Nuclear Factor Of Activated T Research Articles

TNF-α Contributes to Caspase-3 Independent Apoptosis in Neuroblastoma Cells: Role of NFAT

There is increasing evidence that soluble factors in inflammatory central nervous system diseases not only regulate the inflammatory process but also directly influence electrophysiological membrane properties of neurons and astrocytes. In this context, the cytokine TNF-α (tumor necrosis factor-α) has complex injury promoting, as well as protective, effects on neuronal viability. Up-regulated TNF-α expression has also been found in various neurodegenerative diseases such as cerebral malaria, AIDS dementia, Alzheimer's disease, multiple sclerosis, and stroke, suggesting a potential pathogenic role of TNF-α in these diseases as well. We used the neuroblastoma cells SK-N-MC. Transcriptional activity was measured using luciferase reporter gene assays by using lipofectin. We performed cotransfection experiments of NFAT (nuclear factor of activated T cells) promoter constructed with a dominant negative version of NFAT (dn-NFAT). Cell death was performed by MTT (3-(4,5-dimethylthiazol-2-yl)5,5-diphenyltetrazolium bromide) and TUNEL assays. NFAT translocation was confirmed by Western blot. Involvement of NFAT in cell death was assessed by using VIVIT. P53, Fas-L, caspase-3, and caspase-9 expressions were carried out by Western blot. The mechanisms involved in TNF-α-induced cell death were assessed by using microarray analysis. TNF-α causes neuronal cell death in the absence of glia. TNF-α treatment results in nuclear translocation of NFAT through activation of calcineurin in a Ca2+ independent manner. We demonstrated the involvement of FasL/Fas, cytochrome c, and caspase-9 but the lack of caspase-3 activation. NB cell death was absolutely reverted in the presence of VIVIT, and partially diminished by anti-Fas treatment. These data demonstrate that TNF-α promotes FasL expression through NFAT activation in neuroblastoma cells and this event leads to increased apoptosis through independent caspase-3 activation.

TRPC Channels As Effectors of Cardiac Hypertrophy

Transient receptor potential (TRP) channels of multiple subclasses are expressed in the heart, although their functions are only now beginning to emerge, especially for the TRPC subclass that appears to regulate the cardiac hypertrophic response. Although TRP channels permeate many different cations, they are most often ascribed a specific biological function because of Ca(2+) influx, either for microdomain signaling or to reload internal Ca(2+) stores in the endoplasmic reticulum through a store-operated mechanism. However, adult cardiac myocytes arguably do not require store-operated Ca(2+) entry to regulate sarcoplasmic reticulum Ca(2+) levels and excitation-contraction coupling; hence, TRP channels expressed in the heart most likely coordinate signaling within local domains or through direct interaction with Ca(2+)-dependent regulatory proteins. Here, we review the emerging evidence that TRP channels, especially TRPCs, are critical regulators of microdomain signaling in the heart to control pathological hypertrophy in coordination with signaling through effectors such as calcineurin and NFAT (nuclear factor of activated T cells).

Sustained Hemodynamic Stress Disrupts Normal Circadian Rhythms in Calcineurin-Dependent Signaling and Protein Phosphorylation in the Heart

Despite overwhelming evidence of the importance of circadian rhythms in cardiovascular health and disease, little is known regarding the circadian regulation of intracellular signaling pathways controlling cardiac function and remodeling. To assess circadian changes in processes dependent on the protein phosphatase calcineurin, relative to changes in phosphorylation of cardiac proteins, in normal, hypertrophic, and failing hearts. We found evidence of large circadian oscillations in calcineurin-dependent activities in the left ventricle of healthy C57BL/6 mice. Calcineurin-dependent transcript levels and nuclear occupancy of the NFAT (nuclear factor of activated T cells) regularly fluctuated as much as 20-fold over the course of a day, peaking in the morning when mice enter a period of rest. Phosphorylation of the protein phosphatase 1 inhibitor 1 (I-1), a direct calcineurin substrate, and phospholamban, an indirect target, oscillated directly out of phase with calcineurin-dependent signaling. Using a surgical model of cardiac pressure overload, we found that although calcineurin-dependent activities were markedly elevated, the circadian pattern of activation was maintained, whereas, oscillations in phospholamban and I-1 phosphorylation were lost. Changes in the expression of fetal gene markers of heart failure did not mirror the rhythm in calcineurin/NFAT activation, suggesting that these may not be direct transcriptional target genes. Cardiac function in mice subjected to pressure overload was significantly lower in the morning than in the evening when assessed by echocardiography. Normal, opposing circadian oscillations in calcineurin-dependent activities and phosphorylation of proteins that regulate contractility are disrupted in heart failure.

Development of a novel selective inhibitor of the Down syndrome-related kinase Dyrk1A

Dyrk1A (dual-specificity tyrosine-(Y)-phosphorylation-regulated kinase 1A) is a serine/threonine kinase essential for brain development and function, and its excessive activity is considered a pathogenic factor in Down syndrome. The development of potent, selective inhibitors of Dyrk1A would help to elucidate the molecular mechanisms of normal and diseased brains, and may provide a new lead compound for molecular-targeted drug discovery. Here, we report a novel Dyrk1A inhibitor, INDY, a benzothiazole derivative showing a potent ATP-competitive inhibitory effect with IC(50) and K(i) values of 0.24 and 0.18 μM, respectively. X-ray crystallography of the Dyrk1A/INDY complex revealed the binding of INDY in the ATP pocket of the enzyme. INDY effectively reversed the aberrant tau-phosphorylation and rescued the repressed NFAT (nuclear factor of activated T cell) signalling induced by Dyrk1A overexpression. Importantly, proINDY, a prodrug of INDY, effectively recovered Xenopus embryos from head malformation induced by Dyrk1A overexpression, resulting in normally developed embryos and demonstrating the utility of proINDY in vivo.

Microglial phenotype is regulated by activity of the transcription factor, NFAT (nuclear factor of activated T cells).

The transcription factor family, nuclear factor of activated T cells (NFAT), regulates immune cell phenotype. Four different calcium/calmodulin-regulated isoforms have been identified in the periphery, but isoform expression in microglia, the resident immune cells of the CNS, has not been fully defined. In this study microglial NFAT isoform expression and involvement in regulating inflammatory responses in murine primary microglia culture was examined. Western blot analysis demonstrated robust detection of NFATc1 and c2 isoforms in microglia. Electrophoretic mobility shift assays demonstrated increased NFAT-DNA binding from nuclear extracts of lipopolysaccharide (LPS) stimulated microglia. Moreover, LPS-stimulated microglia behaved similarly to T cell receptor agonist antibody-stimulated Jurkat cells demonstrating a transient increase in NFAT-driven luciferase reporter gene expression. LPS-induced NFAT-luciferase activity in microglia was attenuated by pretreatment with tat-VIVIT, a cell-permeable NFAT inhibitory peptide. Furthermore, LPS-mediated secretion of microglial cytokines, TNF-alpha and MCP-1, was decreased by treatment with tat-VIVIT but not with tat-VEET, a negative control peptide. These results demonstrate that NFAT plays a role in regulating proinflammatory responses in cultured murine microglia.

NFATc4 is negatively regulated in miR-133a-mediated cardiomyocyte hypertrophic repression

Activation of NFAT (nuclear factor of activated T cells)-mediated hypertrophic signaling is a major regulatory response to hypertrophic stimuli. A recent study unveiled potential regulatory roles for microRNA-133a (miR-133a) in cardiac hypertrophy. To date, however, no connection has been made between miR-133a and NFAT signaling. In this study, we determined that NFATc4, a hypertrophy-associated mediator, is negatively regulated by miR-133a. Two conserved base-pairing sites between the NFATc4 3'-untranslated region (UTR) and miR-133a were verified. Mutation of these sites in the NFATc4 3'-UTR completely blocked the negative effect of miR-133a on NFATc4, suggesting that NFATc4 is a direct target for miR-133a regulation. Using a gain-of-function approach, we demonstrate that miR-133 significantly reduces the endogenous level of, as well as the hypertrophic stimulus-mediated increase in, NFATc4 gene expression. This latter effect of miR-133a on NFATc4 gene expression was coincided with an attenuated cardiomyocyte hypertrophy induced by an alpha-adrenergic receptor agonist. Conversely, cells treated with miR-133a inhibitor resulted in an increase in NFATc4 expression level. Application of miR-133a had no apparent effect on NFATc4 nuclear localization. We conclude that the negative regulation of NFATc4 expression contributes to miR-133a-mediated hypertrophic repression.

Ca 2+ Cycling and New Therapeutic Approaches for Heart Failure

Received July 2, 2009; accepted October 5, 2009. Heart failure (HF) is a major health problem in Western countries. Despite significant progress in pharmacological and device-based treatment, the disease burden imposed continues to increase, particularly as the population ages. HF incidence approaches 10 per 1000 after age 65 years.1 Congestive HF is the final consequence of diverse cardiovascular disorders, including atherosclerosis, cardiomyopathy, and hypertension. Described as a complex pathophysiological syndrome that involves interactions of the circulatory, neurohormonal, and renal systems, HF is first a disease of the myocardium, although it soon induces defects in other systems. Current treatments for HF, focused on blocking neurohormonal pathways, improve survival, but they do not halt the progression of HF. Late-stage HF has a poor prognosis, and therapeutic options are limited. Faced with these challenges, researchers are exploring novel therapeutic options. Chronic HF is associated with increased sympathetic outflow, which may be compensatory early on, but long-term neurohormonal activation induces significant damage to the heart; in addition, it results in multiple alterations in the β-adrenergic receptor (β-AR) signaling cascade, including receptor downregulation, upregulation of receptor kinases, and increased inhibitory G-protein function.2 The amplitude and velocity of Ca2+ cycling are regulated by a dynamic balance of phosphorylation and dephosphorylation through kinases and phosphatases. Activation of β-ARs stimulates cAMP production and results in protein kinase A (PKA) phosphorylation of key regulators of excitation-contraction coupling, such as L-type Ca2+ channels, phospholamban, troponin I, ryanodine receptors (RyR), myosin-binding protein C, and protein phosphatase inhibitor-1 (I-1; Figure), which leads to increased amplitude and velocity of Ca2+ cycling and increased contractility on a beat-to-beat basis.3 Protein phosphatases PP1 and PP2A counterbalance phosphorylation of these proteins. There is clear evidence that alterations in sarcoplasmic reticulum (SR) Ca2+ cycling are a component of the impaired …

Competitive NFAT Bottleneck For Transcriptional Activation of Endogenous Ca2+ ATPASE (SERCA2) in Adrenergic Hypertrophy

A prominent feature of cardiac hypertrophy and failure is reduced SERCA2 expression and deficient Ca2+ signaling. To uncover a causal relationship between hypertrophy and downregulation of SERCA2, we use neonatal rat cardiac myocytes where SERCA2 transcription can be increased by exposure to 10 nM thapsigargin (TG), which allows cytosolic Ca2+ rise and calmodulin activation of calcineurin (CN). This SERCA2 rise is markedly reduced by cyclosporine (CsA), which inhibits CN phosphatase, and increased by KN-93, a calmodulin activated kinase (CAMKII) inhibitor which relieves CAMKII dependent phosphorylation and inhibition of CN. These CsA and KN-93 effects are also produced on TG enhanced luciferase expression, under the control of an NFAT (Nuclear factor of activated T Cells) dependent promoter. We conclude that NFAT dephosphorylation is a limiting factor for SERCA2 transcription. Exposure of myocytes to PE yields adrenergic hypertrophy, with rise of Atrial Natriuretic Factor (ANF) transcript, protein incorporation of 14C-phenylalanine and fluorescent staining of actin, while SERCA2 is downregulated. The adrenergic response can be reproduced by direct stimulation of protein kinase C (PKC) with phorbol 12-myristate 13-acetate, indicating that inhibition of glycogen synthase kinase (GSK3β) by PKC and consequent reduced NFAT phosphorylation, as well as histone deacetylase (HDAC) phosphorylation by PKC activated MAPK, are involved in the mechanism of adrenergic hypertrophy. The hypertrophy response is markedly reduced by CN inhibition by CsA, indicating that CN dephosphorylation and nuclear import of NFAT play an important role in the development of hypertrophy, and SERCA2 downregulation is produced by competitive utilization of NFAT by the hypertrophy program. Interestingly, CAMKII inhibition limits the development of hypertrophy, emphasizing a rate limiting role of CAMKII dependent phosphorylation and nuclear export of HDAC in the extensive transcriptional hypertrophic program (Supported by 5 R01 HL069830-08).

Regulation of Fas/Fas Ligand-Mediated Apoptosis in Megakaryocytes by Nuclear Factor of Activated T Cells.

Abstract 4023Poster Board III-959Bone marrow megakaryocytes are the precursors of peripheral blood platelets and therefore constitute an integral part of primary haemostasis, thrombosis and wound healing. Platelets have also been implicated in the regulation of inflammation and other immune responses, but the role of megakaryocytes in this context is less clear. The calcineurin-dependent transcription factor NFAT (Nuclear Factor of Activated T cells) is a master regulator of cytokine production in T lymphocytes and therefore central for T cell-dependent immune reactions. We have previously shown that NFAT is strongly expressed in megakaryocytes and is required for the transcription of specific megakaryocytic genes. The biological role of NFAT in megakaryocytes, however, is unknown.Here we show 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 at least partially 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 (FasL), a pro-apoptotic member of the tumor necrosis factor superfamily. Expression of FasL was detectable as early as six 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 FasL expression on megakaryocytes, we performed cocultures of megakaryocytes with Fas-expressing T lymphocytes, in which CMK cells were either left unstimulated or prestimulated 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 FasL or an antagonistic antibody to Fas during the coculture period, indicating involvement of the FasL/Fas apoptosis pathway.These results have several implications. First, they represent the first clear evidence for a biological function of the calcineurin/NFAT pathway in megakaryocytes, namely the regulation of Fas/FasL-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. Our results suggest that this regulatory function may include the induction of Fas/FasL-dependent apoptosis in bystander cells under certain conditions, for example the elimination of activated T cells in inflammatory situations. Disclosures:No relevant conflicts of interest to declare.

A fluorescent polarization-based assay for the identification of disruptors of the RCAN1–calcineurin A protein complex

Calcineurin is a Ca 2+/calmodulin-dependent serine/threonine protein phosphatase involved in many biological processes and developmental programs, including immune response. One of the most studied substrates of calcineurin is the transcription factor NFAT (nuclear factor of activated T cells) responsible for T-cell activation. Different anticalcineurin drugs, such as cyclosporine A and FK506, are the most commonly used immunosuppressants in transplantation therapies. Unfortunately, their mechanism of action, completely blocking the calcineurin phosphatase activity while also requiring continuous administration, bears severe side effects. During recent years, the family of regulators of calcineurin (RCAN) has been described and studied extensively as modulators of calcineurin signaling pathways. The RCAN1 region, spanning amino acids 198 to 218 and responsible for inhibiting the calcineurin–NFAT signaling pathway in vivo, has been identified. An RCAN1-derived peptide spanning this sequence interferes with the calcineurin–NFAT interaction without affecting the general calcineurin phosphatase activity. Here we report the development of an optimized in vitro high-throughput fluorescence polarization assay based on the disruption of the RCAN1 198–218–CnA interaction for identifying molecules with immunosuppressant potential. This approach led us to identify dipyridamole as a disruptor of such interaction. Moreover, three small molecules with a potential immunosuppressive effect were also identified.

TRP channels in agonist-induced proliferation of prostate cancer cells

334 Agonist-induced stimulation of α 1-adrenoreceptors ( α 1-AR) of the cultured primary human prostate cancer (PCa) epithelial cells results in the enhancement of proliferative activity accompanied by the oscillations of the intracellular calcium concentration ([Ca 2+ ] i ), increase of store-independent calcium entry, and activation of the transcription factor NFAT (nuclear factor of activated T cells). Calcium entry in response to the α 1-ARs stimulation is primarily mediated by TRPC6 (transient receptor potential canonical 6) cationic channel: antisense TRPC6 depletion decreases calcium entry, suppresses [Ca 2+ ] i oscillations and slows down proliferation caused by the α 1-AR agonist phenylephrine. On the contrary, stimulation of purinergic P2Y receptors (P2Y-R) leads to complete cessation of proliferative activity of human PCa cells due to enhancement of sustained store-dependent calcium entry, which is mainly mediated by TRPC1 and TRPC4 channels without NFAT involvement. Expression and channel-like function of another member of TRP channels family, cold/menthol-sensitive TRPM8 (melastatin 8), was dependent on the presence of functional androgen receptor and was characteristic of only apical phenotype of PCa epithelial cells. Our results establish TRPC6 and TRPC1/4 channels as important determinants in the control of PCa epithelial cells proliferation and TRPM8 channel as a marker of the PCa stages and androgen dependency.

CaMKII Negatively Regulates Calcineurin–NFAT Signaling in Cardiac Myocytes

Pathological cardiac myocyte hypertrophy is thought to be induced by the persistent increases in intracellular Ca(2+) needed to maintain cardiac function when systolic wall stress is increased. Hypertrophic Ca(2+) binds to calmodulin (CaM) and activates the phosphatase calcineurin (Cn) and CaM kinase (CaMK)II. Cn dephosphorylates cytoplasmic NFAT (nuclear factor of activated T cells), inducing its translocation to the nucleus where it activates antiapoptotic and hypertrophic target genes. Cytoplasmic CaMKII regulates Ca(2+) handling proteins but whether or not it is directly involved in hypertrophic and survival signaling is not known. This study explored the hypothesis that cytoplasmic CaMKII reduces NFAT nuclear translocation by inhibiting the phosphatase activity of Cn. Green fluorescent protein-tagged NFATc3 was used to determine the cellular location of NFAT in cultured neonatal rat ventricular myocytes (NRVMs) and adult feline ventricular myocytes. Constitutively active (CaMKII-CA) or dominant negative (CaMKII-DN) mutants of cytoplasmic targeted CaMKII(deltac) were used to activate and inhibit cytoplasmic CaMKII activity. In NRVM CaMKII-DN (48.5+/-3%, P<0.01 versus control) increased, whereas CaMKII-CA decreased (5.9+/-1%, P<0.01 versus control) NFAT nuclear translocation (Control: 12.3+/-1%). Cn inhibitors were used to show that these effects were caused by modulation of Cn activity. Increasing Ca(2+) increased Cn-dependent NFAT translocation (to 71.7+/-7%, P<0.01) and CaMKII-CA reduced this effect (to 17.6+/-4%). CaMKII-CA increased TUNEL and caspase-3 activity (P<0.05). CaMKII directly phosphorylated Cn at Ser197 in CaMKII-CA infected NRVMs and in hypertrophied feline hearts. These data show that activation of cytoplasmic CaMKII inhibits NFAT nuclear translocation by phosphorylation and subsequent inhibition of Cn.

FcɛRI-induced activation by low antigen concentrations results in nuclear signals in the absence of degranulation

High affinity IgE receptor (FcɛRI)-induced activation of mast cells results in degranulation and generation of leukotrienes and cytokines. FcɛRI-induced mast cell activation was analyzed at a single cell basis using a rat basophilic leukemia (RBL-2H3) cell line transfected with a reporter plasmid containing three tandem NFAT (nuclear factor of activated T cells) binding sites fused to enhanced green fluorescent protein (GFP). Surprisingly, with this sensitive detection system, there is activation of IgE sensitized cells at concentrations of antigen as low as 10 pg/ml, which was 10-fold lower than was detected by degranulation. There were differences in signaling pathways leading to degranulation compared to NFAT-mediated gene activation. Both signaling to NFAT activation and degranulation required Syk and calcineurin. However inhibitors of the phosphatidylinositol 3-kinase pathway blocked degranulation but did not NFAT activation. The results also indicate that NFAT was activated at lower intracellular signals compared to degranulation. Therefore, FcɛRI activation can result in nuclear signals in the absence of the release of mediators.

CD14 regulates the dendritic cell life cycle after LPS exposure through NFAT activation

Toll-like receptors (TLRs) are the best characterized pattern recognition receptors. Individual TLRs recruit diverse combinations of adaptor proteins, triggering signal transduction pathways and leading to the activation of various transcription factors, including nuclear factor kappaB, activation protein 1 and interferon regulatory factors. Interleukin-2 is one of the molecules produced by mouse dendritic cells after stimulation by different pattern recognition receptor agonists. By analogy with the events after T-cell receptor engagement leading to interleukin-2 production, it is therefore plausible that the stimulation of TLRs on dendritic cells may lead to activation of the Ca(2+)/calcineurin and NFAT (nuclear factor of activated T cells) pathway. Here we show that mouse dendritic cell stimulation with lipopolysaccharide (LPS) induces Src-family kinase and phospholipase Cgamma2 activation, influx of extracellular Ca(2+) and calcineurin-dependent nuclear NFAT translocation. The initiation of this pathway is independent of TLR4 engagement, and dependent exclusively on CD14. We also show that LPS-induced NFAT activation via CD14 is necessary to cause the apoptotic death of terminally differentiated dendritic cells, an event that is essential for maintaining self-tolerance and preventing autoimmunity. Consequently, blocking this pathway in vivo causes prolonged dendritic cell survival and an increase in T-cell priming capability. Our findings reveal novel aspects of molecular signalling triggered by LPS in dendritic cells, and identify a new role for CD14: the regulation of the dendritic cell life cycle through NFAT activation. Given the involvement of CD14 in disease, including sepsis and chronic heart failure, the discovery of signal transduction pathways activated exclusively via CD14 is an important step towards the development of potential treatments involving interference with CD14 functions.

It's up to you Egr2

provide new insights into iNKT cell biologyby demonstrating that the gene encoding earlygrowth response 2 (Egr2), a target of thetranscription factor nuclear factor of activatedT (NFAT) cells, has critical functions duringthe ontogeny of this unique cell lineage.iNKT cells derive their name from thecoexpression of NK cell-specific markers,such as the Ly49 receptors and NK1.1, andthe T-cell receptor (TCR). Over 80% of iNKTcells express a canonical or ‘invariant’ TCR,typified by a common TCRa chain rearran-gement (Va14-Ja18 in mice; Va24-Ja18 inhumans) paired with specific TCRb chains(Vb8, 7, 2 in mice; Vb11 in humans).

RNA Interference Targeting STIM1 Suppresses Vascular Smooth Muscle Cell Proliferation and Neointima Formation in the Rat

Our objective was to study the expression and function of stromal interaction molecule 1 (STIM1), an endoplasmic reticulum protein recently identified as the calcium sensor that regulated Ca(2+)-released activated channels in T cells. STIM1 was found to be upregulated in serum-induced proliferating human coronary artery smooth muscle cells (hCASMCs) as well as in the neointima of injured rat carotid arteries. Growth factors-induced proliferation was significantly lower in hCASMC transfected with STIM1 siRNA than in those transfected with scrambled siRNA (increase relative to 0.1% S: 116 +/- 12% and 184 +/- 16%, respectively, P < 0.01). To assess the role of STIM1 in preventing vascular smooth muscle cells (VSMCs) proliferation in vivo, we infected balloon-injured rat carotid arteries with an adenoviral vector expressing a short hairpin (sh) RNA against rat STIM1 mRNA (Ad-shSTIM1). Intima/media ratios reflecting the degree of restenosis were significantly lower in Ad-shSTIM1- infected arteries than in Ad-shLuciferase-infected arteries (0.34 +/- 0.02 vs. 0.92 +/- 0.11, P < 0.006). Finally, we demonstrated that silencing STIM1 prevents activation of the transcription factor NFAT (nuclear factor of activated T cell). In conclusion, STIM1 appears as a major regulator of in vitro and in vivo VSMC proliferation, representing a novel and original pharmacological target for prominent vascular proliferative diseases.

Ca 2+ Signaling Domains Responsible For Cardiac Hypertrophy and Arrhythmias

See related articles, pages 514–521 and 522–530 Ca2+ activates and regulates multiple processes in every cell type. In the mammalian heart, cyclic fluctuations in cytosolic [Ca2+] induce and regulate the strength of cardiac contraction (termed “contractile” [Ca2+]). In addition, changes in Ca2+ appear to be centrally involved in normal and pathological signaling (termed “signaling” [Ca2+]) that regulates myocyte growth, hypertrophy, apoptosis, and necrosis.1 Whether or not contractile and signaling [Ca2+] are derived from common or distinct sources and are constrained to unique cellular microdomains is not established.2 What is clear is that cardiovascular diseases including hypertension and myocardial infarction are associated with alterations in contractile and possibly signaling [Ca2+] that are centrally involved in pathological cardiac hypertrophy, heart failure progression,1 and lethal cardiac arrhythmias.3 Defining the sources of signaling Ca2+ involved in the induction of pathological hypertrophy and the bases of dysregulated contractile [Ca2+] in cardiovascular disease should identify novel ways to treat heart disease. In this issue of Circulation Research , 2 independent reports address fundamental aspects of alterations in signaling and contractile [Ca2+]. Chiang et al4 have studied the idea that Ca2+ influx through voltage operated α1H (CaV3.2) T-type Ca2+ channels (TTCCs) is the source of the signaling [Ca2+] that activates the calcineurin (Cn)-NFAT (nuclear factor of activated T cells) signaling cascade and induces pathological cardiac hypertrophy in pressure overload. In a separate report, Terentyev et al5 explore the idea that microRNA (miR)-1, a muscle-specific microRNA that increases in abundance in cardiac disease,6 causes dysregulated contractile [Ca2+] and induces single cell arrhythmias. These 2 reports are provocative and, if independently confirmed, will have identified novel …

DYRK3 Dual-specificity Kinase Attenuates Erythropoiesis during Anemia

During anemia erythropoiesis is bolstered by several factors including KIT ligand, oncostatin-M, glucocorticoids, and erythropoietin. Less is understood concerning factors that limit this process. Experiments performed using dual-specificity tyrosine-regulated kinase-3 (DYRK3) knock-out and transgenic mice reveal that erythropoiesis is attenuated selectively during anemia. DYRK3 is restricted to erythroid progenitor cells and testes. DYRK3-/- mice exhibited essentially normal hematological profiles at steady state and reproduced normally. In response to hemolytic anemia, however, reticulocyte production increased severalfold due to DYRK3 deficiency. During 5-fluorouracil-induced anemia, both reticulocyte and red cell formation in DYRK3-/- mice were elevated. In short term transplant experiments, DYRK3-/- progenitors also supported enhanced erythroblast formation, and erythropoietic advantages due to DYRK3-deficiency also were observed in 5-fluorouracil-treated mice expressing a compromised erythropoietin receptor EPOR-HM allele. As analyzed ex vivo, DYRK3-/- erythroblasts exhibited enhanced CD71posTer119pos cell formation and 3HdT incorporation. Transgenic pA2gata1-DYRK3 mice, in contrast, produced fewer reticulocytes during hemolytic anemia, and pA2gata1-DYRK3 progenitors were compromised in late pro-erythroblast formation ex vivo. Finally, as studied in erythroid K562 cells, DYRK3 proved to effectively inhibit NFAT (nuclear factor of activated T cells) transcriptional response pathways and to co-immunoprecipitate with NFATc3. Findings indicate that DYRK3 attenuates (and possibly apportions) red cell production selectively during anemia.

Suppressing NFAT Increases VEGF Signaling in Hemangiomas

Infantile hemangiomas represent the most common tumor of endothelial cell (EC) origin, yet the mechanisms regulating hemangioma EC behavior are poorly understood. A new study by Jinnin et al. demonstrates that enhanced VEGFR2 signaling in hemangioma ECs is caused by suppression of NFAT (nuclear factor of activated T cells)-dependent VEGFR1 expression.

Osteomyelosclerosis, Anemia and Extramedullary Hematopoiesis in Mice Deficient for the Transcription Factor NFAT (Nuclear Factor of Activated T Cells) c2

NFAT (Nuclear Factor of Activated T cells) is a family of five calcium-induced, calcineurin-dependent transcription factors, characterized as master regulators of inducible gene expression in T lymphocytes. In various tissues (including bone, cartilage and adipose tissue), NFAT factors have also been found to regulate processes of cellular adaptation and differentiation. NFAT family members are also expressed in human CD34+ hematopoietic progenitor cells as well as in mesenchymal stem cells; however, a biological function of NFAT in these cells, such as a role in hematopoietic differentiation, has not yet been described. To determine a potential role for NFAT in hematopoiesis, we characterized the steady-state hematopoiesis in NFATc2 knockout (KO) mice by analyzing peripheral blood, bone marrow and extramedullary tissue (spleen and liver) of KO and age-matched wildtype (WT) mice by cytology, histopathology as well as flow cytometry. No significant abnormalities were observed in KO mice of young age (6–12 weeks). NFATc2 KO mice of older age (>12 months), however, were found to be anemic, with a significant decrease in red blood cell counts as well as in hemoglobin and hematocrit values, and also showed decreased numbers of peripheral blood platelets as compared to the WT. In contrast, no significant differences between WT and KO were found in total counts of white blood cells or in leukocyte differentials. Femora of NFATc2 KO mice appeared macroscopically pale and, upon elutriation, gave yield to less than 50% of bone marrow cells compared to the WT, indicating bone marrow hypoplasia. Further analysis of bone marrow differentials by morphology and flow cytometry showed a profound decrease in erythroid cells as well as in megakaryocytes. Histopathological examination of KO bone marrow sections revealed evidence of bone marrow sclerosis, as indicated by an increase in bone trabeculae and osteoblasts within the marrow space. In some mice, an increase in reticulin fiber content was also noted. Interestingly, both liver and spleens of NFATc2 KO mice showed clear evidence for extramedullary hematopoiesis, suggesting that displacement of hematopoietic cells by the bone marrow stroma may be a possible cause of the observed cytopenias.These results show that NFATc2 knockout mice over their lifespan develop a phenotype, which clinically resembles human osteomyelofibrosis/-sclerosis. The underlying mechanism for the observed hematopoietic abnormalities has to be defined, in particular the respective contribution of hematopoietic and bone marrow stroma cells. Given that NFAT factors are expressed at considerable levels in both hematopoietic progenitor cells and mesenchymal stem cells, our results should encourage further studies on the yet undefined role of these transcription factors within the bone marrow hematopoietic niche.