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.     

Heparin regulates colon cancer cell growth through p38 mitogen-activated protein kinase signalling

Objectives:  Heparin acts as an extracellular stimulus capable of activating major cell signalling pathways. Thus, we examined the putative mechanisms utilized by heparin to stimulate HT29, SW1116 and HCT116 colon cancer cell growth.
Materials and methods:  Possible participation of the mitogen-activated protein kinase (MAPK) cascade on heparin-induced HT29, SW1116 and HCT116 colon cancer cell growth was evaluated using specific MAPK cascade inhibitors, Western blot analysis, real-time quantitative PCR and FACS apoptosis analysis.
Results:  Treatment with a highly specific p38 kinase inhibitor, SB203580, significantly (50–70%) inhibited heparin-induced colon cancer cell growth, demonstrating that p38 MAPK signalling is involved in their heparin-induced proliferative response. This was shown to be correlated with increased (up to 3-fold) phosphorylation of 181/182 threonine/tyrosine residues on p38 MAP kinase. Furthermore, heparin inhibited cyclin-dependent kinase inhibitor p21 ^{WAF1 / CIP1} and p53 tumour suppressor gene and protein expression up to 2-fold or 1.8-fold, respectively, and stimulated cyclin D1 expression up to 1.8-fold, in these cell lines through a p38-mediated mechanism. On the other hand, treatment with heparin did not appear to affect HT29, SW1116 and HCT116 cell levels of apoptosis.
Conclusions:  This study demonstrates that an extracellular glycosaminoglycan, heparin, finely modulates expression of genes crucial to cell cycle regulation through specific activation of p38 MAP kinase to stimulate colon cancer cell growth.     

Epstein-Barr virus latent membrane protein 2A regulates c-Jun protein through extracellular signal-regulated kinase

Epstein-Barr virus (EBV) latent membrane protein 2A (LMP2A) is widely expressed in both EBV-infected cells and EBV-associated malignancies. However, the function of LMP2A is still veiled. In this study, LMP2A was found to induce the kinase activities of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase/stress-activated protein kinase JNK/SAPK. Furthermore, the downstream effector c-Jun showed hyperphosphorylation under LMP2A expression. The phosphorylation could be inhibited by the ERK pathway inhibitor PD98059, indicating that ERK may contribute to the phosphorylation of c-Jun in LMP2A-expressing cells. The impact on c-Jun phosphorylation by mitogen-activated protein kinase (MAPK) is suggested to increase c-Jun protein stability, and this was also observed in LMP2A-expressing cells by a protein synthesis inhibition assay. Moreover, LMP2A-induced cell invasion was inhibited in the presence of the ERK pathway inhibitor. Taken together, we suggest that LMP2A may exploit MAPK kinases and affect both the phosphorylation and stability of c-Jun protein. Additionally, LMP2A may thereby promote the mobility of the cells. In doing so, it may enhance the mobility of EBV-infected cells and contribute to the metastatic process of malignant cells. Here we demonstrated the first evidence of LMP2A-induced migration and the underlying pathways accounting for it.     

p38 mitogen-activated protein kinase regulates human T cell IL-5 synthesis.

Involvement of p38 mitogen-activated protein (MAP) kinase in human T cell cytokine synthesis was investigated. p38 MAP kinase was clearly induced in human Th cells activated through the TCR. SB203580, a highly selective inhibitor of p38 MAP kinase, inhibited the induction of p38 MAP kinase in human Th cells. Major T cell cytokines, IL-2, IL-4, IL-5, and IFN-gamma, were produced by Der f 2-specific Th clones upon stimulation through the TCR. IL-5 synthesis alone was significantly inhibited by SB203580 in a dose-dependent manner, whereas the production of IL-2, IL-4, and IFN-gamma was not affected. The proliferation of activated T cells was not affected. IL-5 synthesis of human Th clones induced upon stimulation with rIL-2, phorbol ester plus anti-CD28 mAb, and immobilized anti-CD3 mAb plus soluble anti-CD28 mAb was also suppressed by SB203580 in the same concentration response relationship. The results clearly indicated that IL-5 synthesis by human Th cells is dependent on p38 MAP kinase activity, and is regulated distinctly from IL-2, IL-4, and IFN-gamma synthesis. Selective control of IL-5 synthesis will provide a novel treatment devoid of generalized immune suppression for bronchial asthma and atopic dermatitis that are characterized by eosinophilic inflammation.     

TRAF6 promotes myogenic differentiation via the TAK1/p38 mitogen-activated protein kinase and Akt pathways

p38 mitogen-activated protein kinase (MAPK) is an essential kinase involved in myogenic differentiation. Although many substrates of p38 MAPK have been identified, little is known about its upstream activators during myogenic differentiation. TRAF6 is known to function in cytokine signaling during inflammatory responses. However, not much is known about its role in myogenic differentiation and muscle regeneration. We showed here that TRAF6 and its intrinsic ubiquitin E3 ligase activity are required for myogenic differentiation. In mouse myoblasts, knockdown of TRAF6 compromised the p38 MAPK and Akt pathways, while deliberate activation of either pathway rescued the differentiation defect caused by TRAF6 knockdown. TAK1 acted as a key signal transducer downstream of TRAF6 in myogenic differentiation. In vivo, knockdown of TRAF6 in mouse muscles compromised the injury-induced muscle regeneration without impairing macrophage infiltration and myoblast proliferation. Collectively, we demonstrated that TRAF6 promotes myogenic differentiation and muscle regeneration via the TAK1/p38 MAPK and Akt pathways.     

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.     

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.     

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.     

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.     

Opposing roles of the extracellular signal-regulated kinase and p38 mitogen-activated protein kinase cascades in Ras-mediated downregulation of tropomyosin

We showed previously that activated Ras, but not Raf, causes transformation of RIE-1 epithelial cells, demonstrating the importance of Raf-independent pathways in mediating Ras transformation. To assess the mechanism by which Raf-independent effector signaling pathways contribute to Ras-mediated transformation, we recently utilized representational difference analysis to identify genes expressed in a deregulated fashion by activated Ras but not Raf. One gene identified in these analyses encodes for alpha-tropomyosin. Therefore, we evaluated the mechanism by which Ras causes the downregulation of tropomyosin expression. By using RIE-1 cells that harbor inducible expression of activated H-Ras(12V), we determined that the downregulation of tropomyosin expression correlated with the onset of morphological transformation. We found that the reversal of Ras transformation caused by inhibition of extracellular signal-regulated kinase activation corresponded to a restoration of tropomyosin expression. Inhibition of p38 activity in Raf-expressing RIE-1 cells caused both morphological transformation and loss of tropomyosin expression. Thus, a reduction in tropomyosin expression correlated strictly with morphological transformation of RIE-1 cells. However, forced overexpression of tropomyosin in Ras-transformed cells did not reverse morphological or growth transformation, a finding consistent with the possibility that multiple changes in gene expression contribute to Ras transformation. We also determined that tropomyosin expression was low in two human tumor cell lines, DLD-1 and HT1080, that harbor endogenous mutated alleles of ras, but high in transformation-impaired, derivative cell lines in which the mutant ras allele has been genetically deleted. Finally, treatment with azadeoxycytidine restored tropomyosin expression in Ras-transformed RIE-1, HT1080, and DLD-1 cells, suggesting a role for DNA methylation in downregulating tropomyosin expression.     

Activation of p38 mitogen-activated protein kinase promotes epidermal growth factor receptor internalization

Endocytic trafficking plays an important role in the regulation of the epidermal growth factor receptor (EGFR). To address if cellular kinases regulate EGFR internalization, we used anisomycin, a potent activator of kinase cascades in mammalian cells, especially the stress-activated mitogen-activated protein (MAP) kinase subtypes. Here, we report that activation of p38 MAP kinase by anisomycin is sufficient to induce internalization of EGFR. Anisomycin and EGF employ different mechanisms to promote EGFR endocytosis as anisomycin-induced internalization does not require tyrosine kinase activity or ubiquitination of the receptor. In addition, anisomycin treatment did not result in delivery and degradation of EGFR at lysosomes. Incubation with a specific inhibitor of p38, or depletion of endogenous p38 by small interfering RNAs, abolished anisomycin-induced internalization of EGFR while having no effect on transferrin endocytosis, indicating that the effect of p38 activation on EGFR endocytosis is specific. Interestingly, inhibition of p38 activation also abolished endocytosis of EGFR induced by UV radiation. Our results reveal a novel role for p38 in the regulation of EGFR endocytosis and suggest that stimulation of EGFR internalization by p38 might represent a general mechanism to prevent generation of proliferative or anti-apoptotic signals under stress conditions.     

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.     

Epstein-Barr virus latent membrane protein 2 associates with and is a substrate for mitogen-activated protein kinase.

The latent membrane protein 2 (LMP2) of Epstein-Barr virus interferes with B-lymphocyte signal transduction through the immunoglobulin (Ig) receptor. Two isoforms of LMP2 exist and differ only in that one isoform (LMP2a) contains an N-terminal cytoplasmic domain that the other isoform does not. LMP2a is a phosphoprotein that is phosphorylated on tyrosines and serines in the cytoplasmic domain. GST1-119, a glutathione S-transferase (GST) fusion protein containing the 119 amino acids of the cytoplasmic domain, affinity precipitated serine kinase activity from BJAB cell extracts. The affinity-precipitated kinase phosphorylated LMP2a sequences, and kinase activity was increased following induction. Probing of Western immunoblots of affinity-precipitated proteins showed that the Erk1 form of mitogen-activated protein kinase (MAPK) was present. Purified MAPK phosphorylated GST fusion proteins containing the cytoplasmic domain of LMP2a and mutational analyses were used to identify S15 and S102 as the sites of in vitro phosphorylation. A polyclonal rabbit antiserum was prepared against a maltose binding protein-LMP2a cytoplasmic domain fusion protein (MBP1-119) and used to immunoprecipitate LMP2a from the in vitro-immortalized lymphoblastoid B-cell line B95-8CR. LMP2a immunoprecipitates from B95-8CR contained MAPK as a coprecipitated protein. Cross-linking surface Ig on B95-8CR cells failed to induce MAPK activity within the cells. Treatment of B95-8CR with phorbol myristate acetate (PMA) was able to bypass the Ig receptor block and activate MAPK activity. Phosphorylation of LMP2a on serine residues increased after PMA induction. The possible role for LMP2a serine phosphorylation by MAPK in the control of latency is discussed.