Genome-wide identification of the mitogen-activated protein kinase kinases in pear and their functional analysis in response to black spot

The mitogen-activated protein kinase(MAPK)cascade is crucial to plant growth,development,and stress responses.MAPK kinases(MAPKK)play a vital role in linking upstream MAPKK kinases(MAPKKK)with the downstream MAPK.Black spot is one of the most serious fungal diseases of pear which is an important part of the fruit industry in China.The MAPKK genes have been identified in many plants,however,none has been reported in pear(Pyrus bretschneideri).In order to explore whether MAPK gene of pear is related to black spot disease,we designed this experiment.The present study investigated eight putative PbrMAPKK genes obtained from the Chinese white pear genome.The phylogenetic analysis revealed that PbrMAPKK genes were divided into A,B,C,and D groups.These PbrMAPKK genes are randomly distributed on 7 out of 17 chromosomes and mainly originated from the whole-genome duplication(WGD)event.The expression analysis of PbrMAPKK genes in seven pear tissues and the leaves of susceptible and resistant varieties after Alternaria alternata infection by quantitative real-time PCR(qRT-PCR)identified seven candidate genes associated with resistance.Furthermore,virus-induced gene silencing(VIGS)indicated that PbrMAPKK6 gene enhanced resistance to pear black spot disease in pear.

MicroRNA-370-5p inhibits pigmentation and cell proliferation by downregulating mitogen-activated protein kinase kinase kinase 8 expression in sheep melanocytes

In mammals,microRNAs(miRNAs)play key roles in multiple biological processes by regulating the expression of target genes.Studies have found that the levels of miR-370-5p expression differ significantly in the skins of sheep with different hair colors;however,its function remains unclear.In this study,we investigated the roles of miR-370-5p in sheep melanocytes and found that the overexpression of miR-370-5p significantly inhibited cell proliferation(P<0.01),tyrosinase activity(P=0.001)and significantly reduced(P<0.001)melanin production.Functional prediction revealed that the 3′-untranslated region(UTR)of MAP3K8 has a putative miR-370-5p binding site,and the interaction between these two molecules was confirmed using luciferase reporter assays.In situ hybridization assays revealed that MAP3K8 is expressed in the cytoplasm of melanocytes.The results of quantitative RT-PCR and Western blotting analyses revealed that overexpression of miR-370-5p in melanocytes significantly inhibits(P<0.01)MAP3K8 expression via direct targeting of its 3′UTR.Inhibition of MAP3K8 expression by siRNA-MAP3K8 transfection induced a significant inhibition(P<0.01)of melanocyte proliferation and significant reduction(P<0.001)in melanin production,which is consistent with our observations for miR-370-5p.Target gene rescue experiments indicated that the expression of MAP3K8 in melanocytes co-transfected with miR-370-5p and MAP3K8-cDNA(containing sites for the targeted binding to miR-370-5p)was significantly rescued(P≤0.001),which subsequently promoted significant increases in cell proliferation(P<0.001)and melanin production(P<0.01).Collectively,these findings indicate that miR-370-5p plays a functional role in inhibiting sheep melanocyte proliferation and melanogenesis by downregulating the expression of MAP3K8.

Computational Screening of Novel Mitogen-activated Protein Kinase Kinase-1 （MEK1） Inhibitors by Docking and Scoring

The mitogen-activated protein kinase （MAPK） cell signal transduction pathways play a key role in determining the survival of cells. If these pathways can be controlled, they will prohibit the proliferation of cancer cells. To attain this goal, the authors utilize many drugs to interact with mitogen-activated protein kinase kinase-1 （MEK1） in MAPK, and use computer aided drug design （CADD） to analyze the ligand activities of proteins in MEKL The results show that in these drugs, the aromatic group in the terminal of the protein and the PHE209 will induce the stacking force, which is highly related to the actual activities of these drugs.

Optimized new Shengmai powder(优化新生脉散方) inhibits myocardial fibrosis in heart failure by regulating the rat sarcoma/rapidly accelerated fibrosarcoma/mitogen-activated protein kinase kinase/extracellular regulated protein kinases signaling pathway

OBJECTIVE: Exploring the effect of Optimized New Shengmai powder(优化新生脉散方, ONSMP) on myocardial fibrosis in heart failure(HF) based on rat sarcoma(RAS)/rapidly accelerated fibrosarcoma(RAF)/mitogen-activated protein kinase kinase(MEK)/extracellular regulated protein kinases(ERK) signaling pathway. METHODS: Randomized 70 Sprague-Dawley rats into sham(n = 10) and operation(n = 60) groups, then established the HF rat by ligating the left anterior descending branch of the coronary artery. We randomly divided the operation group rats into the model, ONSMP [including low(L), medium(M), and high(H) dose], and enalapril groups. After the 4-week drug intervention, echocardiography examines the cardiac function and calculates the ratios of the whole/left heart to the rat's body weight. Finally, we observed the degree of myocardial fibrosis by pathological sections, determined myocardium collagen(COL) Ⅰ and COL Ⅲ content by enzyme-linked immunosorbent assay, detected the m RNA levels of COL Ⅰ, COL Ⅲ, α-smooth muscle actin(α-SMA), and c-Fos proto-oncogene(c-Fos) by universal real-time, and detected the protein expression of p-RAS, p-RAF, p-MEK1/2, p-ERK1/2, p-ETS-like-1 transcription factor(p-ELK1), p-c-Fos, α-SMA, COL Ⅰ, and COL Ⅲ by Western blot. RESULTS: ONSMP can effectively improve HF rat's cardiac function, decrease cardiac organ coefficient, COL volume fraction, and COL Ⅰ/Ⅲ content, down-regulate the m RNA of COL Ⅰ/Ⅲ, α-SMA and c-Fos, and the protein of p-RAS, p-RAF, p-MEK1/2, p-ERK1/2, p-ELK1, c-Fos, COL Ⅰ/Ⅲ, and α-SMA. CONCLUSIONS: ONSMP can effectively reduce myocardial fibrosis in HF rats, and the mechanism may be related to the inhibition of the RAS/RAF/MEK/ERK signaling pathway.

Overexpression of mitogen-activated protein kinase phosphatase-1 in endothelial cells reduces blood-brain barrier injury in a mouse model of ischemic stroke

Ischemic stroke can cause blood-brain barrier(BBB)injury,which worsens brain damage induced by stroke.Abnormal expression of tight junction proteins in endothelial cells(ECs)can increase intracellular space and BBB leakage.Selective inhibition of mitogen-activated protein kinase,the negative regulatory substrate of mitogen-activated protein kinase phosphatase(MKP)-1,improves tight junction protein function in ECs,and genetic deletion of MKP-1 aggravates ischemic brain injury.However,whether the latter affects BBB integrity,and the cell type-specific mechanism underlying this process,remain unclear.In this study,we established an adult male mouse model of ischemic stroke by occluding the middle cerebral artery for 60 minutes and overexpressed MKP-1 in ECs on the injured side via lentiviral transfection before stroke.We found that overexpression of MKP-1 in ECs reduced infarct volume,reduced the level of inflammatory factors interleukin-1β,interleukin-6,and chemokine C-C motif ligand-2,inhibited vascular injury,and promoted the recovery of sensorimotor and memory/cognitive function.Overexpression of MKP-1 in ECs also inhibited the activation of cerebral ischemia-induced extracellular signal-regulated kinase(ERK)1/2 and the downregulation of occludin expression.Finally,to investigate the mechanism by which MKP-1 exerted these functions in ECs,we established an ischemic stroke model in vitro by depriving the primary endothelial cell of oxygen and glucose,and pharmacologically inhibited the activity of MKP-1 and ERK1/2.Our findings suggest that MKP-1 inhibition aggravates oxygen and glucose deprivation-induced cell death,cell monolayer leakage,and downregulation of occludin expression,and that inhibiting ERK1/2 can reverse these effects.In addition,co-inhibition of MKP-1 and ERK1/2 exhibited similar effects to inhibition of ERK1/2.These findings suggest that overexpression of MKP-1 in ECs can prevent ischemia-induced occludin downregulation and cell death via deactivating ERK1/2,thereby protecting the integrity of BBB,alleviating brain injury,and improving post-stroke prognosis.

Loss of monopolar spindle-binding protein 3B expression promotes colorectal cancer malignant behaviors by activation of target of rapamycin kinase/autophagy signaling

BACKGROUND Monopolar spindle-binding protein 3B(MOB3B)functions as a signal transducer and altered MOB3B expression is associated with the development of human cancers.AIM To investigate the role of MOB3B in colorectal cancer(CRC).METHODS This study collected 102 CRC tissue samples for immunohistochemical detection of MOB3B expression for association with CRC prognosis.After overexpression and knockdown of MOB3B expression were induced in CRC cell lines,changes in cell viability,migration,invasion,and gene expression were assayed.Tumor cell autophagy was detected using transmission electron microscopy,while nude mouse xenograft experiments were performed to confirm the in-vitro results.RESULTS MOB3B expression was reduced in CRC vs normal tissues and loss of MOB3B expression was associated with poor CRC prognosis.Overexpression of MOB3B protein in vitro attenuated the cell viability as well as the migration and invasion capacities of CRC cells,whereas knockdown of MOB3B expression had the opposite effects in CRC cells.At the molecular level,microtubule-associated protein light chain 3 II/I expression was elevated,whereas the expression of matrix metalloproteinase(MMP)2,MMP9,sequestosome 1,and phosphorylated mechanistic target of rapamycin kinase(mTOR)was downregulated in MOB3B-overexpressing RKO cells.In contrast,the opposite results were observed in tumor cells with MOB3B knockdown.The nude mouse data confirmed these in-vitro findings,i.e.,MOB3B expression suppressed CRC cell xenograft growth,whereas knockdown of MOB3B expression promoted the growth of CRC cell xenografts.CONCLUSION Loss of MOB3B expression promotes CRC development and malignant behaviors,suggesting a potential tumor suppressive role of MOB3B in CRC by inhibition of mTOR/autophagy signaling.

Clinical significance of upregulated Rho GTPase activating protein 12 causing resistance to tyrosine kinase inhibitors in hepatocellular carcinoma

BACKGROUND Hepatocellular carcinoma(HCC)is a major health challenge with high incidence and poor survival rates in China.Systemic therapies,particularly tyrosine kinase inhibitors(TKIs),are the first-line treatment for advanced HCC,but resistance is common.The Rho GTPase family member Rho GTPase activating protein 12(ARHGAP12),which regulates cell adhesion and invasion,is a potential therapeutic target for overcoming TKI resistance in HCC.However,no studies on the expression of ARHGAP12 in HCC and its role in resistance to TKIs have been reported.AIM To unveil the expression of ARHGAP12 in HCC,its role in TKI resistance and its potential associated pathways.METHODS This study used single-cell RNA sequencing(scRNA-seq)to evaluate ARHGAP12 mRNA levels and explored its mechanisms through enrichment analysis.CellChat was used to investigate focal adhesion(FA)pathway regulation.We integrated bulk RNA data(RNA-seq and microarray),immunohistochemistry and proteomics to analyze ARHGAP12 mRNA and protein levels,correlating with clinical outcomes.We assessed ARHGAP12 expression in TKI-resistant HCC,integrated conventional HCC to explore its mechanism,identified intersecting FA pathway genes with scRNA-seq data and evaluated its response to TKI and immunotherapy.RESULTS ARHGAP12 mRNA was found to be highly expressed in malignant hepatocytes and to regulate FA.In malignant hepatocytes in high-score FA groups,MDK-[integrin alpha 6(ITGA6)+integrinβ-1(ITGB1)]showed specificity in ligand-receptor interactions.ARHGAP12 mRNA and protein were upregulated in bulk RNA,immunohistochemistry and proteomics,and higher expression was associated with a worse prognosis.ARHGAP12 was also found to be a TKI resistance gene that regulated the FA pathway.ITGB1 was identified as a crossover gene in the FA pathway in both scRNA-seq and bulk RNA.High expression of ARHGAP12 was associated with adverse reactions to sorafenib,cabozantinib and regorafenib,but not to immunotherapy.CONCLUSION ARHGAP12 expression is elevated in HCC and TKI-resistant HCC,and its regulatory role in FA may underlie the TKI-resistant phenotype.

Antagonistic Effects of N-acetylcysteine on Mitogen-activated Protein Kinase Pathway Activation, Oxidative Stress and Inflammatory Responses in Rats with PM2.5 Induced Lung Injuries

Objective To evaluate the antagonistic effects of N-acetylcysteine(NAC)on mitogen-activated protein kinases(MAPK)pathway activation,oxidative stress and inflammatory responses in rats with lung injury induced by fine particulate matter(PM2.5).Methods Forty eight male Wistar rats were randomly divided into six groups:blank control group(C1),water drip control group(C2),PM2.5 exposed group(P),low-dose NAC treated and PM2.5 exposed group(L),middle-dose NAC treated and PM2.5 exposed group(M),and high-dose NAC treated and PM2.5 exposed group(H).PM2.5 suspension(7.5 mg/kg)was administered tracheally once a week for four times.NAC of 125 mg/kg,250 mg/kg and 500 mg/kg was delivered intragastrically to L,M and H group respectively by gavage(10 ml/kg)for six days before PM2.5 exposure.The histopathological changes and human mucin 5 subtype AC(MUC5AC)content in lung tissue of rats were evaluated.We investigated IL-6 in serum and bronchoalveolar lavage fluid(BALF)by Enzyme-linked immunosorbent assay(ELISA),MUC5AC in lung tissue homogenate by ELISA,glutathione peroxidase(GSH-PX)in serum and BALF by spectrophotometry,and the expression of p-ERK1/2,p-JNK1/2 and p-p38 proteins by Western blot.All the measurements were analyzed and compared statistically.Results Lung tissue of rats exposed to PM2.5 showed histological destruction and increased mucus secretion of bronchial epithelial cells.Rats receiving NAC treatment showed less histological destruction and mucus secretion.Of P,L,M and H group,MUC5AC in lung tissue,IL-6 in serum and BALF were higher than controls(C1 and C2)(all P<0.05),with the highest levels found in the P group and a decreasing trend with increase of NAC dose.The activity of GSH-PX in serum and BALF of PM2.5 exposed rats(P,L,M and H)was lower than that of controls(all P<0.05),with higher activities found in NAC treated rats(L,M,and H),and an increasing trend with increase of NAC dose.The expressions of p-ERK1/2,p-JNK1/2 and p-p38 proteins in PM2.5 exposed lung tissue(P,L,M and H)was higher than controls(all P<0.05),with decreased levels and dose dependent downregulation found in NAC treated rats.Conclusion NAC can antagonize major MAPK pathway activation,lung oxidative stress and inflammatory injury induced by PM2.5 in rats.

Association of Common Genetic Variants in Mitogen-activated Protein Kinase Kinase Kinase Kinase 4 with Type 2 Diabetes Mellitus in a Chinese Han Population

Background： A study has identified several novel susceptibility variants of the mitogen-activated protein kinase kinase kinase kinase 4 （MAP4K4） gene for type 2 diabetes mellitus （T2DM） within the German population. Among the variants, five single nucleotide polymorphisms （SNPs） of MAP4K4 （rs1003376, rs1 1674694, rs2236935, rs2236936, and rs6543087） showed significant association with T2DM or diabetes-related quantitative traits. We aimed to evaluate whether common SNPs in the MAP4K4 gene were associated with T2DM in the Chinese population. Methods： Five candidate SNPs were genotyped in 996 patients newly diagnosed with T2DM and in 976 control subjects, using the SNPscanTM method. All subjects were recruited from the Second Affiliated Hospital, Harbin Medical University from October 2010 to September 2013. We evaluated the T2DM risk conferred by individual SNPs and haplotypes using logistic analysis, and the association between the five SNPs and metabolic traits in the subgroups. Results： Of the five variants, SNP rs2236935T/C was significantly associated with T2DM in this study population （odds ratio = 1.293; 95% confidence interval： 1.034-1.619, P= 0.025）. In addition, among the controls, rs1003376 was significantly associated with an increased body mass index （P = 0.045） and homeostatic model assessment-insulin resistance （P = 0.037）. Conclusions： MAP4K4 gene is associated with T2DM in a Chinese Han population, and MAP4K4 gene variants may contribute to the risk toward the development of T2DM.

Wheat kinase TaSnRK2.4 forms a functional module with phosphatase TaPP2C01 and transcription factor TaABF2 to regulate drought response

SNF1-related protein kinase 2(SnRK2)family members are essential components of the plant abscisic acid(ABA)signaling pathway initiated by osmotic stress and triggering a drought stress response.This study characterized the molecular properties of TaSnRK2.4 and its function in mediating adaptation to drought in Triticum aestivum.Transcripts of TaSnRK2.4 were upregulated upon drought and ABA signaling and associated with drought-and ABA-responsive cis-elements ABRE and DRE,and MYB and MYC binding sites in the promoter as indicated by reporter GUS protein staining and activity driven by truncations of the promoter.Yeast two-hybrid,BiFC,and Co-IP assays indicated that TaSnRK2.4 protein interacts with TaPP2C01 and an ABF transcription factor(TF)TaABF2.The results suggested that TaSnRK2.4 forms a functional TaPP2C01-TaSnRK2.4-TaABF2 module with its upstream and downstream partners.Transgene analysis revealed that TaSnRK2.4 and TaABF2 positively regulate drought tolerance whereas TaPP2C01 acts negatively by modulating stomatal movement,osmotic adjustment,reactive oxygen species(ROS)homeostasis,and root morphology.Expression analysis,yeast one-hybrid,and transcriptional activation assays indicated that several osmotic stress-responsive genes,including TaSLAC1-4,TaP5CS3,TaSOD5,TaCAT1,and TaPIN4,are regulated by TaABF2.Transgene analysis verified their functions in positively regulating stomatal movement(TaSLAC1-4),proline accumulation(TaP5CS3),SOD activity(TaSOD5),CAT activity(TaCAT1),and root morphology(TaPIN4).There were high correlations between plant biomass and yield with module transcripts in a wheat variety panel cultivated under drought conditions in the field.Our findings provide insights into understanding plant drought response underlying the SnRK2 signaling pathway in common wheat.

Diabetes and high-glucose could upregulate the expression of receptor for activated C kinase 1 in retina

BACKGROUND Diabetic retinopathy(DR)is a major ocular complication of diabetes mellitus,leading to visual impairment.Retinal pigment epithelium(RPE)injury is a key component of the outer blood retinal barrier,and its damage is an important indicator of DR.Receptor for activated C kinase 1(RACK1)activates protein kinase C-ε(PKC-ε)to promote the generation of reactive oxygen species(ROS)in RPE cells,leading to apoptosis.Therefore,we hypothesize that the activation of RACK1 under hypoxic/high-glucose conditions may promote RPE cell apoptosis by modulating PKC-ε/ROS,thereby disrupting the barrier effect of the outer blood retinal barrier and contributing to the progression of DR.AIM To investigate the role and associated underlying mechanisms of RACK1 in the development of early DR.METHODS In this study,Sprague-Dawley rats and adult RPE cell line-19(ARPE-19)cells were used as in vivo and in vitro models,respectively,to explore the role of RACK1 in mediating PKC-εin early DR.Furthermore,the impact of RACK1 on apoptosis and barrier function of RPE cells was also investigated in the former model.RESULTS Streptozotocin-induced diabetic rats showed increased apoptosis and upregulated expression of RACK1 and PKC-εproteins in RPE cells following a prolonged modeling.Similarly,ARPE-19 cells exposed to high glucose and hypoxia displayed elevated mRNA and protein levels of RACK1 and PKC-ε,accompanied by an increases in ROS production,apoptosis rate,and monolayer permeability.However,silencing RACK1 significantly downregulated the expression of PKC-εand ROS,reduced cell apoptosis and permeability,and protected barrier function.CONCLUSION RACK1 plays a significant role in the development of early DR and might serve as a potential therapeutic target for DR by regulating RPE apoptosis and barrier function.

MicroRNA-298 determines the radio-resistance of colorectal cancer cells by directly targeting human dual-specificity tyrosine(Y)-regulated kinase 1A

BACKGROUND Radiotherapy stands as a promising therapeutic modality for colorectal cancer(CRC);yet,the formidable challenge posed by radio-resistance significantly undermines its efficacy in achieving CRC remission.AIM To elucidate the role played by microRNA-298(miR-298)in CRC radio-resistance.METHODS To establish a radio-resistant CRC cell line,HT-29 cells underwent exposure to 5 gray ionizing radiation that was followed by a 7-d recovery period.The quantification of miR-298 levels within CRC cells was conducted through quantitative RT-PCR,and protein expression determination was realized through Western blotting.Cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and proliferation by clonogenic assay.Radio-induced apoptosis was discerned through flow cytometry analysis.RESULTS We observed a marked upregulation of miR-298 in radio-resistant CRC cells.MiR-298 emerged as a key determinant of cell survival following radiation exposure,as its overexpression led to a notable reduction in radiation-induced apoptosis.Intriguingly,miR-298 expression exhibited a strong correlation with CRC cell viability.Further investigation unveiled human dual-specificity tyrosine(Y)-regulated kinase 1A(DYRK1A)as miR-298’s direct target.CONCLUSION Taken together,our findings underline the role played by miR-298 in bolstering radio-resistance in CRC cells by means of DYRK1A downregulation,thereby positioning miR-298 as a promising candidate for mitigating radioresistance in CRC.

Mitogen-activated protein kinase signaling pathway and invasion and metastasis of gastric cancer

The mortality rate of gastric cancer worldwide is as high as 70%, despite the development of novel therapeutic strategies. One reason for the high mortality is the rapid and uninhibited spread of the disease, such that the majority of patients are diagnosed at a stage when efficient therapeutic treatment is not available. Therefore, in-depth research is needed to investigate the mechanism of gastric cancer metastasis and invasion to improve outcomes and provide biomarkers for early diagnosis. The mitogen-activated protein kinase(MAPK) signaling pathway is widely expressed in multicellular organisms, with critical roles in multiple biological processes, such as cell proliferation, death, differentiation, migration, and invasion. The MAPK pathway typically responds to extracellular stimulation. However, the MAPK pathway is often involved in the occurrence and progression of cancer when abnormally regulated. Many studies have researched the relationship between the MAPK signaling pathway and cancer metastasis and invasion, but little is known about the important roles that the MAPK signaling pathway plays in gastric cancer. Based on an analysis of published data, this review aims to summarize the important role that the MAP kinases play in the invasion and metastasis of gastric cancer and attempts to provide potential directions for further research and clinical treatment.

Electroacupuncture reduces apoptotic index and inhibits p38 mitogen-activated protein kinase signaling pathway in the hippocampus of rats with cerebral ischemia/reperfusion injury

Electroacupuncture attenuates cerebral hypoxia and neuronal apoptosis induced by cerebral ischemia/reperfusion injury.To further identify the involved mechanisms,we assumed that electroacupuncture used to treat cerebral ischemia/reperfusion injury was associated with the p38 mitogen-activated protein kinase（MAPK） signaling pathway.We established rat models of cerebral ischemia/reperfusion injury using the modified Zea-Longa＇s method.At 30 minutes before model establishment,p38 MAPK blocker SB20358 was injected into the left lateral ventricles.At 1.5 hours after model establishment,electroacupuncture was administered at acupoints of Chize（LU5）,Hegu（LI4）,Zusanli（ST36）,and Sanyinjiao（SP6） for 20 minutes in the affected side.Results showed that the combination of EA and SB20358 injection significantly decreased neurologic impairment scores,but no significant differences were determined among different interventional groups.Hematoxylin-eosin staining also showed reduced brain tissue injuries.Compared with the SB20358 group,the cells were regularly arranged,the structures were complete,and the number of viable neurons was higher in the SB20358 ＋ electroacupuncture group.Terminal deoxynucleotidyl transferase（Td T）-mediated d UTP nick-end labeling assay showed a decreased apoptotic index in each group,with a significant decrease in the SB20358 ＋ electroacupuncture group.Immunohistochemistry revealed reduced phosphorylated p38 expression at 3 days in the electroacupuncture group and SB20358 ＋ electroacupuncture group compared with the ischemia/reperfusion group.There was no significant difference in phosphorylated p38 expression between the ischemia/reperfusion group and SB20358 group.These findings confirmed that the electroacupuncture effects on mitigating cerebral ischemia/reperfusion injury are possibly associated with the p38 MAPK signaling pathway.A time period of 3 days could promote the repair of ischemic cerebral nerves.

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: Western 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-1 proteins 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?5 760 vs 122 807±65 515, P= 0.001), ERK-2 (168 471±95 051 vs 120 469±72 874, P<0.001), ERK-3 (118 651±71 513 vs 70 934±68 058,P<0.001), P38 (104 776±51 650 vs 82 930±40 392, P= 0.048) and MEK-1 (116 486±45 725 vs 101 434±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 vs1.24±0.40, P= 0.030). Gastric cancer tissues with either lymph node involvement (2.49±2.91 vs1.03±0.36, P= 0.023; 1.98±1.49vs1.24±0.44, P= 0.036) or serosa invasion (2.39±2.82 vs 1.01±0.35, P= 0.022; 1.95±1.44 vs1.14±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 vs1.23±0.60, P= 0.022; 5.50±5.05 vs1.83±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 MARK signal transduction pathways may play an important role in tumorigenesis and metastatic potential of gastric cancer.

Downregulation of cold-inducible RNA-binding protein activates mitogen-activated protein kinases and impairs spermatoRenic function in mouse testes

Cold-inducible RNA-binding protein （CIRP） is an RNA-binding protein that is expressed in normal testes and downregulated after heat stress caused by cryptorchidism, varicocele or environmental temperatures. The purpose of this study was to investigate the functions of CIRP in the testes. We employed RNAi technique to knock down the expression of CIRP in the testes, and performed haematoxylin and eosin staining to evaluate morphological changes following knockdown. Germ cell apoptosis was examined by terminal deoxynucleotidal transferase-mediated dUTP nick end labelling （TUNEL） assay, and mitogen-activated protein kinase （MAPK） signalling pathways were investigated by Western blotting to determine the possible mechanism of apoptosis. We found that using siRNA is a feasible and reliable method for knocking down gene expression in the testes. Compared to controls, the mean seminiferous tubule diameter （MSTD） and the thickness of the germ cell layers decreased following siRNA treatment, whereas the percentage of apoptotic seminiferous tubules increased. The p44/p42, p38 and SAPK/JNK MAPK pathways were activated after downregulation of CIRP. In conclusion, we discovered that downregulation of CIRP resulted in increased germ cell apoptosis, possibly viathe activation of the p44/p42, p38 and SAPK/JNK MAPK pathways.

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.

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.

Inhibition of p38 mitogen-activated protein kinase may decrease intestinal epithelial cell apoptosis and improve intestinal epithelial barrier function after ischemia- reperf usion injury

AIM: To investigate the role of p38 mitogen-activated protein kinase in rat small intestine after ischemia-reperfusion (I/R)insult and the relationship between activation of p38 MAPK and apoptotic cell death of intestine.METHODS: Ninety Wistar rats were divided randomly into three groups, namely sham-operated group (C), I/R vehicle group (R) and SB203580 pre-treated group(S).In groups R and S, the superior mesenteric artery(SMA)was separated and occluded for 45 min, then released for reperfusion for0.25, 0.5, 1, 2, 6, 12 and 24 h. In group C, SMA was separated without occlusion. Plasma D-lactate levels were examined and histological changes were observed under a light microscope. The activity of p38 MAPK was determined by Western immunoblotting and apoptotic cells were detected by the terminal deoxynucleotidyl transferase (TdT)-mediated dUDP-biotin nick end labeling (TUNEL).RESULTS: Intestinal ischemia followed by reperfusion activated p38 MAPK, and the maximal level of activation (7.3-fold vs sham-operated group) was reached 30 min after I/R. Treatment with SB 203580, a p38 MAPK inhibitor,reduced intestinal apoptosis (26.72±3.39% vs62.50±3.08%in I/R vehicle, P＜0.01) and decreased plasma D-lactate level (0.78±0.15 mmol/L in I/R vehicle vs0.42±0.17 mmol/L in SB-treated group) and improved post-ischemic intestinal histological damage.CONCLUSION: p38 MAPK plays a crucial role in the signal transduction pathway mediating post-ischemic intestinal apoptosis, and inhibition of p38 MAPK may attenuate ischemia-reperfusion injury.

H pylori stimulates proliferation of gastric cancer cells through activating mitogen-activated protein kinase cascade

AIM: To explore the mechanism by which H pylori causes activation of gastric epithelial cells. METHODS: A VacA (+) and CagA (+) standard H pylori line NCTC 11637 and a human gastric adenocarcinoma derived gastric epithelial cell line BGC-823 were applied in the study. MTT assay and 3H-TdR incorporation test were used to detect the proliferation of BGC-823 cells and Western blotting was used to detect the activity and existence of related proteins. RESULTS: Incubation with H pylori extract increased the proliferation of gastric epithelial cells, reflected by both live cell number and DNA synthesis rate. The activity of extracellular signal-regulated protein kinase (ERK) signal transduction cascade increased within 20 min after in- cubation with H pylori extract and appeared to be a sus- tained event. MAPK/ERK kinase (MEK) inhibitor PD98059 abolished the action of H pylori extract on both ERK activity and cell proliferation. Incubation with H pylori extract increased c-Fos expression and SRE-dependent gene expression. H pylori extract caused phosphorylation of several proteins including a protein with molecular size of 97.4 kDa and tyrosine kinase inhibitor genistein inhibited the activation of ERK and the proliferation of cells caused by H pylori extract. CONCLUSION: Biologically active elements in H pylori extract cause proliferation of gastric epithelial cells through activating tyrosine kinase and ERK signal trans- duction cascade.