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.

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.

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.

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.

Glucocorticoid modulation of extracellular signal-regulated protein kinase 1/2 and p38 in human ovarian cancer HO-8910 cells

Objective To investigate the signaling pathway through testing the effects of dexamethasone (Dex) on the activation of the extracellular signal-regulated protein kinase 1/2 (ERK1/2) and p38 kinase (p38) in HO-8910 cells.Methods Activation of the ERK1/2 and p38 was detected by Western blotting using the antibodies against the total ERK1/2 and p38 mitogen-activated protein kinases (MAPKs) protein and the phosphorylated forms of them. Results Dex could suppress the activation of ERK1/2, while enhance the activation of p38 rapidly and strongly in a dose- and time- dependent manner. Neither effect could be blocked by RU486, the antagonist of glucocorticoid receptor (GR).Conclusion Dex has rapid effects on the activation of ERK1/2 and p38, and these effects are not mediated by GR.

Mitogen-activated protein kinase signal pathways play an important role in right ventricular hypertrophy of tetralogy of Fallot

Background Tetralogy of Fallot （TOF） is the most common malformation of children with an incidence of approximately 10% of congenital heart disease patients. There can be a wide spectrum to the severity of the anatomic defects, which include ventricular septal defect, aortic override, right ventricular outflow tract obstruction, and right ventricular hypertrophy. We examined the relationship between right ventricular hypertrophy in patients with TOF and the gene expression of factors in the mitogen-activated protein kinase （MAPK） signal pathway. Methods To gain insight into the characteristic gene（s） involved in molecular mechanisms of right ventricular hypertrophy in TOF, differential mRNA and micro RNA expression profiles were assessed using expression-based micro array technology on right ventricular biopsies from young TOF patients who underwent primary correction and on normal heart tissue. We then analyzed the gene expression of the MAPK signal pathway using reverse transcription-polymerase chain reaction （RT-PCR） in normals and TOF patients. Results Using the micro RNA chip V3.0 and human whole genome oligonucleotide microarray VI.0 to detect the gene expression, we found 1068 genes showing altered expression of at least two-fold in TOF patients compared to the normal hearts, and 47 micro RNAs that showed a significant difference of at least two-fold in TOF patients. We then analyzed these mRNAs and micro RNAs by target gene predicting software Microcosm Targets version 5.0, and determined those mRNA highly relevant to the right ventricular hypertrophy by RT-PCR method. There were obvious differences in the gene expression of factors in the MAPK signal pathway when using RT-PCR, which was consistent to the results of the cDNA microarray.Conclusion The upregulation of genes in the MAPK signal pathway may be the key events that contribute to right ventricular hypertrophy and stunted angiogenesis in patients with TOF.

Effect of cigarette smoke extract on lipopolysaccharide-activated mitogen-activated protein kinase signal transduction pathway in cultured cells

Background Lipopolysaccharide （LPS） forms outer membrane of the wall of Gram-negative cells. LPS can directly cause damage to epithelia of respiratory tract and is the major factor responsible for the chronic inflammation of respiratory passage. The mitogen-activated protein kinase （MAPK） signal transduction pathway of the airway epithelia is intimately associated with the action of LPS. The chronic inflammation of respiratory tract and smoking are interrelated and entwined in the development and progression of chronic lung diseases. This study was designed to examine the effects of cigarette smoke extract （CSE） and LPS on MAPK signal transduction pathway in order to further understand the roles CSE and LPS play in chronic lung inflammation. Methods Cultured primary human epithelial cells of airway were divided into four groups according to the stimulants used： blank control group, LPS-stimulation group, CSE-stimulation group and CSE plus LPS group. Western blotting was employed for the detection of phosphorylation level of extracellular-signal-regulated-kinase （ERK1/2）, p38 MAPK and c-Jun N-terminal kinase （JNK）. The expression of cytokines of MAPK transduction pathway （granulocyte-macrophage colony stimulating factor （GM-CSF） and mRNA of IL-8） in the primary epithelial cells of respiratory tract was also determined. Results Western blotting revealed that the phosphorylation levels of ERK1/2, p38 MAPK and JNK were low and 2 hours after the LPS stimulation, the phosphorylation of ERK1/2, p38 MAPK and JNK were all increased. There was a significant difference in the phosphorylation between the LPS-stimulation group and blank control group （P〈0.05）; no significant difference was found between CSE-stimulation group and blank control group （P〉0.05）; there was a significant difference between CSE ＋ LPS group and blank control group and between CSE ＋ LPS group and LPS group （P〈0.05）. The phosphorylation of CSE-LPS group was higher than that of blank control group but lower than that of LPS group. In blank control group, the expression of IL-8 and GM-CSF mRNA was low in the epithelial cells of airway and the release of IL-8 and GM-CSF was also at a low level. One hour after LPS stimulation, the level of IL-8 mRNA increased （P〈0.05） and reached a peak after 2 hours. On the other hand, GM-CSF mRNA level increased 2 hours after the stimulation （P〈0.05） and reached the highest level 4 hours after the stimulation. Two hours after LPS stimulation, IL-8 and GM-CSF protein level began to rise （P〈0.05）, and the level was the highest 8 hours after the stimulation （P〈0.01）. Stimulation with CSE alone had no effect on the release of IL-8 and GM-CSF and expression of IL-8 mRNA （P〉0.05）, but pre-treatment with CSE could delay the LPS-induced release of IL-8 and GM-CSF and the expression of IL-8 mRNA and its peak was lower. Conclusions LPS stimulation can significantly increase the phosphorylation of ERK1/2, p38 MAPK and JNK in the epithelial cells of airway and activate the MAPK transduction pathway, thereby can activate the downstream signal transduction pathway, and can ultimately result in the release of cytokines by the epithelial cells of airway. CSE can partially abolish the LPS-induced activation of MAPK signal transduction pathway and the expression of cytokines of the pathway, which might contribute to the development and progression of the inflammatory reactions in COPD patients.

Protein tyrosine phosphatase 1B regulates migration of ARPE-19 cells through EGFR/ERK signaling pathway

AIMTo evaluate whether protein tyrosine phosphatase 1B (PTP1B) contributed to initiate human retinal pigment epithelium cells (A)-19 migration and investigate the signaling pathways involved in this process.METHODSARPE-19 cells were cultured and treated with the siRNA-PTP1B. Expression of PTP1B was confirmed by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). AG1478 [a selective inhibitor of epidermal growth factor receptor (EGFR)] and PD98059 (a specific inhibitor of the activation of mitogen-activated protein kinase) were used to help to determine the PTP1B signaling mechanism. Western blot analysis verified expression of EGFR and extracellular signal-regulated kinase (ERK) in ARPE-19 cells. The effect of siRNA-PTP1B on cell differentiation was confirmed by immunostaining for α-smooth muscle actin (α-SMA) and qRT-PCR. Cell migration ability was analyzed by transwell chamber assay.RESULTSThe mRNA levels of PTP1B were reduced by siRNA-PTP1B as determined by qRT-PCR assay. SiRNA-PTP1B activated EGFR and ERK phosphorylation. α-SMA staining and qRT-PCR assay demonstrated that siRNA-PTP1B induced retinal pigment epithelium (RPE) cells to differentiate toward better contractility and motility. Transwell chamber assay proved that PTP1B inhibition improved migration activity of RPE cells. Treatment with AG1478 and PD98059 abolished siRNA-PTP1B-induced activation of EGFR and ERK, α-SMA expression and cell migration.CONCLUSIONPTP1B inhibition promoted myofibroblast differentiation and migration of ARPE-19 cells, and EGFR/ERK signaling pathway played important role in migration process.

Activation of mitogen activated protein kinases via complement receptor type 2

Background Complement receptor type 2 (CR2) is the receptor for C3d and C3dg and for Epstein Barr virus The aim of our study was to explore whether CR2 can independently mediate the activation of mitogen activated protein kinases (MAPKs, including ERK, JNK, and p38MAPK), and to highlight the molecular mechanism of CD 4 + cell deletion in AIDS Methods HOS cells (HOS CR2) and HOS CD4 cells (HOS CD4CR2) stably expressing CR2 were established and then identified by FACS and Western blotting Activation and blocking tests of MAPKs were assessed by Western blot Cell proliferation was determined using Cell Titer 96 Aqueous One Solution Reagent Results FACS results showed that the positive rates of HOS CR2 and HOS CD4CR2 cells were greater than 96%, and Western blot showed that the CR2 expression levels on HOS CR2 and HOS CD4CR2 cells were high Activation and blocking tests of MAPKs (ERK, JNK, and p38MAPK) were carried out in HOS CR2, HOS CD4, and HOS CD4CR2 cells The activation of MAPKs in HOS CR2 cells stimulated with PMA (100 ng/ml) and NHS (10%) was identical The activation of MAPKs increased at 5 minutes, reached a peak at 10 minutes, and decreased to baseline within 30 minutes, all in a time dependent manner; the activation of MAPKs was blocked by anti CR2 McAb, PD98059 (inhibitor of ERK), and Wortmanin (inhibitor of PI 3K), respectively In HOS CD4 cells, MAPKs were activated by HIV gp160 In HOS CD4CR2 cells, MAPK activation was induced by HIV gp160, 10% NHS, and HIV gp160+10%NHS; phosphorylation of p38MAPK was dramatically induced by HIV gp160+NHS, and lasted for 1 hour The cell proliferation results showed that HIV gp160 inhibited the proliferation of HOS CD4 and HOS CD4CR2 cells ( P <0 01) and that NHS enhanced the effect of HIV gp160 ( P <0 01) Conclusions The activation of MAPKs is independently mediated by CR2 and that anti CR2 McAb, PD98059, and Wortmanin block the activation of MAPKs, respectively The results of the signal transduction and cell proliferation assays of HOS CD4CR2 cells show that CR2 plays a role in the pathogenesis of HIV infection, especially in the inhibition of CD 4 + cell proliferation