NFAT Nuclear Translocation Research Articles

Distinct Contributions of Orai1 and TRPC1 to Agonist-Induced [Ca2+]i Signals Determine Specificity of Ca2+-Dependent Gene Expression

Regulation of critical cellular functions, including Ca2+-dependent gene expression, is determined by the temporal and spatial aspects of agonist-induced Ca2+ signals. Stimulation of cells with physiological concentrations of agonists trigger increases [Ca2+]i due to intracellular Ca2+ release and Ca2+ influx. While Orai1-STIM1 channels account for agonist-stimulated [Ca2+]i increase as well as activation of NFAT in cells such as lymphocytes, RBL and mast cells, both Orai1-STIM1 and TRPC1-STIM1 channels contribute to [Ca2+]i increases in human submandibular gland (HSG) cells. However, only Orai1-mediated Ca2+ entry regulates the activation of NFAT in HSG cells. Since both TRPC1 and Orai1 are activated following internal Ca2+ store depletion in these cells, it is not clear how the cells decode individual Ca2+ signals generated by the two channels for the regulation of specific cellular functions. Here we have examined the contributions of Orai1 and TRPC1 to carbachol (CCh)-induced [Ca2+]i signals and activation of NFAT in single cells. We report that Orai1-mediated Ca2+ entry generates [Ca2+]i oscillations at different [CCh], ranging from very low to high. In contrast, TRPC1-mediated Ca2+ entry generates sustained [Ca2+]i elevation at high [CCh] and contributes to frequency of [Ca2+]i oscillations at lower [agonist]. More importantly, the two channels are coupled to activation of distinct Ca2+ dependent gene expression pathways, consistent with the different patterns of [Ca2+]i signals mediated by them. Nuclear translocation of NFAT and NFAT-dependent gene expression display “all-or-none” activation that is exclusively driven by local [Ca2+]i generated by Orai1, independent of global [Ca2+]i changes or TRPC1-mediated Ca2+ entry. In contrast, Ca2+ entry via TRPC1 primarily regulates NFκB-mediated gene expression. Together, these findings reveal that Orai1 and TRPC1 mediate distinct local and global Ca2+ signals following agonist stimulation of cells, which determine the functional specificity of the channels in activating different Ca2+-dependent gene expression pathways.

Modification of STIM1 by O-linked N-Acetylglucosamine (O-GlcNAc) Attenuates Store-operated Calcium Entry in Neonatal Cardiomyocytes

Store-operated calcium entry (SOCE) is a major Ca(2+) signaling pathway responsible for regulating numerous transcriptional events. In cardiomyocytes SOCE has been shown to play an important role in regulating hypertrophic signaling pathways, including nuclear translocation of NFAT. Acute activation of pathways leading to O-GlcNAc synthesis have been shown to impair SOCE-mediated transcription and in diabetes, where O-GlcNAc levels are chronically elevated, cardiac hypertrophic signaling is also impaired. Therefore the goal of this study was to determine whether changes in cardiomyocyte O-GlcNAc levels impaired the function of STIM1, a widely recognized mediator of SOCE. We demonstrated that acute activation of SOCE in neonatal cardiomyocytes resulted in STIM1 puncta formation, which was inhibited in a dose-dependent manner by increasing O-GlcNAc synthesis with glucosamine or inhibiting O-GlcNAcase with thiamet-G. Glucosamine and thiamet-G also inhibited SOCE and were associated with increased O-GlcNAc modification of STIM1. These results suggest that activation of cardiomyocyte O-GlcNAcylation attenuates SOCE via STIM1 O-GlcNAcylation and that this may represent a new mechanism by which increased O-GlcNAc levels regulate Ca(2+)-mediated events in cardiomyocytes. Further, since SOCE is a fundamental mechanism underlying Ca(2+) signaling in most cells and tissues, it is possible that STIM1 represents a nexus linking protein O-GlcNAcylation with Ca(2+)-mediated transcription.

Ca2 + influx through L-type Ca2 + channels and transient receptor potential channels activates pathological hypertrophy signaling

Common cardiovascular diseases such as hypertension and myocardial infarction require that myocytes develop greater than normal force to maintain cardiac pump function. This requires increases in [Ca2+]. These diseases induce cardiac hypertrophy and increases in [Ca2+] are known to be an essential proximal signal for activation of hypertrophic genes. However, the source of “hypertrophic” [Ca2+] is not known and is the topic of this study. The role of Ca2+ influx through L-type Ca2+ channels (LTCC), T-type Ca2+ channels (TTCC) and transient receptor potential (TRP) channels on the activation of calcineurin (Cn)–nuclear factor of activated T cells (NFAT) signaling and myocyte hypertrophy was studied. Neonatal rat ventricular myocytes (NRVMs) and adult feline ventricular myocytes (AFVMs) were infected with an adenovirus containing NFAT–GFP, to determine factors that could induce NFAT nuclear translocation. Four millimolar Ca2+ or pacing induced NFAT nuclear translocation. This effect was blocked by Cn inhibitors. In NRVMs Nifedipine (Nif, LTCC antagonist) blocked high Ca2+-induced NFAT nuclear translocation while SKF-96365 (TRP channel antagonist) and Nickel (Ni, TTCC antagonist) were less effective. The relative potency of these antagonists against Ca2+ induced NFAT nuclear translocation (Nif>SKF-96365>Ni) was similar to their effects on Ca2+ transients and the LTCC current. Infection of NRVM with viruses containing TRP channels also activated NFAT–GFP nuclear translocation and caused myocyte hypertrophy. TRP effects were reduced by SKF-96365, but were more effectively antagonized by Nif. These experiments suggest that Ca2+ influx through LTCCs is the primary source of Ca2+ to activate Cn–NFAT signaling in NRVMs and AFVMs. While TRP channels cause hypertrophy, they appear to do so through a mechanism involving Ca2+ entry via LTCCs.

Leptin-induced Cardiomyocyte Hypertrophy Reveals both Calcium-dependent and Calcium-independent/RhoA-dependent Calcineurin Activation and NFAT Nuclear Translocation

Leptin, a product of the obesity gene, has been shown to produce cardiac hypertrophy. Although leptin's mechanism of action is poorly understood activation of the RhoA/ROCK pathway has been proposed as a contributing mechanism. The Ca2+-dependent phosphatase calcineurin plays a critical role in the hypertrophic program although it is not known whether leptin can activate this signaling pathway or whether there is a relationship between RhoA activation and calcineurin. Accordingly, we determined the effect of leptin on calcineurin activation and assessed the possible role of RhoA. Experiments were performed using cultured neonatal rat ventricular myocytes exposed to 50ng/ml leptin for 24h which resulted in a robust hypertrophic response. Moreover, leptin significantly increased intracellular Ca2+ and Na+ concentrations which was associated with significantly reduced activity of the 3Na+-2K+ATPase. The hypertrophic response to leptin were completely abrogated by both C3 exoenzyme (C3), a RhoA inhibitor as well as the reverse mode 3Na+-1Ca2+ exchange inhibitor KB-R7943 ((2-[2-[4-(4-nitrobenzyloxy)phenyl] ethyl]isothiourea methanesulfonate), however only the effect of the latter was associated with attenuation of intracellular Ca2+ concentrations whereas Ca2+ concentrations were unaffected by C3. Similarly, C3 and KB-R7943 significantly attenuated early leptin-induced increase in calcineurin activity as well as the increase in nuclear translocation of the transcriptional factor nuclear factor of activated T cells. The hypertrophic response to leptin was also associated with increased p38 and ERK1/2 MAPK phosphorylation and increased p38, but not ERK1/2, translocation into nuclei. Both p38 responses as well as hypertrophy were abrogated by KB-R7943 as well as the calcineurin inhibitor FK-506 although ERK1/2 phosphorylation was unaffected. Our study therefore demonstrates a critical role for the calcineurin pathway in mediating leptin-induced hypertrophy. Moreover, we report a novel RhoA-dependent leptin-induced calcineurin activation which acts independently of changes in intracellular Ca2+ concentrations.

Na+/H+ Exchanger 1 Directly Binds to Calcineurin A and Activates Downstream NFAT Signaling, Leading to Cardiomyocyte Hypertrophy

The calcineurin A (CaNA) subunit was identified as a novel binding partner of plasma membrane Na(+)/H(+) exchanger 1 (NHE1). CaN is a Ca(2+)-dependent phosphatase involved in many cellular functions, including cardiac hypertrophy. Direct binding of CaN to the (715)PVITID(720) sequence of NHE1, which resembles the consensus CaN-binding motif (PXIXIT), was observed. Overexpression of NHE1 promoted serum-induced CaN/nuclear factor of activated T cells (NFAT) signaling in fibroblasts, as indicated by enhancement of NFAT promoter activity and nuclear translocation, which was attenuated by NHE1 inhibitor. In neonatal rat cardiomyocytes, NHE1 stimulated hypertrophic gene expression and the NFAT pathway, which were inhibited by a CaN inhibitor, FK506. Importantly, CaN activity was strongly enhanced with increasing pH, so NHE1 may promote CaN/NFAT signaling via increased intracellular pH. Indeed, Na(+)/H(+) exchange activity was required for NHE1-dependent NFAT signaling. Moreover, interaction of CaN with NHE1 and clustering of NHE1 to lipid rafts were also required for this response. Based on these results, we propose that NHE1 activity may generate a localized membrane microdomain with higher pH, thereby sensitizing CaN to activation and promoting NFAT signaling. In cardiomyocytes, such signaling can be a pathway of NHE1-dependent hypertrophy.

Effects of arsenite and UVA-1 radiation on calcineurin signaling

Calcineurin is a Ca2+-dependent serine/threonine phosphatase and the target of the immunosuppressive drugs cyclosporin and tacrolimus, which are used in transplant recipients to prevent rejection. Unfortunately, the therapeutic use of this drugs is complicated by a high incidence of skin malignancy, which has set off a number of studies into the role of calcineurin signaling in skin, particularly with respect to cell cycle control and DNA repair. Both UVA1 radiation and arsenic species are known to promote skin cancer development via production of reactive oxygen species. In light of the well-documented sensitivity of calcineurin to oxidative stress, we examined and compared the effects of UVA1 and arsenite on calcineurin signaling.In this paper, we show that physiologically relevant doses of UVA1 radiation and low micromolar concentrations of arsenite strongly inhibit calcineurin phosphatase activity in Jurkat and skin cells and decrease NFAT nuclear translocation in Jurkat cells. The effects on calcineurin signaling could be partly prevented by inhibition of NADPH oxidase in Jurkat cells or increased dismutation of superoxide in Jurkat and skin cells. In addition, both UVA1 and arsenite decreased NF-κB activity, although at lower concentrations, arsenite enhanced NF-κB activity. These data indicate that UVA1 and arsenite affect a signal transduction route of growingly acknowledged importance in skin and that calcineurin may serve as a potential link between ROS exposure and impaired tumor suppression.

Multiphoton intravital microscopy analysis of signaling in T cells reveals persistent NFAT activation during dynamic APC scanning. (121.21)

Abstract The Ca2+ dependent activation and nuclear shuttling of the cytoplasmic transcription factor NFAT is a hallmark of productive T cell receptor (TCR) triggering. However, the kinetics of NFAT activation and the relationship to the Ca2+ dependent migratory stop signal have not been explored in vivo. We used Multiphoton Intravital Microscopy (MP-IVM) to visualize the subcellular dynamics of a fluorescent reporter of NFAT signaling (NFAT-eGFP) in cytotoxic T cells during their antigen-dependent interactions with various APC in lymph nodes (LNs) and in tumor tissue in vivo. During interactions with peptide-loaded B cells representing optimal APC in LNs, t1/2 of NFAT nuclear translocation was 1 minute. Relocation to the cytoplasm upon contact cessation occurred with a slower t1/2 of ~20 minutes. During its delayed nuclear export, NFAT continued to drive transcription of IFN-gamma. We therefore hypothesized that NFAT may facilitate the integration of TCR signals received through serial APC contacts. To test this in vivo, we performed MP-IVM to visualize contacts between cytotoxic T cells and tumor cells, representing suboptimal APCs. We observed that among T cells with continuously nuclear NFAT, ~30% remained motile while engaging in transient, serial contacts with multiple tumor cells. Hence, NFAT slow off-kinetics may constitute a mechanism of short-term biochemical memory enabling the integration of intermittent TCR signaling into a continuous transcriptional response.

A Cell Permeable Peptide Inhibitor of NFAT Inhibits Macrophage Cytokine Expression and Ameliorates Experimental Colitis

Nuclear factor of activated T cells (NFAT) plays a critical role in the development and function of immune and non-immune cells. Although NFAT is a central transcriptional regulator of T cell cytokines, its role in macrophage specific gene expression is less defined. Previous work from our group demonstrated that NFAT regulates Il12b gene expression in macrophages. Here, we further investigate NFAT function in murine macrophages and determined the effects of a cell permeable NFAT inhibitor peptide 11R-VIVIT on experimental colitis in mice. Treatment of bone marrow derived macrophages (BMDMs) with tacrolimus or 11R-VIVIT significantly inhibited LPS and LPS plus IFN-γ induced IL-12 p40 mRNA and protein expression. IL-12 p70 and IL-23 secretion were also decreased. NFAT nuclear translocation and binding to the IL-12 p40 promoter was reduced by NFAT inhibition. Experiments in BMDMs from IL-10 deficient (Il10 −/−) mice demonstrate that inhibition of IL-12 expression by 11R-VIVIT was independent of IL-10 expression. To test its therapeutic potential, 11R-VIVIT was administered systemically to Il10 −/− mice with piroxicam-induced colitis. 11R-VIVIT treated mice demonstrated significant improvement in colitis compared to mice treated with an inactive peptide. Moreover, decreased spontaneous secretion of IL-12 p40 and TNF in supernatants from colon explant cultures was demonstrated. In summary, NFAT, widely recognized for its role in T cell biology, also regulates important innate inflammatory pathways in macrophages. Selective blocking of NFAT via a cell permeable inhibitory peptide is a promising therapeutic strategy for the treatment of inflammatory bowel diseases.

A Caveolae-Targeted L-Type Ca 2+ Channel Antagonist Inhibits Hypertrophic Signaling Without Reducing Cardiac Contractility

The source of Ca(2+) to activate pathological cardiac hypertrophy is not clearly defined. Ca(2+) influx through the L-type Ca(2+) channels (LTCCs) determines "contractile" Ca(2+), which is not thought to be the source of "hypertrophic" Ca(2+). However, some LTCCs are housed in caveolin-3 (Cav-3)-enriched signaling microdomains and are not directly involved in contraction. The function of these LTCCs is unknown. To test the idea that LTCCs in Cav-3-containing signaling domains are a source of Ca(2+) to activate the calcineurin-nuclear factor of activated T-cell signaling cascade that promotes pathological hypertrophy. We developed reagents that targeted Ca(2+) channel-blocking Rem proteins to Cav-3-containing membranes, which house a small fraction of cardiac LTCCs. Blocking LTCCs within this Cav-3 membrane domain eliminated a small fraction of the LTCC current and almost all of the Ca(2+) influx-induced NFAT nuclear translocation, but it did not reduce myocyte contractility. We provide proof of concept that Ca(2+) influx through LTCCs within caveolae signaling domains can activate "hypertrophic" signaling, and this Ca(2+) influx can be selectively blocked without reducing cardiac contractility.

Down-regulation of interleukin-2 production by CD4+ T cells expressing TIM-3 through suppression of NFAT dephosphorylation and AP-1 transcription

TIM-3 is expressed by TH1 cells and negatively regulates cytokine production by these cells. The aim of the present study was to explore the mechanisms by which IL-2 production is suppressed in TIM-3-expressing T cells. First, the activity of two transcription factors that bind to the IL-2 promoter was examined in Jurkat T cells expressing TIM-3. Both AP-1 and NFAT activity were reduced in TIM-3-expressing cells stimulated with a phorbol ester and a calcium ionophore. At the same time, expression of the AP-1 components, c-Fos and c-Jun, was induced to a lesser extent in stimulated human primary CD4+ T cells expressing high levels of TIM-3 than in those expressing low levels of TIM-3. Furthermore, TIM-3-expression inhibited the stimulation-induced dephosphorylation and nuclear translocation of NFAT in Jurkat T cells and primary CD4+ T cells. Finally, the cytoplasmic tail of TIM-3 was required for the suppression of IL-2 production and for AP-1 and NFAT activation. Taken together, these results suggest that IL-2 production by T cells may be downregulated by TIM-3-mediated signals, leading to suppression of NFAT dephosphorylation and AP-1 transcription.

Novel Modulators of STIM-ORAI Calcium Entry Pathway

In non-excitable cells, NFAT transcription factors are activated in response to store-operated Ca2+ entry (SOCE), which is triggered by depletion of endoplasmic reticulum (ER) Ca2+ stores. The key players in this process - STIM1, a sensor of ER Ca2+ levels, and ORAI1, a plasma membrane Ca2+ channel - were identified in Drosophila RNAi screens. To identify additional modulators of STIM-ORAI Ca2+ pathway in mammalian cells, we performed three successive RNAi screens: (i) a genome-wide RNAi screen for regulators of NFAT nuclear translocation in response to ER store depletion with thapsigargin; (ii) a high-throughput Ca2+ imaging screen in which candidates from the first screen were assessed for effect on SOCE by using a FLEXStation III (FLIPR); (iii) a single-cell Ca2+ analysis of all 97 candidates emerging from the second screen. This third screen yielded 47 strong/moderate and 12 weak candidate modulators. The characterization of some of them shows that they modulate the STIM-ORAI Ca2+ pathway by different mechanisms: (i) controlling membrane organization of ORAI1 and thereby ORAI1 cluster formation; (ii) disruption of STIM-ORAI co-localization; (iii) modulation of STIM1 recruitment to the plasma membrane; (iv) possible post-translational modification of STIM1 and/or ORAI1. In summary, these data demonstrate that additional proteins are required for the proper formation and/or stability of STIM-ORAI1 complex in mammalian cells.

Abstract 9466: Recombinant Mitochondrial Transcriptional Factor A Protein Attenuates Pathological Remodeling in Cardiac Myocytes

Introduction: Mitochondrial transcriptional factor A (TFAM), a nucleoid protein of mitochondrial DNA (mtDNA), is essential not only for transcription and replication but also maintenance of mtDNA. We have previously reported that, in failing myocardium, TFAM expression and the myocardial mtDNA copy number were decreased. Moreover, in myocardial infarction, overexpression of TFAM attenuated the decrease in the mtDNA copy number, ameliorated cardiac remodeling, and markedly improved survival. Since exogenously-administrated recombinant TFAM protein has been shown to enter mitochondria in cultured cells, we examined our hypothesis that recombinant TFAM protein administration attenuates pathological remodeling in cardiac myocytes. Methods and Results: We prepared recombinant human TFAM including mitochondrial targeting signal by glutathione s-transferase fusion protein purification protocol. Recombinant TFAM was successfully recruited into mitochondria of rat neonatal cardiac myocytes, whereas TFAM without mitochondrial targeting signal was observed in nucleus. Treatment with TFAM increased the mtDNA copy number dose-dependently (100 nM: 1.8±0.1-folds), whereas it did not change morphology or population of mitochondria (electron microscopy). To elucidate the mechanism of TFAM on signaling pathways in cardiac hypertrophy, we investigated the effects of TFAM on the nuclear factor of activated T cell (NFAT) signaling, which is a major transcriptional factor regulating pathological hypertrophy and remodeling. TFAM totally abolished NFAT nuclear translocation induced by both angiotensin II (41±3% to 2±1%, p<0.01) and endothelin-1 (43±3% to 3±1%, p<0.01), suppressing respective NFAT transcriptional activity and NFAT-dependent gene expression. TFAM inhibited subsequent morphological hypertrophy of cardiac myocytes (angiotensin II: 1686±37 to 1167±20 μ m 2 , p<0.01, and endothelin-1: 1759±44 to 1206±21 μ m 2 , p<0.01). Conclusion: Recombinant TFAM increases the mtDNA copy number, and attenuates angiotensin II and endothelin-1-induced hypertrophy via inhibiting NFAT signaling. Recombinant TFAM would be an attractive, novel therapeutic strategy for cardiac hypertrophy and remodeling.

Advanced Glycation End Products (AGEs) Induce Apoptosis via a Novel Pathway: INVOLVEMENT OF CA2+ MEDIATED BY INTERLEUKIN-8 PROTEIN

Advanced glycation end products (AGEs) accumulate in diabetic patients due to high blood glucose levels and cause multiple deleterious effects. In this study, we provide evidence that the AGE increased cell death, one such deleterious effect. Methyl glyoxal-coupled human serum albumin (AGE-HSA) induced transcription factors such as NF-κB, NF-AT, and AP-1. AGE acts through its cell surface receptor, RAGE, and degranulates vesicular contents including interleukin-8 (IL-8). The number of RAGEs, as well as the amount of NF-κB activation, is low, but the cell death is higher in neuronal cells upon AGE treatment. Degranulated IL-8 acts through its receptors, IL-8Rs, and induces sequential events in cells: increase in intracellular Ca(2+), activation of calcineurin, dephosphorylation of cytoplasmic NF-AT, nuclear translocation of NF-AT, and expression of FasL. Expressed FasL increases activity of caspases and induces cell death. Although AGE increases the amount of reactive oxygen intermediate, accompanying cell death is not dependent upon reactive oxygen intermediate. AGE induces autophagy, which partially protects cells from cell death. A novel mechanism of AGE-mediated cell death in different cell types, especially in neuronal cells where it is an early event, is provided here. Thus, this study may be important in several age-related neuronal diseases where AGE-induced apoptosis is observed because of high amounts of AGE.

In This Issue

HomeCirculation ResearchVol. 109, No. 5In This Issue Free AccessIn BriefPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessIn BriefPDF/EPUBIn This Issue Originally published19 Aug 2011https://doi.org/10.1161/RES.0b013e31822e8eaaCirculation Research. 2011;109:475Arginase 1 Is Regulated by LXRα (p 492)Pourcet et al identify a new mechanism that promotes the regression of atherosclerotic plaques.Due to its slow life-long progression, the development of atherosclerotic plaques is difficult to control, even with optimal therapy. Therefore, attempts to promote the regression of preformed plaques are particularly attractive. Even though plaque regression is difficult to study, it has been reported that transplantation of atherosclerotic aorta from atherosclerosis-prone apoE-null mice to wild-type nonatherosclerotic mice results in plaque reduction. Using this ingenious model, Pourcet et al now show that plaque regression is accompanied by an increase in the expression of the anti-inflammatory enzyme arginase1 (Arg1). They report that that this increase is regulated by the liver X receptor alpha (LXRα) and that deletion of LXRα prevents the upregulation of Arg1 in regressive lesions. Nevertheless, LXRα did not directly bind to the Arg-1 gene; instead, it increased the expression of IRF8, which in concert with PU.1, stimulated Arg1 gene expression in macrophages. Although previous studies have shown that LXRs prevent inflammation, the work of Pourcet and colleagues reveals a new molecular pathway linking LXRs to plaque regression, raising the possibility that the pathway could be gainfully targeted to reduce inflammation in atherosclerotic lesions and to promote plaque regression.Orai1 and Neointima Formation (p 534)Zhang et al find a new way to prevent abnormal smooth muscle cell growth.Download figureDownload PowerPointAlthough smooth muscle cells in normal blood vessels remain quiescent and contractile, disease or injury can cause these cells to change their phenotype and proliferate, thereby contributing to the formation of atherosclerotic lesions, hypertension, or restenosis after angioplasty. The processes that regulate smooth muscle cell proliferation are not fully understood; however, calcium signaling has been shown to play an essential role. Zhang et al report that Orail1, a protein involved in the generation of calcium release–activated calcium current, is required for the proliferation of smooth muscle cells in rat carotid arteries after balloon injury. They found that Orail1 levels were increased in proliferating smooth muscle cells of the injured artery and that knockdown of Orail1 decreased neointima formation. Because in cultured smooth muscle cells the knockdown of Orail prevented the nuclear translocation of the transcription factor NFAT, the authors suggest that calcium influx via Orail1 promotes cell growth by stimulating the nuclear translocation of NFAT and that Orail1 may be a new drug target for preventing abnormal smooth muscle cell growth.Cell Senescence in Pulmonary Hypertension (p 543)Noureddine et al say that pulmonary hypertension may be due to accelerated aging of smooth muscle cells.Download figureDownload PowerPointBecause chronic obstructive pulmonary disease (COPD) is usually diagnosed in the middle-aged or elderly, it has been long suspected to be a symptom of accelerated aging. Clarifying this relationship, Noureddine et al report that in pulmonary artery smooth muscle cells of COPD patients, the extent of telomere shortening, which is indicative of replicative senescence, is directly related to the severity of pulmonary hypertension. They found that the senescent cells were located near actively dividing cells, and, when the senescent cells were put in culture, they excreted factors that stimulated the growth of normal smooth muscle cells. Thus, not only do some cells in the lung age quickly, they also coax neighboring cells to grow. This causes thickening of the blood vessels and thereby an abnormal increase in blood pressure (pulmonary hypertension). Further understanding of the causes and consequences of pulmonary smooth muscle cell aging could lead to the development of new treatments for COPD, which, despite its status as the 4th leading cause of death in the United States, has no known cure.Written by Aruni Bhatnagar Previous Back to top Next FiguresReferencesRelatedDetails August 19, 2011Vol 109, Issue 5 Advertisement Article InformationMetrics © 2011 American Heart Association, Inc.https://doi.org/10.1161/RES.0b013e31822e8eaa Originally publishedAugust 19, 2011 PDF download Advertisement

Structural requirements for the inhibition of calcium mobilization and mast cell activation by the pyrazole derivative BTP2 (107.16)

Abstract Cytosolic Ca2+ changes in mast cells are central for driving their activation. Thus, pharmacological tools that inhibit this process represent unique strategies for inhibiting mast cell function and their contribution to disease. We find that the pyrazole compound BTP2 inhibited FcϵRI-triggered sustained Ca2+ influx in RBL-2H3 cells and murine BMMCs with little effect on the tyrosine phosphorylation of cellular proteins. It also did not affect FcϵRI-induced phosphorylation of mitogen-activated protein kinases Erk1/2, JNK, and p38 nor downstream c-fos expression. This suggests that BTP2 inhibits Ca2+ mobilization without affecting the FcϵRI signal transduction cascade. In vitro, BTP2 inhibited FcϵRI-triggered degranulation and cytokine secretion, correlating with inhibition of NFAT nuclear translocation. Consistent with this effect, BTP2 inhibited antigen-induced histamine release in vivo. Analysis of structure-function relationships between the BTP2 parent compound and its derivatives implicate a role for the trifluoromethyl group at both carbon positions 3 and 5 of pyrazole in the inhibition of degranulation of BMMCs, with a more dominant effect evoked by the trifluoromethyl group at C3. In conclusion, our studies indicate that pyrazole compounds can provide the molecular framework for the design of specific inhibitors of Ca2+ entry mechanisms and further BTP2 as a medicinal candidate for the treatment of allergic reactions and other mast cell-mediated diseases.

Apolipoprotein A-II Suppressed Concanavalin A-Induced Hepatitis via the Inhibition of CD4 T Cell Function

Con A-induced hepatitis has been used as a model of human autoimmune or viral hepatitis. During the process of identifying immunologically bioactive proteins in human plasma, we found that apolipoprotein A-II (ApoA-II), the second major apolipoprotein of high-density lipoprotein, inhibited the production of IFN-γ by Con A-stimulated mouse and human CD4 T cells. Con A-induced hepatitis was attenuated by the administration of ApoA-II. The beneficial effect of ApoA-II was associated with reduced leukocyte infiltration and decreased production of T cell-related cytokines and chemokines in the liver. ApoA-II inhibited the Con A-induced activation of ERK-MAPK and nuclear translocation of NFAT in CD4 T cells. Interestingly, exacerbated hepatitis was observed in ApoA-II-deficient mice, indicating that ApoA-II plays a suppressive role in Con A-induced hepatitis under physiological conditions. Moreover, the administration of ApoA-II after the onset of Con A-induced hepatitis was sufficient to suppress disease. Thus, the therapeutic effect of ApoA-II could be useful for patients with CD4 T cell-related autoimmune and viral hepatitis.

Enhanced excitation-coupled Ca2+ entry induces nuclear translocation of NFAT and contributes to IL-6 release from myotubes from patients with central core disease

Enhanced excitation-coupled Ca2+ entry induces nuclear translocation of NFAT and contributes to IL-6 release from myotubes from patients with central core disease

A Caveolin Targeted L-type Calcium Channel Antagonist Inhibits Hypertrophic Signaling without Reducing Contractility of Cardiac Myocytes

The source of Ca2+ to activate pathological hypertrophy is not thought to involve the [Ca2+] that activates contraction. We hypothesize that Ca2+ influx through a subpopulation of L-type Ca2+ channels (CaV1.2; ICa,L) localized in caveolin (Cav) containing membrane signaling microdomains locally activates calcium/calmodulin and calcineurin-mediated NFAT nuclear translocation to induce hypertrophy. Rem-GTPase is known to inhibit CaV1.2 which requires a membrane association c-terminal. We truncated the c-terminal of Rem (Rem1-265) which eliminated CaV1.2 inhibition and fused Rem1-265 to a canonical caveolin binding domain to create Rem1-265-Cav. Results: Adenoviral-mediated expression of normal Rem in adult feline myocytes almost fully eliminated ICa,L while non-membrane targeted Rem1-265 had no significant effect on ICa,L. Rem1-265-Cav exhibited a small inhibition (less than 15%) of the normal ICa,L . Myocytes expressing Rem1-265-Cav responded normally to isoproterenol while those infected with normal Rem failed to respond. Myocytes infected with normal Rem had markedly reduced fractional shortening (2.4+/-0.7% resting cell length) while those infected with the truncated Rem1-265 (7.8+/-1.8) and Rem1-265-Cav (5.9+/-1.4) had contractions not significantly smaller than controls (7.4+/-2.1). Sucrose density gradient experiments revealed that Rem1-265-Cav cosedimented with caveolin-3 enriched low-density fractions. These experiments suggest Rem1-265-Cav inhibits caveolin-associated CaV1.2. To investigate the effects of our Rem constructs on NFAT nuclear translocation (hypertrophic signaling) myocytes were also infected with NFAT-GFP. Bath [Ca2+] was elevated to induce NFAT nuclear translocation measured by nuclear to cytoplasmic NFAT-GFP ratio. Normal Rem fully inhibited [Ca2+]-induced nuclear NFAT translocation and truncated Rem1-265 had no effect. Rem1-265-Cav inhibited more than 80% of [Ca2+]-induced NFAT nuclear translocation. Conclusion: These results suggest that CaV1.2 within Cav3 signaling microdomains is a major source of hypertrophic [Ca2+] signaling and it can be blocked with little or no effect on excitation-contraction coupling.

Engineering of the TRPC3 Selectivity Filter Identifies a Unique, Dual Signaling Function of TRPC3 in the Heart

Cardiac TRPC3 has been suggested as a crucial upstream component of Ca2+/calcineurin/nuclear factor of activated T cells (NFAT) signaling. The linkage between TRPC-activity and NFAT nuclear translocation may involve either Ca2+ entry directly through the TRPC pore or promotion of voltage-dependent Ca2+ entry. Here we show that a point mutation in the TRPC3 selectivity filter (E630Q), which disrupts Ca2+ permeability but preserves monovalent permeation, abrogates agonist-induced NFAT translocation in HEK293 cells as well as in murine HL-1 atrial myocytes. The E630Q mutation fully retains the ability to translate activation of phospholipase C-linked stimuli into L-type (CaV1.2) channel-mediated Ca2+ entry, which is effectively isolated from the NFAT pathway. We demonstrate a dichotomy of TRPC-mediated Ca2+ signaling in the heart involving two distinct pathways that are differentially linked to downstream effectors and gene transcription. Coupling of TRPC3 signaling to NFAT activation is mainly initiated by Ca2+ permeation through the TRPC3 pore and recruitment of calcineurin into a TRPC3 signaling microdomain. supported by FWF, P21925-B19.

Enhanced expression of DYRK1A in cardiomyocytes inhibits acute NFAT activation but does not prevent hypertrophy in vivo

The calcineurin and nuclear factor of activated T cells (NFAT) pathway can mediate pro-hypertrophic signalling in the heart. Recently, it has been shown that dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) phosphorylates NFAT, which limits calcineurin/NFAT signal transduction in T cells and hypertrophy in cultured cardiomyocytes. The hypothesis tested in this study was that DYRK1A prevents calcineurin/NFAT-mediated cardiac hypertrophy in vivo. In cultured rat cardiomyocytes, adenovirus-mediated overexpression of DYRK1A antagonized calcineurin-mediated nuclear NFAT translocation and the phenylephrine-induced hypertrophic growth response. To test the ability of DYRK1A to reduce hypertrophic cardiac growth in vivo, we created tetracycline-repressible Dyrk1a transgenic mice to avoid the cardiac developmental defects associated with embryonic DYRK1A expression. However, in the mouse model, histological determination of myocyte diameter, heart weight/body weight ratio, and echocardiographic measurements revealed that myocardial expression of DYRK1A failed to reduce hypertrophy induced via aortic banding or co-expression of calcineurin. This discrepancy is explained, at least in part, by insufficient long-term inhibition of NFAT and the activation of DYRK1A-resistant maladaptive genes in vivo. Isolated augmentation of DYRK1A can be compensated for in vivo, and this may significantly limit anti-hypertrophic interventions aimed at enhancing DYRK1A activity.