Skepinone-L is a selective p38 mitogen-activated protein kinase inhibitor

Until now, a lack of inhibitors with high potency and selectivity in vivo has hampered investigation of the p38 mitogen-activated protein kinase (MAPK) signaling pathway. We describe the design of skepinone-L, which is, to our knowledge, the first ATP-competitive p38 MAPK inhibitor with excellent in vivo efficacy and selectivity. Therefore, skepinone-L is a valuable probe for chemical biology research, and it may foster the development of a unique class of kinase inhibitors.     

Anti-inflammatory effects of a p38 mitogen-activated protein kinase inhibitor during human endotoxemia

The p38 mitogen-activated protein kinase (MAPK) participates in intracellular signaling cascades resulting in inflammatory responses. Therefore, inhibition of the p38 MAPK pathway may form the basis of a new strategy for treatment of inflammatory diseases. However, p38 MAPK activation during systemic inflammation in humans has not yet been shown, and its functional significance in vivo remains unclear. Hence, we exposed 24 healthy male subjects to an i.v. dose of LPS (4 ng/kg), preceded 3 h earlier by orally administered 600 or 50 mg BIRB 796 BS (an in vitro p38 MAPK inhibitor) or placebo. Both doses of BIRB 796 BS significantly inhibited LPS-induced p38 MAPK activation in the leukocyte fraction of the volunteers. Cytokine production (TNF-alpha, IL-6, IL-10, and IL-1R antagonist) was strongly inhibited by both low and high dose p38 MAPK inhibitor. In addition, p38 MAPK inhibition diminished leukocyte responses, including neutrophilia, release of elastase-alpha(1)-antitrypsin complexes, and up-regulation of CD11b with down-regulation of L-selectin. Finally, blocking p38 MAPK decreased C-reactive protein release. These data identify p38 MAPK as a principal mediator of the inflammatory response to LPS in humans. Furthermore, the anti-inflammatory potential of an oral p38 MAPK inhibitor in humans in vivo suggests that p38 MAPK inhibitors may provide a new therapeutic option in the treatment of inflammatory diseases.     

Feedback control of the protein kinase TAK1 by SAPK2a/p38alpha

TAB1, a subunit of the kinase TAK1, was phosphorylated by SAPK2a/p38alpha at Ser423, Thr431 and Ser438 in vitro. TAB1 became phosphorylated at all three sites when cells were exposed to cellular stresses, or stimulated with tumour necrosis factor-alpha (TNF-alpha), interleukin-1 (IL-1) or lipopolysaccharide (LPS). The phosphorylation of Ser423 and Thr431 was prevented if cells were pre-incubated with SB 203580, while the phosphorylation of Ser438 was partially inhibited by PD 184352. Ser423 is the first residue phosphorylated by SAPK2a/p38alpha that is not followed by proline. The activation of TAK1 was enhanced by SB 203580 in LPS-stimulated macrophages, and by proinflammatory cytokines or osmotic shock in epithelial KB cells or embryonic fibroblasts. The activation of TAK1 by TNF-alpha, IL-1 or osmotic shock was also enhanced in embryonic fibroblasts from SAPK2a/p38alpha-deficient mice, while incubation of these cells with SB 203580 had no effect. Our results suggest that TAB1 participates in a SAPK2a/p38alpha-mediated feedback control of TAK1, which not only limits the activation of SAPK2a/p38alpha but synchronizes its activity with other signalling pathways that lie downstream of TAK1 (JNK and IKK).     

Raf-1 is activated by the p38 mitogen-activated protein kinase inhibitor, SB203580

SB203580 (4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imi dazole) is widely used as a specific inhibitor of p38 mitogen-activated protein kinase (MAPK). Here, we report that SB203580 activates the serine/threonine kinase Raf-1 in quiescent smooth muscle cells in a dose-dependent fashion. The concentrations of SB203580 required lie above those necessary to inhibit p38 MAPK and we were unable to detect basal levels of active p38 MAPK. SB203580 does not directly activate Raf-1 in vitro, and fails to activate Ras, MEK, and ERK in intact cells. In vitro, however, SB203580-stimulated Raf-1 activates MEK1 in a coupled assay. We conclude that activation of Raf-1 by SB203580 is not mediated by an inhibition of p38 MAPK, is Ras-independent, and is uncoupled from MEK/ERK signaling.     

The mitogen-activated protein kinase p38 pathway is conserved in metazoans: cloning and activation of p38 of the SAPK2 subfamily from the sponge Suberites domuncula

Our recent data suggest that during auto- and allograft recognition in sponges (Porifera), cytokines are differentially expressed. Since the mitogen-activated protein kinase (MAPK) signal transduction modulates the synthesis and release of cytokines, we intended to identify one key molecule of this pathway. Therefore, a cDNA from the marine sponge Suberites domuncula encoding the MAPK was isolated and analyzed. Its encoded protein is 366 amino acids long (calculated Mr 42 209), has a TGY dual phosphorylation motif in protein kinase subdomain VIII and displays highest overall similarity to the mammalian p38 stress activated protein kinase (SAPK2), one subfamily of MAPKs. The sponge protein was therefore termed p38_SD. The overall homology (identity and similarity) between p38_SD and human p38alpha (CSBP2) kinase is 82%. One feature of the sponge kinase is the absence of threonine at position 106. In human p38alpha MAPK this residue is involved in the interaction with the specific pyridinyl-imidazole inhibitor; T106 is replaced in p38_SD by methionine. Inhibition studies with the respective inhibitor SB 203580 showed that it had no effect on the phosphorylation of the p38 substrate myelin basic protein. A stress responsive kinase Krs_SD similar to mammalian Ste20 kinases, upstream regulators of p38, had already previously been found in S. domuncula. The S. domuncula p38 MAPK is phosphorylated after treatment of the animal in hypertonic medium. In contrast, exposure of cells to hydrogen peroxide, heat shock and ultraviolet light does not cause any phosphorylation of p38. It is concluded that sponges, the oldest and most simple multicellular animals, utilize the conserved p38 MAPK signaling pathway, known to be involved in stress and immune (inflammatory) responses in higher animals.     

Human p38 mitogen-activated protein kinase inhibitor drugs inhibit Plasmodium falciparum replication

We recently demonstrated that human p38 mitogen-activated protein kinase (MAPK) inhibitors reduced in vitro and in vivo replication of the protozoan parasites Toxoplasma gondii and Encephalitozoon cuniculi. In this study, we assessed the efficacy of five p38 MAPK inhibitors to block the replication of Plasmodium falciparum in human erythrocytes cultured ex vivo and demonstrate that the pyridinylimidazole RWJ67657 and the pyrrolobenzimidazole RWJ68198 reduced P. falciparum replication, yielded trophozoites that were greatly diminished in size at 24 h, and that these two agents interfered with stage differentiation. Interestingly, the chloroquine-resistant strain W2 was significantly more sensitive to these drugs than was the chloroquine-sensitive strain HB3. These results suggest that pyridinylimidazoles and pyrrolobenzimidazoles designed to inhibit human p38 MAPK activation can be developed to treat malaria.     

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.     

An inhibitor of p38 mitogen-activated protein kinase protects neonatal cardiac myocytes from ischemia

Cellular ischemia results in activation of a number of kinases, including p38 mitogen-activated protein kinase (MAPK); however, it is not yet clear whether p38 MAPK activation plays a role in cellular damage or is part of a protective response against ischemia. We have developed a model to study ischemia in cultured neonatal rat cardiac myocytes. In this model, two distinct phases of p38 MAPK activation were observed during ischemia. The first phase began within 10 min and lasted less than 1 h, and the second began after 2 h and lasted throughout the ischemic period. Similar to previous studies using in vivo models, the nonspecific activator of p38 MAPK and c-Jun NH2-terminal kinase, anisomycin, protected cardiac myocytes from ischemic injury, decreasing the release of cytosolic lactate dehydrogenase by approximately 25%. We demonstrated, however, that a selective inhibitor of p38 MAPK, SB 203580, also protected cardiac myocytes against extended ischemia in a dose-dependent manner. The protective effect was seen even when the inhibitor was present during only the second, sustained phase of p38 MAPK activation. We found that ischemia induced apoptosis in neonatal rat cardiac myocytes and that SB 203580 reduced activation of caspase-3, a key event in apoptosis. These results suggest that p38 MAPK induces apoptosis during ischemia in cardiac myocytes and that selective inhibition of p38 MAPK could be developed as a potential therapy for ischemic heart disease.     

A novel method to identify protein kinase substrates: eEF2 kinase is phosphorylated and inhibited by SAPK4/p38delta

We have developed a method of general application for identifying putative substrates of protein kinases in cell extracts. Using this procedure, we identified the physiological substrates of several mitogen-activated protein kinase kinases and an authentic substrate of stress-activated protein kinase (SAPK) 2a/p38. A 120 kDa protein was detected in skeletal muscle extracts that was phosphorylated rapidly by SAPK4/p38delta, but poorly by SAPK2/p38, SAPK3/p38gamma, SAPK1/JNK or extracellular signal-regulated kinase 2 (ERK2). It was purified and identified as eukaryotic elongation factor 2 kinase (eEF2K). SAPK4/p38delta phosphorylated eEF2K at Ser359 in vitro, causing its inactivation. eEF2K became phosphorylated at Ser359 and its substrate eEF2 became dephosphorylated (activated) when KB cells were exposed to anisomycin, an agonist that activates all SAPKs, including SAPK4/p38delta. The anisomycin-induced phosphorylation of Ser359 was unaffected by SB 203580, U0126 or rapamycin, and was prevented by overexpression of a catalytically inactive SAPK4/p38delta mutant, suggesting that SAPK4/p38delta may mediate the inhibition of eEF2K by this stress. The phosphorylation of eEF2K at Ser359 was also induced by insulin-like growth factor-1. However, this was blocked by rapamycin, indicating that Ser359 is targeted by at least two signalling pathways.     

p38 mitogen-activated protein kinase/Hog1p regulates translation of the AU-rich-element-bearing MFA2 transcript

AU-rich-element (ARE)-mediated mRNA regulation occurs in Saccharomyces cerevisiae in response to external and internal stimuli through the p38 mitogen-activated protein kinase (MAPK)/Hog1p pathway. We demonstrate that the ARE-bearing MFA2 3' untranslated region (UTR) controls translation efficiency in a p38 MAPK/Hog1p-dependent manner in response to carbon source growth conditions. The carbon source-regulated effect on MFA2 3'-UTR-controlled translation involves the role of conserved ARE binding proteins, the ELAV/TIA-1-like Pub1p, which can interact with the cap/eIF4G complex, and the translation/mRNA stability factor poly(A) binding protein (Pab1p). Pub1p binds the MFA2 3'-UTR in a p38 MAPK/Hog1p-regulated manner in response to carbon source growth conditions. Significantly, the p38 MAPK/Hog1p is also required to modulate Pab1p in response to carbon source. We find that Pab1p can bind the MFA2 3'-UTR in a regulated manner to control MFA2 3'-UTR reporter translation. Binding of full-length Pab1p to the MFA2 3'-UTR correlates with translation repression. Importantly, Pab1p binds the MFA2 3'-UTR only in a PUB1 strain, and correlating with this requirement, Pub1p controls translation repression of MFA2 in a carbon source/Hog1p-regulated manner. These results suggest that the p38 MAPK/Hog1p pathway regulates 3'-UTR-mediated translation by modulating recruitment of Pab1p and Pub1p, which can interact with the translation machinery.     

p38 mitogen-activated protein kinase plays a key role in regulating MAPKAPK2 expression

One of three major families of the mitogen-activated kinases (MAPK), p38 as well as JNK, has been shown to transduce extracellular stress stimuli into cellular responses by phospho-relay cascades. Among p38 families, p38alpha is a widely characterized isoform and the biological phenomena are explained by its kinase activity regulating functions of its downstream substrates. However, its specific contributions to each phenomenon are yet not fully elucidated. For better understanding of the role of MAPKs, especially p38alpha, we utilized newly established mouse fibroblast cell lines originated from a p38alpha null mouse, namely, a parental cell line without p38alpha gene locus, knockout of p38alpha (KOP), Zeosin-resistant (ZKOP), revertant of p38alpha (RKOP), and Exip revertant (EKOP). EKOP is smaller in size but grows faster than the others. Although comparable amounts of ERK and JNK are expressed in each cell line, ERK is highly phosphorylated in EKOP even in normal culture conditions. Serum stimulation after serum starvation led to ERK phosphorylation in RKOP and ZKOP, but not in EKOP as much. On the contrary, relative phosphorylation level of JNK to total JNK in response to UV was low in RKOP. And its phosphorylation as well as total JNK is slightly lower in EKOP. RKOP is less sensitive to UV irradiation as judged by the survival rate. Stress response upon UV or sorbitol stimuli, leading to mitogen activate protein kinase activated kinase 2 (MAPKAPK2) phosphorylation, was only observed in RKOP. Further experiments reveal that MAPKAPK2 expression is largely suppressed in ZKOP and EKOP. Its expression was recovered by re-introduction of p38alpha. The loss of MAPKAPK2 expression accompanied by the defect of p38alpha is confirmed in an embryonic extract prepared from p38alpha null mice. These data demonstrate that p38 signal pathway is regulated not only by phosphorylation but also by modulation of the expression of its component. Together, we have established cell lines that can be used in analyzing the functions of MAPKs, especially p38alpha, and show that p38 is indispensable for MAPKAPK2 expression.     

Role of p38 mitogen-activated protein kinase in thrombus formation

The present study was designed to elucidate the role of p38 mitogen-activated protein kinase (p38) in thrombus formation. We used p38alpha heterozygous (p38alpha+/-) mice and used ferric chloride (FeCl3)-induced carotid artery injury as a model of thrombus formation. The time to thrombotic occlusion induced by FeCl3 in p38alpha+/- mice was prolonged compared to that in wild-type (WT) mice. Platelets prepared from p38alpha+/- mice showed impairment of the aggregatory response to a low concentration of U46619, a thromboxane A2 analogue. Furthermore, platelets prepared from p38alpha+/- mice and activated by U46619 were poorly bound to fibrinogen compared with those from WT mice. Both the expression and activity of tissue factor induced by FeCl3 in WT mice were higher than those in p38alpha+/- mice. These results suggest that p38 plays an important role in thrombus formation by regulating platelet function and tissue factor activity.     

Multiple activation mechanisms of p38alpha mitogen-activated protein kinase

The p38alpha MAPK participates in a variety of biological processes. Activation of p38alpha is mediated by phosphorylation on specific regulatory tyrosine and threonine sites, and the three dual kinases, MAPK kinase 3 (MKK3), MKK4, and MKK6, are known to be the upstream activators of p38alpha. In addition to activation by upstream kinases, p38alpha can autoactivate when interacting with transforming growth factor-beta-activated protein kinase 1-binding protein 1 (TAB1). Here we used MKK3 and MKK6 double knock-out (MKK3/6 DKO) and MKK4/7 DKO mouse embryonic fibroblast (MEF) cells to examine activation mechanisms of p38alpha. We confirmed that the MKK3/6 pathway is a primary mechanism for p38alpha phosphorylation in MEF cells, and we also showed the presence of other p38alpha activation pathways. We show that TAB1-mediated p38alpha phosphorylation in MEF cells did not need MKK3/4/6, and it accounted for a small portion of the total p38alpha phosphorylation that was induced by hyperosmolarity and anisomycin. We observed that a portion of peroxynitrite-induced phospho-p38alpha is associated with an approximately 85-kDa disulfide complex in wild-type MEF cells. Peroxynitrite-induced phosphorylation of p38alpha in the approximately 85-kDa complex is independent from MKK3/6 because only phospho-p38alpha not associated with the disulfide complex was diminished in MKK3/6 DKO cells. In addition, our data suggest interference among different pathways because TAB1 had an inhibitory effect on p38alpha phosphorylation in the peroxynitrite-induced approximately 85-kDa complex. Mutagenesis analysis of the cysteines in p38alpha revealed that no disulfide bond forms between p38alpha and other proteins in the approximately 85-kDa complex, suggesting it is a p38alpha binding partner(s) that forms disulfide bonds, which enable it to bind to p38alpha. Therefore, multiple mechanisms of p38alpha activation exist that can influence each other, be simultaneously activated by a given stimulus, and/or be selectively used by different stimuli in a cell type-specific manner.     

Activation of p38 mitogen-activated protein kinase/cytosolic phospholipase A2 cascade in hydroperoxide-stressed platelets

12-Hydroperoxy-eicosatetraenoic acid (12-HpETE), the main hydroperoxide formed in platelets from arachidonic acid (AA) by 12-lipoxygenase, has been shown to increase the sensitivity of platelets to agonists resulting in increased aggregation. The aim of the present study was to determine the direct effect of low concentrations of 12-HpETE on the signaling pathways leading to AA release from membrane phospholipids and thromboxane A2 (TxA2) formation. Exogenous 12-HpETE activated platelet p38 mitogen-activated protein kinase (p38 MAPK), as assessed by its phosphorylation, at a concentration as low as 100 nM and was much more potent than hydrogen peroxide. Moreover, the incubation of platelets with 100 nM 12-HpETE for 2 min led to the phosphorylation of cytosolic phospholipase A2 (cPLA2). It was associated with a significant decrease in the concentration of AA esterified in phospholipids and an increased concentration of thromboxane B2, the stable catabolite of TxA2. Additionally, decreasing glutathione peroxidase activity pharmacologically favored endogenous 12-HpETE formation and led to an increase in phosphorylated p38 MAPK, while a thiol-reducing agent such as N-acetyl-cysteine fully prevented it. Finally, significant activation of p38 MAPK was also observed in platelets from type 2 diabetic patients with mild hyperglycemia. In conclusion, our data provide a new insight into the mechanism of 12-HpETE-induced platelet priming, suggesting that hydroperoxide-induced p38 MAPK activation could play a relevant role in the exacerbated platelet activation associated with oxidative stress as found in diabetes.