Nuclear Factors Of Activated T-cells Research Articles

Modulatory effects of thymol and carvacrol on inflammatory transcription factors in lipopolysaccharide-treated macrophages

Inflammation is a crucial factor in the pathogenesis of numerous diseases. This study sought to evaluate the effects of thymol and carvacrol, the main components of Thymus vulgaris (thyme) essential oil, on transcription factors regulating inflammation. Lipopolysaccharide (LPS)-stimulated J774.1 mouse macrophages were examined by real time-PCR for interleukin (IL)-1β and tumor necrosis factor (TNF)-α gene expression in the presence of these compounds. Levels of inducible phospho-nuclear factor-κB (pNF-κB) p65, activator protein-1 [AP-1(c-Fos/c-Jun)], and nuclear factors of activated T-cells (NFATs) were also measured using Western blots. Levels of phosphorylation of stress-activated protein kinases (SAPKs)/c-Jun N-terminal kinase (SAPK/JNK), signal transducer, and activator of transcription (STAT-3), p38, IκBα, and NF-κB p65, as well as total levels of IL-1β and TNFα were determined. The results indicated carvacrol significantly reduced both IL-1β and TNFα at the protein and mRNA levels; thymol also significantly reduced IL-1β expression. Western blot analyses of nuclear cell extracts revealed both agents caused significantly decreased expression of c-Fos, NFAT-1, and NFAT-2; decreased expression of c-Jun was only caused by carvacrol. Neither agent inhibited p-NF-κB p65 expression. At the protein level, carvacrol and thymol each caused decreases in inducible phospho-SAPK/JNK and phospho-STAT3 levels, whereas only carvacrol resulted in increased p-p38 levels in the total cell extract. Despite the reduction of phospho-IκBα caused by both agents, p-NF-κB p65 still increased in the presence of carvacrol. Based on these findings, it is concluded that carvacrol and thymol could contribute to reduction of inflammatory responses through modulation of the expression of JNK, STAT-3, AP-1, and NFATs.

A high-throughput chemical screen reveals that harmine-mediated inhibition of DYRK1A increases human pancreatic beta cell replication.

Types 1 and 2 diabetes affect some 380 million people worldwide. Both result ultimately from a deficiency of functional pancreatic insulin-producing beta cells. Beta cells proliferate in humans during a brief temporal window beginning around the time of birth, with peak beta cell labeling indices achieving approximately 2% in first year of life1-4. In embryonic life and after early childhood, beta cell replication rates are very low. While beta cell expansion seems an obvious therapeutic approach to beta cell deficiency, adult human beta cells have proven recalcitrant to such efforts1-8. Hence, there remains an urgent need for diabetes therapeutic agents that can induce regeneration and expansion of adult human beta cells in vivo or ex vivo. Here, we report the results of a high-throughput small molecule screen (HTS) revealing a novel class of human beta cell mitogenic compounds, analogues of the small molecule, harmine. We also define dual specificity tyrosine-regulated kinase-1a (DYRK1A) as the likely target of harmine, and the Nuclear Factors of activated T-cells (NFAT) family of transcription factors as likely mediators of human beta cell proliferation as well as beta cell differentiation. These observations suggest that harmine analogues (“harmalogs”) may have unique therapeutic promise for human diabetes therapy. Enhancing potency and beta cell specificity are important future challenges.

Abstract 1939: Calcineurin/NFAT signaling regulates tumor initiating cell properties in non-small cell lung cancer

Abstract Background: Targeting specific pathways responsible for Tumor Initiating Cells (TIC) maintenance is postulated to be an important therapeutic strategy for more effective control of tumors. Nuclear Factors of Activated T-cells (NFATs), consisting of 4 homologues (NFATc1-c4), are calcium-dependent transcription factors that are activated upon dephosphorylation by the protein phosphatase calcineurin. NFAT is known to be involved in T cell activation and differentiation. Recently, its roles in regulating embryonic stem cell proliferation and cancer survival have been reported. However, the involvement of Calcineurin/NFAT signaling in TIC maintenance remains unknown. Objective: To study the role of Calcineurin/NFAT in regulating TIC properties in non-small cell lung cancer (NSCLC). Methods and results: Analysis of NFAT mRNA expression in a panel of established and early passage patient derived NSCLC cell lines showed NFATc2 and NFATc4 were up-regulated in 92.6% (25 of 27) and 72% (20 of 27) of cancer cells compared to normal bronchial cell line (BEAS-2B), respectively. To study its role in TIC, tumor spheres or flow cytometry-isolated ALDHhigh/CD44high cells were obtained and NFAT expressions and activities were compared between TIC and non-TIC. The expression of NFAT and downstream targets were significantly higher in tumor spheres and ALDHhigh/CD44high cells compared to the corresponding monolayers and ALDHlow/CD44low cells. Correspondingly, luciferase reporter assay also showed higher NFAT activities in tumor spheres compared to monolayers. To study its functional importance, calcineurin and NFAT were inhibited and effects on TIC properties were analyzed. Inhibition of calcineurin by pharmacological inhibitors cyclosporin A (CSA) and FK506 resulted in reduced sphere formation ability, down-regulation of pluripotent genes (SOX2, OCT4, NANOG) and TIC markers (ALDH1A1, CD133, CD166) expression, as well as decreased TIC proportions (ALDHhigh/CD44high and CD133+) by flow cytometry. These effects were verified using siRNA knockdown of PPP3R1 which encodes one of the calcineurin regulatory subunits. Apart from expression changes, stable knockdown of NFATc2 using lentiviral shRNA inhibited in vitro and in vivo tumorigenecity as shown in soft agar colony formation and mice xenografts compared to shRNA control. To explore the clinical relevance of NFAT, NFAT expression levels were analyzed in human resected lung cancers by quantitative PCR and immunohistochemsitry. Results showed that NFATs were significantly up-regulated in clinical NSCLC compared to corresponding normal lung, and higher NFAT expression predicted shorter recurrence free survival. Conclusions: Calcineurin/NFAT signaling is active in both NSCLC and lung TIC. It plays an important role in TIC maintenance. Inhibition of this pathway is a promising approach for long term lung cancer control. Citation Format: Zhi-Jie Xiao, Jing Liu, Vicky Pui-Chi Tin, Maria Pik Wong. Calcineurin/NFAT signaling regulates tumor initiating cell properties in non-small cell lung cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1939. doi:10.1158/1538-7445.AM2014-1939

Nuclear factor of activated T-cells 1 increases sensitivity of v-myb transformed monoblasts to all-trans retinoic acid

Nuclear factors of activated T-cells (NFATs) are important regulators of the cytokine gene expression in activated T-cells. In the last decade, NFATs have been shown to regulate cell cycle, differentiation and apoptosis in cells of various origins revealing their importance for cell homeostasis. In this study, we investigated the effects of NFAT1 on proliferation and differentiation of v-myb-transformed BM2 monoblasts. In contrast to many other leukemic cell lines, BM2 cells do not respond to retinoic acid. However, once overexpressing NFAT1, they became sensitive to all-trans retinoic acid (ATRA). The ATRA-treated BM2NFAT1 cells differentiated along monocyte/macrophage pathway as evidenced by changes in cell morphology, adherence, phagocytic and non-specific esterase activities, reactive oxygen species production, and vimentin expression. Furthermore, overexpressed NFAT1 either alone or in combination with the ATRA-driven signalling pathway deregulated cyclin A and retinoic acid receptor proteins in BM2 cells. Data presented in this study indicate that the NFAT1 and ATRA signalling pathways synergize in control of proliferation and differentiation of BM2 monoblasts.

Overexpression of α-Synuclein Down-Regulates BDNF Expression

Parkinson's disease (PD) is a chronic progressive neurodegenerative movement disorder characterized by the selective loss of nigrostriatal dopaminergic neurons. However, the molecular pathways leading to the dopaminergic neuron degeneration have remained obscure until recently. Reports demonstrated that reduction of brain-derived neurotrophic factor (BDNF) was involved in the etiology and pathogenesis of PD, but its mechanism has not been elucidated. alpha-Synuclein has a causal role in Parkinson's disease, and could interfere with transcriptional regulation of dopamine neurons. In this study, alpha-synuclein overexpression was found to decrease the expression of BDNF, and also to suppress the transactivation of nuclear factors of activated T-cells (NFAT) and cAMP response element binding protein (CREB), both of which regulate BDNF expression. Furthermore, overexpressed alpha-synuclein could associate with protein kinase C (PKC) and impair its activity. Meanwhile glycogen synthase kinase-3beta (GSK3beta) was activated and extracellular signal-regulated protein kinase (ERK) activity was inhibited by overexpression of alpha-synuclein; both of them were downstream kinases of PKC. Therefore, the impaired PKC signal pathway caused by alpha-synuclein overexpression might account at least partially for the down-regulation of BDNF.

Stimulation of NFAT‐dependent transcription activity by agonists and hypoxia in pulmonary arterial smooth muscle cells

Nuclear factors of activated T‐cells (NFATs) are Ca2+‐sensitive transcription factors regulated by calcineurin, and play critical roles in inflammation and cell proliferation. Several previous studies showed that calcineurin/NFAT pathway is involved in the pathogenesis of pulmonary hypertension. We have found recently that NFATc3 is predominantly expressed in rat pulmonary arterial smooth muscle cells (PASMCs), and hNFATc3‐EGFP fusion protein translocates to the nucleus upon stimulation in rat PASMCs. In this study, NFAT‐dependent transcription activity was further examined in primary cultured rat PASMCs using a 4xNFAT‐luciferasereporter. NFAT‐dependent luciferase acitivity was enhanced significantly by ionomycin+PMA, PDGF, ET‐1, and hypoxia (2% O2 for 24 hr). The stimulatory effects of PDGF and hypoxia could be blocked by SKF‐96365 and nifedipine, respectively. Moreover, the effects of ionomycin+PMA and PDGF were further potentiated in PASMCs exposed to hypoxia. Since many agonists and growth factors are involved in hypoxic pulmonary hypertension, these observations suggest that hypoxia may interact synergistically with agonists to enhance NFAT‐dependent gene transcription in PASMCs. (Supported by HL0771835, HL075134)

Hypoxia interacts with NFAT signaling in rat pulmonary arterial smooth muscle cells (PASMCs)

Nuclear factors of activated T‐cells (NFATs) are calcium sensitive transcription factors regulated by calcineurin. They play critical roles in inflammation and cell proliferation. Recent studies showed that calcineurin/NFAT pathway is involved in the pathogenesis of pulmonary hypertension. Here we exposed rats to hypoxia for 3 weeks with daily injection of the calcineurin inhibitor cyclosporin (CsA, 35 mg/kg/day). Right ventricular systolic pressure (CsA treated: 34.3±2.0 mmHg; untreated: 53.9±2.6 mmHg, p<0.05), and right heart hypertrophy were significantly reduced in CsA treated rats. Real‐time RT‐PCR showed that NFATc3 mRNA was predominantly expressed in PA of rats and mice. It was 3‐5 times the amount of other NFAT isoforms, except that it was similar to NFATc1 in mouse PA. NFATc3 and NFATc1 proteins were also detected by western blot. NFATc3 nuclear translocation was examined by expressing hNFATc3‐EGFP fusion protein in rat PASMCs. Confocal images showed that nuclear accumulation of hNFATc3‐EGFP occurred within 5 min and peaked at about 15‐20 min after PASMCs were stimulated by ionomycin and PMA. NFAT‐dependent transcription activity, monitored with a 4xNFAT‐luciferase reporter, was increased 4 folds by ionomycin+PMA. Moreover, the stimulatory effect of ionomycin+PMA was further potentiated in PASMCs exposed to 24 hr hypoxia (2% O2). Since many agonists stimulate PLC to activate IP3‐induced Ca2+ release and PKC similar to ionomycin+PMA, this observation suggests that hypoxia may interact synergistically with agonists to enhance NFAT‐dependent gene transcription in PASMCs.

Rescuing Cardiac Malfunction

See related articles, pages 1473–1482 In response to various hypertrophic stimuli, the heart undergoes alterations in structure and function. One mechanism by which the heart adapts to physiological and pathological stimuli is an increase in size of individual cardiac myocytes, and when those stimuli are removed, the size of the myocytes can decrease. Modulation of cardiac size thus requires a very careful balance between protein synthesis and protein degradation, and any imbalance in these systems could be of potential damage to the heart. Increased protein synthesis in hypertrophied hearts is accompanied by “protein quality control” to eliminate aggregated unfolded proteins, but a failure of this monitoring system eventually leads to cardiac dysfunction.1 Heat shock proteins (HSPs) have chaperone-like properties that can bind to unfolded proteins and prevent their denaturation and aggregation.2 In recent genome surveys of mice, rats, and humans, small-molecular-weight heat shock proteins (sHSPs ≈15 to 30 kDa) have been identified. αB crystalline (CryAB) is a member of the sHSPs that is expressed at high levels in cardiomyocytes.2 CryAB has chaperone-like properties and also binds to both desmin and cytoplasmic actin which helps to maintain cytoskeletal integrity. Previous studies have shown that a mutation in CryAB mimics a form of desmin-related cardiomyopathy.3 Overexpressing CryAB in cultured cardiomyocytes and in transgenic mouse hearts protects against ischemia/reperfusion–induced cell death4,5; on the other hand, double knockout of the small heat shock proteins CryAB/SHSP2 induces abnormal cardiac growth and defective myocardial relaxation.6 In this issue of Circulation Research , Kumarapeli et al7 address and confirm the roles of CryAB in response to …

Calcineurin/NFAT is a common pathway in rat monocrotaline‐ and hypoxia‐induced pulmonary hypertension

The nuclear factors of activated T-cells (NFATs) are calcium sensitive transcription factors regulated by calcineurin. NFATs play critical roles in inflammation and cell proliferation, which are important processes in pulmonary hypertension (PAH) pathogenesis. We hypothesize that NFATs might be the common pathway through which PAH develops. To investigate this hypothesis, we treated monocrotaline (MCT) and hypoxia-induced PAH models with cyclosporine (CsA), a specific calcinurin/NFAT pathway inhibitor, at a dosage of 20 mg/kg/day for 24 days. As a result, right ventricular systolic pressure (RVSP) was suppressed by CsA treatment to 40.5 ± 2.8 mmHg which is otherwise increased to 72.6 ± 4.9 mmHg (p<0.01) in MCT PAH model, compared with CsA treated normal control rat of 24.6 ± 1.7 mmHg. Moreover, right ventricular hypertrophy as reflected by the right ventricle to left ventricle plus septal weight ratio (RV/LV+S) decreased from 0.65 ± 0.04 to 0.38 ± 0.03 (p<0.01) whereas it was 0.28 ± 0.004 in control CsA treated rat. RVSP in hypoxia induced PAH rat was similarly decreased from 51.6% ± 4.3 to 35.1% ± 1.7 (p<0.01) and the RV/LV+S ratio from 0.48 ± 0.01 to 0.35 ± 0.007 (p<0.01) after CsA treatment. Considering the different mechanisms between the MCT- and hypoxia-induced PAH models, we suggest that calcinurin/NFAT pathway is a pivotal common pathway in PAH development.

Intracellular control of developmental and regenerative axon growth.

Axon growth is a highly regulated process that requires stimulating signals from extracellular factors. The extracellular signals are then transduced to regulate coordinately gene expression and local axon assembly. Growth factors, especially neurotrophins that act via receptor tyrosine kinases, have been heavily studied as extracellular factors that stimulate axon growth. Downstream of receptor tyrosine kinases, recent studies have suggested that phosphatidylinositol-3 kinase (PI3K) regulates local assembly of axonal cytoskeleton, especially microtubules, via glycogen synthase kinase 3beta (GSK-3beta) and multiple microtubule binding proteins. The role of extracellular signal regulated kinase (ERK) signalling in regulation of local axon assembly is less clear, but may involve the regulation of local protein translation. Gene expression during axon growth is regulated by transcription factors, among which cyclic AMP response element binding protein and nuclear factors of activated T-cells (NFATs) are known to be required for neurotrophin (NT)-induced axon extension. In addition to growth factors, extracellular matrix molecules and neuronal activity contribute importantly to control axon growth. Increasingly, evidence suggests that these influences act to enhance growth via coordinating with growth factor signalling. Finally, evidence is emerging that developmental versus regenerative axon growth may be mediated by distinct signalling pathways, both at the level of gene transcription and at the level of local axon assembly.

Repression of FasL expression by retinoic acid involves a novel mechanism of inhibition of transactivation function of the nuclear factors of activated T-cells.

Retinoids are potent immune modulators that inhibit Fas ligand (FasL) expression and thereby repress the activation-induced apoptosis of immature thymocytes and T-cell hybridomas. In this study, we demonstrate that all-trans-retinoic acid (all-trans-RA) directly represses the transcriptional activity of the nuclear factors of activated T-cells (NFAT), which is an important transactivator of the FasL promoter. The analysis of reporter constructs containing the FasL promoter and wild-type or mutant NFAT binding-sites indicated that all-trans-RA repression was mediated via an NFAT binding element located in the promoter. A reporter construct comprising the NFAT binding sequence linked to a heterologous SV-40 promoter showed that NFAT transcriptional activity was significantly inhibited by all-trans-RA. Furthermore, all-trans-RA inhibited activation of the distal NFAT binding motif present in the interleukin (IL)-2 promoter, suggesting that the inhibition of NFAT function by all-trans-RA was not specific to the FasL promoter. Gel shift assays corroborated the results of the gene reporter studies by showing that all-trans-RA decreased the NFAT binding to DNA. All-trans-RA blocked translocation of NFATp from the cytosol into the nucleus, which was induced by PMA/ionomycin treatment in HeLa cells transfected with a Flag-tagged NFATp. Taken together, our results indicate that FasL inhibition by all-trans-RA involves a novel mechanism whereby the transcriptional function of NFAT is blocked.