Nuclear Export Of Histone Deacetylase Research Articles

Role of salt-inducible kinase 1 in the activation of MEF2-dependent transcription by BDNF.

Substantial evidence supports a role for myocyte enhancer factor 2 (MEF2)-mediated transcription in neuronal survival, differentiation and synaptic function. In developing neurons, it has been shown that MEF2-dependent transcription is regulated by neurotrophins. Despite these observations, little is known about the cellular mechanisms by which neurotrophins activate MEF2 transcriptional activity. In this study, we examined the role of salt-inducible kinase 1 (SIK1), a member of the AMP-activated protein kinase (AMPK) family, in the regulation of MEF2-mediated transcription by the neurotrophin brain-derived neurotrophic factor (BDNF). We show that BDNF increases the expression of SIK1 in primary cultures of rat cortical neurons through the extracellular signal-regulated kinase 1/2 (ERK1/2)-signaling pathway. In addition to inducing SIK1 expression, BDNF triggers the phosphorylation of SIK1 at Thr182 and its translocation from the cytoplasm to the nucleus of cortical neurons. The effects of BDNF on the expression, phosphorylation and, translocation of SIK1 are followed by the phosphorylation and nuclear export of histone deacetylase 5 (HDAC5). Blockade of SIK activity with a low concentration of staurosporine abolished BDNF-induced phosphorylation and nuclear export of HDAC5 in cortical neurons. Importantly, stimulation of HDAC5 phosphorylation and nuclear export by BDNF is accompanied by the activation of MEF2-mediated transcription, an effect that is suppressed by staurosporine. Consistent with these data, BDNF induces the expression of the MEF2 target genes Arc and Nur77, in a staurosporine-sensitive manner. In further support of the role of SIK1 in the regulation of MEF2-dependent transcription by BDNF, we found that expression of wild-type SIK1 or S577A SIK1, a mutated form of SIK1 which is retained in the nucleus of transfected cells, is sufficient to enhance MEF2 transcriptional activity in cortical neurons. Together, these data identify a previously unrecognized mechanism by which SIK1 mediates the activation of MEF2-dependent transcription by BDNF.

Acute β-Adrenergic Activation Triggers Nuclear Import of Histone Deacetylase 5 and Delays Gq-induced Transcriptional Activation

During hemodynamic stress, catecholamines and neurohumoral stimuli may induce co-activation of G(q)-coupled receptors and β-adrenergic receptors (β-AR), leading to cardiac remodeling. Dynamic regulation of histone deacetylase 5 (HDAC5), a transcriptional repressor, is crucial during stress signaling due to its role in epigenetic control of fetal gene markers. Little is known about its regulation during acute and chronic β-AR stimulation and its cross-interaction with G(q) signaling in adult cardiac myocytes. Here, we evaluate the potential cross-talk between G(q)-driven and β-AR mediated signaling at the level of nucleocytoplasmic shuttling of HDAC5. We show the translocation of GFP-tagged wild type HDAC5 or mutants (S279A and S279D) in response to β-AR or G(q) agonists. Isoproterenol (ISO) or PKA activation results in strong nuclear accumulation of HDAC5 in contrast to nuclear export driven by Ca(2+)-calmodulin protein kinase II and protein kinase D. Moreover, nuclear accumulation of HDAC5 under acute ISO/PKA signaling is dependent on phosphorylation of Ser-279 and can block subsequent G(q)-mediated nuclear HDAC5 export. Intriguingly, the attenuation of G(q)-induced export is abolished after chronic PKA activation, yet nuclear HDAC5 remains elevated. Last, the effect of chronic β-AR signaling on HDAC5 translocation was examined in adult myocytes from a rabbit model of heart failure, where ISO-induced nuclear import is ablated, but G(q)-agonist mediated export is preserved. Acute β-AR/PKA activation protects against hypertrophic signaling by delaying G(q)-mediated transcriptional activation. This serves as a key physiological control switch before allowing genetic reprogramming via HDAC5 nuclear export during more severe stress, such as heart failure.

Nuclear export of histone deacetylase 7 during thymic selection is required for immune self-tolerance

Histone deacetylase 7 (HDAC7) is a T-cell receptor (TCR) signal-dependent regulator of differentiation that is highly expressed in CD4/CD8 double-positive (DP) thymocytes. Here, we examine the effect of blocking TCR-dependent nuclear export of HDAC7 during thymic selection, through expression of a signal-resistant mutant of HDAC7 (HDAC7-ΔP) in thymocytes. We find that HDAC7-ΔP transgenic thymocytes exhibit a profound block in negative thymic selection, but can still undergo positive selection, resulting in the escape of autoreactive T cells into the periphery. Gene expression profiling reveals a comprehensive suppression of the negative selection-associated gene expression programme in DP thymocytes, associated with a defect in the activation of MAP kinase pathways by TCR signals. The consequence of this block in vivo is a lethal autoimmune syndrome involving the exocrine pancreas and other abdominal organs. These experiments establish a novel molecular model of autoimmunity and cast new light on the relationship between thymic selection and immune self-tolerance.

Epac enhances excitation–transcription coupling in cardiac myocytes

Epac is a guanine nucleotide exchange protein that is directly activated by cAMP, but whose cardiac cellular functions remain unclear. It is important to understand cardiac Epac signaling, because it is activated in parallel to classical cAMP-dependent signaling via protein kinase A. In addition to activating contraction, Ca2+ is a key cardiac transcription regulator (excitation–transcription coupling). It is unknown how myocyte Ca2+ signals are decoded in cardiac myocytes to control nuclear transcription. We examine Epac actions on cytosolic ([Ca2+]i) and intranuclear ([Ca2+]n) Ca2+ homeostasis, focusing on whether Epac alters [Ca2+]n and activates a prohypertrophic program in cardiomyocytes. Adult rat cardiomyocytes, loaded with fluo-3 were viewed by confocal microscopy during electrical field stimulation at 1Hz. Acute Epac activation by 8-pCPT increased Ca2+ sparks and diastolic [Ca2+]i, but decreased systolic [Ca2+]i. The effects on diastolic [Ca2+]i and Ca2+ spark frequency were dependent on phospholipase C (PLC), inositol 1,4,5 triphosphate receptor (IP3R) and CaMKII activation. Interestingly, Epac preferentially increased [Ca2+]n during both diastole and systole, correlating with the perinuclear expression pattern of Epac. Moreover, Epac activation induced histone deacetylase 5 (HDAC5) nuclear export, with consequent activation of the prohypertrophic transcription factor MEF2. These data provide the first evidence that the cAMP-binding protein Epac modulates cardiac nuclear Ca2+ signaling by increasing [Ca2+]n through PLC, IP3R and CaMKII activation, and initiates a prohypertrophic program via HDAC5 nuclear export and subsequent activation of the transcription factor MEF2.

MEF2A Binding to the Glut4 Promoter Occurs via an AMPKα2-Dependent Mechanism

The role of AMP-activated protein kinase α2 (AMPKα2) in regulating MEF2A nucleus translocation, nuclear histone deacetylase 5 (HDAC5) association with MEF2, HDAC5 nuclear export, MEF2A binding to the Glut4 promoter, and GLUT4 expression was investigated. This was investigated in muscles from AMPKα2 overexpression (OE) mice, AMPKα2 knockout (KO) mice, and corresponding wild-type (WT) mice that had undertaken a 28-d program of treadmill training by: 1) AMPKα-Thr172 phosphorylation by Western blot, 2) total and nuclear MEF2A by Western blot, 3) nuclear HDAC5 association with MEF2 by coimmunoprecipitation, 4) total and nuclear HDAC5 by Western blot, 5) bound MEF2A at the Glut4 MEF2 cis-element by chromatin immunoprecipitation, and 6) GLUT4 expression by real-time polymerase chain reaction and Western blot. OE or KO of AMPKα2 isoform heightened or attenuated the training-induced increase in nuclear MEF2A content, Glut4 promoter-bound MEF2A. However, OE or KO of the AMPKα2 isoform did not have any effect on the content of nuclear HDAC5 association with MEF2 after 28 d of exercise training, although 35% lower nuclear HDAC5 protein content was found in α2-OE training muscles. Lastly, OE of the α2-isoform was associated with 120% and 155% higher GLUT4 protein and mRNA in training muscles. However, the training-induced increases of GLUT4 protein and mRNA contents were normal in α2-KO muscles despite the reduced AMPK signaling. Exercise training increases the nuclear MEF2A content and binding of MEF2A to their binding sites on the Glut4 gene by an AMPKα2-dependent mechanism, but intracellular signaling molecules other than AMPKα2 are important in regulating training-induced HDAC5 nuclear export. Furthermore, although AMPKα2 mediates the training-induced increase in Glut4 promoter-bound MEF2A, the present data do not support an essential role of AMPKα2 in regulating training-induced GLUT4 expression in skeletal muscle.

Histone modifications and exercise adaptations

The spatial association between genomic DNA and histone proteins within chromatin plays a key role in the regulation of gene expression and is largely governed by post-translational modifications to histone proteins, particularly H3 and H4. These modifications include phosphorylation, acetylation, and mono-, di-, and tri-methylation, and while some are associated with transcriptional repression, acetylation of lysine residues within H3 generally correlates with transcriptional activation. Histone acetylation is regulated by the balance between the activities of histone acetyl transferase (HAT) and histone deacetylase (HDAC). In skeletal muscle, the class II HDACs 4, 5, 7, and 9 play a key role in muscle development and adaptation and have been implicated in exercise adaptations. As just one example, exercise results in the nuclear export of HDACs 4 and 5, secondary to their phosphorylation by CaMKII and AMPK, two kinases that are activated during exercise in response to changes in sarcoplasmic Ca(2+) levels and energy status, in association with increased GLUT4 expression in human skeletal muscle. Unraveling the complexities of the so-called "histone code" before and after exercise is likely to lead to a greater understanding of the regulation of exercise/activity-induced alterations in skeletal muscle gene expression and reinforce the importance of skeletal muscle plasticity in health and disease.

Fluid shear stress stimulates phosphorylation-dependent nuclear export of HDAC5 and mediates expression of KLF2 and eNOS

AbstractFluid shear stress generated by steady laminar blood flow protects vessels from atherosclerosis. Krüppel-like factor 2 (KLF2) and endothelial nitric oxide synthase (eNOS) are fluid shear stress–responsive genes and key mediators in flow anti-inflammatory and antiatherosclerotic actions. However, the molecular mechanisms underlying flow induction of KLF2 and eNOS remain largely unknown. Here, we show a novel role of histone deacetylase 5 (HDAC5) in flow-mediated KLF2 and eNOS expression. We found for the first time that fluid shear stress stimulated HDAC5 phosphorylation and nuclear export in endothelial cells through a calcium/calmodulin-dependent pathway. Consequently, flow induced the dissociation of HDAC5 and myocyte enhancer factor-2 (MEF2) and enhanced MEF2 transcriptional activity, which leads to expression of KLF2 and eNOS. Adenoviral overexpression of a HDAC5 phosphorylation–defective mutant (Ser259/Ser498 were replaced by Ala259/Ala498, HDAC5-S/A), which shows resistance to flow-induced nuclear export, suppressed flow-mediated MEF2 transcriptional activity and expression of KLF2 and eNOS. Importantly, HDAC5-S/A attenuated the flow-inhibitory effect on monocyte adhesion to endothelial cells. Taken together, our results reveal that phosphorylation-dependent derepression of HDAC5 mediates flow-induced KLF2 and eNOS expression as well as flow anti-inflammation, and suggest that HDAC5 could be a potential therapeutic target for the prevention of atherosclerosis.

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

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

Suppression of HDAC nuclear export and cardiomyocyte hypertrophy by novel irreversible inhibitors of CRM1

Histone deacetylase 5 (HDAC5) represses expression of nuclear genes that promote cardiac hypertrophy. Agonism of a variety of G protein coupled receptors (GPCRs) triggers phosphorylation-dependent nuclear export of HDAC5 via the CRM1 nuclear export receptor, resulting in derepression of pro-hypertrophic genes. A cell-based high-throughput screen of a commercial compound collection was employed to identify compounds with the ability to preserve the nuclear fraction of GFP-HDAC5 in primary cardiomyocytes exposed to GPCR agonists. A hit compound potently inhibited agonist-induced GFP-HDAC5 nuclear export in cultured neonatal rat ventricular myocytes (NRVMs). A small set of related compounds was designed and synthesized to evaluate structure-activity relationship (SAR). The results demonstrated that inhibition of HDAC5 nuclear export was a result of compounds irreversibly reacting with a key cysteine residue in CRM1 that is required for its function. CRM1 inhibition by the compounds also resulted in potent suppression of cardiomyocyte hypertrophy. These studies define a novel class of anti-hypertrophic compounds that function through irreversible inhibition of CRM1-dependent nuclear export.

Protein Kinase D Is an Essential Regulator of C. elegans Innate Immunity

Protein kinase D (PKD) mediates signal transduction downstream from phospholipase C and diacylglycerol (DAG). PKDs are activated by hormones and stress in cell lines, but little is known about PKD functions and regulation in vivo. Here, we show that DKF-2, a C. elegans PKD, regulates innate immunity. Animals lacking DKF-2 were hypersensitive to killing by bacteria that are pathogens of C. elegans and humans. DKF-2 induced 85 mRNAs, which encode antimicrobial peptides and proteins that sustain intestinal epithelium. Induction of immune effector mRNAs by DKF-2 proceeded via PMK-1 (p38 Map-kinase)-dependent and -independent pathways. TPA-1, a PKCdelta homolog, regulated activation and functions of DKF-2 in vivo. Therefore, DKF-2 provides a molecular link that couples DAG signaling to regulation of immunity. This intersection between DAG-TPA-1-DKF-2 and PMK-1 pathways enables integrated immune responses to multiple stimuli. Thus, a PKD mobilizes activation of host immune defenses against pathogens by previously unappreciated signaling pathways and mechanisms.

A New Kid on the Block

The formation of blood vessels through the process of angiogenesis is critical in normal vascular development and numerous vascular disorders. The most prominent stimulus for angiogenic processes in endothelial cells is vascular endothelial growth factor (VEGF), which regulates their migration, proliferation, and survival.1 The crucial role for this factor is documented by the lethal phenotype resulting from disruption of a single allele in mice.2,3 Binding of VEGF to its receptors activates several intracellular signaling molecules, among them phospholipase Cγ (PLCγ), protein kinase C (PKC), protein kinase D (PKD), and phosphatidyl-insitol-3 kinase (PI3K).1 The VEF-induced signaling events finally culminate in gene expression changes in the nucleus. See accompanying article on page 1782 A key regulator of gene expression is chromatin structure, which is largely determined by the acetylation status of histones. In general, acetylation of these nucleosomal proteins by histone acetyl transferases (HATs) stimulates transcription, whereas deacetylation by histone deacetylases (HDACs) leads to transcriptional repression. The HDAC family comprises 18 members in humans, which are classified based on their homologies to yeast proteins. …

Activation of Na + /H + Exchanger 1 Is Sufficient to Generate Ca 2+ Signals That Induce Cardiac Hypertrophy and Heart Failure

Activation of the sarcolemmal Na(+)/H(+) exchanger (NHE)1 is increasingly documented as a process involved in cardiac hypertrophy and heart failure. However, whether NHE1 activation alone is sufficient to induce such remodeling remains unknown. We generated transgenic mice that overexpress a human NHE1 with high activity in hearts. The hearts of these mice developed cardiac hypertrophy, contractile dysfunction, and heart failure. In isolated transgenic myocytes, intracellular pH was elevated in Hepes buffer but not in physiological bicarbonate buffer, yet intracellular Na(+) concentrations were higher under both conditions. In addition, both diastolic and systolic Ca(2+) levels were increased as a consequence of Na(+)-induced Ca(2+) overload; this was accompanied by enhanced sarcoplasmic reticulum Ca(2+) loading via Ca(2+)/calmodulin-dependent protein kinase (CaMK)II-dependent phosphorylation of phospholamban. Negative force-frequency dependence was observed with preservation of high Ca(2+), suggesting a decrease in myofibril Ca(2+) sensitivity. Furthermore, the Ca(2+)-dependent prohypertrophic molecules calcineurin and CaMKII were highly activated in transgenic hearts. These effects observed in vivo and in vitro were largely prevented by the NHE1 inhibitor cariporide. Interestingly, overexpression of NHE1 in neonatal rat ventricular myocytes induced cariporide-sensitive nuclear translocation of NFAT (nuclear factor of activated T cells) and nuclear export of histone deacetylase 4, suggesting that increased Na(+)/H(+) exchange activity can alter hypertrophy-associated gene expression. However, in transgenic myocytes, contrary to exclusive translocation of histone deacetylase 4, NFAT only partially translocated to nucleus, possibly because of marked activation of p38, a negative regulator of NFAT signaling. We conclude that activation of NHE1 is sufficient to initiate cardiac hypertrophy and heart failure mainly through activation of CaMKII-histone deacetylase pathway.

Quantitative Control of Adaptive Cardiac Hypertrophy by Acetyltransferase p300

Acetyltransferase p300 is essential for cardiac development and is thought to be involved in cardiac myocyte growth through MEF2- and GATA4-dependent transcription. However, the importance of p300 in the modulation of cardiac growth in vivo is unknown. Pressure overload induced by transverse aortic coarctation, postnatal physiological growth, and human heart failure were associated with large increases in p300. Minimal transgenic overexpression of p300 (1.5- to 3.5-fold) induced striking myocyte and cardiac hypertrophy. Both mortality and cardiac mass were directly related to p300 protein dosage. Heterozygous loss of a single p300 allele reduced pressure overload-induced hypertrophy by approximately 50% and rescued the hypertrophic phenotype of p300 overexpressers. Increased p300 expression had no effect on total histone deacetylase activity but was associated with proportional increases in p300 acetyltransferase activity and acetylation of the p300 substrates histone 3 and GATA-4. Remarkably, a doubling of p300 levels was associated with the de novo acetylation of MEF2. Consistent with this, genes specifically upregulated in p300 transgenic hearts were highly enriched for MEF2 binding sites. Small increments in p300 are necessary and sufficient to drive myocardial hypertrophy, possibly through acetylation of MEF2 and upstream of signals promoting phosphorylation or nuclear export of histone deacetylases. We propose that induction of myocardial p300 content is a primary rate-limiting event in the response to hemodynamic loading in vivo and that p300 availability drives and constrains adaptive myocardial growth. Specific reduction of p300 content or activity may diminish stress-induced hypertrophy and forestall the development of heart failure.

YY1 Protects Cardiac Myocytes from Pathologic Hypertrophy by Interacting with HDAC5

YY1 is a transcription factor that can repress or activate the transcription of a variety of genes. Here, we show that the function of YY1 as a repressor in cardiac myocytes is tightly dependent on its ability to interact with histone deacetylase 5 (HDAC5). YY1 interacts with HDAC5, and overexpression of YY1 prevents HDAC5 nuclear export in response to hypertrophic stimuli and the increase in cell size and re-expression of fetal genes that accompany pathological cardiac hypertrophy. Knockdown of YY1 results in up-regulation of all genes present during fetal development and increases the cell size of neonatal cardiac myocytes. Moreover, overexpression of a YY1 deletion construct that does not interact with HDAC5 results in transcription activation, suggesting that HDAC5 is necessary for YY1 function as a transcription repressor. In support of this relationship, we show that knockdown of HDAC5 results in transcription activation by YY1. Finally, we show that YY1 interaction with HDAC5 is dependent on the HDAC5 phosphorylation domain and that overexpression of YY1 reduces HDAC5 phosphorylation in response to hypertrophic stimuli. Our results strongly suggest that YY1 functions as an antihypertrophic factor by preventing HDAC5 nuclear export and that up-regulation of YY1 in human heart failure may be a protective mechanism against pathological hypertrophy.

Protein Kinase D-dependent Phosphorylation and Nuclear Export of Histone Deacetylase 5 Mediates Vascular Endothelial Growth Factor-induced Gene Expression and Angiogenesis

Vascular endothelial growth factor (VEGF) is essential for normal and pathological angiogenesis. However, the signaling pathways linked to gene regulation in VEGF-induced angiogenesis are not fully understood. Here we demonstrate a critical role of protein kinase D (PKD) and histone deacetylase 5 (HDAC5) in VEGF-induced gene expression and angiogenesis. We found that VEGF stimulated HDAC5 phosphorylation and nuclear export in endothelial cells through a VEGF receptor 2-phospholipase Cgamma-protein kinase C-PKD-dependent pathway. We further showed that the PKD-HDAC5 pathway mediated myocyte enhancer factor-2 transcriptional activation and a specific subset of gene expression in response to VEGF, including NR4A1, an orphan nuclear receptor involved in angiogenesis. Specifically, inhibition of PKD by overexpression of the PKD kinase-negative mutant prevents VEGF-induced HDAC5 phosphorylation and nuclear export as well as NR4A1 induction. Moreover, a mutant of HDAC5 specifically deficient in PKD-dependent phosphorylation inhibited VEGF-mediated NR4A1 expression, endothelial cell migration, and in vitro angiogenesis. These findings suggest that the PKD-HDAC5 pathway plays an important role in VEGF regulation of gene transcription and angiogenesis.

Protein kinases C and D mediate agonist-dependent cardiac hypertrophy through nuclear export of histone deacetylase 5.

A variety of stress signals stimulate cardiac myocytes to undergo hypertrophy. Persistent cardiac hypertrophy is associated with elevated risk for the development of heart failure. Recently, we showed that class II histone deacetylases (HDACs) suppress cardiac hypertrophy and that stress signals neutralize this repressive function by triggering phosphorylation- and CRM1-dependent nuclear export of these chromatin-modifying enzymes. However, the identities of cardiac HDAC kinases have remained unclear. Here, we demonstrate that signaling by protein kinase C (PKC) is sufficient and, in some cases, necessary to drive nuclear export of class II HDAC5 in cardiomyocytes. Inhibition of PKC prevents nucleocytoplasmic shuttling of HDAC5 in response to a subset of hypertrophic agonists. Moreover, a nonphosphorylatable HDAC5 mutant is refractory to PKC signaling and blocks cardiomyocyte hypertrophy mediated by pharmacological activators of PKC. We also demonstrate that protein kinase D (PKD), a downstream effector of PKC, directly phosphorylates HDAC5 and stimulates its nuclear export. These findings reveal a novel function for the PKC/PKD axis in coupling extracellular cues to chromatin modifications that control cellular growth, and they suggest potential utility for small-molecule inhibitors of this pathway in the treatment of pathological cardiac gene expression.