DEXAMETHASONE AND PENTOXIFYLLINE INHIBIT ENDOTOXIN-INDUCED CACHECTIN TUMOR-NECROSIS-FACTOR SYNTHESIS AT SEPARATE POINTS IN THE SIGNALING PATHWAY

The induction of cachectin/tumor necrosis factor (TNF) synthesis by bacterial endotoxins is a process that entails activation at several levels. Cachectin/TNF gene transcription is accelerated, leading to rapid accumulation of mRNA within the macrophage cytosol. In addition, translational derepression occurs, leading to far more efficient message utilization. Through the use of posttranscriptional reporter constructs, we now demonstrate that certain agents capable of inhibiting cachectin/TNF biosynthesis operate through different mechanisms. In RAW 264.7 macrophages, pentoxifylline blocks cachectin/TNF mRNA accumulation but has no effect upon the efficiency of reporter mRNA translation. Dexamethasone, on the other hand, has only a modest effect on cachectin/TNF mRNA accumulation, but strongly impedes translational derepression. Combined application of dexamethasone and pentoxifylline to macrophages causes a greater suppression of cachectin/TNF biosynthesis that can be achieved by either agent alone. These findings suggest that the signaling pathway activated by endotoxin is branched, and that selective inhibition of different parts of the pathway may be achieved through the use of distinct agents

Regulation of PRAK subcellular location by p38 MAP kinases

The p38 mitogen-activated protein kinase (MAPK) pathway plays an important role in cellular responses to inflammatory stimuli and environmental stress. p38 regulated/activated protein kinase (PRAK, also known as mitogen-activated protein kinase activated protein kinase 5 [MAP-KAPK5]) functions downstream of p38alpha and p38beta in mediating the signaling of the p38 pathway. Immunostaining revealed that endogenous PRAK was predominantly localized in the cytoplasm. Interestingly, ectopically expressed PRAK was localized in the nucleus and can be redistributed by coexpression of p38alpha or p38beta to the locations of p38alpha and p38beta. Mutations in the docking groove on p38alpha/p38beta, or the p38-docking site in PRAK, disrupted the PRAK-p38 interaction and impaired the ability of p38alpha and p38beta to redistribute ectopically expressed PRAK, indicating that the location of PRAK could be controlled by its docking interaction with p38alpha and p38beta. Although the majority of PRAK molecules were detected in the cytoplasm, PRAK is consistently shuttling between the cytoplasm and the nucleus. A sequence analysis of PRAK shows that PRAK contains both a putative nuclear export sequence (NES) and a nuclear localization sequence (NLS). The shuttling of PRAK requires NES and NLS motifs in PRAK and can be regulated through cellular activation induced by stress stimuli. The nuclear content of PRAK was reduced after stimulation, which resulted from a decrease in the nuclear import of PRAK and an increase in the nuclear export of PRAK The nuclear import of PRAK is independent from p38 activation, but the nuclear export requires p38-mediated phosphorylation of PRAK Thus, the subcellular distribution of PRAK is determined by multiple factors including its own NES and NLS, docking interactions between PRAK and docking proteins, phosphorylation of PRAK, and cellular activation status. The p38 MAPKs not only regulate PRAK activity and PRAK activation-related translocation, but also dock PRAK to selected subcellular locations in resting cells

Functions and Mechanisms of the p38 MAP Kinase Pathway

通讯作者。Tel: 0592-2189390, E-mail: [email protected][中文文摘]丝裂原活化蛋白激酶家族可以在一系列细胞外刺激下调控细胞的行为。作为该家族的四个亚家族之一,p38亚族在许多生理过程中扮演着重要角色。p38信号通路可以在紫外照射、热击、高渗透压、炎症因子、生长因子等细胞外刺激时被激活,调控细胞分化、细胞周期、炎症反应等多种生理过程。文章重点讨论了p38亚族各个成员的特性、该信号通路的组成部分、调控机制以及生物学功能。另外,还分析了p38与其他信号通路的联系以及对一些生理过程的影响。[英文文摘]The members of the mitogen-activated protein kinase family participate in cellular responses to a wide range of extracellular stimuli. One of the four sub-families, the p38 group MAP kinases, plays a vital role in numerous biological processes. The p38 signaling pathway can be activated in response to many physical, chemical,and biological stresses, including UV light, heat shock, osmotic shock, inflammatory cytokines, and growth factors.The p38 pathway also regulates various physiological processes such as cell differentiation, the cell cycle, and inflammation. This review focuses on the characteristics of each p38 member, the components of the p38 pathway, and the mechanisms and consequences of p38 activation. We also discuss the interplay between the p38 pathway and other signaling pathways

Dual colored mesoporous silica nanoparticles with pH activable rhodamine-lactam for ratiometric sensing of lysosomal acidity

Alteration of lysosome acidity has been implicated in many biological events ranging from apoptosis to cancer metastasis, etc. Mesoporous silica nanoparticles doped with acid activable rhodamine-lactam and fluorescein isothiocyanate (FITC) were developed for ratiometric sensing of lysosomal pH changes in live cells with flow cytometry.NSF China[20802060, 21072162, 30830092, 30921005, 91029304, 81061160512]; Fundamental Research Funds for the Central Universities[2011121020]; 973 program[2009CB522200

Glucocorticoids synergize with IL-1β to induce TLR2 expression via MAP Kinase Phosphatase-1-dependent dual Inhibition of MAPK JNK and p38 in epithelial cells

BACKGROUND: Despite the importance of glucocorticoids in suppressing immune and inflammatory responses, their role in enhancing host immune and defense response against invading bacteria is poorly understood. Toll-like receptor 2 (TLR2) has recently gained importance as one of the major host defense receptors. The increased expression of TLR2 in response to bacteria-induced cytokines has been thought to be crucial for the accelerated immune response and resensitization of epithelial cells to invading pathogens. RESULTS: We show that IL-1β, a key proinflammatory cytokine, greatly up-regulates TLR2 expression in human epithelial cells via a positive IKKβ-IκBα-dependent NF-κB pathway and negative MEKK1-MKK4/7-JNK1/2 and MKK3/6-p38 α/β pathways. Glucocorticoids synergistically enhance IL-1β-induced TLR2 expression via specific up-regulation of the MAP kinase phosphatase-1 that, in turn, leads to dephosphorylation and inactivation of both MAPK JNK and p38, the negative regulators for TLR2 induction. CONCLUSION: These results indicate that glucocorticoids not only suppress immune and inflammatory response, but also enhance the expression of the host defense receptor, TLR2. Thus, our studies may bring new insights into the novel role of glucocorticoids in orchestrating and optimizing host immune and defense responses during bacterial infections and enhance our understanding of the signaling mechanisms underlying the glucocorticoid-mediated attenuation of MAPK

Evidence that age-related changes in p38 MAP kinase contribute to the decreased steroid production by the adrenocortical cells from old rats

The current studies were initiated to investigate whether excessive oxidative stress exerts its antisteroidogenic action through modulation of oxidant-sensitive mitogen-activated protein kinase (MAPK) signaling pathways. Western blot analysis indicated that aging caused increased phosphorylation and activation of rat adrenal p38 MAPK, but not the ERK1/2 or JNK1/2. Lipid peroxidation measurements (an index of cellular oxidative stress) indicated that adrenal membranes from young animals contained only minimal levels of endogenous thiobarbituric acid-reactive substances (TBARS), and exposure of membranes to enzymatic and non-enzymatic pro-oxidants enhanced TBARS formation approximately 12- and 20-fold, respectively. The adrenal membranes from old animals showed much more susceptibility to lipid peroxidation and exhibited roughly 4- to 6-fold higher TBARS formation than young controls both under basal conditions and in response to pro-oxidants. Qualitatively similar results were obtained when lipid peroxide formation was measured using a sensitive FOXRS (ferrous oxidation-xylenol orange-reactive substances) technique. We next tested whether aging-induced excessive oxidative insult alters steroidogenesis through modulation of MAPK signaling pathway. Treatment of adrenocortical cells from old rats with specific p38 MAPK inhibitors restored Bt(2)cAMP-stimulated steroidogenesis similar to 60-70% of the value seen in cells of young animals. Likewise, pretreatment of cells with reactive oxygen species (ROS) scavengers MnTMPyP and N-acetyl cysteine also partially rescued age-induced loss of steroid production. In contrast, simultaneous treatment of cells with ROS scavengers and p38 MAPK inhibitor did not produce any additional effect suggesting that both types of inhibitors exert their stimulatory action through inhibition of p38 MAPK activation. Collectively, these results indicate that p38 MAPK functions as a signaling effector in oxidative stress-induced inhibition of steroidogenesis during aging

Orphan Nuclear Receptor Nur77 Is Involved in Caspase-independent Macrophage Cell Death

Activation-induced cell death in macrophages has been observed, but the mechanism remains largely unknown. Activation-induced cell death in macrophages can be independent from caspases, and the death of activated macrophages can even be triggered by the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (zVAD). Here, we show that this type of macrophage death can occur in the septic mouse model and that toll-like receptor (TLR)-2 or TLR4 signaling is required in this process. We conclude that Nur77 is involved in the macrophage death because Nur77 expression correlates with cell death, and cell death is reduced significantly in Nur77-deficient macrophages. The extracellular signal–regulated kinase pathway, which is downstream of TLR2 or TLR4, and myocyte-specific enhancer binding factor 2 (MEF2) transcription factor activity, which is up-regulated by zVAD, are required for Nur77 induction and macrophage death. Reporter gene analysis suggests that Nap, Ets, Rce, and Sp1 sites in the Nur77 promoter are regulated by TLR4 signaling and that MEF2 sites in the Nur77 promoter are regulated by zVAD treatment. MEF2 transcription factors are constitutively expressed and degraded in macrophages, and zVAD increases MEF2 transcription factor activity by preventing the proteolytic cleavage and degradation of MEF2 proteins. This paper delineates the dual signaling pathways that are required for Nur77 induction in macrophages and demonstrates a role of Nur77 in caspase-independent cell death

Imaging of intracellular acidic compartments with a sensitive rhodamine based fluorogenic pH sensor

The parameters of intracellular acidic compartments are altered in apoptosis, autophagy, cancer metastasis, etc. Low background and selective staining of lysosomes and autophagosomes was achieved with N-(rhodamine 6G)-lactam-ethylenediamine (R6G-EDA) which fluoresces in acidic vesicles via pH mediated ring opening of the intramolecular lactam. Long retention time of R6G-EDA in lysosomes and its exceptional stability against photo-bleaching allow full time tracking of lysosome morphology changes in tumor necrosis factor-alpha (TNF-alpha) triggered cell death, suggesting its utility for acidic vesicle studies in cell biology.NSF China[20802060, 21072162, 30830092, 30921005, 91029304, 81061160512]; Xiamen University[2011121020]; 973 program[2009CB522200