OBJECTIVETo investigate the effects of mitogen activated protein kinase (MAPK) signal transduction pathways on heat shock protein 70 (HSP70) gene expression in endothelial cells exposed to benzo(a)pryene (BaP).

METHODSPorcine aortic endothelial cells were pre-treated or by PD98059 (10 micro mol/L) or SB203580 (20 micro mol/L) for 1 hour, then treated with different concentrations of BaP (0, 0.1, 0.5, 1.0, 5.0 and 10.0 micro mol/L) for 24 hours respectively;Expression levels of three phosphorylated MAPKs [extracellular signal regulated protein kinase (ERK), c-Jun amino-terminal kinase (JNK), and p38] and HSP70 were determined by Western-blot.

RESULTSThe three phosphorylated MAPKs expressional levels especially p-ERK1 had different extents of changes with dose-response relationship under BaP exposure. BaP inhibited the expression of HSP70, which significantly decreased in medium and high dose group (>or= 1.0 micro mol/L) but did not decrease in control group (P < 0.05). Although the inhibitor of ERK (PD98059) could partly weaken the inhibited effects of BaP on HSP70 expression, HSP70 expression levels of endothelial cells pre-treated with PD98059 were still significantly lower than that of control cells (P < 0.05).

CONCLUSIONERK1 pathway might play some roles in HSP70 gene expression in endothelial cells exposed to BaP, and other unknown signal pathways might also have some effects on this process.

Animals ; Benzo(a)pyrene ; toxicity ; Blotting, Western ; Dose-Response Relationship, Drug ; Endothelial Cells ; drug effects ; metabolism ; Enzyme Inhibitors ; pharmacology ; Flavonoids ; pharmacology ; HSP70 Heat-Shock Proteins ; analysis ; Imidazoles ; pharmacology ; JNK Mitogen-Activated Protein Kinases ; MAP Kinase Kinase 4 ; Mitogen-Activated Protein Kinase Kinases ; analysis ; Mitogen-Activated Protein Kinases ; analysis ; antagonists & inhibitors ; Pyridines ; pharmacology ; Signal Transduction ; physiology ; Swine ; p38 Mitogen-Activated Protein Kinases

OBJECTIVETo examine the effect of SH2A gene in cell signal transduction and its subcellular localization.

METHODSRT-PCR method was used to amplify the coding sequence of SH2A gene. Eukaryotic recombined expression vector pcDNA 3.1-SH2A was constructed, and then Bel7402 cell and COS7 cell transfected by liposome. Multiple kinase assay was performed to examine the activity of protein kinase (PKC), mitogen activated protein kinase (MAPK), tyrosine protein kinase (TPK) in the transfected cells. Meantime, pEGFP-SH2A vector was also constructed and the cells transfected with it were examined by fluorescent microscopy.

RESULTSRecombined expression vector pcDNA3.2-SH2A and pEGFP-SH2A contained the coding sequence of SH2A cDNA. In both cell lines expressing SH2A gene, the cytoplasm PKC activity decreased by 40% or so, but no apparent alteration was found in MAPK and TPK activity. SH2A gene was found localized in the cytoplasm of transfected cells under fluorescent microscope.

CONCLUSIONSH2A gene may act as an inhibiting factor in PKC signal transduction, and it is localized in cytoplasm.

Animals ; COS Cells ; Cytoplasm ; chemistry ; Humans ; Membrane Proteins ; analysis ; genetics ; physiology ; Mitogen-Activated Protein Kinases ; metabolism ; Protein Kinase C ; physiology ; Signal Transduction ; physiology ; Transfection

OBJECTIVESTo explorer the change of several signal pathway and their signal after liver transplantation.

METHODSClassified 34 punctured donor liver samples and 10 normal liver samples as A (no rejection) groups, B (mild/moderate acute rejection) groups, C (serious acute rejection) groups, D (chronic rejection/fibrosis) groups and E (control) groups, MAPK, Ras and p53 were performed immunohistochemistry analysis and image analysis. MAPK and Ras were performed in situ hybridizition. Then image analysis was performed.

RESULTSThe protein expression of MAPK, Ras, increase by turns of A, B and C groups (1.42+/-0.28, 3.88+/-0.87, 6.68+/-0.57 in MAPK; 1.27+/-0.12, 2.80+/-0.30, 3.93+/-0.20 in Ras; corresponding), and decrease by turns of D and E groups (1.49+/-0.37, 0.88+/-0.20 in MAPK; 1.47+/-0.21, 1.01+/-0.12 in Ras; corresponding, F=178.39 in MAPK and 320.59 in Ras, groups B, C vs groups A, D, E, P<0.001 in MAPK and Ras), The protein expression of p53 is higher in treated groups (The results of groups A to E are 2.09+/-0.13, 2.39+/-0.11, 2.03+/-0.19, 2.26+/-0.18 and 0.35+/-0.08, corresponding, F=360.08, groups E vs groups A, B, C, D, P<0.001). Expression of MAPK, Ras mRNA is as same as that of protein.

CONCLUSIONThe MAPKs pathway has role in rejection response after liver transplantation. And it seemed that the MAPKs and p53 are one regulation mechanism for protecting the hepatocyte from damage after liver transplantation.

Humans ; Immunohistochemistry ; In Situ Hybridization ; Liver Transplantation ; MAP Kinase Signaling System ; Mitogen-Activated Protein Kinases ; analysis ; Signal Transduction ; physiology ; Tumor Suppressor Protein p53 ; analysis ; ras Proteins ; analysis

Epidermal keratinocyte differentiation is a tightly regulated stepwise process that requires protein kinase C (PKC) activation. Studies on cultured mouse keraitnocytes induced to differentiate with Ca2+ have indirectly implicated the involvement of PKC alpha isoform. When PKC alpha was overexpressed in undifferentiated keratinocytes using adenoviral system, expressions of differentiation markers such as loricrin, filaggrin, keratin 1 (MK1) and keratin 10 (MK10) were increased, and ERK1/2 phosphorylation was concurrently induced without change of other MAPK such as p38 MAPK and JNK1/2. Similarly, transfection of PKC alphakinase active mutant (PKC alpha- CAT) in the undifferentiated keratinocyte, but not PKC beta-CAT, also increased differentiation marker expressions. On the other hand, PKC alphadominant negative mutant (PKC beta-KR) reduced Ca2+ -mediated differentiation marker expressions, while PKC beta-KR did not, suggesting that PKC alphais responsible for keratinocyte differentiation. When downstream pathway of PKC alphain Ca2+ - mediated differentiation was examined, ERK1/2, p38 MAPK and JNK1/2 phosphorylations were increased by Ca2+ shift. Treatment of keratinocytes with PD98059, MEK inhibitor, and SB20358, p38 MAPK inhibitor, before Ca2+ shift induced morphological changes and reduced expressions of differentiation markers, but treatment with SP60012, JNK1/2 inhibitor, did not change at all. Dominant negative mutants of ERK1/2 and p38 MAPK also inhibited the expressions of differentiation marker expressions in Ca2+ shifted cells. The above results indicate that both ERK1/2 and p38 MAPK may be involved in Ca2+- mediated differentiation, and that only ERK1/2 pathway is specific for PKCa-mediated differentiation in mouse keratinocytes.
Animals ; Calcium/pharmacology/physiology ; Cell Differentiation/physiology ; Intermediate Filament Proteins/analysis/metabolism ; Keratinocytes/cytology/*enzymology ; Membrane Proteins/analysis/metabolism ; Mice ; Mitogen-Activated Protein Kinase 1/*metabolism ; Mitogen-Activated Protein Kinase 3/*metabolism ; Phosphorylation ; Protein Kinase C/genetics/*physiology ; Research Support, Non-U.S. Gov't ; p38 Mitogen-Activated Protein Kinases/metabolism

Peroxisome proliferator-activated receptor gamma (PPAR-gamma) is a ligand-activated transcription factor and plays an important role in growth, differentiation, and inflammation in different tissues. In this study, we investigated the effects of 15d-PGJ2, a high-affinity ligand of PPAR-gamma, on dedifferentiation and on inflammatory responses such as COX-2 expression and PGE2 production in rabbit articular chondrocytes with a focus on ERK-1/-2, p38 kinase, and PPAR-gamma activation. We report here that 15d-PGJ2 induced dedifferentiation and/or COX-2 expression and subsequent PGE2 production. 15d-PGJ2 treatment stimulated activation of ERK-1/-2, p38 kinase, and PPAR-gamma. Inhibition of ERK-1/-2 with PD98059 recovered 15d-PGJ2-induced dedifferentiation and enhanced PPAR-gamma activation, whereas inhibition of p38 kinase with SB203580 potentiated dedifferentiation and partially blocked PPAR-gamma activation. Inhibition of ERK-1/-2 and p38 kinase abolished 15d-PGJ2-induced COX-2 expression and subsequent PGE2 production. Our findings collectively suggest that ERK-1/-2 and p38 kinase oppositely regulate 15d-PGJ2-induced dedifferentiation through a PPAR-gamma-dependent mechanism, whereas COX-2 expression and PGE2 production is regulated by ERK-1/-2 through a PPAR-gamma-independent mechanism but not p38 kinase in articular chondrocytes. Additionally, these data suggest that targeted modulation of the PPAR-gamma and mitogen-activated protein kinase pathway may offer a novel approach for therapeutic inhibition of joint tissue degradation.
Animals ; Cartilage, Articular/*cytology ; Cell Differentiation/drug effects ; Chondrocytes/cytology/*drug effects/metabolism ; Cyclooxygenase 2/*analysis ; Dinoprostone/biosynthesis ; Mitogen-Activated Protein Kinase 1/*physiology ; Mitogen-Activated Protein Kinase 3/*physiology ; PPAR gamma/*physiology ; Prostaglandin D2/*analogs & derivatives/pharmacology ; Rabbits ; p38 Mitogen-Activated Protein Kinases/*physiology

In order to study the role of telomerase and extracellular regulated protein kinase (ERK) in drug-resistance of leukemia and ovarian cancer cells, telomeric repeat amplification protocol (TRAP) assay and bioluminescence analysis were used for qualitative analysis or quantitative detection of telomerase activity respectively, and Western blot was used to detect the expression level of phosphorylatedly activated ERK(1) and ERK(2) protein in the parental and drug resistant cells of leukemia and ovarian cancer. In addition, chemotherapy sensitivity to HRT or DDP was evaluated by MTT assay. The difference of cell cycle distribution between parental cell and drug-resistant cell was analyzed by flow cytometry. The results showed that the drug resistant cells were of higher percentage in G(0)/G(1) phase compared with the parental cell lines. Telomerase activity and phosphorylatedly activated ERK(1) and ERK(2) protein expression level were higher in drug-resistant cells than in parental cell. It is suggested that the increasing number of the drug resistant cells in G(0)/G(1) phase may be considered as a sign of drug resistance. The up-regulation of telomerase activity and phosphorylatedly activated ERK(1) and ERK(2) protein expression level may play an important role in drug resistance of leukemia and ovarian cancer cell lines.
Cell Cycle ; Drug Resistance, Neoplasm ; Female ; HL-60 Cells ; Humans ; Leukemia ; drug therapy ; metabolism ; pathology ; Mitogen-Activated Protein Kinase 1 ; analysis ; physiology ; Mitogen-Activated Protein Kinase 3 ; Mitogen-Activated Protein Kinases ; analysis ; physiology ; Ovarian Neoplasms ; drug therapy ; metabolism ; pathology ; Phosphorylation ; Telomerase ; metabolism

BACKGROUNDPulmonary artery smooth muscle cell (PASMC) proliferation plays an important role in pulmonary vessel structural remodelling. At present, the mechanisms related to proliferation of PASMCs are not clear. Focal adhesion kinase (FAK) is a widely expressed nonreceptor protein tyrosine kinase. Recent research indicates that FAK is implicated in signalling pathways which regulate cytoskeletal organization, adhesion, migration, survival and proliferation of cells. Furthermore, there are no reports about the role of FAK in human pulmonary artery smooth muscle cells (HPASMCs). We investigated whether FAK takes part in the intracellular signalling pathway involved in HPASMCs proliferation and apoptosis, by using antisense oligodeoxynucleotides (ODNs) to selectively suppress the expression of FAK protein.

METHODSCultured HPASMCs stimulated by fibronectin (40 microg/ml) were passively transfected with ODNs, sense FAK, mismatch sense and antisense-FAK respectively. Expression of FAK, Jun NH2-terminal kinase (JNK), cyclin-dependent kinase 2 (CDK 2) and caspase-3 proteins were detected by immunoprecipitation and Western blots. Cell cycle and cell apoptosis were analysed by flow cytometry. In addition, cytoplasmic FAK expression was detected by immunocytochemical staining.

RESULTSWhen compared with mismatch sense group, the protein expressions of FAK, JNK and CDK 2 in HPASMCs decreased in antisense-FAK ODNs group and increased in sense-FAK ODNs group significantly. Caspase-3 expression upregulated in HPASMCs when treated with antisense ODNs and downregulated when treated with sense ODNs. When compared with mismatch sense ODNs group, the proportion of cells at G1 phase decreased significantly in sense ODNs group, while the proportion of cells at S phase increased significantly. In contrast, compared with mismatch sense ODNs group, the proportion of cells at G1 phase was increased significantly in antisense-FAK ODNs group. The level of cell apoptosis in antisense-FAK group was higher than in the mismatch sense group and the latter was higher than sense-FAK group. In addition, the sense-FAK ODNs group was strongly stained by immunocytochemistry, whereas the antisense-FAK ODNs group was weakly stained.

CONCLUSIONSThe results suggest that FAK relates to the proliferation of HPASMCs. Antisense-FAK ODNs inhibit HPASMCs proliferation and facilitate their apoptosis. It is possible that FAK via JNK, CDK 2 signalling pathways enhances HPASMCs proliferation and via caspase-3 inhibits HPASMCs apoptosis.

Apoptosis ; CDC2-CDC28 Kinases ; analysis ; Caspase 3 ; Caspases ; analysis ; Cell Cycle ; Cell Proliferation ; drug effects ; Cells, Cultured ; Cyclin-Dependent Kinase 2 ; Focal Adhesion Kinase 1 ; Focal Adhesion Protein-Tyrosine Kinases ; Humans ; Immunohistochemistry ; JNK Mitogen-Activated Protein Kinases ; analysis ; Muscle, Smooth, Vascular ; cytology ; Myocytes, Smooth Muscle ; physiology ; Oligodeoxyribonucleotides, Antisense ; pharmacology ; Protein-Tyrosine Kinases ; analysis ; physiology ; Pulmonary Artery ; cytology

OBJECTIVETo investigate protective effects of hHSS transfection against CCl4 or H2O2.

METHODScDNA coding for hHSS was constructed into eukaryotic vector of pcDNA3.1 and transfected into BEL-7402 hepatoma cells. The expression of hHSS was analyzed with Northern blot.

RESULTSThe growth of the hepatoma cells was remarkably enhanced 24 to 144h after hHSS gene transfection, which suggesting hHSS gene expression could stimulate cells activity. Meantime, incubation of both wild-type and vector-transfected as well as hHSS-transfected cells with CCl4 or H2O2 resulted in severe damage as marked by cell mortality and the rate of apoptosis. However, it appeared that the transfection of hHSS enabled the hepatoma cells to raise obvious resistance against CCl4 and H2O2 injury. Compared the vector cells to the vector-transfected cells, apoptosis ratio were (32.44+/-0.52)% and (25.60+/-0.66)% in which treated with CCl4, while (47.78+/-0.45)% and (37.40+/-0.69)% in which treated with H2O2, t value is 16.82 and 25.20, P<0.01. MAPK phosphorylation was also activated after HSS transfected.

CONCLUSIONThe function of hHSS gene expression could be related to proliferation of cell and protection against free radical damage.

Apoptosis ; drug effects ; Carbon Tetrachloride ; toxicity ; Cytoprotection ; Free Radicals ; Growth Substances ; genetics ; physiology ; Humans ; Hydrogen Peroxide ; toxicity ; Liver Neoplasms ; pathology ; Mitogen-Activated Protein Kinases ; metabolism ; Peptides ; genetics ; physiology ; Phosphorylation ; RNA, Messenger ; analysis ; Transfection

OBJECTIVETo explore the effects and mechanisms of multi-glycoside of Tripterygium wilfordii (GTW) on improving glomerular inflammatory lesion in rats with diabetic nephropathy (DN).

METHODDN model was induced by unilateral nephrectomy and intraperitoneal injection of STZ (35 mg x kg(-1)) twice. The rats were randomly divided into 3 groups, the sham-operated group (Sham group, n = 5), the vehicle-given group (Vehicle group, n = 5 ) and GTW-treated group (GTW group, n = 5). After the model was successfully established, the rats in GTW group were daily oral administrated with GTW suspension (50 mg x kg(-1) x d(-1)), meanwhile, the rats in Vehicle group were daily oral administrated with distilled water (2 mL) for 8 weeks. From the beginning of the administration, all rats were killed 8 weeks later. Blood and renal tissues were collected,and then UAlb, renal function, glomerular morphology characteristics and glomerular macrophages (ED1 + cells) infiltration, as well as the protein expressions of inflammatory cytokines including tumor necrosis factor(TNF)-α and interleukin(IL)-lβ, and the key molecules in p38MAPK signaling pathway including p38 mitogenactivated protein kinase (MAPK), phosphorylated p38 (p-p38MAPK) and transforming growth factor(TGF)-β1 were investigated respectively.

RESULTGTW not only ameliorated the general state of health and body weight,but also attenuated UAlb, glomerulosclerosis, the infiltration of glomerular ED1 + cells and the protein expressions of TNF-α, IL-1β, p-p38MAPK and TGF-β1 in the kidney in DN model rats.

CONCLUSIONBy means of DN model rats, we demonstrated that GTW has the protective effect on renal inflammatory damage in vivo via inhibiting inflammatory cells infiltration and inflammatory cytokines expression. Furthermore, GTW could improve renal inflammatory lesion through down-regulating the expressions of the key signaling molecules in p38MAPK pathway such as p-p38MAPK and TGF-β1 ,and inhibiting the activation of p38MAPK signaling in the kidney.

Animals ; Diabetic Nephropathies ; drug therapy ; Disease Models, Animal ; Glomerulonephritis ; drug therapy ; Glycosides ; pharmacology ; MAP Kinase Signaling System ; drug effects ; Male ; Rats ; Rats, Sprague-Dawley ; Transforming Growth Factor beta1 ; analysis ; Tripterygium ; chemistry ; p38 Mitogen-Activated Protein Kinases ; physiology

OBJECTIVESome research has shown that p38 mitogen-activated protein kinase (p38MAPK) plays important roles in lung injuries induced by various factors. Its expression and role in hyperoxia-induced lung injury remains unknown. This study investigated the expression and role of p38MAPK in hyperoxia-induced lung injury juvenile rat model.

METHODSHyperoxia-induced lung injury rat model was prepared by 90% O(2) exposure. The location and expression of p38MAPK in lung tissues were detected by immunohistochemistry and Western blot respectively. Apoptosis index of lung was evaluated by TUNEL technique. The effect of SB203580, a p38MAPK inhibitor, on the apoptosis index of lung was observed.

RESULTSThe expression of phosphor-p38MAPK increased obviously after hyperoxia. Positive phosphor-p38MAPK cells were mainly distributed in the alveolar, airway epithelial cells, pulmonary vascular endothelium cells and infiltrative inflammatory cells. The apoptosis index of lung also significantly elevated. SB203580 inhibited the activation of p38MAPK, and reduced the apoptosis index of lung.

CONCLUSIONSThe phosphor-p38MAPK increased and was expressed in many kinds of lung cells in lung injury rat model. It may play a role in the induction of apoptosis in hyperoxia-induced lung injury.

Animals ; Apoptosis ; Disease Models, Animal ; Female ; Hyperoxia ; complications ; Imidazoles ; therapeutic use ; Immunoblotting ; Lung Injury ; drug therapy ; enzymology ; etiology ; MAP Kinase Signaling System ; Male ; Phosphorylation ; Pyridines ; therapeutic use ; Rats ; Rats, Wistar ; p38 Mitogen-Activated Protein Kinases ; analysis ; physiology