P38 Mitogen-activated Protein Kinase Levels Research Articles

SB203580, a p38 Inhibitor, Improved Cardiac Function but Worsened Lung Injury and Survival During Escherichia coli Pneumonia in Mice

Supporting its therapeutic application in sepsis, p38 mitogen-activated protein kinase (MAPK) inhibition decreases cardiopulmonary injury and lethality with lipopolysaccharide challenge. However, only one preclinical study has reported the survival effects of a p38 inhibitor (SB203580, 100 mg/kg) during infection. We therefore tested SB203580 in mice (n = 763) challenged with intratracheal Escherichia coli and treated with antibiotics and fluids. Compared with placebo, high dose SB203580 (100 mg/kg) pretreatment increased the hazards ratio of death (95% confidence interval) (3.6 [2.1, 6.1], p < 0.0001). Decreasing doses (10, 1, or 0.1 mg/kg) went from being harmful to having no significant effect (p < 0.0001 for the effect of decreasing dose). At 48 hours, but not 24 hours after E. coli, high and low dose SB203580 pretreatment decreased cardiac phosphorylated p38 MAPK levels and improved cardiac output either (p <or= 0.07). Low dose SB203580 did not alter lung neutrophils significantly but increased lung injury at 48 hours (p = 0.05). High dose decreased lung neutrophils and injury at 24 hours (p = 0.09 and 0.01, respectively) but then increased them at 48 hours (both p <or= 0.01). Lung injury was greater with high versus low dose at 48 hours (p = 0.002). Thus, SB203580 had divergent effects on cardiac and lung function in E. coli challenged mice. Furthermore, high dose worsened survival and low dose did not improve it. Altogether, these findings suggest that clearly defining the risks and benefits of p38 MAPK inhibition is important before such treatment is applied in patients with or at risk of serious infection.

1884 THE MOLECULAR MECHANISMS IN RAT SERTOLI CELL INDUCED BY BISPHENOL A

You have accessJournal of UrologyInfertility: Physiology, Pathophysiology, Basic Research1 Apr 20101884 THE MOLECULAR MECHANISMS IN RAT SERTOLI CELL INDUCED BY BISPHENOL A Koji Chiba, Kohei Yamaguchi, Makoto Ando, and Masato Fujisawa Koji ChibaKoji Chiba More articles by this author , Kohei YamaguchiKohei Yamaguchi More articles by this author , Makoto AndoMakoto Ando More articles by this author , and Masato FujisawaMasato Fujisawa More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2010.02.1837AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Bisphenol A (BPA) is an industrial chemical used primarily to make polycarbonate plastic and epoxy resins – both of which are used in countless applications that make our lives easier, healthier and safer, each and every day. It is well known that exposure to BPA results in impairment of spermatogenesis. BPA not only has estrogenic action but also induces cell apoptosis directly. However, little is known about the signal mechanisms involved in BPA regulation of Sertoli cell (SC) function. This study was designed to evaluate how BPA regulates SC signal molecules and mechanisms. METHODS Purified rat SC was cultured under serum-free conditions and treated with BPA (200ìM) at various time points (30m, 1h, 3h, 6h, 24h). Western blot analysis was used to determine the activation of extracellular signal-related kinases 1 and 2 (ERK1/2), cJun-N-terminal kinase (JNK), p38 mitogen activated protein kinase (MAPK), nuclear factor kappa B (NF-êB), cyclooxygenase-1,2 (COX-1, 2), estrogen receptor-á, â (ER-á, â), androgen receptor (AR), inducible nitric oxide synthase (iNOS), and endothelial nitric oxide synthase (eNOS). The levels of transferrin (TF), prostaglandin E2 (PGE2), prostaglandin F2á (PGF2á) in culture medium were quantified by ELISA. Interleukin (IL)-1â and IL-6 mRNAs were measured by quantitative real-time PCR (QRT-PCR) analysis. RESULTS Compared with control, BPA activated the phosphorylation of ERK1/2 (p-ERK1/2) through 30 min to 6h. IL-1â was up-regulated at 30 min and IL-6 was up-regulated at 1h, 24h. TF was down-regulated at 6h, 24h. There were no significant changes in JNK, p38 MAPK, NF-êB, COX-1, 2, iNOS, and eNOS levels. Furthermore, there were no significant changes in both ER and AR levels. When ERK activity inhibitor (PD98059, 10 μM) was added, up-regulation of IL-1â and IL-6 was inhibited, and down-regulation of TF was also inhibited. CONCLUSIONS In this study, we have revealed that BPA reduces rat Sertoli Cell IL up-regulation and TF down-regulation by activating ERK 1/2. These data have identified that BPA may affect spermatogenesis via ERK1/2, not ER or AR. Although further investigations are necessary to establish the overall, precise SC molecular signaling mechanism response to BPA, our data may provide novel therapeutic targets for interventional use to prevent testicular dysfunction induced by BPA treatment. Kobe, Japan© 2010 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 183Issue 4SApril 2010Page: e732 Advertisement Copyright & Permissions© 2010 by American Urological Association Education and Research, Inc.MetricsAuthor Information Koji Chiba More articles by this author Kohei Yamaguchi More articles by this author Makoto Ando More articles by this author Masato Fujisawa More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Eugenosedin-A amelioration of lipopolysaccharide-induced up-regulation of p38 MAPK, inducible nitric oxide synthase and cyclooxygenase-2

In this study, we investigate the protective effects of eugenosedin-A on p38 mitogen-activated protein kinase (MAPK), inflammatory nitric oxide (NO) and cyclooxygenase-2 (COX-2) pathways in a rat model of endotoxin shock. Rats were pretreated with eugenosedin-A, trazodone, yohimbine (1 mg kg(-1), i.v.), aminoguanidine or ascorbic acid (15 mg kg(-1), i.v.) 30 min before endotoxin challenge. Endotoxaemia was induced by a single i.v. injection of lipopolysaccharide (LPS, 10 mg kg(-1)). In rats not treated with eugenosedin-A, LPS increased plasma concentrations of NO and prostaglandin E(2) (PGE(2)), and levels of p38 MAPK, inducible NO synthase (iNOS) and COX-2 proteins in the liver, lung, aorta and lymphocytes. In the pre-treated rats, eugenosedin-A not only inhibited the LPS-induced NO and PGE(2) levels but also attenuated the LPS-induced increase in p38 MAPK and iNOS levels in the liver, aorta and lymphocytes. Eugenosedin-A also reduced LPS-induced COX-2 proteins in the aorta and lymphocytes. Likewise, aminoguanidine, ascorbic acid, yohimbine and trazodone were also found to decrease NO and PGE(2) concentrations after endotoxin challenge. While aminoguanidine and ascorbic acid also attenuated the LPS-induced increase in p38 MAPK, iNOS and COX-2 proteins in the aorta and lymphocytes, trazodone and yohimbine inhibited only the increase in p38 MAPK, iNOS and COX-2 proteins in lymphocytes. Finally, eugenosedin-A (10(-10)-10(-8) M) significantly inhibited the biphasic response induced by hydrogen peroxide (10(-6)-3 x 10(-5) M) in rat denudated aorta. Taken together, the results of this study indicate that eugenosedin-A, as well as ascorbic acid, can attenuate free-radical-mediated aortic contraction and relaxation. It may therefore be able to reduce the damage caused by septic shock by inhibiting formation of p38 MAPK, iNOS, COX-2 and free radicals.

Engulfment of Axon Debris by Microglia Requires p38 MAPK Activity

The clearance of debris after injuries to the nervous system is a critical step for restoration of the injured neural network. Microglia are thought to be involved in elimination of degenerating neurons and axons in the central nervous system (CNS), presumably restoring a favorable environment after CNS injuries. However, the mechanism underlying debris clearance remains elusive. Here, we establish an in vitro assay system to estimate phagocytosis of axon debris. We employed a Wallerian degeneration model by cutting axons of the cortical explants. The cortical explants were co-cultured with primary microglia or the MG5 microglial cell line. The cortical neurites were then transected. MG5 cells efficiently phagocytosed the debris, whereas primary microglia showed phagocytic activity only when they were activated by lipopolysaccharide or interferon-beta. When MG5 cells or primary microglia were co-cultured with degenerated axons, p38 mitogen-activated protein kinase (MAPK) was activated in these cells. Engulfment of axon debris was blocked by the p38 MAPK inhibitor SB203580, indicating that p38 MAPK is required for phagocytic activity. Receptors that recognize dying cells appeared not to be involved in the process of phagocytosis of the axon debris. In addition, the axons undergoing Wallerian degeneration did not release lactate dehydrogenase, suggesting that degeneration of the severed axons and apoptosis may represent two distinct self-destruction programs. We observed regrowth of the severed neurites after axon debris was removed. This finding suggests that axon debris, in addition to myelin debris, is an inhibitory factor for axon regeneration.

P38 MAPK and MMP-9 cooperatively regulate mucus overproduction in mice exposed to acrolein fog

Objective To evaluate the role of p38 mitogen-activated protein kinase (MAPK) on mice airway inflammation, mucus production and the possible cross-talk between p38 MAPK and matrix metalloproteinase-9 (MMP-9) in mucin protein synthesis. Methods Mice were exposed to 4.0 ppm of acrolein for 21 days with daily intraperitoneal injection of SB203580, a specific inhibitor of p38 MAPK. In control mice, sterile saline was administered instead. On days 7 and 21, mice were sacrificed to examine airway inflammation and mucus production by BALF cell counts, cytokine ELISA, and H&E and AB-PAS staining. The mRNA and protein levels of Muc5ac, p38 MAPK and MMP-9 in the lung were determined by RT-PCR, immunohistochemistry and Western blotting analysis. MMP-9 activity was measured by gelatin zymography. Results Both the numbers of inflammatory cells and mucus-secreting goblet cells were significantly increased in the airways of mice exposed to acrolein as compared to the control mice. Acrolein-increased phosphorylation of p38 MAPK was significantly reduced by SB203580. The airway inflammation and goblet cell hyperplasia after acrolein challenge were also attenuated by SB203580 administration. Moreover, SB203580 treatment decreased the acrolein-induced increase of Muc5ac and MMP-9 expression and MMP-9 activity in airway epithelium. Conclusions The results indicate an important role of p38 MAPK in acrolein-induced airway inflammation and mucus hypersecretion in mice. The cooperation of p38 and MMP-9 may contribute to the mucin overproduction after inflammatory challenge.

Involvement of Rho-kinase in prostaglandin E 1-stimulated VEGF synthesis through stress-activated protein kinase/c- Jun N-terminal kinase in osteoblast-like MC3T3-E1 cells

We have previously shown that prostaglandin E 1 (PGE 1) stimulates the synthesis of vascular endothelial growth factor (VEGF) through p38 mitogen-activated protein (MAP) kinase and stress-activated protein kinase/c- Jun N-terminal kinase (SAPK/JNK) but not p44/p42 MAP kinase in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the involvement of Rho-kinase in the PGE 1-stimulated VEGF synthesis in these cells. PGE 1 induced within 3 min the phosphorylation of myosin phosphatase targeting subunit (MYPT-1), a substrate of Rho-kinase. Y27632 and fasudil, specific inhibitors of Rho-kinase, which attenuated the MYPT-1 phosphorylation, significantly suppressed the PGE 1-stimulated VEGF synthesis. Y27632 and fasudil markedly reduced the PGE 1-induced phosphorylation of SAPK/JNK without affecting the phosphorylation levels of p38 MAP kinase or p44/p42 MAP kinase. These results strongly suggest that Rho-kinase functions at a point upstream of SAPK/JNK and regulates PGE 1-stimulated VEGF synthesis in osteoblasts.

ADENOVIRUS‐MEDIATED FKBP12.6 OVEREXPRESSION INDUCES HYPERTROPHY AND APOPTOSIS IN CULTURED NEONATAL CARDIOMYOCYTES

1. Cardiac ryanodine RyR2 receptors regulate Ca(2+) release from the sarcoplasmic reticulum (SR). FK506 binding protein (FKBP) 12.6 prevents aberrant SR Ca(2+) leakage during diastole, thereby maintaining the integrity of RyR2 function. Previous studies have focused mainly on FKBP12.6 deficiency and so the pathophysiological consequences of FKBP12.6 overexpression remain unclear. Herein, we investigate the effect of FKBP12.6 overexpression on cardiac hypertrophic and apoptotic signalling. 2. Human FKBP12.6 cDNA was cloned into pAdTrack-CMV and the resulting plasmid, along with a control empty plasmid, were transfected into bacteria. The resulting virus, namely Ad-FKBP12.6 containing green fluorescent protein, was propagated and purified. Neonatal rat cardiomyocytes were infected with this virus. Protein and DNA synthesis were measured by [(3)H]-leucine and [(3)H]-thymidine incorporation, respectively. Expression of p38 mitogen-activated protein kinase (MAPK), phosphorylated extracellular signal-regulated kinase 1 or 2 (p-ERK1/2) and Bax were examined by western blotting. 3. Compared with control cells, cardiomyocytes that overexpressed FKBP12.6 became hypertrophic and hyperplastic, with increased levels of both p38 MAPK and p-ERK1/2. At the same time, overexpression of FKBP12.6 induced apoptosis of cardiomyocytes, as determined by both Bax protein expression and DNA fragmentation. Rapamycin treatment downregulated the expression of p-ERK1/2, p38 MAPK and Bax in stimulated cardiomyocytes, with or without FKBP12.6 overexpression, and enhanced protein synthesis, but had no effect on DNA synthesis in cardiomyocytes. 4. In conclusion, FKBP12.6 overexpression may participate in pathophysiological processes through both hypertrophic and apoptotic signalling pathways, leading to cardiomyocyte damage and death.

Epigallocatechin Gallate Stimulates Glucose Uptake Through the Phosphatidylinositol 3-Kinase-Mediated Pathway in L6 Rat Skeletal Muscle Cells

The effect of epigallocatechin gallate (EGCG) on glucose uptake was studied in L6 rat skeletal muscle cells. Glucose uptake assay revealed that EGCG increased glucose uptake >70% compared to control. EGCG-stimulated glucose uptake was blocked by LY294002, an inhibitor of phosphatidylinositol (PI) 3-kinase, which is a major regulatory molecule in glucose uptake pathways. However, AMP-activated protein kinase (AMPK), which is another crucial mediator in independent glucose uptake pathways, did not inhibit EGCG-stimulated glucose uptake by SB203585, a specific inhibitor of the AMPK downstream mediator, p38 mitogen-activated protein kinase (MAPK). We also found that EGCG increased the phosphorylation level of protein kinase B and PI 3-kinase activity, when assessed by PI 3-kinase assay, whereas no increase in the phosphorylation level of AMPK and p38 MAPK was observed. Taken together, these results suggest that EGCG might stimulate glucose uptake, not AMPK-mediated but PI 3-kinase-mediated, in skeletal muscle cells, thereby contributing to glucose homeostasis.

Phosphorylation of Fibroblast Growth Factor (FGF) Receptor 1 at Ser777 by p38 Mitogen-Activated Protein Kinase Regulates Translocation of Exogenous FGF1 to the Cytosol and Nucleus

Exogenous fibroblast growth factor 1 (FGF1) signals through activation of transmembrane FGF receptors (FGFRs) but may also regulate cellular processes after translocation to the cytosol and nucleus of target cells. Translocation of FGF1 occurs across the limiting membrane of intracellular vesicles and is a regulated process that depends on the C-terminal tail of the FGFR. Here, we report that translocation of FGF1 requires activity of the alpha isoform of p38 mitogen-activated protein kinase (MAPK). FGF1 translocation was inhibited after chemical inhibition of p38 MAPK or after small interfering RNA knockdown of p38alpha. Translocation was increased after stimulation of p38 MAPK with anisomycin, mannitol, or H2O2. The activity level of p38 MAPK was not found to affect endocytosis or intracellular sorting of FGF1/FGFR1. Instead, we found that p38 MAPK regulates FGF1 translocation by phosphorylation of FGFR1 at Ser777. The FGFR1 mutation S777A abolished FGF1 translocation, while phospho-mimetic mutations of Ser777 to Asp or Glu allowed translocation to take place and bypassed the requirement for active p38 MAPK. Ser777 in FGFR1 was directly phosphorylated by p38alpha in a cell-free system. These data demonstrate a crucial role for p38alpha MAPK in the regulated translocation of exogenous FGF1 into the cytosol/nucleus, and they reveal a specific role for p38alpha MAPK-mediated serine phosphorylation of FGFR1.

Dietary Genistein Inhibits Metastasis of Human Prostate Cancer in Mice

Dietary genistein has been linked to lower prostate cancer (PCa) mortality. Metastasis is the ultimate cause of death from PCa. Cell detachment and invasion represent early steps in the metastatic cascade. We had shown that genistein inhibits PCa cell detachment and cell invasion in vitro. Genistein-mediated inhibition of activation of focal adhesion kinase (FAK) and of the p38 mitogen-activated protein kinase (MAPK)-heat shock protein 27 (HSP27) pathway has been shown by us to regulate PCa cell detachment and invasion effects, respectively. To evaluate the antimetastatic potential of genistein, we developed an animal model suited to evaluating antimetastatic drug efficacy. Orthotopically implanted human PC3-M PCa cells formed lung micrometastasis by 4 weeks in >80% of inbred athymic mice. Feeding mice dietary genistein before implantation led to blood concentrations similar to those measured in genistein-consuming men. Genistein decreased metastases by 96%, induced nuclear morphometric changes in PC3-M cells indicative of increased adhesion (i.e., decreased detachment) but did not alter tumor growth. Genistein increased tumor levels of FAK, p38 MAPK, and HSP27 "promotility" proteins. However, the ratio of phosphorylated to total protein trended downward, indicating a failure to increase relative amounts of activated protein. This study describes a murine model of human PCa metastasis well suited for testing antimetastatic drugs. It shows for the first time that dietary concentrations of genistein can inhibit PCa cell metastasis. Increases in promotility proteins support the notion of cellular compensatory responses to antimotility effects induced by therapy. Studies of antimetastatic efficacy in man are warranted and are under way.

Inhibition of MAPK Kinase Signaling Pathways Suppressed Renal Cell Carcinoma Growth and Angiogenesis In vivo

The mitogen-activated protein kinase (MAPK) signaling pathways play essential roles in cell proliferation and differentiation. Recent studies also show the activation of MAPK signaling pathways in tumorigenesis, metastasis, and angiogenesis of multiple human malignancies, including renal cell carcinoma (RCC). To assess the role of this pathway in regulating the proliferation and survival of RCC cells, we first examined the expression of MAPK kinase (MKK) and MAPK in clear cell RCC and confirmed the overexpression of MKK1 and extracellular signal-regulated kinase 2 (ERK2) in these tumors. We then tested the effects of pharmacologic inhibition of MKK on human RCC cell lines, both in vitro and in vivo, using anthrax lethal toxin (LeTx), which cleaves and inactivates several MKKs. Western blotting showed that the phosphorylation levels of ERK, c-Jun-NH(2) kinase, and p38 MAPK decreased after 72 h of LeTx treatment. Exposure to LeTx for 72 h reduced cell proliferation by 20% without significant effects on cell cycle distribution and apoptosis. Anchorage-independent growth of RCC cells was dramatically inhibited by LeTx. In vivo studies showed that tumor growth of RCC xenografts could be suppressed by LeTx. Extensive necrosis and decreased tumor neovascularization were observed after LeTx treatment. LeTx also showed direct inhibition of proliferation of endothelial cells in vitro. Our results suggest that suppression of one or more MAPK signaling pathways may inhibit RCC growth through the disruption of tumor vasculature.

Neuron-specific phosphorylation of c-Jun N-terminal kinase increased in the brain of hypoxic preconditioned mice

Accumulated studies have suggested that mitogen-activated protein kinase (MAPK) play a pivotal role in the development of cerebral hypoxic preconditioning (HPC). By using our "auto-hypoxia"-induced HPC mouse model, we have reported increased phosphorylation level of p38 MAPK, and decreased phosphorylation and protein expression levels of extracellular signal regulated kinases 1/2 (ERK1/2) in the brain of HPC mice. In the current study, we investigated the involvement of c-Jun N-terminal kinase (JNK) in the brain of HPC mice. By using Western blot analysis, we found that the phosphorylation levels of JNK at Thr183 and Tyr185 sites (phospho-Thr183/Tyr185 JNK), but not its protein expression, increased significantly (p<0.05, n=6 for each group) both in the hippocampus and frontal cortex of early (H1-H4) and delayed (H5 and H6) HPC mice than that of the normoxic group (H0, n=6). Similarly, enhanced phospho-Thr183/Tyr185 JNK was also observed by immunostaining in the hippocampus and frontal cortex of mice following series of hypoxic exposures (H3 and H6). In addition, we found that phospho-Thr183/Tyr185 JNK predominantly co-localized with a neuron-specific protein, neurogranin, in both the hippocampus and frontal cortex of HPC mice (H3) by using double-labeled immunofluorescence. These results suggest that the increased neuron-specific phosphorylation of JNK at Thr183/Tyr185, not protein expression, might be involved in the development of cerebral HPC of mice.

Topiramate stimulates glucose transport through AMP-activated protein kinase-mediated pathway in L6 skeletal muscle cells

The use of topiramate (TPM) in the treatment of binge-eating disorder, bulimia nervosa, and antipsychotic-induced weight gain has recently increased, however, the exact molecular basis for its effects on body weight reduction and improved glucose homeostasis, is yet to be elucidated. Here we investigated the effect and signaling pathway of TPM on glucose uptake in L6 rat skeletal muscle cells, which account for >70% of glucose disposal in the body. Intriguingly, we found that TPM (10 microM) stimulated the rate of glucose uptake up to twofold increase. And TPM-stimulated glucose transport was inhibited with the overexpression of dominant-negative form of AMP-activated protein kinase (AMPK), an important mediator in glucose transport, implicating that AMPK-mediated pathway is involved. The TPM-stimulated glucose transport was blocked by SB203580, a specific inhibitor of AMPK downstream mediator, p38 mitogen-activated protein kinase (MAPK) protein. LY294002, an inhibitor of phosphatidylinositol (PI) 3-kinase, which is another crucial mediator in independent glucose transport pathway, did not inhibit TPM-stimulated glucose transport. We also found that TPM increased the phosphorylation level of AMPK and p38 MAPK, whereas no effect on the activity of PI 3-kinase of TPM, when assessed by PI 3-kinase assay, was observed. These results together suggest that TPM stimulates glucose transport, not via PI 3-kinase mediated, but via AMPK-mediated pathway in skeletal muscle cells, thereby contributing to the body weight regulation and glucose homeostasis.

Low concentrations of nitric oxide (NO) induced cell death in PC12 cells through activation of p38 mitogen-activated protein kinase (p38 MAPK) but not via extracellular signal-regulated kinases (ERK1/2) or c-Jun N-terminal protein kinase (JNK)

Nitric oxide (NO), a highly reactive gaseous molecule, has been previously reported to induce apoptosis-like cell death even at a low concentration in PC12 cells. In this study, we examined NO-induced activation of members of the mitogen-activated protein kinase (MAPK) family, i.e., p38 MAPK, extracellular signal-regulated kinases (ERK1/2), and c-Jun N-terminal protein kinase (JNK). Following the exposure of PC12 cells to an NO donor, (+)-( E)-4-ethyl-2-[hydroxyimino]-5-nitro-3-hexenamide (NOR3; 100 μM), the phosphorylation level of p38 MAPK increased time dependently from 2 to 6 h, but that of both ERK1/2 and JNK did not. Treatment with a p38 MAPK inhibitor SB203580 partially blocked the NOR3-induced cell death. Neither PD98059, U0126 (inhibitors of ERK1/2) nor SP600125 (a specific inhibitor of JNK) treatments had any significant effect on the NOR3-induced cell death. These findings suggest that the activation of a p38 MAPK pathway, but not that of ERK1/2 or JNK, plays an essential role in the apoptosis-like cell death induced by low concentrations of NO.

Stable expression of EBERs in immortalized nasopharyngeal epithelial cells confers resistance to apoptotic stress

Epstein-Barr virus (EBV) infection is closely associated with the development of nasopharyngeal carcinoma (NPC). The EBV-encoded RNAs (EBERs) are the most abundant EBV transcripts (about 10(7) copies per cell) in EBV infected cells. However, the cellular function of EBER expression, particularly in nasopharyngeal epithelial cells, remains poorly understood. EBERs acquire secondary structures analogous to double-stranded RNA (dsRNA) and may bind to the double-stranded RNA-dependent protein kinase (PKR) and interfere with its function. Activation of PKR involves autophosphorylation resulting in protein synthesis inhibition and cellular apoptosis. Induction of cellular apoptosis by activation of PKR may be an antiviral response adopted by virally infected cells. We have examined the functional properties of EBER expression in an immortalized nasopharyngeal epithelial cell line (NP69). Expression of EBERs was achieved by transfecting the NP69 cells with an EBER-expressing plasmid, pESK10. The EBER-expressing NP69 cells attained a higher growth rate compared to cells transfected with control plasmid (pcDNA3). However, the EBER-expressing NP69 cells did not form colonies in soft agar and were non-tumorigenic in nude mice. To investigate if EBERs may protect the nasopharyngeal epithelial cells from apoptotic insults, we treated the EBER-expressing NP69 cells with a dsRNA analogue, poly(I).poly(C) (pIC), to activate PKR in cells and examined for their responses. Lower level of PKR phosphorylation and elevation of Bcl-2 were observed in EBER-expressing NP69 cells. In addition, other apoptotic markers including the cleaved forms of caspase-3 and poly(ADP)ribose polymerase (PARP) were found to be lower in EBER-expressing NP69 cells after treatment with pIC. Lower phosphorylation levels of p38 MAPK (mitogen-activated protein kinase) and c-jun were also observed in EBER-expressing NP cells. Our results suggest that EBER expression may confer an apoptotic-resistant phenotype in immortalized nasopharyngeal epithelial cells.

P38 MAP kinase activation mediates γ-globin gene induction in erythroid progenitors

Objective Our goal was to determine the role of p38 mitogen-activated protein kinase (MAPK) signaling in fetal hemoglobin (HbF) induction. Two histone deacetylase inhibitors (HDAIs), sodium butyrate (NB), and trichostatin (TSA) and hemin were analyzed. In addition, the effect of direct activation of p38 MAPK on γ-globin gene activity was studied. Method Primary erythroid progenitors derived from peripheral blood mononuclear cell and K562 erythroleukemia cells were analyzed. Cells were grown in NB, TSA, hemin, or anisomycin either alone or in the presence of the p38 MAPK inhibitor SB203580. The effects of the various treatments on γ-globin RNA, HbF, and phosphorylated p38 MAPK levels were measured by RNase protection assay, alkaline denaturation, and Western blot analysis, respectively. A K562 stable line overexpressing constitutively active p38 MAPK was established using MAPK kinase kinase 3 (MKK3) and MKK6, the immediate upstream activators of p38. The direct effect of p38 MAPK overexpression on γ-globin mRNA synthesis was analyzed. Results NB and TSA activated p38 MAPK and increased γ-globin mRNA levels in K562 cells and primary erythroid progenitors. Pretreatment with SB203580 blocked p38 MAPK and γ-globin gene activation. In contrast, no change in p38 activity was observed with hemin inductions. Direct activation of p38 by anisomycin or constitutive overexpression also increased γ-globin mRNA in the absence of HbF inducers in wild-type K562 cells and in the MKK stable lines. Conclusion This study supports a novel role for p38 MAPK in γ-globin regulation in human erythroid progenitors.

Activation of p38 mitogen-activated protein kinase in spinal microglia is a critical link in inflammation-induced spinal pain processing.

We examined the effect of p38 mitogen-activated protein kinase (MAPK) inhibitors in models of nociception and correlated this effect with localization and expression levels of p38 MAPK in spinal cord. There was a rapid increase in phosphorylated p38 MAPK in spinal cord following intrathecal administration of substance P or intradermal injection of formalin. Immunocytochemistry revealed that phosphorylated p38 MAPK-immunoreactive cells were predominantly present in laminae I-IV of the dorsal horn. Double-staining with markers for neurons, microglia, astrocytes and oligodendrocytes unexpectedly revealed co-localization with microglia but not with neurons or other glia. Pretreatment with p38 MAPK inhibitors (SB20358 or SD-282) had no effect on acute thermal thresholds. However, they attenuated hyperalgesia in several nociceptive models associated with spinal sensitization including direct spinal activation (intrathecal substance P) and peripheral tissue inflammation (intraplantar formalin or carrageenan). Spinal sensitization, manifested by enhanced expression of cyclo-oxygenase-2 and inflammation-induced appearance of Fos-positive neurons, was blocked by pretreatment, but not post-treatment, with p38 MAPK inhibitors. Taken together, these results indicate that spinal p38 MAPK is involved in inflammation-induced pain and that activated spinal microglia play a direct role in spinal nociceptive processing.

Involvement of the p38 mitogen-activated protein kinase cascade in hepatocellular carcinoma.

The mitogen-activated protein kinase (MAPK) cascade is activated in response to various extracellular stimuli. The authors investigated the involvement of the p38 MAPK, a member of the MAPK superfamily, cascade in hepatoma cell lines and in human hepatocellular carcinoma (HCC) tissue specimens. Constitutively active mutant of MAPK kinase 6 (MKK6), which is upstream of p38 MAPK, was transfected into the HepG2 and HuH7 human hepatoma cell lines. The constitutive active mutant was constructed by replacing Ser-189 and Thr-193 with Glu. The growth and death of mutant MKK6-transfected hepatoma cells were analyzed by the WST-1 and sub-G1 assays. The surgically resected livers of 20 HCC patients were divided histologically into tumorous (T) and nontumorous (NT) lesions. p38 MAPK activity was analyzed using in vitro kinase assay and MKK6 activity was measured using Western blot analysis. Mutant MKK6 transfection increased p38 MAPK activity, cytochrome c release from the mitochondria to the cytosol, and caspase-3 activity, accompanied by apoptosis. In contrast, SB203580, a p38 MAPK-specific inhibitor, prevented MKK6-induced apoptosis in hepatoma cell lines. In the T lesions of 20 HCC parients, p38 MAPK and MKK6 activities were significantly lower compared with NT lesions (P < 0.05). There was a significant positive correlation between p38 MAPK and MKK6 activity (r = 0.507, P < 0.05). Larger tumors (> 20 mm) exhibited lower levels of p38 MAPK and MKK6 activity than did smaller tumors (P < 0.05). These findings suggested that reduction of the p38 MAPK cascade may account, in part, for the resistance to apoptosis, leading to the unrestricted cell growth of human HCC.

High glucose concentration decreases insulin-like growth factor type 1-mediated mitogen-activated protein kinase activation in bovine retinal endothelial cells

Clinical trials have incontrovertibly demonstrated that the onset and progression of diabetic retinopathy (DR) is influenced by the control of glucose levels in patients. In the present study, we examined the effect of glucose concentration on the responsiveness of bovine retinal endothelial cells (BREC) to insulin-like growth factor type 1 (IGF-1). Retinal endothelial cells were isolated from bovine retina and cultured in 5 or 20 mmol/L glucose with or without 100 ng/mL IGF-1. The level of cell growth and p42/44 and p38 mitogen-activated protein kinase (MAPK) activation was determined using the alamarBlue (Serotech) assay and Western blotting, respectively. IGF-1 significantly enhanced cell growth in BREC exposed to 5 mmol/L glucose but not in cells exposed to high glucose concentrations (20 mmol/L). IGF-1 induced a transient activation of p42/44 MAPK, with peak activation at 15 minutes in cells exposed to 5 mmol/L glucose; however, no increase in p42/44 MAPK was evident at the higher glucose concentration of 20 mmol/L. There was no significant change in the level of p38 MAPK during the time period examined when IGF-1 was also present. However, high glucose concentrations alone increased the level of p38 MAPK after 60 minutes and the level of p42/44 MAPK after only 15 minutes exposure in 20 mmol/L glucose. Thus, BREC exposed to high glucose concentrations are not sensitive to IGF-1 and this is due, at least in part, to a reduced activation of the p42/44 MAPK pathway. Furthermore, the presence of IGF-1 appears to exert a protective effect on the cells in high glucose concentration by preventing progression through the cell cycle. © 2003 Elsevier Inc. All rights reserved.

Characterization of the role of the AMP-activated protein kinase in the stimulation of glucose transport in skeletal muscle cells.

Stimulation of AMP-activated protein kinase (AMPK) in skeletal muscle has been correlated with an increase in glucose transport. Here, we demonstrate that adenoviral-mediated expression of a constitutively active mutant of AMPK alpha leads to activation of glucose transport in a skeletal-muscle cell line, similar to that seen following treatment with 5-amino-imidazolecarboxamide (AICA) riboside, hyperosmotic stress or insulin. In contrast, expression of a dominant-negative form of AMPK blocked the stimulation of glucose transport by both AICA riboside and hyperosmotic stress, but was without effect on either insulin or phorbol-ester-stimulated transport. These results demonstrate that activation of AMPK is sufficient for stimulation of glucose uptake into muscle cells, and is a necessary component of the AICA riboside- and hyperosmotic-stress-induced pathway leading to increased glucose uptake. On the other hand, AMPK is not required in the insulin- or phorbol-ester-mediated pathways. Long-term (5 days) expression of the constitutively active AMPK mutant increased protein expression of GLUT1, GLUT4 and hexokinase II, consistent with previous reports on the chronic treatment of rats with AICA riboside. Expression of constitutively active AMPK had no detectable effect on p38 mitogen-activated protein kinase levels, although interestingly the level of protein kinase B was decreased. These results demonstrate that long-term activation of AMPK is sufficient to cause increased expression of specific proteins in muscle. Our results add further support to the hypothesis that long-term activation of AMPK is involved in the adaptive response of muscle to exercise training.