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 study the changes of telomerase activity and protein expression of phosphorylated (activated) extracellular regulated protein kinases (ERK1 and ERK2) in the course of inhibiting hepatocarcinomatous cell proliferation and inducing cell apoptosis by three kinds of chemotherapy drugs: Harringtonine (HRT), Vincristine (VCR), and Etoposide (Vp16). To discuss the regulative function to hepatocarcinomatous cell apoptosis and interrelation of telomerase and ERK.

METHODSCytotoxicity assay, flow cytometry analysis, telomerase repeat amplification protocol assay (TRAP), bioluminescence analysis, and western blot were used in this experiment.

RESULTSHRT, VCR, and Vp16 could inhibit cell proliferation (0.28% 0.08%, 0.25% 0.16%, 0.24% 0.11%), induce apoptosis (21.12%, 28.83%, 12.30%), inhibit telomerase activity, and down-regulate the protein expression of phosphorylated ERK.

CONCLUSIONSIt might be through ERK signal transduction pathways that chemotherapy drugs down-regulate telomerase activity and induce apoptosis.

Apoptosis ; Carcinoma, Hepatocellular ; drug therapy ; enzymology ; pathology ; Etoposide ; pharmacology ; Harringtonines ; pharmacology ; Humans ; Liver Neoplasms ; drug therapy ; enzymology ; pathology ; Mitogen-Activated Protein Kinase 1 ; antagonists & inhibitors ; physiology ; Mitogen-Activated Protein Kinase 3 ; Mitogen-Activated Protein Kinases ; antagonists & inhibitors ; physiology ; Signal Transduction ; Telomerase ; physiology ; Tumor Cells, Cultured ; Vincristine ; pharmacology

Nitric oxide (NO) seems to play a pivotal role in the vascular endothelial growth factor (VEGF)-induced endothelial cell proliferation. This study was designed to investigate the role and intracellular signal pathway of endothelial nitric oxide synthase (eNOS) activation induced by VEGF. ECV 304 cells were treated with betaVEGF(165) and then cell proliferation, eNOS protein and mRNA expression levels were analyzed to elucidate the functional role of eNOS in cell proliferation induced by VEGF. After exposure of cells to betaVEGF(165) , eNOS activity and cell growth were increased by approximately two-fold in the betaVEGF(165) -treated cells compared to the untreated cells. In addition, VEGF stimulated eNOS expression at both the mRNA and protein levels in a dose-dependent manner. Phosphatidylinositol-3 kinase (PI-3K) inhibitors were used to assess PI-3K involvement in eNOS regulation. LY294002 was found to attenuate VEGF-stimulated eNOS expression. Wortmannin was not as effective as LY294002, but the reduction effect was detectable. Cells activated by VEGF showed increased ERK1/2 levels. Moreover, the VEGF-induced eNOS expression was reduced by the PD98059, MAPK pathway inhibitor. This suggests that eNOS expression might be regulated by PI-3K and the ERK1/2 signaling pathway. In conclusion, betaVEGF(165) induces ECV 304 cell proliferation via the NO produced by eNOS. In addition, eNOS may be regulated by the PI-3K or mitogen-activated protein kinase pathway.
1-Phosphatidylinositol 3-Kinase/*antagonists & inhibitors ; Cell Division/drug effects ; Cell Line ; Endothelial Growth Factors/*metabolism/pharmacology ; Endothelium, Vascular/cytology ; *Gene Expression Regulation, Enzymologic ; Lymphokines/*metabolism/pharmacology ; MAP Kinase Signaling System ; Mitogen-Activated Protein Kinase 1/*antagonists & inhibitors ; Mitogen-Activated Protein Kinase 3 ; Mitogen-Activated Protein Kinases/*antagonists & inhibitors ; Nitric Oxide Synthase/*genetics/metabolism ; Nitric Oxide Synthase Type III ; Signal Transduction ; Vascular Endothelial Growth Factor A ; Vascular Endothelial Growth Factors

OBJECTIVESTo investigate the effect of rhein on endothelial plasminogen activator inhibitor-1 (PAI-1) mRNA expression and protein production induced by transforming growth factor beta1 (TGFbeta1), and to explore the mechanism of the protective action of rhein on endothelial cells.

METHODSA human umbilical endothelium derived cell line (ECV-304) from ATCC was used in this study. The PAI-1 mRNA expression and protein synthesis in the endothelial cells were detected by Northern blot and flow cytometry analysis, respectively. The activity of phospho-p44/p42 MAP kinase induced by TGFbeta1 was determined by immunoprecipitation analysis and western blot.

RESULTSTGFbeta1 rapidly increased PAI-1 mRNA expression in the endothelial cells, and this effect lasted at least 24 hours. The upregulation of PAI-1 mRNA expression induced by TGFbeta1 in endothelial cells was inhibited by rhein in a dose-dependent manner. In addition, rhein inhibited endothelial PAI-1 protein production. Further study revealed that rhein had a significant inhibitory effect on the activity of phospho-p44/p42 MAP kinase induced by TGFbeta1 in human endothelial cells.

CONCLUSIONSOur results showed that rhein may have a protective effect on the endothelial dysfunction by inhibiting overexpression of PAI-1, indicating a way for the treatment of vascular diseases.

Anthraquinones ; pharmacology ; Cells, Cultured ; Dose-Response Relationship, Drug ; Endothelium, Vascular ; drug effects ; metabolism ; Humans ; Mitogen-Activated Protein Kinase 1 ; metabolism ; Mitogen-Activated Protein Kinase 3 ; Mitogen-Activated Protein Kinases ; metabolism ; Plasminogen Activator Inhibitor 1 ; biosynthesis ; genetics ; RNA, Messenger ; analysis ; Transforming Growth Factor beta ; antagonists & inhibitors ; Transforming Growth Factor beta1

Angiotensin II (Ang II), which is an important mediator of both vascular responsiveness and growth, has been shown to induce vascular smooth muscle cell (VSMC) hypertrophy via the activation of a complex series of intracellular signaling events. Heat shock protein 70 (Hsp70) has recently been shown to protect against Ang II-induced hypertension. In this study, we tested the hypothesis that Hsp70 can protect VSMC from Ang II-induced hypertrophy. We treated VSMCs with Ang II to induce hypertrophy and to activate MAPK signaling pathway. We observed that the augmentation of Hsp70 expression inhibited Ang II-stimulated VSMC hypertrophy. This inhibitory effect of Hsp70 appears to be partly due to extracellular signal-regulated kinase (ERK1/2) inactivation, which in turn, may possibly result from the accumulation of MAP kinase phosphatase-1 (MKP-1).
Rats, Sprague-Dawley ; Rats ; RNA, Small Interfering/pharmacology ; Protein-Tyrosine-Phosphatase/metabolism/physiology ; Phosphoprotein Phosphatase/metabolism/physiology ; Muscle, Smooth, Vascular/*cytology/*drug effects ; Mitogen-Activated Protein Kinase 3/antagonists & inhibitors ; Mitogen-Activated Protein Kinase 1/antagonists & inhibitors ; Male ; MAP Kinase Kinase 2/metabolism ; MAP Kinase Kinase 1/metabolism ; Immediate-Early Proteins/metabolism/physiology ; Hypertrophy ; HSP70 Heat-Shock Proteins/antagonists & inhibitors/*pharmacology ; Flavonoids/pharmacology ; Enzyme Stability/drug effects ; Cells, Cultured ; Cell Cycle Proteins/metabolism/physiology ; Aorta/drug effects/pathology ; Animals ; Angiotensin II/*pharmacology

To investigate the mechanism of inhibitory effect of a novel bFGF antagonist peptide isolated from the phage display random heptapeptide library on cell proliferation induced by basic fibroblast growth factor. The effect of P7 on cell morphology was observed under an inverted microscope. Flow cytometry was applied to analyze the effect of P7 on cell cycle progress of bFGF-stimulated cells. The effect of P7 on bFGF-induced activation of MEK and Erk1/2 in MAPK pathway was detected by Western blotting. The results showed that no significant cell morphology change was observed in the range of detected concentrations of P7. Cell cycle analysis showed that P7 decreased S-phase cell population and arrested cell cycle at the G0/G1 phase of bFGF-stimulated cells. The results of MAP kinase activation assay indicated that P7 decreased bFGF-induced MEK and Erk1/2 phosphorylation in a dose-dependent manner. P7 inhibited proliferation of bFGF-stimulated Balb/c 3T3 cells possibly via cell cycle arrest at the G0/G1 phase and down-regulation of signal molecular activation in MAPK pathway.
Animals ; BALB 3T3 Cells ; Cell Cycle ; drug effects ; Cell Proliferation ; drug effects ; Fibroblast Growth Factor 2 ; antagonists & inhibitors ; pharmacology ; MAP Kinase Kinase Kinases ; metabolism ; MAP Kinase Signaling System ; drug effects ; Mice ; Mice, Inbred BALB C ; Mitogen-Activated Protein Kinase 1 ; metabolism ; Mitogen-Activated Protein Kinase 3 ; metabolism ; Peptides ; pharmacology ; Phosphorylation ; Protein Binding

AIMTo study the effects of FAK-ERK1/2 signaling pathway and FAK antisense oligodeoxynucleotides (ODNs) on vascular smooth muscle cell (SMC) migration and adhesion stimulated by fibronectin (FN).

METHODSMigration and adhesion of cultured SMCs were stimulated by different concentrations of FN, FAK, ERK1/2. And their phosphorylation were detected by immunoprecipitation and Western blot. FAK antisense ODNs were transfected into SMCs by cationic lipid to investigate its modulatory effects on tyrosine phosphorylation, SMCs migration and adhesion were also measured by modifing Boyden Chamber and morphological enumeration, respectively.

RESULTSFAK were expressed when SMCs adhesion and migration were successfully simulated by FN (5, 10, 20, 40, 60 micrograms.mL-1), high contents of FAK and ERK1/2 phosphorylation were detected by 20 micrograms.mL-1 FN or more. FAK antisense ODNs were transfected efficiently by cationic lipid. FAK and ERK1/2 phosphorylation were inhibited magnificently after FAK antisense ODNs transfection. Cell migration stimulated by FN 10, 20, 40 and 60 micrograms.mL-1 were reduced by 23.26%, 21.63%, 19.31% and 17.88% respectively (P < 0.05). SMCs adhesive spreading in 5-60 micrograms.mL-1 FN groups were reduced by 17.89%-27.67% (P < 0.05).

CONCLUSIONFAK-ERK1/2 mediated signal transduction play important roles in SMCs migration and adhesion stimulated by extracellular matrix. The process can be inhibited by FAK antisense ODNs effectively.

Animals ; Aorta ; cytology ; Cell Adhesion ; drug effects ; Cell Movement ; drug effects ; Cells, Cultured ; Fibronectins ; pharmacology ; Focal Adhesion Kinase 1 ; Focal Adhesion Protein-Tyrosine Kinases ; Mitogen-Activated Protein Kinase 1 ; biosynthesis ; metabolism ; Mitogen-Activated Protein Kinase 3 ; Mitogen-Activated Protein Kinases ; biosynthesis ; metabolism ; Muscle, Smooth, Vascular ; cytology ; Myocytes, Smooth Muscle ; cytology ; drug effects ; Oligodeoxyribonucleotides, Antisense ; pharmacology ; Protein-Tyrosine Kinases ; antagonists & inhibitors ; genetics ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; Transfection

OBJECTIVETo study the role of extracellular signal-regulated kinase (ERK) in cerebral ischemia and the mechanism of protective effects of U0126 (1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio] butadiene) on ischemic brain.

METHODSMice underwent left middle cerebral artery occlusion (MCAO) by introducing a suture in the lumen. U0126 was injected intravenously through the internal jugular vein. The immuno-activity of phosphorylated ERK1/2 (pERK1/2), phosphorylated mitogen activated protein kinase kinase (pMEK), and phosphorylated Elk-1 (pElk-1) was assessed by Western blot analysis and immunohistochemistry. Interleukin (IL)-1beta mRNA level was measured by ribonuclease protection assay.

RESULTSPhosphorylated ERK1/2 in 2 hours MCAO mice was down-regulated after intravenous injection of U0126. The inhibition was dose dependent and treatment time related. pMEK and pElk-1 were also reduced in a similar fashion after U0126 treatment. IL-1beta mRNA increased after 1 and 2 hours of MCAO. After injection of U0126, it was down-regulated during 1 to 4 hours after MCAO.

CONCLUSIONIntravenous administration of the MEK inhibitor U0126 inhibits pMEK, pERK1/2, and pElk-1 up-regulation induced by cerebral ischemia. The protective effect of U0126 against ischemic injury is probably resulted from the reduction of IL-1beta mRNA via the inhibition of ERK pathway.

Animals ; Butadienes ; pharmacology ; DNA-Binding Proteins ; metabolism ; Enzyme Inhibitors ; pharmacology ; Infarction, Middle Cerebral Artery ; metabolism ; Interleukin-1 ; biosynthesis ; genetics ; Male ; Mice ; Mitogen-Activated Protein Kinase 1 ; metabolism ; Mitogen-Activated Protein Kinase Kinases ; antagonists & inhibitors ; metabolism ; Nitriles ; pharmacology ; Phosphorylation ; Proto-Oncogene Proteins ; metabolism ; RNA, Messenger ; biosynthesis ; genetics ; Signal Transduction ; Transcription Factors ; metabolism ; ets-Domain Protein Elk-1

To explore the intracellular signal pathways for beta-VLDL induced very low density lipoprotein receptor (VLDL-R) transcription up-regulation and their effects on lipid accumulation in macrophages, Western Blot was used to examine phosphorylated ERK1/2 protein and regulated effects by different singal kinase inhibitants. It was found that beta-VLDL induced an increase in ERK1/2 activity in a protein kinase C (PKC)-dependent manner in murine RAW264.7 macrophages. By using different protein kinases inhibitors or activators, it was observed that the effect of beta-VLDL induced VLDL receptor transcription, which was monitored by RT-PCR analysis of VLDL receptor mRNA, was not affected by the inhibitor of p38 kinase and cAMP analog, but extremely abolished by pretreating cells with PD98059, an inhibitor of ERK and GF 109203X, an inhibitor of PKC. These results demonstrated that the PKC-ERK1/2 cascade is the essential signaling pathway by which beta-VLDL activated VLDL-R mRNA expression. Inhibition of the ERK1/2 signaling cascade resulted in suppression of the cellular lipid accumulation induced by beta-VLDL in macrophages.
Cells, Cultured ; Lipoproteins, VLDL ; metabolism ; Macrophages ; cytology ; metabolism ; Mitogen-Activated Protein Kinase 1 ; metabolism ; physiology ; Mitogen-Activated Protein Kinase 3 ; metabolism ; physiology ; Protein Kinase C ; antagonists & inhibitors ; metabolism ; Receptors, LDL ; biosynthesis ; genetics ; Signal Transduction ; Transcription Factors ; metabolism ; Transcription, Genetic ; Up-Regulation

The differentiation of periodontal ligament (PDL) progenitor cells is important for maintaining the homeostasis of PDL tissue and alveolar bone. Vitamin C (VC), a water-soluble nutrient that cannot be biosynthesized by humans, is vital for mesenchymal stem cells differentiation and plays an important role in bone remodeling. Therefore, the objective of this study was to determine the function and mechanism of VC in PDL progenitor cells osteogenic differentiation at the molecular level. We demonstrated that VC could induce the osteogenic differentiation and maturation of PDL progenitor cell without other osteogenic agents. During the process, VC preferentially activated ERK1/2 but did not affect JNK or p38. Co-treatment with ERK inhibitor effectively decreased the Vitamin C-induced expression of Runx2. ERK inhibitor also abrogated Vitamin C-induced the minimized nodules formation. PELP1, a nuclear receptor co-regulator, was up-regulated under VC treatment. PELP1 knockdown inhibited ERK phosphorylation. The overexpression of PELP1 had a positive relationship with Runx2 expression. Taken together, we could make a conclude that VC induces the osteogenic differentiation of PDL progenitor cells via PELP1-ERK axis. Our finding implies that VC may have a potential in the regeneration medicine and application to periodontitis treatment.
Ascorbic Acid ; pharmacology ; Butadienes ; pharmacology ; Cell Differentiation ; drug effects ; Cells, Cultured ; Co-Repressor Proteins ; antagonists & inhibitors ; genetics ; metabolism ; Core Binding Factor Alpha 1 Subunit ; genetics ; metabolism ; Humans ; MAP Kinase Signaling System ; drug effects ; Mitogen-Activated Protein Kinase 1 ; antagonists & inhibitors ; metabolism ; Mitogen-Activated Protein Kinase 3 ; antagonists & inhibitors ; metabolism ; Nitriles ; pharmacology ; Periodontal Ligament ; cytology ; Phosphorylation ; drug effects ; RNA Interference ; RNA, Small Interfering ; metabolism ; Stem Cells ; cytology ; Transcription Factors ; antagonists & inhibitors ; genetics ; metabolism ; Up-Regulation ; drug effects