OBJECTIVETo explore the functional effects of MAPK pathway in the pathogenesis of human osteosarcoma.

METHODSGene microarray (Human Genome U133A, Affymetrix) was used to screen the differential expression of genes involved in MAPK pathway between osteosarcoma cell lines and 3 osteoblastic cell lines. KEGG metabolic pathway analysis was performed among significantly increased or decreased genes using the MATLAB software. Immunohistochemical technique was used to detect the expressions of ERK1/2, JNK and p38 proteins among 48 osteosarcoma and benign 24 osteoblastic tumor samples.

RESULTSUsing an entrance limit of > or = 2.0, 18 differentially expressed MAPK pathway-related genes were selected (10 up-regulated, 8 down-regulated) to mapped to the MAPK pathway of KEGG which are all important node genes. The positive rates of ERK1/2, JNK and p38 proteins were 83.3% (40/48), 72.9% (35/48) and 85.4% (41/48) in osteosarcomas,and 12.5% (3/24), 8.3% (2/24) and 16.7% (4/24) in the control group, respectively. The positive rates and expression intensities were statistically different between the 2 groups (P<0.01).

CONCLUSIONMAPK pathway plays an important role in the pathogenesis of osteosarcoma. ERK, JNK and p38 form an intercoordinating network and regulate the cell proliferation, differentiation, apoptosis, invasion and migration in osteosarcoma.

Adolescent ; Adult ; Aged ; Bone Neoplasms ; genetics ; metabolism ; pathology ; Cell Line, Tumor ; Child ; Female ; Gene Expression Profiling ; Humans ; JNK Mitogen-Activated Protein Kinases ; metabolism ; Male ; Middle Aged ; Mitogen-Activated Protein Kinase 1 ; metabolism ; Mitogen-Activated Protein Kinase 3 ; metabolism ; Mitogen-Activated Protein Kinases ; metabolism ; Oligonucleotide Array Sequence Analysis ; Osteoblastoma ; genetics ; metabolism ; pathology ; Osteosarcoma ; genetics ; metabolism ; pathology ; Signal Transduction ; Young Adult ; p38 Mitogen-Activated Protein Kinases ; metabolism

OBJECTIVETo investigate the effect of interleukin-6 (IL-6) on the human growth hormone (hGH) gene expression in a rat somatotropic pituitary cell line MtT/S.

METHODSThe plasmids containing various lengths of hGH gene 5'-promoter fragments were constructed. Stably transfected MtT/S cells were created by cotransfecting the above plasmids and pcDNA3. 1(+) with DMRIE-C transfection reagent After the administration of these cells with IL-6 and/or various inhibitors of signaling transduction pathways, the luciferase activities in MtT/S cells lysis were assayed to demonstrate the effects of IL-6 on hGH gene promoter activity and possibly involved mechanism.

RESULTSThe 10(3) U/mL IL-6 stimulated GH secretion and synthesis, and promoted the 5'-promoter activity of GH gene in stably transfected MtT/SGL cells with the action of 1.69 times above the control. Among inhibitors of signaling transduction pathways, mitogen-activated protein kinase kinase (MAPKK/MEK) inhibitor PD98059 (40 micromol/L) and p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 (5 micromol/L) completely blocked the stimulatory effect of IL-6. Western blot analysis further confirmed the activation of phosphorylated MEK and p38 MAPK in MtT/SGL cells. Neither over-expression of Pit-1 nor inhibition of Pit-1 expression affected IL-6 induction of hGH promoter activity. A series of deletion constructs of hGH promoter were created to identify the DNA sequence that mediated the effect of IL-6. The results showed that the stimulatory effect of IL-6 was abolished following deletion of the -196 to - 132 bp fragment.

CONCLUSIONSIL-6 promotes GH secretion and synthesis by rat MtT/S somatotroph cells. The stimulatory effect of IL-6 on hGH gene promoter appears to require the activation of MEK and p38 MAPK, and a fragment of promoter sequence that spans the - 196 to - 132 bp of the gene, but may be unlinked with Pit-1 protein.

Animals ; Cell Line ; Gene Expression Regulation ; Human Growth Hormone ; genetics ; metabolism ; Humans ; Interleukin-6 ; genetics ; metabolism ; JNK Mitogen-Activated Protein Kinases ; genetics ; metabolism ; MAP Kinase Signaling System ; physiology ; Mitogen-Activated Protein Kinase Kinases ; genetics ; metabolism ; Promoter Regions, Genetic ; Rats ; Somatotrophs ; cytology ; metabolism ; p38 Mitogen-Activated Protein Kinases ; genetics ; metabolism

OBJECTIVETo study the effect of Rac-MEKK-JNK (Rac-mitogen activated protein kinase kinase kinase-C-jun N-terminal protein kinase) signal pathway on heat shock-induced hsp90 beta gene expression and the impact of Hsp90 on the regulation of the pathway.

METHODSDN-Rac, DN-MEKK or DN-JNK were cotransfected with hsp90 beta CAT reporter plasmid beta 3.1 into Jurkat or LETPa-2 cells individually, the CAT mRNA expression was then determined quantitatively by competitive RT-PCR based system. Western blot was carried out to detect the expression level and phosphorylation of c-Jun in Jurkat and LETPa-2 cells that were transfected with DN-Rac, DN-MEKK or DN-JNK. By in vitro kinase activity assay and Western blot, the effect of geldnamycin (GA) on heat induced JNK activity were evaluated.

RESULTSIn Jurkat cell transfected with DN-Rac, DN-MEKK or DN-JNK, heat shock induced relative CAT mRNA expression level was decreased to (72.8 +/- 5)%, (60 +/- 13.2)% and (47.7 +/- 12.1)% of the control respectively; while in LETPa-2 cell hsp90 beta 3.1 reporter gene expression was accordingly suppressed to (16.17 +/- 5.1)%, (50.2 +/- 8.7)% and (47.5 +/- 10)% of control. C-Jun expression and phosphorylation were inhibited by the transfection of either one of DN-Rac, DN-MEKK or DN-JNK. With GA treatment, heat shock induced JNK activity was repressed, while the expression level of JNK or c-Jun was not obviously changed.

CONCLUSIONSRac-MEKK-JNK pathway promotes heat shock induced hsp90 beta gene expression and hsp90 may participate in the regulation of heat shock activated Rac-MEKK-JNK signal pathway in both Jurkat and LETPa-2 cells.

Benzoquinones ; Cell Line, Tumor ; Genes, Reporter ; HSP90 Heat-Shock Proteins ; biosynthesis ; genetics ; Hot Temperature ; Humans ; JNK Mitogen-Activated Protein Kinases ; Lactams, Macrocyclic ; Leukemia, T-Cell ; pathology ; Mitogen-Activated Protein Kinase Kinases ; physiology ; Mitogen-Activated Protein Kinases ; physiology ; Protein Kinase C ; physiology ; Quinones ; pharmacology ; Signal Transduction ; Transfection

OBJECTIVETo investigate the expression of Smad4 in non-small cell lung cancer (NSCLC), its correlation with MAPK (mitogen activated protein kinase) and their clinical significance in NSCLC.

METHODSWestern blotting and RT-PCR were employed to test 42 resected lung cancers and normal lung tissues for the expression of Smad4. Imunohistochemistry was used to detect Smad4 and subtribes of MAPK in 71 paraffin samples.

RESULTSThe level of protein and mRNA expression of Smad4 in lung cancer tissues were 0.2092 +/- 0.1308 and 0.3986 +/- 0. 1982, respectively, lower than those in normal tissues (0.7852 +/- 0.4386 and 1.1206 +/- 0.6772, P < 0.05). The expression of p38, ERK1 and Smad4 was associated with TNM staging (P = 0.000, 0.000 and 0.005, respectively) and JNK1 with tumor location (P = 0.028) and staging (P = 0.000). There was a correlation between p38 and Smad4 (P = 0.000). The expression of Smad4 (P = 0.0001), p38 (P = 0.0000) and JNK1 (P = 0.0208), tumor differentiation (P = 0.0059) and staging (P = 0.0000) were significantly correlated with prognosis of NSCLC by univariate analysis. Smad4 (P = 0.019), p38 (P = 0.044), tumor differentiation (P = 0.003), and staging (P = 0.020) were correlated with prognosis tested by multivariable analysis. Taking p38 and Smad4 together, we found that the negative expression of p38 and positive expression of Smad4 were associated with a better prognosis of NSCLC (P = 0.000).

CONCLUSIONSmad4 could be of importance for the initiation and development of NSCLC. There is a significant correlation between main proteins of TGF-beta/smad4 and those of ras-MAPK signal transduction pathways. The expression of Smad4 is inhibited by p38. Smad4, as well as p38, tumor differentiation and staging can be used as prognostic factors of NSCLC.

Adult ; Aged ; Blotting, Western ; Carcinoma, Non-Small-Cell Lung ; metabolism ; pathology ; Cell Differentiation ; Female ; Humans ; Lung Neoplasms ; genetics ; metabolism ; pathology ; Male ; Middle Aged ; Mitogen-Activated Protein Kinase 3 ; genetics ; metabolism ; Mitogen-Activated Protein Kinase 8 ; genetics ; metabolism ; Mitogen-Activated Protein Kinases ; genetics ; metabolism ; Neoplasm Staging ; Prognosis ; RNA, Messenger ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Signal Transduction ; Smad4 Protein ; genetics ; metabolism ; physiology ; p38 Mitogen-Activated Protein Kinases ; genetics ; metabolism

OBJECTIVETo investigate the effect of cytoskeleton reorganization inhibition with RNA interference on the activation of extracellular signal-regulated kinase (ERK1/2) in primary osteoblasts induced by fluid shear stress (FSS).

METHODSBALB/c mouse primary cultured osteoblasts were isolated by enzyme digestion technique. Osteoblasts were treated with LIM domain kinase 2 (LIM-2) specific siRNA or negative control siRNA, and then were loaded or unloaded by FSS of 1.2 Pa for 0, 5, 15, 30 and 60 min, respectively. The Western blotting was performed to detect the protein expression levels of P-ERK1/2 and ERK1/2, respectively. Two-way ANOVA and one-way ANOVA were used in data analysis.

RESULTSFSS loading for different time (0, 5, 15, 30, 60 min) treated with negative RNA inteference had significant effect on the levels of P-ERK/ERK ratio (0.047 ± 0.031, 0.253 ± 0.137, 0.390 ± 0.155, 0.613 ± 0.123, 0.680 ± 0.108, respectively, P < 0.01). Statistical analysis showed that there was significant interaction between FSS and cytoskeleton reorganization inhibition treated with RNA inteference on the levels of P-ERK/ERK ratio (P < 0.01). The levels of P-ERK/ERK ratio increased when osteoblasts were loaded for 5 - 15 min (0.623 ± 0.129 and 0.623 ± 0.064, respectively, P < 0.05) and returned to baseline at 30 min (0.333 ± 0.086), and then reached the peak at 60 min (0.667 ± 0.064, P < 0.01).

CONCLUSIONSFSS could activate ERK1/2 rapidly in primary cultured osteoblasts. Cytoskeleton reorganization inhibition treated with RNA interference speeded-up the activation of ERK1/2 by FSS, which could increase the sensitivity of ERK1/2 to FSS.

Animals ; Cells, Cultured ; Cytoskeleton ; metabolism ; physiology ; Lim Kinases ; genetics ; metabolism ; Mechanotransduction, Cellular ; Mice ; Mice, Inbred BALB C ; Mitogen-Activated Protein Kinase 1 ; metabolism ; Mitogen-Activated Protein Kinase 3 ; metabolism ; Osteoblasts ; cytology ; enzymology ; Phosphorylation ; RNA Interference ; RNA, Small Interfering ; Stress, Mechanical

Rubella virus (RV), a member of the family Togaviridae, can induce apoptosis of host cells in vitro. Protein kinases of the Ras-Raf-MEK-ERK pathway and PI3K-Akt pathway play essential roles in virus multiplication, cell survival and apoptosis. Proteins p53 and TAp63 that bind to specific DNA sequences stimulate Bax in a manner to produce functional pores that facilitate release of mitochondrial cytochrome c and downstream caspase activation. In this review, the molecular mechanisms of RV-induced cell apoptosis, including RV-infected cell lines, pathological changes in cell components and apoptosis signaling pathways are summarized.
Apoptosis ; Humans ; MAP Kinase Signaling System ; Mitogen-Activated Protein Kinases ; genetics ; metabolism ; Rubella ; genetics ; metabolism ; physiopathology ; virology ; Rubella virus ; genetics ; physiology

OBJECTIVETo investigate the effects of protein tyrosine phosphatase-SHP2 and dual-specificity MAPK phosphatase-MKP5 on the activation of MAPKs and cell invasion induced by P2Y purinergic receptor in human prostate cancer cell lines with different metastatic potentials.

METHODSThe wide type (-wt) SHP2, mutant type (-cs) SHP2 and wide type (-wt) MKP5 cDNA expression vectors were constructed and stably transfected into 1E8 cells (highly metastatic) and/or 2B4 cells (non-metastatic). The tyrosine phosphorylation of SHP2 was examined by immunoprecipitation. The activation of ERK1/2 and p38 induced by P2Y receptor agonist ATP was analyzed by Western blot with phospho-specific antibodies against the dually phosphorylated, active forms of ERK1/2 and p38. The in-vitro invasive ability through Matrigel was measured by boyden-chamber assay.

RESULTSATP induced significant SHP2 phosphorylation, which was stronger and lasted longer in 1E8 than in 2B4. SHP2-wt enhanced the ERK1/2 activation induced by ATP in 2B4 cells, while SHP2-cs delayed and decreased this effect in 1E8 cells. Both SHP2-wt and SHP2-cs had no obvious influence on p38 activation. ATP stimulated cell invasion of both 1E8 and 2B4, while transfection of SHP2-wt into 2B4 cells further increased the invasive-stimulating ability of ATP (18.7% increase compared with ATP treatment alone). Transfection of SHP2-cs into 1E8 cells, however, antagonized the invasive-stimulating ability of ATP (40.9% decrease compared with ATP treated group). Up-regulation of MKP5-wt inhibited phosphorylation of p38 by ATP and reduced cell invasion stimulated by ATP (22.4% and 28.7% decrease compared with ATP treated group of 1E8 and 2B4, respectively).

CONCLUSIONSBoth SHP2 and MKP5 play some roles in P2Y receptor-mediated activation of MEK/ERK, p38 signaling pathways and prostate cancer invasion. SHP2 positively regulates ERK activation and prostate cancer invasion, whereas MKP5 inhibits the invasion by suppressing p38 activation.

Adenosine Triphosphate ; pharmacology ; Cell Line, Tumor ; DNA, Complementary ; genetics ; Dual-Specificity Phosphatases ; Genetic Vectors ; Humans ; Intracellular Signaling Peptides and Proteins ; genetics ; metabolism ; Male ; Mitogen-Activated Protein Kinase 1 ; metabolism ; Mitogen-Activated Protein Kinase 3 ; metabolism ; Mitogen-Activated Protein Kinase Phosphatases ; Neoplasm Invasiveness ; Phosphorylation ; Prostatic Neoplasms ; metabolism ; pathology ; Protein Tyrosine Phosphatase, Non-Receptor Type 11 ; Protein Tyrosine Phosphatases ; genetics ; metabolism ; Receptors, Purinergic P2 ; physiology ; Signal Transduction ; Transfection ; p38 Mitogen-Activated Protein Kinases ; metabolism

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

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

OBJECTIVETo discuss the role of early growth response factor (Egr)-1 and it's upstream signaling pathway in the development of silicosis.

METHODSThe expression and localization of Egr-1 were analyzed by immunofluorescence and in-situ hybridization. The activity of Egr-1 was observed in treated cells by using a reporter plasmid and EMSA, the activity of ERK1/2 in RAW264.7 incubated with SiO(2) by using a kinase assay, and further by using a kinase inhibitor assay to investigate the role of upstream kinase in the signal pathway of the activation of Egr-1.

RESULTSThe obvious increase of expression and transcription of Egr-1 was observed shortly after being treated by silica and its activity increased abruptly. There was an increase of the activity of ERK1/2 in RAW264.7 cells treated, which reached a peak at 30 minutes. The expression and transcription of Egr-1 decreased maniferstly after using kinase inhibitors.

CONCLUSIONEgr-1 expression can be induced by silica dioxide in RAW264.7 cells, and the ERK1/2, p38 kinases may take part in this process which suggest the pathway of SiO(2), ERK1/2, p38 and Egr-1 may play an important role in the development of silicosis.

Animals ; Butadienes ; pharmacology ; Cells, Cultured ; Early Growth Response Protein 1 ; biosynthesis ; genetics ; physiology ; Enzyme Inhibitors ; pharmacology ; Gene Expression Regulation ; Macrophages ; metabolism ; Mice ; Mitogen-Activated Protein Kinase 1 ; metabolism ; Mitogen-Activated Protein Kinase 3 ; metabolism ; Nitriles ; pharmacology ; RNA, Messenger ; biosynthesis ; genetics ; Signal Transduction ; Silicon Dioxide ; pharmacology ; p38 Mitogen-Activated Protein Kinases ; metabolism