Restoration of Corticosteroid Sensitivity by p38 Mitogen Activated Protein Kinase Inhibition in Peripheral Blood Mononuclear Cells from Severe Asthma

Background

Severe asthma accounts for a small number of asthmatics but represents a disproportionate cost to health care systems. The underlying mechanism in severe asthma remains unknown but several mechanisms are likely to be involved because of a very heterogeneous profile. We investigated the effects of a p38MAPK inhibitor in corticosteroid sensitivity in peripheral blood mononuclear cells (PBMCs) from severe asthmatics and the profile of its responders.

Methodology/Principal Findings

Corticosteroid sensitivity was determined by measuring dexamethasone inhibition of CD3/28 and TNF-α induced IL-8 production in PBMCs by using ELISA. PBMCs from severe asthmatics were relatively less sensitive to dexamethasone (Dex) as compared to those of non-severe asthmatics and healthy volunteers. The IC₅₀ values of Dex negatively correlated with decreased glucocorticoid receptor (GR) nuclear translocation assessed using immunocytochemistry (r = −0.65; p<0.0005) and with decreased FEV₁ (% predicted) (r = 0.6; p<0.0005). A p38α/β inhibitor (SB203580) restored Dex-sensitivity in a subpopulation of severe asthma that was characterized by a defective GR nuclear translocation, clinically by lower FEV₁ and higher use of oral prednisolone. We also found that SB203580 partially inhibited GR phosphorylation at serine 226, resulting in increased GR nuclear translocation in IL-2/IL-4 treated corticosteroid insensitive U937s.

Conclusions/Significance

p38MAPKα/β is involved in defective GR nuclear translocation due to phosphorylation at Ser226 and this will be a useful biomarker to identify responders to p38MAPKα/β inhibitor in the future.     

蓖麻毒素对肝癌细胞有丝分裂原激活蛋白激酶的影响

为进一步探讨蓖麻毒素的毒作用机理 ,采用 Western印迹和免疫组化方法研究蓖麻毒素对人肝癌细胞内磷酸化状态和有丝分裂原激活蛋白激酶 (MAPK)的影响 .其结果表明 :蓖麻毒素能够影响细胞内的磷酸化状态 ,并能激活细胞内的有丝分裂原激活蛋白激酶 ,对 MAPK的激活有时间依赖性和剂量依赖性 . MAPK信号传导途径参与了蓖麻毒素对肝癌细胞的毒作用 .     

AICAR Enhances the Phagocytic Ability of Macrophages towards Apoptotic Cells through P38 Mitogen Activated Protein Kinase Activation Independent of AMP-Activated Protein Kinase

Recent studies have suggested that 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR) increases macrophage phagocytosis through adenosine monophosphate-activated protein kinase (AMPK). However, little information is available on the effects of AICAR on the clearance of apoptotic cells by macrophages, known as efferocytosis, which is essential in maintaining tissue homeostasis and resolving inflammation. AICAR increased p38 MAPK activation and the phagocytosis of apoptotic cells by macrophages, which were inhibited by the p38 MAPK inhibitor, SB203580, the TGF-beta-activated kinase 1 (TAK1) inhibitor, (5Z)-7-oxozeaenol, and siRNA-mediated knock-down of p38α. AICAR increased phosphorylation of Akt, but the inhibition of PI3K/Akt activity using {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 did not affect the AICAR-induced changes in efferocytosis in macrophages. {"type":"entrez-protein","attrs":{"text":"CGS15943","term_id":"875345334","term_text":"CGS15943"}}CGS15943, a non-selective adenosine receptor antagonist, did not affect AICAR-induced changes in efferocytosis, but dipyridamole, an adenosine transporter inhibitor, diminished the AICAR-mediated increases in efferocytosis. AICAR-induced p38 MAPK phosphorylation was not inhibited by the AMPK inhibitor, compound C, or siRNA-mediated knock-down of AMPKα1. Inhibition of AMPK using compound C or 5’-iodotubercidin did not completely block AICAR-mediated increases in efferocytosis. Furthermore, AICAR also increased the removal of apoptotic neutrophils or thymocytes in mouse lungs. These results reveal a novel mechanism by which AICAR increases macrophage-mediated phagocytosis of apoptotic cells and suggest that AICAR may be used to treat efferocytosis-related inflammatory conditions.     

Mitogen Activated Protein Kinase Activated Protein Kinase 2 Regulates Actin Polymerization and Vascular Leak in Ventilator Associated Lung Injury

Mechanical ventilation, a fundamental therapy for acute lung injury, worsens pulmonary vascular permeability by exacting mechanical stress on various components of the respiratory system causing ventilator associated lung injury. We postulated that MK2 activation via p38 MAP kinase induced HSP25 phosphorylation, in response to mechanical stress, leading to actin stress fiber formation and endothelial barrier dysfunction. We sought to determine the role of p38 MAP kinase and its downstream effector MK2 on HSP25 phosphorylation and actin stress fiber formation in ventilator associated lung injury. Wild type and MK2^−/− mice received mechanical ventilation with high (20 ml/kg) or low (7 ml/kg) tidal volumes up to 4 hrs, after which lungs were harvested for immunohistochemistry, immunoblotting and lung permeability assays. High tidal volume mechanical ventilation resulted in significant phosphorylation of p38 MAP kinase, MK2, HSP25, actin polymerization, and an increase in pulmonary vascular permeability in wild type mice as compared to spontaneous breathing or low tidal volume mechanical ventilation. However, pretreatment of wild type mice with specific p38 MAP kinase or MK2 inhibitors abrogated HSP25 phosphorylation and actin polymerization, and protected against increased lung permeability. Finally, MK2^−/− mice were unable to phosphorylate HSP25 or increase actin polymerization from baseline, and were resistant to increases in lung permeability in response to HV_T MV. Our results suggest that p38 MAP kinase and its downstream effector MK2 mediate lung permeability in ventilator associated lung injury by regulating HSP25 phosphorylation and actin cytoskeletal remodeling.     

Comparison of Peptide Array Substrate Phosphorylation of c-Raf and Mitogen Activated Protein Kinase Kinase Kinase 8

Kinases are pivotal regulators of cellular physiology. The human genome contains more than 500 putative kinases, which exert their action via the phosphorylation of specific substrates. The determinants of this specificity are still only partly understood and as a consequence it is difficult to predict kinase substrate preferences from the primary structure, hampering the understanding of kinase function in physiology and prompting the development of technologies that allow easy assessment of kinase substrate consensus sequences. Hence, we decided to explore the usefulness of phosphorylation of peptide arrays comprising of 1176 different peptide substrates with recombinant kinases for determining kinase substrate preferences, based on the contribution of individual amino acids to total array phosphorylation. Employing this technology, we were able to determine the consensus peptide sequences for substrates of both c-Raf and Mitogen Activated Protein Kinase Kinase Kinase 8, two highly homologous kinases with distinct signalling roles in cellular physiology. The results show that although consensus sequences for these two kinases identified through our analysis share important chemical similarities, there is still some sequence specificity that could explain the different biological action of the two enzymes. Thus peptide arrays are a useful instrument for deducing substrate consensus sequences and highly homologous kinases can differ in their requirement for phosphorylation events.     

植物病原真菌的MAPK基因及其功能

叙述在植物病原真菌中促分裂原活化蛋白激酶 (Mitogenactivatedproteinkinase,MAPK)基因的种类和特征 ,概括了MAPK基因在植物病原真菌生长发育、胁迫反应和侵染、致病过程中的作用及其研究现状 ,讨论了进行植物病原真菌MAPK基因研究的意义及重点研究的课题 ,并根据最新研究进展 ,提出了植物病原真菌MAPK基因研究的发展前景。     

OS-2 Mitogen Activated Protein Kinase Regulates the Clock-Controlled Gene ccg-1 in Neurospora crassa

OS-2 MAP kinase is involved in osmoadaptation in Neurospora crassa. Clock-controlled genes ccg-1, bli-3, and con-10 were induced by osmotic stress in an OS-2 dependent manner. In contrast, osmotic stress did not affect the expression of clock genes frq, wc-1, and wc-2 or of clock-controlled genes ccg-2 and bli-4. These results suggest that OS-2 participates in the regulation of certain circadian-clock output genes.     

Rottlerin-Mediated Inhibition of Chlamydia trachomatis Growth and Uptake of Sphingolipids Is Independent of p38-Regulated/Activated Protein Kinase (PRAK)

We previously found that rottlerin, a plant-derived small molecule compound, profoundly inhibited Chlamydia trachomatis growth and blocked sphingolipid trafficking from host cell Golgi into chlamydial inclusions. Since the p38-regulated/activated protein kinase (PRAK) is a known target of rottlerin and is activated in Chlamydia trachomatis-infected cells, we investigated the potential role of this kinase in rottlerin-mediated anti-chlamydial activity. However, we found that a PRAK-specific inhibitor failed to inhibit chlamydial growth, suggesting that the kinase activity of PRAK may not be required for chlamydial intracellular replication. This conclusion was supported by the observation that chlamydial organisms replicated equally well in mouse embryonic fibroblast cells with or without PRAK. Moreover, neither the PRAK inhibitor nor PRAK deficiency altered host sphingolipid trafficking into chlamydial inclusions. Finally, rottlerin maintained its anti-chlamydial activity in PRAK-deficient cells. Together, these observations have demonstrated that PRAK is not required for either the rottlerin-mediated anti-chlamydial activity or rottlerin inhibition of sphingolipid trafficking, suggesting that rottlerin may achieve its inhibitory role by targeting other host factors.     

Coordinated Regulation of Radioadaptive Response by Protein Kinase C and p38 Mitogen-Activated Protein Kinase

Eukaryotic cells are known to have an inducible or adaptive response that enhances radioresistance after a low priming dose of radiation. This radioadaptive response seems to present a novel cellular defense mechanism. However, its molecular processing and signaling mechanisms are largely unknown. Here, we studied the role of protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) in the expression of radioadaptive response in cultured mouse cells. Protein immunoblot analysis using isoform-specific antibodies showed an immediate activation of PKC-alpha upon X-irradiation as indicated by a translocation from cytosol to membrane. A low priming dose caused a prolonged translocation, while a nonadaptive high dose dramatically downregulated the total PKC level. Low-dose X-rays also activated the p38 MAPK. The activation of p38 MAPK and resistance to chromosome aberration formation were blocked by SB203580, an inhibitor of p38 MAPK, and Calphostin C, an inhibitor of PKC. Furthermore, it was demonstrated that p38 MAPK was physically associated with delta1 isoform of phospholipase C (PLC-delta1), which hydrolyzed phosphatidylinositol bisphosphate into diacylglycerol, an activator of PKC, and that SB203580 also blocked the activation of PKC-alpha. These results indicate the presence of a novel mechanism for coordinated regulation of adaptive response to low-dose X-rays by a nexus of PKC-alpha/p38 MAPK/PLC-delta1 circuitry feedback signaling pathway with its breakage operated by downregulation of labile PKC-alpha at high doses or excess stimuli.     

A Computer‐Based Model for the Regulation of Mitogen Activated Protein Kinase (MAPK) Activation

Abstract

Computer simulations and mathematical modeling of biological processes are becoming increasingly popular, and yet the complexity of the biochemical systems or the differences between experimental setups make it very difficult to establish a standard formula for these modeling projects. Before we can start using computer‐based models for predictions or targeted experiment designs, it is very important to establish a reliable model on which those predictions can be based and experimentally tested. Here we attempt to present a computer model for the mitogen‐activated protein kinase (MAPK) signaling cascade which is consistent with previously published experimental results. In this study we have focused our attention to a generic MAPK ERK (extracellular signal‐regulated kinase) pathway activated by epidermal growth factor (EGF) in an attempt to understand how receptors may achieve different activation kinetics of the MAPK signaling. We successfully show that the level of receptor expression is one key determinant in this regulation, and that the binding affinity of the active receptor to adaptor proteins can have a small but albeit direct effect on the downstream activation.     

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 p38α heterozygous (p38α+/?) 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 p38α+/? mice was prolonged compared to that in wild-type (WT) mice. Platelets prepared from p38α+/? mice showed impairment of the aggregatory response to a low concentration of U46619, a thromboxane A₂ analogue. Furthermore, platelets prepared from p38α+/? 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 p38α+/? mice. These results suggest that p38 plays an important role in thrombus formation by regulating platelet function and tissue factor activity.     

Mitogen Activated Protein kinase signal transduction pathways in the prostate

The biochemistry of the mitogen activated protein kinases ERK, JNK, and p38 have been studied in prostate physiology in an attempt to elucidate novel mechanisms and pathways for the treatment of prostatic disease. We reviewed articles examining mitogen-activated protein kinases using prostate tissue or cell lines. As with other tissue types, these signaling modules are links/transmitters for important pathways in prostate cells that can result in cellular survival or apoptosis. While the activation of the ERK pathway appears to primarily result in survival, the roles of JNK and p38 are less clear. Manipulation of these pathways could have important implications for the treatment of prostate cancer and benign prostatic hypertrophy.     

植物丝裂原活化蛋白激酶级联信号转导通路研究进展

丝裂原活化蛋白激酶(MAPK)是酵母、动物和植物等真核生物中普遍存在和高度保守的一类信号转导通路,由MAPKKK、MAPKK和MAPK等3部分组成,在应对生物非生物胁迫、激素、细胞分裂调控及植物生长发育等过程中发挥重要作用。该文对近年来国内外有关MAPK级联通路的组成、在植株体内的生物学功能以及MAPK通路的失活进行了概述,旨在为今后MAPK通路介导的信号转导机制的研究提供参考依据。     

p38蛋白激酶在热损伤诱导单核细胞Raw264.7凋亡中的作用

为探讨ｐ３８蛋白激酶在热损伤诱导单核细胞株Ｒａｗ ２６４．７凋亡中的调控作用．应用流式细胞检测术、透射电镜技术、ＤＮＡ 凝胶电泳、Ｗｅｓｔｅｒｎ ｂｌｏｔ、激酶活性测定检测ｐ３８蛋白激酶在热损伤诱导细胞凋亡中的作用．结果显示,热损伤后５ ｍ ｉｎ,ｐ３８即被激活,３０ ｍ ｉｎ 时达到最高水平,然后逐渐下降,２ ｈ 达基底水平;热损伤后３ ｈ 细胞凋亡率开始明显增高,ｐ３８蛋白激酶活性增高是细胞凋亡发生之前的事件;进一步观察了ｐ３８特异性抑制剂ＦＨＰＩ对细胞凋亡的影响,发现ＦＨＰＩ可以抑制热损伤诱导的细胞凋亡．上述结果提示,ｐ３８参与介导热损伤诱导的Ｒａｗ ２６４．７细胞凋亡．     

Reactivating Kinase/p38 Phosphorylates τ Protein In Vitro

Abstract: Neurofibrillary tangles, one of the major pathological hallmarks of Alzheimer-diseased brains, consist primarily of aggregated paired helical filaments (PHFs) of hyperphosphorylated τ protein. τ from normal brain and especially from foetal brain is also phosphorylated on some of the sites phosphorylated in PHFs, mainly at serines or threonines followed by prolines. A number of protein kinases can phosphorylate τ in vitro; those that require or accept prolines include GSK3 and members of the mitogen-activated protein (MAP) kinase family, ERK1, ERK2, and SAP kinase-β/JNK. In this report, we show that another member of the MAP kinase family, the stress-activated kinase p38/RK, can phosphorylate τ in vitro. Western blots with phosphorylation-sensitive antibodies showed that p38, like ERK2 and SAP kinase-β/JNK, phosphorylated τ at sites found phosphorylated physiologically (Thr¹⁸¹, Ser²⁰², Thr²⁰⁵, and Ser³⁹⁶) and also at Ser⁴²², which is phosphorylated in neurofibrillary tangles but not in normal adult or foetal brain. These findings support the possibility that cellular stress might contribute to τ hyperphosphorylation during the formation of PHFs, and hence, to the development of τ pathology.