Unmethylated CpG oligodeoxynucleotides (CpG ODNs) activate immune cells to produce immune mediators. This study demonstrates that in murine macrophage RAW 264.7 cells, CpG ODN-mediated matrix metalloproteinase-9 (MMP-9) expression is regulated at transcriptional level and requires de novo protein synthesis. Inhibition of ERK and p38 MAPK, but not JNK, results in significant decrease of CpG ODN-induced MMP-9 expression. We found that endosomal maturation inhibitors, chloroquine and bafilomycin A, block CpG ODN-induced ERK and p38 MAPK activation and the subsequent MMP-9 expression. We also observed that CpG ODN induces NF-kappa B activation and NF-kappa B is a downstream target of p38 MAPK. Taken together, our data demonstrate that CpG ODN triggers MMP-9 expression via TLR-9 dependent ERK and p38 MAPK activation followed by NF-kappa B activation.
Animals ; Cell Line ; Enzyme Activation/drug effects ; Enzyme Induction/drug effects ; Matrix Metalloproteinase 9/*biosynthesis ; Mice ; Mitogen-Activated Protein Kinase 1/antagonists & inhibitors/metabolism ; Mitogen-Activated Protein Kinase 3/antagonists & inhibitors/metabolism ; Mitogen-Activated Protein Kinases/*metabolism ; NF-kappa B/*metabolism ; Oligodeoxyribonucleotides/*pharmacology ; Signal Transduction/drug effects ; Toll-Like Receptor 9/antagonists & inhibitors/*metabolism ; p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors/metabolism

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

We stimulated preadipocyte of mice with calcium acetate, p38 mitogen-activated protein kinase (p38 MAPK) inhibitor SB203580, the paralysors and excitomotors of calcium channel. Then we detected expression level of preadipocyte differentiation's marker genes and calcium signal related acceptor genes by real-time PCR, and determined intracellular free Ca2+ concentration ([Ca2+]i]) with Fura-2/AM, intracellular lipid accumulation by oil red O staining. Our aim was to investigate the potential mechanism between calcium signal and preadipocyte differentiation. The results indicated that the paralysors and excitomotors of calcium channel changed the expression level of lipoprotein lipase (LPL), peroxisome proliferators-activated receptor gamma (PPARgamma), fatty acid synthetase (FAS), and the lipid accumulation, markedly. Compared with exocellular Ca2+'s decrease, inhibited intracellular Ca2+'s liberation can promoted preadipocyte differentiation (P < 0.01), and compared with intracellular Ca2+'s increase, promoted exocellular Ca2+'s ingest inhibited preadipocyte differentiation (P < 0.01). SB203580 degraded [Ca2+]i, promoted differentiation marker genes' expression and lipid accumulation in preadipocyte (P < 0.01). But calcium signal didn't have effects to vitamin D receptor (VDR) and extracellular Ca2+-sensing receptor (CaSR)'s expression. It indicated that calcium signal may effect preadipocyte different and lipid accumulation by p38 MAPK pathway.
Adipocytes ; cytology ; Animals ; Calcium ; metabolism ; Calcium Signaling ; drug effects ; Cell Differentiation ; physiology ; Cells, Cultured ; Imidazoles ; pharmacology ; Lipids ; biosynthesis ; Mice ; Pyridines ; pharmacology ; p38 Mitogen-Activated Protein Kinases ; antagonists & inhibitors

OBJECTIVETo study the effect of p38 on the cycloheximide (CHX)-induced HL-60 cell death through mitochondria pathway.

METHODSInhibition of p38 pathway was by SB203580 (SB). Four groups were set up: control, SB only, CHX only and SB + CHX. Sub-diploid cell ratio was detected by PI staining flow cytometry at 6, 9, 12, 18, 24 h time points, and apoptotic cell ratio by Annexin V-FITC/PI double staining flow cytometry at 6 h and 18 h time points. High J-aggregate cells were evaluated by the J-aggregate contents, measurement of the J-aggregate (FL2) and J-monomer (FL1) by JC-1 flow cytometry, calculation of the delta psi m by FL2/FL1 and analysis of the delta psi m changes at 18 h time points.

RESULTSThe sub-diploid cell ratio in CHX group was significantly higher than that in control group at 6 h time point, and the ratio in SB + CHX group was significantly higher than that in CHX group at 9 h time point. At 18 h time point the apoptotic cell ratios in both CHX and SB + CHX groups were significantly higher than those in control group (P < 0.01). There was no significant difference of apoptotic cell ratio between CHX group and SB + CHX group (P > 0.05). At 18 h time point the necrotic cell ratios in both CHX and SB + CHX groups were significantly higher than that in control group (P < 0.01); and that in SB + CHX group was significantly higher than that in CHX group (P < 0.01). The high J-aggregate cell ratios in CHX and SB + CHX groups were significantly lower than that in control group (P < 0.05), and that was signficantly lower in SB + CHX group than in CHX group (P < 0.01). For the FL2/FL1 value (delta psi m) CHX group (0.17 +/- 0.01) and SB + CHX group (0.05 +/- 0.003) were significantly higher than control group (0.38 +/- 0.02) (P < 0.01), and SB + CHX group was significantly lower than CHX group (P < 0.01).

CONCLUSIONCHX can induce HL-60 cell apoptosis and the cell mitochondria depolarization, and the latter was intensified by inhibition of the p38 pathway. p38 pathway may related to the cell necrosis in the cycloheximide-induced HL-60 cell apoptosis model. s

Apoptosis ; drug effects ; Cycloheximide ; pharmacology ; HL-60 Cells ; Humans ; Membrane Potentials ; Mitochondria ; physiology ; p38 Mitogen-Activated Protein Kinases ; antagonists & inhibitors ; metabolism

NMDA-induced excitotoxicity cause severe neuronal damage including apoptosis and necrosis. The present study was aimed to evaluate the proportion of NMDA-induced apoptosis of rat cortical neurons and discover signal transduction mechanism. Caspase inhibitor and lactate dehydrogenase (LDH) assay were used to study the NMDA-induced apoptosis. To explore the involved signal pathways, the primary culture of rat cortical neurons were pretreated by the inhibitors of three MAPK pathways, extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK. With 2 h of NMDA treatment, cellular apoptosis was measured by caspase-3 activity, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL) and Annexin V staining. The results showed that: (1) Caspase-dependent apoptosis accounted for 22.49% in NMDA-induced neuronal death; (2) Pretreatment with p38 MAPK inhibitor SB203580 (10 μmol/L) significantly decreased NMDA-mediated caspase-3 activity by 30.43% (P < 0.05). However, ERK inhibitor PD98059 (20 μmol/L) or JNK inhibitor SP600125 (20 μmol/L) did not influence caspase-3 activity; (3) Pretreatment with SB203580 significantly reduced the number of NMDA-induced TUNEL-positive cells by 33.10% (P < 0.05). PD98059 (20 μmol/L) or SP600125 (20 μmol/L) did not show obvious effect; (4) Pretreatment with SB203580 (10 μmol/L) significantly reduced the number of NMDA-induced early apoptotic neurons by 55.56% (P < 0.05). Also, SP600125 (20 μmol/L) significantly decreased the amount of late apoptotic/dead cells by 67.59% (P < 0.05). There was no effect of PD98059 (20 μmol/L). These results indicate that: (1) NMDA induces neuronal apoptosis besides necrosis; (2) p38 MAPK, but not JNK and ERK, is involved in NMDA-induced neuronal apoptosis, and inhibition of the apoptotic signaling pathway contributes to neuroprotection; (3) JNK activation might contribute to NMDA-induced neuronal necrosis rather than apoptosis.
Animals ; Anthracenes ; pharmacology ; Apoptosis ; Caspase 3 ; metabolism ; Cells, Cultured ; Extracellular Signal-Regulated MAP Kinases ; antagonists & inhibitors ; Imidazoles ; pharmacology ; JNK Mitogen-Activated Protein Kinases ; antagonists & inhibitors ; MAP Kinase Signaling System ; N-Methylaspartate ; pharmacology ; Neurons ; cytology ; Primary Cell Culture ; Pyridines ; pharmacology ; Rats ; p38 Mitogen-Activated Protein Kinases ; antagonists & inhibitors

OBJECTIVETo investigate the potential molecular mechanism underlying down-regulation of albumin expression induced by lipopolysaccharide (LPS) in rat hepatocytes.

METHODSThe albumin mRNA expression and albumin protein levels in the supernatant were observed at 0, 2, 8, 12 and 24 hours after 1 micro g/ml LPS treatment. The albumin mRNA expression was determined by RT-PCR, and albumin protein levels were assayed with EIA in the supernatant of hepatocytes pretreated with specific inhibitors of extracellular signal-regulated kinase (PD98059) and p38 kinase (SB203580).

RESULTSThe albumin mRNA expression was reduced to 70% of baseline value, meanwhile, the albumin protein concentration in the supernatant was reduced by 50% compared with the controls after 24 hours of LPS treatment. Pretreatment with PD98059 and SB203580 did significantly inhibit the reduction of albumin by LPS at the given dose.

CONCLUSIONThe results indicate that LPS can induce the reduction of albumin synthesis by down-regulation of albumin mRNA expression at transcription level, and the process may be related to the signal transducation of extracellular signal-regulated kinase and p38 kinase.

Albumins ; genetics ; metabolism ; Animals ; Calcium-Calmodulin-Dependent Protein Kinases ; antagonists & inhibitors ; Dose-Response Relationship, Drug ; Down-Regulation ; Enzyme Inhibitors ; pharmacology ; Enzyme-Linked Immunosorbent Assay ; Flavonoids ; pharmacology ; Hepatocytes ; drug effects ; metabolism ; Imidazoles ; pharmacology ; Lipopolysaccharides ; toxicity ; Male ; Mitogen-Activated Protein Kinases ; antagonists & inhibitors ; Pyridines ; pharmacology ; RNA, Messenger ; genetics ; metabolism ; Rats ; Reverse Transcriptase Polymerase Chain Reaction ; Time Factors ; p38 Mitogen-Activated Protein Kinases

BACKGROUNDMucus hypersecretion in the respiratory tract and goblet cell metaplasia in the airway epithelium contribute to the morbidity and mortality associated with airway inflammatory diseases. This study aimed to examine the effect and mechanisms of simvastatin on airway mucus hypersecretion in rats treated with lipopolysaccharide (LPS).

METHODSMucus hypersecretion in rat airways was induced by intra-tracheal instillation of LPS. Rats treated with or without LPS were administered intra-peritoneally simvastatin (5 and 20 mg/kg) for 4 days. Expression of Muc5ac, RhoA and mitogen-activated protein kinases (MAPK) p38 in lung were detected by real-time polymerase chain reaction (PCR), immunohistochemistry or Western blotting. Tumor necrosis factor (TNF)-alpha and IL-8 in bronchoalveolar lavage fluid (BALF) were assayed by an enzyme-linked lectin assay and enzyme linked immunosorbent assay (ELISA).

RESULTSSimvastatin attenuated LPS-induced goblet cell hyperplasia in bronchial epithelium and Muc5ac hypersecretion at both the gene and protein levels in lung (P <0.05). Moreover, simvastatin inhibited neutrophil accumulation and the increased concentration of TNF-alpha and IL-8 in BALF follows LPS stimulation (P < 0.05). The higher dose of simvastatin was associated with a more significant reduction in Muc5ac mRNA expression, neutrophil accumulation and inflammatory cytokine release. Simultaneously, the increased expression of RhoA and p38 MAPK were observed in LPS-treated lung (P <0.05). Simvastatin inhibited the expression of RhoA and p38 phosphorylation in lung following LPS stimulation (P < 0.05). However, the increased expression of p38 protein in LPS-treated lung was not affected by simvastatin administration.

CONCLUSIONSSimvastatin attenuates airway mucus hypersecretion and pulmonary inflammatory damage induced by LPS. The inhibitory effect of simvastatin on airway mucus hypersecretion may be through, at least in part, the suppression of neutrophil accumulation and inflammatory cytokine release via inactivation of RhoA and p38 signaling pathway.

Animals ; Hydroxymethylglutaryl-CoA Reductase Inhibitors ; pharmacology ; Lipopolysaccharides ; toxicity ; Male ; Mucin 5AC ; secretion ; Rats ; Rats, Sprague-Dawley ; Respiratory Mucosa ; drug effects ; secretion ; Simvastatin ; pharmacology ; p38 Mitogen-Activated Protein Kinases ; antagonists & inhibitors ; rhoA GTP-Binding Protein ; antagonists & inhibitors

OBJECTIVETo investigate the effect(s) of interleukin-1beta (IL-1beta) on the activity of human growth hormone (hGH) gene promoter in rat pituitary GH3 cells and the molecular mechanism.

METHODSThe method of luciferase reporter gene was used. We firstly established stable GH3 cell line which contains hGH gene promoter -484-30 bp and luciferase reporter gene. After treating these cells with IL-1beta or IL-1beta plus various signaling transduction inhibitors, the concentration of GH in the medium and lysate of GH3 cells and luciferase activities in GH3 cells were measured to reflect the effect of IL-1beta on secretion and synthesis of GH and the promoter activity of the hGH gene and the molecular mechanism. Results IL-1beta (10-10(4)U/ml) increased secretion and synthesis of GH. IL-1beta at levels of 10(2)-10(4) U/ml promoted the luciferase expression in stable GH3 cells, and the maximal action was 1.61 times of the control (P < 0.001). Among the inhibitors of intracellular signaling transduction pathways, mitogen-activated protein kinases (MAPK) inhibitor PD98059 (40 micromol/L) and p38 MAPK inhibitor SB203580 (5 micromol/L) completely blocked the stimulatory effect of IL-1beta, and phosphoinositide 3-kinase (PI3-K) inhibitor LY294002 (10 micromol/L) partly blocked the induction of IL-1beta. Neither overexpression of Pit-1 nor inhibiting Pit-1 expression affected IL-1beta induction of hGH promoter activity. The stimulatory effect of IL-1beta was abolished following deletion of the -196 to -132 bp fragment.

CONCLUSIONSIL-1beta increases the activity of hGH gene promoter in rat pituitary GH3 cells. This stimulatory effect of IL-1beta appears to require the intracellular MAPK, p38 MAPK, and PI3-K dependent signaling pathways. The effect of IL-1beta requires the promoter sequence that spans the -196 to -132 bp fragment of the gene, but it is unrelated to Pit-1 protein.

Animals ; Cell Line ; Chromones ; pharmacology ; Flavonoids ; pharmacology ; Genes, Reporter ; Growth Hormone ; biosynthesis ; genetics ; Humans ; Interleukin-1 ; pharmacology ; Luciferases ; metabolism ; Mitogen-Activated Protein Kinases ; antagonists & inhibitors ; Morpholines ; pharmacology ; Phosphatidylinositol 3-Kinases ; antagonists & inhibitors ; Pituitary Gland ; cytology ; enzymology ; metabolism ; Rats ; Signal Transduction ; p38 Mitogen-Activated Protein Kinases

This study was purposed to explore the changes of possible angiogenetic factors other than VEGF after inhibition of NHE1 and their related mechanisms. The K562 cells were treated by NHE1 specific inhibitor cariporide, the angiogenesis factors after inhibition of NHE1 were screened by using protein chip, the IL-8 expression level after cariporide treatment was detected by real-time quantitative PCR; the K562 cells with stable interference of NHE1 were constructed, the IL-8 expression level after interference of NHE1 was detected by real-time quantitative PCR; the p38 phosphorylation level in K562 cells treated with cariporide was detected by Western blot. After treatment of K562 cells with p38 inhibitor SB203580, the IL-8 expression level was decreased by real-time quantitative PCR. The results of protein chip showed that IL-8 expression decreased after cariporide treatment. Real-time quantitative PCR confirmed this inhibitory effect. The p38 phosphorylation level increased after cariporide treatment. The down-regulation of IL-8 expression induced by cariporide treatment was partially restored after K562 cells were treated with p38 inhibitor SB203580. It is concluded that the inhibition of NHE1 can inhibit IL-8 expression through up-regulation of p38 phosphorylation.
Cation Transport Proteins ; antagonists & inhibitors ; Down-Regulation ; Guanidines ; pharmacology ; Humans ; Imidazoles ; pharmacology ; Interleukin-8 ; metabolism ; K562 Cells ; Phosphorylation ; drug effects ; Pyridines ; pharmacology ; Sodium-Hydrogen Exchanger 1 ; Sodium-Hydrogen Exchangers ; antagonists & inhibitors ; Sulfones ; pharmacology ; p38 Mitogen-Activated Protein Kinases ; metabolism

Ultraviolet B (UVB) irradiation of skin induces an acute inflammation. Cyclooxygenase-2 (COX-2) protein plays key roles in acute inflammation in UVB-irradiated keratinocyte cell line HaCaT. Recently, curcumin has been regarded as a promising anti-inflammatory agent due to its ability to inhibit COX-2 expression. However, it remains largely unknown whether curcumin inhibits the UVB-induced COX-2 expression in HaCaT cells. This study was undertaken to clarify the effect of curcumin on the expression of COX-2 in UVB- irradiated HaCaT cells and further determined the molecular mechanisms associated with this process. In this study, we have found that the expression of COX-2 mRNA and protein were up-regulated in UVB-irradiated HaCaT cells in a dose- and time-dependent manner. Interestingly, treatment with curcumin strongly inhibited COX-2 mRNA and protein expressions in UVB-irradiated HaCaT cells. Notably, there was effective inhibition by curcumin on UVB-induced activations of p38 MAPK and JNK in HaCaT cells. The DNA binding activity of AP-1 transcription factor was also markedly decreased with curcumin treatment in UVB-irradiated HaCaT cells. These results collectively suggest that curcumin may inhibit COX- 2 expression by suppressing p38 MAPK and JNK activities in UVB-irradiated HaCaT cells. We propose that curcumin may be applied as an effective and novel sunscreen drug for the protection of photoinflammation.
Curcumin/*pharmacology ; Enzyme Activation/drug effects/radiation effects ; Enzyme Inhibitors/pharmacology ; Humans ; JNK Mitogen-Activated Protein Kinases/antagonists & inhibitors/*metabolism ; Keratinocytes/cytology/*drug effects/*radiation effects ; Prostaglandin-Endoperoxide Synthase/*metabolism ; Research Support, Non-U.S. Gov't ; Transcription Factor AP-1/*metabolism ; Ultraviolet Rays ; p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors/*metabolism