Increased expression of mitogen-activated protein kinase and its upstream regulating signal in human gastric cancer

AIM: To investigate the expression of mitogen-activated protein kinases (MAPKs) and its upstream protein kinase in human gastric cancer and to evaluate the relationship between protein levels and clinicopathological parameters.METHODS: Westem blot was used to measure the expression of extracellular signal-regulated kinase (ERK)-1, ERK-2, ERK-3,p38 and mitogen or ERK activated protein kinaseMEK-1proteins in surgically resected gastric carcinoma, adjacent normal mucosa and metastatic lymph nodes from 42 patients.Immunohistochemistry was employed for their localization.RESULTS: Compared with normal tissues, the protein levels of ERK-1 (integral optical density value 159 526±65 760 vs122 807±65 515, P = 0.001), ERK-2 (168 471±95 051 vs120 469±72 874, P＜0.001), ERK-3 (118 651±71 513 vs70 934±68 058, P＜0.001), P38 (104 776±51 650 vs82 930±40 392, P= 0.048) and MEK-1(116 486±45 725 vs101 434±49 387, P = 0.027) were increased in gastric cancer tissues. Overexpression of ERK-3 was correlated to TNM staging [average ratio of integral optic density (IOD)tumor:IODnormal in TNM Ⅰ, Ⅱ, Ⅲ, Ⅳ tumors was 1.43±0.34,5.08±3.74, 4.99±1.08, 1.44±1.02, n = 42, P = 0.023] and serosa invasion (4.31±4.34 vs 2.00±2.03, P = 0.037). In poorly differentiated cancers (n = 33), the protein levels of ERK-1 and ERK-2 in stage Ⅲ and Ⅳ tumors were higher than those in stage Ⅰ and Ⅱ tumors (2.64±3.01 vs 1.01±0.33,P = 0.022; 2.05±1.54 vs 1.24±0.40, P = 0.030). Gastric cancer tissues with either lymph node involvement (2.49±2.91vs 1.03±0.36, P= 0.023; 1.98±1.49 vs 1.24±0.44, P= 0.036)or serosa invasion (2.39±2.82 vs 1.01±0.35, P = 0.022;1.95±1.44 vs 1.14±0.36, P = 0.015) expressed higher protein levels of ERK-1 and ERK-2. In Borrmann Ⅱ tumors,expression of ERK-2 and ERK-3 was increased compared with Borrmann Ⅲ tumors (2.57±1.86 vs 1.23±0.60, P = 0.022;5.50±5.05 vs 1.83±1.21, P = 0.014). Borrmann Ⅳ tumors expressed higher p38 protein levels. No statistically significant difference in expression of MAPKs was found when stratified to tumor size or histological grade (P＞0.05).Protein levels of ERK-2, ERK-3 and MEK-1 in metastatic lymph nodes were 2-7 folds higher than those in adjacent normal mucosa. The immunohistochemistry demonstrated that ERK-1, ERK-2, ERK-3, p38 and MEK-1 proteins were mainly localized in cytoplasm. The expression of MEK-1 in gastric cancer cells metastasized to lymph nodes was higher than that of the primary site.CONCLUSION: MAPKs, particularly ERK subclass are overexpressed in the majority of gastric cancers.Overexpression of ERKs is correlated to TNM staging, serosa invasion, and lymph node involvement. The overexpression of p38 most likely plays a prominent role in certain morphological subtypes of gastric cancers. MEK-1 is also overexpressed in gastric cancer, particularly in metastatic lymph nodes. Upregulation of MAPK signal transduction pathways may play an important role in tumorigenesis and metastatic potential of gastric cancer.

Physiological roles of mitogen-activated-protein-kinase-activated p38-regulated/activated protein kinase

Mitogen-activated protein kinases(MAPKs)are a family of proteins that constitute signaling pathways involved in processes that control gene expression,cell division, cell survival,apoptosis,metabolism,differentiation and motility.The MAPK pathways can be divided into conventional and atypical MAPK pathways.The first group converts a signal into a cellular response through a relay of three consecutive phosphorylation events exerted by MAPK kinase kinases,MAPK kinase,and MAPK.Atypical MAPK pathways are not organized into this three-tiered cascade.MAPK that belongs to both conventional and atypical MAPK pathways can phosphorylate both non-protein kinase substrates and other protein kinases.The latter are referred to as MAPK-activated protein kinases.This review focuses on one such MAPK-activated protein kinase,MAPK-activated protein kinase 5(MK5)or p38-regulated/activated protein kinase(PRAK).This protein is highly conserved throughout the animal kingdom and seems to be the target of both conventional and atypical MAPK pathways.Recent findings on the regulation of the activity and subcellular localization,bona fide interaction partners and physiological roles of MK5/PRAK are discussed.

Mechanisms of extracellular signal-regulated kinase/cAMP response element-binding protein/brain-derived neurotrophic factor signal transduction pathway in depressive disorder

The extracellular signal-regulated kinase/cAMP response element-binding protein/brain-derived neurotrophic factor signal transduction pathway plays an important role in the mechanism of action of antidepressant drugs and has dominated recent studies on the pathogenesis of depression. In the present review we summarize the known roles of extracellular signal-regulated kinase, cAMP response element-binding protein and brain-derived neurotrophic factor in the pathogenesis of depression and in the mechanism of action of antidepressant medicines. The extracellular signal-regulated kinase/cAMP response element-binding protein/brain-derived neurotrophic factor pathway has potential to be used as a biological index to help diagnose depression, and as such it is considered as an important new target in the treatment of depression.

Ceramide from sphingomyelin hydrolysis differentially mediates mitogen-activated protein kinases (MAPKs) activation following cerebral ischemia in rat hippocampal CA1 subregion

Objective： To explore the role that ceramide plays in the activation of mitogen-activated protein kinases （MAPKs） during cerebral ischemia and reperfusion. Methods： Rats were subjected to ischemia by the fourvessel occlusion （4-VO） method. The sphingomyelinase inhibitor TPCK was administered to the CA1 subregion of the rat hippocampus before inducing ischemia. Western blot was used to examine the activity of extracellular- signal regulated kinase （ERK） and c-Jun N-terminal protein kinase （JNK） using antibodies against ERK, JNK and diphosphorylated ERK and JNK. Results： At lh reperfusion post-ischemia, JNK reached its peak activity while ERK was undergoing a sharp inactivation （P 〈 0.05）. The level of diphosphorylated JNK was significantly reduced but the sharp inactivation of ERK was visibly reversed （P 〈 0.05） by the sphingomyelinase inhibitor. Conclusion： The ceramide signaling pathway is up-regulated through sphingomyelin hydrolysis in brain ischemia, promoting JNK activation and suppressing ERK activation, culminating in the ischemic lesion.

Mitogen activated protein kinase signaling pathways participate in the active principle region of Buyang Huanwu decoction-induced differentiation of bone marrow mesenchymal stem cells

Our preliminary studies confirmed that an active principle region of Buyang Huanwu decoction, comprising alkaloid, polysaccharide, aglycon, glucoside and volatile oil, can induce bone marrow mesenchymal stem cell differentiation into neurons. Mitogen-activated protein kinase signaling was identified as one of the key pathways underlying this differentiation process. The present study shows phosphorylated extracellular signal-regulated protein kinase and phosphorylated p38 protein expression was increased after differentiation. Cellular signaling pathway blocking agents, PD98059 and SB203580, inhibited extracellular signal-regulated protein kinase and p38 in mitogen-activated protein kinase signaling pathways respectively, mRNA and protein expression of the neuronal marker, neuron specific enolase, and neural stem cell marker, nestin, were decreased in bone marrow mesenchymal stem cells after treatment with the active principle region of Buyang Huanwu decoction. Experimental findings indicate that, extracellular signal-regulated protein kinase and p38 in mitogen-activated protein kinase signaling pathways participate in bone marrow mesenchymal stem cell differentiation into neuron-like cells, induced by the active principle region of Buyang Huanwu decoction.

Influence of electroacupuncture on mitogen-activated protein kinase signal transduction in a rat model of cerebral ischemia/reperfusion

Following electroacupuncture at Baihui （DU 20） and Dazhui （DU 14） in a rat model of cerebral ischemia/reperfusion, extracellular-signal-regulated kinase expression in cerebral cortex and corpus striatum, serum glutathione reductase, glutathione peroxidase activity, and serum glutathione content were elevated, and neurobehavioral scores improved. However, these effects were antagonized by mitogen-activated protein kinase inhibitor PD98059. Results indicated that electroacupuncture reversed free radical chain reactions and oxidative stress injury caused by cerebral ischemia/reperfusion, thereby providing neuroprotection. This process could correlate with the mitogen-activated protein kinase signal transduction pathway.

Bacteroides fragilis enterotoxin upregulates heme oxygenase-1 in dendritic cells via reactive oxygen species-,mitogen-activated protein kinase-,and Nrf2-dependent pathway

BACKGROUND Enterotoxigenic Bacteroides fragilis(ETBF)causes colitis and diarrhea,and is considered a candidate pathogen in inflammatory bowel diseases as well as colorectal cancers.These diseases are dependent on ETBF-secreted toxin(BFT).Dendritic cells(DCs)play an important role in directing the nature of adaptive immune responses to bacterial infection and heme oxygenase-1(HO-1)is involved in the regulation of DC function.AIM To investigate the role of BFT in HO-1 expression in DCs.METHODS Murine DCs were generated from specific pathogen-free C57BL/6 and Nrf2−/−knockout mice.DCs were exposed to BFT,after which HO-1 expression and the related signaling factor activation were measured by quantitative RT-PCR,EMSA,fluorescent microscopy,immunoblot,and ELISA.RESULTS HO-1 expression was upregulated in DCs stimulated with BFT.Although BFT activated transcription factors such as NF-κB,AP-1,and Nrf2,activation of NF-κB and AP-1 was not involved in the induction of HO-1 expression in BFT-exposed DCs.Instead,upregulation of HO-1 expression was dependent on Nrf2 activation in DCs.Moreover,HO-1 expression via Nrf2 in DCs was regulated by mitogenactivated protein kinases such as ERK and p38.Furthermore,BFT enhanced the production of reactive oxygen species(ROS)and inhibition of ROS production resulted in a significant decrease of phospho-ERK,phospho-p38,Nrf2,and HO-1 CONCLUSION These results suggest that signaling pathways involving ROS-mediated ERK and p38 mitogen-activated protein kinases-Nrf2 activation in DCs are required for HO-1 induction during exposure to ETBF-produced BFT.

Differential activation of mitogen-activated protein kinases by γ-irradi-ation in IEC-6 cells: Role of intracellular Ca^(2+)

Abstract Objective:To explore the effects of γ-irradiation on mitogen-activatedprotein kinases(MAPKs) and role intracellular calcium in this event in intestinal epithelial cell line 6(IEC-6 cells).Methods:After cultured rat IIEC-6 cells with or without the pretreatment of intracellular Ca^2+ chelator were exposed to γ-ir-radiation of 6 Gy, the total and phosphorylated MAPKs in the cells were determined with Western blotting and apoptosis was examined with flow cytometry.Activities of Extracellular signal-regulated protein kinase (ERK) and p38 MAPK were determined by using immuoprecipitation followed by Western blotting.Results:In response to γ-irradiation,phosphorylation of ERK was not significantly observed ,while the levels of phos-phorylated c-Jun NH2-terminal kinase(JNK) and p38 MAPK were increased in 30 min and reached the peak 2h after exposure to 6Gy γ-irradiation,though the cell viability was significantly lowered 12h.On the other hand, no obvious changes were seen in the total protein levels of ERK, JNK and p38 MAPK.Chelation of in-tracellular Ca^2+ almost completely suppressed the JNK and p38 MAPK phosphorylation induced by γ-irradiation is a potent activator for JNK and p38 MAPK, and Ca^2+ mobilized from intracellular stores plays an important role in the activation of MAPKs and the induction of apoptosis in IEC-6 cells.

Influence of Ren and Du meridian electro-acupuncture on neural stem cell proliferation and extracellular signal-regulated kinase pathway in a rat model of focal cerebral ischemia injury

BACKGROUND： Studies have shown that electro-acupuncture at the Ren meridian could improve proliferation of subventricular zone neural stem cells in cerebral-ischemic rats. However, there are few reports on the influence of electro-acupuncture at the Du meridian on neural stem cell proliferation. OBJECTIVE： To observe the influence of electro-acupuncture at Ren and Du meridians on neural stem cell proliferation in the subventricular zone and altered signal transduction in cerebral ischemia rats. DESIGN, TIME AND SETTING： A randomized, controlled, animal experiment was performed at the Laboratory of Human Anatomy, Medical College of Sun Yat-sen University from May 2006 to February 2008. MATERIALS： Mouse anti-rat bromodeoxyuridine （BrdU） monoclonal antibody was provided by Sigma, USA; mouse anti-rat nestin monoclonal antibody and extracellular signal-regulated protein kinase （ERK） specific inhibitor PD98059 were provided by Calbiochem, Germany; acupuncture needle was provided by Suzhou Acupuncture Supplies, China. METHODS： A total of 126 rats were randomly assigned to four groups： model （n = 36）, Du meridian （n = 36）, Ren/Du meridian （n = 36）, and Ren/Du meridian ＋ PD98059 （n = 18）. Rats in the Ren/Du meridian ＋ PD98059 group were observed on days 7 （n = 6） and 14 （n = 12） after cerebral ischemia injury. Rats in the model, Du meridian, and Ren/Du meridian groups were observed on days 7, 14, and 28 after cerebral ischemia injury, with 12 rats per group at each time point. Thread occlusion was used to establish middle cerebral artery occlusion models. Electro-acupuncture was performed at Renzhong （DU 26） and Baihui （DU 20） acupoints in the Du meridian group, as well as Chengjiang （RN 24）, Guanyuan （RN 4）, Renzhong, and Baihuiacupoints in the Ren/Du meridian and Ren/Du meridian ＋ PD98059 groups 2 days after model establishment. In addition, electro-acupuncture stimulation with disperse-dense waves was performed, with 30 Hz disperse wave, 100 Hz dense wave, and 5 V intensity for 20 minutes. Rats in the Ren/Du meridian ＋ PD98059 group were treated with 0.2 pg PD98059 injection into the subventricular zone, 2 pL per rat. Rats in the model group were not treated with electro-acupuncture. MAIN OUTCOME MEASURES： BrdU/nestin immunofluorescent staining was used to detect proliferating neural stem cells in the subventricular zone of cerebral ischemia rats; Western blot was used to determine phosphorylated ERK1 and 2 （pERK1/2） expression in the subventricular zone. RESULTS： On days 14 and 28 after cerebral ischemia, there were significantly more BrdU-positive and BrdU/nestin-positive cells in the Ren/Du meridian group compared with the Du meridian group （P 〈 0.05）. PD98059 decreased the number of BrdU-positive and BrdU/nestin-positive cells induced by electro-acupuncture at the/：ten and Du meridians （P 〈 0.05）. On days 7, 14, and 28 after treatment, pERK1/2 expression was significantly greater in the Du meridian and Ren/Du meridian groups compared with the model group （P 〈 0.05）. The promoting effect of electro-acupuncture at Ren and Du meridians on ERK1/2 phosphorylation was superior to electro-acupuncture at the Du meridian alone on day 14 after model induction （P 〈 0.05）. However, PD98059 completely abolished the promoting effect of electro-acupuncture at Ren/Du meridians on pERK1/2 expression （P 〈 0.05）. CONCLUSION： Electro-acupuncture at Ren and Du meridians increased proliferation of subventricular zone neural stem cells, which was related to activation of the ERK pathway in a rat model of cerebral ischemia injury.

XAF1 mediates apoptosis through an extracellular signal-regulated kinase pathway in colon cancer

Background:XIAP-associated factor 1(XAF1)negatively regulates the function of the X-linked inhibitor of apoptosis protein(XIAP),a member of the IAP family that exerts antiapoptotic effects.The extracellular signal-regulated kinase(ERK)pathway is thought to increase cell proliferation and to protect cells from apoptosis.The aim of the study was to investigate the correlation between the ERK1/2 signaling pathway and XAF1 in colon cancer.Methods:Four human colon cancer cell lines,HCT1116 and Lovo(wildtype p53),DLD1 and SW1116(mutant p53),were used.Lovo stable transfectants with XAF1 sense and antisense were established.The effects of dominant-negative MEK1(DN-MEK1)and MEK-specific inhibitor U0126 on the ERK signaling pathway and expression of XAF1 and XIAP proteins were determined.The transcription activity of core XAF1 promoter was assessed by dual luciferase reporter assay.Cell proliferation was measured by MTT assay.Apoptosis was determined by Hoechst 33258 staining.Results:U0126 increased the expression of XAF1 in a time-and dose-dependent manner.A similar result was obtained in cells transfected with DN-MEK1 treatment.Conversely,the expression of XIAP was down-regulated.Activity of the putative promoter of the XAF1 gene was significantly increased by U0126 treatment and DN-MEK1 transient transfection.rhEGF-stimulated phosphorylation of ERK appeared to have little or no effect on XAF1 expression.Overexpression of XAF1 was more sensitive to U0126-induced apoptosis,whereas down-regulation of XAF1 by antisense reversed U0126-induced inhibition of cell proliferation.Conclusions:XAF1 expression was up-regulated by inhibition of the ERK1/2 pathway through transcriptional regulation,which required de novo protein synthesis.The results suggest that XAF1 mediates apoptosis induced by the ERK1/2 pathway in colon cancer.

Mitogen-activated protein kinase phosphatase 1 protects PC12 cells from amyloid beta-induced neurotoxicity

The mitogen-activated protein kinase（MAPK） signaling pathway plays an important role in the regulation of cell growth, proliferation, differentiation, transformation and death. Mitogen-activated protein kinase phosphatase 1（MKP1） has an inhibitory effect on the p38 MAPK and JNK pathways, but it is unknown whether it plays a role in Aβ-induced oxidative stress and neuronal inflammation. In this study, PC12 cells were infected with MKP1 sh RNA, MKP1 lentivirus or control lentivirus for 12 hours, and then treated with 0.1, 1, 10 or 100 μM amyloid beta 42（Aβ42）. The cell survival rate was measured using the cell counting kit-8 assay. MKP1, tumor necrosis factor-alpha（TNF-α） and interleukin-1β（IL-1β） m RNA expression levels were analyzed using quantitative real time-polymerase chain reaction. MKP1 and phospho-c-Jun N-terminal kinase（JNK） expression levels were assessed using western blot assay. Reactive oxygen species（ROS） levels were detected using 2′,7′-dichlorofluorescein diacetate. Mitochondrial membrane potential was measured using flow cytometry. Superoxide dismutase activity and malondialdehyde levels were evaluated using the colorimetric method. Lactate dehydrogenase activity was measured using a microplate reader. Caspase-3 expression levels were assessed by enzyme-linked immunosorbent assay. Apoptosis was evaluated using the terminal deoxynucleotidyl transferase d UTP nick end labeling method. MKP1 overexpression inhibited Aβ-induced JNK phosphorylation and the increase in ROS levels. It also suppressed the Aβ-induced increase in TNF-α and IL-1β levels as well as apoptosis in PC12 cells. In contrast, MKP1 knockdown by RNA interference aggravated Aβ-induced oxidative stress, inflammation and cell damage in PC12 cells. Furthermore, the JNK-specific inhibitor SP600125 abolished this effect of MKP1 knockdown on Aβ-induced neurotoxicity. Collectively, these results show that MKP1 mitigates Aβ-induced apoptosis, oxidative stress and neuroinflammation by inhibiting the JNK signaling pathway, thereby playing a neuroprotective role.

Mitogen-Activated Protein Kinase Pathways Following Traumatic Brain Injury

The mechanisms underlying the secondary or delayed cell death in the hippocampus and cerebral hemisphere after traumatic brain injury (TBI) have been poorly understood. Recent data suggesting that TBI may have relationship with both an inflammatory and a neurodegenerative factors are also presented. Mitogen-activated protein kinases (MAPK), which play a crucial role in signal transduction, are activated by phosphorylation in response to a variety of mitogenic signals. In this article, we review the clinical and experimental evidence for brain damage after TBI. In addition, the MAPK pathways, closely involved in signal transduction after TBI, which could therefore be a new and potentially effective therapeutic target in TBI. Further investigations are therefore necessary to better understand cerebral traumatic damage and delineate the best practice strategies needed to improve the patient outcomes after TBI.

Interleukin-1 beta up-regulates tissue inhibitor of matrix metalloproteinase-1 mRNA and phosphorylation of c-jun N-terminal kinase and p38 in hepatic stellate cells

瞄准：学习在 interleukin-1beta (IL-1beta ) 之间的关系矩阵 metalloproteinase-1 (TIMMP-1 ) 的起来调整的织物禁止者 mRNA 表示和两 c-jun N 终端激酶(JNK ) 和在老鼠的 p38 的磷酸化肝的星形细胞(HSC ) 。方法：RT-PCR 被执行在老鼠 HSC 测量 TIMMP-1 mRNA 的表示。西方的污点被执行在老鼠 HSC 测量 IL-1beta-induced JNK 和 p38 活动。结果：TIMMP-1 mRNA 表示(1.191+/-0.079 ) 比在控制组(0.545+/-0.091 )(P【0.01 ) 为 24 h 是有 IL-1beta (10 ng/mL ) 的许多更高的术后疗法。IL-1beta 以一种时间依赖者方式激活 JNK 和 p38。在有为 0， 5， 15， 30， 60 和 120 min 的 IL-1beta 的刺激以后， JNK 活动分别地是 0.982+/-0.299,1.501+/-0.720， 2.133+/-0.882， 3.360+/-0.452， 2.181+/-0.789，和 1.385+/-0.368。在在 15 min (P【0.01 ) 的 JNK 活动有有效差量， 30 min (P【0.01 ) 和在 0 min 的与那相比的 60 min (P【0.01 ) 。p38 活动分别地是在 6 个次点(0， 5， 15， 30， 60 和 120 min ) 的 1.061+/-0.310,2.050+/-0.863， 2.380+/-0.573， 2.973+/-0.953， 2.421+/-0.793，和 1.755+/-0.433。在在 5 点的 p38 活动有有效差量 min ( P【0.05 )， 15 min ( P【0.01 )， 30 min ( P【0.01 )和在在 3 减少的 0 min.TIMMP-1 mRNA 表示 trended 的与那相比的 60 min ( P【0.01 )与 SP600125 的不同集中组织 pretreated ( 10 micromol/L ， 1.022+/-0.113 ；20 micromol/L， 0.869+/-0.070；40 micromol/L， 0.666+/-0.123 ) 。他们的减少都是重要的(P【0.05， P【0.01， P【0.01 ) 与控制组相比(没有 SP600125 处理， 1.163+/-0.107 ) 。在其它， 3 与 SB203580 的不同集中组织 pretreated (10 micromol/L， 1.507+/-0.099；20 micromol/L， 1.698+/-0.107；40 micromol/L， 1.857+/-0.054 ) ， TIMMP-1 mRNA 的表示增加了。他们的层次比在控制组的那些高(没有 SB203580 处理， 1.027+/-0.061 ) 与重要统计意义(P【0.01 ) 。结论：IL-1beta 在老鼠 HSC 由起来调整的 TIMMP-1 mRNA 表示在肝的纤维变性上有一个直接行动。JNK 和 p38 激活 mitogen 的蛋白质家族 ases (MAPK ) 涉及 IL-1beta-induced TIMMP-1 基因表示，并且在这进程起一个不同作用，显示 p38 和 JNK 小径合作地调停在老鼠 HSC 的 TIMP-1 mRNA 表示。

Serine-threonine protein kinase activation may be an effective target for reducing neuronal apoptosis after spinal cord injury

The signaling mechanisms underlying ischemia-induced nerve cell apoptosis are poorly understood. We investigated the effects of apoptosis-related signal transduction pathways following ischemic spinal cord injury, including extracellular signal-regulated kinase（ERK）, serine-threonine protein kinase（Akt） and c-Jun N-terminal kinase（JNK） signaling pathways. We established a rat model of acute spinal cord injury by inserting a catheter balloon in the left subclavian artery for 25 minutes. Rat models exhibited notable hindlimb dysfunction. Apoptotic cells were abundant in the anterior horn and central canal of the spinal cord. The number of apoptotic neurons was highest 48 hours post injury. The expression of phosphorylated Akt（pAkt） and phosphorylated ERK（p-ERK） increased immediately after reperfusion, peaked at 4 hours（p-Akt） or 2 hours（p-ERK）, decreased at 12 hours, and then increased at 24 hours. Phosphorylated JNK expression reduced after reperfusion, increased at 12 hours to near normal levels, and then showed a downward trend at 24 hours. Pearson linear correlation analysis also demonstrated that the number of apoptotic cells negatively correlated with p-Akt expression. These findings suggest that activation of Akt may be a key contributing factor in the delay of neuronal apoptosis after spinal cord ischemia, particularly at the stage of reperfusion, and thus may be a target for neuronal protection and reduction of neuronal apoptosis after spinal cord injury.

Imbalanced expression of mitogen-activated protein kinase phosphatase-1 and phosphorylated extracellular signal-regulated kinases in lung squamous cell carcinoma

Objective:Mitogen-activated protein kinases (MAPKs) are correlated with a more malignant phenotype in many cancers.This study was designed to evaluate the predictive value of the expression of MAPK phosphatase-1 (MKP-1) and phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK 1/2),as the key regulatory mechanism of the MAPKs,in lung squamous cell carcinoma (SCC).Methods:We assessed the expressions of MKP-1 and p-ERK 1/2 in twenty subjects at different differentiation degree of SCC and five normal lungs by immunohistochemistry and real-time reverse transcriptase polymerase chain reaction (RT-PCR) analysis.Results:Immunohistochemistry and real-time RT-PCR assay showed that the expression of MKP-1 was gradually decreased as tissue type went from normal lung tissues to increasingly undifferentiated carcinoma,and it was negatively correlated with tumor differentiation (P<0.01).However,the expression of p-ERK 1/2 or ERK 1/2 was gradually increased as tissue type went from normal lung tissues to increasingly undifferentiated carcinoma,and it was positively correlated with tumor differentiation (P<0.01).Conclusions:Our data indicates the relevance of MKP-1 and p-ERK 1/2 in SCC as a potential positive and negative prognostic factor.The imbalanced expression of MKP-1 and p-ERK 1/2 may play a role in the development of SCC and these two molecules may be new targets for the therapy and prognosis of SCC.

Effect of oxymatrine on the p38 mitogen-activated protein kinases signalling pathway in rats with CCl4 induced hepatic fibrosis

Background Recent studies have suggested that p38 mitogen-activated protein kinases （MAPK） signalling pathway plays an important role in hepatic fibrosis. This study explored the antifibrotic effect of oxymatrine on tetrachloromethane induced liver fibrosis in rats and its modulation on the p38 MAPK signalling pathway. Methods One hundred and twenty healthy male Sprague-Dawley rats were randomly assigned to six groups： normal （n=20）, induced fibrosis （n=20）, colchicine （n=20） and three treatment groups of oxymatrine （n=20x3）. We obesrved changes in deposition of collagen, hyaluronic acid （HA）, laminin （LN）, collagen type IV （CIV）, procollagen III （PCIll） and hydroxyproline （Hyp）, a-smooth muscle actin （α-SMA） and phosphor-p38 （pp38）. Results The relative indicators of changes in histopathology, HA, LN, CIV, PCIII, Hyp, a-SMA and pp38 were raised significantly in the induced fibrosis group （P〈0.01 vs normal group）. The semiquantitative hepatic fibrosis staging scores of middle dose group and high dose group were decreased （P 〈0.05 and P 〈0.01 respectively vs the induced fibrosis group）, as was the average area of collagen in rats＇ liver, the concentrations of serum HA, LN, CIV, PCIII and liver tissue homogenate Hyp. The gene expression of α-SMA mRNA was considerably decreased in the treated animals, as was the protein espression of pp38 protein. Conclusions Oxymatrine is effective in reducing the production and deposition of collagen in the liver tissue of experimental rats in ways which relate to modulating the fibrogenic signal transduction via p38 MAPK signalling pathway.

Calcitonin gene-related peptide induces proliferation and monocyte chemoattractant protein-1 expression via extracellular signal-regulated kinase activation in rat osteoblasts

Background Calcitonin gene-related peptide （CGRP）, a sensory neuropeptide, affects osteoblast proliferation and bone formation. However, the mechanisms are not fully understood. Monocyte chemoattractant protein-1 （MCP-1） is a chemokine that stimulates the migration of monocytes and plays important roles in regulating bone remolding during fracture repair. In this study, we investigated the effects of CGRP on proliferation and MCP-1 expression in cultured rat osteoblasts. Methods Primary rat osteoblasts were isolated from fetal rats calvariae. Cells were exposed to gradient concentrations （10^-9 to 10^-7 mol/L） of CGRP. Protein and mRNA levels of MCP-1 were quantified by Western blotting and semiquantitative reverse transcdption-polymerase chain reaction, respectively. The protein level of MCP-1 was investigated and compared in cell culture media by enzyme linked immunosorbent assay （ELISA）. Phospho-extracellular signal-regulated kinase （ERK） expression was detected by Western blotting. Cell proliferative activity was measured by 3-（4,5-dimethylthiazol-2-yl）-2, 5-diphenyl tetrazolium bromide （MTT） and BrdU assay. The effects of MAPK/ERK kinase （MEK）-inhibitor U0126 on CGRP-induced MCP-1 expression in primary rat osteoblasts were examined. Results CGRP effectively enhanced primary rat osteoblast proliferation and led to significant increases in the expression of MCP-1 mRNA and protein in time- and dose-dependent manners. CGRP activated the ERK pathway. Pretreatment of cultured rat osteoblasts with MEK inhibitor U0126 resulted in dose-dependent inhibitions of CGRP-induced MCP-1 mRNA and protein levels. Thus, CGRP promoted cell proliferation and stimulated MCP-1 expression in cultured rat osteoblasts. Conclusion These studies document novel links between CGRP and MCP-1 and illuminate the effects of CGRP in regulating bone remodeling.

Marsdenia tenacissima extract induces G_0/G_1 cell cycle arrest in human esophageal carcinoma cells by inhibiting mitogen-activated protein kinase(MAPK) signaling pathway

Marsdenia tenacissima extract(MTE, trade name: Xiao-Ai-Ping injection) is an extract of a single Chinese plant medicine. It has been used for the treatment of cancer in China for decades, especially for esophageal cancer and other cancers in the digestive tract. In the present study, the potential mechanism for MTE's activity in esophageal cancer was explored. The effects of MTE on the proliferation of human esophageal cancer cells(KYSE150 and Eca-109) were investigated by the MTT assay, the Brd U(bromodeoxyuridine) incorporation immunofluorescence assay, and flow cytometric analysis. MTE inhibited cell proliferation through inducing G0/G1 cell cycle arrest in KYSE150 and Eca-109. Western blot analysis was employed to determine protein levels in the MTE treated cells. Compared with the control cells, the expression levels of the cell cycle regulatory proteins cyclin D1/D2/D3, cyclin E1, CDK2/4/6(CDK: cyclin dependent kinase), and p-Rb were decreased significantly in the cells treated with MTE at 40 mg·m L-1. In addition, MTE had an inhibitory effect on the MAPK(mitogen-activated protein kinase) signal transduction pathway, including ERK(extracellular signal-regulated kinase), JNK(c-Jun N-terminal kinase), and p38 MAPK. Moreover, MTE showed little additional effects on the regulation of cyclin D1/D3, CDK4/6, and p-Rb when the ERK pathway was already inhibited by the specific ERK inhibitor U0126. In conclusion, these data suggest that MTE inhibits human esophageal cancer cell proliferation through regulation of cell cycle regulatory proteins and the MAPK signaling pathways, which is probably mediated by the inhibition of ERK activation.

Mnk kinase pathway: Cellular functions and biological outcomes

The mitogen-activated protein kinase(MAPK) interacting protein kinases 1 and 2(Mnk1 and Mnk2) play important roles in controlling signals involved in mRNA translation. In addition to the MAPKs(p38 or Erk), multiple studies suggest that the Mnk kinases can be regulated by other known kinases such as Pak2 and/or other unidentified kinases by phosphorylation of residues distinct from the sites phosphorylated by the MAPKs. Several studies have established multiple Mnk protein targets, including PSF, heterogenous nuclear ribonucleoprotein A1, Sprouty 2 and have lead to the identification of distinct biological functions and substrate specificity for the Mnk kinases. In this review we discuss the pathways regulating the Mnk kinases, their known substrates as well as the functional consequences of engagement of pathways controlled by Mnk kinases. These kinases play an important role in mRNA translation via their regulation of eukaryotic initiation factor 4E(eIF4E) and their functions have important implications in tumor biology as well as the regulation of drug resistance to anti-oncogenic therapies. Other studies have identified a role for the Mnk kinases in cap-independent mRNA translation, suggesting that the Mnk kinases can exert important functional effects independently of the phosphorylation of eIF4 E. The role of Mnk kinases in inflammation and inflammationinduced malignancies is also discussed.

Evodiamine induces extrinsic and intrinsic apoptosis of ovarian cancer cells via the mitogen-activated protein kinase/phosphatidylinositol-3-kinase/protein kinase B signaling pathways

OBJECTIVE: To explore the effects of evodiamine on ovarian cancer cells and the mechanisms underlying such effects.METHODS: Human ovarian cancer cells HO-8910 PM were treated with evodiamine at 0, 1.25,2.5, and 5 μM for 1-4 d. 3-(4,5-Dimethiylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) assay was used to detect the growth inhibition rate of evodiamine-treated HO-8910 PM cells. The cell cycle was observed via propidium iodide(PI) staining. Apoptosis induction was assessed via Annexin V-fluorescein isothiocyanate/propidium iodide(Annexin V-FITC/PI) double staining assay. To verify the mechanism of apoptosis, caspase-dependent apoptotic pathway-related protein was detected by Western blot analysis. The expression levels of mitogen-activated protein kinase(MAPK)and/or phosphatidylinositol-3-kinase(PI3K)/pro-tein kinase B(Akt) pathway-related proteins were also investigated.RESULTS: Evodiamine significantly inhibited the proliferation of HO-8910 PM cells in a dose- and time-dependent manner. Evodiamine induced G2/M arrest with an increase of cyclin B1 level, and promoted cell apoptosis with a decrease of B cell lymphoma/lewkmia-2(Bcl-2) and an increase of Bcl-2-associated X protein(Bax) level. In addition,evodiamine treatment led to the activation of caspase-8, caspase-9, and caspase-3 and the cleavage of poly(ADP-ribose)-polymerase(PARP). Evodiamine targeted the MAPK and/or PI3K/Akt pathways by reducing the expression and activity of PI3 K, Akt, and extracellular signal-regulated kinase mitogen-activated protein kinase(ERK1/2 MAPK)and the activity of p38 MAPK.CONCLUSION: Evodiamine can inhibit the growth of ovarian cancer cells by G2/M arrest and intrinsic and extrinsic apoptosis. In addition, evodiamine-induced PI3K/Akt, ERK1/2 MAPK, and p38 MAPK signaling may be involved in cell death.