Effects Of Mitogen-activated Protein Kinase Research Articles

Enhanced cell death effects of MAP kinase inhibitors in propyl gallate-treated lung cancer cells are related to increased ROS levels and GSH depletion

Propyl gallate (PG) has an anti-growth effect in lung cancer cells. The present study investigated the effects of mitogen-activated protein kinase (MAPK; MEK, JNK, and p38) inhibitors on PG-treated Calu-6 and A549 lung cancer cells in relation to cell death as well as reactive oxygen species (ROS) and glutathione (GSH) levels. PG induced cell death in both Calu-6 and A549 lung cancer cells at 24 h, which was accompanied by loss of mitochondrial membrane potential (MMP; ΔΨm). All of the tested MAPK inhibitors increased cell death in both PG-treated lung cancer cell lines. In particular, MEK inhibitor strongly enhanced cell death and MMP (ΔΨm) loss in PG-treated Calu-6 cells and p38 inhibitor had the same effects in A549 cells as well. PG increased ROS levels and caused GSH depletion in both cell lines at 24 h. MAPK inhibitors increased O2•- levels and GSH depletion in PG-treated Calu-6 cells, and JNK and p38 inhibitors increased ROS levels and GSH depletion in PG-treated A549 cells. In conclusion, MAPK inhibitors increased cell death in PG-treated Calu-6 and A549 lung cancer cells. Enhanced cell death and GSH depletion in Calu-6 cells caused by the MEK inhibitor were related to increased O2•- levels, and the effects of the p38 inhibitor in A549 cells were correlated with increased general ROS levels.

Mitogen-activated protein kinases are involved in cucurbitacin D-induced antitumor effects on adult T-cell leukemia cells

Adult T cell leukemia (ATL) is an aggressive and malignant blood disease. We previously reported that steroid-structured cucurbitacin D (CuD) induces apoptosis in ATL cells. In this study, we investigated the effects of mitogen-activated protein kinase (MAPK) signaling inhibitors on CuD-induced cell death in peripheral blood lymphocytes (PBLs) isolated from ATL/acute lymphoblastic leukemia (ALL) patients and two human leukemia cell lines (MT-1 and MT-4). PBLs were isolated from an ATL/ALL patient as well as from a healthy donor. Cell surface markers were examined using flow cytometry. Serum cytokine levels were estimated using LEGENDplex or analyzed at the Center for Clinical and Translational Research of Kyushu University Hospital. Cell proliferation was assessed using the Cell Titer-Glo luminescent cell viability assay. Protein expression was determined by western blotting. PBLs from patients highly expressed CD4 and CD5. Serum from the patient contained high levels of interleukin (IL)-8, IL-10, IL-18, and interferon-γ compared to the healthy donor. CuD-induced cell death was enhanced by the mitogen-activated protein kinase kinase (MEK)1/2 inhibitor U0126. However, a c-Jun N-terminal kinase (JNK) inhibitor prevented CuD-induced cell death. Immunoblot analyses revealed that CuD reduced the phosphorylation of extracellular signal-regulated kinase (ERK), p38, and JNK, and co-treatment with CuD and U0126 did not affect the phosphorylation of ERK. MEK1/2 and p38 inhibitors enhanced CuD-induced cell death, and U0126 enhanced the CuD-induced de-phosphorylation of ERK in MT-1 and MT-4 cells. We conclude that CuD reduces ERK activation, resulting in enhanced antitumor effects on leukemic cells.

GC-Derived EVs Enriched with MicroRNA-675-3p Contribute to the MAPK/PD-L1-Mediated Tumor Immune Escape by Targeting CXXC4

MicroRNAs (miRNAs) delivered by gastric cancer (GC)-secreted extracellular vesicles (GC-EVs) are associated with the immune escape in GC. Microarray analysis based on the GEO: GSE112369 dataset identified the presence of poorly expressed CXXC finger protein 4 (CXXC4) in GC, which was validated in clinical samples of GC patients. Moreover, prediction based on TargetScan analysis demonstrated the putative miR-675-3p binding site in the 3′ UTR region of CXXC4. Thereby, our study aims to determine the role of GC-EV-encapsulated miR-675-3p in GC. First, CXXC4 was found to be negatively correlated with programmed cell death 1 ligand 1 (PD-L1). The effects of mitogen-activated protein kinase (MAPK) signaling on GC were evaluated using activator of the MAPK pathway. The overexpression of CXXC4 led to a downregulated MAPK signaling pathway, thus decreasing PD-L1 expression to augment the proliferation and activation of T cells co-cultured with GC HGC-27 cells. GC-EV-encapsulated miR-675-3p negatively regulated the expression of its target gene CXXC4. GC-EV-encapsulated miR-675-3p increased PD-L1 expression to stimulate the immune escape in vitro and EV-encapsulated miR-675-3p accelerated cisplatin resistance in vivo. Collectively, the aforementioned findings present a mechanism in which EV-mediated miR-675-3p upregulates PD-L1 expression, promoting immune escape in GC.

Expression and significance of MAPK/ERK in the specimens and cells of epithelial ovarian cancer

Objective: To detect phosphorylated-extracellular signal-regulated kinase (p-ERK1/2) protein expression in epithelial ovarian cancer and cell lines, and to examine the effects of mitogen-activated protein kinase (MAPK) kinase (MEK) inhibitor AZD6244 on cell proliferation, apoptosis as well as cell cycle of ovarian cancer cells. To explore the function and significance of MAPK/extracellular signal-regulated kinase (ERK) signaling pathway in the development of ovarian cancer. Methods: (1) A total of 104 cases of patients with ovarian cancer who accepted the treatment of gynecological surgery and being confirmed by pathological examination in First Affiliated Hospital, Dalian Medical University from January 2004 to December 2013 were selected. The expressions of p-ERK1/2 protein were detected by immunohistochemistry in ovarian cancer specimens, and the relationship between the expressions of p-ERK1/2 and the clinical features of patients was analyzed. (2) p-ERK1/2 and other related proteins were determined by western blot in various ovarian cancer cells, including SKOV3, OV2008, C13, A2780S, A2780CP, OVCAR4, OVCAR5, OVCAR8 and CAOV3 treated with or without MEK inhibitor. The cellular proliferation, apoptosis and cell cycle of ovarian cancer cells after treatment with MEK inhibitor were analyzed by methyl thiazolyl tetrazolium (MTT) assay and flow cytometry, respectively. Results: (1) The immunohistochemical method showed that p-ERK1/2 between low grade serous carcinoma and clear cell carcinoma were not significantly higher expressed (P>0.05) . However, a lower level of the p-ERK1/2 expression were observed among high grade serous carcinoma, mucinous carcinoma and endometrioid carcinoma (all P<0.05) . There was no significant correlation between the protein expression of p-ERK1/2 and patients' age, pathological stage of surgery, and preoperative serum CA(125) level (P>0.05). (2) Western blot showed that the protein p-ERK1/2 was widely expressed in various ovarian cancer cell lines such as SKOV3, OV2008, C13, A2780S, A2780CP, OVCAR4, OVCAR5, OVCAR8 and CAOV3. After treatment with AZD6244 (5, 10 μmol/L), the level of p-ERK1/2 in OVCAR5 and OVCAR8 decreased significantly in dose-dependent manner. Additionally, we found a reduction of the expression level of cyclin D1, caspase-3 and appeared cleaved poly adenosine diphosphate ribose polymerase (PARP) in OVCAR5 and OVCAR8, compared with control groups. MTT assays showed that OVCAR5, OVCAR8 and A2780S were differently inhibited in the dose-dependent manner after being treated with different concentrations of AZD6244 (0, 2.5, 5, 10, 25, 50 and 100 μmol/L, all P<0.05). Further tested by flow cytometry, the results showed that AZD6244 (5, 10 μmol/L) was able to induce the apoptosis of OVCAR5, OVCAR8 and A2780S, as well as G(0)/G(1) phase arrest, both in a dose-dependent manner (P<0.05). Conclusions: As the main active and functional unit of MAPK/ERK signaling pathway, p-ERK1/2 protein is expressed in both the tissues and various ovarian cancer cell lines. AZD6244 could down-regulated the expression of p-ERK1/2 in ovarian cancer cells, accompanied by the decreased proliferation and increased cell apoptosis of ovarian cancer cells. In conclusion, MAPK/ERK signaling pathway might play a role in the development and progression of ovarian cancer, and may be provide a novel option for molecular targeted therapies of the disease.

In Vitro and in Vivo Effect of Mapk Signal Transduction Pathway Inhibitors on Echinococcus multilocularis

To evaluate the effect of mitogen-activated protein kinase (MAPK) signal transduction pathway inhibitors against alveolar echinococcosis in vitro and in vivo, Echinococcus multilocularis metacestode cysts and protoscolices were obtained from infected mice. Protein chip technology was utilized to screen for key highly expressed target proteins in the MAPK pathway in this parasite and their corresponding inhibitors. Four-week-old Balb/c female mice used for the in vivo experiment underwent inoculation of E. multilocularis by intraperitoneal injection, as well as intragastric administration of MAPK inhibitors for 6 wk. We included 6 groups of mice: a phosphate-buffered saline (PBS) group (negative control); an albendazole-treated group (positive group); and 4 experimental groups treated with TRx0237 mesylate, GDC-0994, pifithrin-β hydrobromide, or Selonsertib. Echinococcus multilocularis protoscolices were collected and cultured in 1066 medium with penicillin/streptomycin and 10% fetal bovine serum. The in vitro experiment included a PBS group (negative control), a dimethyl sulfoxide-treated group (solvent group), and 4 inhibitor-treated groups as in the in vivo experiment (experimental groups). Each inhibitor group received 4 drug concentrations (5, 30, 55, and 80 μM), and the experiment was performed in triplicate per sample. Fluorescence microscopy was used to evaluate the survival rate of the protoscolices every 48 hr beginning from the first 24 hr. The same grouping was used to evaluate cytotoxicity on E. multilocularis germinal cells and L02 cells. The average weights of E. multilocularis metacestode cyst tissue from each group of the in vivo experiment were 873 mg (PBS), 335 mg (albendazole), 323 mg (TRx0237 mesylate), 420 mg (GDC-0994), 340 mg (pifithrin-β hydrobromide), and 642 mg (Selonsertib). Results showed albendazole, TRx0237 mesylate, and pifithrin-β hydrobromide had significant inhibitory effects on inhibition of E. multilocularis. We found a positive correlation between drug concentrations and the inhibitory effects seen in the in vitro experiment, with the differences in contrast with the control group becoming statistically significant after 72 hr of treatment ( P < 0.05). The inhibition rates of TRx0237 mesylate to germinal cells by drug concentration were 23.73, 46.59, 74.71, and 77.44%. Other drugs had no effect on germinal cells. All the inhibitors had low toxicity on L02 cells. Inhibitors of the MAPK signal transduction pathway showed significant inhibitory effects on E. multilocularis, suggesting these may be potential candidates for the treatment of alveolar echinococcosis.

Crosstalk between mitogen-activated protein kinase inhibitors and transforming growth factor-β signaling results in variable activation of human dermal fibroblasts.

Fibroblast activation is a key step in the establishment of skin fibrosis induced by acute injury, and it is characterized by the differentiation of plastic resident tissue fibroblasts into contractile, extracellular matrix-secreting myofibroblasts. As fibroblast activation must be regulated in vivo, fibroblasts receive signals from the surrounding environment that initiate their fibrotic program. Thus, the present study investigated the effects of mitogen-activated protein kinase (MAPK) signaling pathways on fibroblast activation. It was demonstrated in primary human dermal fibroblasts that small molecule-mediated inhibition of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) potentiated fibroblast activation, and that small molecule-mediated inhibition of p38 antagonized fibroblast activation. ERK and JNK inhibition cooperatively enhanced fibroblast activation mediated by treatment with exogenous transforming growth factor (TGF)-β1, and p38 inhibition antagonized ERK inhibitor-mediated or JNK inhibitor-mediated fibroblast activation. Transcript analysis demonstrated that ERK and JNK inhibitor-mediated fibroblast activation was accompanied by distinct changes in the expression of TGF-β-associated ligands and receptors, and that p38 inhibitor-mediated antagonism of fibroblast activation was accompanied by a distinct expression paradigm of TGF-β-associated genes, including upregulation of beta-glycan. ERK inhibitor-mediated and JNK inhibitor-mediated fibroblast activation was partially antagonized by small molecule-mediated inhibition of TGF-β receptor (R)1, indicating that these mechanisms of fibroblast activation are partially dependent on TGF-β/TGF-βR signaling. These data collectively demonstrate and provide partial explanations of the varied effects and pathway dependencies of MAPK inhibitor-mediated effects on fibroblast activation.

Influence of hypothyroidism on testicular mitochondrial oxidative stress by activating the p38 mitogen-activated protein kinase and c-Jun NH2-terminal kinase signaling pathways in rats.

Thyroid hormone deficiency can impair testicular function. However, knowledge of the effects of mitogen-activated protein kinase (MAPK) pathways on testicular mitochondrial oxidative damage induced by hypothyroidism is still rudimentary. This study aims to explore the possible mechanisms of testicular mitochondrial oxidative damage in hypothyroidism rats. Wistar male rats were randomly divided into control (C), low- (L), and high-hypothyroidism (H) groups (1 ml/100 g body weights (BWs)/day 0, 0.001% and 0.1% propylthiouracil, respectively) by intragastric gavage for 60 days. Blood samples were collected to measure the levels of serum triiodothyronine (T3), thyroxine (T4), and thyroid stimulating hormone (TSH). Testicular mitochondrial homogenates were used to measure the activities of superoxide dismutase (SOD), catalase (CAT), and Ca2+-ATPase as well as protein and mRNA expression of androgen receptor (AR), p38 MAPK, and c-Jun NH2-terminal kinase (JNK). Results showed that the BWs, testes weights, and levels of T3 and T4 were all significantly decreased and the testes coefficient and level of TSH were significantly increased in the H group. There were significant decreases in SOD activity in the H group as well as decreases in CAT and Ca2+-ATPase activities in the L and H groups. Additionally, protein expression of AR decreased significantly and protein expression of phosphorylated p38MAPK and JNK increased significantly in the H group. Therefore, the study suggests that hypothyroidism could affect male reproductive function by disturbing expression of AR, changing the activity of Ca2+-ATPase, inducing oxidative stress and then leading to activation of p38MAPK and JNK signaling in the testicular mitochondria.

MAPK Pathway Involved in Epidermal Terminal Differentiation of Normal Human Epidermal Keratinocytes.

ObjectiveTo investigate the effect of mitogen-activated protein kinase (MAPK) signaling pathway in epidermal terminal differentiation.MethodsThe MAPK pathways (p38, ERK1/2, JNK) were inhibited by SB203580, PD98059, and SP600125 in normal human epidermal keratinocytes (NHEKs), respectively. Western blotting assays were performed to detect expression of filaggrin and differentiation-related proteins. The mRNA expressions of differentiation-related proteins were detected by real-time quantitative PCR (qRT-PCR).ResultsInhibition of MAPK pathway by SB203580, PD98059, and SP600125 resulted in significant reduction of filaggrin expression in NHEKs. Inhibition of the p38 MAPK pathway decreased the expression of differentiation-related proteins (cytokeratin 5, cytokeratin 14, ST14, and SPRR3), Akt, and NF-κB. Inhibition of JNK also suppressed expression of cytokeratin 14, SPRR3, Akt, and NF-κB. However, inhibition of ERK1/2 merely decreased expression of SPRR3 and Akt.ConclusionMAPK pathways regulates epidermal terminal differentiation in NHEKs. The p38 signaling pathway plays an especially important role.

Effects of MAPKs inhibitors on GSH metabolic enzymes in rat hepatocytes

To investigate the effects of mitogen-activated protein kinases (MAPKs) inhibitors on glutathione (GSH) metabolism, and to explore the pathway related to GSH metabolism. BRL rat hepatocytes were treated by c-Jun NH2-terminal kinase (JNK),p38, and extracellular signal-regulated kinase 1/2 (ERK1/2) inhibitors:SP600125, SB203580 and PD98659, respectively, for 24 h. MTT method was used to measure hepatocytes viability. The content of GSH was determined by high performance liquid chromatography. The protein expressions of JNK and phosphorylated JNK (p-JNK) was tested by Luminex method. Activities of GSH metabolic enzymes were detected by commercial kits. Hepatocytes vitality was inhibited when the concentrations of SP600125, SB203580 and PD98659 were higher than 10 μmol/L, 20 μmol/L, and 40 μmol/L, respectively; SP600125 decreased the content of GSH in hepatocytes, while SB203580 and PD98659 had no effect. SP600125 reduced p-JNK protein expression, and enhanced GSH-Px activity significantly. JNK MAPK pathway takes part in the GSH metabolism in hepatocytes.

Effect of mitogen-activated protein kinase signaling pathway on apoptosis induced by chloroacetic acid in human normal bronchial epithelial 16HBE cells

Objective: The aim of this study was to investigate the effect of mitogen-activated protein kinase (MAPK) signaling pathway on apoptosis induced by chloroacetic acid in human normal bronchial epithelial 16HBE cells. Methods: 16HBE cells were exposed to 0.5, 1.0, 1.5, 2.0, 2.5, 3.0 and 3.5 mmol/L chloroacetic acid for 24 h in vitro. The cytotoxicity induced by chloroacetic acid was assessed by CCK-8 and LDH assays. Cell apoptosis was detected by Annexin V-FITC and PI staining. The protein expression levels of phosphorylation of p38, ERK1/2 and JNK were determined by western blotting. 16HBE cells were pretreated with MAPK signaling pathway specific inhibitors including SB203580, U0126 and SP600125 for 1 h, and these cells were subsequently treated with 2.5 mmol/L chloroacetic acid for 24 h. The expressions of p-p38, p-ERK1/2 and p-JNK as well as the changes of cell viability and apoptosis were measured after pretreated with inhibitors for 1 h. Results: The cell viability by CCK-8 and LDH methods gradually reduced in a dose-dependent manner when chloroacetic acid concentrations elevated (P<0.05) , and their correlation coefficients were -0.902 and -0.825, respectively. The detection efficiency of CCK-8 assay significantly increased compared with LDH assay (P<0.05) . The cell apoptosis rates, which were (17.2±4.0) %, (24.6± 4.2) %, (39.3 ± 5.7) % in 1.5, 2.0, 2.5 mmol/L chloroacetic acid-treated groups, were higher than that of the control group[ (5.6 ± 3.0) %] (P<0.05) . There was a time-or dose-dependent change in the protein expressions of p-p38, p-ERK1/2 and p-JNK. Compared with the control, the levels of p-p38 had 2.1 and 2.6-fold increases in 16 and 24 h treated groups (P<0.01) , while the levels of p-ERK1/2 distinctly decreased by 37% and 52% (P<0.01) . In comparison with the control group, the expressions of p-p38 had 1.9 and 2.6-fold increases in 1.5 and 2.5 mmol/L treatment groups (P<0.01) , whereas the expressions of p-ERK1/2 significantly decreased by 40% and 50% (P<0.01) . No significant change was observed in p-JNK protein expression between the chloroacetic acid-treated and control groups. In comparison with the vehicle control and the exposed group, p-p38, p-ERK1/2, p-JNK protein expressions significantly declined in the inhibitor controls and inhibitor groups. Compared with the controls, the cell survival rates had significant reductions of 28%, 18%, 36% and 26% respectively in chloroacetic acid treated group, SB203580 group, U0126 group and SP600125 group, and the apoptosis rates in the abovementioned groups were 7, 4, 8 and 7 times. Compared with chloroacetic acid-treated group, the cell viability increased by 14% in SB203580 group and decreased by 11% in U0126 group, and the cell apoptosis rates decreased by 36% in SB203580 group and increased by 18% in U0126 group (P<0.05) . But no significant changes were observed in cell viability and apoptosis between SP600125 and chloroacetic acid-treated group. Conclusion: Chloroacetic acid might activate p38 MAPK signaling pathway and inhibit ERK1/2 MAPK signaling pathway. The signaling pathways of p38 and ERK1/2 MAPK are involved in 16HBE cell apoptosis induced by chloroacetic acid, but JNK is not involved in chloroacetic acid-induced 16HBE cell apoptosis.

Erk1/2 activation and modulation of STAT3 signaling in oral cancer.

Constitutive activation of the signal transducer and activator of transcription 3(STAT3) signaling pathway possesses confirmed oncogenic potential in oral squamous cell carcinoma(OSCC). Crosstalk with other molecular pathways contributes to STAT3 regulation in cancer. The effects of mitogen-activated protein kinases(MAPKs) and particularly extracellular signal-regulated kinase 1/2(Erk1/2) on STAT3 signaling in OSCC have not been thoroughly investigated. The present study examined the effects of Erk1/2 modulation on STAT3 signaling and cell growth in OSCC cells. Constitutive expression levels of phosphorylated (tyrosine and serine) and total STAT3, Erk1/2 and cyclinD1 were assessed in OSCC cell lines. Erk1/2 modulation was achieved by pharmacological agents; siRNA silencing against Erk1/2 was also performed. Cell proliferation and viability were assessed. Erk1/2 inhibition with either U0126 treatment or specific siRNA silencing resulted in decreases in p-serSTAT3 and cyclinD1 levels and increases in p-tyr STAT3 in OSCC cells. Moreover, Erk1/2 inhibition resulted in a dose-dependent reduction in OSCC cell growth and viability. Erk1/2 induction had the opposite effects. Taken together, these results are supportive of an active crosstalk between the oncogenic Erk1/2 and STAT3 pathways in OSCC, the significance of which requires further investigation.

Ethanol-induced apoptosis in human liver adenocarcinoma cells (SK-Hep1): Fas- and mitochondria-mediated pathways and interaction with MAPK signaling system

For studying molecular mechanisms regulating the fate of ethanol-treated hepatocytes, involvement of Fas in ethanol-induced apoptosis was examined in human liver adenocarcinoma (SK-Hep1) cells in which the function of Fas-associated death domain (FADD) protein was knocked down by transfection. In FADD-knocked down cells, while ethanol-induced increase in generation of reactive oxygen species (ROS) was unaffected, apoptosis was significantly suppressed, demonstrating the involvement of Fas in ethanol-induced hepatocyte apoptosis more directly than in the past reports. On the other hand, effects of mitogen-activated protein kinase (MAPK), which is well known to determine the fate of various cells, on ethanol-induced apoptosis have not been examined in SK-Hep1 cells. Of three major MAPKs, only p38 MAPK and JNK were found activated by 200mM ethanol treatment. When cells were incubated with inhibitors of p38 MAPK and JNK, ethanol-induced apoptosis was decreased while ROS generation was unaffected, and examination of pro-apoptotic Bax and anti-apoptotic Bcl-2 levels showed decrease of the former and increase of the latter. We concluded that oxidative stress inflicted by ROS triggered Fas-mediated and mitochondria-mediated apoptotic pathways in ethanol-treated SK-Hep1 cells, and that p38 MAPK and JNK were promoting mitochondrial pathway, suggesting interaction between apoptosis and MAPK signaling systems.

Effects of mitogen-activated protein kinases on neurotoxicity of local anesthetics

Background The potential toxicity is a well-known side effect of local anesthetics (LA) which is widely used in nerve block and analgesia.The mechanisms of local anesthetic toxicity were further investigated.Objective This review summarized the recent studies examining the mitogen-activated protein kinases (MAPKs) signaling pathway and their regulation,and discussed the effects of MAPKs on neurotoxicity of LA.Content Accumulating evidences indicate that neurotoxicity of LA is related to apoptosis.MAPKs,a serine-threonine protein kinase family,play a crucial role in apoptosis of cells.So that the effects of MAPKs signaling pathway on neurotoxicity of LA is becoming a hot issue to be researched.And the impact of extracellular signalregulated kinase (ERK),c-Jun N-terminal kinase (JNK),p38MAPK are more concerned.Trend The MAPKs signaling pathway and their regulation play an important part in the underlying mechanisms of local anesthetic neurotoxicity which could provide evidences and theory for clinical use of LA. Key words: Local anesthetics; Neurotoxicity; Mitogen-activated protein kinases

The effects of mitogen-activated protein kinases on the genes expression of the pulmonary surfactants from alveolar epithelial cells during ventilation induced lung injury in rats

Objective To investigate the effects of mitogen-activated protein kinases (MAPK) on the genes expression of the pulmonary surfactants from alveolar epithelial cells during ventilation induced lung injury in rats.Methods Anesthetized male SD rats were randomized into six groups:① Control group(C) received no mechanical ventilation.② Low-volume ventilation(LV) group received mechanical ventilation with Vt=6 ml/kg,RR=40 bmp,I∶E=1∶2,FiO2=21％,positive end-expiratory pressure =0.③ Hyperventilation(HV) group received mechanical ventilation with the same respiratory parameters as the LV group and the Vt was 40 ml/kg.The inhibitors of MAPK including the JNK's:SP600125,the ERK's:PD98059 and the p38's:SB203580 were pretreated 30 min before ventilation just like in the HV group.④ SP600125 group (SP600125 + HV group).⑤ PD98059 group (PD98059 + HV group).⑥ SB203580 group (SB203580 + HV group).After 4 h ventilation,the animals in each group were killed and the lungs were removed for detecting the expression of SP-A,B,C and RTI40 mRNA by RT-PCR.Results ① Compared with group C,the expression of RTI40 mRNA of lung tissue increased significantly in the LV group and the HV group (P＜0.05),but the expression of SP-A,SP-C mRNA decreased in the HV group,SP600125 group and SB203580 group (P＜0.05),while there was no significant difference in the expression of SP-A、C mRNA in LV groups and PD98059 group (P＜0.05).② Compared with the HV group,the SP-A,C gene expressions of lung tissue in SP600125 group,PD98059 group and SB203580 group increased significantly (P＜0.05),but RTI40 gene expressions decreased(P＜0.05).③ The SP-A,C gene expressions of lung tissue in PD98059 group increased significantly than those of SP600125 group and SB203580 group.④ The SP-B gene expression had no difference in all groups.Conclusions The results of our study show that mechanical ventilation inhibited the expressions of the SP-A and SP-C,but upregulated RTI40 gene expressions.The inhibitors of MAPK attenuated the changes of SP-A,C and RTI40 mRNA during the process of high volume ventilation,which indicates that MAPK,at least in part,may play a role in the functions of Ⅰ and Ⅱ alveolar epithelial cells in the lung injury induced by mechanical ventilation. Key words: Ventilation induced lung injury; Pulmonary surfactant; Genes expression; Mitogen-activated protein kinases

Matrix metalloproteinase-13 expression in IL-1β-treated chondrocytes by activation of the p38 MAPK/c-Fos/AP-1 and JAK/STAT pathways

Matrix metalloproteinase-13 (MMP-13, mammalian collagenase) degrades the cartilage matrix in pathological conditions such as osteoarthritis. Here, to establish the signaling pathway to MMP-13 induction, effects of mitogen-activated protein kinase (MAPK) pathway and the possibility of some other signaling pathways involved are investigated in interleukin-1β (IL-1β)-treated human chondrosarcoma cell line, SW1353 cells. IL-1β (10 ng/mL) treatment induced MMP-13 in SW1353 cells, with concomitant activation of nuclear factor-κB, activator protein-1 (AP-1) and MAPKs, including extracellular signal-regulated protein kinase, p38 MAPK and c-Jun N-terminal kinase. Among these MAPKs, only p38 MAPK inhibitor (SB203580) blocked MMP-13 induction and AP-1 activation in IL-1β-treated SW1353 cells. SB203580 also inhibited c-Fos translocation to the nucleus (but not c-Jun). Importantly, IL-1β treatment induced Janus kinase 2 (JAK2) and signal transducer and activator of transcription 1/2 (STAT1/2) activation. The JAK2 inhibitor (AG490) blocked STAT1/2 activation as well as MMP-13 induction in IL-1β-treated SW1353 cells. STAT1/2 siRNA transfection also reduced MMP-13 expression levels. Thus, from the present study, it is concluded that p38 MAPK/c-Fos/AP-1 and JAK2/STAT1/2 are involved in MMP-13 induction of IL-1β-treated human chondrocytes, SW1353 cells. Blocking these signaling pathways may have chondroprotective effects in cartilage degeneration.

The Effects of Mitogen-Activated Protein Kinase Inhibitors or Small Interfering RNAs on Gallic Acid-Induced HeLa Cell Death in Relation to Reactive Oxygen Species and Glutathione

Gallic acid (GA) is widely distributed in various plants and foods and has various biological properties including anticancer effects. In this study, we investigated the effects of mitogen-activated protein kinase (MAPK) [MAP 20 kinase or ERK kinase (MEK), c-Jun N-terminal kinase (JNK), or p38)] inhibitors or small interfering RNAs (siRNAs) on GA-induced HeLa cell death in relation to reactive oxygen species (ROS) and glutathione (GSH) levels. GA dose dependently inhibited the growth of HeLa cells via apoptosis and/or necrosis at 24 h, which was accompanied by the loss of mitochondrial membrane potential (MMP; ΔΨ(m)). Treatment with 70 μM GA increased the ROS level including O(2)(•-) and significantly induced GSH depletion in HeLa cells. GA decreased the activity of extracellular signal-regulated kinase (ERK) at 24 h, whereas it increased that of JNK at the same time. While the MEK inhibitor or ERK siRNA did not affect cell growth and death in 70 μM GA-treated HeLa cells at 24 h, JNK and p38 inhibitors enhanced cell growth inhibition and death in these cells. Additionally, p38 siRNA administration augmented growth inhibition, death, and MMP (ΔΨ(m)) loss in 70 μM GA-treated HeLa cells. In relation to ROS and GSH levels, JNK and p38 inhibitors increased ROS levels, and GSH-depleted cell numbers in GA-treated HeLa cells. Moreover, p38 siRNA increased O(2)(•-) levels and GSH depletion in GA-treated HeLa cells. Each MAPK inhibitor and siRNA differentially affected ROS and GSH levels in HeLa control cells. Conclusively, JNK and p38 inhibitors and p38 siRNA enhanced growth inhibition and cell death in GA-treated HeLa cells, which were to some extent related to GSH depletion and ROS levels, especially O(2)(•-).

The extracellular signal-regulated kinase was promoted by pyrroloquinoline quinine in cultured Schwann cells

To investigate the effect of mitogen-activated protein kinase (MEK) kinase cascade, extracellular signal-regulated kinase (ERK1/2) signal pathway on Schwann cells proliferation promoted by Pyrroloquinoline Quinine (PQQ) and its molecular mechanisms. Schwann cells were cultured and purified in vitro. The purity was identified by S-100. Different time and concentration of PQQ was added into culture medium. The expression of ERK1/2 and phosphorylated-ERK1/2 was detected by western blot. The expression of p-ERK1/2 after blocking of MEK signal pathway by specific inhibitor PD98059 was detected by western blot. Morphological change was observed in PQQ treated Schwann cells. 1-500 nmol/L PQQ could up-regulate the expression of p-ERK1/2, and 1000 nmol/L had no effects, while 10 000 nmol/L exhibited inhibitory effect (P < 0.05). p-ERK1/2 increased to peak 1 h after PQQ added, and this up-regulation of p-ERK1/2 was inhibited by PD98059 (P < 0.05). PQQ could affect morphology of Schwann cells and activation of ERK1/2. MEK inhibitor PD98059 could, block this activation. It suggests that MEK/ERK signal pathway should be involved in Schwann cells proliferation promoted by PQQ.

The MEK inhibitor PD98059 attenuates growth inhibition and death in gallic acid-treated Calu-6 lung cancer cells by preventing glutathione depletion

Gallic acid (GA) is widely distributed in various plants and foods and has various biological effects. In this study, we investigated the effects of mitogen-activated protein kinase (MEK, JNK or p38) inhibitors on GA-induced Calu-6 lung cancer cell death in relation to reactive oxygen species (ROS) and glutathione (GSH) levels. GA inhibited the growth of Calu-6 cells and induced apoptosis and/or necrosis accompanied by the loss of mitochondrial membrane potential (MMP; Δψm). ROS levels and the number of GSH-depleted cells were observed to be increased at 24 h. MEK inhibitor suppressed cell growth inhibition, death, MMP (Δψm) loss and GSH depletion induced by GA, but failed to suppress the increase in ROS levels. JNK inhibitor also somewhat suppressed cell growth inhibition, MMP (Δψm) loss and GSH depletion induced by GA, and limited the increase in ROS levels. By contrast, p38 inhibitor mildly enhanced GA-induced cell growth inhibition, MMP (Δψm) loss and the increase in ROS levels. In conclusion, MEK inhibitor suppressed GA-induced cell growth inhibition and death in Calu-6 cells. This was related to the prevention of GSH depletion.

The effects of mitogen-activated protein kinase on EGF-induced expression of integrin α_5 in human RPE cells

Background Human retinal pigment epithelial(RPE) cells play a critical role in the pathogenesis of proliferative vitreoretinopathy(PVR) and other related ocular diseases.Research demonstrated that epidermal growth factor(EGF) stimulates activation of RPE cells and therefore causes PVR,and integrin α_5 mediates the adhesion of cells and EGF.Objective This study aims to investigate the role of mitogen-activated protein kinase(MAPK) in regulation of EGF on integrin α_5 expression in human RPE cells.MethodsHuman RPE cells strain(CRL2302) was cultured in DMEM/F12 with 10% calf serum and passaged in 0.25% trypsin.Cultured cells were divided into DMEM/F12control group,20μmol/L PD98059 +10 ng/mL EGF＋DMEM/F12(PD98059) group and 10 ng/mL EGF＋DMEM/F12(EGF) group.The expression of integrin α_5 protein in CRL2302 cells was detected by RT-PCR and calculated as α_5 mRNA/β-actin mRNA,and the expression of integrin α_5 mRNA in CRL2302 cells was detected evaluated by flow cytometry.The MAPK phosphorylation level in each group of human RPE cells was tested by Western blot.ResultsThe expression value of integrin α_5 mRNA was 0.93±0.06 in the control group,1.06±0.07 in PD98059 group and 1.97±0.09 in EGF group,showing a significant difference among the three groups(F=458.896,P<0.01).The fluorescence intensity of integrin α5 protein in CRL2302 cells after 24 hours was 1.94±0.22,4.56±0.25,2.39± 0.14 in three groups respectively with a significant difference(F=21.05,P<0.05).After 30 minutes of culture,Western blot result showed that the strongest phosphorylation levels of ERK1/2 activation in EGF group and the weakest phosphorylation levels of ERK1/2 activation in the control group;While that in PD98059 group was significantly stronger than control group and weaker than EGF group(F=143.14,P<0.01).ConclusionEGF stimulates activation of ERK1/2 pathway in human RPE cells and the expressions of integrin α_5 mRNA and protein in human RPE cells in vitro. Key words: RPE cell; epidermal growth factor; integrin α_5; mitogen-activated protein kinase

The effects of MAPK inhibitors on antimycin A-treated calf pulmonary arterial endothelial cells in relation to cell death, reactive oxygen species and glutathione

Antimycin A (AMA) inhibits succinate oxidase and the mitochondrial electron transport chain between cytochrome b and c. Here, we report on the effects of mitogen-activated protein kinase (MAPK) inhibitors on AMA-treated calf pulmonary artery endothelial cells (CPAEC) in relation to cell death, reactive oxygen species (ROS) and glutathione (GSH). AMA inhibited the growth of CPAEC and also induced cell death, which was accompanied by the loss of mitochondrial membrane potential (MMP; ΔΨm). AMA increased ROS levels including O2·-, and depleted GSH cell numbers in CPAEC. All the MAPK (MEK, JNK, p38) inhibitors enhanced cell growth inhibition and death by AMA, and appeared to augment ROS but not O2·- levels in AMA-treated CPAEC. MEK and p38 inhibitors did not increase the number of GSH-depleted cells in AMA-treated CPAEC, but JNK inhibitor significantly did. Each MAPK inhibitor affected cell growth, death, ROS and GSH levels differently in comparison to control CPAEC. In conclusion, the MAPK inhibitors enhanced cell growth inhibition and death by AMA. Changes in ROS and GSH levels by AMA and/or MAPK inhibitors affected growth and death in CPAEC.