p38γ Mitogen-activated Protein Kinase Signals through Phosphorylating Its Phosphatase PTPH1 in Regulating Ras Protein Oncogenesis and Stress Response

Phosphatase plays a crucial role in determining cellular fate by inactivating its substrate kinase, but it is not known whether a kinase can vice versa phosphorylate its phosphatase to execute this function. Protein-tyrosine phosphatase H1 (PTPH1) is a specific phosphatase of p38γ mitogen-activated protein kinase (MAPK) through PDZ binding, and here, we show that p38γ is also a PTPH1 kinase through which it executes its oncogenic activity and regulates stress response. PTPH1 was identified as a substrate of p38γ by unbiased proteomic analysis, and its resultant phosphorylation at Ser-459 occurs in vitro and in vivo through their complex formation. Genetic and pharmacological analyses showed further that Ser-459 phosphorylation is directly regulated by Ras signaling and is important for Ras, p38γ, and PTPH1 oncogenic activity. Moreover, experiments with physiological stimuli revealed a novel stress pathway from p38γ to PTPH1/Ser-459 phosphorylation in regulating cell growth and cell death by a mechanism dependent on cellular environments but independent of canonical MAPK activities. These results thus reveal a new mechanism by which a MAPK regulates Ras oncogenesis and stress response through directly phosphorylating its phosphatase.     

Molecular analysis of RANKL-independent cell fusion of osteoclast-like cells induced by TNF-alpha, lipopolysaccharide, or peptidoglycan

Focusing on the final step of osteoclastogenesis, we studied cell fusion from tartrate-resistant acid phosphatase (TRAP)-positive mononuclear cells into multinuclear cells. TRAP-positive mononuclear cells before generation of multinuclear cells by cell fusion were differentiated from RAW264.7 cells by treatment with receptor activator of nuclear factor kappa B ligand (RANKL), and then the cells were treated with lipopolysaccharide (LPS), followed by culturing for further 12 h. LPS-induced cell fusion even in the absence of RANKL. Similarly, tumor necrosis factor (TNF)-alpha and peptidoglycan (PGN) induced cell fusion, but M-CSF did not. The cell fusion induced by RANKL, TNF-alpha, and LPS was specifically blocked by osteoprotegerin (OPG), anti-TNF-alpha antibody, and polymyxin B, respectively. LPS- and PGN-induced cell fusion was partly inhibited by anti-TNF-alpha antibody but not by OPG. When TRAP-positive mononuclear cells fused to yield multinuclear cells, phosphorylation of Akt, Src, extracellular signal-regulated kinase (ERK), p38MAPK (p38), and c-Jun NH2-terminal kinase (JNK) was observed. The specific chemical inhibitors LY294002 (PI3K), PP2 (Src), U0126 (MAPK-ERK kinase (MEK)/ERK), and SP600125 (JNK) effectively suppressed cell fusion, although SB203580 (p38) did not. mRNA of nuclear factor of activated T-cells c1 (NFATc1) and dendritic cell-specific transmembrane protein (DC-STAMP) during the cell fusion was quantified, however, there was no obvious difference among the TRAP-positive mononuclear cells treated with or without M-CSF, RANKL, TNF-alpha, LPS, or PGN. Collectively, RANKL, TNF-alpha, LPS, and PGN induced cell fusion of osteoclasts through their own receptors. Subsequent activation of signaling pathways involving PI3K, Src, ERK, and JNK molecules was required for the cell fusion. Although DC-STAMP is considered to be a requisite for cell fusion of osteoclasts, cell fusion-inducing factors other than DC-STAMP might be necessary for the cell fusion.     

Phosphatidylinositol ether lipid analogues that inhibit AKT also independently activate the stress kinase, p38alpha, through MKK3/6-independent and -dependent mechanisms

Previously, we identified five active phosphatidylinositol ether lipid analogues (PIAs) that target the pleckstrin homology domain of Akt and selectively induce apoptosis in cancer cells with high levels of Akt activity. To examine specificity, PIAs were screened against a panel of 29 purified kinases. No kinase was inhibited, but one isoform of p38, p38alpha, was uniformly activated 2-fold. Molecular modeling of p38alpha revealed the presence of two regions that could interact with PIAs, one in the activation loop and a heretofore unappreciated region in the upper lobe that resembles a pleckstrin homology domain. In cells, two phases of activation were observed, an early phase that was independent of the upstream kinase MKK3/6 and inhibited by the p38 inhibitor SB203580 and a latter phase that was coincident with MKK3/6 activation. In short term xenograft experiments that employed immunohistochemistry and immunoblotting, PIA administration increased phosphorylation of p38 but not MKK3/6 in tumors in a statistically significant manner. Although PIAs rapidly activated p38 with similar time and dose dependence as Akt inhibition, p38 activation and Akt inhibition were independent events induced by PIAs. Using SB203580 or p38alpha(-/-) cells, we showed that p38alpha is not required for PIA-induced apoptosis but is required for H(2)O(2)- and anisomycin-induced apoptosis. Nonetheless, activation of p38a contributes to PIA-induced apoptosis, because reconstitution of p38a into p38alpha(-/-) cells increased apoptosis. These studies indicate that p38alpha is activated by PIAs through a novel mechanism and show that p38alpha activation contributes to PIA-induced cell death. Independent modulation of Akt and p38alpha could account for the profound cytotoxicity of PIAs.     

p38 differentially regulates ERK,p21, and mitogenic signalling in two pancreatic carcinoma cell lines

Whereas the p38 MAP kinase has largely been associated with anti-proliferative functions, several observations have indicated that it may also have positive effects on proliferation. In hepatocytes, we have found that p38 has opposing effects on DNA synthesis when activated by EGF and HGF. Here we have studied the function of p38 in EGF- and HGF-induced DNA synthesis in the two pancreatic carcinoma cell lines AsPC-1 and Panc-1. In Panc-1 cells, the MEK inhibitor PD98059 reduced EGF- and HGF-induced DNA synthesis, while the p38 inhibitor SB203580 strongly increased the basal DNA synthesis and reduced expression of the cyclin-dependent kinase inhibitor (CDKI) p21. In contrast, in AsPC-1 cells, EGF- and HGF-induced DNA synthesis was not significantly reduced by PD98059 but was inhibited by SB203580. Treatment with SB203580 amplified the sustained ERK phosphorylation induced by these growth factors and caused a marked upregulation of the expression of p21, which could be blocked by PD98059. These results suggest that while DNA synthesis in Panc-1 cells is enhanced by ERK and strongly suppressed by p38, in AsPC-1 cells, p38 exerts a pro-mitogenic effect through MEK/ERK-dependent downregulation of p21. Thus, p38 may have suppressive or stimulatory effects on proliferation depending on the cell type, due to differential cross-talk between the p38 and MEK/ERK pathways.     

Inhibition of p38 mitogen-activated protein kinase reduces TNF-induced activation of NF-kappaB, elicits caspase activity, and enhances cytotoxicity

Among other cellular responses, tumor necrosis factor (TNF) induces different forms of cell death and the activation of the p38 mitogen-activated protein kinase (MAPK). The influence of p38 MAPK activation on TNF-induced apoptosis or necrosis is controversially discussed. Here, we demonstrate that pharmacological inhibition of p38 MAPK enhances TNF-induced cell death in murine fibroblast cell lines L929 and NIH3T3. Furthermore, overexpression of dominant-negative versions of p38 MAPK or its upstream kinase MKK6 led to increased cell death in L929 cells. While overexpression of the p38 isoforms alpha and beta did not protect L929 cells from TNF-induced toxicity, overexpression of constitutively active MKK6 decreased TNF-induced cell death. Although the used inhibitors of p38 MAPK decreased the phosphorylation of the survival kinase PKB/Akt, this effect could be ruled out as cause of the observed sensitization to TNF-induced cytotoxicity. Finally, we demonstrate that the nuclear factor kappaB (NF-kappaB)-dependent gene expression, shown as an example for the anti-apoptotic gene cellular inhibitor of apoptosis (c-IAP2), was reduced by p38 MAPK inhibition. In consequence, we found that inhibition of p38 MAPK led to the activation of the executioner caspase-3.     

O-linked β-N-acetylglucosamine supports p38 MAPK activation by high glucose in glomerular mesangial cells

Hyperglycemia augments flux through the hexosamine biosynthetic pathway and subsequent O-linkage of single β-N-acetyl-d-glucosamine moieties to serine and threonine residues on cytoplasmic and nuclear proteins (O-GlcNAcylation). Perturbations in this posttranslational modification have been proposed to promote glomerular matrix accumulation in diabetic nephropathy, but clear evidence and mechanism are lacking. We tested the hypothesis that O-GlcNAcylation enhances profibrotic signaling in rat mesangial cells. An adenovirus expressing shRNA directed against O-GlcNAc transferase (OGT) markedly reduced basal and high-glucose-stimulated O-GlcNAcylation. Interestingly, O-GlcNAc depletion prevented high-glucose-induced p38 mitogen-activated protein kinase (MAPK) and c-Jun NH(2)-terminal kinase phosphorylation. Downstream of p38, O-GlcNAc controlled the expression of plasminogen activator inhibitor-1, fibronectin, and transforming growth factor-β, important factors in matrix accumulation in diabetic nephropathy. Treating mesangial cells with thiamet-G, a highly selective inhibitor of O-GlcNAc-specific hexosaminidase (O-GlcNAcase), increased O-GlcNAcylation and p38 phosphorylation. The high-glucose-stimulated kinase activity of apoptosis signal-regulating kinase 1 (ASK1), an upstream MAPK kinase kinase for p38 that is negatively regulated by Akt, was inhibited by OGT shRNA. Akt Thr(308) and Ser(473) phosphorylation were enhanced following OGT shRNA expression in high-glucose-exposed mesangial cells, but high-glucose-induced p38 phosphorylation was not attenuated by OGT shRNA in cells pretreated with the phosphatidylinositol 3-kinase inhibitor LY-294002. OGT shRNA also reduced high-glucose-stimulated reactive oxygen species (ROS) formation. In contrast, diminished O-GlcNAcylation caused elevated ERK phosphorylation and PKCδ membrane translocation. Thus, O-GlcNAcylation is coupled to profibrotic p38 MAPK signaling by high glucose in part through Akt and possibly through ROS.     

Vibrio vulnificus-induced death of Jurkat T-cells requires activation of p38 mitogen-activated protein kinase by NADPH oxidase-derived reactive oxygen species

Vibrio vulnificus, a pathogenic bacterium causing primary septicemia, exhibited cytotoxicity towards Jurkat cells of T-lymphocytes through intracellular reactive oxygen species (ROS) production. Pretreatment of Jurkat T-cells with diphenyleneiodonium chloride (DPI) abolished V. vulnificus-induced ROS generation and bacterial ability to cause cell death. Jurkat T-cells expressing dominant-negative protein of Rac subunit of NADPH oxidase (NOX) did not show increased ROS production and cell death by V. vulnificus. Vibrio vulnificus also triggered phosphorylation of mitogen-activated protein kinases (MAPKs) including p38 and ERK1/2 in Jurkat T-cells. Experiments using inhibitors or small interfering RNAs for each MAPK showed that both MAPKs are involved in V. vulnificus-induced cell death. DPI only blocked the phosphorylation of p38 MAPK in Jurkat T-cells exposed by V. vulnificus. This study demonstrates that V. vulnificus induces death of Jurkat T-cells via ROS-dependent activation of p38 MAPK, and that NOX plays a major role in ROS generation in V. vulnificus-exposed cells.     

Apoptotic signaling in dopamine-induced cell death: the role of oxidative stress, p38 mitogen-activated protein kinase, cytochrome c and caspases

Oxidative stress generated by dopamine (DA) oxidation could be one of the factors underlying the selective vulnerability of nigral dopaminergic neurons in Parkinson's diseases. Here we show that DA induces apoptosis in SH-SY5Y neuroblastoma cells demonstrated by activation of caspase-9 and caspase-3, cleavage of poly(ADP-ribose) polymerase as well as nuclear condensation. We also show that p38 mitogen-activated protein kinase is activated within 10 min of DA treatment, which precedes the onset of apoptosis because the potent p38 kinase inhibitor SB203580 protects against DA-induced cell death as well as against caspase-9 and caspase-3 activation. In addition, the antioxidant N-acetyl-L-cysteine (NAC) effectively blocks DA-induced p38 kinase activation, caspase-9 and caspase-3 cleavage and subsequent apoptosis, indicating that DA triggers apoptosis via a signaling pathway that is initiated by the generation of reactive oxygen species (ROS). Dopamine exerts its toxicity principally intracellularly as the DA uptake inhibitor, nomifensine significantly reduces DA-induced cell death as well as activation of p38 kinase and caspase-3. Furthermore, DA induces mitochondrial cytochrome c release, which is dependent on p38 kinase activation and precedes the cleavage of caspases. These observations indicate that DA induces apoptosis primarily by generating ROS, p38 kinase activation, cytochrome c release followed by caspase-9 and caspase-3 activation.     

The p38/RK mitogen-activated protein kinase pathway regulates interleukin-6 synthesis response to tumor necrosis factor.

Tumour necrosis factor (TNF) is a pleiotropic cytokine, the activities of which include effects on gene expression, cell growth and cell death. The biological signalling mechanisms which are responsible for these TNF effects remain largely unknown. Here we demonstrate that the stress-responsive p38 mitogen-activated protein (MAP) kinase is involved in TNF-induced cytokine expression. TNF Treatment of cell activated the p38 MAP kinase pathway, as revealed by increased phosphorylation of p38 MAP kinase itself, activation of the substrate protein MAPKAP kinase-2, and culminating in the phosphorylation of the heat shock protein 27 (hsp27). Pretreatment of cells with the highly specific p38 MAP kinase inhibitor SB203580 completely blocked this TNF-induced activation of MAPKAP kinase-2 and hsp27 phosphorylation. Under the same conditions, SB203580 also completely inhibited TNF-induced synthesis of interleukin (IL)-6 and expression of a reporter gene that was driven by a minimal promoter containing two NF-Kappa B elements. However, neither TNF-induced DNA binding of TNF-Kappa B nor TNF-induced phosphorylation of its subunits was modulated by SB203580, suggesting that NF-Kappa B is not a direct target for the p38 MAP kinase pathway. Interestingly, TNF-induced cytotoxicity was not affected by SB203580, indicating that p38 MAP kinase might be an interesting target to interfere selectively with TNF-induced gene activation.     

Curcumin attenuates glutamate-induced HT22 cell death by suppressing MAP kinase signaling

Glutamate induces cell death by upsetting the cellular redox homeostasis, termed oxidative glutamate toxicity, in a mouse hippocampal cell line, HT22. Extracellular signal-regulated kinases (ERK) 1/2 are known key players in this process. Here we characterized the roles of both MAP kinases and cell cycle regulators in mediating oxidative glutamate toxicity and the neuroprotective mechanisms of curcumin in HT22 cells. c-Jun N-terminal kinase (JNK) and p38 kinase were activated during the glutamate-induced HT22 cell death, but at a later stage than ERK activation. Treatment with a JNK inhibitor, SP600125, or a p38 kinase inhibitor, SB203580, partly attenuated this cell death. Curcumin, a natural inhibitor of JNK signaling, protected the HT22 cells from glutamate-induced death at nanomolar concentrations more efficiently than SP600125. These doses of curcumin affected neither the level of intracellular glutathione nor the level of reactive oxygen species, but inactivated JNK and p38 significantly. Moreover, curcumin markedly upregulated a cell-cycle inhibitory protein, p21cip1, and downregulated cyclin D1 levels, which might help the cell death prevention. Our results suggest that curcumin has a neuroprotective effect against oxidative glutamate toxicity by inhibiting MAP kinase signaling and influencing cell-cycle regulation.     

p38 alpha MAPK inhibits JNK activation and collaborates with IkappaB kinase 2 to prevent endotoxin-induced liver failure

Activation of c-Jun amino-terminal kinase (JNK) facilitates tumour necrosis factor (TNF)-induced cell death. The p38 mitogen-activated protein kinase pathway is induced by TNF stimulation, but it has not been implicated in TNF-induced cell death. Here, we show that hepatocyte-specific ablation of p38alpha in mice results in excessive activation of JNK in the liver after in vivo challenge with bacterial lipopolysaccharide (LPS). Despite increased JNK activity, p38alpha-deficient hepatocytes were not sensitive to LPS/TNF toxicity showing that JNK activation was not sufficient to mediate TNF-induced liver damage. By contrast, LPS injection caused liver failure in mice lacking both p38alpha and IkappaB kinase 2 (IKK2) in hepatocytes. Therefore, when combined with partial nuclear factor-kappaB inhibition, p38alpha deficiency sensitizes the liver to cytokine-induced damage. Collectively, these results reveal a new function of p38alpha in collaborating with IKK2 to protect the liver from LPS/TNF-induced failure by controlling JNK activation.     

Nuclear Localization of p38 MAPK in Response to DNA Damage

p38 MAP kinase (MAPK) is activated in response to environmental stress, cytokines and DNA damage, and mediates death, cell differentiation and cell cycle checkpoints. The intracellular localization of p38 MAPK upon activation remains unclear, and may depend on the stimulus. We show here that activation of p38 MAPK by stimuli that induce DNA double strand breaks (DSBs), but not other stimuli, leads to its nuclear translocation. In addition, naturally occurring DSBs generated through V(D)J recombination in immature thymocytes also promote nuclear accumulation of p38 MAPK. Nuclear translocation of p38 MAPK does not require its catalytic activity, but is induced by a conformational change of p38 MAPK triggered by phosphorylation within the active site. The selective nuclear accumulation of p38 MAPK in response to DNA damage could be a mechanism to facilitate the phosphorylation of p38 MAPK nuclear targets for the induction of a G2/M cell cycle checkpoint and DNA repair.