Histone Deacetylase Isoforms Research Articles

Abstract T P69: Stroke Induces HDAC4 Nuclear Shuttling in Neurons

Background: Nuclear shuttling of class II histone deacetylases (HDACs) in neurons regulates apoptosis and facilitates neuronal recovery in response to injury. The present study examined the temporal profile of the subcellular localization of class II HDACs (4 and 5) in the peri-infarct cortex during brain recovery after stroke. Methods: Adult male Wistar rats were subjected to permanent middle cerebral artery occlusion (MCAo) and were sacrificed at 2, 7 and 14 days after MCAo. Sham operated rats were used as a control (n=6/group). To identify phenotypes of HDAC immunoreactive cells, double immunohistochemistry was performed with antibodies against each HDAC isoform (4 or 5) along with MAP-2 (a marker of dendrites), p-NFH (a marker of axons), APC (a marker of oligodendrocytes) or GFAP (a marker for astrocytes). Results: In cortical neurons of sham operated rats, HDACs 4 and 5 immunoreactivity was mainly localized to the cytoplasm and dendrites, but not axons. Stroke significantly (p<0.05) increased the percentage of neurons with nuclear HDAC4 immunoreactivity in the peri-infarct cortex at 2 (55 ± 2%), 7 (65 ± 5%) and 14 (76 ± 2%) days after MCAo compared to sham (27 ± 3%). Stroke-induced nuclear expression of HDAC4 was accompanied by a significant (p<0.05) decrease in HDAC4 staining in neuronal dendrites over the same period (7 ± 1% at 2d, 7 ± 2% at 7d, and 6 ± 1% at 14d vs. 19 ± 2% in sham). Neurons with nuclear HDAC4 were not TUNEL positive, indicating that these cells are not apoptotic. In parallel, augmentation of neurons expressing nuclear HDAC4 was significantly correlated to an increase in MAP-2 immunoreactive density after stroke (r=0.7, p<0.05). Stroke-induced nuclear shuttling of HDAC4 is specific to neurons, since HDAC4 was not detected in nuclei of GFAP+ astrocytes or APC+ oligodendrocytes. Neuronal nuclear HDAC4 localization was not associated with a change in Histone H3 lysine 9 acetylation or methylation levels. Unlike HDAC4, stroke did not alter nuclear localization of HDAC5 in neurons. Conclusion: Our data show that stroke induces HDAC4 nuclear shuttling in neurons with increased dendritic density in the peri-infarct cortex, suggesting a role for HDAC4 in promoting neuronal remodeling after stroke.

Ribosomal Protein S6, a Target of Rapamycin, Is Involved in the Regulation of rRNA Genes by Possible Epigenetic Changes in Arabidopsis

The target of rapamycin (TOR) kinase pathway regulates various biological processes, including translation, synthesis of ribosomal proteins, and transcription of rRNA. The ribosomal protein S6 (RPS6) is one of the well known downstream components of the TOR pathway. Ribosomal proteins have been known to have diverse functions in regulating cellular metabolism as well as protein synthesis. So far, however, little is known about other possible role(s) of RPS6 in plants, besides being a component of the 40 S ribosomal subunit and acting as a target of TOR. Here, we report that RPS6 may have a novel function via interaction with histone deacetylase 2B (AtHD2B) that belongs to the plant-specific histone deacetylase HD2 family. RPS6 and AtHD2B were localized to the nucleolus. Co-expression of RPS6 and AtHD2B caused a change in the location of both RPS6 and AtHD2B to one or several nucleolar spots. ChIP analysis suggests that RPS6 directly interacts with the rRNA gene promoter. Protoplasts overexpressing both AtHD2B and RPS6 exhibited down-regulation of pre-18 S rRNA synthesis with a concomitant decrease in transcription of some of the ribosomal proteins, suggesting their direct role in ribosome biogenesis and plant development. This is consistent with the mutation in rps6b that results in reduction in 18 S rRNA transcription and decreased root growth. We propose that the interaction between RPS6 and AtHD2B brings about a change in the chromatin structure of rDNA and thus plays an important role in linking TOR signaling to rDNA transcription and ribosome biogenesis in plants.

Energy-optimised pharmacophore approach to identify potential hotspots during inhibition of Class II HDAC isoforms

Histone deacetylases (HDACs) are conjugated enzymes that modulate chromatin architecture by deacetylating lysine residues on the histone tails leading to transcriptional repression. Pharmacological interventions of these enzymes with small molecule inhibitors called Histone deacetylase inhibitors (HDACi) have shown enhanced acetylation of the genome and are hence emerging as potential targets at the clinic. Type-specific inhibition of Class II HDACs has shown enhanced therapeutic benefits against developmental and neurodegenerative disorders. However, the structural identity of class-specific isoforms limits the potential of their inhibitors in precise targeting of their enzymes. Diverse strategies have been implemented to recognise the features in HDAC enzymes which may help in identifying isoform specificity factors. This work attempts a computational approach that combines in silico docking and energy-optimised pharmacophore (E-pharmacophore) mapping of 18 known HDAC inhibitors and has identified structural variations that regulate their interactions against the six Class II HDAC enzymes considered for the study. This combined approach establishes that inhibitors possessing higher number of aromatic rings in different structural regions might function as potent inhibitors, while inhibitors with scarce ring structures might point to compromised potency. This would aid the rationale for chemical optimisation and design of isoform selective HDAC inhibitors with enhanced affinity and therapeutic efficiency.

Divergent Kinetics Differentiate the Mechanism of Action of Two HDAC Inhibitors

Histone deacetylases (HDACs) play diverse roles in many diseases including cancer, sarcopenia, and Alzheimer's. Different isoforms of HDACs appear to play disparate roles in the cell and are associated with specific diseases; as such, a substantial effort has been made to develop isoform-selective HDAC inhibitors. Our group focused on developing HDAC1/HDAC2-specific inhibitors as a cancer therapeutic. In the course of characterizing the mechanism of inhibition of a novel HDAC1/2-selective inhibitor, it was determined that it did not exhibit classical Michaelis-Menten kinetic behavior; this result is in contrast to the seminal HDAC inhibitor SAHA. Enzymatic assays, along with a newly developed binding assay, were used to determine the rates of binding and the affinities of both the HDAC1/2-selective inhibitor and SAHA. The mechanism of action studies identified a potential conformational change required for optimal binding by the selective inhibitor. A model of this putative conformational change is proposed.

Four-and-a-half LIM domains proteins are novel regulators of the protein kinase D pathway in cardiac myocytes

PKD (protein kinase D) is a serine/threonine kinase implicated in multiple cardiac roles, including the phosphorylation of the class II HDAC5 (histone deacetylase isoform 5) and thereby de-repression of MEF2 (myocyte enhancer factor 2) transcription factor activity. In the present study we identify FHL1 (four-and-a-half LIM domains protein 1) and FHL2 as novel binding partners for PKD in cardiac myocytes. This was confirmed by pull-down assays using recombinant GST-fused proteins and heterologously or endogenously expressed PKD in adult rat ventricular myocytes or NRVMs (neonatal rat ventricular myocytes) respectively, and by co-immunoprecipitation of FHL1 and FHL2 with GFP–PKD1 fusion protein expressed in NRVMs. In vitro kinase assays showed that neither FHL1 nor FHL2 is a PKD1 substrate. Selective knockdown of FHL1 expression in NRVMs significantly inhibited PKD activation and HDAC5 phosphorylation in response to endothelin 1, but not to the α1-adrenoceptor agonist phenylephrine. In contrast, selective knockdown of FHL2 expression caused a significant reduction in PKD activation and HDAC5 phosphorylation in response to both stimuli. Interestingly, neither intervention affected MEF2 activation by endothelin 1 or phenylephrine. We conclude that FHL1 and FHL2 are novel cardiac PKD partners, which differentially facilitate PKD activation and HDAC5 phosphorylation by distinct neurohormonal stimuli, but are unlikely to regulate MEF2-driven transcriptional reprogramming.

Histone deacetylase isoforms regulate innate immune responses by deacetylating mitogen-activated protein kinase phosphatase-1

The MAPK pathway mediates TLR signaling during innate immune responses. We discovered previously that MKP-1 is acetylated, enhancing its interaction with its MAPK substrates and deactivating TLR signaling. As HDACs modulate inflammation by deacetylating histone and nonhistone proteins, we hypothesized that HDACs may regulate LPS-induced inflammation by deacetylating MKP-1. We found that mouse macrophages expressed a subset of HDAC isoforms (HDAC1, HDAC2, and HDAC3), which all interacted with MKP-1. Genetic silencing or pharmacologic inhibition of HDAC1, -2, and -3 increased MKP-1 acetylation in cells. Furthermore, knockdown or pharmacologic inhibition of HDAC1, -2, and -3 decreased LPS-induced phosphorylation of the MAPK member p38. Also, pharmacologic inhibition of HDAC did not decrease MAPK signaling in MKP-1 null cells. Finally, inhibition of HDAC1, -2, and -3 decreased LPS-induced expression of TNF-α, IL-1β, iNOS (NOS2), and nitrite synthesis. Taken together, our results show that HDAC1, -2, and -3 deacetylate MKP-1 and that this post-translational modification increases MAPK signaling and innate immune signaling. Thus, HDAC1, -2, and -3 isoforms are potential therapeutic targets in inflammatory diseases.

Class I HDACs regulate angiotensin II-dependent cardiac fibrosis via fibroblasts and circulating fibrocytes

Fibrosis, which is defined as excessive accumulation of fibrous connective tissue, contributes to the pathogenesis of numerous diseases involving diverse organ systems. Cardiac fibrosis predisposes individuals to myocardial ischemia, arrhythmias and sudden death, and is commonly associated with diastolic dysfunction. Histone deacetylase (HDAC) inhibitors block cardiac fibrosis in pre-clinical models of heart failure. However, which HDAC isoforms govern cardiac fibrosis, and the mechanisms by which they do so, remains unclear. Here, we show that selective inhibition of class I HDACs potently suppresses angiotensin II (Ang II)-mediated cardiac fibrosis by targeting two key effector cell populations, cardiac fibroblasts and bone marrow-derived fibrocytes. Class I HDAC inhibition blocks cardiac fibroblast cell cycle progression through derepression of the genes encoding the cyclin-dependent kinase (CDK) inhibitors, p15 and p57. In contrast, class I HDAC inhibitors block agonist-dependent differentiation of fibrocytes through a mechanism involving repression of ERK1/2 signaling. These findings define novel roles for class I HDACs in the control of pathological cardiac fibrosis. Furthermore, since fibrocytes have been implicated in the pathogenesis of a variety of human diseases, including heart, lung and kidney failure, our results suggest broad utility for isoform-selective HDAC inhibitors as anti-fibrotic agents that function, in part, by targeting these circulating mesenchymal cells.

Towards selective inhibition of histone deacetylase isoforms: what has been achieved, where we are and what will be next.

Histone deacetylases (HDACs) are widely studied targets for the treatment of cancer and other diseases. Up to now, over twenty HDAC inhibitors have entered clinical studies and two of them have already reached the market, namely the hydroxamic acid derivative SAHA (vorinostat, Zolinza) and the cyclic depsipeptide FK228 (romidepsin, Istodax) that have been approved for the treatment of cutaneous T-cell lymphoma (CTCL). A common aspect of the first HDAC inhibitors is the absence of any particular selectivity towards specific isozymes. Some of molecules resulted to be “pan”-HDAC inhibitors, while others are class I selective. In the meantime, the knowledge of HDAC biology has continuously progressed. Key advances in the structural biology of various isozymes, reliable molecular homology models as well as suitable biological assays have provided new tools for drug discovery activities. This Minireview aims at surveying these recent developments as well as the design, synthesis and biological characterization of isoform-selective derivatives.

Combination Of Selective HDAC Inhibitors That Target Specific HDAC Isoforms Synergistically Inhibits T Cell Lymphoma

BackgroundTargeting the epigenetic apparatus has become a successful treatment paradigm for the treatment of T-cell lymphomas (TCL), as demonstrated by the FDA approval of two histone deacetylase (HDAC) inhibitors, romidepsin and vorinostat. Romidepsin and vorinostat are pan-HDAC inhibitors, and their lack of enzyme specificity may be associated with toxicities that limit their clinical application. There is emerging evidence that the 11 classical HDAC isoforms, HDAC1-11, play different roles in oncogenesis, depending on the disease context. For example, HDAC3 has been strongly implicated in the survival of diffuse large B cell lymphoma, by interacting with STAT3. In T cells, HDAC1 and HDAC2 suppress lymphomagenesis in a dosage dependent manner, and are required to maintain the survival of TCL. Our hypothesis is that if different HDAC isoforms are involved in distinct cellular functions, then isoform selective HDAC inhibitors may be active in select subtypes of lymphoma, and that unique combinations of isofrom selective inhibitors could be used to more precisely target the relevant biology. MethodsCytotoxicity of isoform selective and nonselective HDAC inhibitors was determined in a panel of T cell lymphoma cell lines using a High Throughput Screening approach. Growth inhibition was determined using Cell TiterGlo that measures ATP produced by live and proliferating cells. The concentrations required to reach 50% of inhibition (IC50) were calculated from cells treated for 48 hours, using the CalcuSyn software. Cell death was determined by flow cytometry, using both Annexin V and propidium iodide as the probes. Finally, drug : drug synergy was determined by calculating relative risk ratio (RRR), as published previously. Values below 1 indicates synergy, and the smaller the RRR values the higher the level of synergy. ResultsIsoform selective HDAC inhibitors as a rule were much less potent than nonselective HDAC inhibitors in their ability to inhibit the growth of TCL cells (Table 1). However, two HDAC1/2 selective inhibitors, BRD2283 and BRD6597, demonstrated IC50 values comparable to vorinostat in the H9 cell line, but not in any other cell lines. This may be due to unique epigenetic features in H9 that are sufficiently sensitive to the inhibition of only HDAC1 and 2. In contrast, other cell lines may have complex epigenetic abnormalities that are not sensitive to the inhibition of only HDAC1 and 2, but were nevertheless sensitive to inhibition of a broader spectrum of HDAC isoforms. To further confirm that inhibiting a broader array of HDAC enzymes may lead to more efficient growth inhibition, we investigated the effect of combining the HDAC1/2 inhibitor BRD2283 and the selective HDAC3 inhibitor repligen-136 on TCL cell growth. Figure 1 demonstrates that repligen-136 at the concentration of 4uM produced only 30% growth inhibition at 48-hour exposure. BRD2283 as a single agent produced 20, 30, and 65% of growth inhibition at the concentration of 0.5, 1, and 2uM, respectively. BRD2283 at these concentrations, combined with the mildly active repligen-136 at 4uM, produced 80, 90, and 95% of growth inhibition, respectively. The calculated RRR values were 0.35, 0.20, and 0.13, respectively, highly significant for synergistic drug : drug interaction. These results serve as a proof of principle that inhibition of a narrow spectrum of isoform specific HDAC enzymes may be sufficient to inhibit select lymphoma characterized by 'uncomplicated' epigenetic apparatus. In contrast, lymphoma with 'complicated' epigenetic abnormalities may require simultaneous inhibition of multiple HDAC isoforms, which may be achieved by combining HDAC inhibitors that target different HDAC enzymes. The combination approach, as compared to a single, non-selective HDAC inhibitor, may allow for more opportunity of personalized cancer treatment based on improved understanding of the precise epigenetic abnormalities in the lymphoma. We have begun a broader combinatorial screening approach to explore what specific set of isoform selective inhibitors recapitulate, or perhaps even improve upon, the activity seen with the pan HDAC inhibitors. [Display omitted] [Display omitted] C-: Negative controlRep: Repligen-1362283: BRD2283 RRR: Relative risk ratio Disclosures:No relevant conflicts of interest to declare.

HDAC6 Inhibition Increases CD20 Level and Improves The Efficacy Of Anti-CD20 Monoclonal Antibodies

CD20, an integrate membrane protein expressed on the surface of normal and malignant B-cells is widely used as a molecular target for monoclonal antibodies (mAbs) in the therapy of non-Hodgkin's lymphomas and chronic lymphocytic leukemia (CLL). Accumulating evidence indicates that CD20 can be modulated at several levels, both transcriptional and posttranscriptional and its up-regulation would result in increased efficacy of anti-CD20 mAbs. CD20 antigen has been reported to be regulated epigenetically e.g. by histone deacetylases (HDACs). The results of our preliminary experiments show that use of non-selective HDAC inhibitors as well as blocking the activity of a single HDAC isoform - HDAC6 leads to up-regulation of CD20 protein in B-cell lymphoma cell lines and increase of the efficacy of therapy with anti-CD20 mAbs. Since HDAC6 is engaged mainly in the acetylation of non-histone substrates and the observed up-regulation of CD20 molecule does not seem to rely on transcriptional mechanisms we postulate that HDAC6 is engaged in processes of CD20 trafficking or/and degradation. CD20 being a membrane bound protein is most probably undergoing endocytosis. However, this process and the role of HDAC6 in its regulation has not been explored so far.The aim of this study was to understand how the inhibition of HDAC6 activity influences CD20 level in normal and malignant B-cells. We wanted to determine the mechanisms underlying this phenomena.This study required use of B-cell lymphoma cell lines as well as lymphocytes infected with Epstein-Barr virus and normal B- lymphocytes. Several HDAC pan-inhibitors and HDAC6 inhibitors were tested. To assess the membrane level of CD20 antigen, FITC-anti-CD20 staining was performed followed by cytometric analysis. The influence of HDACi on total level of CD20 protein was assessed in Western blotting. The complement-dependent cytotoxicity (CDC) assay was performed using rituximab and ofatumumab as well as human serum as a source of complement. Cell cytotoxicity was assessed by propidium iodide staining followed by cytometric analysis. The influence of HDAC inhibition on the transcription of CD20 was examined by qRT-PCR using SyBR Green and hydrolysis probes. The activity of CD20 promoter after inhibition of HDAC was assessed in Dual Luciferase Assay. The colocalisation of CD20 with other proteins that may influence its trafficking/degradation was assessed using immunocytochemistry with specific antibodies and observed under confocal microscope.The results of our study strongly suggest that combining HDACi with anti-CD20 antibodies can be an effective therapeutic modality for patients suffering from B-cell malignancies. Extensive experiments aiming at determining what factors are engaged in the regulation of CD20 by HDAC6 are planned. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Silencing Histone Deacetylase–Specific Isoforms Enhances Expression of Pluripotency Genes in Bovine Fibroblasts

Histone deacetylases (HDACs) catalyze deacetylation of histones that results in altered transcriptional activity. Inhibitors of HDACs have been shown to induce transcriptional changes that contribute positively to reprogramming somatic cells either by nuclear transfer or inducing a pluripotent state. However, the exact molecular mechanisms whereby HDAC inhibitors function and the specificity of the HDAC isoforms in cell reprogramming are not yet fully understood. Herein, we report the ability of individual isoform-specific HDACs to modulate endogenous expression of pluripotency-associated genes in bovine somatic cells. This in vitro study showed that a transient selective depletion of HDACs resulted in elevated mRNA levels of Oct-4, Sox2, and Nanog. In particular, we found that inhibition of specific HDAC isoforms using small interfering (si) RNA significantly increased expression of Nanog, a key factor required for totipotency induced by somatic cell nuclear transfer and for maintaining pluripotency in embryonic and induced pluripotent stem cells. Our study suggests that this gene might be the most susceptible to HDAC activity inhibition. Moreover, a regulatory role of the class III HDAC, SIRT3, on an Oct4-Sox2-Nanog transcriptional network was revealed. We observed the upregulation of pluripotency-related genes by depletion of SIRT3. SIRT3 is localized to mitochondria and is associated with energy metabolism processes, suggesting metabolic changes may be linked to reprogramming in bovine fibroblasts. In conclusion, we show that targeting selective HDACs can potentially be useful to enhance reprogramming and that sirtuins may play a pivotal role in somatic cell reprogramming by upregulating an Oct4-Sox2-Nanog transcriptional network. Dedifferentiating donor somatic cells by upregulating developmentally important genes through specific knockdown of epigenetic targets, in particular HDACs, may provide a path to improving livestock cloning and the in vitro production of pluripotent cells.

A chimeric SERM-histone deacetylase inhibitor approach to breast cancer therapy.

Breast cancer remains a significant cause of death in women, and few therapeutic options exist for estrogen receptor negative (ER (-)) cancers. Epigenetic reactivation of target genes using histone deacetylase (HDAC) inhibitors has been proposed in ER (-) cancers to resensitize to therapy using selective estrogen receptor modulators (SERMs) that are effective in ER (+) cancer treatment. Based upon preliminary studies in ER (+) and ER (-) breast cancer cells treated with combinations of HDAC inhibitors and SERMs, hybrid drugs, termed SERMostats, were designed with computational guidance. Assay for inhibition of four type I HDAC isoforms and antagonism of estrogenic activity in two cell lines yielded a SERMostat with 1-3 μM potency across all targets. The superior hybrid caused significant cell death in ER (-) human breast cancer cells and elicited cell death at the same concentration as the parent SERM in combination treatment and at an earlier time point.

The expression of histone deacetylase 4 is associated with prednisone poor-response in childhood acute lymphoblastic leukemia

This study aimed at the identification of histone deacetylase (HDAC) isoforms relevant for childhood acute lymphoblastic leukemia (ALL). Expression of HDAC1-11 was determined in 93 primary ALL and eight healthy donor samples. HDAC1, HDAC2 and HDAC8 showed significantly higher expressions in ALL samples. Correlation analysis of HDAC expression with clinicopathological parameters revealed that high HDAC1, HDAC2, HDAC4 and HDAC11 levels were significantly associated with unfavorable prognostic factors. Particularly, high HDAC4 expression was associated with high initial leukocyte count, T cell ALL and prednisone poor-response. siRNA-mediated inhibition of HDAC4 sensitized a T-ALL cell line to etoposide-induced cell death. In conclusion, our data point to HDAC4 as drug target in childhood ALL, especially in prednisone poor-responders.

S03 * ALCOHOL, CANCER, HEPATITIS AND FATTY LIVER: INTERACTIONS AND CO-MORBIDITIES

S03 * ALCOHOL, CANCER, HEPATITIS AND FATTY LIVER: INTERACTIONS AND CO-MORBIDITIES

Inhibition of HDAC2 Protects the Retina From Ischemic Injury

Protein acetylation is an essential mechanism in regulating transcriptional and inflammatory events. Studies have shown that nonselective histone deacetylase (HDAC) inhibitors can protect the retina from ischemic injury in rats. However, the role of specific HDAC isoforms in retinal degenerative processes remains obscure. The purpose of this study was to investigate the role of HDAC2 isoform in a mouse model of ischemic retinal injury. Localization of HDAC2 in mice retinas was evaluated by immunohistochemical analyses. To investigate whether selective reduction in HDAC2 activity can protect the retina from ischemic injury, Hdac2⁺/⁻ mice were utilized. Electroretinographic (ERG) and morphometric analyses were used to assess retinal function and morphology. Our results demonstrated that HDAC2 is primarily localized in nuclei in inner nuclear and retinal ganglion cell layers, and HDAC2 activity accounted for approximately 35% of the total activities of HDAC1, 2, 3, and 6 in the retina. In wild-type mice, ERG a- and b-waves from ischemic eyes were significantly reduced when compared to pre-ischemia baseline values. Morphometric examination of these eyes revealed significant degeneration of inner retinal layers. In Hdac2⁺/⁻ mice, ERG a- and b-waves from ischemic eyes were significantly greater than those measured in ischemic eyes from wild-type mice. Morphologic measurements demonstrated that Hdac2⁺/⁻ mice exhibit significantly less retinal degeneration than wild-type mice. This study demonstrated that suppressing HDAC2 expression can effectively reduce ischemic retinal injury. Our results support the idea that the development of selective HDAC2 inhibitors may provide an efficacious treatment for ischemic retinal injury.

Class I histone deacetylase activity is required for proliferation of renal epithelial cells

The process of renal regeneration after acute kidney injury is thought to recapitulate renal development, and proliferation of renal proximal tubular cells (RPTCs) is a critical step in the regenerative response. Recent studies indicate that class I histone deacetylases (HDACs) are required for embryonic kidney gene expression, growth, and differentiation. The role and underlying mechanisms of class I HDAC activation in RPTC proliferation, however, remain unclear. In this study, we used cultured RPTCs to examine this issue since four class I HDAC isoforms (1, 2, 3, and 8) are abundantly expressed in this cell type. Blocking class I HDAC activity with a highly selective inhibitor, MS-275, induced global histone H3 hyperacetylation, reduced RPTC proliferation, and diminished expression of cyclin D1 and proliferating cell nuclear antigen. Silencing HDAC1, 3, or 8 with small interfering RNA resulted in similar biological effects. Activation of epidermal growth factor receptor (EGFR) and signal transducers and activators of transcription 3 (STAT3) was required for RPTC proliferation, and STAT3 functioned downstream of EGFR. Treatment with MS-275 or knockdown of HDAC1, 3, or 8 suppressed EGFR expression and phosphorylation, and silencing HDAC1 and 3 also reduced STAT3 phosphorylation. However, HDAC2 downregulation did not affect RPTC proliferation and phosphorylation of EGFR and STAT3. Collectively, these data reveal a critical role of class I HDACs in mediating proliferation of renal epithelial cells through activation of the EGFR/STAT3 signaling pathway.

Spirulina platensis inhibits lipopolysaccharide‐induced inflammation through the repression of histone deacetylases in RAW 264.7 macrophages

We observed a potent anti‐inflammatory effect of Spirulina platensis (SP), an edible blue‐green alga, in vitro and ex vivo. To gain a mechanistic insight into the anti‐inflammatory effect of SP organic extract (SPE), we examined the effect of SPE on the expression of histone deacetylases (HDAC) 1 to 11 in lipopolysaccharide (LPS)‐activated RAW 264.7 macrophages. When macrophages were treated with LPS alone (100 ng/ml), SPE alone (100 μg/ml), or SPE+LPS, mRNA and protein levels of most HDAC isoforms were significantly decreased. However, one striking difference was that HDAC1 and HDAC3 protein levels were markedly decreased by SPE but LPS had a minimal effect. The potent repression of HDAC by SPE was evidenced by a marked increase in histone 3 acetylation to a similar extent to that of 25 nM trichostatin A (TSA), a pan HDAC inhibitor. When LPSactivated macrophages were treated with TSA (25 nM), proinflammatory cytokine expression including tumor necrosis factor α and interleukin 1β was significantly decreased but the reduction was less than that of SPE (50 or 100 μg/ml). The repressive effect of SPE on HDAC is likely cell type‐specific because this effect was also observed in 3T3‐L1 preadipocytes but not in HepG2 and Caco‐2 cells. In conclusion, SPE exerts its anti‐inflammatory effect at least in part by a HDAC‐dependent mechanism via the repression of HDAC1 and HDAC3 in macrophages.Grant Funding Source: NIH R21AT005152

920 EXPRESSION OF HISTONE DEACETYLASES 1,2 AND 3 IN UROTHELIAL BLADDER CANCER

You have accessJournal of UrologyBladder Cancer: Basic Research (II)1 Apr 2013920 EXPRESSION OF HISTONE DEACETYLASES 1,2 AND 3 IN UROTHELIAL BLADDER CANCER Cédric Poyet, Bastian Jentsch, Thomas Hermanns, Peter Wild, Martin Schmidtpeter, Daniel Schweckendieck, Tullio Sulser, Holger Moch, and Glen Kristiansen Cédric PoyetCédric Poyet Zürich, Switzerland More articles by this author , Bastian JentschBastian Jentsch Zürich, Switzerland More articles by this author , Thomas HermannsThomas Hermanns Zürich, Switzerland More articles by this author , Peter WildPeter Wild Zürich, Switzerland More articles by this author , Martin SchmidtpeterMartin Schmidtpeter Zürich, Switzerland More articles by this author , Daniel SchweckendieckDaniel Schweckendieck Zürich, Switzerland More articles by this author , Tullio SulserTullio Sulser Zürich, Switzerland More articles by this author , Holger MochHolger Moch Zürich, Switzerland More articles by this author , and Glen KristiansenGlen Kristiansen Zürich, Switzerland More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2013.02.497AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Superficial urothelial bladder cancer recurs in around 70 % after transurethral resection and 10-20 % of them show progression into muscle-invasive bladder cancer (MIBC). Due to the worse prognosis of MIBC, there is a high interest in identifying markers that can diagnose superficial cancers with a high risk of progression. Histone deacetylases (HDACs) are involved in different cellbiological processes like proliferation and differentiation and are known to be overexpressed in some cancer tumors. A range of much more potent, structurally diverse HDAC inhibitors can inhibit tumor growth and have been already clinically validated in cancer patients with hematologic malignancies. According to the literature only few data for HDAC expression in urothelial cancer is available. Ki-67 overexpression in urothelial cancer and its association with worse prognosis has been shown in several studies. METHODS In this study we aimed to evaluate the protein expression of class I histone deacetylases (HDAC) in urothelial bladder cancer. 189 primary urothelial bladder cancer were included in a tissue microarray. Median follow-up of the cohort was 6.7 years (3 to 36 years). The final TMA contents 105 pTa tumors, 71 pT1 and 13 pT2 or higher. Expression of class I HDAC isoforms 1, 2 and 3 and Ki67 was assessed by immunohistochemistry. To study statistical associations between clinicopathologic and immunohistochemical data chi-Square test and Spearman-correlation were used. Progression free survival curves were calculated using the KaplanûMeier method with significance evaluated by 2-sided log-rank statistics. P < 0.05 was considered significant. RESULTS A significant association of Ki67 expression was found with t-stage, grading and progression free survival. This reveals the comparability to other constructed urothelial TMAs. HDAC were highly expressed in about 40 to 60 % of all cases (HDAC-1 40 %, HDAC-2 44 %, HDAC-3 59 %). Furthermore we observed a significant overexpression of HDAC-2 in higher staged or higher graded tumors. However, there was no significant association with progression free survival. CONCLUSIONS HDAC-2 was associated with more aggressive pathological parameters like t-stage and grading. In contrary to Ki67, a relevant prognostic value for HDAC 1-3 could not be demonstrated. However, the high expression levels of HDACs in urothelial bladder cancer might still be indicative for a treatment response to HDAC inhibitors which ought to be evaluated in further studies. © 2013 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 189Issue 4SApril 2013Page: e378 Advertisement Copyright & Permissions© 2013 by American Urological Association Education and Research, Inc.MetricsAuthor Information Cédric Poyet Zürich, Switzerland More articles by this author Bastian Jentsch Zürich, Switzerland More articles by this author Thomas Hermanns Zürich, Switzerland More articles by this author Peter Wild Zürich, Switzerland More articles by this author Martin Schmidtpeter Zürich, Switzerland More articles by this author Daniel Schweckendieck Zürich, Switzerland More articles by this author Tullio Sulser Zürich, Switzerland More articles by this author Holger Moch Zürich, Switzerland More articles by this author Glen Kristiansen Zürich, Switzerland More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Class I HDAC inhibition blocks cocaine-induced plasticity by targeted changes in histone methylation

Induction of histone acetylation in the nucleus accumbens (NAc), a key brain reward region, promotes cocaine-induced alterations in gene expression. Histone deacetylases (HDACs) tightly regulate the acetylation of histone tails, but little is known about the functional specificity of different HDAC isoforms in the development and maintenance of cocaine-induced plasticity, and prior studies of HDAC inhibitors report conflicting effects on cocaine-elicited behavioral adaptations. Here, we demonstrate that specific and prolonged blockade of HDAC1 in NAc of mice increased global levels of histone acetylation, but also induced repressive histone methylation and antagonized cocaine-induced changes in behavior, an effect mediated in part via a chromatin-mediated suppression of GABAA receptor subunit expression and inhibitory tone on NAc neurons. Our findings suggest a novel mechanism by which prolonged and selective HDAC inhibition can alter behavioral and molecular adaptations to cocaine and inform the development of novel therapeutics for cocaine addiction.

Unique functional roles for class I and class II histone deacetylases in central nervous system development and function

Non-specific pharmacological inhibition of the histone deacetylase (HDAC) family of enzymes has largely beneficial effects in a variety of diverse contexts including cancer, cognitive function, and neurodegeneration. This review will discuss the role of individual HDAC isoforms in brain function during development and in the adult. Importantly class I and class II HDACs exhibit distinct cellular and subcellular expression patterns and utilize different signaling pathways to influence their substrates. Moreover, dissociable phenotypic outcomes emerge following manipulation of individual HDACs in the brain. To date, pharmacological inhibitors capable of targeting individual HDACs have proven difficult to develop, an obstacle that must be overcome to unlock the substantial clinical promise of manipulating endogenous HDAC isoforms in the central nervous system.