OBJECTIVETo study the role of MEKK2 on the production of IL-2 in Jurkat cells stimulated by PHA/anti-CD28 antibody.

METHODSThe MEKK2 and JNK kinase activities were measured in both dominant negative MEKK2 Jurkat (dnMEKK2 Jurkat) cells and parental Jurkat cells. The AP(1) and IL-2 promotor activities were measured by luciferase activity assay. The IL-2 mRNA and protein were detected by RT-PCR and Western blot.

RESULTSAfter stimulation by PHA/anti-CD28, JNK was activated in parental Jurkat cells but not in dnMEKK2 Jurkat cells. The luciferase report gene activities of AP1 and IL-2 promotors were increased by 4- and 5-folds in parental cells whereas only by 1 fold in dnMEKK2 Jurkat cells. The level of IL-2 mRNA and IL-2 protein were increased in parental Jurkat cells but not in dnMEKK2 Jurkat cells.

CONCLUSIONMEKK2 plays an important role on the production of IL-2 in Jurkat cell stimulated with PHA/anti-CD28 antibody. It is a potential drug target for the treatment of GVHD and autoimmune disease.

Humans ; Interleukin-2 ; biosynthesis ; genetics ; Jurkat Cells ; MAP Kinase Kinase 4 ; metabolism ; MAP Kinase Kinase Kinase 2 ; metabolism ; physiology

OBJECTIVETo explore the correlation between expression of mitogen-activated protein kinase kinase 4 (MKK4) and metastasis of oral squamous cell carcinoma (OSCC).

METHODSExpression levels of MKK4 mRNA and protein were examined in surgically resected oral squamous cell carcinoma specimens and corresponding lymph nodes by semi-quantitative reverse Transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry.

RESULTSThe expression of MKK4 in 48 cases of metastasis of lymph node group was significantly higher than 27 cases of without metastasis of lymph node group in 75 cases of paraffin-embedded OSCC samples (P < 0.05). The expression of MKK4 in 48 cases of metastatic lymph node lesions was higher than 48 cases of primary site of OSCC (P < 0.05). There was no correlation between the expression of MKK4 and primary site, size of tumor, histological grade ( P > 0.05). The expression of MKK4 mRNA in 16 cases of metastasis of lymph node group was significantly higher than 22 cases of without metastasis of lymph node group in 38 cases of fresh OSCC samples (P < 0.01). The expression of MKK4 in 6 cases of matched metastatic lymph node lesions was higher than 16 cases of primary tumour (P < 0.05)

CONCLUSIONThe up-regulation of MKK4 protein and mRNA may be related with the invasion and metastasis of OSCC. MKK4 maybe played a promoting role in the progression and metastasis of OSCC.

Carcinoma, Squamous Cell ; Humans ; Immunohistochemistry ; Lymphatic Metastasis ; MAP Kinase Kinase 4 ; Mouth Neoplasms ; RNA, Messenger

OBJECTIVETo analyze the expression of MAP2K4 and vimentin in human endometrial carcinoma (EC) and their association with the clinicopathological features and prognosis of the patients.

METHODSMAP2K4 and vimentin expressions were detected immunohistochemically in paraffin-embedded tissue sections from 128 patients with EC, and the correlation of MAP2K4 and vimentin expressions with the clinicopathological factors of the patients was analyzed.

RESULTSMAP2K4 and vimentin proteins were positively expressed in 49 (38.3%) and 83 (64.8%) of the patients, respectively. A positive expression of MAP2K4 was negatively correlated with FIGO stage of the tumor (P=0.010) and lymph node status (P=0.016); a positive expression of vimentin was positively correlated with FIGO stage of the tumor (P=0.025), histological grades (P=0.017), depth of myometrial invasion (P=0.044) and lymph node status (P=0.032). MAP2K4 was inversely associated with vimentin expression in EC(r=-0.598, P<0.001). Patients positive for MAP2K4 tended to have a higher overall survival rate (P=0.002), and those positive for vimentin tended to have a lower overall survival rate (P=0.007); patients positive for MAP2K4 but negative for vimentin had the longest survival time, while those negative for MAP2K4 and positive for vimentin had lowest survival rate (P=0.004).

CONCLUSIONDetection of MAP2K4 and vimentin might help in early diagnosis and prognostic evaluation of patients with EC.

Endometrial Neoplasms ; metabolism ; pathology ; Female ; Humans ; MAP Kinase Kinase 4 ; metabolism ; Prognosis ; Survival Rate ; Vimentin ; metabolism

OBJECTIVETo study the effects of 50 Hz power-frequency magnetic fields on signal transduction pathway of stress-activated protein kinase(SAPK), and explore the cellular signal transduction mechanism of the biological effects induced by power-frequency magnetic fields.

METHODSChinese hamster lung(CHL) cell line was exposed to power-frequency magnetic fields with two intensities for different exposure durations. The cytoplasmic protein was extracted and the phosphorylated portion of SAPK and SEK1/MKK4 was measured with Western blotting analysis. The SAPK enzymatic activity was measured by the solid-phase kinase assay in cells exposed to power-frequency magnetic fields for 15 min.

RESULTSBoth 0.4 mT and 0.8 mT power-frequency magnetic fields could enhance the phosphorylation of SAPK in a time-relative course manner, and reached the maximum extent at 15 min, with an increase of 20% and 17% respectively. The solid-phase kinase assay showed that the enzymatic activities of SAPK were also increased, which were 2.9 +/- 0.4 and 2.1 +/- 0.9 times of control respectively. However, the duration of SAPK phosphorylation induced by 0.8 mT was longer than that of 0.4 mT, while the duration and extent of SAPK dephosphorylation was remarkably shorter than that of 0.4 mT. The power-frequency magnetic fields under equal conditions could not phosphorylate(activate) the SEK1/MKK4.

CONCLUSIONPower-frequency magnetic fields could activate the SAPK, but not SEK1/MKK4. It is suggested that power-frequency magnetic fields may activate SAPK signal transduction pathway through a kinase other than SEK1/MKK4. The activation mechanism of SAPK of power-frequency magnetic fields needs to be identified in more detail.

Animals ; Cell Line ; Cricetinae ; Cricetulus ; Enzyme Activation ; radiation effects ; Lung ; metabolism ; radiation effects ; MAP Kinase Kinase 4 ; metabolism ; MAP Kinase Signaling System ; physiology ; radiation effects ; Magnetics ; Phosphorylation

OBJECTIVETo investigate the mechanism of intense noise-induced apoptosis of vestibular hair cells in guinea pigs and the effect of phosphorylated c-Jun N-terminal kinase (JNK) signal transduction pathway in intense noise-induced apoptosis of vestibular hair cells.

METHODSThirty-two guinea pigs were randomly and equally divided into 1, 5, and 15 d experimental groups and control group. The guinea pigs in the experimental groups were exposed to 4 kHz narrow-band noise at 120 dB SPL for 4 h and then subjected to measurement of auditory brainstem response at 1, 5, or 15 d after noise exposure. In each group, four guinea pigs were used to prepare paraffin sections of vestibular hair cells, and the rest for extraction of total protein from vestibular hair cells. The apoptosis of vestibular hair cells was detected by terminal deoxynucleotidyl transferase (TdT)-mediated d-UTP nick-end labeling (TUNEL). The expression levels of p-JNK and pc-Jun were measured by immunohistochemistry and Western blot.

RESULTSTUNEL-positive cells were found in the vestibular hair cells in the experimental groups, most in the 1 d experimental group and least in the 15 d experimental group, but no positive cells were found in the control group. The immunohistochemical results showed that p-JNK and pc-Jun were detected in the cell nuclei in the experimental groups, but no p-JNK- and pc-Jun-positive cells were found in the control group. The Western blot showed that p-JNK and pc-Jun were increased and activated quickly at 1d after noise exposure, reached the peak levels at 5 d after noise exposure, and were then decreased gradually, but they were still at relatively high levels at 15 d after noise exposure.

CONCLUSIONIntense noise can cause injury to vestibular hair cells by inducing cell apoptosis, and p-JNK marks the activation of JNK signal transduction pathway, suggesting that JNK signal transduction pathway plays an important role in intense noise-induced apoptosis of vestibular hair cells in guinea pigs.

Animals ; Apoptosis ; Guinea Pigs ; Hair Cells, Vestibular ; cytology ; MAP Kinase Kinase 4 ; metabolism ; Noise ; adverse effects ; Phosphorylation ; Signal Transduction

Recent studies have demonstrated that human periodontal ligament cells express receptor activation of nuclear factor kappaB ligand (RANKL) which enhances the bone resorbing activity of osteoclasts differentiated from hematopoietic preosteoclasts. The purpose of this study is to determine the effects of p38 MAPK and JNK kinase upon regulating RANKL and OPG in response to IL-1beta(1 ng/ml) in rat periodontal ligament cells. Soluble RANKL was measured by immunoassay. The effects of p38 MAPK on RANKL and OPG expression was determined by RT-PCR. The results were as follows: 1. Periodontal ligament cells which stimulated by IL-1beta increased soluble RANKL synthesis by dose-dependent pattern. 2. p38 MAP kinase inhibitor (SB203580) showed regulation of soluble RANKL expression by dose-dependent manners. 3. p38 MAP kinase inhibitor (SB203580) regulated the expression of RANKL, but it dose regulate the expresseion of OPG. 4. JNK (c-jun NH2-terminal kinase) inhibitor (PD98059) did not regulate mRANKL and mOPG. These results suggested that p38 MAPK play a significant role in RANKL gene expression.
Animals ; Gene Expression ; Humans ; Immunoassay ; MAP Kinase Kinase 4 ; Osteoclasts ; p38 Mitogen-Activated Protein Kinases* ; Periodontal Ligament* ; Rats*

Epstein-Barr virus (EBV) latent infection transforms B lymphocytes into proliferating lymphoblastoid cell lines (LCLs). EBV latent infection membrane protein 1 (LMP1) is required for EBV-mediated B lymphocyte transformation, and LMP1-induced NF-kappaB activation is essential for LCL survival. Previously, it was reported that the level of reactive oxygen species (ROS) and the expression of apoptosis signal-regulating kinase 1 (ASK1) are elevated in EBV-positive Burkitt's lymphoma (BL) cells, the potential role of ASK1 in LMP1-induced NF-kappaB activation was thus investigated in this study. In EBV-positive BL cells, ASK1 was highly expressed and activated. In addition, TRAF6-ASK1 interaction was significantly increased in EBV-positive BL cells. Interestingly, the expression of LMP1 alone facilitated ASK1 activation. The expression of a dominant negative ASK1 mutant (ASK1KM) strongly blocked LMP1-induced NF-kappaB activation. Furthermore, LMP1-induced NF-kappaB activation was significantly reduced in ASK1 knock out (ASK1-/-) mouse embryonic fibroblasts (MEFs). Taken together, these results demonstrate that ASK1 is activated by LMP1 and is critical for LMP1-induced NF-kappaB activation.
Animals ; B-Lymphocytes ; Burkitt Lymphoma ; Cell Line ; Fibroblasts ; Herpesvirus 4, Human ; Lymphocyte Activation ; MAP Kinase Kinase Kinase 5 ; Membrane Proteins ; Mice ; NF-kappa B ; Reactive Oxygen Species

In the present study, we were to screen the specific microRNA (miRNA) of exercise-induced muscle damage (EIMD) and assess the EIMD-specific miRNAs-regulated target of sarcolemmal damage in rats. Twenty-four male Sprague-Dawley (SD) rats were randomly divided into 3 groups, which included sedentary (C), 24 h post-exercise (E24) and 48 h post-exercise (E48) groups. Rat EIMD model was established by an acute eccentric exercise, i.e., a downhill running treatment at -16º gradient. EIMD characteristics were verified by Evans blue dye staining, differentially expressed miRNAs were detected by microarray assay, EIMD-specific miRNAs expressions were further validated by real-time quantitative RT-PCR (RT-qPCR), and targets of the miRNAs were predicted based on mRNA expressions of associated proteins and related pathway core molecules of sarcolemmal damage. Two EIMD-specific expressed miRNAs, including miR-206-3p and miR-139-3p, were found in the study. There was a significantly negative correlation (P < 0.05) between miR-206-3p expression and dystrophin (r = -0.68), utrophin (r = -0.64), JNK (r = -0.62) or ERK1 (r = -0.68) respectively, but no correlation was found between miR-139-3p and these biomolecules. The results suggest that: i) the expression profile of miRNAs in rat is significantly affected by EIMD, ii) miR-206-3p and miR-139-3p are the EIMD-specific miRNAs, and iii) miR-206-3p may control sarcolemmal damage by regulating dystrophin, utrophin, JNK and ERK1.
Animals ; Dystrophin ; genetics ; MAP Kinase Kinase 4 ; genetics ; MAP Kinase Signaling System ; Male ; MicroRNAs ; genetics ; Physical Conditioning, Animal ; adverse effects ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Real-Time Polymerase Chain Reaction ; Running ; Sarcolemma ; pathology ; Utrophin ; genetics

AIMTo investigate the effect of diazoxide preconditioning and the role of ERK and JNK in cellular signaling during diazoxide preconditioning protection in isolated spontaneous hypertension rat (SHR) hearts.

METHODSHearts were isolated from male SHR rats, and perfused on a Langendorff apparatus. Five groups were considered (n = 6). Con: after 40 min perfusion the hearts were submitted to 25 min ischemia followed by 30 min reperfusion. IP: the hearts were preconditioned with 2 periods of 5 min ischemia and 10 min reperfusion prior to 25 min ischemia. DP: the hearts were preconditioned with 2 periods of 10 min K-H solution with 50 micromol x L(-1) diazoxide and 5 min K-H solution reperfusion prior to 25 min ischemia. 5-HD: perfuse with 100 micromol x L(-1) 5-HD (a special mitochondrial ATP sensitive potassium channel blocker) for 10 min followed by 30 min K-H solution perfusion before 25 min ischemia. 5-HD + DP: 100 micromol x L(-1) 5-HD was given for 10 min before diazoxide preconditioning.

RESULTSDuring reperfusion, comparing with Con group, the recoveries of left ventricle developed pressure (LVDP), + dP/dt(max), - dP/dt(max) and left ventricle end diastolic pressure (LVEDP) were improved in IP and DP groups (P < 0.01 vs Con). At the end of reperfusion, compared with Con group, the expression of ERK in myocardium were higher in IP and DP groups (P < 0.01 vs Con), there was no significance between 5-HD and Con group, but 5-HD couldn't inhibit the expression of ERK induced by diazoxide preconditioning. The expression of JNK in IP and DP groups were decreased (P < 0.05 vs Con), this effect could been inhibited by 5-HD.

CONCLUSIONThese results indicated that diazoxide preconditioning could mimic ischemic preconditioning, the activation of ERK expression and the declining of JNK expression involved in diazoxide preconditioning in isolated SHR hearts.

Animals ; Diazoxide ; pharmacology ; In Vitro Techniques ; MAP Kinase Kinase 4 ; metabolism ; MAP Kinase Signaling System ; Male ; Myocardial Ischemia ; metabolism ; physiopathology ; Myocardial Reperfusion Injury ; metabolism ; physiopathology ; Myocardium ; metabolism ; Rats ; Rats, Inbred SHR

A stable hepatoma cell line(Hep G2 cell) insulin resistance model was established and used to analyze the effect of effective components of Mori Folium in alleviating insulin resistance,and preliminary explore the mechanism for alleviating insulin resistance. The Hep G2 insulin action concentration and the duration of action were investigated using the glucose oxidase method(GOD-POD method) to establish a stable Hep G2 insulin resistance model. Normal control group,model group,Mori Folium polysaccharide group,Mori Folium flavonoid group and rosiglitazone group were divided to determine the glucose consumption. The effect of Mori Folium effective components on Hep G2 insulin resistance was analyzed. The mRNA expressions of JNK,IRS-1 and PDX-1 in each group were detected by Real-time quantitative PCR(qRT-PCR). The protein expressions of p-JNK,IRS-1 and PDX-1 were detected by Western blot. And the mechanism of effective components of Mori Folium in alleviating insulin resistance was investigated. The results showed that the glucose consumption was significantly decreased in the insulin resistance cells after incubation with 25. 0 mg·L-1 insulin for 36 h(P<0. 01),and the model was relatively stable within 36 h. Mori Folium polysaccharides and flavonoids all alleviated insulin resistance,among which Mori Folium flavonoids had better effect in alleviating Hep G2 insulin resistance(P<0. 05). The qRT-PCR analysis showed that Mori Folium polysaccharides and flavonoids could inhibit JNK and IRS-1 mRNA expressions,while enhancing PDX-1 mRNA expression. Western blot analysis displayed that Mori Folium polysaccharides and flavonoids could inhibit p-JNK and IRS-1 protein expressions,while enhancing PDX-1 protein expression. Mori Folium polysaccharides and flavonoids can alleviate insulin resistance in Hep G2 cells,and its mechanism may be the alleviation of insulin resistance by inhibiting JNK signaling pathway.
Drugs, Chinese Herbal ; pharmacology ; Glucose ; Hep G2 Cells ; Homeodomain Proteins ; metabolism ; Humans ; Insulin ; Insulin Receptor Substrate Proteins ; metabolism ; Insulin Resistance ; MAP Kinase Kinase 4 ; metabolism ; MAP Kinase Signaling System ; Morus ; chemistry ; Plant Leaves ; chemistry ; Trans-Activators ; metabolism