HDAC2 Protein Levels Research Articles

The histone deacetylase Hdac1 regulates inflammatory signalling in intestinal epithelial cells

BackgroundIt has recently been found that both nuclear epithelial-expressed histone deacetylases Hdac1 and Hdac2 are important to insure intestinal homeostasis and control the mucosal inflammatory response in vivo. In addition, HDAC inhibitors modulate epithelial cell inflammatory responses in cancer cells. However, little is known of the specific role of different HDAC, notably Hdac1, in the regulation of inflammatory gene expression in intestinal epithelial cells (IEC).MethodsWe investigated the role of Hdac1 in non-transformed IEC-6 rat cells infected with lentiviral vectors expressing specific Hdac1 shRNAs, to suppress Hdac1 expression. Proliferation was assessed by cell counting. Deacetylase activity was measured with a colorimetric HDAC assay. Cells were treated with IL-1β and/or the JQ1 bromodomain acetyl-binding inhibitor. Nuclear protein levels of Hdac1, Hdac2, phosphorylated or unphosphorylated NF-κB p65 or C/EBPβ, and NF-κB p50 and actin were determined by Western blot. Chemokine and acute phase protein expression was assessed by semi-quantitative RT-PCR analysis. Secreted cytokine and chemokine levels were assessed with a protein array. Chromatin immunoprecipitation experiments were done to assess RNA polymerase II recruitment.ResultsReduced Hdac1 protein levels led to Hdac2 protein increases and decreased cell proliferation. Hdac1 depletion prolonged nuclear IL-1β-induced phosphorylation of NF-κB p65 protein on Ser536 as opposed to total p65, and of C/EBPβ on Ser105. In addition, semi-quantitative RT-PCR analysis revealed three patterns of expression caused by Hdac1 depletion, namely increased basal and IL-1β-stimulated levels (Hp, Kng1), increased IL-1β-stimulated levels (Cxcl2) and decreased basal levels with normal IL-1β induction levels (Ccl2, Ccl5, Cxcl1, C3). Secreted cytokine and chemokine measurements confirmed that Hdac1 played roles both as an IL-1β signalling repressor and activator. Hdac1 depletion did not alter the JQ1 dependent inhibition of basal and IL-1β-induced inflammatory gene expression. Hdac1 depletion led to decreased basal levels of RNA polymerase II enrichment on the Ccl2 promoter, as opposed to the Gapdh promoter, correlating with decreased Ccl2 basal mRNA expression.ConclusionsHdac1 is a major nuclear HDAC controlling IL-1β-dependent inflammatory response in IEC, notably by regulating gene-specific transcriptional responses. Hdac1 may be important in restricting basal and inflammatory-induced gene levels to defined ranges of expression.Electronic supplementary materialThe online version of this article (doi:10.1186/s12950-014-0043-2) contains supplementary material, which is available to authorized users.

Abstract 20587: Myocyte-Specific Over-Expression of Hdac4 Promotes Myocardial Ischemia and Reperfusion Injury

Background: Histone deacetylases (HDACs) have been recently demonstrated to play a critical role in modulating myocardial protection and cardiomyocyte survival. However, the specific HDAC in mediating myocardial ischemia/reperfusion injury is currently unknown. Objective: The goal of this study is sought to define whether cardiac specific over-expression of HDAC4 would exacerbate myocardial ischemia and reperfusion injury. Methods: We created myocyte-specific HDAC4 transgenic mice, in which cDNA encoding HDAC4 was cloned into an expression vector encoding alpha-myosine heavy chain (the α-MHC promoter). Langendorff isovolumetrically perfused hearts from wild type (WT) control (n=5) and α-MHC-HDAC4 mice (n=5) were subjected to 30 minutes of ischemia followed by 30 minutes of reperfusion. Infarct size was measured by triphenyl tetrazolium chloride staining. HDAC4 protein and activity in myocardium were determined. Results: The HDAC4 protein levels in the hearts of α-MHC-HDAC4 mice were significantly higher than those of control wild type. HDAC activity was elevated in MHC-HDAC4 mice as compared with wild type mice. In adult mice, there was no significant difference in the heart weight-body weight ratio between α-MHC-HDAC and wild-type mice. As compared to wild type mice, myocyte-specific over-expression of HDAC4 resulted in an elevation of left ventricular end-diastolic pressure (LVEDP) and reduction in the recovery of rate and pressure products (RPP) at the end of reperfusion. Furthermore, over-expression of HDAC4 increased myocardial infarct size as compared to wild type mice. Conclusions: These findings indicate that specific over-expression of HDAC4 exacerbates myocardial ischemia and reperfusion injury.

Melatonin prevents neonatal dexamethasone induced programmed hypertension: Histone deacetylase inhibition

Adulthood hypertension can be programmed by corticosteroid exposure in early life. Oxidative stress, epigenetic regulation by histone deacetylases (HDACs), and alterations of renin–angiotensin system (RAS) are involved in the developmental programming of hypertension. We examined whether melatonin prevented neonatal dexamethasone (DEX)-induced programmed hypertension and how melatonin prevented these processes. We also examined whether HDAC inhibition by trichostatin A (TSA, a HDAC inhibitor) had similar effects. Male offspring were assigned to 5 groups (n=6/group): control, DEX, melatonin, DEX+melatonin, and DEX+TSA. Male rat pups were injected i.p. with DEX on day 1 (0.5mg/kg BW), day 2 (0.3mg/kg BW), and day 3 (0.1mg/kg BW) after birth. Melatonin was administered in drinking water at the dose of 0.01% during the lactation period. The DEX+TSA group received DEX and 0.5mg/kg TSA subcutaneous injection once daily for 1 week. All rats were killed at 16 weeks of age. Neonatal DEX exposure induced hypertension in male offspring at 16 weeks of age, which melatonin prevented. Neonatal DEX exposure decreased gene expression related to apoptosis, nephrogenesis, RAS, and sodium transporters. Yet DEX treatment increased protein levels of HDAC-1, -2, and -3 in the kidney. Melatonin therapy preserved the decreases of gene expression and decreased HDACs. Similarly, HDAC inhibition prevented DEX-induced programmed hypertension. In conclusion, melatonin therapy exerts a long-term protection against neonatal DEX-induced programmed hypertension. Its beneficial effects include alterations of RAS components and inhibition of class I HDACs. Given that the similar protective effects of melatonin and TSA, melatonin might inhibit HDACs to epigenetic regulation of hypertension-related genes to prevent programmed hypertension.

Do epigenetic events take place in the vastus lateralis of patients with mild chronic obstructive pulmonary disease?

Muscle dysfunction is a major comorbidity in Chronic Obstructive Pulmonary Disease (COPD). Several biological mechanisms including epigenetic events regulate muscle mass and function in models of muscle atrophy. Investigations conducted so far have focused on the elucidation of biological mechanisms involved in muscle dysfunction in advanced COPD. We assessed whether the epigenetic profile may be altered in the vastus lateralis of patients with mild COPD, normal body composition, and mildly impaired muscle function and exercise capacity. In vastus lateralis (VL) of mild COPD patients with well-preserved body composition and in healthy age-matched controls, expression of DNA methylation, muscle-enriched microRNAs, histone acetyltransferases (HTAs) and deacetylases (HDACs), protein acetylation, small ubiquitin-related modifier (SUMO) ligases, and muscle structure were explored. All subjects were clinically evaluated. Compared to healthy controls, in the VL of mild COPD patients, muscle function and exercise capacity were moderately reduced, DNA methylation levels did not differ, miR-1 expression levels were increased and positively correlated with both forced expiratory volume in one second (FEV1) and quadriceps force, HDAC4 protein levels were increased, and muscle fiber types and sizes were not different. Moderate skeletal muscle dysfunction is a relevant feature in patients with mild COPD and preserved body composition. Several epigenetic events are differentially expressed in the limb muscles of these patients, probably as an attempt to counterbalance the underlying mechanisms that alter muscle function and mass. The study of patients at early stages of their disease is of interest as they are a target for timely therapeutic interventions that may slow down the course of the disease and prevent the deleterious effects of major comorbidities.

Histone deacetylase 1 plays an important role in brown adipogenesis (817.4)

Induction of brown adipocytes in white fat plays an important role in the regulation of energy homeostasis. Here, we have investigated the role of HDAC1 in brown adipogenesis. The RNA and protein levels of HDAC1 are lower in mice brown compared to white adipose tissue, in brown adipose tissue of mice exposed to cold compared to that of mice residing in room temperature, and reduced during brown adipocyte differentiation. Overexpression of HDAC1 inhibits, whereas genetic deletion of HDAC1 results in further enhancement of isoproterenol‐stimulated expression of uncoupling protein 1 (UCP1) and other BAT‐specific genes, including peroxisome proliferative activated receptor γ coactivator 1α (PGC1α), PGC1β, PR domain containing 16 (PRDM16), type 2 deiodinase (DIO2), fibroblast growth factor 21 (FGF21) and myelin protein zero‐like 2 (Eva1) in brown adipocyte cell lines BAT‐1 and HIB1B. Chromatin immunoprecipitation assay indicates that histone H3 and H4 acetylation is significantly increased at the UCP1 promoter in β‐adrenergic stimulated brown adipocytes, which is also associated with increased active histone markers H3 lysine 4 tri‐methylation, and H3 lysine 27 acetylation. Thus, our data suggest that epigenetics plays an important role in brown adipogenesis and thermogenesis, and identify HDAC1 as an important regulator during this process.Grant Funding Source: NIHR01HL107500

Effects of histone deacetylase inhibitors on amygdaloid histone acetylation and neuropeptide Y expression: a role in anxiety-like and alcohol-drinking behaviours

Recent studies have demonstrated the involvement of epigenetic mechanisms in psychiatric disorders, including alcoholism. Here, we investigated the effects of histone deacetylase (HDAC) inhibitor, trichostatin A (TSA) on amygdaloid HDAC-induced histone deacetylation and neuropeptide Y (NPY) expression and on anxiety-like and alcohol-drinking behaviours in alcohol-preferring (P) and -non-preferring (NP) rats. It was found that P rats displayed higher anxiety-like and alcohol-drinking behaviours, higher amygdaloid nuclear, but not cytosolic, HDAC activity, which was associated with increased HDAC2 protein levels and deficits in histone acetylation and NPY expression in the central (CeA) and medial nucleus of amygdala (MeA), as compared to NP rats. TSA treatment attenuated the anxiety-like and alcohol-drinking behaviours, with concomitant reductions in amygdaloid nuclear, but not cytosolic HDAC activity, and HDAC2, but not HDAC4, protein levels in the CeA and MeA of P rats, without effect in NP rats. TSA treatment also increased global histone acetylation (H3-K9 and H4-K8) and NPY expression in the CeA and MeA of P, but not in NP rats. Histone H3 acetylation within the NPY promoter was also innately lower in the amygdala of P rats compared with NP rats; which was normalized by TSA treatment. Voluntary ethanol intake in P, but not NP rats, produced anxiolytic effects and decreased the HDAC2 levels and increased histone acetylation in the CeA and MeA. These results suggest that higher HDAC2 expression-related deficits in histone acetylation may be involved in lower NPY expression in the amygdala of P rats, and operative in controlling anxiety-like and alcohol-drinking behaviours.

Tumor Suppressor MiR-29b Targets HDAC4 and Modulates Vorinostat Activity In Multiple Myeloma Cells

MicroRNAs (miRNAs) are short non-coding RNAs that control gene expression at the post-transcriptional level by inducing mRNA decay or translation repression. A subclass of miRNAs, named epi-miRNAs, is known to exert anti-tumor activity by targeting effectors of the epigenetic machinery. We recently demonstrated a key role for the tumor suppressor miR-29b in reducing the global DNA methylation of multiple myeloma (MM) cells through the targeting of DNA-methyltransferases. In silico search of additional miR-29b targets contributing to clarify its role as epi-miRNA unveiled the class II histone deacetylase HDAC4. Since histone deacetylases represent promising molecular targets for cancer treatment, we sought to characterize HDAC4 expression and function and its regulation by miR-29b in MM cells. HDAC4 protein levels and enzymatic activity were found up-regulated in a panel of 11 MM cell lines as compared to peripheral blood mononuclear cells from healthy donors. Moreover, the analysis of HDAC4 mRNA levels in a microarray dataset consisting of 4 healthy controls, 55 MM and 29 plasma cell leukemia patients indicated significant over-expression in cancer samples, suggesting that high HDAC4 expression might be involved in MM pathogenesis. Notably, the analysis of miRNA/mRNA paired expression in the same microarray dataset revealed an inverse correlation between miR-29b and HDAC4 mRNA, strengthening the relevance of miR-29b as a key regulator of HDAC4. Synthetic miR-29b mimics transfected in MM cells (SKMM1 and NCI-H929) down-regulated HDAC4 mRNA and protein levels and inhibited the 3’UTR of HDAC4 cloned in a luciferase reporter vector, whereas failed to regulate a 3’UTR devoid of two predicted miR-29b target sites. On the other hand, lentiviral-mediated overexpression of HDAC4 strongly inhibited miR-29b expression. These results underscore a negative feedback loop occurring between miR-29b and its target HDAC4 in MM cells. Through loss of function experiments, we assessed the functional significance of HDAC4 in MM cells. Stable silencing of HDAC4 by shRNAs induced growth inhibition, caspase 3/7-dependent apoptosis and autophagy in U266 and KMS11 cells, which occurred together to miR-29b up-regulation. Interestingly, the pan-HDAC inhibitor vorinostat also triggered apoptosis and autophagy in MM cells, along with the induction of miR-29b and the down-regulation of HDAC4 and other miR-29b-canonical targets like CDK6, MCL-1 and Sp1. miR-29b itself was able to promote autophagy, as assessed by beclin-1 up-regulation and LC3A/B proteolytic cleavage in miR-29b mimics-transfected MM cells, which was abrogated by constitutive expression of HDAC4. Finally, we provided evidence that miR-29b over-expression potentiated, whereas its stable inhibition dampened, apoptosis and autophagy triggered by vorinostat. In conclusion, our findings shed light on the oncogenic role of HDAC4, which can be targeted through miR-29b-based therapeutic approaches, and identify miR-29b as a relevant effector of vorinostat activity in MM cells. Disclosures: No relevant conflicts of interest to declare.

Inhibition of histone deacetylases protects septic mice from lung and splenic apoptosis

BackgroundEpigenetic programming, dynamically regulated by histone acetylation, may play a key role in the pathophysiology of sepsis. We examined whether histone deacetylase (HDAC) can contribute to sepsis-associated inflammation and apoptosis. Materials and methodsPolymicrobial sepsis was induced by cecal ligation and puncture (CLP) in BALB/c mice. An intraperitoneal injection of CG200745 (10 mg/kg), a novel broad-spectrum HDAC inhibitor, or valproic acid (500 mg/kg), a predominant inhibitor of class I HDACs, was given 3 h before surgery. ResultsHDAC1, HDAC2, and HDAC3 protein levels were decreased in lungs after CLP. Furthermore, CLP-induced sepsis increased both histone H3 and H4 acetylation levels in lungs. When CG200745 was given, apoptosis induction was strongly suppressed in lungs and spleens of septic mice. This antiapoptotic effect of CG200745 was not accompanied by upregulation of antiapoptotic and downregulation of proapoptotic Bcl-2 family member proteins. Treatment with CG200745 failed to inhibit elevated levels of serum cytokines and prevent lung inflammation in septic mice. Valproic acid also showed antiapoptotic but not anti-inflammatory effects in septic mice. ConclusionsThese findings imply that HDAC inhibitors are a unique agent to prevent cell apoptosis in sepsis at their doses that do not improve inflammatory features, indicating that septic inflammation and apoptosis may not necessarily be essential for one another's existence. This study also represents the first report that CLP-induced sepsis downregulates HDACs. Nevertheless, the data with HDAC inhibitors suggest that imbalance in histone acetylation may play a contributory role in expression or repression of genes involved in septic cell apoptosis.

S31 * EPIGENETIC MECHANISMS OF ALCOHOLISM: FROM PRENATAL STAGE TO ADULTHOOD

S31 * EPIGENETIC MECHANISMS OF ALCOHOLISM: FROM PRENATAL STAGE TO ADULTHOOD

Abstract 3658: Sulforaphane suppresses 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced mouse epidermal JB6 P+ cell transformation through epigenetic re-activation of Nrf2.

Abstract Sulforaphane (SFN), an isothiocyanate in cruciferous vegetables including broccoli, cabbage, cauliflower, Chinese cabbage, and watercress, has strong anti-cancer/cancer chemopreventive potentials. Transcription factor nuclear factor erythroid 2 p45 (NF-E2)-related factor 2 (Nrf2) plays an important role in regulating the expressions of cellular antioxidant and detoxification enzymes. In this study, we investigated the preventive effect of SFN on tumor promoter-induced transformation in mouse skin epidermal JB6 promotion-sensitive (JB6 P+) cells, which might be involved with the activation of Nrf2. After 5 days pre-treatment, the results showed that SFN significantly inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced neoplastic transformation. SFN enhanced nuclear translocation of Nrf2 and increased the Nrf2 downstream target enzymes including HO-1, NQO1, and UGT1A1 at both the transcriptional and protein levels. Comparing to shMock-transfected cells, TPA effectively induced colony formation in shNrf2 knockdown transfected cells were more resistant to the inhibitory effects of SFN. SFN's induction of Nrf2, HO1, and NQO1 protein expressions in shNrf2-transfected cells were lower than those in shMock-transfected cells. Epigenetics study showed that SFN suppresses methyltion ratio of the first fifteen CpGs in the promoter of Nrf2 gene in JB6 P+ cells utilizing bisulfite sequencing. Furthermore, SFN strongly reduced the protein expressions of DNA methyl transferases (DNMTs) including DNMT1, DNMT3a, and DNMT3b. In terms of histone deacetylases (HDACs), SFN not only inhibited total HDAC activity but also decreased the protein levels of HDAC1, HDAC2, HDAC3, and HDAC4, significantly. Taken together, these findings suggest that epigenetic re-activation of Nrf2 with subsequent induction of Nrf2 downstream target genes by SFN may provide insights into the chemopreventive mechanism of SFN against tumor promoter-induced mouse skin carcinogenesis. Citation Format: Zheng-Yuan Su, Limin Shu, Chengyue Zhang, Tin Oo Khor, Jong Hun Lee, Tien-Yuan Wu, Allan H. Conney, Ah-Ng Tony Kong. Sulforaphane suppresses 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced mouse epidermal JB6 P+ cell transformation through epigenetic re-activation of Nrf2. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3658. doi:10.1158/1538-7445.AM2013-3658

Nuclear Calcium Signaling Regulates Nuclear Export of a Subset of Class IIa Histone Deacetylases following Synaptic Activity

In neurons, dynamic changes in the subcellular localization of histone deacetylases (HDACs) are thought to contribute to signal-regulated gene expression. Here we show that in mouse hippocampal neurons, synaptic activity-dependent nucleo-cytoplasmic shuttling is a common feature of all members of class IIa HDACs, which distinguishes them from other classes of HDACs. Nuclear calcium, a key regulator in neuronal gene expression, is required for the nuclear export of a subset of class IIa HDACs. We found that inhibition of nuclear calcium signaling using CaMBP4 or increasing the nuclear calcium buffering capacity by means of expression of a nuclear targeted version of parvalbumin (PV.NLS-mC) led to a build-up of HDAC4 and HDAC5 in the cell nucleus, which in the case of PV.NLS-mC can be reversed by nuclear calcium transients triggered by bursts of action potential firing. A similar nuclear accumulation of HDAC4 and HDAC5 was observed in vivo in the mouse hippocampus following stereotaxic delivery of recombinant adeno-associated viruses expressing either CaMBP4 or PV.NLS-mC. The modulation of HDAC4 activity either by RNA interference-mediated reduction of HDAC4 protein levels or by expression of a constitutively nuclear localized mutant of HDAC4 leads to changes in the mRNA levels of several nuclear calcium-regulated genes with known functions in acquired neuroprotection (atf3, serpinb2), memory consolidation (homer1, arc), and the development of chronic pain (ptgs2, c1qc). These results identify nuclear calcium as a regulator of nuclear export of HDAC4 and HDAC5. The reduction of nuclear localized HDACs represents a novel transcription-promoting pathway stimulated by nuclear calcium.

Aberrant Histone Deacetylase2–Mediated Histone Modifications and Synaptic Plasticity in the Amygdala Predisposes to Anxiety and Alcoholism

Epigenetic mechanisms have been implicated in psychiatric disorders, including alcohol dependence. However, the epigenetic basis and role of specific histone deacetylase (HDAC) isoforms in the genetic predisposition to anxiety and alcoholism is unknown. We measured amygdaloid HDAC activity, levels of HDAC isoforms, and histone H3 acetylation in selectively bred alcohol-preferring (P) and -nonpreferring (NP) rats. We employed HDAC2 small interfering RNA infusion into the central nucleus of amygdala (CeA) of P rats to determine the causal role of HDAC2 in anxiety-like and alcohol-drinking behaviors. Chromatin immunoprecipitation analysis was performed to examine the histone acetylation status of brain-derived neurotrophic factor (Bdnf) and activity-regulated cytoskeleton associated protein (Arc) genes. Golgi-Cox staining was performed to measure dendritic spine density. We found that P rats innately display higher nuclear HDAC activity and HDAC2 but not HDAC 1, 3, 4, 5, and 6 protein levels and lower acetylation of H3-K9 but not H3-K14, in the CeA and medial nucleus of amygdala compared with NP rats. Acute ethanol exposure decreased amygdaloid HDAC activity and HDAC2 protein levels, increased global and gene (Bdnf and Arc)-specific histone acetylation, and attenuated anxiety-like behaviors in P rats but had no effects in NP rats. The HDAC2 knockdown in the CeA attenuated anxiety-like behaviors and voluntary alcohol but not sucrose consumption in P rats and increased histone acetylation of Bdnf and Arc with a resultant increase in protein levels that correlated with increased dendritic spine density. These novel data demonstrate the role of HDAC2-mediated epigenetic mechanisms in anxiety and alcoholism.

Plant Polyphenols and Oxidative Metabolites of the Herbal Alkenylbenzene Methyleugenol Suppress Histone Deacetylase Activity in Human Colon Carcinoma Cells

Evidence has been provided that diet and environmental factors directly influence epigenetic mechanisms associated with cancer development in humans. The inhibition of histone deacetylase (HDAC) activity and the disruption of the HDAC complex have been recognized as a potent strategy for cancer therapy and chemoprevention. In the present study, we investigated whether selected plant constituents affect HDAC activity or HDAC1 protein status in the human colon carcinoma cell line HT29. The polyphenols (−)-epigallocatechin-3-gallate (EGCG) and genistein (GEN) as well as two oxidative methyleugenol (ME) metabolites were shown to inhibit HDAC activity in intact HT29 cells. Concomitantly, a significant decrease of the HDAC1 protein level was observed after incubation with EGCG and GEN, whereas the investigated ME metabolites did not affect HDAC1 protein status. In conclusion, dietary compounds were found to possess promising HDAC-inhibitory properties, contributing to epigenetic alterations in colon tumor cells, which should be taken into account in further risk/benefit assessments of polyphenols and alkenylbenzenes.

Expression level of histone deacetylase 2 correlates with occurring of chronic obstructive pulmonary diseases

Chronic obstructive pulmonary disease (COPD) is associated with chronic severe airway inflammation and causes increasing global health problems. New biological markers for COPD prediction and prognosis are urgently necessary. Previous studies indicate that histone deacetylases (HDACs) play essential roles in COPD. This study is to investigate if HDAC2 levels can be used as a promising, easily detected biomarker of COPD. In this paper, 49 COPD patients were enrolled and 42 healthy individuals (smokers or non-smokers) were used as healthy controls. Human bronchial epithelial cells derived from non-smokers, smokers, or COPD patients were grown in primary cultures. Total proteins were harvested from lung tissues or bronchial epithelial cells and then subjected to immunoblot analyses of HDAC2, HDAC3, and HDAC5. Quantitative RT-PCR analysis of HDAC2, HDAC3, and HDAC5 mRNA levels in tissues or cells were also preformed. We found that among the three HDAC proteins, the mRNA and protein levels of HDAC2, but not HDAC3 and HDAC5, in the tissues or cultured cells from patients have a significant correlation with development and prognosis of COPD. These results suggested that HDAC2 levels may serve as a promising, easily detected biomarker of COPD.

Abstract 259: Histone Deacetylase1 and 2 Are Increased in Myofibroblasts/Fibroblasts After MI

Myofibroblast activation after myocardial infarction (MI) is one of the major mechanisms of myocardial fibrosis and subsequent heart failure. Elucidating the mechanisms of myofibroblast activation will provide a strategy to treat heart failure. Histone Deacetylases (HDAC) were reported to regulate myofibroblast differentiation and extracellular matrix deposition in idiopathic pulmonary fibrosis. Furthermore, HDAC inhibitors were shown to retards myocardial remodeling and improve cardiac function after MI. Therefore, here we examined whether HDAC1/2 are up-regulated after MI, and identify which myocardial cell type is predominately expressing HDAC. MI was created in rats by ligation of the left coronary artery. First, we assessed expression of HDAC 1 and 2 following MI in heart sections. Both HDACs uniformly expressed in myocardium in right ventricular (LV) and left ventricular (LV) in sham hearts. On the other hand, we observed up-regulation of both HDAC1 and 2 in the infarcted LV following MI. In addition, HDAC1 and 2 were co-localized with fibroblast marker Vimentin in the infarcted area. Next, we performed western blot analysis to investigate protein levels of HDAC1 and HDAC2 in right ventricular (RV), septum (S) and LV (infarcted and remote area). We found up-regulation of HDAC1 and HDAC2 only in the infarcted area compared to sham rats. Altogether these results suggest HDAC1 and 2 play a role in fibrosis following MI.

3,3′-Diindolylmethane, but not indole-3-carbinol, inhibits histone deacetylase activity in prostate cancer cells

Increased consumption of cruciferous vegetables is associated with a reduced risk of developing prostate cancer. Indole-3-carbinol (I3C) and 3,3′-diindolylmethane (DIM) are phytochemicals derived from cruciferous vegetables that have shown promise in inhibiting prostate cancer in experimental models. Histone deacetylase (HDAC) inhibition is an emerging target for cancer prevention and therapy. We sought to examine the effects of I3C and DIM on HDACs in human prostate cancer cell lines: androgen insensitive PC-3 cells and androgen sensitive LNCaP cells. I3C modestly inhibited HDAC activity in LNCaP cells by 25% but no inhibition of HDAC activity was detected in PC-3 cells. In contrast, DIM significantly inhibited HDAC activity in both cell lines by as much as 66%. Decreases in HDAC activity correlated with increased expression of p21, a known target of HDAC inhibitors. DIM treatment caused a significant decrease in the expression of HDAC2 protein in both cancer cell lines but no significant change in the protein levels of HDAC1, HDAC3, HDAC4, HDAC6 or HDAC8 was detected. Taken together, these results show that inhibition of HDAC activity by DIM may contribute to the phytochemicals' anti-proliferative effects in the prostate. The ability of DIM to target aberrant epigenetic patterns, in addition to its effects on detoxification of carcinogens, may make it an effective chemopreventive agent by targeting multiple stages of prostate carcinogenesis.

Glucose depletion activates mmu-miR-466h-5p expression through oxidative stress and inhibition of histone deacetylation

MicroRNAs (miRNAs) are involved in the regulation of multiple cellular processes. Changes of miRNA expression have been linked to the development of various diseases including cancer, but the molecular events leading to these changes at different physiological conditions are not well characterized. Here we examined the intracellular events responsible for the miR-466h-5p activation in mouse cells exposed to glucose deprivation. MiR-466h-5p is a member of the miR-297-669 cluster located in intron 10 of Sfmbt2 gene on mouse chromosome 2 and has a pro-apoptotic role. We showed that the time-dependant activation of miR-466h-5p, miR-669c and the Sfmbt2 gene followed the inhibition of histone deacetylation caused by glucose deprivation-induced oxidative stress. This oxidative stress causes the accumulation of reactive oxygen species (ROS) and depletion of reduced glutathione (GSH) that together inhibited histone deacetylases (HDACs) activity, reduced protein levels of HDAC2 and increased acetylation in miR-466h-5p promoter region, which led to the activation of this miRNA. Based on this study and previous work, we suggest a possible role of miR-466h-5p (and miR 297-669 cluster) in the cells during toxic metabolites accumulation. Improved characterization of the molecular events that lead to the activation of miR-466h-5p may provide a better understanding of the relation between cellular environment and miRNA activation.

Methamphetamine causes differential alterations in gene expression and patterns of histone acetylation/hypoacetylation in the rat nucleus accumbens.

Methamphetamine (METH) addiction is associated with several neuropsychiatric symptoms. Little is known about the effects of METH on gene expression and epigenetic modifications in the rat nucleus accumbens (NAC). Our study investigated the effects of a non-toxic METH injection (20 mg/kg) on gene expression, histone acetylation, and the expression of the histone acetyltransferase (HAT), ATF2, and of the histone deacetylases (HDACs), HDAC1 and HDAC2, in that structure. Microarray analyses done at 1, 8, 16 and 24 hrs after the METH injection identified METH-induced changes in the expression of genes previously implicated in the acute and longterm effects of psychostimulants, including immediate early genes and corticotropin-releasing factor (Crf). In contrast, the METH injection caused time-dependent decreases in the expression of other genes including Npas4 and cholecystokinin (Cck). Pathway analyses showed that genes with altered expression participated in behavioral performance, cell-to-cell signaling, and regulation of gene expression. PCR analyses confirmed the changes in the expression of c-fos, fosB, Crf, Cck, and Npas4 transcripts. To determine if the METH injection caused post-translational changes in histone markers, we used western blot analyses and identified METH-mediated decreases in histone H3 acetylated at lysine 9 (H3K9ac) and lysine 18 (H3K18ac) in nuclear sub-fractions. In contrast, the METH injection caused time-dependent increases in acetylated H4K5 and H4K8. The changes in histone acetylation were accompanied by decreased expression of HDAC1 but increased expression of HDAC2 protein levels. The histone acetyltransferase, ATF2, showed significant METH-induced increased in protein expression. These results suggest that METH-induced alterations in global gene expression seen in rat NAC might be related, in part, to METH-induced changes in histone acetylation secondary to changes in HAT and HDAC expression. The causal role that HATs and HDACs might play in METH-induced gene expression needs to be investigated further.

S09 * ALCOHOLISM: TRANSCRIPTIONAL/EPIGENETIC ADAPTATIONS IN ANIMAL AND HUMAN BRAIN * S09.1 * EPIGENETIC BASIS FOR ANXIETY AND ALCOHOLISM

S09 * ALCOHOLISM: TRANSCRIPTIONAL/EPIGENETIC ADAPTATIONS IN ANIMAL AND HUMAN BRAIN * S09.1 * EPIGENETIC BASIS FOR ANXIETY AND ALCOHOLISM

High Mobility Group Box 1 Release from Hepatocytes during Ischemia and Reperfusion Injury Is Mediated by Decreased Histone Deacetylase Activity

The mobilization and extracellular release of nuclear high mobility group box-1 (HMGB1) by ischemic cells activates inflammatory pathways following liver ischemia/reperfusion (I/R) injury. In immune cells such as macrophages, post-translational modification by acetylation appears to be critical for active HMGB1 release. Hyperacetylation shifts its equilibrium from a predominant nuclear location toward cytosolic accumulation and subsequent release. However, mechanisms governing its release by parenchymal cells such as hepatocytes are unknown. In this study, we found that serum HMGB1 released following liver I/R in vivo is acetylated, and that hepatocytes exposed to oxidative stress in vitro also released acetylated HMGB1. Histone deacetylases (HDACs) are a family of enzymes that remove acetyl groups and control the acetylation status of histones and various intracellular proteins. Levels of acetylated HMGB1 increased with a concomitant decrease in total nuclear HDAC activity, suggesting that suppression in HDAC activity contributes to the increase in acetylated HMGB1 release after oxidative stress in hepatocytes. We identified the isoforms HDAC1 and HDAC4 as critical in regulating acetylated HMGB1 release. Activation of HDAC1 was decreased in the nucleus of hepatocytes undergoing oxidative stress. In addition, HDAC1 knockdown with siRNA promoted HMGB1 translocation and release. Furthermore, we demonstrate that HDAC4 is shuttled from the nucleus to cytoplasm in response to oxidative stress, resulting in decreased HDAC activity in the nucleus. Together, these findings suggest that decreased nuclear HDAC1 and HDAC4 activities in hepatocytes following liver I/R is a mechanism that promotes the hyperacetylation and subsequent release of HMGB1.