N-methyl-D-aspartate receptors mediate diphosphorylation of extracellular signal-regulated kinases through Src family tyrosine kinases and Ca^2＋/calmodulin-dependent protein kinase Ⅱ in rat hippocampus after cerebral ischemia

Objective： Extracellular signal-regulated kinases （ERKs） can be activated by calcium signals. In this study, we investigated whether calcium-dependent kinases were involved in ERKs cascade activation after global cerebral ischemia. Methods Cerebral ischemia was induced by four-vessel occlusion, and the calcium-dependent proteins were detected by immunoblot. Results Lethal-simulated ischemia significantly resulted in ERKs activation in N-methyl-D-aspartate （NMDA） receptor-dependent manner, accompanying with differential upregulation of Src kinase and Ca^2＋/calmodulin-dependent protein kinase Ⅱ （CaMKⅡ） activities. With the inhibition of Src family tyrosine kinases or CaMKⅡ by administration of PP2 or KN62, the phosphorylation of ERKs was impaired dramatically during post-ischemia recovery. However, ischemic challenge also repressed ERKs activity when Src kinase was excessively activated. Conclusions Src family tyrosine kinases and CaMKⅡ might be involved in the activation of ERKs mediated by NMDA receptor in response to acute ischemic stimuli in vivo, but the intense activation of Src kinase resulted from ischemia may play a reverse role in the ERKs cascade.

Involvement of MAPK/ERK kinase-ERK pathway in exogenous bFGF-induced Egr-1 binding activity enhancement in anoxia-reoxygenation injured astrocytes

Objective Intravenous administration of basic fibroblast growth factor （bFGF） is effective to reduce the volume of cerebral infract due to ischemia. This study was designed to investigate the molecular mechanism, especially the signal transduction pathways, involved in this protective role of bFGF. Methods Anoxia-reoxygenation treated atrocytes were used to study the role of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase （MAPK/ERK kinase, MEK）-ERK signaling pathway after exogenous bFGF administration by Western blot. Electrophoretic mobile shift assay was used to detect the binding activity of early growth response factor-1 （Egr-1）, an important transcription factor for endogenous bFGF. Results bFGF could protect some signal transduction proteins from the oxygen-derived free radicals induced degradation. ERK1/2 was activated and involved in Egr-1 binding activity enhancement induced by exogenous bFGF. Conclusion MEK-ERK MAPK cascade may be an important signal transduction pathway contributed to bFGF induced enhancement of Egr-1 binding activity in anoxia-reoxygenation injured astrocytes.

Components of the mitogen-activated protein kinase cascade are activated in hepatic cells by Echinococcus multilocularis metacestode

AIM： To explore the effect of Echinococcusmultilocularis on the activation of mitogen-activated protein kinase （MAPK） signaling pathways and on livercell proliferation.METHODS： Changes in the phosphorylation of MAPKs and proliferating cell nuclear antigen （PCNA）expression were measured in the liver of patients withalveolar echinococcosis （AE）. MAPKs, MEK1/2 [MAPK/extracellular signal-regulated protein kinase （ERK）kinase] and ribosomal S6 kinase （RSK） phosphorylationwere detected in primary cultures of rat hepatocytesin contact in vitro with （1） E. multilocu/aris vesicle fluid（EmF）, （2）E. multilocularis-conditioned medium （EmCM）.RESULTS： In the liver of AE patients, ERK 1/2 andp38 MAPK were activated and PCNA expression wasincreased, especially in the vicinity of the metacestode.Upon exposure to EmF, p38, c-Jun N-terminal kinase（JNK） and ERK1/2 were also activated in hepatocytesin vitro, as well as MEK1/2 and RSK, in the absenceof any toxic effect. Upon exposure to EmCM, only JNKwas up-regulated.CONCLUSION： Previous studies have demonstratedan influence of the host on the MAPK cascade inE. multilocularis. Our data suggest that the reverse,i.e. parasite-derived signals efficiently acting onMAPK signaling pathways in host liver ceils, is actuallyoperating.

Down-regulation of extracellular signal-regulated kinase 1/2 activity in P-glycoprotein-mediated multidrug resistant hepatocellular carcinoma cells

AIM： To study the expression and phosphorylation of extracellular signal-regulated kinase （ERK） i and ERK2 in multidrug resistant （MDR） hepatocellular carcinoma （HCC） cells.METHODS： MDR HCC cell lines, HepG2/adriamycin （ADM） and SMMC7721/ADM, were developed by exposing parental cells to stepwise increasing concentrations of ADM. MTT assay was used to determine drug sensitivity. Flow cytometry was employed to analyze cell cycle distribution and measure cell P-glycoprotein （P-gp） and multidrug resistant protein 1 （MRP1） expression levels. ERK1 and ERK2 mRNA expression lev-ls were measured by quantitative real-time PCR （QRTPCR）. Expression and phosphorylation of ERK1 and ERK2 were analyzed by Western blot.RESULTS： MTT assay showed that HepG2/ADM andSMMC7721/ADM were resistant not only to ADM, but also to multiple anticancer drugs. The P-gp expression was over 10-fold higher in HepG2/ADM cells than in HepG2 cells （8.92% ±0.22% vs 0.88% ± 0.05%, P 〈 0.001） and over 4-fold higher in SMMC7721/ADM cells than in SMMC7721 cells （7.37% ± 0.26% vs 1.74% ± 0.25%, P 〈 0.001）. However, the MRP1 expression was not significantly higher in HepG2/ADM and SMMC7721/ADM cells than in parental cells. In addition, the percentage of MDR HepG2/ADM and SMMC7721/ADM cells was significantly decreased in the G0/G1 phase and increased in the the S phase or G2/M phase. QRT-PCR analysis demonstrated that the ERK1 and ERK2 mRNA expression increased apparently in HepG2/ADM cells and decreased significantly in SMMC7721/ADM cells. Compared with the expression of parental cells, ERK1 and ERK2 protein expressions were markedly decreased in SMMC7721/ADM cells. However, ERK2 protein expression was markedly increased while ERK1 protein expression had no significant change in HepG2/ADM cells. Phosphorylation of ERK1 and ERK2 was markedly decreased in both HepG2/ADM and SMMC7721/ADM MDR cells.CONCLUSION： ERK1 and ERK2 activities are downregulated in P-gp-mediated MDR HCC cells. ERK1 or ERK2 might be a potential drug target for circumventing MDR HCC cells,

Regulation of Hepatitis C Virus Replication and Gene Expression by the MAPK-ERK Pathway

The mitogen activated protein kinases-extracellular signal regulated kinases （MAPK-ERK） pathway is involved in regulation of multiple cellular processes including the cell cycle. In the present study using a Huh7 cell line Conl with an HCV replicon, we have shown that the MAPK-ERK pathway plays a significant role in the modulation of HCV replication and protein expression and might influence IFN-a signalling. Epithelial growth factor （EGF） was able to stimulate ERK activation and decreased HCV RNA load while a MAPK-ERK pathway inhibitor U0126 led to an elevated HCV RNA load and higher NS5A protein amounts in Conl cells. It could be further demonstrated that the inhibition of the MAPK-ERK pathway facilitated the translation directed by the HCV internal ribosome entry site. Consistently, a U0126 treatment enhanced activity of the HCV reporter replicon in transient transfeetion assays. Thus, the MAPK-ERK pathway plays an important role in the regulation of HCV gene expression and replication. In addition, cyclin-dependent kinases （CDKs） downstream of ERK may also be involved in the modulation of HCV replication since roscovitine, an inhibitor of CDKs had a similar effect to that of U0126. Modulation of the cell cycle progression by cell cycle inhibitor or RNAi resulted consistently in changes of HCV RNA levels. Further, the replication of HCV replicon in Conl cells was inhibited by IFN-~z. The inhibitory effect of IFN-CZ could be partly reversed by pre-incubation of Con-1 cells with inhibitors of the MAPK-ERK pathway and CDKs. It could be shown that the MAPK-ERK inhibitors are able to partially modulate the expression of interferon-stimulated genes.

Study on the Expression and Significance of TGF-β1, p-ERK1/2and K-ras in Colorectal Cancer Using Tissue Microarray Technique

OBJECTIVE This study aimed to explore the expression and significance of transforming growth factor β1（TGF-β1）,extracellular signal-regulated kinases 1/2 （ERK1/2）, and K-ras in colorectal cancer （CRC） using tissue microarray technology.METHODS The expressions of TGF-β1, ERK1/2, and K-ras in colon cancer cells taken from the specimens of 92 CRC patients （stage Ⅰ： 16 cases, stage Ⅱ： 28 cases, stage Ⅲ： 24 cases, and stage Ⅳ：24 cases） were analyzed using tissue microarray technology and immunohistochemistry, and compared with those of 20 normal colon tissue samples.RESULTS High immunoreactive scores （IRS） of TGF-β1,p-ERK1/2, and K-ras protein in CRC were obtained, which were 66.3% （61/92）, 59.8% （55/92）, and 48.9% （45/92）, respectively, and those in normal epithelial cells of colon were 10% （2/20）, 20% （4/20）, and 30% （6/20）, respectively （P 〈 0.05）. The expressions of TGF-β1 and ERK1/2 in CRC at stage Ⅰwere 37.5% and 31.3%,respectively, and those in CRC at stage Ⅳ were 83.3% and79.3%, respectively, with statistically significant differences. No significant relationship was found between K-ras expression and tumor stages （P〉0.05）.CONCLUSION High level expressions of TGF-β1 and ERK1/2 are closely related to the clinical stages of colon cancer and crosstalk may exist between the 2 signal pathways.

MAPK在介导人肺泡巨噬细胞模型噬菌中的作用

目的 探讨MAPK信号传导途径在介导人肺泡巨噬细胞吞噬细菌中的作用。方法使用Ficoll—Hypaque密度梯度法将外周血单核细胞分离自13名健康志愿者外周血，经2μg／L粒细胞-巨噬细胞集落刺激因子诱导培养12d成单核细胞源性巨噬细胞（GM-MO），即肺泡巨噬细胞研究替代细胞模型。用共聚焦荧光显微镜检测GMMO吞噬荧光标记的金黄色葡萄球菌；使用多功能酶标仪检测p38MAPK、ERK、JNK特异性抑制剂对GM-MO吞噬金黄色葡萄球菌量的影响。以细胞吞噬抑制剂细胞松弛素D为阳性对照。结果GM—MD吞噬金黄色葡萄球菌呈时间依赖，4h后呈逐渐饱和状态。以吞噬反应4h为观测终点，共聚焦荧光显微镜检测显示绝大部分金黄色葡萄球菌均被吞噬进细胞内。使用细胞松弛素D后，GM-MO对金黄色葡萄球菌的吞噬几乎全部被抑制，其荧光值为（4259士869）RFU；p38aMAPK特异性抑制剂Gw856553随着浓度的升高可抑制GM—MD对金黄色葡萄球菌的吞噬，并呈浓度依赖性：浓度为10μmol／L时荧光值为（18290±5491）RFU，浓度为10μmol／L时荧光值为（16802±6440）RFU，与空白对照[（19489士5450）RFU3相比差异均有统计学意义。而ERK抑制剂U0126和JNK抑制剂SP600125在各实验浓度下（10^-8～10mol／L）均对GM-M0吞噬金黄色葡萄球菌无影响。结论p38MAPK途径可能参与了介导人肺泡巨噬细胞对细菌的吞噬作用，而ERK及JNK信号传导途径与此无关。

Activation of extracellular signal-regulated kinase in the anterior cingulate cortex contributes to the induction of long-term potentiation in rats

Objective To explore the role of the extracellular signal-regulated kinase （ERK）/cAMP response element binding protein （CREB） pathway in the induction of long-term potentiation （LTP） in the anterior cingulate cortex （ACC） that may be implicated in pain-related negative emotion. Methods LTP of field potential was recorded in ACC slice and the expressions of phospho-ERK （pERK） and phospho-CREB （pCREB） were examined using immunohistochemistry method. Results LTP could be induced stably in ACC slice by high frequency stimulation （2-train, 100 Hz, 1 s）, while APv （an antagonist of NMDA receptor） could block the induction of LTP in the ACC, indicating that LTP in this experiment was NMDA receptor-dependent. Bath application of PD98059 （50 μmol/L）, a selective MEK inhibitor, at 30 min before tetanic stimulation could completely block the induction of LTP. Moreover, the protein level of pERK in the ACC was transiently increased after LTP induction, starting at 5 rain and returning to basal at 1 h after tetanic stimulation. The protein level of pCREB was also increased after LTP induction. The up-regulation in pERK and pCREB expressions could be blocked by pretreatment of PD98059. Double immunostaining showed that after LTP induction, most pERK was co-localized with pCREB. Conclusion NMDA receptor and ERK-CREB pathway are necessary for the induction of LTP in rat ACC and may play important roles in pain emotion.

Busuishengxue ranules mediate their effects upon non-severe aplastic anemia via mitogen-activated protein kinase/extracellular signal-regulated kinase pathway

OBJECTIVE:To observe the clinical efficacy of Busuishengxue granules on non-severe aplastic anemia(NSAA)and investigate its effect on the mitogen-activated protein kinase/extracellular signal-regulated kinase(MAPK/ERK)pathway.METHODS:Sixty NSAA patients were divided equally into two groups.Subjects in the experimental group were treated with Busuishengxue granules,and the control group with Zaizaoshengxue tablets.The treatment course was 6 months and cu-rative efficacy was compared between the two groups as well as with 10 healthy individuals.Flow cytometry(FCM)was used to detect the intracellular concentration of Ca2+([Ca2+]i).Western blotting was employed to detect the expression of enzymes in the MAPK/ERK pathway.RESULTS:The efficacy of Busuishengxue granules was significantly better than that of Zaizaoshengxue tablets(P<0.05).Before treatment,expression of JNK,phospho-ERK 1/2 and p-JNK was higher,and[Ca2+]i higher,than that of the control group(P<0.05).After treatment with Busuishengxue granules,expression of all enzymes related to signal transduction pathways in the blood cells of NSSA patients were altered to different degrees.CONCLUSION:Busuishengxue granules had a better effect with regard to improving symptom scores,increasing the number of blood leukocytes,and increasing hemoglobin levels than Zaizaoshengxue tablets,and they differed slightly in terms of increasing the number of platelets.

Research progress of the role and mechanism of extracellular signal-regulated protein kinase 5(ERK5) pathway in pathological pain

Extracellular signal-regulated protein kinase 5 （ERK5）, also known as big mitogen-activated protein kinase 1 （MAPK1）, is an important member of ERK family, which is a subfamily of the large MAPK family. ERK5 is expressed in many tissues, including the dorsal root ganglion （DRG） neurons and the spinal cord. In this review, we focus on elaborating ERK5-associated pathway in pathological pain, in which the ERK5/CREB （cyclic adenosine monophos- phate （cAMP）-response element-binding protein） pathway plays a crucial role in the transduction of pain signal and contributes to pain hypersensitivity. ERK5 activation in the spinal dorsal horn occurs mainly in microglia. The activation of ERK5 can be mediated by N-methyI-D-aspartate （NMDA） receptors. We also elaborate the relationship between ERK5 activation and nerve growth factor-tyrosine kinase A （NGF-TrkA）, and the connection between ERK5 activation and brain-derived neurotrophic factor （BDNF） in pathological pain in detail.