Increased Phosphorylation Of P38 MAPK Research Articles

Neuroprotective effects of constituents of the root bark of Dictamnus dasycarpus in mouse hippocampal cells

Glutamate-induced oxidative injury causes neuronal degeneration related to many central nervous system diseases, such as Parkinson's disease, Alzheimer's disease, epilepsy and ischemia. The bioassay-guided fractionation of the EtOH extract of the root bark of Dictamnus dasycarpus Trucz. provided one neuroprotective limonoid, obacunone, together with a degraded limonoid, fraxinellone and two alkaloids, dictamine and haplopine. At concentrations of 100-150 microM, obacunone showed the potent neuroprotective effects on glutamateinduced neurotoxicity and induced the expression of heme oxygenase (HO)-1 in the mouse hippocampal HT22 cells. In addition, we found that obacunone increased p38 MAPK phosphorylation and induced HO-1 expression via p38 MAPK pathway. These results suggest that obacunone isolated from the root bark of D. dasycarpus increases cellular resistance to glutamate-induced oxidative injury in mouse hippocampal HT22 cells, presumably through the p38 MAPK pathway-dependent HO-1 expression. These results suggest that obacunone could be the effective candidates for the treatment of ROS-related neurological diseases.

The anti-apoptotic effect of alpha-lipoic acid against neointimal hyperplasia via induction of Nur77-mediation.

e13624 Background: Neointimal hyperplasia of vascular smooth muscle cells (VSMCs) is caused by increased proliferation and decreased apoptosis. We previously reported that alpha-lipoic acid (ALA), a naturally occurring antioxidant, prevents neointimal hyperplasia through its anti-inflammatory property. Nuclear receptor Nur77 (NR4A1; NGFI-B/TR3) induces apoptosis of various cancer cells and also inhibits VSMC proliferation. However, it is unknown whether Nur77-mediated apoptotic pathway is involved in the inhibitory effect of ALA on neointimal hyperplasia. We explored the hypothesis that ALA protects against neointimal hyperplasia through the induction of Nur77-mediated apoptosis in VSMCs. Methods: VSMCs were isolated from the thoracic aorta of 4-week-old male Sprague–Dawley (SD) rats and were treated with ALA (2 mmol/L). Results: ALA treatment induced VSMC apoptosis, as evidenced by increases in Bax expression, cytochrome c release and caspase-3 activation. Also, ALA enhanced the expression and cytoplasmic localization of Nur77, resulting in interaction of Nur77 with Bcl-2, which triggers Nur77-mediated apoptosis. siRNA-mediated downregulation of Nur77 diminished the pro-apoptotic effect of ALA on VSMCs. Moreover, ALA increased p38 MAPK phosphorylation and inhibition of p38 MAPK abolished ALA-induced apoptosis and cytoplasimc localization of Nur77. Adenovirus-mediated transfer of shRNA against Nur77 diminished inhibitory effect of ALA on neointimal hyperplasia. Conclusions: Collectively, these data indicate that ALA induced cell apoptosis through induction of p38 MAPK/Nur77-mediation. This study also suggests that stimulation of Nur77-mediated apoptotic pathway in VSMCs can be a potential therapeutic strategy to treatment of cancer. No significant financial relationships to disclose.

Abstract 4018: Crosstalk between the pleiotrophin-anaplastic lymphoma kinase axis and the protein tyrosine phosphatase ζ signaling pathways

Abstract Pleiotrophin (PTN) is a heparin-binding growth factor that can support tumor growth, invasion, angiogenesis, and metastasis. The mechanism of action (receptor signaling pathway) responsible for mediating PTN's biological effects, however, is still controversial. Here we show that an antibody targeting the ligand-binding domain of anaplastic lymphoma kinase (ALK) is capable of blocking the signaling effects of PTN in a glioblastoma cell line that expresses high levels of ALK. This corroborates recent findings by Stylianou, et al. (2009), who showed disruption of PTN activity with a single chain variable IgG fragment that binds to the ALK ligand-binding domain. Furthermore, expression of protein tyrosine phosphatase receptor ζ (Ptprz) in cells resulted in crosstalk with the PTN-ALK signaling axis at the level of p38 MAPK. While PTN increased p38 MAPK phosphorylation, the presence of Ptprz decreased its phosphorylation, both at its basal as well as PTN-enhanced levels. Enhancement of p38 MAPK phosphorylation by PTN is possibly mediated through activation of MKK4, as it was phosphorylated in the presence of PTN. On the other hand, the mechanism for Ptprz-mediated suppression of p38 MAPK is not yet clear, as there was no change in phosphorylation of MKK3, MKK4, or MKK6 after its over-expression. These findings suggest that ALK is playing a direct role in PTN activity, and that the Ptprz signaling pathway may act in a separate but inter-connecting fashion. Parallel targeting of both the ALK and Ptprz signaling pathway may be necessary for the treatment of tumors (glioblastoma) expressing both membrane proteins. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4018.

EGCG protects against oxLDL‐induced endothelial dysfunction by inhibiting LOX‐1‐mediated signaling

Lectin‐like oxidized low‐density lipoprotein receptor‐1 (LOX‐1), originally identified as the major receptor for oxidized low‐density lipoprotein (oxLDL) in endothelial cells, plays a major role in the pathology of vascular diseases. In this study, we hypothesize that the most abundant polyphenolic compound in tea, epigallocatechin‐3‐gallate (EGCG), can down‐regulate parameters of endothelial dysfunction by modulating LOX‐1‐regulated cell signaling. In cultured human umbilical vein endothelial cells (HUVECs), exposure to oxLDL increased the expression of LOX‐1 at the RNA and protein levels. OxLDL rapidly activated the membrane translocation of Rac1 and p47phox and the subsequent induction of reactive oxygen species generation. It also increased p38MAPK phosphorylation and decreased the phosphorylation of Akt as well as nuclear translocation of NF‐κB. In addition, oxLDL diminished the expression of endothelial NO synthase, enhanced the expression of endothelin‐1 and adhesion molecules, and increased the adherence of monocytic THP‐1 cells to HUVECs. Pretreatment with EGCG, however, exerted significant cytoprotective effects. Results from this study may provide insight into a possible molecular mechanism by which EGCG suppresses oxLDL‐mediated vascular endothelial dysfunction. (This study was supported by grants from the National Science Council; NSC98‐2320‐B‐039‐020‐MY3).

Role of TGF-β1 and MAP Kinases in the Antiproliferative Effect of Aspirin in Human Vascular Smooth Muscle Cells

BackgroundWe aimed to test the antiproliferative effect of acetylsalicylic acid (ASA) on vascular smooth muscle cells (VSMC) from bypass surgery patients and the role of transforming growth factor beta 1 (TGF-β1).Methodology/Principal FindingsVSMC were isolated from remaining internal mammary artery from patients who underwent bypass surgery. Cell proliferation and DNA fragmentation were assessed by ELISA. Protein expression was assessed by Western blot. ASA inhibited BrdU incorporation at 2 mM. Anti-TGF-β1 was able to reverse this effect. ASA (2 mM) induced TGF-β1 secretion; however it was unable to induce Smad activation. ASA increased p38MAPK phosphorylation in a TGF-β1-independent manner. Anti-CD105 (endoglin) was unable to reverse the antiproliferative effect of ASA. Pre-surgical serum levels of TGF-β1 in patients who took at antiplatelet doses ASA were assessed by ELISA and remained unchanged.Conclusions/Significance In vitro antiproliferative effects of aspirin (at antiinflammatory concentration) on human VSMC obtained from bypass patients are mediated by TGF-β1 and p38MAPK. Pre-surgical serum levels of TGF- β1 from bypass patients who took aspirin at antiplatelet doses did not change.

EGCG protects against oxidized LDL-induced endothelial dysfunction by inhibiting LOX-1-mediated signaling

Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), originally identified as the major receptor for oxidized low-density lipoprotein (oxLDL) in endothelial cells, plays a major role in the pathology of vascular diseases. Green tea consumption is associated with reduced cardiovascular mortality in some epidemiological studies. In the present study, we hypothesized that the most abundant polyphenolic compound in tea, epigallocatechin-3-gallate (EGCG), can downregulate parameters of endothelial dysfunction by modulating LOX-1-regulated cell signaling. In cultured human umbilical vein endothelial cells (HUVECs), exposure to oxLDL (130 microg/ml), which led to an increase in LOX-1 expression at the RNA and protein levels, was abrogated by addition of EGCG or DPI, a well-known inhibitor of flavoproteins, suggesting the involvement of NADPH oxidase. Furthermore, oxLDL rapidly activated the membrane translocation of Rac-1 and p47phox and the subsequent induction of ROS generation, which was suppressed markedly by pretreatment with EGCG or anti-LOX-1 monoclonal antibody. OxLDL also increased p38 MAPK phosphorylation and decreased phosphorylation of the amino-terminal region of Akt, with maximal induction at about 30 min, and NF-kappaB phosphorylation within 1 h, resulting in redox-sensitive signaling. In addition, oxLDL diminished the expression of endothelial nitric oxide synthase (eNOS), enhanced the expression of endothelin-1 and adhesion molecules (ICAM, E-selectin, and monocyte chemoattractant protein-1), and increased the adherence of monocytic THP-1 cells to HUVECs. Pretreatment with EGCG, however, exerted significant cytoprotective effects in all events. These data suggest that EGCG inhibits the oxLDL-induced LOX-1-mediated signaling pathway, at least in part, by inhibiting NADPH oxidase and consequent ROS-enhanced LOX-1 expression, which contributes to further ROS generation and the subsequent activation of NF-kappaB via the p38 MAPK pathway. Results from this study may provide insight into a possible molecular mechanism by which EGCG suppresses oxLDL-mediated vascular endothelial dysfunction.

Influence of thiopental on MAPK and NF-κB activation in A549 cells

Abstract Thiopental (TP) along with being an intravenous anaesthetic is frequently used in high doses to treat increased intracranial hypertension. Long-term treatments result in its accumulation in the lungs and an increased incidence of nosocomial infections. Since TP has been shown to modulate TNF-induced inflammatory pathways in lymphocytes, we hypothesised that TP may modulate pro-inflammatory events in human pulmonary epithelial cells. We investigated the potential influence of TP on MAPK and NF-κB signal transduction pathways in the absence and presence of TNF. TP at 1 mg/ml concentrations produced a 10-fold increase in p38 activation as assessed by Western blotting using monoclonal antibodies specific for double phosphorylated, thereby activated form of p38. This increase in p38 MAPK phosphorylation was evident as early as 10 minutes (4.5-fold), peaked at 60 minutes (11.3-fold) with return towards baseline (3.6-fold) in 2 hours. This response was specific for p38, since there was no evidence for activation of NF-κB pathway by TP as judged from Western blot analysis of steady-state IκB levels. TNF activated both p38 (2.4–11.4-fold increases) and depleted IκB (64–35% of control) in a concentration dependent manner (0.1–10 ng/ml). Interestingly, in the presence of TP, TNF did not produce a further increase in p38 activation. In addition, TP tended to attenuate TNF-induced NF-κB activation. These data suggest that high dose TP has the capacity to specifically alter p38MAPK activation and to modulate TNF signalling in epithelial cells, which might have implications to immunological responses in the lung.

YC-1 enhances natural killer cell differentiation from hematopoietic stem cells

NK cells play crucial roles in innate immunity and adaptive immunity. The detailed mechanisms, however, governing NK cell development remains unclear. In this study, we report that YC-1 significantly enhances NK cell populations differentiated from human umbilical cord blood hematopoietic stem cells (HSCs). NK cells increased by YC-1 display both phenotypic and functional features of fully mature NK (mNK) cells, but YC-1 does not affect the activation of mNK cells. YC-1 did not affect cGMP production and phosphorylation of STAT-5 which is essential for IL-15R signaling. On the other hand, YC-1 increased p38 MAPK phosphorylation during NK cell differentiation. Furthermore, p38 inhibitor SB203580 inhibited the differentiation of NK cells enhanced by YC-1. Taken together, these data suggest that YC-1 enhances NK cell differentiation through the activation of p38 MAPK which is involved in NK cell differentiation.

OSU-DY7, a Novel D-Tyrosinol Derivative, Mediates Cytotoxicity in Chronic Lymphocytic Leukemia and Lymphoblastic Lymphoma through p38 Mitogen-Activated Protein Kinase Pathway.

Abstract 3778Poster Board III-714 IntroductionChronic lymphocytic leukemia (CLL) is a most common form of adult leukemia with minimal treatment options. Drug resistance and associated immune deregulation limit use of current therapies, thus warranting need for alternative therapy development. Our laboratories recently identified a novel D-tyrosinol-derived compound targeting p38 MAPK pathway, OSU-DY7 [(R)-2-amino-3-(4-heptyloxy-phenyl)-propan-1-ol]. Here we demonstrate the efficacy of OSU-DY7 for lymphocytic cell lines and primary B cells from CLL patients. Materials and MethodsWe tested the pre-clinical efficacy of OSU-DY7 in primary CLL B cells and B cell lines representing CLL (MEC-1), ALL (697), and lymphoblastic lymphoma (Raji and Ramos). ResultsThe cytotoxicity of OSU-DY7 was both dose- and time-dependent. The IC50 of OSU-DY7 at 24 hrs for MEC-1, 697, Raji, Ramos and primary B-CLL cells were 2.40 μM, 10.39 μM, 6.04 μM, 3.75 μM and 3.58 μM respectively. OSU-DY7 induced activation of caspase-3 and poly (adenocine diphosphate-ribose) polymerase (PARP) cleavage. Pancaspase inhibitor Z-VAD-FMK partially rescued OSU-DY7-induced cytotoxicity in CLL B cells and cell lines. Interestingly, OSU-DY7 mediated-cytotoxicity was associated with increased phosphorylation of p38 MAPK and its downstream target protein MAPKAPK2 in both lymphocytic cell lines and primary B-CLL cells. Compared with control group, the ratio of p-p38MAPK (Thr180Tyr182) versus p38MAPK in Raji cells treated with 2 μM, 4 μM and 8 μM of OSU-DY7 increased 2.2, 4.7 and 11 fold respectively (p<0.0001). Similar increase in p38MAPK phosphorylation was also observed in six CLL patient samples after treatment with OSU-DY7 2 μM, 4 μM and 8 μM for 24 hours (p=0.0004). Moreover, SB202190, a specific p38 MAPK inhibitor, decreased MAPKAPK2 protein level with concomitant rescue of the cells from the OSU-DY7 mediated-cytotoxicity. Thus pretreatment of Raji and primary CLL B cells with SB202190 reduced the OSU-DY7 induced cytotoxicity by 36.7% and 24.3% respectively by 24hrs (Raji:p<0.0001; primary CLL B cells: p<0.005 ). Furthermore, SB202190 rescued OSU-DY7 down regulated survivin protein by ∼2 fold (p<0.0001, n=3) and mRNA levels by 2.4 fold (95% CI: 1.56∼3.84 fold, p=0.0026, n=3) in Raji cells indicating a role for p38MAPK dependent regulation of survivin by OSU-DY7. ConclusionsThis study provides an evidence for a role of OSU-DY7 in p38 MAPK dependent activation and survivin down regulation associated with apoptosis in lymphocytic cells, thus warranting further development of this novel agent for alternative therapy for lymphocytic malignancies. [This work was supported by D. Warren Brown Foundation and Leukemia and Lymphoma Society] Disclosures:No relevant conflicts of interest to declare.

Engineered Early Embryonic Cardiac Tissue Increases Cardiomyocyte Proliferation by Cyclic Mechanical Stretch via p38-MAP Kinase Phosphorylation

Cardiomyocyte (CM) transplantation is one therapeutic option for cardiac repair. Studies suggest that fetal CMs display the best cell type for cardiac repair, which can finitely proliferate, integrate with injured host myocardium, and restore cardiac function. We have recently developed an engineered early embryonic cardiac tissue (EEECT) using embryonic cardiac cells and have shown that EEECT contractile properties and cellular proliferative response to cyclic mechanical stretch stimulation mimic developing fetal myocardium. However, it remains unknown whether cyclic mechanical stretch-mediated high cellular proliferation activity within EEECT reflects CM or non-CM population. Studies have shown that p38-mitogen-activated protein kinase (p38MAPK) plays an important role in both cyclic mechanical stretch stimulation and cellular proliferation. Therefore, in the present study, we tested the hypothesis that cyclic mechanical stretch (0.5 Hz, 5% strain for 48 h) specifically increases EEECT CM proliferation mediated by p38MAPK activity. Cyclic mechanical stretch increased CM, but not non-CM, proliferation and increased p38MAPK phosphorylation. Treatment of EEECT with the p38MAPK inhibitor, SB202190, reduced CM proliferation. The negative CM proliferation effects of SB202190 were not reversed by concurrent stretch stimulation. Results suggest that immature CM proliferation within EEECT can be positively regulated by mechanical stretch and negatively regulated by p38MAPK inhibition.

Epidermal growth factor competes with EGF receptor inhibitors to induce cell death in EGFR-overexpressing tumor cells

Epidermal growth factor receptor (EGFR) signaling plays an important role in cell growth and differentiation. Mutations in the EGFR gene and EGFR gene amplifications have been associated with increased responsiveness to selective EGFR tyrosine kinase inhibitors (EGFR-TKIs). By contrast, EGF may also stimulate apoptosis in tumor cells, depending on EGFR and Her2 ( erbB-2) expression levels. In the present study, we investigated cellular responses after EGFR activation by EGF, or inhibition by cetuximab and gefitinib. EGF treatment induced a near-immediate increase in p38 MAPK phosphorylation together with inactivation of ERK1/2. In contrast, gefitinib- and cetuximab-induced phosphorylation of p38 MAPK was much delayed, and gefitinib also induced a delayed activation of ERK1/2. EGF induced progressive cell death of A431 cells with prolonged treatment, whereas cetuximab- or gefitinib-treated cells showed temporary growth arrest and subsequent re-growth. Moreover, in combination treatment experiments, cetuximab or gefitinib competitively inhibited EGF-induced cell death. Normal WI38-VA13 cells did not display any noticeable changes in cell proliferation in response to EGF, gefitinib or cetuximab. EGF-induced death signaling is apparently irreversible: EGF induced significant EGFR phosphorylation/internalization and activated caspase-3, -8 and -9, effects that were not observed in cetuximab- or gefitinib-treated cells. Collectively, these results indicate that EGF may be a more potent cytotoxic agent than EGFR blockers in EGFR-overexpressing cancer cells.

Lithium action on glucose transport in insulin‐resistant rat skeletal muscle: roles of insulin signaling and p38 MAPK

In insulin‐sensitive rat skeletal muscle, lithium activates glucose transport via a mechanism involving p38 mitogen‐activated protein kinase (p38 MAPK). The mechanism underlying lithium action on glucose transport in insulin‐resistant rat skeletal muscle is unknown. We therefore assessed the effects of lithium on basal and insulin‐stimulated glucose transport, insulin signaling (insulin receptor (IR), Akt, and glycogen synthase kinase‐3 (GSK‐3)), and p38 MAPK in soleus muscle from obese Zucker rats. Lithium (10 mM LiCl) increased basal glucose transport by 49% (p&lt;0.05). Lithium did not induce basal IR tyrosine phosphorylation, but did enhance Akt ser473 phosphorylation (40%) and GSK‐3β ser9 phosphorylation (88%). Lithium potentiated insulin action on glucose transport (74%), Akt ser473 phosphorylation (39%), and GSK‐3β ser9 phosphorylation (36%), and elicited robust increases in p38 MAPK phosphorylation both in the absence (100%) or presence (88%) of insulin. Selective inhibition of p38 MAPK (using 10 µM A304000) completely blocked basal activation of glucose transport by lithium, and significantly reduced (42%) the lithium‐induced enhancement of insulin‐stimulated glucose transport. These results indicate that lithium enhances basal and insulin‐stimulated glucose transport and distal insulin signaling in insulin‐resistant skeletal muscle of obese Zucker rats via engagement of p38 MAPK.

Heat stress upregulates chaperone heat shock protein 70 and antioxidant manganese superoxide dismutase through reactive oxygen species (ROS), p38MAPK, and Akt

Chinese hamster lung fibroblasts V79 cells were treated with heat stress for 4 weeks with short duration (15 min) heat shock every alternate day in culture. It was observed that Hsp 70 and the antioxidant enzyme MnSOD became overexpressed during the chronic heat stress period. Both p38 MAPK and Akt became phosphorylated by chronic heat stress exposure. Simultaneous exposure to SB203580, a potent and specific p38MAPK inhibitor drastically inhibited the phosphorylation of p38MAPK and Akt. Furthermore, exposure to SB203580 also blocked the increase in Hsp70 and MnSOD levels and the elevated SOD activity brought about by chronic heat stress. Heat shock factor 1 (HSF1) transcriptional activity and nuclear translocation of HSF1 were prominently augmented by chronic heat stress, and this amplification is markedly reduced by concomitant exposure to SB203580. Also, activations of p38MAPK and Akt and upregulations of Hsp70 and MnSOD were observed on exposure to heat shock for a single exposure of longer duration (40 min). siRNA against p38MAPK notably reduced Akt phosphorylation by single exposure to heat stress and drastically diminished the rise in Hsp70 and MnSOD levels. Similarly, siRNA against Akt also eliminated the augmentation in Hsp70 and MnSOD levels but p38MAPK levels remained unaffected. Heat stress produced reactive oxygen species (ROS) in V79 fibroblasts. N-acetyl cysteine blocked the increase in phosphorylation of p38MAPK, amplification of Hsp70, and MnSOD levels by heat stress. Therefore, we conclude that heat stress-activated p38MAPK which in turn activated Akt. Akt acted downstream of p38MAPK to increase Hsp70 and MnSOD levels.Concise summary: Thermal injury of the skin over a long period of time has been associated with development of cancerous lesions. Also, in many cancers, the cytoprotective genes Hsp70 and MnSOD have been found to be overexpressed. Therefore, we considered it important to identify the signaling elements upstream of the upregulated survival genes in heat stress. We conclude that heat stress activated p38MAPK which in turn activated Akt. Akt mediated an augmentation in Hsp70 and MnSOD levels working downstream of p38MAPK.

Sonic Hedgehog signaling in astrocytes is dependent on p38 mitogen‐activated protein kinase and G‐protein receptor kinase 2

The molecular determinants of Sonic Hedgehog (Shh) signaling in mammalian cells and, in particular, those of the CNS are unclear. Here we report that primary cortical astrocyte cultures are highly responsive to both Shh protein and Hh Agonist 1.6, a selective, small molecule Smoothened agonist. Both agonists produced increases in mRNA expression of Shh-regulated gene targets, Gli-1 and Patched in a cyclopamine- and forskolin-sensitive manner. Using this model we show for the first time that Shh pathway activation mediates rapid increases in p38 MAPK phosphorylation, without altering phosphorylation of either extracellular-signal-regulated kinases or c-jun N-terminal kinases. Selective inhibition of p38 MAPK significantly attenuated Shh-dependent up-regulation of Gli-1, inter-alpha trypsin inhibitor and thrombomodulin mRNA, however did not affect expression of insulin-like growth factor 2 or a novel Shh target, membrane-associated guanylate kinase p55 subfamily member 6. Using RNAi and a constitutively-active mutant we show that Shh signaling to p38 MAPK and subsequent Gli-1 transcription requires G-protein receptor kinase 2. Taken together, these findings provide evidence for a central role of G-protein receptor kinase 2-dependent p38 MAPK activity in regulating Shh-mediated gene transcription in astrocytes.

P38-MAPK is involved in restoration of the lost protection of preconditioning by nicorandil in vivo

Nicorandil, a selective mitochondrial K ATP channel opener, reinstates the waned protection after multiple cycles of preconditioning. In this study, we determined the signal transduction activated in heart after 3 or 8 cycles of preconditioning and prolonged ischemia in rabbits treated with placebo or nicorandil. In a first series (eight groups) we evaluated the (%) infarct to risk ratio after 30 min ischemia/3 h reperfusion and in a second series (six groups), we assessed the intracellular levels of cyclic GMP (c-GMP), protein kinase C (PKC) activity and p38-mitogen activated protein kinase (p38-MAPK) phosphorylation from heart samples taken during the long ischemia. Cardioprotection by 3 cycles of preconditioning (11.7 ± 3.8% vs 45.9 ± 5.2% in the control, P < 0.001) was lost after 8 cycles (43.9 ± 5.1%, P = NS vs control). Nicorandil restored it to the levels of classic preconditioning (13.7 ± 2.4% vs 40.8 ± 3.5% in respective controls, P < 0.001). This was reversed by the p38-MAPK inhibitor SB203580 (48.8 ± 5.1%) which had no protective effect in the control group (44.6 ± 5.8%). In the placebo-treated rabbits, intracellular c-GMP and PKC were increased only in the group subjected to 3 cycles of preconditioning. Despite that nicorandil equalizes the intracellular levels of c-GMP, PKC and activated p38-MAPK at the long ischemia, specific alterations of p38-MAPK phosphorylation differentiate the protected groups. Our data delineate the signal transduction mechanism mediating the beneficial effect of nicorandil and imply that the recapture of the lost protection is due to a dynamic process of the intracellular mediators accompanied by an increase in p38-MAPK phosphorylation and not to an instantaneous event.

Tumor Necrosis Factor-α Induces Insulin Resistance in Endothelial Cells via a p38 Mitogen-Activated Protein Kinase-Dependent Pathway

Chronic inflammation contributes to vascular insulin resistance and endothelial dysfunction. Systemic infusion of TNF-alpha abrogates insulin's action to enhance skeletal muscle microvascular perfusion. In skeletal muscle TNF-alpha induces insulin resistance via the p38 MAPK pathway. To examine whether p38 MAPK also regulates TNF-alpha-induced vascular insulin resistance, bovine aortic endothelial cells (bAECs) were incubated+/-TNF-alpha (5 ng/ml) for 6 h in the presence or absence of SB203580 (p38 MAPK specific inhibitor, 10 microM) after serum starvation for 10 h. For the last 30 min, cells were treated+/-1 nM insulin, and insulin receptor substrate (IRS)-1, Akt, endothelial nitric oxide synthase (eNOS), p38 MAPK, ERK1/2, c-Jun N-terminal kinase, and AMP-activated protein kinase (AMPK) phosphorylation, and eNOS activity were measured. TNF-alpha increased p38 MAPK phosphorylation, potently stimulated IRS-1 serine phosphorylation, and blunted insulin-stimulated IRS-1 tyrosine and Akt phosphorylation and eNOS activity. TNF-alpha also potently stimulated the phosphorylation of ERK1/2 and AMPK. Treatment with SB203580 decreased p38 MAPK phosphorylation back to the baseline and restored insulin sensitivity of IRS-1 tyrosine and Akt phosphorylation and eNOS activity in TNF-alpha-treated bAECs without affecting TNF-alpha-induced ERK1/2 and AMPK phosphorylation. We conclude that in cultured bAECs, TNF-alpha induces insulin resistance in the phosphatidylinositol 3-kinase/Akt/eNOS pathway via a p38 MAPK-dependent mechanism and enhances ERK1/2 and AMPK phosphorylation independent of the p38 MAPK pathway. This differential modulation of TNF-alpha's actions by p38 MAPK suggests that p38 MAPK plays a key role in TNF-alpha-mediated vascular insulin resistance and may contribute to the generalized endothelial dysfunction seen in type 2 diabetes mellitus and the cardiometabolic syndrome.

Peroxisome Proliferator Activator Receptor γ Coactivator-1 Expression Is Reduced in Obesity

Peroxisome proliferator activator receptor-gamma coactivator 1 (PGC-1) is a major candidate gene for diabetes-related metabolic phenotypes, contributing to decreased expression of nuclear-encoded mitochondrial genes in muscle and adipose tissue. We have demonstrated that muscle expression of PGC-1alpha and -beta is reduced in both genetic (Lep(ob)/Lep(ob)) and acquired obesity (high fat diet). In C57BL6 mice, muscle PGC-1alpha expression decreased by 43% (p < 0.02) after 1 week of a high fat diet and persisted more than 11 weeks. In contrast, PGC-1alpha reductions were not sustained in obesity-resistant A/J mice. To identify mediators of obesity-linked reductions in PGC-1, we tested the effects of cellular nutrients in C2C12 myotubes. Although overnight exposure to high insulin, glucose, glucosamine, or amino acids had no effect, saturated fatty acids potently reduced PGC-1alpha and -beta mRNA expression. Palmitate decreased PGC-1alpha and -beta expression by 38% (p = 0.01) and 53% (p = 0.006); stearate similarly decreased expression of PGC-1alpha and -beta by 22% (p = 0.02) and 39% (p = 0.02). These effects were mediated at a transcriptional level, as indicated by an 11-fold reduction of PGC-1alpha promoter activity by palmitate and reversal of effects by histone deacetylase inhibition. Palmitate also (a) reduced expression of tricarboxylic acid cycle and oxidative phosphorylation mitochondrial genes and (b) reduced oxygen consumption. These effects were reversed by overexpression of PGC-1alpha or -beta, indicating PGC-1 dependence. Palmitate effects also required p38 MAPK, as demonstrated by 1) palmitate-induced increase in p38 MAPK phosphorylation, 2) reversal of palmitate effects on PGC-1 and mitochondrial gene expression by p38 MAPK inhibitors, and 3) reversal of palmitate effects by small interfering RNA-mediated decreases in p38alpha MAPK. These data indicate that obesity and saturated fatty acids decrease PGC-1 and mitochondrial gene expression and function via p38 MAPK-dependent transcriptional pathways.

Dietary red palm oil reduces ischaemia–reperfusion injury in rats fed a hypercholesterolaemic diet

We have previously shown that dietary red palm oil (RPO) supplementation improves functional recovery in hearts subjected to ischaemia-reperfusion. However, little knowledge exists concerning the effects of RPO supplementation of a high-cholesterol diet on ischaemia-reperfusion injury. The signalling mechanisms responsible for RPO's effects in the presence of cholesterol also remain to be elucidated. Therefore, the aim of the present study was to examine the effects of RPO, given with a high-cholesterol diet, on mitogen-activated protein kinase (MAPK) phosphorylation and apoptosis. Long-Evans rats were fed a control diet, a control diet containing 2% cholesterol, or a control diet containing 2% cholesterol and 7 g RPO per kg (CRPO) for 5 weeks. Hearts were excised and mounted on an isolated working heart perfusion apparatus. Cardiac function was measured after which hearts were freeze-clamped and used to assess MAPK phosphorylation and to evaluate apoptosis. Cholesterol supplementation caused a poor aortic output (AO) recovery compared with the control group (35.5 (sem 6.2) v. 55.4 (sem 2.5) %), but when RPO was added, the percentage AO increased significantly. The cholesterol group's poor AO was associated with a significant increase in p38-MAPK phosphorylation, whereas the CRPO-supplemented group showed as significant reduction in p38-MAPK phosphorylation when compared with the cholesterol-supplemented group. This significant reduction in p38-MAPK was also associated with reduced apoptosis as indicated by significant reductions in caspase-3 and poly(ADP-ribose) polymerase cleavage.

P2X7 receptor mediated phosphorylation of p38MAP kinase in the hippocampus

This study was designed to explore the effect of P2X7 receptor (P2X7R) activation on the expression of p38 MAP kinase (p38 MAPK) enzyme in hippocampal slices of wild-type (WT) and P2X7R −/− mice using the Western blot technique and to clarify its role in P2X7 receptor mediated [ 3H]glutamate release. ATP (1 mM) and the P2X7R agonist BzATP (100 μM) significantly increased p38 MAPK phosphorylation in WT mice, and these effects were absent in the hippocampal slices of P2X7R −/− mice. Both ATP- and BzATP-induced p38 MAPK phosphorylations were sensitive to the p38 MAP kinase inhibitor, SB203580 (1 μM). ATP elicited [ 3H]glutamate release from hippocampal slices, which was significantly attenuated by SB203580 (1 μM) but not by the extracellular signal-regulated kinase (ERK1/2) inhibitor, PD098095 (10 μM). Consequently, we suggest that P2X7Rs and p38 MAPK are involved in the stimulatory effect of ATP on glutamate release in the hippocampal slices of WT mice.

Lignan compounds and 4,4′-dihydroxybiphenyl protect C2C12 cells against damage from oxidative stress

Lignan compounds are known to have various biological activities, especially antioxidative effects. We investigated whether lignan compounds show antioxidative activity in myoblast C2C12 cells. Among 14 lignan compounds investigated, two lignans containing two phenolic functional groups, namely Gomisin J and GR-12, prevented hydrogen peroxide (H 2O 2)-induced cell death. A simple compound, 4,4′-dihydroxybiphenyl, which was found to be a common component of Gomisin J and GR-12, also largely prevented H 2O 2-induced cell death and almost completely prevented H 2O 2-induced increases in p38 MAPK phosphorylation. Our present results provide a useful in vitro system for clarifying the molecular mechanisms of lignan-mediated antioxidative effects and evaluating lead molecules toward the development of therapeutic drugs.