Contrasting actions of prolonged mitogen-activated protein kinase activation on cell survival

Activation of the ERK mitogen-activated protein kinase pathway has been implicated in pro-survival and cellular protective mechanisms, so that chronic ERK activation may be a useful therapeutic strategy. Here, we further explored the consequences of prolonged ERK activation following expression of constitutively active form of MEK, MEK-EE, in cardiac myocytes. We confirmed that chronic MEK-EE overexpression halved myocyte death following glucose deprivation, but surprisingly this was not associated with preserved intracellular ATP levels. Whilst activities of a number of antioxidant enzymes were not altered upon MEK-EE expression, paradoxically Cu/Zn superoxide dismutase activity was almost halved upon MEK-EE expression. When we then exposed myocytes to the superoxide generator menadione, we observed significantly higher death of MEK-EE expressing myocytes. Pre-incubation with U0126 inhibited menadione-induced death. Our results are the first to show that MEK-ERK signalling can act to increase or decrease cell survival, the outcome depending on the form of stress stimulus encountered.     

Dehydroepiandrosterone negatively regulates the p38 mitogen-activated protein kinase pathway by a novel mitogen-activated protein kinase phosphatase

Dehydroepiandrosterone-sulfate, the sulfated form of dehydroepiandrosterone, is the most abundant steroid in young adults, but gradually declines with aging. In humans, the clinical application of dehydroepiandrosterone targeting some collagen diseases, such as systemic lupus erythematosus, as an adjunctive treatment has been applied in clinical trial. Here, we report that dehydroepiandrosterone may negatively regulate the mitogen-activated protein kinase pathway in humans via a novel dual specificity protein phosphatase, DDSP (dehydroepiandrosterone-enhanced dual specificity protein phosphatase). DDSP is highly homologous to LCPTP/HePTP, a tissue-specific protein tyrosine phosphatase (PTP) which negatively regulates both ERK and p38-mitogen-activated protein kinase, and is transcribed from the PTPN7 locus by alternative splicing. Although previous reports have shown that the mRNA expression of the LCPTP/HePTP gene was inducible by extracellular signals such as T-cell antigen receptor stimulation, reverse transcribed (RT)-PCR experiments using specific sets of primers suggested that the expression of LCPTP/HePTP was constitutive while the actual inducible sequence was that of DDSP. Furthermore DDSP was widely distributed among different types of human tissues and specifically interacted with p38-mitogen-activated protein kinase. This inducible negative regulation of the p38-mitogen-activated protein kinase-dependent pathway may help to clarify the broad range of dehydroepiandrosterone actions, thereby aiding the development of new preventive or adjunctive applications for human diseases.     

A rit GTPase-p38 mitogen-activated protein kinase survival pathway confers resistance to cellular stress

Cells mobilize diverse signaling cascades to protect against stress-mediated injury. Ras family GTPases play a pivotal role in cell fate determination, serving as molecular switches to control the integration of multiple signaling pathways. p38 mitogen-activated protein kinase (MAPK) signaling serves as a critical fulcrum in this process, regulating networks that stimulate cellular apoptosis but also have the capacity to promote cell survival. However, relatively little is known concerning this functional dichotomy, particularly the regulation of p38-dependent survival pathways. Here, we demonstrate that the Rit GTPase promotes cell survival by directing an unexpected p38 MAPK-dependent AKT survival pathway. Following stress exposure, Rit small hairpin RNA interference (shRNAi)-treated cells display increased apoptosis and selective disruption of p38 MAPK signaling, while expression of constitutively activated Rit promotes p38-AKT-dependent cell survival. Rit, but not Ras or Rap GTPases, can associate with, and is critical for, stress-mediated activation of the scaffolded p38-MK2-HSP27-AKT prosurvival signaling complex. Together, our studies establish Rit as a central regulator of a p38 MAPK-dependent signaling cascade that functions as a critical cellular survival mechanism in response to stress.     

The proteasome inhibitor bortezomib enhances ATRA-induced differentiation of neuroblastoma cells via the JNK mitogen-activated protein kinase pathway

Neuroblastoma (NB) is the most common extracranial solid tumor in childhood. Differentiated human NBs are associated with better outcome and lower stage; induction of differentiation is considered to be therapeutically advantageous. All-trans retinoic acid (ATRA) has been shown to induce the differentiation of neuroblastoma (NB) cell lines. The proteasome inhibitor bortezomib inhibits cell growth and angiogenesis in NBs. Here, we investigated the synergistic effect between bortezomib and ATRA in inducing NB cell differentiation in different NB cell lines. Bortezomib combined with ATRA had a significantly enhanced antiproliferative effect. This inhibition was characterized by a synergistic increase in neuronal differentiation. At the same time, the combination therapy showed little neuronal toxicity which was assessed in primary cultures of rat cerebellar granule cells by the MTT assay, PI staining. The combination of bortezomib and ATRA triggered increased differentiation through the activation of proteins, including RARα, RARβ, RARγ, p-JNK and p21, compared with ATRA treatment alone. Using JNK inhibitor SP600125 to block JNK-dependent activity, the combination therapy-induced neuronal differentiation was partially attenuated. In addition, p21 shRNA had no effect on the combination therapy-induced neuronal differentiation. The in vivo antitumor activities were examined in human NB cell xenografts and GFP-labeled human NB cell xenografts. Treatment of human NB cell CHP126-bearing nude mice with ATRA plus bortezomib resulted in more significant tumor growth inhibition than mice treated with either drug alone. These findings provide the rationale for the development of a new therapeutic strategy for NB based on the pharmacological combination of ATRA and bortezomib.     

EhMAPK, the mitogen-activated protein kinase from Entamoeba histolytica is associated with cell survival

Mitogen Activated Protein Kinases (MAPKs) are a class of serine/threonine kinases that regulate a number of different cellular activities including cell proliferation, differentiation, survival and even death. The pathogen Entamoeba histolytica possess a single homologue of a typical MAPK gene (EhMAPK) whose identification was previously reported by us but its functional implications remained unexplored. EhMAPK, the only mitogen-activated protein kinase from the parasitic protist Entamoeba histolytica with Threonine-X-Tyrosine (TXY) phosphorylation motif was cloned, expressed in E. coli and functionally characterized under different stress conditions. The expression profile of EhMAPK at the protein and mRNA level remained similar among untreated, heat shocked and hydrogen peroxide-treated samples in all cases of dose and time. But a significant difference was obtained in the phosphorylation status of the protein in response to different stresses. Heat shock at 43°C or 0.5 mM H(2)O(2) treatment enhanced the phosphorylation status of EhMAPK and augmented the kinase activity of the protein whereas 2.0 mM H(2)O(2) treatment induced dephosphorylation of EhMAPK and loss of kinase activity. 2.0 mM H(2)O(2) treatment reduced parasite viability significantly but heat shock and 0.5 mM H(2)O(2) treatment failed to adversely affect E. histolytica viability. Therefore, a distinct possibility that activation of EhMAPK is associated with stress survival in E. histolytica is seen. Our study also gives a glimpse of the regulatory mechanism of the protein under in vivo conditions. Since the parasite genome lacks any typical homologue of mammalian MEK, the dual specificity kinases which are the upstream activators of MAPK, indications of the existence of some alternate regulatory mechanisms of the EhMAPK activity is perceived. These may include the autophosphorylation activity of the protein itself in combination with some upstream phosphatases which are not yet identified.     

Catalytic reaction pathway for the mitogen-activated protein kinase ERK2

The structural, functional, and regulatory properties of the mitogen-activated protein kinases (MAP kinases) have long attracted considerable attention owing to the critical role that these enzymes play in signal transduction. While several MAP kinase X-ray crystal structures currently exist, there is by comparison little mechanistic information available to correlate the structural data with the known biochemical properties of these molecules. We have employed steady-state kinetic and solvent viscosometric techniques to characterize the catalytic reaction pathway of the MAP kinase ERK2 with respect to the phosphorylation of a protein substrate, myelin basic protein (MBP), and a synthetic peptide substrate, ERKtide. A minor viscosity effect on k(cat) with respect to the phosphorylation of MBP was observed (k(cat) = 10 +/- 2 s(-1), k(cat)(eta) = 0.18 +/- 0.05), indicating that substrate processing occurs via slow phosphoryl group transfer (12 +/- 4 s(-1)) followed by the faster release of products (56 +/- 4 s(-1)). At an MBP concentration extrapolated to infinity, no significant viscosity effect on k(cat)/K(m(ATP)) was observed (k(cat)/K(m(ATP)) = 0.2 +/- 0.1 microM(-1) s(-1), k(cat)/K(m(ATP))(eta) = -0.08 +/- 0.04), consistent with rapid-equilibrium binding of the nucleotide. In contrast, at saturating ATP, a full viscosity effect on k(cat)/K(m) for MBP was apparent (k(cat)/K(m(MBP)) = 2.4 +/- 1 microM(-1) s(-1), k(cat)/K(m(MBP))(eta) = 1.0 +/- 0.1), while no viscosity effect was observed on k(cat)/K(m) for the phosphorylation of ERKtide (k(cat)/K(m(ERKtide)) = (4 +/- 2) x 10(-3) microM(-1) s(-1), k(cat)/K(m(ERKtide))(eta) = -0.02 +/- 0.02). This is consistent with the diffusion-limited binding of MBP, in contrast to the rapid-equilibrium binding of ERKtide, to form the ternary Michaelis complex. Calculated values for binding constants show that the estimated value for K(d(MBP)) (/= 1.5 mM). The dramatically higher catalytic efficiency of MBP in comparison to that of ERKtide ( approximately 600-fold difference) is largely attributable to the slow dissociation rate of MBP (/=56 s(-1)), from the ERK2 active site.     

The mitogen-activated protein kinase cascade promotes myoblast cell survival by stabilizing the cyclin-dependent kinase inhibitor,p21WAF1 protein

During myogenesis, proliferating myoblasts withdraw from the cell cycle and are either eliminated by programmed cell death or differentiate into mature myotubes. Previous studies indicate that mitogen-activated protein kinase (MAPK) activity is significantly induced with the onset of terminal differentiation of C2 myoblasts. We have investigated the part played by the MAPK pathway in the differentiation of C2 myoblasts. Specific activation of MAPK by expression of an active Raf1-estrogen receptor chimera protein reduced significantly the number of myoblasts undergoing programmed cell death in the differentiation medium. Activation of Raf1 prevented the proteolytic activation of the proapoptotic caspase 9-protein during differentiation. The antiapoptotic function of Raf1 correlated with accumulation of the p21WAF1 protein resulting from its increased stability. Antisense expression of p21 was used to determine whether the p21WAF1 protein mediated the antiapoptotic activity of Raf1. Reduction of p21WAF1 protein in muscle cells abolished the antiapoptotic activity of the MAPK pathway. We conclude that MAPK contributes to muscle differentiation by preventing apoptotic cell death of differentiating myoblasts and that this activity is mediated by stabilization of the p21WAF1 protein.     

Carbon monoxide has anti-inflammatory effects involving the mitogen-activated protein kinase pathway

The stress-inducible protein heme oxygenase-1 provides protection against oxidative stress. The anti-inflammatory properties of heme oxygenase-1 may serve as a basis for this cytoprotection. We demonstrate here that carbon monoxide, a by-product of heme catabolism by heme oxygenase, mediates potent anti-inflammatory effects. Both in vivo and in vitro, carbon monoxide at low concentrations differentially and selectively inhibited the expression of lipopolysaccharide-induced pro-inflammatory cytokines tumor necrosis factor-alpha, interleukin-1beta, and macrophage inflammatory protein-1beta and increased the lipopolysaccharide-induced expression of the anti-inflammatory cytokine interleukin-10. Carbon monoxide mediated these anti-inflammatory effects not through a guanylyl cyclase-cGMP or nitric oxide pathway, but instead through a pathway involving the mitogen-activated protein kinases. These data indicate the possibility that carbon monoxide may have an important protective function in inflammatory disease states and thus has potential therapeutic uses.     

Correlation of mitogen-activated protein kinase activities with cell survival and apoptosis in porcine granulosa cells

The regulation of granulosa cell survival and death is critical for determining the fate of ovarian follicles. Mitogen-activated protein kinases (MAPKs) play central roles in various cellular responses, but the relationship between MAPK activities and granulosa cell survival as well as death is poorly understood. The present study examines the roles of the extracellular signal-regulated kinase (ERK) and p38 MAPK activities in porcine granulosa cells in response to survival factors and oxidative stress. Cell survival and apoptosis were evaluated by Trypan blue staining, DNA fragmentation, and chromatin staining with Hoechst 33342. Cell survival induced by serum or by follicle-stimulating hormone (FSH) was inhibited when ERK activity was attenuated with PD98059, which led to the induction of apoptosis. The p38 inhibitor SB203580 significantly decreased the cell survival evoked by FSH, but not by serum. Even in the presence of 10% serum, H(2)O(2) caused apoptosis, indicating that H(2)O (2) may be an atretogenic factor or its mediator. Interestingly, this induction of apoptosis was also prevented by SB203580, suggesting that p38 is involved in an apoptotic pathway induced by H(2)O (2) as well as in a survival pathway evoked by FSH in granulosa cells. These results indicate that whereas ERK activity is critical to the survival of granulosa cells, p38 activity contributes to their survival or apoptosis depending on the stimulus.     

The role of phosphatidylinositide-3-kinase in basal mitogen-activated protein kinase activity and cell survival

Phosphatidylinositide-3-OH-kinase (PI 3-kinase) is an upstream activator of p42/p44 mitogen-activated protein kinase (MAPK), but the role of PI 3-kinase-dependent MAPK remains obscure. Here we demonstrate that in a variety of different cell types, PI 3-kinase inhibition results in an inhibition of MAPK in unstimulated cells but does not interfere with growth factor-, or TPA-induced MAPK activity. Furthermore, inhibition of either PI 3-kinase or MEK/MAPK results in cell death in serum-starved cells. We concluded that basal, but not induced MAPK activity is mediated by PI 3-kinase and that this PI 3-kinase-mediated MEK/MAPK activity is essential for cell survival in quiescent cells.     

Unusual chemokine receptor antagonism involving a mitogen-activated protein kinase pathway

Antagonism of chemokines on chemokine receptors constitutes a new regulatory principle in inflammation. Eotaxin (CCL11), an agonist for CCR3 and an attractant of eosinophils, basophils, and Th2 lymphocytes, was shown to act as an antagonist for CCR2, which is widely expressed on leukocytes and is essential for inflammatory responses. In this report we provide direct evidence for a novel mechanism how chemokine receptor function can be arrested by endogenous ligands. We show that binding of eotaxin to CCR2 stimulates the mitogen-activated protein kinases extracellular signal-regulated kinase 1/2 (ERK1/2). Activation of the mitogen-activated protein kinase kinase 1/2-ERK pathway is indispensable for eotaxin-mediated attenuation of CCR2 function, as inhibition of ERK phosphorylation abolishes the arresting effect. ERK is also activated by CCR2 agonists, e.g., monocyte chemoattractant protein-1 (CCL2). However, the involved pathways are different, although in either case coupling of CCR2 to pertussis toxin-sensitive heterotrimeric G proteins is necessary. The results are in agreement with the view that CCR2 could assume different activation states depending on the ligand it encounters. With respect to actin polymerization and calcium mobilization, the different activation states lead to agonistic and antagonistic responses. It is conceivable that the intracellular signal transduction pathway that is activated by eotaxin could cause an attenuation of proinflammatory responses mediated by CCR2.     

The neuroprotective agent, valproic acid, regulates the mitogen-activated protein kinase pathway through modulation of protein kinase A signalling in Dictyostelium discoideum

Activation of the mitogen-activated protein kinase (MAPK) cascade gives rise to a neuroprotective effect in a variety of cell types. The bipolar disorder treatment, valproic acid (VPA), increases the activity of this pathway by modulating extracellular signal-regulated kinase 2 (ERK2) phosphorylation through an unknown mechanism. To investigate the molecular basis of this effect, we have used the biomedical model system Dictyostelium discoideum to dissect this signalling pathway. We find that, similar to mammalian systems, VPA causes a transient increase in the activation of the MAPK signalling pathway, as shown by ERK2 phosphorylation. We show that the MAP kinase and phosphatase, protein kinase A (PKA) and glycogen synthase kinase signalling pathways all function in controlling the levels of phospho-ERK2 (pERK2). We find that VPA induces elevated pERK2 levels through attenuation of the PKA signalling pathway. Interestingly, pERK2 levels are also controlled by another bipolar disorder drug, lithium, providing a common effect of these two drugs. This work therefore suggests a conserved pathway in eukaryotes that is targeted by neuroprotective and bipolar disorder drugs and allows us to propose a model for this neuroprotective effect.