Acetaldehyde ; pharmacology ; Animals ; Cells, Cultured ; Extracellular Signal-Regulated MAP Kinases ; antagonists & inhibitors ; Flavonoids ; pharmacology ; Hepatocytes ; cytology ; physiology ; Rats

OBJECTIVETo analyze the expression and functions of ERK (extracellular regulated kinase) in Fas-mediated apoptosis in gastric carcinoma cell line SGC-7901 and to elucidate the potential significance of this signaling pathway in tumor progression.

METHODSRadioisotope labeling and Western blotting with special anti-ERK antibody were used to check ERK activity in SGC-7901 cell line after anti-Fas antibody treatment. Apoptosis induced by several treatment factors was evaluated by FACS can flow cytometer.

RESULTSERK activity increased and reached the peak at 30 min after treatment with anti-Fas antibody and decreased in PD98059 pretreated group. The number of sub-G(1) cell was 30.5% +/- 2.6% in PD98059 pretreated group, which was higher than anti-Fas treatment group and control group, respectively.

CONCLUSIONIn gastric cancer cell line SGC-7901, Fas-induced ERK activation may suppress Fas-mediated apoptosis. Inhibition of ERK may enhance the sensitivity of SGC-7901 cells to Fas-mediated apoptosis. Fas-induced ERK activation may confer gastric cancer cells ability to escape the immune surveillance.

Apoptosis ; physiology ; Cell Line, Tumor ; Extracellular Signal-Regulated MAP Kinases ; antagonists & inhibitors ; metabolism ; Fas Ligand Protein ; metabolism ; Humans ; Signal Transduction ; Stomach Neoplasms ; pathology

Embryonic stem (ES) cells hold great promise in regenerative medicine and it is an urgent task to understand the underlying molecular mechanisms that control ES cell fate choice between self-renewal and differentiation. In mouse ES cells, extrinsic leukemia inhibitory factor (LIF) and bone morphogenetic protein (BMP) signaling pathways play pivotal roles in maintaining the self-renewal status under serum and feeder free culture conditions. Intrinsic extracellular-signal regulated kinase (ERK) activity is also important in determining mouse ES cell fate-low ERK activity keeps mouse ES cell self-renewal while high ERK activity drives differentiation. We recently found that while LIF signaling augments ERK activity, BMP signaling inhibits ERK activity in mouse ES cells via direct upregulation of an ERK phosphatase-dual-specificity phosphatase 9. The cooperative effects of LIF and BMP signaling keep appropriate ERK activity and maintain mouse ES cell self-renewal (Li et al., 2012). These findings shed light on how extrinsic signals converge to intrinsic signaling molecules to regulate cell fate determination. This perspective summarizes our recent new findings and discusses the current unsolved questions and future directions.
Animals ; Bone Morphogenetic Proteins ; metabolism ; Embryonic Stem Cells ; enzymology ; metabolism ; Extracellular Signal-Regulated MAP Kinases ; antagonists & inhibitors ; metabolism ; Humans ; Mice ; Signal Transduction

The aim of this study was to explore the molecular mechanisms of the effect of low intensity pulsed ultrasound (LIPUS) on human primary macrophage functions. Macrophage phagocytosis was analyzed using fluorescein isothiocyanate (FITC)-labelled Escherichia coli (E.Coli); focal complex and extracellular matrix metalloproteinase inducer (EMMPRIN) were observed by fluorescence microscopy; the secretion of metalloproteinases (MMPs) was examined by gelatin zymography, and the expressions of EMMPRIN and extracellular signal-regulated kinases (ERKs) were detected by Western blot. The results indicated that LIPUS accelerated macrophages to phagocytose E.Coli (29.81+/-0.36 vs 18.00+/-0.78), promoted the protein expressions of EMMPRIN and MMPs, increased the level of protein tyrosine phosphorylation, and induced the phosphorylation of ERKs. Furthermore, the above functions were only found in adherent macrophages, and were inhibited or decreased by mitogen activated protein kinase kinase (MAPK kinase, MEK) inhibitor PD98059 and RGD (Arg-Gly-Asp peptide), one of main integrin recognition sequences. It is concluded that the effect of LIPUS on macrophages depends on cell adhesion, and relates to integrin-MEK-ERK pathway.
Basigin ; metabolism ; Extracellular Signal-Regulated MAP Kinases ; antagonists & inhibitors ; metabolism ; Humans ; Macrophages ; cytology ; immunology ; radiation effects ; Matrix Metalloproteinases ; metabolism ; Phagocytosis ; radiation effects ; Phosphorylation ; Ultrasonics

OBJECTIVE: To investigate the role of focal adhesion kinase (FAK) in extracellular signal-regulated kinase (ERK) signaling pathway mediated invadsion of trophoblasts. METHODS: We established a human extravillous cytotrophoblasts in vitro invasion model. Different concentrations of herbimycin A(FAK inhibitor)and PD98059 (ERK inhibitor) were given to observe the influence on the growth of trophoblast cells, FAK, ERK phosphorylation, and trophoblast invasion abilities. RESULTS: The expression of phosphorylated FAK in the extravillous cytotrophoblasts (EVCT) was inhibited by herbimycin A in a concentration-dependent manner and expression of phosphorylated ERK1/2 was also partially reduced. PD98059 had no effect on the expression of phosphorylated FAK. Herbimycin A and PD98059 suppressed the in vitro invasion of EVCT to various degrees. CONCLUSION ERK signaling pathway may be the common pathway for many invasive signals,and play a key role in the regulation of trophoblast invasion.
Benzoquinones ; pharmacology ; Cell Division ; physiology ; Cell Movement ; physiology ; Extracellular Signal-Regulated MAP Kinases ; antagonists & inhibitors ; metabolism ; Flavonoids ; pharmacology ; Focal Adhesion Protein-Tyrosine Kinases ; antagonists & inhibitors ; metabolism ; Humans ; Lactams, Macrocyclic ; pharmacology ; Phosphorylation ; Rifabutin ; analogs & derivatives ; Signal Transduction ; physiology ; Trophoblasts ; cytology ; physiology

BACKGROUNDNumerous studies have shown that reducing the level of tumor necrosis factor-alpha (TNFα) through the use of anti-TNF antibodies or soluble TNF receptor is a safe and efficacious treatment to inflammatory diseases such as rheumatoid arthritis. Therefore, novel approaches to achieve this outcome are desired. The aim of this study was to investigate the characterization of a small molecule inhibitor, Y316, which blocks TNF mRNA upregulation and TNF production by lipopolysaccharides (LPS) stimulated monocytes.

METHODSPeripheral blood mononuclear cells (PBMC) from healthy volunteers were plated in 24-well plates and stimulated with LPS (1 µg/ml), phorbol-12-myristate-13-acetate (PMA) (100 ng/ml), zymosan (10 µg/ml) and Tsst (100 ng/ml). Supernatants were collected after 4-hour culture at 37°C, and quantitative determination of TNFα, interleukin-1β (IL-1β), IL-6, IL-8, IL-10 and IL-2 production in the supernatants was performed by colorimetric enzyme-linked immunosorbent assay (ELISA). Total RNA of PBMC was isolated and cytokine mRNA quantitation was performed by using a RNA level measuring kit (R & D Systems). PBMC were pretreated with Y316 (10 µmol/L, 1 µmol/L, 0.1 µmol/L, 0.01 µol/L and 0.001 µmol/L) or dimethyl sulfoxide at 37°C for 10 minutes, and then stimulated with LPS or PMA, protein concentrations of p44.42, IKBα, P38 and Jun NH2-terminal kinase were determined by Western blotting. Cyclic adenosine-3',5'-monophosphate (cAMP) of PBMC was measured by enzyme immunoassay kit (Amersham Pharmacia Biotech).

RESULTSY316 blocked TNF production and inhibited the upregulation of TNF mRNA levels in response to LPS, and also prevented the production of IL-1 and IL-6. In contrast, Y316 augmented the production of IL-10 in LPS-stimulated monocytes. Y316 failed to prevent the production of IL-2 and TNF in antigen-stimulated T cells, suggesting that its effects may be cell-type specific. Y316 prevented the phosphorylation and activation of the MAPK, ERK, and therefore appeared to mediate its effects on TNF by acting at an early point in the signaling cascade induced in response to LPS. There was no effect of Y316 on cAMP levels either alone or in the presence of LPS.

CONCLUSIONSY316 appears to be a small molecule inhibiting TNF production, which may act via a novel mechanism. Identification of the target of Y316 may lead to the development of alternative strategies for achieving selective cytokine inhibition.

Anti-Inflammatory Agents ; pharmacology ; Extracellular Signal-Regulated MAP Kinases ; metabolism ; Humans ; Interleukin-1 ; antagonists & inhibitors ; biosynthesis ; Interleukin-6 ; antagonists & inhibitors ; biosynthesis ; Lipopolysaccharides ; pharmacology ; Monocytes ; drug effects ; immunology ; Phosphorylation ; Tumor Necrosis Factor-alpha ; antagonists & inhibitors ; biosynthesis

OBJECTIVETo demonstrate the effects and mechanisms of Qifu decoction( QFD) on renal interstitial fibrosis (RIF) in model rats with yang-deficiency syndrome.

METHODThe rats were randomly divided into 3 groups, the Sham group (Group A), the Model group (Group B), the Qifu decoction group (Group C) and the Enalapril group (Group D). The RIF model was established by adenine administrated and unilateral ureteral obstruction (UUO) of the left ureter. After the model was successfully established, the rats in Group C and D were administrated with QFD or the Enalapril suspension,while the rats in Group A and B were administrated with distilled water. All rats were administrated for 3 weeks. Before administration and at the end of week 1, 2 and 3, the rats were weighted, and 24 h urinary protein excretion (Upro), urinary β2-microglobulin (Uβ2-MG) and urinary N-acetyl-D-glucosaminidase (NAG) were examined, respectively. All rats were killed after administration for 3 weeks. Blood and renal tissues were collected, renal morphology and tubulointerstitial morphology were evaluated, respectively. Serum cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), blood urea nitrogen (BUN), serum creatinine (Scr) and uric acid (UA) were detected, respectively. The protein expressions of E-cadherin, α-smooth muscle actin(α-SMA), transforming growth factor-β1 (TGF-β1), onnective tissue growth factor (CTGF) extracellular signal-regulated protein kinase 1/2(ERK1/2) and phosphorylated-ERK1/2 (p-ERK1/2) in kidney were evaluated, respectively.

RESULTQFD ameliorated serum cAMP level and the rate of cAMP/cGMP, attenuated urinary β2-MG level, NAG level and renal tubulointerstitial fibrosis, increased E-cadherin protein expression, and reduced α-SMA, TGF-β1, CTGF and p-ERK1/2 protein expressions in the kidney. However, QFD had no influence on renal function in vivo. In addition, these effects were better than those of the model rats treated by Enalapril.

CONCLUSIONQFD could alleviate yang-deficiency parameters, as well as urinary β2-MG level and NAG level in model rats induced by adenine administration and UUO. Moreover, QFD could improve EMT and RIF by up-regulating E-cadherin protein expression, and down-regulating α-SMA, TGF-β1, CTGF and p-ERK1/2 protein expressions, the key molecular in ERK1/2 signaling pathway.

Animals ; Drugs, Chinese Herbal ; pharmacology ; Extracellular Signal-Regulated MAP Kinases ; antagonists & inhibitors ; Fibrosis ; Kidney ; drug effects ; pathology ; Kidney Diseases ; drug therapy ; pathology ; MAP Kinase Signaling System ; drug effects ; Male ; Rats ; Rats, Sprague-Dawley ; Ureteral Obstruction ; complications ; Yang Deficiency ; complications

OBJECTIVETo observe the effect of ischemic postconditioning on phosphatidylinositol-3-OH kinase (PI3K) and extracellular signal-regulated protein kinase 1/2(ERK1/2) in rats after hepatic ischemia reperfusion in rats and investigate the mechanism of ischemic postcoditioning of the liver.

METHODSThree cycles of 1 min-off-1 min-on ischemic postconditioning regime were used in a rat model of 70% hepatic ischemia-reperfusion injury. The changes in the liver function, hepatocyte apoptosis, phosphorylation of Akt and ERK1/2 were assessed in rats treated with sham operation, LY294002+sham operation (LY+S), PD98059+sham operation (PD+S), ischemia reperfusion (IR), ischemic postconditioning (IPO), LY294002+ ischemic postconditioning (LY+IPO), or PD98059+ischemic postconditioning (PD+IPO).

RESULTSIschemic postconditioning significantly alleviated hepatic ischemia-reperfusion-induced liver function injury and hepatocyte apoptosis and increased phosphorylation of Akt and ERK1/2. LY294002 and PD98059 antagonized the effects of ischemic postconditioning in the liver.

CONCLUSIONActivation of PI3K and ERK1/2 may mediate the protective effect of ischemic postconditioning against hepatic ischemia- reperfusion injury in rats.

Animals ; Chromones ; pharmacology ; Extracellular Signal-Regulated MAP Kinases ; metabolism ; Flavonoids ; pharmacology ; Ischemic Postconditioning ; Liver ; blood supply ; Male ; Morpholines ; pharmacology ; Phosphatidylinositol 3-Kinases ; antagonists & inhibitors ; metabolism ; Rats ; Rats, Sprague-Dawley ; Reperfusion Injury ; metabolism

OBJECTIVETo investigate the possible molecular mechanisms of heme oxygenase-1 (HO-1) induction by quercetin using rat primary hepatocytes.

METHODSSprague-Dawley rat primary hepatocytes were isolated using a two-step collagenase perfusion technique and treated with quercetin at various doses (25 - 200 mumol/L) and times (2 - 12 h). To investigate the roles of various signaling pathways, the hepatocytes were pre-treated with 50 mumol/L quercetin plus an extracellular signal-regulated kinase (ERK) inhibitor (PD98059 at 10 mumol/L), a p38 inhibitor (SB203580 at 10 mumol/L), a c-Jun N-terminal kinase inhibitor (SP600125 at 10 mumol/L) or a phosphatidylinositol 3-kinase inhibitor (Wortmannin at 1 mumol/L) for 12 h. Changes in the mRNA and protein levels of HO-1 and nuclear factor, E-2 related factor 2 (Nrf2) were detected by RT-PCR and western blotting.

RESULTSAfter 4 - 12 h of treatment with quercetin at all concentrations, the HO-1 mRNA level in hepatocytes had increased significantly (vs. untreated control cells; all P less than 0.01). The quercetin-induced HO-1 expression and Nrf2 translocation into the nucleolus was inhibited by PD98059.

CONCLUSIONQuercetin may induce HO-1 expression via the ERK/Nrf2 signaling transduction pathway.

Animals ; Cells, Cultured ; Enzyme Inhibitors ; pharmacology ; Extracellular Signal-Regulated MAP Kinases ; antagonists & inhibitors ; Heme Oxygenase (Decyclizing) ; metabolism ; Hepatocytes ; drug effects ; metabolism ; NF-E2-Related Factor 2 ; metabolism ; Primary Cell Culture ; Quercetin ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; drug effects

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