NF-AT DNA Binding Activity Research Articles

PKC θ-mediated Ca2+/NF-AT signalling pathway may be involved in T-cell immunosuppression in coal-burning arsenic-poisoned population

Arsenic poisoning is a worldwide endemic disease that affects thousands of people. Growing evidence from animal, cell, and human studies indicates that arsenic has deleterious effects on the immune system. The present investigation is a population-based study that observed changes in the proliferation of human T-cells and IL-2 and INF-γ mRNA expression. Our results show that coal-burning arsenic can cause T-cell immunosuppression in the population, and participates in the occurrence and development of arsenic poisoning. In addition, we analyzed the intracellular calcium index, expression of protein kinase C theta (PKC θ) and phosphorylated PKC θ, and the DNA-binding activity of NF-AT in peripheral blood mononuclear cells (PBMCs). Our analysis demonstrates that the PKC θ-mediated Ca2+/NF-AT signalling pathway may be involved in the T-cell immunosuppression of coal-burning arsenic-poisoned population. This study provides important data for a mechanistic understanding of endemic arsenic poisoning.

Role of Ca/CaN/NFAT signaling in IL-4 expression by splenic lymphocytes exposed to phthalate (2-ethylhexyl) ester in spleen lymphocytes

The aims of present study were to investigate the effect of phthalate (2-ethylhexyl) ester (DEHP) and mono-(2-ethylhexyl) phthalate (MEHP) on Th1/Th2 balance signaling for interleukin 4 (IL-4) expression in splenic lymphocytes, and contribution of MEHP to any hypothesized changes in vitro. Primary splenic lymphocytes were exposed to DEHP/MEHP. ELISA and Western blotting were used to detect proteins. Confocal-microscopy was used to examine nuclear translocation. Nuclear factor of activated T cells (NFAT) DNA binding activity was examined by electrophoretic mobility-shift assay. DEHP significantly increased IL-4 and interferon gamma (IFN-γ) level, and reduced Th1/Th2 ratio (reflected by IFN-γ/IL-4) with 5 μg/L Concanavalin A (ConA) treatment. While MEHP reduced Th1/Th2 ratio (represented by IFN-γ/IL-6). IL-4 mRNA was significantly increased by DEHP but not by MEHP after PMA and Ion treatment. DEHP significantly inhibited NFATp protein in cytosol and nucleus. DEHP augmented nuclear translocation of NFATc in transfected EL4 cells and NFAT DNA-binding activity. DEHP-mediated enhancement of calcium-dependent phosphatase calcineurin (CaN) protein, and NFAT and IL-4 expression were abrogated by calcium antagonist verapamil and CaN inhibitor tarcolimus. Ca(2+)/calmodulin antagonist chlorpromazine significantly suppressed IL-4 and CaN production with no NFAT mRNA change. Our study suggests that DEHP and MEHP impact Th1/Th2 balance by modulating different cytokines. DEHP-affected IL-4 expression through Ca/CaN/NFAT signaling pathway, but no effect was discovered for MEHP.

DNA binding activity of nuclear factor of activated T cells in mononuclear cells from renal transplant patients with and without BK virus viruria

ObjectivesRenal transplant patients receive calcineurin inhibitors to suppress the calcineurin-nuclear factor of activated T cells (NFAT) pathway. The DNA binding activity of NFAT and its relationship to the reactivation of BK virus (BKV) has not been evaluated in renal transplant patients. Patients and MethodsThe DNA binding activity of NFAT cytoplasmic 1 (NFATc1) was measured by enzyme-linked immunosorbent assay in peripheral blood mononuclear cells from 26 renal transplant patients and 26 healthy controls. At the same time, their urinary BKV viral load was measured by real-time polymerase chain reaction. ResultsThe activity of NFATc1 was lower in renal transplant patients without BKV viruria [BKV (−)] than in healthy controls, while it trended to be higher in renal transplant patients with BKV viruria [BKV (+)] than in BKV (−) patients. The tacrolimus blood levels did not differ between BKV (+) and BKV (−) renal transplant patients or correlate with NFATc1 activity. ConclusionNFATc1 DNA binding activity was lower in renal transplant patients without BKV viruria than in those who were BKV (+). However, there was no relationship between tacrolimus blood levels and NFATc1 activity in renal transplant patients.

상백피에 의한 MC/9 비만세포의 활성 억제 조절 연구

ABSTRACT Objective : Morus alba Linne Root Bark (MRAL) is a medicinal herb in Korean Medicine, known for its anti-inflammatory and anti-allergic properties. However, its me chanisms of action and the cellular targets have not yet been found and the study was developed to investigate the allergic suppressive effect of MRAL. The purpose of this study is to investigate the allergic suppressive effects of MRAL on activation of MC/9 mast cells.Methods : Cytotoxic activity of MRAL (50, 100, 200, 400 µg/mL) on MC/9 mast cells measured using EZ-Cytox cell viability assay kit (WST reagent). The levels of interleuk in-5 (IL-5), IL-13 and IL-4, IL-5, IL-6, IL-13 mRNA expression were measured by enzyme-linked immunosorbent as say (ELISA) and real-time PCR respectively. The expression of transcription factors such as G ATA-1, GATA-2, NFAT, AP-1 and NF-κB p65 DNA binding activity were measured by western blot and electrop horesis mobility shift assay (EMSA).Results : Our results indicated that MRAL (50 µg/mL, 100 µg/mL) significantly inhibited PMA/Ionomycin-induced production of IL-5 and IL-13 and the expr ession of IL-4, IL-5, IL-6 and IL-13 mRNA in MC/9 mast cells. Moreover, MRAL (50 µg/mL, 100 µg/mL) inhibited PMA/Ionomycin-induced GATA-1, GATA-2, NFAT-1, NFAT-2, c-Fos protein expression and NF-κB p65 DNA binding activity in MC/9 mast cells.Conclusions : In conclusion, we suspect the anti-allergenic activities of MRAL, may be related to the regulation of transcription factors GATA-1, GATA-2, NFAT-1, NFAT-2, c-Fos and NF-κB p65 DNA binding assay causing inhibition of Th2 cytokines IL-5 and IL-13 in mast cells.Key words : Morus alba Linne Root Bark (MRAL), MC/9 mast cell line, Anti-allergy, Transcription factor, Th2 cytokines

Ethanol Inhibits Lipid Raft‐Dependent TCR Signaling and IL‐2 Expression: Potential Mechanism of Alcohol‐Induced Immune Suppression

Alcohol abuse has long‐term deleterious effects on the immune system, and results in reduced function of CD4+T lymphocytes, which regulate both innate and adaptive immunity. T lymphocyte activation via T‐cell receptor (TCR) involves the plasma membrane lipid raft‐dependent aggregation of proteins into the immunological signalosome, which in turn, triggers a signaling cascade resulting in interleukin‐2 (IL‐2) production. IL‐2 is a critical cytokine that regulates the proliferation/clonal expansion of activated T‐cells, and is essential for an effective immune response.The present work examines the mechanisms underlying ethanol‐induced dysfunction of CD4+T lymphocytes, and demonstrates for the first time, that physiologically relevant concentrations of ethanol inhibit lipid raft‐dependent TCR signaling and decrease production of IL‐2 in both primary and cultured human CD4+T lymphocytes. Specifically, ethanol substantially down‐regulates lipid raft colocalization and activation/phosphorylation of Lck, ZAP70 and LAT, resulting in a marked decrease in DNA‐binding activity of NFAT and IL‐2 mRNA and protein. Importantly, inhibition of ethanol metabolism prevented these inhibitory effects. Overall, our findings have clinical relevance in alcohol abuse‐associated immune dysfunction, especially in conditions such as HIV and HCV infection where alcohol abuse is a known comorbidity.

Calcineurin-NFAT signaling is involved in phenylephrine-induced vascular smooth muscle cell proliferation

Catecholamine-induced vascular smooth muscle cell (VSMC) proliferation is one of the major events in the pathogenesis of atherosclerosis and vascular remodeling. The calcineurin-NFAT pathway plays a role in regulating growth and differentiation in various cell types. We investigated whether the calcineurin-NFAT pathway was involved in the regulation of phenylephrine-induced VSMC proliferation. Proliferation of VSMC was measured using an MTT assay and cell counts. Localization of NFATc1 was detected by immunofluorescence staining. NFATc1-DNA binding was determined by EMSA and luciferase activity analyses. NFATc1 and calcineurin levels were assayed by immunoprecipitation. Phenylephrine (PE, an alpha(1)-adrenoceptor agonist) increased VSMC proliferation and cell number. Prazosin (an alpha(1)-adrenoceptor antagonist), cyclosporin A (CsA, an inhibitor of calcineurin) and chelerythrine (an inhibitor of PKC) decreased PE-induced proliferation and cell number. Additional treatment of VSMC with CsA or chelerythrine further inhibited proliferation and cell number in the chelerythrine-pretreatment group and the CsA-pretreatment group. CsA and chelerythrine alone had no effect on either absorbance or cell number. CsA decreased PE-induced calcineurin levels and activity. NFATc1 was translocated from the cytoplasm to the nucleus upon treatment with PE. This translocation was reversed by CsA. CsA decreased the PE-induced NFATc1 level in the nucleus. PE increased NFAT's DNA binding activity and NFAT-dependent reporter gene expression. CsA blocked these effects. CsA partially suppresses PE-induced VSMC proliferation by inhibiting calcineurin activity and NFATc1 nuclear translocation. The calcineurin-NFATc1 pathway is involved in the hyperplastic growth of VSMC induced by phenylephrine.

Characterization of isoquinolinium salt derivatives as potent inhibitors of transcription factor NFAT (33.20)

Abstract Transcription factor NFAT plays critical roles in gene expression during the immune response. NFAT activation is regulated by calcineurin (CaN), a target of the immunosuppressive drugs such as cyclosporin A (CsA) and FK-506. CsA blocks phosphatase activity of CaN, which results in inactivation of NFAT. In search for novel immunosuppressive drug candidates, we have screened an organic chemical library and identified several isoquinolinium salt derivatives as novel inhibitors of NFAT. These compounds inhibited proliferation of peripheral blood mononuclear cell (PBMC) and T-lymphocytes by a mixed-lymphocyte reaction. Their inhibitory effect was specific to transcription factor NFAT, but in contrast with CsA, they neither affect the phosphatase activity of CaN nor nuclear localization of NFAT upon ionomycin stimulation. The DNA binding assay reveals that these compounds strongly attenuated DNA binding activity of NFAT. Taken together, these results indicate that these novel compounds inhibit NFAT activity without any significant change in CaN activity, which is a different mechanism from that of CaA, and suggest as potential candidates for immunosuppression

Regulation of nerve growth factor in the heart: The role of the calcineurin–NFAT pathway

A heightened sympathetic tone accelerates the development of lethal arrhythmias after myocardial infarction (MI) and the progression of heart failure (HF). Cardiomyocytes control their local neural milieu by expression of nerve growth factor (NGF), which triggers sympathetic neural growth (sympathetic nerve sprouting: SNS). The molecular mechanisms that regulate NGF expression are largely unknown. During HF or MI the myocytes are exposed to increased mechanical load and adrenergic stimulation. Both stimuli induce myocyte hypertrophy. The angiotensin-II–calcineurin–NFAT (nuclear factor of activated t-cells) pathway is a well characterized signaling cascade in the pathogenesis of myocyte hypertrophy. The present study aims to investigate the molecular mechanisms by which mechanical stretch and/or alpha-1-adrenergic stimulation affect NGF expression in neonatal rat ventricular myocytes. Both stimuli resulted in a down-regulation of NGF gene and protein expression. Angiotensin-II type 1 receptor blockade with losartan blunted the stretch-induced NGF down-regulation. Specific calcineurin inhibition with cyclosporine A and FK506 or NFAT inhibition with 11R-VIVIT reversed the stretch or alpha-1-adrenergic induced decrease of NGF. Calcineurin over-expression increased NFAT-DNA binding activity and decreased NGF expression. The magnitude of NGF decrease was sufficient to reduce neurite outgrowth of cultured sympathetic neurons. In conclusion, mechanical stretch and alpha-1-adrenergic stimulation contribute to a decrease of cardiomyocyte NGF expression via the calcineurin–NFAT pathway. To evaluate if the calcineurin–NFAT is critically involved in the pathogenesis of SNS further in-vivo studies in models of HF and MI are required. Nevertheless, the calcineurin–NFAT pathway may provide promising starting points for new pharmacological strategies to prevent SNS in the heart.

Modulations of the calcineurin/NF-AT pathway in skeletal muscle atrophy

Calcineurin has been proposed to regulate skeletal muscle hypertrophy, while its relevance to the pathogenesis of muscle atrophy is unknown. The present study was aimed to investigate if perturbations of the calcineurin pathway may be involved in causing skeletal muscle atrophy in two different experimental conditions: cancer cachexia (rats bearing the AH-130 hepatoma), and hyperglycemia (rats treated with streptozotocin). Calcineurin expression in the gastrocnemius was comparable between tumor hosts and controls. By contrast, besides unchanged calcineurin mRNA levels, those of protein were lower in diabetic animals than in controls. The DNA-binding activity of the transcription factors NF-AT and MEF-2 was analysed as an indirect measure of calcineurin activity in vivo. The nuclear translocation of both factors was similar in tumor hosts and controls. Consistently with the reduced calcineurin protein levels, NF-AT DNA-binding activity significantly decreased in the gastrocnemius of diabetic rats compared to controls. Finally, muscle wasting correction afforded in the AH-130 hosts by pentoxifylline or interleukin-15 was not paralleled by changes of calcineurin mRNA levels, while treatment of diabetic animals with dehydroepiandrosterone partially prevented calcineurin down-regulation. These results suggest that modulations of calcineurin activity may be involved in the pathogenesis of muscle wasting in diabetes though not in cancer cachexia.

Suberosin inhibits proliferation of human peripheral blood mononuclear cells through the modulation of the transcription factors NF-AT and NF-kappaB.

Extracts of Plumbago zeylanica containing suberosin exhibit anti-inflammatory activity. We purified suberosin from such extracts and studied its effects on a set of key regulatory events in the proliferation of human peripheral blood mononuclear cells (PBMC) stimulated by phytohemagglutinin (PHA). Proliferation of PBMC in culture was measured by uptake of 3H-thymidine; production of cytokines and cyclins by Western blotting and RT-PCR. Transcription factors NF-AT and NF-kappaB were assayed by immunocytochemistry and EMSA. Suberosin suppressed PHA-induced PBMC proliferation and arrested cell cycle progression from the G1 transition to the S phase. Suberosin suppressed, in activated PBMC, transcripts of interleukin-2 (IL-2), interferon-gamma (IFN-gamma), and cyclins D3, E, A, and B. DNA binding activity and nuclear translocation of NF-AT and NF-kappaB induced by PHA were blocked by suberosin. Suberosin decreased the rise in intracellular Ca2+ concentration ([Ca2+]i) in PBMC stimulated with PHA. Suberosin did not affect phosphorylation of p38 and JNK but did reduce activation of ERK in PHA-treated PBMC. Pharmacological inhibitors of NF-kappaB, NF-AT, and ERK decreased expression of mRNA for the cyclins, IL-2, and IFN-gamma and cell proliferation in PBMC activated by PHA. The inhibitory effects of suberosin on PHA-induced PBMC proliferation, were mediated, at least in part, through reduction of [Ca2+]i, ERK, NF-AT, and NF-kappaB activation, and early gene expression in PBMC including cyclins and cytokines, and arrest of cell cycle progression in the cells. Our observations provide an explanation for the anti-inflammatory activity of P. zeylanica.

Transcriptional Regulation by Foxp3 Is Associated with Direct Promoter Occupancy and Modulation of Histone Acetylation

Regulatory T cells (T(reg)) express Foxp3, a forkhead family member that is necessary and sufficient for T(reg) lineage choice and function. Ectopic expression of Foxp3 in non-T(reg) leads to repression of the interleukin 2 (IL-2) and interferon gamma (IFNgamma) genes, gain of suppressor function, and induction of genes such as CD25, GITR, and CTLA-4, but the mode by which Foxp3 enforces this program is unclear. Using chromatin immunoprecipitation, we have demonstrated that Foxp3 binds to the endogenous IL-2 and IFNgamma loci in T cells, but only after T cell receptor stimulation. This activation-induced Foxp3 binding was abrogated by cyclosporin A, suggesting a role for the phosphatase calcineurin in Foxp3 function. We have also shown that binding of Foxp3 to the IL-2 and IFNgamma genes induces active deacetylation of histone H3, a process that inhibits chromatin remodeling and opposes gene transcription. Conversely, binding of Foxp3 to the GITR, CD25, and CTLA-4 genes results in increased histone acetylation. These data indicate that Foxp3 may regulate transcription through direct chromatin remodeling and show that Foxp3 function is influenced by signals from the TCR.

Post-prandial endothelial dysfunction in hypertriglyceridemic subjects: Molecular mechanisms and gene expression studies

Objective Triglyceride-rich lipoproteins (TGRLs) are a cardiovascular risk factor and induce endothelial dysfunction. In the present study, we investigated the effects of post-prandial TGRLs from type IV hyperlipidemic subjects on endothelial activation addressing the effects of the lipoproteins on intracellular pathways and gene expression. Methods Thirty fasted hypertriglyceridemic patients were given an oral fat load (OFL) and blood samples were collected before the OFL (T0) and 2, 4, 6 and 8 h thereafter. Endothelial function, determined as flow-mediated dilatation of the brachial artery, was assessed at the same time points. TGRLs were isolated at T0 and T4 (PP-TGRL) for in vitro studies. Results Compared with TGRLs, PP-TGRLs induced to a larger extent phosphorylation of p38 MAPK, CREB and IKB-α in human endothelial cells and increased the DNA binding activity of CREB, NFAT and NF-κB. Furthermore, PP-TRGLs upregulated the expression of several pro-inflammatory genes including vascular cell adhesion molecule-1 (VCAM-1), PECAM-1, ELAM-1, intercellular adhesion molecule-1 (ICAM-1), P-selectin, MCP-1, interleukin-6 (IL-6), TLR-4, CD40, ADAMTS1 and PAI-1. Conclusion These effects may relate to the severe impairment of endothelial function seen during the post-prandial phase in hypertriglyceridemic patients.

Defective nuclear translocation of nuclear factor of activated T cells and extracellular signal-regulated kinase underlies deficient IL-2 gene expression in Wiskott-Aldrich syndrome

Proliferation and IL-2 production in response to T-cell receptor ligation are impaired in patients with Wiskott-Aldrich syndrome (WAS). The transcription factors nuclear factor-kappaB (NF-kappaB), nuclear factor of activated T cells (NF-AT), and activating protein-1 (AP-1) play a critical role in IL-2 gene expression. To investigate the mechanisms of impaired IL-2 production after T-cell receptor ligation in T cells deficient in WAS protein (WASP). T cells from WASP-/- mice were stimulated with anti-CD3 and anti-CD28. Nuclear NF-kappaB, NF-AT, and AP-1 DNA-binding activity was examined by electroshift mobility assay. NF-ATp dephosphorylation and nuclear localization were examined by Western blot and indirect immunofluorescence. Phosphorylation of the mitogen-activated protein kinases Erk and Jnk, and of their nuclear substrates Elk-1 and c-Jun, was examined by Western blot. Expression of mRNA for IL-2 and the NF-kappaB-dependent gene A20 and of the AP-1 components c-fos and c-Jun was examined by quantitative RT-PCR. Nuclear translocation and activity of NF-kappaB were normal in T cells from WASP-/- mice. In contrast, NF-ATp dephosphorylation and nuclear localization, nuclear AP-1 binding activity, and expression of c-fos, but not c-Jun, were all impaired. Phosphorylation of Jnk, c-Jun, and Erk were normal. However, nuclear translocation of phosphorylated Erk and phosphorylation of its nuclear substrate Elk1, which activates the c-fos promoter, were impaired. These results suggest that WASP is essential for NF-ATp activation, and for nuclear translocation of p-Erk, Elk1 phosphorylation, and c-fos gene expression in T cells. These defects underlie defective IL-2 expression and T-cell proliferation in WAS.

Inhibition of interleukin-4 production in activated T cells via down-regulation of NF-AT DNA binding activity by apigenin, a flavonoid present in dietary plants

Apigenin, a flavonoid present in many dietary plants, exhibits anti-allergic activity and inhibits IL-4 production by basophils and mast cells. However, the mechanism by which apigenin suppresses IL-4 production especially in T cells remains unclear. In this study, the effect of apigenin and its underlying mechanism on IL-4 production were investigated in activated T cells. Apigenin significantly inhibited IL-4 production in both EL4 T thymoma cells and primary lymph node cells. Apigenin also inhibited IL-4 gene promoter activity in EL4 cells transiently transfected with IL-4 gene promoter constructs, but this inhibitory effect was impaired in EL4 cells transfected with an IL-4 promoter construct deleted of NF-AT binding sites. In addition, apigenin inhibited NF-AT DNA binding activities, indicating that apigenin may inhibit IL-4 production in T cells via down-regulation of NF-AT DNA binding activity.

Cyclosporin A Affects Signaling Events Differentially in Human Gingival Fibroblasts

Gingival overgrowth is a common side-effect of the administration of cyclosporin A (CSA), phenytoin, and calcium blockers. To identify the signaling mechanisms possibly involved in the overgrowth, we examined how CSA affects the activities of MAP kinases and transcription factors in human gingival fibroblasts (HGF). The HGF were treated with CSA and TNF-alpha or PDGF. DNA-binding activity of NFAT, NFkappaB, and AP-1 transcription factors was determined by gel shift assay, and JNK, p38, and ERK1 and ERK2 activation was assessed by Western blot analysis of immunoprecipitates. The CSA inhibited NFAT, NFkappaB, and p38 and JNK activities; however, ERK1 and ERK2 were not affected significantly. AP-1 activity increased approximately 4.5-fold. Our results indicate that CSA affects signaling molecules in HGF differently from other cell types, and that a CSA-induced increase in AP-1 activity may affect the expression of fibrogenic molecules in gingiva and promote gingival overgrowth.

Blockade of Nuclear Factor of Activated T Cells Activation Signaling Suppresses Balloon Injury-induced Neointima Formation in a Rat Carotid Artery Model

We have previously reported that nuclear factor of activated T cells (NFATs) play an important role in the regulation of vascular smooth muscle cell migration and proliferation by receptor tyrosine kinase and G protein-coupled receptor agonists, platelet-derived growth factor-BB and thrombin, respectively. To understand the role of NFATs in vascular disease, we have now studied the involvement of these transcription factors in neointima formation in a rat carotid artery balloon injury model. The levels of NFATc1 in injured right common carotid arteries were increased at 72 h, 1 week, and 2 weeks after balloon injury compared with its levels in uninjured left common carotid arteries. Intraperitoneal injection of cyclosporine A (CsA), a pharmacological inhibitor of the calcineurin-NFAT activation pathway, suppressed balloon injury-induced neointima formation by 40%. Similarly, adenoviral-mediated expression of GFPVIVIT, a competent peptide inhibitor of the calcineurin-NFAT activation pathway, in injured arteries also reduced neointima formation by about 40%. Furthermore, CsA and GFPVIVIT attenuated balloon injury-induced neointimal smooth muscle cell proliferation as determined by bromodeoxyuridine staining. Platelet-derived growth factor-BB induced the expression of COX-2 in cultured VSMC in a time- and NFAT-dependent manner. COX-2 expression was also increased in the right common carotid artery in a time-dependent manner after balloon injury as compared with its levels in uninjured left common carotid artery and both CsA and GFPVIVIT negated this response. Together these results for the first time demonstrate that NFATs play a critical role in neointima formation via induction of expression of COX-2.

Impaired AP‐1 dimers and NFAT complex formation in immature thymocytes during in vivo glucocorticoid‐induced apoptosis

Ca2+-regulated nuclear factor of activated T cell (NFAT) family members are transcription factors crucial for the expression of various cytokine and other immunoregulatory genes. Moreover, NFAT transcription factors are involved in the regulation of development, maturation and selection of thymocytes. Typically, the NFAT complex is made up of NFATc (NFATc1-4) protein and activator protein-1 (AP-1) transcription factor. AP-1 is a dimer consisting of two Jun proteins (homodimers) or Jun and Fos proteins (heterodimers). We have previously reported that NFAT DNA-binding activity significantly decreases in the thymus during glucocorticoid-induced apoptosis. In this study, we demonstrate that the expression and phosphorylation status of the NFAT proteins do not change during glucocorticoid-induced apoptosis. This suggests that glucocorticoids do not disturb a signal transduction pathway leading to the activation of NFATc proteins in thymocytes. Although the levels of particular Jun and Fos proteins do not decrease after glucocorticoid administration, the formation or DNA-binding activity of some AP-1 dimers is specifically abolished. Thus, the observed inhibition of NFAT transcription factor activity during glucocorticoid-induced apoptosis is likely to be a consequence of this perturbation or the lack of a proper AP-1 component.

Positive feedback regulation of PLCgamma1/Ca(2+) signaling by PKCtheta in restimulated T cells via a Tec kinase-dependent pathway.

PKCtheta plays an essential role in activation of mature T cells. Here, we report that the TCR/CD28-induced tyrosine phosphorylation and activation of PLCgamma1 was significantly impaired in PKCtheta (-/-) primary, restimulated T cells. Consistent with this finding, receptor-induced Ca(2+) mobilization, NF-AT DNA-binding activity and the membrane translocation of PKCalpha, a PLCgamma1-dependent conventional PKC, were also markedly reduced in the same cells. Moreover, a dominant-negative PLCgamma1 mutant blocked the PKCtheta-induced activation of an AP-1 reporter gene in Jurkat and primary cells. Regulation of PLCgamma1 signaling by PKCtheta required the tyrosine kinase Tec since a dominant-negative Tec mutant blocked PKCtheta-induced AP-1 (but not NF-kappaB) activation. In addition, wild-type Tec, but not Itk or Rlk, potently activated AP-1. Furthermore, Tec was found to constitutively associate with PKCtheta, an interaction that like AP-1 activation required the pleckstrin-homology domain of Tec. These findings define a novel PKCtheta-initiated pathway that regulates Ca(2+) signaling and AP-1 activation via Tec and PLCgamma1. Moreover, they identify Tec as a key point downstream of PKCtheta, where TCR- and PKCtheta-induced signaling pathways, leading to AP-1 versus NF-kappaB activation, diverge in T cells.

Exposure to 4- tert-octylphenol, an environmentally persistent alkylphenol, enhances interleukin-4 production in T cells via NF-AT activation

4- tert-Octylphenol (OP) is a representative endocrine disruptor that may have adverse effects on human health. The influence of this compound on allergic immune responses remains unclear. In this study, we have examined the effects of OP on production of interleukin-4 (IL-4), a pro-inflammatory cytokine closely associated with allergic immune responses. OP significantly enhanced IL-4 production in antigen-primed T cells in a dose-dependent manner. Treatment with OP in vivo resulted in significant increase of IL-4 production in T cells and of IgE levels in sera of antigen-primed mice. Furthermore, OP enhanced the activation of IL-4 gene promoter in EL4 T cells transiently transfected with IL-4 promoter/reporter constructs, and the enhancing effect mapped to a region in the IL-4 promoter containing binding sites for nuclear factor of activated T cell (NF-AT). Activation of T cells by phorbol-12-myristate-13-acetate (PMA) resulted in markedly enhanced binding activities to the NF-AT site, which significantly increased upon addition of OP, indicating that the transcription factor NF-AT was involved in the enhancing effect of OP on IL-4 production. The enhancement of IL-4 production by OP was blocked by FK506, a calcineurin inhibitor, but not by the estrogen receptor (ER) antagonist ICI 182 780. FK506 inhibited the NF-AT–DNA binding activity and IL-4 gene promoter activity enhanced by OP in a dose-dependent manner. These findings demonstrate that OP enhances IL-4 production in T cells via the stimulation of calcineurin-dependent NF-AT activation.

CARD11 mediates factor-specific activation of NF-kappaB by the T cell receptor complex.

NF-kappaB is a critical target of signaling downstream of the T cell receptor (TCR) complex, but how TCR signaling activates NF-kappaB is poorly understood. We have developed an expression cloning strategy that can identify catalytic and noncatalytic molecules that participate in different pathways of NF-kappaB activation. Screening of a mouse thymus cDNA library yielded CARD11, a membrane-associated guanylate kinase (MAGUK) family member containing CARD, PDZ, SH3 and GUK domains. Using a CARD-deleted variant of CARD11 and RNA interference (RNAi), we demonstrate that CARD11 mediates NF-kappaB activation by alphaCD3/alphaCD28 cross-linking and PMA/ionomycin treatment, but not by TNFalpha or dsRNA. CARD11 is not required for TCR-mediated induction of NFAT or AP-1. CARD11 functions upstream of the IkappaB-kinase (IKK) complex and cooperates with Bcl10 in a CARD domain-dependent manner. RNAi-rescue experiments suggest that the CARD, coiled-coil, SH3 and GUK domains of CARD11 are critical for its signaling function. These results implicate CARD11 in factor- specific activation of NF-kappaB by the TCR complex and establish a role for a MAGUK family member in antigen receptor signaling.