas stimulation of biosynthesis and surface expression of tTG by the MEK1/ERK module promotes and sustains cell-matrix adhesion of untransformed cells. Contrasting effects of Ras/Raf oncogenes and their immediate downstream signaling module, MEK1/ERK, on tTG expression are consistent with adhesive function of surface tTG.     

Contribution of the ERK5/MEK5 pathway to Ras/Raf signaling and growth control.

The activity of the catalytic domain of the orphan MAP kinase ERK5 is increased by Ras but not Raf-1 in cells, which suggests that ERK5 might mediate Raf-independent signaling by Ras. We found that Raf-1 does contribute to Ras activation of ERK5 but in a manner that does not correlate with Raf-1 catalytic activity. A clue to the mechanism of action of Raf-1 on ERK5 comes from the observation that endogenous Raf-1 binds to endogenous ERK5, suggesting the involvement of regulatory protein-protein interactions. This interaction is specific because Raf-1 binds only to ERK5 and not ERK2 or SAPK. Finally, we demonstrate the ERK5/MEK5 pathway is required for Raf-dependent cellular transformation and that a constitutively active form of MEK5, MEK5DD, synergizes with Raf to transform NIH 3T3 cells. These observations suggest that ERK5 plays a large role in Raf-1-mediated signal transduction.     

The Raf/MEK/ERK pathway can govern drug resistance,apoptosis and sensitivity to targeted therapy

The effects of the Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR signaling pathways on proliferation, drug resistance, prevention of apoptosis and sensitivity to signal transduction inhibitors were examined in FL/ΔAkt-1:ER*(Myr⁺) + ΔRaf-1:AR cells which are conditionally-transformed to grow in response to Raf and Akt activation. Drug resistant cells were isolated from FL/ΔAkt-1:ER*(Myr⁺) + ΔRaf-1:AR cells in the presence of doxorubicin. Activation of Raf-1, in the drug resistant FL/ΔAkt-1:ER*(Myr⁺) + ΔRaf-1:AR cells, increased the IC₅₀ for doxorubicin 80-fold, whereas activation of Akt-1, by itself, had no effect on the doxorubicin IC₅₀. However, Akt-1 activation enhanced cell proliferation and clonogenicity in the presence of chemotherapeutic drugs. Thus the Raf/MEK/ERK pathway had profound effects on the sensitivity to chemotherapeutic drugs, and Akt-1 activation was required for the long-term growth of these cells as well as resistance to chemotherapeutic drugs. The effects of doxorubicin on the induction of apoptosis in the drug resistant cells were enhanced by addition of either mTOR and MEK inhibitors. These results indicate that targeting the Raf/MEK/ERK and PI3K/Akt/mTOR pathways may be an effective approach for therapeutic intervention in drug resistant cancers that have mutations activating these cascades.     

Signaling crossroads: the function of Raf kinase inhibitory protein in cancer, the central nervous system and reproduction

The Raf kinase inhibitory protein 1 (RKIP-1) and its orthologs are conserved throughout evolution and widely expressed in eukaryotic organisms. In its non-phosphorylated form RKIP-1 negatively regulates the Raf/MEK/ERK pathway by interfering with the activity of Raf-1. In its phosphorylated state, RKIP-1 dissociates from Raf-1 and inhibits GRK-2, a negative regulator of G-protein coupled receptors (GPCRs). Available data indicate that the phosphorylation of RKIP-1 by PKC can stimulate both the Raf/MEK/ERK and GPCR pathways. RKIP-1 has also been implicated as a negative regulator of the NF-kappaB pathway. Recent studies have shown that phosphorylated RKIP-1 binds to the centrosomal and kinetochore regions of metaphase chromosomes, where it may be involved in regulating the partitioning of chromosomes and the progression through mitosis. The collective evidence indicates that RKIP-1 regulates the activity and mediates the crosstalk between several important cellular signaling pathways. A variety of ablative interventions suggest that reduced RKIP-1 function may influence metastasis, angiogenesis, resistance to apoptosis, and genome integrity. Attenuation of RKIP-1 may also affect cardiac and neurological functions, spermatogenesis, sperm decapacitation, and reproductive behavior. In this review, the role of RKIP-1 in cellular signaling, and especially its functions revealed using a mouse knockout model, are discussed.     

Raf-1 kinase activation is uncoupled from downstream MEK/ERK pathway in cells treated with Src tyrosine kinase inhibitor PP2

Recently we demonstrated that PP2 (4-amino-5-(4-chloro-phenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine), a potent and selective inhibitor of the Src-family tyrosine kinase, markedly enhanced Ras-independent activation of Raf-1 by the combination of phorbol myristate acetate (PMA) and hydrogen peroxide (H(2)O(2)). We report here that Raf-1 knockdown cells were significantly more sensitive to treatment of PP2 than control cells. This PP2-induced growth inhibition was found to be linked to decreased ERK and p38 activity. Interestingly, the growth of Sprouty knockdown cells appeared to be inhibited at earlier time points of PP2 treatment when compared with control cells. Unexpectedly, siRNA-mediated knockdown of Spry2, which is known to modulate the Ras/Raf/MAPK signal through feedback regulation, resulted in decreased Raf-1 kinase activity. PP2 had limited effect on the ability of PMA/H(2)O(2) to induce significant phosphorylation of MEK/ERK proteins in both Spry2 knockdown and control cells, indicating that PP2-mediated activation of Raf-1 did not potentiate signaling through the downstream MEK/ERK pathway. Taken together our results suggest that Raf-1 signaling may be bypassed in PP2-treated cells by uncoupling from downstream MEK/ERK pathway.     

The Raf/MEK/ERK pathway can govern drug resistance,apoptosis and sensitivity to targeted therapy

The effects of the Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR signaling pathways on proliferation, drug resistance, prevention of apoptosis and sensitivity to signal transduction inhibitors were examined in FL/ΔAkt-1:ER*(Myr⁺) + ΔRaf-1:AR cells which are conditionally-transformed to grow in response to Raf and Akt activation. Drug resistant cells were isolated from FL/ΔAkt-1:ER*(Myr⁺) + ΔRaf-1:AR cells in the presence of doxorubicin. Activation of Raf-1, in the drug resistant FL/ΔAkt-1:ER*(Myr⁺) + ΔRaf-1:AR cells, increased the IC₅₀ for doxorubicin 80-fold, whereas activation of Akt-1, by itself, had no effect on the doxorubicin IC₅₀. However, Akt-1 activation enhanced cell proliferation and clonogenicity in the presence of chemotherapeutic drugs. Thus the Raf/MEK/ERK pathway had profound effects on the sensitivity to chemotherapeutic drugs, and Akt-1 activation was required for the long-term growth of these cells as well as resistance to chemotherapeutic drugs. The effects of doxorubicin on the induction of apoptosis in the drug resistant cells were enhanced by addition of either mTOR and MEK inhibitors. These results indicate that targeting the Raf/MEK/ERK and PI3K/Akt/mTOR pathways may be an effective approach for therapeutic intervention in drug resistant cancers that have mutations activating these cascades.     

ERK and Beyond: Insights from B-Raf and Raf-1 Conditional Knockouts

The Raf/MEK/ERK cascade is a highly conserved signal transduction module whose activation reportedly results in a plethora of physiological outcomes. Depending on the cell type or the stimulus used, the pathway has been implicated in proliferation, differentiation, survival, and migration. Their wide range of activities renders the component of the Raf/MEK/ERK pathway prime candidates for molecule-targeted therapies, in particular, but not exclusively, in the context of cancer. Ras, Raf and MEK inhibitors have been developed, and some of them are in advanced clinical trials. Somewhat surprising in view of all this interest, our understanding of the fundamental biology of the ERK pathway in vivo is still scanty. Its investigation has been hampered by the fact that conventional targeting of many of these genes results in embryonic lethality. Recently, we and others have generated mouse strains that allow the conditional ablation of the genes coding for Raf-1, B-Raf and MEK-1. We are using these tools to identify the essential biological functions of these kinases, and to understand how the ERK pathway is wired in vivo. Here, we discuss some of the surprises yielded by the analysis of the role of B-Raf and Raf-1 and of their downstream effectors.