Oxygen glucose deprivation inhibits the growth and ERK phosphorylation of neural progenitor cells in vitro

The neurogenic regions such as subventricular zone of the lateral ventricles could become ischemic in some clinical situations due to the blockage of blood vessels by blood clots. Hence the aim of this study is to investigate the effects of OGD on the growth of neural progenitor cells and the phosphorylation of ERK, which plays an important role in the growth of these cells. Oxygen glucose deprivation (OGD) for 4 h decreased the growth of neural progenitor cells in vitro and also decreased the phosphorylation of extracellular signal regulated kinase (ERK). Inhibition of the ERK pathway for 4 h using U0126 (10 μM) also decreased the growth of progenitor cells. These data suggest that a decline in the phospho-ERK content might decrease the growth of progenitor cells following OGD.     

Down-regulation of dual-specificity phosphatase 6, a negative regulator of oncogenic ERK signaling,by ACA-28 induces apoptosis in NIH/3T3 cells overexpressing HER2/ErbB2

Dual-specificity phosphatase 6 (DUSP6) is a key negative feedback regulator of the member of the RAS-ERK MAPK signaling pathway that is associated with cellular proliferation and differentiation. Deterioration of DUSP6 expression could therefore result in deregulated growth activity. We have previously discovered ACA-28, a novel anticancer compound with a unique property to stimulate ERK phosphorylation and induce apoptosis in ERK-active melanoma cells. However, the mechanism of cancer cell-specific-apoptosis by ACA-28 remains obscure. Here, we investigated the involvement of DUSP6 in the mechanisms of the ACA-28-mediated apoptosis by using the NIH/3T3 cells overexpressing HER2/ErbB2 (A4-15 cells), as A4-15 exhibited higher ERK phosphorylation and are more susceptible to ACA-28 than NIH/3T3. We showed that A4-15 exhibited high DUSP6 protein levels, which require ERK activation. Notably, the silencing of the DUDSP6 gene by siRNA inhibited proliferation and induced apoptosis in A4-15, but not in NIH/3T3, indicating that A4-15 requires high DUSP6 expression for growth. Importantly, ACA-28 preferentially down-regulated the DUSP6 protein and proliferation in A4-15 via the proteasome, while it stimulated ERK phosphorylation. Collectively, the up-regulation of DUSP6 may exert a growth-promoting role in cancer cells overexpressing HER2. DUSP6 down-regulation in ERK-active cancer cells might have the potential as a novel cancer measure.     

急性冷热应激对大鼠脊髓和脊神经节ERK信号转导的影响

ERK是细胞内MAPK信号转导系统的重要蛋白激酶,参与多种刺激的应激反应。本实验建立冷热应激模型,应用免疫组织化学和Western blot方法,研究冷热应激刺激对大鼠脊髓和脊神经节ERK的激活作用。结果显示:(1)正常脊髓非磷酸化ERK1(pan-ERK1)阳性细胞分布于RexedIII-X层,而I-II层没有免疫反应,胞浆着色,没有核膜断裂;磷酸化ERK1/2(p-ERK1/2)阳性细胞见于灰质Rexed各层(I-X),散在分布。免疫产物位于胞浆核周区,少见于细胞核,核膜有断裂。脊神经节p-ERK1/2免疫反应见于大、中、小型神经元,定位于细胞核;没有pan-ERK1免疫反应。(2)20min热应激后,RexedII层p-ERK1/2阳性细胞数明显增多,核周着色区增大,核膜常有断裂,脊神经节阳性细胞数没有明显变化;10min热应激和体温(38℃)应激后,脊髓和脊神经节阳性细胞数和核着色没有显著变化。(3)20min冷应激后,脊髓II层p-ERK1/2阳性细胞数轻度减少,核周着色区变窄,核膜有断裂,脊神经节阳性细胞数没有变化;10min冷应激后阳性细胞数没有明显变化。(4)Western blot显示,热应激和冷应激分别提高和降低了胸段脊髓p-ERK1/2的蛋白表达水平。本研究证实,ERK存在于脊髓灰质各层和脊神经节各型神经元,热应激提高了ERK激活水平而冷应激则相反,提示MAPK信号转导激活具有应激类型和应激强度的依赖性,参与了感觉神经元的功能调节。     

Grp78表达对人肝细胞癌细胞外信号调节激酶1/2活性和表达的影响

目的 通过检测葡萄糖调节蛋白78(Grp78)、细胞外信号调节激酶(ERK1/2)、磷酸化ERK1/2在肝细胞癌手术切除组织中的表达,并在SMMC-7721细胞中干预Grp78的表达,探讨Grp78对ERK1/2激酶的调控作用。 方法 应用免疫组织化学方法（IHC）和免疫印迹法检测47例肝细胞癌手术切除组织样本中Grp78,ERK1/2的表达和ERK1/2磷酸化水平;在高表达Grp78的肝细胞癌细胞系SMMC-7721中,通过用小RNA干扰Grp78的表达来探讨Grp78表达水平,改变对ERK1/2活性及表达的影响。 结果 在肝细胞癌组织样本中,Grp78的表达情况与ERK1/2和磷酸化ERK1/2的表达明显呈正相关。在SMMC-7721细胞中Grp78高表达促进ERK1/2的磷酸化,应用RNA干扰特异性下调Grp78高表达细胞中Grp78水平可以抑制ERK1/2的磷酸化。 结论 Grp78参与调解ERK1/2信号通路,并且在肝细胞癌临床治疗方面可能成为潜在的治疗靶点。     

Regulation of ERK2 phosphorylation by histamine in splenocytes

Histamine is implicated in allergic disease and asthma and ERK1/2 is involved in allergic inflammation including Th2 differentiation and proliferation. This study was designed to study the effects of histamine on ERK1/2 phosphorylation in splenocytes. C57/BL6 splenocytes were treated with different concentrations of histamine (10^?4 to 10^?11 M). Histamine (10^?4 M) increased ERK2 phosphorylation. There was, however, no significant effect seen at other concentrations (10^?11 to 10^?6 M). Surprisingly, H1 receptor agonist β-histine (10^?5 M), H2 agonist amthamine (10^?5 M), H3 agonist methimepip (10^?6 M), and H4 agonist 4-methyl histamine (10^?6 M), all increased ERK2 phosphorylation. H1R antagonist pyrilamine (10^?6 M), H2R antagonist ranitidine (10^?5 M), H3/H4R antagonist thioperamide (10^?6 M), and H3R antagonist clobenpropit (10^?5 M) inhibited histamine-mediated ERK2 phosphorylation suggesting that all four histamine receptor subtypes played some role in this phosphorylation. Because tumor necrosis factor-α (TNF-α) causes phosphorylation of ERK1/2, we investigated whether histamine acted via secretion of TNF-α to affect ERK1/2 phosphorylation. As a consequence, TNF-α knockout mice were used and we found that there was inhibition of ERK1 and ERK2 phosphorylation by H2, H3, and H4 agonists. This was in contrast to the wild-type splenocytes where histamine augmented the phosphorylation of ERK2 via H2, H3, and H4 receptors. In TNF-α knockout mice histamine did not affect the phosphorylation of ERK2 via H1 receptors. The results suggested that histamine indirectly caused the ERK2 phosphorylation via its effects on the secretion of TNF-α and these effects were mediated via H1, H2, H3, and H4 receptors.     

葛根素通过ERK1/2信号通路促进内皮细胞增殖

目的:体外观察葛根素对人脐静脉内皮细胞(HUVEC)的促增殖作用,并对其机制进行初步探讨。方法:采用四甲基偶氮唑蓝(MTT)法检测不同浓度葛根素对HUVEC细胞增殖的影响;流式细胞术检测细胞凋亡;Western blot检测p-ERK1/2蛋白表达水平。结果:葛根素能剂量依赖性促进HUVEC增殖,在80μM效果最明显,且作用36h时,能显著减少早期凋亡,激活ERK1/2磷酸化。结论:葛根素素对人脐静脉内皮细胞有明显的促进增殖作用,其作用可能与ERK1/2的活化有关。     

The role of ERK5 in T-cell signalling

The mitogen-activated protein kinase (MAPK) ERK5 plays an important role in mammary epithelial proliferation, endothelial cell survival and normal embryonic development. In nonhaematopoietic cells, mitogenic and stress signals activate the ERK5 cascade. Here, we investigated the role of the ERK5 pathway in T-cell activation and show that primary and leukaemic T cells express ERK5, whose activating phosphorylation is induced by antibodies against CD3 but not by phorbol myristate acetate treatment. ERK5 localized in the cytosol and nucleus in quiescent and activated T cells. In the latter, ERK5 phosphorylation was mainly observed in the nucleus. Selective activation of the ERK5 cascade by transfecting constitutively active MEK5 and wildtype ERK5 induced a reporter gene driven by the IL-2 promoter while barely affecting CD69 expression. These results suggest a new role for the ERK5 cascade in intracellular signalling in T cells.     

Cytokines induce tight junction disassembly in airway cells via an EGFR-dependent MAPK/ERK1/2-pathway

Epithelial barrier permeability is altered in inflammatory respiratory disorders by a variety of noxious agents through modifications of the epithelial cell structure that possibly involve tight junction (TJ) organization. To evaluate in vitro whether pro-inflammatory cytokines involved in the pathogenesis of respiratory disorders could alter TJ organization and epithelial barrier integrity, and to characterize the signal transduction pathway involved Calu-3 airway epithelial cells were exposed to TNF-a, IL-4 and IFN-g to assess changes in: (a) TJ assembly, that is, occludin and zonula occludens (ZO)-1 expression and localization, evaluated by confocal microscopy; (b) apoptotic activity, quantified using terminal transferase deoxyuridine triphosphate nick-end labeling staining; (c) epithelial barrier integrity, detected as transmembrane electrical resistance and expressed as G(T) values; (d) epidermal growth factor receptor (EGFR)-dependent mitogenactivated protein (MAP) kinase (MAPK)/extracellular signal-regulated kinases (ERK)1/2 phosphorylation, assessed by western blotting. Exposure to cytokines for 48 h induced a noticeable downregulation of the TJ transmembrane proteins. The degree ZO-1 and occludin colocalization was 62±2% in control cultures and significantly decreased in the presence of TNF-a (47±3%), IL-4 (43±1%) and INF-g (35±3%). Although no apoptosis induction was detected following exposure to cytokines, changes in the epithelial barrier integrity were observed, with a significant enhancement in paracellular conductance. G(T) values were, respectively, 1.030±0.0, 1.300±0.04, 1.260±0.020 and 2.220±0.015 (mS/cm2)1000 in control cultures and in those exposed to TNF-a, IFN-g and IL-4. The involvement of EGFR-dependent MAPK/ERK1/2 signaling pathway in cytokine-induced damage was demonstrated by a significant increase in threonine/tyrosine phosphorylation of ERK1/2, already detectable after 5 min incubation. All these cytokine-induced changes were markedly prevented when Calu-3 cells were cultured in the presence of an EGFR inhibitor (AG1478, 1 μM) or a MAP kinase inhibitor (U0126, 25 μM). In conclusion, cytokine-induced epithelial injury includes TJ disassembly and epithelial barrier permeability alteration and involves the EGFR-dependent MAPK/ERK1/2 signaling pathway.     

生长抑制蛋白4核定位信号和PHD指状结构域共同调控丝裂原活化蛋白激酶通路中ERK1的激活

目的探索生长抑制蛋白4(inhibitor of growth 4,ING4)的核定位信号(nuclear localization sig-nal,NLS)和PHD指状结构域(plant homeodomain)在刺激丝裂原活化蛋白激酶(MAPK)细胞信号通路成员ERK1磷酸化中的作用。方法通过构建ING4的NLS和PHD结构域突变或缺失重组质粒,将上述质粒分别与ERK1表达载体共转染HEK-293细胞,Western印迹检测ERK1总蛋白和磷酸化ERK1蛋白水平。结果在HEK-293细胞中,ING4蛋白分别缺失NLS和PHD结构域后,其对MAPK细胞信号通路成员ERK1磷酸化的促进作用均降低,突变ING4的NLS结构域中两个重要位点Lys-Lys(KK)和Arg-Ala-Arg-Ser-Lys(RARSK)并不改变对ERK1激活的能力,但突变ING4 PHD结构域中RKKK位点则改变了调控ERK1磷酸化的能力。结论ING4的NLS和PHD指状结构域同时参与了刺激MAPK细胞信号通路成员ERK1的激活。     

High nuclear expression of phosphorylated extracellular signal–regulated kinase in tumor cells in colorectal glands is associated with poor outcome in colorectal cancer

Extracellular signal–regulated kinase (ERK) is a major downstream transducer of Ras and plays an important role in transducing extracellular signals to the nuclei of cells. It is located in both the cytoplasm and the nucleus of cells. The nuclear localization of phosphorylated or activated ERK is involved in the invasive behavior of tumor cells. We studied the association between Ras mutation/ERK activation and the prognosis of patients with colorectal cancer. We analyzed 126 surgically resected colorectal cancer specimens for K-Ras mutation using direct sequencing. Activation/phosphorylation of ERK was assayed by immunohistochemistry with tissue microarray, and the staining intensity was analyzed using a semiquantitative scoring system. K-Ras mutations were detected in 32.5% (41/126) of the colorectal tumors. Colorectal glands are important functional organs in colorectal tissue and form the origin of colorectal carcinomas. Tissue microarray immunohistochemistry tests showed that tumors in colorectal cancer specimens were significantly stained for phospho-ERK (100%; 126/126), whereas nonneoplastic colorectal glands mainly showed faint phosphorylated ERK staining. High nuclear phospho-ERK expression in tumors was associated with highly invasive cancer stage and T status of the disease. Kaplan-Meier analysis showed that nuclear but not cytoplasmic phosphorylated ERK expression correlated with the patients' overall survival rate (P = .039). Colorectal adenomas including tubular adenomas and tubulovillous adenomas mainly showed weak cytoplasmic phospho-ERK expression. Our results suggest that immunohistologic analysis of phosphorylated ERK expression in colorectal glands may aid the diagnosis of colorectal cancer and that nuclear phosphorylated ERK might be a valuable prognostic marker for colorectal cancer.     

14,15-EET通过EGFR和p44/42 MAPK信号通道诱导肿瘤细胞增殖

目的探讨14,15-EET促进肿瘤细胞增殖的机制。方法Tca-8113培养于含10%小牛血清的DMEM,必要时更换无血清DMEM使其进入静止期。用14,15-EET和/或抑制剂处理后收集细胞蛋白,用Western印迹检测EGFR和p44/42MAPK(ERK1/ERK2)的磷酸化水平。结果14,15-EET以剂量依赖性方式刺激EGFR和p44/42MAPK(ERK1/ERK2)蛋白磷酸化,该效应能够被特异性的EGFR抑制剂AG1478阻断。MTT分析显示,AG1478能够完全取消14,15-EET诱导的Tca-8113细胞增殖效应。结论EGFR的活化是花生四烯酸细胞色素P450表氧化酶代谢产物14,15-EET丝裂原信号途径中的关键事件;在该途径中EGFR的活化是p44/42MAPK(ERK1/ERK2)活化的上游事件。     

Reversal of Multidrug Resistance by Gefitinib Via RAF1/ERK Pathway in Pancreatic Cancer Cell Line

Pancreatic cancer is a devastating malignancy, characterized by intrinsic or acquired resistance to conventional chemotherapies. Recent evidences suggest an involvement of tyrosine kinase pathway in the regulation of multidrug resistance (MDR) protein gene expression. The aim of this study was to test whether gefitinib, an epidermal growth factor receptor tyrosine kinase inhibitor could regulate the MDR protein gene expression and sensitize the resistant cancer cells to chemotherapy. The gene expression of MDR proteins (MRP1, MRP2, MRP3, and PGP) were evaluated by quantitative RT‐PCR, and expression levels of various tyrosine kinases were investigated by quantitative RT‐PCR and Western Blot in pancreatic cancer cell line. MTT assay was used for evaluating the effect of chemotherapeutic agents. Chemotherapeutics induced drug resistance by regulating the gene expression of MDR proteins (MRP1, MRP2, and MRP3), and increased the gene expression of RAF1/ERK and the phosphorylation of ERK in pancreatic cancer Bxpc‐3 cells. Gefitinib caused an inhibition of p‐ERK tyrosine kinase activation in a dose‐dependent manner, and reversed gemcitabine‐induced RAF1/ERK gene expression and p‐ERK activation. In addition, a reversal of MDR proteins gene expression was achieved by gefitinib, which sensitized resistant cells to gemcitabine. This study demonstrated that MDR of Bxpc‐3 cell is involved in the RAF1/ERK tyrosine kinase pathway. Gefitinib reverses the MDR protein gene expression and restores sensitivity of resistant cells to gemcitabine via RAF1/ERK signaling pathway. Combination of gefitinib with conventional chemotherapeutic agents may offer a new approach for the treatment of patients with pancreatic cancer. Anat Rec, 2012. © 2012 Wiley Periodicals, Inc.     

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。     

Oleanolic acid suppresses the proliferation of human bladder cancer by Akt/mTOR/S6K and ERK1/2 signaling

Oleanolic acid has significant pharmacological activities, such as anti-tumor, regulating blood sugar level and liver protection, which are more effective compared with free aglyconeoleanolic acid. However, it is still unknown if oleanolic acid affects the proliferation of human bladder cancer. We utilized T24 cells to study the effect of oleanolic acid on the proliferation and apoptosis of human bladder cancer. In this study, we found that the anti-cancer effect of oleanolic acid significantly suppressed cell proliferation and increased apoptosis and caspase-3 activity of T24 cells. Furthermore, Akt, mTOR and S6K protein expression was greatly inhibited in T24 cells under oleanolic acid treatment. Meanwhile, ERK1/2 of phosphorylation protein expression was significantly promoted by oleanolic acid treatment. Taken together, we provided evidences that oleanolic acid was Akt/mTOR/S6K and ERK1/2 signaling-targeting anti-tumor agent. These findings represent new evidences that oleanolic acid suppresses the proliferation of human bladder cancer by Akt/mTOR/S6K and ERK1/2 signaling, and oleanolic acid may be used to prevent human bladder cancer.     

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。     

Apoptosis induced by paclitaxel via Bcl-2, Bax and caspases 3 and 9 activation in NB4 human leukaemia cells is not modulated by ERK inhibition

We have studied the role of pivotal bio-molecules involved in signalling of cytotoxic effects induced by paclitaxel (Ptx) on acute promyelocytic human leukaemia NB4 cells. A time-dependent increase in cell death and DNA cleavage was observed after 30 μM Ptx treatment. Cell death induction by Ptx proceeds mainly as programmed cell death as shown by annexin V-FITC, reaching up to 30% of apoptotic cells after 24 h. Significant reductions of p53, changes in Bax and Bcl-2 and activation of caspases 3 and 9 were observed as the treatment was applied for long times. Ptx treatments produced NFkB depletion with expression levels abolished at 19 h what could be involved in reduction of survival signals. Phosphorylation of intracellular kinases showed that pERK1/2 decreased significantly at 19 h of Ptx treatment. When these cells were preincubated for 90 min with 20 μM PD98059, 2′-amino-3′-methoxyflavone, an inhibitor of ERK phosphorylation, a slight reduction of cell viability was observed in comparison to that produced by Ptx alone. Pretreatment with PD98059 neither activated caspases nor significantly increased the apoptotic effect of Ptx. Taken together, our data reveal that the inhibition of ERK phosphorylation does not seem to be an essential pathway for bursting an increased induction of apoptosis by Ptx. Decrease of p53 and Bcl-2, fragmentation of DNA, increase of Bax and, finally, activation of caspases 3 and 9 in NB4 leukaemia cells make the apoptotic process induced by Ptx irreversible. Application of Ptx in leukaemia cells shows therefore a promising potential with particular effects on different leukaemia cell types.     

番泻苷B抑制STAT3和ERK1/2磷酸化对A549细胞生长侵袭及裸鼠成瘤的影响

目的　探讨番泻苷B对A549细胞生长、侵袭及裸鼠成瘤的影响及机制。 方法　采用0、5、10、20 μM番泻苷B处理非小细胞肺癌A549细胞,将细胞随机分为4组进行后续实验,Brdu染色检测各组细胞增殖;Hoechst染色检测细胞凋亡;划痕实验检测细胞迁移;Transwell实验检测细胞侵袭;蛋白免疫印迹检测ki67、PCNA 、cl-caspase-3、cl-caspase-9、VEGF、N-cadherin和E-cadherin蛋白表达水平,STAT3和ERK1/2的磷酸化情况;建立荷瘤小鼠模型,检测肿瘤重量,免疫组化检测Ki67和VEGF表达。 结果　与Control组相比较,各番泻苷B剂量组Brdu阳性细胞数量、侵袭细胞数明显减少（P<0.05）,细胞凋亡率明显上升（P<0.05）,细胞划痕愈合率降低（P<0.05）,ki67、PCNA、VEGF、N-cadherin蛋白水平明显降低（P<0.05）,cl-caspase-3、cl-caspase-9、E-cadherin蛋白水平明显升高（P<0.05）,STAT3和ERK1/2的磷酸化水平均明显降低（P<0.05）,降低荷瘤小鼠肿瘤重量与Ki67和VEGF表达水平（P<0.05）。 结论　番泻苷B抑制STAT3、ERK1/2磷酸化对A549细胞体内外生长有抑制作用。