Tesis sobre el tema "NFATc [Nuclear Factor of Activated T-cell]"

L’expression du gène codant pour la chaîne1 du collagène I (Col1a1) dans les tendons nécessite la coopération des éléments cis-activateurs TSE1, TSE2 et d’éléments localisés entre – 1537 et – 220 pb du promoteur proximal de Col1a1. Mon travail de thèse avait pour but d’identifier les facteurs de transcription qui, en se liant à ces séquences, activent le promoteur de Col1a1 dans les tendons. J’ai montré (1) que scleraxis (SCX), dont l’expression est spécifique des tendons, active le promoteur de Col1a1 en se liant à TSE2 sous forme d��hétérodimère SCX/E47 ; (2) que des facteurs de transcription NFATc sont exprimés dans les fibroblastes tendineux, peuvent se lier à TSE1 et augmenter l’expression de Col1a1 ; et (3) que la protéine Egr2 active le promoteur de Col1a1 probablement en se liant à un élément localisé entre – 1537 et – 220 pb. L’ensemble de ces résultats suggère que l’expression du gène Col1a1 dans les fibroblastes tendineux nécessite la coopération de SCX et de NFATc avec Egr2.

Les leucémies aigües lymphobastiques (LAL) représentent un tiers des leucémies et constituent le cancer pédiatrique le plus fréquent chez l’enfant. Les LAL de type T (LAL-T)sont caractérisées par l’expansion anormale de progéniteurs de lymphocytes T. Aujourd’hui,la réponse curative aux traitements est proche de 80% chez l’enfant et 50% chez l’adulte. La rechute reste donc fréquente et souvent de mauvais pronostic. Pour ces raisons,l’identification de nouvelles voies de signalisation en vue de développer de nouvelles stratégies thérapeutiques est cruciale afin d’améliorer le traitement des LAL-T.Les résultats précédents du laboratoire ont révélé l’activation soutenue de la voie calcineurine (Cn)/NFAT dans des échantillons humains de lymphomes et de LAL, ainsi que dans des modèles murins de ces pathologies. Le laboratoire a ensuite montré que Cn est intrinsèquement requise pour la capacité des cellules leucémiques de LAL-T à propager la maladie (activité LIC « Leukemia Initiating Cells ») dans un modèle murin de LAL-T induit parun allèle activé de NOTCH1 (ICN1). Puisque l’inhibition pharmacologique de Cn induit de nombreux effets secondaires, la recherche de cibles thérapeutiques en aval de Cn constitue un axe de recherche important. J’ai participé à une étude du laboratoire montrant que l’expression à la surface cellulaire de CXCR4 est régulée par Cn et requise pour la migration des cellules de LAL-T, mais non suffisante pour rétablir le potentiel de ré-initiation suggérant que d’autres effecteurs doivent être impliqués dans cette activité.Les facteurs de transcription NFAT (NFAT1, NFAT2 et NFAT4) sont des effecteurs importants de Cn en réponse à la signalisation calcique lors du développement des thymocytes, mais également dans les lymphocytes T. L’essentiel de ce travail de thèse a utilisé des LAL-T induites par ICN1 dans lesquelles l’inactivation génique des trois facteurs NFAT par recombinaison homologue. Nous avons ainsi montré que (i) les facteurs NFAT sont requis en aval de Cn pour le potentiel LIC des LAL-T-ICN1 in vivo, (ii) leur inactivation altère la survie, la prolifération et la migration des cellules de LAL-T in vitro, (iii) NFAT1,NFAT2 et NFAT4 ont une fonction largement redondante dans les LAL-T. Nous avons également par une approche transcriptomique identifié deux gènes dont l’expression estsous contrôle des facteurs NFAT et impliqués dans la régulation de la survie et de la prolifération des LAL-T in vitro : CDKN1A et MAFB.Tout comme la voie Cn/NFAT, les CaMKs sont des protéines kinases activées en aval de la signalisation calcique dans les lymphocytes T. Nous avons montré par une approche pharmacologique que l’inhibition des CaMKs dans les LAL-T-ICN1 in vitro altère la survie etla prolifération des cellules leucémiques. L’inhibition spécifique par une approche d’ARN interférence de deux isoenzymes CaMKIIγ et CaMKIIδ suggèrent que ces protéines jouent dans le maintien des cellules leucémiques in vitro
T cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy of T cell progenitors. Despite initial response to chemotherapy, relapses remain frequent in children and adults. Previous results identify sustained activation of Calcineurin (Cn)/NFAT signaling pathway in human T-ALL and murine T-ALL models. Importantly, they also demonstrated Cn is essential for T-ALL Leukemia Initiating Cells (LIC) activity in a murine model of T-ALL induced by an activated allele of NOTCH1 (ICN1). Since pharmacologic inhibition of Cn induces side effects, we aim to identify downstream effectors involved in T-ALL. NFAT (Nuclear Factor of Activated T cells) factors play crucial roles downstream Cn during development and activation of T cells. To address their role in T-ALL, we generated mouse ICN1-induced T-ALL in which NFAT genes can be inactivated either single or in combination following Cre-mediated gene deletion. We demonstrated that (i) NFAT factors are required downstream Cn for LIC activity in T-ALL in vivo (ii) ex vivo NFAT factors deletion alters survival, proliferation and migration of T-ALL (iii) NFAT1, 2 and 4 have a largely redundant function in T-ALL. Moreover, the NFAT-dependant transcriptome allowed to identify important targets (CDKN1A, MAFB) involved in T-ALL survival and proliferation in vitro. Calmodulin-dependant kinases (CaMK) are kinases activated by calcium signaling in T cells. We showed that pharmacologic inhibition of CaMKs in ICN1-induced T-ALL alters survival and proliferation of T-ALL in vitro. Beside, specific inhibition by RNA interference of CaMKIIg and CaMKIId suggests a putative role of these kinases in T-ALL maintenance

Understanding the transcriptional mechanisms that control hematopoiesis and the interaction between hematopoietic stem cells and the bone marrow (BM) microenvironment in vivo is of considerable interest. The calcineurin-dependent transcription factor NFAT (Nuclear Factor of Activated T cells) is known as master regulator of cytokine production in T lymphocytes and therefore central for T cell-dependent immune reactions, but has also been shown to regulate a process of differentiation and tissue adaptation in various cell types. The activation of NFAT is dependent on the calcium level within the cell. In resting cells, calcium levels are low and NFAT is cytoplasmic and inactive. A sustained increase in the internal calcium concentration within an external stimuli leads to activation of the calcium-dependent calcineurin, followed by dephosphorylation and nuclear translocation of NFAT. We have previously shown that NFATc2, a member of the NFAT family, is expressed in CD34+ hematopoietic stem cells (HSC). A mouse model harboring NFATc2 deficiency provides the opportunity for in vivo investigation of the role of NFATc2 in hematopoiesis. Our recent observations showed that aged mice lacking the transcription factor NFATc2 develop peripheral blood anemia and thrombocytopenia, BM hypoplasia and extramedullary hematopoiesis in spleen and liver. The proliferation and differentiation of NFATc2-deficient hematopoietic stem cells ex vivo, however, was found to be intact. It remained therefore unclear whether the disturbed hematopoiesis in NFATc2-deficient mice was caused by the hematopoietic or the stroma component of the BM hematopoietic niche. In the current study we dissected the relative contribution of hematopoietic and stroma cells to the phenotype of the NFATc2-deficent mice by transplanting immuno-magnetically purified NFATc2-deficient (KO) HSCs to lethally irradiated wild type (WT) mice, and vice versa. After a post-transplantation period of 6-8 months, peripheral blood, BM as well as spleen and liver of the transplanted animals were analyzed and compared to WT and KO mice transplanted with control cells. Transplantation of NFATc2-deficient HSCs into WT recipients (KO WT) induced similar hematological abnormalities as those occurring in non-transplanted KO mice or in KO mice transplanted with KO cells (KO KO). Compared to WT mice transplanted with WT cells (WT WT), KO WT mice showed evidence of anemia, thrombocytopenia and a significantly reduced number of hematopoietic cells in their BM. Likewise, KO WT mice developed clear signs of extramedullary hematopoiesis in spleen and liver, which was not the case in WT WT control animals. In addition to the hematopoietic abnormalities, transplantation of NFATc2-deficient HSC also induced osteogenic abnormalities such as BM sclerosis and fibrosis in WT mice. This phenomenon was rather subtle and of incomplete penetrance, but never seen in mice transplanted with WT cells. These data demonstrate for the first time, that the NFATc2 transcription factor directly regulates the intrinsic function of hematopoietic stem cells in vivo. However, the transcriptional targets for NFAT in these cells are yet unknown. In addition to hematopoietic stem cells, NFATc2 has been shown to be expressed in a lineage-specific manner during myeloid differentiation and, notably, is maintained during megakaryopoiesis while it is suppressed during the differentiation of neutrophils. Bone marrow megakaryocytes are the precursors of peripheral blood platelets and therefore constitute an integral part of primary hemostasis, thrombosis and wound healing. The biological role of NFAT in megakaryocytes is unknown. We have recently shown that NFATc2 is not necessary for megakaryocytic differentiation. On the other hand, recent evidence suggests that NFATc2 is required for the transcription of specific megakaryocytic genes. In this study, we showed that activation of the calcineurin/NFAT pathway in either primary megakaryocytes or CMK megakaryocytic cells forces the cells to go into apoptosis. Cell death in megakaryocytes is induced by treating the cells with the calcium ionophore ionomycin and suppressed by either the pan-caspase inhibitor zVAD or the calcineurin inhibitor cyclosporin A (CsA). Ionomycin stimulation of megakaryocytes leads to the expression of Fas Ligand (FASLG), a pro-apoptotic member of the tumor necrosis factor superfamily. Expression of FASLG was detectable as early as four hours after stimulation on the membrane of ionomycin-treated megakaryocytes, was augmented in cells stably overexpressing NFATc2, and was suppressed in cells either pretreated with CsA or expressing the specific peptide inhibitor of NFAT, VIVIT. To investigate the physiological relevance of FASLG expression on megakaryocytes, we performed co-cultures of megakaryocytes with Fas-expressing T-lymphocytes, in which CMK cells were left either unstimulated or pre-stimulated with ionomycin and then added to Jurkat cells. The presence of ionomycin-stimulated CMK cells, but not of unstimulated cells or cells stimulated in the presence of CsA, significantly induced apoptosis in Jurkat cells. Overexpression of NFATc2 in CMK cells enhanced their potency to induce apoptosis in Jurkat cells, while cells expressing VIVIT were less effective. Apoptosis induction of Jurkat cells by stimulated CMK cells was partially blocked by the presence of either a neutralizing antibody against FASLG or an antagonistic antibody to Fas during the co-culture period, indicating involvement of the FASLG/Fas apoptosis pathway. These results represent the first clear evidence for a biological function of the calcineurin/NFAT pathway in megakaryocytes, namely the regulation of Fas/FASLG-dependent apoptosis. Second, they underline that the biological role of megakaryocytes is not restricted to the production of proteins and other cellular structures for platelet assembly, but that this population of cells fulfills an independent regulatory function in the context of the surrounding tissue. Finally, we have identified by RNA sequencing analysis of NFATc2-expressing and -deficient cells, the entire set of genes which is induced by NFATc2 in stimulated megakaryocytes. Functional pathway analysis suggests an involvement of NFATc2 in pro-inflammatory pathways in these cells. The significance of these findings has to be addressed in further studies.

Ca2+-dependent transcription is a fundamental process by which neurons translate activation experience into cellular level adaptations. The nuclear factor of activated T-cells (NFAT) family of proteins comprise four Ca2+/CaN-dependent transcription factors that are widely expressed throughout virtually all tissues. Within neurons, NFAT dependent signaling is critical for axonal development, regulation of synapse number and efficacy, and survival. Furthermore, NFAT is implicated in activity dependent regulation of genes involved in synaptic transmission, learning and memory, mood, and pain sensation. NFAT is activated upon elevations in intracellular Ca2+, which results in CaN -dependent dephosphorylation of multiple serine residues within an N-terminal regulatory region. NFAT dephosphorylation permits NFAT translocation to the nucleus, where it can regulate gene expression, frequently co-operatively with other transcription factors, including AP-1 and MEF2. NFAT activation is opposed or terminated by several kinases, including CK1 and GSK3. Despite the importance of NFAT proteins as regulators of Ca2+-dependent transcription, little is known about the regulation and function of specific NFAT isoforms within neurons. In Aim 1 of this thesis I characterized the differential activation of NFATc3 and NFATc4 in DRG neurons. While NFATc3 rapidly translocates the nucleus upon Ca2+-influx through voltage-gated calcium channels, NFATc4 remained remarkably intransient. Modular substitution of NFATc3 regulatory elements increased the rate or retention of NFATc4, whereas converse substitutions of NFATc4 regulatory elements into NFATc3 decreased NFATc3 nuclear translocation. The activation of NFATc4 appears to be inhibited by preferential phosphorylation by kinases, such as GSK3, which counteract CaN-dependent dephosphorylation. In Aim 2 I investigated the role of NFATc3 in hippocampal neurons. While the majority of NFAT reports in neurons have focused on NFATc4, my data suggest that NFATc3 is the predominantly expressed isoform in hippocampal neurons and is critical for depolarization-induced NFAT target gene expression. I further characterized NFATc3 KO mice in a battery of behavioral assays to test whether loss of NFATc3 expression would affect the baseline anxiety/depression state of the animal, or if NFATc3 was critical for learning and memory. Taken together, my data suggest that NFATc3 is important for NFAT-dependent gene expression in central and peripheral neurons and that distinct regulation of NFAT isoforms within neurons may underlie isoform-specific effects on gene expression.

Angiogenesis is the formation of new blood vessels from pre-existing ones. Unregulated angiogenesis is associated with several diseases such as diabetic retinopathy and tumour growth. Many signal transduction pathways have been implicated in the regulation of angiogenesis such as p38 mitogen-activated protein kinase (MAPK), phosphatidylinositol-3 kinase (PI3K), extracellular signal-related kinase 1/2 (Erk1/2) and of particular interest the calcineurin/nuclear factor of activated T-cell (NFAT) pathway. Inhibition of calcineurin activity by the drug cyclopsorin A (CsA) has been shown to inhibit processes required for successful angiogenesis such as in vitro cell migration, tube formation and additionally attenuates corneal angiogenesis in vivo. CsA is associated with severe side effects and therefore the identification of an endogenous regulator of this pathway would be beneficial. One possibility is the plasma membrane calcium ATPases (PMCAs). These high affinity calcium extrusion pumps have been shown to interact with calcineurin in mammalian cells and cardiomyocytes and down-regulate the calcineurin/NFAT pathway. This is hypothesised to be due to the interaction between the two proteins which maintains calcineurin in a low calcium micro-environment generated by the calcium removal function of the pump. Interestingly, PMCA4 has been shown to interact with calcineurin in endothelial cells. The aim of our study was to further our understanding of PMCA4s regulation of the calcineurin/NFAT pathway specifically in endothelial cells and establish if PMCA4 has a role in the regulation of angiogenesis. ‘Gain of function’ by adenoviral over-expression of PMCA4 and ‘loss of function’ by either si-RNA mediated knockdown of PMCA4 or isolation of PMCA4-/- MLEC were used as models. Over-expression of PMCA4 in HUVEC resulted in inhibition of the calcineurin/NFAT pathway with the opposite result occurring in the case of the knockout of PMCA4, identifying PMCA4 as a negative-regulator of the calcineurin/NFAT pathway in endothelial cells. Over-expression of PMCA4 significantly attenuated VEGF-induced protein and mRNA expression of the pro-angiogenic proteins RCAN1.4 and Cox-2, endothelial cell migration and in vitro and in vivo tube formation with the opposite result occurring in knockdown or knockout studies, confirming PMCA4 as a down-regulator of angiogenesis. Interestingly, over-expression or knockdown of PMCA4 had no effect on VEGF-induced HUVEC proliferation or Erk1/2 phopshorylation proposing PMCA4 may be a potential inhibitor of angiogenesis without compromising cell survival. Disruption of the interaction between PMCA4 and calcineurin by generation and ectopic expression of an adenovirus encoding the region of PMCA4 that interacts with calcineurin (428-651) (Ad-ID4) resulted in an increase in NFAT activity, RCAN1.4 protein expression and in vitro tube formation. These results identify the mechanism of PMCA4s inhibitory effect of the calcineurin/NFAT pathway and consequently angiogenesis is a result of the interaction between the two proteins. The novel findings of this study establish PMCA4 as a negative-regulator of the calcineurin/NFAT pathway in endothelial cells and angiogenesis. These results are far reaching and highlight a potential role for PMCA4 as a therapeutic target in a variety of diseases that are associated with pathological angiogenesis.

Les lymphocytes constituent un modèle original de cellules somatiques puisqu’ils sont capables de réactiver la télomérase lorsqu’ils sont stimulés. Nous avons montré que les lymphocytes, en culture prolongée et soumis à des stimulations itératives par la PHA, présentent une diminution progressive de l’activité télomérasique interrompue à chaque stimulation par une augmentation transitoire. Ces variations sont corrélées positivement aux variations de hTERT et de la longueur des télomères. Les foyers γ-H2AX et 53BP1 et leur localisation au niveau des télomères augmentent lors du vieillissement cellulaire. Nous montrons un dysfonctionnement des télomères au cours de la sénescence lymphocytaire in vitro résultant d’une érosion accrue des télomères et d’une diminution de l’expression des protéines qui les coiffent. Le mécanisme des variations précoces de l’expression de hTERT lors de l’activation lymphocytaire restaient à comprendre. Les conséquences du traitement des lymphocytes par différents immunosuppresseurs agissant tous de façon directe ou indirecte sur l’activation de NFAT suggéraient le rôle de NFAT dans la régulation transcriptionnelle de hTERT. Nous avons montré i) 5 éléments de réponse potentiels pour NFAT au niveau du promoteur de hTERT, ii) l’activation in vitro du promoteur de hTERT par NFAT essentiellement via un site consensus localisé dans le coeur du promoteur de hTERT en position -40 et une synergie fonctionnelle entre NFAT et SP1, iii) la liaison directe de NFAT sur le promoteur de hTERT via ce site consensus in vivo. Ainsi, NFAT1 régule la transcription de hTERT et est impliqué dans l’activation de la télomérase lors de la stimulation lymphocytaire
Lymphocytes are an example of somatic cells capable to induce telomerase activity when stimulated. We showed that lymphocytes, during long-term culture and repeated PHA stimulations, present a progressive drop in telomerase activity interrupted at each stimulation by a transitory increase. These variations are positively correlated with hTERT and telomere length variations. γ-H2AX and 53BP1 foci and their localization on telomeres increase with cell aging. We show a telomere dysfunction during in vitro lymphocyte senescence resulting from an excessive telomere shortening and a decrease in shelterin content. The mechanism involved in early variations of hTERT expression during lymphocyte activation remained to be understood. Consequences of lymphocyte treatment with different immunosuppressors, all acting directly or indirectly on NFAT activation, suggested a role for NFAT in the regulation of hTERT transcription. Five putative responsive elements for NFAT were identified in the hTERT promoter. We showed that NFAT activates in vitro the hTERT promoter mainly via a consensus site localized in the promoter core at position -40 and a functional synergy between NFAT and SP1. Furthermore, NFAT1 binds directly to the endogenous hTERT promoter via this consensus site in vivo. Thus, NFAT positively regulates the hTERT transcription and we propose its implication in telomerase activation during lymphocyte stimulation

博士
國立陽明大學
微生物及免疫學研究所
96
Abstract The promyelocytic leukemia protein (PML) is a tumor suppressor. It is involved in many different physiological functions but the complicated action mechanisms are not yet fully understood. PML localizes in the nucleus to form speckles called PML nuclear bodies, containing regulatory proteins, such as p53, Daxx, CREB binding protein (CBP) and small ubiquitin-related modifier(SUMO-1). Nuclear factor of activated T cell (NFAT) is an important transcription factor, regulates many key T cell functions. In this study, we found that NFAT is an unexpected partner of PML: PML specifically enhanced the transcription activation of NFAT in T cells and 293 cells. In PML-null mouse embryonic fibroblasts, no transcription activity of NFAT could be detected, while PML expression restored NFAT activation. In constrast, PML was not regulated for NF-�羠 transcription. Sumoylation of PML was essential for PML to promote the transcription activity of NFAT. The additive effect of PML was not due to sequestration of Daxx by PML. There was a selective requirement of PML isoform in NFAT activation. PML-I and PML-VI, but not PML-IV, enhanced NFAT transactivation. The potential interaction between NFAT and PML was demonstrated by co-immunoprecipitation of NFATc with PML-I, PML-IV, or PML-VI. GST pull down assay further illustrated a direct binding between the NFAT-homology region (NHR) of NFATc and the RING finger, B-boxes of PML. Inside nucleus of activated T cells, NFATc was found co-localized with PML speckles. Even though PML overexpression did not increase nuclear localization of NFATc, knockdown of PML was moderately reduced nuclear presence of NFAT. Some, but not all, of NFAT activity-mediated genes expression were regulated by PML. The interaction of PML with NFATc in vivo was further confirmed by chromatin immunoprecipitation (ChIP) and DNA affinity precipitation assay (DAPA) analysis. Knockdown of PML was also associated with reduced promoter binding of NFAT and CBP. It is suggested that part of mechanisms of underlying enhancement of NFATc transactivation mediated by PML is due to stabilization of NFAT-CBP transcriptional complex. The unexpected coupling of PML with NFAT reveals a novel mechanism underlying the diverse physiological functions of PML.

碩士
國立臺灣大學
藥理學研究所
91
Background Cytosolic calcium overloading occurs in atrial fibrillation tissue. We therefore investigated the calcium-activated calcineurin-nuclear factor of activated T-cell (NFAT) pathway in atrial tissue in a porcine model of atrial fibrillation. Methods and Results Atrial fibrillation was induced in adult pigs by rapid atrial pacing at a rate of 600 per minute. Atrial tissue from the left atrium was obtained 6 weeks after implantation of the pacemaker (pacing for 4 weeks and atrial fibrillation without pacing for 2 weeks). We found that calcineurin enzyme activity increased significantly in pigs with atrial fibrillation when compared to control pigs (0.143OD620 ± 0.013 vs 0.038OD620 ± 0.028, p < 0.01). We found that both NFATc3 and NFATc4, the down stream effectors of calcineurin, increased significantly in the nuclei in atrial fibrillation tissue using immunoblotting. Translocation of NFATc3 and NFATc4 into the nuclei was also demonstrated in atrial tissue microsections using immunohistochemistry. The electrophoretic mobility shift assay further demonstrated that nuclear extracts from atrial fibrillation tissue had a significantly larger binding capacity for NFAT specific oligonucleotide probes. Conclusion Our results demonstrated that calcineurin activity was increased in atrial fibrillation tissue with subsequent NFATc3 and NFATc4 translocation into the nucleus. Activation of this signal transduction pathway may play an important role in the pathogenesis of atrial fibrillation.