Résumé
RSK2 is a downstream signaling protein of ERK1 and ERK2 and plays a key role in physiological homeostasis. For this reason, RSK2 is a highly conserved protein among the p90RSK family members. In its location in the signaling pathway, RSK2 is a kinase just upstream of transcription and epigenetic factors, and a few kinases involved in cell cycle regulation and protein synthesis. Moreover, activation of RSK2 by growth factors is directly involved in cell proliferation, anchorage-independent cell transformation and cancer development. Direct evidences regarding the etiological roles of RSK2 in cancer development in humans have been published by our research group illustrating that elevated total- and phospho-RSK2 protein levels mediated by ERK1 and ERK2 are higher in skin cancer tissues compared to normal skin tissues. Notably, it has been shown that RSK2 ectopic expression in JB6 Cl41 cells induces cell proliferation and anchorage-independent cell transformation. Importantly, knockdown of RSK2 suppresses Ras-mediated foci formation and anchorage-independent colony growth of cancer cells. Kaempferol is a one of the natural compounds showing selectivity in inhibiting RSK2 activity in epidermal growth factor-induced G1/S cell cycle transition and cell transformation. Thus, ERKs/RSK2 signaling axis is an important target signaling molecule in chemoprevention.
Sujets)
Humains , Axis , Carcinogenèse , Cycle cellulaire , Prolifération cellulaire , Transformation cellulaire néoplasique , Chimioprévention , Épigénomique , Homéostasie , Protéines et peptides de signalisation intercellulaire , Phosphotransferases , Peau , Tumeurs cutanéesRésumé
The present study focuses on the neuroprotective effect of glycyrrhizic acid (GA, a major compound separated from Glycyrrhiza Radix, which is a crude Chinese traditional drug) against glutamate-induced cytotoxicity in differentiated PC12 (DPC12) cells. The results showed that GA treatment improved cell viability and ameliorated abnormal glutamate-induced alterations in mitochondria in DPC12 cells. GA reversed glutamate-suppressed B-cell lymphoma 2 levels, inhibited glutamate-enhanced expressions of Bax and cleaved caspase 3, and reduced cytochrome C (Cyto C) release. Exposure to glutamate strongly inhibited phosphorylation of AKT (protein kinase B) and extracellular signal-regulated kinases (ERKs); however, GA pretreatment enhanced activation of ERKs but not AKT. The presence of PD98059 (a mitogen-activated protein/extracellular signal-regulated kinase kinase [MEK] inhibitor) but not LY294002 (a phosphoinositide 3-kinase [PI3K] inhibitor) diminished the potency of GA for improving viability of glutamate-exposed DPC12 cells. These results indicated that ERKs and mitochondria-related pathways are essential for the neuroprotective effect of GA against glutamate-induced toxicity in DPC12 cells. The present study provides experimental evidence supporting GA as a potential therapeutic agent for use in the treatment of neurodegenerative diseases.
Sujets)
Animaux , Rats , Anti-inflammatoires/usage thérapeutique , Acide glutamique/toxicité , Acide glycyrrhizique/usage thérapeutique , Neuroprotecteurs/usage thérapeutique , /effets des médicaments et des substances chimiques , Transduction du signal/effets des médicaments et des substances chimiques , Apoptose/effets des médicaments et des substances chimiques , /isolement et purification , Différenciation cellulaire/effets des médicaments et des substances chimiques , Survie cellulaire/effets des médicaments et des substances chimiques , 4H-1-Benzopyran-4-ones/pharmacologie , Cytochromes c/effets des médicaments et des substances chimiques , Antienzymes/pharmacologie , Flavonoïdes/pharmacologie , Système de signalisation des MAP kinases/effets des médicaments et des substances chimiques , Mitochondries/effets des médicaments et des substances chimiques , Morpholines/pharmacologie , /classification , /cytologie , Protéines proto-oncogènes c-akt/effets des médicaments et des substances chimiques , /isolement et purification , /isolement et purificationRésumé
Objective:Hepatic stellate cells (HSCs) are the principal cells responsible for the development of hepatic fibrosis and cirrhosis.During the fibrotic process,HSCs undergo proliferation and transdifferentiation from a quiescent to myofibroblast-like phenotype.The fate of myofibroblastlike HSCs includes apoptosis or reversion back to a quiescent phenotype.The mechanisms involved in the apoptotic process of HSCs have yet to be determined.The purpose of the present study is to determine the effects of extracellular signal-regulated kinases (ERKs) phosphorylation on the apoptosis of HSCs induced by staurosporine.Methods:We used Western blot and flow cytometry to detect the expression level of ERK and cell apoptosis status in four rat hepatic stellate cell lines (CFSC-8B,-2G,-3H and -5H).Results:Each hepatic stellate cell line had a distinct morphology consistent with their expression level of α-SMA and that CFSC-8B cells had the highest α-SMA expression.Although all four cell types expressed similar levels of ERK1/2,phosphorylation levels were significantly higher in CFSC8B and CFSC-2G than in CFSC-3H and CFSC-5H cells.When CFSC-8B ceils (high ERK1/2 phosphorylation) and CFSC-5H cells (low ERK1/2 phosphorylation) were employed to examine staurosporine-induced apoptosis,CFSC-8B cells were significantly more sensitive.Staurosporine further increased ERK1/2 phosphorylation in both cell lines.Conclusion:ERK1/2 phosphorylation in HSCs determines the sensitivity of HSCs to staurosporine-induced apoptosis.
Résumé
Objective To investigate the effect of extracellular signal-regulated kinases (ERKs) on cell cycle regulation after ischemia. Methods Ischemic model was induced by photochemistry method. Animals were divided randomly into cerebral ischemia groups (control and treatment groups) and sham group. Rats in treatment group were subjected to U0126 solution injection at 30 rain pre-ischemia through caudal veins, and animals in control group were subjected to identical volume DMSO solution without U0126. Positive immunostaining for CyclinD1 and CyclinE were detected by immunohistofluorescence method. Expressions of phosphorylated ERK1/2 (pERK1/2), CyclinD1, and CyclinE proteins were examined by Western blot in ischemic slide of brain cortex. Semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the expression of transcription factor E2F mRNA in ischemic slide of brain cortex. Results The numbers of CyclinD1 and CyclinE positive cells were highly decreased in the U0126-treated group (P<0.05 vs vehicle-treated group). Expression of pERK1/2 protein in the ischemic group was significantly higher than that in the U0126-treated group, which peaked at 4h, and decreased to the baseline at 12 after ischemia. While the expression levels of CyclinD1 and CyclinE in the U0126-treated group were increased at 6h post injury, peaked at 12 after injury (P<0.05 vs that in vehicle-treated group). In addition, expression of E2F mRNA in the vehicle-treated group was significantly higher than those in the sham-operated group and the U0126-treated group (P<0.05). Conclusions ERK pathway plays a very important role in cerebral ischemia. Inhibiting ERK1/2 phosphorylation post-ischemia reduces the expressions of CyclinD1, CyclinE and E2F, which indicates that ERK can affect cell cycle regulation.
Résumé
Recent data have shown the importance of oxidative stresses in the pathogenesis of inflammatory bowel disease, crohn's disease and ulcerative colitis. H2O2, reactive oxygen species (ROS) donor, has been reported to act as a signaling molecule involved in a variety of cellular functions such as apoptosis and proliferation. In the present study, we investigated viability of cultured ileal smooth muscle cells (ISMC) after stimulation with H2O2. Trypan blue method revealed that the cell viability of ISMC treated with 1 mM H2O2 was not different from that of controls at up to 2 h time point, while treatment of ISMC with 1 mM H2O2 for 48 h finally induced significant decrease in the cell viability. Therefore, we evaluated whether H2O2 was capable of ERKs activation in ISMC for the short-term exposure and examined whether tyrosine kinase was involved in the process of ERK activation by H2O2 in ISMC. We also investigated the effects of H2O2 on activation of SAPK/JNK and p38 MAP kinase in ISMC. Thus, ISMC were cultured and exposed to H2O2, and western blot analysis was performed with phospho- specific MAP kinase antibodies. Robust activation of ERK occurred within 30 min of 1 mM H2O2 treatment. H2O2-induced ERK activation was attenuated by a tyrosine kinase inhibitor, genistein, indicating that tyrosine kinase was probably involved in the ERK activation by H2O2. H2O2 was a moderate activator of SAPK/JNK, while p38 MAP kinase was not activated by H2O2. We suggest that ERK activation induced by short-term H2O2 treatment plays a critical role in cellular protection in the early stage of response to oxidative stress. The present study suggests the necessity of identification of MAPK signaling pathways affected by ROS, since it could ultimately elucidate cellular consequences involved in initiation and perpetuation of intestinal tissue damage in the diseases such as crohn's disease and ulcerative colitis, resulted from excessive ROS.
Sujets)
Humains , Anticorps , Apoptose , Technique de Western , Survie cellulaire , Rectocolite hémorragique , Maladie de Crohn , Génistéine , Maladies inflammatoires intestinales , Muscles lisses , Myocytes du muscle lisse , Stress oxydatif , p38 Mitogen-Activated Protein Kinases , Phosphotransferases , Protein-tyrosine kinases , Espèces réactives de l'oxygène , Donneurs de tissus , Bleu de trypanRésumé
Objective To investigate possible intracellular s ignal molecules involved in TGF-?-induced airway smooth muscle cell proli feration in rats. Methods The cultured airway smooth muscle cells were divide d into 3 groups: control group (20 mL?L -1FCS/DMEM), 10 ?g?L -1 TGF-?1 group and 10?g?L -1 TGF-?1 /U-0126 (1 ? mol?L -1) group. The proliferation of ASMCs was detected by MTT. Exp ression of phospho-p42/p44 extracellular signal-regulated kinase (ERKs) with i mmunocytochemistry were examined in different groups. A values were detected by image analysis. Results By MTT, A values of 10?g?L -1 TGF- ?1 group (0.36?0.043) were significantly higher than those of control grou p (0.126?0.052, t=5.44,7.62, P