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.     

Pyridinylimidazole p38 mitogen-activated protein kinase inhibitors block intracellular Toxoplasma gondii replication

Toxoplasma gondii is a medically important, obligate intracellular parasite. Little is known regarding factors that regulate its replication within cells. Such knowledge would further understanding of T. gondii pathogenesis, and might lead to novel therapeutic strategies. Mitogen-activated protein kinases (MAPKs) govern diverse cellular processes including proliferation and differentiation. We now show that treatment of T. gondii-infected cells with SB203580 or SB202190, substituted pyridinylimidazoles that are potent inhibitors of human p38 MAPK, inhibits intracellular T. gondii replication. Several independent experimental approaches suggest that the anti-proliferative effects of pyridinylimidazoles depend on direct action on tachyzoites, not the host cell: (i) selective inhibition of host p38 MAPK using recombinant adenoviruses had little effect on tachyzoite replication, (ii) pyridinylimidazole-treated tachyzoites developed abnormal morphology suggesting defective parasite division, and (iii) pyridinylimidazole-resistant mutant tachyzoites were developed through culture in progressively higher drug concentrations. We hypothesise that pyridinylimidazoles target a human p38 MAPK homologue in tachyzoites that regulates their replication. Phylogenetic data suggest that T. gondii likely encodes a p38 MAPK homologue, but such a homologue is absent from the incomplete Toxoplasma genomic data base. As all eukaryotic pathogens, including agents of malaria, leishmaniasis and trypanosomiasis encode endogenous MAPKs, drugs inhibiting endogenous MAPK activation may represent a novel, potentially broadly-acting class of anti-parasitic agents. Pyridinylimidazoles also represent tools to elucidate factors governing intracellular tachyzoite replication.     

Murine coronavirus replication-induced p38 mitogen-activated protein kinase activation promotes interleukin-6 production and virus replication in cultured cells

Analyses of mitogen-activated protein kinases (MAPKs) in a mouse hepatitis virus (MHV)-infected macrophage-derived J774.1 cell line showed activation of two MAPKs, p38 MAPK and c-Jun N-terminal kinase (JNK), but not of extracellular signal-regulated kinase (ERK). Activation of MAPKs was evident by 6 h postinfection. However, UV-irradiated MHV failed to activate MAPKs, which demonstrated that MHV replication was necessary for their activation. Several other MHV-permissive cell lines also showed activation of both p38 MAPK and JNK, which indicated that the MHV-induced stress-kinase activation was not restricted to any particular cell type. The upstream kinase responsible for activating MHV-induced p38 MAPK was the MAPK kinase 3. Experiments with a specific inhibitor of p38 MAPK, SB 203580, demonstrated that MHV-induced p38 MAPK activation resulted in the accumulation of interleukin-6 (IL-6) mRNAs and an increase in the production of IL-6, regardless of MHV-induced general host protein synthesis inhibition. Furthermore, MHV production was suppressed in SB 203580-treated cells, demonstrating that activated p38 MAPK played a role in MHV replication. The reduced MHV production in SB 203580-treated cells was, at least in part, due to a decrease in virus-specific protein synthesis and virus-specific mRNA accumulation. Interestingly, there was a transient increase in the amount of phosphorylation of the translation initiation factor 4E (eIF4E) in infected cells, and this eIF4E phosphorylation was p38 MAPK dependent; it is known that phosphorylated eIF4E enhances translation rates of cap-containing mRNAs. Furthermore, the upstream kinase responsible for eIF4E phosphorylation, MAPK-interacting kinase 1, was also phosphorylated and activated in response to MHV infection. Our data suggested that host cells, in response to MHV replication, activated p38 MAPK, which subsequently phosphorylated eIF4E to efficiently translate certain host proteins, including IL-6, during virus-induced severe host protein synthesis inhibition. MHV utilized this p38 MAPK-dependent increase in eIF4E phosphorylation to promote virus-specific protein synthesis and subsequent progeny virus production. Enhancement of virus-specific protein synthesis through virus-induced eIF4E activation has not been reported in any other viruses.     

Hepatitis B virus X protein activates the p38 mitogen-activated protein kinase pathway in dedifferentiated hepatocytes

Hepatitis B virus X protein (pX) is implicated in hepatocarcinogenesis by an unknown mechanism. Employing a cellular model linked to pX-mediated transformation, we investigated the role of the previously reported Stat3 activation by pX in hepatocyte transformation. Our model is composed of a differentiated hepatocyte (AML12) 3pX-1 cell line that undergoes pX-dependent transformation and a dedifferentiated hepatocyte (AML12) 4pX-1 cell line that does not exhibit transformation by pX. We report that pX-dependent Stat3 activation occurs only in non-pX-transforming 4pX-1 cells and conclude that Stat3 activation is not linked to pX-mediated transformation. Maximum Stat3 transactivation requires Ser727 phosphorylation, mediated by mitogenic pathway activation. Employing dominant negative mutants and inhibitors of mitogenic pathways, we demonstrate that maximum, pX-dependent Stat3 transactivation is inhibited by the p38 mitogen-activated protein kinase (MAPK)-specific inhibitor SB 203580. Using transient-transreporter and in vitro kinase assays, we demonstrate for the first time that pX activates the p38 MAPK pathway only in 4pX-1 cells. pX-mediated Stat3 and p38 MAPK activation is Ca(2+) and c-Src dependent, in agreement with the established cellular action of pX. Importantly, pX-dependent activation of p38 MAPK inactivates Cdc25C by phosphorylation of Ser216, thus initiating activation of the G(2)/M checkpoint, resulting in 4pX-1 cell growth retardation. Interestingly, pX expression in the less differentiated hepatocyte 4pX-1 cells activates signaling pathways known to be active in regenerating hepatocytes. These results suggest that pX expression in the infected liver effects distinct mitogenic pathway activation in less differentiated versus differentiated hepatocytes.     

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.     

Active mutants of the human p38alpha mitogen-activated protein kinase

Mitogen-activated protein (MAP) kinases compose a family of serine/threonine kinases that function in many signal transduction pathways and affect various cellular phenotypes. Despite the abundance of available data, the exact role of each MAP kinase is not completely defined, in part because of the inability to activate MAP kinase molecules individually and specifically. Based on activating mutations found in the yeast MAP kinase p38/Hog1 (Bell, M., Capone, R., Pashtan, I., Levitzki, A., and Engelberg, D. (2001) J. Biol. Chem. 276, 25351-25358), we designed and constructed single and multiple mutants of human MAP kinase p38alpha. Single (p38D176A, p38F327L, and p38F327S) and subsequent double (p38D176A/F327L and p38D176A/F327S) mutants acquired high intrinsic activity independent of any upstream regulation and reached levels of 10 and 25%, respectively, in reference to the dually phosphorylated wild type p38alpha. The active p38 mutants have retained high specificity toward p38 substrates and were inhibited by the specific p38 inhibitors SB-203580 and PD-169316. We also show that similar mutations can render p38gamma active as well. Based on the available structures of p38 and ERK2, we have analyzed the p38 mutants and identified a hydrophobic core stabilized by three aromatic residues, Tyr-69, Phe-327, and Trp-337, in the vicinity of the L16 loop region. Upon activation, a segment of the L16 loop, including Phe-327, becomes disordered. Structural analysis suggests that the active p38 mutants emulate the conformational changes imposed naturally by dual phosphorylation, namely, destabilization of the hydrophobic core. Essentially, the hydrophobic core is an inherent stabilizer that maintains low basal activity level in unphosphorylated p38.     

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.     

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.     

Vaccinia virus protein A52R activates p38 mitogen-activated protein kinase and potentiates lipopolysaccharide-induced interleukin-10

Vaccinia virus (VV) has many mechanisms to suppress and modulate the host immune response. The VV protein A52R was previously shown to act as an intracellular inhibitor of nuclear factor kappaB (NFkappaB) signaling by Toll-like receptors (TLRs). Co-immunoprecipitation studies revealed that A52R interacted with both tumor necrosis factor receptor-associated factor 6 (TRAF6) and interleukin-1 receptor-associated kinase 2 (IRAK2). The effect of A52R on signals other than NFkappaB was not determined. Here, we show that A52R does not inhibit TLR-induced p38 or c-Jun amino N-terminal kinase (JNK) mitogen activating protein (MAP) kinase activation. Rather, A52R could drive activation of these kinases. Two lines of evidence suggested that the A52R/TRAF6 interaction was critical for these effects. First, A52R-induced p38 MAP kinase activation was inhibited by overexpression of the TRAF domain of TRAF6, which sequestered A52R and inhibited its interaction with endogenous TRAF6. Second, a truncated version of A52R, which interacted with IRAK2 and not TRAF6, was unable to activate p38. Because interleukin 10 (IL-10) production is strongly p38-dependent, we examined the effect of A52R on IL-10 gene induction. A52R was found to be capable of inducing the IL-10 promoter through a TRAF6-dependent mechanism. Furthermore, A52R enhanced lipopolysaccharide/TLR4-induced IL-10 production, while inhibiting the TLR-induced NFkappaB-dependent genes IL-8 and RANTES. These results show that although A52R inhibits NFkappaB activation by multiple TLRs it can simultaneously activate MAP kinases. A52R-mediated enhancement of TLR-induced IL-10 may be important to virulence, given the role of IL-10 in immunoregulation.     

Essential role of p38 mitogen-activated protein kinase in contact hypersensitivity

The present study was designed to elucidate the role of p38 mitogen-activated protein kinase (p38) in the pathogenesis of inflammation, using a mouse contact hypersensitivity (CHS) model induced by 2,4-dinitro-1-fluorobenzene (DNFB). Ear swelling was induced by challenge with DNFB, accompanied by infiltration of mononuclear cells, neutrophils, and eosinophils and a marked increase in mRNA levels of cytokines such as interleukin (IL)-2, interferon (IFN)-gamma, IL-4, IL-5, IL-1beta, IL-18, and tumor necrosis factor-alpha in the challenged ear skin. Both ear swelling and the number of infiltrated cells in DNFB-challenged ear skin were significantly inhibited by treatment with SB202190, a p38 inhibitor. Furthermore, the DNFB-induced expression of all cytokines except IL-4 was significantly inhibited by treatment with SB202190. Ribonuclease protection assay revealed that the mRNA levels of chemokines such as IP-10 and MCP-1 in ear skin were markedly increased at 24 h after challenge with DNFB. The induction of these chemokines was significantly inhibited by treatment with SB202190. In p38alpha +/- mice, both ear swelling and infiltration of cells induced by DNFB were reduced compared with those in wild-type mice. However, induction of cytokines by DNFB was also observed in p38alpha +/- mice, although the induction of IFN-gamma, IL-5, and IL-18 was typically reduced compared with that in wild-type mice. Challenge with DNFB slightly induced IP-10 and MCP-1 mRNA in p38alpha +/- mice, with weaker signals than those in SB202190-treated wild-type mice. These results suggest that p38 plays a key role in CHS and is an important target for the treatment of CHS.     

Growth regulation via p38 mitogen-activated protein kinase in developing liver

During normal development in the rat, hepatocytes undergo marked changes in the rate of proliferation. We have previously observed transient G(1) growth arrest at term, re-activation of proliferation immediately after birth, and a gradual transition to the quiescent adult hepatocyte phenotype after postnatal day 4. We hypothesized that these changes in proliferation are due in part to growth inhibitory effects mediated by the p38 mitogen-activated protein kinase pathway. p38 kinase activity measurements showed an inverse relationship with hepatocyte proliferation during the perinatal and postnatal transitions, whereas p38 content remained constant. Anisomycin activated the p38 pathway in fetal hepatocyte cultures while inducing growth inhibition that was sensitive to the p38 inhibitor, SB203580. Activation of p38 in these cultures, via transient transfection with a constitutively active form of its upstream kinase MKK6, also inhibited DNA synthesis as well as reducing cyclin D1 content. Transfection with inactive MKK6 did neither. Furthermore, MKK6-induced growth arrest was sensitive to SB203580. Finally, administration of SB203580 to near-term fetal rats in utero abrogated the transient hepatocyte growth arrest that occurs at term. These findings indicate a role for the p38 mitogen-activated protein kinase pathway in the physiological regulation of hepatocyte proliferation during normal development in the rat.     

Activation of TRAF5 and TRAF6 signal cascades negatively regulates the latent replication origin of Epstein-Barr virus through p38 mitogen-activated protein kinase

Latent Epstein-Barr virus (EBV) is maintained by the virus replication origin oriP that initiates DNA replication with the viral oriP-binding factor EBNA1. However, it is not known whether oriP's replicator activity is regulated by virus proteins or extracellular signals. By using a transient replication assay, we found that a low level of expression of viral signal transduction activator latent membrane protein 1 (LMP1) suppressed oriP activity. The binding site of the tumor necrosis factor receptor-associated factor (TRAF) of LMP1 was essential for this suppressive effect. Activation of the TRAF signal cascade by overexpression of TRAF5 and/or TRAF6 also suppressed oriP activity. Conversely, blocking of TRAF signaling with dominant negative mutants of TRAF5 and TRAF6, as well as inhibition of a downstream signal mediator p38 MAPK, released the LMP1-induced oriP suppression. Furthermore, activation of TRAF6 signal cascade by lipopolysaccharides (LPS) resulted in loss of EBV from Burkitt's lymphoma cell line Akata, and inhibition of p38 MAPK abolished the suppressive effect of LPS. These results suggested that the level of oriP activity is regulated by LMP1 and extracellular signals through TRAF5- and TRAF6-mediated signal cascades.     

Differential activation of p38 mitogen-activated protein kinase isoforms depending on signal strength

We have investigated the ability of the mitogen-activated protein kinase (MAPK) kinase MKK6 to activate different members of the p38 subfamily of MAPKs and found that some MKK6 mutants can efficiently activate p38alpha but not p38gamma. In contrast, a constitutively active MKK6 mutant activated both p38 MAPK isoforms to similar extents. The same results were obtained upon co-expression in Xenopus oocytes and in vitro using either MKK6 immunoprecipitates from transfected cells or bacterially produced recombinant proteins. We also found that the preferential activation of p38alpha by MKK6 correlated with more efficient binding of MKK6 to p38alpha than to p38gamma. Furthermore, increasing concentrations of constitutively active MKK6 differentially activated either p38alpha alone (low MKK6 activity) or both p38alpha and p38gamma (high MKK6 activity), both in vitro and in injected oocytes. The determinants for selectivity are located at the carboxyl-terminal lobe of p38 MAPKs but do not correspond to the activation loop or common docking sequences. We also showed that different stimuli can induce different levels of endogenous MKK6 activity that correlate with differential activation of p38 MAPKs. Our results suggest that the level of MKK6 activity triggered by a given stimulus may determine the pattern of downstream p38 MAPK activation in the particular response.     

Stimulation of glucose transport by AMP-activated protein kinase via activation of p38 mitogen-activated protein kinase

Activation of AMP-activated protein kinase (AMPK) has been recently demonstrated to be associated with 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR)-stimulated glucose transport mediated by both GLUT1 and GLUT4 transporters. However, signaling events upstream and downstream of AMPK are unknown. Here we report that 1) p38 mitogen-activated protein kinase (MAPK) and mitogen-activated protein kinase kinase 3 (MKK3) were activated by AICAR in Clone 9 cells, which express only the GLUT1 transporters, and 2) activation of p38 was required for AICAR-stimulated glucose transport since treatment of the cells with p38 inhibitor SB203580 or overexpression of dominant negative p38 mutant inhibited glucose transport. Moreover, we found that overexpression of the constitutively active form of AMPK mutant also resulted in a significant activation of p38, and inhibition of p38 activity by SB203580 did not affect AICAR-stimulated activation of AMPK. These findings demonstrate that AICAR-stimulated activation of p38 is indeed mediated by AMPK, and the p38 MAPK cascade is downstream of AMPK in the signaling pathway of AICAR-stimulated glucose transport in Clone 9 cells.     

p38 mitogen-activated protein kinase regulates oscillation of chick pineal circadian clock

Extracellular signal-regulated kinase (ERK) and p38 are members of the mitogen-activated protein kinase (MAPK) family, and in some cases these kinases serve for closely related cellular functions within a cell. In a wide range of animal clock structures, ERK plays an important role in the circadian time-keeping mechanism. Here we found that immunoreactivity to p38 protein was uniformly distributed among cells in the chick pineal gland. On the other hand, a constant level of activated p38 was detected over the day, predominantly in the follicular and parafollicular pinealocytes that are potential circadian clock-containing cells. Chronic application of SB203580, a selective and reversible inhibitor of p38, to the cultured chick pineal cells markedly lengthened the period of the circadian rhythm of the melatonin release (up to 28.7 h). Noticeably, despite no significant temporal change of activated p38 level, a 4-h pulse treatment with SB203580 delayed the phase of the rhythm only when delivered during the subjective day. These results indicate a time-of-day-specific role of continuously activated p38 in the period length regulation of the chick pineal clock and suggest temporally separated regulation of the clock by two MAPKs, nighttime-activated ERK and daytime-working p38.