OBJECTIVETo determine the role of extracellular signal-regulated kinase (ERK)1/2 during focal cerebral ischemia.

METHODSLeft middle cerebral artery occlusion (MCAO) was undergone after the introduction of a nylon suture to the left internal carotid artery in 70 male adult CD-1 mice. ERK 1/2 phosphorylation was detected using Western blot analysis, and the morphological feature was determined by immunohistochemistry. An ERK pathway inhibitor, 1,4-diamino-2,3-dicyano-1,4-bis[2-amino-phenylthio] butadiene (U0126), was administered intravenously 20 minutes before MCAO, and the neurological deficit levels and the infarct volumes were measured 24 hours after MCAO.

RESULTSPhosphorylated ERK 1/2 (pERK 1/2) activity increased after 30 minutes of MCAO and peaked at 2 hours. The immunohistochemical study displayed a large number of pERK 1/2 positive cells in the ischemic basal ganglion and surrounding cortex. Double-labeled fluorescent staining identified the pERK1/2 positive cells as neurons or astrocytes. In U0126 treated mice which had undergone 24 hours of MCAO, the neurological deficit levels and the infarct volumes were 44.6% and 45.8% respectively, less than those of the control mice.

CONCLUSIONSERK plays an important role in focal cerebral ischemia and inhibition of the ERK pathway can help protect against ischemic brain injury, which may provide a therapeutic approach for cerebral ischemia.

Animals ; Basal Ganglia ; pathology ; Brain Ischemia ; metabolism ; pathology ; physiopathology ; Butadienes ; pharmacology ; Cerebral Cortex ; pathology ; Immunohistochemistry ; Male ; Mice ; Mitogen-Activated Protein Kinases ; antagonists & inhibitors ; physiology ; Nitriles ; pharmacology ; Phosphorylation

OBJECTIVETo study the inhibitory effect of the dual usage of BEZ235 and U0126, the inhibitor of phosphatidyl inositol-3-kinase/protein kinase B pathway and extracellular regulated proteinkinase/mitogen-activated protein kinase pathway, respectively, on cell proliferation.

METHODSPhosphatase and tensin homolog knockout mouse embryonic fibroblast (PTEN-/-MEF) cell lines were used as the cellular model for malignant tumors. BEZ235, the dual inhibitor of phosphatidyl inositol-3-kinase and mammalian target of rapamycin, and U0126, the inhibitor of mitogen-activated protein kinase were used to treat the cells individually and in a combination manner. The inhibitory effects to cell proliferation were monitored by MTT.

RESULTSBoth BEZ235 and U0126 suppressed PTEN knockout cell proliferation, and their half inhibitory concentrations were 6.257 nmol/L and 22.85 μmol/L, respectively. However, the combination treatment of the two drugs showed antagonistic rather than synergistic effect on cell proliferation.

CONCLUSIONBEZ235 and U0126 are not suitable for a combined target therapy regimen.

Animals ; Butadienes ; pharmacology ; Cell Line ; Cell Proliferation ; drug effects ; Drug Antagonism ; Fibroblasts ; drug effects ; Imidazoles ; pharmacology ; Mice ; Mice, Knockout ; Nitriles ; pharmacology ; Phosphatidylinositol 3-Kinase ; antagonists & inhibitors ; pharmacology ; Quinolines ; pharmacology

The present study was to explore the effects of insulin on proliferation of skeletal myoblast cells in rats. Separated and cultured primary skeletal myoblast cells from rats were treated by insulin. By means of the incorporation of (3)H-TdR, BrdU assay and MTT assay, the proliferation of skeletal myoblast cells was detected. Western blot was used to check the phosphorylation of Akt and ERK of myoblast cells. The results showed that insulin significantly promoted the incorporation of (3)H-TdR into cultured skeletal myoblast cells in a dose-dependent manner. MTT assay and BrdU assay also showed insulin promoted the proliferation of skeletal myoblast cells. The promotion of skeletal myoblast cells proliferation by insulin was inhibited by PI3K inhibitor wortmannin or MEK inhibitor U0126, and the same phenomenon was shown in L6 and C2C12 cells. Also, insulin increased the phosphorylation of Akt and ERK in myoblast cells. These results suggest that insulin may promote proliferation of skeletal myoblast cells through PI3K/Akt and MEK/ERK pathways.
Androstadienes ; pharmacology ; Animals ; Butadienes ; pharmacology ; Cell Proliferation ; Cells, Cultured ; Enzyme Inhibitors ; pharmacology ; Insulin ; pharmacology ; MAP Kinase Signaling System ; Myoblasts, Skeletal ; cytology ; drug effects ; Nitriles ; pharmacology ; Phosphatidylinositol 3-Kinases ; metabolism ; Phosphorylation ; Proto-Oncogene Proteins c-akt ; metabolism ; Rats

OBJECTIVETo investigate the regulation of leptin on insulin secretion and expression of ATP-sensitive potassium channel subunit sulfonulurea receptor 1 (SUR1) mRNA, and to determine whether the effects of leptin are mediated through known intracellular signaling transduction.

METHODSPancreatic islets were isolated by the collagenase method from male SD rats. The purified islets were incubated with different concentrations of leptin for 2 h in the presence of different concentrations of glucose. Insulin release was measured using radioimmunoassay. Expression of SUR1 mRNA was detected by RT-PCR.

RESULTSIn the presence of leptin 2 nmol/L, insulin release was significantly inhibited at either 11.1 or 16.7 mmol/L glucose concentration (both P < 0.05), but insulin release was not altered at glucose of 5.6 mmol/L physiological concentration. The dose-response experiment showed that the maximal effect of leptin on insulin secretion achieved at 2 nmol/L. Exposure of islets to 2 nmol/L leptin induced a significant increase of SUR1 transcription levels by 71% (P < 0.01) at 11.1 mmol/L glucose and by 56% (P < 0.05) at 16.7 mmol/L glucose concentration. Selective phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor wortmannin significantly prevented the leptin effect on insulin secretion and SUR1 mRNA expression.

CONCLUSIONSRegulatory effects of leptin on insulin secretion could be biphasic at different concentrations of glucose and leptin. The stimulatory regulation of SUR1 transcription levels may be mediated through activation of PI 3-kinase pathway, which may be a possible mechanism of leptin in regulating insulin secretion.

Animals ; Butadienes ; pharmacology ; Cells, Cultured ; Dose-Response Relationship, Drug ; Insulin ; secretion ; Islets of Langerhans ; drug effects ; metabolism ; Leptin ; pharmacology ; Male ; Nitriles ; pharmacology ; Phosphatidylinositol 3-Kinases ; physiology ; Potassium Channels, Inwardly Rectifying ; genetics ; RNA, Messenger ; analysis ; Rats ; Rats, Sprague-Dawley

BACKGROUNDInvasive aspergillosis (IA), which is mainly caused by Aspergillus fumigatus (A. fumigatus), is a major cause of morbidity and mortality in immunocompromised patients. Despite considerable progress in currently available antifungals the mortality still remains high in critically ill patients. U0126 which is a highly selective inhibitor of MEK1 and MEK2 in the RAF/MEK/ERK pathway in mammalian cells has been demonstrated to have an anti-proliferative role in cancer cells. The purpose of this study was to explore the role of U0126 on growth inhibition and activation of mitogen-activated protein kinases (MAPKs) in A. fumigatus.

METHODSGermination percentage and hyphae growth in A. fumigatus treated with U0126 were observed and compared with untreated controls. Western blotting analysis was used to detect changes in activation of SakA, MpkA and MpkB.

RESULTSU0126 inhibited germination and hyphae growth in A. fumigatus and enhanced the phosphorylation of SakA and MpkA under oxidative stress. U0126 at 10 µmol/L did not block the activation of MpkB during nitrogen starvation stress.

CONCLUSIONU0126 shows promise as an antifungal candidate and the MAPK pathway may be a possible antifungal drug target for A. fumigatus.

Aspergillus fumigatus ; drug effects ; enzymology ; growth & development ; Butadienes ; pharmacology ; Enzyme Activation ; drug effects ; Enzyme Inhibitors ; pharmacology ; MAP Kinase Signaling System ; drug effects ; Mitogen-Activated Protein Kinases ; metabolism ; Nitriles ; pharmacology

OBJECTIVETo determine the effect of MEK inhibitor (U0126) on donor testes from ischemia-reperfusion injury after orthotopic testicular transplantation in rats.

METHODSThe rats were divided into 7 groups, Group 1: normal control; Group 2: cold perfusion control; Group 3: sham operation control; Group 4: transplanted for 30 min; Group 5: transplanted for 1 week; Group 6: transplanted for 30 min with pretreatment of U0126; Group 7: transplanted for 1 week with pretreatment of U0126. The orthotopic testicular transplantation model was established with cuff. The levels of ERK1, ERK2, pERK1 and pERK2 of donor testes were evaluated; the change of histology and gonadal hormones were measured as well.

RESULTGroup 1, 2 and 3 had no significant differences in all results (P>0.05). The levels of ERK1, ERK2, pERK1 and pERK2 in Group 4 were significantly increased compared with Group 1 (P<0.05), the levels of ERK1 and ERK2 in Group 6 were not different from those of Group 4 (P >0.05), but the levels of pERK1 and pERK2 in Group 6 were lower than those in Group 4 significantly(P <0.05), the histological changes in Group 6 were similar to Group 1 but milder than that in Group 4. The histological injury was more severe in Group 5 than that in Group 7, and the levels of gonadal hormones in Group 5 were lower than those in Group 7 (P <0.05) which remained at the normal levels.

CONCLUSIONU0126 has a protective effect on the donor testes in a short period through inhibiting expression of pERK1/2 activated by testicular transplantation.

Animals ; Butadienes ; pharmacology ; therapeutic use ; Enzyme Inhibitors ; pharmacology ; therapeutic use ; Extracellular Signal-Regulated MAP Kinases ; antagonists & inhibitors ; Male ; Nitriles ; pharmacology ; therapeutic use ; Random Allocation ; Rats ; Rats, Inbred Lew ; Reperfusion Injury ; prevention & control ; Testis ; blood supply ; transplantation

The present study was aimed to investigate the pathways, by which IL-27 regulates the expression of adherent molecule Mac-1, chemotactic factor receptor fMLP-R and pro-inflammatory cytokine IL-1beta in human neutrophils. Highly purified human neutrophils were isolated from peripheral blood using Ficoll-Hypaque gradients centrifugation and erythrocyte lysis. The mRNA expression of IL-27 receptor components (WSX-1/TCCR and gp130) in human neutrophils was detected by reverse transcription polymerase chain reaction (RT-PCR). After incubation with IL-27 and specific inhibitors (p38 MAPK inhibitor SB203580, PI3K inhibitor LY294002 and ERK inhibitor U0126), the mRNA levels of fMLP-R and IL-1beta were determined by real time RT-PCR, and the adherent molecule Mac-1 expression in human neutrophils was determined by flow cytometry. The IL-1beta level in culture supernatant of human neutrophils was assayed by radioimmunoassay. The results showed that IL-27 receptor components (WSX-1/TCCR and gp130) were constitutively expressed in human neutrophils. IL-27 down-regulated Mac-1 expression in human neutrophils (p<0.05). After incubation with specific inhibitors, SB203580, not LY294002 and U0126, inhibited the down-regulation of Mac-1 expression by IL-27. However, IL-27 up-regulated the mRNA expression of fMLP-R and IL-1beta, and increased the release of IL-1beta (p<0.05). Interestingly, LY294002, not SB203580 and U0126, inhibited the up-regulation of fMLP-R and IL-1beta by IL-27. It is concluded that the IL-27 may regulate the expression of Mac-1, fMLP-R and IL-1beta in human neutrophils through p38 MAPK and PI3K signal pathways.
Butadienes ; pharmacology ; Chromones ; pharmacology ; Down-Regulation ; Humans ; Imidazoles ; pharmacology ; Interleukin-1beta ; metabolism ; Interleukins ; metabolism ; Macrophage-1 Antigen ; metabolism ; Morpholines ; pharmacology ; Neutrophils ; metabolism ; Nitriles ; pharmacology ; Phosphatidylinositol 3-Kinases ; metabolism ; Pyridines ; pharmacology ; Receptors, Formyl Peptide ; metabolism ; Signal Transduction ; Up-Regulation ; p38 Mitogen-Activated Protein Kinases ; metabolism

Cholesterol is one of major components of cell membrane and plays a role in vesicular trafficking and cellular signaling. We investigated the effects of cholesterol on matrix metalloproteinase-2 (MMP-2) activation in human dermal fibroblasts. We found that tissue inhibitor of matrix metalloproteinase-2 (TIMP-2) expression and active form MMP-2 (64 kD) were dose-dependently increased by methyl-beta-cyclodextrin (MbetaCD), a cholesterol depletion agent. In contrast, cholesterol depletion-induced TIMP-2 expression and MMP-2 activation were suppressed by cholesterol repletion. Then we investigated the regulatory mechanism of TIMP-2 expression by cholesterol depletion. We found that the phosphorylation of JNK as well as ERK was significantly increased by cholesterol depletion. Moreover, cholesterol depletion-induced TIMP-2 expression and MMP-2 activation was significantly decreased by MEK inhibitor U0126, and JNK inhibitor SP600125, respectively. While a low dose of recombinant TIMP-2 (100 ng/ml) increased the level of active MMP-2 (64 kD), the high dose of TIMP-2 (> or = 200 ng/ml) decreased the level of active MMP-2 (64 kD). Taken together, we suggest that the induction of TIMP-2 by cholesterol depletion leads to the conversion of proMMP-2 (72 kD) into active MMP-2 (64 kD) in human dermal fibroblasts.
Anthracenes/pharmacology ; Butadienes/pharmacology ; Cells, Cultured ; Child ; Child, Preschool ; Cholesterol/metabolism/*physiology ; Cyclodextrins/pharmacology ; Enzyme Inhibitors/pharmacology ; Extracellular Signal-Regulated MAP Kinases/antagonists & inhibitors/physiology ; Fibroblasts/*drug effects/*metabolism/ultrastructure ; Humans ; Immunoblotting ; Immunoprecipitation ; JNK Mitogen-Activated Protein Kinases/antagonists & inhibitors/physiology ; Matrix Metalloproteinase 2/*metabolism ; Microscopy, Electron, Transmission ; Nitriles/pharmacology ; Tissue Inhibitor of Metalloproteinase-2/*metabolism

OBJECTIVETo study the regulatory effect of Helicobacter pylori CagA protein on gastrin promoter and the related signaling pathways as to further elucidate the mechanism of the development and progression of human gastric carcinoma.

METHODSAfter pcDNA3.1ZEO(-)/CagAand PGL/GP were identified by double restriction enzyme digestion, PCR and sequencing, the gastric cancer cell lines AGS and SGC-7901 cells were co-transfected with pcDNA3.1ZEO(-)/CagA and PGL/GP for 48 h. Alternatively, AGS and SGC-7901 cells were transfected by PGL/GP for 36 h later, and infected with Helicobacter pylori for additional 12 h. Meanwhile, the transfected and infected cells were treated using the JAK2 signaling pathway inhibitor AG490 and the ERK signaling pathway inhibitor U0126. The untreated cells and empty-vector-transfected cells were used as the control. Finally, luciferase activity was detected using the luciferase reporter assay system in transfected and infected cells. The levels of gastrin mRNA was determined by TaqMan® real-time quantitative PCR.

RESULTSAfter co-transfection with pcDNA3.1ZEO(-)/CagA and PGL/GP, the activities of luciferase were increased by 251.3, 106.1 and 2.4 times in AGS cells and 35.8, 22.7 and 13.4 times in SGC-7901 cells, respectively, as compared with that of the control, pcDNA3.1 ZEO(-)/CagA + PGL3/Basic and pcDNA3.1 ZEO(-) + PGL/GP groups. The activities of luciferase in PGL/GP transfection and HP infection group were also increased by 1673.2, 33.5, 1.4 times in AGS cells and 1180.2, 72.2 and 1.5 times in SGC-7901 cells, respectively, as compared with that of the control, PGL3/Basic + HP and PGL/GP groups. There were statistically significant differences between them (P < 0.05), which suggested that the transcription activity of gastrin promoter increased significantly. But after adding the inhibitor AG490 and U0126, respectively, the activities of luciferase were significantly decreased by 95.7% (U0126) and 33.0% (AG490) in co-transfected AGS cells and 94.8% (U0126) and 86.2% (AG490) in co-transfected SGC-7901 cells with pcDNA3.1ZEO(-)/CagA and PGL/GP (P < 0.05). In the PGL/GP transfection and HP infection group, the activities of luciferase were significantly decreased by 24.6% (U0126) and 25.8% (AG490) in AGS cells and 57.3% (U0126) and 14.1% (AG490) after adding the inhibitor AG490 and U0126, respectively (P < 0.05). The results showed that the gastrin promoter activities were significantly inhibited. The gastrin mRNA levels were 3.0 and 4.5 times higher in HP-infected AGS and SGC-7901 cells, respectively, than that in the control groups. In the cells transfected with pcDNA3.1ZEO(-)/CagA, the gastrin mRNA levels were raised 10.8 and 2.3 times (AGS cells) and 10.9 and 16.2 times (SGC-7901 cells), respectively, as compared with that of control and pcDNA3.1ZEO(-) groups. All of the differences were statistically significant (P < 0.05).

CONCLUSIONThese results suggest that CagA may activate the gastrin promoter and up-regulate the expression of gastrin gene, and CagA is one of the important proteins in regulating gastrin gene expression. The ERK/MAPK and JAK/STAT signaling pathways may be involved in the controlling of gastrin gene expression by CagA.

Antigens, Bacterial ; genetics ; metabolism ; Antineoplastic Agents ; pharmacology ; Bacterial Proteins ; genetics ; metabolism ; Butadienes ; pharmacology ; Cell Line, Tumor ; Enzyme Inhibitors ; pharmacology ; Gastrins ; biosynthesis ; genetics ; Gene Expression Regulation, Neoplastic ; Genetic Vectors ; Helicobacter Infections ; Helicobacter pylori ; isolation & purification ; Humans ; Nitriles ; pharmacology ; Promoter Regions, Genetic ; RNA, Messenger ; metabolism ; Stomach Neoplasms ; metabolism ; pathology ; virology ; Transfection ; Tyrphostins ; pharmacology ; Up-Regulation

PURPOSE: Imbalances between osteogenic and adipogenic differentiation leads to diseases such as osteoporosis. The aim of our study was to demonstrate the differences in extracellular signal-regulated kinase (ERK) phosphorylation during both adipogenesis and osteogenesis of human bone marrow-derived stem cells (BMSCs). MATERIALS AND METHODS: Using troglitazone, GW9662 and U0126, we investigated their role in hBMSC differentiation to adipogenic and osteogenic fates. RESULTS: ERK1/2 inhibition by U0126 suppressed proliferator-activated receptor (PPAR)gamma expression and lipid accumulation, while it decreased the mRNA expression of adipogenic genes (lipoprotein lipase, PPARgamma, and adipocyte protein) and osteogenic genes (type I collagen and osteopontin). ERK phosphorylation was transient and decreased during adipogenesis, whereas it occurred steadily during osteogenesis. Troglitazone, a PPARgamma agonist, induced adipogenesis by inhibiting ERK phosphorylation even in an osteogenic medium, suggesting that ERK signaling needs to be shut off in order to proceed with adipose cell commitment. Cell proliferation was greatly increased in osteogenesis but was not changed during adipogenesis, indicating that ERK might play different roles in cellular proliferation and differentiation between the two committed cell types. CONCLUSION: The duration and magnitude of ERK activation might be a crucial factor for the balance between adipogenesis and osteogenesis in human bone marrow-derived stem cells.
Adipogenesis/*drug effects/genetics ; Adult ; Anilides/pharmacology ; Bone Marrow Cells/*cytology/drug effects/metabolism ; Butadienes/pharmacology ; Cell Differentiation/drug effects ; Cells, Cultured ; Chromans/pharmacology ; Extracellular Signal-Regulated MAP Kinases/*metabolism ; Female ; Humans ; Male ; Middle Aged ; Nitriles/pharmacology ; Osteogenesis/*drug effects/genetics ; PPAR gamma/agonists/antagonists & inhibitors ; Phosphorylation/drug effects ; Reverse Transcriptase Polymerase Chain Reaction ; Stem Cells/*cytology/drug effects/*metabolism ; Thiazolidinediones/pharmacology