Role Of P38 MAPK Research Articles

Astaxanthin attenuates glucose-induced liver injury in largemouth bass: role of p38MAPK and PI3K/Akt signaling pathways

BackgroundAstaxanthin (ASX) has been documented to exert beneficial influence on various processes in fish. Largemouth bass (Micropterus salmoides) serves as a common model for studying glucose-induced liver disease, making it imperative to investigate the regulatory mechanisms underlying its liver health.MethodsLargemouth bass were fed with a control diet (CON), a high carbohydrate diet (HC), or a HC diet supplemented astaxanthin (HCA) for 8-weeks, followed by the glucose tolerance test (GTT). Primary hepatocytes were treated with low glucose and high glucose combined with different concentrations of astaxanthin for 48 h. The histopathology, enzymology, transcriptomics, molecular biology and cell biology were combined to investigate the mechanism of liver injury.ResultsThis study provides evidence for the protective effects of ASX against growth performance reduction and hepatic liver injure in largemouth bass fed HC diet. In GTT, HCA diet exhibited an improvement in glucose tolerance following glucose loading. Although HCA diet did not restore the expression of insulin resistance-related genes in livers at different time during the GTT, the addition of ASX in the long-term HC diet did improve the insulin resistance pathway by regulating the PTP1B/PI3K/Akt signaling pathway. Hepatic transcriptome analyses showed that ASX plays an essential role in the modulation of glucose homeostasis in response to treated with HC diet. In in vitro study, ASX treatment resulted in an exaltation in cell viability and a reduction in the rate of cell apoptosis and reactive oxygen species (ROS). Additionally, astaxanthin was observed to improve apoptosis induced by high-glucose via p38MAPK/bcl-2/caspase-3 signaling pathway.ConclusionsAstaxanthin exhibited a protective effect against apoptosis by regulating p38MAPK/bcl-2/caspase-3 pathway, and ameliorated insulin resistance by activating the PTP1B/PI3K/Akt pathway. This study elucidated the mechanism of astaxanthin in the liver injury of largemouth bass from a new perspective and provided a new target for the treatment of insulin resistance.Graphical

P38 MAPK and Radiotherapy: Foes or Friends?

Over the last 30 years, the study of the cellular response to ionizing radiation (IR) has increased exponentially. Among the various signaling pathways affected by IR, p38 MAPK has been shown to be activated both in vitro and in vivo, with involvement in key processes triggered by IR-mediated genotoxic insult, such as the cell cycle, apoptosis or senescence. However, we do not yet have a definitive clue about the role of p38 MAPK in terms of radioresistance/sensitivity and its potential use to improve current radiotherapy. In this review, we summarize the current knowledge on this family of MAPKs in response to IR as well as in different aspects related to radiotherapy, such as their role in the control of REDOX, fibrosis, and in the radiosensitizing effect of several compounds.

P38 MAPK-dependent phosphorylation of TFEB promotes monocyte-to-macrophage differentiation.

The transcription factor EB (TFEB) regulates energy homeostasis and cellular response to a wide variety of stress conditions, including nutrient deprivation, oxidative stress, organelle damage, and pathogens. Here we identify S401 as a novel phosphorylation site within the TFEB proline-rich domain. Phosphorylation of S401 increases significantly in response to oxidative stress, UVC light, growth factors, and LPS, whereas this increase is prevented by p38 MAPK inhibition or depletion, revealing a new role for p38 MAPK in TFEB regulation. Mutation of S401 in THP1 cells demonstrates that the p38 MAPK/TFEB pathway plays a particularly relevant role during monocyte differentiation into macrophages. TFEB-S401A monocytes fail to upregulate the expression of multiple immune genes in response to PMA-induced differentiation, including critical cytokines, chemokines, and growth factors. Polarization of M0 macrophages into M1 inflammatory macrophages is also aberrant in TFEB-S401A cells. These results indicate that TFEB-S401 phosphorylation links differentiation signals to the transcriptional control of monocyte differentiation.

P38 MAPK Activity Is Required to Prevent Hyperactivation of NLRP3 Inflammasome.

Inflammation contributes to the pathogenesis and morbidity of wide spectrum of human diseases. The inflammatory response must be actively controlled to prevent bystander damage to tissues. Yet, the mechanisms controlling excessive inflammatory responses are poorly understood. NLRP3 inflammasome plays an important role in innate immune response to cellular infection or stress. Its activation must be tightly regulated because uncontrolled inflammasome activation is associated with a number of human diseases. p38 MAPK signaling plays an essential role in the regulation of inflammation. The role of p38 MAPK in inflammatory response associated with the expression of proinflammatory molecules is known. However, the anti-inflammatory functions of p38 MAPK are largely unknown. In this study, we show that pharmacologic inhibition or genetic deficiency of p38 MAPK leads to hyperactivation of NLRP3 inflammasome, resulting in enhanced Caspase 1 activation and IL-1β and IL-18 production. The deficiency of p38 MAPK activity induced an increase of cytosolic Ca2+ and excessive mitochondrial Ca2+ uptake, leading to exacerbation of mitochondrial damage, which was associated with hyperactivation of NLRP3 inflammasome. In addition, mice with deficiency of p38 MAPK in granulocytes had evidence of in vivo hyperactivation of NLRP3 inflammasome and were more susceptible to LPS-induced sepsis compared with wild-type mice. Our results suggest that p38 MAPK negatively regulates NLRP3 inflammasome through control of Ca2+ mobilization. Hyperactivity of inflammasome in p38-deficient mice causes lung inflammation and increased susceptibility to septic shock.

Ethanol-induced lymphatic endothelial cell permeability via MAP-kinase regulation.

Chronic alcohol alters the immune system enhancing the susceptibility to inflammation, bacterial, and viral infections in alcohol users. We have shown that alcohol causes increased permeability of mesenteric lymphatic vessels in alcohol-fed rats. The mechanisms of alcohol-induced lymphatic leakage are unknown. Endothelial cell monolayer permeability is controlled by junctional proteins complexes called tight junctions (TJ) and adherens junctions (AJ), and each can be regulated by MAPK activation. We hypothesize that ethanol induces lymphatic endothelial cell (LEC) permeability via disruption of LEC TJ through MAPK activation. An in vitro model of rat LECs was used. Ethanol-supplemented medium was added at concentrations of 0, 25, and 50 mM to confluent cells. Resistance-based barrier function, transwell permeability, cell viability, TJ, AJ, and MAPK protein activity, TJ and AJ gene expressions, and the role of p38 MAPK in barrier function regulation were measured. Ethanol increased the permeability of LECs compared to controls that was not associated with decreased cell viability. LECs treated with 50 mM ethanol showed an increase in phosphorylated levels of p38. No significant changes in TJ and AJ gene or protein expressions were observed after ethanol treatment. p38 inhibition prevented ethanol-induced increases in permeability. These findings suggest that p38 may play a role in the regulation of ethanol-induced LEC permeability, but altered permeability may not be associated with decreased TJ or AJ protein expression. Further investigation into junctional protein localization is needed to better understand the effects of ethanol on lymphatic endothelial cell-to-cell contacts and hyperpermeability.

Molecular and hormonal changes caused by long-term use of high dose pregabalin on testicular tissue: the role of p38 MAPK, oxidative stress and apoptosis.

In 1990, pregabalin was introduced as a novel antiepileptic drug that acts by binding selectively to the alpha-2-delta subunits of voltage-gated calcium channels resulting in increasing neuronal GABA levels and inhibiting the release of exciting neurotransmitters. The aim of our study is to assess the hazardous effects of prolonged high-dose pregabalin (like that abused by addicts) on testes and to clarify the potential causative mechanisms. The current study was conducted on 70 adult male Wistar albino rats which were divided into 7 groups. In our study we evaluated the effect of pregabalin, at concentrations 150 and 300mg/kg/day for 90 days, on hormones; FSH, LH, testosterone and prolactin secretion. Our study also evaluated the expression of apoptosis-related genes BAX and BCL2 in testicular tissue in addition to the western blotted analysis of p38 Mitogen activated protein kinases (p38 MAPK). The levels of reduced glutathione, malondialdehyde and superoxide dismutase were also measured. Pregabalin decreased testosterone level while FSH, LH and prolactin showed a significant increase. It also produced genotoxicity through reversal of the BAX/BCL2 ratio; increased p38 MAPK level and induction of oxidative stress markers. The concomitant administration of vitamin E significantly reduced all the previously mentioned biochemical and hormonal adverse effects caused by pregabalin. Pregabalin can adversely affect male fertility particularly in addicts and patients who are being treated with it for long periods as those suffering from neuropathies and seizures. Antioxidants like vitamin E could have a role in amelioration.

Stretch, scratch, and stress: Suppressors and supporters of senescence in human fetal membranes

IntroductionThroughout gestation, amnion membranes undergo mechanical and or physiological stretch, scratch, or stress which is withstood by repairing and remodeling processes to protect the growing fetus. At term, increased oxidative stress (OS) activates p38MAPK, induces senescence, and inflammation contributing to membrane dysfunction to promote labor. However, the signaling initiated by stretch and scratch is still unclear. This study compares the induction of p38MAPK mediated senescence by stretch, scratch, and stress in human amnion epithelial cells (AECs). MethodsPrimary AECs from term, not-in-labor, fetal membranes were cultured using the following conditions (N = 3); 1) CellFlex chambers with or without 20% biaxial stretch, 2) 8-well coverslips with or without scratch, and 3) cells exposed to cigarette smoke extract (CSE) inducing OS. p38MAPK (Western blot or immunocytochemistry), senescence activation, and inflammation (matrix metalloproteinases 9 [MMP9] activity-ELISA) were determined in cells exposed to various conditions. T-test and One-Way ANOVA was used to assess significance. ResultsBiological membrane extension, mimicked by 20% biaxial stretch of AEC, maintained an epithelial morphology and activated P-p38MAPK (P = 0.02) compared to the non-stretch controls, but did not induce senescence or MMP9 activation. AEC scratches were healed within 40-hrs, which included proliferation, migration, and cellular transitions aided by p38MAPK activation but not senescence. CSE induced OS increased p38MAPK (P = 0.018) activation, senescence (P = 0.019), and MMP9 (P = 0.02). ConclusionPhysiologic stretch and scratch experienced during gestation can cause p38MAPK activation without causing senescence or inflammation. This may be indicative of p38MAPK's role in tissue remodeling during pregnancy. Overwhelming OS, experienced at term, results in P-p38MAPK mediated senescence and inflammation to disrupt membrane remodeling.

MON-512 The Anti-Cancer Agent, Homoharringtonine, Induces the Sodium Iodide Symporter (NIS) Gene Expression in Culture Cells from Papillary Thyroid Cancer, as Well as Non-Thyroid Cancers

BACKGROUND: The therapeutic effect of thyroid cancer by radioiodide treatment is dependent on enhancement of NIS expression by TSH, especially its much greater magnitude in target tissue(s) compared to other healthy tissues. We preliminarily found that a protein synthesis inhibitor cycloheximide (CHX) markedly enhanced NIS mRNA expression, followed by increased iodide uptake, in several cancer cell lines, though CHX is highly toxic for clinical use. Aims: To evaluate the possibility of clinical application of such a pathway to enhance NIS expression, we tried another weak protein synthesis inhibitor, homoharringtonine (HHT), a natural plant alkaloid already utilized as an anti-leukemia agent, in several human cancer cell lines, including thyroid cancer. Methods: BHP 2-7 papillary thyroid cancer cells, MCF7 breast cancer cells, and MKN gastric cancer cells were treated with HHT and/or a p38 inhibitor, and harvested for quantitative RT-PCR of NIS. Results: HHT significantly induced the NIS mRNA expression in all of the cell lines tested, up to 298-fold in BHP cells, 38-fold in MCF7 cells, and 235-fold in MKN cells. Time course experiments indicated a biphasic induction of NIS in BHP cells with two peaks at 48 hours and 96 hours, with the EC50 of 664 ng/mL and 767 ng/mL, respectively. In contrast, NIS induction by HHT was monophasic in MCF7 cells at 24 hours with EC50 of 24.6 ng/mL, as well as MKN cells at 96 hours with EC50 of 255.7 ng/mL. Roles of p38 MAPK in the NIS induction has been reported previously, however, p38 inhibitors, SB239063 (10μM), as well as ML3403 (30μM), did not significantly reduce the NIS expression in HHT-treated BHP cells. Conclusion: These results indicated that HHT has a potential to enhance NIS expression in some NIS-expressing cancer tissues, including papillary thyroid cancer, although its functionality and efficacy are to be validated. The heterogeneity of response in the NIS expression to HHT in three cell lines could be due to differential mechanisms of NIS gene regulation in different tissues.

Elevated p38MAPK activity promotes neural stem cell aging.

Age-progressive neural stem cell (NSC) dysfunction leads to impaired neurogenesis, cognitive decline and the onset of age-related neurodegenerative pathologies. p38MAPK signalling pathway limits stem cell activity during aging in several tissues. Its role in NSCs remains controversial. In this work, we show that p38MAPK activity increases in NSCs with age in the subventricular zone (SVZ) and its pharmacological inhibition is sufficient to rejuvenate their activity in vitro. These data reveal a cell-autonomous role for p38MAPK increase in decreasing NSC homeostasis with age. This information shed light in the role of p38MAPK in NSC aging.

The roles of p38 MAPK\u2009\u2192\u2009COX2 and NF-\u03baB\u2009\u2192\u2009COX2 signal pathways in age-related testosterone reduction

In our study, we explored changes in the redox status and inflammatory response in the testes of the SAMP8 model of varying ages (2, 4, 8, 10 months old) compared with control mice SAMR1 by the methods of immunohistochemical staining, Western blotting, RT-PCR and Luminex multi-analyte cytokine profiling. We found that as ROS and inflammation levels increased during aging, steroidogenic enzymes (StAR and P450scc) reduced and led to the decline of testosterone production eventually. The pathways of P38 MAPK → COX2 and NF-κB → COX2 were detected by using specific inhibitors of SB203580 and Bay 11-7082 in isolated Leydig cells. These results indicated that activation of both p38 MAPK → COX2 and NF-κB → COX2 signaling pathways are functionally linked to the oxidative stress response and chronic inflammation during aging, and mediate their inhibitory effects on testosterone production.

Regulation of MAP kinase-mediated endothelial dysfunction in hyperglycemia via arginase I and eNOS dysregulation

Emerging evidence suggests that arginase contributes to endothelial dysfunction in diabetes. Intracellular signaling pathways, which interplay between arginase and eNOS enzyme activity leading to the development of endothelial dysfunction in hyperglycemia are not fully understood. Here, we analyzed the possible involvement of hyperglycemia (HG) induced arginase expression in eNOS protein regulation and activity and also the impact of arginase inhibition on eNOS activity. Furthermore, the roles of p38 MAPK and Erk1/2 phosphorylation in upregulation of arginase expression and eNOS dysregulation in endothelial cells (ECs) under hyperglycemia were evaluated. Protein analysis showed a concurrent increase in arginase I expression and decrease in eNOS expression and phosphorylation at Ser1177 under HG conditions. There was no simultaneous change in phosphorylation of eNOS at Thr495 in HG. Arginase inhibition prevented increased arginase activity, restored impaired NO bioavailability and reduced superoxide anion generation. Inhibition of MAP-kinases demonstrated that, unlike Erk1/2, p38 MAPK is an upstream activator in a signaling cascade leading to increased arginase I in HG conditions. P38 MAPK protein expression and phosphorylation were increased in response to HG. In the presence of a p38 MAPK inhibitor, HG-induced arginase expression was blunted. Although Erk1/2 was activated in HG, increased arginase expression was not blocked by co-treatment with an Erk1/2 inhibitor. Activation of both, p38 MAPK and Erk1/2 in HG, induced a downregulation in eNOS activity. Hence, applying MAPK inhibitors increased eNOS phosphorylation in HG.In conclusion, these findings demonstrate contributions of arginase I in the development of endothelial cell dysfunction under HG conditions via impaired eNOS regulation, which maybe mediated by p38 MAPK.

Role of P38 MAPK in Bile Formation and Cholestasis

Role of P38 MAPK in Bile Formation and Cholestasis

Role of P38 mitogen-activated protein kinase on Cx43 phosphorylation in cerebral vasospasm after subarachnoid hemorrhage

Objectives: Cx43 phosphorylation is involved in the pathogenesis of cerebral vasospasm (CVS) after subarachnoid hemorrhage (SAH). However, the exact phosphorylation mechanism of Cx43 in CVS is not fully elucidated. Thus, we examined the role of P38MAPK in CVS and Cx43 phosphorylation, using a double hemorrhage rat model.Methods: Sprague–Dawley rats weighing 300–350 g were grouped into sham, SAH, vehicle, and SAH + SB203580. SAH was induced by double injecting blood into the prechiasmatic cisterns. Neurological score was measured with the Garcia scoring system, and the diameters of basilar arteries and the expression of pCx43, pP38MAPK, and P38MAPK proteins were measured through pressure myograph measurement and Western blot analysis, respectively.Results: The neurological scores remarkably decreased after SAH but remarkably improved after SB203580 was used. The results of pressure myograph analysis on the SAH and vehicle groups showed the considerable narrowing of the basilar arteries in comparison with that of the sham group. By contrast, the arterial diameters in the SAH + SB203580 group were much larger than those observed in the SAH and vehicle groups. Moreover, the P38MAPK expression in the sham group had no substantial change in contrast to the SAH and vehicle groups, and pCx43 and pP38MAPK increased in the SAH and vehicle groups. Meanwhile, the SAH + SB203580 group showed marked decrease in Cx43 and P38MAPK phosphorylation levels relative to the SAH and vehicle groups.Conclusions: P38MAPK pathway facilitates the development of CVS through the upregulation of Cx43 phosphorylation.

Systematic review of p38 mitogen‐activated kinase and its functional role in reproductive tissues

Oxidative stress (OS) plays a role in uterine tissue remodeling during pregnancy and parturition. While p38 MAPK is an OS-response kinase, a precise functional role is unknown. Therefore, we conducted a systematic review of literature on p38 MAPK expression, activation, and function in reproductive tissues throughout pregnancy and parturition, published between January 1980 and August 2017, using four electronic databases (Web of Science, PubMed, Medline, and CoCHRANE). We identified 418 reports; 108 were selected for full-text evaluation and 74 were included in final review. p38 MAPK was investigated using feto-maternal primary or immortalized cells, tissue explants, and animal models. Western blot was most commonly used to report phosphorylated (active) p38 MAPK. Human placenta (27), chorioamniotic membranes (14), myometrium (13), decidua (8), and cervix (1) were the studied tissues. p38 MAPK's functions were tissue and gestational age dependent. Isoform specificity was hardly reported. p38 MAPK activity was induced by ROS or proinflammatory cytokines to promote cell signaling linked to cell fate, primed uterus, ripened cervix, and proinflammatory cytokine/chemokine production. In 35years, reports on p38 MAPK's role during pregnancy and parturition are scarce and current literature is insufficient to provide a comprehensive description of p38 MAPK's mechanistic role during pregnancy and parturition.

Inhibition of p38 MAPK activity leads to cell type-specific effects on the molecular circadian clock and time-dependent reduction of glioma cell invasiveness

BackgroundThe circadian clock is the basis for biological time keeping in eukaryotic organisms. The clock mechanism relies on biochemical signaling pathways to detect environmental stimuli and to regulate the expression of clock-controlled genes throughout the body. MAPK signaling pathways function in both circadian input and output pathways in mammals depending on the tissue; however, little is known about the role of p38 MAPK, an established tumor suppressor, in the mammalian circadian system. Increased expression and activity of p38 MAPK is correlated with poor prognosis in cancer, including glioblastoma multiforme; however, the toxicity of p38 MAPK inhibitors limits their clinical use. Here, we test if timed application of the specific p38 MAPK inhibitor VX-745 reduces glioma cell invasive properties in vitro.MethodsThe levels and rhythmic accumulation of active phosphorylated p38 MAPK in different cell lines were determined by western blots. Rhythmic luciferase activity from clock gene luciferase reporter cells lines was used to test the effect of p38 MAPK inhibition on clock properties as determined using the damped sine fit and Levenberg–Marquardt algorithm. Nonlinear regression and Akaike’s information criteria were used to establish rhythmicity. Boyden chamber assays were used to measure glioma cell invasiveness following time-of-day-specific treatment with VX-745. Significant differences were established using t-tests.ResultsWe demonstrate the activity of p38 MAPK cycles under control of the clock in mouse fibroblast and SCN cell lines. The levels of phosphorylated p38 MAPK were significantly reduced in clock-deficient cells, indicating that the circadian clock plays an important role in activation of this pathway. Inhibition of p38 MAPK activity with VX-745 led to cell-type-specific period changes in the molecular clock. In addition, phosphorylated p38 MAPK levels were rhythmic in HA glial cells, and high and arrhythmic in invasive IM3 glioma cells. We show that inhibition of p38 MAPK activity in IM3 cells at the time of day when the levels are normally low in HA cells under control of the circadian clock, significantly reduced IM3 invasiveness.ConclusionsGlioma treatment with p38 MAPK inhibitors may be more effective and less toxic if administered at the appropriate time of the day.

Tumor p38MAPK signaling enhances breast carcinoma vascularization and growth by promoting expression and deposition of pro-tumorigenic factors.

The breast carcinoma microenvironment strikingly influences cancer progression and response to therapy. Various cell types in the carcinoma microenvironment show significant activity of p38 mitogen-activated protein kinase (MAPK), although the role of p38MAPK in breast cancer progression is still poorly understood. The present study examined the contribution of tumor p38MAPK to breast carcinoma microenvironment and metastatic capacity. Inactivation of p38MAPK signaling in metastatic breast carcinoma cells was achieved by forced expression of the kinase-inactive mutant of p38/MAPK14 (a dominant-negative p38, dn-p38). Disruption of tumor p38MAPK signaling reduced growth and metastases of breast carcinoma xenografts. Importantly, dn-p38 markedly decreased tumor blood-vessel density and lumen sizes. Mechanistic studies revealed that p38 controls expression of pro-angiogenic extracellular factors such as matrix protein Fibronectin and cytokines VEGFA, IL8, and HBEGF. Tumor-associated fibroblasts enhanced tumor growth and vasculature as well as increased expression of the pro-angiogenic factors. These effects were blunted by dn-p38. Metadata analysis showed elevated expression of p38 target genes in breast cancers and this was an unfavorable marker of disease recurrence and poor-outcome. Thus, our study demonstrates that tumor p38MAPK signaling promotes breast carcinoma growth, invasive and metastatic capacities. Importantly, p38 enhances carcinoma vascularization by facilitating expression and deposition of pro-angiogenic factors. These results argue that p38MAPK is a valuable target for anticancer therapy affecting tumor vasculature. Anti-p38 drugs may provide new therapeutic strategies against breast cancer, including metastatic disease.

Aspergillus fumigatus-induced early inflammatory response in pulmonary microvascular endothelial cells: Role of p38 MAPK and inhibition by silibinin

Human invasive pulmonary aspergillosis (IPA) is a serious infectious disease mainly caused by Aspergillus fumigatus (A. fumigatus). Pulmonary microvascular endothelial cells (PMVECs) are important ones in the human lung tissue. However, it remains unclear about the role of PMVECs in IPA. In the present study, we cocultured PMVECs with A. fumigatus. We observed that A. fumigatus induced dose- and time-dependent increases of interleukin 6 (IL-6), interleukin 1β (IL-1β) and intercellular adhesion molecule 1 (ICAM-1) concentration in the cultures. Significant increases in IL-6, IL-1β, E-selectin, and ICAM-1 mRNA expression were also observed in the cultures treated with A. fumigatus. While preincubation with SB203580 (10μM) did not cause significant changes in IL-6, IL-1β and ICAM-1 concentration in the cocultures, significant IL-6, IL-1β and ICAM-1 concentration decreases were observed in the cocultures preincubated with SB203580 (20μM). Neither SP600125 (10-20μM) nor PD98059 (10-20μM) caused significant changes in IL-6, IL-1β and ICAM-1 concentration in the cocultures. PCR results also showed that SB203580 (20μM) (neither SP600125 (20μM) nor PD98059 (20μM)) preincubation significantly decreased IL-6, IL-1β, E-selectin and ICAM-1 mRNA expression in the cocultures. In addition, significant p38 MAPK phosphorylation increase was observed in the PMVECs cultures treated with A. fumigatus. Furthermore, silibinin pre-treatment and post-treatment were observed to significantly down-regulate mRNA and protein expression of proinflammatory factors and adhesion molecules in the cocultures. Finally, we observed that silibinin significantly inhibited A. fumigatus-induced p38 MAPK activation in PMVECs. Our results indicated that PMVECs might participate in IPA early inflammation which is mediated by p38 MAPK. Silibinin may inhibit A. fumigatus-induced inflammation in PMVECs through p38 MAPK.

Role of p38 MAPK and NF-κB signaling in chromatin decondensation and acquisition of cytokine competence during T cell activation

Abstract A proper immune response is hallmarked by the activation and clonal proliferation of antigen specific T cells. Antigen presentation to the T cell receptor (TCR) leads to the production of two signaling molecules, IP3 and DAG. While IP3 induces calcium flux, DAG activates Protein Kinase C (PKC). The activation of these pathways results in the decondensation of chromatin and the production of interleukin-2 (IL-2), both of which are required for the clonal proliferation of activated T cells. Previous work in our lab demonstrated that the IP3 pathway is sufficient for chromatin decondensation. In this study, we show that this pathway is not sufficient to make cells respond to IL-2. Furthermore, we investigate the role of the DAG pathway, specifically the role of the downstream molecules p38 MAPK and NF-κB, in the decondensation of chromatin and the competence to respond to IL-2 signaling. We found that neither of these molecules play a role in chromatin decondensation and that NF-κB may play a role in IL-2 competence. Collectively, these data, along with our previous findings, suggest that chromatin decondensation and competence to respond to IL-2 may be controlled by IP3 and DAG signaling respectively.

Involvement of the α/β Isoform of p38 MAP Kinase in Chemotactic Responses of Human Eosinophils to Eotaxin (CCL11) and RANTES (CCL5)

Eosinophils are the principal effector cells for allergic inflammation in a variety of diseases, in which they contribute to tissue damage and remodelling processes via the secretion of cytotoxic granular proteins and cytokines. The intracellular mechanisms that control the activation, recruitment and survival of eosinophils are fundamental in understanding these disease processes. Phosphoinositide 3-kinase (PI3K) has been shown previously to be essential for eosinophil chemotactic responses to some stimuli but not others. Human blood neutrophils have been shown to utilize two antagonistic signalling pathways for chemotaxis: PI3K and p38 mitogen-activated protein kinase (p38 MAPK). In the present study, the role of p38 MAPK in chemotactic responses of an eosinophil-differentiated myeloid leukaemia cell line (EOL-1) and human peripheral blood eosinophils to a range of stimuli - platelet-activating factor (PAF), eotaxin 1 (CCL11), RANTES (CCL5), interleukin 8 (IL8, CXCL8) and IL16 - was explored through the use of the p38 MAPK α/β isoform inhibitor, SB 203580. SB 203580 caused significant inhibition of chemotactic responses of both EOL-1 cells and blood eosinophils to eotaxin 1 and RANTES (≥75% inhibition at 1 μM SB 203580, p<0.01) but had no effect on the migration induced by PAF and IL16 (<25%) and little or no effect on responses to IL8. Responses to PAF - but not eotaxin - have been shown previously to be suppressed by PI3K inhibition. The complementary pattern of inhibition observed in the present study provides evidence that distinct PI3K-dependent and p38 MAPK-dependent chemoattractants may also exist for eosinophils.

TNFα alters occludin and cerebral endothelial permeability: Role of p38MAPK.

Occludin is a key tight junction (TJ) protein in cerebral endothelial cells (CECs) playing an important role in modulating blood-brain barrier (BBB) functions. This protein (65kDa) has been shown to engage in many signaling pathways and phosphorylation by both tyrosine and threonine kinases. Despite yet unknown mechanisms, pro-inflammatory cytokines and endotoxin (lipopolysaccharides, LPS) may alter TJ proteins in CECs and BBB functions. Here we demonstrate the responses of occludin in an immortalized human cerebral endothelial cell line (hCMEC/D3) to stimulation by TNFα (10 ng/mL), IL-1β (10 ng/mL) and LPS (100 ng/mL). Exposing cells to TNFα resulted in a rapid and transient upward band-shift of occludin, suggesting of an increase in phosphorylation. Exposure to IL-1β produced significantly smaller effects and LPS produced almost no effects on occludin band-shift. TNFα also caused transient stimulation of p38MAPK and ERK1/2 in hCMEC/D3 cells, and the occludin band-shift induced by TNFα was suppressed by SB202190, an inhibitor for p38MAPK, and partly by U0126, the MEK1/2-ERK1/2 inhibitor. Cells treated with TNFα and IL-1β but not LPS for 24 h resulted in a significant (p < 0.001) decrease in the expression of occludin, and the decrease could be partially blocked by SB202190, the inhibitor for p38MAPK. Treatment with TNFα also altered cell morphology and enhanced permeability of the CEC layer as measured by the FITC-dextran assay and the trans-endothelial electrical resistances (TEER). However, treatment with SB202190 alone could not effectively reverse the TNFα -induced morphology changes or the enhanced permeability changes. These results suggest that despite effects of TNFα on p38MAPK-mediated occludin phosphorylation and expression, these changes are not sufficient to avert the TNFα-induced alterations on cell morphology and permeability.