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Sample records for cardiomyocyte nfat activation

  1. Angiotensin II and norepinephrine activate specific calcineurin-dependent NFAT transcription factor isoforms in cardiomyocytes.

    PubMed

    Lunde, Ida G; Kvaløy, Heidi; Austbø, Bjørg; Christensen, Geir; Carlson, Cathrine R

    2011-11-01

    Norepinephrine (NE) and angiotensin II (ANG II) are primary effectors of the sympathetic adrenergic and the renin-angiotensin-aldosterone systems, mediating hypertrophic, apoptotic, and fibrotic events in the myocardium. As NE and ANG II have been shown to affect intracellular calcium in cardiomyocytes, we hypothesized that they activate the calcium-sensitive, prohypertrophic calcineurin-nuclear factor of activated T-cell (NFATc) signaling pathway. More specifically, we have investigated isoform-specific activation of NFAT in NE- and ANG II-stimulated cardiomyocytes, as it is likely that each of the four calcineurin-dependent isoforms, c1-c4, play specific roles. We have stimulated neonatal ventriculocytes from C57/B6 and NFAT-luciferase reporter mice with ANG II or NE and quantified NFAT activity by luciferase activity and phospho-immunoblotting. ANG II and NE increased calcineurin-dependent NFAT activity 2.4- and 1.9-fold, measured as luciferase activity after 24 h of stimulation, and induced protein synthesis, measured by radioactive leucine incorporation after 24 and 72 h. To optimize measurements of NFAT isoforms, we examined the specificity of NFAT antibodies on peptide arrays and by immunoblotting with designed blocking peptides. Western analyses showed that both effectors activate NFATc1 and c4, while NFATc2 activity was regulated by NE only, as measured by phospho-NFAT levels. Neither ANG II nor NE activated NFATc3. As today's main therapies for heart failure aim at antagonizing the adrenergic and renin-angiotensin-aldosterone systems, understanding their intracellular actions is of importance, and our data, through validating a method for measuring myocardial NFATs, indicate that ANG II and NE activate specific NFATc isoforms in cardiomyocytes.

  2. Sumoylation-independent activation of Calcineurin-NFAT-signaling via SUMO2 mediates cardiomyocyte hypertrophy

    PubMed Central

    Bernt, Alexander; Rangrez, Ashraf Y.; Eden, Matthias; Jungmann, Andreas; Katz, Sylvia; Rohr, Claudia; Müller, Oliver J.; Katus, Hugo A.; Sossalla, Samuel T.; Williams, Tatjana; Ritter, Oliver; Frank, Derk; Frey, Norbert

    2016-01-01

    The objective of this study was to identify unknown modulators of Calcineurin (Cn)-NFAT signaling. Measurement of NFAT reporter driven luciferase activity was therefore utilized to screen a human cardiac cDNA-library (~107 primary clones) in C2C12 cells through serial dilutions until single clones could be identified. This extensive screening strategy culminated in the identification of SUMO2 as a most efficient Cn-NFAT activator. SUMO2-mediated activation of Cn-NFAT signaling in cardiomyocytes translated into a hypertrophic phenotype. Prohypertrophic effects were also observed in mice expressing SUMO2 in the heart using AAV9 (Adeno-associated virus), complementing the in vitro findings. In addition, increased SUMO2-mediated sumoylation in human cardiomyopathy patients and in mouse models of cardiomyopathy were observed. To decipher the underlying mechanism, we generated a sumoylation-deficient SUMO2 mutant (ΔGG). Surprisingly, ΔGG replicated Cn-NFAT-activation and the prohypertrophic effects of native SUMO2, both in vitro and in vivo, suggesting a sumoylation-independent mechanism. Finally, we discerned a direct interaction between SUMO2 and CnA, which promotes CnA nuclear localization. In conclusion, we identified SUMO2 as a novel activator of Cn-NFAT signaling in cardiomyocytes. In broader terms, these findings reveal an unexpected role for SUMO2 in cardiac hypertrophy and cardiomyopathy, which may open the possibility for therapeutic manipulation of this pathway. PMID:27767176

  3. Na(+)/H(+) exchanger 1 directly binds to calcineurin A and activates downstream NFAT signaling, leading to cardiomyocyte hypertrophy.

    PubMed

    Hisamitsu, Takashi; Nakamura, Tomoe Y; Wakabayashi, Shigeo

    2012-08-01

    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.

  4. Cyclic GMP/PKG-dependent inhibition of TRPC6 channel activity and expression negatively regulates cardiomyocyte NFAT activation Novel mechanism of cardiac stress modulation by PDE5 inhibition.

    PubMed

    Koitabashi, Norimichi; Aiba, Takeshi; Hesketh, Geoffrey G; Rowell, Janelle; Zhang, Manling; Takimoto, Eiki; Tomaselli, Gordon F; Kass, David A

    2010-04-01

    Increased cyclic GMP from enhanced synthesis or suppressed catabolism (e.g. PDE5 inhibition by sildenafil, SIL) activates protein kinase G (PKG) and blunts cardiac pathological hypertrophy. Suppressed calcineurin (Cn)-NFAT (nuclear factor of activated T-cells) signaling appears to be involved, though it remains unclear how this is achieved. One potential mechanism involves activation of Cn/NFAT by calcium entering via transient receptor potential canonical (TRPC) channels (notably TRPC6). Here, we tested the hypothesis that PKG blocks Cn/NFAT activation by modifying and thus inhibiting TRPC6 current to break the positive feedback loop involving NFAT and NFAT-dependent TRPC6 upregulation. TRPC6 expression rose with pressure-overload in vivo, and angiotensin (ATII) or endothelin (ET1) stimulation in neonatal and adult cardiomyocytes in vitro. 8Br-cGMP and SIL reduced ET1-stimulated TRPC6 expression and NFAT dephosphorylation (activity). TRPC6 upregulation was absent if its promoter was mutated with non-functional NFAT binding sites, whereas constitutively active NFAT triggered TRPC6 expression that was not inhibited by SIL. PKG phosphorylated TRPC6, and both T70 and S322 were targeted. Both sites were functionally relevant, as 8Br-cGMP strongly suppressed current in wild-type TRPC6 channels, but not in those with phospho-silencing mutations (T70A, S322A or S322Q). NFAT activation and increased protein synthesis stimulated by ATII or ET1 was blocked by 8Br-cGMP or SIL. However, transfection with T70A or S322Q TRPC6 mutants blocked this inhibitory effect, whereas phospho-mimetic mutants (T70E, S322E, and both combined) suppressed NFAT activation. Thus PDE5-inhibition blocks TRPC6 channel activation and associated Cn/NFAT activation signaling by PKG-dependent channel phosphorylation.

  5. O-GlcNAc signaling is essential for NFAT-mediated transcriptional reprogramming during cardiomyocyte hypertrophy.

    PubMed

    Facundo, Heberty T; Brainard, Robert E; Watson, Lewis J; Ngoh, Gladys A; Hamid, Tariq; Prabhu, Sumanth D; Jones, Steven P

    2012-05-15

    The regulation of cardiomyocyte hypertrophy is a complex interplay among many known and unknown processes. One specific pathway involves the phosphatase calcineurin, which regulates nuclear translocation of the essential cardiac hypertrophy transcription factor, nuclear factor of activated T-cells (NFAT). Although metabolic dysregulation is frequently described during cardiac hypertrophy, limited insights exist regarding various accessory pathways. One metabolically derived signal, beta-O-linked N-acetylglucosamine (O-GlcNAc), has emerged as a highly dynamic posttranslational modification of serine and threonine residues regulating physiological and stress processes. Given the metabolic dysregulation during hypertrophy, we hypothesized that NFAT activation is dependent on O-GlcNAc signaling. Pressure overload-induced hypertrophy (via transverse aortic constriction) in mice or treatment of neonatal rat cardiac myocytes with phenylephrine significantly enhanced global O-GlcNAc signaling. NFAT-luciferase reporter activity revealed O-GlcNAc-dependent NFAT activation during hypertrophy. Reversal of enhanced O-GlcNAc signaling blunted cardiomyocyte NFAT-induced changes during hypertrophy. Taken together, these results demonstrate a critical role of O-GlcNAc signaling in NFAT activation during hypertrophy and provide evidence that O-GlcNAc signaling is coordinated with the onset and progression of cardiac hypertrophy. This represents a potentially significant and novel mechanism of cardiac hypertrophy, which may be of particular interest in future in vivo studies of hypertrophy.

  6. Dexamethasone promotes hypertrophy of H9C2 cardiomyocytes through calcineurin B pathway, independent of NFAT activation.

    PubMed

    Sangeetha, K N; Lakshmi, B S; Niranjali Devaraj, S

    2016-01-01

    Metabolic syndrome-induced cardiac hypertrophy is a global concern leading to an increase in the morbidity and mortality of patients, with the signalling mechanism associated with them still unclear. The present study attempts to understand the metabolic syndrome-associated cardiac hypertrophy through an in vitro model using external stimuli well known for inducing metabolic disorders, i.e. dexamethasone (DEX), a synthetic glucocorticoid. DEX (0.1 and 1 μM) promoted cardiac hypertrophy in H9C2 cells at 4 days of treatment as evidenced through increased cell size and protein content. A significant induction in foetal gene reprogramming was observed, confirming the establishment of hypertrophy. Moreover, the hypertrophic response at 4 days was perceived to be physiological at 0.1 μM and pathological at 1 μM based on α-MHC and IGF1R expression, but complete inhibition in the PKB/AKT expression confirmed it to be pathological hypertrophy at both the concentrations (0.1 and 1 μM). The present study reports for the first time the mechanistic insights into DEX-mediated hypertrophy. It is hypothesized to be orchestrated through the activation of AT1R that is involved in the alteration of the cardiac isoform of SERCA2 expression perturbing the calcium homeostasis. This leads to the activation of calcineurin B, independent of NFAT involvement, which in coordination with ROS induces the activation of JNK of the MAPK signalling. PMID:26511233

  7. Na(+)/H (+) exchanger isoform 1 induced osteopontin expression in cardiomyocytes involves NFAT3/Gata4.

    PubMed

    Mlih, Mohamed; Abdulrahman, Nabeel; Gadeau, Alain-Pierre; Mohamed, Iman A; Jaballah, Maiy; Mraiche, Fatima

    2015-06-01

    Osteopontin (OPN), a multifunctional glycophosphoprotein, has been reported to contribute to the development and progression of cardiac remodeling and hypertrophy. Cardiac-specific OPN knockout mice were protected against hypertrophy and fibrosis mediated by Ang II. Recently, transgenic mice expressing the active form of the Na(+)/H(+) exchanger isoform 1 (NHE1) developed spontaneous hypertrophy in association with elevated levels of OPN. The mechanism by which active NHE1 induces OPN expression and contributes to the hypertrophic response remains unclear. To validate whether expression of the active form of NHE1 induces OPN, cardiomyocytes were stimulated with Ang II, a known inducer of both OPN and NHE1. Ang II induced hypertrophy and increased OPN protein expression (151.6 ± 28.19 %, P < 0.01) and NHE1 activity in H9c2 cardiomyoblasts. Ang II-induced hypertrophy and OPN protein expression were regressed in the presence of an NHE1 inhibitor, EMD 87580, or a calcineurin inhibitor, FK506. In addition, our results indicated that activation of NHE1-induced NFAT3 translocation into the nucleus and a significant activation of the transcription factor Gata4 (NHE1: 149 ± 28 % of control, P < 0.05). NHE1-induced activation of Gata4 was inhibited by FK506. In summary, our results suggest that activation of NHE1 induces hypertrophy through the activation of NFAT3/Gata4 and OPN expression. PMID:25758355

  8. Activation of NFAT signaling in podocytes causes glomerulosclerosis.

    PubMed

    Wang, Yinqiu; Jarad, George; Tripathi, Piyush; Pan, Minggui; Cunningham, Jeanette; Martin, Daniel R; Liapis, Helen; Miner, Jeffrey H; Chen, Feng

    2010-10-01

    Mutant forms of TRPC6 can activate NFAT-dependent transcription in vitro via calcium influx and activation of calcineurin. The same TRPC6 mutants can cause FSGS, but whether this involves an NFAT-dependent mechanism is unknown. Here, we generated mice that allow conditional induction of NFATc1. Mice with NFAT activation in nascent podocytes in utero developed proteinuria and glomerulosclerosis postnatally, resembling FSGS. NFAT activation in adult mice also caused progressive proteinuria and FSGS. Ultrastructural studies revealed podocyte foot process effacement and deposition of extracellular matrix. NFAT activation did not initially affect expression of podocin, synaptopodin, and nephrin but reduced their expression as glomerular injury progressed. In contrast, we observed upregulation of Wnt6 and Fzd9 in the mutant glomeruli before the onset of significant proteinuria, suggesting a potential role for Wnt signaling in the pathogenesis of NFAT-induced podocyte injury and FSGS. These results provide in vivo evidence for the involvement of NFAT signaling in podocytes, proteinuria, and glomerulosclerosis. Furthermore, this study suggests that NFAT activation may be a key intermediate step in the pathogenesis of mutant TRPC6-mediated FSGS and that suppression of NFAT activity may contribute to the antiproteinuric effects of calcineurin inhibitors.

  9. Leptin-induced cardiomyocyte hypertrophy reveals both calcium-dependent and calcium-independent/RhoA-dependent calcineurin activation and NFAT nuclear translocation.

    PubMed

    Rajapurohitam, Venkatesh; Izaddoustdar, Farzad; Martinez-Abundis, Eduardo; Karmazyn, Morris

    2012-12-01

    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 Ca(2+)-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 50 ng/ml leptin for 24h which resulted in a robust hypertrophic response. Moreover, leptin significantly increased intracellular Ca(2+) 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(+)-1Ca(2+) 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 Ca(2+) concentrations whereas Ca(2+) 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

  10. A fluorescence-based assay to monitor transcriptional activity of NFAT in living cells.

    PubMed

    Rinne, Andreas; Blatter, Lothar A

    2010-09-01

    Ca(2+)-sensitive NFAT (nuclear factor of activated T-cells) transcription factors are implicated in many pathophysiological processes in different cell types. The precise control of activation varies with NFAT isoform and cell type. Here we present feasibility of an in vivo assay (NFAT-RFP) that reports transcriptional activity of NFAT via expression of red fluorescent protein (RFP) in individual cells. This new tool allows continuous monitoring of transcriptional activity of NFAT in a physiological context in living cells. Furthermore, NFAT-RFP can be used simultaneously with NFAT-GFP fusion proteins to monitor transcriptional activity and subcellular localization of NFAT in the same cell.

  11. Regulation of different human NFAT isoforms by neuronal activity.

    PubMed

    Vihma, Hanna; Luhakooder, Mirjam; Pruunsild, Priit; Timmusk, Tõnis

    2016-05-01

    Nuclear factor of activated T-cells (NFAT) is a family of transcription factors comprising four calcium-regulated members: NFATc1, NFATc2, NFATc3, and NFATc4. Upon activation by the calcium-dependent phosphatase calcineurin (CaN), NFATs translocate from cytosol to the nucleus and regulate their target genes, which in the nervous system are involved in axon growth, synaptic plasticity, and neuronal survival. We have shown previously that there are a number of different splice variants of NFAT genes expressed in the brain. Here, we studied the subcellular localizations and transactivation capacities of alternative human NFAT isoforms in rat primary cortical or hippocampal neurons in response to membrane depolarization and compared the induced transactivation levels in neurons to those obtained from HEK293 cells in response to calcium signaling. We confirm that in neurons the translocation to the nucleus of all NFAT isoforms is reliant on the activity of CaN. However, our results suggest that both the regulation of subcellular localization and transcriptional activity of NFAT proteins in neurons is isoform specific. We show that in primary hippocampal neurons NFATc2 isoforms have very fast translocation kinetics, whereas NFATc4 isoforms translocate relatively slowly to the nucleus. Moreover, we demonstrate that the strongest transcriptional activators in HEK293 cells are NFATc1 and NFATc3, but in neurons NFATc3 and NFATc4 lead to the highest induction, and NFATc2 and NFATc1 display isoform-specific transcription activation capacities. Altogether, our results indicate that the effects of calcium signaling on the action of NFAT proteins are isoform-specific and can differ between cell types. We show that the effects of calcium signaling on the action of NFAT proteins are isoform-specific and differ between cell types. Although nuclear localization of all NFAT isoforms in neurons requires calcineurin, the subcellular distributions, neuronal activity-induced nuclear

  12. Annexin A7 deficiency potentiates cardiac NFAT activity promoting hypertrophic signaling

    SciTech Connect

    Voelkl, Jakob; Alesutan, Ioana; Pakladok, Tatsiana; Viereck, Robert; Feger, Martina; Mia, Sobuj; Schönberger, Tanja; Noegel, Angelika A.; Gawaz, Meinrad; Lang, Florian

    2014-02-28

    Highlights: • Cardiac Anxa7 expression was up-regulated following TAC. • The hypertrophic response following TAC was augmented in Anxa7-deficient mice. • Silencing of Anxa7 increased indicators of HL-1 cardiomyocytes hypertrophy. • Silencing of Anxa7 induced Nfatc1 nuclear translocation. • Silencing of Anxa7 enhanced NFAT-dependent transcriptional activity. - Abstract: Annexin A7 (Anxa7) is a cytoskeletal protein interacting with Ca{sup 2+} signaling which in turn is a crucial factor for cardiac remodeling following cardiac injury. The present study explored whether Anxa7 participates in the regulation of cardiac stress signaling. To this end, mice lacking functional Anxa7 (anxa7{sup −/−}) and wild-type mice (anxa7{sup +/+}) were investigated following pressure overload by transverse aortic constriction (TAC). In addition, HL-1 cardiomyocytes were silenced with Anxa7 siRNA and treated with isoproterenol. Transcript levels were determined by quantitative RT-PCR, transcriptional activity by luciferase reporter assay and protein abundance by Western blotting and confocal microscopy. As a result, TAC treatment increased the mRNA and protein levels of Anxa7 in wild-type mice. Moreover, TAC increased heart weight to body weight ratio and the cardiac mRNA levels of αSka, Nppb, Col1a1, Col3a1 and Rcan1, effects more pronounced in anxa7{sup −/−} mice than in anxa7{sup +/+} mice. Silencing of Anxa7 in HL-1 cardiomyocytes significantly increased nuclear localization of Nfatc1. Furthermore, Anxa7 silencing increased NFAT-dependent transcriptional activity as well as αSka, Nppb, and Rcan1 mRNA levels both, under control conditions and following β-adrenergic stimulation by isoproterenol. These observations point to an important role of annexin A7 in the regulation of cardiac NFAT activity and hypertrophic response following cardiac stress conditions.

  13. Oxytocin-stimulated NFAT transcriptional activation in human myometrial cells.

    PubMed

    Pont, Jason N A; McArdle, Craig A; López Bernal, Andrés

    2012-10-01

    Oxytocin (OXT) is a peptide hormone that binds the OXT receptor on myometrial cells, initiating an intracellular signaling cascade, resulting in accumulation of intracellular calcium and smooth muscle contraction. In other systems, an elevation of intracellular Ca(2+) stimulates nuclear translocation of the transcription factor, nuclear factor of activated T cells (NFAT), which is transcriptionally active in arterial and ileal smooth muscle. Here we have investigated the role of NFAT in the mechanism of action of OXT. Human myometrial cells expressed all five NFAT isoforms (NFATC1-C4 and -5). Myometrial cells were transduced with a recombinant adenovirus expressing a NFATC1-EFP reporter, and a semi-automated imaging system was used to monitor effects of OXT on reporter localization in live cells. OXT induced a concentration-dependent nuclear translocation of NFATC1-EFP in a reversible manner, which was inhibited by OXT antagonists and calcineurin inhibitors. Pulsatile stimulation with OXT caused intermittent, pulse-frequency-dependent, nuclear translocation of NFATC1-EFP, which was more efficient than sustained stimulation. OXT induced nuclear translocation of endogenous NFAT that was transcriptionally active, because OXT stimulated activity of a NFAT-response element-luciferase reporter and induced calcineurin-NFAT dependent expression of RGS2, RCAN1, and PTGS2 (COX2) mRNA. Furthermore, OXT-dependent transcription was dependent on protein neosynthesis; cycloheximide abolished RGS2 transcription but augmented RCAN1 and COX2 transcriptional readouts. This study identifies a novel signaling mechanism within the myometrium, whereby calcineurin-NFAT signaling mediates OXT-induced transcriptional activity. Furthermore, we show NFATC1-EFP is responsive to pulses of OXT, a mechanism by which myometrial cells could decode OXT pulse frequency.

  14. Nuclear factor of activated T-cells (NFAT) plays a role in SV40 infection

    SciTech Connect

    Manley, Kate; O'Hara, Bethany A.; Atwood, Walter J.

    2008-03-01

    Recent evidence highlighted a role for the transcription factor, nuclear factor of activated T-cells (NFAT), in the transcription of the human polyomavirus JCV. Here we show that NFAT is also important in the transcriptional control of the related polyomavirus, Simian Virus 40 (SV40). Inhibition of NFAT activity reduced SV40 infection of Vero, 293A, and HeLa cells, and this block occurred at the stage of viral transcription. Both NFAT3 and NFAT4 bound to the SV40 promoter through {kappa}B sites located within the 72 bp repeated enhancer region. In Vero cells, NFAT was involved in late transcription, but in HeLa and 293A cells both early and late viral transcription required NFAT activity. SV40 large T-Ag was found to increase NFAT activity and provided a positive feedback loop to transactivate the SV40 promoter.

  15. Annexin A7 deficiency potentiates cardiac NFAT activity promoting hypertrophic signaling.

    PubMed

    Voelkl, Jakob; Alesutan, Ioana; Pakladok, Tatsiana; Viereck, Robert; Feger, Martina; Mia, Sobuj; Schönberger, Tanja; Noegel, Angelika A; Gawaz, Meinrad; Lang, Florian

    2014-02-28

    Annexin A7 (Anxa7) is a cytoskeletal protein interacting with Ca(2+) signaling which in turn is a crucial factor for cardiac remodeling following cardiac injury. The present study explored whether Anxa7 participates in the regulation of cardiac stress signaling. To this end, mice lacking functional Anxa7 (anxa7(-/-)) and wild-type mice (anxa7(+/+)) were investigated following pressure overload by transverse aortic constriction (TAC). In addition, HL-1 cardiomyocytes were silenced with Anxa7 siRNA and treated with isoproterenol. Transcript levels were determined by quantitative RT-PCR, transcriptional activity by luciferase reporter assay and protein abundance by Western blotting and confocal microscopy. As a result, TAC treatment increased the mRNA and protein levels of Anxa7 in wild-type mice. Moreover, TAC increased heart weight to body weight ratio and the cardiac mRNA levels of αSka, Nppb, Col1a1, Col3a1 and Rcan1, effects more pronounced in anxa7(-/-) mice than in anxa7(+/+) mice. Silencing of Anxa7 in HL-1 cardiomyocytes significantly increased nuclear localization of Nfatc1. Furthermore, Anxa7 silencing increased NFAT-dependent transcriptional activity as well as αSka, Nppb, and Rcan1 mRNA levels both, under control conditions and following β-adrenergic stimulation by isoproterenol. These observations point to an important role of annexin A7 in the regulation of cardiac NFAT activity and hypertrophic response following cardiac stress conditions. PMID:24508799

  16. Syndecan-4 is essential for development of concentric myocardial hypertrophy via stretch-induced activation of the calcineurin-NFAT pathway.

    PubMed

    Finsen, Alexandra V; Lunde, Ida G; Sjaastad, Ivar; Østli, Even K; Lyngra, Marianne; Jarstadmarken, Hilde O; Hasic, Almira; Nygård, Ståle; Wilcox-Adelman, Sarah A; Goetinck, Paul F; Lyberg, Torstein; Skrbic, Biljana; Florholmen, Geir; Tønnessen, Theis; Louch, William E; Djurovic, Srdjan; Carlson, Cathrine R; Christensen, Geir

    2011-01-01

    Sustained pressure overload leads to compensatory myocardial hypertrophy and subsequent heart failure, a leading cause of morbidity and mortality. Further unraveling of the cellular processes involved is essential for development of new treatment strategies. We have investigated the hypothesis that the transmembrane Z-disc proteoglycan syndecan-4, a co-receptor for integrins, connecting extracellular matrix proteins to the cytoskeleton, is an important signal transducer in cardiomyocytes during development of concentric myocardial hypertrophy following pressure overload. Echocardiographic, histochemical and cardiomyocyte size measurements showed that syndecan-4(-/-) mice did not develop concentric myocardial hypertrophy as found in wild-type mice, but rather left ventricular dilatation and dysfunction following pressure overload. Protein and gene expression analyses revealed diminished activation of the central, pro-hypertrophic calcineurin-nuclear factor of activated T-cell (NFAT) signaling pathway. Cardiomyocytes from syndecan-4(-/-)-NFAT-luciferase reporter mice subjected to cyclic mechanical stretch, a hypertrophic stimulus, showed minimal activation of NFAT (1.6-fold) compared to 5.8-fold increase in NFAT-luciferase control cardiomyocytes. Accordingly, overexpression of syndecan-4 or introducing a cell-permeable membrane-targeted syndecan-4 polypeptide (gain of function) activated NFATc4 in vitro. Pull-down experiments demonstrated a direct intracellular syndecan-4-calcineurin interaction. This interaction and activation of NFAT were increased by dephosphorylation of serine 179 (pS179) in syndecan-4. During pressure overload, phosphorylation of syndecan-4 was decreased, and association between syndecan-4, calcineurin and its co-activator calmodulin increased. Moreover, calcineurin dephosphorylated pS179, indicating that calcineurin regulates its own binding and activation. Finally, patients with hypertrophic myocardium due to aortic stenosis had increased

  17. NFAT5 Is Activated by Hypoxia: Role in Ischemia and Reperfusion in the Rat Kidney

    PubMed Central

    Villanueva, Sandra; Suazo, Cristian; Santapau, Daniela; Pérez, Francisco; Quiroz, Mariana; Carreño, Juan E.; Illanes, Sebastián; Lavandero, Sergio; Michea, Luis; Irarrazabal, Carlos E.

    2012-01-01

    The current hypothesis postulates that NFAT5 activation in the kidney's inner medulla is due to hypertonicity, resulting in cell protection. Additionally, the renal medulla is hypoxic (10–18 mmHg); however there is no information about the effect of hypoxia on NFAT5. Using in vivo and in vitro models, we evaluated the effect of reducing the partial pressure of oxygen (PO2) on NFAT5 activity. We found that 1) Anoxia increased NFAT5 expression and nuclear translocation in primary cultures of IMCD cells from rat kidney. 2) Anoxia increased transcriptional activity and nuclear translocation of NFAT5 in HEK293 cells. 3) The dose-response curve demonstrated that HIF-1α peaked at 2.5% and NFAT5 at 1% of O2. 4) At 2.5% of O2, the time-course curve of hypoxia demonstrated earlier induction of HIF-1α gene expression than NFAT5. 5) siRNA knockdown of NFAT5 increased the hypoxia-induced cell death. 6) siRNA knockdown of HIF-1α did not affect the NFAT5 induction by hypoxia. Additionally, HIF-1α was still induced by hypoxia even when NFAT5 was knocked down. 7) NFAT5 and HIF-1α expression were increased in kidney (cortex and medulla) from rats subjected to an experimental model of ischemia and reperfusion (I/R). 7) Experimental I/R increased the NFAT5-target gene aldose reductase (AR). 8) NFAT5 activators (ATM and PI3K) were induced in vitro (HEK293 cells) and in vivo (I/R kidneys) with the same timing of NFAT5. 8) Wortmannin, which inhibits ATM and PI3K, reduces hypoxia-induced NFAT5 transcriptional activation in HEK293 cells. These results demonstrate for the first time that NFAT5 is induced by hypoxia and could be a protective factor against ischemic damage. PMID:22768306

  18. Calcineurin-NFAT activation and DSCR-1 auto-inhibitory loop: how is homoeostasis regulated?

    PubMed

    Minami, Takashi

    2014-04-01

    Calcineurin-nuclear factor of activated T cells (NFAT) signalling plays a critical role not only in the immune and nervous systems, but also in cardiovascular development and pathological endothelial cell activation during angiogenesis or inflammation. Studies in NFAT-null mice demonstrated that there is high redundancy between functions of the different NFAT family members. Deletion of only one NFAT causes mild phenotypes, but compound deletions of multiple NFAT family members leads to severe abnormalities in multiple organ systems. Genome-wide transcription analysis revealed that many NFAT target genes are related to cell growth and inflammation, whereas the gene most strongly induced by NFAT in endothelial cells is an auto-inhibitory molecule, Down syndrome critical region (DSCR)-1. The NFAT-DSCR-1 signalling axis may vary depending on the cell-type or signal dosage level under the microenvironment. In the endothelium, stable expression of the DSCR-1 short isoform attenuates septic inflammatory shock, tumour growth and tumour metastasis to lung. Moreover, dysfunction of DSCR-1 and the NFAT priming kinase, DYRK1A, prevents NFAT nuclear occupancy. This change in NFAT nuclear localization is responsible for many of the features of Down syndrome. Thus, fine-tuning of the NFAT-DSCR-1 negative feedback loop may enable therapeutic manipulation in vasculopathic diseases.

  19. Calcineurin-NFAT activation and DSCR-1 auto-inhibitory loop: how is homoeostasis regulated?

    PubMed

    Minami, Takashi

    2014-04-01

    Calcineurin-nuclear factor of activated T cells (NFAT) signalling plays a critical role not only in the immune and nervous systems, but also in cardiovascular development and pathological endothelial cell activation during angiogenesis or inflammation. Studies in NFAT-null mice demonstrated that there is high redundancy between functions of the different NFAT family members. Deletion of only one NFAT causes mild phenotypes, but compound deletions of multiple NFAT family members leads to severe abnormalities in multiple organ systems. Genome-wide transcription analysis revealed that many NFAT target genes are related to cell growth and inflammation, whereas the gene most strongly induced by NFAT in endothelial cells is an auto-inhibitory molecule, Down syndrome critical region (DSCR)-1. The NFAT-DSCR-1 signalling axis may vary depending on the cell-type or signal dosage level under the microenvironment. In the endothelium, stable expression of the DSCR-1 short isoform attenuates septic inflammatory shock, tumour growth and tumour metastasis to lung. Moreover, dysfunction of DSCR-1 and the NFAT priming kinase, DYRK1A, prevents NFAT nuclear occupancy. This change in NFAT nuclear localization is responsible for many of the features of Down syndrome. Thus, fine-tuning of the NFAT-DSCR-1 negative feedback loop may enable therapeutic manipulation in vasculopathic diseases. PMID:24505143

  20. Transcription factor NFAT1 controls allergic contact hypersensitivity through regulation of activation induced cell death program

    PubMed Central

    Kwon, Ho-Keun; Kim, Gi-Cheon; Hwang, Ji Sun; Kim, Young; Chae, Chang-Suk; Nam, Jong Hee; Jun, Chang-Duk; Rudra, Dipayan; Surh, Charles D.; Im, Sin-Hyeog

    2016-01-01

    Allergic contact hypersensitivity (CHS) is an inflammatory skin disease mediated by allergen specific T cells. In this study, we investigated the role of transcription factor NFAT1 in the pathogenesis of contact hypersensitivity. NFAT1 knock out (KO) mice spontaneously developed CHS-like skin inflammation in old age. Healthy young NFAT1 KO mice displayed enhanced susceptibility to hapten-induced CHS. Both CD4+ and CD8+ T cells from NFAT1 KO mice displayed hyper-activated properties and produced significantly enhanced levels of inflammatory T helper 1(Th1)/Th17 type cytokines. NFAT1 KO T cells were more resistant to activation induced cell death (AICD), and regulatory T cells derived from these mice showed a partial defect in their suppressor activity. NFAT1 KO T cells displayed a reduced expression of apoptosis associated BCL-2/BH3 family members. Ectopic expression of NFAT1 restored the AICD defect in NFAT1 KO T cells and increased AICD in normal T cells. Recipient Rag2−/− mice transferred with NFAT1 KO T cells showed more severe CHS sensitivity due to a defect in activation induced hapten-reactive T cell apoptosis. Collectively, our results suggest the NFAT1 plays a pivotal role as a genetic switch in CD4+/CD8+ T cell tolerance by regulating AICD process in the T cell mediated skin inflammation. PMID:26777750

  1. Expression, localisation and functional activation of NFAT-2 in normal human skin, psoriasis, and cultured keratocytes.

    PubMed

    Al-Daraji, Wael I; Malak, Tamer T; Prescott, Richard J; Abdellaoui, Adel; Ali, Mahmud M; Dabash, Tarek; Zelger, Bettina G; Zelger, Bernhard

    2009-06-18

    Ciclosporin A (CsA) is widely utilized for the treatment of inflammatory skin diseases such as psoriasis. The therapeutic effects of CsA are thought to be mediated via its immunosuppressive action on infiltrating lymphocytes in skin lesions. CsA and tacrolimus block T cell activation by inhibiting the phosphatase calcineurin and preventing translocation from the cytoplasm to the nucleus of the transcription factor Nuclear Factor of Activated T cells (NFAT). As calcineurin and NFAT 1 have been shown to be functionally active in cultured human keratocytes, expression of other NFAT family members such as NFAT-2 and possible functional activation was investigated in human keratocytes. RT-PCR and Western Analysis were used to investigate the presence of NFAT-2 mRNA and protein in human keratocytes. Tissue culture of human keratocytes and immunostaining of cells on coverslips and confocal microscopy were used to assess the degree of nuclear localisation of NFAT-2 in cultured cells. Keratome biopsies were taken from patients with psoriasis (lesional and non-lesional skin) and normal skin and immunohistochemistry was used to assess the NFAT-2 localisation in these biopsies using a well characterized anti-NFAT-2 antibody. The NFAT-2 mRNA and protein expression was demonstrated using RT-PCR and Western blotting. Moreover, the expression of NFAT-2 in normal skin, non-lesional and lesional psoriasis showed a striking basal staining suggesting a role for NFAT-2 in keratocytes proliferation. A range of cell types in the skin express NFAT-2. The expression of NFAT-2 in human keratocytes and response to different agonists provides perhaps a unique opportunity to examine the regulation, subcellular localization and kinetics of translocation of different NFATs in primary cultured human cells. In these experiments the author assessed the expression, localization of NFAT-2 in cultured human keratocytes and measured the degree of nuclear localisaion of NFAT-2 using immunofluorescence

  2. NFAT-133 increases glucose uptake in L6 myotubes by activating AMPK pathway.

    PubMed

    Thakkar, Chandni S; Kate, Abhijeet S; Desai, Dattatraya C; Ghosh, Asit Ranjan; Kulkarni-Almeida, Asha A

    2015-12-15

    NFAT-133 is an aromatic compound with cinammyl alcohol moiety, isolated from streptomycetes strain PM0324667. We have earlier reported that NFAT-133 increases insulin stimulated glucose uptake in L6 myotubes using a PPARγ independent mechanism and reduces plasma or blood glucose levels in diabetic mice. Here we investigated the effects of NFAT-133 on cellular signaling pathways leading to glucose uptake in L6 myotubes. Our studies demonstrate that NFAT-133 increases glucose uptake in a dose- and time-dependent manner independent of the effects of insulin. Treatment with Akti-1/2, wortmannin and increasing concentrations of insulin had no effect on NFAT-133 mediated glucose uptake. NFAT-133 induced glucose uptake is completely mitigated by Compound C, an AMPK inhibitor. Further, the kinases upstream of AMPK activation namely; LKB-1 and CAMKKβ are not involved in NFAT-133 mediated AMPK activation nor does the compound NFAT-133 have any effect on AMPK enzyme activity. Further analysis confirmed that NFAT-133 indirectly activates AMPK by reducing the mitochondrial membrane potential and increasing the ratio of AMP:ATP.

  3. NFAT-133 increases glucose uptake in L6 myotubes by activating AMPK pathway.

    PubMed

    Thakkar, Chandni S; Kate, Abhijeet S; Desai, Dattatraya C; Ghosh, Asit Ranjan; Kulkarni-Almeida, Asha A

    2015-12-15

    NFAT-133 is an aromatic compound with cinammyl alcohol moiety, isolated from streptomycetes strain PM0324667. We have earlier reported that NFAT-133 increases insulin stimulated glucose uptake in L6 myotubes using a PPARγ independent mechanism and reduces plasma or blood glucose levels in diabetic mice. Here we investigated the effects of NFAT-133 on cellular signaling pathways leading to glucose uptake in L6 myotubes. Our studies demonstrate that NFAT-133 increases glucose uptake in a dose- and time-dependent manner independent of the effects of insulin. Treatment with Akti-1/2, wortmannin and increasing concentrations of insulin had no effect on NFAT-133 mediated glucose uptake. NFAT-133 induced glucose uptake is completely mitigated by Compound C, an AMPK inhibitor. Further, the kinases upstream of AMPK activation namely; LKB-1 and CAMKKβ are not involved in NFAT-133 mediated AMPK activation nor does the compound NFAT-133 have any effect on AMPK enzyme activity. Further analysis confirmed that NFAT-133 indirectly activates AMPK by reducing the mitochondrial membrane potential and increasing the ratio of AMP:ATP. PMID:26546724

  4. Disparate effects of serum on basal and evoked NFAT activity in primary astrocyte cultures.

    PubMed

    Furman, Jennifer L; Artiushin, Irina A; Norris, Christopher M

    2010-01-29

    In astrocytes, the Ca(2+)-dependent protein phosphatase calcineurin (CN) strongly regulates neuro-immune/inflammatory cascades through activation of the transcription factor, nuclear factor of activated T cells (NFAT). While primary cell cultures provide a useful model system for investigating astrocytic CN/NFAT signaling, variable results may arise both within and across labs because of differences in culture conditions. Here, we determined the extent to which serum and cell confluency affect basal and evoked astrocytic NFAT activity in primary cortical astrocyte cultures. Cells were grown to either approximately 50% or >90% confluency, pre-loaded with an NFAT-luciferase reporter construct, and maintained for 16 h in medium with or without 10% fetal bovine serum (FBS). NFAT-dependent luciferase expression was then measured 5h after treatment with vehicle alone to assess basal NFAT activity, or with Ca(2+) mobilizers and IL-1 beta to assess evoked activity. The results revealed significantly higher levels of basal NFAT activity in FBS-containing medium, regardless of cell confluency. Conversely, evoked NFAT activation was significantly lower in serum-containing medium, with an even greater inhibition observed in confluent cultures. Application of 10% FBS to serum-free astrocyte cultures quickly evoked a roughly seven-fold increase in NFAT activity that was significantly reduced by co-delivery of neutralizing agents for IL-1 beta, TNFalpha, and/or IFN gamma, suggesting that serum occludes evoked NFAT activation through a cytokine-based mechanism. Together, the results demonstrate that the presence of serum and cell confluency have a major impact on CN/NFAT signaling in primary astrocyte cultures and therefore must be taken into consideration when using this model system.

  5. Band-pass processing in a GPCR signaling pathway selects for NFAT transcription factor activation.

    PubMed

    Sumit, M; Neubig, R R; Takayama, S; Linderman, J J

    2015-11-01

    Many biological processes are rhythmic and proper timing is increasingly appreciated as being critical for development and maintenance of physiological functions. To understand how temporal modulation of an input signal influences downstream responses, we employ microfluidic pulsatile stimulation of a G-protein coupled receptor, the muscarinic M3 receptor, in single cells with simultaneous real-time imaging of both intracellular calcium and NFAT nuclear localization. Interestingly, we find that reduced stimulation with pulses of ligand can give more efficient transcription factor activation, if stimuli are timed appropriately. Our experiments and computational analyses show that M3 receptor-induced calcium oscillations form a low pass filter while calcium-induced NFAT translocation forms a high pass filter. The combination acts as a band-pass filter optimized for intermediate frequencies of stimulation. We demonstrate that receptor desensitization and NFAT translocation rates determine critical features of the band-pass filter and that the band-pass may be shifted for different receptors or NFAT dynamics. As an example, we show that the two NFAT isoforms (NFAT4 and NFAT1) have shifted band-pass windows for the same receptor. While we focus specifically on the M3 muscarinic receptor and NFAT translocation, band-pass processing is expected to be a general theme that applies to multiple signaling pathways.

  6. Dynamic assembly of a membrane signaling complex enables selective activation of NFAT by Orai1.

    PubMed

    Kar, Pulak; Samanta, Krishna; Kramer, Holger; Morris, Otto; Bakowski, Daniel; Parekh, Anant B

    2014-06-16

    NFAT-dependent gene expression is essential for the development and function of the nervous, immune, and cardiovascular systems and kidney, bone, and skeletal muscle. Most NFAT protein resides in the cytoplasm because of extensive phosphorylation, which masks a nuclear localization sequence. Dephosphorylation by the Ca(2+)-calmodulin-activated protein phosphatase calcineurin triggers NFAT migration into the nucleus. In some cell types, NFAT can be activated by Ca(2+) nanodomains near open store-operated Orai1 and voltage-gated Ca(2+) channels in the plasma membrane. How local Ca(2+) near Orai1 is detected and whether other Orai channels utilize a similar mechanism remain unclear. Here, we report that the paralog Orai3 fails to activate NFAT. Orai1 is effective in activating gene expression via Ca(2+) nanodomains because it participates in a membrane-delimited signaling complex that forms after store depletion and brings calcineurin, via the scaffolding protein AKAP79, to calmodulin tethered to Orai1. By contrast, Orai3 interacts less well with AKAP79 after store depletion, rendering it ineffective in activating NFAT. A channel chimera of Orai3 with the N terminus of Orai1 was able to couple local Ca(2+) entry to NFAT activation, identifying the N-terminal domain of Orai1 as central to Ca(2+) nanodomain-transcription coupling. The formation of a store-dependent signaling complex at the plasma membrane provides for selective activation of a fundamental downstream response by Orai1.

  7. Dual effect of lithium on NFAT5 activity in kidney cells

    PubMed Central

    Küper, Christoph; Beck, Franz-Xaver; Neuhofer, Wolfgang

    2015-01-01

    Lithium salts are used widely for treatment of bipolar and other mental disorders. Lithium therapy is accompanied frequently by renal side effects, such as nephrogenic diabetes insipidus or chronic kidney disease (CKD), but the molecular mechanisms underlying these effects are still poorly understood. In the present study we examined the effect of lithium on the activity of the osmosensitive transcriptional activator nuclear factor of activated T cells 5 (NFAT5, also known as TonEBP), which plays a key role in renal cellular osmoprotection and urinary concentrating ability. Interestingly, we found different effects of lithium on NFAT5 activity, depending on medium osmolality and incubation time. When cells were exposed to lithium for a relative short period (24 h), NFAT5 activity was significantly increased, especially under isosmotic conditions, resulting in an enhanced expression of the NFAT5 target gene heat shock protein 70 (HSP70). Further analysis revealed that the increase of NFAT5 activity depended primarily on an enhanced activity of the c-terminal transactivation domain (TAD), while NFAT5 protein abundance was largely unaffected. Enhanced activity of the TAD is probably mediated by lithium-induced inhibitory phosphorylation of glycogen synthase kinase 3β (GSK-3β), which is in accordance with previous studies. When cells were exposed to lithium for a longer period (96 h), cellular NFAT5 activity and subsequently expression of HSP70 significantly decreased under hyperosmotic conditions, due to diminished NFAT5 protein abundance, also resulting from GSK-3β inhibition. Taken together, our results provide evidence that lithium has opposing effects on NFAT5 activity, depending on environmental osmolality and exposure duration. The potential impacts of these observations on the diverse effects of lithium on kidney function are discussed. PMID:26441681

  8. Dual effect of lithium on NFAT5 activity in kidney cells.

    PubMed

    Küper, Christoph; Beck, Franz-Xaver; Neuhofer, Wolfgang

    2015-01-01

    Lithium salts are used widely for treatment of bipolar and other mental disorders. Lithium therapy is accompanied frequently by renal side effects, such as nephrogenic diabetes insipidus or chronic kidney disease (CKD), but the molecular mechanisms underlying these effects are still poorly understood. In the present study we examined the effect of lithium on the activity of the osmosensitive transcriptional activator nuclear factor of activated T cells 5 (NFAT5, also known as TonEBP), which plays a key role in renal cellular osmoprotection and urinary concentrating ability. Interestingly, we found different effects of lithium on NFAT5 activity, depending on medium osmolality and incubation time. When cells were exposed to lithium for a relative short period (24 h), NFAT5 activity was significantly increased, especially under isosmotic conditions, resulting in an enhanced expression of the NFAT5 target gene heat shock protein 70 (HSP70). Further analysis revealed that the increase of NFAT5 activity depended primarily on an enhanced activity of the c-terminal transactivation domain (TAD), while NFAT5 protein abundance was largely unaffected. Enhanced activity of the TAD is probably mediated by lithium-induced inhibitory phosphorylation of glycogen synthase kinase 3β (GSK-3β), which is in accordance with previous studies. When cells were exposed to lithium for a longer period (96 h), cellular NFAT5 activity and subsequently expression of HSP70 significantly decreased under hyperosmotic conditions, due to diminished NFAT5 protein abundance, also resulting from GSK-3β inhibition. Taken together, our results provide evidence that lithium has opposing effects on NFAT5 activity, depending on environmental osmolality and exposure duration. The potential impacts of these observations on the diverse effects of lithium on kidney function are discussed. PMID:26441681

  9. Selective NFAT targeting in T cells ameliorates GvHD while maintaining antitumor activity.

    PubMed

    Vaeth, Martin; Bäuerlein, Carina A; Pusch, Tobias; Findeis, Janina; Chopra, Martin; Mottok, Anja; Rosenwald, Andreas; Beilhack, Andreas; Berberich-Siebelt, Friederike

    2015-01-27

    Graft-versus-host disease (GvHD) is a life-threatening immunological complication after allogenic hematopoietic stem cell transplantation (allo-HCT). The intrinsic graft-versus-leukemia (GvL) effect, however, is the desirable curative benefit. Patients with acute GvHD are treated with cyclosporine A (CsA) or tacrolimus (FK506), which not only often causes severe adverse effects, but also interferes with the anticipated GvL. Both drugs inhibit calcineurin, thus at first suppressing activation of the nuclear factor of activated T cells (NFAT). Therefore, we explored the specific contribution of individual NFAT factors in donor T cells in animal models of GvHD and GvL. Ablation of NFAT1, NFAT2, or a combination of both resulted in ameliorated GvHD, due to reduced proliferation, target tissue homing, and impaired effector function of allogenic donor T cells. In contrast, the frequency of Foxp3(+) regulatory T (Treg) cells was increased and NFAT-deficient Tregs were fully protective in GvHD. CD8(+) T-cell recall response and, importantly, the beneficial antitumor activity were largely preserved in NFAT-deficient effector T cells. Thus, specific inhibition of NFAT opens an avenue for an advanced therapy of GvHD maintaining protective GvL.

  10. Nuclear factor of activated T cells (NFAT) in pearl oyster Pinctada fucata: molecular cloning and functional characterization.

    PubMed

    Huang, Xian-De; Wei, Guo-jian; Zhang, Hua; He, Mao-Xian

    2015-01-01

    Nuclear factor of activated T cells (NFAT) plays an important role in nonimmune cells and also in T cells and many other cells of the immune system, by regulating the expression of a variety of genes involved in the immune response, organ development, developmental apoptosis and angiogenesis. In the present study, the NFAT homology gene, PfNFAT, from the pearl oyster Pinctada fucata was cloned and its genomic structure and promoter were analyzed. PfNFAT encodes a putative protein of 1226 amino acids, and contains a highly conserved Rel homology region (RHR) with DNA-binding specificity, and a regulatory domain (NFAT homology region, NHR) containing a potent transactivation domain (TAD). The PfNFAT gene consists of 12 exons and 11 introns, and its promoter contains potential binding sites for transcription factors such as NF-κB (Nuclear factor κB), STATx (signal transducer and activator of transcription), AP-1 (activator protein-1) and Sox-5/9 (SRY type HMG box-5/9), MyoD (Myogenic Differentiation Antigen) and IRF (Interferon regulatory factor). Comparison and phylogenetic analysis revealed that PfNFAT shows high identity with other invertebrate NFAT, and clusters with the NFAT5 subgroup. Furthermore, gene expression analysis revealed that PfNFAT is involved in the immune response to lipopolysaccharide (LPS) and Polyinosinic-polycytidylic acid (poly I:C) stimulation and in the nucleus inserting operation. The study of PfNFAT may increase understanding of molluscan innate immunity.

  11. Activation of HuR downstream of p38 MAPK promotes cardiomyocyte hypertrophy.

    PubMed

    Slone, Samuel; Anthony, Sarah R; Wu, Xiaoqing; Benoit, Joshua B; Aube, Jeffrey; Xu, Liang; Tranter, Michael

    2016-11-01

    The RNA binding protein Human antigen R (HuR) interacts with specific AU-rich domains in target mRNAs and is highly expressed in many cell types, including cardiomyocytes. However, the role of HuR in cardiac physiology is largely unknown. Our results show that HuR undergoes cytoplasmic translocation, indicative of its activation, in hypertrophic cardiac myocytes. Specifically, HuR cytoplasmic translocation is significantly increased in NRVMs (neonatal rat ventricular myocytes) following treatment with phenylephrine or angiotensin II, agonists of two independent Gαq-coupled GPCRs known to induce hypertrophy. This Gq-mediated HuR activation is dependent on p38 MAP kinase, but not canonical Gq-PKC signaling. Furthermore, we show that HuR activation is necessary for Gq-mediated hypertrophic growth of NRVMs as siRNA-mediated knockdown of HuR inhibits hypertrophy as measured by cell size and expression of ANF (atrial natriuretic factor). Additionally, HuR overexpression is sufficient to induce hypertrophic cell growth. To decipher the downstream mechanisms by which HuR translocation promotes cardiomyocyte hypertrophy, we assessed the role of HuR in the transcriptional activity of NFAT (nuclear factor of activated T cells), the activation of which is a hallmark of cardiac hypertrophy. Using an NFAT-luciferase reporter assay, we show an acute inhibition of NFAT transcriptional activity following pharmacological inhibition of HuR. In conclusion, our results identify HuR as a novel mediator of cardiac hypertrophy downstream of the Gq-p38 MAPK pathway, and suggest modulation of NFAT activity as a potential mechanism.

  12. Combination therapy for KIT-mutant mast cells: targeting constitutive NFAT and KIT activity.

    PubMed

    Macleod, Alison C; Klug, Lillian R; Patterson, Janice; Griffith, Diana J; Beadling, Carol; Town, Ajia; Heinrich, Michael C

    2014-12-01

    Resistant KIT mutations have hindered the development of KIT kinase inhibitors for treatment of patients with systemic mastocytosis. The goal of this research was to characterize the synergistic effects of a novel combination therapy involving inhibition of KIT and calcineurin phosphatase, a nuclear factor of activated T cells (NFAT) regulator, using a panel of KIT-mutant mast cell lines. The effects of monotherapy or combination therapy on the cellular viability/survival of KIT-mutant mast cells were evaluated. In addition, NFAT-dependent transcriptional activity was monitored in a representative cell line to evaluate the mechanisms responsible for the efficacy of combination therapy. Finally, shRNA was used to stably knockdown calcineurin expression to confirm the role of calcineurin in the observed synergy. The combination of a KIT inhibitor and a calcineurin phosphatase inhibitor (CNPI) synergized to reduce cell viability and induce apoptosis in six distinct KIT-mutant mast cell lines. Both KIT inhibitors and CNPIs were found to decrease NFAT-dependent transcriptional activity. NFAT-specific inhibitors induced similar synergistic apoptosis induction as CNPIs when combined with a KIT inhibitor. Notably, NFAT was constitutively active in each KIT-mutant cell line tested. Knockdown of calcineurin subunit PPP3R1 sensitized cells to KIT inhibition and increased NFAT phosphorylation and cytoplasmic localization. Constitutive activation of NFAT appears to represent a novel and targetable characteristic of KIT-mutant mast cell disease. Our studies suggest that combining KIT inhibition with NFAT inhibition might represent a new treatment strategy for mast cell disease.

  13. Constitutive nuclear localization of NFAT in Foxp3+ regulatory T cells independent of calcineurin activity.

    PubMed

    Li, Qiuxia; Shakya, Arvind; Guo, Xiaohua; Zhang, Hongbo; Tantin, Dean; Jensen, Peter E; Chen, Xinjian

    2012-05-01

    Foxp3 plays an essential role in conferring suppressive functionality to CD4(+)/Foxp3(+) regulatory T cells (Tregs). Although studies showed that Foxp3 has to form cooperative complexes with NFAT to bind to target genes, it remains unclear whether NFAT is available in the nucleus of primary Tregs for Foxp3 access. It is generally believed that NFAT in resting cells resides in the cytoplasm, and its nuclear translocation depends on calcineurin (CN) activation. We report that a fraction of NFAT protein constitutively localizes in the nucleus of primary Tregs, where it selectively binds to Foxp3 target genes. Treating Tregs with CN inhibitor does not induce export of NFAT from the nucleus, indicating that its nuclear translocation is independent of CN activity. Consistently, Tregs are resistant to CN inhibitors in the presence of IL-2 and continue to proliferate in response to anti-CD3 stimulation, whereas proliferation of non-Tregs is abrogated by CN inhibitors. In addition, PMA, which activates other transcription factors required for T cell activation but not NFAT, selectively induces Treg proliferation in the absence of ionomycin. TCR interaction with self-MHC class II is not required for PMA-induced Treg proliferation. Tregs expanded by PMA or in the presence of CN inhibitors maintain Treg phenotype and functionality. These findings shed light on Treg biology, paving the way for strategies to selectively activate Tregs.

  14. Dependence on nuclear factor of activated T-cells (NFAT) levels discriminates conventional T cells from Foxp3+ regulatory T cells.

    PubMed

    Vaeth, Martin; Schliesser, Ulrike; Müller, Gerd; Reissig, Sonja; Satoh, Kazuki; Tuettenberg, Andrea; Jonuleit, Helmut; Waisman, Ari; Müller, Martin R; Serfling, Edgar; Sawitzki, Birgit S; Berberich-Siebelt, Friederike

    2012-10-01

    Several lines of evidence suggest nuclear factor of activated T-cells (NFAT) to control regulatory T cells: thymus-derived naturally occurring regulatory T cells (nTreg) depend on calcium signals, the Foxp3 gene harbors several NFAT binding sites, and the Foxp3 (Fork head box P3) protein interacts with NFAT. Therefore, we investigated the impact of NFAT on Foxp3 expression. Indeed, the generation of peripherally induced Treg (iTreg) by TGF-β was highly dependent on NFAT expression because the ability of CD4(+) T cells to differentiate into iTreg diminished markedly with the number of NFAT family members missing. It can be concluded that the expression of Foxp3 in TGF-β-induced iTreg depends on the threshold value of NFAT rather than on an individual member present. This is specific for iTreg development, because frequency of nTreg remained unaltered in mice lacking NFAT1, NFAT2, or NFAT4 alone or in combination. Different from expectation, however, the function of both nTreg and iTreg was independent on robust NFAT levels, reflected by less nuclear NFAT in nTreg and iTreg. Accordingly, absence of one or two NFAT members did not alter suppressor activity in vitro or during colitis and transplantation in vivo. This scenario emphasizes an inhibition of high NFAT activity as treatment for autoimmune diseases and in transplantation, selectively targeting the proinflammatory conventional T cells, while keeping Treg functional.

  15. Nuclear translocation of nuclear factor of activated T cells (NFAT) as a quantitative pharmacodynamic parameter for tacrolimus.

    PubMed

    Maguire, Orla; Tornatore, Kathleen M; O'Loughlin, Kieran L; Venuto, Rocco C; Minderman, Hans

    2013-12-01

    Nuclear factor of activated T cells (NFAT) is a family of transcription factors involved in regulating the immune response. The canonical NFAT pathway is calcium-dependent and upon activation, NFAT is dephosphorylated by the phosphatase, calcineurin. This results in its translocation from the cytoplasm to the nucleus and transcription of downstream target genes that include the cytokines IL-2, IL-10, and IFNγ. Calcineurin inhibitors including tacrolimus inhibit the NFAT pathway and are used as immunosuppressants in transplant settings to prevent graft rejection. There is, as yet, no direct means to monitor tacrolimus pharmacodynamics. In this study, a rapid, quantitative, image cytometry-based measurement of nuclear translocation of NFAT1 is used to evaluate NFAT activation in T cells and its tacrolimus-induced inhibition. A strong dose-dependent correlation between NFAT1 inhibition and tacrolimus dose is demonstrated in vitro. Time kinetic analysis of NFAT1 inhibition in plasma from stable renal transplant recipients before and after an in vivo dose with tacrolimus correlated with the expected pharmacokinetic profile of tacrolimus. This was further corroborated by analysis of patients' autologous CD4 and CD8 T cells. This is the first report to show that the measurement of NFAT1 activation potential by nuclear translocation can be used as a direct, sensitive, reproducible and quantitative pharmacodynamic readout for tacrolimus action. These results, and the rapid turnaround time for this assay, warrant its evaluation in a larger clinical setting to assess its role in therapeutic drug monitoring of calcineurin inhibitors.

  16. Herpesviral G protein-coupled receptors activate NFAT to induce tumor formation via inhibiting the SERCA calcium ATPase.

    PubMed

    Zhang, Junjie; He, Shanping; Wang, Yi; Brulois, Kevin; Lan, Ke; Jung, Jae U; Feng, Pinghui

    2015-03-01

    G protein-coupled receptors (GPCRs) constitute the largest family of proteins that transmit signal to regulate an array of fundamental biological processes. Viruses deploy diverse tactics to hijack and harness intracellular signaling events induced by GPCR. Herpesviruses encode multiple GPCR homologues that are implicated in viral pathogenesis. Cellular GPCRs are primarily regulated by their cognate ligands, while herpesviral GPCRs constitutively activate downstream signaling cascades, including the nuclear factor of activated T cells (NFAT) pathway. However, the roles of NFAT activation and mechanism thereof in viral GPCR tumorigenesis remain unknown. Here we report that GPCRs of human Kaposi's sarcoma-associated herpesvirus (kGPCR) and cytomegalovirus (US28) shortcut NFAT activation by inhibiting the sarcoplasmic reticulum calcium ATPase (SERCA), which is necessary for viral GPCR tumorigenesis. Biochemical approaches, entailing pharmacological inhibitors and protein purification, demonstrate that viral GPCRs target SERCA2 to increase cytosolic calcium concentration. As such, NFAT activation induced by vGPCRs was exceedingly sensitive to cyclosporine A that targets calcineurin, but resistant to inhibition upstream of ER calcium release. Gene expression profiling identified a signature of NFAT activation in endothelial cells expressing viral GPCRs. The expression of NFAT-dependent genes was up-regulated in tumors derived from tva-kGPCR mouse and human KS. Employing recombinant kGPCR-deficient KSHV, we showed that kGPCR was critical for NFAT-dependent gene expression in KSHV lytic replication. Finally, cyclosporine A treatment diminished NFAT-dependent gene expression and tumor formation induced by viral GPCRs. These findings reveal essential roles of NFAT activation in viral GPCR tumorigenesis and a mechanism of "constitutive" NFAT activation by viral GPCRs.

  17. Focal adhesion kinase regulates the activity of the osmosensitive transcription factor TonEBP/NFAT5 under hypertonic conditions

    PubMed Central

    Neuhofer, Wolfgang; Küper, Christoph; Lichtnekert, Julia; Holzapfel, Konstantin; Rupanagudi, Khader V.; Fraek, Maria-Luisa; Bartels, Helmut; Beck, Franz-Xaver

    2014-01-01

    TonEBP/NFAT5 is a major regulator of the urinary concentrating process and is essential for the osmoadaptation of renal medullary cells. Focal adhesion kinase (FAK) is a mechanosensitive non-receptor protein tyrosine kinase expressed abundantly in the renal medulla. Since osmotic stress causes cell shrinkage, the present study investigated the contribution of FAK on TonEBP/NFAT5 activation. Osmotic stress induced time-dependent activation of FAK as evidenced by phosphorylation at Tyr-397, and furosemide reduces FAK Tyr-397 phosphorylation in the rat renal medulla. Both pharmacological inhibition of FAK and siRNA-mediated knockdown of FAK drastically reduced TonEBP/NFAT5 transcriptional activity and target gene expression in HEK293 cells. This effect was not mediated by impaired nuclear translocation or by reduced transactivating activity of TonEBP/NFAT5. However, TonEBP/NFAT5 abundance under hypertonic conditions was diminished by 50% by FAK inhibition or siRNA knockdown of FAK. FAK inhibition only marginally reduced transcription of the TonEBP/NFAT5 gene. Rather, TonEBP/NFAT5 mRNA stability was diminished significantly by FAK inhibition, which correlated with reduced reporter activity of the TonEBP/NFAT5 mRNA 3′ untranslated region (3′-UTR). In conclusion, FAK is a major regulator of TonEBP/NFAT5 activity by increasing its abundance via stabilization of the mRNA. This in turn, depends on the presence of the TonEBP/NFAT5 3′-UTR. PMID:24772088

  18. Isoform- and tissue-specific regulation of the Ca(2+)-sensitive transcription factor NFAT in cardiac myocytes and heart failure.

    PubMed

    Rinne, Andreas; Kapur, Nidhi; Molkentin, Jeffery D; Pogwizd, Steven M; Bers, Donald M; Banach, Kathrin; Blatter, Lothar A

    2010-06-01

    Nuclear factors of activated T cells (NFATs) are Ca(2+)-sensitive transcription factors that have been implicated in hypertrophy, heart failure (HF), and arrhythmias. Cytosolic NFAT is activated by dephosphorylation by the Ca(2+)-sensitive phosphatase calcineurin, resulting in translocation to the nucleus, which is opposed by kinase activity, rephosphorylation, and nuclear export. Four different NFAT isoforms are expressed in the heart. The activation and regulation of NFAT in adult cardiac myocytes, which may depend on the NFAT isoform and cell type, are not fully understood. This study compared basal localization, import, and export of NFATc1 and NFATc3 in adult atrial and ventricular myocytes to identify isoform- and tissue-specific regulatory mechanisms of NFAT activation under physiological conditions and in HF. NFAT-green fluorescent protein fusion proteins and NFAT immunocytochemistry were used to analyze NFAT regulation in adult cat and rabbit myocytes. NFATc1 displayed basal nuclear localization in atrial and ventricular myocytes, an effect that was attenuated by reducing intracellular Ca(2+) concentration and inhibiting calcineurin, and enhanced by the inhibition of nuclear export. In contrast, NFATc3 was localized to the cytoplasm but could be driven to the nucleus by angiotensin II and endothelin-1 stimulation in atrial, but not ventricular, cells. Inhibition of nuclear export (by leptomycin B) facilitated nuclear localization in both cell types. Ventricular myocytes from HF rabbits showed increased basal nuclear localization of endogenous NFATc3 and reduced responsiveness of NFAT translocation to phenylephrine stimulation. In control myocytes, Ca(2+) overload, leading to spontaneous Ca(2+) waves, induced substantial translocation of NFATc3 to the nucleus. We conclude that the activation of NFAT in adult cardiomyocytes is isoform and tissue specific and is tightly controlled by nuclear export. NFAT is activated in myocytes from HF animals and may be

  19. Fermentation, Isolation, Structure, and antidiabetic activity of NFAT-133 produced by Streptomyces strain PM0324667

    PubMed Central

    2011-01-01

    Type-2 diabetes is mediated by defects in either insulin secretion or insulin action. In an effort to identify extracts that may stimulate glucose uptake, similar to insulin, a high throughput-screening assay for measuring glucose uptake in skeletal muscle cells was established. During the screening studies to discover novel antidiabetic compounds from microbial resources a Streptomyces strain PM0324667 (MTCC 5543, the Strain accession number at Institute of Microbial Technology, Chandigarh, India), an isolate from arid soil was identified which expressed a secondary metabolite that induced glucose uptake in L6 skeletal muscle cells. By employing bioactivity guided fractionation techniques, a tri-substituted simple aromatic compound with anti-diabetic potential was isolated. It was characterized based on MS and 2D NMR spectral data and identified as NFAT-133 which is a known immunosuppressive agent that inhibits NFAT-dependent transcription in vitro. Our investigations revealed the antidiabetic potential of NFAT-133. The compound induced glucose uptake in differentiated L6 myotubes with an EC50 of 6.3 ± 1.8 μM without activating the peroxisome proliferator-activated receptor-γ. Further, NFAT-133 was also efficacious in vivo in diabetic animals and reduced systemic glucose levels. Thus it is a potential lead compound which can be considered for development as a therapeutic for the treatment of type-2 diabetes. We have reported herewith the isolation of the producer microbe, fermentation, purification, in vitro, and in vivo antidiabetic activity of the compound. PMID:22104600

  20. Fermentation, Isolation, Structure, and antidiabetic activity of NFAT-133 produced by Streptomyces strain PM0324667.

    PubMed

    Kulkarni-Almeida, Asha A; Brahma, Manoja K; Padmanabhan, Prabhu; Mishra, Prabhu D; Parab, Rajashri R; Gaikwad, Nitin V; Thakkar, Chandni S; Tokdar, Pradipta; Ranadive, Prafull V; Nair, Amrutha S; Damre, Anagha A; Bahirat, Umakant A; Deshmukh, Nitin J; Doshi, Lalit S; Dixit, Amol V; George, Saji D; Vishwakarma, Ram A; Nemmani, Kumar Vs; Mahajan, Girish B

    2011-11-21

    Type-2 diabetes is mediated by defects in either insulin secretion or insulin action. In an effort to identify extracts that may stimulate glucose uptake, similar to insulin, a high throughput-screening assay for measuring glucose uptake in skeletal muscle cells was established. During the screening studies to discover novel antidiabetic compounds from microbial resources a Streptomyces strain PM0324667 (MTCC 5543, the Strain accession number at Institute of Microbial Technology, Chandigarh, India), an isolate from arid soil was identified which expressed a secondary metabolite that induced glucose uptake in L6 skeletal muscle cells. By employing bioactivity guided fractionation techniques, a tri-substituted simple aromatic compound with anti-diabetic potential was isolated. It was characterized based on MS and 2D NMR spectral data and identified as NFAT-133 which is a known immunosuppressive agent that inhibits NFAT-dependent transcription in vitro. Our investigations revealed the antidiabetic potential of NFAT-133. The compound induced glucose uptake in differentiated L6 myotubes with an EC50 of 6.3 ± 1.8 μM without activating the peroxisome proliferator-activated receptor-γ. Further, NFAT-133 was also efficacious in vivo in diabetic animals and reduced systemic glucose levels. Thus it is a potential lead compound which can be considered for development as a therapeutic for the treatment of type-2 diabetes. We have reported herewith the isolation of the producer microbe, fermentation, purification, in vitro, and in vivo antidiabetic activity of the compound.

  1. The involvement of NFAT transcriptional activity suppression in SIRT1-mediated inhibition of COX-2 expression induced by PMA/Ionomycin.

    PubMed

    Jia, Yu-Yan; Lu, Jie; Huang, Yue; Liu, Guang; Gao, Peng; Wan, Yan-Zhen; Zhang, Ran; Zhang, Zhu-Qin; Yang, Rui-Feng; Tang, Xiaoqiang; Xu, Jing; Wang, Xu; Chen, Hou-Zao; Liu, De-Pei

    2014-01-01

    SIRT1, a class III histone deacetylase, acts as a negative regulator for many transcription factors, and plays protective roles in inflammation and atherosclerosis. Transcription factor nuclear factor of activated T cells (NFAT) has been previously shown to play pro-inflammatory roles in endothelial cells. Inhibition of NFAT signaling may be an attractive target to regulate inflammation in atherosclerosis. However, whether NFAT transcriptional activity is suppressed by SIRT1 remains unknown. In this study, we found that SIRT1 suppressed NFAT-mediated transcriptional activity. SIRT1 interacted with NFAT, and the NHR and RHR domains of NFAT mediated the interaction with SIRT1. Moreover, we found that SIRT1 primarily deacetylated NFATc3. Adenoviral over-expression of SIRT1 suppressed PMA and calcium ionophore Ionomycin (PMA/Io)-induced COX-2 expression in human umbilical vein endothelial cells (HUVECs), while SIRT1 RNAi reversed the effects in HUVECs. Moreover, inhibition of COX-2 expression by SIRT1 in PMA/Io-treated HUVECs was largely abrogated by inhibiting NFAT activation. Furthermore, SIRT1 inhibited NFAT-induced COX-2 promoter activity, and reduced NFAT binding to the COX-2 promoter in PMA/Io-treated HUVECs. These results suggest that suppression of NFAT transcriptional activity is involved in SIRT1-mediated inhibition of COX-2 expression induced by PMA/Io, and that the negative regulatory mechanisms of NFAT by SIRT1 may contribute to its anti-inflammatory effects in atherosclerosis.

  2. DYRK1A Is a Novel Negative Regulator of Cardiomyocyte Hypertrophy*

    PubMed Central

    Kuhn, Christian; Frank, Derk; Will, Rainer; Jaschinski, Christoph; Frauen, Robert; Katus, Hugo A.; Frey, Norbert

    2009-01-01

    Activation of the phosphatase calcineurin and its downstream targets, transcription factors of the NFAT family, results in cardiomyocyte hypertrophy. Recently, it has been shown that the dual specificity tyrosine (Y) phosphorylation-regulated kinase 1A (DYRK1A) is able to antagonize calcineurin signaling by directly phosphorylating NFATs. We thus hypothesized that DYRK1A might modulate the hypertrophic response of cardiomyocytes. In a model of phenylephrine-induced hypertrophy, adenovirus-mediated overexpression of DYKR1A completely abrogated the hypertrophic response and significantly reduced the expression of the natriuretic peptides ANF and BNP. Furthermore, DYRK1A blunted cardiomyocyte hypertrophy induced by overexpression of constitutively active calcineurin and attenuated the induction of the hypertrophic gene program. Conversely, knockdown of DYRK1A, utilizing adenoviruses encoding for a specific synthetic miRNA, resulted in an increase in cell surface area accompanied by up-regulation of ANF- mRNA. Similarly, treatment of cardiomyocytes with harmine, a specific inhibitor of DYRK1A, revealed cardiomyocyte hypertrophy on morphological and molecular level. Moreover, constitutively active calcineurin led to robust induction of an NFAT-dependent luciferase reporter, whereas DYRK1A attenuated calcineurin-induced reporter activation in cardiomyocytes. Conversely, both knockdown and pharmacological inhibition of DYRK1A significantly augmented the effect of calcineurin in this assay. In summary, we identified DYRK1A as a novel negative regulator of cardiomyocyte hypertrophy. Mechanistically, this effect appears to be mediated via inhibition of NFAT transcription factors. PMID:19372220

  3. Activation of the Ca{sup 2+}/calcineurin/NFAT{sub 2} pathway controls smooth muscle cell differentiation

    SciTech Connect

    Larrieu, Daniel; Thiebaud, Pierre; Duplaa, Cecile; Sibon, Igor; Theze, Nadine; Lamaziere, Jean-Marie Daniel . E-mail: jean-marie.d-lamaziere@bordeaux.inserm.fr

    2005-10-15

    Cellular mechanisms controlling smooth muscle cells (SMCs) phenotypic modulation are largely unknown. Intracellular Ca{sup 2+} movements are essential to ensure SMC functions; one of the roles of Ca{sup 2+} is to regulate calcineurin, which in turn induces nuclear localization of the nuclear factor of activated T-cell (NFAT). In order to investigate, during phenotypic differentiation of SMCs, the effect of calcineurin inhibition on NFAT{sub 2} nuclear translocation, we used a culture model of SMC differentiation in serum-free conditions. We show that the treatment of cultured SMC with the calcineurin inhibitor cyclosporine A induced their dedifferentiation while preventing their differentiation. These findings suggest that nuclear translocation of NFAT{sub 2} is dependent of calcineurin activity during the in vitro SMC differentiation kinetic and that the nuclear presence of NFAT{sub 2} is critical in the acquisition and maintenance of SMC differentiation.

  4. Expression of nuclear factor of activated T cells (NFAT) and downstream muscle-specific proteins in ground squirrel skeletal and heart muscle during hibernation.

    PubMed

    Zhang, Yichi; Storey, Kenneth B

    2016-01-01

    The thirteen-lined ground squirrel (Ictidomys tridecemlineatus) undergoes remarkable adaptive changes during hibernation. Interestingly, skeletal muscle remodelling occurs during the torpor-arousal cycle of hibernation to prevent net muscle loss despite inactivity. Reversible cardiomyocyte hypertrophy occurs in cardiac muscle, allowing the heart to preserve cardiac output during hibernation, while avoiding chronic maladaptive hypertrophy post-hibernation. We propose that calcium signalling proteins [calcineurin (Cn), calmodulin (CaM), and calpain], the nuclear factor of activated T cell (NFAT) family of transcription factors, and the NFAT targets myoferlin and myomaker contribute significantly to adaptations taking place in skeletal and cardiac muscle during hibernation. Protein-level analyses were performed over several conditions: euthermic room temperature (ER), euthermic cold room (EC), entrance into (EN), early (ET), and late torpor (LT) time points, in addition to early (EA), interbout (IA), and late arousal (LA) time points using immunoblotting and DNA-protein interaction (DPI) enzyme-linked immunosorbent assay (ELISAs). In skeletal and cardiac muscle, NFATc2 protein levels were elevated during torpor. NFATc4 increased throughout the torpor-arousal cycle in both tissues, and NFATc1 showed this trend in cardiac muscle only. NFATc3 showed an elevation in DNA-binding activity but not expression during torpor. Myoferlin protein levels dramatically increased during torpor in both skeletal and cardiac muscle. Myomaker levels also increased significantly in cardiac muscle during torpor. Cardiac Cn levels remained stable, whereas CaM and calpain decreased throughout the torpor-arousal cycle. Activation and/or upregulation of NFATc2, c3, myoferlin, and myomaker at torpor could be part of a stress-response mechanism to preserve skeletal muscle mass, whereas CaM and calpain appear to initiate the rapid reversal of cardiac hypertrophy during arousal through

  5. Nickel differentially regulates NFAT and NF-{kappa}B activation in T cell signaling

    SciTech Connect

    Saito, Rumiko; Hirakawa, Satoshi; Ohara, Hiroshi; Yasuda, Makoto; Yamazaki, Tomomi; Nishii, Shigeaki; Aiba, Setsuya

    2011-08-01

    Nickel is a potent hapten that induces contact hypersensitivity in human skin. While nickel induces the maturation of dendritic cells via NF-{kappa}B and p38 MAPK activation, it also exerts immunosuppressive effects on T cells through an unknown mechanism. To elucidate the molecular mechanisms of its effects on T cells, we examined the effects of NiCl{sub 2} on mRNA expression in human CD3+ T cells stimulated with CD3 and CD28 antibodies. Using a DNA microarray and Gene Ontology, we identified 70 up-regulated (including IL-1{beta}, IL-6 and IL-8) and 61 down-regulated (including IL-2, IL-4, IL-10 and IFN-{gamma}) immune responsive genes in NiCl{sub 2}-treated T cells. The DNA microarray results were verified using real-time PCR and a Bio-Plex{sup TM} suspension protein array. Suppression of IL-2 and IFN-{gamma} gene transcription by NiCl{sub 2} was also confirmed using Jurkat T cells transfected with IL-2 or IFN-{gamma} luciferase reporter genes. To explore the NiCl{sub 2}-regulated signaling pathway, we examined the binding activity of nuclear proteins to NFAT, AP-1, and NF-{kappa}B consensus sequences. NiCl{sub 2} significantly and dose-dependently suppressed NFAT- and AP-1-binding activity, but augmented NF-{kappa}B-binding activity. Moreover, NiCl{sub 2} decreased nuclear NFAT expression in stimulated T cells. Using Jurkat T cells stimulated with PMA/ionomycin, we demonstrated that NiCl{sub 2} significantly suppressed stimulation-evoked cytosolic Ca{sup 2+} increases, suggesting that NiCl{sub 2} regulates NFAT signals by acting as a blocker of Ca{sup 2+} release-activated Ca{sup 2+} (CRAC) channels. These data showed that NiCl{sub 2} decreases NFAT and increases NF-{kappa}B signaling in T cells. These results shed light on the effects of nickel on the molecular regulation of T cell signaling. - Graphical Abstract: Nickel suppresses stimulation-evoked cytosolic Ca{sup 2+} increase, which results in the suppression of NFAT signals. On the other hand, Ni rather

  6. Immune-suppressive activity of punicalagin via inhibition of NFAT activation

    SciTech Connect

    Lee, Sang-Ik; Kim, Byoung-Soo; Kim, Kyoung-Shin; Lee, Samkeun; Shin, Kwang-Soo; Lim, Jong-Soon

    2008-07-11

    Since T cell activation is central to the development of autoimmune diseases, we screened a natural product library comprising 1400 samples of medicinal herbal extracts, to identify compounds that suppress T cell activity. Punicalagin (PCG) isolated from the fruit of Punica granatum was identified as a potent immune suppressant, based on its inhibitory action on the activation of the nuclear factor of activated T cells (NFAT). PCG downregulated the mRNA and soluble protein expression of interleukin-2 from anti-CD3/anti-CD28-stimulated murine splenic CD4+ T cells and suppressed mixed leukocytes reaction (MLR) without exhibiting cytotoxicity to the cells. In vivo, the PCG treatment inhibited phorbol 12-myristate 13-acetate (PMA)-induced chronic ear edema in mice and decreased CD3+ T cell infiltration of the inflamed tissue. These results suggest that PCG could be a potential candidate for the therapeutics of various immune pathologies.

  7. The calcineurin/NFAT pathway is activated in diagnostic breast cancer cases and is essential to survival and metastasis of mammary cancer cells.

    PubMed

    Quang, C Tran; Leboucher, S; Passaro, D; Fuhrmann, L; Nourieh, M; Vincent-Salomon, A; Ghysdael, J

    2015-02-26

    Nuclear factor of activated T cells 1 (NFAT1) expression has been associated with increased migratory/invasive properties of mammary tumor-derived cell lines in vitro. It is unknown, however, if NFAT activation actually occurs in breast cancer cases and whether the calcineurin/NFAT pathway is important to mammary tumorigenesis. Using a cohort of 321 diagnostic cases of the major subgroup of breast cancer, we found Cn/NFAT pathway activated in ER(-)PR(-)HER2(-) triple-negative breast cancer subtype, whereas its prevalence is less in other subgroups. Using a small hairpin RNA-based gene expression silencing approach in murine mammary tumor cell line (4T1), we show that not only NFAT1 but also NFAT2 and their upstream activator Cn are essential to the migratory and invasive properties of mammary tumor cells. We also demonstrate that Cn, NFAT1 and NFAT2 are essential to the tumorigenic and metastatic properties of these cells in mice, a phenotype which coincides with increased apoptosis in vivo. Finally, global gene expression analyses identified several NFAT-deregulated genes, many of them being previously associated with mammary tumorigenesis. In particular, we identified the gene encoding a disintegrin and metalloproteinase with thrombonspondin motifs 1, as being a potential direct target of NFAT1. Thus, our results show that the Cn/NFAT pathway is activated in diagnostic cases of breast cancers and is essential to the tumorigenic and metastatic potential of mammary tumor cell line. These results suggest that pharmacological inhibition of the Cn/NFAT pathway at different levels could be of therapeutical interest for breast cancer patients.

  8. NFATc2 recruits cJun homodimers to an NFAT site to synergistically activate interleukin-2 transcription.

    PubMed

    Walters, Ryan D; Drullinger, Linda F; Kugel, Jennifer F; Goodrich, James A

    2013-11-01

    Transcription of interleukin-2 (IL-2), a pivotal cytokine in the mammalian immune response, is induced by NFAT and AP-1 transcriptional activators in stimulated T cells. NFATc2 and cJun drive high levels of synergistic human IL-2 transcription, which requires a unique interaction between the C-terminal activation domain of NFATc2 and cJun homodimers. Here we studied the mechanism by which this interaction contributes to synergistic activation of IL-2 transcription. We found that NFATc2 can recruit cJun homodimers to the -45 NFAT element, which lacks a neighboring AP-1 site. The bZip domain of cJun is sufficient to interact with the C-terminal activation domain of NFATc2 in the absence of DNA and this interaction is inhibited by AP-1 DNA. When the -45 NFAT site was replaced by either an NFAT/AP-1 composite site or a single AP-1 site the specificity for cJun homodimers in synergistically activating IL-2 transcription was lost, and cJun/cFos heterodimers strongly activated transcription. These studies support a model in which IL-2 transcriptional synergy is mediated by the unique recruitment of a cJun homodimer to the -45 NFAT site by NFATc2, where it acts as a co-activator for IL-2 transcription.

  9. Virion encapsidated HIV-1 Vpr induces NFAT to prime non-activated T cells for productive infection.

    PubMed

    Höhne, Kristin; Businger, Ramona; van Nuffel, Anouk; Bolduan, Sebastian; Koppensteiner, Herwig; Baeyens, Ann; Vermeire, Jolien; Malatinkova, Eva; Verhasselt, Bruno; Schindler, Michael

    2016-07-01

    The majority of T cells encountered by HIV-1 are non-activated and do not readily allow productive infection. HIV-1 Vpr is highly abundant in progeny virions, and induces signalling and HIV-1 LTR transcription. We hence hypothesized that Vpr might be a determinant of non-activated T-cell infection. Virion-delivered Vpr activated nuclear factor of activated T cells (NFAT) through Ca(2+) influx and interference with the NFAT export kinase GSK3β. This leads to NFAT translocation and accumulation within the nucleus and was required for productive infection of unstimulated primary CD4(+) T cells. A mutagenesis approach revealed correlation of Vpr-mediated NFAT activation with its ability to enhance LTR transcription and mediate cell cycle arrest. Upon NFAT inhibition, Vpr did not augment resting T-cell infection, and showed reduced G2/M arrest and LTR transactivation. Altogether, Vpr renders unstimulated T cells more permissive for productive HIV-1 infection and stimulates activation of productively infected as well as virus-exposed T cells. Therefore, it could be involved in the establishment and reactivation of HIV-1 from viral reservoirs and might have an impact on the levels of immune activation, which are determinants of HIV-1 pathogenesis.

  10. Virion encapsidated HIV-1 Vpr induces NFAT to prime non-activated T cells for productive infection

    PubMed Central

    Höhne, Kristin; Businger, Ramona; van Nuffel, Anouk; Bolduan, Sebastian; Koppensteiner, Herwig; Baeyens, Ann; Vermeire, Jolien; Malatinkova, Eva; Verhasselt, Bruno; Schindler, Michael

    2016-01-01

    The majority of T cells encountered by HIV-1 are non-activated and do not readily allow productive infection. HIV-1 Vpr is highly abundant in progeny virions, and induces signalling and HIV-1 LTR transcription. We hence hypothesized that Vpr might be a determinant of non-activated T-cell infection. Virion-delivered Vpr activated nuclear factor of activated T cells (NFAT) through Ca2+ influx and interference with the NFAT export kinase GSK3β. This leads to NFAT translocation and accumulation within the nucleus and was required for productive infection of unstimulated primary CD4+ T cells. A mutagenesis approach revealed correlation of Vpr-mediated NFAT activation with its ability to enhance LTR transcription and mediate cell cycle arrest. Upon NFAT inhibition, Vpr did not augment resting T-cell infection, and showed reduced G2/M arrest and LTR transactivation. Altogether, Vpr renders unstimulated T cells more permissive for productive HIV-1 infection and stimulates activation of productively infected as well as virus-exposed T cells. Therefore, it could be involved in the establishment and reactivation of HIV-1 from viral reservoirs and might have an impact on the levels of immune activation, which are determinants of HIV-1 pathogenesis. PMID:27383627

  11. Virion encapsidated HIV-1 Vpr induces NFAT to prime non-activated T cells for productive infection.

    PubMed

    Höhne, Kristin; Businger, Ramona; van Nuffel, Anouk; Bolduan, Sebastian; Koppensteiner, Herwig; Baeyens, Ann; Vermeire, Jolien; Malatinkova, Eva; Verhasselt, Bruno; Schindler, Michael

    2016-07-01

    The majority of T cells encountered by HIV-1 are non-activated and do not readily allow productive infection. HIV-1 Vpr is highly abundant in progeny virions, and induces signalling and HIV-1 LTR transcription. We hence hypothesized that Vpr might be a determinant of non-activated T-cell infection. Virion-delivered Vpr activated nuclear factor of activated T cells (NFAT) through Ca(2+) influx and interference with the NFAT export kinase GSK3β. This leads to NFAT translocation and accumulation within the nucleus and was required for productive infection of unstimulated primary CD4(+) T cells. A mutagenesis approach revealed correlation of Vpr-mediated NFAT activation with its ability to enhance LTR transcription and mediate cell cycle arrest. Upon NFAT inhibition, Vpr did not augment resting T-cell infection, and showed reduced G2/M arrest and LTR transactivation. Altogether, Vpr renders unstimulated T cells more permissive for productive HIV-1 infection and stimulates activation of productively infected as well as virus-exposed T cells. Therefore, it could be involved in the establishment and reactivation of HIV-1 from viral reservoirs and might have an impact on the levels of immune activation, which are determinants of HIV-1 pathogenesis. PMID:27383627

  12. Dephosphorylation of the nuclear factor of activated T cells (NFAT) transcription factor is regulated by an RNA-protein scaffold complex.

    PubMed

    Sharma, Sonia; Findlay, Gregory M; Bandukwala, Hozefa S; Oberdoerffer, Shalini; Baust, Beate; Li, Zhigang; Schmidt, Valentina; Hogan, Patrick G; Sacks, David B; Rao, Anjana

    2011-07-12

    Nuclear factor of activated T cells (NFAT) proteins are Ca(2+)-regulated transcription factors that control gene expression in many cell types. NFAT proteins are heavily phosphorylated and reside in the cytoplasm of resting cells; when cells are stimulated by a rise in intracellular Ca(2+), NFAT proteins are dephosphorylated by the Ca(2+)/calmodulin-dependent phosphatase calcineurin and translocate to the nucleus to activate target gene expression. Here we show that phosphorylated NFAT1 is present in a large cytoplasmic RNA-protein scaffold complex that contains a long intergenic noncoding RNA (lincRNA), NRON [noncoding (RNA) repressor of NFAT]; a scaffold protein, IQ motif containing GTPase activating protein (IQGAP); and three NFAT kinases, casein kinase 1, glycogen synthase kinase 3, and dual specificity tyrosine phosphorylation regulated kinase. Combined knockdown of NRON and IQGAP1 increased NFAT dephosphorylation and nuclear import exclusively after stimulation, without affecting the rate of NFAT rephosphorylation and nuclear export; and both NRON-depleted T cells and T cells from IQGAP1-deficient mice showed increased production of NFAT-dependent cytokines. Our results provide evidence that a complex of lincRNA and protein forms a scaffold for a latent transcription factor and its regulatory kinases, and support an emerging consensus that lincRNAs that bind transcriptional regulators have a similar scaffold function.

  13. The calcineurin-NFAT pathway controls activity-dependent circadian gene expression in slow skeletal muscle

    PubMed Central

    Dyar, Kenneth A.; Ciciliot, Stefano; Tagliazucchi, Guidantonio Malagoli; Pallafacchina, Giorgia; Tothova, Jana; Argentini, Carla; Agatea, Lisa; Abraham, Reimar; Ahdesmäki, Miika; Forcato, Mattia; Bicciato, Silvio; Schiaffino, Stefano; Blaauw, Bert

    2015-01-01

    Objective Physical activity and circadian rhythms are well-established determinants of human health and disease, but the relationship between muscle activity and the circadian regulation of muscle genes is a relatively new area of research. It is unknown whether muscle activity and muscle clock rhythms are coupled together, nor whether activity rhythms can drive circadian gene expression in skeletal muscle. Methods We compared the circadian transcriptomes of two mouse hindlimb muscles with vastly different circadian activity patterns, the continuously active slow soleus and the sporadically active fast tibialis anterior, in the presence or absence of a functional skeletal muscle clock (skeletal muscle-specific Bmal1 KO). In addition, we compared the effect of denervation on muscle circadian gene expression. Results We found that different skeletal muscles exhibit major differences in their circadian transcriptomes, yet core clock gene oscillations were essentially identical in fast and slow muscles. Furthermore, denervation caused relatively minor changes in circadian expression of most core clock genes, yet major differences in expression level, phase and amplitude of many muscle circadian genes. Conclusions We report that activity controls the oscillation of around 15% of skeletal muscle circadian genes independently of the core muscle clock, and we have identified the Ca2+-dependent calcineurin-NFAT pathway as an important mediator of activity-dependent circadian gene expression, showing that circadian locomotor activity rhythms drive circadian rhythms of NFAT nuclear translocation and target gene expression. PMID:26629406

  14. Angular-type furocoumarins from the roots of Angelica atropurpurea and their inhibitory activity on the NFAT signal transduction pathway.

    PubMed

    Nagasawa, Azumi; Sakasai, Mitsuyoshi; Sakaguchi, Daishi; Moriwaki, Shigeru; Nishizawa, Yoshinori; Kitahara, Takashi

    2014-12-01

    One new (1) and two known angular-type (2,3) furocoumarins, isoarchangelicin (1), archangelicin (2), and 2'-angeloyl-3'-isovaleryl vaginate (3), were isolated from the roots of Angelica atropurpurea. The structure of the new compound was established on the basis of one- and two-dimensional NMR spectra and other spectroscopic studies. The inhibitory activity of these three compounds and a deacylated form of archangelicin (4) on the nuclear factor of activated T cells (NFAT) signal transduction pathway was tested by NFAT-responsive luciferase reporter gene assay in cultured cells. Although 4 did not exhibit inhibitory activity on NFAT signaling, 1-3 exhibited dose-dependent inhibition with IC50 values of 16.5 (1), 9.0 (2), and 9.2 (3) μM.

  15. Anabolic steroids activate calcineurin-NFAT signaling and thereby increase myotube size and reduce denervation atrophy.

    PubMed

    Qin, Weiping; Pan, Jiangping; Wu, Yong; Bauman, William A; Cardozo, Christopher

    2015-01-01

    Anabolic androgens have been shown to reduce muscle loss due to immobilization, paralysis and many other medical conditions, but the molecular basis for these actions is poorly understood. We have recently demonstrated that nandrolone, a synthetic androgen, slows muscle atrophy after nerve transection associated with down-regulation of regulator of calcineurin 2 (RCAN2), a calcineurin inhibitor, suggesting a possible role of calcineurin-NFAT signaling. To test this possibility, rat gastrocnemius muscle was analyzed at 56 days after denervation. In denervated muscle, calcineurin activity declined and NFATc4 was excluded from the nucleus and these effects were reversed by nandrolone. Similarly, nandrolone increased calcineurin activity and nuclear NFATc4 levels in cultured L6 myotubes. Nandrolone also induced cell hypertrophy that was blocked by cyclosporin A or overexpression of RCAN2. Finally protection against denervation atrophy by nandrolone in rats was blocked by cyclosporin A. These results demonstrate for the first time that nandrolone activates calcineurin-NFAT signaling, and that such signaling is important in nandrolone-induced cell hypertrophy and protection against paralysis-induced muscle atrophy.

  16. SH3BP2 is an activator of NFAT activity and osteoclastogenesis

    SciTech Connect

    Lietman, Steven A. Yin Lihong; Levine, Michael A.

    2008-07-11

    Heterozygous activating mutations in exon 9 of SH3BP2 have been found in most patients with cherubism, an unusual genetic syndrome characterized by excessive remodeling of the mandible and maxilla due to spontaneous and excessive osteoclastic bone resorption. Osteoclasts differentiate after binding of sRANKL to RANK induces a number of downstream signaling effects, including activation of the calcineurin/NFAT (nuclear factor of activated T cells) pathway. Here, we have investigated the functional significance of SH3BP2 protein on osteoclastogenesis in the presence of sRANKL. Our results indicate that SH3BP2 both increases nuclear NFATc1 in sRANKL treated RAW 264.7 preosteoclast cells and enhances expression of tartrate resistant acid phosphatase (TRAP), a specific marker of osteoclast differentiation. Moreover, overexpression of SH3BP2 in RAW 264.7 cells potentiates sRANKL-stimulated phosphorylation of PLC{gamma}1 and 2, thus providing a mechanistic pathway for the rapid translocation of NFATc1 into the nucleus and increased osteoclastogenesis in cherubism.

  17. Activation of NFAT signaling establishes a tumorigenic microenvironment through cell autonomous and non-cell autonomous mechanisms.

    PubMed

    Tripathi, P; Wang, Y; Coussens, M; Manda, K R; Casey, A M; Lin, C; Poyo, E; Pfeifer, J D; Basappa, N; Bates, C M; Ma, L; Zhang, H; Pan, M; Ding, L; Chen, F

    2014-04-01

    NFAT (the nuclear factor of activated T cells) upregulation has been linked to cellular transformation intrinsically, but it is unclear whether and how tissue cells with NFAT activation change the local environment for tumor initiation and progression. Direct evidence showing NFAT activation initiates primary tumor formation in vivo is also lacking. Using inducible transgenic mouse systems, we show that tumors form in a subset of, but not all, tissues with NFATc1 activation, indicating that NFAT oncogenic effects depend on cell types and tissue contexts. In NFATc1-induced skin and ovarian tumors, both cells with NFATc1 activation and neighboring cells without NFATc1 activation have significant upregulation of c-Myc and activation of Stat3. Besides known and suspected NFATc1 targets, such as Spp1 and Osm, we have revealed the early upregulation of a number of cytokines and cytokine receptors, as key molecular components of an inflammatory microenvironment that promotes both NFATc1(+) and NFATc1(-) cells to participate in tumor formation. Cultured cells derived from NFATc1-induced tumors were able to establish a tumorigenic microenvironment, similar to that of the primary tumors, in an NFATc1-dependent manner in nude mice with T-cell deficiency, revealing an addiction of these tumors to NFATc1 activation and downplaying a role for T cells in the NFATc1-induced tumorigenic microenvironment. These findings collectively suggest that beyond the cell autonomous effects on the upregulation of oncogenic proteins, NFATc1 activation has non-cell autonomous effects through the establishment of a promitogenic microenvironment for tumor growth. This study provides direct evidence for the ability of NFATc1 in inducing primary tumor formation in vivo and supports targeting NFAT signaling in anti-tumor therapy.

  18. NFAT as cancer target: Mission possible?

    PubMed Central

    Qin, Jiang-Jiang; Nag, Subhasree; Wang, Wei; Zhou, Jianwei; Zhang, Wei-Dong; Wang, Hui; Zhang, Ruiwen

    2014-01-01

    The NFAT signaling pathway regulates various aspects of cellular functions; NFAT acts as a calcium sensor, integrating calcium signaling with other pathways involved in development and growth, immune response, and inflammatory response. The NFAT family of transcription factors regulates diverse cellular functions such as cell survival, proliferation, migration, invasion, and angiogenesis. The NFAT isoforms are constitutively activated and overexpressed in several cancer types wherein they transactivate downstream targets that play important roles in cancer development and progression. Though the NFAT family has been conclusively proved to be pivotal in cancer progression, the different isoforms play distinct roles in different cellular contexts. In this review, our discussion is focused on the mechanisms that drive activation of the various NFAT isoforms in cancer. Additionally, we analyze the potential of NFAT as a valid target for cancer prevention and therapy. PMID:25072963

  19. NFAT as cancer target: mission possible?

    PubMed

    Qin, Jiang-Jiang; Nag, Subhasree; Wang, Wei; Zhou, Jianwei; Zhang, Wei-Dong; Wang, Hui; Zhang, Ruiwen

    2014-12-01

    The NFAT signaling pathway regulates various aspects of cellular functions; NFAT acts as a calcium sensor, integrating calcium signaling with other pathways involved in development and growth, immune response, and inflammatory response. The NFAT family of transcription factors regulates diverse cellular functions such as cell survival, proliferation, migration, invasion, and angiogenesis. The NFAT isoforms are constitutively activated and overexpressed in several cancer types wherein they transactivate downstream targets that play important roles in cancer development and progression. Though the NFAT family has been conclusively proved to be pivotal in cancer progression, the different isoforms play distinct roles in different cellular contexts. In this review, our discussion is focused on the mechanisms that drive the activation of various NFAT isoforms in cancer. Additionally, we analyze the potential of NFAT as a valid target for cancer prevention and therapy.

  20. Leishmania infantum-chagasi activates SHP-1 and reduces NFAT5/TonEBP activity in the mouse kidney inner medulla.

    PubMed

    Zhou, Xiaoming; Wang, Hong; Koles, Nancy L; Zhang, Aihong; Aronson, Naomi E

    2014-09-01

    Visceral leishmaniasis patients have been reported to have a urine concentration defect. Concentration of urine by the renal inner medulla is essentially dependent on a transcription factor, NFAT5/TonEBP, because it activates expression of osmoprotective genes betaine/glycine transporter 1 (BGT1) and sodium/myo-inositol transporter (SMIT), and water channel aquaporin-2, all of which are imperative for concentrating urine. Leishmania parasites evade macrophage immune defenses by activating protein tyrosine phosphatases, among which SHP-1 is critical. We previously demonstrated that SHP-1 inhibits tonicity-dependent activation of NFAT5/TonEBP in HEK293 cells through screening a genome-wide small interfering (si) RNA library against phosphatases (Zhou X, Gallazzini M, Burg MB, Ferraris JD. Proc Natl Acad Sci USA 107: 7072-7077, 2010). We sought to examine whether Leishmania can activate SHP-1 and inhibit NFAT5/TonEBP activity in the renal inner medulla in a murine model of visceral leishmaniasis by injection of female BALB/c mice with a single intravenous dose of 5 × 10(5) L. chagasi metacyclic promastigotes. We found that SHP-1 is expressed in the kidney inner medulla. L. chagasi activates SHP-1 with an increase in stimulatory phosphorylation of SHP-1-Y536 in the region. L. chagasi reduces expression of NFAT5/TonEBP mRNA and protein as well as expression of its targeted genes: BGT1, SMIT, and aquaporin-2. The culture supernatant from L. chagasi metacyclic promastigotes increases SHP-1 protein abundance and potently inhibits NFAT5 transcriptional activity in mIMCD3 cells. However, L. chagasi in our animal model has no significant effect on urinary concentration. We conclude that L. chagasi, most likely through its secreted virulence factors, activates SHP-1 and reduces NFAT5/TonEBP gene expression, which leads to reduced NFAT5/TonEBP transcriptional activity in the kidney inner medulla. PMID:24990897

  1. Identification of two nuclear factor of activated T-cells (NFAT)-response elements in the 5'-upstream regulatory region of the ET-1 promoter.

    PubMed

    Strait, Kevin A; Stricklett, Peter K; Kohan, Rachel M; Kohan, Donald E

    2010-09-10

    Collecting duct-derived ET-1 regulates salt excretion and blood pressure. We have reported the presence of an inner medullary collecting duct (IMCD)-specific enhancer region in the 5'-upstream ET-1 promoter (Strait, K. A., Stricklett, P. K., Kohan, J. L., Miller, M. B., and Kohan, D. E. (2007) Am. J. Physiol. Renal Physiol. 293, F601-F606). The current studies provide further characterization of the ET-1 5'-upstream distal promoter to identify the IMCD-specific enhancer elements. Deletion studies identified two regions of the 5'-upstream ET-1 promoter, -1725 to -1319 bp and -1319 to -1026 bp, which were required for maximal promoter activity in transfected rat IMCD cells. Transcription factor binding site analysis of these regions identified two consensus nuclear factor of activated T-cells (NFAT) binding sites at -1263 and -1563. EMSA analysis using nuclear extracts from IMCD cells showed that both the -1263 and the -1563 NFAT sites in the ET-1 distal promoter competed for NFAT binding to previously identified NFAT sites in the IL-2 and TNF genes. Gel supershift analysis showed that each of the NFAT binding sites in the ET-1 promoter bound NFAT proteins derived from IMCD nuclear extracts, but they selectively bound different NFAT isoforms; ET-1263 bound NFATc1, whereas ET-1563 bound NFATc3. Site-directed mutagenesis of either the ET-1263 or the ET-1563 sites prevented NFAT binding and reduced ET-1 promoter activity. Thus, NFAT appears to be an important regulator of ET-1 transcription in IMCD cells, and thus, it may play a role in controlling blood pressure through ET-1 regulation of renal salt excretion.

  2. Depressed phosphatidic acid-induced contractile activity of failing cardiomyocytes.

    PubMed

    Tappia, Paramjit S; Maddaford, Thane G; Hurtado, Cecilia; Panagia, Vincenzo; Pierce, Grant N

    2003-01-10

    The effects of phosphatidic acid (PA), a known inotropic agent, on Ca(2+) transients and contractile activity of cardiomyocytes in congestive heart failure (CHF) due to myocardial infarction were examined. In control cells, PA induced a significant increase (25%) in active cell shortening and Ca(2+) transients. The phospholipase C (PLC) inhibitor, 2-nitro-4-carboxyphenyl N,N-diphenylcarbonate, blocked the positive inotropic action induced by PA, indicating that PA induces an increase in contractile activity and Ca(2+) transients through stimulation of PLC. Conversely, in failing cardiomyocytes there was a loss of PA-induced increase in active cell shortening and Ca(2+) transients. PA did not alter resting cell length. Both diastolic and systolic [Ca(2+)] were significantly elevated in the failing cardiomyocytes. In vitro assessment of the cardiac sarcolemmal (SL) PLC activity revealed that the impaired failing cardiomyocyte response to PA was associated with a diminished stimulation of SL PLC activity by PA. Our results identify an important defect in the PA-PLC signaling pathway in failing cardiomyocytes, which may have significant implications for the depressed contractile function during CHF.

  3. Pseudoephedrine inhibits T-cell activation by targeting NF-κB, NFAT and AP-1 signaling pathways.

    PubMed

    Fiebich, Bernd L; Collado, Juan A; Stratz, Cristian; Valina, Christian; Hochholzer, Willibald; Muñoz, Eduardo; Bellido, Luz M

    2012-02-01

    Pseudoephedrine (PSE) is a stereoisomer of ephedrine that is commonly used as a nasal decongestant in combination with other anti-inflammatory drugs for the symptomatic treatment of some common pathologies such as common cold. Herein, we describe for the first time the effects of PSE on T-cell activation events. We found that PSE inhibits interleukin-2 (IL-2) and tumor necrosis factor (TNF) alpha-gene transcription in stimulated Jurkat cells, a human T-cell leukemia cell line. To further characterize the inhibitory mechanisms of PSE at the transcriptional level, we examined the transcriptional activities of nuclear factor kappa B (NF-κB), nuclear factor of activated T cells (NFAT), and activator protein-1 (AP-1) transcription factors and found that PSE inhibited NF-κB-dependent transcriptional activity without affecting either the phosphorylation, the degradation of the cytoplasmic NF-κB inhibitory protein, IκBα or the DNA-binding activity. However, phosphorylation of the p65/RelA subunit was clearly inhibited by PSE in stimulated cells. In addition, PSE inhibited the transcriptional activity of NFAT without interfering with the calcium-induced NFAT dephosphorylation event, which represents the major signaling pathway for its activation. NFAT cooperates with c-Jun, a compound of the AP-1 complex, to activate target genes, and we also found that PSE inhibited both JNK activation and AP-1 transcriptional activity. These findings provide new mechanistic insights into the potential immunomodulatory activities of PSE and highlight their potential in designing novel therapeutic strategies to manage inflammatory diseases.

  4. Calcineurin/NFAT signalling inhibits myeloid haematopoiesis.

    PubMed

    Fric, Jan; Lim, Clarice X F; Koh, Esther G L; Hofmann, Benjamin; Chen, Jinmiao; Tay, Hock Soon; Mohammad Isa, Siti Aminah Bte; Mortellaro, Alessandra; Ruedl, Christiane; Ricciardi-Castagnoli, Paola

    2012-04-01

    Nuclear factor of activated T cells (NFAT) comprises a family of transcription factors that regulate T cell development, activation and differentiation. NFAT signalling can also mediate granulocyte and dendritic cell (DC) activation, but it is unknown whether NFAT influences their development from progenitors. Here, we report a novel role for calcineurin/NFAT signalling as a negative regulator of myeloid haematopoiesis. Reconstituting lethally irradiated mice with haematopoietic stem cells expressing an NFAT-inhibitory peptide resulted in enhanced development of the myeloid compartment. Culturing bone marrow cells in media supplemented with Flt3-L in the presence of the calcineurin/NFAT inhibitor Cyclosporin A increased numbers of differentiated DC. Global gene expression analysis of untreated DC and NFAT-inhibited DC revealed differential expression of transcripts that regulate cell cycle and apoptosis. In conclusion, these results provide evidence that calcineurin/NFAT signalling negatively regulates myeloid lineage development. The finding that inhibition of NFAT enhances myeloid development provides a novel insight into understanding how the treatment with drugs targeting calcineurin/NFAT signalling influence the homeostasis of the innate immune system.

  5. Distinct activation properties of the nuclear factor of activated T-cells (NFAT) isoforms NFATc3 and NFATc4 in neurons.

    PubMed

    Ulrich, Jason D; Kim, Man-Su; Houlihan, Patrick R; Shutov, Leonid P; Mohapatra, Durga P; Strack, Stefan; Usachev, Yuriy M

    2012-11-01

    The Ca(2+)/calcineurin-dependent transcription factor NFAT (nuclear factor of activated T-cells) is implicated in regulating dendritic and axonal development, synaptogenesis, and neuronal survival. Despite the increasing appreciation for the importance of NFAT-dependent transcription in the nervous system, the regulation and function of specific NFAT isoforms in neurons are poorly understood. Here, we compare the activation of NFATc3 and NFATc4 in hippocampal and dorsal root ganglion neurons following electrically evoked elevations of intracellular Ca(2+) concentration ([Ca(2+)](i)). We find that NFATc3 undergoes rapid dephosphorylation and nuclear translocation that are essentially complete within 20 min, although NFATc4 remains phosphorylated and localized to the cytosol, only exhibiting nuclear localization following prolonged (1-3 h) depolarization. Knocking down NFATc3, but not NFATc4, strongly diminished NFAT-mediated transcription induced by mild depolarization in neurons. By analyzing NFATc3/NFATc4 chimeras, we find that the region containing the serine-rich region-1 (SRR1) mildly affects initial NFAT translocation, although the region containing the serine-proline repeats is critical for determining the magnitude of NFAT activation and nuclear localization upon depolarization. Knockdown of glycogen synthase kinase 3β (GSK3β) significantly increased the depolarization-induced nuclear localization of NFATc4. In contrast, inhibition of p38 or mammalian target of rapamycin (mTOR) kinases had no significant effect on nuclear import of NFATc4. Thus, electrically evoked [Ca(2+)](i) elevation in neurons rapidly and strongly activates NFATc3, whereas activation of NFATc4 requires a coincident increase in [Ca(2+)](i) and suppression of GSK3β, with differences in the serine-proline-containing region giving rise to these distinct activation properties of NFATc3 and NFATc4.

  6. Calcineurin/NFAT Activation-Dependence of Leptin Synthesis and Vascular Growth in Response to Mechanical Stretch

    PubMed Central

    Soudani, Nadia; Ghantous, Crystal M.; Farhat, Zein; Shebaby, Wassim N.; Zibara, Kazem; Zeidan, Asad

    2016-01-01

    Background and Aims: Hypertension and obesity are important risk factors of cardiovascular disease. They are both associated with high leptin levels and have been shown to promote vascular hypertrophy, through the RhoA/ROCK and ERK1/2 phosphorylation. Calcineurin/NFAT activation also induces vascular hypertrophy by upregulating various genes. This study aimed to decipher whether a crosstalk exists between the RhoA/ROCK pathway, Ca2+/calcineurin/NFAT pathway, and ERK1/2 phosphorylation in the process of mechanical stretch-induced vascular smooth muscle cell (VSMC) hypertrophy and leptin synthesis. Methods and Results: Rat portal vein (RPV) organ culture was used to investigate the effect of mechanical stretch and exogenous leptin (3.1 nM) on VSMC hypertrophy and leptin synthesis. Results showed that stretching the RPV significantly upregulated leptin secretion, mRNA, and protein expression, which were inhibited by the calcium channel blocker nifedipine (10 μM), the selective calcineurin inhibitor FK506 (1 nM), and the ERK1/2 inhibitor PD98059 (1 μM). The transcription inhibitor actinomycin D (0.1 μM) and the translation inhibitor cycloheximide (1 mM) significantly decreased stretch-induced leptin protein expression. Mechanical stretch or leptin caused an increase in wet weight changes and protein synthesis, considered as hypertrophic markers, while they were inhibited by FK506 (0.1 nM; 1 nM). In addition, stretch or exogenous leptin significantly increased calcineurin activity and MCIP1 expression whereas leptin induced NFAT nuclear translocation in VSMCs. Moreover, in response to stretch or exogenous leptin, the Rho inhibitor C3 exoenzyme (30 ng/mL), the ROCK inhibitor Y-27632 (10 μM), and the actin depolymerization agents Latrunculin B (50 nM) and cytochalasin D (1 μM) reduced calcineurin activation and NFAT nuclear translocation. ERK1/2 phosphorylation was inhibited by FK506 and C3. Conclusions: Mechanical stretch-induced VSMC hypertrophy and leptin

  7. Prolonged CD154 Expression on Pediatric Lupus CD4 T Cells Correlates with Increased CD154 Transcription, Increased NFAT Activity, and Glomerulonephritis

    PubMed Central

    Mehta, Jay; Genin, Anna; Brunner, Michael; Scalzi, Lisabeth V; Mishra, Nilamadhab; Beukelman, Timothy; Cron, Randy Q

    2010-01-01

    Objective To assess CD154 expression in pediatric lupus and explore a transcriptional mechanism explaining dysregulated CD154 expression. Methods Cell surface CD154 expression was examined, pre- and post-activation, on peripheral blood CD4 T cells from 29 children with lupus and matched controls by flow cytometry. CD154 expression was correlated with clinical features, laboratory parameters, and treatments received. Increased CD154 expression on lupus CD4 T cells was correlated with CD154 message and transcription rates by real-time RT-PCR and nuclear run-on assays, respectively. NFAT transcriptional activity and NFAT mRNA levels in lupus CD4 T cells were explored by reporter gene analysis and real-time RT-PCR, respectively. Results CD154 surface protein levels were increased 1.44-fold on lupus CD4 T cells compared to controls at one day post-activation ex vivo. This increase correlated clinically with the presence of nephritis and elevated erythrocyte sedimentation rate. Increased CD154 protein also correlated with increased CD154 mRNA levels and rates of CD154 transcription, particularly at later time-points post-T cell activation. Reporter gene analyses revealed a trend for increased NFAT, but decreased AP-1 and similar NFκB, activity in lupus CD4 T cell compared to controls. Moreover, NFAT1 and, in particular, NFAT2 mRNA levels were notably increased in lupus CD4 T cells compared to controls. Conclusion Following activation, cell surface CD154 is increased on pediatric lupus CD4 T cells compared to controls, and this correlates with the presence of nephritis, increased CD154 transcription rates, and NFAT activity. These results suggest that NFAT/calcineurin inhibitors, such as tacrolimus and cyclosporine, may be beneficial in treating lupus nephritis. PMID:20506525

  8. Nerve growth factor (NGF) regulates activity of nuclear factor of activated T-cells (NFAT) in neurons via the phosphatidylinositol 3-kinase (PI3K)-Akt-glycogen synthase kinase 3β (GSK3β) pathway.

    PubMed

    Kim, Man-Su; Shutov, Leonid P; Gnanasekaran, Aswini; Lin, Zhihong; Rysted, Jacob E; Ulrich, Jason D; Usachev, Yuriy M

    2014-11-01

    The Ca(2+)/calcineurin-dependent transcription factor nuclear factor of activated T-cells (NFAT) plays an important role in regulating many neuronal functions, including excitability, axonal growth, synaptogenesis, and neuronal survival. NFAT can be activated by action potential firing or depolarization that leads to Ca(2+)/calcineurin-dependent dephosphorylation of NFAT and its translocation to the nucleus. Recent data suggest that NFAT and NFAT-dependent functions in neurons can also be potently regulated by NGF and other neurotrophins. However, the mechanisms of NFAT regulation by neurotrophins are not well understood. Here, we show that in dorsal root ganglion sensory neurons, NGF markedly facilitates NFAT-mediated gene expression induced by mild depolarization. The effects of NGF were not associated with changes in [Ca(2+)]i and were independent of phospholipase C activity. Instead, the facilitatory effect of NGF depended on activation of the PI3K/Akt pathway downstream of the TrkA receptor and on inhibition of glycogen synthase kinase 3β (GSK3β), a protein kinase known to phosphorylate NFAT and promote its nuclear export. Knockdown or knockout of NFATc3 eliminated this facilitatory effect. Simultaneous monitoring of EGFP-NFATc3 nuclear translocation and [Ca(2+)]i changes in dorsal root ganglion neurons indicated that NGF slowed the rate of NFATc3 nuclear export but did not affect its nuclear import rate. Collectively, our data suggest that NGF facilitates depolarization-induced NFAT activation by stimulating PI3K/Akt signaling, inactivating GSK3β, and thereby slowing NFATc3 export from the nucleus. We propose that NFAT serves as an integrator of neurotrophin action and depolarization-driven calcium signaling to regulate neuronal gene expression.

  9. Identification and evolution of an NFAT gene involving Branchiostoma belcheri innate immunity.

    PubMed

    Song, Xiaojun; Hu, Jing; Jin, Ping; Chen, Liming; Ma, Fei

    2013-10-01

    The Nuclear Factor of Activated T cells (NFAT) plays an important role in innate and adaptive immunity, but no NFAT genes have yet been identified in amphioxus species. Here we identified and characterized an NFAT-like gene from Branchiostoma belcheri, and also studied extensively the evolutionary history of NFAT family genes. We found that the amphioxus genome contains an AmphiNFAT gene encoding an NFAT homolog. The AmphiNFAT gene was found to be involved in the innate immune response to LPS stimulation in B. belcheri and was ubiquitously and differentially expressed in all investigated tissues. The NFAT family genes were present in a common ancestor with cnidaria, and NFAT1-4 paralogs were lost early in Branchiostoma and Strongylocentrotus genomes. We discovered that NFAT family genes underwent strong purifying selection. Taken together, our findings provide an insight into the innate immune response of amphioxus and the evolution of the NFAT gene family.

  10. Identification and evolution of an NFAT gene involving Branchiostoma belcheri innate immunity.

    PubMed

    Song, Xiaojun; Hu, Jing; Jin, Ping; Chen, Liming; Ma, Fei

    2013-10-01

    The Nuclear Factor of Activated T cells (NFAT) plays an important role in innate and adaptive immunity, but no NFAT genes have yet been identified in amphioxus species. Here we identified and characterized an NFAT-like gene from Branchiostoma belcheri, and also studied extensively the evolutionary history of NFAT family genes. We found that the amphioxus genome contains an AmphiNFAT gene encoding an NFAT homolog. The AmphiNFAT gene was found to be involved in the innate immune response to LPS stimulation in B. belcheri and was ubiquitously and differentially expressed in all investigated tissues. The NFAT family genes were present in a common ancestor with cnidaria, and NFAT1-4 paralogs were lost early in Branchiostoma and Strongylocentrotus genomes. We discovered that NFAT family genes underwent strong purifying selection. Taken together, our findings provide an insight into the innate immune response of amphioxus and the evolution of the NFAT gene family. PMID:23657135

  11. Inhibiting the Calcineurin-NFAT (Nuclear Factor of Activated T Cells) Signaling Pathway with a Regulator of Calcineurin-derived Peptide without Affecting General Calcineurin Phosphatase Activity*S⃞

    PubMed Central

    Mulero, Ma Carme; Aubareda, Anna; Orzáez, Mar; Messeguer, Joaquim; Serrano-Candelas, Eva; Martínez-Hoyer, Sergio; Messeguer, Àngel; Pérez-Payá, Enrique; Pérez-Riba, Mercè

    2009-01-01

    Calcineurin phosphatase plays a crucial role in T cell activation. Dephosphorylation of the nuclear factors of activated T cells (NFATs) by calcineurin is essential for activating cytokine gene expression and, consequently, the immune response. Current immunosuppressive protocols are based mainly on calcineurin inhibitors, cyclosporine A and FK506. Unfortunately, these drugs are associated with severe side effects. Therefore, immunosuppressive agents with higher selectivity and lower toxicity must be identified. The immunosuppressive role of the family of proteins regulators of calcineurin (RCAN, formerly known as DSCR1) which regulate the calcineurin-NFAT signaling pathway, has been described recently. Here, we identify and characterize the minimal RCAN sequence responsible for the inhibition of calcineurin-NFAT signaling in vivo. The RCAN-derived peptide spanning this sequence binds to calcineurin with high affinity. This interaction is competed by a peptide spanning the NFAT PXIXIT sequence, which binds to calcineurin and facilitates NFAT dephosphorylation and activation. Interestingly, the RCAN-derived peptide does not inhibit general calcineurin phosphatase activity, which suggests that it may have a specific immunosuppressive effect on the calcineurin-NFAT signaling pathway. As such, the RCAN-derived peptide could either be considered a highly selective immunosuppressive compound by itself or be used as a new tool for identifying innovative immunosuppressive agents. We developed a low throughput assay, based on the RCAN1-calcineurin interaction, which identifies dipyridamole as an efficient in vivo inhibitor of the calcineurin-NFAT pathway that does not affect calcineurin phosphatase activity. PMID:19189965

  12. PKC-α contributes to high NaCl-induced activation of NFAT5 (TonEBP/OREBP) through MAPK ERK1/2.

    PubMed

    Wang, Hong; Ferraris, Joan D; Klein, Janet D; Sands, Jeff M; Burg, Maurice B; Zhou, Xiaoming

    2015-01-15

    High NaCl in the renal medullary interstitial fluid powers the concentration of urine but can damage cells. The transcription factor nuclear factor of activated T cells 5 (NFAT5) activates the expression of osmoprotective genes. We studied whether PKC-α contributes to the activation of NFAT5. PKC-α protein abundance was greater in the renal medulla than in the cortex. Knockout of PKC-α reduced NFAT5 protein abundance and expression of its target genes in the inner medulla. In human embryonic kidney (HEK)-293 cells, high NaCl increased PKC-α activity, and small interfering RNA-mediated knockdown of PKC-α attenuated high NaCl-induced NFAT5 transcriptional activity. Expression of ERK1/2 protein and phosphorylation of ERK1/2 were higher in the renal inner medulla than in the cortex. Knockout of PKC-α decreased ERK1/2 phosphorylation in the inner medulla, as did knockdown of PKC-α in HEK-293 cells. Also, knockdown of ERK2 reduced high NaCl-dependent NFAT5 transcriptional activity in HEK-293 cells. Combined knockdown of PKC-α and ERK2 had no greater effect than knockdown of either alone. Knockdown of either PKC-α or ERK2 reduced the high NaCl-induced increase of NFAT5 transactivating activity. We have previously found that the high NaCl-induced increase of phosphorylation of Ser(591) on Src homology 2 domain-containing phosphatase 1 (SHP-1-S591-P) contributes to the activation of NFAT5 in cell culture, and here we found high levels of SHP-1-S591-P in the inner medulla. PKC-α has been previously shown to increase SHP-1-S591-P, which raised the possibility that PKC-α might be acting through SHP-1. However, we did not find that knockout of PKC-α in the renal medulla or knockdown in HEK-293 cells affected SHP-1-S591-P. We conclude that PKC-α contributes to high NaCl-dependent activation of NFAT5 through ERK1/2 but not through SHP-1-S591. PMID:25391900

  13. Nuclear presence of nuclear factor of activated T cells (NFAT) c3 and c4 is required for Toll-like receptor-activated innate inflammatory response of monocytes/macrophages.

    PubMed

    Minematsu, Hiroshi; Shin, Mike J; Celil Aydemir, Ayse B; Kim, Kyung-Ok; Nizami, Saqib A; Chung, Gook-Jin; Lee, Francis Young-In

    2011-11-01

    Nuclear factor of activated T cells (NFATs) are crucial transcription factors that tightly control proinflammatory cytokine expression for adaptive immunity in T and B lymphocytes. However, little is known about the role of NFATs for innate immunity in macrophages. In this study, we report that NFAT is required for Toll-like receptor (TLR)-initiated innate immune responses in bone marrow-derived macrophages (BMMs). All TLR ligand stimulation including LPS, a TLR4 ligand, and Pam(3)CSK(4), a TLR1/2 ligand, induced expression of TNF which was inhibited by VIVIT, an NFAT-specific inhibitor peptide. BMMs from NFATc4 knock-out mouse expressed less TNF than wild type. Despite apparent association between NFAT and TNF, LPS did not directly activate NFAT based on NFAT-luciferase reporter assay, whereas NF-κB was inducibly activated by LPS. Instead, macrophage exhibited constitutive NFAT activity which was not increased by LPS and was decreased by VIVIT. Immunocytochemical examination of NFATc1-4 of BMMs exhibited nuclear localization of NFATc3/c4 regardless of LPS stimulation. LPS stimulation did not cause nuclear translocation of NFATc1/c2. Treatment with VIVIT resulted in nuclear export of NFATc3/c4 and inhibited TLR-activated TNF expression, suggesting that nuclear residence of NFATc is required for TLR-related innate immune response. Chromatin immunoprecipitation (ChIP) assay using anti-RNA polymerase II (PolII) antibody suggested that VIVIT decreased PolII binding to TNF gene locus, consistent with VIVIT inhibition of LPS-induced TNF mRNA expression. This study identifies a novel paradigm of innate immune regulation rendered by NFAT which is a well known family of adaptive immune regulatory proteins.

  14. NFAT activation by membrane potential follows a calcium pathway distinct from other activity-related transcription factors in skeletal muscle cells.

    PubMed

    Valdés, Juan Antonio; Gaggero, Eduardo; Hidalgo, Jorge; Leal, Nancy; Jaimovich, Enrique; Carrasco, M Angélica

    2008-03-01

    Depolarization of skeletal muscle cells triggers intracellular Ca2+ signals mediated by ryanodine and inositol 1,4,5-trisphosphate (IP3) receptors. Previously, we have reported that K+-induced depolarization activates transcriptional regulators ERK, cAMP response element-binding protein, c-fos, c-jun, and egr-1 through IP3-dependent Ca2+ release, whereas NF-kappa B activation is elicited by both ryanodine and IP3 receptor-mediated Ca2+ signals. We have further shown that field stimulation with electrical pulses results in an NF-kappa B activation increase dependent of the amount of pulses and independent of their frequency. In this work, we report the results obtained for nuclear factor of activated T cells (NFAT)-mediated transcription and translocation generated by both K+ and electrical stimulation protocols in primary skeletal muscle cells and C2C12 cells. The Ca2+ source for NFAT activation is through release by ryanodine receptors and extracellular Ca2+ entry. We found this activation to be independent of the number of pulses within a physiological range of stimulus frequency and enhanced by long-lasting low-frequency stimulation. Therefore, activation of the NFAT signaling pathway differs from that of NF-kappa B and other transcription factors. Calcineurin enzyme activity correlated well with the relative activation of NFAT translocation and transcription using different stimulation protocols. Furthermore, both K+-induced depolarization and electrical stimulation increased mRNA levels of the type 1 IP3 receptor mediated by calcineurin activity, which suggests that depolarization may regulate IP3 receptor transcription. These results confirm the presence of at least two independent pathways for excitation-transcription coupling in skeletal muscle cells, both dependent on Ca2+ release and triggered by the same voltage sensor but activating different intracellular release channels. PMID:18184878

  15. The azetidine derivative, KHG26792 protects against ATP-induced activation of NFAT and MAPK pathways through P2X7 receptor in microglia.

    PubMed

    Kim, Eun-A; Cho, Chang Hun; Kim, Jiae; Hahn, Hoh-Gyu; Choi, Soo Young; Yang, Seung-Ju; Cho, Sung-Woo

    2015-12-01

    Azetidine derivatives are of interest for drug development because they may be useful therapeutic agents. However, their mechanisms of action remain to be completely elucidated. Here, we have investigated the effects of 3-(naphthalen-2-yl(propoxy)methyl)azetidine hydrochloride (KHG26792) on ATP-induced activation of NFAT and MAPK through P2X7 receptor in the BV-2 mouse microglial cell line. KHG26792 decreased ATP-induced TNF-α release from BV-2 microglia by suppressing, at least partly, P2X7 receptor stimulation. KHG26792 also inhibited the ATP-induced increase in IL-6, PGE2, NO, ROS, CXCL2, and CCL3. ATP induced NFAT activation through P2X7 receptor, with KHG26792 reducing the ATP-induced NFAT activation. KHG26792 inhibited an ATP-induced increase in iNOS protein and ERK phosphorylation. KHG26792 prevented an ATP-induced increase in MMP-9 activity through the P2X7 receptor as a result of degradation of TIMP-1 by cathepsin B. Our data provide mechanistic insights into the role of KHG26792 in the inhibition of TNF-α produced via P2X7 receptor-mediated activation of NFAT and MAPK pathways in ATP-treated BV-2 cells. This study highlights the potential use of KHG26792 as a therapeutic agent for the many diseases of the CNS related to activated microglia.

  16. Calcineurin Aβ regulates NADPH oxidase (Nox) expression and activity via nuclear factor of activated T cells (NFAT) in response to high glucose.

    PubMed

    Williams, Clintoria R; Gooch, Jennifer L

    2014-02-21

    Hypertrophy is an adaptive response that enables organs to appropriately meet increased functional demands. Previously, we reported that calcineurin (Cn) is required for glomerular and whole kidney hypertrophy in diabetic rodents (Gooch, J. L., Barnes, J. L., Garcia, S., and Abboud, H. E. (2003). Calcineurin is activated in diabetes and is required for glomerular hypertrophy and ECM accumulation. Am. J. Physiol. Renal Physiol. 284, F144-F154; Reddy, R. N., Knotts, T. L., Roberts, B. R., Molkentin, J. D., Price, S. R., and Gooch, J. L. (2011). Calcineurin Aβ is required for hypertrophy but not matrix expansion in the diabetic kidney. J. Cell Mol. Med. 15, 414-422). Because studies have also implicated the reactive oxygen species-generating enzymes NADPH oxidases (Nox) in diabetic kidney responses, we tested the hypothesis that Nox and Cn cooperate in a common signaling pathway. First, we examined the role of the two main isoforms of Cn in hypertrophic signaling. Using primary kidney cells lacking a catalytic subunit of Cn (CnAα(-/-) or CnAβ(-/-)), we found that high glucose selectively activates CnAβ, whereas CnAα is constitutively active. Furthermore, CnAβ but not CnAα mediates hypertrophy. Next, we found that chronic reactive oxygen species generation in response to high glucose is attenuated in CnAβ(-/-) cells, suggesting that Cn is upstream of Nox. Consistent with this, loss of CnAβ reduces basal expression and blocks high glucose induction of Nox2 and Nox4. Inhibition of nuclear factor of activated T cells (NFAT), a CnAβ-regulated transcription factor, decreases Nox2 and Nox4 expression, whereas NFAT overexpression increases Nox2 and Nox4, indicating that the CnAβ/NFAT pathway modulates Nox. These data reveal that the CnAβ/NFAT pathway regulates Nox and plays an important role in high glucose-mediated hypertrophic responses in the kidney.

  17. Phagocytosis-dependent activation of a TLR9–BTK–calcineurin–NFAT pathway co-ordinates innate immunity to Aspergillus fumigatus

    PubMed Central

    Herbst, Susanne; Shah, Anand; Mazon Moya, Maria; Marzola, Vanessa; Jensen, Barbara; Reed, Anna; Birrell, Mark A; Saijo, Shinobu; Mostowy, Serge; Shaunak, Sunil; Armstrong-James, Darius

    2015-01-01

    Transplant recipients on calcineurin inhibitors are at high risk of invasive fungal infection. Understanding how calcineurin inhibitors impair fungal immunity is a key priority for defining risk of infection. Here, we show that the calcineurin inhibitor tacrolimus impairs clearance of the major mould pathogen Aspergillus fumigatus from the airway, by inhibiting macrophage inflammatory responses. This leads to defective early neutrophil recruitment and fungal clearance. We confirm these findings in zebrafish, showing an evolutionarily conserved role for calcineurin signalling in neutrophil recruitment during inflammation. We find that calcineurin–NFAT activation is phagocytosis dependent and collaborates with NF-κB for TNF-α production. For yeast zymosan particles, activation of macrophage calcineurin–NFAT occurs via the phagocytic Dectin-1–spleen tyrosine kinase pathway, but for A. fumigatus, activation occurs via a phagosomal TLR9-dependent and Bruton's tyrosine kinase-dependent signalling pathway that is independent of MyD88. We confirm the collaboration between NFAT and NF-κB for TNF-α production in primary alveolar macrophages. These observations identify inhibition of a newly discovered macrophage TLR9–BTK–calcineurin–NFAT signalling pathway as a key immune defect that leads to organ transplant-related invasive aspergillosis. PMID:25637383

  18. Angiotensin II activates the calcineurin/NFAT signaling pathway and induces cyclooxygenase-2 expression in rat endometrial stromal cells.

    PubMed

    Abraham, Florencia; Sacerdoti, Flavia; De León, Romina; Gentile, Teresa; Canellada, Andrea

    2012-01-01

    Cyclooxygenase (COX)-2, the inducible isoform of cyclooxygenase, plays a role in the process of uterine decidualization and blastocyst attachment. On the other hand, overexpression of COX-2 is involved in the proliferation of the endometrial tissue during endometriosis. Deregulation of the renin-angiotensin-system plays a role in the pathophysiology of endometriosis and pre-eclampsia. Angiotensin II increases intracellular Ca(2+) concentration by targeting phospholypase C-gamma in endometrial stromal cells (ESC). A key element of the cellular response to Ca(2+) signals is the activity of the Ca(2+)- and calmodulin-dependent phosphatase calcineurin. Our first aim was to study whether angiotensin II stimulated Cox-2 gene expression in rat ESC and to analyze whether calcineurin activity was involved. In cells isolated from non-pregnant uteri, COX-2 expression--both mRNA and protein--was induced by co-stimulation with phorbol ester and calcium ionophore (PIo), as well as by angiotensin II. Pretreatment with the calcineurin inhibitor cyclosporin A inhibited this induction. We further analyzed the role of the calcineurin/NFAT signaling pathway in the induction of Cox-2 gene expression in non-pregnant rat ESC. Cyclosporin A abolished NFATc1 dephosphorylation and translocation to the nucleus. Cyclosporin A also inhibited the transcriptional activity driven by the Cox-2 promoter. Exogenous expression of the peptide VIVIT -specific inhibitor of calcineurin/NFAT binding- blocked the activation of Cox-2 promoter and the up-regulation of COX-2 protein in these cells. Finally we analyzed Cox-2 gene expression in ESC of early-pregnant rats. COX-2 expression--both mRNA and protein--was induced by stimulation with PIo as well as by angiotensin II. This induction appears to be calcineurin independent, since it was not abrogated by cyclosporin A. In conclusion, angiotensin II induced Cox-2 gene expression by activating the calcineurin/NFAT signaling pathway in endometrial stromal

  19. Angiotensin II Activates the Calcineurin/NFAT Signaling Pathway and Induces Cyclooxygenase-2 Expression in Rat Endometrial Stromal Cells

    PubMed Central

    Abraham, Florencia; Sacerdoti, Flavia; De León, Romina; Gentile, Teresa; Canellada, Andrea

    2012-01-01

    Cyclooxygenase (COX)-2, the inducible isoform of cyclooxygenase, plays a role in the process of uterine decidualization and blastocyst attachment. On the other hand, overexpression of COX-2 is involved in the proliferation of the endometrial tissue during endometriosis. Deregulation of the renin-angiotensin-system plays a role in the pathophysiology of endometriosis and pre-eclampsia. Angiotensin II increases intracellular Ca2+ concentration by targeting phospholypase C-gamma in endometrial stromal cells (ESC). A key element of the cellular response to Ca2+ signals is the activity of the Ca2+- and calmodulin-dependent phosphatase calcineurin. Our first aim was to study whether angiotensin II stimulated Cox-2 gene expression in rat ESC and to analyze whether calcineurin activity was involved. In cells isolated from non-pregnant uteri, COX-2 expression -both mRNA and protein- was induced by co-stimulation with phorbol ester and calcium ionophore (PIo), as well as by angiotensin II. Pretreatment with the calcineurin inhibitor cyclosporin A inhibited this induction. We further analyzed the role of the calcineurin/NFAT signaling pathway in the induction of Cox-2 gene expression in non-pregnant rat ESC. Cyclosporin A abolished NFATc1 dephosphorylation and translocation to the nucleus. Cyclosporin A also inhibited the transcriptional activity driven by the Cox-2 promoter. Exogenous expression of the peptide VIVIT -specific inhibitor of calcineurin/NFAT binding- blocked the activation of Cox-2 promoter and the up-regulation of COX-2 protein in these cells. Finally we analyzed Cox-2 gene expression in ESC of early-pregnant rats. COX-2 expression -both mRNA and protein- was induced by stimulation with PIo as well as by angiotensin II. This induction appears to be calcineurin independent, since it was not abrogated by cyclosporin A. In conclusion, angiotensin II induced Cox-2 gene expression by activating the calcineurin/NFAT signaling pathway in endometrial stromal cells of

  20. Transcriptional regulation of the c-Myc promoter by NFAT1 involves negative and positive NFAT-responsive elements.

    PubMed

    Mognol, Giuliana P; de Araujo-Souza, Patricia S; Robbs, Bruno K; Teixeira, Leonardo K; Viola, Joao P B

    2012-03-01

    A number of physiological processes in both normal and cancer cells are regulated by the proto-oncogene c-Myc. Among them, processes such as cell cycle regulation, apoptosis, angiogenesis and metastasis are also controlled by the nuclear factor of activated T cells (NFAT) family of transcription factors. It is already known that NFAT upregulates c-Myc expression by binding to an element located in the minimal c-Myc promoter. However, the importance of other NFAT sites in the context of the full promoter has not been evaluated. In this work, we demonstrate that the regulation of c-Myc by NFAT1 is more complex than previously conceived. In addition to the proximal site, NFAT1 directly binds to distal sites in the c-Myc promoter with different affinities. Promoter deletions and site-directed mutagenesis of NFAT binding sites in HEK293T cells suggest that in NFAT1-mediated transactivation, some NFAT elements are negative and dominant and others are positive and recessive. Furthermore, we demonstrate that cooperation with partner proteins, such as p300, enhances NFAT1-mediated transactivation of the c-Myc promoter. At last, the newly identified sites are also responsive to NFAT2 in HEK293T cells. However, in NIH3T3 cells, the regulation mediated by NFAT proteins is not dependent on the known NFAT sites, including the site previously described. Thus, our data suggest that the contribution of NFAT to the regulation of c-Myc expression may depend on a balance between the binding to positive and negative NFAT-responsive elements and cooperation with transcriptional cofactors, which may differ according to the context and/or cell type.

  1. Interleukin-7 induces HIV replication in primary naive T cells through a nuclear factor of activated T cell (NFAT)-dependent pathway

    SciTech Connect

    Managlia, Elizabeth Z. . E-mail: lalharth@rush.edu

    2006-07-05

    Interleukin (IL)-7 plays several roles critical to T cell maturation, survival, and homeostasis. Because of these functions, IL-7 is under investigation as an immune-modulator for therapeutic use in lymphopenic clinical conditions, including HIV. We reported that naive T cells, typically not permissive to HIV, can be productively infected when pre-treated with IL-7. We evaluated the mechanism by which IL-7-mediates this effect. IL-7 potently up-regulated the transcriptional factor NFAT, but had no effect on NF{kappa}B. Blocking NFAT activity using a number of reagents, such as Cyclosporin A, FK-506, or the NFAT-specific inhibitor known as VIVIT peptide, all markedly reduced IL-7-mediated induction of HIV replication in naive T cells. Additional neutralization of cytokines present in IL-7-treated cultures and/or those that have NFAT-binding sequences within their promotors indicated that IL-10, IL-4, and most significantly IFN{gamma}, all contribute to IL-7-induction of HIV productive replication in naive T cells. These data clarify the mechanism by which IL-7 can overcome the block to HIV productive infection in naive T cells, despite their quiescent cell status. These findings are relevant to the treatment of HIV disease and understanding HIV pathogenesis in the naive CD4+ T cell compartment, especially in light of the vigorous pursuit of IL-7 as an in vivo immune modulator.

  2. Regulation of Akt/PKB activity by P21-activated kinase in cardiomyocytes.

    PubMed

    Mao, Kai; Kobayashi, Satoru; Jaffer, Zahara M; Huang, Yuan; Volden, Paul; Chernoff, Jonathan; Liang, Qiangrong

    2008-02-01

    Akt/PKB is a critical regulator of cardiac function and morphology, and its activity is governed by dual phosphorylation at active loop (Thr308) by phosphoinositide-dependent protein kinase-1 (PDK1) and at carboxyl-terminal hydrophobic motif (Ser473) by a putative PDK2. P21-activated kinase-1 (Pak1) is a serine/threonine protein kinase implicated in the regulation of cardiac hypertrophy and contractility and was shown previously to activate Akt through an undefined mechanism. Here we report Pak1 as a potential PDK2 that is essential for Akt activity in cardiomyocytes. Both Pak1 and Akt can be activated by multiple hypertrophic stimuli or growth factors in a phosphatidylinositol-3-kinase (PI3K)-dependent manner. Pak1 overexpression induces Akt phosphorylation at both Ser473 and Thr308 in cardiomyocytes. Conversely, silencing or inactivating Pak1 gene diminishes Akt phosphorylation in vitro and in vivo. Purified Pak1 can directly phosphorylate Akt only at Ser473, suggesting that Pak1 may be a relevant PDK2 responsible for AKT Ser473 phosphorylation in cardiomyocytes. In addition, Pak1 protects cardiomyocytes from cell death, which is blocked by Akt inhibition. Our results connect two important regulators of cellular physiological functions and provide a potential mechanism for Pak1 signaling in cardiomyocytes. PMID:18054038

  3. Application of intact cell-based NFAT-β-lactamase reporter assay for Pasteurella multocida toxin-mediated activation of calcium signaling pathway

    PubMed Central

    Luo, Shuhong; Ho, Mengfei; Wilson, Brenda A.

    2009-01-01

    Pasteurella multocida toxin (PMT) stimulates and subsequently uncouples phospholipase C β1 (PLCβ1) signal transduction through its selective action on the alpha subunit of the Gq protein. Here, we describe the application of an NFAT-β-lactamase reporter assay as a functional readout for PMT-induced activation of the Gq-protein-coupled PLCβ1-IP3-Ca2+ signaling pathway. Use of the NFAT-β-lactamase reporter assay with a cell-permeable fluorogenic substrate provides high sensitivity due to the absence of endogenous β-lactamase activity in mammalian cells. This assay system was optimized for cell density, dose and time exposure of PMT stimulation. It is suited for quantitative characterization of PMT activity in mammalian cells and for use as a high-throughput screening method for PMT deletion and point mutants suitable for vaccine development. This method has application for diagnostic screening of clinical isolates of toxinogenic P. multocida. PMID:18190943

  4. The effect of nuclear factor of activated T-cells (NFAT) in kidney I/R mediated by C5a/C5aR.

    PubMed

    Zhang, Ze-Ying; Wu, Yang-Qian; Luo, Heng; Liu, Dong-Xu

    2015-01-01

    To investigate the relationship between NFAT and C5a/C5aR in C5a/C5aR-mediated kidney Ischemia/reperfusion (I/R) injury, the rats' NRK-52E cell line was used in this study and was distributed into 4 groups, I: the normal control (NC), II: the ischemia/reperfusion (I/R) injury cell model (MG), III: the ischemia/reperfusion (I/R) injury cell model treated with C5a (50 nmol/l) (MG + C5a), IV: the ischemia/reperfusion (I/R) injury cell model treated with C5aR antagonist (2.5 μmol/l) (MG + anti-C5aR). Reverse transcription polymerase chain reaction (RT-PCR), western blot, immunofluorescence and flow cytometry were performed. Nuclear Factor Activated T Cell (NFAT), tumor necrosis factor-α (TNF-α) and interleukin (IL-6) were detected in this study. The results of immunofluorescence showed that NFAT had a nuclear translocation phenomenon during the study. The RT-PCR and WB data indicated that the expression of TNF-α and IL-6 in group III were higher than any other groups. Apoptosis in group III was much serious than other groups. All the results in this study showed that NFAT plays an important role in ischemia/reperfusion injury, it can be induced to up-regulate the inflammatory factor TNF-α and IL-6 by the complement system member C5a/C5aR.

  5. Nuclear factor of activated T cell (NFAT) transcription proteins regulate genes involved in adipocyte metabolism and lipolysis

    SciTech Connect

    Holowachuk, Eugene W. . E-mail: geneh@telenet.net

    2007-09-21

    NFAT involvement in adipocyte physiological processes was examined by treatment with CsA and/or GSK3{beta} inhibitors (Li{sup +} or TZDZ-8), which prevent or increase NFAT nuclear translocation, respectively. CsA treatment reduced basal and TNF{alpha}-induced rates of lipolysis by 50%. Adipocytes preincubated with Li{sup +} or TZDZ-8 prior to CsA and/or TNF{alpha}, exhibited enhanced basal rates of lipolysis and complete inhibition of CsA-mediated decreased rates of lipolysis. CsA treatment dramatically reduced the mRNA levels of adipocyte-specific genes (aP2, HSL, PPAR{gamma}, ACS and Adn), compared with control or TNF{alpha}-treatment, whereas Li{sup +} pretreatment blocked the inhibitory effects of CsA, and mRNA levels of aP2, HSL, PPAR{gamma}, and ACS were found at or above control levels. NFAT nuclear localization, assessed by EMSA, confirmed that CsA or Li{sup +} treatments inhibited or increased NFAT nuclear translocation, respectively. These results show that NFAT proteins in mature adipocytes participate in the transcriptional control of genes involved in adipocyte metabolism and lipolysis.

  6. Blueberry polyphenols prevent cardiomyocyte death by preventing calpain activation and oxidative stress.

    PubMed

    Louis, Xavier Lieben; Thandapilly, Sijo Joseph; Kalt, Wilhelmina; Vinqvist-Tymchuk, Melinda; Aloud, Basma Milad; Raj, Pema; Yu, Liping; Le, Hoa; Netticadan, Thomas

    2014-08-01

    The purpose of this study was to examine the efficacy of an aqueous wild blueberry extract and five wild blueberry polyphenol fractions on an in vitro model of heart disease. Adult rat cardiomyocytes were pretreated with extract and fractions, and then exposed to norepinephrine (NE). Cardiomyocyte hypertrophy, cell death, oxidative stress, apoptosis and cardiomyocyte contractile function as well as the activities of calpain, superoxide dismutase (SOD) and catalase (CAT) were measured in cardiomyocytes treated with and without NE and blueberry fraction (BF). Four of five blueberry fractions prevented cell death and cardiomyocyte hypertrophy induced by NE. Total phenolic fraction was used for all further analysis. The NE-induced increase in oxidative stress, nuclear condensation, calpain activity and lowering of SOD and CAT activities were prevented upon pretreatment with BF. Reduced contractile function was also significantly improved with BF pretreatment. Blueberry polyphenols prevent NE-induced adult cardiomyocyte hypertrophy and cell death. The protective effects of BF may be in part attributed to a reduction in calpain activity and oxidative stress.

  7. Potent Anti-Inflammatory Activity of Ursolic Acid, a Triterpenoid Antioxidant, Is Mediated through Suppression of NF-κB, AP-1 and NF-AT

    PubMed Central

    Checker, Rahul; Sandur, Santosh K.; Sharma, Deepak; Patwardhan, Raghavendra S.; Jayakumar, S.; Kohli, Vineet; Sethi, Gautam; Aggarwal, Bharat B.; Sainis, Krishna B.

    2012-01-01

    Background Ursolic acid (UA), a pentacyclic triterpenoid carboxylic acid, is the major component of many plants including apples, basil, cranberries, peppermint, rosemary, oregano and prunes and has been reported to possess antioxidant and anti-tumor properties. These properties of UA have been attributed to its ability to suppress NF-κB (nuclear factor kappa B) activation. Since NF-κB, in co-ordination with NF-AT (nuclear factor of activated T cells) and AP-1(activator protein-1), is known to regulate inflammatory genes, we hypothesized that UA might exhibit potent anti-inflammatory effects. Methodology/Principal Findings The anti-inflammatory effects of UA were assessed in activated T cells, B cells and macrophages. Effects of UA on ERK, JNK, NF-κB, AP-1 and NF-AT were studied to elucidate its mechanism of action. In vivo efficacy of UA was studied using mouse model of graft-versus-host disease. UA inhibited activation, proliferation and cytokine secretion in T cells, B cells and macrophages. UA inhibited mitogen-induced up-regulation of activation markers and co-stimulatory molecules in T and B cells. It inhibited mitogen-induced phosphorylation of ERK and JNK and suppressed the activation of immunoregulatory transcription factors NF-κB, NF-AT and AP-1 in lymphocytes. Treatment of cells with UA prior to allogenic transplantation significantly delayed induction of acute graft-versus-host disease in mice and also significantly reduced the serum levels of pro-inflammatory cytokines IL-6 and IFN-γ. UA treatment inhibited T cell activation even when added post-mitogenic stimulation demonstrating its therapeutic utility as an anti-inflammatory agent. Conclusions/Significance The present study describes the detailed mechanism of anti-inflammatory activity of UA. Further, UA may find application in the treatment of inflammatory disorders. PMID:22363615

  8. TIM-3 Suppresses Anti-CD3/CD28-Induced TCR Activation and IL-2 Expression through the NFAT Signaling Pathway.

    PubMed

    Tomkowicz, Brian; Walsh, Eileen; Cotty, Adam; Verona, Raluca; Sabins, Nina; Kaplan, Fred; Santulli-Marotto, Sandy; Chin, Chen-Ni; Mooney, Jill; Lingham, Russell B; Naso, Michael; McCabe, Timothy

    2015-01-01

    TIM-3 (T cell immunoglobulin and mucin-domain containing protein 3) is a member of the TIM family of proteins that is preferentially expressed on Th1 polarized CD4+ and CD8+ T cells. Recent studies indicate that TIM-3 serves as a negative regulator of T cell function (i.e. T cell dependent immune responses, proliferation, tolerance, and exhaustion). Despite having no recognizable inhibitory signaling motifs, the intracellular tail of TIM-3 is apparently indispensable for function. Specifically, the conserved residues Y265/Y272 and surrounding amino acids appear to be critical for function. Mechanistically, several studies suggest that TIM-3 can associate with interleukin inducible T cell kinase (ITK), the Src kinases Fyn and Lck, and the p85 phosphatidylinositol 3-kinase (PI3K) adaptor protein to positively or negatively regulate IL-2 production via NF-κB/NFAT signaling pathways. To begin to address this discrepancy, we examined the effect of TIM-3 in two model systems. First, we generated several Jurkat T cell lines stably expressing human TIM-3 or murine CD28-ECD/human TIM-3 intracellular tail chimeras and examined the effects that TIM-3 exerts on T cell Receptor (TCR)-mediated activation, cytokine secretion, promoter activity, and protein kinase association. In this model, our results demonstrate that TIM-3 inhibits several TCR-mediated phenotypes: i) NF-kB/NFAT activation, ii) CD69 expression, and iii) suppression of IL-2 secretion. To confirm our Jurkat cell observations we developed a primary human CD8+ cell system that expresses endogenous levels of TIM-3. Upon TCR ligation, we observed the loss of NFAT reporter activity and IL-2 secretion, and identified the association of Src kinase Lck, and PLC-γ with TIM-3. Taken together, our results support the conclusion that TIM-3 is a negative regulator of TCR-function by attenuating activation signals mediated by CD3/CD28 co-stimulation.

  9. TIM-3 Suppresses Anti-CD3/CD28-Induced TCR Activation and IL-2 Expression through the NFAT Signaling Pathway

    PubMed Central

    Tomkowicz, Brian; Walsh, Eileen; Cotty, Adam; Verona, Raluca; Sabins, Nina; Kaplan, Fred; Santulli-Marotto, Sandy; Chin, Chen-Ni; Mooney, Jill; Lingham, Russell B.; Naso, Michael; McCabe, Timothy

    2015-01-01

    TIM-3 (T cell immunoglobulin and mucin-domain containing protein 3) is a member of the TIM family of proteins that is preferentially expressed on Th1 polarized CD4+ and CD8+ T cells. Recent studies indicate that TIM-3 serves as a negative regulator of T cell function (i.e. T cell dependent immune responses, proliferation, tolerance, and exhaustion). Despite having no recognizable inhibitory signaling motifs, the intracellular tail of TIM-3 is apparently indispensable for function. Specifically, the conserved residues Y265/Y272 and surrounding amino acids appear to be critical for function. Mechanistically, several studies suggest that TIM-3 can associate with interleukin inducible T cell kinase (ITK), the Src kinases Fyn and Lck, and the p85 phosphatidylinositol 3-kinase (PI3K) adaptor protein to positively or negatively regulate IL-2 production via NF-κB/NFAT signaling pathways. To begin to address this discrepancy, we examined the effect of TIM-3 in two model systems. First, we generated several Jurkat T cell lines stably expressing human TIM-3 or murine CD28-ECD/human TIM-3 intracellular tail chimeras and examined the effects that TIM-3 exerts on T cell Receptor (TCR)-mediated activation, cytokine secretion, promoter activity, and protein kinase association. In this model, our results demonstrate that TIM-3 inhibits several TCR-mediated phenotypes: i) NF-kB/NFAT activation, ii) CD69 expression, and iii) suppression of IL-2 secretion. To confirm our Jurkat cell observations we developed a primary human CD8+ cell system that expresses endogenous levels of TIM-3. Upon TCR ligation, we observed the loss of NFAT reporter activity and IL-2 secretion, and identified the association of Src kinase Lck, and PLC-γ with TIM-3. Taken together, our results support the conclusion that TIM-3 is a negative regulator of TCR-function by attenuating activation signals mediated by CD3/CD28 co-stimulation. PMID:26492563

  10. A critical role of cardiac fibroblast-derived exosomes in activating renin angiotensin system in cardiomyocytes.

    PubMed

    Lyu, Linmao; Wang, Hui; Li, Bin; Qin, Qingyun; Qi, Lei; Nagarkatti, Mitzi; Nagarkatti, Prakash; Janicki, Joseph S; Wang, Xing Li; Cui, Taixing

    2015-12-01

    Chronic activation of the myocardial renin angiotensin system (RAS) elevates the local level of angiotensin II (Ang II) thereby inducing pathological cardiac hypertrophy, which contributes to heart failure. However, the precise underlying mechanisms have not been fully delineated. Herein we report a novel paracrine mechanism between cardiac fibroblasts (CF)s and cardiomyocytes whereby Ang II induces pathological cardiac hypertrophy. In cultured CFs, Ang II treatment enhanced exosome release via the activation of Ang II receptor types 1 (AT1R) and 2 (AT2R), whereas lipopolysaccharide, insulin, endothelin (ET)-1, transforming growth factor beta (TGFβ)1 or hydrogen peroxide did not. The CF-derived exosomes upregulated the expression of renin, angiotensinogen, AT1R, and AT2R, downregulated angiotensin-converting enzyme 2, and enhanced Ang II production in cultured cardiomyocytes. In addition, the CF exosome-induced cardiomyocyte hypertrophy was blocked by both AT1R and AT2R antagonists. Exosome inhibitors, GW4869 and dimethyl amiloride (DMA), inhibited CF-induced cardiomyocyte hypertrophy with little effect on Ang II-induced cardiomyocyte hypertrophy. Mechanistically, CF exosomes upregulated RAS in cardiomyocytes via the activation of mitogen-activated protein kinases (MAPKs) and Akt. Finally, Ang II-induced exosome release from cardiac fibroblasts and pathological cardiac hypertrophy were dramatically inhibited by GW4869 and DMA in mice. These findings demonstrate that Ang II stimulates CFs to release exosomes, which in turn increase Ang II production and its receptor expression in cardiomyocytes, thereby intensifying Ang II-induced pathological cardiac hypertrophy. Accordingly, specific targeting of Ang II-induced exosome release from CFs may serve as a novel therapeutic approach to treat cardiac pathological hypertrophy and heart failure.

  11. An Altered gp100 Peptide Ligand with Decreased Binding by TCR and CD8α Dissects T Cell Cytotoxicity from Production of Cytokines and Activation of NFAT.

    PubMed

    Schaft, Niels; Coccoris, Miriam; Drexhage, Joost; Knoop, Christiaan; de Vries, I Jolanda M; Adema, Gosse J; Debets, Reno

    2013-01-01

    Altered peptide ligands (APLs) provide useful tools to study T cell activation and potentially direct immune responses to improve treatment of cancer patients. To better understand and exploit APLs, we studied the relationship between APLs and T cell function in more detail. Here, we tested a broad panel of gp100280-288 APLs with respect to T cell cytotoxicity, production of cytokines, and activation of Nuclear Factor of Activated T cells (NFAT) by human T cells gene-engineered with a gp100-HLA-A2-specific TCRαβ. We demonstrated that gp100-specific cytotoxicity, production of cytokines, and activation of NFAT were not affected by APLs with single amino acid substitutions, except for an APL with an amino acid substitution at position 3 (APL A3), which did not elicit any T cell response. A gp100 peptide with a double amino acid mutation (APL S4S6) elicited T cell cytotoxicity and production of IFNγ, and to a lesser extent TNFα, IL-4, and IL-5, but not production of IL-2 and IL-10, or activation of NFAT. Notably, T cell receptor (TCR)-mediated functions showed decreases in sensitivities for S4S6 versus gp100 wild-type (wt) peptide, which were minor for cytotoxicity but at least a 1000-fold more prominent for the production of cytokines. TCR-engineered T cells did not bind A3-HLA-A2, but did bind S4S6-HLA-A2 although to a lowered extent compared to wt peptide-HLA-A2. Moreover, S4S6-induced T cell function demonstrated an enhanced dependency on CD8α. Taken together, most gp100 APLs functioned as agonists, but A3 and S4S6 peptides acted as a null ligand and partial agonist, respectively. Our results further suggest that TCR-mediated cytotoxicity can be dissected from production of cytokines and activation of NFAT, and that the agonist potential of peptide mutants relates to the extent of binding by TCR and CD8α. These findings may facilitate the design of APLs to advance the study of T cell activation and their use for therapeutic applications. PMID:24027572

  12. An Altered gp100 Peptide Ligand with Decreased Binding by TCR and CD8α Dissects T Cell Cytotoxicity from Production of Cytokines and Activation of NFAT

    PubMed Central

    Schaft, Niels; Coccoris, Miriam; Drexhage, Joost; Knoop, Christiaan; de Vries, I. Jolanda M.; Adema, Gosse J.; Debets, Reno

    2013-01-01

    Altered peptide ligands (APLs) provide useful tools to study T cell activation and potentially direct immune responses to improve treatment of cancer patients. To better understand and exploit APLs, we studied the relationship between APLs and T cell function in more detail. Here, we tested a broad panel of gp100280–288 APLs with respect to T cell cytotoxicity, production of cytokines, and activation of Nuclear Factor of Activated T cells (NFAT) by human T cells gene-engineered with a gp100-HLA-A2-specific TCRαβ. We demonstrated that gp100-specific cytotoxicity, production of cytokines, and activation of NFAT were not affected by APLs with single amino acid substitutions, except for an APL with an amino acid substitution at position 3 (APL A3), which did not elicit any T cell response. A gp100 peptide with a double amino acid mutation (APL S4S6) elicited T cell cytotoxicity and production of IFNγ, and to a lesser extent TNFα, IL-4, and IL-5, but not production of IL-2 and IL-10, or activation of NFAT. Notably, T cell receptor (TCR)-mediated functions showed decreases in sensitivities for S4S6 versus gp100 wild-type (wt) peptide, which were minor for cytotoxicity but at least a 1000-fold more prominent for the production of cytokines. TCR-engineered T cells did not bind A3-HLA-A2, but did bind S4S6-HLA-A2 although to a lowered extent compared to wt peptide-HLA-A2. Moreover, S4S6-induced T cell function demonstrated an enhanced dependency on CD8α. Taken together, most gp100 APLs functioned as agonists, but A3 and S4S6 peptides acted as a null ligand and partial agonist, respectively. Our results further suggest that TCR-mediated cytotoxicity can be dissected from production of cytokines and activation of NFAT, and that the agonist potential of peptide mutants relates to the extent of binding by TCR and CD8α. These findings may facilitate the design of APLs to advance the study of T cell activation and their use for therapeutic applications. PMID:24027572

  13. How do kinases contribute to tonicity-dependent regulation of the transcription factor NFAT5?

    PubMed Central

    Zhou, Xiaoming

    2016-01-01

    NFAT5 plays a critical role in maintaining the renal functions. Its dis-regulation in the kidney leads to or is associated with certain renal diseases or disorders, most notably the urinary concentration defect. Hypertonicity, which the kidney medulla is normally exposed to, activates NFAT5 through phosphorylation of a signaling molecule or NFAT5 itself. Hypotonicity inhibits NFAT5 through a similar mechanism. More than a dozen of protein and lipid kinases have been identified to contribute to tonicity-dependent regulation of NFAT5. Hypertonicity activates NFAT5 by increasing its nuclear localization and transactivating activity in the early phase and protein abundance in the late phase. The known mechanism for inhibition of NFAT5 by hypotonicity is a decrease of nuclear NFAT5. The present article reviews the effect of each kinase on NFAT5 nuclear localization, transactivation and protein abundance, and the relationship among these kinases, if known. Cyclosporine A and tacrolimus suppress immune reactions by inhibiting the phosphatase calcineurin-dependent activation of NFAT1. It is hoped that this review would stimulate the interest to seek explanations from the NFAT5 regulatory pathways for certain clinical presentations and to explore novel therapeutic approaches based on the pathways. On the basic science front, this review raises two interesting questions. The first one is how these kinases can specifically signal to NFAT5 in the context of hypertonicity or hypotonicity, because they also regulate other cellular activities and even opposite activities in some cases. The second one is why these many kinases, some of which might have redundant functions, are needed to regulate NFAT5 activity. This review reiterates the concept of signaling through cooperation. Cells need these kinases working in a coordinated way to provide the signaling specificity that is lacking in the individual one. Redundancy in regulation of NFAT5 is a critical strategy for cells to

  14. NFAT signaling in osteoblasts regulates the hematopoietic niche in the bone microenvironment.

    PubMed

    Sesler, Cheryl L; Zayzafoon, Majd

    2013-01-01

    Osteoblasts support hematopoietic cell development, including B lymphopoiesis. We have previously shown that the nuclear factor of activated T cells (NFAT) negatively regulates osteoblast differentiation and bone formation. Interestingly, in smooth muscle, NFAT has been shown to regulate the expression of vascular cellular adhesion molecule-1 (VCAM-1), a mediator of cell adhesion and signaling during leukocyte development. To examine whether NFAT signaling in osteoblasts regulates hematopoietic development in vivo, we generated a mouse model expressing dominant-negative NFAT driven by the 2.3 kb fragment of the collagen-αI promoter to disrupt NFAT activity in osteoblasts (dnNFAT(OB)). Bone histomorphometry showed that dnNFAT(OB) mice have significant increases in bone volume (44%) and mineral apposition rate (131%) and decreased trabecular thickness (18%). In the bone microenvironment, dnNFAT(OB) mice displayed a significant increase (87%) in Lineage(-)cKit(+)Sca-1(+) (LSK) cells and significant decreases in B220(+)CD19(-)IgM(-) pre-pro-B cells (41%) and B220(+)CD19(+)IgM(+) immature B cells (40%). Concurrent with these findings, LSK cell differentiation into B220(+) cells was inhibited when cocultured on differentiated primary osteoblasts harvested from dnNFAT(OB) mice. Gene expression and protein levels of VCAM-1 in osteoblasts decreased in dnNFAT(OB) mice compared to controls. These data suggest that osteoblast-specific NFAT activity mediates early B lymphopoiesis, possibly by regulating VCAM-1 expression on osteoblasts.

  15. TRPC6 channel activation promotes neonatal glomerular mesangial cell apoptosis via calcineurin/NFAT and FasL/Fas signaling pathways

    PubMed Central

    Soni, Hitesh; Adebiyi, Adebowale

    2016-01-01

    Glomerular mesangial cell (GMC) proliferation and death are involved in the pathogenesis of glomerular disorders. The mechanisms that control GMC survival are poorly understood, but may include signal transduction pathways that are modulated by changes in intracellular Ca2+ ([Ca2+]i) concentration. In this study, we investigated whether activation of the canonical transient receptor potential (TRPC) 6 channels and successive [Ca2+]i elevation alter neonatal GMC survival. Hyperforin (HF)-induced TRPC6 channel activation increased [Ca2+]i concentration, inhibited proliferation, and triggered apoptotic cell death in primary neonatal pig GMCs. HF-induced neonatal GMC apoptosis was not associated with oxidative stress. However, HF-induced TRPC6 channel activation stimulated nuclear translocation of the nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1). HF also increased cell death surface receptor Fas ligand (FasL) level and caspase-8 activity in the cells; effects mitigated by [Ca2+]i chelator BAPTA, calcineurin/NFAT inhibitor VIVIT, and TRPC6 channel knockdown. Accordingly, HF-induced neonatal GMC apoptosis was attenuated by BAPTA, VIVIT, Fas blocking antibody, and a caspase-3/7 inhibitor. These findings suggest that TRPC6 channel-dependent [Ca2+]i elevation and the ensuing induction of the calcineurin/NFAT, FasL/Fas, and caspase signaling cascades promote neonatal pig GMC apoptosis. PMID:27383564

  16. The adaptor protein 3BP2 associates with VAV guanine nucleotide exchange factors to regulate NFAT activation by the B-cell antigen receptor.

    PubMed

    Foucault, Isabelle; Le Bras, Séverine; Charvet, Céline; Moon, Chéol; Altman, Amnon; Deckert, Marcel

    2005-02-01

    Engagement of the B-cell antigen receptor (BCR) activates kinases of the Src and Syk families and signaling complexes assembled by adaptor proteins, which dictate B-cell fate and function. The adaptor 3BP2/SH3BP2, an Abl Src homology domain 3 (SH3)-binding and Syk-kinases interacting protein, exhibits positive regulatory roles in T, natural killer (NK), and basophilic cells. However, its involvement in BCR signaling is completely unknown. Here we show that 3BP2 is tyrosine phosphorylated following BCR aggregation on B lymphoma cells, and that 3BP2 is a substrate for Syk and Fyn, but not Btk. To further explore the function of 3BP2 in B cells, we screened a yeast 2-hybrid B-lymphocyte library and found 3BP2 as a binding partner of Vav proteins. The interaction between 3BP2 and Vav proteins involved both constitutive and inducible mechanisms. 3BP2 also interacted with other components of the BCR signaling pathway, including Syk and phospholipase C gamma (PLC-gamma). Furthermore, overexpression and RNAi blocking experiments showed that 3BP2 regulated BCR-mediated activation of nuclear factor of activated T cells (NFATs). Finally, evidence was provided that 3BP2 functionally cooperates with Vav proteins and Rho GTPases to activate NFATs. Our results show that 3BP2 may regulate BCR-mediated gene activation through Vav proteins.

  17. Ischemia induces nuclear NOX2 expression in cardiomyocytes and subsequently activates apoptosis.

    PubMed

    Meischl, C; Krijnen, P A J; Sipkens, J A; Cillessen, S A G M; Muñoz, I Gámez; Okroj, M; Ramska, M; Muller, A; Visser, C A; Musters, R J P; Simonides, W S; Hack, C E; Roos, D; Niessen, H W M

    2006-06-01

    In previous work we have demonstrated increased expression of NOX2 in cardiomyocytes of infarcted human hearts. In the present manuscript we investigated the functional role of NOX2 in ischemically challenged H9c2 cells, a rat cardiomyoblast cell line, and adult rat cardiomyocytes. Expression of NOX2 in H9c2 cells was confirmed by RT-PCR. In Western-blot experiments, increased NOX2 expression was detected during ischemia, which was inhibited by transcription and translation inhibitors. Surprisingly, under ischemia, in addition to an increased cytosolic expression, NOX2 was localized mainly in the nucleus of apoptotic cardiomyocytes, where it colocalized with nitrotyrosine residues and activated caspase 3. Inhibition of reactive-oxygen-species generation with the flavoenzyme inhibitor diphenylene iodonium (DPI) and the NADPH-oxidase inhibitor apocynin led to a significantly decreased induction of apoptosis as assessed by quantification of caspase-3 activity and by TUNEL analysis. These results demonstrate that NOX2 is expressed in the nucleus of cardiomyocytes during apoptosis and that it likely participates in proapoptotic signaling. To the best of our knowledge, this is the first demonstration of nuclear NOX2 expression and its involvement in cardiomyocyte apoptosis.

  18. Inhibition of chymotryptic-like standard proteasome activity exacerbates doxorubicin-induced cytotoxicity in primary cardiomyocytes.

    PubMed

    Spur, Eva-Margarete; Althof, Nadine; Respondek, Dorota; Klingel, Karin; Heuser, Arnd; Overkleeft, Hermen S; Voigt, Antje

    2016-04-15

    The anthracycline doxorubicin (DOX) is a potent anticancer agent for multiple myeloma (MM). A major limitation of this drug is the induction of death in cardiomyocytes leading to heart failure. Here we report on the role of the ubiquitin-proteasome system (UPS) as a critical surveillance pathway for preservation of cell vitality counteracting DOX treatment. Since in addition to DOX also suppression of proteasome activity is a rational therapeutic strategy for MM, we examined how small molecular compounds with clinically relevant proteasome subunit specificity affect DOX cytotoxicity. We found that during DOX-treatment, the activity of the β5 standard proteasome subunit is crucial for limiting off-target cytotoxicity in primary cardiomyocytes. In contrast, we demonstrate that the β5 equivalent LMP7 of the immunoproteasome represents a safe target for subunit-specific inhibitors in DOX-exposed cardiomyocytes. Neither inhibition of LMP7 in primary cardiomyocytes nor genetic ablation of LMP7 in heart tissue influenced the development of DOX cardiotoxicity. Our results indicate that as compared to compounds like carfilzomib, which target both the β5 standard proteasome and the LMP7 immunoproteasome subunit, immunoproteasome-specific inhibitors with known anti-tumor capacity for MM cells might be advantageous for reducing cardiomyocyte death, when a combination therapy with DOX is envisaged.

  19. Doxorubicin-mediated Apoptosis in Glioma Cells Requires NFAT3

    PubMed Central

    Gopinath, Sreelatha; Vanamala, Sravan K.; Gujrati, Meena; Klopfenstein, Jeffrey D.; Dinh, Dzung H.; Rao, Jasti S.

    2009-01-01

    Nuclear Factor of Activated T cells (NFAT), a family of transcription factors, has been implicated in many cellular processes, including some cancers. For the first time, the present study characterizes the role of NFAT3 in doxorubicin (DOX) mediated apoptosis, migration, and invasion in SNB19 and U87 glioma cells. This study demonstrates specific knockdown of NFAT3 results in a dramatic inhibition of the apoptotic effect, induced by DOX, and favors cell survival. Inhibition of NFAT3 activation by shNFAT3 (shNF3) significantly downregulated TNF-α induction, its receptor TNFR1, caspase 10, caspase 3 and PARP, abrogating DOX-mediated apoptosis in glioma cells. DOX treatment resulted in NFAT3 translocation to the nucleus. Similarly, shNF3 treatment in SNB19 and U87 cells reversed DOX-induced inhibition of cell migration and invasion as determined by wound healing and matrigel invasion assays. Taken together, these results indicate that NFAT3 is a prerequisite for the induction of DOX-mediated apoptosis in glioma cells. PMID:19784808

  20. NFAT2 Regulates Generation of Innate-Like CD8+ T Lymphocytes and CD8+ T Lymphocytes Responses

    PubMed Central

    Pachulec, Emilia; Neitzke-Montinelli, Vanessa; Viola, João P. B.

    2016-01-01

    Nuclear factor of activated T cells (NFAT) 2 null mutant mice die in utero of cardiac failure, precluding analysis of the role of NFAT2 in lymphocyte responses. Only the NFAT2−/−/Rag-1−/− chimeric mice model gave insight into the role of NFAT2 transcription factor in T lymphocyte development, activation, and differentiation. As reports are mainly focused on the role of NFAT2 in CD4+ T lymphocytes activation and differentiation, we decided to investigate NFAT2’s impact on CD8+ T lymphocyte responses. We report that NFAT2 is phosphorylated and inactive in the cytoplasm of naive CD8+ T cells, and upon TCR stimulation, it is dephosphorylated and translocated into the nucleus. To study the role of NFAT2 in CD8+ T responses, we employed NFAT2fl/flCD4-Cre mice with NFAT2 deletion specifically in T cells. Interestingly, the absence of NFAT2 in T cells resulted in increased percentage of non-conventional innate-like CD8+ T cells. These cells were CD122+, rapid producer of interferon gamma (IFN-γ) and had characteristics of conventional memory CD8+ T cells. We also observed an expansion of PLZF+ expressing CD3+ thymocyte population in the absence of NFAT2 and increased IL-4 production. Furthermore, we found that CD8+ T lymphocytes deficient in NFAT2 had reduced activation, proliferation, and IFN-γ and IL-2 production at suboptimal TCR strength. NFAT2 absence did not significantly influence differentiation of CD8+ T cells into cytotoxic effector cells but reduced their IFN-γ production. This work documents NFAT2 as a negative regulator of innate-like CD8+ T cells development. NFAT2 is required for complete CD8+ T cell responses at suboptimal TCR stimulation and regulates IFN-γ production by cytotoxic CD8+ T cells in vitro. PMID:27766099

  1. Balanced interactions of calcineurin with AKAP79 regulate Ca2+-calcineurin-NFAT signaling.

    PubMed

    Li, Huiming; Pink, Matthew D; Murphy, Jonathan G; Stein, Alexander; Dell'Acqua, Mark L; Hogan, Patrick G

    2012-02-19

    In hippocampal neurons, the scaffold protein AKAP79 recruits the phosphatase calcineurin to L-type Ca(2+) channels and couples Ca(2+) influx to activation of calcineurin and of its substrate, the transcription factor NFAT. Here we show that an IAIIIT anchoring site in human AKAP79 binds the same surface of calcineurin as the PxIxIT recognition peptide of NFAT, albeit more strongly. A modest decrease in calcineurin-AKAP affinity due to an altered anchoring sequence is compatible with NFAT activation, whereas a further decrease impairs activation. Counterintuitively, increasing calcineurin-AKAP affinity increases recruitment of calcineurin to the scaffold but impairs NFAT activation; this is probably due to both slower release of active calcineurin from the scaffold and sequestration of active calcineurin by 'decoy' AKAP sites. We propose that calcineurin-AKAP79 scaffolding promotes NFAT signaling by balancing strong recruitment of calcineurin with its efficient release to communicate with NFAT.

  2. NFAT targets signaling molecules to gene promoters in pancreatic β-cells.

    PubMed

    Lawrence, Michael C; Borenstein-Auerbach, Nofit; McGlynn, Kathleen; Kunnathodi, Faisal; Shahbazov, Rauf; Syed, Ilham; Kanak, Mazhar; Takita, Morihito; Levy, Marlon F; Naziruddin, Bashoo

    2015-02-01

    Nuclear factor of activated T cells (NFAT) is activated by calcineurin in response to calcium signals derived by metabolic and inflammatory stress to regulate genes in pancreatic islets. Here, we show that NFAT targets MAPKs, histone acetyltransferase p300, and histone deacetylases (HDACs) to gene promoters to differentially regulate insulin and TNF-α genes. NFAT and ERK associated with the insulin gene promoter in response to glucagon-like peptide 1, whereas NFAT formed complexes with p38 MAPK (p38) and Jun N-terminal kinase (JNK) upon promoters of the TNF-α gene in response to IL-1β. Translocation of NFAT and MAPKs to gene promoters was calcineurin/NFAT dependent, and complex stability required MAPK activity. Knocking down NFATc2 expression, eliminating NFAT DNA binding sites, or interfering with NFAT nuclear import prevented association of MAPKs with gene promoters. Inhibiting p38 and JNK activity increased NFAT-ERK association with promoters, which repressed TNF-α and enhanced insulin gene expression. Moreover, inhibiting p38 and JNK induced a switch from NFAT-p38/JNK-histone acetyltransferase p300 to NFAT-ERK-HDAC3 complex formation upon the TNF-α promoter, which resulted in gene repression. Histone acetyltransferase/HDAC exchange was reversed on the insulin gene by p38/JNK inhibition in the presence of glucagon-like peptide 1, which enhanced gene expression. Overall, these data indicate that NFAT directs signaling enzymes to gene promoters in islets, which contribute to protein-DNA complex stability and promoter regulation. Furthermore, the data suggest that TNF-α can be repressed and insulin production can be enhanced by selectively targeting signaling components of NFAT-MAPK transcriptional/signaling complex formation in pancreatic β-cells. These findings have therapeutic potential for suppressing islet inflammation while preserving islet function in diabetes and islet transplantation.

  3. Impaired ALDH2 activity decreases the mitochondrial respiration in H9C2 cardiomyocytes.

    PubMed

    Mali, Vishal R; Deshpande, Mandar; Pan, Guodong; Thandavarayan, Rajarajan A; Palaniyandi, Suresh S

    2016-02-01

    Reactive oxygen species (ROS)-mediated reactive aldehydes induce cellular stress. In cardiovascular diseases such as ischemia-reperfusion injury, lipid-peroxidation derived reactive aldehydes such as 4-hydroxy-2-nonenal (4HNE) are known to contribute to the pathogenesis. 4HNE is involved in ROS formation, abnormal calcium handling and more importantly defective mitochondrial respiration. Aldehyde dehydrogenase (ALDH) superfamily contains NAD(P)(+)-dependent isozymes which can detoxify endogenous and exogenous aldehydes into non-toxic carboxylic acids. Therefore we hypothesize that 4HNE afflicts mitochondrial respiration and leads to cell death by impairing ALDH2 activity in cultured H9C2 cardiomyocyte cell lines. H9C2 cardiomyocytes were treated with 25, 50 and 75 μM 4HNE and its vehicle, ethanol as well as 25, 50 and 75 μM disulfiram (DSF), an inhibitor of ALDH2 and its vehicle (DMSO) for 4 h. 4HNE significantly decreased ALDH2 activity, ALDH2 protein levels, mitochondrial respiration and mitochondrial respiratory reserve capacity, and increased 4HNE adduct formation and cell death in cultured H9C2 cardiomyocytes. ALDH2 inhibition by DSF and ALDH2 siRNA attenuated ALDH2 activity besides reducing ALDH2 levels, mitochondrial respiration and mitochondrial respiratory reserve capacity and increased cell death. Our results indicate that ALDH2 impairment can lead to poor mitochondrial respiration and increased cell death in cultured H9C2 cardiomyocytes.

  4. Cell cycle and apoptosis regulation by NFAT transcription factors: new roles for an old player

    PubMed Central

    Mognol, G P; Carneiro, F R G; Robbs, B K; Faget, D V; Viola, J P B

    2016-01-01

    The NFAT (nuclear factor of activated T cells) family of transcription factors consists of four Ca2+-regulated members (NFAT1–NFAT4), which were first described in T lymphocytes. In addition to their well-documented role in T lymphocytes, where they control gene expression during cell activation and differentiation, NFAT proteins are also expressed in a wide range of cells and tissue types and regulate genes involved in cell cycle, apoptosis, angiogenesis and metastasis. The NFAT proteins share a highly conserved DNA-binding domain (DBD), which allows all NFAT members to bind to the same DNA sequence in enhancers or promoter regions. The same DNA-binding specificity suggests redundant roles for the NFAT proteins, which is true during the regulation of some genes such as IL-2 and p21. However, it has become increasingly clear that different NFAT proteins and even isoforms can have unique functions. In this review, we address the possible reasons for these distinct roles, particularly regarding N- and C-terminal transactivation regions (TADs) and the partner proteins that interact with these TADs. We also discuss the genes regulated by NFAT during cell cycle regulation and apoptosis and the role of NFAT during tumorigenesis. PMID:27100893

  5. Cell cycle and apoptosis regulation by NFAT transcription factors: new roles for an old player.

    PubMed

    Mognol, G P; Carneiro, F R G; Robbs, B K; Faget, D V; Viola, J P B

    2016-04-21

    The NFAT (nuclear factor of activated T cells) family of transcription factors consists of four Ca(2+)-regulated members (NFAT1-NFAT4), which were first described in T lymphocytes. In addition to their well-documented role in T lymphocytes, where they control gene expression during cell activation and differentiation, NFAT proteins are also expressed in a wide range of cells and tissue types and regulate genes involved in cell cycle, apoptosis, angiogenesis and metastasis. The NFAT proteins share a highly conserved DNA-binding domain (DBD), which allows all NFAT members to bind to the same DNA sequence in enhancers or promoter regions. The same DNA-binding specificity suggests redundant roles for the NFAT proteins, which is true during the regulation of some genes such as IL-2 and p21. However, it has become increasingly clear that different NFAT proteins and even isoforms can have unique functions. In this review, we address the possible reasons for these distinct roles, particularly regarding N- and C-terminal transactivation regions (TADs) and the partner proteins that interact with these TADs. We also discuss the genes regulated by NFAT during cell cycle regulation and apoptosis and the role of NFAT during tumorigenesis.

  6. Cell cycle and apoptosis regulation by NFAT transcription factors: new roles for an old player.

    PubMed

    Mognol, G P; Carneiro, F R G; Robbs, B K; Faget, D V; Viola, J P B

    2016-01-01

    The NFAT (nuclear factor of activated T cells) family of transcription factors consists of four Ca(2+)-regulated members (NFAT1-NFAT4), which were first described in T lymphocytes. In addition to their well-documented role in T lymphocytes, where they control gene expression during cell activation and differentiation, NFAT proteins are also expressed in a wide range of cells and tissue types and regulate genes involved in cell cycle, apoptosis, angiogenesis and metastasis. The NFAT proteins share a highly conserved DNA-binding domain (DBD), which allows all NFAT members to bind to the same DNA sequence in enhancers or promoter regions. The same DNA-binding specificity suggests redundant roles for the NFAT proteins, which is true during the regulation of some genes such as IL-2 and p21. However, it has become increasingly clear that different NFAT proteins and even isoforms can have unique functions. In this review, we address the possible reasons for these distinct roles, particularly regarding N- and C-terminal transactivation regions (TADs) and the partner proteins that interact with these TADs. We also discuss the genes regulated by NFAT during cell cycle regulation and apoptosis and the role of NFAT during tumorigenesis. PMID:27100893

  7. Ca²⁺ signal-induced cardiomyocyte hypertrophy through activation of myocardin.

    PubMed

    Li, Man; Wang, Nan; Gong, Hui-Qin; Li, Wei-Zong; Liao, Xing-Hua; Yang, Xiao-Long; He, Hong-Peng; Cao, Dong-Sun; Zhang, Tong-Cun

    2015-02-15

    Hypertrophic growth of cardiomyocytes in response to pressure overload is an important stage during the development of many cardiac diseases. Ca(2+) overload as well as subsequent activation of Ca(2+) signaling pathways has been reported to induce cardiac hypertrophy. Myocardin, a transcription cofactor of serum response factor (SRF), is a key transducer of hypertrophic signals. However, the direct role of myocardin in Ca(2+) signal-induced cardiomyocyte hypertrophy has not been explained clearly. In the present study, we discovered that embryonic rat heart-derived H9c2 cells responded to the stimulation of calcium ionophore A23187 with a cell surface area enlargement and an increased expression of cardiac hypertrophy marker genes. Increased Ca(2+) also induces an organization of sarcomeres in neonatal rat cardiomyocytes, as revealed by α-actinin staining. Increased Ca(2+) could upregulate the expression of myocardin. Knockdown of myocardin by shRNA attenuates hypertrophic responses triggered by increased intracellular Ca(2+), suggesting that Ca(2+) signals induce cardiomyocyte hypertrophy partly through activation of myocardin. Furthermore, A23187 treatment directly activates myocardin promoter, chelation of Ca(2+) by EGTA inhibits this activation and knockdown of myocardin expression using shRNA also abrogates A23187-induced ANF and SK-α-actin promoter activity. CSA (calcineurin inhibitor) and KN93 (CaMKII inhibitor) inhibit A23187-induced the increase in myocardin expression. These results suggest that myocardin plays a critical role in Ca(2+) signal-induced cardiomyocyte hypertrophy, which may serve as a novel mechanism that is important for cardiac hypertrophy.

  8. Ca²⁺ signal-induced cardiomyocyte hypertrophy through activation of myocardin.

    PubMed

    Li, Man; Wang, Nan; Gong, Hui-Qin; Li, Wei-Zong; Liao, Xing-Hua; Yang, Xiao-Long; He, Hong-Peng; Cao, Dong-Sun; Zhang, Tong-Cun

    2015-02-15

    Hypertrophic growth of cardiomyocytes in response to pressure overload is an important stage during the development of many cardiac diseases. Ca(2+) overload as well as subsequent activation of Ca(2+) signaling pathways has been reported to induce cardiac hypertrophy. Myocardin, a transcription cofactor of serum response factor (SRF), is a key transducer of hypertrophic signals. However, the direct role of myocardin in Ca(2+) signal-induced cardiomyocyte hypertrophy has not been explained clearly. In the present study, we discovered that embryonic rat heart-derived H9c2 cells responded to the stimulation of calcium ionophore A23187 with a cell surface area enlargement and an increased expression of cardiac hypertrophy marker genes. Increased Ca(2+) also induces an organization of sarcomeres in neonatal rat cardiomyocytes, as revealed by α-actinin staining. Increased Ca(2+) could upregulate the expression of myocardin. Knockdown of myocardin by shRNA attenuates hypertrophic responses triggered by increased intracellular Ca(2+), suggesting that Ca(2+) signals induce cardiomyocyte hypertrophy partly through activation of myocardin. Furthermore, A23187 treatment directly activates myocardin promoter, chelation of Ca(2+) by EGTA inhibits this activation and knockdown of myocardin expression using shRNA also abrogates A23187-induced ANF and SK-α-actin promoter activity. CSA (calcineurin inhibitor) and KN93 (CaMKII inhibitor) inhibit A23187-induced the increase in myocardin expression. These results suggest that myocardin plays a critical role in Ca(2+) signal-induced cardiomyocyte hypertrophy, which may serve as a novel mechanism that is important for cardiac hypertrophy. PMID:25485719

  9. Calcineurin-NFAT Signaling Controls Somatic Cell Reprogramming in a Stage-Dependent Manner.

    PubMed

    Sun, Ming; Liao, Bing; Tao, Yu; Chen, Hao; Xiao, Feng; Gu, Junjie; Gao, Shaorong; Jin, Ying

    2016-05-01

    Calcineurin-NFAT signaling is critical for early lineage specification of mouse embryonic stem cells and early embryos. However, its roles in somatic cell reprogramming remain unknown. Here, we report that calcineurin-NFAT signaling has a dynamic activity and plays diverse roles at different stages of reprogramming. At the early stage, calcineurin-NFAT signaling is transiently activated and its activation is required for successful reprogramming. However, at the late stage of reprogramming, activation of calcineurin-NFAT signaling becomes a barrier for reprogramming and its inactivation is critical for successful induction of pluripotency. Mechanistically, calcineurin-NFAT signaling contributes to the reprogramming through regulating multiple early events during reprogramming, including mesenchymal to epithelial transition (MET), cell adhesion and emergence of SSEA1(+) intermediate cells. Collectively, this study reveals for the first time the important roles of calcineurin-NFAT signaling during somatic cell reprogramming and provides new insights into the molecular regulation of reprogramming.

  10. NFAT5 promotes proliferation and migration of lung adenocarcinoma cells in part through regulating AQP5 expression

    SciTech Connect

    Guo, Kai; Jin, Faguang

    2015-09-25

    The osmoregulated transcription factor nuclear factor of activated T-cells 5(NFAT5), has been found to play important roles in the development of many kinds of human cancers, including breast cancer, colon carcinoma, renal cell carcinoma and melanoma. The aim of the present study was to determine whether NFAT5 is involved in the proliferation and migration of lung adenocarcinoma cells. We found that NFAT5 was upregulated in lung adenocarcinoma cells and knockdown of NFAT5 decreased proliferation and migration of the cells, accompanied by a significant reduction in the expression of AQP5. AQP5 was upregulated in lung adenocarcinoma cells and knockdown of AQP5 also inhibited proliferation and migration of the cells as knockdown of NFAT5 did. Moreover, overexpression of NFAT5 promoted proliferation and migration of lung adenocarcinoma cells, accompanied by a significant increase in the expression of AQP5. These results indicate that NFAT5 plays important roles in proliferation and migration of human lung adenocarcinoma cells through regulating AQP5 expression, providing a new therapeutic option for lung adenocarcinoma therapy. - Highlights: • NFAT5 expression is higher in lung adenocarcinoma cells compared with normal cells. • NFAT5 knockdown decreases proliferation and migration of lung adenocarcinoma cells. • Knockdown of NFAT5 reduces AQP5 expression in human lung adenocarcinoma cells. • Overexpression of NFAT5 promotes proliferation and migration of lung adenocarcinoma cells. • Overexpression of NFAT5 increases AQP5 expression in human lung adenocarcinoma cells.

  11. NFAT1 promotes intratumoral neutrophil infiltration by regulating IL8 expression in breast cancer.

    PubMed

    Kaunisto, Aura; Henry, Whitney S; Montaser-Kouhsari, Laleh; Jaminet, Shou-Ching; Oh, Eun-Yeong; Zhao, Li; Luo, Hongbo R; Beck, Andrew H; Toker, Alex

    2015-06-01

    NFAT transcription factors are key regulators of gene expression in immune cells. In addition, NFAT1-induced genes play diverse roles in mediating the progression of various solid tumors. Here we show that NFAT1 induces the expression of the IL8 gene by binding to its promoter and leading to IL8 secretion. Thapsigargin stimulation of breast cancer cells induces IL8 expression in an NFAT-dependent manner. Moreover, we show that NFAT1-mediated IL8 production promotes the migration of primary human neutrophils in vitro and also promotes neutrophil infiltration in tumor xenografts. Furthermore, expression of active NFAT1 effectively suppresses the growth of nascent and established tumors by a non cell-autonomous mechanism. Evaluation of breast tumor tissue reveals that while the levels of NFAT1 are similar in tumor cells and normal breast epithelium, cells in the tumor stroma express higher levels of NFAT1 compared to normal stroma. Elevated levels of NFAT1 also correlate with increased neutrophil infiltrate in breast tumors. These data point to a mechanism by which NFAT1 orchestrates the communication between breast cancer cells and host neutrophils during breast cancer progression.

  12. Proteins secreted by embryonic stem cells activate cardiomyocytes through ligand binding pathways.

    PubMed

    LaFramboise, W A; Petrosko, P; Krill-Burger, J M; Morris, D R; McCoy, A R; Scalise, D; Malehorn, D E; Guthrie, R D; Becich, M J; Dhir, R

    2010-03-10

    Human embryonic stem cells (hESC) underlie embryogenesis but paracrine signals associated with the process are unknown. This study was designed to 1) profile native proteins secreted by undifferentiated hESC and 2) determine their biological effects on primary neonatal cardiomyocytes. We utilized multi-analyte, immunochemical assays to characterize media conditioned by undifferentiated hESC versus unconditioned media. Expression profiling was performed on cardiomyocytes subjected to these different media conditions and altered transcripts were mapped to critical pathways. Thirty-two of 109 proteins were significantly elevated in conditioned media ranging in concentration from thrombospondin (57.2+/-5.0 ng/ml) to nerve growth factor (7.4+/-1.2pg/ml) and comprising chemokines, cytokines, growth factors, and proteins involved in cell adhesion and extracellular matrix remodeling. Conditioned media induced karyokinesis, cytokinesis and proliferation in mono- and binucleate cardiomyocytes. Pathway analysis revealed comprehensive activation of the ROCK 1 and 2 G-protein coupled receptor (GPCR) pathway associated with cytokinesis, and the RAS/RAF/MEK/ERK receptor tyrosine kinase (RTK) and JAK/STAT-cytokine pathway involved in cell cycle progression. These results provide a partial database of proteins secreted by pluripotent hESC that potentiate cell division in cardiomyocytes via a paracrine mechanism suggesting a potential role for these stem cell factors in cardiogenesis and cardiac repair.

  13. The calcineurin-NFAT pathway negatively regulates megakaryopoiesis.

    PubMed

    Zaslavsky, Alexander; Chou, Stella T; Schadler, Keri; Lieberman, Allyson; Pimkin, Maxim; Kim, Yeo Jung; Baek, Kwan-Hyuck; Aird, William C; Weiss, Mitchell J; Ryeom, Sandra

    2013-04-18

    The calcium regulated calcineurin-nuclear factor of activated T cells (NFAT) pathway modulates the physiology of numerous cell types, including hematopoietic. Upon activation, calcineurin dephosphorylates NFAT family transcription factors, triggering their nuclear entry and activation or repression of target genes. NFATc1 and c2 isoforms are expressed in megakaryocytes. Moreover, human chromosome 21 (Hsa21) encodes several negative regulators of calcineurin-NFAT, candidates in the pathogenesis of Down syndrome (trisomy 21)-associated transient myeloproliferative disorder and acute megakaryoblastic leukemia. To investigate the role of calcineurin-NFAT in megakaryopoiesis, we examined wild-type mice treated with the calcineurin inhibitor cyclosporin A and transgenic mice expressing a targeted single extra copy of Dscr1, an Hsa21-encoded calcineurin inhibitor. Both murine models exhibited thrombocytosis with increased megakaryocytes and megakaryocyte progenitors. Pharmacological or genetic inhibition of calcineurin in mice caused accumulation of megakaryocytes exhibiting enhanced 5-bromo-2'-deoxyuridine uptake and increased expression of messenger RNAs encoding CDK4 and G1 cyclins, which promote cell division. Additionally, human megakaryocytes with trisomy 21 show increased proliferation and decreased NFAT activation compared with euploid controls. Our data indicate that inhibition of calcineurin-NFAT drives proliferation of megakaryocyte precursors by de-repressing genes that drive cell division, providing insights into mechanisms of normal megakaryopoiesis and megakaryocytic abnormalities that accompany Down syndrome.

  14. TGF-β-Smad2 dependent activation of CDC 25A plays an important role in cell proliferation through NFAT activation in metastatic breast cancer cells.

    PubMed

    Sengupta, Suman; Jana, Samir; Bhattacharyya, Arindam

    2014-02-01

    In late stages of cancer, TGF-β promotes the metastasis process by enhancing the invasiveness of cancer cells and inducing the epithelial-to-mesenchymal transition (EMT), a process that is concomitantly associated with breast cancer metastasis. Metastasis comprises of multiple steps with the regulation of complex network of signaling. Metastasis is associated with both the EMT and cell proliferation, but yet it has not been clearly distinguished how the balance between the cell proliferation and EMT is maintained together. Recently, it has been accounted that a transcription factor, NFAT has an important role for switching tumor suppressive to progressive effect of TGF-β and NFAT has a role in TGF-β mediated EMT by regulating N-cadherin. CDC 25A phosphatase, an important cell cycle regulator is overexpressed in breast cancer. Our results demonstrate that TGF-β regulating the CDC 25A in a Smad2 dependent way, translocates NFAT to nucleus and NFAT in co-operation with Smad2 promotes the tumor progression by upregulating the CDK2, CDK4, and cyclin E. This result signifies that TGF-β by regulating NFAT in different ways maintains the balance between EMT and cell proliferation mechanism concurrently during the late stage of breast cancer.

  15. Activation of calcium-sensing receptor increases TRPC3 expression in rat cardiomyocytes

    SciTech Connect

    Feng, Shan-Li; Sun, Ming-Rui; Li, Ting-Ting; Yin, Xin; Xu, Chang-Qing; Sun, Yi-Hua

    2011-03-11

    Research highlights: {yields} Calcium-sensing receptor (CaR) activation stimulates TRP channels. {yields} CaR promoted transient receptor potential C3 (TRPC3) expression. {yields} Adult rat ventricular myocytes display capacitative calcium entry (CCE), which was operated by TRPCs. {yields} TRPC channels activation induced by CaR activator sustained the increased [Ca{sup 2+}]{sub i} to evoke cardiomyocytes apoptosis. -- Abstract: Transient receptor potential (TRP) channels are expressed in cardiomyocytes, which gate a type of influx of extracellular calcium, the capacitative calcium entry. TRP channels play a role in mediating Ca{sup 2+} overload in the heart. Calcium-sensing receptors (CaR) are also expressed in rat cardiac tissue and promote the apoptosis of cardiomyocytes by Ca{sup 2+} overload. However, data about the link between CaR and TRP channels in rat heart are few. In this study, reverse transcriptase polymerase chain reaction (RT-PCR) and Western blotting were used to examine the expression of the TRP canonical proteins TRPC1 and TRPC3 in adult and neonatal rat cardiomyocytes. Laser scan confocal microscopy was used to detect intracellular [Ca{sup 2+}]{sub i} levels in isolated adult rat ventricular myocytes. The results showed that, in adult rat cardiomyocytes, the depletion of Ca{sup 2+} stores in the endoplasmic/sarcoplasmic reticulum (ER/SR) by thapsigargin induced a transient increase in [Ca{sup 2+}]{sub i} in the absence of [Ca{sup 2+}]{sub o} and the subsequent restoration of [Ca{sup 2+}]{sub o} sustained the increased [Ca{sup 2+}]{sub i} for a few minutes, whereas, the persisting elevation of [Ca{sup 2+}]{sub i} was reduced in the presence of the TRPC inhibitor SKF96365. The stimulation of CaR by its activator gadolinium chloride (GdCl{sub 3}) or spermine also resulted in the same effect and the duration of [Ca{sup 2+}]{sub i} increase was also shortened in the absence of [Ca{sup 2+}]{sub o}. In adult and neonatal rat cardiomyocytes, GdCl{sub 3

  16. Labdane diterpenes protect against anoxia/reperfusion injury in cardiomyocytes: involvement of AKT activation

    PubMed Central

    Cuadrado, I; Fernández-Velasco, M; Boscá, L; de las Heras, B

    2011-01-01

    Several labdane diterpenes exert anti-inflammatory and cytoprotective actions; therefore, we have investigated whether these molecules protect cardiomyocytes in an anoxia/reperfusion (A/R) model, establishing the molecular mechanisms involved in the process. The cardioprotective activity of three diterpenes (T1, T2 and T3) was studied in the H9c2 cell line and in isolated rat cardiomyocyte subjected to A/R injury. In both cases, treatment with diterpenes T1 and T2 protected from A/R-induced apoptosis, as deduced by a decrease in the percentage of apoptotic and caspase-3 active positive cells, a decrease in the Bcl-2/Bax ratio and an increase in the expression of antiapoptotic proteins. Analysis of cell survival signaling pathways showed that diterpenes T1 and T2 added after A/R increased phospho-AKT and phospho-ERK 1/2 levels. These cardioprotective effects were lost when AKT activity was pharmacologically inhibited. Moreover, the labdane-induced cardioprotection involves activation of AMPK, suggesting a role for energy homeostasis in their mechanism of action. Labdane diterpenes (T1 and T2) also exerted cardioprotective effects against A/R-induced injury in isolated cardiomyocytes and the mechanisms involved activation of specific survival signals (PI3K/AKT pathways, ERK1/2 and AMPK) and inhibition of apoptosis. PMID:22071634

  17. Astragaloside IV attenuates apoptosis of hypertrophic cardiomyocyte through inhibiting oxidative stress and calpain-1 activation.

    PubMed

    Mei, Meng; Tang, Futian; Lu, Meili; He, Xin; Wang, Hongxin; Hou, Xuwei; Hu, Jin; Xu, Chonghua; Han, Ronghui

    2015-11-01

    Calpain-1 activation and oxidative stress are two critical factors contributing to apoptosis of hypertrophic cardiomyocyte. Astragaloside IV (ASIV) exhibits protective effect against various heart diseases. The present study was designed to investigate whether the inhibitory effect of ASIV on isoproterenol (ISO)-induced apoptosis of hypertrophic cardiomyocyte was associated with the anti-oxidation and calpain-1 inhibition. Hypertrophy, apoptosis, mitochondrial oxidative stress and calpain-1 expression were measured in the heart tissue of Sprague-Dawley (SD) rats and H9C2 cells treated with ISO alone or combination with ASIV. The results showed that ASIV attenuated apoptotic rate, increased Bcl-2 expression, decreased Bax expression, ameliorated the integrity of mitochondrial structure and improved mitochondrial membrane potential (MMP). Moreover, ASIV combination reduced both calpain-1 protein expression and calpain activity, down-regulated mitochondrial NOX4 (mito-NOX4) expression, increased activity of mitochondrial superoxide dismutase (mito-SOD) and mitochondrial catalase (mito-CAT) compared to ISO treated alone. The results suggested that ASIV exerted anti-apoptosis effect on ISO-induced hypertrophic cardiomyocyte by attenuating oxidative stress and calpain-1 activation. PMID:26433482

  18. Upregulation of phase II enzymes through phytochemical activation of Nrf2 protects cardiomyocytes against oxidant stress.

    PubMed

    Reuland, Danielle J; Khademi, Shadi; Castle, Christopher J; Irwin, David C; McCord, Joe M; Miller, Benjamin F; Hamilton, Karyn L

    2013-03-01

    Increased production of reactive oxygen species has been implicated in the pathogenesis of cardiovascular disease (CVD), and enhanced endogenous antioxidants have been proposed as a mechanism for regulating redox balance. Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is a transcriptional regulator of phase II antioxidant enzymes, and activation of Nrf2 has been suggested to be an important step in attenuating oxidative stress associated with CVD. A well-defined combination of five widely studied medicinal plants derived from botanical sources (Bacopa monniera, Silybum marianum (milk thistle), Withania somnifera (Ashwagandha), Camellia sinensis (green tea), and Curcuma longa (turmeric)) has been shown to activate Nrf2 and induce phase II enzymes through the antioxidant response element. The purpose of these experiments was to determine if treatment of cardiomyocytes with this phytochemical composition, marketed as Protandim, activates Nrf2, induces phase II detoxification enzymes, and protects cardiomyocytes from oxidant-induced apoptosis in a Nrf2-dependent manner. In cultured HL-1 cardiomyocytes, phytochemical treatment was associated with nuclear accumulation of Nrf2, significant induction of phase II enzymes, and concomitant protection against hydrogen peroxide-induced apoptosis. The protection against oxidant stress was abolished when Nrf2 was silenced by shRNA, suggesting that our phytochemical treatment worked through the Nrf2 pathway. Interestingly, phytochemical treatment was found to be a more robust activator of Nrf2 than oxidant treatment, supporting the use of the phytochemicals as a potential treatment to increase antioxidant defenses and protect heart cells against an oxidative challenge.

  19. NFAT regulates calcium-sensing receptor-mediated TNF production

    SciTech Connect

    abdullah, huda ismail; Pedraza, Paulina L.; Hao, Shoujin; Rodland, Karin D.; McGiff, John C.; Ferreri, Nicholas R.

    2006-05-01

    Because nuclear factor of activated T cells (NFAT) has been implicated in TNF production as well as osmoregulation and salt and water homeostasis, we addressed whether calcium-sensing receptor (CaR)-mediated TNF production in medullary thick ascending limb (mTAL) cells was NFAT dependent. TNF production in response to addition of extracellular Ca2+ (1.2 mM) was abolished in mTAL cells transiently transfected with a dominant-negative CaR construct (R796W) or pretreated with the phosphatidylinositol phospholipase C (PI-PLC) inhibitor U-73122. Cyclosporine A (CsA), an inhibitor of the serine/threonine phosphatase calcineurin, and a peptide ligand, VIVIT, that selectively inhibits calcineurin-NFAT signaling, also prevented CaR-mediated TNF production. Increases in calcineurin activity in cells challenged with Ca2+ were inhibited after pretreatment with U-73122 and CsA, suggesting that CaR activation increases calcineurin activity in a PI-PLC-dependent manner. Moreover, U-73122, CsA, and VIVIT inhibited CaR-dependent activity of an NFAT construct that drives expression of firefly luciferase in transiently transfected mTAL cells. Collectively, these data verify the role of calcineurin and NFAT in CaR-mediated TNF production by mTAL cells. Activation of the CaR also increased the binding of NFAT to a consensus oligonucleotide, an effect that was blocked by U-73122 and CsA, suggesting that a calcineurin- and NFAT-dependent pathway increases TNF production in mTAL cells. This mechanism likely regulates TNF gene transcription as U-73122, CsA, and VIVIT blocked CaR-dependent activity of a TNF promoter construct. Elucidating CaR-mediated signaling pathways that regulate TNF production in the mTAL will be crucial to understanding mechanisms that regulate extracellular fluid volume and salt balance.

  20. NFAT2 mediates high glucose-induced glomerular podocyte apoptosis through increased Bax expression

    SciTech Connect

    Li, Ruizhao; Zhang, Li; Shi, Wei; Zhang, Bin; Liang, Xinling; Liu, Shuangxin; Wang, Wenjian

    2013-04-15

    Background: Hyperglycemia promotes podocyte apoptosis and plays a key role in the pathogenesis of diabetic nephropathy. However, the mechanisms that mediate hyperglycemia-induced podocyte apoptosis is still far from being fully understood. Recent studies reported that high glucose activate nuclear factor of activated T cells (NFAT) in vascular smooth muscle or pancreatic β-cells. Here, we sought to determine if hyperglycemia activates NFAT2 in cultured podocyte and whether this leads to podocyte apoptosis. Meanwhile, we also further explore the mechanisms of NFAT2 activation and NFAT2 mediates high glucose-induced podocyte apoptosis. Methods: Immortalized mouse podocytes were cultured in media containing normal glucose (NG), or high glucose (HG) or HG plus cyclosporine A (a pharmacological inhibitor of calcinerin) or 11R-VIVIT (a special inhibitor of NFAT2). The activation of NFAT2 in podocytes was detected by western blotting and immunofluorescence assay. The role of NFAT2 in hyperglycemia-induced podocyte apoptosis was further evaluated by observing the inhibition of NFAT2 activation by 11R-VIVIT using flow cytometer. Intracellular Ca{sup 2+} was monitored in HG-treated podcocytes using Fluo-3/AM. The mRNA and protein expression of apoptosis gene Bax were measured by real time-qPCR and western blotting. Results: HG stimulation activated NFAT2 in a time- and dose-dependent manner in cultured podocytes. Pretreatment with cyclosporine A (500 nM) or 11R-VIVIT (100 nM) completely blocked NFAT2 nuclear accumulation. Meanwhile, the apoptosis effects induced by HG were also abrogated by concomitant treatment with 11R-VIVIT in cultured podocytes. We further found that HG also increased [Ca{sup 2+}]i, leading to activation of calcineurin, and subsequent increased nuclear accumulation of NFAT2 and Bax expression in cultured podocytes. Conclusion: Our results identify a new finding that HG-induced podocyte apoptosis is mediated by calcineurin/NFAT2/Bax signaling pathway

  1. NFAT1 transcription factor regulates cell cycle progression and cyclin E expression in B lymphocytes.

    PubMed

    Teixeira, Leonardo K; Carrossini, Nina; Sécca, Cristiane; Kroll, José E; DaCunha, Déborah C; Faget, Douglas V; Carvalho, Lilian D S; de Souza, Sandro J; Viola, João P B

    2016-09-01

    The NFAT family of transcription factors has been primarily related to T cell development, activation, and differentiation. Further studies have shown that these ubiquitous proteins are observed in many cell types inside and outside the immune system, and are involved in several biological processes, including tumor growth, angiogenesis, and invasiveness. However, the specific role of the NFAT1 family member in naive B cell proliferation remains elusive. Here, we demonstrate that NFAT1 transcription factor controls Cyclin E expression, cell proliferation, and tumor growth in vivo. Specifically, we show that inducible expression of NFAT1 inhibits cell cycle progression, reduces colony formation, and controls tumor growth in nude mice. We also demonstrate that NFAT1-deficient naive B lymphocytes show a hyperproliferative phenotype and high levels of Cyclin E1 and E2 upon BCR stimulation when compared to wild-type B lymphocytes. NFAT1 transcription factor directly regulates Cyclin E expression in B cells, inhibiting the G1/S cell cycle phase transition. Bioinformatics analysis indicates that low levels of NFAT1 correlate with high expression of Cyclin E1 in different human cancers, including Diffuse Large B-cell Lymphomas (DLBCL). Together, our results demonstrate a repressor role for NFAT1 in cell cycle progression and Cyclin E expression in B lymphocytes, and suggest a potential function for NFAT1 protein in B cell malignancies.

  2. De novo cardiomyocytes from within the activated adult heart after injury

    PubMed Central

    Smart, Nicola; Bollini, Sveva; Dubé, Karina N.; Vieira, Joaquim M.; Zhou, Bin; Davidson, Sean; Yellon, Derek; Riegler, Johannes; Price, Anthony N.; Lythgoe, Mark F.; Pu, William T.; Riley, Paul R.

    2013-01-01

    A significant bottleneck in cardiovascular regenerative medicine is the identification of a viable source of stem/progenitor cells that could contribute new muscle after ischaemic heart disease and acute myocardial infarction1. A therapeutic ideal—relative to cell transplantation—would be to stimulate a resident source, thus avoiding the caveats of limited graft survival, restricted homing to the site of injury and host immune rejection. Here we demonstrate in mice that the adult heart contains a resident stem or progenitor cell population, which has the potential to contribute bona fide terminally differentiated cardiomyocytes after myocardial infarction. We reveal a novel genetic label of the activated adult progenitors via re-expression of a key embryonic epicardial gene, Wilm’s tumour 1 (Wt1), through priming by thymosin β4, a peptide previously shown to restore vascular potential to adult epicardium-derived progenitor cells2 with injury. Cumulative evidence indicates an epicardial origin of the progenitor population, and embryonic reprogramming results in the mobilization of this population and concomitant differentiation to give rise to de novo cardiomyocytes. Cell transplantation confirmed a progenitor source and chromosome painting of labelled donor cells revealed transdifferentiation to a myocyte fate in the absence of cell fusion. Derived cardiomyocytes are shown here to structurally and functionally integrate with resident muscle; as such, stimulation of this adult progenitor pool represents a significant step towards residentcell-based therapy in human ischaemic heart disease. PMID:21654746

  3. Role of the stress-activated protein kinases in endothelin-induced cardiomyocyte hypertrophy.

    PubMed Central

    Choukroun, G; Hajjar, R; Kyriakis, J M; Bonventre, J V; Rosenzweig, A; Force, T

    1998-01-01

    The signal transduction pathways governing the hypertrophic response of cardiomyocytes are not well defined. Constitutive activation of the stress-activated protein kinase (SAPK) family of mitogen-activated protein (MAP) kinases or another stress-response MAP kinase, p38, by overexpression of activated mutants of various components of the pathways is sufficient to induce a hypertrophic response in cardiomyocytes, but it is not clear what role these pathways play in the response to physiologically relevant hypertrophic stimuli. To determine the role of the SAPKs in the hypertrophic response, we used adenovirus-mediated gene transfer of SAPK/ERK kinase-1 (KR) [SEK-1(KR)], a dominant inhibitory mutant of SEK-1, the immediate upstream activator of the SAPKs, to block signal transmission down the SAPK pathway in response to the potent hypertrophic agent, endothelin-1 (ET-1). SEK-1(KR) completely inhibited ET-1-induced SAPK activation without affecting activation of the other MAP kinases implicated in the hypertrophic response, p38 and extracellular signal-regulated protein kinases (ERK)-1/ERK-2. Expression of SEK-1(KR) markedly inhibited the ET-1-induced increase in protein synthesis. In contrast, the MAPK/ERK kinase inhibitor, PD98059, which blocks ERK activation, and the p38 inhibitor, SB203580, had no effect on ET-1-induced protein synthesis. ET-1 also induced a significant increase in atrial natriuretic factor mRNA expression as well as in the percentage of cells with highly organized sarcomeres, responses which were also blocked by expression of SEK-1(KR). In summary, inhibiting activation of the SAPK pathway abrogated the hypertrophic response to ET-1. These data are the first demonstration that the SAPKs are necessary for the development of agonist-induced cardiomyocyte hypertrophy, and suggest that in response to ET-1, they transduce critical signals governing the hypertrophic response. PMID:9769323

  4. Cardiac peroxisome proliferator-activated receptor-γ expression is modulated by oxidative stress in acutely infrasound-exposed cardiomyocytes.

    PubMed

    Pei, Zhaohui; Meng, Rongsen; Zhuang, Zhiqiang; Zhao, Yiqiao; Liu, Fangpeng; Zhu, Miao-Zhang; Li, Ruiman

    2013-12-01

    The aim of the present study was to examine the effects of acute infrasound exposure on oxidative damage and investigate the underlying mechanisms in rat cardiomyocytes. Neonatal rat cardiomyocytes were cultured and exposed to infrasound for several days. In the study, the expression of CAT, GPx, SOD1, and SOD2 and their activities in rat cardiomyocytes in infrasound exposure groups were significantly decreased compared to those in the various time controls, along with significantly higher levels of O2 (-) and H2O2. Decreased cardiac cell viability was not observed in various time controls. A significant reduction in cardiac cell viability was observed in the infrasound group compared to the control, while significantly increased cardiac cell viability was observed in the infrasound exposure and rosiglitazone pretreatment group. Compared to the control, rosiglitazone significantly upregulated CAT, GPx, SOD1, and SOD2 expression and their activities in rat cardiomyocytes exposed to infrasound, while the levels of O2 (-) or H2O2 were significantly decreased. A potential link between a significant downregulation of PPAR-γ expression in rat cardiomyocytes in the infrasound group was compared to the control and infrasound-induced oxidative stress. These findings indicate that infrasound can induce oxidative damage in rat cardiomyocytes by inactivating PPAR-γ. PMID:23632742

  5. Balanced interactions of calcineurin with AKAP79 regulate Ca2+-calcineurin-NFAT signaling

    SciTech Connect

    Li, Huiming; Pink, Matthew D; Murphy, Jonathan G; Stein, Alexander; Dell,; Acqua, Mark L; Hogan, Patrick G

    2012-04-30

    In hippocampal neurons, the scaffold protein AKAP79 recruits the phosphatase calcineurin to L-type Ca2+ channels and couples Ca2+ influx to activation of calcineurin and of its substrate, the transcription factor NFAT. Here we show that an IAIIIT anchoring site in human AKAP79 binds the same surface of calcineurin as the PxIxIT recognition peptide of NFAT, albeit more strongly. A modest decrease in calcineurin-AKAP affinity due to an altered anchoring sequence is compatible with NFAT activation, whereas a further decrease impairs activation. Counterintuitively, increasing calcineurin-AKAP affinity increases recruitment of calcineurin to the scaffold but impairs NFAT activation; this is probably due to both slower release of active calcineurin from the scaffold and sequestration of active calcineurin by 'decoy' AKAP sites. We propose that calcineurin-AKAP79 scaffolding promotes NFAT signaling by balancing strong recruitment of calcineurin with its efficient release to communicate with NFAT.

  6. NOD1 Activation Induces Cardiac Dysfunction and Modulates Cardiac Fibrosis and Cardiomyocyte Apoptosis

    PubMed Central

    Fernández-Velasco, María; Prieto, Patricia; Terrón, Verónica; Benito, Gemma; Flores, Juana M.; Delgado, Carmen; Zaragoza, Carlos; Lavin, Begoña; Gómez-Parrizas, Mónica; López-Collazo, Eduardo; Martín-Sanz, Paloma; Boscá, Lisardo

    2012-01-01

    The innate immune system is responsible for the initial response of an organism to potentially harmful stressors, pathogens or tissue injury, and accordingly plays an essential role in the pathogenesis of many inflammatory processes, including some cardiovascular diseases. Toll like receptors (TLR) and nucleotide-binding oligomerization domain-like receptors (NLRs) are pattern recognition receptors that play an important role in the induction of innate immune and inflammatory responses. There is a line of evidence supporting that activation of TLRs contributes to the development and progression of cardiovascular diseases but less is known regarding the role of NLRs. Here we demonstrate the presence of the NLR member NOD1 (nucleotide-binding oligomerization domain containing 1) in the murine heart. Activation of NOD1 with the specific agonist C12-iEDAP, but not with the inactive analogue iE-Lys, induces a time- and dose-dependent cardiac dysfunction that occurs concomitantly with cardiac fibrosis and apoptosis. The administration of iEDAP promotes the activation of the NF-κB and TGF-β pathways and induces apoptosis in whole hearts. At the cellular level, both native cardiomyocytes and cardiac fibroblasts expressed NOD1. The NLR activation in cardiomyocytes was associated with NF-κB activation and induction of apoptosis. NOD1 stimulation in fibroblasts was linked to NF-κB activation and to increased expression of pro-fibrotic mediators. The down-regulation of NOD1 by specific siRNAs blunted the effect of iEDAP on the pro-fibrotic TGF-β pathway and cell apoptosis. In conclusion, our report uncovers a new pro-inflammatory target that is expressed in the heart, NOD1. The specific activation of this NLR induces cardiac dysfunction and modulates cardiac fibrosis and cardiomyocyte apoptosis, pathological processes involved in several cardiac diseases such as heart failure. PMID:23028889

  7. Methylglyoxal increases cardiomyocyte ischemia-reperfusion injury via glycative inhibition of thioredoxin activity

    PubMed Central

    Wang, Xiao-Liang; Lau, Wayne B.; Yuan, Yue-Xing; Wang, Ya-Jing; Yi, Wei; Christopher, Theodore A.; Lopez, Bernard L.; Liu, Hui-Rong

    2010-01-01

    Diabetes mellitus (DM) is closely related to cardiovascular morbidity and mortality, but the specific molecular basis linking DM with increased vulnerability to cardiovascular injury remains incompletely understood. Methylglyoxal (MG), a precursor to advanced glycation end products (AGEs), is increased in diabetic patient plasma, but its role in diabetic cardiovascular complications is unclear. Thioredoxin (Trx), a cytoprotective molecule with antiapoptotic function, has been demonstrated to be vulnerable to glycative inhibition, but whether Trx is glycatively inhibited by MG, thus contributing to increased cardiac injury, has never been investigated. Cultured H9c2 cardiomyocytes were treated with MG (200 μM) for 6 days. The following were determined pre- and post-simulated ischemia-reperfusion (SI-R; 8 h of hypoxia followed by 3 h of reoxygenation): cardiomyocyte death/apoptosis, Trx expression and activity, AGE formation, Trx-apoptosis-regulating kinase-1 (Trx-ASK1) complex formation, and p38 mitogen-activated protein kinase (MAPK) phosphorylation and activity. Compared with vehicle, MG significantly increased SI-R-induced cardiomyocyte LDH release and apoptosis (P < 0.01). Prior to SI-R, Trx activity was reduced in MG-treated cells, but Trx expression was increased moderately. Moreover, Trx-ASK1 complex formation was reduced, and both p38 MAPK activity and phosphorylation were increased. To investigate the effects of MG on Trx directly, recombinant human Trx (hTrx) was incubated with MG in vitro. Compared with vehicle, MG incubation markedly increased CML formation (a glycation footprint) and inhibited Trx activity. Finally, glycation inhibitor aminoguanidine administration during MG treatment of cultured cells reduced AGE formation, increased Trx activity, restored Trx-ASK1 interaction, and reduced p38 MAPK phosphorylation and activity, caspase-3 activation, and LDH release (P < 0.01). We demonstrated for the first time that methylglyoxal sensitized cultured

  8. Genome-wide approaches reveal functional vascular endothelial growth factor (VEGF)-inducible nuclear factor of activated T cells (NFAT) c1 binding to angiogenesis-related genes in the endothelium.

    PubMed

    Suehiro, Jun-ichi; Kanki, Yasuharu; Makihara, Chihiro; Schadler, Keri; Miura, Mai; Manabe, Yuuka; Aburatani, Hiroyuki; Kodama, Tatsuhiko; Minami, Takashi

    2014-10-17

    VEGF is a key regulator of endothelial cell migration, proliferation, and inflammation, which leads to activation of several signaling cascades, including the calcineurin-nuclear factor of activated T cells (NFAT) pathway. NFAT is not only important for immune responses but also for cardiovascular development and the pathogenesis of Down syndrome. By using Down syndrome model mice and clinical patient samples, we showed recently that the VEGF-calcineurin-NFAT signaling axis regulates tumor angiogenesis and tumor metastasis. However, the connection between genome-wide views of NFAT-mediated gene regulation and downstream gene function in the endothelium has not been studied extensively. Here we performed comprehensive mapping of genome-wide NFATc1 binding in VEGF-stimulated primary cultured endothelial cells and elucidated the functional consequences of VEGF-NFATc1-mediated phenotypic changes. A comparison of the NFATc1 ChIP sequence profile and epigenetic histone marks revealed that predominant NFATc1-occupied peaks overlapped with promoter-associated histone marks. Moreover, we identified two novel NFATc1 regulated genes, CXCR7 and RND1. CXCR7 knockdown abrogated SDF-1- and VEGF-mediated cell migration and tube formation. siRNA treatment of RND1 impaired vascular barrier function, caused RhoA hyperactivation, and further stimulated VEGF-mediated vascular outgrowth from aortic rings. Taken together, these findings suggest that dynamic NFATc1 binding to target genes is critical for VEGF-mediated endothelial cell activation. CXCR7 and RND1 are NFATc1 target genes with multiple functions, including regulation of cell migration, tube formation, and barrier formation in endothelial cells.

  9. Activity-dependent Transcriptional Regulation of M-type (Kv7) K+ Channels by AKAP79/150-mediated NFAT Actions

    PubMed Central

    Zhang, Jie; Shapiro, Mark S.

    2013-01-01

    Summary M-type K+ channels, encoded by the KCNQ2-5 (Kv7) gene family, play key roles in regulation of neuronal excitability; however, less is known about the mechanisms controlling their transcriptional expression. Here, we discovered a novel mechanism regulating KCNQ2/3 transcriptional expression by neuronal activity in rodent neurons, involving activation of calcineurin and Nuclear Factor of Activated T-cells (NFAT) transcription factors, orchestrated by A-kinase-anchoring protein (AKAP)79/150. The signal requires Ca2+ influx through L-type Ca2+ channels and both local and global Ca2+ elevations. We postulate increased M-channel expression to act as a negative-feedback to suppress hyper-excitability of neurons, demonstrated by profoundly up-regulated KCNQ2/3 transcription in hippocampi from wild-type mice after drug-induced seizures, an effect nearly eliminated in AKAP150−/− mice. Thus, we suggest a distinct role of AKAP79/150 and the complex it organizes in activity-dependent M-channel transcription, which may potentially serve throughout the nervous system to limit over-excitability associated with disease states such as epilepsy. PMID:23259949

  10. Expression, fermentation and purification of a predicted intrinsically disordered region of the transcription factor, NFAT5.

    PubMed

    DuMond, Jenna F; He, Yi; Burg, Maurice B; Ferraris, Joan D

    2015-11-01

    Hypertonicity stimulates Nuclear Factor of Activated T-cells 5 (NFAT5) nuclear localization and transactivating activity. Many transcription factors are known to contain intrinsically disordered regions (IDRs) which become more structured with local environmental changes such as osmolality, temperature and tonicity. The transactivating domain of NFAT5 is predicted to be intrinsically disordered under normal tonicity, and under high NaCl, the activity of this domain is increased. To study the binding of co-regulatory proteins at IDRs a cDNA construct expressing the NFAT5 TAD was created and transformed into Escherichia coli cells. Transformed E. coli cells were mass produced by fermentation and extracted by cell lysis to release the NFAT5 TAD. The NFAT5 TAD was subsequently purified using a His-tag column, cation exchange chromatography as well as hydrophobic interaction chromatography and then characterized by mass spectrometry (MS). PMID:26256058

  11. Immunosuppressive Activity of Daphnetin, One of Coumarin Derivatives, Is Mediated through Suppression of NF-κB and NFAT Signaling Pathways in Mouse T Cells

    PubMed Central

    Liu, Yan; Xu, Sisi; Huang, Guoren; Xiong, Ying; Zhang, Shuang; Xu, Linli; Deng, Xuming; Guan, Shuang

    2014-01-01

    Daphnetin, a plant-derived dihydroxylated derivative of coumarin, is an effective compound extracted from a plant called Daphne Korean Nakai. Coumarin derivates were known for their antithrombotic, anti-inflammatory, and antioxidant activities. The present study was aimed to determine the immunosuppressive effects and the underlying mechanisms of daphnetin on concanavalin A (ConA) induced T lymphocytes in mice. We showed that, in vitro, daphnetin suppressed ConA-induced splenocyte proliferation, influenced production of the cytokines and inhibited cell cycle progression through the G0/G1 transition. The data also revealed that daphnetin could down-regulate activation of ConA induced NF-κB and NFAT signal transduction pathways in mouse T lymphocyte. In vivo, daphnetin treatment significantly inhibited the 2, 4- dinitrofluorobenzene (DNFB) -induced delayed type hypersensitivity (DTH) reactions in mice. Collectively, daphnetin had strong immunosuppressive activity both in vitro and in vivo, suggesting a potential role for daphnetin as an immunosuppressive agent, and established the groundwork for further research on daphnetin. PMID:24800925

  12. Ligand activation of cannabinoid receptors attenuates hypertrophy of neonatal rat cardiomyocytes.

    PubMed

    Lu, Yan; Akinwumi, Bolanle C; Shao, Zongjun; Anderson, Hope D

    2014-11-01

    : Endocannabinoids are bioactive amides, esters, and ethers of long-chain polyunsaturated fatty acids. Evidence suggests that activation of the endocannabinoid pathway offers cardioprotection against myocardial ischemia, arrhythmias, and endothelial dysfunction of coronary arteries. As cardiac hypertrophy is a convergence point of risk factors for heart failure, we determined a role for endocannabinoids in attenuating endothelin-1-induced hypertrophy and probed the signaling pathways involved. The cannabinoid receptor ligand anandamide and its metabolically stable analog, R-methanandamide, suppressed hypertrophic indicators including cardiomyocyte enlargement and fetal gene activation (ie, the brain natriuretic peptide gene) elicited by endothelin-1 in isolated neonatal rat ventricular myocytes. The ability of R-methanandamide to suppress myocyte enlargement and fetal gene activation was mediated by CB2 and CB1 receptors, respectively. Accordingly, a CB2-selective agonist, JWH-133, prevented only myocyte enlargement but not brain natriuretic peptide gene activation. A CB1/CB2 dual agonist with limited brain penetration, CB-13, inhibited both hypertrophic indicators. CB-13 activated AMP-activated protein kinase (AMPK) and, in an AMPK-dependent manner, endothelial nitric oxide synthase (eNOS). Disruption of AMPK signaling, using compound C or short hairpinRNA knockdown, and eNOS inhibition using L-NIO abolished the antihypertrophic actions of CB-13. In conclusion, CB-13 inhibits cardiomyocyte hypertrophy through AMPK-eNOS signaling and may represent a novel therapeutic approach to cardioprotection. PMID:24979612

  13. Ets-1 facilitates nuclear entry of NFAT proteins and their recruitment to the IL-2 promoter.

    PubMed

    Tsao, Hsiao-Wei; Tai, Tzong-Shyuan; Tseng, William; Chang, Hui-Hsin; Grenningloh, Roland; Miaw, Shi-Chuen; Ho, I-Cheng

    2013-09-24

    E26 transformation-specific sequence 1 (Ets-1), the prototype of the ETS family of transcription factors, is critical for the expression of IL-2 by murine Th cells; however, its mechanism of action is still unclear. Here we show that Ets-1 is also essential for optimal production of IL-2 by primary human Th cells. Although Ets-1 negatively regulates the expression of Blimp1, a known suppressor of IL-2 expression, ablation of B lymphocyte-induced maturation protein 1 (Blimp1) does not rescue the expression of IL-2 by Ets-1-deficient Th cells. Instead, Ets-1 physically and functionally interacts with the nuclear factor of activated T-cells (NFAT) and is required for the recruitment of NFAT to the IL-2 promoter. In addition, Ets-1 is located in both the nucleus and cytoplasm of resting Th cells. Nuclear Ets-1 quickly exits the nucleus in response to calcium-dependent signals and competes with NFAT proteins for binding to protein components of noncoding RNA repressor of NFAT complex (NRON), which serves as a cytoplasmic trap for phosphorylated NFAT proteins. This nuclear exit of Ets-1 precedes rapid nuclear entry of NFAT and Ets-1 deficiency results in impaired nuclear entry, but not dephosphorylation, of NFAT proteins. Thus, Ets-1 promotes the expression of IL-2 by modulating the activity of NFAT.

  14. Lipoprotein lipase activity in rat cardiomyocytes is stimulated by insulin and dexamethasone.

    PubMed Central

    Ewart, H S; Carroll, R; Severson, D L

    1997-01-01

    Lipoprotein lipase (LPL) activity was studied in rat cardiomyocytes after overnight culture (16 h) in the presence of insulin (100 nM) and/or dexamethasone (100 nM). Insulin in combination with dexamethasone (INS/DEX) increased heparin-releasable LPL activity by 71% over the control level (566+/-85 versus 331+/-48 nmol/h.mg cell protein). This was accompanied by a 61% increase in total cellular LPL activity (914+/-89 versus 567+/-64 nmol/h.mg cell protein). The increase in LPL activity occurred at sub-nanomolar concentrations of the hormones, but neither hormone was effective alone. LPL protein mass, quantified by ELISA, was the same in both control and INS/DEX-treated cells (27.7 versus 28.6 ng/mg cell protein, respectively), thus LPL specific activity in cardiomyocytes was increased by INS/DEX treatment (0.113 versus 0.069 mU/ng LPL protein). These findings emphasize the importance of hormonal interactions in the regulation of LPL in heart tissue. PMID:9359413

  15. Sulforaphane prevents rat cardiomyocytes from hypoxia/reoxygenation injury in vitro via activating SIRT1 and subsequently inhibiting ER stress

    PubMed Central

    Li, Yun-peng; Wang, Shu-lin; Liu, Bei; Tang, Lu; Kuang, Rong-ren; Wang, Xian-bao; Zhao, Cong; Song, Xu-dong; Cao, Xue-ming; Wu, Xiang; Yang, Ping-zhen; Wang, Li-zi; Chen, Ai-hua

    2016-01-01

    Aim: Sulforaphane (SFN), a natural dietary isothiocyanate, is found to exert beneficial effects for cardiovascular diseases. This study aimed to investigate the mechanisms underlying the protective effects of SFN in a model of myocardial hypoxia/reoxygenation (H/R) injury in vitro. Methods: Cultured neonatal rat cardiomyocytes pretreated with SFN were subjected to 3-h hypoxia followed by 3-h reoxygenation. Cell viability and apoptosis were detected. Caspase-3 activity and mitochondrial membrane potential (ΔΨm) was measured. The expression of ER stress-related apoptotic proteins were analyzed with Western blot analyses. Silent information regulator 1 (SIRT1) activity was determined with SIRT1 deacetylase fluorometric assay kit. Results: SFN (0.1–5 μmol/L) dose-dependently improved the viability of cardiomyocytes, diminished apoptotic cells and suppressed caspase-3 activity. Meanwhile, SFN significantly alleviated the damage of ΔΨm and decreased the expression of ER stress-related apoptosis proteins (GRP78, CHOP and caspase-12), elevating the expression of SIRT1 and Bcl-2/Bax ratio in the cardiomyocytes. Co-treatment of the cardiomyocytes with the SIRT1-specific inhibitor Ex-527 (1 μmol/L) blocked the SFN-induced cardioprotective effects. Conclusion: SFN prevents cardiomyocytes from H/R injury in vitro most likely via activating SIRT1 pathway and subsequently inhibiting the ER stress-dependent apoptosis. PMID:26775664

  16. Band-pass processing in a GPCR signaling pathway selects for NFAT transcription factor activation†

    PubMed Central

    Sumit, M.; Neubig, R. R.; Takayama, S.; Linderman, J. J.

    2015-01-01

    Many biological processes are rhythmic and proper timing is increasingly appreciated as being critical for development and maintenance of physiological functions. To understand how temporal modulation of an input signal influences downstream responses, we employ microfluidic pulsatile stimulation of a G-Protein coupled receptor, the muscarinic M3 receptor, in single cells with simultaneous real-time imaging of both intracellular calcium and NFAT nuclear localization. Interestingly, we find that reduced stimulation with pulses of ligand can give more efficient transcription factor activation, if stimuli are timed appropriately. Our experiments and computational analyses show that M3 receptor-induced calcium oscillations form a low pass filter while calcium-induced NFAT translocation forms a high pass filter. The combination acts as a band-pass filter optimized for intermediate frequencies of stimulation. We demonstrate that receptor desensitization and NFAT translocation rates determine critical features of the band-pass filter and that the band-pass may be shifted for different receptors or NFAT dynamics. As an example, we show that the two NFAT isoforms (NFAT4 and NFAT1) have shifted band-pass windows for the same receptor. While we focus specifically on the M3 muscarinic receptor and NFAT translocation, band-pass processing is expected to be a general theme that applies to multiple signaling pathways. PMID:26374065

  17. Hypertonic-induced lamin A/C synthesis and distribution to nucleoplasmic speckles is mediated by TonEBP/NFAT5 transcriptional activator

    SciTech Connect

    Favale, Nicolas O.; Sterin Speziale, Norma B.; Fernandez Tome, Maria C.

    2007-12-21

    Lamin A/C is the most studied nucleoskeletal constituent. Lamin A/C expression indicates cell differentiation and is also a structural component of nuclear speckles, which are involved in gene expression regulation. Hypertonicity has been reported to induce renal epithelial cell differentiation and expression of TonEBP (NFAT5), a transcriptional activator of hypertonicity-induced gene transcription. In this paper, we investigate the effect of hypertonicity on lamin A/C expression in MDCK cells and the involvement of TonEBP. Hypertonicity increased lamin A/C expression and its distribution to nucleoplasm with speckled pattern. Microscopy showed codistribution of TonEBP and lamin A/C in nucleoplasmic speckles, and immunoprecipitation demonstrated their interaction. TonEBP silencing caused lamin A/C redistribution from nucleoplasmic speckles to the nuclear rim, followed by lamin decrease, thus showing that hypertonicity induces lamin A/C speckles through a TonEBP-dependent mechanism. We suggest that lamin A/C speckles could serve TonEBP as scaffold thus favoring its role in hypertonicity.

  18. Photocontrol of Voltage-Gated Ion Channel Activity by Azobenzene Trimethylammonium Bromide in Neonatal Rat Cardiomyocytes

    PubMed Central

    Frolova, Sheyda R.; Gaiko, Olga; Tsvelaya, Valeriya A.; Pimenov, Oleg Y.; Agladze, Konstantin I.

    2016-01-01

    The ability of azobenzene trimethylammonium bromide (azoTAB) to sensitize cardiac tissue excitability to light was recently reported. The dark, thermally relaxed trans- isomer of azoTAB suppressed spontaneous activity and excitation propagation speed, whereas the cis- isomer had no detectable effect on the electrical properties of cardiomyocyte monolayers. As the membrane potential of cardiac cells is mainly controlled by activity of voltage-gated ion channels, this study examined whether the sensitization effect of azoTAB was exerted primarily via the modulation of voltage-gated ion channel activity. The effects of trans- and cis- isomers of azoTAB on voltage-dependent sodium (INav), calcium (ICav), and potassium (IKv) currents in isolated neonatal rat cardiomyocytes were investigated using the whole-cell patch-clamp technique. The experiments showed that azoTAB modulated ion currents, causing suppression of sodium (Na+) and calcium (Ca2+) currents and potentiation of net potassium (K+) currents. This finding confirms that azoTAB-effect on cardiac tissue excitability do indeed result from modulation of voltage-gated ion channels responsible for action potential. PMID:27015602

  19. NFAT5 regulates transcription of the mouse telomerase reverse transcriptase gene

    SciTech Connect

    Fujiki, Tsukasa; Udono, Miyako; Kotake, Yojiro; Yamashita, Makiko; Shirahata, Sanetaka; Katakura, Yoshinori

    2010-12-10

    We aimed to clarify the transcription-regulation mechanisms of the mouse telomerase reverse transcriptase gene (mTERT). First, we searched for the promoter region required for transcriptional activation of mTERT and identified an enhancer cis-element (named mTERT-EE) located between - 200 and - 179 bp of the mouse TERT gene (mTERT). EMSA results suggested that nuclear factor of activated T cells (NFAT) member proteins bind to mTERT-EE. We then identified NFAT5 as the factor binding to mTERT-EE and found that it activates the transcription of the mTERT core promoter. The results that siRNA directed against NFAT5 significantly reduced mTERT expression and mTERT core promoter activity and that the expressions of NFAT5 and mTERT were well correlated in various mouse tissues except liver suggest that NFAT5 dominantly and directly regulates mTERT expression. To clarify their functionality further, we investigated the effect of hypertonic stress, a known stimulus affecting the expression and transcriptional activity of NFAT5, on mTERT expression. The result indicated that hypertonic stress activates mTERT transcription via the activation and recruitment of NFAT5 to the mTERT promoter. These results provide useful information about the transcription-regulation mechanisms of mTERT.

  20. NFAT transcription factors regulate survival, proliferation, migration, and differentiation of neural precursor cells.

    PubMed

    Serrano-Pérez, María C; Fernández, Miriam; Neria, Fernando; Berjón-Otero, Mónica; Doncel-Pérez, Ernesto; Cano, Eva; Tranque, Pedro

    2015-06-01

    The study of factors that regulate the survival, proliferation, and differentiation of neural precursor cells (NPCs) is essential to understand neural development as well as brain regeneration. The Nuclear Factor of Activated T Cells (NFAT) is a family of transcription factors that can affect these processes besides playing key roles during development, such as stimulating axonal growth in neurons, maturation of immune system cells, heart valve formation, and differentiation of skeletal muscle and bone. Interestingly, NFAT signaling can also promote cell differentiation in adults, participating in tissue regeneration. The goal of the present study is to evaluate the expression of NFAT isoforms in NPCs, and to investigate its possible role in NPC survival, proliferation, migration, and differentiation. Our findings indicate that NFAT proteins are active not only in neurogenic brain regions such as hippocampus and subventricular zone (SVZ), but also in cultured NPCs. The inhibition of NFAT activation with the peptide VIVIT reduced neurosphere size and cell density in NPC cultures by decreasing proliferation and increasing cell death. VIVIT also decreased NPC migration and differentiation of astrocytes and neurons from NPCs. In addition, we identified NFATc3 as a predominant NFAT isoform in NPC cultures, finding that a constitutively-active form of NFATc3 expressed by adenoviral infection reduces NPC proliferation, stimulates migration, and is a potent inducer of NPC differentiation into astrocytes and neurons. In summary, our work uncovers active roles for NFAT signaling in NPC survival, proliferation and differentiation, and highlights its therapeutic potential for tissue regeneration.

  1. Activation of Sonic hedgehog signaling in ventricular cardiomyocytes exerts cardioprotection against ischemia reperfusion injuries

    PubMed Central

    Paulis, Ludovit; Fauconnier, Jeremy; Cazorla, Olivier; Thireau, Jérome; Soleti, Raffaella; Vidal, Bastien; Ouillé, Aude; Bartholome, Marion; Bideaux, Patrice; Roubille, François; Le Guennec, Jean-Yves; Andriantsitohaina, Ramaroson; Martínez, M. Carmen; Lacampagne, Alain

    2015-01-01

    Sonic hedgehog (SHH) is a conserved protein involved in embryonic tissue patterning and development. SHH signaling has been reported as a cardio-protective pathway via muscle repair–associated angiogenesis. The goal of this study was to investigate the role of SHH signaling pathway in the adult myocardium in physiological situation and after ischemia-reperfusion. We show in a rat model of ischemia-reperfusion that stimulation of SHH pathway, either by a recombinant peptide or shed membranes microparticles harboring SHH ligand, prior to reperfusion reduces both infarct size and subsequent arrhythmias by preventing ventricular repolarization abnormalities. We further demonstrate in healthy animals a reduction of QTc interval mediated by NO/cGMP pathway leading to the shortening of ventricular cardiomyocytes action potential duration due to the activation of an inward rectifying potassium current sharing pharmacological and electrophysiological properties with ATP-dependent potassium current. Besides its effect on both angiogenesis and endothelial dysfunction we demonstrate here a novel cardio-protective effect of SHH acting directly on the cardiomyocytes. This emphasizes the pleotropic effect of SHH pathway as a potential cardiac therapeutic target. PMID:25613906

  2. Rapid fusion between mesenchymal stem cells and cardiomyocytes yields electrically active, non-contractile hybrid cells

    PubMed Central

    Shadrin, Ilya Y.; Yoon, Woohyun; Li, Liqing; Shepherd, Neal; Bursac, Nenad

    2015-01-01

    Cardiac cell therapies involving bone marrow-derived human mesenchymal stem cells (hMSCs) have shown promising results, although their mechanisms of action are still poorly understood. Here, we investigated direct interactions between hMSCs and cardiomyocytes in vitro. Using a genetic Ca2+ indicator gCaMP3 to efficiently label hMSCs in co-cultures with neonatal rat ventricular myocytes (NRVMs), we determined that 25–40% of hMSCs (from 4 independent donors) acquired periodic Ca2+ transients and cardiac markers through spontaneous fusion with NRVMs. Sharp electrode and voltage-clamp recordings in fused cells showed action potential properties and Ca2+ current amplitudes in between those of non-fused hMSCs and NRVMs. Time-lapse video-microscopy revealed the first direct evidence of active fusion between hMSCs and NRVMs within several hours of co-culture. Application of blebbistatin, nifedipine or verapamil caused complete and reversible inhibition of fusion, suggesting potential roles for actomyosin bridging and Ca2+ channels in the fusion process. Immunostaining for Cx43, Ki67, and sarcomeric α-actinin showed that fused cells remain strongly coupled to surrounding NRVMs, but downregulate sarcomeric structures over time, acquiring a non-proliferative and non-contractile phenotype. Overall, these results describe the phenotype and mechanisms of hybrid cell formation via fusion of hMSCs and cardiomyocytes with potential implications for cardiac cell therapy. PMID:26159124

  3. Integrated Analysis of Contractile Kinetics, Force Generation, and Electrical Activity in Single Human Stem Cell-Derived Cardiomyocytes

    PubMed Central

    Kijlstra, Jan David; Hu, Dongjian; Mittal, Nikhil; Kausel, Eduardo; van der Meer, Peter; Garakani, Arman; Domian, Ibrahim J.

    2015-01-01

    Summary The quantitative analysis of cardiomyocyte function is essential for stem cell-based approaches for the in vitro study of human cardiac physiology and pathophysiology. We present a method to comprehensively assess the function of single human pluripotent stem cell-derived cardiomyocyte (hPSC-CMs) through simultaneous quantitative analysis of contraction kinetics, force generation, and electrical activity. We demonstrate that statistical analysis of movies of contracting hPSC-CMs can be used to quantify changes in cellular morphology over time and compute contractile kinetics. Using a biomechanical model that incorporates substrate stiffness, we calculate cardiomyocyte force generation at single-cell resolution and validate this approach with conventional traction force microscopy. The addition of fluorescent calcium indicators or membrane potential dyes allows the simultaneous analysis of contractility and calcium handling or action potential morphology. Accordingly, our approach has the potential for broad application in the study of cardiac disease, drug discovery, and cardiotoxicity screening. PMID:26626178

  4. Estrogen-related receptor gamma regulates dopaminergic neuronal phenotype by activating GSK3β/NFAT signaling in SH-SY5Y cells.

    PubMed

    Lim, Juhee; Choi, Hueng-Sik; Choi, Hyun Jin

    2015-05-01

    The orphan nuclear receptor estrogen-related receptor gamma (ERRγ) is highly expressed in the nervous system during embryogenesis and in adult brains, but its physiological role in neuronal development remains unknown. In this study, we evaluated the relevance of ERRγ in regulating dopaminergic (DAergic) phenotype and the corresponding signaling pathway. We used retinoic acid (RA) to differentiate human neuroblastoma SH-SY5Y cells. RA induced neurite outgrowth of SH-SY5Y cells with an increase in DAergic neuron-like properties, including up-regulation of tyrosine hydroxylase, dopamine transporter, and vesicular monoamine transporter 2. ERRγ, but not ERRα, was up-regulated by RA, and participated in RA effect on SH-SY5Y cells. ERRγ over-expression enhanced mature DAergic neuronal phenotype with neurite outgrowth as with RA treatment; and RA-induced increase in DAergic phenotype was attenuated by silencing ERRγ expression. ERRγ appears to have a crucial role in morphological and functional regulation of cells that is selective for DAergic neurons. Polo-like kinase 2 was up-regulated in ERRγ-over-expressing SH-SY5Y cells, which was involved in phosphorylation of glycogen synthase kinase 3β and resulting downstream activation of nuclear factor of activated T cells. The likely involvement of ERRγ in regulating the DAergic neuronal phenotype makes this orphan nuclear receptor a novel target for understanding DAergic neuronal differentiation. We propose the relevance of estrogen-related receptor gamma (ERRγ) in regulating dopaminergic neuronal phenotype: ERRγ is up-regulated by retinoic acid in SH-SY5Y cells, and enhances dopaminergic phenotypes and induces neurite outgrowth; Polo-like kinase 2 (PLK2) and glycogen synthase kinase 3 beta/nuclear factor of activated T cells (GSK3β/NFAT) signaling are responsible for the ERRγ effect. Our findings provide the first insights into the role of ERRγ in the brain, as a novel approach toward understanding

  5. Sildenafil prevents the up-regulation of transient receptor potential canonical channels in the development of cardiomyocyte hypertrophy

    SciTech Connect

    Kiso, Hironori; Ohba, Takayoshi; Iino, Kenji; Sato, Kazuhiro; Terata, Yutaka; Murakami, Manabu; Ono, Kyoichi; Watanabe, Hiroyuki; Ito, Hiroshi

    2013-07-05

    Highlights: •Transient receptor potential canonical (TRPC1, 3 and 6) are up-regulated by ET-1. •Sildenafil inhibited hypertrophic responses (BNP, Ca entry, NFAT activation). •Sildenafil suppressed TRPC1, 3 and 6 expression. -- Abstract: Background: Transient receptor potential canonical (TRPCs) channels are up-regulated in the development of cardiac hypertrophy. Sildenafil inhibits TRPC6 activation and expression, leading to the prevention of cardiac hypertrophy. However, the effects of sildenafil on the expression of other TRPCs remain unknown. We hypothesized that in addition to its effects of TRPC6, sildenafil blocks the up-regulation of other TRPC channels to suppress cardiomyocyte hypertrophy. Methods and results: In cultured neonatal rat cardiomyocytes, a 48 h treatment with 10 nM endothelin (ET)-1 induced hypertrophic responses characterized by nuclear factor of activated T cells activation and enhancement of brain natriuretic peptide expression and cell surface area. Co-treatment with sildenafil (1 μM, 48 h) inhibited these ET-1-induced hypertrophic responses. Although ET-1 enhanced the gene expression of TRPCs, sildenafil inhibited the enhanced gene expression of TRPC1, C3 and C6. Moreover, co-treatment with sildenafil abolished the augmentation of SOCE in the hypertrophied cardiomyocytes. Conclusions: These results suggest that sildenafil inhibits cardiomyocyte hypertrophy by suppressing the up-regulation of TRPC expression.

  6. UCR1C is a novel activator of phosphodiesterase 4 (PDE4) long isoforms and attenuates cardiomyocyte hypertrophy

    PubMed Central

    Wang, Li; Burmeister, Brian T.; Johnson, Keven R.; Baillie, George S.; Karginov, Andrei V.; Skidgel, Randal A.; O’Bryan, John P.; Carnegie, Graeme K.

    2015-01-01

    Hypertrophy increases the risk of heart failure and arrhythmia. Prevention or reversal of the maladaptive hypertrophic phenotype has thus been proposed to treat heart failure. Chronic β-adrenergic receptor (β-AR) stimulation induces cardiomyocyte hypertrophy by elevating 3′, 5′-cyclic adenosine monophosphate (cAMP) levels and activating downstream effectors such protein kinase A (PKA). Conversely, hydrolysis of cAMP by phosphodiesterases (PDEs) spatiotemporally restricts cAMP signaling. Here, we demonstrate that PDE4, but not PDE3, is critical in regulating cardiomyocyte hypertrophy, and may represent a potential target for preventing maladaptive hypertrophy. We identify a sequence within the upstream conserved region 1 of PDE4D, termed UCR1C, as a novel activator of PDE4 long isoforms. UCR1C activates PDE4 in complex with A-Kinase anchoring protein (AKAP)-Lbc resulting in decreased PKA signaling facilitated by AKAP-Lbc. Expression of UCR1C in cardiomyocytes inhibits hypertrophy in response to chronic β-AR stimulation. This effect is partially due to inhibition of nuclear PKA activity, which decreases phosphorylation of the transcription factor cAMP response element-binding protein (CREB). In conclusion, PDE4 activation by UCR1C attenuates cardiomyocyte hypertrophy by specifically inhibiting nuclear PKA activity. PMID:25683917

  7. Impaired NFAT and NFκB activation are involved in suppression of CD40 ligand expression by Δ{sup 9}-tetrahydrocannabinol in human CD4{sup +} T cells

    SciTech Connect

    Ngaotepprutaram, Thitirat; Kaplan, Barbara L.F.; Kaminski, Norbert E.

    2013-11-15

    We have previously reported that Δ{sup 9}-tetrahydrocannabinol (Δ{sup 9}-THC), the main psychoactive cannabinoid in marijuana, suppresses CD40 ligand (CD40L) expression by activated mouse CD4{sup +} T cells. CD40L is involved in pathogenesis of many autoimmune and inflammatory diseases. In the present study, we investigated the molecular mechanism of Δ{sup 9}-THC-mediated suppression of CD40L expression using peripheral blood human T cells. Pretreatment with Δ{sup 9}-THC attenuated CD40L expression in human CD4{sup +} T cells activated by anti-CD3/CD28 at both the protein and mRNA level, as determined by flow cytometry and quantitative real-time PCR, respectively. Electrophoretic mobility shift assays revealed that Δ{sup 9}-THC suppressed the DNA-binding activity of both NFAT and NFκB to their respective response elements within the CD40L promoter. An assessment of the effect of Δ{sup 9}-THC on proximal T cell-receptor (TCR) signaling induced by anti-CD3/CD28 showed significant impairment in the rise of intracellular calcium, but no significant effect on the phosphorylation of ZAP70, PLCγ1/2, Akt, and GSK3β. Collectively, these findings identify perturbation of the calcium-NFAT and NFκB signaling cascade as a key mechanistic event by which Δ{sup 9}-THC suppresses human T cell function. - Highlights: • Δ{sup 9}-THC attenuated CD40L expression in activated human CD4+ T cells. • Δ{sup 9}-THC suppressed DNA-binding activity of NFAT and NFκB. • Δ{sup 9}-THC impaired elevation of intracellular Ca2+. • Δ{sup 9}-THC did not affect phosphorylation of ZAP70, PLCγ1/2, Akt, and GSK3β.

  8. Aβ mediates Sigma receptor degradation via CaN/NFAT pathway.

    PubMed

    Fang, Min; Zhang, Pei; Zhao, Yanxin; Jin, Aiping; Liu, Xueyuan

    2016-01-01

    Sigma receptor is an endoplasmic reticulum protein and belongs to non-opioid receptor. Increasing evidence shows that Sigma receptor activation can significantly attenuate AD induced neurological dysfunction and the functional deficiency of Sigma receptor plays an important role in the Aβ induced neuronal loss. This study aimed to investigate the influence of extracellular accumulation of Aβ on the Sigma receptor expression. Our results showed the increase in extracellular Aβ had little influence on the mRNA expression of Sigma receptor, but gradually reduced its protein expression. Co-immunoprecipitation was employed to evaluate the interaction of Sigma receptor with other proteins. Results showed BIP could bind to Sigma receptor to affect the ubiquitination of Sigma receptor. Further investigation showed there was a NFAT binding site at the promoter of BIP. Then, Western blot assay was performed to detect NFAT expression. Results showed extracellular Aβ affected the nuclear translocation of NFAT and the CaN activity of NFAT also increased with the accumulation of extracellular Aβ. In this study, NFAT-BIP luciferase reporter gene system was constructed. Results showed NFAT was able to regulate the transcription of BIP. Thus, we speculate that extracellular Aβ accumulation may activate CaN/NFAT signaling pathway to induce chaperone BIP expression, which results in Sigma receptor ubiquitination and its degradation. PMID:27648137

  9. Aβ mediates Sigma receptor degradation via CaN/NFAT pathway.

    PubMed

    Fang, Min; Zhang, Pei; Zhao, Yanxin; Jin, Aiping; Liu, Xueyuan

    2016-01-01

    Sigma receptor is an endoplasmic reticulum protein and belongs to non-opioid receptor. Increasing evidence shows that Sigma receptor activation can significantly attenuate AD induced neurological dysfunction and the functional deficiency of Sigma receptor plays an important role in the Aβ induced neuronal loss. This study aimed to investigate the influence of extracellular accumulation of Aβ on the Sigma receptor expression. Our results showed the increase in extracellular Aβ had little influence on the mRNA expression of Sigma receptor, but gradually reduced its protein expression. Co-immunoprecipitation was employed to evaluate the interaction of Sigma receptor with other proteins. Results showed BIP could bind to Sigma receptor to affect the ubiquitination of Sigma receptor. Further investigation showed there was a NFAT binding site at the promoter of BIP. Then, Western blot assay was performed to detect NFAT expression. Results showed extracellular Aβ affected the nuclear translocation of NFAT and the CaN activity of NFAT also increased with the accumulation of extracellular Aβ. In this study, NFAT-BIP luciferase reporter gene system was constructed. Results showed NFAT was able to regulate the transcription of BIP. Thus, we speculate that extracellular Aβ accumulation may activate CaN/NFAT signaling pathway to induce chaperone BIP expression, which results in Sigma receptor ubiquitination and its degradation.

  10. Inhibition of FOXP3/NFAT Interaction Enhances T Cell Function after TCR Stimulation.

    PubMed

    Lozano, Teresa; Villanueva, Lorea; Durántez, Maika; Gorraiz, Marta; Ruiz, Marta; Belsúe, Virginia; Riezu-Boj, José I; Hervás-Stubbs, Sandra; Oyarzábal, Julen; Bandukwala, Hozefa; Lourenço, Ana R; Coffer, Paul J; Sarobe, Pablo; Prieto, Jesús; Casares, Noelia; Lasarte, Juan J

    2015-10-01

    Regulatory T cell (Treg) activity is modulated by a cooperative complex between the transcription factor NFAT and FOXP3, a lineage specification factor for Tregs. FOXP3/NFAT interaction is required to repress expression of IL-2, upregulate expression of the Treg markers CTLA4 and CD25, and confer suppressor function to Tregs. However, FOXP3 is expressed transiently in conventional CD4(+) T cells upon TCR stimulation and may lead to T cell hyporesponsiveness. We found that a short synthetic peptide able to inhibit FOXP3/NFAT interaction impaired suppressor activity of conventional Tregs in vitro. Specific inhibition of FOXP3/NFAT interaction with this inhibitory peptide revealed that FOXP3 downregulates NFAT-driven promoter activity of CD40L and IL-17. Inhibition of FOXP3/NFAT interaction upregulated CD40L expression on effector T cells and enhanced T cell proliferation and IL-2, IFN-γ, IL-6, or IL-17 production in response to TCR stimulation. The inhibitory peptide impaired effector T cell conversion into induced Tregs in the presence of TGF-β. Moreover, in vivo peptide administration showed antitumor efficacy in mice bearing Hepa129 or TC1 tumor cells when combined with sorafenib or with an antitumor vaccine, respectively. Our results suggest that inhibition of NFAT/FOXP3 interaction might improve antitumor immunotherapies.

  11. Aβ mediates Sigma receptor degradation via CaN/NFAT pathway

    PubMed Central

    Fang, Min; Zhang, Pei; Zhao, Yanxin; Jin, Aiping; Liu, Xueyuan

    2016-01-01

    Sigma receptor is an endoplasmic reticulum protein and belongs to non-opioid receptor. Increasing evidence shows that Sigma receptor activation can significantly attenuate AD induced neurological dysfunction and the functional deficiency of Sigma receptor plays an important role in the Aβ induced neuronal loss. This study aimed to investigate the influence of extracellular accumulation of Aβ on the Sigma receptor expression. Our results showed the increase in extracellular Aβ had little influence on the mRNA expression of Sigma receptor, but gradually reduced its protein expression. Co-immunoprecipitation was employed to evaluate the interaction of Sigma receptor with other proteins. Results showed BIP could bind to Sigma receptor to affect the ubiquitination of Sigma receptor. Further investigation showed there was a NFAT binding site at the promoter of BIP. Then, Western blot assay was performed to detect NFAT expression. Results showed extracellular Aβ affected the nuclear translocation of NFAT and the CaN activity of NFAT also increased with the accumulation of extracellular Aβ. In this study, NFAT-BIP luciferase reporter gene system was constructed. Results showed NFAT was able to regulate the transcription of BIP. Thus, we speculate that extracellular Aβ accumulation may activate CaN/NFAT signaling pathway to induce chaperone BIP expression, which results in Sigma receptor ubiquitination and its degradation. PMID:27648137

  12. Aβ mediates Sigma receptor degradation via CaN/NFAT pathway

    PubMed Central

    Fang, Min; Zhang, Pei; Zhao, Yanxin; Jin, Aiping; Liu, Xueyuan

    2016-01-01

    Sigma receptor is an endoplasmic reticulum protein and belongs to non-opioid receptor. Increasing evidence shows that Sigma receptor activation can significantly attenuate AD induced neurological dysfunction and the functional deficiency of Sigma receptor plays an important role in the Aβ induced neuronal loss. This study aimed to investigate the influence of extracellular accumulation of Aβ on the Sigma receptor expression. Our results showed the increase in extracellular Aβ had little influence on the mRNA expression of Sigma receptor, but gradually reduced its protein expression. Co-immunoprecipitation was employed to evaluate the interaction of Sigma receptor with other proteins. Results showed BIP could bind to Sigma receptor to affect the ubiquitination of Sigma receptor. Further investigation showed there was a NFAT binding site at the promoter of BIP. Then, Western blot assay was performed to detect NFAT expression. Results showed extracellular Aβ affected the nuclear translocation of NFAT and the CaN activity of NFAT also increased with the accumulation of extracellular Aβ. In this study, NFAT-BIP luciferase reporter gene system was constructed. Results showed NFAT was able to regulate the transcription of BIP. Thus, we speculate that extracellular Aβ accumulation may activate CaN/NFAT signaling pathway to induce chaperone BIP expression, which results in Sigma receptor ubiquitination and its degradation.

  13. Electrical field stimulation induces cardiac fibroblast proliferation through the calcineurin-NFAT pathway.

    PubMed

    Chen, Qing-Qing; Zhang, Wei; Chen, Xiang-Fan; Bao, Yun-Jian; Wang, Jing; Zhu, Wei-Zhong

    2012-12-01

    Most cardiac diseases are associated with fibrosis. Calcineurin (CaN) is regulated by Ca(2+)/calmodulin (CaM). The CaN-NFAT (nuclear factor of activated T cell) pathway is involved in the process of cardiac diseases, such as cardiac hypertrophy, but its effect on myocardial fibrosis remains unclear. The present study investigates whether the CaN-NFAT pathway is involved in cardiac fibroblast (CF) proliferation induced by electrical field stimulation (EFS), which recently became a popular treatment for heart failure and cardiac tissue engineering. CF proliferation was evaluated by a cell survival assay (MTT) and cell counts. Myocardial fibrosis was assessed by collagen I and collagen III protein expression. Green fluorescent protein (GFP)-tagged NFAT was used to detect NFAT nuclear translocation. CF proliferation, myocardial fibrosis, CaN activity, and NFAT nuclear translocation were enhanced by EFS. More importantly, these effects were abolished by CaN inhibitors, dominant negative CaN (DN-CaN), and CaN gene silenced with siRNA. Furthermore, buffering intracellular Ca(2+) with BAPTA-AM and blocking Ca(2+) influx with nifedipine suppressed EFS-induced increase in intracellular Ca(2+) and CF proliferation. These results suggested that the CaN-NFAT pathway mediates CF proliferation, and that the CaN-NFAT pathway might be a possible therapeutic target for EFS-induced myocardial fibrosis and cardiac tissue engineering.

  14. Streptococcal M1 protein triggers chemokine formation, neutrophil infiltration, and lung injury in an NFAT-dependent manner.

    PubMed

    Zhang, Songen; Zhang, Su; Garcia-Vaz, Eliana; Herwald, Heiko; Gomez, Maria F; Thorlacius, Henrik

    2015-06-01

    Streptococcus pyogenes of the M1 serotype can cause STSS, which is associated with significant morbidity and mortality. The purpose of the present study was to examine the role of NFAT signaling in M1 protein-induced lung injury. NFAT-luc mice were treated with the NFAT inhibitor A-285222 before administration of the M1 protein. Neutrophil infiltration, edema, and CXC chemokines were quantified in the lung, 4 h after challenge with the M1 protein. Flow cytometry was used to determine Mac-1 expression. Challenge with the M1 protein increased NFAT-dependent transcriptional activity in the lung, spleen, and liver in NFAT-luc mice. Administration of the NFAT inhibitor A-285222 abolished M1 protein-evoked NFAT activation in the lung, spleen, and liver. M1 protein challenge induced neutrophil recruitment, edema, and CXC chemokine production in the lung, as well as up-regulation of Mac-1 on circulating neutrophils. Inhibition of NFAT activity attenuated M1 protein-induced neutrophil infiltration by 77% and edema formation by 50% in the lung. Moreover, administration of A-285222 reduced M1 protein-evoked pulmonary formation of CXC chemokine >80%. In addition, NFAT inhibition decreased M1 protein-triggered Mac-1 up-regulation on neutrophils. These findings indicate that NFAT signaling controls pulmonary infiltration of neutrophils in response to streptococcal M1 protein via formation of CXC chemokines and neutrophil expression of Mac-1. Thus, the targeting of NFAT activity might be a useful way to ameliorate lung injury in streptococcal infections.

  15. Activity, inhibition, and induction of cytochrome P450 2J2 in adult human primary cardiomyocytes.

    PubMed

    Evangelista, Eric A; Kaspera, Rüdiger; Mokadam, Nahush A; Jones, J P; Totah, Rheem A

    2013-12-01

    Cytochrome P450 2J2 plays a significant role in the epoxidation of arachidonic acid to signaling molecules important in cardiovascular events. CYP2J2 also contributes to drug metabolism and is responsible for the intestinal clearance of ebastine. However, the interaction between arachidonic acid metabolism and drug metabolism in cardiac tissue, the main expression site of CYP2J2, has not been examined. Here we investigate an adult-derived human primary cardiac cell line as a suitable model to study metabolic drug interactions (inhibition and induction) of CYP2J2 in cardiac tissue. The primary human cardiomyocyte cell line demonstrated similar mRNA-expression profiles of P450 enzymes to adult human ventricular tissue. CYP2J2 was the dominant isozyme with minor contributions from CYP2D6 and CYP2E1. Both terfenadine and astemizole oxidation were observed in this cell line, whereas midazolam was not metabolized suggesting lack of CYP3A activity. Compared with recombinant CYP2J2, terfenadine was hydroxylated in cardiomyocytes at a similar K(m) value of 1.5 μM. The V(max) of terfenadine hydroxylation in recombinant enzyme was found to be 29.4 pmol/pmol P450 per minute and in the cells 6.0 pmol/pmol P450 per minute. CYP2J2 activity in the cell line was inhibited by danazol, astemizole, and ketoconazole in submicromolar range, but also by xenobiotics known to cause cardiac adverse effects. Of the 14 compounds tested for CYP2J2 induction, only rosiglitazone increased mRNA expression, by 1.8-fold. This cell model can be a useful in vitro model to investigate the role of CYP2J2-mediated drug metabolism, arachidonic acid metabolism, and their association to drug induced cardiotoxicity. PMID:24021950

  16. Parallel activation of Ca(2+)-induced survival and death pathways in cardiomyocytes by sorbitol-induced hyperosmotic stress.

    PubMed

    Chiong, M; Parra, V; Eisner, V; Ibarra, C; Maldonado, C; Criollo, A; Bravo, R; Quiroga, C; Contreras, A; Vicencio, J M; Cea, P; Bucarey, J L; Molgó, J; Jaimovich, E; Hidalgo, C; Kroemer, G; Lavandero, S

    2010-08-01

    Hyperosmotic stress promotes rapid and pronounced apoptosis in cultured cardiomyocytes. Here, we investigated if Ca(2+) signals contribute to this response. Exposure of cardiomyocytes to sorbitol [600 mosmol (kg water)(-1)] elicited large and oscillatory intracellular Ca(2+) concentration increases. These Ca(2+) signals were inhibited by nifedipine, Cd(2+), U73122, xestospongin C and ryanodine, suggesting contributions from both Ca(2+) influx through voltage dependent L-type Ca(2+) channels plus Ca(2+) release from intracellular stores mediated by IP(3) receptors and ryanodine receptors. Hyperosmotic stress also increased mitochondrial Ca(2+) levels, promoted mitochondrial depolarization, reduced intracellular ATP content, and activated the transcriptional factor cyclic AMP responsive element binding protein (CREB), determined by increased CREB phosphorylation and electrophoretic mobility shift assays. Incubation with 1 mM EGTA to decrease extracellular [Ca(2+)] prevented cardiomyocyte apoptosis induced by hyperosmotic stress, while overexpression of an adenoviral dominant negative form of CREB abolished the cardioprotection provided by 1 mM EGTA. These results suggest that hyperosmotic stress induced by sorbitol, by increasing Ca(2+) influx and raising intracellular Ca(2+) concentration, activates Ca(2+) release from stores and causes cell death through mitochondrial function collapse. In addition, the present results suggest that the Ca(2+) increase induced by hyperosmotic stress promotes cell survival by recruiting CREB-mediated signaling. Thus, the fate of cardiomyocytes under hyperosmotic stress will depend on the balance between Ca(2+)-induced survival and death pathways. PMID:20454859

  17. Ulinastatin protects cardiomyocytes against ischemia‑reperfusion injury by regulating autophagy through mTOR activation.

    PubMed

    Xiao, Jian; Zhu, Xiaoyan; Ji, Guangyu; Yang, Qian; Kang, Bo; Zhao, Jianquan; Yao, Feng; Wu, Lihui; Ni, Xin; Wang, Zhinong

    2014-10-01

    Autophagy is significant in myocardial ischemia-reperfusion (IR) injury. Ulinastatin has been demonstrated to protect cardiomyocytes against IR through inducing anti-inflammatory effects. However, whether ulinastatin has an anti‑autophagic effect is yet to be elucidated. The present study aimed to investigate the effect of ulinastatin on the regulation of autophagy during IR injury. Cardiomyocytes of neonatal rats were randomly divided into control, hypoxia-reoxygenation (HR) and ulinastatin groups. In order to investigate whether mammalian target of rapamycin (mTOR) is involved in mediating the protective effect of ulinastatin, cells were treated with the mTOR inhibitor, rapamycin 30 min prior to ulinastatin treatment. To demonstrate the anti-autophagic effect of ulinastatin in vivo, a rat IR model was established. Ulinastatin (1x104 U/kg body weight) was administered 30 min prior to the induction of IR via peritoneal injection. Light chain 3 (LC3), phosphorylated (p)‑mTOR, p‑protein kinase B (Akt) and p‑P70S6 kinase (p‑P70S6K) protein expression were assessed using western blot analysis. In addition, cell vitality, myocardial infarct size and lactate dehydrogenase (LDH) levels were measured. LC3‑Ⅱ protein expression was found to be downregulated, while p‑Akt, p‑mTOR and p‑P70S6K protein expression were observed to be upregulated by ulinastatin. In addition, cell vitality was found to increase and LDH was observed to decrease in the ulinastatin group compared with the HR group in vitro. Furthermore, rapamycin was found to attenuate the myocardial protective effect that is induced by ulinastatin. In vivo, ulinastatin was found to downregulate LC3‑Ⅱ protein expression, and reduce myocardium infarct size and LDH serum levels. These findings indicate that ulinastatin exhibits a myocardial protective effect against IR injury by regulating autophagy through mTOR activation.

  18. A cell permeable peptide inhibitor of NFAT inhibits macrophage cytokine expression and ameliorates experimental colitis.

    PubMed

    Elloumi, Houda Z; Maharshak, Nitsan; Rao, Kavitha N; Kobayashi, Taku; Ryu, Hyungjin S; Mühlbauer, Marcus; Li, Fengling; Jobin, Christian; Plevy, Scott E

    2012-01-01

    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.

  19. Generation of a conditional knockout allele for the NFAT5 gene in mice.

    PubMed

    Küper, Christoph; Beck, Franz-Xaver; Neuhofer, Wolfgang

    2014-01-01

    The osmosensitive transcription factor nuclear factor of activated T-cells 5 (NFAT5), also known as tonicity enhancer element binding protein (TonEBP) plays a crucial role in protection of renal medullary cells against hyperosmotic stress, urinary concentration, the adaptive immune response, and other physiological systems. Since it is also important for development, conventional homozygous-null mutations result in perinatal death, which hinders the analysis of NFAT5 function in specific tissues in vivo. Here we describe the generation of mice with a conditional-null allele, in which loxP sites are inserted around exon 4. Mice harboring the floxed allele (NFAT5(flx) ) were mated to a strain expressing a tamoxifen-inducible derivative of the Cre-recombinase (Cre (+)) under the control of the ubiqitinC promoter. The resultant homozygous conditional knockout mice (Cre (+) NFAT5 (flx/flx) ) are viable, fertile, and show normal expression of NFAT5 and NFAT5 target genes, indicating that the conditional alleles retain their wild-type function. Induction of Cre-mediated recombination by administration of tamoxifen in 8-week-old mice resulted in a decrease in NFAT5 expression of about 70-90% in all tested tissues (renal cortex, renal outer medulla, renal inner medulla, heart, lung, spleen, skeletal muscle). Accordingly, the expression of the NFAT5 target genes aldose reductase and heat shock protein 70 in the renal medulla was also significantly decreased. Mice harboring this conditional knockout allele should be useful in future studies for gaining a better understanding of tissue and cell-type specific functions of NFAT5 in adult animals under physiological and pathophysiological conditions. PMID:25601839

  20. Tumor suppressor gene ING3 induces cardiomyocyte hypertrophy via inhibition of AMPK and activation of p38 MAPK signaling.

    PubMed

    Wang, Jiaojiao; Liu, Zhiping; Feng, Xiaojun; Gao, Si; Xu, Suowen; Liu, Peiqing

    2014-11-15

    Cardiac hypertrophy, an adaptive growth process that occurs in response to various pathophysiological stimuli, constitutes an important risk factor for the development of heart failure. However, the molecular mechanisms that regulate this cardiac growth response are not completely understood. Here we revealed that ING3 (inhibitor of growth family, member 3), a type II tumor suppressor, plays a critical role in the regulation of cardiac hypertrophy. ING3 expression was present in relatively high abundance in the heart, and was prominently upregulated in hypertrophic agonists angiotensin II (Ang II), phenylephrine (PE), or isoproterenol (ISO)-stimulated cardiomyocytes and in hearts of rat undergoing abdominal aortic constriction (AAC) surgery. In cardiomyocytes, overexpression of ING3 caused an increase in ANP, BNP and β-MHC mRNA levels and cell surface area, while depletion of ING3 attenuated PE-induced cardiomyocyte hypertrophy. Mechanistically, we have demonstrated that overexpression of ING3 could inactivate the AMPK and activate the canonical p38 MAPK signaling. Remarkably, AMPK agonist AICAR or p38 MAPK inhibitor SB203580 abrogated ING3-induced hypertrophic response in cardiomyocytes. In summary, our data disclose a novel role of ING3 as an inducer of pathological cardiac hypertrophy, suggesting that silencing of ING3 may be explored as a potential therapeutic target in preventing cardiac hypertrophy.

  1. Integrative genomics identifies DSCR1 (RCAN1) as a novel NFAT-dependent mediator of phenotypic modulation in vascular smooth muscle cells.

    PubMed

    Lee, Monica Y; Garvey, Sean M; Baras, Alex S; Lemmon, Julia A; Gomez, Maria F; Schoppee Bortz, Pamela D; Daum, Guenter; LeBoeuf, Renee C; Wamhoff, Brian R

    2010-02-01

    Vascular smooth muscle cells (SMCs) display remarkable phenotypic plasticity in response to environmental cues. The nuclear factor of activated T-cells (NFAT) family of transcription factors plays a critical role in vascular pathology. However, known functional NFAT gene targets in vascular SMCs are currently limited. Publicly available whole-genome expression array data sets were analyzed to identify differentially expressed genes in human, mouse and rat SMCs. Comparison between vehicle and phenotypic modulatory stimuli identified 63 species-conserved, upregulated genes. Integration of the 63 upregulated genes with an in silico NFAT-ome (a species-conserved list of gene promoters containing at least one NFAT binding site) identified 18 putative NFAT-dependent genes. Further intersection of these 18 potential NFAT target genes with a mouse in vivo vascular injury microarray identified four putative NFAT-dependent, injury-responsive genes. In vitro validations substantiated the NFAT-dependent role of Cyclooxygenase 2 (COX2/PTGS2) in SMC phenotypic modulation and uncovered Down Syndrome Candidate Region 1 (DSCR1/RCAN1) as a novel NFAT target gene in SMCs. We show that induction of DSCR1 inhibits calcineurin/NFAT signaling through a negative feedback mechanism; DSCR1 overexpression attenuates NFAT transcriptional activity and COX2 protein expression, whereas knockdown of endogenous DSCR1 enhances NFAT transcriptional activity. Our integrative genomics approach illustrates how the combination of publicly available gene expression arrays, computational databases and empirical research methods can answer specific questions in any cell type for a transcriptional network of interest. Herein, we report DSCR1 as a novel NFAT-dependent, injury-inducible, early gene that may serve to negatively regulate SMC phenotypic switching.

  2. Unexpected anti-hypertrophic responses to low-level stimulation of protease-activated receptors in adult rat cardiomyocytes.

    PubMed

    Fender, Anke C; Pavic, Goran; Drummond, Grant R; Dusting, Gregory J; Ritchie, Rebecca H

    2014-10-01

    Activators of protease-activated receptors PAR-1 and PAR-2 such as thrombin and synthetic hexapeptides promote hypertrophy of isolated neonatal cardiomyocytes at pathological concentrations. Since PAR-activating proteases often show dual actions at low vs. high concentrations, the potential hypertrophic effects of low-level PAR activation were examined. In H9c2 cardiomyoblasts, messenger RNA (mRNA) expression of the hypertrophic marker atrial natriuretic peptide (ANP) was significantly increased only by higher concentrations of thrombin, trypsin or the synthetic PAR-2 agonist SLIGRL. The dual PAR-1/PAR-2 agonist SFLLRN did not influence basal ANP mRNA expression in H9c2 cells. Low concentration of thrombin or trypsin (up to 0.1 U/mL) or of the synthetic ligands SFLLRN and SLIGRL (1 μM); however, all suppressed ANP mRNA expression stimulated by angiotensin II (Ang II). The PAR-1 selective ligand TFLLRN exerted a comparable effect as SFLLRN. In adult rat cardiomyocytes, protein synthesis determined by [(3)H]phenylalanine incorporation was not increased by various PAR agonists at concentrations tenfold lower than conventionally used to study PAR function in vitro (10 μM for SFLLRN or SLIGRL, 0.1 U/mL for thrombin or trypsin). The positive control endothelin-1 (ET-1, 60 nM) however significantly increased protein synthesis in adult rat cardiomyocytes. Addition of low concentrations of PAR agonists to cardiomyocytes treated with ET-1 or Ang II suppressed [(3)H]phenylalanine incorporation induced by the hypertrophic stimuli. The inhibitory effect of SFLLRN effect was partially reversed by the PAR-1 antagonist RWJ56110. These findings suggest that physiological concentrations of PAR activators may suppress hypertrophy, in contrast to the pro-hypertrophic effects evident at high concentrations. PAR-1 and PAR-2 may dynamically control cardiomyocyte growth, with the net effect critically dependent upon local agonist concentrations. The precise significance of proposed

  3. Isoflurane induced cognitive impairment in aged rats through hippocampal calcineurin/NFAT signaling

    SciTech Connect

    Ni, Cheng; Li, Zhengqian; Qian, Min; Zhou, Yang; Wang, Jun; Guo, Xiangyang

    2015-05-15

    Calcineurin (CaN) over-activation constrains synaptic plasticity and memory formation. Upon CaN activation, NFAT imports into the nucleus and guides its downstream genes, which also affect neuronal and synaptic function. Aberrant CaN/NFAT signaling involves in neurotoxicity and cognitive impairment in neurological disorders such as Alzheimer's disease, but its role in postoperative cognitive dysfunction (POCD) remains uninvestigated. Inhaled anesthetic isoflurane facilitates the development of POCD, and the present study investigated the role of CaN/NFAT signaling in isoflurane induced cognitive impairment of aged rats, and the therapeutic effects of CaN inhibitor cyclosporine A (CsA). The results indicated that hippocampal CaN activity increased and peaked at 6 h after isoflurane exposure, and NFAT, especially NFATc4, imported into the nucleus following CaN activation. Furthermore, phamacological inhibition of CaN by CsA markedly attenuated isoflurane induced aberrant CaN/NFATc4 signaling in the hippocampus, and rescued relevant spatial learning and memory impairment of aged rats. Overall, the study suggests hippocampal CaN/NFAT signaling as the upstream mechanism of isoflurane induced cognitive impairment, and provides potential therapeutic target and possible treatment methods for POCD. - Highlights: • Isoflurane induces hippocampal calcineurin activation. • Isoflurane induces hippocampal NFAT, especially NFATc4, nuclear import. • Cyclosporine A attenuates isoflurane induced aberrant calcineurin/NFAT signaling. • Cyclosporine A rescues isoflurane induced cognitive impairment. • Calcineurin/NFAT signaling is the upstream mechanism of isoflurane induced synaptic dysfunction and cognitive impairment.

  4. Parathyroid hormone induction of cyclooxygenase-2 in murine osteoblasts: role of the calcium-calcineurin-NFAT pathway.

    PubMed

    Huang, Hechang; Chikazu, Daichi; Voznesensky, Olga S; Herschman, Harvey R; Kream, Barbara E; Drissi, Hicham; Pilbeam, Carol C

    2010-04-01

    Murine MC3T3-E1 and MC-4 cells were stably transfected with -371/+70 bp of the murine cyclooxygenase-2 (COX-2) promoter fused to a luciferase reporter (Pluc371) or with Pluc371 carrying site-directed mutations. Mutations were made in (1) the cAMP response element (CRE) at -57/-52 bp, (2) the activating protein-1 (AP-1)-binding site at -69/-63 bp, (3) the nuclear factor of activated T-cells (NFAT)-binding site at -77/-73 bp, and (4) both the AP-1 and NFAT sites, which comprise a composite consensus sequence for NFAT/AP-1. Single mutation of CRE, AP-1, or NFAT sites decreased parathyroid hormone (PTH)-stimulated COX-2 promoter activity 40% to 60%, whereas joint mutation of NFAT and AP-1 abrogated the induction. On electrophoretic mobility shift analysis, PTH stimulated binding of phosphorylated CREB to an oligonucleotide spanning the CRE and binding of NFATc1, c-Fos, and c-Jun to an oligonucleotide spanning the NFAT/AP-1 composite site. Mutation of the NFAT site was less effective than mutation of the AP-1 site in competing binding to the composite element, suggesting that cooperative interactions of NFATc1 and AP-1 are more dependent on NFAT than on AP-1. Both PTH and forskolin, an activator of adenylyl cyclase, stimulated NFATc1 nuclear translocation. PTH- and forskolin-stimulated COX-2 promoter activity was inhibited 56% to 80% by calcium chelation or calcineurin inhibitors and 60% to 98% by protein kinase A (PKA) inhibitors. These results indicate an important role for the calcium-calcineurin-NFAT signaling pathway in the PTH induction of COX-2 and suggest that cross-talk between the cAMP/PKA pathway and the calcium-calcineurin-NFAT pathway may play a role in other functions of PTH in osteoblasts.

  5. NFAT5 in cellular adaptation to hypertonic stress - regulations and functional significance.

    PubMed

    Cheung, Chris Yk; Ko, Ben Cb

    2013-01-01

    The Nuclear Factor of Activated T Cells-5 (NFAT5), also known as OREBP or TonEBP, is a member of the nuclear factors of the activated T cells family of transcription factors. It is also the only known tonicity-regulated transcription factor in mammals. NFAT5 was initially known for its role in the hypertonic kidney inner medulla for orchestrating a genetic program to restore the cellular homeostasis. Emerging evidence, however, suggests that NFAT5 might play a more diverse functional role, including a pivotal role in blood pressure regulation and the development of autoimmune diseases. Despite the growing significance of NFAT5 in physiology and diseases, our understanding of how its activity is regulated remains very limited. Furthermore, how changes in tonicities are converted into functional outputs via NFAT5 remains elusive. Therefore, this review aims to summarize our current knowledge on the functional roles of NFAT5 in osmotic stress adaptation and the signaling pathways that regulate its activity. PMID:23618372

  6. Crystal Structure of NFAT Bound to the HIV-1 LTR Tandem κB Enhancer Element

    SciTech Connect

    Bates, Darren L.; Barthel, Kristen K.B.; Wu, Yongqing; Kalhor, Reza; Stroud, James C.; Giffin, Michael J.; Chen, Lin

    2008-05-27

    Here, we have determined the crystal structure of the DNA binding domain of NFAT bound to the HIV-1 long terminal repeat (LTR) tandem {kappa}B enhancer element of 3.05 {angstrom} resolution. NFAT binds as a dimer to the upstream {kappa}B site (Core II), but as a monomer to the 3' end of the downstream {kappa}B site (Core I). The DNA shows a significant bend near the 5' end of Core I, where a lysine residue from NFAT bound to the 3' end of Core II inserts into the minor groove and seems to cause DNA bases to flip out. Consistent with this structural feature, the 5' end of Core I become hypersensitive to dimethylsulfate in the in vivo footprinting upon transcriptional activation of the HIV-1 LTR. Our studies provide a basis for futher investigating the functional mechanism of NFAT in HIV-1 transcription and replication.

  7. Resveratrol inhibits doxorubicin-induced cardiotoxicity via sirtuin 1 activation in H9c2 cardiomyocytes

    PubMed Central

    Liu, Mi-Hua; Shan, Jian; Li, Jian; Zhang, Yuan; Lin, Xiao-Long

    2016-01-01

    Doxorubicin (DOX) is an efficient drug used in cancer therapy; however, it can induce severe cytotoxicity, which limits its clinical application. In the present study, the effects of resveratrol (RES) on sirtuin 1 (SIRT1) activation in mediating DOX-induced cytotoxicity in H9c2 cardiac cells was investigated. H9c2 cells were exposed to 5 µM DOX for 24 h to establish a model of DOX cardiotoxicity. Apoptosis of H9c2 cardiomyocytes was assessed using the MTT assay and Hoechst nuclear staining. The results demonstrated that pretreating H9c2 cells with RES prior to the exposure of DOX resulted in increased cell viability and a decreased quantity of apoptotic cells. Western blot analysis demonstrated that DOX decreased the expression level of SIRT1. These effects were significantly alleviated by co-treatment with RES. In addition, the results demonstrated that DOX administration amplified forkhead box O1 (FoxO1) and P53 expression levels in H9c2 cells. RES was also found to protect against DOX-induced increases of FoxO1 and P53 expression levels in H9c2 cells. Furthermore, the protective effects of RES were arrested by the SIRT1 inhibitor nicotinamide. In conclusion, the results demonstrated that RES protected H9c2 cells against DOX-induced injuries via SIRT1 activation. PMID:27446329

  8. E-NTPDase 3 (ATP diphosphohydrolase) from cardiomyocytes, activity and expression are modulated by thyroid hormone.

    PubMed

    Barreto-Chaves, Maria Luiza M; Carneiro-Ramos, Marcela Sorelli; Cotomacci, Guilherme; Júnior, Marconi Barbosa Coutinho; Sarkis, João José Freitas

    2006-06-01

    Degradation of adenine nucleotides by myocardial cells occurs, in part, by a cascade of surface-located enzymes converting ATP into adenosine that has important implications for the regulation of the nucleotide/nucleoside ratio modulating the cardiac functions. Thyroid hormones have profound effects on cardiovascular system, as observed in hypo- and hyperthyroidism. Combined biochemical parameters and gene expression analysis approaches were used to investigate the influence of tri-iodothyronine (T3) on ATP and ADP hydrolysis by isolated myocytes. Cultures of cardiomyocytes were submitted to increasing doses of T3 for 24h. Enzymatic activity and expression were evaluated. T3 (0.1 nM) caused an increase in ATP and ADP hydrolysis. Experiments with specific inhibitors suggest the involvement of an NTPDase, which was confirmed by an increase in NTPDase 3 messenger RNA (mRNA) levels. Since T3 promotes an increase in the contractile protein, leading to cardiac hypertrophy, it is tempting to postulate that the increase in ATP hydrolysis and the decrease in the extracellular levels signify an important factor for prevention of excessive contractility. PMID:16584835

  9. Placental TonEBP/NFAT5 osmolyte regulation in an ovine model of intrauterine growth restriction.

    PubMed

    Arroyo, Juan A; Garcia-Jones, Pastora; Graham, Amanda; Teng, Cecilia C; Battaglia, Frederick C; Galan, Henry L

    2012-03-01

    TonEBP/NFAT5 (the tonicity-responsive enhancer binding protein/nuclear factor of activated T cells) modulates cellular response to osmotic changes by accumulating inositol and sorbitol inside the cells. Our objective was to assess placental osmolytes, TonEBP/NFAT5 RNA and protein expression, and signaling molecules across gestation between control and intrauterine growth restriction (IUGR) ovine pregnancies. Pregnant sheep were placed in hyperthermic conditions to induce IUGR. Placental tissues were collected at 55, 95, and 130 days gestational age (dGA) to measure inositol, sorbitol, TonEBP/NFAT5 (NFAT5), sodium-dependent myo-inositol transporter (SMIT; official symbol SLC5A3), aldose reductase (AR), and NADPH (official symbol DE-CR1). Placental weight was reduced in IUGR compared to controls at 95 and 130 dGA. Osmolyte concentrations were similar between control and IUGR placentas, but both groups demonstrated a significant decrease in inositol concentration and an increase in sorbitol concentration with advancing gestation. Cytosolic NFAT5 protein decreased significantly from 55 to 95 dGA in both groups, and nuclear NFAT5 protein increased only at 130 dGA in the IUGR group, but no differences were seen between groups for either cytosolic or nuclear NFAT5 protein concentrations. DE-CR1 concentrations were similar between groups and increased significantly with advancing gestational age. AR was lowest at 55dGA, and SLC5A3 increased with advancing gestational age. We conclude that both placental osmolytes inositol and sorbitol (and their corresponding proteins SLC5A3 and AR) change with gestational age and are regulated, at least in part, by NFAT5 and DE-CR1 (NADPH). The inverse relationship between each osmolyte across gestation (e.g., inositol higher in early gestation and sorbitol higher in late gestation) may reflect nutritional needs that change across gestation. PMID:22190709

  10. AMPK activation restores the stimulation of glucose uptake in an in vitro model of insulin-resistant cardiomyocytes via the activation of protein kinase B.

    PubMed

    Bertrand, Luc; Ginion, Audrey; Beauloye, Christophe; Hebert, Alexandre D; Guigas, Bruno; Hue, Louis; Vanoverschelde, Jean-Louis

    2006-07-01

    Diabetic hearts are known to be more susceptible to ischemic disease. Biguanides, like metformin, are known antidiabetic drugs that lower blood glucose concentrations by decreasing hepatic glucose production and increasing glucose disposal in muscle. Part of these metabolic effects is thought to be mediated by the activation of AMP-activated protein kinase (AMPK). In this work, we studied the relationship between AMPK activation and glucose uptake stimulation by biguanides and oligomycin, another AMPK activator, in both insulin-sensitive and insulin-resistant cardiomyocytes. In insulin-sensitive cardiomyocytes, insulin, biguanides and oligomycin were able to stimulate glucose uptake with the same efficiency. Stimulation of glucose uptake by insulin or biguanides was correlated to protein kinase B (PKB) or AMPK activation, respectively, and were additive. In insulin-resistant cardiomyocytes, where insulin stimulation of glucose uptake was greatly reduced, biguanides or oligomycin, in the absence of insulin, induced a higher stimulation of glucose uptake than that obtained in insulin-sensitive cells. This stimulation was correlated with the activation of both AMPK and PKB and was sensitive to the phosphatidylinositol-3-kinase/PKB pathway inhibitors. Finally, an adenoviral-mediated expression of a constitutively active form of AMPK increased both PKB phosphorylation and glucose uptake in insulin-resistant cardiomyocytes. We concluded that AMPK activators, like biguanides and oligomycin, are able to restore glucose uptake stimulation, in the absence of insulin, in insulin-resistant cardiomyocytes via the additive activation of AMPK and PKB. Our results suggest that AMPK activation could restore normal glucose metabolism in diabetic hearts and could be a potential therapeutic approach to treat insulin resistance.

  11. NFAT5-mediated expression of S100A4 contributes to proliferation and migration of renal carcinoma cells.

    PubMed

    Küper, Christoph; Beck, Franz-Xaver; Neuhofer, Wolfgang

    2014-01-01

    The osmosensitive transcription factor nuclear factor of activated T-cells (NFAT) 5, also known as tonicity enhancer binding protein (TonEBP), has been associated with the development of a variety of tumor entities, among them breast cancer, colon carcinoma, and melanoma. The aim of the present study was to determine whether NFAT5 is also involved in the development of renal cell carcinoma (RCC). The most common type of RCC, the clear cell RCC, originates from the proximal convoluted tubule. We tested our hypothesis in the clear cell RCC cell line CaKi-1 and the non-cancerous proximal tubule cell line HK-2, as control. Basal expression of NFAT5 and NFAT5 activity in CaKi-1 cells was several times higher than in HK-2 cells. Osmotic stress induced an increased NFAT5 activity in both CaKi-1 and HK-2 cells, again with significantly higher activities in CaKi-1 cells. Analysis of NFAT5-regulating signaling pathways in CaKi-1 cells revealed that inhibition of the MAP kinases p38, c-Jun-terminal kinase (JNK) and extracellular regulated kinase (ERK) and of the focal adhesion kinase (FAK) partially blunted NFAT5 activity. FAK and ERK were both constitutively active, even under isotonic conditions, which may contribute to the high basal expression and activity of NFAT5 in CaKi-1 cells. In contrast, the MAP kinases p38 and JNK were inactive under isotonic conditions and became activated under osmotic stress conditions, indicating that p38 and JNK mediate upregulation of NFAT5 activity under these conditions. siRNA-mediated knockdown of NFAT5 in CaKi-1 cells reduced the expression of S100A4, a member of the S100 family of proteins, which promotes metastasis. Knockdown of NFAT5 was accompanied by a significant decrease in proliferation and migration activity. Taken together, our results indicate that NFAT5 induces S100A4 expression in CaKi-1 cells, thereby playing an important role in RCC proliferation and migration. PMID:25152734

  12. TNF-α contributes to caspase-3 independent apoptosis in neuroblastoma cells: role of NFAT.

    PubMed

    Alvarez, Susana; Blanco, Almudena; Fresno, Manuel; Muñoz-Fernández, Ma Ángeles

    2011-01-27

    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 Ca(2+) 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.

  13. Origin of Cardiomyocytes in the Adult Heart

    PubMed Central

    Leri, Annarosa; Rota, Marcello; Pasqualini, Francesco S.; Goichberg, Polina; Anversa, Piero

    2014-01-01

    This review article discusses the mechanisms of cardiomyogenesis in the adult heart. They include the reentry of cardiomyocytes into the cell cycle; dedifferentiation of preexisting cardiomyocytes which assume an immature replicating cell phenotype; transdifferentiation of hematopoietic stem cells into cardiomyocytes; and cardiomyocytes derived from activation and lineage specification of resident cardiac stem cells. The recognition of the origin of cardiomyocytes is of critical importance for the development of strategies capable of enhancing the growth response of the myocardium; in fact, cell therapy for the decompensated heart has to be based on the acquisition of this fundamental biological knowledge. PMID:25552694

  14. T-plastin expression downstream to the calcineurin/NFAT pathway is involved in keratinocyte migration.

    PubMed

    Brun, Cécilia; Demeaux, Agathe; Guaddachi, Frédéric; Jean-Louis, Francette; Oddos, Thierry; Bagot, Martine; Bensussan, Armand; Jauliac, Sébastien; Michel, Laurence

    2014-01-01

    Cutaneous wound healing requires keratinocyte proliferation, migration and differentiation to restore the barrier function of the skin. The calcineurin/nuclear factor of activated-T-cell (NFAT) signaling pathway has been recently shown to be involved in keratinocyte growth, differentiation and migration. It is induced by an increased intracellular calcium rate and its inhibition results in decreased capacities of keratinocytes to migrate. Nevertheless, the link between calcineurin activation and keratinocyte migration remains unknown. Recently, Orai1, a pore subunit of a store-operated calcium channel that favors calcium influx, was shown to play a critical role to control proliferation and migration of basal keratinocytes. Of interest, the actin-bundling T-plastin is crucial in cell motility through cross-linking to actin filament and its synthesis was shown to be induced by calcium influx and regulated by the calcineurin/NFAT pathway in tumor Sezary cells. We investigated herein the role of the calcineurin/NFAT pathway-dependent T-plastin in keratinocyte migration, by quantifying T-plastin expression in keratinocytes and by analyzing their migration under calcineurin inhibition or knockdown of NFAT2 or T-plastin. We did confirm the role of the calcineurin/NFAT pathway in keratinocyte migration as shown by their decreased capacities to migrate after FK506 treatment or siNFAT2 transfection in both scratching and Boyden assays. The expression of NFAT2 and T-plastin in keratinocytes was decreased under FK506 treatment, suggesting that T-plastin plays a role in keratinocyte migration downstream to the calcineurin/NFAT pathway. Accordingly, siRNA knockdown of T-plastin expression also decreased their migration capacities. Actin lamellipodia formation as well as FAK and β6-integrin expression were also significantly decreased after treatment with FK506 or siRNA, reinforcing that NFAT2-dependent T-plastin expression plays a role in keratinocyte migration. These results

  15. Astragaloside IV inhibits doxorubicin-induced cardiomyocyte apoptosis mediated by mitochondrial apoptotic pathway via activating the PI3K/Akt pathway.

    PubMed

    Jia, Yuanyuan; Zuo, Daiying; Li, Zengqiang; Liu, Hanmo; Dai, Zhengning; Cai, Jiayi; Pang, Lili; Wu, Yingliang

    2014-01-01

    Doxorubicin (DOX) is a widely used antitumor drug whose application is seriously limited by its cardiotoxicity. Mitochondria-mediated cardiomyocyte apoptosis plays a critical role in DOX-induced cardiotoxicity (DIC). The aim of the present study was to investigate the protective effect of astragaloside IV (3-O-beta-D-xylopyranosyl-6-O-beta-D-glucopyranosyl-cycloastragenol, AS-IV), a pure saponin isolated from Astragalus membranaceus, against DOX-induced cardiomyocyte apoptosis in primary cultured neonatal rat cardiomyocytes. Immunocytochemistry and Microculture Tetrazolium (MTT) assays showed that AS-IV significantly reduced DOX-induced cardiomyocyte loss. Additionally, AS-IV markedly ameliorated DOX-caused cardiomyocyte dysfunction via restoring the beating cell ratio and beating rate in cardiomyocytes. Furthermore, AS-IV substantially reduced the mitochondrial reactive oxygen species (ROS) production and lactate dehydrogenase (LDH), creatine kinase-MB isoenzyme (CK-MB) and cytochrome c (CytC) release, and restored the reduced ATP level, succinate dehydrogenase (SDH) and ATP synthase activities induced by DOX, suggesting that AS-IV significantly attenuated DOX-induced mitochondrial damage and dysfunction. It was further observed that DOX-induced cardiomyocyte apoptosis, as qualitatively evaluated by Hoechst 33258 staining and accurately quantified by flow cytometry, was markedly inhibited by AS-IV. Western blot analysis manifested that AS-IV significantly inhibited the activation of mitochondrial apoptotic pathway (MAP) via inducing the phosphorylation of Akt and Bad. Furthermore, phosphatidylinositol 3-kinase (PI3K) inhibitor 2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride (LY294002) remarkably inhibited the anti-apoptotic effect of AS-IV. Moreover, AS-IV didn't compromise the antitumor activity of DOX. Taken together, our findings indicate that AS-IV ameliorates DIC, and this beneficial effect appears to be dependent on the activation of the PI3K

  16. NFAT5 promotes proliferation and migration of lung adenocarcinoma cells in part through regulating AQP5 expression.

    PubMed

    Guo, Kai; Jin, Faguang

    2015-09-25

    The osmoregulated transcription factor nuclear factor of activated T-cells 5(NFAT5), has been found to play important roles in the development of many kinds of human cancers, including breast cancer, colon carcinoma, renal cell carcinoma and melanoma. The aim of the present study was to determine whether NFAT5 is involved in the proliferation and migration of lung adenocarcinoma cells. We found that NFAT5 was upregulated in lung adenocarcinoma cells and knockdown of NFAT5 decreased proliferation and migration of the cells, accompanied by a significant reduction in the expression of AQP5. AQP5 was upregulated in lung adenocarcinoma cells and knockdown of AQP5 also inhibited proliferation and migration of the cells as knockdown of NFAT5 did. Moreover, overexpression of NFAT5 promoted proliferation and migration of lung adenocarcinoma cells, accompanied by a significant increase in the expression of AQP5. These results indicate that NFAT5 plays important roles in proliferation and migration of human lung adenocarcinoma cells through regulating AQP5 expression, providing a new therapeutic option for lung adenocarcinoma therapy.

  17. Reconciling depressed Ca2+ sparks occurrence with enhanced RyR2 activity in failing mice cardiomyocytes.

    PubMed

    Ruiz-Hurtado, Gema; Li, Linwei; Fernández-Velasco, María; Rueda, Angélica; Lefebvre, Florence; Wang, Yueyi; Mateo, Philippe; Cassan, Cécile; Gellen, Barnabas; Benitah, Jean Pierre; Gómez, Ana María

    2015-10-01

    Abnormalities in cardiomyocyte Ca2+ handling contribute to impaired contractile function in heart failure (HF). Experiments on single ryanodine receptors (RyRs) incorporated into lipid bilayers have indicated that RyRs from failing hearts are more active than those from healthy hearts. Here, we analyzed spontaneous Ca2+ sparks (brief, localized increased in [Ca2+]i) to evaluate RyR cluster activity in situ in a mouse post-myocardial infarction (PMI) model of HF. The cardiac ejection fraction of PMI mice was reduced to ∼30% of that of sham-operated (sham) mice, and their cardiomyocytes were hypertrophied. The [Ca2+]i transient amplitude and sarcoplasmic reticulum (SR) Ca2+ load were decreased in intact PMI cardiomyocytes compared with those from sham mice, and spontaneous Ca2+ sparks were less frequent, whereas the fractional release and the frequency of Ca2+ waves were both increased, suggesting higher RyR activity. In permeabilized cardiomyocytes, in which the internal solution can be controlled, Ca2+ sparks were more frequent in PMI cells (under conditions of similar SR Ca2+ load), confirming the enhanced RyR activity. However, in intact cells from PMI mice, the Ca2+ sparks frequency normalized by the SR Ca2+ load in that cell were reduced compared with those in sham mice, indicating that the cytosolic environment in intact cells contributes to the decrease in Ca2+ spark frequency. Indeed, using an internal "failing solution" with less ATP (as found in HF), we observed a dramatic decrease in Ca2+ spark frequency in permeabilized PMI and sham myocytes. In conclusion, our data show that, even if isolated RyR channels show more activity in HF, concomitant alterations in intracellular media composition and SR Ca2+ load may mask these effects at the Ca2+ spark level in intact cells. Nonetheless, in this scenario, the probability of arrhythmogenic Ca2+ waves is enhanced, and they play a potential role in the increase in arrhythmia events in HF patients.

  18. Cardiomyocyte VEGFR-1 activation by VEGF-B induces compensatory hypertrophy and preserves cardiac function after myocardial infarction.

    PubMed

    Zentilin, Lorena; Puligadda, Uday; Lionetti, Vincenzo; Zacchigna, Serena; Collesi, Chiara; Pattarini, Lucia; Ruozi, Giulia; Camporesi, Silvia; Sinagra, Gianfranco; Pepe, Martino; Recchia, Fabio A; Giacca, Mauro

    2010-05-01

    Mounting evidence indicates that the function of members of the vascular endothelial growth factor (VEGF) family extends beyond blood vessel formation. Here, we show that the prolonged intramyocardial expression of VEGF-A(165) and VEGF-B(167) on adeno-associated virus-mediated gene delivery determined a marked improvement in cardiac function after myocardial infarction in rats, by promoting cardiac contractility, preserving viable cardiac tissue, and preventing remodeling of the left ventricle (LV) over time. Consistent with this functional outcome, animals treated with both factors showed diminished fibrosis and increased contractile myocardium, which were more pronounced after expression of the selective VEGF receptor-1 (VEGFR-1) ligand VEGF-B, in the absence of significant induction of angiogenesis. We found that cardiomyocytes expressed VEGFR-1, VEGFR-2, and neuropilin-1 and that, in particular, VEGFR-1 was specifically up-regulated in hypoxia and on exposure to oxidative stress. VEGF-B exerted powerful antiapoptotic effect in both cultured cardiomyocytes and after myocardial infarction in vivo. Finally, VEGFR-1 activation by VEGF-B was found to elicit a peculiar gene expression profile proper of the compensatory, hypertrophic response, consisting in activation of alphaMHC and repression of betaMHC and skeletal alpha-actin, and an increase in SERCA2a, RYR, PGC1alpha, and cardiac natriuretic peptide transcripts, both in cultured cardiomyocytes and in infarcted hearts. The finding that VEGFR-1 activation by VEGF-B prevents loss of cardiac mass and promotes maintenance of cardiac contractility over time has obvious therapeutic implications.

  19. Isoflurane induced cognitive impairment in aged rats through hippocampal calcineurin/NFAT signaling.

    PubMed

    Ni, Cheng; Li, Zhengqian; Qian, Min; Zhou, Yang; Wang, Jun; Guo, Xiangyang

    2015-05-15

    Calcineurin (CaN) over-activation constrains synaptic plasticity and memory formation. Upon CaN activation, NFAT imports into the nucleus and guides its downstream genes, which also affect neuronal and synaptic function. Aberrant CaN/NFAT signaling involves in neurotoxicity and cognitive impairment in neurological disorders such as Alzheimer's disease, but its role in postoperative cognitive dysfunction (POCD) remains uninvestigated. Inhaled anesthetic isoflurane facilitates the development of POCD, and the present study investigated the role of CaN/NFAT signaling in isoflurane induced cognitive impairment of aged rats, and the therapeutic effects of CaN inhibitor cyclosporine A (CsA). The results indicated that hippocampal CaN activity increased and peaked at 6 h after isoflurane exposure, and NFAT, especially NFATc4, imported into the nucleus following CaN activation. Furthermore, phamacological inhibition of CaN by CsA markedly attenuated isoflurane induced aberrant CaN/NFATc4 signaling in the hippocampus, and rescued relevant spatial learning and memory impairment of aged rats. Overall, the study suggests hippocampal CaN/NFAT signaling as the upstream mechanism of isoflurane induced cognitive impairment, and provides potential therapeutic target and possible treatment methods for POCD.

  20. EGCG Blocked Phenylephrin-Induced Hypertrophy in H9C2 Cardiomyocytes, by Activating AMPK-Dependent Pathway.

    PubMed

    Cai, Yi; Zhao, Li; Qin, Yuan; Wu, Xiao-Qian

    2015-05-01

    AMP-activated protein kinase (AMPK) is a key regulator of energy metabolism. Previous studies have shown that activation of AMPK results in suppression of cardiac myocyte hypertrophy via inhibition of the p70S6 kinase (p70S6K) and eukaryotic elongation factor-2 (eEF2) signaling pathways. Epigallocatechin-3-gallate (EGCG), the major polyphenol found in green tea, possesses multiple protective effects on the cardiovascular system including cardiac hypertrophy. However, the molecular mechanisms has not been well investigated. In this study, we found that EGCG could significantly reduce natriuretic peptides type A (Nppa), brain natriuretic polypeptide (BNP) mRNA expression and decrease cell surface area in H9C2 cardiomyocytes stimulated with phenylephrine (PE). Moreover, we showed that AMPK is activated in H9C2 cardiomyocytes by EGCG, and AMPK-dependent pathway participates in the inhibitory effects of EGCG on cardiac hypertrophy. Taken together, our findings provide the first evidence that the effect of EGCG against cardiac hypertrophy may be attributed to its activation on AMPK-dependent signaling pathway, suggesting the therapeutic potential of EGCG on the prevention of cardiac remodeling in patients with pressure overload hypertrophy. PMID:25954124

  1. NFAT is required for spontaneous pulmonary hypertension in superoxide dismutase 1 knockout mice.

    PubMed

    Ramiro-Diaz, Juan Manuel; Nitta, Carlos H; Maston, Levi D; Codianni, Simon; Giermakowska, Wieslawa; Resta, Thomas C; Gonzalez Bosc, Laura V

    2013-05-01

    Elevated reactive oxygen species are implicated in pulmonary hypertension (PH). Superoxide dismutase (SOD) limits superoxide bioavailability, and decreased SOD activity is associated with PH. A decrease in SOD activity is expected to increase superoxide and reduce hydrogen peroxide levels. Such an imbalance of superoxide/hydrogen peroxide has been implicated as a mediator of nuclear factor of activated T cells (NFAT) activation in epidermal cells. We have shown that NFATc3 is required for chronic hypoxia-induced PH. However, it is unknown whether NFATc3 is activated in the pulmonary circulation in a mouse model of decreased SOD1 activity and whether this leads to PH. Therefore, we hypothesized that an elevated pulmonary arterial superoxide/hydrogen peroxide ratio activates NFATc3, leading to PH. We found that SOD1 knockout (KO) mice have elevated pulmonary arterial wall superoxide and decreased hydrogen peroxide levels compared with wild-type (WT) littermates. Right ventricular systolic pressure (RVSP) was elevated in SOD1 KO and was associated with pulmonary arterial remodeling. Vasoreactivity to endothelin-1 was also greater in SOD1 KO vs. WT mice. NFAT activity and NFATc3 nuclear localization were increased in pulmonary arteries from SOD1 KO vs. WT mice. Administration of A-285222 (selective NFAT inhibitor) decreased RVSP, arterial wall thickness, vasoreactivity, and NFAT activity in SOD1 KO mice to WT levels. The SOD mimetic, tempol, also reduced NFAT activity, NFATc3 nuclear localization, and RVSP to WT levels. These findings suggest that an elevated superoxide/hydrogen peroxide ratio activates NFAT in pulmonary arteries, which induces vascular remodeling and increases vascular reactivity leading to PH.

  2. Peroxisomes in cardiomyocytes and the peroxisome / peroxisome proliferator-activated receptor-loop.

    PubMed

    Colasante, Claudia; Chen, Jiangping; Ahlemeyer, Barbara; Baumgart-Vogt, Eveline

    2015-03-01

    It is well established that the heart is strongly dependent on fatty acid metabolism. In cardiomyocytes there are two distinct sites for the β-oxidisation of fatty acids: the mitochondrion and the peroxisome. Although the metabolism of these two organelles is believed to be tightly coupled, the nature of this relationship has not been fully investigated. Recent research has established the significant contribution of mitochondrial function to cardiac ATP production under normal and pathological conditions. In contrast, limited information is available on peroxisomal function in the heart. This is despite these organelles harbouring metabolic pathways that are potentially cardio-protective, and findings that patients with peroxisomal diseases, such as adult Refsum´s disease, can develop heart failure. In this article, we provide a comprehensive overview on the current knowledge of peroxisomes and the regulation of lipid metabolism by PPARs in cardiomyocytes. We also present new experimental evidence on the differential expression of peroxisome-related genes in the heart chambers and demonstrate that even a mild peroxisomal biogenesis defect (Pex11α-/-) can induce profound alterations in the cardiomyocyte´s peroxisomal compartment and related gene expression, including the concomitant deregulation of specific PPARs. The possible impact of peroxisomal dysfunction in the heart is discussed and a model for the modulation of myocardial metabolism via a peroxisome/PPAR-loop is proposed.

  3. Suppression of Bim by microRNA-19a may protect cardiomyocytes against hypoxia-induced cell death via autophagy activation.

    PubMed

    Gao, Yan-Hua; Qian, Ju-Ying; Chen, Zhang-Wei; Fu, Ming-Qiang; Xu, Jian-Feng; Xia, Yan; Ding, Xue-Feng; Yang, Xiang-Dong; Cao, Yuan-Yuan; Zou, Yun-Zeng; Ren, Jun; Sun, Ai-Jun; Ge, Jun-Bo

    2016-08-22

    Microvascular obstruction (MO), one of unfavorable complications of percutaneous coronary intervention (PCI), is responsible for the lost benefit of reperfusion therapy. Determination of microRNA-19a, a member of the miR-17-92 cluster, using quantitative real-time polymerase chain reaction (PCR) revealed notably down-regulated microRNA-19a, in myocardium with MO. Nonetheless, the role of miR-19a in MO and the underlying mechanism remains to be elucidated. To this end, an in vitro microembolization model in cardiomyocytes was used. Our data revealed that hypoxic exposure prompted cardiomyocyte apoptosis in a time-dependent manner accompanied by reduced miR-19a. miR-19a overexpression clearly ameliorated hypoxia-induced cell death (necrosis and apoptosis), at least in part, through switching on autophagy. Further dual-luciferase reporter assay and immunoblotting studies demonstrated that miR-19a-induced cytoprotection might be achieved in part through modulation of the specific target Bcl-2 interacting mediator of cell death, Bim, an apoptotic activator. Bim sufficiently interfered with miR-19a-induced LC3 conversion and increased cardiomyocyte apoptosis under hypoxia. Moreover, cardiomyocytes pretreated with 3-methyladenine conferred resistance to the cytoprotective effect of miR-19a and displayed notably increased TUNEL staining and caspase-3 activity. In conclusion, miR-19a protected cardiomyocytes against hypoxia-induced lethality at least in part via Bim suppression and subsequently autophagy activation.

  4. The potential role of NFAT5 and osmolarity in peritoneal injury.

    PubMed

    Seeger, Harald; Kitterer, Daniel; Latus, Joerg; Alscher, Mark Dominik; Braun, Niko; Segerer, Stephan

    2015-01-01

    A rise in osmotic concentration (osmolarity) activates the transcription factor Nuclear Factor of Activated T Cells 5 (NFAT5, also known as Tonicity-responsive Enhancer Binding Protein, TonEBP). This is part of a regulatory mechanism of cells adjusting to environments of high osmolarity. Under physiological conditions these are particularly important in the kidney. Activation of NFAT5 results in the modulation of various genes including some which promote inflammation. The osmolarity increases in patients with renal failure. Additionally, in peritoneal dialysis the cells of the peritoneal cavity are repeatedly exposed to a rise and fall in osmotic concentrations. Here we review the current information about NFAT5 activation in uremic patients and patients on peritoneal dialysis. We suggest that high osmolarity promotes injury in the "uremic" milieu, which results in inflammation locally in the peritoneal membrane, but most likely also in the systemic circulation. PMID:26495302

  5. NFAT5 Is Up-Regulated by Hypoxia: Possible Implications in Preeclampsia and Intrauterine Growth Restriction.

    PubMed

    Dobierzewska, Aneta; Palominos, Macarena; Irarrazabal, Carlos E; Sanchez, Marianela; Lozano, Mauricio; Perez-Sepulveda, Alejandra; Monteiro, Lara J; Burmeister, Yara; Figueroa-Diesel, Horacio; Rice, Gregory E; Illanes, Sebastian E

    2015-07-01

    During gestation, low oxygen environment is a major determinant of early placentation process, while persistent placental hypoxia leads to pregnancy-related complications such as preeclampsia (PE) and intrauterine growth restriction (IUGR). PE affects 5%-8% of all pregnancies worldwide and is a cause of maternal and fetal morbidity and mortality. During placental development, persistent hypoxia due to poor trophoblast invasion and reduced uteroplacental perfusion leads to maternal endothelial dysfunction and clinical manifestation of PE. Here we hypothesized that nuclear factor of activated T cells-5 (NFAT5), a well-known osmosensitive renal factor and recently characterized hypoxia-inducible protein, is also activated in vivo in placentas of PE and IUGR complications as well as in the in vitro model of trophoblast hypoxia. In JAR cells, low oxygen tension (1% O2) induced NFAT5 mRNA and increased its nuclear abundance, peaking at 16 h. This increase did not occur in parallel with the earlier HIF1A induction. Real-time PCR and Western blot analysis confirmed up-regulation of NFAT5 mRNA and NFAT5 nuclear content in human preeclamptic placentas and in rabbit placentas of an experimentally induced IUGR model, as compared with the control groups. In vitro lambda protein phosphatase (lambda PPase) treatment revealed that increased abundance of NFAT5 protein in nuclei of either JAR cells (16 h of hypoxia) or PE and IUGR placentas is at least partially due to NFAT5 phosphorylation. NFAT5 downstream targets aldose reductase (AR) and sodium-myo-inositol cotransporter (SMIT; official symbol SLC5A3) were not significantly up-regulated either in JAR cells exposed to hypoxia or in placentas of PE- and IUGR-complicated pregnancies, suggesting that hypoxia-dependent activation of NFAT5 serves as a separate function to its tonicity-dependent stimulation. In conclusion, we propose that NFAT5 may serve as a novel marker of placental hypoxia and ischemia independently of HIF1A. PMID

  6. Leptin suppresses non-apoptotic cell death in ischemic rat cardiomyocytes by reduction of iPLA{sub 2} activity

    SciTech Connect

    Takatani-Nakase, Tomoka Takahashi, Koichi

    2015-07-17

    Caspase-independent, non-apoptotic cell death is an important therapeutic target in myocardial ischemia. Leptin, an adipose-derived hormone, is known to exhibit cytoprotective effects on the ischemic heart, but the mechanisms are poorly understood. In this research, we found that pretreatment of leptin strongly suppressed ischemic-augmented nuclear shrinkage and non-apoptotic cell death on cardiomyocytes. Leptin was also shown to significantly inhibit the activity of iPLA{sub 2}, which is considered to play crucial roles in non-apoptotic cell death, resulting in effective prevention of ischemia-induced myocyte death. These findings provide the first evidence of a protective mechanism of leptin against ischemia-induced non-apoptotic cardiomyocyte death. - Highlights: • Myocardial ischemia-model induces in caspase-independent, non-apoptotic cell death. • Leptin strongly inhibits ischemic-augmented non-apoptotic cell death. • Leptin reduces iPLA{sub 2} activity, leading to avoidance of non-apoptotic cell death.

  7. NFAT restricts osteochondroma formation from entheseal progenitors

    PubMed Central

    Tsang, Kelly; He, Lizhi; Garcia, Roberto A.; Ermann, Joerg; Mizoguchi, Fumitaka; Zhang, Minjie; Aliprantis, Antonios O.

    2016-01-01

    Osteochondromas are common benign osteocartilaginous tumors in children and adolescents characterized by cartilage-capped bony projections on the surface of bones. These tumors often cause pain, deformity, fracture, and musculoskeletal dysfunction, and they occasionally undergo malignant transformation. The pathogenesis of osteochondromas remains poorly understood. Here, we demonstrate that nuclear factor of activated T cells c1 and c2 (NFATc1 and NFATc2) suppress osteochondromagenesis through individual and combinatorial mechanisms. In mice, conditional deletion of NFATc1 in mesenchymal limb progenitors, Scleraxis-expressing (Scx-expressing) tendoligamentous cells, or postnatally in Aggrecan-expressing cells resulted in osteochondroma formation at entheses, the insertion sites of ligaments and tendons onto bone. Combinatorial deletion of NFATc1 and NFATc2 gave rise to larger and more numerous osteochondromas in inverse proportion to gene dosage. A population of entheseal NFATc1- and Aggrecan-expressing cells was identified as the osteochondroma precursor, previously believed to be growth plate derived or perichondrium derived. Mechanistically, we show that NFATc1 restricts the proliferation and chondrogenesis of osteochondroma precursors. In contrast, NFATc2 preferentially inhibits chondrocyte hypertrophy and osteogenesis. Together, our findings identify and characterize a mechanism of osteochondroma formation and suggest that regulating NFAT activity is a new therapeutic approach for skeletal diseases characterized by defective or exaggerated osteochondral growth. PMID:27158674

  8. Calcineurin/NFAT signaling and innate host defence: a role for NOD1-mediated phagocytic functions

    PubMed Central

    2014-01-01

    The calcineurin/nuclear factor of activated T cells (NFATs) signaling pathway plays a central role in T cell mediated adaptive immune responses, but a number of recent studies demonstrated that calcineurin/NFAT signaling also plays a key role in the control of the innate immune response by myeloid cells. Calcineurin inhibitors, such as cyclosporine A (CsA) and tacrolimus (FK506), are commonly used in organ transplantation to prevent graft rejection and in a variety of immune diseases. These immunosuppressive drugs have adverse effects and significantly increase host’s susceptibility towards bacterial or fungal infections. Recent studies highlighted the role of NFAT signaling in fungal infection and in the control of the pattern recognition receptor nucleotide-binding oligomerization domain-containing protein 1 (NOD1), which predominantly senses invasive Gram-negative bacteria and mediates neutrophil phagocytic functions. This review summarises some of the current knowledge concerning the role of NFAT signaling in the innate immune response and the recent advances on NFAT-dependent inhibition of NOD1-mediated innate immune response caused by CsA, which may contribute to sensitizing transplant recipients to bacterial infection. PMID:24479879

  9. Optogenetic Control of Calcium Oscillation Waveform Defines NFAT as an Integrator of Calcium Load.

    PubMed

    Hannanta-Anan, Pimkhuan; Chow, Brian Y

    2016-04-27

    It is known that the calcium-dependent transcription factor NFAT initiates transcription in response to pulsatile loads of calcium signal. However, the relative contributions of calcium oscillation frequency, amplitude, and duty cycle to transcriptional activity remain unclear. Here, we engineer HeLa cells to permit optogenetic control of intracellular calcium concentration using programmable LED arrays. This approach allows us to generate calcium oscillations of constant peak amplitude, in which frequency is varied while holding duty cycle constant, or vice versa. Using this setup and mathematical modeling, we show that NFAT transcriptional activity depends more on duty cycle, defined as the proportion of the integrated calcium concentration over the oscillation period, than on frequency alone. This demonstrates that NFAT acts primarily as a signal integrator of cumulative load rather than a frequency-selective decoder. This approach resolves a fundamental question in calcium encoding and demonstrates the value of optogenetics for isolating individual dynamical components of larger signaling behaviors. PMID:27135540

  10. Calcium and calcineurin-NFAT signaling regulate granulocyte-monocyte progenitor cell cycle via Flt3-L.

    PubMed

    Fric, Jan; Lim, Clarice X F; Mertes, Alexandra; Lee, Bernett T K; Viganò, Elena; Chen, Jinmiao; Zolezzi, Francesca; Poidinger, Michael; Larbi, Anis; Strobl, Herbert; Zelante, Teresa; Ricciardi-Castagnoli, Paola

    2014-12-01

    Maintenance of myeloid progenitor cells is controlled by complex regulatory mechanisms and is orchestrated by multiple different transcription factors. Here, we report that the activation of the transcription factor nuclear factor of activated T cells (NFAT) by calcium-sensing protein calcineurin inhibits the proliferation of myeloid granulocyte-monocyte progenitors (GMPs). Myeloid progenitor subtypes exhibit variable sensitivity to induced Ca(2+) entry and consequently display differential engagement of the calcineurin-NFAT pathway. This study shows that inhibition of the calcineurin-NFAT pathway enhances the proliferation of GMPs both in vitro and in vivo and demonstrates that calcineurin-NFAT signaling in GMPs is initiated by Flt3-L. Inhibition of the calcineurin-NFAT pathway modified expression of the cell cycle regulation genes Cdk4, Cdk6, and Cdkn1a (p21), thus enabling rapid cell cycle progression specifically in GMPs. NFAT inhibitor drugs are extensively used in the clinic to restrict the pathological activation of lymphoid cells, and our data reveal for the first time that these therapies also exert potent effects on maintenance of the myeloid cell compartment through specific regulation of GMP proliferation.

  11. Calcium and Calcineurin-NFAT Signaling Regulate Granulocyte-Monocyte Progenitor Cell Cycle via Flt3-L

    PubMed Central

    Fric, Jan; Lim, Clarice XF; Mertes, Alexandra; Lee, Bernett TK; Viganò, Elena; Chen, Jinmiao; Zolezzi, Francesca; Poidinger, Michael; Larbi, Anis; Strobl, Herbert; Zelante, Teresa; Ricciardi-Castagnoli, Paola

    2014-01-01

    Abstract Maintenance of myeloid progenitor cells is controlled by complex regulatory mechanisms and is orchestrated by multiple different transcription factors. Here, we report that the activation of the transcription factor nuclear factor of activated T cells (NFAT) by calcium-sensing protein calcineurin inhibits the proliferation of myeloid granulocyte–monocyte progenitors (GMPs). Myeloid progenitor subtypes exhibit variable sensitivity to induced Ca2+ entry and consequently display differential engagement of the calcineurin-NFAT pathway. This study shows that inhibition of the calcineurin-NFAT pathway enhances the proliferation of GMPs both in vitro and in vivo and demonstrates that calcineurin-NFAT signaling in GMPs is initiated by Flt3-L. Inhibition of the calcineurin-NFAT pathway modified expression of the cell cycle regulation genes Cdk4, Cdk6, and Cdkn1a (p21), thus enabling rapid cell cycle progression specifically in GMPs. NFAT inhibitor drugs are extensively used in the clinic to restrict the pathological activation of lymphoid cells, and our data reveal for the first time that these therapies also exert potent effects on maintenance of the myeloid cell compartment through specific regulation of GMP proliferation. Stem Cells 2014;32:3232–3244 PMID:25100642

  12. NFAT transcription factor regulation by urocortin II in cardiac myocytes and heart failure.

    PubMed

    Walther, Stefanie; Awad, Sawsan; Lonchyna, Vassyl A; Blatter, Lothar A

    2014-03-01

    Urocortin II (UcnII), a cardioactive peptide with beneficial effects in normal and failing hearts, is also arrhythmogenic and prohypertrophic. We demonstrated that cardiac effects are mediated by a phosphatidylinositol-3 kinase (PI3K)/Akt kinase (Akt)/endothelial nitric oxide synthase (eNOS)/nitric oxide (NO) signaling pathways. Nuclear factor of activated T-cells (NFAT) transcription factors play a key role in the regulation of gene expression in cardiac development, maintenance of an adult differentiated cardiac phenotype, and remodeling processes in cardiac hypertrophy and heart failure (HF). We tested the hypothesis that UcnII differentially regulates NFAT activity in cardiac myocytes from both normal and failing hearts through the PI3K/Akt/eNOS/NO pathway. Isoforms NFATc1 and NFATc3 revealed different basal subcellular distribution in normal and HF rabbit ventricular myocytes with a nuclear NFATc1 and a cytosolic localization of NFATc3. However, in HF, the nuclear localization of NFATc1 was less pronounced, whereas the nuclear occupancy of NFATc3 was increased. In normal myocytes, UcnII induced nuclear export of NFATc1 and attenuated NFAT-dependent transcriptional activity but did not affect the distribution of NFATc3. In HF UcnII facilitated nuclear export of both isoforms and reduced transcriptional activity. NFAT regulation was mediated by a PI3K/Akt/eNOS/NO signaling cascade that converged on the activation of several kinases, including glycogen synthase kinase-3β (GSK3β), c-Jun NH2-terminal kinase (JNK), p38 mitogen-activated kinase (p38), and PKG, resulting in phosphorylation, deactivation, and nuclear export of NFAT. In conclusion, while NFATc1 and NFATc3 reveal distinct subcellular distribution patterns, both are regulated by the UcnII-PI3K/Akt/eNOS/NO pathway that converges on the activation of NFAT kinases and NFAT inactivation. The data reconcile cardioprotective and prohypertrophic UcnII effects mediated by different NFAT isoforms.

  13. Rcan1-1L overexpression induces mitochondrial autophagy and improves cell survival in angiotensin II-exposed cardiomyocytes

    SciTech Connect

    Duan, Hongyan; Li, Yongqiang; Yan, Lijie; Yang, Haitao; Wu, Jintao; Qian, Peng; Li, Bing; Wang, Shanling

    2015-07-01

    Mitochondrial autophagy is an important adaptive stress response and can be modulated by various key molecules. A previous study found that the regulator of calcineurin 1-1L (Rcan1-1L) may regulate mitochondrial autophagy and cause mitochondria degradation in neurocytes. However, the effect of Rcan1-1L on cardiomyocytes has not been determined. In the present study, we aimed to investigate the role of Rcan1-1L in angiotensin II (Ang II)-exposed human cardiomyocytes. Above all, Human adult cardiac myocytes (HACMs) were exposed to 200 nmol/L Ang II for 4 days. Enhanced H{sub 2}O{sub 2} production, cytochrome C release and mitochondrial permeability were observed in these cells, which were blocked by valsartan. Consistently, Ang II exposure significantly reduced cardiomyocyte viability. However, transfection of Rcan1-1L vector promoted cell viability and ameliorated the apoptosis caused by Ang II. Rcan1-1L clearly promoted mitochondrial autophagy in HACMs, with elevated autophagy protein (ATG) 5 and light chain 3 (LC3) expression. Transient mitochondrial biogenesis and reduced cytochrome C release was also induced by Rcan1-1L. Additionally, Rcan1-1L significantly inhibited calcineurin/nuclear factor of activated T cells (NFAT) signaling. We thus conclude that Rcan1-1L may play a protective role in Ang II-treated cardiomyocytes through the induction of mitochondrial autophagy, and may be an alternative method of cardiac protection. - Highlights: • Transfection of Rcan1-1L into HACMs promoted cell viability and reduced apoptosis. • Transfection of Rcan1-1L promoted mitochondrial autophagy in HACMs. • Rcan1-1L inhibited the calcineurin/nuclear factor of activated T cells signaling.

  14. Tanshinone IIA Prevents Leu27IGF-II-Induced Cardiomyocyte Hypertrophy Mediated by Estrogen Receptor and Subsequent Akt Activation.

    PubMed

    Weng, Yueh-Shan; Wang, Hsueh-Fang; Pai, Pei-Ying; Jong, Gwo-Ping; Lai, Chao-Hung; Chung, Li-Chin; Hsieh, Dennis Jine-Yuan; HsuanDay, Cecilia; Kuo, Wei-Wen; Huang, Chih-Yang

    2015-01-01

    IGF-IIR plays important roles as a key regulator in myocardial pathological hypertrophy and apoptosis, which subsequently lead to heart failure. Salvia miltiorrhiza Bunge (Danshen) is a traditional Chinese medicinal herb used to treat cardiovascular diseases. Tanshinone IIA is an active compound in Danshen and is structurally similar to 17[Formula: see text]-estradiol (E[Formula: see text]. However, whether tanshinone IIA improves cardiomyocyte survival in pathological hypertrophy through estrogen receptor (ER) regulation remains unclear. This study investigates the role of ER signaling in mediating the protective effects of tanshinone IIA on IGF-IIR-induced myocardial hypertrophy. Leu27IGF-II (IGF-II analog) was shown in this study to specifically activate IGF-IIR expression and ICI 182,780 (ICI), an ER antagonist used to investigate tanshinone IIA estrogenic activity. We demonstrated that tanshinone IIA significantly enhanced Akt phosphorylation through ER activation to inhibit Leu27IGF-II-induced calcineurin expression and subsequent NFATc3 nuclear translocation to suppress myocardial hypertrophy. Tanshinone IIA reduced the cell size and suppressed ANP and BNP, inhibiting antihypertrophic effects induced by Leu27IGF-II. The cardioprotective properties of tanshinone IIA that inhibit Leu27IGF-II-induced cell hypertrophy and promote cell survival were reversed by ICI. Furthermore, ICI significantly reduced phospho-Akt, Ly294002 (PI3K inhibitor), and PI3K siRNA significantly reduced the tanshinone IIA-induced protective effect. The above results suggest that tanshinone IIA inhibited cardiomyocyte hypertrophy, which was mediated through ER, by activating the PI3K/Akt pathway and inhibiting Leu27IGF-II-induced calcineurin and NFATC3. Tanshinone IIA exerted strong estrogenic activity and therefore represented a novel selective ER modulator that inhibits IGF-IIR signaling to block cardiac hypertrophy. PMID:26621443

  15. Intermedilysin induces EGR-1 expression through calcineurin/NFAT pathway in human cholangiocellular carcinoma cells

    SciTech Connect

    Susilowati, Heni; Okamura, Hirohiko; Hirota, Katsuhiko; Shono, Masayuki; Yoshida, Kaya; Murakami, Keiji; Tabata, Atsushi; Nagamune, Hideaki; Haneji, Tatsuji; Miyake, Yoichiro

    2011-01-07

    Research highlights: {yields} ILY leads to the accumulation of [Ca{sup 2+}]i in the nucleus in HuCCT1 cells. {yields} ILY induced activation of NFAT1 through a calcineurin-dependent pathway. {yields} Calcineuri/NFAT pathway is involved in EGR-1 expression in response to ILY treatment. -- Abstract: Intermedilysin (ILY) is a cholesterol-dependent cytolysin produced by Streptococcus intermedius, which is associated with human brain and liver abscesses. Although intrahepatic bile duct cells play a valuable role in the pathogenesis of liver abscess, the molecular mechanism of ILY-treated intrahepatic bile duct cells remains unknown. In this study, we report that ILY induced a nuclear accumulation of intracellular calcium ([Ca{sup 2+}]i) in human cholangiocellular cells HuCCT1. We also demonstrate that 10 ng/ml ILY induced NFAT1 dephosphorylation and its nuclear translocation in HuCCT1 cells. In contrast to the result that ILY induced NF-{kappa}B translocation in human hepatic HepG2 cells, ILY did not affect NF-{kappa}B localization in HuCCT1 cells. Dephosphorylation and nuclear translocation of NFAT1 caused by ILY were prevented by [Ca{sup 2+}]i calcium chelator, BAPTA/AM, and calcineurin inhibitors, cyclosporine A and tacrolimus. ILY induced early growth response-1 (EGR-1) expression and it was inhibited by the pre-treatment with cyclosporine A, indicating that the calcineurin/NFAT pathway was involved in EGR-1 expression in response to ILY. ILY-induced calcineurin/NFAT1 activation and sequential EGR-1 expression might be related to the pathogenesis of S. intermedius in human bile duct cells.

  16. Zinc pyrithione salvages reperfusion injury by inhibiting NADPH oxidase activation in cardiomyocytes.

    PubMed

    Kasi, Viswanath; Bodiga, Sreedhar; Kommuguri, Upendra Nadh; Sankuru, Suneetha; Bodiga, Vijaya Lakshmi

    2011-07-01

    Zinc pyrithione (ZPT), has a strong anti-apoptotic effect when administered just before reperfusion. Because oxidative stress has been proposed to contribute to myocardial reperfusion injury, we tested whether ZPT can reduce the production of reactive oxygen species during reoxygenation in cultured neonatal rat cardiac myocytes and evaluated the role of NADPH oxidase in hypoxia/reoxygenation (H/R) injury. The cells were subjected to 8h of simulated ischemia, followed by either 30 min or 16 h of reoxygenation. ZPT when started just before reoxygenation significantly reduced superoxide generation, LDH release and improved cell survival compared to H/R. Attenuation of the ROS production by ZPT paralleled its capacity to prevent pyknotic nuclei formation. In addition, ZPT reversed the H/R-induced expression of NOX2 and p47(phox) phosphorylation indicating that ZPT directly protects cardiomyocytes from reperfusion injury by a mechanism that attenuates NADPH oxidase mediated intracellular oxidative stress. PMID:21651898

  17. Cardiomyocytes In Vitro Adhesion Is Actively Influenced by Biomimetic Synthetic Peptides for Cardiac Tissue Engineering

    PubMed Central

    Huerta-Cantillo, Rocio; Comisso, Marina; Danesin, Roberta; Ghezzo, Francesca; Naso, Filippo; Gastaldello, Alessandra; Schittullo, Eleonora; Buratto, Edward; Spina, Michele; Gerosa, Gino; Dettin, Monica

    2012-01-01

    Scaffolds for tissue engineering must be designed to direct desired events such as cell attachment, growth, and differentiation. The incorporation of extracellular matrix-derived peptides into biomaterials has been proposed to mimic biochemical signals. In this study, three synthetic fragments of fibronectin, vitronectin, and stromal-derived factor-1 were investigated for the first time as potential adhesive sequences for cardiomyocytes (CMs) compared to smooth muscle cells. CMs are responsive to all peptides to differing degrees, demonstrating the existence of diverse adhesion mechanisms. The pretreatment of nontissue culture well surfaces with the (Arginine-Glycine-Aspartic Acid) RGD sequence anticipated the appearance of CMs' contractility compared to the control (fibronectin-coated well) and doubled the length of cell viability. Future prospects are the inclusion of these sequences into biomaterial formulation with the improvement in cell adhesion that could play an important role in cell retention during dynamic cell seeding. PMID:22011064

  18. PTD-mediated intracellular delivery of mutant NFAT minimum DNA binding domain inhibited the proliferation of T cells.

    PubMed

    Liu, Xia; Zhao, Qianqian; Peng, Xin; Xia, Sheng; Shen, Weihong; Zong, Yangyong; Cheng, Jing; Wu, Weijiang; Zhang, Miaomiao; Du, Fengyi; Xu, Wenrong; Qian, Hui; Shao, Qixiang

    2014-03-01

    The nuclear factor of activated T cell (NFAT) family of calcium-regulated transcription factors plays a key role in the development and function of the immune system. Calcineurin, a protein phosphatase, activates NFAT by dephosphorylation. The activated NFAT is translocated into the nucleus, where it up-regulates the expression of interleukin 2 (IL-2) and other target genes. Calcineurin inhibitors such as cyclosporine A (CsA) and FK506 are effective immunosuppressant drugs and dramatically increase the success rate of organ transplantation procedures. However, since calcineurin is expressed in most tissues in the body and calcineurin inhibition alters many cellular processes besides immune cell activation, the therapeutic use of calcineurin inhibitors is limited by serious side effects. Thus inhibiting NFAT by other mechanisms such as blocking its binding to DNA could be a more selective and safer approach to target NFAT for therapeutic applications. In peripheral T cells, productive immune responses are dependent upon the cooperative binding of the NFAT/AP-1 transcriptional complex to the promoter regions of genes such as interleukin-2 (IL-2), while NFAT in the absence of AP-1 leads to T cell anergy. Protein transduction domains (PTDs) are able to penetrate cell membranes and can be used to transport exogenous proteins across the cell and nuclear membranes. In this study, we constructed a fusion protein of PTD and a minimum DNA binding domain of human NFAT1 (PTD-ΔNFATminiDBD), which contains two mutations (R466A and T533G) in the AP-1 binding sites. The delivery and functions of this fusion protein in T cells were investigated. The results indicated that PTD-ΔNFATminiDBD could be effectively delivered into T cells and transported into the nucleus. PTD-ΔNFATminiDBD attenuated IL-2 production in T cells and then inhibited T cell proliferation, likely through competing against endogenous NFAT for binding to the IL-2 gene promoter. These results demonstrated that

  19. MicroRNA-128 inhibition attenuates myocardial ischemia/reperfusion injury-induced cardiomyocyte apoptosis by the targeted activation of peroxisome proliferator-activated receptor gamma

    PubMed Central

    ZENG, XIAO CONG; LI, LANG; WEN, HONG; BI, QI

    2016-01-01

    The aim of the present study was to investigate the effects of microRNA (miR)-128 inhibition on the targeted activation of peroxisome proliferator-activated receptor gamma (PPARG) and on cardiomyocyte apoptosis induced by myocardial ischemia/reperfusion (I/R) injury. In vitro, the expression of PPARG was detected by reverse transcription-quantitative polymerase chain reaction and western blotting in neonatal rat ventricular myocytes (NRVMs) and HEK293 cells transfected with the mimics or inhibitors of miR-128 or control RNA. Luciferase reporter assays were used to identify whether PPARG is a direct target of miR-128. In vivo, miR-128 was knocked down via ear vein injection of antagomir-128 in a rabbit myocardial I/R injury model. Western blotting investigated the activation of Akt [phosphorylated (p)-Akt] and the expression of total-Akt, PPARG and myeloid leukemia cell differentiation protein-1 (Mcl-1) in the myocardium. Cardiomyocyte apoptosis was examined with transmission electron microscropy and terminal deoxynucleotidyl transferase dUTP nick end labeling staining. PPARG mRNA and protein were downregulated in NRVMs transfected with miR-128 mimics, but upregulated by antagomir-128 compared with control. This indicates that PPARG is a direct miR-128 target. Activation of Akt (p-Akt), Mcl-1 and PPARG expression in the myocardium were increased by miR-128 inhibition. Furthermore, miR-128 antagomirs significantly reduced apoptosis in hearts subjected to I/R injury, which was blocked by the PPARG inhibitor GW9662. In conclusion, miR-128 inhibition attenuated I/R injury-induced cardiomyocyte apoptosis by the targeted activation of PPARG signaling. PMID:27150726

  20. Novel fluorescence resonance energy transfer-based reporter reveals differential calcineurin activation in neonatal and adult cardiomyocytes.

    PubMed

    Bazzazi, Hojjat; Sang, Lingjie; Dick, Ivy E; Joshi-Mukherjee, Rosy; Yang, Wanjun; Yue, David T

    2015-09-01

    Novel fluorescence resonance energy transfer-based genetically encoded reporters of calcineurin are constructed by fusing the two subunits of calcineurin with P2A-based linkers retaining the expected native conformation of calcineurin. Calcineurin reporters display robust responses to calcium transients in HEK293 cells. The sensor responses are correlated with NFATc1 translocation dynamics in HEK293 cells. The sensors are uniformly distributed in neonatal myocytes and respond efficiently to single electrically evoked calcium transients and show cumulative activation at frequencies of 0.5 and 1 Hz. In adult myocytes, the calcineurin sensors appear to be localized to the cardiac z-lines, and respond to cumulative calcium transients at frequencies of 0.5 and 1 Hz. The phosphatase calcineurin is a central component of many calcium signalling pathways, relaying calcium signals from the plasma membrane to the nucleus. It has critical functions in a multitude of systems, including immune, cardiac and neuronal. Given the widespread importance of calcineurin in both normal and pathological conditions, new tools that elucidate the spatiotemporal dynamics of calcineurin activity would be invaluable. Here we develop two separate genetically encoded fluorescence resonance energy transfer (FRET)-based sensors of calcineurin activation, DuoCaN and UniCaN. Both sensors showcase a large dynamic range and rapid response kinetics, differing primarily in the linker structure between the FRET pairs. Both sensors were calibrated in HEK293 cells and their responses correlated well with NFAT translocation to the nucleus, validating the biological relevance of the sensor readout. The sensors were subsequently expressed in neonatal rat ventricular myocytes and acutely isolated adult guinea pig ventricular myocytes. Both sensors demonstrated robust responses in myocytes and revealed kinetic differences in calcineurin activation during changes in pacing rate for neonatal versus adult myocytes

  1. Novel fluorescence resonance energy transfer-based reporter reveals differential calcineurin activation in neonatal and adult cardiomyocytes.

    PubMed

    Bazzazi, Hojjat; Sang, Lingjie; Dick, Ivy E; Joshi-Mukherjee, Rosy; Yang, Wanjun; Yue, David T

    2015-09-01

    Novel fluorescence resonance energy transfer-based genetically encoded reporters of calcineurin are constructed by fusing the two subunits of calcineurin with P2A-based linkers retaining the expected native conformation of calcineurin. Calcineurin reporters display robust responses to calcium transients in HEK293 cells. The sensor responses are correlated with NFATc1 translocation dynamics in HEK293 cells. The sensors are uniformly distributed in neonatal myocytes and respond efficiently to single electrically evoked calcium transients and show cumulative activation at frequencies of 0.5 and 1 Hz. In adult myocytes, the calcineurin sensors appear to be localized to the cardiac z-lines, and respond to cumulative calcium transients at frequencies of 0.5 and 1 Hz. The phosphatase calcineurin is a central component of many calcium signalling pathways, relaying calcium signals from the plasma membrane to the nucleus. It has critical functions in a multitude of systems, including immune, cardiac and neuronal. Given the widespread importance of calcineurin in both normal and pathological conditions, new tools that elucidate the spatiotemporal dynamics of calcineurin activity would be invaluable. Here we develop two separate genetically encoded fluorescence resonance energy transfer (FRET)-based sensors of calcineurin activation, DuoCaN and UniCaN. Both sensors showcase a large dynamic range and rapid response kinetics, differing primarily in the linker structure between the FRET pairs. Both sensors were calibrated in HEK293 cells and their responses correlated well with NFAT translocation to the nucleus, validating the biological relevance of the sensor readout. The sensors were subsequently expressed in neonatal rat ventricular myocytes and acutely isolated adult guinea pig ventricular myocytes. Both sensors demonstrated robust responses in myocytes and revealed kinetic differences in calcineurin activation during changes in pacing rate for neonatal versus adult myocytes

  2. Na+/H+ exchanger isoform 1 induced cardiomyocyte hypertrophy involves activation of p90 ribosomal s6 kinase.

    PubMed

    Jaballah, Maiy; Mohamed, Iman A; Alemrayat, Bayan; Al-Sulaiti, Fatima; Mlih, Mohamed; Mraiche, Fatima

    2015-01-01

    Studies using pharmacological and genetic approaches have shown that increased activity/expression of the Na+/H+ exchanger isoform 1 (NHE1) play a critical role in the pathogenesis of cardiac hypertrophy. Despite the importance of NHE1 in cardiac hypertrophy, severe cerebrovascular side effects were associated with the use of NHE1 inhibitors when administered to patients with myocardial infarctions. p90 ribosomal S6 Kinase (RSK), a downstream regulator of the mitogen-activated protein kinase pathway, has also been implicated in cardiac hypertrophy. We hypothesized that RSK plays a role in the NHE1 induced cardiomyocyte hypertrophic response. Infection of H9c2 cardiomyoblasts with the active form of the NHE1 adenovirus induced hypertrophy and was associated with an increase in the phosphorylation of RSK (P<0.05). Parameters of hypertrophy such as cell area, protein content and atrial natriuretic mRNA expression were significantly reduced in H9c2 cardiomyoblasts infected with active NHE1 in the presence of dominant negative RSK (DN-RSK) (P<0.05). These results confirm that NHE1 lies upstream of RSK. Increased phosphorylation and activation of GATA4 at Ser261 was correlated with increased RSK phosphorylation. This increase was reversed upon inhibition of RSK or NHE1. These findings demonstrate for the first time that the NHE1 mediated hypertrophy is accounted for by increased activation and phosphorylation of RSK, which subsequently increased the phosphorylation of GATA4; eventually activating fetal gene transcriptional machinery. PMID:25830299

  3. AKAP5 keeps L-type channels and NFAT on their toes.

    PubMed

    Navedo, Manuel F; Hell, Johannes W

    2014-06-12

    In this issue of Cell Reports, Murphy et al. and Dittmer et al. present exciting new insight into the regulation of Ca2+ influx via the L-type Ca2+ channel Cav1.2 and how increased Ca2+ influx translates into localized activation of the nuclear transcription factor NFAT upon depolarization in neurons.

  4. Elatoside C protects against hypoxia/reoxygenation-induced apoptosis in H9c2 cardiomyocytes through the reduction of endoplasmic reticulum stress partially depending on STAT3 activation.

    PubMed

    Wang, Min; Meng, Xiang-bao; Yu, Ying-li; Sun, Gui-bo; Xu, Xu-dong; Zhang, Xiao-po; Dong, Xi; Ye, Jing-xue; Xu, Hui-bo; Sun, Yi-fan; Sun, Xiao-bo

    2014-12-01

    Endoplasmic reticulum (ER) stress-induced apoptosis has been suggested to contribute to myocardial ischemia-reperfusion (I/R) injury. Elatoside C is one of the major triterpenoid compounds isolated from Aralia elata that is known to be cardioprotective. However, its effects on I/R injury to cardiac myocytes have not been clarified. This study aimed to investigate the possible protective effect of Elatoside C against hypoxia/reoxygenation (H/R)-induced H9c2 cardiomyocyte injury and its underlying mechanisms. H9c2 cardiomyocytes were subjected to H/R in the presence of Elatoside C. Our results showed that Elatoside C (25 μM) treatment provided significant protection against H/R-induced cell death, as evidenced by improved cell viability, maintained mitochondrial membrane potential, diminished mitochondrial ROS, and reduced apoptotic cardiomyocytes (P < 0.05). These changes were associated with the inhibition of ER stress-associated apoptosis markers (GRP78, CHOP, Caspase-12 and JNK), as well as the increased phosphorylation of STAT3 and an increased Bcl2/Bax ratio. Moreover, these effects of Elatoside C were prevented by the STAT3 inhibitor Stattic. Taken together, these results suggested that Elatoside C can alleviate H/R-induced cardiomyocyte apoptosis most likely by activating the STAT3 pathways and reducing ER stress-associated apoptosis. PMID:25326083

  5. Antisense-mediated loss of calcium homoeostasis endoplasmic reticulum protein (CHERP; ERPROT213-21) impairs Ca2+ mobilization, nuclear factor of activated T-cells (NFAT) activation and cell proliferation in Jurkat T-lymphocytes.

    PubMed Central

    O'Rourke, Flavia A; LaPlante, Janice M; Feinstein, Maurice B

    2003-01-01

    We recently discovered a novel gene on chromosome 19p13.1 and its product, an integral endoplasmic reticulum (ER) membrane protein, termed CHERP (calcium homoeostasis endoplasmic reticulum protein). A monoclonal antibody against its C-terminal domain inhibits Ins(1,4,5) P (3)-induced Ca(2+) release from ER membrane vesicles of many cell types, and an antisense-mediated knockdown of CHERP in human erythroleukemia (HEL) cells greatly impaired Ca(2+) mobilization by thrombin. In the present paper, we explore further CHERP's function in Jurkat T-lymphocytes. Confocal laser immunofluorescence microscopy showed that CHERP was co-localized with the Ins(1,4,5) P (3) receptor throughout the cytoplasmic and perinuclear region, as previously found in HEL cells. Transfection of Jurkat cells with a lac I-regulated mammalian expression vector containing CHERP antisense cDNA caused a knockdown of CHERP and impaired the rise of cytoplasmic Ca(2+) (measured by fura-2 acetoxymethyl ester fluorescence) caused by phytohaemagglutinin (PHA) and thrombin. A 50% fall of CHERP decreased the PHA-induced rise of the cytoplasmic free Ca(2+) concentration ([Ca(2+)](i)), but Ca(2+) influx was unaffected. Greater depletion of CHERP (>70%) did not affect the concentration of Ins(1,4,5) P (3) receptors, but diminished the rise of [Ca(2+)](i) in response to PHA to activation and translocation of the key transcription factor NFAT (nuclear factor of activated T-cells) from cytoplasm to nucleus was suppressed. Furthermore, cell proliferation was greatly slowed (as in HEL cells) along with a 60% decrease in cyclin D1, a key regulator of progression through the G(1) phase of the cell cycle. These findings

  6. Protection of cardiomyocytes from the hypoxia-mediated injury by a peptide targeting the activator of G-protein signaling 8.

    PubMed

    Sato, Motohiko; Hiraoka, Masahiro; Suzuki, Hiroko; Sakima, Miho; Mamun, Abdullah Al; Yamane, Yukiko; Fujita, Takayuki; Yokoyama, Utako; Okumura, Satoshi; Ishikawa, Yoshihiro

    2014-01-01

    Signaling via heterotrimeric G-protein is involved in the development of human diseases including ischemia-reperfusion injury of the heart. We previously identified an ischemia-inducible G-protein activator, activator of G-protein signaling 8 (AGS8), which regulates Gβγ signaling and plays a key role in the hypoxia-induced apoptosis of cardiomyocytes. Here, we attempted to intervene in the AGS8-Gβγ signaling process and protect cardiomyocytes from hypoxia-induced apoptosis with a peptide that disrupted the AGS8-Gβγ interaction. Synthesized AGS8-peptides, with amino acid sequences based on those of the Gβγ-binding domain of AGS8, successfully inhibited the association of AGS8 with Gβγ. The AGS8-peptide effectively blocked hypoxia-induced apoptosis of cardiomyocytes, as determined by DNA end-labeling and an increase in cleaved caspase-3. AGS8-peptide also inhibited the change in localization/permeability of channel protein connexin 43, which was mediated by AGS8-Gβγ under hypoxia. Small compounds that inhibit a wide range of Gβγ signals caused deleterious effects in cardiomyocytes. In contrast, AGS8-peptide did not cause cell damage under normoxia, suggesting an advantage inherent in targeted disruption of the AGS8-Gβγ signaling pathway. These data indicate a pivotal role for the interaction of AGS8 with Gβγ in hypoxia-induced apoptosis of cardiomyocytes, and suggest that targeted disruption of the AGS8-Gβγ signal provides a novel approach for protecting the myocardium against ischemic injury.

  7. Peroxisome Proliferator-Activated Receptor α Reduces Endothelin-1-Caused Cardiomyocyte Hypertrophy by Inhibiting Nuclear Factor-κB and Adiponectin

    PubMed Central

    Jen, Hsu-Lung; Liu, Po-Len; Chen, Jaw-Wen; Lin, Shing-Jong

    2016-01-01

    Peroxisome proliferator-activated receptor α (PPARα) plays a role in the pathogenesis of cardiac hypertrophy, although its underlying mechanism remains unclear. The purpose of this study was to evaluate the effect of PPARα activation on endothelin-1- (ET-1-) caused cardiomyocyte hypertrophy and explore its underlying mechanisms. Human cardiomyocytes (HCMs) were cultured with or without ET-1, whereafter the inhibitory effects of fenofibrate, a PPARα activator, on cell size and adiponectin protein were tested. We examined the activation of extracellular signal-regulated kinase (ERK) and p38 proteins caused by ET-1 and the inhibition of the ERK and p38 pathways on ET-1-induced cell size and adiponectin expression. Moreover, we investigated the interaction of PPARα with adiponectin and nuclear factor-κB (NF-κB) by electrophoretic mobility shift assays and coimmunoprecipitation. ET-1 treatment significantly increased cell size, suppressed PPARα expression, and enhanced the expression of adiponectin. Pretreatment with fenofibrate inhibited the increase in cell size and enhancement of adiponectin expression. ET-1 significantly activated the ERK and p38 pathways, whereas PD98059 and SB205380, respectively, inhibited them. Our results suggest that activated PPARα can decrease activation of adiponectin and NF-κB and inhibit ET-1-induced cardiomyocyte hypertrophy. PMID:27807394

  8. Caveolin-3 Overexpression Attenuates Cardiac Hypertrophy via Inhibition of T-type Ca2+ Current Modulated by Protein Kinase Cα in Cardiomyocytes*

    PubMed Central

    Markandeya, Yogananda S.; Phelan, Laura J.; Woon, Marites T.; Keefe, Alexis M.; Reynolds, Courtney R.; August, Benjamin K.; Hacker, Timothy A.; Roth, David M.; Patel, Hemal H.; Balijepalli, Ravi C.

    2015-01-01

    Pathological cardiac hypertrophy is characterized by subcellular remodeling of the ventricular myocyte with a reduction in the scaffolding protein caveolin-3 (Cav-3), altered Ca2+ cycling, increased protein kinase C expression, and hyperactivation of calcineurin/nuclear factor of activated T cell (NFAT) signaling. However, the precise role of Cav-3 in the regulation of local Ca2+ signaling in pathological cardiac hypertrophy is unclear. We used cardiac-specific Cav-3-overexpressing mice and in vivo and in vitro cardiac hypertrophy models to determine the essential requirement for Cav-3 expression in protection against pharmacologically and pressure overload-induced cardiac hypertrophy. Transverse aortic constriction and angiotensin-II (Ang-II) infusion in wild type (WT) mice resulted in cardiac hypertrophy characterized by significant reduction in fractional shortening, ejection fraction, and a reduced expression of Cav-3. In addition, association of PKCα and angiotensin-II receptor, type 1, with Cav-3 was disrupted in the hypertrophic ventricular myocytes. Whole cell patch clamp analysis demonstrated increased expression of T-type Ca2+ current (ICa, T) in hypertrophic ventricular myocytes. In contrast, the Cav-3-overexpressing mice demonstrated protection from transverse aortic constriction or Ang-II-induced pathological hypertrophy with inhibition of ICa, T and intact Cav-3-associated macromolecular signaling complexes. siRNA-mediated knockdown of Cav-3 in the neonatal cardiomyocytes resulted in enhanced Ang-II stimulation of ICa, T mediated by PKCα, which caused nuclear translocation of NFAT. Overexpression of Cav-3 in neonatal myocytes prevented a PKCα-mediated increase in ICa, T and nuclear translocation of NFAT. In conclusion, we show that stable Cav-3 expression is essential for protecting the signaling mechanisms in pharmacologically and pressure overload-induced cardiac hypertrophy. PMID:26170457

  9. Structural heterogeneity promotes triggered activity, reflection and arrhythmogenesis in cardiomyocyte monolayers

    PubMed Central

    Auerbach, David S; Grzȩda, Krzysztof R; Furspan, Philip B; Sato, Priscila Y; Mironov, Sergey; Jalife, José

    2011-01-01

    Abstract Patients with structural heart disease are predisposed to arrhythmias by incompletely understood mechanisms. We hypothesized that tissue expansions promote source-to-sink mismatch leading to early after-depolarizations (EADs) and reflection of impulses in monolayers of well-polarized neonatal rat ventricular cardiomyocytes. We traced electrical propagation optically in patterned monolayers consisting of two wide regions connected by a thin isthmus. Structural heterogeneities provided a substrate for EADs, retrograde propagation along the same pathway (reflection) and reentry initiation. Reflection always originated during the action potential (AP) plateau at the distal expansion. To determine whether increased sodium current (INa) would promote EADs, we employed adenoviral transfer of Nav1.5 (Ad-Nav1.5). Compared with uninfected and adenoviral expression of green fluorescent protein (Ad-GFP; viral control), Ad-Nav1.5 significantly increased Nav1.5 protein expression, peak and persistent INa density, AP upstroke velocity, AP duration, conduction velocity and EAD incidence, as well as reflection incidence (29.2%, n = 48 vs. uninfected, 9.4%, n = 64; and Ad-GFP, 4.8%, n = 21). Likewise, the persistent INa agonist veratridine (0.05–3 μm) prolonged the AP, leading to EADs and reflection. Reflection led to functional reentry distally and bigeminal and trigeminal rhythms proximally. Reflection was rare in the absence of structural heterogeneities. Computer simulations demonstrated the importance of persistent INa in triggering reflection and predicted that the gradient between the depolarizing cells at the distal expansion and the repolarizing cells within the isthmus enabled retrograde flow of depolarizing electrotonic current to trigger EADs and reflection. A combination of a substrate (structural heterogeneity) and a trigger (increased persistent INa and EADs) promotes reflection and arrhythmogenesis. PMID:21486795

  10. ANG II promotes IGF-IIR expression and cardiomyocyte apoptosis by inhibiting HSF1 via JNK activation and SIRT1 degradation

    PubMed Central

    Huang, C-Y; Kuo, W-W; Yeh, Y-L; Ho, T-J; Lin, J-Y; Lin, D-Y; Chu, C-H; Tsai, F-J; Tsai, C-H; Huang, C-Y

    2014-01-01

    Hypertension-induced cardiac hypertrophy and apoptosis are major characteristics of early-stage heart failure. Our previous studies found that the activation of insulin-like growth factor receptor II (IGF-IIR) signaling was critical for hypertensive angiotensin II (ANG II)-induced cardiomyocyte apoptosis. However, the detailed mechanism by which ANG II regulates IGF-IIR in heart cells remains elusive. In this study, we found that ANG II activated its downstream kinase JNK to increase IGF-IIR expression through the ANG II receptor angiotensin type 1 receptor. JNK activation subsequently led to sirtuin 1 (SIRT1) degradation via the proteasome, thus preventing SIRT1 from deacetylating heat-shock transcription factor 1 (HSF1). The resulting increase in the acetylation of HSF1 impaired its ability to bind to the IGF-IIR promoter region (nt −748 to −585). HSF1 protected cardiomyocytes by acting as a repressor of IGF-IIR gene expression, and ANG II diminished this HSF1-mediated repression through enhanced acetylation, thus activating the IGF-IIR apoptosis pathway. Taken together, these results suggest that HSF1 represses IGF-IIR gene expression to protect cardiomyocytes. ANG II activates JNK to degrade SIRT1, resulting in HSF1 acetylation, which induces IGF-IIR expression and eventually results in cardiac hypertrophy and apoptosis. HSF1 could be a valuable target for developing treatments for cardiac diseases in hypertensive patients. PMID:24786827

  11. Signal-dependent repression of DUSP5 by class I HDACs controls nuclear ERK activity and cardiomyocyte hypertrophy.

    PubMed

    Ferguson, Bradley S; Harrison, Brooke C; Jeong, Mark Y; Reid, Brian G; Wempe, Michael F; Wagner, Florence F; Holson, Edward B; McKinsey, Timothy A

    2013-06-11

    Cardiac hypertrophy is a strong predictor of morbidity and mortality in patients with heart failure. Small molecule histone deacetylase (HDAC) inhibitors have been shown to suppress cardiac hypertrophy through mechanisms that remain poorly understood. We report that class I HDACs function as signal-dependent repressors of cardiac hypertrophy via inhibition of the gene encoding dual-specificity phosphatase 5 (DUSP5) DUSP5, a nuclear phosphatase that negatively regulates prohypertrophic signaling by ERK1/2. Inhibition of DUSP5 by class I HDACs requires activity of the ERK kinase, mitogen-activated protein kinase kinase (MEK), revealing a self-reinforcing mechanism for promotion of cardiac ERK signaling. In cardiac myocytes treated with highly selective class I HDAC inhibitors, nuclear ERK1/2 signaling is suppressed in a manner that is absolutely dependent on DUSP5. In contrast, cytosolic ERK1/2 activation is maintained under these same conditions. Ectopic expression of DUSP5 in cardiomyocytes results in potent inhibition of agonist-dependent hypertrophy through a mechanism involving suppression of the gene program for hypertrophic growth. These findings define unique roles for class I HDACs and DUSP5 as integral components of a regulatory signaling circuit that controls cardiac hypertrophy.

  12. The cooperation of CREB and NFAT is required for PTHrP-induced RANKL expression in mouse osteoblastic cells.

    PubMed

    Park, Hyun-Jung; Baek, Kyunghwa; Baek, Jeong-Hwa; Kim, Hyung-Ryong

    2015-03-01

    Parathyroid hormone-related protein (PTHrP) is known to induce the expression of receptor activator of NF-κB ligand (RANKL) in stromal cells/osteoblasts. However, the signaling pathways involved remain controversial. In the present study, we investigated the role of cAMP/protein kinase A (PKA) and calcineurin/NFAT pathways in PTHrP-induced RANKL expression in C2C12 and primary cultured mouse calvarial cells. PTHrP-mediated induction of RANKL expression was significantly inhibited by H89 and FK506, an inhibitor of PKA and calcineurin, respectively. PTHrP upregulated CREB phosphorylation and the transcriptional activity of NFAT. Knockdown of CREB or NFATc1 blocked PTHrP-induced RANKL expression. PTHrP increased the activity of the RANKL promoter reporter that contains approximately 2 kb mouse RANKL promoter DNA sequences. Insertions of mutations in CRE-like element or in NFAT-binding element abrogated PTHrP-induced RANKL promoter activity. Chromatin immunoprecipitation assays showed that PTHrP increased the binding of CREB and NFATc1/NFATc3 to their cognate binding elements in the RANKL promoter. Inhibition of cAMP/PKA and its downstream ERK activity suppressed PTHrP-induced expression and transcriptional activity of NFATc1. CREB knockdown prevented PTHrP induction of NFATc1 expression. Furthermore, NFATc1 and CREB were co-immunoprecipitated. Mutations in CRE-like element completely blocked NFATc1-induced transactivation of the RANKL promoter reporter; however, mutations in NFAT-binding element partially suppressed CREB-induced RANKL promoter activity. Overexpression of CREB increased NFATc1 binding to the RANKL promoter and vice versa. These results suggest that PTHrP-induced RANKL expression depends on the activation of both cAMP/PKA and calcineurin/NFAT pathways, and subsequently, CREB and NFAT cooperate to transactivate the mouse RANKL gene.

  13. CaMKII Negatively Regulates Calcineurin-NFAT Signaling in Cardiac Myocytes

    PubMed Central

    MacDonnell, Scott M.; Weisser-Thomas, Jutta; Kubo, Hajime; Hanscome, Marie; Liu, Qinghang; Jaleel, Naser; Berretta, Remus; Chen, Xiongwen; Brown, Joan H.; Sabri, Abdel-Karim; Molkentin, Jeffery D.; Houser, Steven R.

    2009-01-01

    Rationale Pathologic cardiac myocyte hypertrophy is thought to be induced by the persistent increases in intracellular Ca2+ needed to maintain cardiac function when systolic wall stress is increased. Hypertrophic Ca2+ binds to calmodulin (CaM) and activates the phosphatase calcineurin (Cn) and CaM kinase (CaMKII). Cn dephosphorylates cytoplasmic nuclear factor of activated T-cells (NFAT), inducing its translocation to the nucleus where it activates anti-apoptotic and hypertrophic target genes. Cytoplasmic CaMKII regulates Ca2+ handling proteins but whether or not it is directly involved in hypertrophic and survival signaling is not known. Objective This study explored the hypothesis that cytoplasmic CaMKII reduces NFAT nuclear translocation by inhibiting the phosphatase activity of Cn. Methods and Results GFP-tagged NFATc3 was used to determine the cellular location of NFAT in cultured neonatal rat ventricular myocytes (NRVM) and adult feline ventricular myocytes. Constitutively active (CaMKII-CA) or dominant negative (CaMKII-DN) mutants of cytoplasmic targeted CaMKIIδc were used to activate and inhibit cytoplasmic CaMKII activity. In NRVM CaMKII-DN (48.5±3%, P<0.01 vs control) increased while CaMKII-CA decreased (5.9±1%, P<0.01 vs 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 Ca2+ 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 NRVM and in hypertrophied feline hearts. Conclusion These data show that activation of cytoplasmic CaMKII inhibits NFAT nuclear translocation by phosphorylation and subsequent inhibition of Cn. PMID:19608982

  14. Identification of novel regulatory NFAT and TFII-I binding elements in the calbindin-D28k promoter in response to serum deprivation.

    PubMed

    Hajibeigi, Asghar; Dioum, Elhadji M; Guo, Jianfei; Öz, Orhan K

    2015-09-25

    Calbindin-D28k, a key regulator of calcium homeostasis plays a cytoprotective role in various tissues. We used serum free (SFM) and charcoal stripped serum (csFBS) culture media as models of cellular stress to modulate calbindin D28k expression and identify regulatory cis-elements and trans-acting factors in kidney and beta cells. The murine calbindin-D28k promoter activity was significantly upregulated under SFM or csFBS condition. Promoter analysis revealed evolutionary conserved regulatory cis-elements and deletion of 23 nt from +117/+139 as critical for basal transcription. Bioinformatics analysis of the promoter revealed conserved NFAT and TFII regulators elements. Forced expression of NFAT stimulated promoter activity. Inhibition of NFAT transcriptional activity by FK506 attenuated calbindin-D28k expression. TFII-I was shown to be necessary for basal promoter activity and to act cooperatively with NFAT. Using chromatin immunoprecipitation (ChIP) assays, NFAT was shown to bind to both proximal and distal promoter regions. ChIP assays also revealed recruitment of TFII to the -36/+139 region. Knockdown of TFII-I decreased promoter activity. In summary, calbindin-D28k expression during serum deprivation is partly regulated by NFAT and TF-II. This regulation may be important in vivo during ischemia and growth factor withdrawal to regulate cellular function and maintenance.

  15. FK506 ameliorates podocyte injury in type 2 diabetic nephropathy by down-regulating TRPC6 and NFAT expression.

    PubMed

    Ma, Ruixia; Liu, Liqiu; Jiang, Wei; Yu, Yanjuan; Song, Haifeng

    2015-01-01

    Diabetic nephropathy (DN) is the leading cause of end-stage renal failure, and podocyte injury plays a major role in the development of DN. In this study, we investigated whether tacrolimus (FK506), an immunosuppressor, can attenuate podocyte injury in a type 2 diabetic mellitus (T2DM) rat model with DN. Transmission electron microcopy was used to morphologically evaluate renal injury. The urinary albumin (UAL), creatinine clearance rate (Ccr) and major biochemical parameters, including glucose, insulin, serum creatinine (Scr), urea nitrogen, total cholesterol (CHO) and triglyceride (TG), were examined 12 weeks after the administration of FK506. The expressions of the canonical transient receptor potential 6 (TRPC6), nuclear factor of activated T-cells (NFAT) and nephrin were detected by Western blotting and qPCR. In the rat model of DN, the expressions of TRPC6 and NFAT were significantly elevated compared with the normal rat group; however, the treatment with FK506 normalized the increased expression of TRPC6 and NFAT and attenuated podocyte ultrastructure injury. UAL, Ccr and the biochemical parameters were also improved by the use of FK506. In cell experiments, FK506 improved the decreased expression of nephrin and suppressed the elevated expression of both TRPC6 and NFAT caused by high glucose in accordance with TRPC6 blocker U73122. Our results demonstrated that FK506 could ameliorate podocyte injury in T2DM, which may be related to suppressed expressions of TRPC6 and NFAT.

  16. Endothelial RSPO3 Controls Vascular Stability and Pruning through Non-canonical WNT/Ca(2+)/NFAT Signaling.

    PubMed

    Scholz, Beate; Korn, Claudia; Wojtarowicz, Jessica; Mogler, Carolin; Augustin, Iris; Boutros, Michael; Niehrs, Christof; Augustin, Hellmut G

    2016-01-11

    The WNT signaling enhancer R-spondin3 (RSPO3) is prominently expressed in the vasculature. Correspondingly, embryonic lethality of Rspo3-deficient mice is caused by vessel remodeling defects. Yet the mechanisms underlying vascular RSPO3 function remain elusive. Inducible endothelial Rspo3 deletion (Rspo3-iECKO) resulted in perturbed developmental and tumor vascular remodeling. Endothelial cell apoptosis and vascular pruning led to reduced microvessel density in Rspo3-iECKO mice. Rspo3-iECKO mice strikingly phenocopied the non-canonical WNT signaling-induced vascular defects of mice deleted for the WNT secretion factor Evi/Wls. An endothelial screen for RSPO3 and EVI/WLS co-regulated genes identified Rnf213, Usp18, and Trim30α. RNF213 targets filamin A and NFAT1 for proteasomal degradation attenuating non-canonical WNT/Ca(2+) signaling. Likewise, USP18 and TRIM5α inhibited NFAT1 activation. Consequently, NFAT protein levels were decreased in endothelial cells of Rspo3-iECKO mice and pharmacological NFAT inhibition phenocopied Rspo3-iECKO mice. The data identify endothelial RSPO3-driven non-canonical WNT/Ca(2+)/NFAT signaling as a critical maintenance pathway of the remodeling vasculature.

  17. The lncRNA NRON modulates HIV-1 replication in a NFAT-dependent manner and is differentially regulated by early and late viral proteins.

    PubMed

    Imam, Hasan; Bano, Aalia Shahr; Patel, Paresh; Holla, Prasida; Jameel, Shahid

    2015-03-02

    A majority of the human genome is transcribed into noncoding RNAs, of which the functions of long noncoding RNAs (lncRNAs) are poorly understood. Many host proteins and RNAs have been characterized for their roles in HIV/AIDS pathogenesis, but there is only one lncRNA, NEAT1, which is shown to affect the HIV-1 life cycle. We profiled 90 disease-related lncRNAs and found NRON (noncoding repressor of Nuclear Factor of Activated T cells [NFAT]) to be one of several lncRNAs whose expression was significantly altered following HIV-1 infection. The regulation of NRON expression during the HIV-1 life cycle was complex; its levels were reduced by the early viral accessory protein Nef and increased by the late protein Vpu. Consequently, Nef and Vpu also modulated activity of the transcription factor NFAT. The knockdown of NRON enhanced HIV-1 replication through increased activity of NFAT and the viral LTR. Using siRNA-mediated NFAT knockdown, we show the effects of NRON on HIV-1 replication to be mediated by NFAT, and the viral Nef and Vpu proteins to modulate NFAT activity through their effects on NRON. These findings add the lncRNA, NRON to the vast repertoire of host factors utilized by HIV for infection and persistence.

  18. Clusterin: a protective mediator for ischemic cardiomyocytes?

    PubMed

    Krijnen, P A J; Cillessen, S A G M; Manoe, R; Muller, A; Visser, C A; Meijer, C J L M; Musters, R J P; Hack, C E; Aarden, L A; Niessen, H W M

    2005-11-01

    We examined the relationship between clusterin and activated complement in human heart infarction and evaluated the effect of this protein on ischemic rat neonatal cardiomyoblasts (H9c2) and isolated adult ventricular rat cardiomyocytes as in vitro models of acute myocardial infarction. Clusterin protects cells by inhibiting complement and colocalizes with complement on jeopardized human cardiomyocytes after infarction. The distribution of clusterin and complement factor C3d was evaluated in the infarcted human heart. We also analyzed the protein expression of clusterin in ischemic H9c2 cells. The binding of endogenous and purified human clusterin on H9c2 cells was analyzed by flow cytometry. Furthermore, the effect of clusterin on the viability of ischemically challenged H9c2 cells and isolated adult ventricular rat cardiomyocytes was analyzed. In human myocardial infarcts, clusterin was found on scattered, morphologically viable cardiomyocytes within the infarcted area that were negative for complement. In H9c2 cells, clusterin was rapidly expressed after ischemia. Its expression was reduced after reperfusion. Clusterin bound to single annexin V-positive or annexin V and propidium iodide-positive H9c2 cells. Clusterin inhibited ischemia-induced death in H9c2 cells as well as in isolated adult ventricular rat cardiomyocytes in the absence of complement. We conclude that ischemia induces the upregulation of clusterin in ischemically challenged, but viable, cardiomyocytes. Our data suggest that clusterin protects cardiomyocytes against ischemic cell death via a complement-independent pathway.

  19. Cutting edge: tubulin α functions as an adaptor in NFAT-importin β interaction.

    PubMed

    Ishiguro, Kazuhiro; Ando, Takafumi; Maeda, Osamu; Watanabe, Osamu; Goto, Hidemi

    2011-03-01

    Upon T cell stimulation, NFAT is dephosphorylated by calcineurin, leading to nuclear translocation via NFAT-importin β interaction. Whereas the process of NFAT dephosphorylation has been well researched, the molecular mechanism of NFAT-importin β interaction remains unknown. In contrast to NF-κB and STAT, no importin α family members have been reported as adaptor proteins for NFAT. Our study shows that tubulin α, but not tubulin β, binds to the N-terminal region of NFAT containing the regulatory and Rel homology domains. Importin β interacts with the NFAT-tubulin α complex rather than NFAT or tubulin α alone, resulting in cotranslocation of NFAT and tubulin α into the nucleus. Furthermore, the interaction is suppressed by acetate-induced tubulin α acetylation at lysine 40. In conclusion, tubulin α functions as an adaptor in NFAT-importin β interaction, and this function is regulated by acetate-induced acetylation.

  20. Identifying panaxynol, a natural activator of nuclear factor erythroid-2 related factor 2 (Nrf2) from American ginseng as a suppressor of inflamed macrophage-induced cardiomyocyte hypertrophy

    PubMed Central

    Qu, Chen; Li, Bin; Lai, Yimu; Li, Hechu; Windust, Anthony; Hofseth, Lorne J.; Nagarkatti, Mitzi; Nagarkatti, Prakash; Wang, Xing Li; Tang, Dongqi; Janicki, Joseph S.; Tian, Xingsong; Cui, Taixing

    2015-01-01

    Ethnopharmacological relevance American ginseng is capable of ameliorating cardiac dysfunction and activating Nrf2, a master regulator of antioxidant defense, in the heart. This study was designed to isolate compounds from American ginseng and to determine those responsible for the Nrf2-mediated resolution of inflamed macrophage-induced cardiomyocyte hypertrophy. Materials and methods A standardized crude extract of American ginseng was supplied by the National Research Council of Canada, Institute for National Measurement Standards. A bioassay-based fractionization of American ginseng was performed to identify the putative substances which could activate Nrf2-mediated suppression of pro-inflammatory cytokine expression in macrophages and macrophage-mediated pro-hypertrophic growth in cardiomyocytes. Results A hexane fraction of an anti-inflammatory crude extract of American ginseng was found to be most effective in suppressing the inflammatory responses in macrophages. Preparative, reverse-phase HPLC and a comparative analysis by analytical scale LC–UV/MS revealed the hexane fraction contains predominantly C17 polyacetylenes and linolenic acid. Panaxynol, one of the major polyacetylenes, was found to be a potent Nrf2 activator. Panaxynol posttranscriptionally activated Nrf2 by inhibiting Kelch-like ECH-associated protein (Keap) 1-mediated degradation without affecting the binding of Keap1 and Nrf2. Moreover, panaxynol suppressed a selected set of cytokine expression via the activation of Nrf2 while minimally regulating nuclear factor-kappa B (NF-κB)-mediated cytokine expression in macrophages. It also dramatically inhibited the inflamed macrophage-mediated cardiomyocyte death and hypertrophy by activating Nrf2 in macrophages. Conclusions These results demonstrate that American ginseng-derived panaxynol is a specific Nrf2 activator and panaxynol-activated Nrf2 signaling is at least partly responsible for American ginseng-induced health benefit in the heart. PMID

  1. Identification of lineariifolianoid A as a novel dual NFAT1 and MDM2 inhibitor for human cancer therapy

    PubMed Central

    Qin, Jiang-Jiang; Sarkar, Sushanta; Voruganti, Sukesh; Agarwal, Rajesh; Wang, Wei; Zhang, Ruiwen

    2016-01-01

    Abstract There is an increasing interest in development of novel anticancer agents that target oncogenes. We have recently discovered that nuclear factor of activated T cells 1 (NFAT1) is a novel regulator of the Mouse Double Minute 2 (MDM2) oncogene and the NFAT1-MDM2 pathway has been implicated in human cancer development and progression, justifying that targeting the NFAT1-MDM2 pathway could be a novel strategy for discovery and development of novel cancer therapeutics. The present study was designed to examine the anticancer activity and underlying mechanisms of action of lineariifolianoid A (LinA), a novel natural product inhibitor of the NFAT1-MDM2 pathway. The cytotoxicity of LinA was first tested in various human cancer cell lines in comparison with normal cell lines. The results showed that the breast cancer cells were highly sensitive to LinA treatment. We next demonstrated the effects of LinA on cell proliferation, colony formation, cell cycle progression, and apoptosis in breast cancer MCF7 and MDA-MB-231 cells, in dose-dependent and p53-independent manners. LinA also inhibited the migration and invasion of these cancer cells. Our mechanistic studies further indicated that its anticancer activities were attributed to its inhibitory effects on the NFAT1-MDM2 pathway and modulatory effects on the expression of key proteins involved in cell cycle progression, apoptosis, and DNA damage. In summary, LinA is a novel NFAT1-MDM2 inhibitor and may be developed as a preventive and therapeutic agent against human cancer. PMID:27533941

  2. Regulation of gene expression by NFAT transcription factors in hibernating ground squirrels is dependent on the cellular environment.

    PubMed

    Zhang, Yichi; Storey, Kenneth B

    2016-09-01

    Calcineurin is a calmodulin-stimulated phosphatase that regulates the nuclear translocation of nuclear factor of activated T cell (NFAT) c1-4 through dephosphorylation. We believe that this mechanism plays various roles in the remodeling and maintenance of Ictidomys tridecemlineatus skeletal muscle. During hibernation, bouts of torpor and arousal take place, and squirrels do not lose muscle mass despite being inactive. Protein expression of Ca(2+) signaling proteins were studied using immunoblotting. A DNA-protein interaction ELISA technique was created to test the binding of NFATs in the nucleus to DNA probes containing the NFAT response element under environmental conditions reflective of those during hibernation. Calcineurin protein levels increased by 3.08-fold during torpor (compared to euthermic control), whereas calpain1 levels also rose by 3.66-fold during torpor. Calmodulin levels were elevated upon entering torpor. NFATc4 binding to DNA showed a 1.4-fold increase during torpor, and we found that this binding was further enhanced when 600 nM of Ca(2+) was supplemented. We also found that decreasing the temperature of ELISAs resulted in progressive decreases in the binding of NFATs c1, c3, and c4 to DNA. In summary, calmodulin and calpain1 appear to activate calcineurin and NFATc4 during torpor. NFAT binding to target promoters is affected by intranuclear [Ca(2+)] and environmental temperatures. Therefore, Ca(2+) signaling and temperature changes play key roles in regulation of the NFAT-calcineurin pathway in skeletal muscle of hibernating 13-lined ground squirrels over the torpor-arousal cycle, and they may contribute to the avoidance of disuse-induced muscle atrophy that occurs naturally in these animals.

  3. Regulation of gene expression by NFAT transcription factors in hibernating ground squirrels is dependent on the cellular environment.

    PubMed

    Zhang, Yichi; Storey, Kenneth B

    2016-09-01

    Calcineurin is a calmodulin-stimulated phosphatase that regulates the nuclear translocation of nuclear factor of activated T cell (NFAT) c1-4 through dephosphorylation. We believe that this mechanism plays various roles in the remodeling and maintenance of Ictidomys tridecemlineatus skeletal muscle. During hibernation, bouts of torpor and arousal take place, and squirrels do not lose muscle mass despite being inactive. Protein expression of Ca(2+) signaling proteins were studied using immunoblotting. A DNA-protein interaction ELISA technique was created to test the binding of NFATs in the nucleus to DNA probes containing the NFAT response element under environmental conditions reflective of those during hibernation. Calcineurin protein levels increased by 3.08-fold during torpor (compared to euthermic control), whereas calpain1 levels also rose by 3.66-fold during torpor. Calmodulin levels were elevated upon entering torpor. NFATc4 binding to DNA showed a 1.4-fold increase during torpor, and we found that this binding was further enhanced when 600 nM of Ca(2+) was supplemented. We also found that decreasing the temperature of ELISAs resulted in progressive decreases in the binding of NFATs c1, c3, and c4 to DNA. In summary, calmodulin and calpain1 appear to activate calcineurin and NFATc4 during torpor. NFAT binding to target promoters is affected by intranuclear [Ca(2+)] and environmental temperatures. Therefore, Ca(2+) signaling and temperature changes play key roles in regulation of the NFAT-calcineurin pathway in skeletal muscle of hibernating 13-lined ground squirrels over the torpor-arousal cycle, and they may contribute to the avoidance of disuse-induced muscle atrophy that occurs naturally in these animals. PMID:27344571

  4. Blockade of Astrocytic Calcineurin/NFAT Signaling Helps to Normalize Hippocampal Synaptic Function and Plasticity in a Rat Model of Traumatic Brain Injury

    PubMed Central

    Furman, Jennifer L.; Sompol, Pradoldej; Kraner, Susan D.; Pleiss, Melanie M.; Putman, Esther J.; Dunkerson, Jacob; Mohmmad Abdul, Hafiz; Roberts, Kelly N.; Scheff, Stephen W.

    2016-01-01

    Increasing evidence suggests that the calcineurin (CN)-dependent transcription factor NFAT (Nuclear Factor of Activated T cells) mediates deleterious effects of astrocytes in progressive neurodegenerative conditions. However, the impact of astrocytic CN/NFAT signaling on neural function/recovery after acute injury has not been investigated extensively. Using a controlled cortical impact (CCI) procedure in rats, we show that traumatic brain injury is associated with an increase in the activities of NFATs 1 and 4 in the hippocampus at 7 d after injury. NFAT4, but not NFAT1, exhibited extensive labeling in astrocytes and was found throughout the axon/dendrite layers of CA1 and the dentate gyrus. Blockade of the astrocytic CN/NFAT pathway in rats using adeno-associated virus (AAV) vectors expressing the astrocyte-specific promoter Gfa2 and the NFAT-inhibitory peptide VIVIT prevented the injury-related loss of basal CA1 synaptic strength and key synaptic proteins and reduced the susceptibility to induction of long-term depression. In conjunction with these seemingly beneficial effects, VIVIT treatment elicited a marked increase in the expression of the prosynaptogenic factor SPARCL1 (hevin), especially in hippocampal tissue ipsilateral to the CCI injury. However, in contrast to previous work on Alzheimer's mouse models, AAV-Gfa2-VIVIT had no effects on the levels of GFAP and Iba1, suggesting that synaptic benefits of VIVIT were not attributable to a reduction in glial activation per se. Together, the results implicate the astrocytic CN/NFAT4 pathway as a key mechanism for disrupting synaptic remodeling and homeostasis in the hippocampus after acute injury. SIGNIFICANCE STATEMENT Similar to microglia, astrocytes become strongly “activated” with neural damage and exhibit numerous morphologic/biochemical changes, including an increase in the expression/activity of the protein phosphatase calcineurin. Using adeno-associated virus (AAV) to inhibit the calcineurin

  5. Hypoxia-inducible factor 1 mediates hypoxia-induced cardiomyocyte lipid accumulation by reducing the DNA binding activity of peroxisome proliferator-activated receptor {alpha}/retinoid X receptor

    SciTech Connect

    Belanger, Adam J.; Luo Zhengyu; Vincent, Karen A.; Akita, Geoffrey Y.; Cheng, Seng H.; Gregory, Richard J.; Jiang Canwen

    2007-12-21

    In response to cellular hypoxia, cardiomyocytes adapt to consume less oxygen by shifting ATP production from mitochondrial fatty acid {beta}-oxidation to glycolysis. The transcriptional activation of glucose transporters and glycolytic enzymes by hypoxia is mediated by hypoxia-inducible factor 1 (HIF-1). In this study, we examined whether HIF-1 was involved in the suppression of mitochondrial fatty acid {beta}-oxidation in hypoxic cardiomyocytes. We showed that either hypoxia or adenovirus-mediated expression of a constitutively stable hybrid form (HIF-1{alpha}/VP16) suppressed mitochondrial fatty acid metabolism, as indicated by an accumulation of intracellular neutral lipid. Both treatments also reduced the mRNA levels of muscle carnitine palmitoyltransferase I which catalyzes the rate-limiting step in the mitochondrial import of fatty acids for {beta}-oxidation. Furthermore, adenovirus-mediated expression of HIF-1{alpha}/VP16 in cardiomyocytes under normoxic conditions also mimicked the reduction in the DNA binding activity of peroxisome proliferator-activated receptor {alpha} (PPAR{alpha})/retinoid X receptor (RXR), in the presence or absence of a PPAR{alpha} ligand. These results suggest that HIF-1 may be involved in hypoxia-induced suppression of fatty acid metabolism in cardiomyocytes by reducing the DNA binding activity of PPAR{alpha}/RXR.

  6. AM404 inhibits NFAT and NF-κB signaling pathways and impairs migration and invasiveness of neuroblastoma cells.

    PubMed

    Caballero, Francisco J; Soler-Torronteras, Rafael; Lara-Chica, Maribel; García, Victor; Fiebich, Bernd L; Muñoz, Eduardo; Calzado, Marco A

    2015-01-01

    N-Arachidonoylphenolamine (AM404), a paracetamol lipid metabolite, is a modulator of the endocannabinoid system endowed with pleiotropic activities. AM404 is a dual agonist of the Transient Receptor Potential Vanilloid type 1 (TRPV1) and the Cannabinoid Receptor type 1 (CB₁) and inhibits anandamide (AEA) transport and degradation. In addition, it has been shown that AM404 also exerts biological activities through TRPV1- and CB₁ -independent pathways. In the present study we have investigated the effect of AM404 in the NFAT and NF-κB signaling pathways in SK-N-SH neuroblastoma cells. AM404 inhibited NFAT transcriptional activity through a CB₁- and TRPV1-independent mechanism. Moreover, AM404 inhibited both the expression of COX-2 at transcriptional and post-transcriptional levels and the synthesis of PGE₂. AM404 also inhibited NF-κB activation induced by PMA/Ionomycin in SK-N-SH cells by targeting IKKβ phosphorylation and activation. We found that Cot/Tlp-2 induced NFAT and COX-2 transcriptional activities were inhibited by AM404. NFAT inhibition paralleled with the ability of AM404 to inhibit MMP-1, -3 and -7 expression, cell migration and invasion in a cell-type specific dependent manner. Taken together, these data reveal that paracetamol, the precursor of AM404, can be explored not only as an antipyretic and painkiller drug but also as a co-adjuvant therapy in inflammatory and cancer diseases.

  7. HIV-1 gp120 induces NFAT nuclear translocation in resting CD4+ T-cells

    SciTech Connect

    Cicala, Claudia . E-mail: ccicala@nih.gov; Arthos, James; Censoplano, Nina; Cruz, Catherine; Chung, Eva; Martinelli, Elena; Lempicki, Richard A.; Natarajan, Ven; VanRyk, Donald; Daucher, Marybeth; Fauci, Anthony S.

    2006-02-05

    The replication of human immunodeficiency virus (HIV) in CD4+ T-cells is strongly dependent upon the state of activation of infected cells. Infection of sub-optimally activated cells is believed to play a critical role in both the transmission of virus and the persistence of CD4+ T-cell reservoirs. There is accumulating evidence that HIV can modulate signal-transduction pathways in a manner that may facilitate replication in such cells. We previously demonstrated that HIV gp120 induces virus replication in resting CD4+ T cells isolated from HIV-infected individuals. Here, we show that in resting CD4+ T-cells, gp120 activates NFATs and induces their translocation into the nucleus. The HIV LTR encodes NFAT recognition sites, and NFATs may play a critical role in promoting viral replication in sub-optimally activated cells. These observations provide insight into a potential mechanism by which HIV is able to establish infection in resting cells, which may have implications for both transmission of HIV and the persistence of viral reservoirs.

  8. A Cross-Talk Between NFAT and NF-κB Pathways is Crucial for Nickel-Induced COX-2 Expression in Beas-2B Cells

    PubMed Central

    Cai, T.; Li, X.; Ding, J.; Luo, W.; Li, J.; Huang, C.

    2013-01-01

    Cyclooxygenase-2 (COX-2) is a critical enzyme implicated in chronic inflammation-associated cancer development. Our studies have shown that the exposure of Beas-2B cells, a human bronchial epithelial cell line, to lung carcinogenic nickel compounds results in increased COX-2 expression. However, the signaling pathways leading to nickel-induced COX-2 expression are not well understood. In the current study, we found that the exposure of Beas-2B cells to nickel compounds resulted in the activation of both nuclear factor of activated T cell (NFAT) and nuclear factor-κB (NF-κB). The expression of COX-2 induced upon nickel exposure was inhibited by either a NFAT pharmacological inhibitor or the knockdown of NFAT3 by specific siRNA. We further found that the activation of NFAT and NF-κB was dependent on each other. Since our previous studies have shown that NF-κB activation is critical for nickel-induced COX-2 expression in Beas-2B cells exposed to nickel compounds under same experimental condition, we anticipate that there might be a cross-talk between the activation of NFAT and NF-κB for the COX-2 induction due to nickel exposure in Beas-2B cells. Furthermore, we showed that the scavenging of reactive oxygen species (ROS) by introduction of mitochondrial catalase inhibited the activation of both NFAT and NF-κB, and the induction of COX-2 due to nickel exposure. Taken together, our results defining the evidence showing a key role of the cross-talk between NFAT and NF-κB pathways in regulating nickel-induced COX-2 expression, further provide insight into the understanding of the molecular mechanisms linking nickel exposure to its lung carcinogenic effects. PMID:21486220

  9. Foxd3 suppresses NFAT-mediated differentiation to maintain self-renewal of embryonic stem cells

    PubMed Central

    Zhu, Lili; Zhang, Shiyue; Jin, Ying

    2014-01-01

    Pluripotency-associated transcription factor Foxd3 is required for maintaining pluripotent cells. However, molecular mechanisms underlying its function are largely unknown. Here, we report that Foxd3 suppresses differentiation induced by calcineurin–NFAT signaling to maintain the ESC identity. Mechanistically, Foxd3 interacts with NFAT proteins and recruits co-repressor Tle4, a member of the Tle repressor family highly expressed in undifferentiated ESCs, to suppress NFATc3's transcriptional activities. Furthermore, global transcriptome analysis shows that Foxd3 and NFATc3 co-regulate a set of differentiation-associated genes in ESCs. Collectively, our study establishes a molecular and functional link between a pluripotency-associated factor and an important ESC differentiation-inducing pathway. Subject Categories Development & Differentiation; Stem Cells PMID:25378483

  10. Toward label-free Raman-activated cell sorting of cardiomyocytes derived from human embryonic stem cells

    NASA Astrophysics Data System (ADS)

    Pascut, Flavius C.; Goh, Huey T.; George, Vinoj; Denning, Chris; Notingher, Ioan

    2011-04-01

    Raman micro-spectroscopy (RMS) has been recently proposed for label-free phenotypic identification of human embryonic stem cells (hESC)-derived cardiomyocytes. However, the methods used for measuring the Raman spectra led to acquisition times of minutes per cell, which is prohibitive for rapid cell sorting applications. In this study we evaluated two measurement strategies that could reduce the measurement time by a factor of more than 100. We show that sampling individual cells with a laser beam focused to a line could eliminate the need of cell raster scanning and achieve high prediction accuracies (>95% specificity and >96% sensitivity) with acquisition times ~5 seconds per cell. However, the use of commercially-available higher power lasers could potentially lead to sorting speeds of ~10 cells per s. This would start to progress RMS to the field of cell sorting for applications such as enrichment and purification of hESC-derived cardiomyocytes.

  11. Interleukin-13-induced MUC5AC expression is regulated by a PI3K–NFAT3 pathway in mouse tracheal epithelial cells

    SciTech Connect

    Yan, Fugui; Li, Wen; Zhou, Hongbin; Wu, Yinfang; Ying, Songmin; Chen, Zhihua; Shen, Huahao

    2014-03-28

    Highlights: • IL-13 specifically induced NFAT3 activation in mouse tracheal epithelial cells. • CsA and LY294002 significantly blocked IL-13-induced MUC5AC production. • The PI3K–NFAT3 pathway is positively involved in IL-13-induced MUC5AC production. - Abstract: Interleukin-13 (IL-13) plays a critical role in asthma mucus overproduction, while the mechanisms underlying this process are not fully elucidated. Previous studies showed that nuclear factor of activated T cells (NFAT) is involved in the pathogenesis of asthma, but whether it can directly regulate IL-13-induced mucus (particularly MUC5AC) production is still not clear. Here we showed that IL-13 specifically induced NFAT3 activation through promoting its dephosphorylation in air–liquid interface (ALI) cultures of mouse tracheal epithelial cells (mTECs). Furthermore, both Cyclosporin A (CsA, a specific NFAT inhibitor) and LY294002 (a Phosphoinositide 3-kinase (PI3K) inhibitor) significantly blocked IL-13-induced MUC5AC mRNA and protein production through the inhibition of NFAT3 activity. We also confirmed that CsA could not influence the forkhead Box A2 (Foxa2) and mouse calcium dependent chloride channel 3 (mClca3) expression in IL-13-induced MUC5AC production, which both are known to be important in IL-13-stimulated mucus expression. Our study is the first to demonstrate that the PI3K–NFAT3 pathway is positively involved in IL-13-induced mucus production, and provided novel insights into the molecular mechanism of asthma mucus hypersecretion.

  12. In Vitro Inhibition of NFAT5-Mediated Induction of CCL2 in Hyperosmotic Conditions by Cyclosporine and Dexamethasone on Human HeLa-Modified Conjunctiva-Derived Cells

    PubMed Central

    Baudouin, Christophe; Gard, Carole; Brignole-Baudouin, Françoise

    2016-01-01

    Purpose To investigate the pro-inflammatory intracellular mechanisms induced by an in vitro model of dry eye disease (DED) on a Hela-modified conjunctiva-derived cells in hyperosmolarity (HO) stress conditions. This study focused on CCL2 induction and explored the implications of the nuclear factor of activated T-cells 5 (NFAT5) as well as mitogen-activated protein kinases (MAPK) and nuclear factor kappa B (NFĸB). This work was completed by an analysis of the effects of cyclosporine A (CsA), dexamethasone (Dex) and doxycycline (Dox) on HO-induced CCL2 and NFAT5 induction. Methods A human HeLa-modified conjunctiva-derived cell line was cultured in NaCl-hyperosmolar medium for various exposure times. Cellular viability, CCL2 secretion, NFAT5 and CCL2 gene expression, and intracytoplasmic NFAT5 were assessed using the Cell Titer Blue® assay, enzyme-linked immunosorbent assay (ELISA), RT-qPCR and immunostaining, respectively. In selected experiments, inhibitors of MAPKs or NFκB, therapeutic agents or NFAT5 siRNAs were added before the hyperosmolar stimulations. Results HO induced CCL2 secretion and expression as well as NFAT5 gene expression and translocation. Adding NFAT5-siRNA before hyperosmolar stimulation led to a complete inhibition of CCL2 induction and to a decrease in cellular viability. p38 MAPK (p38), c-Jun NH2-terminal kinase (JNK) and NFĸB inhibitors, CsA and Dex induced a partial inhibition of HO-induced CCL2, while Dox and extracellular signal-regulated kinase (ERK) inhibitor did not. Dex also induced a partial inhibition of HO-induced NFAT5 gene expression but not CsA or Dox. Conclusions These in vitro results suggest a potential role of CCL2 in DED and highlight the crucial role of NFAT5 in the pro-inflammatory effect of HO on HeLa-modified conjunctiva-derived cells, a rarely studied cellular type. This inflammatory pathway involving NFAT5 and CCL2 could offer a promising target for developing new therapies to treat DED, warranting further

  13. Directed Differentiation of Zebrafish Pluripotent Embryonic Cells to Functional Cardiomyocytes.

    PubMed

    Xiao, Yao; Gao, Maomao; Gao, Luna; Zhao, Yu; Hong, Qiang; Li, Zhigang; Yao, Jing; Cheng, Hanhua; Zhou, Rongjia

    2016-09-13

    A cardiomyocyte differentiation in vitro system from zebrafish embryos remains to be established. Here, we have determined pluripotency window of zebrafish embryos by analyzing their gene-expression patterns of pluripotency factors together with markers of three germ layers, and have found that zebrafish undergoes a very narrow period of pluripotency maintenance from zygotic genome activation to a brief moment after oblong stage. Based on the pluripotency and a combination of appropriate conditions, we established a rapid and efficient method for cardiomyocyte generation in vitro from primary embryonic cells. The induced cardiomyocytes differentiated into functional and specific cardiomyocyte subtypes. Notably, these in vitro generated cardiomyocytes exhibited typical contractile kinetics and electrophysiological features. The system provides a new paradigm of cardiomyocyte differentiation from primary embryonic cells in zebrafish. The technology provides a new platform for the study of heart development and regeneration, in addition to drug discovery, disease modeling, and assessment of cardiotoxic agents. PMID:27569061

  14. Glucocorticoid-induced TNF receptor expression by T cells is reciprocally regulated by NF-kappaB and NFAT.

    PubMed

    Zhan, Yifan; Gerondakis, Steve; Coghill, Elise; Bourges, Dorothee; Xu, Yuekang; Brady, Jamie L; Lew, Andrew M

    2008-10-15

    Although the transcription factor Foxp3 is implicated in regulating glucocorticoid-induced TNF receptor (GITR) expression in the T regulatory cell lineage, little is known about how GITR is transcriptionally regulated in conventional T cells. In this study, we provide evidence that TCR-mediated GITR expression depends on the ligand affinity and the maturity of conventional T cells. A genetic dissection of GITR transcriptional control revealed that of the three transcription factors downstream of the classical NF-kappaB pathway (RelA, cRel, and NF-kappaB1), RelA is a critical positive regulator of GITR expression, although cRel and NF-kappaB1 also play a positive regulatory role. Consistent with this finding, inhibiting NF-kappaB using Bay11-7082 reduces GITR up-regulation. In contrast, NFAT acts as a negative regulator of GITR expression. This was evidenced by our findings that agents suppressing NFAT activity (e.g., cyclosporin A and FK506) enhanced TCR-mediated GITR expression, whereas agents enhancing NFAT activity (e.g., lithium chloride) suppressed TCR-mediated GITR up-regulation. Critically, the induction of GITR was found to confer protection to conventional T cells from TCR-mediated apoptosis. We propose therefore that two major transcriptional factors activated downstream of the TCR, namely, NF-kappaB and NFAT, act reciprocally to balance TCR-mediated GITR expression in conventional T cells, an outcome that appears to influence cell survival.

  15. High-salt intake induces cardiomyocyte hypertrophy in rats in response to local angiotensin II type 1 receptor activation.

    PubMed

    Katayama, Isis A; Pereira, Rafael C; Dopona, Ellen P B; Shimizu, Maria H M; Furukawa, Luzia N S; Oliveira, Ivone B; Heimann, Joel C

    2014-10-01

    Many studies have shown that risk factors that are independent of blood pressure (BP) can contribute to the development of cardiac hypertrophy (CH). Among these factors, high-salt (HS) intake was prominent. Although some studies have attempted to elucidate the role of salt in the development of this disease, the mechanisms by which salt acts are not yet fully understood. Thus, the aim of this study was to better understand the mechanisms of CH and interstitial fibrosis (IF) caused by HS intake. Male Wistar rats were divided into 5 groups according to diet [normal salt (NS; 1.27% NaCl) or HS (8% NaCl)] and treatment [losartan (LOS) (HS+LOS group), hydralazine (HZ) (HS+HZ group), or N-acetylcysteine (NAC) (HS+NAC group)], which was given in the drinking water. Tail-cuff BP, transverse diameter of the cardiomyocyte, IF, angiotensin II type 1 receptor (AT1) gene and protein expression, serum aldosterone, cardiac angiotensin II, cardiac thiobarbituric acid-reactive substances, and binding of conformation-specific anti-AT1 and anti-angiotensin II type 2 receptor (AT2) antibodies in the 2 ventricles were measured. Based on the left ventricle transverse diameter data, the primary finding was the occurrence of significant BP-independent CH in the HS+HZ group (96% of the HS group) and a partial or total prevention of such hypertrophy via treatment with NAC or LOS (81% and 67% of the HS group, respectively). The significant total or partial prevention of IF using all 3 treatments (HS+HZ, 27%; HS+LOS, 27%; and HS+NAC, 58% of the HS group, respectively), and an increase in the AT1 gene and protein expression and activity in groups that developed CH, confirmed that CH occurred via the AT1 in this experimental model. Thus, this study unveiled some relevant previously unknown mechanisms of CH induced by chronic HS intake in Wistar rats. The link of oxidative stress with CH in our experimental model is very interesting and stimulates further evaluation for its full comprehension.

  16. NFATc2 (NFAT1) assists BCR-mediated anergy in anti-insulin B cells.

    PubMed

    Bonami, Rachel H; Wolfle, William T; Thomas, James W; Kendall, Peggy L

    2014-12-01

    NFAT transcription factors play critical roles in both the activation and repression of T and B lymphocyte responses. To understand the role of NFATc2 (NFAT1) in the maintenance of tolerance for anti-insulin B cells, functionally inactive NFATc2 (NFATc2(-/-)) was introduced into C57BL/6 mice that harbor anergic anti-insulin 125Tg B cells. The production and peripheral maturation of anti-insulin B cells into follicular and marginal zone subsets was not altered by the absence of functional NFATc2. Surface B cell receptor expression levels, important for tonic signaling and altered by anergy, were not altered in any spleen B cell subset. The levels of anti-insulin antibodies were not different in 125Tg/B6/NFATc2(-/-) mice and the anti-insulin response remained silenced following T cell dependent immunization. However, studies addressing in vitro proliferation reveal the anergic state of 125Tg B cells is relieved in 125Tg/B6/NFATc2(-/-) B cells in response to BCR stimulation. In contrast, anergy is not released in 125Tg/B6/NFATc2(-/-) B cells following stimulation with anti-CD40. The relief of anergy to BCR stimulation in 125Tg/B6/NFATc2(-/-) B cells is associated with increased transcription of both NFATc1 and NFATc3 while expression of these NFATs does not change in anti-IgM stimulated 125Tg/B6/NFATc2(+/+) B cells. The data suggest that NFATc2 plays a subtle and selective role in maintaining anergy for BCR stimulation by repressing the transcription of other NFAT family members. PMID:24507801

  17. DYRK1A Controls HIV-1 Replication at a Transcriptional Level in an NFAT Dependent Manner

    PubMed Central

    Booiman, Thijs; Loukachov, Vladimir V.; van Dort, Karel A.; van ’t Wout, Angélique B.; Kootstra, Neeltje A.

    2015-01-01

    Background Transcription of the HIV-1 provirus is regulated by both viral and host proteins and is very important in the context of viral latency. In latently infected cells, viral gene expression is inhibited as a result of the sequestration of host transcription factors and epigenetic modifications. Results In our present study we analyzed the effect of host factor dual specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A) on HIV-1 replication. We show that DYRK1A controls HIV-1 replication by regulating provirus transcription. Downregulation or inhibition of DYRK1A increased LTR-driven transcription and viral replication in cell lines and primary PBMC. Furthermore, inhibition of DYRK1A resulted in reactivation of latent HIV-1 provirus to a similar extent as two commonly used broad-spectrum HDAC inhibitors. We observed that DYRK1A regulates HIV-1 transcription via the Nuclear Factor of Activated T-cells (NFAT) by promoting its translocation from the nucleus to the cytoplasm. Therefore, inhibition of DYRK1A results in increased nuclear levels of NFAT and increased NFAT binding to the viral LTR and thus increasing viral transcription. Conclusions Our data indicate that host factor DYRK1A plays a role in the regulation of viral transcription and latency. Therefore, DYRK1A might be an attractive candidate for therapeutic strategies targeting the viral reservoir. PMID:26641855

  18. Tert-butylhydroquinone attenuates the ethanol-induced apoptosis of and activates the Nrf2 antioxidant defense pathway in H9c2 cardiomyocytes

    PubMed Central

    SHI, XIAOJING; LI, YANG; HU, JUN; YU, BO

    2016-01-01

    Tert-butylhydroquinone (tBHQ), an inducer of nuclear factor erythroid 2-related factor 2 (Nrf2), has been demonstrated to attenuate oxidative stress-induced injury and the apoptosis of human neural stem cells and other cell types. However, whether tBHQ is able to exert a protective effect against oxidative stress and the apoptosis of cardiomyocytes has not yet been determined. Thus, the objective of the present study was to determine whether tBHQ protects H9c2 cardiomyocytes against ethanol-induced apoptosis. For this purpose, four sets of experiments were performed under standard culture conditions as follows: i) untreated control cells; ii) cell treatment with 200 mM ethanol; iii) cell treatment with 5 µM tBHQ; and iv) cell pre-treatment with 5 µM tBHQ for 24 h, followed by medium change and co-culture with 200 mM ethanol containing 5 µM tBHQ for a further 24 h. The viability of the cardiomyocytes was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The levels of intracellular reactive oxygen species (ROS) and apoptosis were assessed by flow cytometry. Protein expression was measured by western blot analysis, and Nrf2 nuclear localization was observed by immunofluorescence. Exposure to ethanol led to a decrease in the protein expression of Nrf2 and its downstream antioxidant enzymes, accompanied by an increase in ROS generation and in the apoptosis of H9c2 cells. Pre-treatment with tBHQ significantly prevented the H9c2 cells from undergoing ethanol-induced apoptosis. tBHQ also increased the expression of B-cell lymphoma-2 (Bcl-2), whereas Bcl-2-associated X protein (Bax) expression was decreased. tBHQ promoted Nrf2 nuclear localization and increased the expression of Nrf2, superoxide dismutase (SOD), catalase (CAT) and heme oxygenase-1 (HO-1), and simultaneously inhibited the ethanol-induced overproduction of intracellular ROS. Therefore, tBHQ confers protection against the ethanol-induced apoptosis of and activates the

  19. Tert-butylhydroquinone attenuates the ethanol-induced apoptosis of and activates the Nrf2 antioxidant defense pathway in H9c2 cardiomyocytes.

    PubMed

    Shi, Xiaojing; Li, Yang; Hu, Jun; Yu, Bo

    2016-07-01

    induced apoptosis of and activates the Nrf2 antioxidant pathway in H9c2 cardiomyocytes. PMID:27220726

  20. MicroRNAs involved in the mitogen-activated protein kinase cascades pathway during glucose-induced cardiomyocyte hypertrophy.

    PubMed

    Shen, E; Diao, Xuehong; Wang, Xiaoxia; Chen, Ruizhen; Hu, Bing

    2011-08-01

    Cardiac hypertrophy is a key structural feature of diabetic cardiomyopathy in the late stage of diabetes. Recent studies show that microRNAs (miRNAs) are involved in the pathogenesis of cardiac hypertrophy in diabetic mice, but more novel miRNAs remain to be investigated. In this study, diabetic cardiomyopathy, characterized by hypertrophy, was induced in mice by streptozotocin injection. Using microarray analysis of myocardial tissue, we were able to identify changes in expression in 19 miRNA, of which 16 miRNAs were further validated by real-time PCR and a total of 3212 targets mRNA were predicted. Further analysis showed that 31 GO functions and 16 KEGG pathways were enriched in the diabetic heart. Of these, MAPK signaling pathway was prominent. In vivo and in vitro studies have confirmed that three major subgroups of MAPK including ERK1/2, JNK, and p38, are specifically upregulated in cardiomyocyte hypertrophy during hyperglycemia. To further explore the potential involvement of miRNAs in the regulation of glucose-induced cardiomyocyte hypertrophy, neonatal rat cardiomyocytes were exposed to high glucose and transfected with miR-373 mimic. Overexpression of miR-373 decreased the cell size, and also reduced the level of its target gene MEF2C, and miR-373 expression was regulated by p38. Our data highlight an important role of miRNAs in diabetic cardiomyopathy, and implicate the reliability of bioinformatics analysis in shedding light on the mechanisms underlying diabetic cardiomyopathy.

  1. The Correlation of PPARα Activity and Cardiomyocyte Metabolism and Structure in Idiopathic Dilated Cardiomyopathy during Heart Failure Progression

    PubMed Central

    Czarnowska, E.; Domal-Kwiatkowska, D.; Reichman-Warmusz, E.; Bierla, J. B.; Sowinska, A.; Ratajska, A.; Goral-Radziszewska, K.; Wojnicz, R.

    2016-01-01

    This study aimed to define relationship between PPARα expression and metabolic-structural characteristics during HF progression in hearts with DCM phenotype. Tissue endomyocardial biopsy samples divided into three groups according to LVEF ((I) 45–50%, n = 10; (II) 30–40%, n = 15; (III) <30%, n = 15; and control (donor hearts, >60%, n = 6)) were investigated. The PPARα mRNA expression in the failing hearts was low in Group (I), high in Group (II), and comparable to that of the control in Group (III). There were analogous changes in the expression of FAT/CD36 and CPT-1 mRNA in contrast to continuous overexpression of GLUT-4 mRNA and significant increase of PDK-4 mRNA in Group (II). In addition, significant structural changes of cardiomyocytes with glycogen accumulation were accompanied by increased expression of PPARα. For the entire study population with HF levels of FAT/CD36 mRNA showed a strong tendency of negative correlation with LVEF. In conclusion, PPARα elevated levels may be a direct cause of adverse remodeling, both metabolic and structural. Thus, there is limited time window for therapy modulating cardiac metabolism and protecting cardiomyocyte structure in failing heart. PMID:26981112

  2. NFAT5 induction by the pre–T-cell receptor serves as a selective survival signal in T-lymphocyte development

    PubMed Central

    Berga-Bolaños, Rosa; Alberdi, Maria; Buxadé, Maria; Aramburu, José; López-Rodríguez, Cristina

    2013-01-01

    The Rel-like transcription factors nuclear factor kappa B (NF-κB) and the calcineurin-dependent nuclear factor of activated T cells (NFATc) control specific points of thymocyte maturation. Thymocytes also express a distinct member of the Rel family, the calcineurin-independent, osmostress response regulator NFAT5. Here we show that IKKβ regulates the expression of NFAT5 in thymocytes, which in turn contributes to the survival of T-cell receptor αβ thymocytes and the transition from the β-selection checkpoint to the double-positive stage in an osmostress-independent manner. NFAT5-deficient thymocytes had normal expression and proximal signaling of the pre–T-cell receptor but exhibited a partial defect in β-chain allelic exclusion and increased apoptosis. Further analysis showed that NFAT5 regulated the expression of the prosurvival factors A1 and Bcl2 and attenuated the proapoptotic p53/Noxa axis. These findings position NFAT5 as a target of the IKKβ/NF-κB pathway in thymocytes and as a downstream effector of the prosurvival role of the pre–T-cell receptor. PMID:24043824

  3. NFAT5 induction by the pre-T-cell receptor serves as a selective survival signal in T-lymphocyte development.

    PubMed

    Berga-Bolaños, Rosa; Alberdi, Maria; Buxadé, Maria; Aramburu, José; López-Rodríguez, Cristina

    2013-10-01

    The Rel-like transcription factors nuclear factor kappa B (NF-κB) and the calcineurin-dependent nuclear factor of activated T cells (NFATc) control specific points of thymocyte maturation. Thymocytes also express a distinct member of the Rel family, the calcineurin-independent, osmostress response regulator NFAT5. Here we show that IKKβ regulates the expression of NFAT5 in thymocytes, which in turn contributes to the survival of T-cell receptor αβ thymocytes and the transition from the β-selection checkpoint to the double-positive stage in an osmostress-independent manner. NFAT5-deficient thymocytes had normal expression and proximal signaling of the pre-T-cell receptor but exhibited a partial defect in β-chain allelic exclusion and increased apoptosis. Further analysis showed that NFAT5 regulated the expression of the prosurvival factors A1 and Bcl2 and attenuated the proapoptotic p53/Noxa axis. These findings position NFAT5 as a target of the IKKβ/NF-κB pathway in thymocytes and as a downstream effector of the prosurvival role of the pre-T-cell receptor.

  4. Mammalian heart renewal by pre-existing cardiomyocytes.

    PubMed

    Senyo, Samuel E; Steinhauser, Matthew L; Pizzimenti, Christie L; Yang, Vicky K; Cai, Lei; Wang, Mei; Wu, Ting-Di; Guerquin-Kern, Jean-Luc; Lechene, Claude P; Lee, Richard T

    2013-01-17

    Although recent studies have revealed that heart cells are generated in adult mammals, the frequency of generation and the source of new heart cells are not yet known. Some studies suggest a high rate of stem cell activity with differentiation of progenitors to cardiomyocytes. Other studies suggest that new cardiomyocytes are born at a very low rate, and that they may be derived from the division of pre-existing cardiomyocytes. Here we show, by combining two different pulse-chase approaches--genetic fate-mapping with stable isotope labelling, and multi-isotope imaging mass spectrometry--that the genesis of cardiomyocytes occurs at a low rate by the division of pre-existing cardiomyocytes during normal ageing, a process that increases adjacent to areas of myocardial injury. We found that cell cycle activity during normal ageing and after injury led to polyploidy and multinucleation, but also to new diploid, mononucleate cardiomyocytes. These data reveal pre-existing cardiomyocytes as the dominant source of cardiomyocyte replacement in normal mammalian myocardial homeostasis as well as after myocardial injury.

  5. Chronic intermittent hypoxia induces cardiac hypertrophy by impairing autophagy through the adenosine 5'-monophosphate-activated protein kinase pathway.

    PubMed

    Xie, Sheng; Deng, Yan; Pan, Yue-Ying; Ren, Jie; Jin, Meng; Wang, Yu; Wang, Zhi-Hua; Zhu, Die; Guo, Xue-Ling; Yuan, Xiao; Shang, Jin; Liu, Hui-Guo

    2016-09-15

    Autophagy is tightly regulated to maintain cardiac homeostasis. Impaired autophagy is closely associated with pathological cardiac hypertrophy. However, the relationship between autophagy and cardiac hypertrophy induced by chronic intermittent hypoxia (CIH) is not known. In the present study, we measured autophagy-related genes and autophagosomes during 10 weeks of CIH in rats, and 6 days in H9C2 cardiomyocytes, and showed that autophagy was impaired. This conclusion was confirmed by the autophagy flux assay. We detected significant hypertrophic changes in myocardium with impaired autophagy. Rapamycin, an autophagy enhancer, attenuated the cardiac hypertrophy induced by CIH. Moreover, silencing autophagy-related gene 5 (ATG5) exerted the opposite effect. The role of adenosine monophosphate-activated protein kinase (AMPK) in regulating autophagy under CIH was confirmed using AICAR to upregulate this enzyme and restore autophagy flux. Restoring autophagy by AICAR or rapamycin significantly reversed the hypertrophic changes in cardiomyocytes. To investigate the mechanism of autophagy impairment, we compared phospho (p)-AMPK, p-Akt, cathepsin D, and NFAT3 levels, along with calcineurin activity, between sham and CIH groups. CIH activated calcineurin, and inhibited AMPK and AMPK-mediated autophagy in an Akt- and NFAT3-independent manner. Collectively, these data demonstrated that impaired autophagy induced by CIH through the AMPK pathway contributed to cardiac hypertrophy. PMID:27412517

  6. Chronic intermittent hypoxia induces cardiac hypertrophy by impairing autophagy through the adenosine 5'-monophosphate-activated protein kinase pathway.

    PubMed

    Xie, Sheng; Deng, Yan; Pan, Yue-Ying; Ren, Jie; Jin, Meng; Wang, Yu; Wang, Zhi-Hua; Zhu, Die; Guo, Xue-Ling; Yuan, Xiao; Shang, Jin; Liu, Hui-Guo

    2016-09-15

    Autophagy is tightly regulated to maintain cardiac homeostasis. Impaired autophagy is closely associated with pathological cardiac hypertrophy. However, the relationship between autophagy and cardiac hypertrophy induced by chronic intermittent hypoxia (CIH) is not known. In the present study, we measured autophagy-related genes and autophagosomes during 10 weeks of CIH in rats, and 6 days in H9C2 cardiomyocytes, and showed that autophagy was impaired. This conclusion was confirmed by the autophagy flux assay. We detected significant hypertrophic changes in myocardium with impaired autophagy. Rapamycin, an autophagy enhancer, attenuated the cardiac hypertrophy induced by CIH. Moreover, silencing autophagy-related gene 5 (ATG5) exerted the opposite effect. The role of adenosine monophosphate-activated protein kinase (AMPK) in regulating autophagy under CIH was confirmed using AICAR to upregulate this enzyme and restore autophagy flux. Restoring autophagy by AICAR or rapamycin significantly reversed the hypertrophic changes in cardiomyocytes. To investigate the mechanism of autophagy impairment, we compared phospho (p)-AMPK, p-Akt, cathepsin D, and NFAT3 levels, along with calcineurin activity, between sham and CIH groups. CIH activated calcineurin, and inhibited AMPK and AMPK-mediated autophagy in an Akt- and NFAT3-independent manner. Collectively, these data demonstrated that impaired autophagy induced by CIH through the AMPK pathway contributed to cardiac hypertrophy.

  7. Myoferlin regulation by NFAT in muscle injury, regeneration and repair

    PubMed Central

    Demonbreun, Alexis R.; Lapidos, Karen A.; Heretis, Konstantina; Levin, Samantha; Dale, Rodney; Pytel, Peter; Svensson, Eric C.; McNally, Elizabeth M.

    2010-01-01

    Ferlin proteins mediate membrane-fusion events in response to Ca2+. Myoferlin, a member of the ferlin family, is required for normal muscle development, during which it mediates myoblast fusion. We isolated both damaged and intact myofibers from a mouse model of muscular dystrophy using laser-capture microdissection and found that the levels of myoferlin mRNA and protein were increased in damaged myofibers. To better define the components of the muscle-injury response, we identified a discreet 1543-bp fragment of the myoferlin promoter, containing multiple NFAT-binding sites, and found that this was sufficient to drive high-level myoferlin expression in cells and in vivo. This promoter recapitulated normal myoferlin expression in that it was downregulated in healthy myofibers and was upregulated in response to myofiber damage. Transgenic mice expressing GFP under the control of the myoferlin promoter were generated and GFP expression in this model was used to track muscle damage in vivo after muscle injury and in muscle disease. Myoferlin modulates the response to muscle injury through its activity in both myoblasts and mature myofibers. PMID:20571050

  8. Myoferlin regulation by NFAT in muscle injury, regeneration and repair.

    PubMed

    Demonbreun, Alexis R; Lapidos, Karen A; Heretis, Konstantina; Levin, Samantha; Dale, Rodney; Pytel, Peter; Svensson, Eric C; McNally, Elizabeth M

    2010-07-15

    Ferlin proteins mediate membrane-fusion events in response to Ca(2+). Myoferlin, a member of the ferlin family, is required for normal muscle development, during which it mediates myoblast fusion. We isolated both damaged and intact myofibers from a mouse model of muscular dystrophy using laser-capture microdissection and found that the levels of myoferlin mRNA and protein were increased in damaged myofibers. To better define the components of the muscle-injury response, we identified a discreet 1543-bp fragment of the myoferlin promoter, containing multiple NFAT-binding sites, and found that this was sufficient to drive high-level myoferlin expression in cells and in vivo. This promoter recapitulated normal myoferlin expression in that it was downregulated in healthy myofibers and was upregulated in response to myofiber damage. Transgenic mice expressing GFP under the control of the myoferlin promoter were generated and GFP expression in this model was used to track muscle damage in vivo after muscle injury and in muscle disease. Myoferlin modulates the response to muscle injury through its activity in both myoblasts and mature myofibers.

  9. Dynamic DNA methylation orchestrates cardiomyocyte development, maturation and disease.

    PubMed

    Gilsbach, Ralf; Preissl, Sebastian; Grüning, Björn A; Schnick, Tilman; Burger, Lukas; Benes, Vladimir; Würch, Andreas; Bönisch, Ulrike; Günther, Stefan; Backofen, Rolf; Fleischmann, Bernd K; Schübeler, Dirk; Hein, Lutz

    2014-01-01

    The heart is a highly specialized organ with essential function for the organism throughout life. The significance of DNA methylation in shaping the phenotype of the heart remains only partially known. Here we generate and analyse DNA methylomes from highly purified cardiomyocytes of neonatal, adult healthy and adult failing hearts. We identify large genomic regions that are differentially methylated during cardiomyocyte development and maturation. Demethylation of cardiomyocyte gene bodies correlates strongly with increased gene expression. Silencing of demethylated genes is characterized by the polycomb mark H3K27me3 or by DNA methylation. De novo methylation by DNA methyltransferases 3A/B causes repression of fetal cardiac genes, including essential components of the cardiac sarcomere. Failing cardiomyocytes partially resemble neonatal methylation patterns. This study establishes DNA methylation as a highly dynamic process during postnatal growth of cardiomyocytes and their adaptation to pathological stress in a process tightly linked to gene regulation and activity. PMID:25335909

  10. Dynamic DNA methylation orchestrates cardiomyocyte development, maturation and disease.

    PubMed

    Gilsbach, Ralf; Preissl, Sebastian; Grüning, Björn A; Schnick, Tilman; Burger, Lukas; Benes, Vladimir; Würch, Andreas; Bönisch, Ulrike; Günther, Stefan; Backofen, Rolf; Fleischmann, Bernd K; Schübeler, Dirk; Hein, Lutz

    2014-10-22

    The heart is a highly specialized organ with essential function for the organism throughout life. The significance of DNA methylation in shaping the phenotype of the heart remains only partially known. Here we generate and analyse DNA methylomes from highly purified cardiomyocytes of neonatal, adult healthy and adult failing hearts. We identify large genomic regions that are differentially methylated during cardiomyocyte development and maturation. Demethylation of cardiomyocyte gene bodies correlates strongly with increased gene expression. Silencing of demethylated genes is characterized by the polycomb mark H3K27me3 or by DNA methylation. De novo methylation by DNA methyltransferases 3A/B causes repression of fetal cardiac genes, including essential components of the cardiac sarcomere. Failing cardiomyocytes partially resemble neonatal methylation patterns. This study establishes DNA methylation as a highly dynamic process during postnatal growth of cardiomyocytes and their adaptation to pathological stress in a process tightly linked to gene regulation and activity.

  11. Dynamic DNA methylation orchestrates cardiomyocyte development, maturation and disease

    PubMed Central

    Gilsbach, Ralf; Preissl, Sebastian; Grüning, Björn A.; Schnick, Tilman; Burger, Lukas; Benes, Vladimir; Würch, Andreas; Bönisch, Ulrike; Günther, Stefan; Backofen, Rolf; Fleischmann, Bernd K.; Schübeler, Dirk; Hein, Lutz

    2014-01-01

    The heart is a highly specialized organ with essential function for the organism throughout life. The significance of DNA methylation in shaping the phenotype of the heart remains only partially known. Here we generate and analyse DNA methylomes from highly purified cardiomyocytes of neonatal, adult healthy and adult failing hearts. We identify large genomic regions that are differentially methylated during cardiomyocyte development and maturation. Demethylation of cardiomyocyte gene bodies correlates strongly with increased gene expression. Silencing of demethylated genes is characterized by the polycomb mark H3K27me3 or by DNA methylation. De novo methylation by DNA methyltransferases 3A/B causes repression of fetal cardiac genes, including essential components of the cardiac sarcomere. Failing cardiomyocytes partially resemble neonatal methylation patterns. This study establishes DNA methylation as a highly dynamic process during postnatal growth of cardiomyocytes and their adaptation to pathological stress in a process tightly linked to gene regulation and activity. PMID:25335909

  12. Cooperative involvement of NFAT and SnoN mediates transforming growth factor-β (TGF-β) induced EMT in metastatic breast cancer (MDA-MB 231) cells.

    PubMed

    Sengupta, Suman; Jana, Samir; Biswas, Subir; Mandal, Palash Kumar; Bhattacharyya, Arindam

    2013-12-01

    Epithelial to mesenchymal transition (EMT) is a secondary phenomenon concomitantly associated with the tumor progression. The regulatory signals and mechanistic details of EMT are not fully elucidated. Here, we shared a TGF-β mediated mechanism of EMT in breast cancer (MDA-MB 231) cells. Initial exposure of TGF-β for 48 h, enhanced the rate of cell proliferation and associated with EMT of MDA-MB 231 cells. The EMT was characterized by observing the increased N-cadherin, fibronectin, Snail expression and associated with the morphological change with a reduced E-cadherin expression. NFAT, a transcription factor, alters tumor suppressive function of TGF-β towards tumor progression. Up regulation of NFAT, coupled with a foremost translocation of one oncogenic protein SnoN from cytoplasm to nucleus was noticed during this TGF-β mediated EMT. Silencing of NFAT also showed the inhibition of TGF-β mediated EMT characterized by down regulation of N-cadherin and associated with reduced expression of SnoN. In addition, it was also observed that NFAT sequestering the Smad3 prevents the proteasome mediated degradation of SnoN and this SnoN has a role on the regulation of MMP-2, MMP-9 activity. Increased Smad3-SnoN interaction and proteasome mediated degradation of SnoN were detected after silencing of NFAT with a reduced MMP-2, MMP-9 activity. All of these observations provide a fresh mechanism in which by a twofold involvement of NFAT and SnoN plays a crucial role in TGF-β mediated EMT by recruiting the effector molecules N-cadherin and MMP-2, MMP-9.

  13. Transcriptomic and genetic studies identify NFAT5 as a candidate gene for cocaine dependence

    PubMed Central

    Fernàndez-Castillo, N; Cabana-Domínguez, J; Soriano, J; Sànchez-Mora, C; Roncero, C; Grau-López, L; Ros-Cucurull, E; Daigre, C; van Donkelaar, M M J; Franke, B; Casas, M; Ribasés, M; Cormand, B

    2015-01-01

    Cocaine reward and reinforcing effects are mediated mainly by dopaminergic neurotransmission. In this study, we aimed at evaluating gene expression changes induced by acute cocaine exposure on SH-SY5Y-differentiated cells, which have been widely used as a dopaminergic neuronal model. Expression changes and a concomitant increase in neuronal activity were observed after a 5 μM cocaine exposure, whereas no changes in gene expression or in neuronal activity took place at 1 μM cocaine. Changes in gene expression were identified in a total of 756 genes, mainly related to regulation of transcription and gene expression, cell cycle, adhesion and cell projection, as well as mitogen-activeated protein kinase (MAPK), CREB, neurotrophin and neuregulin signaling pathways. Some genes displaying altered expression were subsequently targeted with predicted functional single-nucleotide polymorphisms (SNPs) in a case–control association study in a sample of 806 cocaine-dependent patients and 817 controls. This study highlighted associations between cocaine dependence and five SNPs predicted to alter microRNA binding at the 3′-untranslated region of the NFAT5 gene. The association of SNP rs1437134 with cocaine dependence survived the Bonferroni correction for multiple testing. A functional effect was confirmed for this variant by a luciferase reporter assay, with lower expression observed for the rs1437134G allele, which was more pronounced in the presence of hsa-miR-509. However, brain volumes in regions of relevance to addiction, as assessed with magnetic resonance imaging, did not correlate with NFAT5 variation. These results suggest that the NFAT5 gene, which is upregulated a few hours after cocaine exposure, may be involved in the genetic predisposition to cocaine dependence. PMID:26506053

  14. Transcriptomic and genetic studies identify NFAT5 as a candidate gene for cocaine dependence.

    PubMed

    Fernàndez-Castillo, N; Cabana-Domínguez, J; Soriano, J; Sànchez-Mora, C; Roncero, C; Grau-López, L; Ros-Cucurull, E; Daigre, C; van Donkelaar, M M J; Franke, B; Casas, M; Ribasés, M; Cormand, B

    2015-01-01

    Cocaine reward and reinforcing effects are mediated mainly by dopaminergic neurotransmission. In this study, we aimed at evaluating gene expression changes induced by acute cocaine exposure on SH-SY5Y-differentiated cells, which have been widely used as a dopaminergic neuronal model. Expression changes and a concomitant increase in neuronal activity were observed after a 5 μM cocaine exposure, whereas no changes in gene expression or in neuronal activity took place at 1 μM cocaine. Changes in gene expression were identified in a total of 756 genes, mainly related to regulation of transcription and gene expression, cell cycle, adhesion and cell projection, as well as mitogen-activeated protein kinase (MAPK), CREB, neurotrophin and neuregulin signaling pathways. Some genes displaying altered expression were subsequently targeted with predicted functional single-nucleotide polymorphisms (SNPs) in a case-control association study in a sample of 806 cocaine-dependent patients and 817 controls. This study highlighted associations between cocaine dependence and five SNPs predicted to alter microRNA binding at the 3'-untranslated region of the NFAT5 gene. The association of SNP rs1437134 with cocaine dependence survived the Bonferroni correction for multiple testing. A functional effect was confirmed for this variant by a luciferase reporter assay, with lower expression observed for the rs1437134G allele, which was more pronounced in the presence of hsa-miR-509. However, brain volumes in regions of relevance to addiction, as assessed with magnetic resonance imaging, did not correlate with NFAT5 variation. These results suggest that the NFAT5 gene, which is upregulated a few hours after cocaine exposure, may be involved in the genetic predisposition to cocaine dependence. PMID:26506053

  15. TAK1 Regulates Myocardial Response to Pathological Stress via NFAT, NFκB, and Bnip3 Pathways.

    PubMed

    Li, Lei; Chen, Yi; Li, Jing; Yin, Haifeng; Guo, Xiaoyun; Doan, Jessica; Molkentin, Jeffery D; Liu, Qinghang

    2015-11-13

    TAK1 (TGFβ-activated kinase-1) signaling is essential in regulating a number of important biological functions, including innate immunity, inflammatory response, cell growth and differentiation, and myocardial homeostasis. The precise role of TAK1 in the adult heart under pathological conditions remains largely unknown. Importantly, we observed that TAK1 is upregulated during compensatory hypertrophy but downregulated in end-stage heart failure. Here we generated transgenic mice with inducible expression of an active TAK1 mutant (TAK1ΔN) in the adult heart. TAK1ΔN transgenic mice developed greater cardiac hypertrophy compared with control mice after transverse aortic constriction (TAC), which was largely blocked by ablation of calcineurin Aβ. Expression of TAK1ΔN also promoted NFAT (nuclear factor of activated T-cells) transcriptional activity in luciferase reporter mice at baseline, which was further enhanced after TAC. Our results revealed that activation of TAK1 promoted adaptive cardiac hypertrophy through a cross-talk between calcineurin-NFAT and IKK-NFκB pathways. More significantly, adult-onset inducible expression of TAK1ΔN protected the myocardium from adverse remodeling and heart failure after myocardial infarction or long-term pressure overload, by preventing cardiac cell death and fibrosis. Mechanistically, TAK1 exerts its cardioprotective effect through activation of NFAT/NFκB, downregulation of Bnip3, and inhibition of cardiac cell death.

  16. The neuropeptide catestatin promotes vascular smooth muscle cell proliferation through the Ca{sup 2+}-calcineurin-NFAT signaling pathway

    SciTech Connect

    Guo, Xiaoxia; Zhou, Chunyan; Sun, Ningling

    2011-04-22

    Highlights: {yields} Catestatin stimulates proliferation of vascular smooth muscle cells in a dose-dependent manner. {yields} Catestatin provokes sustained increase in intracellular Ca{sup 2+}. {yields} Catestatin produces increased activation of calcineurin and promotes NFATc1 translocation into the nucleus. -- Abstract: The Chromogranin A-derived neuropeptide catestatin is an endogenous nicotinic cholinergic antagonist that acts as a pleiotropic hormone. Since catestatin shares several functions with other members derived from the chromogranin/secretogranin protein family and other neuropeptides which exert proliferative effects on vascular smooth muscle cells (VSMCs), we therefore hypothesized that catestatin would regulate VSMC proliferation. The present study demonstrates that catestatin caused a dose-dependent induction of proliferation in rat aortic smooth muscle cells and furthermore evoked a sustained increase in intracellular calcium. This subsequently leaded to enhanced activation of the Ca{sup 2+}/calmodulin-dependent phosphatase, calcineurin and resulted in an activation of the Ca{sup 2+}-dependent transcription factor, nuclear factor of activated T cells (NFAT), initiating transcription of proliferative genes. In addition, cyclosporin A (CsA), a potent inhibitor of calcineurin, abrogated catestatin-mediated effect on VSMCs, indicating that the calcineurin-NFAT signaling is strongly required for catestatin-induced growth of VSMCs. The present study establishes catestatin as a novel proliferative cytokine on vascular smooth muscle cells and this effect is mediated by the Ca{sup 2+}-calcineurin-NFAT signaling pathway.

  17. Assessment of costimulation and coinhibition in a triple parameter T cell reporter line: Simultaneous measurement of NF-κB, NFAT and AP-1.

    PubMed

    Jutz, Sabrina; Leitner, Judith; Schmetterer, Klaus; Doel-Perez, Iago; Majdic, Otto; Grabmeier-Pfistershammer, Katharina; Paster, Wolfgang; Huppa, Johannes B; Steinberger, Peter

    2016-03-01

    Engagement of the T cell receptor complex reprograms T cells for proliferation, cytokine production and differentiation towards effector cells. This process depends on activating costimulatory signals and is counteracted by coinhibitory molecules. Three transcription factors, namely NF-κB, NFAT and AP-1, have a major role in inducing the transcriptional program that is required for T cell activation and differentiation. Here we describe the generation of a triple parameter reporter based on the human Jurkat T cell line, where response elements for NF-κB, NFAT and AP-1 drive the expression of the fluorescent proteins CFP, eGFP and mCherry, respectively. The emission spectra of these proteins allow simultaneous assessment of NF-κB, NFAT and AP-1 activity in response to stimulation. Ligation of the TCR complex induced moderate reporter activity, which was strongly enhanced upon coengagement of the costimulatory receptors CD2 or CD28. Moreover, we have generated and tested triple parameter reporter cells that harbor costimulatory and inhibitory receptors not endogenously expressed in the Jurkat cells. In these experiments we could show that engagement of the costimulatory molecule 4-1BB enhances NF-κB and AP-1 activity, whereas coinhibition via PD-1 or BTLA strongly reduced the activation of NF-κB and NFAT. Engagement of BTLA significantly inhibited AP-1, whereas PD-1 had little effect on the activation of this transcription factor. Our triple parameter reporter T cell line is an excellent tool to assess the effect of costimulatory and coinhibitory receptors on NF-κB, NFAT and AP-1 activity and has a wide range of applications beyond the evaluation of costimulatory pathways.

  18. Regulation of ITAM adaptor molecules and their receptors by inhibition of calcineurin-NFAT signalling during late stage osteoclast differentiation

    SciTech Connect

    Zawawi, M.S.F.; Dharmapatni, A.A.S.S.K.; Cantley, M.D.; McHugh, K.P.; Haynes, D.R.; Crotti, T.N.

    2012-10-19

    Highlights: Black-Right-Pointing-Pointer Calcineurin/NFAT inhibitors FK506 and VIVIT treated human PBMC derived osteoclasts in vitro. Black-Right-Pointing-Pointer Differential regulation of ITAM receptors and adaptor molecules by calcineurin/NFAT inhibitors. Black-Right-Pointing-Pointer FK506 and VIVIT suppress ITAM factors during late phase osteoclast differentiation. -- Abstract: Osteoclasts are specialised bone resorptive cells responsible for both physiological and pathological bone loss. Osteoclast differentiation and activity is dependent upon receptor activator NF-kappa-B ligand (RANKL) interacting with its receptor RANK to induce the transcription factor, nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 1 (NFATc1). The immunoreceptor tyrosine-based activation motif (ITAM)-dependent pathway has been identified as a co-stimulatory pathway in osteoclasts. Osteoclast-associated receptor (OSCAR) and triggering receptor expressed in myeloid cells (TREM2) are essential receptors that pair with adaptor molecules Fc receptor common gamma chain (FcR{gamma}) and DNAX-activating protein 12 kDa (DAP12) respectively to induce calcium signalling. Treatment with calcineurin-NFAT inhibitors, Tacrolimus (FK506) and the 11R-VIVIT (VIVIT) peptide, reduces NFATc1 expression consistent with a reduction in osteoclast differentiation and activity. This study aimed to investigate the effects of inhibiting calcineurin-NFAT signalling on the expression of ITAM factors and late stage osteoclast genes including cathepsin K (CathK), Beta 3 integrin ({beta}3) and Annexin VIII (AnnVIII). Human peripheral blood mononuclear cells (PBMCs) were differentiated with RANKL and macrophage-colony stimulating factor (M-CSF) over 10 days in the presence or absence of FK506 or VIVIT. Osteoclast formation (as assessed by tartrate resistant acid phosphatase (TRAP)) and activity (assessed by dentine pit resorption) were significantly reduced with treatment. Quantitative real

  19. KMUP-1 Attenuates Endothelin-1-Induced Cardiomyocyte Hypertrophy through Activation of Heme Oxygenase-1 and Suppression of the Akt/GSK-3β, Calcineurin/NFATc4 and RhoA/ROCK Pathways.

    PubMed

    Liou, Shu-Fen; Hsu, Jong-Hau; Chen, You-Ting; Chen, Ing-Jun; Yeh, Jwu-Lai

    2015-01-01

    The signaling cascades of the mitogen activated protein kinase (MAPK) family, calcineurin/NFATc4, and PI3K/Akt/GSK3, are believed to participate in endothelin-1 (ET-1)-induced cardiac hypertrophy. The aim of this study was to investigate whether KMUP-1, a synthetic xanthine-based derivative, prevents cardiomyocyte hypertrophy induced by ET-1 and to elucidate the underlying mechanisms. We found that in H9c2 cardiomyocytes, stimulation with ET-1 (100 nM) for 4 days induced cell hypertrophy and enhanced expressions of hypertrophic markers, including atrial natriuretic peptide and brain natriuretic peptide, which were all inhibited by KMUP-1 in a dose-dependent manner. In addition, KMUP-1 prevented ET-1-induced intracellular reactive oxygen species generation determined by the DCFH-DA assay in cardiomyocytes. KMUP-1 also attenuated phosphorylation of ERK1/2 and Akt/GSK-3β, and activation of calcineurin/NFATc4 and RhoA/ROCK pathways induced by ET-1. Furthermore, we found that the expression of heme oxygenase-1 (HO-1), a stress-response enzyme implicated in cardio-protection, was up-regulated by KMUP-1. Finally, KMUP-1 attenuated ET-1-stimulated activator protein-1 DNA binding activity. In conclusion, KMUP-1 attenuates cardiomyocyte hypertrophy induced by ET-1 through inhibiting ERK1/2, calcineurin/NFATc4 and RhoA/ROCK pathways, with associated cardioprotective effects via HO-1 activation. Therefore, KMUP-1 may have a role in pharmacological therapy of cardiac hypertrophy. PMID:26056815

  20. The Calcineurin-NFAT-Angiopoietin 2 signaling axis in lung endothelium is critical for the establishment of lung metastases

    PubMed Central

    Minami, Takashi; Jiang, Shuying; Schadler, Keri; Suehiro, Jun-ichi; Osawa, Tsuyoshi; Oike, Yuichi; Miura, Mai; Naito, Makoto; Kodama, Tatsuhiko; Ryeom, Sandra

    2013-01-01

    SUMMARY The pre-metastatic niche is a pre-determined site of metastases, awaiting the influx of tumor cells. However, regulation of the angiogenic switch at these sites has not been examined. Here we demonstrate that the calcineurin-NFAT pathway is activated specifically in lung endothelium prior to the detection of tumor cells that preferentially metastasize to the lung. Upregulation of the calcineurin pathway via deletion of its endogenous inhibitor Dscr-1 leads to a significant increase in lung metastasis due to increased expression of a newly identified NFAT target, Angiopoietin (Ang)-2. Increased VEGF levels specifically in the lung and not other organ microenvironments triggers a threshold of calcineurin-NFAT signaling that transactivates Ang2 in lung endothelium. Further, we demonstrate that overexpression of DSCR-1 or the Ang-2 receptor, soluble Tie2, prevents activation of the lung endothelium inhibiting lung metastases in our mouse models. Our studies provide insights into mechanisms underlying angiogenesis in the pre-metastatic niche and offers new targets for lung metastases. PMID:23954784

  1. Investigating mitochondrial metabolism in contracting HL-1 cardiomyocytes following hypoxia and pharmacological HIF activation identifies HIF-dependent and independent mechanisms of regulation.

    PubMed

    Ambrose, Lucy J A; Abd-Jamil, Amira H; Gomes, Renata S M; Carter, Emma E; Carr, Carolyn A; Clarke, Kieran; Heather, Lisa C

    2014-11-01

    Hypoxia is a consequence of cardiac disease and downregulates mitochondrial metabolism, yet the molecular mechanisms through which this occurs in the heart are incompletely characterized. Therefore, we aimed to use a contracting HL-1 cardiomyocyte model to investigate the effects of hypoxia on mitochondrial metabolism. Cells were exposed to hypoxia (2% O2) for 6, 12, 24, and 48 hours to characterize the metabolic response. Cells were subsequently treated with the hypoxia inducible factor (HIF)-activating compound, dimethyloxalylglycine (DMOG), to determine whether hypoxia-induced mitochondrial changes were HIF dependent or independent, and to assess the suitability of this cultured cardiac cell line for cardiovascular pharmacological studies. Hypoxic cells had increased glycolysis after 24 hours, with glucose transporter 1 and lactate levels increased 5-fold and 15-fold, respectively. After 24 hours of hypoxia, mitochondrial networks were more fragmented but there was no change in citrate synthase activity, indicating that mitochondrial content was unchanged. Cellular oxygen consumption was 30% lower, accompanied by decreases in the enzymatic activities of electron transport chain (ETC) complexes I and IV, and aconitase by 81%, 96%, and 72%, relative to controls. Pharmacological HIF activation with DMOG decreased cellular oxygen consumption by 43%, coincident with decreases in the activities of aconitase and complex I by 26% and 30%, indicating that these adaptations were HIF mediated. In contrast, the hypoxia-mediated decrease in complex IV activity was not replicated by DMOG treatment, suggesting HIF-independent regulation of this complex. In conclusion, 24 hours of hypoxia increased anaerobic glycolysis and decreased mitochondrial respiration, which was associated with changes in ETC and tricarboxylic acid cycle enzyme activities in contracting HL-1 cells. Pharmacological HIF activation in this cardiac cell line allowed both HIF-dependent and independent

  2. Evaluation of calcineurin/NFAT inhibitor selectivity in primary human Th cells using bar-coding and phospho-flow cytometry.

    PubMed

    Frischbutter, Stefan; Schultheis, Katherine; Pätzel, Michael; Radbruch, Andreas; Baumgrass, Ria

    2012-11-01

    Small molecular inhibitors are excellent tools for manipulating cell reactions. They are widely used in scientific research to study molecular mechanisms of cells under physiological and pathophysiological conditions as well as in clinical applications to treat patients. However, their selectivity is often not well known. Moreover, it can vary according to cell types and the analysis methods used. Therefore, it is usually not possible to make comparisons between the data presented in the literature. Here we analyzed the selectivity of five chosen inhibitors of calcineurin/NFAT activation under the same conditions. Using a combination of fluorescent cell barcoding and phospho-specific flow cytometry we studied the inhibition of activation of NF-κBp65 and MAPK pathways in stimulated primary human Th cells. This semi-high throughput approach enabled us to demonstrate that (i) CsA and NCI3 are around 5 to 10- and 20-fold less potent, respectively, at inhibiting phosphorylation of NF-κBp65 and p38 than activation of NFAT, (ii) AM404 is at least 15-fold selective for NFAT but already toxic at concentrations above 40 μM, (iii) INCA6 is not selective at all, and (iv) BTP1 is at least 100-fold selective for inhibition of NFAT activation relative to NF-κBp65, p38 and ERK1/2 phosphorylation. Altogether, our results not only show the applicability of a semi-high throughput inhibitor test system but also that BTP1 is the most selective inhibitor of calcineurin/NFAT activation among the studied inhibitors under the used conditions.

  3. Calcium-sensing receptor induces rat neonatal ventricular cardiomyocyte apoptosis

    SciTech Connect

    Sun Yihua; Liu Meina; Li Hong; Shi Sa; Zhao Yajun; Wang Rui; Xu Changqing . E-mail: syh200415@yahoo.com.cn

    2006-12-01

    The calcium-sensing receptor (CaSR) exists in many tissues, and its expression has been identified in rat cardiac tissue. However, Physiological importance and pathophysiological involvement of CaSR in homeostatic regulation of cardiac function are unclear. To investigate the relation of CaSR and apoptosis in cardiomyocytes, we examined the role of the CaSR activator gadolinium chloride (GdCl{sub 3}) in rat neonatal ventricular cardiomyocytes. Expression of the CaSR protein was observed by Western blot. The apoptotic ratio of rat neonatal ventricular cardiomyocytes was measured with flow cytometry and immunofluorescence techniques. A laser scan confocal microscope was used to detect the intracellular concentration of calcium ([Ca{sup 2+}]{sub i}) in rat neonatal ventricular cardiomyocytes using the acetoxymethyl ester of fluo-3 (fluo-3/(AM)) as a fluorescent dye. The results showed that GdCl{sub 3} increased the phosphorylation of extracellular signal-regulated protein kinase (ERK), c-Jun NH{sub 2}-terminal protein kinases (JNK), and p38. GdCl{sub 3} also activated caspase 9 and increased apoptosis in myocyte by increasing [Ca{sup 2+}]{sub i}. In conclusion, these results suggest that CaSR promotes cardiomyocyte apoptosis in rat neonatal ventricular cardiomyocytes through activation of mitogen-activated protein kinases and caspase 9 signaling pathways.

  4. Evaluation of bupropion hydrochloride developmental cardiotoxic effects in chick cardiomyocyte micromass culture and stem cell derived cardiomyocyte systems.

    PubMed

    Shaikh Qureshi, W M; Latif, Muhammad Liaque; Parker, Terry L; Pratten, Margaret K

    2014-10-01

    The use of antidepressant drug bupropion hydrochloride (BPN) during pregnancy results in increased cardiovascular anomalies. In this study, BPN developmental cardiotoxic effects in in vitro system were evaluated using chick cardiomyocyte micromass (MM) culture system and mouse embryonic stem cell derived cardiomyocyte (ESDC) system. In MM system, the cardiomyocyte contractile activity significantly decreased only at BPN 200 μM, while in ESDC system BPN concentration above 75 μM resulted in decreased contractile activity. The increase in drug concentration also affected the cardiomyocyte viability and total cellular protein content in both systems, but in ESDC system the cell viability failed to attain significant difference. The drug failed to induce reactive oxygen species production in both systems, but has affected the cardiac connexin43 expression especially in MM system. We observed that BPN showed developmental cardiotoxic effects irrespective of the stage of cardiac development in both in vitro systems.

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

    PubMed

    Pei, Xiucong; Duan, Zhiwen; Ma, Mingyue; Zhang, Yuming; Guo, Li

    2014-01-01

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

  6. Calcium and mitochondrial metabolism in ceramide-induced cardiomyocyte death.

    PubMed

    Parra, Valentina; Moraga, Francisco; Kuzmicic, Jovan; López-Crisosto, Camila; Troncoso, Rodrigo; Torrealba, Natalia; Criollo, Alfredo; Díaz-Elizondo, Jessica; Rothermel, Beverly A; Quest, Andrew F G; Lavandero, Sergio

    2013-08-01

    Ceramides are important intermediates in the biosynthesis and degradation of sphingolipids that regulate numerous cellular processes, including cell cycle progression, cell growth, differentiation and death. In cardiomyocytes, ceramides induce apoptosis by decreasing mitochondrial membrane potential and promoting cytochrome-c release. Ca(2+) overload is a common feature of all types of cell death. The aim of this study was to determine the effect of ceramides on cytoplasmic Ca(2+) levels, mitochondrial function and cardiomyocyte death. Our data show that C2-ceramide induces apoptosis and necrosis in cultured cardiomyocytes by a mechanism involving increased Ca(2+) influx, mitochondrial network fragmentation and loss of the mitochondrial Ca(2+) buffer capacity. These biochemical events increase cytosolic Ca(2+) levels and trigger cardiomyocyte death via the activation of calpains.

  7. Calcium and mitochondrial metabolism in ceramide-induced cardiomyocyte death

    PubMed Central

    Parra, Valentina; Moraga, Francisco; Kuzmicic, Jovan; López-Crisosto, Camila; Troncoso, Rodrigo; Torrealba, Natalia; Criollo, Alfredo; Díaz-Elizondo, Jessica; Rothermel, Beverly A.; Quest, Andrew F.G.; Lavandero, Sergio

    2014-01-01

    Ceramides are important intermediates in the biosynthesis and degradation of sphingolipids that regulatenumerous cellular processes, including cell cycle progression, cell growth, differentiation and death. In cardiomyocytes, ceramides induce apoptosis by decreasing mitochondrial membrane potential and promoting cytochrome-c release. Ca2+ overload is a common feature of all types of cell death. The aim of this study was to determine the effect of ceramides on cytoplasmic Ca2+ levels, mitochondrial function and cardiomyocyte death. Our data show that C2-ceramide induces apoptosis and necrosis in cultured cardiomyocytes by a mechanism involving increased Ca2+ influx, mitochondrial network fragmentation and loss of the mitochondrial Ca2+ buffer capacity. These biochemical events increase cytosolic Ca2+ levels and trigger cardiomyocyte death via the activation of calpains. PMID:23602992

  8. Calcium and mitochondrial metabolism in ceramide-induced cardiomyocyte death.

    PubMed

    Parra, Valentina; Moraga, Francisco; Kuzmicic, Jovan; López-Crisosto, Camila; Troncoso, Rodrigo; Torrealba, Natalia; Criollo, Alfredo; Díaz-Elizondo, Jessica; Rothermel, Beverly A; Quest, Andrew F G; Lavandero, Sergio

    2013-08-01

    Ceramides are important intermediates in the biosynthesis and degradation of sphingolipids that regulate numerous cellular processes, including cell cycle progression, cell growth, differentiation and death. In cardiomyocytes, ceramides induce apoptosis by decreasing mitochondrial membrane potential and promoting cytochrome-c release. Ca(2+) overload is a common feature of all types of cell death. The aim of this study was to determine the effect of ceramides on cytoplasmic Ca(2+) levels, mitochondrial function and cardiomyocyte death. Our data show that C2-ceramide induces apoptosis and necrosis in cultured cardiomyocytes by a mechanism involving increased Ca(2+) influx, mitochondrial network fragmentation and loss of the mitochondrial Ca(2+) buffer capacity. These biochemical events increase cytosolic Ca(2+) levels and trigger cardiomyocyte death via the activation of calpains. PMID:23602992

  9. Comparative Analysis of Telomerase Activity in CD117+CD34+ Cardiac Telocytes with Bone Mesenchymal Stem Cells, Cardiac Fibroblasts and Cardiomyocytes

    PubMed Central

    Li, Yuan-Yuan; Lu, Shan-Shan; Xu, Ting; Zhang, Hong-Qi; Li, Hua

    2015-01-01

    Background: This study characterized the cardiac telocyte (TC) population both in vivo and in vitro, and investigated its telomerase activity related to mitosis. Methods: Using transmission electron microscopy and a phase contrast microscope, the typical morphological features of cardiac TCs were observed; by targeting the cell surface proteins CD117 and CD34, CD117+CD34+ cardiac TCs were sorted via flow cytometry and validated by immunofluorescence based on the primary cell culture. Then the optimized basal nutrient medium for selected population was examined with the cell counting kit 8. Under this conditioned medium, the process of cell division was captured, and the telomerase activity of CD117+CD34+ cardiac TCs was detected in comparison with bone mesenchymal stem cells (BMSCs), cardiac fibroblasts (CFBs), cardiomyocytes (CMs). Results: Cardiac TCs projected characteristic telopodes with thin segments (podomers) in alternation with dilation (podoms). In addition, 64% of the primary cultured cardiac TCs were composed of CD117+CD34+ cardiac TCs; which was verified by immunofluorescence. In a live cell imaging system, CD117+CD34+ cardiac TCs were observed to enter into cell division in a short time, followed by an significant invagination forming across the middle of the cell body. Using a real-time quantitative telomeric-repeat amplification assay, the telomerase concentration in CD117+CD34+ cardiac TCs was obviously lower than in BMSCs and CFBs, and significantly higher than in CMs. Conclusions: Cardiac TCs represent a unique cell population and CD117+CD34+ cardiac TCs have relative low telomerase activity that differs from BMSCs, CFBs and CMs and thus they might play an important role in maintaining cardiac homeostasis. PMID:26168836

  10. Simple non-invasive analysis of embryonic stem cell-derived cardiomyocytes beating in vitro

    NASA Astrophysics Data System (ADS)

    Radaszkiewicz, Katarzyna Anna; Sýkorová, Dominika; Karas, Pavel; Kudová, Jana; Kohút, Lukáš; Binó, Lucia; Večeřa, Josef; Víteček, Jan; Kubala, Lukáš; Pacherník, Jiří

    2016-02-01

    The analysis of digital video output enables the non-invasive screening of various active biological processes. For the monitoring and computing of the beating parameters of cardiomyocytes in vitro, CB Analyser (cardiomyocyte beating analyser) software was developed. This software is based on image analysis of the video recording of beating cardiomyocytes. CB Analyser was tested using cardiomyocytes derived from mouse embryonic stem cells at different stages of cardiomyogenesis. We observed that during differentiation (from day 18), the beat peak width decreased, which corresponded to the increased speed of an individual pulse. However, the beating frequency did not change. Further, the effects of epinephrine modulating mature cardiomyocyte functions were tested to validate the CB Analyser analysis. In conclusion, data show that CB Analyser is a useful tool for evaluating the functions of both developing and mature cardiomyocytes under various conditions in vitro.

  11. Cardioprotective activity of urocortin by preventing caspase-independent, non-apoptotic death in cultured neonatal rat cardiomyocytes exposed to ischemia

    SciTech Connect

    Takatani-Nakase, Tomoka; Takahashi, Koichi

    2010-11-12

    Research highlights: {yields} Ischemia induces high level of iPLA{sub 2} resulting in caspase-independent myocyte death. {yields} Urocortin causes iPLA{sub 2} down-regulation leading to avoidance of non-apoptotic death. {yields} The survival-promoting effect of urocortin is abrogated by CRH receptor antagonist. -- Abstract: Caspase-independent, non-apoptotic cell death in ischemic heart disease is considered to be one of the important therapeutic targets, however, the detailed mechanisms of this cell death process are not clear. In this study, we investigated the mechanisms of non-apoptotic cell death in cultured neonatal rat cardiomyocytes during ischemia, and the cardioprotection by preventing the mechanisms. We found that ischemia caused elevation of the phospholipase A{sub 2} (iPLA{sub 2}) expression in the myocytes, leading to distinctive non-apoptotic nuclear shrinkage, and cell death. Moreover, we investigated whether the potent cardioprotective corticotropin-releasing hormone (CRH), urocortin, which had been less focused on non-apoptotic cell death, inhibits the ischemic myocyte death. Ischemia-augmented nuclear shrinkage of the myocytes was suppressed by the pretreatment of {approx}10 nM urocortin before the cells were exposed to ischemia. Urocortin could significantly suppress the expression and activity of iPLA{sub 2}, resulting in preventing the ischemia-induced cell death. The survival-promoting effect of urocortin was abrogated by the CRH receptor antagonist astressin. These findings provide the first evidence linking the targets of the urocortin-mediated cardioprotection to the suppression of the caspase-independent, non-apoptotic death in cardiac myocytes exposed to ischemia.

  12. ZAK induces cardiomyocyte hypertrophy and brain natriuretic peptide expression via p38/JNK signaling and GATA4/c-Jun transcriptional factor activation.

    PubMed

    Hsieh, You-Liang; Tsai, Ying-Lan; Shibu, Marthandam Asokan; Su, Chia-Chi; Chung, Li-Chin; Pai, Peiying; Kuo, Chia-Hua; Yeh, Yu-Lan; Viswanadha, Vijaya Padma; Huang, Chih-Yang

    2015-07-01

    Cardiomyocyte hypertrophy is an adaptive response of heart to various stress conditions. During the period of stress accumulation, transition from physiological hypertrophy to pathological hypertrophy results in the promotion of heart failure. Our previous studies found that ZAK, a sterile alpha motif and leucine zipper containing kinase, was highly expressed in infarcted human hearts and demonstrated that overexpression of ZAK induced cardiac hypertrophy. This study evaluates, cellular events associated with the expression of two doxycycline (Dox) inducible Tet-on ZAK expression systems, a Tet-on ZAK WT (wild-type), and a Tet-on ZAK DN (mutant, Dominant-negative form) in H9c2 myoblast cells; Tet-on ZAK WT was found to increase cell size and hypertrophic marker BNP in a dose-dependent manner. To ascertain the mechanism of ZAK-mediated hypertrophy, expression analysis with various inhibitors of the related upstream and downstream proteins was performed. Tet-on ZAK WT expression triggered the p38 and JNK pathway and also activated the expression and nuclear translocation of p-GATA4 and p-c-Jun transcription factors, without the involvement of p-ERK or NFATc3. However, Tet-on ZAK DN showed no effect on the p38 and JNK signaling cascade. The results showed that the inhibitors of JNK1/2 and p38 significantly suppressed ZAK-induced BNP expression. The results show the role of ZAK and/or the ZAK downstream events such as JNK and p38 phosphorylation, c-Jun, and GATA-4 nuclear translocation in cardiac hypertrophy. ZAK and/or the ZAK downstream p38, and JNK pathway could therefore be potential targets to ameliorate cardiac hypertrophy symptoms in ZAK-overexpressed patients. PMID:25869677

  13. Carbonic anhydrase inhibition prevents and reverts cardiomyocyte hypertrophy

    PubMed Central

    Alvarez, Bernardo V; Johnson, Danielle E; Sowah, Daniel; Soliman, Daniel; Light, Peter E; Xia, Ying; Karmazyn, Morris; Casey, Joseph R

    2007-01-01

    Hypertrophic cardiomyocyte growth contributes substantially to the progression of heart failure. Activation of the plasma membrane Na+–H+ exchanger (NHE1) and Cl−–HCO3− exchanger (AE3) has emerged as a central point in the hypertrophic cascade. Both NHE1 and AE3 bind carbonic anhydrase (CA), which activates their transport flux, by providing H+ and HCO3−, their respective transport substrates. We examined the contribution of CA activity to the hypertrophic response of cultured neonatal and adult rodent cardiomyocytes. Phenylephrine (PE) increased cell size by 37 ± 2% and increased expression of the hypertrophic marker, atrial natriuretic factor mRNA, twofold in cultured neonatal rat cardiomyocytes. Cell size was also increased in adult cardiomyocytes subjected to angiotensin II or PE treatment. These effects were associated with increased expression of cytosolic CAII protein and the membrane-anchored isoform, CAIV. The membrane-permeant CA inhibitor, 6-ethoxyzolamide (ETZ), both prevented and reversed PE-induced hypertrophy in a concentration-dependent manner in neonate cardiomyocytes (IC50 = 18 μm). ETZ and the related CA inhibitor methazolamide prevented hypertrophy in adult cardiomyocytes. In addition, ETZ inhibited transport activity of NHE1 and the AE isoform, AE3, with respective EC50 values of 1.2 ± 0.3 μm and 2.7 ± 0.3 μm. PE significantly increased neonatal cardiomyocyte Ca2+ transient frequency from 0.33 ± 0.4 Hz to 0.77 ± 0.04 Hz following 24 h treatment; these Ca2+-handling abnormalities were completely prevented by ETZ (0.28 ± 0.07 Hz). Our study demonstrates a novel role for CA in mediating the hypertrophic response of cardiac myocytes to PE and suggests that CA inhibition represents an effective therapeutic approach towards mitigation of the hypertrophic phenotype. PMID:17124262

  14. Functional expression of the hyperpolarization-activated, non-selective cation current If in immortalized HL-1 cardiomyocytes

    PubMed Central

    Sartiani, Laura; Bochet, Pascal; Cerbai, Elisabetta; Mugelli, Alessandro; Fischmeister, Rodolphe

    2002-01-01

    HL-1 cells are adult mouse atrial myocytes induced to proliferate indefinitely by SV40 large T antigen. These cells beat spontaneously when confluent and express several adult cardiac cell markers including the outward delayed rectifier K+ channel. Here, we examined the presence of a hyperpolarization-activated If current in HL-1 cells using the whole-cell patch-clamp technique on isolated cells enzymatically dissociated from the culture at confluence. Cell membrane capacitance (Cm) ranged from 5 to 53 pF. If was detected in about 30 % of the cells and its occurrence was independent of the stage of the culture. If maximal slope conductance was 89.7 ± 0.4 pS pF−1 (n = 10). If current in HL-1 cells showed typical characteristics of native cardiac If current: activation threshold between −50 and −60 mV, half-maximal activation potential of −83.1 ± 0.7 mV (n = 50), reversal potential at −20.8 ± 1.5 mV (n = 10), time-dependent activation by hyperpolarization and blockade by 4 mm Cs+. In half of the cells tested, activation of adenylyl cyclase by the forskolin analogue L858051 (20 μm) induced both a ≈6 mV positive shift of the half-activation potential and a ≈37 % increase in the fully activated If current. RT-PCR analysis of the hyperpolarization-activated, cyclic nucleotide-gated channels (HCN) expressed in HL-1 cells demonstrated major contributions of HCN1 and HCN2 channel isoforms to If current. Cytosolic Ca2+ oscillations in spontaneously beating HL-1 cells were measured in Fluo-3 AM-loaded cells using a fast-scanning confocal microscope. The oscillation frequency ranged from 1.3 to 5 Hz and the spontaneous activity was stopped in the presence of 4 mm Cs+. Action potentials from HL-1 cells had a triangular shape, with an overshoot at +15 mV and a maximal diastolic potential of −69 mV, i.e. more negative than the threshold potential for If activation. In conclusion, HL-1 cells display a hyperpolarization-activated If current which might

  15. EDF-1 downregulates the CaM/Cn/NFAT signaling pathway during adipogenesis

    SciTech Connect

    López-Victorio, Carlos J.; Velez-delValle, Cristina; Beltrán-Langarica, Alicia; Kuri-Harcuch, Walid

    2013-03-01

    Highlights: ► EDF-1 participates early adipogenesis in 3T3F442A cells induced with Staurosporine/Dexamethasone. ► EDF-1 associates with CaM and Cn, most likely inactivating Cn. ► EDF-1/CaM complex seems to prevent NFATc1 activation by Cn. ► EDF-1 regulates the Cn/CaM/NFATc1 pathway during adipogenesis. ► EDF-1 may regulate the activation of Cn through a complex formation with CaM. - Abstract: The endothelial differentiation factor-1 (EDF-1) is a calmodulin binding protein that regulates calmodulin-dependent enzymes. In endothelial cells, this factor can form a protein complex with calmodulin. We analyzed the relationship between this factor and the members of calmodulin/calcineurin/nuclear factor of activated T-cells (NFAT) signaling pathway during adipogenesis of 3T3-F442A cells. We found that the expression of edf1 is upregulated during early adipogenesis, whereas that of calcineurin gene is lowered, suggesting that this pathway should be downregulated to allow for adipogenesis to occur. We also found that EDF-1 associates with calmodulin and calcineurin, most likely inactivating calcineurin. Our results showed that EDF-1 inactivates the calmodulin/calcineurin/NFAT pathway via sequestration of calmodulin, during early adipogenesis, and we propose a mechanism that negatively regulates the activation of calcineurin through a complex formation between EDF-1 and calmodulin. This finding raises the possibility that modulating this pathway might offer some alternatives to regulate adipose biology.

  16. Pro-survival function of MEF2 in cardiomyocytes is enhanced by β-blockers

    PubMed Central

    Hashemi, S; Salma, J; Wales, S; McDermott, JC

    2015-01-01

    β1-Adrenergic receptor (β1-AR) stimulation increases apoptosis in cardiomyocytes through activation of cAMP/protein kinase A (PKA) signaling. The myocyte enhancer factor 2 (MEF2) proteins function as important regulators of myocardial gene expression. Previously, we reported that PKA signaling directly represses MEF2 activity. We determined whether (a) MEF2 has a pro-survival function in cardiomyocytes, and (b) whether β-adrenergic/PKA signaling modulates MEF2 function in cardiomyocytes. Initially, we observed that siRNA-mediated gene silencing of MEF2 induces cardiomyocyte apoptosis as indicated by flow cytometry. β1-AR activation by isoproterenol represses MEF2 activity and promotes apoptosis in cultured neonatal cardiomyocytes. Importantly, β1-AR mediated apoptosis was abrogated in cardiomyocytes expressing a PKA-resistant form of MEF2D (S121/190A). We also observed that a β1-blocker, Atenolol, antagonizes isoproterenol-induced apoptosis while concomitantly enhancing MEF2 transcriptional activity. β-AR stimulation modulated MEF2 cellular localization in cardiomyocytes and this effect was reversed by β-blocker treatment. Furthermore, Kruppel-like factor 6, a MEF2 target gene in the heart, functions as a downstream pro-survival factor in cardiomyocytes. Collectively, these data indicate that (a) MEF2 has an important pro-survival role in cardiomyocytes, and (b) β-adrenergic signaling antagonizes the pro-survival function of MEF2 in cardiomyocytes and β-blockers promote it. These observations have important clinical implications that may contribute to novel strategies for preventing cardiomyocyte apoptosis associated with heart pathology. PMID:27551452

  17. Effects of short-chain acyl-CoA dehydrogenase on cardiomyocyte apoptosis.

    PubMed

    Zeng, Zhenhua; Huang, Qiuju; Shu, Zhaohui; Liu, Peiqing; Chen, Shaorui; Pan, Xuediao; Zang, Linquan; Zhou, Sigui

    2016-07-01

    Short-chain acyl-CoA dehydrogenase (SCAD), a key enzyme of fatty acid β-oxidation, plays an important role in cardiac hypertrophy. However, its effect on the cardiomyocyte apoptosis remains unknown. We aimed to determine the role of SCAD in tert-butyl hydroperoxide (tBHP)-induced cardiomyocyte apoptosis. The mRNA and protein expression of SCAD were significantly down-regulated in the cardiomyocyte apoptosis model. Inhibition of SCAD with siRNA-1186 significantly decreased SCAD expression, enzyme activity and ATP content, but obviously increased the content of free fatty acids. Meanwhile, SCAD siRNA treatment triggered the same apoptosis as cardiomyocytes treated with tBHP, such as the increase in cell apoptotic rate, the activation of caspase3 and the decrease in the Bcl-2/Bax ratio, which showed that SCAD may play an important role in primary cardiomyocyte apoptosis. The changes of phosphonate AMP-activated protein kinase α (p-AMPKα) and Peroxisome proliferator-activated receptor α (PPARα) in cardiomyocyte apoptosis were consistent with that of SCAD. Furthermore, PPARα activator fenofibrate and AMPKα activator AICAR treatment significantly increased the expression of SCAD and inhibited cardiomyocyte apoptosis. In conclusion, for the first time our findings directly demonstrated that SCAD may be as a new target to prevent cardiomyocyte apoptosis through the AMPK/PPARα/SCAD signal pathways. PMID:26989860

  18. The NPM-ALK tyrosine kinase mimics TCR signalling pathways, inducing NFAT and AP-1 by RAS-dependent mechanisms.

    PubMed

    Turner, Suzanne D; Yeung, Debra; Hadfield, Kathryn; Cook, Simon J; Alexander, Denis R

    2007-04-01

    Nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) expression is associated with the lymphoid malignancy anaplastic large cell lymphoma (ALCL) and results from a t(2;5) chromosomal translocation. We show that NPM-ALK induces Ras activation and phosphorylation of the ERK MAP Kinase consistent with activation of the Ras-MAP Kinase pathway. Furthermore, we demonstrate that activation of Ras is necessary for inducing transcription via NFAT/AP-1 composite transcriptional binding sites. This activity is dependent on NPM-ALK forming complexes with proteins that bind to autophosphorylated tyrosine residues at positions 156, 567 and 664, associated with binding to IRS-1, Shc and PLCgamma, respectively. Specifically, NPM-ALK activates transcription from the TRE promoter element, an AP-1 binding region, an activity dependent on both Ras and Shc activity. Our results show that NPM-ALK mimics activated T-cell receptor signalling by inducing pathways associated with the activation of NFAT/AP-1 transcription factors that bind to promoter elements found in a broad array of cytokine genes.

  19. Angiotensin II contributes to podocyte injury by increasing TRPC6 expression via an NFAT-mediated positive feedback signaling pathway.

    PubMed

    Nijenhuis, Tom; Sloan, Alexis J; Hoenderop, Joost G J; Flesche, Jan; van Goor, Harry; Kistler, Andreas D; Bakker, Marinka; Bindels, Rene J M; de Boer, Rudolf A; Möller, Clemens C; Hamming, Inge; Navis, Gerjan; Wetzels, Jack F M; Berden, Jo H M; Reiser, Jochen; Faul, Christian; van der Vlag, Johan

    2011-10-01

    The transient receptor potential channel C6 (TRPC6) is a slit diaphragm-associated protein in podocytes involved in regulating glomerular filter function. Gain-of-function mutations in TRPC6 cause hereditary focal segmental glomerulosclerosis (FSGS), and several human acquired proteinuric diseases show increased glomerular TRPC6 expression. Angiotensin II (AngII) is a key contributor to glomerular disease and may regulate TRPC6 expression in nonrenal cells. We demonstrate that AngII regulates TRPC6 mRNA and protein levels in cultured podocytes and that AngII infusion enhances glomerular TRPC6 expression in vivo. In animal models for human FSGS (doxorubicin nephropathy) and increased renin-angiotensin system activity (Ren2 transgenic rats), glomerular TRPC6 expression was increased in an AngII-dependent manner. TRPC6 expression correlated with glomerular damage markers and glomerulosclerosis. We show that the regulation of TRPC6 expression by AngII and doxorubicin requires TRPC6-mediated Ca(2+) influx and the activation of the Ca(2+)-dependent protein phosphatase calcineurin and its substrate nuclear factor of activated T cells (NFAT). Accordingly, calcineurin inhibition by cyclosporine decreased TRPC6 expression and reduced proteinuria in doxorubicin nephropathy, whereas podocyte-specific inducible expression of a constitutively active NFAT mutant increased TRPC6 expression and induced severe proteinuria. Our findings demonstrate that the deleterious effects of AngII on podocytes and its pathogenic role in glomerular disease involve enhanced TRPC6 expression via a calcineurin/NFAT positive feedback signaling pathway. PMID:21839714

  20. Myricitrin Attenuates High Glucose-Induced Apoptosis through Activating Akt-Nrf2 Signaling in H9c2 Cardiomyocytes.

    PubMed

    Zhang, Bin; Chen, Yaping; Shen, Qiang; Liu, Guiyan; Ye, Jingxue; Sun, Guibo; Sun, Xiaobo

    2016-01-01

    Hyperglycemia, as well as diabetes mellitus, has been shown to trigger cardiac cell apoptosis. We have previously demonstrated that myricitrin prevents endothelial cell apoptosis. However, whether myricitrin can attenuate H9c2 cell apoptosis remains unknown. In this study, we established an experiment model in H9c2 cells exposed to high glucose. We tested the hypothesis that myricitrin may inhibit high glucose (HG)-induced cardiac cell apoptosis as determined by TUNEL staining. Furthermore, myricitrin promoted antioxidative enzyme production, suppressed high glucose-induced reactive oxygen species (ROS) production and decreased mitochondrial membrane potential (MMP) in H9c2 cells. This agent significantly inhibited apoptotic protein expression, activated Akt and facilitated the transcription of NF-E2-related factor 2 (Nrf2)-mediated protein (heme oxygenase-1 (HO-1) and quinone oxidoreductase 1 (NQO-1) expression as determined by Western blotting. Significantly, an Akt inhibitor (LY294002) or HO-1 inhibitor (ZnPP) not only inhibited myricitrin-induced HO-1/NQO-1 upregulation but also alleviated its anti-apoptotic effects. In summary, these observations demonstrate that myricitrin activates Nrf2-mediated anti-oxidant signaling and attenuates H9c2 cell apoptosis induced by high glucose via activation of Akt signaling. PMID:27399653

  1. Collagen regulates transforming growth factor-β receptors of HL-1 cardiomyocytes through activation of stretch and integrin signaling.

    PubMed

    Lu, Yen-Yu; Lin, Yung-Kuo; Kao, Yu-Hsun; Chung, Cheng-Chih; Yeh, Yung-Hsin; Chen, Shih-Ann; Chen, Yi-Jen

    2016-10-01

    The extracellular matrix (ECM) and transforming growth factor-β (TGF)-β are important in cardiac fibrosis, however, the effects of the ECM on TGF‑β signaling remain to be fully elucidated. The aims of the present study were to evaluate the role of collagen in TGF‑β signaling and examine the underlying mechanisms. In the present study, western blot analysis was used to examine TGF‑β signaling in HL‑1 cells treated with and without (control) type I collagen (10 µg/ml), which was co‑administered with either an anti‑β1 integrin antibody (10 µg/ml) or a stretch‑activated channel inhibitor (gadolinium; 50 µM). Cell proliferation and adhesion assays were used to investigate the roles of integrin, mechanical stretch and mitogen‑activated protein kinases (MAPKs) on cell proliferation and adhesion. The type I collagen (10 µg/ml)‑treated HL‑1 cells were incubated with or without anti‑β1 integrin antibody (10 µg/ml), gadolinium (50 µM) or inhibitors of p38 (SB203580; 3 µM), extracellular signal‑regulated kinase (ERK; PD98059; 50 µM) and c‑Jun N‑terminal kinase (JNK; SP600125; 50 µM). Compared with the control cells, the collagen‑treated HL‑1 cells had lower expression levels of type I and type II TGF‑β receptors (TGFβRI and TGFβRII), with an increase in phosphorylated focal adhesion kinase (FAK), p38 and ERK1/2, and a decrease in JNK. Incubation with the anti‑β1 integrin antibody reversed the collagen‑induced downregulation of the expression of TGFβRII and phosphorylated FAK. Gadolinium downregulated the expression levels of TGFβRI and small mothers against decapentaplegic (Smad)2/3, and decreased the levels of phosphorylated p38, ERK1/2 and JNK. In addition, gadolinium reversed the collagen‑induced activation of p38 and ERK1/2. In the presence of gadolinium and anti‑β1 integrin antibody, collagen regulated the expression levels of TGFβRI, TGFβRII and Smad2/3, but did not alter the phosphorylation

  2. Overexpression of the Large-Conductance, Ca2+-Activated K+ (BK) Channel Shortens Action Potential Duration in HL-1 Cardiomyocytes

    PubMed Central

    Stimers, Joseph R.; Song, Li; Rusch, Nancy J.; Rhee, Sung W.

    2015-01-01

    Long QT syndrome is characterized by a prolongation of the interval between the Q wave and the T wave on the electrocardiogram. This abnormality reflects a prolongation of the ventricular action potential caused by a number of genetic mutations or a variety of drugs. Since effective treatments are unavailable, we explored the possibility of using cardiac expression of the large-conductance, Ca2+-activated K+ (BK) channel to shorten action potential duration (APD). We hypothesized that expression of the pore-forming α subunit of human BK channels (hBKα) in HL-1 cells would shorten action potential duration in this mouse atrial cell line. Expression of hBKα had minimal effects on expression levels of other ion channels with the exception of a small but significant reduction in Kv11.1. Patch-clamped hBKα expressing HL-1 cells exhibited an outward voltage- and Ca2+-sensitive K+ current, which was inhibited by the BK channel blocker iberiotoxin (100 nM). This BK current phenotype was not detected in untransfected HL-1 cells or in HL-1 null cells sham-transfected with an empty vector. Importantly, APD in hBKα-expressing HL-1 cells averaged 14.3 ± 2.8 ms (n = 10), which represented a 53% reduction in APD compared to HL-1 null cells lacking BKα expression. APD in the latter cells averaged 31.0 ± 5.1 ms (n = 13). The shortened APD in hBKα-expressing cells was restored to normal duration by 100 nM iberiotoxin, suggesting that a repolarizing K+ current attributed to BK channels accounted for action potential shortening. These findings provide initial proof-of-concept that the introduction of hBKα channels into a cardiac cell line can shorten APD, and raise the possibility that gene-based interventions to increase hBKα channels in cardiac cells may hold promise as a therapeutic strategy for long QT syndrome. PMID:26091273

  3. Mechanical stimulation of polycystin-1 induces human osteoblastic gene expression via potentiation of the calcineurin/NFAT signalling axis

    PubMed Central

    Georgopoulou, Urania; Adamopoulos, Christos; Basdra, Efthimia K.; Papavassiliou, Athanasios G.

    2016-01-01

    Mechanical forces trigger biological responses in bone cells that ultimately control osteoblastogenesis and bone remodelling. Although several mechanosensors have been postulated, the precise mechanotransduction pathway remains obscure as the initial mechanosensing event has not yet been identified. Studies in kidney cells have shown that polycystin-1 (PC1), via its extracellular N-terminal part, may function as an “antenna-like” protein providing a linkage between environmental cues and their conversion into biochemical responses that regulate various cellular processes via the calcineurin/ NFAT pathway. Here we explored the involvement of PC1 in mechanical load (stretching)-induced signalling cascades that control osteoblastogenesis/bone formation. FACS and TransAM Transcription Factor ELISA analyses employing extracts from primary human osteoblast-like, PC1 expressing cells subjected to mechanical stretching (0-6 h) revealed that unphosphorylated/DNA-binding competent NFATc1 increased at 0.5-1 h and returned to normal at 6 h. In accordance with the activation mechanism of NFATc1, stretching of cultured cells pre-treated with cyclosporin A (CsA, a specific inhibitor of the calcineurin/NFAT pathway) abrogated the observed decrease in the abundance of the cytoplasmic pNFATc1 (phosphorylated/inactive) species. Furthermore, stretching of osteoblastic cells pre-treated with an antibody against the mechanosensing N-terminal part of PC1 also abrogated the observed decrease in the cytoplasmic levels of the inactive pNFATc1 species. Importantly, under similar conditions (pre-incubation of stretched cells with the inhibitory anti-PC1 antibody), the expression of the key osteoblastic, NFATc1-target gene runx2 decreased compared to untreated cells. Therefore PC1 acts as chief mechanosensing molecule that modulates osteoblastic gene transcription and hence bone-cell differentiation through the calcineurin/NFAT signalling cascade. PMID:23014991

  4. Trypanosoma cruzi infection disturbs mitochondrial membrane potential and ROS production rate in cardiomyocytes

    PubMed Central

    Gupta, Shivali; Bhatia, Vandanajay; Wen, Jian-jun; Wu, Yewen; Huang, Ming-He; Garg, Nisha Jain

    2009-01-01

    In this study, we investigated the role of Trypanosoma cruzi invasion and inflammatory processes in reactive oxygen species (ROS) production in mouse atrial cardiomyocyte line (HL-1) and primary adult rat ventricular cardiomyocytes. Cardiomyocytes were incubated with T. cruzi (Tc) trypomastigotes, Tc lysate (TcTL) or Tc secreted proteins (TcSP) for 0-72 h, and ROS measured by amplex red assay. Cardiomyocytes infected by T. cruzi (but not those incubated with TcTL or TcSP) exhibited a linear increase in ROS production during 2-48 h post-infection (max.18-fold increase) which was further enhanced by recombinant cytokines (IL-1β, TNF-α and IFN-γ). We observed no increase in NADPH oxidase, xanthine oxidase, and myeloperoxidase activities, and specific inhibitor of these enzymes did not block the increased rate of ROS production in infected cardiomyocytes. Instead, the mitochondrial membrane potential was perturbed, and resulted in inefficient electron transport chain (ETC) activity, and enhanced electron leakage and ROS formation in infected cardiomyocytes. HL-1 rho (ρ) cardiomyocytes lacked a functional ETC, and exhibited no increase in ROS formation in response to T. cruzi. Together, these results demonstrate that invasion by T. cruzi and inflammatory milieu affect mitochondrial integrity and contribute to electron transport chain inefficiency and ROS production in cardiomyocytes. PMID:19686837

  5. Glucocorticoid Induced Leucine Zipper inhibits apoptosis of cardiomyocytes by doxorubicin

    SciTech Connect

    Aguilar, David; Strom, Joshua; Chen, Qin M.

    2014-04-01

    Doxorubicin (Dox) is an indispensable chemotherapeutic agent for the treatment of various forms of neoplasia such as lung, breast, ovarian, and bladder cancers. Cardiotoxicity is a major concern for patients receiving Dox therapy. Previous work from our laboratory indicated that glucocorticoids (GCs) alleviate Dox-induced apoptosis in cardiomyocytes. Here we have found Glucocorticoid-Induced Leucine Zipper (GILZ) to be a mediator of GC-induced cytoprotection. GILZ was found to be induced in cardiomyocytes by GC treatment. Knocking down of GILZ using siRNA resulted in cancelation of GC-induced cytoprotection against apoptosis by Dox treatment. Overexpressing GILZ by transfection was able to protect cells from apoptosis induced by Dox as measured by caspase activation, Annexin V binding and morphologic changes. Western blot analyses indicate that GILZ overexpression prevented cytochrome c release from mitochondria and cleavage of caspase-3. When bcl-2 family proteins were examined, we found that GILZ overexpression causes induction of the pro-survival protein Bcl-xL. Since siRNA against Bcl-xL reverses GC induced cytoprotection, Bcl-xL induction represents an important event in GILZ-induced cytoprotection. Our data suggest that GILZ functions as a cytoprotective gene in cardiomyocytes. - Highlights: • Corticosteroids act as a cytoprotective agent in cardiomyocytes • Corticosteroids induce GILZ expression in cardiomyocytes • Elevated GILZ results in resistance against apoptosis induced by doxorubicin • GILZ induces Bcl-xL protein without inducing Bcl-xL mRNA.

  6. Glucocorticoid signaling in the heart: A cardiomyocyte perspective.

    PubMed

    Oakley, Robert H; Cidlowski, John A

    2015-09-01

    Heart failure is one of the leading causes of death in the Western world. Glucocorticoids are primary stress hormones that regulate a vast array of biological processes, and synthetic derivatives of these steroids have been mainstays in the clinic for the last half century. Abnormal levels of glucocorticoids are known to negatively impact the cardiovascular system; however, surprisingly little is known about the direct role of glucocorticoid signaling in the heart. The actions of glucocorticoids are mediated classically by the glucocorticoid receptor (GR). In certain cells, such as cardiomyocytes, glucocorticoid occupancy and activation of the mineralocorticoid receptor (MR) may also contribute to the observed response. Recently, there has been a surge of reports investigating the in vivo function of glucocorticoid signaling in the heart using transgenic mice that specifically target GR or MR in cardiomyocytes. Results from these studies suggest that GR signaling in cardiomyocytes is critical for the normal development and function of the heart. In contrast, MR signaling in cardiomyocytes participates in the development and progression of cardiac disease. In the following review, we discuss these genetic mouse models and the new insights they are providing into the direct role cardiomyocyte glucocorticoid signaling plays in heart physiology and pathophysiology. This article is part of a Special Issue entitled 'Steroid Perspectives'.

  7. On the presence of serotonin in mammalian cardiomyocytes.

    PubMed

    Pönicke, Klaus; Gergs, Ulrich; Buchwalow, Igor B; Hauptmann, Steffen; Neumann, Joachim

    2012-06-01

    Pleiotropic effects of serotonin (5-HT) in the cardiovascular system are well documented. However, it remains to be elucidated, whether 5-HT is present in adult mammalian cardiomyocytes. To address this issue, we investigated the levels of 5-HT in blood, plasma, platelets, cardiac tissue, and cardiomyocytes from adult mice and for comparison in human right atrial tissue. Immunohistochemically, 5-HT was hardly found in mouse cardiac tissue, but small amounts could be detected in renal preparations, whereas adrenal preparations revealed a strong positive immunoreaction for 5-HT. Using a sensitive HPLC detection system, 5-HT was also detectable in the mouse heart and human atrium. Furthermore, we could identify 5-HT in isolated cardiomyocytes from adult mice. These findings were supported by detection of the activity of 5-HT-forming enzymes-tryptophan hydroxylase and aromatic L-amino acid decarboxylase-in isolated cardiomyocytes from adult mice and by inhibition of these enzymes with p-chlorophenylalanine and 3-hydroxybenzyl hydrazine. Addition of the first intermediate of 5-HT generation, that is 5-hydroxytryptophan, enhanced the 5-HT level and inhibition of monoamine oxidase by tranylcypromine further increased the level of 5-HT. Our findings reveal the presence and synthesis of 5-HT in cardiomyocytes of the mammalian heart implying that 5-HT may play an autocrine and/or paracrine role in the heart. PMID:22367115

  8. Inhibition of Receptor Interacting Protein Kinases Attenuates Cardiomyocyte Hypertrophy Induced by Palmitic Acid.

    PubMed

    Zhao, Mingyue; Lu, Lihui; Lei, Song; Chai, Hua; Wu, Siyuan; Tang, Xiaoju; Bao, Qinxue; Chen, Li; Wu, Wenchao; Liu, Xiaojing

    2016-01-01

    Palmitic acid (PA) is known to cause cardiomyocyte dysfunction. Cardiac hypertrophy is one of the important pathological features of PA-induced lipotoxicity, but the mechanism by which PA induces cardiomyocyte hypertrophy is still unclear. Therefore, our study was to test whether necroptosis, a receptor interacting protein kinase 1 and 3 (RIPK1 and RIPK3-) dependent programmed necrosis, was involved in the PA-induced cardiomyocyte hypertrophy. We used the PA-treated primary neonatal rat cardiac myocytes (NCMs) or H9c2 cells to study lipotoxicity. Our results demonstrated that cardiomyocyte hypertrophy was induced by PA treatment, determined by upregulation of hypertrophic marker genes and cell surface area enlargement. Upon PA treatment, the expression of RIPK1 and RIPK3 was increased. Pretreatment with the RIPK1 inhibitor necrostatin-1 (Nec-1), the PA-induced cardiomyocyte hypertrophy, was attenuated. Knockdown of RIPK1 or RIPK3 by siRNA suppressed the PA-induced myocardial hypertrophy. Moreover, a crosstalk between necroptosis and endoplasmic reticulum (ER) stress was observed in PA-treated cardiomyocytes. Inhibition of RIPK1 with Nec-1, phosphorylation level of AKT (Ser473), and mTOR (Ser2481) was significantly reduced in PA-treated cardiomyocytes. In conclusion, RIPKs-dependent necroptosis might be crucial in PA-induced myocardial hypertrophy. Activation of mTOR may mediate the effect of necroptosis in cardiomyocyte hypertrophy induced by PA. PMID:27057269

  9. B cell receptor induced Fc receptor-like 5 expression is mediated by multiple signaling pathways converging on NF-κB and NFAT.

    PubMed

    Damdinsuren, Bazarragchaa; Dement-Brown, Jessica; Li, Huifang; Tolnay, Mate

    2016-05-01

    Fc receptor-like (FCRL) proteins are novel regulators of the B cell response to antigen. Human FCRL5 binds intact IgG and modifies the strength of antigen receptor (BCR) signaling. Altering FCRL5 expression could therefore regulate the B cell response to antigen. In this study, we found that FCRL5 expression is induced specifically upon BCR stimulation and dissected the molecular mechanism. FCRL5 mRNA and cell surface protein expression required prolonged BCR stimulation and de novo protein synthesis. Using chemical inhibitors and activators, we identified roles for several signaling pathways, indicating a complex mechanism. Specifically, the PI3K/AKT, JNK, PKC and IKK2-dependent classical NF-κB pathways were involved in induced FCRL5 expression. Furthermore, induced FCRL5 expression required elevation of intracellular Ca(++) and was partially blocked by cyclosporine A, a calcineurin inhibitor. The importance of the transcription factors NF-κB, NFAT and CREB-binding protein was revealed based on sensitivity to inhibitors. Using reporter gene assays, we showed that the core FCRL5 promoter was sufficient to drive induced gene expression. Mutations of two predicted NF-κB sites or an NFAT site in the core promoter abrogated induced gene expression, suggesting direct regulation of the FCRL5 gene by NF-κB and NFAT. In support, we detected binding of NF-κB and NFAT family proteins to oligonucleotides corresponding to the predicted sites. We propose that the identified intricate mechanism serves to ensure that FCRL5 is expressed on B cells at a precise time following antigen encounter, with potential implications regarding regulation of the B cell response.

  10. Upregulated autophagy protects cardiomyocytes from oxidative stress-induced toxicity.

    PubMed

    Dutta, Debapriya; Xu, Jinze; Kim, Jae-Sung; Dunn, William A; Leeuwenburgh, Christiaan

    2013-03-01

    Autophagy is a cellular self-digestion process that mediates protein quality control and serves to protect against neurodegenerative disorders, infections, inflammatory diseases and cancer. Current evidence suggests that autophagy can selectively remove damaged organelles such as the mitochondria. Mitochondria-induced oxidative stress has been shown to play a major role in a wide range of pathologies in several organs, including the heart. Few studies have investigated whether enhanced autophagy can offer protection against mitochondrially-generated oxidative stress. We induced mitochondrial stress in cardiomyocytes using antimycin A (AMA), which increased mitochondrial superoxide generation, decreased mitochondrial membrane potential and depressed cellular respiration. In addition, AMA augmented nuclear DNA oxidation and cell death in cardiomyocytes. Interestingly, although oxidative stress has been proposed to induce autophagy, treatment with AMA did not result in stimulation of autophagy or mitophagy in cardiomyocytes. Our results showed that the MTOR inhibitor rapamycin induced autophagy, promoted mitochondrial clearance and protected cardiomyocytes from the cytotoxic effects of AMA, as assessed by apoptotic marker activation and viability assays in both mouse atrial HL-1 cardiomyocytes and human ventricular AC16 cells. Importantly, rapamycin improved mitochondrial function, as determined by cellular respiration, mitochondrial membrane potential and morphology analysis. Furthermore, autophagy induction by rapamycin suppressed the accumulation of ubiquitinylated proteins induced by AMA. Inhibition of rapamycin-induced autophagy by pharmacological or genetic interventions attenuated the cytoprotective effects of rapamycin against AMA. We propose that rapamycin offers cytoprotection against oxidative stress by a combined approach of removing dysfunctional mitochondria as well as by degrading damaged, ubiquitinated proteins. We conclude that autophagy induction by

  11. Upregulated autophagy protects cardiomyocytes from oxidative stress-induced toxicity

    PubMed Central

    Dutta, Debapriya; Xu, Jinze; Kim, Jae-Sung; Dunn, Jr., William A.; Leeuwenburgh, Christiaan

    2013-01-01

    Autophagy is a cellular self-digestion process that mediates protein quality control and serves to protect against neurodegenerative disorders, infections, inflammatory diseases and cancer. Current evidence suggests that autophagy can selectively remove damaged organelles such as the mitochondria. Mitochondria-induced oxidative stress has been shown to play a major role in a wide range of pathologies in several organs, including the heart. Few studies have investigated whether enhanced autophagy can offer protection against mitochondrially-generated oxidative stress. We induced mitochondrial stress in cardiomyocytes using antimycin A (AMA), which increased mitochondrial superoxide generation, decreased mitochondrial membrane potential and depressed cellular respiration. In addition, AMA augmented nuclear DNA oxidation and cell death in cardiomyocytes. Interestingly, although oxidative stress has been proposed to induce autophagy, treatment with AMA did not result in stimulation of autophagy or mitophagy in cardiomyocytes. Our results showed that the MTOR inhibitor rapamycin induced autophagy, promoted mitochondrial clearance and protected cardiomyocytes from the cytotoxic effects of AMA, as assessed by apoptotic marker activation and viability assays in both mouse atrial HL-1 cardiomyocytes and human ventricular AC16 cells. Importantly, rapamycin improved mitochondrial function, as determined by cellular respiration, mitochondrial membrane potential and morphology analysis. Furthermore, autophagy induction by rapamycin suppressed the accumulation of ubiquitinylated proteins induced by AMA. Inhibition of rapamycin-induced autophagy by pharmacological or genetic interventions attenuated the cytoprotective effects of rapamycin against AMA. We propose that rapamycin offers cytoprotection against oxidative stress by a combined approach of removing dysfunctional mitochondria as well as by degrading damaged, ubiquitinated proteins. We conclude that autophagy induction by

  12. Lead induces COX-2 expression in glial cells in a NFAT-dependent, AP-1/NFκB-independent manner.

    PubMed

    Wei, Jinlong; Du, Kejun; Cai, Qinzhen; Ma, Lisha; Jiao, Zhenzhen; Tan, Jinrong; Xu, Zhou; Li, Jingxia; Luo, Wenjin; Chen, Jingyuan; Gao, Jimin; Zhang, Dongyun; Huang, Chuanshu

    2014-11-01

    Epidemiologic studies have provided solid evidence for the neurotoxic effect of lead for decades of years. In view of the fact that children are more vulnerable to the neurotoxicity of lead, lead exposure has been an urgent public health concern. The modes of action of lead neurotoxic effects include disturbance of neurotransmitter storage and release, damage of mitochondria, as well as induction of apoptosis in neurons, cerebrovascular endothelial cells, astroglia and oligodendroglia. Our studies here, from a novel point of view, demonstrates that lead specifically caused induction of COX-2, a well known inflammatory mediator in neurons and glia cells. Furthermore, we revealed that COX-2 was induced by lead in a transcription-dependent manner, which relayed on transcription factor NFAT, rather than AP-1 and NFκB, in glial cells. Considering the important functions of COX-2 in mediation of inflammation reaction and oxidative stress, our studies here provide a mechanistic insight into the understanding of lead-associated inflammatory neurotoxicity effect via activation of pro-inflammatory NFAT3/COX-2 axis. PMID:25193092

  13. Berberine Sulfate Attenuates Osteoclast Differentiation through RANKL Induced NF-κB and NFAT Pathways.

    PubMed

    Zhou, Lin; Song, Fangming; Liu, Qian; Yang, Mingli; Zhao, Jinmin; Tan, Renxiang; Xu, Jun; Zhang, Ge; Quinn, Julian M W; Tickner, Jennifer; Xu, Jiake

    2015-11-13

    Osteoporosis, a metabolic bone disease, is characterized by an excessive formation and activation of osteoclasts. Anti-catabolic treatment using natural compounds has been proposed as a potential therapeutic strategy against the osteoclast related osteolytic diseases. In this study, the activity of berberine sulfate (an orally available form of berberine) on osteoclast differentiation and its underlying molecular mechanisms of action were investigated. Using bone marrow macrophages (BMMs) derived osteoclast culture system, we showed that berberine sulfate at the dose of 0.25, 0.5 and 1 μM significantly inhibited the formation of osteoclasts. Notably, berberine sulfate at these doses did not affect the BMM viability. In addition, we observed that berberine sulfate inhibited the expression of osteoclast marker genes, including cathepsin K (Ctsk), nuclear factor of activated T cells cytoplasmic 1 (NFATc1), tartrate resistant acid phosphatase (TRAcP, Acp5) and Vacuolar-type H+-ATPase V0 subunit D2 (V-ATPase d2). Luciferase reporter gene assay and Western blot analysis further revealed that berberine sulfate inhibits receptor for activation of nuclear factor ligand (RANKL)-induced NF-κB and NFAT activity. Taken together, our results suggest that berberine sulfate is a natural compound potentially useful for the treatment of osteoporosis.

  14. Pathways involved in interleukin-1β-mediated murine cardiomyocyte apoptosis.

    PubMed

    Shen, Yi; Qin, Jie; Bu, Peili

    2015-04-01

    Accumulating evidence suggests that interleukin-1 (IL-1) signaling plays an essential role in the pathogenesis of heart failure by inducing cardiomyocyte apoptosis, but the mechanisms of this process are poorly defined. We further explored these molecular pathways. We isolated cardiomyocytes from neonatal mice and then cultured and stimulated them with murine IL-1β in vitro. Cell apoptotic ratios were measured by means of flow cytometry. Expression of effector molecules was analyzed by means of enzyme-linked immunosorbent assay, Western blotting, and real-time quantitative polymerase chain reaction. The results showed that IL-1β induced murine cardiomyocyte apoptosis through a release of cytochrome c into cytoplasm and through caspase 3 activation. Simultaneously, IL-1β signaling promoted expression of endonuclease G and high-temperature requirement protein A2 messenger RNA. Survivin and X-linked inhibitors of apoptosis protein (IAP), members of the IAP family, were inhibited on the messenger RNA level during IL-1β-mediated cardiomyocyte apoptosis. We found that IL-1β signaling during cardiomyocyte apoptosis in vitro induced the activation of caspase-dependent and caspase-independent pathways, and inhibited IAPs. Understanding the molecular mechanisms involved in IL-1β-mediated cardiomyocyte apoptosis might assist in the design of therapeutic approaches to protect cardiomyocyte function and prevent heart failure. PMID:25873819

  15. Coordinating cardiomyocyte interactions to direct ventricular chamber morphogenesis.

    PubMed

    Han, Peidong; Bloomekatz, Joshua; Ren, Jie; Zhang, Ruilin; Grinstein, Jonathan D; Zhao, Long; Burns, C Geoffrey; Burns, Caroline E; Anderson, Ryan M; Chi, Neil C

    2016-06-29

    Many organs are composed of complex tissue walls that are structurally organized to optimize organ function. In particular, the ventricular myocardial wall of the heart comprises an outer compact layer that concentrically encircles the ridge-like inner trabecular layer. Although disruption in the morphogenesis of this myocardial wall can lead to various forms of congenital heart disease and non-compaction cardiomyopathies, it remains unclear how embryonic cardiomyocytes assemble to form ventricular wall layers of appropriate spatial dimensions and myocardial mass. Here we use advanced genetic and imaging tools in zebrafish to reveal an interplay between myocardial Notch and Erbb2 signalling that directs the spatial allocation of myocardial cells to their proper morphological positions in the ventricular wall. Although previous studies have shown that endocardial Notch signalling non-cell-autonomously promotes myocardial trabeculation through Erbb2 and bone morphogenetic protein (BMP) signalling, we discover that distinct ventricular cardiomyocyte clusters exhibit myocardial Notch activity that cell-autonomously inhibits Erbb2 signalling and prevents cardiomyocyte sprouting and trabeculation. Myocardial-specific Notch inactivation leads to ventricles of reduced size and increased wall thickness because of excessive trabeculae, whereas widespread myocardial Notch activity results in ventricles of increased size with a single-cell-thick wall but no trabeculae. Notably, this myocardial Notch signalling is activated non-cell-autonomously by neighbouring Erbb2-activated cardiomyocytes that sprout and form nascent trabeculae. Thus, these findings support an interactive cellular feedback process that guides the assembly of cardiomyocytes to morphologically create the ventricular myocardial wall and more broadly provide insight into the cellular dynamics of how diverse cell lineages organize to create form.

  16. Coordinating cardiomyocyte interactions to direct ventricular chamber morphogenesis.

    PubMed

    Han, Peidong; Bloomekatz, Joshua; Ren, Jie; Zhang, Ruilin; Grinstein, Jonathan D; Zhao, Long; Burns, C Geoffrey; Burns, Caroline E; Anderson, Ryan M; Chi, Neil C

    2016-06-30

    Many organs are composed of complex tissue walls that are structurally organized to optimize organ function. In particular, the ventricular myocardial wall of the heart comprises an outer compact layer that concentrically encircles the ridge-like inner trabecular layer. Although disruption in the morphogenesis of this myocardial wall can lead to various forms of congenital heart disease and non-compaction cardiomyopathies, it remains unclear how embryonic cardiomyocytes assemble to form ventricular wall layers of appropriate spatial dimensions and myocardial mass. Here we use advanced genetic and imaging tools in zebrafish to reveal an interplay between myocardial Notch and Erbb2 signalling that directs the spatial allocation of myocardial cells to their proper morphological positions in the ventricular wall. Although previous studies have shown that endocardial Notch signalling non-cell-autonomously promotes myocardial trabeculation through Erbb2 and bone morphogenetic protein (BMP) signalling, we discover that distinct ventricular cardiomyocyte clusters exhibit myocardial Notch activity that cell-autonomously inhibits Erbb2 signalling and prevents cardiomyocyte sprouting and trabeculation. Myocardial-specific Notch inactivation leads to ventricles of reduced size and increased wall thickness because of excessive trabeculae, whereas widespread myocardial Notch activity results in ventricles of increased size with a single-cell-thick wall but no trabeculae. Notably, this myocardial Notch signalling is activated non-cell-autonomously by neighbouring Erbb2-activated cardiomyocytes that sprout and form nascent trabeculae. Thus, these findings support an interactive cellular feedback process that guides the assembly of cardiomyocytes to morphologically create the ventricular myocardial wall and more broadly provide insight into the cellular dynamics of how diverse cell lineages organize to create form. PMID:27357797

  17. Identification of genes directly regulated by the intrinsic circadian clock within the cardiomyocyte

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Circadian rhythms in cardiovascular physiology (e.g., heart rate, cardiac output) and pathophysiology (e.g., arrhythmias) are firmly established. These phenomena have been attributed primarily to circadian rhythms in neurohumoral influences (e.g., sympathetic activity). Nevertheless, cardiomyocytes ...

  18. Tonicity-independent regulation of the osmosensitive transcription factor TonEBP (NFAT5).

    PubMed

    Halterman, Julia A; Kwon, H Moo; Wamhoff, Brian R

    2012-01-01

    Tonicity-responsive enhancer binding protein (TonEBP/nuclear factor of activated T-cells 5 [NFAT5]) is a Rel homology transcription factor classically known for its osmosensitive role in regulating cellular homeostasis during states of hypo- and hypertonic stress. A recently growing body of research indicates that TonEBP is not solely regulated by tonicity, but that it can be stimulated by various tonicity-independent mechanisms in both hypertonic and isotonic tissues. Physiological and pathophysiological stimuli such as cytokines, growth factors, receptor and integrin activation, contractile agonists, ions, and reactive oxygen species have been implicated in the positive regulation of TonEBP expression and activity in diverse cell types. These new data demonstrate that tonicity-independent stimulation of TonEBP is critical for tissue-specific functions like enhanced cell survival, migration, proliferation, vascular remodeling, carcinoma invasion, and angiogenesis. Continuing research will provide a better understanding as to how these and other alternative TonEBP stimuli regulate gene expression in both health and disease. PMID:21998140

  19. Two Closely Spaced Tyrosines Regulate NFAT Signaling in B Cells via Syk Association with Vav▿

    PubMed Central

    Chen, Chih-Hong; Martin, Victoria A.; Gorenstein, Nina M.; Geahlen, Robert L.; Post, Carol Beth

    2011-01-01

    Activated Syk, an essential tyrosine kinase in B cell signaling, interacts with Vav guanine nucleotide exchange factors and regulates Vav activity through tyrosine phosphorylation. The Vav SH2 domain binds Syk linker B by an unusual recognition of two closely spaced Syk tyrosines: Y342 and Y346. The binding affinity is highest when both Y342 and Y346 are phosphorylated. An investigation in B cells of the dependence of Vav phosphorylation and NFAT activation on phosphorylation of Y342 and Y346 finds that cellular response levels match the relative binding affinities of the Vav1 SH2 domain for singly and doubly phosphorylated linker B peptides. This key result suggests that the uncommon recognition determinant of these two closely spaced tyrosines is a limiting factor in signaling. Interestingly, differences in affinities for binding singly and doubly phosphorylated peptides are reflected in the on rate, not the off rate. Such a control mechanism would be highly effective for regulating binding among competing Syk binding partners. The nuclear magnetic resonance (NMR) structure of Vav1 SH2 in complex with a doubly phosphorylated linker B peptide reveals diverse conformations associated with the unusual SH2 recognition of two phosphotyrosines. NMR relaxation indicates compensatory changes in loop fluctuations upon binding, with implications for nonphosphotyrosine interactions of Vav1 SH2. PMID:21606197

  20. Matrix metalloproteinase-2 in oncostatin M-induced sarcomere degeneration in cardiomyocytes.

    PubMed

    Fan, Xiaohu; Hughes, Bryan G; Ali, Mohammad A M; Chan, Brandon Y H; Launier, Katherine; Schulz, Richard

    2016-07-01

    Cardiomyocyte dedifferentiation may be an important source of proliferating cardiomyocytes facilitating cardiac repair. Cardiomyocyte dedifferentiation and proliferation induced by oncostatin-M (OSM) is characterized by sarcomere degeneration. However, the mechanism underlying sarcomere degeneration remains unclear. We hypothesized that this process may involve matrix metalloproteinase-2 (MMP-2), a key protease localized at the sarcomere in cardiomyocytes. We tested the hypothesis that MMP-2 is involved in the sarcomere degeneration that characterizes cardiomyocyte dedifferentiation. Confocal immunofluorescence and biochemical methods were used to explore the role of MMP-2 in OSM-induced dedifferentiation of neonatal rat ventricular myocytes (NRVM). OSM caused a concentration- and time-dependent loss of sarcomeric α-actinin and troponin-I in NRVM. Upon OSM-treatment, the mature sarcomere transformed to a phenotype resembling a less-developed sarcomere, i.e., loss of sarcomeric proteins and Z-disk transformed into disconnected Z bodies, characteristic of immature myofibrils. OSM dose dependently increased MMP-2 activity. Both the pan-MMP inhibitor GM6001 and the selective MMP-2 inhibitor ARP 100 prevented sarcomere degeneration induced by OSM treatment. OSM also induced NRVM cell cycling and increased methyl-thiazolyl-tetrazolium (MTT) staining, preventable by MMP inhibition. These results suggest that MMP-2 mediates sarcomere degeneration in OSM-induced cardiomyocyte dedifferentiation and thus potentially contributes to cardiomyocyte regeneration.

  1. NAF-1 antagonizes starvation-induced autophagy through AMPK signaling pathway in cardiomyocytes.

    PubMed

    Du, Xiaohong; Xiao, Renjie; Xiao, Fan; Chen, Yong; Hua, Fuzhou; Yu, Shuchun; Xu, Guohai

    2015-07-01

    NAF-1 (nutrient-deprivation autophagy factor-1), an autophagy-related gene-related (ATG) protein, has been implicated in the autophagic pro-survival response. However, its role in autophagy has not been examined in the cardiomyocytes. In this study, we found that nutritional stress (NS) induced by glucose deprivation strongly induced autophagy in cultured neonatal rat cardiomyocytes, which was associated with NAF-1 down-regulation in cardiomyocytes under NS conditions. Furthermore, we demonstrate that ectopic expression of NAF-1 was sufficient to inhibit autophagy in cardiomyocytes under glucose deprivation conditions. Moreover, results of the co-immunoprecipitation assay indicate that NAF-1 antagonized autophagy by promoting the interaction between Beclin1 and Bcl-2 in NS-induced cardiomyocytes. Importantly, our results indicate that overexpression of NAF-1 significantly inhibited AMPK activity and protected cardiomyocytes from NS-induced cell death. Taken together, these data show that ectopic expression of NAF-1 antagonizes the degree of autophagy in cardiomyocytes and enhances cell survival during starvation conditions.

  2. Excitation model of pacemaker cardiomyocytes of cardiac conduction system

    NASA Astrophysics Data System (ADS)

    Grigoriev, M.; Babich, L.

    2015-11-01

    Myocardium includes typical and atypical cardiomyocytes - pacemakers, which form the cardiac conduction system. Excitation from the atrioventricular node in normal conditions is possible only in one direction. Retrograde direction of pulses is impossible. The most important prerequisite for the work of cardiomyocytes is the anatomical integrity of the conduction system. Changes in contractile force of the cardiomyocytes, which appear periodically, are due to two mechanisms of self-regulation - heterometric and homeometric. Graphic course of the excitation pulse propagation along the heart muscle more accurately reveals the understanding of the arrhythmia mechanism. These models have the ability to visualize the essence of excitation dynamics. However, they do not have the proper forecasting function for result estimation. Integrative mathematical model enables further investigation of general laws of the myocardium active behavior, allows for determination of the violation mechanism of electrical and contractile function of cardiomyocytes. Currently, there is no full understanding of the topography of pacemakers and ionic mechanisms. There is a need for the development of direction of mathematical modeling and comparative studies of the electrophysiological arrangement of cells of atrioventricular connection and ventricular conduction system.

  3. Innervating sympathetic neurons regulate heart size and the timing of cardiomyocyte cell cycle withdrawal.

    PubMed

    Kreipke, R E; Birren, S J

    2015-12-01

    Sympathetic drive to the heart is a key modulator of cardiac function and interactions between heart tissue and innervating sympathetic fibres are established early in development. Significant innervation takes place during postnatal heart development, a period when cardiomyocytes undergo a rapid transition from proliferative to hypertrophic growth. The question of whether these innervating sympathetic fibres play a role in regulating the modes of cardiomyocyte growth was investigated using 6-hydroxydopamine (6-OHDA) to abolish early sympathetic innervation of the heart. Postnatal chemical sympathectomy resulted in rats with smaller hearts, indicating that heart growth is regulated by innervating sympathetic fibres during the postnatal period. In vitro experiments showed that sympathetic interactions resulted in delays in markers of cardiomyocyte maturation, suggesting that changes in the timing of the transition from hyperplastic to hypertrophic growth of cardiomyocytes could underlie changes in heart size in the sympathectomized animals. There was also an increase in the expression of Meis1, which has been linked to cardiomyocyte cell cycle withdrawal, suggesting that sympathetic signalling suppresses cell cycle withdrawal. This signalling involves β-adrenergic activation, which was necessary for sympathetic regulation of cardiomyocyte proliferation and hypertrophy. The effect of β-adrenergic signalling on cardiomyocyte hypertrophy underwent a developmental transition. While young postnatal cardiomyocytes responded to isoproterenol (isoprenaline) with a decrease in cell size, mature cardiomyocytes showed an increase in cell size in response to the drug. Together, these results suggest that early sympathetic effects on proliferation modulate a key transition between proliferative and hypertrophic growth of the heart and contribute to the sympathetic regulation of adult heart size.

  4. Calcineurin/NFAT signaling in osteoblasts regulates bone mass.

    PubMed

    Winslow, Monte M; Pan, Minggui; Starbuck, Michael; Gallo, Elena M; Deng, Lei; Karsenty, Gerard; Crabtree, Gerald R

    2006-06-01

    Development and repair of the vertebrate skeleton requires the precise coordination of bone-forming osteoblasts and bone-resorbing osteoclasts. In diseases such as osteoporosis, bone resorption dominates over bone formation, suggesting a failure to harmonize osteoclast and osteoblast function. Here, we show that mice expressing a constitutively nuclear NFATc1 variant (NFATc1(nuc)) in osteoblasts develop high bone mass. NFATc1(nuc) mice have massive osteoblast overgrowth, enhanced osteoblast proliferation, and coordinated changes in the expression of Wnt signaling components. In contrast, viable NFATc1-deficient mice have defects in skull bone formation in addition to impaired osteoclast development. NFATc1(nuc) mice have increased osteoclastogenesis despite normal levels of RANKL and OPG, indicating that an additional NFAT-regulated mechanism influences osteoclastogenesis in vivo. Calcineurin/NFATc signaling in osteoblasts controls the expression of chemoattractants that attract monocytic osteoclast precursors, thereby coupling bone formation and bone resorption. Our results indicate that NFATc1 regulates bone mass by functioning in both osteoblasts and osteoclasts. PMID:16740479

  5. Nuclear Factor of Activated T Cells Is Activated in the Endothelium of Retinal Microvessels in Diabetic Mice

    PubMed Central

    Zetterqvist, Anna V.; Blanco, Fabiana; Öhman, Jenny; Kotova, Olga; Berglund, Lisa M.; de Frutos Garcia, Sergio; Al-Naemi, Raed; Wigren, Maria; McGuire, Paul G.; Gonzalez Bosc, Laura V.; Gomez, Maria F.

    2015-01-01

    The pathogenesis of diabetic retinopathy (DR) remains unclear but hyperglycemia is an established risk factor. Endothelial dysfunction and changes in Ca2+ signaling have been shown to precede the onset of DR. We recently demonstrated that high extracellular glucose activates the Ca2+/calcineurin-dependent transcription factor NFAT in cerebral arteries and aorta, promoting the expression of inflammatory markers. Here we show, using confocal immunofluorescence, that NFAT is expressed in the endothelium of retinal microvessels and is readily activated by high glucose. This was inhibited by the NFAT blocker A-285222 as well as by the ectonucleotidase apyrase, suggesting a mechanism involving the release of extracellular nucleotides. Acute hyperglycemia induced by an IP-GTT (intraperitoneal glucose tolerance test) resulted in increased NFATc3 nuclear accumulation and NFAT-dependent transcriptional activity in retinal vessels of NFAT-luciferase reporter mice. In both Akita (Ins2+/−) and streptozotocin- (STZ-) induced diabetic mice, NFAT transcriptional activity was elevated in retinal vessels. In vivo inhibition of NFAT with A-285222 decreased the expression of OPN and ICAM-1 mRNA in retinal vessels, prevented a diabetes driven downregulation of anti-inflammatory IL-10 in retina, and abrogated the increased vascular permeability observed in diabetic mice. Results identify NFAT signaling as a putative target for treatment of microvascular complications in diabetes. PMID:25918731

  6. Nuclear factor of activated T-cells 5 increases intestinal goblet cell differentiation through an mTOR/Notch signaling pathway

    PubMed Central

    Zhou, Yuning; Wang, Qingding; Weiss, Heidi L.; Evers, B. Mark

    2014-01-01

    The intestinal mucosa undergoes a continual process of proliferation, differentiation, and apoptosis that is regulated by multiple signaling pathways. Previously, we have shown that the nuclear factor of activated T-cells 5 (NFAT5) is involved in the regulation of intestinal enterocyte differentiation. Here we show that treatment with sodium chloride (NaCl), which activates NFAT5 signaling, increased mTORC1 repressor regulated in development and DNA damage response 1 (REDD1) protein expression and inhibited mTOR signaling; these alterations were attenuated by knockdown of NFAT5. Knockdown of NFAT5 activated mammalian target of rapamycin (mTOR) signaling and significantly inhibited REDD1 mRNA expression and protein expression. Consistently, overexpression of NFAT5 increased REDD1 expression. In addition, knockdown of REDD1 activated mTOR and Notch signaling, whereas treatment with mTOR inhibitor rapamycin repressed Notch signaling and increased the expression of the goblet cell differentiation marker mucin 2 (MUC2). Moreover, knockdown of NFAT5 activated Notch signaling and decreased MUC2 expression, while overexpression of NFAT5 inhibited Notch signaling and increased MUC2 expression. Our results demonstrate a role for NFAT5 in the regulation of mTOR signaling in intestinal cells. Importantly, these data suggest that NFAT5 participates in the regulation of intestinal homeostasis via the suppression of mTORC1/Notch signaling pathway. PMID:25057011

  7. Aberrant activation of nuclear factor of activated T cell 2 in lamina propria mononuclear cells in ulcerative colitis

    PubMed Central

    Shih, Tsung-Chieh; Hsieh, Sen-Yung; Hsieh, Yi-Yueh; Chen, Tse-Chin; Yeh, Chien-Yu; Lin, Chun-Jung; Lin, Deng-Yn; Chiu, Cheng-Tang

    2008-01-01

    AIM: To investigate the role of nuclear factor of activated T cell 2 (NFAT2), the major NFAT protein in peripheral T cells, in sustained T cell activation and intractable inflammation in human ulcerative colitis (UC). METHODS: We used two-dimensional gel-electrophoresis, immunohistochemistry, double immunohistochemical staining, and confocal microscopy to inspect the expression of NFAT2 in 107, 15, 48 and 5 cases of UC, Crohn’s disease (CD), non-specific colitis, and 5 healthy individuals, respectively. RESULTS: Up-regulation with profound nucleo-translocation/activation of NFAT2 of lamina propria mononuclear cells (LPMC) of colonic mucosa was found specifically in the affected colonic mucosa from patients with UC, as compared to CD or NC (P < 0.001, Kruskal-Wallis test). Nucleo-translocation/activation of NFAT2 primarily occurred in CD8+T, but was less prominent in CD4+ T cells or CD20+B cells. It was strongly associated with the disease activity, including endoscopic stage (τ = 0.2145, P = 0.0281) and histologic grade (τ = 0.4167, P < 0.001). CONCLUSION: We disclose for the first time the nucleo-translocation/activatin of NFAT2 in lamina propria mononuclear cells in ulcerative colitis. Activation of NFAT2 was specific for ulcerative colitis and highly associated with disease activity. Since activation of NFAT2 is implicated in an auto-regulatory positive feedback loop of sustained T-cell activation and NFAT proteins play key roles in the calcium/calcineurin signaling pathways, our results not only provide new insights into the mechanism for sustained intractable inflammation, but also suggest the calcium-calcineurin/NFAT pathway as a new therapeutic target for ulcerative colitis. PMID:18350607

  8. [SOI-nanowire biosensor for the detection of D-NFAT 1 protein].

    PubMed

    Malsagova, K A; Ivanov, Yu D; Pleshakova, T O; Kozlov, A F; Krohin, N V; Kaysheva, A L; Shumov, I D; Popov, V P; Naumova, O V; Fomin, B I; Nasimov, D A

    2015-01-01

    The nanowire (NW) detection is one of fast-acting and high-sensitive methods allowing to reveal potentially relevant protein molecules. A NW biosensor based on the silicon-on-insulator (SOI)-structures was used for biospecific label-free detection of NFAT 1 (D-NFAT 1) oncomarker in real time. For this purpose, SOI-nanowires (NWs) were modified with aptamers against NFAT 1 used as molecular probes. It was shown that using this biosensor it is possible to reach the sensitivity of ~10(-15) M. This sensitivity was comparable with that of the NW biosensor with immobilized antibodies used as macromolecular probes. The results demonstrate promising approaches used to form the sensor elements for high-sensitive disease diagnostics.

  9. Probing the role of septins in cardiomyocytes.

    PubMed

    Ahuja, Preeti; Perriard, Evelyne; Trimble, William; Perriard, Jean-Claude; Ehler, Elisabeth

    2006-05-15

    Heart growth in the embryo is achieved by division of differentiated cardiomyocytes. Around birth, cardiomyocytes stop dividing and heart growth occurs only by volume increase of the individual cells. Cardiomyocytes seem to lose their capacity for cytokinesis at this developmental stage. Septins are GTP-binding proteins that have been shown to be involved in cytokinesis from yeast to vertebrates. We wanted to determine whether septin expression patterns can be correlated to the cessation of cytokinesis during heart development. We found significant levels of expression only for SEPT2, SEPT6, SEPT7 and SEPT9 in heart, in a developmentally regulated fashion, with high levels in the embryonic heart, downregulation around birth and no detectable expression in the adult. In dividing embryonic cardiomyocytes, all septins localize to the cleavage furrow. We used drugs to probe for the functional interactions of SEPT2 in dividing embryonic cardiomyocytes. Differences in the effects on subcellular septin localization in cardiomyocytes were observed, depending whether a Rho kinase (ROCK) inhibitor was used or whether actin and myosin were targeted directly. Our data show a tight correlation of high levels of septin expression and the ability to undergo cytokinesis in cardiomyocytes. In addition, we were able to dissect the different contributions of ROCK signaling and the actomyosin cytoskeleton to septin localization to the contractile ring using cardiomyocytes as an experimental system.

  10. Evidence for Cardiomyocyte Renewal in Humans

    SciTech Connect

    Bergmann, O; Bhardwaj, R D; Bernard, S; Zdunek, S; Barnabe-Heider, F; Walsh, S; Zupicich, J; Alkass, K; Buchholz, B A; Druid, H; Jovinge, S; Frisen, J

    2008-10-14

    It has been difficult to establish whether we are limited to the heart muscle cells we are born with or if cardiomyocytes are generated also later in life. We have taken advantage of the integration of {sup 14}C, generated by nuclear bomb tests during the Cold War, into DNA to establish the age of cardiomyocytes in humans. We report that cardiomyocytes renew, with a gradual decrease from 1% turning over annually at the age of 20 to 0.3% at the age of 75. Less than 50% of cardiomyocytes are exchanged during a normal lifespan. The capacity to generate cardiomyocytes in the adult human heart suggests that it may be rational to work towards the development of therapeutic strategies aiming to stimulate this process in cardiac pathologies.

  11. Evidence for cardiomyocyte renewal in humans.

    PubMed

    Bergmann, Olaf; Bhardwaj, Ratan D; Bernard, Samuel; Zdunek, Sofia; Barnabé-Heider, Fanie; Walsh, Stuart; Zupicich, Joel; Alkass, Kanar; Buchholz, Bruce A; Druid, Henrik; Jovinge, Stefan; Frisén, Jonas

    2009-04-01

    It has been difficult to establish whether we are limited to the heart muscle cells we are born with or if cardiomyocytes are generated also later in life. We have taken advantage of the integration of carbon-14, generated by nuclear bomb tests during the Cold War, into DNA to establish the age of cardiomyocytes in humans. We report that cardiomyocytes renew, with a gradual decrease from 1% turning over annually at the age of 25 to 0.45% at the age of 75. Fewer than 50% of cardiomyocytes are exchanged during a normal life span. The capacity to generate cardiomyocytes in the adult human heart suggests that it may be rational to work toward the development of therapeutic strategies aimed at stimulating this process in cardiac pathologies.

  12. Evidence for cardiomyocyte renewal in humans.

    PubMed

    Bergmann, Olaf; Bhardwaj, Ratan D; Bernard, Samuel; Zdunek, Sofia; Barnabé-Heider, Fanie; Walsh, Stuart; Zupicich, Joel; Alkass, Kanar; Buchholz, Bruce A; Druid, Henrik; Jovinge, Stefan; Frisén, Jonas

    2009-04-01

    It has been difficult to establish whether we are limited to the heart muscle cells we are born with or if cardiomyocytes are generated also later in life. We have taken advantage of the integration of carbon-14, generated by nuclear bomb tests during the Cold War, into DNA to establish the age of cardiomyocytes in humans. We report that cardiomyocytes renew, with a gradual decrease from 1% turning over annually at the age of 25 to 0.45% at the age of 75. Fewer than 50% of cardiomyocytes are exchanged during a normal life span. The capacity to generate cardiomyocytes in the adult human heart suggests that it may be rational to work toward the development of therapeutic strategies aimed at stimulating this process in cardiac pathologies. PMID:19342590

  13. Leukemia Inhibitory Factor Enhances Endogenous Cardiomyocyte Regeneration after Myocardial Infarction

    PubMed Central

    Kanda, Masato; Nagai, Toshio; Takahashi, Toshinao; Liu, Mei Lan; Kondou, Naomichi; Naito, Atsuhiko T.; Akazawa, Hiroshi; Sashida, Goro; Iwama, Atsushi; Komuro, Issei; Kobayashi, Yoshio

    2016-01-01

    Cardiac stem cells or precursor cells regenerate cardiomyocytes; however, the mechanism underlying this effect remains unclear. We generated CreLacZ mice in which more than 99.9% of the cardiomyocytes in the left ventricular field were positive for 5-bromo-4-chloro-3-indolyl-β-d-galactoside (X-gal) staining immediately after tamoxifen injection. Three months after myocardial infarction (MI), the MI mice had more X-gal-negative (newly generated) cells than the control mice (3.04 ± 0.38/mm2, MI; 0.47 ± 0.16/mm2, sham; p < 0.05). The cardiac side population (CSP) cell fraction contained label-retaining cells, which differentiated into X-gal-negative cardiomyocytes after MI. We injected a leukemia inhibitory factor (LIF)-expression construct at the time of MI and identified a significant functional improvement in the LIF-treated group. At 1 month after MI, in the MI border and scar area, the LIF-injected mice had 31.41 ± 5.83 X-gal-negative cardiomyocytes/mm2, whereas the control mice had 12.34 ± 2.56 X-gal-negative cardiomyocytes/mm2 (p < 0.05). Using 5-ethynyl-2'-deoxyurinide (EdU) administration after MI, the percentages of EdU-positive CSP cells in the LIF-treated and control mice were 29.4 ± 2.7% and 10.6 ± 3.7%, respectively, which suggests that LIF influenced CSP proliferation. Moreover, LIF activated the Janus kinase (JAK)signal transducer and activator of transcription (STAT), mitogen-activated protein kinase/extracellular signal-regulated (MEK)extracellular signal-regulated kinase (ERK), and phosphatidylinositol 3-kinase (PI3K)–AKT pathways in CSPs in vivo and in vitro. The enhanced green fluorescent protein (EGFP)-bone marrow-chimeric CreLacZ mouse results indicated that LIF did not stimulate cardiogenesis via circulating bone marrow-derived cells during the 4 weeks following MI. Thus, LIF stimulates, in part, stem cell-derived cardiomyocyte regeneration by activating cardiac stem or precursor cells. This approach may represent a novel therapeutic

  14. Leukemia Inhibitory Factor Enhances Endogenous Cardiomyocyte Regeneration after Myocardial Infarction.

    PubMed

    Kanda, Masato; Nagai, Toshio; Takahashi, Toshinao; Liu, Mei Lan; Kondou, Naomichi; Naito, Atsuhiko T; Akazawa, Hiroshi; Sashida, Goro; Iwama, Atsushi; Komuro, Issei; Kobayashi, Yoshio

    2016-01-01

    Cardiac stem cells or precursor cells regenerate cardiomyocytes; however, the mechanism underlying this effect remains unclear. We generated CreLacZ mice in which more than 99.9% of the cardiomyocytes in the left ventricular field were positive for 5-bromo-4-chloro-3-indolyl-β-d-galactoside (X-gal) staining immediately after tamoxifen injection. Three months after myocardial infarction (MI), the MI mice had more X-gal-negative (newly generated) cells than the control mice (3.04 ± 0.38/mm2, MI; 0.47 ± 0.16/mm2, sham; p < 0.05). The cardiac side population (CSP) cell fraction contained label-retaining cells, which differentiated into X-gal-negative cardiomyocytes after MI. We injected a leukemia inhibitory factor (LIF)-expression construct at the time of MI and identified a significant functional improvement in the LIF-treated group. At 1 month after MI, in the MI border and scar area, the LIF-injected mice had 31.41 ± 5.83 X-gal-negative cardiomyocytes/mm2, whereas the control mice had 12.34 ± 2.56 X-gal-negative cardiomyocytes/mm2 (p < 0.05). Using 5-ethynyl-2'-deoxyurinide (EdU) administration after MI, the percentages of EdU-positive CSP cells in the LIF-treated and control mice were 29.4 ± 2.7% and 10.6 ± 3.7%, respectively, which suggests that LIF influenced CSP proliferation. Moreover, LIF activated the Janus kinase (JAK)signal transducer and activator of transcription (STAT), mitogen-activated protein kinase/extracellular signal-regulated (MEK)extracellular signal-regulated kinase (ERK), and phosphatidylinositol 3-kinase (PI3K)-AKT pathways in CSPs in vivo and in vitro. The enhanced green fluorescent protein (EGFP)-bone marrow-chimeric CreLacZ mouse results indicated that LIF did not stimulate cardiogenesis via circulating bone marrow-derived cells during the 4 weeks following MI. Thus, LIF stimulates, in part, stem cell-derived cardiomyocyte regeneration by activating cardiac stem or precursor cells. This approach may represent a novel therapeutic

  15. Effect of heat shock pretreatment on apoptosis and metallothionein expression in rat cardiomyocytes

    PubMed Central

    Zhang, Xian; Sha, Ming-Lei; Yao, Yu-Ting; Da, Jia; Ni, Xiu-Shi

    2015-01-01

    To investigate the effect of heat shock pretreatment on apoptosis and mitochondrial metallothionein (MT) expression in rat cardiomyocytes. In vitro cultured H9C2 cells were randomly divided into three groups: control, hydrogen peroxide (H2O2) injury, and H2O2 injury after heat shock pretreatment (n = 6 per group). Cardiomyocyte apoptosis and caspase-3 activity were assayed after treatment. Mitochondrial cytochrome (cyt) c and MT expression was assayed by Western blotting. Compared with the control group, the H2O2 injury group had a growing number of apoptotic cardiomyocytes (P < 0.01) and significantly elevated caspase-3 activity (P < 0.01) with markedly increased mitochondrial cyt c and MT expression (P < 0.01). After heat shock pretreatment, the numbers of apoptotic and necrotic cardiomyocytes (P < 0.01) and the caspase-3 activity significantly declined (P < 0.01), while mitochondrial cyt c and MT expression continued to increase (P < 0.01) compared with the H2O2 injury group. Heat shock pretreatment inhibits cardiomyocyte apoptosis, which may have a protective effect on cardiomyocytes by increasing the expression of myocardial protective MT and reducing the release of mitochondrial cyt c. PMID:26221315

  16. GLP1 protects cardiomyocytes from palmitate-induced apoptosis via Akt/GSK3b/b-catenin pathway.

    PubMed

    Ying, Ying; Zhu, Huazhang; Liang, Zhen; Ma, Xiaosong; Li, Shiwei

    2015-12-01

    Activation of apoptosis in cardiomyocytes by saturated palmitic acids contributes to cardiac dysfunction in diabetic cardiomyopathy. Beta-catenin (b-catenin) is a transcriptional regulator of several genes involved in survival/anti-apoptosis. However, its role in palmitate-induced cardiomyocyte apoptosis remains unclear. Glucagon-like peptide 1 (GLP1) has been shown to exhibit potential cardioprotective properties. This study was designed to evaluate the role of b-catenin signalling in palmitate-induced cardiomyocyte apoptosis and the molecular mechanism underlying the protective effects of GLP1 on palmitate-stressed cardiomyocytes. Exposure of neonatal rat cardiomyocytes to palmitate increased the fatty acid transporter CD36-mediated intracellular lipid accumulation and cardiomyocyte apoptosis, decreased accumulation and nuclear translocation of active b-catenin, and reduced expression of b-catenin target protein survivin and BCL2. These detrimental effects of palmitate were significantly attenuated by GLP1 co-treatment. However, the anti-apoptotic effects of GLP1 were markedly abolished when b-catenin was silenced with a specific short hairpin RNA. Furthermore, analysis of the upstream molecules and mechanisms responsible for GLP1-associated cardiac protection revealed that GLP1 restored the decreased phosphorylation of protein kinase B (Akt) and glycogen synthase kinase-3b (GSK3b) in palmitate-stimulated cardiomyocytes. In contrast, inhibition of Akt with an Akt-specific inhibitor MK2206 or blockade of GLP1 receptor (GLP1R) with a competitive antagonist exendin-(9-39) significantly abrogated the GLP1-mediated activation of GSK3b/b-catenin signalling, leading to increased apoptosis in palmitate-stressed cardiomyocytes. Collectively, our results demonstrated for the first time that the attenuated b-catenin signalling may contribute to palmitate-induced cardiomyocyte apoptosis, while GLP1 can protect cardiomyocytes from palmitate-induced apoptosis through

  17. GLP1 protects cardiomyocytes from palmitate-induced apoptosis via Akt/GSK3b/b-catenin pathway

    PubMed Central

    Ying, Ying; Zhu, Huazhang; Liang, Zhen; Ma, Xiaosong; Li, Shiwei

    2015-01-01

    Activation of apoptosis in cardiomyocytes by saturated palmitic acids contributes to cardiac dysfunction in diabetic cardiomyopathy. Beta-catenin (b-catenin) is a transcriptional regulator of several genes involved in survival/anti-apoptosis. However, its role in palmitate-induced cardiomyocyte apoptosis remains unclear. Glucagon-like peptide 1 (GLP1) has been shown to exhibit potential cardioprotective properties. This study was designed to evaluate the role of b-catenin signalling in palmitate-induced cardiomyocyte apoptosis and the molecular mechanism underlying the protective effects of GLP1 on palmitate-stressed cardiomyocytes. Exposure of neonatal rat cardiomyocytes to palmitate increased the fatty acid transporter CD36-mediated intracellular lipid accumulation and cardiomyocyte apoptosis, decreased accumulation and nuclear translocation of active b-catenin, and reduced expression of b-catenin target protein survivin and BCL2. These detrimental effects of palmitate were significantly attenuated by GLP1 co-treatment. However, the anti-apoptotic effects of GLP1 were markedly abolished when b-catenin was silenced with a specific short hairpin RNA. Furthermore, analysis of the upstream molecules and mechanisms responsible for GLP1-associated cardiac protection revealed that GLP1 restored the decreased phosphorylation of protein kinase B (Akt) and glycogen synthase kinase-3b (GSK3b) in palmitate-stimulated cardiomyocytes. In contrast, inhibition of Akt with an Akt-specific inhibitor MK2206 or blockade of GLP1 receptor (GLP1R) with a competitive antagonist exendin-(9–39) significantly abrogated the GLP1-mediated activation of GSK3b/b-catenin signalling, leading to increased apoptosis in palmitate-stressed cardiomyocytes. Collectively, our results demonstrated for the first time that the attenuated b-catenin signalling may contribute to palmitate-induced cardiomyocyte apoptosis, while GLP1 can protect cardiomyocytes from palmitate-induced apoptosis through

  18. Zinc pyrithione inhibits caspase-3 activity, promotes ErbB1-ErbB2 heterodimerization and suppresses ErbB2 downregulation in cardiomyocytes subjected to ischemia/reperfusion.

    PubMed

    Bodiga, Vijaya Lakshmi; Thokala, Sandhya; Vemuri, Praveen Kumar; Bodiga, Sreedhar

    2015-12-01

    Heart tissue becomes zinc-depleted and the capacity to mobilize labile zinc is diminished, indicating zinc dyshomeostasis during ischemia/reperfusion (I/R). Apparently, zinc pyrithione restores the basal zinc levels during I/R and prevents apoptosis by activating phosphatidyl inositol-3-kinase/Akt and targeting mitochondrial permeability transition. Receptor tyrosine kinases of the ErbB family (ErbB1 to ErbB4) are cell surface proteins that can regulate cell growth, proliferation and survival. Previous studies have shown that zinc pyrithione-induced activation of PI3kinase/Akt requires ErbB2 expression. On the other hand, while I/R decreases ErbB2 levels causing cardiomyocyte dysfunction and cell death, zinc pyrithione restores ErbB2 levels and maintains cardiomyocyte function. H9c2 cells expressed all the four ErbBs, although the expression of ErbB1 and ErbB2 were higher compared to ErbB3 and ErbB4. Hypoxia/Reoxygenation (H/R) had opposing effects on the mRNA expression of ErbB1 and ErbB2. ErbB2 mRNA levels were enhanced, but corresponding ErbB2 protein levels decreased after reoxygenation. H/R induced the degradation of ErbB2 in caspase-3 dependent manner, with the formation of a 25kDa fragment. This fragment could be detected after H/R only upon treatment of the cells with a proteasomal inhibitor, ALLN, suggesting that caspase-mediated cleavage of 185kDa ErbB2 results in C-terminal cleavage and formation of 25kDa fragment, which is further degraded by proteasome. Heterodimerization and phosphorylation of ErbB2/ErbB1 which decreased upon reoxygenation, was promoted by zinc pyrithione. Zinc pyrithione effectively suppressed the caspase activation, decreased the proteolytic cleavage of ErbB2, enhanced the phosphorylation and activation of ErbB1-ErbB2 complexes and improved the cell survival after hypoxia/reoxygenation. PMID:26436560

  19. Pyrroloquinoline quinone inhibits oxygen/glucose deprivation-induced apoptosis by activating the PI3K/AKT pathway in cardiomyocytes.

    PubMed

    Xu, Feng; Yu, Haixia; Liu, Jinyao; Cheng, Lu

    2014-01-01

    The purposes of this study were to examine the protective effect of pyrroloquinoline quinone (PQQ) on oxygen/glucose deprivation (OGD)-induced injury to H9C2 rat cardiomyocytes and to investigate the mechanism. Using H9C2 cells cultured in vitro, we examined changes in cell viability with an MTT assay at 12, 24, and 48 h after injury induced by OGD. Various concentrations of PQQ (1, 10, and 100 μM) were added, and the effect of PQQ on cell viability after OGD was assessed using the MTT assay. Thus, the optimal concentration of PQQ for the protection of cardiomyocytes against oxygen and glucose deprivation injury was determined. We also used flow cytometry analysis to examine the effect of PQQ on H9C2 cells with OGD-induced injury. The molecular probe 2',7'-dichlorofluorescin diacetate was used to label the H9C2 cells, and flow cytometry was used to detect the effect of PQQ on reactive oxygen species (ROS) content. After labeling the H9C2 cells using a mitochondrial green fluorescent probe (Mito-Tracker Green), we measured the change in the mitochondrial content of PQQ-treated H9C2 cells. Western blotting was used to examine the effect of PQQ on the phosphatidylinositol 3-kinase (PI3K)/Akt pathway in the H9C2 cells. The results of the MTT assay showed that 48 h of OGD significantly injured the H9C2 cells (p < 0.01) and that treatment with 100 μM PQQ effectively decreased the level of OGD-induced injury (p < 0.01). The results of the flow cytometry analysis showed that PQQ significantly reduced apoptosis in H9C2 cells subjected to OGD (p < 0.05). In addition, OGD significantly increased the ROS level in H9C2 cells (p < 0.01), and PQQ significantly inhibited this increase (p < 0.05). The results of the Mito-Tracker Green staining suggested that PQQ effectively inhibited the decrease in mitochondrial content caused by OGD (p < 0.05). Western blot analysis showed that PQQ partially reversed the decrease in Akt phosphorylation that was caused by OGD (p

  20. Calcineurin/NFAT pathway mediates wear particle-induced TNF-α release and osteoclastogenesis from mice bone marrow macrophages in vitro

    PubMed Central

    Liu, Feng-xiang; Wu, Chuan-long; Zhu, Zhen-an; Li, Mao-qiang; Mao, Yuan-qing; Liu, Ming; Wang, Xiao-qing; Yu, De-gang; Tang, Ting-ting

    2013-01-01

    Aim: To investigate the roles of the calcineurin/nuclear factor of activated T cells (NFAT) pathway in regulation of wear particles-induced cytokine release and osteoclastogenesis from mouse bone marrow macrophages in vitro. Methods: Osteoclasts were induced from mouse bone marrow macrophages (BMMs) in the presence of 100 ng/mL receptor activator of NF-κB ligand (RANKL). Acridine orange staining and MTT assay were used to detect the cell viability. Osteoclastogenesis was determined using TRAP staining and RT-PCR. Bone pit resorption assay was used to examine osteoclast phenotype. The expression and cellular localization of NFATc1 were examined using RT-PCR and immunofluorescent staining. The production of TNFα was analyzed with ELISA. Results: Titanium (Ti) or polymethylmethacrylate (PMMA) particles (0.1 mg/mL) did not significantly change the viability of BMMs, but twice increased the differentiation of BMMs into mature osteoclasts, and markedly increased TNF-α production. The TNF-α level in the PMMA group was significantly higher than in the Ti group (96 h). The expression of NFATc1 was found in BMMs in the presence of the wear particles and RANKL. In bone pit resorption assay, the wear particles significantly increased the resorption area and total number of resorption pits in BMMs-seeded ivory slices. Addition of 11R-VIVIT peptide (a specific inhibitor of calcineurin-mediated NFAT activation, 2.0 μmol/L) did not significantly affect the viability of BMMs, but abolished almost all the wear particle-induced alterations in BMMs. Furthermore, VIVIT reduced TNF-α production much more efficiently in the PMMA group than in the Ti group (96 h). Conclusion: Calcineurin/NFAT pathway mediates wear particles-induced TNF-α release and osteoclastogenesis from BMMs. Blockade of this signaling pathway with VIVIT may provide a promising therapeutic modality for the treatment of periprosthetic osteolysis. PMID:24056707

  1. Polydatin attenuates cardiac hypertrophy through modulation of cardiac Ca2+ handling and calcineurin-NFAT signaling pathway.

    PubMed

    Ding, Wenwen; Dong, Ming; Deng, Jianxin; Yan, Dewen; Liu, Yun; Xu, Teng; Liu, Jie

    2014-09-01

    Polydatin (PD), a resveratrol glucoside extracted from the perennial herbage Polygonum cuspidatum, has been suggested to have wide cardioprotective effects. This study aimed to explore the direct antihypertrophic role of PD in cultured neonatal rat ventricular myocytes (NRVMs) and its therapeutic effects against pressure overload (PO)-induced hypertrophic remodeling and heart failure. Furthermore, we investigated the mechanisms underlying the actions of PD. Treatment of NRVMs with phenylephrine for 72 h induced myocyte hypertrophy, where the cell surface area and protein levels of atrial natriuretic peptide and β-myosin heavy chain (β-MHC) were significantly increased. The amplitude of systolic Ca(2+) transient was increased, and sarcoplasmic reticulum Ca(2+) recycling was prolonged. Concomitantly, calcineurin activity was increased and NFAT protein was imported into the nucleus. PD treatment restored Ca(2+) handling and inhibited calcineurin-NFAT signaling, thus attenuating the hypertrophic remodeling in NRVMs. PO-induced cardiac hypertrophy was produced by transverse aortic constriction (TAC) in C57BL/6 mice, where the left ventricular posterior wall thickness and heart-to-body weight ratio were significantly increased. The cardiac function was increased at 5 wk of TAC, but significantly decreased at 13 wk of TAC. The amplitude of Ca(2+) transient and calcineurin activity were increased at 5 wk of TAC. PD treatment largely abolished TAC-induced hypertrophic remodeling by inhibiting the Ca(2+)-calcineurin pathway. Surprisingly, PD did not inhibit myocyte contractility despite that the amplitude of Ca(2+) transient was decreased. The cardiac function remained intact at 13 wk of TAC. In conclusion, PD is beneficial against PO-induced cardiac hypertrophy and heart failure largely through inhibiting the Ca(2+)-calcineurin pathway without compromising cardiac contractility. PMID:25015961

  2. Repression of cyclin D1 expression is necessary for the maintenance of cell cycle exit in adult mammalian cardiomyocytes.

    PubMed

    Tane, Shoji; Kubota, Misae; Okayama, Hitomi; Ikenishi, Aiko; Yoshitome, Satoshi; Iwamoto, Noriko; Satoh, Yukio; Kusakabe, Aoi; Ogawa, Satoko; Kanai, Ayumi; Molkentin, Jeffery D; Nakamura, Kazuomi; Ohbayashi, Tetsuya; Takeuchi, Takashi

    2014-06-27

    The hearts of neonatal mice and adult zebrafish can regenerate after injury through proliferation of preexisting cardiomyocytes. However, adult mammals are not capable of cardiac regeneration because almost all cardiomyocytes exit their cell cycle. Exactly how the cell cycle exit is maintained and how many adult cardiomyocytes have the potential to reenter the cell cycle are unknown. The expression and activation levels of main cyclin-cyclin-dependent kinase (CDK) complexes are extremely low or undetectable at adult stages. The nuclear DNA content of almost all cardiomyocytes is 2C, indicating the cell cycle exit from G1-phase. Here, we induced expression of cyclin D1, which regulates the progression of G1-phase, only in differentiated cardiomyocytes of adult mice. In these cardiomyocytes, S-phase marker-positive cardiomyocytes and the expression of main cyclins and CDKs increased remarkably, although cyclin B1-CDK1 activation was inhibited in an ATM/ATR-independent manner. The phosphorylation pattern of CDK1 and expression pattern of Cdc25 subtypes suggested that a deficiency in the increase in Cdc25 (a and -b), which is required for M-phase entry, inhibited the cyclin B1-CDK1 activation. Finally, analysis of cell cycle distribution patterns showed that >40% of adult mouse cardiomyocytes reentered the cell cycle by the induction of cyclin D1. The cell cycle of these binucleated cardiomyocytes was arrested before M-phase, and many mononucleated cardiomyocytes entered endoreplication. These data indicate that silencing the cyclin D1 expression is necessary for the maintenance of the cell cycle exit and suggest a mechanism that involves inhibition of M-phase entry.

  3. A Modular Enhancer Is Differentially Regulated by GATA and NFAT Elements That Direct Different Tissue-Specific Patterns of Nucleosome Positioning and Inducible Chromatin Remodeling▿

    PubMed Central

    Bert, Andrew G.; Johnson, Brett V.; Baxter, Euan W.; Cockerill, Peter N.

    2007-01-01

    We investigated alternate mechanisms employed by enhancers to position and remodel nucleosomes and activate tissue-specific genes in divergent cell types. We demonstrated that the granulocyte-macrophage colony-stimulating factor (GM-CSF) gene enhancer is modular and recruits different sets of transcription factors in T cells and myeloid cells. The enhancer recruited distinct inducible tissue-specific enhanceosome-like complexes and directed nucleosomes to different positions in these cell types. In undifferentiated T cells, the enhancer was activated by inducible binding of two NFAT/AP-1 complexes which disrupted two specifically positioned nucleosomes (N1 and N2). In myeloid cells, the enhancer was remodeled by GATA factors which constitutively displaced an upstream nucleosome (N0) and cooperated with inducible AP-1 elements to activate transcription. In mast cells, which express both GATA-2 and NFAT, these two pathways combined to activate the enhancer and generate high-level gene expression. At least 5 kb of the GM-CSF locus was organized as an array of nucleosomes with fixed positions, but the enhancer adopted different nucleosome positions in T cells and mast cells. Furthermore, nucleosomes located between the enhancer and promoter were mobilized upon activation in an enhancer-dependent manner. These studies reveal that distinct tissue-specific mechanisms can be used either alternately or in combination to activate the same enhancer. PMID:17283044

  4. Matrix Metalloproteinases in Primary Culture of Cardiomyocytes.

    PubMed

    Bildyug, N B; Voronkina, I V; Smagina, L V; Yudintseva, N M; Pinaev, G P

    2015-10-01

    The highly organized contractile apparatus of cardiomyocytes in heart tissue allows for their continuous contractility, whereas extracellular matrix components are synthesized and spatially organized by fibroblasts and endothelial cells. However, reorganization of the cardiomyocyte contractile apparatus occurs upon their 2D cultivation, which is accompanied by transient loss of their contractility and acquired capability of extracellular matrix synthesis (Bildyug, N. B., and Pinaev, G. P. (2013) Tsitologiya, 55, 713-724). In this study, matrix metalloproteinases were investigated at different times of cardiomyocyte 2D cultivation and 3D cultivation in collagen gels. It was found that cardiomyocytes in 2D culture synthesize matrix metalloproteinases MMP-2 and MMP-9, wherein their amount varies with the cultivation time. The peak MMP-9 amount is at early cultivation time, when the reorganization of cardiomyocyte contractile apparatus occurs, and the MMP-2 peak precedes the recovery of the initial organization of their contractile apparatus. Upon cardiomyocyte cultivation in 3D collagen gels, in which case their contractile apparatus does not rearrange, a steady small amount of MMP-2 and MMP-9 is observed. These data indicate that the cardiomyocyte contractile apparatus reorganization in culture is associated with synthesis and spatial organization of their own extracellular matrix.

  5. Real-time monitoring of hypertrophy in HL-1 cardiomyocytes by impedance measurements reveals different modes of growth.

    PubMed

    Bloch, Laura; Ndongson-Dongmo, Bernadin; Kusch, Angelika; Dragun, Duska; Heller, Regine; Huber, Otmar

    2016-10-01

    Hypertrophic growth is a response of the heart to increased mechanical load or physiological stress. Thereby, cardiomyocytes grow in length and/or width to maintain cardiac pump function. Major signaling pathways involved in cardiomyocyte growth and remodeling have been identified during recent years including calcineurin-NFAT and PI3K-Akt signaling. Modulation of these pathways is of certain interest for therapeutic treatment of cardiac hypertrophy. However, quantification and characterization of hypertrophy in response to different stimuli or modulators is difficult. This study aims to test different read-out systems for detection and quantification of differences in hypertrophic growth in response to prohypertrophic stimuli. Real-time impedance measurements allowed the detection of distinct differences in hypertrophic growth in response to endothelin, norepinephrine, phenylephrine or BIO, which were not observable by other methods such as flow cytometry. Endothelin treatment induced a rapid and strong peak in the impedance signal concomitant with a massive reorientation of the actin cytoskeleton. Changes in expression of hypertrophy-associated genes were detected and stabilization of β-catenin was identified as a common response to all hypertrophic stimuli used in this study. Hypertrophic growth was blocked by the PI3K/mTOR inhibitor PI-103.

  6. 5-Methoxytryptophan-dependent protection of cardiomyocytes from heart ischemia reperfusion injury.

    PubMed

    Chou, Hsiu-Chuan; Chan, Hong-Lin

    2014-02-01

    5-Methoxytryptophan (5-MTP), a catabolic product of tryptophan, can block Cox-2 overexpression in cancer cells as well as suppress cancer cell growth, migration and invasion. The aim of this study was to in vitro examine whether 5-MTP is able to reduce reactive oxygen species (ROS)-induced heart ischemia reperfusion injury and activate the cardiomyocyte's damage surveillance systems. Accordingly, rattus cardiomyocytes were treated with H2O2 as a heart ischemia reperfusion model prior to incubation with/without 5-MTP and proteomic analysis was performed to investigate the physiologic protection of 5-MTP in H2O2-induced ischemia reperfusion in cardiomyocyte. Our data demonstrated that 5-MTP treatment does protect cardiomyocyte in the ROS-induced ischemia reperfusion model. 5-MTP has also been shown to significantly facilitate cell migration and wound healing via cytoskeletal regulations. Additionally, two-dimensional differential gel electrophoresis (2D-DIGE) combined matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF/TOF MS) analysis showed that 5-MTP might modulate growth-associated proteins, cytoskeleton regulation, redox regulation and protein folding to stimulate wound healing as well as prevent these ischemia reperfusion-damaged cardiomyocytes from cell death through maintaining cellular redox-balance and reducing ER-stress. To our knowledge, we report for the first time the cell repair mechanism of 5-MTP against ischemia reperfusion-damage in cardiomyocytes based on cell biology and proteomic analysis. PMID:24384558

  7. Role of salubrinal in protecting cardiomyocytes from doxorubicin-induced apoptosis.

    PubMed

    Gong, N; Wu, J H; Liang, Z S; Jiang, W H; Wang, X W

    2015-01-01

    We determined whether salubrinal can protect cardio-myocytes from doxorubicin-induced apoptosis and explored the related mechanisms to provide experimental evidence for exploring novel drug candidates to decrease cardiac toxicity. Neonatal rat cardiomyocytes were isolated, cultured in vitro, and pretreated with salubrinal (10, 20, or 40 μM) to observe their response to doxorubicin-induced cell apoptosis. Lactate dehydrogenase assay, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling staining, and flow cytometry were used to assess the extent of cardiomyocyte apoptosis. Fluorescent probes conjugated with 2',7'-dichlorofluorescein diacetate and a chemiluminescence assay were used to detect the pro-duction of reactive oxygen species. Western blotting was employed to quantify expression levels of cleaved caspase-3, cytosolic cytochrome c, and B-cell lymphoma-extra large (Bcl-xL). The mechanisms of salubrinal-related functions were also explored. Salubrinal effectively inhibited doxorubicin-induced reactive oxygen species production and nicotinamide adenine dinucleotide phosphate oxidase activation, decreased the levels of cleaved caspase-3 and cytosol cytochrome c, and increased Bcl-xL expression, thereby protecting cardiomyocytes from doxorubicin-induced apoptosis. Furthermore, salubrinal was found to protect cardiomyocytes by decreasing the dephosphorylation of eukaryotic translation initiation factor 2α (eIF2α). Salubrinal can protect cardiomyocytes from doxorubicin-induced apoptosis through its effects on eIF2α. It possibly ameliorates cardiac toxicity and can be used in clinical practice. PMID:26505387

  8. MiR-25 protects cardiomyocytes against oxidative damage by targeting the mitochondrial calcium uniporter.

    PubMed

    Pan, Lei; Huang, Bi-Jun; Ma, Xiu-E; Wang, Shi-Yi; Feng, Jing; Lv, Fei; Liu, Yuan; Liu, Yi; Li, Chang-Ming; Liang, Dan-Dan; Li, Jun; Xu, Liang; Chen, Yi-Han

    2015-03-10

    MicroRNAs (miRNAs) are a class of small non-coding RNAs, whose expression levels vary in different cell types and tissues. Emerging evidence indicates that tissue-specific and -enriched miRNAs are closely associated with cellular development and stress responses in their tissues. MiR-25 has been documented to be abundant in cardiomyocytes, but its function in the heart remains unknown. Here, we report that miR-25 can protect cardiomyocytes against oxidative damage by down-regulating mitochondrial calcium uniporter (MCU). MiR-25 was markedly elevated in response to oxidative stimulation in cardiomyocytes. Further overexpression of miR-25 protected cardiomyocytes against oxidative damage by inactivating the mitochondrial apoptosis pathway. MCU was identified as a potential target of miR-25 by bioinformatical analysis. MCU mRNA level was reversely correlated with miR-25 under the exposure of H2O2, and MCU protein level was largely decreased by miR-25 overexpression. The luciferase reporter assay confirmed that miR-25 bound directly to the 3' untranslated region (UTR) of MCU mRNA. MiR-25 significantly decreased H2O2-induced elevation of mitochondrial Ca2+ concentration, which is likely to be the result of decreased activity of MCU. We conclude that miR-25 targets MCU to protect cardiomyocytes against oxidative damages. This finding provides novel insights into the involvement of miRNAs in oxidative stress in cardiomyocytes.

  9. NFAT isoforms play distinct roles in TNFα-induced retinal leukostasis.

    PubMed

    Bretz, Colin A; Savage, Sara R; Capozzi, Megan E; Suarez, Sandra; Penn, John S

    2015-11-03

    The objective of this study was to determine the role of individual NFAT isoforms in TNFα-induced retinal leukostasis. To this end, human retinal microvascular endothelial cells (HRMEC) transfected with siRNA targeting individual NFAT isoforms were treated with TNFα, and qRT-PCR was used to examine the contribution of each isoform to the TNFα-induced upregulation of leukocyte adhesion proteins. This showed that NFATc1 siRNA increased ICAM1 expression, NFATc2 siRNA reduced CX3CL1, VCAM1, SELE, and ICAM1 expression, NFATc3 siRNA increased CX3CL1 and SELE expression, and NFATc4 siRNA reduced SELE expression. Transfected HRMEC monolayers were also treated with TNFα and assayed using a parallel plate flow chamber, and both NFATc2 and NFATc4 knockdown reduced TNFα-induced cell adhesion. The effect of isoform-specific knockdown on TNFα-induced cytokine production was also measured using protein ELISAs and conditioned cell culture medium, and showed that NFATc4 siRNA reduced CXCL10, CXCL11, and MCP-1 protein levels. Lastly, the CN/NFAT-signaling inhibitor INCA-6 was shown to reduce TNFα-induced retinal leukostasis in vivo. Together, these studies show a clear role for NFAT-signaling in TNFα-induced retinal leukostasis, and identify NFATc2 and NFATc4 as potentially valuable therapeutic targets for treating retinopathies in which TNFα plays a pathogenic role.

  10. NFAT isoforms play distinct roles in TNFα-induced retinal leukostasis

    PubMed Central

    Bretz, Colin A.; Savage, Sara R.; Capozzi, Megan E.; Suarez, Sandra; Penn, John S.

    2015-01-01

    The objective of this study was to determine the role of individual NFAT isoforms in TNFα-induced retinal leukostasis. To this end, human retinal microvascular endothelial cells (HRMEC) transfected with siRNA targeting individual NFAT isoforms were treated with TNFα, and qRT-PCR was used to examine the contribution of each isoform to the TNFα-induced upregulation of leukocyte adhesion proteins. This showed that NFATc1 siRNA increased ICAM1 expression, NFATc2 siRNA reduced CX3CL1, VCAM1, SELE, and ICAM1 expression, NFATc3 siRNA increased CX3CL1 and SELE expression, and NFATc4 siRNA reduced SELE expression. Transfected HRMEC monolayers were also treated with TNFα and assayed using a parallel plate flow chamber, and both NFATc2 and NFATc4 knockdown reduced TNFα-induced cell adhesion. The effect of isoform-specific knockdown on TNFα-induced cytokine production was also measured using protein ELISAs and conditioned cell culture medium, and showed that NFATc4 siRNA reduced CXCL10, CXCL11, and MCP-1 protein levels. Lastly, the CN/NFAT-signaling inhibitor INCA-6 was shown to reduce TNFα-induced retinal leukostasis in vivo. Together, these studies show a clear role for NFAT-signaling in TNFα-induced retinal leukostasis, and identify NFATc2 and NFATc4 as potentially valuable therapeutic targets for treating retinopathies in which TNFα plays a pathogenic role. PMID:26527057

  11. Influence of the availability of iron during hypoxia on the genes associated with apoptotic activity and local iron metabolism in rat H9C2 cardiomyocytes and L6G8C5 skeletal myocytes.

    PubMed

    Dziegala, Magdalena; Kasztura, Monika; Kobak, Kamil; Bania, Jacek; Banasiak, Waldemar; Ponikowski, Piotr; Jankowska, Ewa A

    2016-10-01

    The differential availability of iron during hypoxia is presumed to affect the functioning of cardiac and skeletal myocytes. Rat H9C2 cardiomyocytes and L6G8C5 myocytes were cultured for 48 h in normoxic or hypoxic conditions at the optimal, reduced or increased iron concentration. The mRNA expression levels of markers of apoptosis [B‑cell lymphoma‑2 (Bcl2; inhibition) and Bcl‑2‑activated X protein (Bax; induction)], atrophy (Atrogin), glycolysis (pyruvate kinase 2; PKM2) and iron metabolism [transferrin receptor 1 (TfR1; iron importer), ferroportin 1 (FPN1; iron exporter), ferritin heavy chain (FTH; iron storage protein) and hepcidin (HAMP; iron regulator)] were determined using reverse transcription‑quantitative polymerase chain reaction, and cell viability was measured using an tetrazolium reduction assay. Cardiomyocytes and myocytes, when exposed to hypoxia, demonstrated an increased Bax/Bcl‑2 gene expression ratio (P<0.05). Additional deferoxamine (DFO) treatment resulted in further increases in Bax/Bcl‑2 in each cell type (P<0.001 each) and this was associated with the 15% loss in viability. The analogous alterations were observed in both cell types upon ammonium ferric citrate (AFC) treatment during hypoxia; however, the increased Bax/Bcl‑2 ratio and associated viability loss was lower compared with that in case of DFO treatment (P<0.05 each). Under hypoxic conditions, myocytes demonstrated an increased expression of PKM2 (P<0.01). Additional DFO treatment caused an increase in the mRNA expression levels of PKM2 and Atrogin‑1 (P<0.001 and P<0.05, respectively), whereas AFC treatment caused an increased mRNA expression of PKM2 (P<0.01) and accompanied decreased mRNA expression of Atrogin‑1 (P<0.05). The expression augmentation of PKM2 during hypoxia was greater upon low iron compared with that of ferric salt treatment (P<0.01). Both cell types upon DFO during hypoxia demonstrated the increased expression of TfR1

  12. Influence of the availability of iron during hypoxia on the genes associated with apoptotic activity and local iron metabolism in rat H9C2 cardiomyocytes and L6G8C5 skeletal myocytes.

    PubMed

    Dziegala, Magdalena; Kasztura, Monika; Kobak, Kamil; Bania, Jacek; Banasiak, Waldemar; Ponikowski, Piotr; Jankowska, Ewa A

    2016-10-01

    The differential availability of iron during hypoxia is presumed to affect the functioning of cardiac and skeletal myocytes. Rat H9C2 cardiomyocytes and L6G8C5 myocytes were cultured for 48 h in normoxic or hypoxic conditions at the optimal, reduced or increased iron concentration. The mRNA expression levels of markers of apoptosis [B‑cell lymphoma‑2 (Bcl2; inhibition) and Bcl‑2‑activated X protein (Bax; induction)], atrophy (Atrogin), glycolysis (pyruvate kinase 2; PKM2) and iron metabolism [transferrin receptor 1 (TfR1; iron importer), ferroportin 1 (FPN1; iron exporter), ferritin heavy chain (FTH; iron storage protein) and hepcidin (HAMP; iron regulator)] were determined using reverse transcription‑quantitative polymerase chain reaction, and cell viability was measured using an tetrazolium reduction assay. Cardiomyocytes and myocytes, when exposed to hypoxia, demonstrated an increased Bax/Bcl‑2 gene expression ratio (P<0.05). Additional deferoxamine (DFO) treatment resulted in further increases in Bax/Bcl‑2 in each cell type (P<0.001 each) and this was associated with the 15% loss in viability. The analogous alterations were observed in both cell types upon ammonium ferric citrate (AFC) treatment during hypoxia; however, the increased Bax/Bcl‑2 ratio and associated viability loss was lower compared with that in case of DFO treatment (P<0.05 each). Under hypoxic conditions, myocytes demonstrated an increased expression of PKM2 (P<0.01). Additional DFO treatment caused an increase in the mRNA expression levels of PKM2 and Atrogin‑1 (P<0.001 and P<0.05, respectively), whereas AFC treatment caused an increased mRNA expression of PKM2 (P<0.01) and accompanied decreased mRNA expression of Atrogin‑1 (P<0.05). The expression augmentation of PKM2 during hypoxia was greater upon low iron compared with that of ferric salt treatment (P<0.01). Both cell types upon DFO during hypoxia demonstrated the increased expression of TfR1

  13. Prostaglandin F2α-F-prostanoid receptor regulates CXCL8 expression in endometrial adenocarcinoma cells via the calcium–calcineurin–NFAT pathway

    PubMed Central

    Sales, Kurt J.; Maldonado-Pérez, David; Grant, Vivien; Catalano, Rob D.; Wilson, Martin R.; Brown, Pamela; Williams, Alistair R.W.; Anderson, Richard A.; Thompson, E. Aubrey; Jabbour, Henry N.

    2009-01-01

    Pro-inflammatory mediators, like prostaglandin (PG) and chemokines, promote tumourigenesis by enhancing cell proliferation, migration of immune cells and recruitment of blood vessels. Recently we showed elevated expression of the chemokine (C-X-C motif) receptor 2 (CXCR2) in endometrial adenocarcinomas localized to neutrophils and neoplastic epithelial and vascular cells. Furthermore we found that PGF2α-F-prostanoid (FP) receptor regulates the expression of the CXCR2 ligand CXCL1, to promote neutrophil chemotaxis in endometrial adenocarcinomas. In the present study we identified another CXCR2 ligand, CXCL8 as a target for PGF2α-FP receptor signalling which enhances epithelial cell proliferation in endometrial adenocarcinoma cells in vitro and in nude mice in vivo. We found that PGF2α-FP receptor interaction induces CXCL8 expression in endometrial adenocarcinoma cells via the protein kinase C–calcium–calcineurin–NFAT signaling pathway. Promoter analysis revealed that CXCL8 transcriptional activation by PGF2α signaling is mediated by cooperative interactions between the AP1 and NFAT binding sites. Furthermore, PGF2α via the FP receptor induced the expression of the regulator of calcineurin 1 isoform 4 (RCAN1-4) via the calcineurin/NFAT pathway in a reciprocal manner to CXCL8. Using an adenovirus to overexpress RCAN1-4, we found that RCAN1-4 is a negative regulator of CXCL8 expression in endometrial adenocarcinoma cells. Taken together our data have elucidated the molecular and cellular mechanism whereby PGF2α regulates CXCL8 expression via the FP receptor in endometrial adenocarcinomas and have highlighted RCAN1-4 as a negative regulator of CXCL8 expression which may be exploited therapeutically to inhibit CXCL8-mediated tumour development. PMID:19819266

  14. Regulation of the pro-inflammatory cytokine osteopontin by GIP in adipocytes - A role for the transcription factor NFAT and phosphodiesterase 3B

    SciTech Connect

    Omar, Bilal; Banke, Elin; Guirguis, Emilia; Aakesson, Lina; Manganiello, Vincent; Lyssenko, Valeriya; Groop, Leif; Gomez, Maria F.; Degerman, Eva

    2012-09-07

    Highlights: Black-Right-Pointing-Pointer GIP stimulates lipogenesis and osteopontin expression in primary adipocytes. Black-Right-Pointing-Pointer GIP-induced osteopontin expression is NFAT-dependent. Black-Right-Pointing-Pointer Osteopontin expression is PDE3-dependent. Black-Right-Pointing-Pointer Osteopontin expression is increased in PDE3B KO mice. -- Abstract: The incretin - glucose-dependent insulinotropic polypeptide (GIP) - and the pro-inflammatory cytokine osteopontin are known to have important roles in the regulation of adipose tissue functions. In this work we show that GIP stimulates lipogenesis and osteopontin expression in primary adipocytes. The GIP-induced increase in osteopontin expression was inhibited by the NFAT (the transcription factor nuclear factor of activated T-cells) inhibitor A-285222. Also, the NFAT kinase glycogen synthase kinase (GSK) 3 was upregulated by GIP. To test whether cAMP might be involved in GIP-mediated effects on osteopontin a number of strategies were used. Thus, the {beta}3-adrenergic receptor agonist CL316,243 stimulated osteopontin expression, an effects which was mimicked by OPC3911, a specific inhibitor of phosphodiesterase 3. Furthermore, treatment of phosphodiesterase 3B knock-out mice with CL316,243 resulted in a dramatic upregulation of osteopontin in adipose tissue which was not the case in wild-type mice. In summary, we delineate mechanisms by which GIP stimulates osteopontin in adipocytes. Given the established link between osteopontin and insulin resistance, our data suggest that GIP by stimulating osteopontin expression, also could promote insulin resistance in adipocytes.

  15. Effect of biophysical cues on reprogramming to cardiomyocytes.

    PubMed

    Sia, Junren; Yu, Pengzhi; Srivastava, Deepak; Li, Song

    2016-10-01

    Reprogramming of fibroblasts to cardiomyocytes offers exciting potential in cell therapy and regenerative medicine, but has low efficiency. We hypothesize that physical cues may positively affect the reprogramming process, and studied the effects of periodic mechanical stretch, substrate stiffness and microgrooved substrate on reprogramming yield. Subjecting reprogramming fibroblasts to periodic mechanical stretch and different substrate stiffness did not improve reprogramming yield. On the other hand, culturing the cells on microgrooved substrate enhanced the expression of cardiomyocyte genes by day 2 and improved the yield of partially reprogrammed cells at day 10. By combining microgrooved substrate with an existing optimized culture protocol, yield of reprogrammed cardiomyocytes with striated cardiac troponin T staining and spontaneous contractile activity was increased. We identified the regulation of Mkl1 activity as a new mechanism by which microgroove can affect reprogramming. Biochemical approach could only partially recapitulate the effect of microgroove. Microgroove demonstrated an additional effect of enhancing organization of sarcomeric structure, which could not be recapitulated by biochemical approach. This study provides insights into new mechanisms by which topographical cues can affect cellular reprogramming.

  16. Transient receptor potential channel 1/4 reduces subarachnoid hemorrhage-induced early brain injury in rats via calcineurin-mediated NMDAR and NFAT dephosphorylation

    PubMed Central

    Wang, Zhong; Wang, Yibin; Tian, Xiaodi; Shen, Haitao; Dou, Yang; Li, Haiying; Chen, Gang

    2016-01-01

    Transient receptor potential channel 1/4 (TRPC1/4) are considered to be related to subarachnoid hemorrhage (SAH)-induced cerebral vasospasm. In this study, a SAH rat model was employed to study the roles of TRPC1/4 in the early brain injury (EBI) after SAH. Primary cultured hippocampal neurons were exposed to oxyhemoglobin to mimic SAH in vitro. The protein levels of TRPC1/4 increased and peaked at 5 days after SAH in rats. Inhibition of TRPC1/4 by SKF96365 aggravated SAH-induced EBI, such as cortical cell death (by TUNEL staining) and degenerating (by FJB staining). In addition, TRPC1/4 overexpression could increase calcineurin activity, while increased calcineurin activity could promote the dephosphorylation of N-methyl-D-aspartate receptor (NMDAR). Calcineurin antagonist FK506 could weaken the neuroprotection and the dephosphorylation of NMDAR induced by TRPC1/4 overexpression. Contrarily, calcineurin agonist chlorogenic acid inhibited SAH-induced EBI, even when siRNA intervention of TRPC1/4 was performed. Moreover, calcineurin also could lead to the nuclear transfer of nuclear factor of activated T cells (NFAT), which is a transcription factor promoting the expressions of TRPC1/4. TRPC1/4 could inhibit SAH-induced EBI by supressing the phosphorylation of NMDAR via calcineurin. TRPC1/4-induced calcineurin activation also could promote the nuclear transfer of NFAT, suggesting a positive feedback regulation of TRPC1/4 expressions. PMID:27641617

  17. Transient receptor potential channel 1/4 reduces subarachnoid hemorrhage-induced early brain injury in rats via calcineurin-mediated NMDAR and NFAT dephosphorylation.

    PubMed

    Wang, Zhong; Wang, Yibin; Tian, Xiaodi; Shen, Haitao; Dou, Yang; Li, Haiying; Chen, Gang

    2016-01-01

    Transient receptor potential channel 1/4 (TRPC1/4) are considered to be related to subarachnoid hemorrhage (SAH)-induced cerebral vasospasm. In this study, a SAH rat model was employed to study the roles of TRPC1/4 in the early brain injury (EBI) after SAH. Primary cultured hippocampal neurons were exposed to oxyhemoglobin to mimic SAH in vitro. The protein levels of TRPC1/4 increased and peaked at 5 days after SAH in rats. Inhibition of TRPC1/4 by SKF96365 aggravated SAH-induced EBI, such as cortical cell death (by TUNEL staining) and degenerating (by FJB staining). In addition, TRPC1/4 overexpression could increase calcineurin activity, while increased calcineurin activity could promote the dephosphorylation of N-methyl-D-aspartate receptor (NMDAR). Calcineurin antagonist FK506 could weaken the neuroprotection and the dephosphorylation of NMDAR induced by TRPC1/4 overexpression. Contrarily, calcineurin agonist chlorogenic acid inhibited SAH-induced EBI, even when siRNA intervention of TRPC1/4 was performed. Moreover, calcineurin also could lead to the nuclear transfer of nuclear factor of activated T cells (NFAT), which is a transcription factor promoting the expressions of TRPC1/4. TRPC1/4 could inhibit SAH-induced EBI by supressing the phosphorylation of NMDAR via calcineurin. TRPC1/4-induced calcineurin activation also could promote the nuclear transfer of NFAT, suggesting a positive feedback regulation of TRPC1/4 expressions. PMID:27641617

  18. Transient receptor potential channel 1/4 reduces subarachnoid hemorrhage-induced early brain injury in rats via calcineurin-mediated NMDAR and NFAT dephosphorylation.

    PubMed

    Wang, Zhong; Wang, Yibin; Tian, Xiaodi; Shen, Haitao; Dou, Yang; Li, Haiying; Chen, Gang

    2016-01-01

    Transient receptor potential channel 1/4 (TRPC1/4) are considered to be related to subarachnoid hemorrhage (SAH)-induced cerebral vasospasm. In this study, a SAH rat model was employed to study the roles of TRPC1/4 in the early brain injury (EBI) after SAH. Primary cultured hippocampal neurons were exposed to oxyhemoglobin to mimic SAH in vitro. The protein levels of TRPC1/4 increased and peaked at 5 days after SAH in rats. Inhibition of TRPC1/4 by SKF96365 aggravated SAH-induced EBI, such as cortical cell death (by TUNEL staining) and degenerating (by FJB staining). In addition, TRPC1/4 overexpression could increase calcineurin activity, while increased calcineurin activity could promote the dephosphorylation of N-methyl-D-aspartate receptor (NMDAR). Calcineurin antagonist FK506 could weaken the neuroprotection and the dephosphorylation of NMDAR induced by TRPC1/4 overexpression. Contrarily, calcineurin agonist chlorogenic acid inhibited SAH-induced EBI, even when siRNA intervention of TRPC1/4 was performed. Moreover, calcineurin also could lead to the nuclear transfer of nuclear factor of activated T cells (NFAT), which is a transcription factor promoting the expressions of TRPC1/4. TRPC1/4 could inhibit SAH-induced EBI by supressing the phosphorylation of NMDAR via calcineurin. TRPC1/4-induced calcineurin activation also could promote the nuclear transfer of NFAT, suggesting a positive feedback regulation of TRPC1/4 expressions.

  19. Cytoprotective and Cytotoxic Effects of Rice Bran Extracts in Rat H9c2(2-1) Cardiomyocytes

    PubMed Central

    Tan, Xian Wen; Bhave, Mrinal; Fong, Alan Yean Yip; Matsuura, Eiji; Kobayashi, Kazuko; Shen, Lian Hua; Hwang, Siaw San

    2016-01-01

    This study was aimed at preliminarily assessing the cytoprotective and antioxidative effects of rice bran extracts (RBEs) from a Sarawak local rice variety (local name: “BJLN”) and a commercial rice variety, “MR219,” on oxidative stress in rat H9c2(2-1) cardiomyocytes. The cardiomyocytes were incubated with different concentrations of RBE and hydrogen peroxide (H2O2), respectively, to identify their respective IC50 values and safe dose ranges. Two nonlethal and close-to-IC50 doses of RBE were selected to evaluate their respective effects on H2O2 induced oxidative stress in cardiomyocytes. Both RBEs showed dose-dependent cytotoxicity effects on cardiomyocytes. H2O2 induction of cardiomyocytes pretreated with RBE further revealed the dose-dependent cytoprotective and antioxidative effects of RBE via an increase in IC50 values of H2O2. Preliminary analyses of induction effects of RBE and H2O2 on cellular antioxidant enzyme, catalase (CAT), also revealed their potential in regulating these activities and expression profile of related gene on oxidative stress in cardiomyocytes. Pretreated cardiomyocytes significantly upregulated the enzymatic activity and expression level of CAT under the exposure of H2O2 induced oxidative stress. This preliminary study has demonstrated the potential antioxidant effects of RBE in alleviating H2O2-mediated oxidative injuries via upregulation in enzymatic activities and expression levels of CAT. PMID:27239253

  20. Cytoprotective and Cytotoxic Effects of Rice Bran Extracts in Rat H9c2(2-1) Cardiomyocytes.

    PubMed

    Tan, Xian Wen; Bhave, Mrinal; Fong, Alan Yean Yip; Matsuura, Eiji; Kobayashi, Kazuko; Shen, Lian Hua; Hwang, Siaw San

    2016-01-01

    This study was aimed at preliminarily assessing the cytoprotective and antioxidative effects of rice bran extracts (RBEs) from a Sarawak local rice variety (local name: "BJLN") and a commercial rice variety, "MR219," on oxidative stress in rat H9c2(2-1) cardiomyocytes. The cardiomyocytes were incubated with different concentrations of RBE and hydrogen peroxide (H2O2), respectively, to identify their respective IC50 values and safe dose ranges. Two nonlethal and close-to-IC50 doses of RBE were selected to evaluate their respective effects on H2O2 induced oxidative stress in cardiomyocytes. Both RBEs showed dose-dependent cytotoxicity effects on cardiomyocytes. H2O2 induction of cardiomyocytes pretreated with RBE further revealed the dose-dependent cytoprotective and antioxidative effects of RBE via an increase in IC50 values of H2O2. Preliminary analyses of induction effects of RBE and H2O2 on cellular antioxidant enzyme, catalase (CAT), also revealed their potential in regulating these activities and expression profile of related gene on oxidative stress in cardiomyocytes. Pretreated cardiomyocytes significantly upregulated the enzymatic activity and expression level of CAT under the exposure of H2O2 induced oxidative stress. This preliminary study has demonstrated the potential antioxidant effects of RBE in alleviating H2O2-mediated oxidative injuries via upregulation in enzymatic activities and expression levels of CAT. PMID:27239253

  1. Transcriptional Regulation of BK Virus by Nuclear Factor of Activated T Cells▿

    PubMed Central

    Jordan, Joslynn A.; Manley, Kate; Dugan, Aisling S.; O'Hara, Bethany A.; Atwood, Walter J.

    2010-01-01

    The human polyomavirus BK virus (BKV) is a common virus for which 80 to 90% of the adult population is seropositive. BKV reactivation in immunosuppressed patients or renal transplant patients is the primary cause of polyomavirus-associated nephropathy (PVN). Using the Dunlop strain of BKV, we found that nuclear factor of activated T cells (NFAT) plays an important regulatory role in BKV infection. Luciferase reporter assays and chromatin immunoprecipitation assays demonstrated that NFAT4 bound to the viral promoter and regulated viral transcription and infection. The mutational analysis of the NFAT binding sites demonstrated complex functional interactions between NFAT, c-fos, c-jun, and the p65 subunit of NF-κB that together influence promoter activity and viral growth. These data indicate that NFAT is required for BKV infection and is involved in a complex regulatory network that both positively and negatively influences promoter activity and viral infection. PMID:19955309

  2. Hydrogen peroxide-induced necrotic cell death in cardiomyocytes is independent of matrix metalloproteinase-2.

    PubMed

    Ali, Mohammad A M; Kandasamy, Arulmozhi D; Fan, Xiaohu; Schulz, Richard

    2013-09-01

    Matrix metalloproteinase-2 (MMP-2) is well known to proteolyse both extracellular and intracellular proteins. Reactive oxygen species activate MMP-2 at both transcriptional and post-translational levels, thus MMP-2 activation is considered an early event in oxidative stress injury. Although hydrogen peroxide is widely used to trigger oxidative stress-induced cell death, the type of cell death (apoptosis vs. necrosis) in cardiomyocytes is still controversial depending on the concentration used and the exposure time. We carefully investigated the mode of cell death in neonatal rat cardiomyocytes induced by different concentrations (50-500 μM) of hydrogen peroxide at various time intervals after exposure and determined whether MMP-2 is implicated in hydrogen peroxide-induced cardiomyocyte death. Treating cardiomyocytes with hydrogen peroxide led to elevated MMP-2 level/activity with maximal effects seen at 200 μM. Hydrogen peroxide caused necrotic cell death by disrupting the plasmalemma as evidenced by the release of lactate dehydrogenase in a concentration- and time-dependent manner as well as the necrotic cleavage of PARP-1. The absence of both caspase-3 cleavage/activation and apoptotic cleavage of PARP-1 illustrated the weak contribution of apoptosis. Pre-treatment with selective MMP inhibitors did not protect against hydrogen peroxide-induced necrosis. In conclusion hydrogen peroxide increases MMP-2 level/activity in cardiomyocytes and induces necrotic cell death, however, the later effect is MMP-2 independent.

  3. HMGB1 mediates hyperglycaemia-induced cardiomyocyte apoptosis via ERK/Ets-1 signalling pathway.

    PubMed

    Wang, Wen-Ke; Lu, Qing-Hua; Zhang, Jia-Ning; Wang, Ben; Liu, Xiang-Juan; An, Feng-Shuang; Qin, Wei-Dong; Chen, Xue-Ying; Dong, Wen-Qian; Zhang, Cheng; Zhang, Yun; Zhang, Ming-Xiang

    2014-11-01

    Apoptosis is a key event involved in diabetic cardiomyopathy. The expression of high mobility group box 1 protein (HMGB1) is up-regulated in diabetic mice. However, the molecular mechanism of high glucose (HG)-induced cardiomyocyte apoptosis remains obscure. We aimed to determine the role of HMGB1 in HG-induced apoptosis of cardiomyocytes. Treating neonatal primary cardiomyocytes with HG increased cell apoptosis, which was accompanied by elevated levels of HMGB1. Inhibition of HMGB1 by short-hairpin RNA significantly decreased HG-induced cell apoptosis by reducing caspase-3 activation and ratio of Bcl2-associated X protein to B-cell lymphoma/leukemia-2 (bax/bcl-2). Furthermore, HG activated E26 transformation-specific sequence-1 (Ets-1), and HMGB1 inhibition attenuated HG-induced activation of Ets-1 via extracellular signal-regulated kinase 1/2 (ERK1/2) signalling. In addition, inhibition of Ets-1 significantly decreased HG-induced cardiomyocyte apoptosis. Similar results were observed in streptozotocin-treated diabetic mice. Inhibition of HMGB1 by short-hairpin RNA markedly decreased myocardial cell apoptosis and activation of ERK and Ets-1 in diabetic mice. In conclusion, inhibition of HMGB1 may protect against hyperglycaemia-induced cardiomyocyte apoptosis by down-regulating ERK-dependent activation of Ets-1.

  4. Isolation and Physiological Analysis of Mouse Cardiomyocytes

    PubMed Central

    Roth, Gretchen M.; Bader, David M.; Pfaltzgraff, Elise R.

    2014-01-01

    Cardiomyocytes, the workhorse cell of the heart, contain exquisitely organized cytoskeletal and contractile elements that generate the contractile force used to pump blood. Individual cardiomyocytes were first isolated over 40 years ago in order to better study the physiology and structure of heart muscle. Techniques have rapidly improved to include enzymatic digestion via coronary perfusion. More recently, analyzing the contractility and calcium flux of isolated myocytes has provided a vital tool in the cellular and sub-cellular analysis of heart failure. Echocardiography and EKGs provide information about the heart at an organ level only. Cardiomyocyte cell culture systems exist, but cells lack physiologically essential structures such as organized sarcomeres and t-tubules required for myocyte function within the heart. In the protocol presented here, cardiomyocytes are isolated via Langendorff perfusion. The heart is removed from the mouse, mounted via the aorta to a cannula, perfused with digestion enzymes, and cells are introduced to increasing calcium concentrations. Edge and sarcomere detection software is used to analyze contractility, and a calcium binding fluorescent dye is used to visualize calcium transients of electrically paced cardiomyocytes; increasing understanding of the role cellular changes play in heart dysfunction. Traditionally used to test drug effects on cardiomyocytes, we employ this system to compare myocytes from WT mice and mice with a mutation that causes dilated cardiomyopathy. This protocol is unique in its comparison of live cells from mice with known heart function and known genetics. Many experimental conditions are reliably compared, including genetic or environmental manipulation, infection, drug treatment, and more. Beyond physiologic data, isolated cardiomyocytes are easily fixed and stained for cytoskeletal elements. Isolating cardiomyocytes via perfusion is an extremely versatile method, useful in studying cellular changes

  5. BMPER regulates cardiomyocyte size and vessel density in vivo

    PubMed Central

    Willis, Monte S.; Dyer, Laura A.; Ren, Rongqin; Lockyer, Pamela; Moreno-Miralles, Isabel; Schisler, Jonathan C.; Patterson, Cam

    2016-01-01

    Background BMPER, an orthologue of Drosophila melanogaster Crossveinless-2, is a secreted factor that regulates bone morphogenetic protein activity in endothelial cell precursors and during early cardiomyocyte differentiation. Although previously described in the heart, the role of BMPER in cardiac development and function remain unknown. Methods BMPER-deficient hearts were phenotyped histologically and functionally using echocardiography and Doppler analysis. Since BMPER −/− mice die perinatally, adult BMPER +/− mice were challenged to pressure-overload-induced cardiac hypertrophy and hindlimb ischemia to determine changes in angiogenesis and regulation of cardiomyocyte size. Results We identify for the first time the cardiac phenotype associated with BMPER haploinsufficiency. BMPER messenger RNA and protein are present in the heart during cardiac development through at least E14.5 but is lost by E18.5. BMPER +/− ventricles are thinner and less compact than sibling wild-type hearts. In the adult, BMPER +/− hearts present with decreased anterior and posterior wall thickness, decreased cardiomyocyte size and an increase in cardiac vessel density. Despite these changes, BMPER +/− mice respond to pressure-overload-induced cardiac hypertrophy challenge largely to the same extent as wild-type mice. Conclusion BMPER appears to play a role in regulating both vessel density and cardiac development in vivo; however, BMPER haploinsufficiency does not result in marked effects on cardiac function or adaptation to pressure overload hypertrophy. PMID:23200275

  6. Cardiomyocyte specific deletion of PP2A causes cardiac hypertrophy

    PubMed Central

    Li, Lei; Fang, Chao; Xu, Di; Xu, Yidan; Fu, Heling; Li, Jianmin

    2016-01-01

    Cardiac hypertrophy is a common pathological alteration in heart disease, which has been reported to be connected with serine/threonine protein phosphatases that control the dephosphorylation of a variety of cardiac proteins. Herein, we generated protein phosphatase type 2A knockout expressing a tamoxifen-inducible Cre recombinase protein fused to two mutant estrogen-receptor ligand-binding domains (MerCreMer) under the control of the a-myosin heavy chain promoter. Cardiac function of mice was determined by echocardiography. Decrease in PP2A activity leads to increased cardiomyocyte hypertrophy and fibrosis. Loss of PP2ACα leads to the heart failure, including the changes of EF, FS, LV, ANP and BNP. On the molecular level, knockout mice shows increased expression of B55a and B56e at 60 days after tamoxifen injection. Additionally, the regulation of the Akt/GSK3β/β-catenin pathway is severely disturbed in knockout mice. In conclusion, cardiomyocyte specific deletion of PP2A gene causes the cardiac hypertrophy. We will use the knockout mice to generate a type of cardiomyocyte hypertrophy mouse model with myocardial fibrosis. PMID:27186301

  7. Calcium-mediated histone modifications regulate alternative splicing in cardiomyocytes

    PubMed Central

    Sharma, Alok; Nguyen, Hieu; Geng, Cuiyu; Hinman, Melissa N.; Luo, Guangbin; Lou, Hua

    2014-01-01

    In cardiomyocytes, calcium is known to control gene expression at the level of transcription, whereas its role in regulating alternative splicing has not been explored. Here we report that, in mouse primary or embryonic stem cell-derived cardiomyocytes, increased calcium levels induce robust and reversible skipping of several alternative exons from endogenously expressed genes. Interestingly, we demonstrate a calcium-mediated splicing regulatory mechanism that depends on changes of histone modifications. Specifically, the regulation occurs through changes in calcium-responsive kinase activities that lead to alterations in histone modifications and subsequent changes in the transcriptional elongation rate and exon skipping. We demonstrate that increased intracellular calcium levels lead to histone hyperacetylation along the body of the genes containing calcium-responsive alternative exons by disrupting the histone deacetylase-to-histone acetyltransferase balance in the nucleus. Consequently, the RNA polymerase II elongation rate increases significantly on those genes, resulting in skipping of the alternative exons. These studies reveal a mechanism by which calcium-level changes in cardiomyocytes impact on the output of gene expression through altering alternative pre-mRNA splicing patterns. PMID:25368158

  8. Cation dyshomeostasis and cardiomyocyte necrosis: the Fleckenstein hypothesis revisited

    PubMed Central

    Borkowski, Brian J.; Cheema, Yaser; Shahbaz, Atta U.; Bhattacharya, Syamal K.; Weber, Karl T.

    2011-01-01

    An ongoing loss of cardiomyocytes to apoptotic and necrotic cell death pathways contributes to the progressive nature of heart failure. The pathophysiological origins of necrotic cell loss relate to the neurohormonal activation that accompanies acute and chronic stressor states and which includes effector hormones of the adrenergic nervous system. Fifty years ago, Albrecht Fleckenstein and coworkers hypothesized the hyperadrenergic state, which accompanies such stressors, causes cardiomyocyte necrosis based on catecholamine-initiated excessive intracellular Ca2+ accumulation (EICA), and mitochondrial Ca2+ overloading in particular, in which the ensuing dysfunction and structural degeneration of these organelles leads to necrosis. In recent years, two downstream factors have been identified which, together with EICA, constitute a signal–transducer–effector pathway: (i) mitochondria-based induction of oxidative stress, in which the rate of reactive oxygen metabolite generation exceeds their rate of detoxification by endogenous antioxidant defences; and (ii) the opening of the mitochondrial inner membrane permeability transition pore (mPTP) followed by organellar swelling and degeneration. The pathogenesis of stress-related cardiomyopathy syndromes is likely related to this pathway. Other factors which can account for cytotoxicity in stressor states include: hypokalaemia; ionized hypocalcaemia and hypomagnesaemia with resultant elevations in parathyroid hormone serving as a potent mediator of EICA; and hypozincaemia with hyposelenaemia, which compromise antioxidant defences. Herein, we revisit the Fleckenstein hypothesis of EICA in leading to cardiomyocyte necrosis and the central role played by mitochondria. PMID:21398641

  9. Signaling Pathways and Gene Regulatory Networks in Cardiomyocyte Differentiation

    PubMed Central

    Parikh, Abhirath; Wu, Jincheng; Blanton, Robert M.

    2015-01-01

    Strategies for harnessing stem cells as a source to treat cell loss in heart disease are the subject of intense research. Human pluripotent stem cells (hPSCs) can be expanded extensively in vitro and therefore can potentially provide sufficient quantities of patient-specific differentiated cardiomyocytes. Although multiple stimuli direct heart development, the differentiation process is driven in large part by signaling activity. The engineering of hPSCs to heart cell progeny has extensively relied on establishing proper combinations of soluble signals, which target genetic programs thereby inducing cardiomyocyte specification. Pertinent differentiation strategies have relied as a template on the development of embryonic heart in multiple model organisms. Here, information on the regulation of cardiomyocyte development from in vivo genetic and embryological studies is critically reviewed. A fresh interpretation is provided of in vivo and in vitro data on signaling pathways and gene regulatory networks (GRNs) underlying cardiopoiesis. The state-of-the-art understanding of signaling pathways and GRNs presented here can inform the design and optimization of methods for the engineering of tissues for heart therapies. PMID:25813860

  10. Stimulation of protein synthesis by phosphatidic acid in rat cardiomyocytes.

    PubMed

    Xu, Y J; Yau, L; Yu, L P; Elimban, V; Zahradka, P; Dhalla, N S

    1996-12-13

    Phosphatidic acid (PA) was observed to stimulate protein synthesis in adult cardiomyocytes in a time- and concentration-dependent manner. The maximal stimulation in protein synthesis (142 +/- 12% vs 100% as the control) was achieved at 10 microM PA within 60 min and was inhibited by actinomycin D (107 +/- 4% of the control) or cycloheximide (105 +/- 6% of the control). The increase in protein synthesis due to PA was attenuated or abolished by preincubation of cardiomyocytes with a tyrosine kinase inhibitor, genistein (94 +/- 9% of the control), phospholipase C inhibitors 2-nitro-4-carboxyphenyl N,N-diphenyl carbamate or carbon-odithioic acid O-(octahydro-4,7-methanol-1H-inden-5-yl (101 +/- 6 and 95 +/- 5% of the control, respectively), protein kinase C inhibitors staurosporine or polymyxin B (109 +/- 3 and 93 +/- 3% of the control), and chelators of extracellular and intracellular free Ca2+ EGTA or BAPTA/AM (103 +/- 6 and 95 +/- 6% of the control, respectively). PA at different concentrations (0.1 to 100 microM) also caused phosphorylation of a cell surface protein of approximately 24 kDa. In addition, mitogen-activated protein kinase was stimulated by PA in a concentration-dependent manner; maximal stimulation (217 +/- 6% of the control) was seen at 10 microM PA. These data suggest that PA increases protein synthesis in adult rat cardiomyocytes and thus may play an important role in the development of cardiac hypertrophy.

  11. Regulation of nucleocytoplasmic trafficking of transcription factor OREBP/TonEBP/NFAT5.

    PubMed

    Tong, Edith H Y; Guo, Jin-Jun; Huang, Ai-Long; Liu, Han; Hu, Chang-Deng; Chung, Stephen S M; Ko, Ben C B

    2006-08-18

    The osmotic response element-binding protein (OREBP), also known as tonicity enhancer-binding protein (TonEBP) or NFAT5, regulates the hypertonicity-induced expression of a battery of genes crucial for the adaptation of mammalian cells to extracellular hypertonic stress. The activity of OREBP/TonEBP is regulated at multiple levels, including nucleocytoplasmic trafficking. OREBP/TonEBP protein can be detected in both the cytoplasm and nucleus under isotonic conditions, although it accumulates exclusively in the nucleus or cytoplasm when subjected to hypertonic or hypotonic challenges, respectively. Using immunocytochemistry and green fluorescent protein fusions, the protein domains that determine its subcellular localization were identified and characterized. We found that OREBP/TonEBP nuclear import is regulated by a nuclear localization signal. However, under isotonic conditions, nuclear export of OREBP/TonEBP is mediated by a CRM1-dependent, leucine-rich canonical nuclear export sequence (NES) located in the N terminus. Disruption of NES by site-directed mutagenesis yielded a mutant OREBP/TonEBP protein that accumulated in the nucleus under isotonic conditions but remained a target for hypotonicity-induced nuclear export. More importantly, a putative auxiliary export domain distal to the NES was identified. Disruption of the auxiliary export domain alone is sufficient to abolish the nuclear export of OREBP/TonEBP induced by hypotonicity. By using bimolecular fluorescence complementation assay, we showed that CRM1 interacts with OREBP/TonEBP, but not with a mutant protein deficient in NES. Our findings provide insight into how nucleocytoplasmic trafficking of OREBP/TonEBP is regulated by changes in extracellular tonicity. PMID:16782704

  12. CaMKII in addition to MLCK contributes to phosphorylation of regulatory light chain in cardiomyocytes.

    PubMed

    Eikemo, Hilde; Moltzau, Lise Román; Hussain, Rizwan I; Nguyen, Cam H T; Qvigstad, Eirik; Levy, Finn Olav; Skomedal, Tor; Osnes, Jan-Bjørn

    2016-02-26

    The aim was to identify kinase activities involved in the phosphorylation of regulatory light chain (RLC) in situ in cardiomyocytes. In electrically stimulated rat cardiomyocytes, phosphatase inhibition by calyculin A unmasked kinase activities evoking an increase of phosphorylated RLC (P-RLC) from about 16% to about 80% after 80 min. The phosphorylation rate in cardiomyocytes was reduced by about 40% by the myosin light chain kinase (MLCK) inhibitor, ML-7. In rat ventricular muscle strips, calyculin A induced a positive inotropic effect that correlated with P-RLC levels. The inotropic effect and P-RLC elevation were abolished by ML-7 treatment. The kinase activities phosphorylating RLC in cardiomyocytes were reduced by about 60% by the non-selective kinase inhibitor staurosporine and by about 50% by the calmodulin antagonist W7. W7 eliminated the inhibitory effect of ML-7, suggesting that the cardiac MLCK is Ca(2+)/calmodulin (CaM)-dependent. The CaM-dependent kinase II (CaMKII) inhibitor KN-93 attenuated the calyculin A-induced RLC phosphorylation by about 40%, indicating a contribution from CaMKII. The residual phosphorylation in the presence of W7 indicated that also CaM-independent kinase activities might contribute. RLC phosphorylation was insensitive to protein kinase C inhibition. In conclusion, in addition to MLCK, CaMKII phosphorylates RLC in cardiomyocytes. Involvement of other kinases cannot be excluded.

  13. Cardiomyocyte marker expression in a human lymphocyte cell line using mouse cardiomyocyte extract.

    PubMed

    Vojdani, Zahra; Tavakolinejad, Sima; Talaei-Khozani, Tahereh; Esmaeilpour, Tahereh; Rasooli, Manuchehr

    2011-03-01

    Cell transplantation shows potential for the treatment of cardiac diseases. Embryonic stem cells, cord blood and mesenchymal stem cells have been suggested as sources for transplantation therapy. Because of some technical limitations with the use of stem cells, transdifferentiation of fully differentiated cells is a potentially useful alternative. We investigated whether human peripheral blood cells could transdifferentiate into cardiomyocyte. Transdifferentiation was induced in a human B lymphocyte cell line (Raji). Cardiomyocyte extract was prepared from adult mouse cardiomyocytes. The cells were treated with 5-aza-2-deoxycytidine and trichostatin A, permeabilized with streptolysin O, and exposed to the mouse cardiomyocyte extract. They were cultured for 10 days, 3 weeks and 4 weeks. Cardiomyocyte markers were detected with immunohistochemistry and flow cytometry. Immunocytochemistry revealed that some cells expressed myosin heavy chain, α-actinin and cardiac troponin T after 3 and 4 weeks. Flow cytometry confirmed these data. In cells exposed to trichostatin A and 5-aza-2-deoxycytidine and permeabilized in the presence of the cardiomyocyte extract, troponin T expression was seen in 3.53% of the cells and 3.11% of them expressed α-actinin. After exposure to the cardiomyocyte extract, some permeabilized cells adhered to the plate loosely; however, the morphology did not change significantly, and they continued to show a rounded shape after 4 weeks. Our treated lymphocytes expressed cardiomyocyte markers. Our results suggest that lymphocytes may be useful in future research as a source of cells for reprogramming procedures.

  14. Humid heat exposure induced oxidative stress and apoptosis in cardiomyocytes through the angiotensin II signaling pathway.

    PubMed

    Wang, Xiaowu; Yuan, Binbin; Dong, Wenpeng; Yang, Bo; Yang, Yongchao; Lin, Xi; Gong, Gu

    2015-05-01

    Exposure to humid heat stress leads to the initiation of serious physiological dysfunction that may result in heat-related diseases, including heat stroke, heat cramp, heat exhaustion, and even death. Increasing evidences have shown that the humid heat stress-induced dysfunction of the cardiovascular system was accompanied with severe cardiomyocyte injury; however, the precise mechanism of heat stress-induced injury of cardiomyocyte remains unknown. In the present study, we hypothesized that humid heat stress promoted oxidative stress through the activation of angiotensin II (Ang II) in cardiomyocytes. To test our hypothesis, we established mouse models of humid heat stress. Using the animal models, we found that Ang II levels in serum were significantly up-regulated and that the Ang II receptor AT1 was increased in cardiomyocytes. The antioxidant ability in plasma and heart tissues which was detected by the ferric reducing/antioxidant power assay was also decreased with the increased ROS production under humid heat stress, as was the expression of antioxidant genes (SOD2, HO-1, GPx). Furthermore, we demonstrated that the Ang II receptor antagonist, valsartan, effectively relieved oxidative stress, blocked Ang II signaling pathway and suppressed cardiomyocyte apoptosis induced by humid heat stress. In addition, overexpression of antioxidant genes reversed cardiomyocyte apoptosis induced by Ang II. Overall, these results implied that humid heat stress increased oxidative stress and caused apoptosis of cardiomyocytes through the Ang II signaling pathway. Thus, targeting the Ang II signaling pathway may provide a promising approach for the prevention and treatment of cardiovascular diseases caused by humid heat stress.

  15. Bone marrow stromal cells as an inducer for cardiomyocyte differentiation from mouse embryonic stem cells.

    PubMed

    Yue, Fengming; Johkura, Kohei; Tomotsune, Daihachiro; Shirasawa, Sakiko; Yokoyama, Tadayuki; Nagai, Mika; Sasaki, Katsunori

    2010-09-20

    Bone marrow stromal cells (BMSCs) secrete soluble factors and display varied cell-biological functions. To confirm the ability and efficiency of BMSCs to induce embryonic stem cells (ESCs) into cardiomyocytes, mouse embryoid bodies (EBs) were co-cultured with rat BMSCs. After about 10 days, areas of rhythmically contracting cells in more solid aggregates became evident with bundle-like structures formed along borders between EB outgrowth and BMSC layer. ESC-derived cardiomyocytes exhibited sarcomeric striations when stained with troponin I (Trop I), organized in separated bundles. Besides, the staining for connexin 43 was detected in cell-cell junctions, which demonstrated that ESC-derived cardiomyocytes were coupled by gap junction in culture. The related genes of cardiomyocytes were found in these beating and no-beating EBs co-cultured with BMSCs. In addition, an improved efficiency of cardiomyocyte differentiation from ESC-BMSC co-culture was found in the serum-free medium: 5-fold up-regulation in the number of beating area compared with the serum medium. Effective cardiac differentiation was also recognized in transfer filter assay and in condition medium obtained from BMSC culture. A clear increase in the expression of cardiac genes and TropI protein confirmed further cardiac differentiation by BMP4 and Retinoic Acid (RA) treatment. These results demonstrate that BMSCs can induce cardiomyocyte differentiation from ESCs through soluble factors and enhance it with BMP4 or RA treatment. Serum-free ESC-BMSC co-culture represents a defined in vitro model for identifying the cardiomyocyte-inducing activity from BMSCs and, in addition, a straightforward experimental system for assessing clinical applications. PMID:20801009

  16. Copper reverses cardiomyocyte hypertrophy through vascular endothelial growth factor-mediated reduction in the cell size.

    PubMed

    Zhou, Yang; Jiang, Youchun; Kang, Y James

    2008-07-01

    Previous studies have shown that dietary copper supplementation reversed heart hypertrophy induced by pressure overload in a mouse model. The present study was undertaken to understand the cellular basis of copper-induced regression of cardiac hypertrophy. Primary cultures of neonatal rat cardiomyocytes were treated with phenylephrine (PE) at a final concentration of 100 microM in cultures for 48 h to induce cellular hypertrophy. The hypertrophied cardiomyocytes were exposed to copper sulfate at a final concentration of 5 microM in cultures for additional 24 h. This copper treatment reduced the size of the hypertrophied cardiomyocytes, as measured by flow cytometry, protein content in cells, cell volume and cardiomyocyte hypertrophy markers including beta-myosin heavy chain protein, skeletal alpha-actin, and atrial natriuretic peptide. Cell cycle analysis and cell sorting of p-histone-3 labeled cardiomyocytes indicated that cell division was not involved in the copper-induced regression of cardiomyocyte hypertrophy. Copper also inhibited PE-induced apoptosis, determined by a TUNEL assay. Because copper stimulates vascular endothelial growth factor (VEGF) production through activation of hypoxia-inducible transcription factor, an anti-VEGF antibody at a final concentration of 2 ng/ml in cultures was used and shown to blunt copper-induced regression of cell hypertrophy. Conversely, VEGF alone at a final concentration of 0.2 microg/ml reversed cell hypertrophy as the same as copper did. This study demonstrates that both copper and VEGF reduce the size of hypertrophied cardiomyocytes, and copper regression of cardiac hypertrophy is VEGF-dependent. PMID:18495151

  17. The electrophysiological development of cardiomyocytes.

    PubMed

    Liu, Jie; Laksman, Zachary; Backx, Peter H

    2016-01-15

    The generation of human cardiomyocytes (CMs) from human pluripotent stem cells (hPSCs) has become an important resource for modeling human cardiac disease and for drug screening, and also holds significant potential for cardiac regeneration. Many challenges remain to be overcome however, before innovation in this field can translate into a change in the morbidity and mortality associated with heart disease. Of particular importance for the future application of this technology is an improved understanding of the electrophysiologic characteristics of CMs, so that better protocols can be developed and optimized for generating hPSC-CMs. Many different cell culture protocols are currently utilized to generate CMs from hPSCs and all appear to yield relatively “developmentally” immature CMs with highly heterogeneous electrical properties. These hPSC-CMs are characterized by spontaneous beating at highly variable rates with a broad range of depolarization-repolarization patterns, suggestive of mixed populations containing atrial, ventricular and nodal cells. Many recent studies have attempted to introduce approaches to promote maturation and to create cells with specific functional properties. In this review, we summarize the studies in which the electrical properties of CMs derived from stem cells have been examined. In order to place this information in a useful context, we also review the electrical properties of CMs as they transition from the developing embryo to the adult human heart. The signal pathways involved in the regulation of ion channel expression during development are also briefly considered. PMID:26788696

  18. The electrophysiological development of cardiomyocytes.

    PubMed

    Liu, Jie; Laksman, Zachary; Backx, Peter H

    2016-01-15

    The generation of human cardiomyocytes (CMs) from human pluripotent stem cells (hPSCs) has become an important resource for modeling human cardiac disease and for drug screening, and also holds significant potential for cardiac regeneration. Many challenges remain to be overcome however, before innovation in this field can translate into a change in the morbidity and mortality associated with heart disease. Of particular importance for the future application of this technology is an improved understanding of the electrophysiologic characteristics of CMs, so that better protocols can be developed and optimized for generating hPSC-CMs. Many different cell culture protocols are currently utilized to generate CMs from hPSCs and all appear to yield relatively “developmentally” immature CMs with highly heterogeneous electrical properties. These hPSC-CMs are characterized by spontaneous beating at highly variable rates with a broad range of depolarization-repolarization patterns, suggestive of mixed populations containing atrial, ventricular and nodal cells. Many recent studies have attempted to introduce approaches to promote maturation and to create cells with specific functional properties. In this review, we summarize the studies in which the electrical properties of CMs derived from stem cells have been examined. In order to place this information in a useful context, we also review the electrical properties of CMs as they transition from the developing embryo to the adult human heart. The signal pathways involved in the regulation of ion channel expression during development are also briefly considered.

  19. Ethanol enhances arsenic-induced cyclooxygenase-2 expression via both NFAT and NF-κB signalings in colorectal cancer cells.

    PubMed

    Wang, Lei; Hitron, John Andrew; Wise, James T F; Son, Young-Ok; Roy, Ram Vinod; Kim, Donghern; Dai, Jin; Pratheeshkumar, Poyil; Zhang, Zhuo; Xu, Mei; Luo, Jia; Shi, Xianglin

    2015-10-15

    Arsenic is a known carcinogen to humans, and chronic exposure to environmental arsenic is a worldwide health concern. As a dietary factor, ethanol carries a well-established risk for malignancies, but the effects of co-exposure to arsenic and ethanol on tumor development are not well understood. In the present study, we hypothesized that ethanol would enhance the function of an environmental carcinogen such as arsenic through increase in COX-2 expression. Our in vitro results show that ethanol enhanced arsenic-induced COX-2 expression. We also show that the increased COX-2 expression associates with intracellular ROS generation, up-regulated AKT signaling, with activation of both NFAT and NF-κB pathways. We demonstrate that antioxidant enzymes have an inhibitory effect on arsenic/ethanol-induced COX-2 expression, indicating that the responsive signaling pathways from co-exposure to arsenic and ethanol relate to ROS generation. In vivo results also show that co-exposure to arsenic and ethanol increased COX-2 expression in mice. We conclude that ethanol enhances arsenic-induced COX-2 expression in colorectal cancer cells via both the NFAT and NF-κB pathways. These results imply that, as a common dietary factor, ethanol ingestion may be a compounding risk factor for arsenic-induced carcinogenesis/cancer development.

  20. Simultaneous Assessment of Cardiomyocyte DNA Synthesis and Ploidy: A Method to Assist Quantification of Cardiomyocyte Regeneration and Turnover.

    PubMed

    Richardson, Gavin D

    2016-01-01

    Although it is accepted that the heart has a limited potential to regenerate cardiomyocytes following injury and that low levels of cardiomyocyte turnover occur during normal ageing, quantification of these events remains challenging. This is in part due to the rarity of the process and the fact that multiple cellular sources contribute to myocardial maintenance. Furthermore, DNA duplication within cardiomyocytes often leads to a polyploid cardiomyocyte and only rarely leads to new cardiomyocytes by cellular division. In order to accurately quantify cardiomyocyte turnover discrimination between these processes is essential. The protocol described here employs long term nucleoside labeling in order to label all nuclei which have arisen as a result of DNA replication and cardiomyocyte nuclei identified by utilizing nuclei isolation and subsequent PCM1 immunolabeling. Together this allows the accurate and sensitive identification of the nucleoside labeling of the cardiomyocyte nuclei population. Furthermore, 4',6-diamidino-2-phenylindole labeling and analysis of nuclei ploidy, enables the discrimination of neo-cardiomyocyte nuclei from nuclei which have incorporated nucleoside during polyploidization. Although this method cannot control for cardiomyocyte binucleation, it allows a rapid and robust quantification of neo-cardiomyocyte nuclei while accounting for polyploidization. This method has a number of downstream applications including assessing the potential therapeutics to enhance cardiomyocyte regeneration or investigating the effects of cardiac disease on cardiomyocyte turnover and ploidy. This technique is also compatible with additional downstream immunohistological techniques, allowing quantification of nucleoside incorporation in all cardiac cell types. PMID:27285379

  1. Simultaneous Assessment of Cardiomyocyte DNA Synthesis and Ploidy: A Method to Assist Quantification of Cardiomyocyte Regeneration and Turnover

    PubMed Central

    Richardson, Gavin D.

    2016-01-01

    Although it is accepted that the heart has a limited potential to regenerate cardiomyocytes following injury and that low levels of cardiomyocyte turnover occur during normal ageing, quantification of these events remains challenging. This is in part due to the rarity of the process and the fact that multiple cellular sources contribute to myocardial maintenance. Furthermore, DNA duplication within cardiomyocytes often leads to a polyploid cardiomyocyte and only rarely leads to new cardiomyocytes by cellular division. In order to accurately quantify cardiomyocyte turnover discrimination between these processes is essential. The protocol described here employs long term nucleoside labeling in order to label all nuclei which have arisen as a result of DNA replication and cardiomyocyte nuclei identified by utilizing nuclei isolation and subsequent PCM1 immunolabeling. Together this allows the accurate and sensitive identification of the nucleoside labeling of the cardiomyocyte nuclei population. Furthermore, 4′,6-diamidino-2-phenylindole labeling and analysis of nuclei ploidy, enables the discrimination of neo-cardiomyocyte nuclei from nuclei which have incorporated nucleoside during polyploidization. Although this method cannot control for cardiomyocyte binucleation, it allows a rapid and robust quantification of neo-cardiomyocyte nuclei while accounting for polyploidization. This method has a number of downstream applications including assessing the potential therapeutics to enhance cardiomyocyte regeneration or investigating the effects of cardiac disease on cardiomyocyte turnover and ploidy. This technique is also compatible with additional downstream immunohistological techniques, allowing quantification of nucleoside incorporation in all cardiac cell types. PMID:27285379

  2. Role of cardiomyocyte circadian clock in myocardial metabolic adaptation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Marked circadian rhythmicities in cardiovascular physiology and pathophysiology exist. The cardiomyocyte circadian clock has recently been linked to circadian rhythms in myocardial gene expression, metabolism, and contractile function. For instance, the cardiomyocyte circadian clock is essential f...

  3. Essential role of STIM1 in the development of cardiomyocyte hypertrophy

    SciTech Connect

    Ohba, Takayoshi; Watanabe, Hiroyuki; Murakami, Manabu; Sato, Takako; Ono, Kyoichi; Ito, Hiroshi

    2009-11-06

    Store-operated Ca{sup 2+} entry (SOCE) through transient receptor potential (TRP) channels is important in the development of cardiac hypertrophy. Recently, stromal interaction molecule 1 (STIM1) was identified as a key regulator of SOCE. In this study, we examined whether STIM1 is involved in the development of cardiomyocyte hypertrophy. RT-PCR showed that cultured rat cardiomyocytes constitutively expressed STIM1. Endothelin-1 (ET-1) treatment for 48 h enhanced TRPC1 expression, SOCE, and nuclear factor of activated T cells activation without upregulating STIM1. However, the knockdown of STIM1 suppressed these effects, thereby preventing a hypertrophic response. These results suggest that STIM1 plays an essential role in the development of cardiomyocyte hypertrophy.

  4. Iron induces protection and necrosis in cultured cardiomyocytes: Role of reactive oxygen species and nitric oxide.

    PubMed

    Munoz, Juan Pablo; Chiong, Mario; García, Lorena; Troncoso, Rodrigo; Toro, Barbra; Pedrozo, Zully; Diaz-Elizondo, Jessica; Salas, Daniela; Parra, Valentina; Núñez, Marco T; Hidalgo, Cecilia; Lavandero, Sergio

    2010-02-15

    We investigate here the role of reactive oxygen species and nitric oxide in iron-induced cardiomyocyte hypertrophy or cell death. Cultured rat cardiomyocytes incubated with 20 microM iron (added as FeCl(3)-Na nitrilotriacetate, Fe-NTA) displayed hypertrophy features that included increased protein synthesis and cell size, plus realignment of F-actin filaments along with sarcomeres and activation of the atrial natriuretic factor gene promoter. Incubation with higher Fe-NTA concentrations (100 microM) produced cardiomyocyte death by necrosis. Incubation for 24 h with Fe-NTA (20-40 microM) or the nitric oxide donor Delta-nonoate increased iNOS mRNA but decreased iNOS protein levels; under these conditions, iron stimulated the activity and the dimerization of iNOS. Fe-NTA (20 microM) promoted short- and long-term generation of reactive oxygen species, whereas preincubation with l-arginine suppressed this response. Preincubation with 20 microM Fe-NTA also attenuated the necrotic cell death triggered by 100 microM Fe-NTA, suggesting that these preincubation conditions have cardioprotective effects. Inhibition of iNOS activity with 1400 W enhanced iron-induced ROS generation and prevented both iron-dependent cardiomyocyte hypertrophy and cardioprotection. In conclusion, we propose that Fe-NTA (20 microM) stimulates iNOS activity and that the enhanced NO production, by promoting hypertrophy and enhancing survival mechanisms through ROS reduction, is beneficial to cardiomyocytes. At higher concentrations, however, iron triggers cardiomyocyte death by necrosis. PMID:19969068

  5. Caffeine induces cardiomyocyte hypertrophy via p300 and CaMKII pathways.

    PubMed

    Shi, Liang; Xu, Hao; Wei, Jinhong; Ma, Xingfeng; Zhang, Jianbao

    2014-09-25

    Caffeine is commonly utilized to trigger intracellular calcium in cardiomyocyte. It is well accepted that caffeine could induce cardiac arrhythmia, but it is not clear with regard of its impacts on the cardiac function. This article presents a recent study concerning the effects of caffeine on the cardiomyocyte hypertrophy and the associated signal pathway. The experimental results showed that the total protein contents, the surface area of cardiomyocyte and β-myosin heavy chain (β-MHC) expression increased in ventricular myocytes of neonatal Sprague-Dawley (SD) rats after 24h caffeine incubation. It is also observed that the basal intracellular calcium (Ca(2+)) level has increased, while the amplitude of Ca(2+) oscillation and Ca(2+) content have decreased in sarcoplasmic reticulum (SR). The caffeine-induced myocyte enhancer factor-2 (MEF2) expression and hypertrophy can be completely abolished by the inhibition of cardiac ryanodine receptor (RyR2), as well as KN93 and curcumin treatments. Meanwhile, the amplitude of Ca(2+) oscillation and the Ca(2+) content of SR in the completely-inhibited group have reached the physiological level. These results suggest that the caffeine-induced cardiomyocyte hypertrophy established the connection between Ca(2+) release from SR and cytosol that activates CaMKII and p300, which in turn enhances the expression of MEF2 that promotes cardiomyocyte hypertrophy.

  6. Mitochondrial fission is required for cardiomyocyte hypertrophy mediated by a Ca2+-calcineurin signaling pathway.

    PubMed

    Pennanen, Christian; Parra, Valentina; López-Crisosto, Camila; Morales, Pablo E; Del Campo, Andrea; Gutierrez, Tomás; Rivera-Mejías, Pablo; Kuzmicic, Jovan; Chiong, Mario; Zorzano, Antonio; Rothermel, Beverly A; Lavandero, Sergio

    2014-06-15

    Cardiomyocyte hypertrophy has been associated with diminished mitochondrial metabolism. Mitochondria are crucial organelles for the production of ATP, and their morphology and function are regulated by the dynamic processes of fusion and fission. The relationship between mitochondrial dynamics and cardiomyocyte hypertrophy is still poorly understood. Here, we show that treatment of cultured neonatal rat cardiomyocytes with the hypertrophic agonist norepinephrine promotes mitochondrial fission (characterized by a decrease in mitochondrial mean volume and an increase in the relative number of mitochondria per cell) and a decrease in mitochondrial function. We demonstrate that norepinephrine acts through α1-adrenergic receptors to increase cytoplasmic Ca(2+), activating calcineurin and promoting migration of the fission protein Drp1 (encoded by Dnml1) to mitochondria. Dominant-negative Drp1 (K38A) not only prevented mitochondrial fission, it also blocked hypertrophic growth of cardiomyocytes in response to norepinephrine. Remarkably, an antisense adenovirus against the fusion protein Mfn2 (AsMfn2) was sufficient to increase mitochondrial fission and stimulate a hypertrophic response without agonist treatment. Collectively, these results demonstrate the importance of mitochondrial dynamics in the development of cardiomyocyte hypertrophy and metabolic remodeling.

  7. Mitochondrial fission is required for cardiomyocyte hypertrophy mediated by a Ca2+-calcineurin signaling pathway

    PubMed Central

    Pennanen, Christian; Parra, Valentina; López-Crisosto, Camila; Morales, Pablo E.; del Campo, Andrea; Gutierrez, Tomás; Rivera-Mejías, Pablo; Kuzmicic, Jovan; Chiong, Mario; Zorzano, Antonio; Rothermel, Beverly A.; Lavandero, Sergio

    2014-01-01

    ABSTRACT Cardiomyocyte hypertrophy has been associated with diminished mitochondrial metabolism. Mitochondria are crucial organelles for the production of ATP, and their morphology and function are regulated by the dynamic processes of fusion and fission. The relationship between mitochondrial dynamics and cardiomyocyte hypertrophy is still poorly understood. Here, we show that treatment of cultured neonatal rat cardiomyocytes with the hypertrophic agonist norepinephrine promotes mitochondrial fission (characterized by a decrease in mitochondrial mean volume and an increase in the relative number of mitochondria per cell) and a decrease in mitochondrial function. We demonstrate that norepinephrine acts through α1-adrenergic receptors to increase cytoplasmic Ca2+, activating calcineurin and promoting migration of the fission protein Drp1 (encoded by Dnml1) to mitochondria. Dominant-negative Drp1 (K38A) not only prevented mitochondrial fission, it also blocked hypertrophic growth of cardiomyocytes in response to norepinephrine. Remarkably, an antisense adenovirus against the fusion protein Mfn2 (AsMfn2) was sufficient to increase mitochondrial fission and stimulate a hypertrophic response without agonist treatment. Collectively, these results demonstrate the importance of mitochondrial dynamics in the development of cardiomyocyte hypertrophy and metabolic remodeling. PMID:24777478

  8. Mitochondrial fission is required for cardiomyocyte hypertrophy mediated by a Ca2+-calcineurin signaling pathway.

    PubMed

    Pennanen, Christian; Parra, Valentina; López-Crisosto, Camila; Morales, Pablo E; Del Campo, Andrea; Gutierrez, Tomás; Rivera-Mejías, Pablo; Kuzmicic, Jovan; Chiong, Mario; Zorzano, Antonio; Rothermel, Beverly A; Lavandero, Sergio

    2014-06-15

    Cardiomyocyte hypertrophy has been associated with diminished mitochondrial metabolism. Mitochondria are crucial organelles for the production of ATP, and their morphology and function are regulated by the dynamic processes of fusion and fission. The relationship between mitochondrial dynamics and cardiomyocyte hypertrophy is still poorly understood. Here, we show that treatment of cultured neonatal rat cardiomyocytes with the hypertrophic agonist norepinephrine promotes mitochondrial fission (characterized by a decrease in mitochondrial mean volume and an increase in the relative number of mitochondria per cell) and a decrease in mitochondrial function. We demonstrate that norepinephrine acts through α1-adrenergic receptors to increase cytoplasmic Ca(2+), activating calcineurin and promoting migration of the fission protein Drp1 (encoded by Dnml1) to mitochondria. Dominant-negative Drp1 (K38A) not only prevented mitochondrial fission, it also blocked hypertrophic growth of cardiomyocytes in response to norepinephrine. Remarkably, an antisense adenovirus against the fusion protein Mfn2 (AsMfn2) was sufficient to increase mitochondrial fission and stimulate a hypertrophic response without agonist treatment. Collectively, these results demonstrate the importance of mitochondrial dynamics in the development of cardiomyocyte hypertrophy and metabolic remodeling. PMID:24777478

  9. Activation of Transient Receptor Potential Canonical 3 (TRPC3)-mediated Ca2+ Entry by A1 Adenosine Receptor in Cardiomyocytes Disturbs Atrioventricular Conduction*

    PubMed Central

    Sabourin, Jessica; Antigny, Fabrice; Robin, Elodie; Frieden, Maud; Raddatz, Eric

    2012-01-01

    Although the activation of the A1-subtype of the adenosine receptors (A1AR) is arrhythmogenic in the developing heart, little is known about the underlying downstream mechanisms. The aim of this study was to determine to what extent the transient receptor potential canonical (TRPC) channel 3, functioning as receptor-operated channel (ROC), contributes to the A1AR-induced conduction disturbances. Using embryonic atrial and ventricular myocytes obtained from 4-day-old chick embryos, we found that the specific activation of A1AR by CCPA induced sarcolemmal Ca2+ entry. However, A1AR stimulation did not induce Ca2+ release from the sarcoplasmic reticulum. Specific blockade of TRPC3 activity by Pyr3, by a dominant negative of TRPC3 construct, or inhibition of phospholipase Cs and PKCs strongly inhibited the A1AR-enhanced Ca2+ entry. Ca2+ entry through TRPC3 was activated by the 1,2-diacylglycerol (DAG) analog OAG via PKC-independent and -dependent mechanisms in atrial and ventricular myocytes, respectively. In parallel, inhibition of the atypical PKCζ by myristoylated PKCζ pseudosubstrate inhibitor significantly decreased the A1AR-enhanced Ca2+ entry in both types of myocytes. Additionally, electrocardiography showed that inhibition of TRPC3 channel suppressed transient A1AR-induced conduction disturbances in the embryonic heart. Our data showing that A1AR activation subtly mediates a proarrhythmic Ca2+ entry through TRPC3-encoded ROC by stimulating the phospholipase C/DAG/PKC cascade provide evidence for a novel pathway whereby Ca2+ entry and cardiac function are altered. Thus, the A1AR-TRPC3 axis may represent a potential therapeutic target. PMID:22692208

  10. Dataset of integrin-linked kinase protein: Protein interactions in cardiomyocytes identified by mass spectrometry.

    PubMed

    Traister, Alexandra; Lu, Mingliang; Coles, John G; Maynes, Jason T

    2016-06-01

    Using hearts from mice overexpressing integrin linked kinase (ILK) behind the cardiac specific promoter αMHC, we have performed immunoprecipitation and mass spectrometry to identify novel ILK protein:protein interactions that regulate cardiomyocyte activity and calcium flux. Integrin linked kinase complexes were captured from mouse heart lysates using a commercial antibody, with subsequent liquid chromatography tandem mass spectral analysis. Interacting partners were identified using the MASCOT server, and important interactions verified using reverse immunoprecipitation and mass spectrometry. All ILK interacting proteins were identified in a non-biased manner, and are stored in the ProteomeXchange Consortium via the PRIDE partner repository (reference ID PRIDE: PXD001053). The functional role of identified ILK interactions in cardiomyocyte function and arrhythmia were subsequently confirmed in human iPSC-cardiomyocytes. PMID:27408918

  11. FAK Forms a Complex with MEF2 to Couple Biomechanical Signaling to Transcription in Cardiomyocytes.

    PubMed

    Cardoso, Alisson Campos; Pereira, Ana Helena Macedo; Ambrosio, Andre Luis Berteli; Consonni, Silvio Roberto; Rocha de Oliveira, Renata; Bajgelman, Marcio Chain; Dias, Sandra Martha Gomes; Franchini, Kleber Gomes

    2016-08-01

    Focal adhesion kinase (FAK) has emerged as a mediator of mechanotransduction in cardiomyocytes, regulating gene expression during hypertrophic remodeling. However, how FAK signaling is relayed onward to the nucleus is unclear. Here, we show that FAK interacts with and regulates myocyte enhancer factor 2 (MEF2), a master cardiac transcriptional regulator. In cardiomyocytes exposed to biomechanical stimulation, FAK accumulates in the nucleus, binds to and upregulates the transcriptional activity of MEF2 through an interaction with the FAK focal adhesion targeting (FAT) domain. In the crystal structure (2.9 Å resolution), FAT binds to a stably folded groove in the MEF2 dimer, known to interact with regulatory cofactors. FAK cooperates with MEF2 to enhance the expression of Jun in cardiomyocytes, an important component of hypertrophic response to mechanical stress. These findings underscore a connection between the mechanotransduction involving FAK and transcriptional regulation by MEF2, with potential relevance to the pathogenesis of cardiac disease. PMID:27427476

  12. Cholesterol Depletion Alters Cardiomyocyte Subcellular Signaling and Increases Contractility

    PubMed Central

    McIntosh, Victoria J.; Abou Samra, Abdul B.; Mohammad, Ramzi M.; Lasley, Robert D.

    2016-01-01

    Membrane cholesterol levels play an important factor in regulating cell function. Sarcolemmal cholesterol is concentrated in lipid rafts and caveolae, which are flask-shaped invaginations of the plasma membrane. The scaffolding protein caveolin permits the enrichment of cholesterol in caveolae, and caveolin interactions with numerous proteins regulate their function. The purpose of this study was to determine whether acute reductions in cardiomyocyte cholesterol levels alter subcellular protein kinase activation, intracellular Ca2+ and contractility. Methods: Ventricular myocytes, isolated from adult Sprague Dawley rats, were treated with the cholesterol reducing agent methyl-β-cyclodextrin (MβCD, 5 mM, 1 hr, room temperature). Total cellular cholesterol levels, caveolin-3 localization, subcellular, ERK and p38 mitogen activated protein kinase (MAPK) signaling, contractility, and [Ca2+]i were assessed. Results: Treatment with MβCD reduced cholesterol levels by ~45 and shifted caveolin-3 from cytoskeleton and triton-insoluble fractions to the triton-soluble fraction, and increased ERK isoform phosphorylation in cytoskeletal, cytosolic, triton-soluble and triton-insoluble membrane fractions without altering their subcellular distributions. In contrast the primary effect of MβCD was on p38 subcellular distribution of p38α with little effect on p38 phosphorylation. Cholesterol depletion increased cardiomyocyte twitch amplitude and the rates of shortening and relaxation in conjunction with increased diastolic and systolic [Ca2+]i. Conclusions: These results indicate that acute reductions in membrane cholesterol levels differentially modulate basal cardiomyocyte subcellular MAPK signaling, as well as increasing [Ca2+]i and contractility. PMID:27441649

  13. Coordinated regulation of differentiation and proliferation of embryonic cardiomyocytes by a jumonji (Jarid2)-cyclin D1 pathway.

    PubMed

    Nakajima, Kuniko; Inagawa, Masayo; Uchida, Chiharu; Okada, Kumiko; Tane, Shoji; Kojima, Mizuyo; Kubota, Misae; Noda, Masatsugu; Ogawa, Satoko; Shirato, Haruki; Sato, Michio; Suzuki-Migishima, Rika; Hino, Toshiaki; Satoh, Yukio; Kitagawa, Masatoshi; Takeuchi, Takashi

    2011-05-01

    In general, cell proliferation and differentiation show an inverse relationship, and are regulated in a coordinated manner during development. Embryonic cardiomyocytes must support embryonic life by functional differentiation such as beating, and proliferate actively to increase the size of the heart. Therefore, progression of both proliferation and differentiation is indispensable. It remains unknown whether proliferation and differentiation are related in these embryonic cardiomyocytes. We focused on abnormal phenotypes, such as hyperproliferation, inhibition of differentiation and enhanced expression of cyclin D1 in cardiomyocytes of mice with mutant jumonji (Jmj, Jarid2), which encodes the repressor of cyclin D1. Analysis of Jmj/cyclin D1 double mutant mice showed that Jmj was required for normal differentiation and normal expression of GATA4 protein through cyclin D1. Analysis of transgenic mice revealed that enhanced expression of cyclin D1 decreased GATA4 protein expression and inhibited the differentiation of cardiomyocytes in a CDK4/6-dependent manner, and that exogenous expression of GATA4 rescued the abnormal differentiation. Finally, CDK4 phosphorylated GATA4 directly, which promoted the degradation of GATA4 in cultured cells. These results suggest that CDK4 activated by cyclin D1 inhibits differentiation of cardiomyocytes by degradation of GATA4, and that initiation of Jmj expression unleashes the inhibition by repression of cyclin D1 expression and allows progression of differentiation, as well as repression of proliferation. Thus, a Jmj-cyclin D1 pathway coordinately regulates proliferation and differentiation of cardiomyocytes.

  14. An integrated mechanism of cardiomyocyte nuclear Ca(2+) signaling.

    PubMed

    Ibarra, Cristián; Vicencio, Jose Miguel; Varas-Godoy, Manuel; Jaimovich, Enrique; Rothermel, Beverly A; Uhlén, Per; Hill, Joseph A; Lavandero, Sergio

    2014-10-01

    In cardiomyocytes, Ca(2+) plays a central role in governing both contraction and signaling events that regulate gene expression. Current evidence indicates that discrimination between these two critical functions is achieved by segregating Ca(2+) within subcellular microdomains: transcription is regulated by Ca(2+) release within nuclear microdomains, and excitation-contraction coupling is regulated by cytosolic Ca(2+). Accordingly, a variety of agonists that control cardiomyocyte gene expression, such as endothelin-1, angiotensin-II or insulin-like growth factor-1, share the feature of triggering nuclear Ca(2+) signals. However, signaling pathways coupling surface receptor activation to nuclear Ca(2+) release, and the phenotypic responses to such signals, differ between agonists. According to earlier hypotheses, the selective control of nuclear Ca(2+) signals by activation of plasma membrane receptors relies on the strategic localization of inositol trisphosphate receptors at the nuclear envelope. There, they mediate Ca(2+) release from perinuclear Ca(2+) stores upon binding of inositol trisphosphate generated in the cytosol, which diffuses into the nucleus. More recently, identification of such receptors at nuclear membranes or perinuclear sarcolemmal invaginations has uncovered novel mechanisms whereby agonists control nuclear Ca(2+) release. In this review, we discuss mechanisms for the selective control of nuclear Ca(2+) signals with special focus on emerging models of agonist receptor activation.

  15. An integrated mechanism of cardiomyocyte nuclear Ca2+ signaling

    PubMed Central

    Ibarra, Cristián; Vicencio, Jose Miguel; Varas-Godoy, Manuel; Jaimovich, Enrique; Rothermel, Beverly A.; Uhlén, Per; Hill, Joseph A.; Lavandero, Sergio

    2015-01-01

    In cardiomyocytes, Ca2+ plays a central role in governing both contraction and signaling events that regulate gene expression. Current evidence indicates that discrimination between these two critical functions is achieved by segregating Ca2+ within subcellular microdomains: transcription is regulated by Ca2+ release within nuclear microdomains, and excitation–contraction coupling is regulated by cytosolic Ca2+. Accordingly, a variety of agonists that control cardiomyocyte gene expression, such as endothelin-1, angiotensin-II or insulin-like growth factor-1, share the feature of triggering nuclear Ca2+ signals. However, signaling pathways coupling surface receptor activation to nuclear Ca2+ release, and the phenotypic responses to such signals, differ between agonists. According to earlier hypotheses, the selective control of nuclear Ca2+ signals by activation of plasma membrane receptors relies on the strategic localization of inositol trisphosphate receptors at the nuclear envelope. There, they mediate Ca2+ release from perinuclear Ca2+ stores upon binding of inositol trisphosphate generated in the cytosol, which diffuses into the nucleus. More recently, identification of such receptors at nuclear membranes or perinuclear sarcolemmal invaginations has uncovered novel mechanisms whereby agonists control nuclear Ca2+ release. In this review, we discuss mechanisms for the selective control of nuclear Ca2+ signals with special focus on emerging models of agonist receptor activation. PMID:24997440

  16. Notch1 signaling stimulates proliferation of immature cardiomyocytes.

    PubMed

    Collesi, Chiara; Zentilin, Lorena; Sinagra, Gianfranco; Giacca, Mauro

    2008-10-01

    The identification of the molecular mechanisms controlling cardiomyocyte proliferation during the embryonic, fetal, and early neonatal life appears of paramount interest in regard to exploiting this information to promote cardiac regeneration. Here, we show that the proliferative potential of neonatal rat cardiomyocytes is powerfully stimulated by the sustained activation of the Notch pathway. We found that Notch1 is expressed in proliferating ventricular immature cardiac myocytes (ICMs) both in vitro and in vivo, and that the number of Notch1-positive cells in the heart declines with age. Notch1 expression in ICMs paralleled the expression of its Jagged1 ligand on non-myocyte supporting cells. The inhibition of Notch signaling in ICMs blocked their proliferation and induced apoptosis; in contrast, its activation by Jagged1 or by the constitutive expression of its activated form using an adeno-associated virus markedly stimulated proliferative signaling and promoted ICM expansion. Maintenance or reactivation of Notch signaling in cardiac myocytes might represent an interesting target for innovative regenerative therapy. PMID:18824567

  17. An integrated mechanism of cardiomyocyte nuclear Ca(2+) signaling.

    PubMed

    Ibarra, Cristián; Vicencio, Jose Miguel; Varas-Godoy, Manuel; Jaimovich, Enrique; Rothermel, Beverly A; Uhlén, Per; Hill, Joseph A; Lavandero, Sergio

    2014-10-01

    In cardiomyocytes, Ca(2+) plays a central role in governing both contraction and signaling events that regulate gene expression. Current evidence indicates that discrimination between these two critical functions is achieved by segregating Ca(2+) within subcellular microdomains: transcription is regulated by Ca(2+) release within nuclear microdomains, and excitation-contraction coupling is regulated by cytosolic Ca(2+). Accordingly, a variety of agonists that control cardiomyocyte gene expression, such as endothelin-1, angiotensin-II or insulin-like growth factor-1, share the feature of triggering nuclear Ca(2+) signals. However, signaling pathways coupling surface receptor activation to nuclear Ca(2+) release, and the phenotypic responses to such signals, differ between agonists. According to earlier hypotheses, the selective control of nuclear Ca(2+) signals by activation of plasma membrane receptors relies on the strategic localization of inositol trisphosphate receptors at the nuclear envelope. There, they mediate Ca(2+) release from perinuclear Ca(2+) stores upon binding of inositol trisphosphate generated in the cytosol, which diffuses into the nucleus. More recently, identification of such receptors at nuclear membranes or perinuclear sarcolemmal invaginations has uncovered novel mechanisms whereby agonists control nuclear Ca(2+) release. In this review, we discuss mechanisms for the selective control of nuclear Ca(2+) signals with special focus on emerging models of agonist receptor activation. PMID:24997440

  18. Sequential activation of alpha-actin genes during avian cardiogenesis: vascular smooth muscle alpha-actin gene transcripts mark the onset of cardiomyocyte differentiation

    PubMed Central

    1988-01-01

    The expression of cytoplasmic beta-actin and cardiac, skeletal, and smooth muscle alpha-actins during early avian cardiogenesis was analyzed by in situ hybridization with mRNA-specific single-stranded DNA probes. The cytoplasmic beta-actin gene was ubiquitously expressed in the early chicken embryo. In contrast, the alpha-actin genes were sequentially activated in avian cardiac tissue during the early stages of heart tube formation. The accumulation of large quantities of smooth muscle alpha-actin transcripts in epimyocardial cells preceded the expression of the sarcomeric alpha-actin genes. The accumulation of skeletal alpha-actin mRNAs in the developing heart lagged behind that of cardiac alpha-actin by several embryonic stages. At Hamburger- Hamilton stage 12, the smooth muscle alpha-actin gene was selectively down-regulated in the heart such that only the conus, which subsequently participates in the formation of the vascular trunks, continued to express this gene. This modulation in smooth muscle alpha- actin gene expression correlated with the beginning of coexpression of sarcomeric alpha-actin transcripts in the epimyocardium and the onset of circulation in the embryo. The specific expression of the vascular smooth muscle alpha-actin gene marks the onset of differentiation of cardiac cells and represents the first demonstration of coexpression of both smooth muscle and striated alpha-actin genes within myogenic cells. PMID:3204121

  19. Redox mechanisms of cardiomyocyte mitochondrial protection

    PubMed Central

    Bartz, Raquel R.; Suliman, Hagir B.; Piantadosi, Claude A.

    2015-01-01

    Oxidative and nitrosative stress are primary contributors to the loss of myocardial tissue in insults ranging from ischemia/reperfusion injury from coronary artery disease and heart transplantation to sepsis-induced myocardial dysfunction and drug-induced myocardial damage. This cell damage caused by oxidative and nitrosative stress leads to mitochondrial protein, DNA, and lipid modifications, which inhibits energy production and contractile function, potentially leading to cell necrosis and/or apoptosis. However, cardiomyocytes have evolved an elegant set of redox-sensitive mechanisms that respond to and contain oxidative and nitrosative damage. These responses include the rapid induction of antioxidant enzymes, mitochondrial DNA repair mechanisms, selective mitochondrial autophagy (mitophagy), and mitochondrial biogenesis. Coordinated cytoplasmic to nuclear cell-signaling and mitochondrial transcriptional responses to the presence of elevated cytoplasmic oxidant production, e.g., H2O2, allows nuclear translocation of the Nfe2l2 transcription factor and up-regulation of downstream cytoprotective genes such as heme oxygenase-1 which generates physiologic signals, such as CO that up-regulates Nfe212 gene transcription. Simultaneously, a number of other DNA binding transcription factors are expressed and/or activated under redox control, such as Nuclear Respiratory Factor-1 (NRF-1), and lead to the induction of genes involved in both intracellular and mitochondria-specific repair mechanisms. The same insults, particularly those related to vascular stress and inflammation also produce elevated levels of nitric oxide, which also has mitochondrial protein thiol-protective functions and induces mitochondrial biogenesis through cyclic GMP-dependent and perhaps other pathways. This brief review provides an overview of these pathways and interconnected cardiac repair mechanisms. PMID:26578967

  20. Murine gammaherpesvirus M2 protein induction of IRF4 via the NFAT pathway leads to IL-10 expression in B cells.

    PubMed

    Rangaswamy, Udaya S; Speck, Samuel H

    2014-01-01

    Reactivation of the gammaherpesviruses Epstein-Barr virus (EBV), Kaposi's sarcoma-associated herpesvirus (KSHV) and murine gammaherpesvirus 68 (MHV68) from latently infected B cells has been linked to plasma cell differentiation. We have previously shown that the MHV68 M2 protein is important for virus reactivation from B cells and, when expressed alone in primary murine B cells, can drive B cell differentiation towards a pre-plasma cell phenotype. In addition, expression of M2 in primary murine B cells leads to secretion of high levels of IL-10 along with enhanced proliferation and survival. Furthermore, the absence of M2 in vivo leads to a defect in the appearance of MHV68 infected plasma cells in the spleen at the peak of MHV68 latency. Here, employing an inducible B cell expression system, we have determined that M2 activates the NFAT pathway in a Src kinase-dependent manner--leading to induction of the plasma cell-associated transcription factor, Interferon Regulatory Factor-4 (IRF4). Furthermore, we show that expression of IRF4 alone in a B cell line up-regulates IL-10 expression in culture supernatants, revealing a novel role for IRF4 in B cell induced IL-10. Consistent with the latter observation, we show that IRF4 can regulate the IL-10 promoter in B cells. In primary murine B cells, addition of cyclosporine (CsA) resulted in a significant decrease in M2-induced IL-10 levels as well as IRF4 expression, emphasizing the importance of the NFAT pathway in M2- -mediated induction of IL-10. Together, these studies argue in favor of a model wherein M2 activation of the NFAT pathway initiates events leading to increased levels of IRF4--a key player in plasma cell differentiation--which in turn triggers IL-10 expression. In the context of previous findings, the data presented here provides insights into how M2 facilitates plasma cell differentiation and subsequent virus reactivation.

  1. Murine Gammaherpesvirus M2 Protein Induction of IRF4 via the NFAT Pathway Leads to IL-10 Expression in B Cells

    PubMed Central

    Rangaswamy, Udaya S.; Speck, Samuel H.

    2014-01-01

    Reactivation of the gammaherpesviruses Epstein-Barr virus (EBV), Kaposi's sarcoma-associated herpesvirus (KSHV) and murine gammaherpesvirus 68 (MHV68) from latently infected B cells has been linked to plasma cell differentiation. We have previously shown that the MHV68 M2 protein is important for virus reactivation from B cells and, when expressed alone in primary murine B cells, can drive B cell differentiation towards a pre-plasma cell phenotype. In addition, expression of M2 in primary murine B cells leads to secretion of high levels of IL-10 along with enhanced proliferation and survival. Furthermore, the absence of M2 in vivo leads to a defect in the appearance of MHV68 infected plasma cells in the spleen at the peak of MHV68 latency. Here, employing an inducible B cell expression system, we have determined that M2 activates the NFAT pathway in a Src kinase-dependent manner – leading to induction of the plasma cell-associated transcription factor, Interferon Regulatory Factor-4 (IRF4). Furthermore, we show that expression of IRF4 alone in a B cell line up-regulates IL-10 expression in culture supernatants, revealing a novel role for IRF4 in B cell induced IL-10. Consistent with the latter observation, we show that IRF4 can regulate the IL-10 promoter in B cells. In primary murine B cells, addition of cyclosporine (CsA) resulted in a significant decrease in M2-induced IL-10 levels as well as IRF4 expression, emphasizing the importance of the NFAT pathway in M2- mediated induction of IL-10. Together, these studies argue in favor of a model wherein M2 activation of the NFAT pathway initiates events leading to increased levels of IRF4 – a key player in plasma cell differentiation – which in turn triggers IL-10 expression. In the context of previous findings, the data presented here provides insights into how M2 facilitates plasma cell differentiation and subsequent virus reactivation. PMID:24391506

  2. Increased expression of estrogen-related receptor β during adaptation of adult cardiomyocytes to sustained hypoxia

    PubMed Central

    Cunningham, Kathryn F; Beeson, Gyda C; Beeson, Craig C; McDermott, Paul J

    2016-01-01

    Estrogen-related Receptors (ERR) are members of the steroid hormone receptor superfamily of transcription factors that regulate expression of genes required for energy metabolism including mitochondrial biogenesis, fatty acid oxidation and oxidative phosphorylation. While ERRα and EPPγ isoforms are known to share a wide array of target genes in the adult myocardium, the function of ERRβ has not been characterized in cardiomyocytes. The purpose of this study was to determine the role of ERRβ in regulating energy metabolism in adult cardiomyocytes in primary culture. Adult feline cardiomyocytes were electrically stimulated to contract in either hypoxia (0.5% O2) or normoxia (21% O2). As compared to baseline values measured in normoxia, ERRβ mRNA levels increased significantly after 8 hours of hypoxia and remained elevated over 24 h. Conversely, ERRβ mRNA decreased to normoxic levels after 4 hours of reoxygenation. Hypoxia increased expression of the α and β isoforms of Peroxisome Proliferator-Activated Receptor γ Coactivator-1 (PGC-1) mRNA by 6-fold and 3-fold, respectively. Knockdown of ERRβ expression via adenoviral-mediated delivery of ERRβ shRNA blocked hypoxia-induced increases in PGC-1β mRNA, but not PGC-1α mRNA. Loss of ERRβ had no effect on mtDNA content as measured after 24 h of hypoxia. To determine whether loss of ERRβ affected mitochondrial function, oxygen consumption rates (OCR) were measured in contracting versus quiescent cardiomyocytes in normoxia. OCR was significantly lower in contracting cardiomyocytes expressing ERRβ shRNA than scrambled shRNA controls. Maximal OCR also was reduced by ERRβ knockdown. In conclusion: 1) hypoxia increases in ERRβ mRNA expression in contracting cardiomyocytes; 2) ERRβ is required for induction of the PGC-1β isoform in response to hypoxia; 3) ERRβ expression is required to sustain OCR in normoxic conditions. PMID:27335690

  3. Light Chain Amyloid Fibrils Cause Metabolic Dysfunction in Human Cardiomyocytes

    SciTech Connect

    McWilliams-Koeppen, Helen P.; Foster, James S.; Hackenbrack, Nicole; Ramirez-Alvarado, Marina; Donohoe, Dallas; Williams, Angela; Macy, Sallie; Wooliver, Craig; Wortham, Dale; Morrell-Falvey, Jennifer; Foster, Carmen M.; Kennel, Stephen J.; Wall, Jonathan S.

    2015-09-22

    Light chain (AL) amyloidosis is the most common form of systemic amyloid disease, and cardiomyopathy is a dire consequence, resulting in an extremely poor prognosis. AL is characterized by the production of monoclonal free light chains that deposit as amyloid fibrils principally in the heart, liver, and kidneys causing organ dysfunction. We have studied the effects of amyloid fibrils, produced from recombinant λ6 light chain variable domains, on metabolic activity of human cardiomyocytes. The data indicate that fibrils at 0.1 μM, but not monomer, significantly decrease the enzymatic activity of cellular NAD(P)H-dependent oxidoreductase, without causing significant cell death. The presence of amyloid fibrils did not affect ATP levels; however, oxygen consumption was increased and reactive oxygen species were detected. Confocal fluorescence microscopy showed that fibrils bound to and remained at the cell surface with little fibril internalization. Ultimately, these data indicate that AL amyloid fibrils severely impair cardiomyocyte metabolism in a dose dependent manner. These data suggest that effective therapeutic intervention for these patients should include methods for removing potentially toxic amyloid fibrils.

  4. Light Chain Amyloid Fibrils Cause Metabolic Dysfunction in Human Cardiomyocytes

    DOE PAGES

    McWilliams-Koeppen, Helen P.; Foster, James S.; Hackenbrack, Nicole; Ramirez-Alvarado, Marina; Donohoe, Dallas; Williams, Angela; Macy, Sallie; Wooliver, Craig; Wortham, Dale; Morrell-Falvey, Jennifer; et al

    2015-09-22

    Light chain (AL) amyloidosis is the most common form of systemic amyloid disease, and cardiomyopathy is a dire consequence, resulting in an extremely poor prognosis. AL is characterized by the production of monoclonal free light chains that deposit as amyloid fibrils principally in the heart, liver, and kidneys causing organ dysfunction. We have studied the effects of amyloid fibrils, produced from recombinant λ6 light chain variable domains, on metabolic activity of human cardiomyocytes. The data indicate that fibrils at 0.1 μM, but not monomer, significantly decrease the enzymatic activity of cellular NAD(P)H-dependent oxidoreductase, without causing significant cell death. The presencemore » of amyloid fibrils did not affect ATP levels; however, oxygen consumption was increased and reactive oxygen species were detected. Confocal fluorescence microscopy showed that fibrils bound to and remained at the cell surface with little fibril internalization. Ultimately, these data indicate that AL amyloid fibrils severely impair cardiomyocyte metabolism in a dose dependent manner. These data suggest that effective therapeutic intervention for these patients should include methods for removing potentially toxic amyloid fibrils.« less

  5. Analysis of cardiomyocyte movement in the developing murine heart

    SciTech Connect

    Hashimoto, Hisayuki; Yuasa, Shinsuke; Tabata, Hidenori; Tohyama, Shugo; Seki, Tomohisa; Egashira, Toru; Hayashiji, Nozomi; Hattori, Fumiyuki; Kusumoto, Dai; Kunitomi, Akira; Takei, Makoto; Kashimura, Shin; Yozu, Gakuto; Shimojima, Masaya; Motoda, Chikaaki; Muraoka, Naoto; Nakajima, Kazunori; Sakaue-Sawano, Asako; Miyawaki, Atsushi; Fukuda, Keiichi

    2015-09-04

    The precise assemblage of several types of cardiac precursors controls heart organogenesis. The cardiac precursors show dynamic movement during early development and then form the complicated heart structure. However, cardiomyocyte movements inside the newly organized mammalian heart remain unclear. We previously established the method of ex vivo time-lapse imaging of the murine heart to study cardiomyocyte behavior by using the Fucci (fluorescent ubiquitination-based cell cycle indicator) system, which can effectively label individual G1, S/G2/M, and G1/S-transition phase nuclei in living cardiomyocytes as red, green, and yellow, respectively. Global analysis of gene expression in Fucci green positive ventricular cardiomyocytes confirmed that cell cycle regulatory genes expressed in G1/S, S, G2/M, and M phase transitions were upregulated. Interestingly, pathway analysis revealed that many genes related to the cell cycle were significantly upregulated in the Fucci green positive ventricular cardiomyocytes, while only a small number of genes related to cell motility were upregulated. Time-lapse imaging showed that murine proliferating cardiomyocytes did not exhibit dynamic movement inside the heart, but stayed on site after entering the cell cycle. - Highlights: • We directly visualized cardiomyocyte movement inside the developing murine heart. • Cell cycle related genes were upregulated in the proliferating cardiomyocytes. • Time-lapse imaging revealed that proliferating murine cardiomyocytes stayed in place. • Murine ventricular cardiomyocytes proliferate on site during development.

  6. NFAT5/STAT3 interaction mediates synergism of high salt with IL-17 towards induction of VEGF-A expression in breast cancer cells

    PubMed Central

    Amara, Suneetha; Alotaibi, Dalal; Tiriveedhi, Venkataswarup

    2016-01-01

    Chronic inflammation has been considered an important player in cancer proliferation and progression. High salt (sodium chloride) levels have been considered a potent inducer of chronic inflammation. In the present study, the synergistic role of high salt with interleukin (IL)-17 towards induction of the inflammatory and angiogenic stress factor vascular endothelial growth factor (VEGF)-A was investigated. Stimulation of MCF-7 breast cancer cells with high salt (0.2 M NaCl) and sub-minimal IL-17 (1 ng/ml) enhanced the expression of VEGF-A (2.9 and 2.6-fold, respectively, P<0.05) compared with untreated cells. Furthermore, co-treatment with both high salt and sub-minimal IL-17 led to a 5.9-fold increase in VEGF-A expression (P<0.01), thus suggesting a synergistic role of these factors. VEGF-A promoter analysis and specific small interfering RNA knock-down of transcription factors revealed that high salt induced VEGF-A expression through nuclear factor of activated T-cells (NFAT)5, while IL-17 induced VEGF-A expression via signal transducer and activator of transcription (STAT)3 signaling mechanisms. Treatment of normal human aortic endothelial cells with the supernatant of activated MCF-7 cells enhanced cell migration and induced expression of migration-specific factors, including vascular cell adhesion protein, β1 integrin and cluster of differentiation 31. These data suggest that high salt levels synergize with pro-inflammatory IL-17 to potentially induce cancer progression and metastasis through VEGF-A expression. Therefore, low-salt diet, anti-NFAT5 and anti-STAT3 therapies may provide novel avenues for enhanced efficiency of the current cancer therapy. PMID:27446373

  7. Klotho inhibits angiotensin II-induced cardiomyocyte hypertrophy through suppression of the AT1R/beta catenin pathway.

    PubMed

    Yu, Liangzhu; Meng, Wei; Ding, Jieqiong; Cheng, Menglin

    2016-04-29

    Myocardial hypertrophy is an independent risk factor for cardiac morbidity and mortality. The antiaging protein klotho reportedly possesses a protective role in cardiac diseases. However, the precise mechanisms underlying the cardioprotective effects of klotho remain unknown. This study was aimed to determine the effects of klotho on angiotensin II (Ang II)-induced hypertrophy in neonatal rat cardiomyocytes and the possible mechanism of actions. We found that klotho significantly inhibited Ang II-induced hypertrophic growth of neonatal cardiomyocytes, as evidenced by decreased [(3)H]-Leucine incorporation, cardiomyocyte surface area and β-myosin heavy chain (β-MHC) mRNA expression. Meanwhile, klotho inhibited Ang II-stimulated activation of the Wnt/β-catenin pathway in cardiomyocytes, as evidenced by decreased protein expression of active β-catenin, downregulated protein and mRNA expression of the β-catenin target genes c-myc and cyclin D1, and increased β-catenin phosphorylation. Inhibition of the Wnt/β-catenin pathway by the specific inhibitor XAV939 markedly attenuated Ang II-induced cardiomyocyte hypertrophy. The further study revealed that klotho treatment significantly downregulated protein expression of Ang II receptor type I (AT1R) but not type II (AT2R). The AT1R antagonist losartan inhibited Ang II-stimulated activation of the Wnt/β-catenin pathway and cardiomyocyte hypertrophy. Our findings suggest that klotho inhibits Ang II-induced cardiomyocyte hypertrophy through suppression of the AT1R/β-catenin signaling pathway, which may provide new insights into the mechanism underlying the protective effects of klotho in heart diseases, and raise the possibility that klotho may act as an endogenous antihypertrophic factor by inhibiting the Ang II signaling pathway. PMID:26970306

  8. Negative elongation factor controls energy homeostasis in cardiomyocytes.

    PubMed

    Pan, Haihui; Qin, Kunhua; Guo, Zhanyong; Ma, Yonggang; April, Craig; Gao, Xiaoli; Andrews, Thomas G; Bokov, Alex; Zhang, Jianhua; Chen, Yidong; Weintraub, Susan T; Fan, Jian-Bing; Wang, Degeng; Hu, Yanfen; Aune, Gregory J; Lindsey, Merry L; Li, Rong

    2014-04-10

    Negative elongation factor (NELF) is known to enforce promoter-proximal pausing of RNA polymerase II (Pol II), a pervasive phenomenon observed across multicellular genomes. However, the physiological impact of NELF on tissue homeostasis remains unclear. Here, we show that whole-body conditional deletion of the B subunit of NELF (NELF-B) in adult mice results in cardiomyopathy and impaired response to cardiac stress. Tissue-specific knockout of NELF-B confirms its cell-autonomous function in cardiomyocytes. NELF directly supports transcription of those genes encoding rate-limiting enzymes in fatty acid oxidation (FAO) and the tricarboxylic acid (TCA) cycle. NELF also shares extensively transcriptional target genes with peroxisome proliferator-activated receptor α (PPARα), a master regulator of energy metabolism in the myocardium. Mechanistically, NELF helps stabilize the transcription initiation complex at the metabolism-related genes. Our findings strongly indicate that NELF is part of the PPARα-mediated transcription regulatory network that maintains metabolic homeostasis in cardiomyocytes. PMID:24656816

  9. DNA hydroxymethylation controls cardiomyocyte gene expression in development and hypertrophy

    PubMed Central

    Greco, Carolina M.; Kunderfranco, Paolo; Rubino, Marcello; Larcher, Veronica; Carullo, Pierluigi; Anselmo, Achille; Kurz, Kerstin; Carell, Thomas; Angius, Andrea; Latronico, Michael V. G.; Papait, Roberto; Condorelli, Gianluigi

    2016-01-01

    Methylation at 5-cytosine (5-mC) is a fundamental epigenetic DNA modification associated recently with cardiac disease. In contrast, the role of 5-hydroxymethylcytosine (5-hmC)—5-mC's oxidation product—in cardiac biology and disease is unknown. Here we assess the hydroxymethylome in embryonic, neonatal, adult and hypertrophic mouse cardiomyocytes, showing that dynamic modulation of hydroxymethylated DNA is associated with specific transcriptional networks during heart development and failure. DNA hydroxymethylation marks the body of highly expressed genes as well as distal regulatory regions with enhanced activity. Moreover, pathological hypertrophy is characterized by a shift towards a neonatal 5-hmC distribution pattern. We also show that the ten-eleven translocation 2 (TET2) enzyme regulates the expression of key cardiac genes, such as Myh7, through 5-hmC deposition on the gene body and at enhancers. Thus, we provide a genome-wide analysis of 5-hmC in the cardiomyocyte and suggest a role for this epigenetic modification in heart development and disease. PMID:27489048

  10. Protein kinase Cα inhibits myocardin-induced cardiomyocyte hypertrophy through the promotion of myocardin phosphorylation.

    PubMed

    Li, Weizong; Wang, Nan; Li, Man; Gong, Huiqin; Liao, Xinghua; Yang, Xiaolong; Zhang, Tongcun

    2015-09-01

    Myocardin plays a key role in the development of cardiac hypertrophy. However, the upstream signals that control the stability and transactivity of myocardin remain to be fully understood. The expression of protein kinase Cα (PKCα) also induces cardiac hypertrophy. An essential downstream molecule of PKCα, extracellular signal-regulated kinase 1/2, was reported to negatively regulate the activities of myocardin. But, the effect of cooperation between PKCα and myocardin and the potential molecular mechanism by which PKCα regulates myocardin-mediated cardiac hypertrophy are unclear. In this study, a luciferase assay was performed using H9C2 cells transfected with expression plasmids for PKCα and myocardin. Surprisingly, the results showed that PKCα inhibited the transcriptional activity of myocardin. PKCα inhibited myocardin-induced cardiomyocyte hypertrophy, demonstrated by the decrease in cell surface area and fetal gene expression, in cardiomyocyte cells overexpressing PKCα and myocardin. The potential mechanism underlying the inhibition effect of PKCα on the function of myocardin is further explored. PKCα directly promoted the basal phosphorylation of endogenous myocardin at serine and threonine residues. In myocardin-overexpressing cardiomyocyte cells, PKCα induced the excessive phosphorylation of myocardin, resulting in the degradation of myocardin and a transcriptional suppression of hypertrophic genes. These results demonstrated that PKCα inhibits myocardin-induced cardiomyocyte hypertrophy through the promotion of myocardin phosphorylation. PMID:26206583

  11. Cardiomyocyte Overexpression of FABP4 Aggravates Pressure Overload-Induced Heart Hypertrophy

    PubMed Central

    Zhang, Ji; Qiao, Congzhen; Chang, Lin; Guo, Yanhong; Fan, Yanbo; Villacorta, Luis; Chen, Y. Eugene; Zhang, Jifeng

    2016-01-01

    Fatty acid binding protein 4 (FABP4) is a member of the intracellular lipid-binding protein family, responsible for the transportation of fatty acids. It is considered to express mainly in adipose tissues, and be strongly associated with inflammation, obesity, diabetes and cardiovasculardiseases. Here we report that FABP4 is also expressed in cardiomyocytes and plays an important role in regulating heart function under pressure overload. We generated heart-specific transgenic FABP4 (FABP4-TG) mice using α myosin-heavy chain (α-MHC) promoter and human FABP4 sequence, resulting in over-expression of FABP4 in cardiomyocytes. The FABP4-TG mice displayed normal cardiac morphology and contractile function. When they were subjected to the transverse aorta constriction (TAC) procedure, the FABP4-TG mice developed more cardiac hypertrophy correlated with significantly increased ERK phosphorylation, compared with wild type controls. FABP4 over-expression in cardiomyocytes activated phosphor-ERK signal and up-regulate the expression of cardiac hypertrophic marker genes. Conversely, FABP4 induced phosphor-ERK signal and hypertrophic gene expressions can be markedly inhibited by an ERK inhibitor PD098059 as well as the FABP4 inhibitor BMS309403. These results suggest that FABP4 over-expression in cardiomyocytes can aggravate the development of cardiac hypertrophy through the activation of ERK signal pathway. PMID:27294862

  12. Cardiomyocyte Overexpression of FABP4 Aggravates Pressure Overload-Induced Heart Hypertrophy.

    PubMed

    Zhang, Ji; Qiao, Congzhen; Chang, Lin; Guo, Yanhong; Fan, Yanbo; Villacorta, Luis; Chen, Y Eugene; Zhang, Jifeng

    2016-01-01

    Fatty acid binding protein 4 (FABP4) is a member of the intracellular lipid-binding protein family, responsible for the transportation of fatty acids. It is considered to express mainly in adipose tissues, and be strongly associated with inflammation, obesity, diabetes and cardiovasculardiseases. Here we report that FABP4 is also expressed in cardiomyocytes and plays an important role in regulating heart function under pressure overload. We generated heart-specific transgenic FABP4 (FABP4-TG) mice using α myosin-heavy chain (α-MHC) promoter and human FABP4 sequence, resulting in over-expression of FABP4 in cardiomyocytes. The FABP4-TG mice displayed normal cardiac morphology and contractile function. When they were subjected to the transverse aorta constriction (TAC) procedure, the FABP4-TG mice developed more cardiac hypertrophy correlated with significantly increased ERK phosphorylation, compared with wild type controls. FABP4 over-expression in cardiomyocytes activated phosphor-ERK signal and up-regulate the expression of cardiac hypertrophic marker genes. Conversely, FABP4 induced phosphor-ERK signal and hypertrophic gene expressions can be markedly inhibited by an ERK inhibitor PD098059 as well as the FABP4 inhibitor BMS309403. These results suggest that FABP4 over-expression in cardiomyocytes can aggravate the development of cardiac hypertrophy through the activation of ERK signal pathway. PMID:27294862

  13. Mechanisms by which calcium receptor stimulation modifies electromechanical coupling in isolated ventricular cardiomyocytes.

    PubMed

    Schreckenberg, Rolf; Dyukova, Elena; Sitdikova, Guzel; Abdallah, Yaser; Schlüter, Klaus-Dieter

    2015-02-01

    The calcium-sensing receptor (CaR) is widely expressed throughout the entire cardiovascular system and is capable of activating signaling pathways in different cells. Alongside calcium, the CaR also responds to physiological polycations such as putrescine underlining a participation in physiological and pathophysiological processes. Here, we aimed to determine mechanisms as to how CaR activation affects the contractile responsiveness of ventricular cardiomyocytes under basal and stimulated conditions. For that purpose, cardiac myocytes from 3-month-old male Wistar rats were isolated, and the acute effects of an antagonist (NPS2390), agonists (putrescine and gadolinium), or of downregulation of the CaR by siRNA on cell shortening were recorded in a cell-edge-detection system. In addition, experiments were performed on muscle stripes and Langendorff preparations. Mechanistic insights were taken from calcium transients of beating fura-2 AM-loaded cardiomyocytes and western blots. Isolated ventricular cardiomyocytes constitutively express CaR. The expression in the atria is less pronounced. Acute inhibition of CaR reduced basal cell shortening of ventricular myocytes at nearly physiological levels of extracellular calcium. Inhibition of CaR strongly reduced contractility of ventricular muscle stripes but not of atria. Activation of CaR by putrescine and gadolinium influences the contractile responsiveness of isolated cardiomyocytes. Increased calcium mobilization from the sarcoplasmic reticulum via an IP3-dependent mechanism was responsible for amplified systolic calcium transients and a subsequent improvement in cell shortening. Alongside with these effects, activation of CaR increased relaxation velocity of the cells. In conclusion, ventricular CaR expression affects contractile parameters of ventricular heart muscle cells and modifies electromechanical coupling of cardiomyocytes.

  14. Tyrosine phosphorylation of RACK1 triggers cardiomyocyte hypertrophy by regulating the interaction between p300 and GATA4.

    PubMed

    Suzuki, Hidetoshi; Katanasaka, Yasufumi; Sunagawa, Yoichi; Miyazaki, Yusuke; Funamoto, Masafumi; Wada, Hiromichi; Hasegawa, Koji; Morimoto, Tatsuya

    2016-09-01

    The zinc finger protein GATA4 is a transcription factor involved in cardiomyocyte hypertrophy. It forms a functional complex with the intrinsic histone acetyltransferase (HAT) p300. The HAT activity of p300 is required for the acetylation and transcriptional activity of GATA4, as well as for cardiomyocyte hypertrophy and the development of heart failure. In the present study, we have identified Receptor for Activated Protein Kinase C1 (RACK1) as a novel GATA4-binding protein using tandem affinity purification and mass spectrometry analyses. We found that exogenous RACK1 repressed phenylephrine (PE)-induced hypertrophic responses, such as myofibrillar organization, increased cell size, and hypertrophy-associated gene transcription, in cultured cardiomyocytes. RACK1 physically interacted with GATA4 and the overexpression of RACK1 reduced PE-induced formation of the p300/GATA4 complex and the acetylation and DNA binding activity of GATA4. In response to hypertrophic stimulation in cultured cardiomyocytes and in the hearts of hypertensive heart disease model rats, the tyrosine phosphorylation of RACK1 was increased, and the binding between GATA4 and RACK1 was reduced. In addition, the tyrosine phosphorylation of RACK1 was required for the disruption of the RACK1/GATA4 complex and for the formation of the p300/GATA4 complex. These findings demonstrate that RACK1 is involved in p300/GATA4-dependent hypertrophic responses in cardiomyocytes and is a promising therapeutic target for heart failure. PMID:27208796

  15. Elastic interactions synchronize beating in cardiomyocytes.

    PubMed

    Cohen, Ohad; Safran, Samuel A

    2016-07-13

    Motivated by recent experimental results, we study theoretically the synchronization of the beating phase and frequency of two nearby cardiomyocyte cells. Each cell is represented as an oscillating force dipole in an infinite, viscoelastic medium and the propagation of the elastic signal within the medium is predicted. We examine the steady-state beating of two nearby cells, and show that elastic interactions result in forces that synchronize the phase and frequency of beating in a manner that depends on their mutual orientation. The theory predicts both in-phase and anti-phase steady-state beating depending on the relative cell orientations, as well as how synchronized beating varies with substrate elasticity and the inter-cell distance. These results suggest how mechanics plays a role in cardiac efficiency, and may be relevant for the design of cardiomyocyte based micro devices and other biomedical applications.

  16. Elastic interactions synchronize beating in cardiomyocytes.

    PubMed

    Cohen, Ohad; Safran, Samuel A

    2016-07-13

    Motivated by recent experimental results, we study theoretically the synchronization of the beating phase and frequency of two nearby cardiomyocyte cells. Each cell is represented as an oscillating force dipole in an infinite, viscoelastic medium and the propagation of the elastic signal within the medium is predicted. We examine the steady-state beating of two nearby cells, and show that elastic interactions result in forces that synchronize the phase and frequency of beating in a manner that depends on their mutual orientation. The theory predicts both in-phase and anti-phase steady-state beating depending on the relative cell orientations, as well as how synchronized beating varies with substrate elasticity and the inter-cell distance. These results suggest how mechanics plays a role in cardiac efficiency, and may be relevant for the design of cardiomyocyte based micro devices and other biomedical applications. PMID:27352146

  17. Characterizing functional stem cell–cardiomyocyte interactions

    PubMed Central

    Bursac, Nenad; Kirkton, Robert D; McSpadden, Luke C; Liau, Brian

    2010-01-01

    Despite the progress in traditional pharmacological and organ transplantation therapies, heart failure still afflicts 5.3 million Americans. Since June 2000, stem cell-based approaches for the prevention and treatment of heart failure have been pursued in clinics with great excitement; however, the exact mechanisms of how transplanted cells improve heart function remain elusive. One of the main difficulties in answering these questions is the limited ability to directly access and study interactions between implanted cells and host cardiomyocytes in situ. With the growing number of candidate cell types for potential clinical use, it is becoming increasingly more important to establish standardized, well-controlled in vitro and in situ assays to compare the efficacy and safety of different stem cells in cardiac repair. This article describes recent innovative methodologies to characterize direct functional interactions between stem cells and cardiomyocytes, aimed to facilitate the rational design of future cell-based therapies for heart disease. PMID:20017697

  18. Reciprocal regulation of transcription factors and PLC isozyme gene expression in adult cardiomyocytes.

    PubMed

    Singal, Tushi; Dhalla, Naranjan S; Tappia, Paramjit S

    2010-06-01

    By employing a pharmacological approach, we have shown that phospholipase C (PLC) activity is involved in the regulation of gene expression of transcription factors such as c-Fos and c-Jun in cardiomyocytes in response to norepinephrine (NE). However, there is no information available regarding the identity of specific PLC isozymes involved in the regulation of c-Fos and c-Jun or on the involvement of these transcription factors in PLC isozyme gene expression in adult cardiomyocytes. In this study, transfection of cardiomyocytes with PLC isozyme specific siRNA was found to prevent the NE-mediated increases in the corresponding PLC isozyme gene expression, protein content and activity. Unlike PLC gamma(1) gene, silencing of PLC beta(1), beta(3) and delta(1) genes with si RNA prevented the increases in c-Fos and c-Jun gene expression in response to NE. On the other hand, transfection with c-Jun si RNA suppressed the NE-induced increase in c-Jun as well as PLC beta(1), beta(3) and delta(1) gene expression, but had no effect on PLC gamma(1) gene expression. Although transfection of cardiomyocytes with c-Fos si RNA prevented NE-induced expression of c-Fos, PLC beta(1) and PLC beta(3) genes, it did not affect the increases in PLC delta(1) and PLC gamma(1) gene expression. Silencing of either c-Fos or c-Jun also depressed the NE-mediated increases in PLC beta(1), beta(3) and gamma(1) protein content and activity in an isozyme specific manner. Furthermore, silencing of all PLC isozymes as well as of c-Fos and c-Jun resulted in prevention of the NE-mediated increase in atrial natriuretic factor gene expression. These findings, by employing gene silencing techniques, demonstrate that there occurs a reciprocal regulation of transcription factors and specific PLC isozyme gene expression in cardiomyocytes.

  19. Direct Cardiomyocyte Reprogramming: A New Direction for Cardiovascular Regenerative Medicine

    PubMed Central

    Yi, B. Alexander; Mummery, Christine L.; Chien, Kenneth R.

    2013-01-01

    The past few years have seen unexpected new developments in direct cardiomyocyte reprogramming. Direct cardiomyocyte reprogramming potentially offers an entirely novel approach to cardiovascular regenerative medicine by converting cardiac fibroblasts into functional cardiomyocytes in situ. There is much to be learned, however, about the mechanisms of direct reprogramming in order that the process can be made more efficient. Early efforts have suggested that this new technology can be technically challenging. Moreover, new methods of inducing heart reprogramming will need to be developed before this approach can be translated to the bedside. Despite this, direct cardiomyocyte reprogramming may lead to new therapeutic options for sufferers of heart disease. PMID:24003244

  20. Cardiomyocyte Death: Insights from Molecular and Microstructural Magnetic Resonance Imaging

    PubMed Central

    Berry, Natalia C.

    2011-01-01

    Cardiomyocytes can die via necrosis, apoptosis, and autophagy. Although the molecular signals and pathways underlying these processes have been well elucidated, the pathophysiology of cardiomyocyte death remains incompletely understood. This review describes the development and application of novel imaging techniques to detect and characterize cardiomyocyte death noninvasively in vivo. It focuses on molecular and microstructural magnetic resonance images (MRIs) and their respective abilities to image cellular events such as apoptosis, inflammation, and myofiber architecture. These in vivo imaging techniques have the potential to provide novel insights into the mechanisms of cardiomyocyte death and to help guide the development of novel cardioprotective therapies. PMID:21298427

  1. TGF-β receptor type II costameric localization in cardiomyocytes and host cell TGF-β response is disrupted by Trypanosoma cruzi infection.

    PubMed

    Calvet, Claudia Magalhães; Silva, Tatiana Araújo; DE Melo, Tatiana Galvão; DE Araújo-Jorge, Tânia Cremonini; Pereira, Mirian Claudia DE Souza

    2016-05-01

    Transforming growth factor beta (TGF-β) cytokine is involved in Chagas disease establishment and progression. Since Trypanosoma cruzi can modulate host cell receptors, we analysed the TGF-β receptor type II (TβRII) expression and distribution during T. cruzi - cardiomyocyte interaction. TβRII immunofluorescent staining revealed a striated organization in cardiomyocytes, which was co-localized with vinculin costameres and enhanced (38%) after TGF-β treatment. Cytochalasin D induced a decrease of 45·3% in the ratio of cardiomyocytes presenting TβRII striations, demonstrating an association of TβRII with the cytoskeleton. Western blot analysis showed that cytochalasin D significantly inhibited Smad 2 phosphorylation and fibronectin stimulation after TGF-β treatment in cardiomyocytes. Trypanosoma cruzi infection elicited a decrease of 79·8% in the frequency of cardiomyocytes presenting TβRII striations, but did not interfere significantly in its expression. In addition, T. cruzi-infected cardiomyocytes present a lower response to exogenous TGF-β, showing no enhancement of TβRII striations and a reduction of phosphorylated Smad 2, with no significant difference in TβRII expression when compared to uninfected cells. Together, these results suggest that the co-localization of TβRII with costameres is important in activating the TGF-β signalling cascade, and that T. cruzi-derived cytoskeleton disorganization could result in altered or low TGF-β response in infected cardiomyocytes.

  2. Early administration of nifedipine protects against angiotensin II-induced cardiomyocyte hypertrophy through regulating CaMKII-SERCA2a pathway and apoptosis in rat cardiomyocytes.

    PubMed

    Luo, Ji; Zhang, Wei-dong; Du, Yi-meng

    2016-04-01

    The calcium channel blocker (CCB), nifedipine, is a more effective treatment for early- than late-stage cardiac hypertrophy. We investigated the effects of early- and late-stage nifedipine administration on calcium homeostasis, CaMKII (Ca(2+) /calmodulin-dependent protein kinase II) activity and apoptosis of cardiomyocytes under hypertrophic stimulation with angiotensin II (AngII). Primary rat cardiomyocytes were divided into five treatment groups: AK, AngII plus the CaMKII inhibitor, KN-93; AN-1 (early-stage), AngII plus nifedipine × 48 h; AN-2 (late-stage), AngII × 48 h, then AngII plus nifedipine × 48 h; C, untreated; and A, AngII × 48 h. The t1/2β [time required for intracellular Ca(2+) concentration ([Ca(2+) ]i) to decline to one half of the peak value] decreased; however, CaMKII and SERCA2a (sarcoplasmic reticulum Ca(2+) -ATPase 2a) activities increased in the AN-1 group compared with the AK group. In the AN-2 group compared with the AN-1 group, CaMKII activity, t1/2α [time required for [Ca(2+) ]i to increase from the bottom to one half of peak value], t1/2β, and apoptosis increased. These results indicate that the timing of CCB administration affects the calcium concentration and apoptosis of hypertrophic cardiomyocytes through the CaMKII-SERCA2a signalling pathway, thereby influencing the drug's protective activity against cardiomyocyte hypertrophy. PMID:26968727

  3. Dexamethasone Increases αvβ3 Integrin Expression and Affinity through a Calcineurin/NFAT Pathway

    PubMed Central

    Faralli, Jennifer A.; Gagen, Debjani; Filla, Mark S.; Crotti, Tania N.; Peters, Donna M.

    2013-01-01

    The purpose of this study was to determine how dexamethasone (DEX) regulates the expression and activity of αvβ3 integrin. FACS analysis showed that DEX treatment induced expression of an activated αvβ3 integrin. Its expression remained high as long as DEX was present and continued following DEX removal. FACS analysis showed that the upregulation of αvβ3 integrin was the result of an increase in the expression of the β3 integrin subunit. By real time qPCR, DEX treatment induced a 6.2-fold increase (p<0.04) in β3 integrin mRNA by day 2 compared to control and remained elevated for 6 days of treatment and then an additional 10 days once the DEX was removed. The increase in β3 integrin mRNA levels required only 1 day of DEX treatment to increase levels for 4 days in the absence of DEX. In contrast, DEX did not alter β1 integrin mRNA or protein levels. The DEX-induced upregulation of β3 integrin mRNA was partly due to an increase in its half-life to 60.7 h from 22.5 h in control cultures (p<0.05) and could be inhibited by RU486 and cycloheximide, suggesting that DEX-induced de novo protein synthesis of an activation factor was needed. The calcineurin inhibitors cyclosporin A (CsA) and FK506 inhibited the DEX induced increase in β3 integrin mRNA. In summary, the DEX-induced increase in β3 integrin is a secondary glucocorticoid response that results in prolonged expression of αvβ3 integrin and the upregulation of the β3 integrin subunit through the calcineurin/NFAT pathway. PMID:24100160

  4. Hydrogen sulfide improves cardiomyocytes electrical remodeling post ischemia/reperfusion injury in rats.

    PubMed

    Sun, Ying-Gang; Wang, Xin-Yan; Chen, Xiu; Shen, Cheng-Xing; Li, Yi-Gang

    2015-01-01

    Hydrogen sulfide (H2S), produced by cystanthionine-γ-lysase (CSE) in the cardiovascular system, is an endogenous gaseous mediator exerting pronounced physiological effects as the third gasotransmitter in addition to nitric oxide (NO) and carbon monoxide (CO). Accumulating evidence indicated that H2S could mediate the cardioprotective effects in myocardial ischemia model. Ventricular arrhythmia is the most important risk factor for cardiac mortality and sudden death after acute myocardial infarction (AMI). The potential impact of H2S on cardiomyocytes electrical remodeling post ischemic insult is not fully explored now. Present study investigated the role of H2S on cardiomyocytes electrical remodeling in rats with ischemia/reperfusion injury. H2S concentration was reduced and arrhythmia score was increased in this model. CSE mRNA level was also upregulated in the ischemic myocardium. Exposure to exogenous NaHS reduced the action potential duration (APD), inhibited L-type Ca(2+) channels and activated K(ATP) channels in cardiomyocytes isolated from ischemic myocardium Exogenous H2S application improves electrical remodeling in cardiomyocytes isolated from ischemic myocardium. These results indicated that reduced H2S level might be linked to ischemia/reperfusion induced arrhythmias. PMID:25755736

  5. Hydrogen sulfide improves cardiomyocytes electrical remodeling post ischemia/reperfusion injury in rats

    PubMed Central

    Sun, Ying-Gang; Wang, Xin-Yan; Chen, Xiu; Shen, Cheng-Xing; Li, Yi-Gang

    2015-01-01

    Hydrogen sulfide (H2S), produced by cystanthionine-γ-lysase (CSE) in the cardiovascular system, is an endogenous gaseous mediator exerting pronounced physiological effects as the third gasotransmitter in addition to nitric oxide (NO) and carbon monoxide (CO). Accumulating evidence indicated that H2S could mediate the cardioprotective effects in myocardial ischemia model. Ventricular arrhythmia is the most important risk factor for cardiac mortality and sudden death after acute myocardial infarction (AMI). The potential impact of H2S on cardiomyocytes electrical remodeling post ischemic insult is not fully explored now. Present study investigated the role of H2S on cardiomyocytes electrical remodeling in rats with ischemia/reperfusion injury. H2S concentration was reduced and arrhythmia score was increased in this model. CSE mRNA level was also upregulated in the ischemic myocardium. Exposure to exogenous NaHS reduced the action potential duration (APD), inhibited L-type Ca2+ channels and activated KATP channels in cardiomyocytes isolated from ischemic myocardium Exogenous H2S application improves electrical remodeling in cardiomyocytes isolated from ischemic myocardium. These results indicated that reduced H2S level might be linked to ischemia/reperfusion induced arrhythmias. PMID:25755736

  6. Complete restoration of multiple dystrophin isoforms in genetically corrected Duchenne muscular dystrophy patient-derived cardiomyocytes.

    PubMed

    Zatti, Susi; Martewicz, Sebastian; Serena, Elena; Uno, Narumi; Giobbe, Giovanni; Kazuki, Yasuhiro; Oshimura, Mitsuo; Elvassore, Nicola

    2014-01-01

    Duchenne muscular dystrophy (DMD)-associated cardiac diseases are emerging as a major cause of morbidity and mortality in DMD patients, and many therapies for treatment of skeletal muscle failed to improve cardiac function. The reprogramming of patients' somatic cells into pluripotent stem cells, combined with technologies for correcting the genetic defect, possesses great potential for the development of new treatments for genetic diseases. In this study, we obtained human cardiomyocytes from DMD patient-derived, induced pluripotent stem cells genetically corrected with a human artificial chromosome carrying the whole dystrophin genomic sequence. Stimulation by cytokines was combined with cell culturing on hydrogel with physiological stiffness, allowing an adhesion-dependent maturation and a proper dystrophin expression. The obtained cardiomyocytes showed remarkable sarcomeric organization of cardiac troponin T and α-actinin, expressed cardiac-specific markers, and displayed electrically induced calcium transients lasting less than 1 second. We demonstrated that the human artificial chromosome carrying the whole dystrophin genomic sequence is stably maintained throughout the cardiac differentiation process and that multiple promoters of the dystrophin gene are properly activated, driving expression of different isoforms. These dystrophic cardiomyocytes can be a valuable source for in vitro modeling of DMD-associated cardiac disease. Furthermore, the derivation of genetically corrected, patient-specific cardiomyocytes represents a step toward the development of innovative cell and gene therapy approaches for DMD.

  7. Complete restoration of multiple dystrophin isoforms in genetically corrected Duchenne muscular dystrophy patient–derived cardiomyocytes

    PubMed Central

    Zatti, Susi; Martewicz, Sebastian; Serena, Elena; Uno, Narumi; Giobbe, Giovanni; Kazuki, Yasuhiro; Oshimura, Mitsuo; Elvassore, Nicola

    2014-01-01

    Duchenne muscular dystrophy (DMD)–associated cardiac diseases are emerging as a major cause of morbidity and mortality in DMD patients, and many therapies for treatment of skeletal muscle failed to improve cardiac function. The reprogramming of patients’ somatic cells into pluripotent stem cells, combined with technologies for correcting the genetic defect, possesses great potential for the development of new treatments for genetic diseases. In this study, we obtained human cardiomyocytes from DMD patient–derived, induced pluripotent stem cells genetically corrected with a human artificial chromosome carrying the whole dystrophin genomic sequence. Stimulation by cytokines was combined with cell culturing on hydrogel with physiological stiffness, allowing an adhesion-dependent maturation and a proper dystrophin expression. The obtained cardiomyocytes showed remarkable sarcomeric organization of cardiac troponin T and α-actinin, expressed cardiac-specific markers, and displayed electrically induced calcium transients lasting less than 1 second. We demonstrated that the human artificial chromosome carrying the whole dystrophin genomic sequence is stably maintained throughout the cardiac differentiation process and that multiple promoters of the dystrophin gene are properly activated, driving expression of different isoforms. These dystrophic cardiomyocytes can be a valuable source for in vitro modeling of DMD-associated cardiac disease. Furthermore, the derivation of genetically corrected, patient-specific cardiomyocytes represents a step toward the development of innovative cell and gene therapy approaches for DMD. PMID:26015941

  8. Effects of epinephrine on angiogenesis-related gene expressions in cultured rat cardiomyocytes

    PubMed Central

    Liu, Henry; Sangkum, Lisa; Liu, Geoffrey; Green, Michael; Li, Marilyn; Kaye, Alan

    2016-01-01

    Abstract Epinephrine is often used for the treatment of patients with heart failure, low cardiac output and cardiac arrest. It can acutely improve hemodynamic parameters; however, it does not seem to improve longer term clinical outcomes. Therefore, we hypothesized that epinephrine may induce unfavorable changes in gene expression of cardiomyocyte. Thus, we investigated effects of epinephrine exposure on the mediation or modulation of gene expression of cultured cardiomyocytes at a genome-wide scale. Our investigation revealed that exposure of cardiomyocytes to epinephrine in an in vitro environment can up-regulate the expression of angiopoietin-2 gene (+2.1 times), and down-regulate the gene expression of neuregulin 1 (−3.7 times), plasminogen activator inhibitor-1 (−2.4 times) and SPARC-related modular calcium-binding protein-2 (−4.5 times). These changes suggest that epinephrine exposure may induce inhibition of angiogenesis-related gene expressions in cultured rat cardiomyocytes. The precise clinical significance of these changes in gene expression, which was induced by epinephrine exposure, warrants further experimental and clinical investigations.

  9. Role of CAPE on cardiomyocyte protection via connexin 43 regulation under hypoxia

    PubMed Central

    Chen, Chien-Cheng; Kuo, Chan-Yen; Chen, Rong-Fu

    2016-01-01

    Background: Cardiomyocyte under hypoxia cause cell death or damage is associated with heart failure. Gap junction, such as connexin 43 play a role in regulation of heart function under hypoxia. Caffeic acid phenethyl ester (CAPE) has been reported as an active component of propolis, has antioxidative, anti-inflammatory antiproliferative and antineoplastic biological properties. Aims: Connexin 43 appear to have a critical role in heart failure under hypoxia, there has been considerable interest in identifying the candidate component or compound to reduce cell death. Methods: In this study, we used human cardiomyocyte as a cell model to study the role of connexin 43 in hypoxia- incubated human cardiomyocyte in absence or presence of CAPE treatment. Results: Results showed that hypoxia induced connexin 43 expression, but not altered in connexin 40. Interestingly, CAPE attenuates hypoxia-caused connexin 43 down-regulation and cell death or cell growth inhibition. Conclusion: We suggested that reduction of cell death in cardiomyocytes by CAPE is associated with an increase in connexin 43 expression. PMID:27766024

  10. Transfusion effects on cardiomyocyte growth and proliferation in fetal sheep following chronic anemia

    PubMed Central

    Jonker, Sonnet S.; Scholz, Thomas D.; Segar, Jeffrey L.

    2011-01-01

    Chronic fetal anemia results in significant cardiac remodeling. The capacity to reverse these effects is unknown. We examined the effects of transfusion on cardiomyocyte adaptations following chronic anemia in fetal sheep subjected to daily hemorrhage beginning at 109d gestation age (GA; term ∼145d). Following 10 days of anemia, one group was euthanized for comparison to age-matched controls. A separate group of anemic fetuses was transfused with red blood cells at 119d GA for comparison to controls at 129d GA. Anemia significantly increased the heart-to-body weight ratio, an effect partially ameliorated following transfusion. Cardiomyocyte dimensions were similar among all groups, suggesting an absence of hypertrophy. The percentages of mono- and binucleated cardiomyocytes were similar between groups at 119d GA, though the percentage of binucleated cells was significantly less in transfused fetuses compared to controls at 129d GA. Protein levels of mitogen activated protein kinases and protein kinase B were similar between controls and their respective intervention groups, except for a significant increase in phosphorylated c-Jun N-terminal kinase 1/2 (JNK1/2) in transfused fetuses. Thus, cardiomyocyte proliferation but not hypertrophy contributes to cardiac enlargement during fetal anemia. Transfusion results in slowing but not cessation of cardiac growth following anemia. PMID:21386752

  11. Paeoniflorin inhibits doxorubicin-induced cardiomyocyte apoptosis by downregulating microRNA-1 expression

    PubMed Central

    LI, JIAN-ZHE; TANG, XIU-NENG; LI, TING-TING; LIU, LI-JUAN; YU, SHU-YI; ZHOU, GUANG-YU; SHAO, QING-RUI; SUN, HUI-PING; WU, CHENG; YANG, YANG

    2016-01-01

    Doxorubicin (DOX) is an effective anthracycline anti-tumor antibiotic. Because of its cardiotoxicity, the clinical application of DOX is limited. Paeoniflorin (PEF), a monoterpene glucoside extracted from the dry root of Paeonia, is reported to exert multiple beneficial effects on the cardiovascular system. The present study was designed to explore the protective effect of PEF against DOX-induced cardiomyocyte apoptosis and the underlying mechanism. In cultured H9c2 cells, PEF (100 µmol/l) was added for 2 h prior to exposure to DOX (5 µmol/l) for 24 h. Cell viability, creatine kinase activity, cardiomyocyte apoptosis, intracellular reactive oxygen species (ROS) levels, and the expression of microRNA-1 (miR-1) and B-cell lymphoma 2 (Bcl-2) were measured following treatment with PEF and/or DOX. The results showed that treatment with DOX notably induced cardiomyocyte apoptosis, concomitantly with enhanced ROS generation, upregulated miR-1 expression and downregulated Bcl-2 expression. These effects of DOX were significantly inhibited by pretreatment of the cells with PEF. These results suggest that the inhibitory effect of PEF on DOX-induced cardiomyocyte apoptosis may be associated with downregulation of miR-1 expression via a reduction in ROS generation. PMID:27284328

  12. RNA-Seq Reveals a Role for NFAT-Signaling in Human Retinal Microvascular Endothelial Cells Treated with TNFα

    PubMed Central

    Penn, John S.

    2015-01-01

    TNFα has been identified as playing an important role in pathologic complications associated with diabetic retinopathy and retinal inflammation, such as retinal leukostasis. However, the transcriptional effects of TNFα on retinal microvascular endothelial cells and the different signaling pathways involved are not yet fully understood. In the present study, RNA-seq was used to profile the transcriptome of human retinal microvascular endothelial cells (HRMEC) treated for 4 hours with TNFα in the presence or absence of the NFAT-specific inhibitor INCA-6, in order to gain insight into the specific effects of TNFα on RMEC and identify any involvement of NFAT signaling. Differential expression analysis revealed that TNFα treatment significantly upregulated the expression of 579 genes when compared to vehicle-treated controls, and subsequent pathway analysis revealed a TNFα-induced enrichment of transcripts associated with cytokine-cytokine receptor interactions, cell adhesion molecules, and leukocyte transendothelial migration. Differential expression analysis comparing TNFα-treated cells to those co-treated with INCA-6 revealed 10 genes whose expression was significantly reduced by the NFAT inhibitor, including those encoding the proteins VCAM1 and CX3CL1 and cytokines CXCL10 and CXCL11. This study identifies the transcriptional effects of TNFα on HRMEC, highlighting its involvement in multiple pathways that contribute to retinal leukostasis, and identifying a previously unknown role for NFAT-signaling downstream of TNFα. PMID:25617622

  13. Depletion of PHD3 Protects Heart from Ischemia/Reperfusion Injury by Inhibiting Cardiomyocyte Apoptosis

    PubMed Central

    Xie, Liang; Pi, Xinchun; Wang, Zhongjing; He, Jun; Willis, Monte S.; Patterson, Cam

    2015-01-01

    PHD3, a member of a family of Prolyl-4 Hydroxylase Domain (PHD) proteins, has long been considered a pro-apoptotic protein. Although the pro-apoptotic effect of PHD3 requires its prolyl hydroxylase activity, it may be independent of HIF-1α, the common substrate of PHDs. PHD3 is highly expressed in the heart, however, its role in cardiomyocyte apoptosis remains unclear. This study was undertaken to determine whether inhibition or depletion of PHD3 inhibits cardiomyocyte apoptosis and attenuates myocardial injury induced by ischemia-reperfusion (I/R). PHD3 knockout mice and littermate controls were subjected to left anterior descending (LAD) coronary artery ligation for 40 minutes followed by reperfusion. Histochemical analysis using Evan’s Blue, triphenyl-tetrazolium chloride and TUNEL staining, demonstrated that myocardial injury and cardiomyocyte apoptosis induced I/R injury were significantly attenuated in PHD3 knockout mice. PHD3 knockout mice exhibited no changes in HIF-1α protein level, the expression of some HIF target genes or the myocardium capillary density at physiological condition. However, depletion of PHD3 further enhanced the induction of HIF-1α protein at hypoxic condition and increased expression of HIF-1α inhibited cardiomyocyte apoptosis induced by hypoxia. In addition, it has been demonstrated that PHD3 plays an important role in ATR/Chk1/p53 pathway. Consistently, a prolyl hydroxylase inhibitor or depletion of PHD3 significantly inhibits the activation of Chk1 and p53 in cardiomyocytes and the subsequent apoptosis induced by doxorubicin, hydrogen peroxide or hypoxia/re-oxygenation. Taken together, these data suggest that depletion of PHD3 leads to increased stabilization of HIF-1α and inhibition of DNA damage response, both of which may contribute to the cardioprotective effect seen with depletion of PHD3. PMID:25633836

  14. NPR3 protects cardiomyocytes from apoptosis through inhibition of cytosolic BRCA1 and TNF-α

    PubMed Central

    Lin, Dong; Chai, Yubo; Izadpanah, Reza; Braun, Stephen E.; Alt, Eckhard

    2016-01-01

    ABSTRACT Natriuretic peptide receptor 3 (NPR3) is a clearance receptor by binding and internalizing natriuretic peptides (NPs) for ultimate degradation. Patients with cardiac failure show elevated NPs. NPs are linked to poor long-term survival because of their apoptotic effects. However, the underling mechanisms have not been identified yet. Here we report the role of NPR3 in anti-apoptosis via the breast cancer type 1 susceptibility protein (BRCA1) and tumor necrosis factor α (TNF-α ). To demonstrate a role for NPR3 in apoptosis, stable H9C2 cardiomyocyte cell lines using shRNA to knockdown NPR3 were generated. The activities of caspase-3, 8, and 9 were significantly increased in NPR3 knockdown H9C2 cardiomyocytes. Knockdown of NPR3 increased the expression of BRCA1. Also NPR3 knockdown remarkably increased the activity of cAMP response element-binding protein (CREB), a positive regulatory element for BRCA1 expression. BRCA1 showed dispersed nuclear localization in non-cardiomyocytes while predominantly cytoplasmic localization in H9C2 cells. Meanwhile, NPR3 knockdown significantly increased TNF-α gene expression. These data show that NPR3 knockdown in H9C2 cells triggered both extrinsic and intrinsic apoptotic pathways. NPR3 protects cardiomyocytes from apoptosis through inhibition of cytosolic BRCA1 and TNF-α, which are regulators of apoptosis. Our studies demonstrate anti-apoptosis role of NPR3 in protecting cardiomyocytes and establish the first molecular link between NP system and programmed cell death. PMID:27494651

  15. Inhibition of type 2A secretory phospholipase A2 reduces death of cardiomyocytes in acute myocardial infarction.

    PubMed

    van Dijk, Annemieke; Krijnen, Paul A J; Vermond, Rob A; Pronk, Amanda; Spreeuwenberg, Marieke; Visser, Frans C; Berney, Richard; Paulus, Walter J; Hack, C Erik; van Milligen, Florine J; Niessen, Hans W M

    2009-06-01

    During acute myocardial infarction (AMI), ischemia leads to necrotic areas surrounded by border zones of reversibly damaged cardiomyocytes, showing membrane flip-flop. During reperfusion type IIA secretory phopholipase A(2) (sPLA(2)-IIA) induces direct cell-toxicity and facilitates binding of other inflammatory mediators on these cardiomyocytes. Therefore, we hypothesized that the specific sPLA(2)-IIA-inhibitor PX-18 would reduce cardiomyocyte death and infarct size in vivo. Wistar rats were treated with PX-18 starting minutes after reperfusion, and at day 1 and 2 post AMI. After 28 days hearts were analyzed. Furthermore, the effect of PX-18 on membrane flip-flop and apoptosis was investigated in vitro. PX-18 significantly inhibited sPLA(2)-IIA activity and reduced infarct size (reduction 73 +/- 9%, P < 0.05), compared to the vehicle-treated group, without impairing wound healing. In vitro, PX-18 significantly reduced reversible membrane flip-flop and apoptosis in cardiomyocytes. However, no sPLA(2)-IIA activity could be detected, suggesting that PX-18 also exerted a protective effect independent of sPLA(2)-IIA. In conclusion, PX-18 is a potent therapeutic to reduce infarct size by inhibiting sPLA(2)-IIA, and possibly also by inhibiting apoptosis of cardiomyocytes in a sPLA(2)-IIA independent manner.

  16. SIRT1 Functions as an Important Regulator of Estrogen-Mediated Cardiomyocyte Protection in Angiotensin II-Induced Heart Hypertrophy

    PubMed Central

    Shen, Tao; Ding, Ling; Ruan, Yang; Qin, Weiwei; Lin, Yajun; Xi, Chao; Lu, Yonggang; Dou, Lin; Zhu, Yuping; Cao, Yuan; Man, Yong; Bian, Yunfei; Wang, Shu; Xiao, Chuanshi; Li, Jian

    2014-01-01

    Background. Sirtuin 1 (SIRT1) is a member of the sirtuin family, which could activate cell survival machinery and has been shown to be protective in regulation of heart function. Here, we determined the mechanism by which SIRT1 regulates Angiotensin II- (AngII-) induced cardiac hypertrophy and injury in vivo and in vitro. Methods. We analyzed SIRT1 expression in the hearts of control and AngII-induced mouse hypertrophy. Female C57BL/6 mice were ovariectomized and pretreated with 17β-estradiol to measure SIRT1 expression. Protein synthesis, cardiomyocyte surface area analysis, qRT-PCR, TUNEL staining, and Western blot were performed on AngII-induced mouse heart hypertrophy samples and cultured neonatal rat ventricular myocytes (NRVMs) to investigate the function of SIRT1. Results. SIRT1 expression was slightly upregulated in AngII-induced mouse heart hypertrophy in vivo and in vitro, accompanied by elevated cardiomyocyte apoptosis. SIRT1 overexpression relieves AngII-induced cardiomyocyte hypertrophy and apoptosis. 17β-Estradiol was able to protect cardiomyocytes from AngII-induced injury with a profound upregulation of SIRT1 and activation of AMPK. Moreover, estrogen receptor inhibitor ICI 182,780 and SIRT1 inhibitor niacinamide could block SIRT1's protective effect. Conclusions. These results indicate that SIRT1 functions as an important regulator of estrogen-mediated cardiomyocyte protection during AngII-induced heart hypertrophy and injury. PMID:25614777

  17. Panax quinquefolium saponin attenuates cardiomyocyte apoptosis induced by thapsigargin through inhibition of endoplasmic reticulum stress

    PubMed Central

    Liu, Mi; Xue, Mei; Wang, Xiao-Reng; Tao, Tian-Qi; Xu, Fei-Fei; Liu, Xiu-Hua; Shi, Da-Zhuo

    2015-01-01

    Background Endoplasmic reticulum (ER) stress-related apoptosis is involved in the pathophysiology of many cardiovascular diseases, and Panax quinquefolium saponin (PQS) is able to inhibit excessive ER stress-related apoptosis of cardiomyocytes following hypoxia/reoxygenation and myocardial infarction. However, the pathway by which PQS inhibits the ER stress-related apoptosis is not well understood. To further investigate the protective effect of PQS against ER stress-related apoptosis, primary cultured cardiomyocytes were stimulated with thapsigargin (TG), which is widely used to model cellular ER stress, and it could induce apoptotic cell death in sufficient concentration. Methods Primary cultured cardiomyocytes from neonatal rats were exposed to TG (1 µmol/L) treatment for 24 h, following PQS pre-treatment (160 µg/mL) for 24 h or pre-treatment with small interfering RNA directed against protein kinase-like endoplasmic reticulum kinase (Si-PERK) for 6 h. The viability and apoptosis rate of cardiomyocytes were detected by cell counting kit-8 and flow cytometry respectively. ER stress-related protein expression, such as glucose-regulated protein 78 (GRP78), calreticulin, PERK, eukaryotic translation initiation factor 2α (eIF2α), activating transcription factor 4 (ATF4), and C/EBP homologous protein (CHOP) were assayed by western blotting. Results Both PQS pre-treatment and PERK knockdown remarkably inhibited the cardiomyocyte apoptosis induced by TG, increased cell viability, decreased phosphorylation of both PERK and eIF2α, and decreased protein levels of both ATF4 and CHOP. There was no statistically significant difference between PQS pre-treatment and PERK knockdown in the cardioprotective effect. Conclusions Our data indicate that the PERK-eIF2α-ATF4-CHOP pathway of ER stress is involved in the apoptosis induced by TG, and PQS might prevent TG-induced cardiomyocyte apoptosis through a mechanism involving the suppression of this pathway. These findings

  18. Mitochondrial Remodeling in Mice with Cardiomyocyte-Specific Lipid Overload

    PubMed Central

    Elezaby, Aly; Sverdlov, Aaron L.; Tu, Vivian H.; Soni, Kanupriya; Luptak, Ivan; Qin, Fuzhong; Liesa, Marc; Shirihai, Orian S.; Rimer, Jamie; Schaffer, Jean E.; Colucci, Wilson S.; Miller, Edward J.

    2014-01-01

    Background Obesity leads to metabolic heart disease (MHD) that is associated with a pathologic increase in myocardial fatty acid (FA) uptake and impairment of mitochondrial function. The mechanism of mitochondrial dysfunction in MHD, which results in oxidant production and decreased energetics, is poorly understood but may be related to excess FAs. Determining the effects of cardiac FA excess on mitochondria can be hindered by the systemic sequelae of obesity. Mice with cardiomyocyte-specific overexpression of the fatty acid transport protein FATP1 have increased cardiomyocyte FA uptake and develop MHD in the absence of systemic lipotoxicity, obesity or diabetes. We utilized this model to assess 1) the effect of cardiomyocyte lipid accumulation on mitochondrial structure and energetic function and 2) the role of lipid-driven transcriptional regulation, signaling, toxic metabolite accumulation, and mitochondrial oxidative stress in lipid-induced MHD. Methods Cardiac lipid species, lipid-dependent signaling, and mitochondrial structure / function were examined from FATP1 mice. Cardiac structure and function were assessed in mice overexpressing both FATP1 and mitochondrial-targeted catalase. Results FATP1 hearts exhibited a net increase (+12%) in diacylglycerol, with increases in several very long-chain diacylglycerol species (+160-212%, p<0.001) and no change in ceramide, sphingomyelin, or acylcarnitine content. This was associated with an increase in phosphorylation of PKCα and PKCδ, and a decrease in phosphorylation of AKT and expression of CREB, PGC1α, PPARα and the mitochondrial fusion genes MFN1, MFN2 and OPA1. FATP1 overexpression also led to marked decreases in mitochondrial size (-49%, p<0.01), complex II-driven respiration (-28.6%, p<0.05), activity of isolated complex II (-62%, p=0.05), and expression of complex II subunit B (SDHB) (-60% and -31%, p<0.01) in the absence of change in ATP synthesis. Hydrogen peroxide production was not increased in FATP1

  19. A role for Gcn5 in cardiomyocyte differentiation of rat mesenchymal stem cells.

    PubMed

    Li, Li; Zhu, Jing; Tian, Jie; Liu, Xiaoyan; Feng, Chuan

    2010-12-01

    MSCs possess the capacity of self-renewal and potential of differentiation into various kinds of specialized tissue cells including myocardiocytes. From self-renewing to oriented differentiation, chromatin is remodeled into heritable states that allow activation or maintain the repression of regulatory genes, which means specific genes in self-renewing switched off and specific genes in oriented differentiation activated (Bernstein et al. Cell 125:315-326, 2006). These epigenetic states are established and controlled largely by specific patterns of histone posttranslational modifications, in particular, histone acetylation (Li Nat Rev Genet 3:662-673, 2002). In cardiomyocyte differentiation of rat MSCs, we focused on Gcn5, which linked a known transcriptional coactivator with catalytic histone acetyltransferase activity (Brownell et al. Cell 84:843-851, 1996). To clarify participatory in vivo role of Gcn5, using an RNA interference (RNAi) strategy employing shRNA to specifically knockdown Gcn5 expression in MSCs, we found that HAT activity altered dynamically depended on the inhibition of Gcn5 during MSCs differentiation. Chromatin immunoprecipitation (ChIP) assay showed the increased binding of acetyl histone H3 to the early cardiomyocyte-specific genes GATA4 and NKx2.5 promoters in cardiomyocyte differentiation of MSCs by 5-azacytidine inducing, whereas the decreased binding with lower Gcn5 expression. Cell ultrastructure analysis revealed that MSCs induced by 5-azacytidine possess morphological characteristics of cardiomyocyte cells. The shape of MSCs transfected by Gcn5 RNAi was similar to normal MSCs, but the chromatin showed heavy electron-density and a hard-packed structure. This intermediate state of chromatin may be an inactive part of MSCs differentiation. These results demonstrate that Gcn5, possessing acetyltransferase activity, is involved in regulating chromatin configuration around GATA4 and NKx2.5 in cardiomyocyte differentiation of rat MSCs by

  20. Altered calcium handling and increased contraction force in human embryonic stem cell derived cardiomyocytes following short term dexamethasone exposure.

    PubMed

    Kosmidis, Georgios; Bellin, Milena; Ribeiro, Marcelo C; van Meer, Berend; Ward-van Oostwaard, Dorien; Passier, Robert; Tertoolen, Leon G J; Mummery, Christine L; Casini, Simona

    2015-11-27

    One limitation in using human pluripotent stem cell derived cardiomyocytes (hPSC-CMs) for disease modeling and cardiac safety pharmacology is their immature functional phenotype compared with adult cardiomyocytes. Here, we report that treatment of human embryonic stem cell derived cardiomyocytes (hESC-CMs) with dexamethasone, a synthetic glucocorticoid, activated glucocorticoid signaling which in turn improved their calcium handling properties and contractility. L-type calcium current and action potential properties were not affected by dexamethasone but significantly faster calcium decay, increased forces of contraction and sarcomeric lengths, were observed in hESC-CMs after dexamethasone exposure. Activating the glucocorticoid pathway can thus contribute to mediating hPSC-CMs maturation.