Decreased Histone Deacetylase Activity Research Articles

Release of Neuronal HMGB1 by Ethanol through Decreased HDAC Activity Activates Brain Neuroimmune Signaling

Neuroimmune gene induction is involved in many brain pathologies including addiction. Although increased expression of proinflammatory cytokines has been found in ethanol-treated mouse brain and rat brain slice cultures as well as in post-mortem human alcoholic brain, the mechanisms remain elusive. High-mobility group box 1 (HMGB1) protein is a nuclear protein that has endogenous cytokine-like activity. We previously found increased HMGB1 in post-mortem alcoholic human brain as well as in ethanol treated mice and rat brain slice cultures. The present study investigated the mechanisms for ethanol-induced release of HMGB1 and neuroimmune activation in a model of rat hippocampal-entorhinal cortex (HEC) brain slice cultures. Ethanol exposure triggered dose-dependent HMGB1 release, predominantly from neuronal cells. Inhibitors of histone deacetylases (HDACs) promoted nucleocytoplasmic mobilization of HDAC1/4 and HMGB1 resulting in increased total HMGB1 and acetylated HMGB1 release. Similarly, ethanol treatment was found to induce the translocation of HDAC1/4 and HMGB1 proteins from nuclear to cytosolic fractions. Furthermore, ethanol treatment reduced HDAC1/4 mRNA and increased acetylated HMGB1 release into the media. These results suggest decreased HDAC activity may be critical in regulating acetylated HMGB1 release from neurons in response to ethanol. Ethanol and HMGB1 treatment increased mRNA expression of proinflammatory cytokines TNFα and IL-1β as well as toll-like receptor 4 (TLR4). Targeting HMGB1 or microglial TLR4 by using siRNAs to HMGB1 and TLR4, HMGB1 neutralizing antibody, HMGB1 inhibitor glycyrrhizin and TLR4 antagonist as well as inhibitor of microglial activation all blocked ethanol-induced expression of proinflammatory cytokines TNFα and IL-1β. These results support the hypothesis that ethanol alters HDACs that regulate HMGB1 release and that danger signal HMGB1 as endogenous ligand for TLR4 mediates ethanol-induced brain neuroimmune signaling through activation of microglial TLR4. These findings provide new therapeutic targets for brain neuroimmune activation and alcoholism.

Activity and expression of histone acetylases and deacetylases in inflammatory phenotypes of asthma

Histone acetyltransferases (HATs) and histone deacetylases (HDACs) regulate gene expression, yet differences in the activity of these enzymes in the inflammatory phenotypes of asthma are unknown. We hypothesized that neutrophilic asthma (NA) would be associated with increased HAT and decreased HDAC activity. To investigate total HAT/HDAC activity and gene expression in isolated blood monocytes and sputum macrophages from healthy and patients with asthma. Peripheral blood and induced sputum were collected from adults with asthma (n = 52) and healthy controls (n = 9). Sputum inflammatory cell counts were performed and asthma inflammatory phenotypes were classified according to sputum eosinophil and neutrophil cut-off's of > 3% and > 61% respectively. Peripheral blood monocytes were isolated (n = 61) and sputum macrophages were isolated from a subgroup of patients with asthma (n = 14), using immunomagnetic cell separation. RNA and nuclear proteins were extracted and quantified. Enzyme activity was assessed using fluorescent assays and gene expression of EP300, KAT2B, CREBBP, and HDACs 1, 2 and 3 were measured by qPCR. There was a significant inverse association between blood monocyte HAT and HDAC activity (r = -0.58, P < 0.001). NA was associated with increased blood monocyte HAT enzyme activity (P = 0.02), decreased HDAC activity (P = 0.03), and increased HAT: HDAC ratio (P < 0.01) compared with eosinophilic asthma. There were no differences in gene expression of EP300, KAT2B, CREBBP, or HDACs 1, 2 and 3 in blood monocytes from subjects with asthma or inflammatory phenotypes of asthma. There was no effect of inhaled corticosteroid use, poor asthma control, or asthma severity on HAT/HDAC activities. Sputum macrophages had increased expression of KAT2B in eosinophilic compared with paucigranulocytic asthma. Neutrophilic airway inflammation is associated with increased HAT and reduced HDAC activity in blood monocytes, demonstrating further systemic manifestations relating to the altered inflammatory gene transcription profile of neutrophilic asthma.

Biophysical regulation of epigenetic state and cell reprogramming

Biochemical factors can help reprogram somatic cells into pluripotent stem cells, yet the role of biophysical factors during reprogramming is unknown. Here, we show that biophysical cues, in the form of parallel microgrooves on the surface of cell-adhesive substrates, can replace the effects of small-molecule epigenetic modifiers and significantly improve reprogramming efficiency. The mechanism relies on the mechanomodulation of the cells' epigenetic state. Specifically, decreased histone deacetylase activity and upregulation of the expression of WD repeat domain 5 (WDR5)--a subunit of H3 methyltranferase--by microgrooved surfaces lead to increased histone H3 acetylation and methylation. We also show that microtopography promotes a mesenchymal-to-epithelial transition in adult fibroblasts. Nanofibrous scaffolds with aligned fibre orientation produce effects similar to those produced by microgrooves, suggesting that changes in cell morphology may be responsible for modulation of the epigenetic state. These findings have important implications in cell biology and in the optimization of biomaterials for cell-engineering applications.

The novel HDAC inhibitor NDACI054 sensitizes human cancer cells to radiotherapy

Background and purposeInhibition of histone deacetylases (HDACs) has preclinically and clinically shown promise to overcome radio- and chemoresistance of tumor cells. NDACI054 is a novel HDAC inhibitor, which has been evaluated here for its effects on cell survival and radiosensitization of human tumor cell lines from different origins cultured under more physiological three-dimensional (3D), extracellular matrix (ECM)-based conditions. Material and methodsA549 lung, DLD-1 colorectal, MiaPaCa2 pancreatic and UT-SCC15 head and neck squamous cell carcinoma cells were treated with increasing NDACI054 concentrations (0–50nM, 24h) either alone or in combination with X-rays (single dose, 0–6Gy). Subsequently, 3D clonogenic cell survival, HDAC activity, histone H3 acetylation, apoptosis, residual DNA damage (γH2AX/p53BP1 foci assay 24h post irradiation) and phosphorylation kinetics of Ataxia telangiectasia mutated (ATM), DNA-dependent protein kinase (DNA-PK), Caspase-3 and Poly(ADP-ribose)-Polymerase 1 (PARP 1) cleavage were analyzed. ResultsNDACI054 potently decreased HDAC activity with concomitant increase in acetyl-histone H3 levels, mediated significant cytotoxicity and radiosensitization. These effects were accompanied by a significant increase of residual γH2AX/p53BP1-positive foci, slightly elevated levels of Caspase-3 and PARP 1 cleavage but no induction of apoptosis. ConclusionsOur data show potent antisurvival and radiosensitizing effects of the novel HDAC inhibitor NDACI054 encouraging further preclinical examinations on this compound for future clinical use.

Multi-targeted DATS prevents tumor progression and promotes apoptosis in ectopic glioblastoma xenografts in SCID mice via HDAC inhibition

Glioblastoma, the most lethal brain tumor, remains incurable despite aggressive chemotherapy and surgical interventions. New chemotherapeutics for glioblastoma have been explored in preclinical models and some agents have reached the clinical setting. However, success rates are not significant. Previous investigations involving diallyl trisulfide (DATS), a garlic compound, indicated significant anti-cancer effects in glioblastoma in vitro. DATS has also been shown to inhibit histone deacetylase activity and impede glioblastoma tumor progression. We hypothesized that DATS would block ectopic U87MG tumor by multiple pro-apoptotic pathways via inhibiting histone deacetylase (HDAC). To prove this, we developed ectopic U87MG tumors in SCID mice and treated them daily with intraperitoneal injections of DATS for 7 days. Results indicated that DATS (10 μg/kg-10 mg/kg) dose-dependently reduced tumor mass and number of mitotic cells within tumors. Histological and biochemical assays demonstrated that DATS reduced mitosis in tumors, decreased HDAC activity, increased acetylation of H3 and H4, inhibited cell cycle progression, decreased pro-tumor markers (e.g., survivin, Bcl-2, c-Myc, mTOR, EGFR, VEGF), promoted apoptotic factors (e.g., bax, mcalpian, active caspase-3), and induced DNA fragmentation. Our data also demonstrated an increase in p21Waf1 expression, which correlated with increased p53 expression and MDM2 degradation following DATS treatment. Finally, histological assessment and enzyme assays showed that even the highest dose of DATS did not negatively impact hepatic function. Collectively, our results clearly demonstrated that DATS could be an effective therapeutic agent in preventing tumor progression and inducing apoptosis in human glioblastoma in vivo, without impairing hepatic function.

Molecular mechanism of sphingosine-1-phosphate action in Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is a lethal muscle-wasting disease. Studies in Drosophila showed that genetic increase of the levels of the bioactive sphingolipid sphingosine-1-phosphate (S1P) or delivery of 2-acetyl-5-tetrahydroxybutyl imidazole (THI), an S1P lyase inhibitor, suppresses dystrophic muscle degeneration. In the dystrophic mouse (mdx), upregulation of S1P by THI increases regeneration and muscle force. S1P can act as a ligand for S1P receptors and as a histone deacetylase (HDAC) inhibitor. Because Drosophila has no identified S1P receptors and DMD correlates with increased HDAC2 levels, we tested whether S1P action in muscle involves HDAC inhibition. Here we show that beneficial effects of THI treatment in mdx mice correlate with significantly increased nuclear S1P, decreased HDAC activity and increased acetylation of specific histone residues. Importantly, the HDAC2 target microRNA genes miR-29 and miR-1 are significantly upregulated, correlating with the downregulation of the miR-29 target Col1a1 in the diaphragm of THI-treated mdx mice. Further gene expression analysis revealed a significant THI-dependent decrease in inflammatory genes and increase in metabolic genes. Accordingly, S1P levels and functional mitochondrial activity are increased after THI treatment of differentiating C2C12 cells. S1P increases the capacity of the muscle cell to use fatty acids as an energy source, suggesting that THI treatment could be beneficial for the maintenance of energy metabolism in mdx muscles.

Epigenetic Regulation of Opioid-Induced Hyperalgesia, Dependence, and Tolerance in Mice

Repeated administration of opioids such as morphine induces persistent behavioral changes including opioid-induced hyperalgesia (OIH), tolerance, and physical dependence. In the current work we explored how the balance of histone acetyltransferase (HAT) versus histone deacetylase (HDAC) might regulate these morphine-induced changes. Nociceptive thresholds, analgesia, and physical dependence were assessed during and for a period of several weeks after morphine exposure. To probe the roles of histone acetylation, the HAT inhibitor curcumin or a selective HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) was administered daily to groups of animals. Histone acetylation in spinal cord was assessed by Western blot and immunohistochemistry. Concurrent administration of curcumin with morphine for 4 days significantly reduced development of opioid-induced mechanical allodynia, thermal hyperalgesia, tolerance, and physical dependence. Conversely, the HDAC inhibitor SAHA enhanced these responses. Interestingly, SAHA treatment after the termination of opioid administration sustained these behavioral changes for at least 4 weeks. Histone H3 acetylation in the dorsal horn of the spinal cord was increased after chronic morphine treatment, but H4 acetylation was unchanged. Moreover, we observed a decrease in HDAC activity in the spinal cords of morphine-treated mice while overall HAT activity was unchanged, suggesting a shift toward a state of enhanced histone acetylation. PerspectiveThe current study indicates that epigenetic mechanisms play a crucial role in opioid-induced long-lasting neuroplasticity. These results provide new sight into understanding the mechanisms of opioid-induced neuroplasticity and suggest new strategies to limit opioid abuse potential and increase the value of these drugs as analgesics.

Evidence for a link between histone deacetylation and Ca2+ homoeostasis in sphingosine-1-phosphate lyase-deficient fibroblasts

Embryonic fibroblasts from S1P (sphingosine-1-phosphate) lyase-deficient mice [Sgpl1-/- MEFs (mouse embryonic fibroblasts)] are characterized by intracellular accumulation of S1P, elevated cytosolic [Ca2+]i and enhanced Ca2+ storage. Since S1P, produced by sphingosine kinase 2in the nucleus of MCF-7 cells, inhibited HDACs (histone deacetylases) [Hait, Allegood, Maceyka, Strub, Harikumar, Singh, Luo, Marmorstein, Kordula, Milstein et al. (2009) Science 325, 1254-1257], in the present study we analysed whether S1P accumulated in the nuclei of S1P lyase-deficient MEFs and caused HDAC inhibition. Interestingly, nuclear concentrations of S1P were disproportionally elevated in Sgpl1-/- MEFs. HDAC activity was reduced, acetylation of histone 3-Lys9 was increased and the HDAC-regulated gene p21 cyclin-dependent kinase inhibitor was up-regulated in these cells. Furthermore, the expression of HDAC1 and HDAC3 was reduced in Sgpl1-/- MEFs. In wild-type MEFs, acetylation of histone 3-Lys9 was increased by the S1P lyase inhibitor 4-deoxypyridoxine. The non-specific HDAC inhibitor trichostatin A elevated basal [Ca2+]i and enhanced Ca2+ storage, whereas the HDAC1/2/3 inhibitor MGCD0103 elevated basal [Ca2+]i without influence on Ca2+ storage in wild-type MEFs. Overexpression of HDAC1 or HDAC2 reduced the elevated basal [Ca2+]i in Sgpl1-/- MEFs. Taken together, S1P lyase-deficiency was associated with elevated nuclear S1P levels, reduced HDAC activity and down-regulation of HDAC isoenzymes. The decreased HDAC activity in turn contributed to the dysregulation of Ca2+ homoeostasis, particularly to the elevated basal [Ca2+]i, in Sgpl1-/- MEFs.

Grape seed proanthocyanidins reactivate silenced tumor suppressor genes in human skin cancer cells by targeting epigenetic regulators

Grape seed proanthocyanidins (GSPs) have been shown to have anti-skin carcinogenic effects in in vitro and in vivo models. However, the precise epigenetic molecular mechanisms remain unexplored. This study was designed to investigate whether GSPs reactivate silenced tumor suppressor genes following epigenetic modifications in skin cancer cells. For this purpose, A431 and SCC13 human squamous cell carcinoma cell lines were used as in vitro models. The effects of GSPs on DNA methylation, histone modifications and tumor suppressor gene expressions were studied in these cell lines using enzyme activity assays, western blotting, dot-blot analysis and real-time polymerase chain reaction (RT-PCR). We found that treatment of A431 and SCC13 cells with GSPs decreased the levels of: (i) global DNA methylation, (ii) 5-methylcytosine, (iii) DNA methyltransferase (DNMT) activity and (iv) messenger RNA (mRNA) and protein levels of DNMT1, DNMT3a and DNMT3b in these cells. Similar effects were noted when these cancer cells were treated identically with 5-aza-2′-deoxycytidine, an inhibitor of DNA methylation. GSPs decreased histone deacetylase activity, increased levels of acetylated lysines 9 and 14 on histone H3 (H3-Lys 9 and 14) and acetylated lysines 5, 12 and 16 on histone H4, and reduced the levels of methylated H3-Lys 9. Further, GSP treatment resulted in re-expression of the mRNA and proteins of silenced tumor suppressor genes, RASSF1A, p16INK4a and Cip1/p21. Together, this study provides a new insight into the epigenetic mechanisms of GSPs and may have significant implications for epigenetic therapy in the treatment/prevention of skin cancers in humans.

Histone acetylation of the htr3a gene in the prefrontal cortex of Wistar rats regulates ethanol-seeking behavior.

Previous reports showed that decreased histone deacetylase activity significantly potentiated the rewarding effects of psychostimulants, and that encoding of the 5-HT3 receptor by the htr3a gene was related to ethanol-seeking behavior. However, the effects of a histone deacetylase inhibitor on ethanol-seeking behavior and epigenetic regulation of htr3a mRNA expression after chronic ethanol exposure are not fully understood. Using quantitative reverse transcription-polymerase chain reaction and chromatin immunoprecipitation analysis, we investigated the effects of chronic ethanol exposure and its interaction with a histone deacetylase inhibitor on histone-acetylation-mediated changes in htr3a mRNA expression in the htr3a promoter region. The conditioned place preference procedure was used to evaluate ethanol-seeking behavior. Chronic exposure to ethanol effectively elicited place conditioning. In the prefrontal cortex, the acetylation of H3K9 and htr3a mRNA expression in the htr3a promoter region were significantly higher in the ethanol group than in the saline group. The histone deacetylase inhibitor sodium butyrate potentiated the effects of ethanol on htr3a mRNA expression and enhanced ethanol-induced conditioned place preferences. These results suggest that ethanol upregulates htr3a levels through mechanisms involving H3K9 acetylation, and that histone acetylation may be a therapeutic target for treating ethanol abuse.

Long-acting β2 agonists and corticosteroids restore the reduction of histone deacetylase activity and inhibit H2O2-induced mediator release from alveolar macrophages

BackgroundTreatment of COPD with a combination of long-acting β2 agonists and corticosteroids is currently used worldwide. The mechanisms of the anti-inflammatory effects and their associations with histone deacetylase (HDAC) activity remain unclear. MethodsHuman alveolar macrophages were isolated and stimulated with H2O2 in the presence of varying concentrations of long-acting β2 agonists and corticosteroids. Supernatants were collected for IL-8 and MMP-9 measurements. Cell lysates were analyzed for HDAC (mainly HDAC1/HDAC2) activity. Quantitative real-time PCR was performed to determine the levels of IL-8 and MMP-9 mRNA. ResultsBoth long-acting β2 agonists, salmeterol and formoterol, and corticosteroids, fluticasone and budesonide, showed anti-inflammatory effects to a certain extent on H2O2-induced IL-8 and MMP-9 release in alveolar macrophages. Combinations of long-acting β2 agonists and corticosteroids exerted greater effects to suppress mediator release, and both transcription and translation of IL-8 and MMP-9 were inhibited. It seemed that the levels of IL-8 and MMP-9 after H2O2 stimulation were inversely associated with the activity of HDAC. H2O2 stimulation resulted in a significant decrease in HDAC activity and was associated with an increase in mediator release. In contrast, treatment with long-acting β2 agonists, corticosteroids or theophylline restored the H2O2-induced decrease in HDAC activity and inhibited mediator release. ConclusionCombinations of long-acting β2 agonists and corticosteroids exerted greater effects on the suppression of mediator release in relation to the enhancement of HDAC activity. This supports at least in part the likely contribution of anti-inflammatory effects of long-acting β2 agonists and corticosteroids to the clinical benefits seen in COPD patients.

Abstract 4079: Proanthocyanidins reactivate silenced tumor suppressor genes p16INK4 a and Cip1/p21 by reducing DNA methylation and increasing histone acetylation in human non-small cell lung cancer cells

Abstract Non-small cell lung cancer (NSCLC) represents α80% of all types of lung cancer and includes squamous cell carcinoma, adenocarcinoma and large-cell carcinomas. The risk of NSCLC is greatly affected by epigenetic modulations in lung cells. Cancer epigenetics deal with important processes including DNA methylation and histone modification patterns that can either activate oncogenes or inactivate tumor suppressor genes, the crucial steps in tumor development. The study of epigenetic regulations has become a new target for early detection, risk assessment, prognosis and cancer therapy. Therefore, to develop more effective therapeutic agents for lung cancer, we sought to determine the effect of grape seed proanthocyanidins (GSPs), which have been shown to have anti-carcinogenic effects in some tumor models, on epigenetic modulation in NSCLC cells. For this purpose two genetically different NSCLC cell lines, A549 (p53 wild type) and H1299 (p53 mutant), were selected and treated with lower concentrations of GSPs (5, 10, 20µg/ml) for six days. The status of DNA global methylation patterns was determined by immunostaining using a monoclonal antibody specific to 5-methylcytosine (5-mC) and global DNA methylation assay. Treatment of cells with GSPs resulted in a dose-dependent decrease in 5-mC-positive cells as compared to non-GSPs-treated controls. These data were verified by dot-blot analysis. Global methylation levels of DNA as obtained from quantitative DNA methylation assay reduced to 36±5% in A549 cells and 31±5% in H1299 cells with the treatment of GSPs. The expression levels of the DNA methyltransferases, e.g., DNMT1, DNMT3a and DNMT3b, were higher in non-GSPs treated cells and reduced with GSPs treatment as analyzed by western blotting and real-time PCR. The hypermethylation of DNA in NSCLC cells may lead to silencing of tumor suppressor genes, thereby contributing to the risk of lung cancer. Therefore, we analyzed the methylation status of the various tumor suppressor genes, like p16INK4a, Cip1/p21 and RASSF1A through methylation specific PCR and the relative repression levels of their mRNA were analyzed using real-time PCR, which indicated that the DNA methylation levels of these tumor suppressor genes were reduced in GSPs treated cells, and that resulted in re-expression of the mRNA and proteins of silenced tumor suppressor genes compared to non-GSPs treated cells. GSPs decreased histone deacetylase activity and increased the levels of histone acetylation which included the acetylated lysine 9 and 14 on histone H3 (H3-Lys 9 and 14) and acetylated lysine 5 and 12 on histone H4 but decreased the levels of methylated H3-Lys 9. Together, our study provides new insight into the epigenetic mechanism of action of GSPs that may have important implications for epigenetic therapy of lung cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4079. doi:1538-7445.AM2012-4079

Chemotherapy-induced cognitive impairment is associated with decreases in cell proliferation and histone modifications

BackgroundIn this study, we examined the effects of cyclophosphamide, methothrexate, and 5-Fluorouracil (CMF) drug combination on various aspects of learning and memory. We also examined the effects of CMF on cell proliferation and chromatin remodeling as possible underlying mechanisms to explain chemotherapy-associated cognitive dysfunction. Twenty-four adult female Wistar rats were included in the study and had minimitter implantation for continuous activity monitoring two weeks before the chemotherapy regimen was started. Once baseline activity data were collected, rats were randomly assigned to receive either CMF or saline injections given intraperitoneally. Treatments were given once a week for a total of 4 weeks. Two weeks after the last injection, rats were tested in the water maze for spatial learning and memory ability as well as discrimination learning. Bromodeoxyuridine (BrdU) injection was given at 100 mg/Kg intraperitoneally 4 hours prior to euthanasia to determine hippocampal cell proliferation while histone acetylation and histone deacetylase activity was measured to determine CMF effects on chromatin remodeling.ResultsOur data showed learning and memory impairment following CMF administration independent of the drug effects on physical activity. In addition, CMF-treated rats showed decreased hippocampal cell proliferation, associated with increased histone acetylation and decreased histone deacetylase activity.ConclusionsThese results suggest the negative consequences of chemotherapy on brain function and that anti-cancer drugs can adversely affect the self-renewal potential of neural progenitor cells and also chromatin remodeling in the hippocampus. The significance of our findings lie on the possible usefulness of animal models in addressing the clinical phenomenon of 'chemobrain.'

Smoking and Asthma

The purpose of this review is to describe the current understanding of the prevalence and adverse effects of cigarette smoking and secondhand smoke (SHS) in asthmatics in terms of patient outcomes and response to inhaled corticosteroids. We searched the English biomedical literature via PubMed, Embase, and Scopus using the terms "smoking and asthma," "secondhand smoke and asthma," "environmental tobacco smoke and asthma," and "smoking/secondhand smoke and corticosteroids." We also reviewed reference lists of identified articles for relevant citations. In asthmatic patients who smoke, disease control is poorer than in asthmatic nonsmokers. Of all forms of SHS, maternal exposure seems to have the largest impact on asthma by increasing the frequency and severity of the disease and decreasing lung function. Asthmatic children exposed to multiple household smokers face an increased risk for respiratory illness-related absences from school, and these effects persist during adolescence but weaken during adulthood. Airway mucosal permeability is increased in smokers, which could lead to increased clearance of inhaled corticosteroids from the airways. Smokers also have decreased histone deacetylase activity, which is necessary for corticosteroids to fully suppress cytokine production, and can lead to corticosteroid resistance. Cigarette smoking and SHS in asthmatics lead to detrimental effects in patient outcomes and effectiveness of steroid therapy.

Hyperoxia Impairs Alveolar Formation and Induces Senescence Through Decreased Histone Deacetylase Activity and Up-Regulation of p21 in Neonatal Mouse Lung

Alveolar development comprises the transition of lung architecture from saccules to gas-exchange units during late gestation and early postnatal development. Exposure to hyperoxia disrupts developmental signaling pathways and causes alveolar hypoplasia as seen in bronchopulmonary dysplasia affecting preterm human newborns. Expanding literature suggests that epigenetic changes caused by environmental triggers during development may lead to heritable changes in gene expression. Given recent data on altered histone deacetylase (HDAC) activity in lungs of humans and animal models with airspace enlargement/emphysema, we hypothesized that alveolar hypoplasia from hyperoxia exposure in neonatal mice is a consequence of cell cycle arrest and reduced HDAC activity and up-regulation of the cyclin-dependent kinase inhibitor, p21. We exposed newborn mice to hyperoxia and compared lung morphologic and epigenetic changes to room air controls. Furthermore, we pretreated a subgroup of animals with the macrolide antibiotic azithromycin (AZM), known to possess antiinflammatory properties. Our results showed that hyperoxia exposure resulted in alveolar hypoplasia and was associated with decreased HDAC1 and HDAC2 and increased p53 and p21 expression. Furthermore, AZM did not confer protection against hyperoxia-induced alveolar changes. These findings suggest that alveolar hypoplasia caused by hyperoxia is mediated by epigenetic changes affecting cell cycle regulation/senescence during lung development.

Biophysical Regulation of Histone Acetylation in Mesenchymal Stem Cells

Histone deacetylation and acetylation are catalyzed by histone deacetylase (HDAC) and histone acetyltransferase, respectively, which play important roles in the regulation of chromatin remodeling, gene expression, and cell functions. However, whether and how biophysical cues modulate HDAC activity and histone acetylation is not well understood. Here, we tested the hypothesis that microtopographic patterning and mechanical strain on the substrate regulate nuclear shape, HDAC activity, and histone acetylation. Bone marrow mesenchymal stem cells (MSCs) were cultured on elastic membranes patterned with parallel microgrooves 10 μm wide that kept MSCs aligned along the axis of the grooves. Compared with MSCs on an unpatterned substrate, MSCs on microgrooves had elongated nuclear shape, a decrease in HDAC activity, and an increase of histone acetylation. To investigate anisotropic mechanical sensing by MSCs, cells on the elastic micropatterned membranes were subjected to static uniaxial mechanical compression or stretch in the direction parallel or perpendicular to the microgrooves. Among the four types of loads, compression or stretch perpendicular to the microgrooves caused a decrease in HDAC activity, accompanied by the increase in histone acetylation and slight changes of nuclear shape. Knocking down nuclear matrix protein lamin A/C abolished mechanical strain-induced changes in HDAC activity. These results demonstrate that micropattern and mechanical strain on the substrate can modulate nuclear shape, HDAC activity, and histone acetylation in an anisotropic manner and that nuclear matrix mediates mechanotransduction. These findings reveal a new mechanism, to our knowledge, by which extracellular biophysical signals are translated into biochemical signaling events in the nucleus, and they will have significant impact in the area of mechanobiology and mechanotransduction.

Differential effects of sulforaphane on histone deacetylases, cell cycle arrest and apoptosis in normal prostate cells versus hyperplastic and cancerous prostate cells

Sulforaphane (SFN) is an isothiocyanate derived from cruciferous vegetables such as broccoli. The ability of SFN to inhibit histone deacetylase (HDAC) enzymes may be one mechanism by which it acts as a chemoprevention agent. The ability of a chemopreventive agent to specifically cause cytotoxicity in cancer and not normal cells is an important factor in determining its safety and clinical relevance. We characterized the effects of SFN in normal (PrEC), benign hyperplasia (BPH1) and cancerous (LnCap and PC3) prostate epithelial cells. We observed that 15 μM SFN selectively induced cell cycle arrest and apoptosis in BPH1, LnCap and PC3 cells but not PrEC cells. SFN treatment also selectively decreased HDAC activity, and Class I and II HDAC proteins, increased acetylated histone H3 at the promoter for P21, induced p21 expression and increased tubulin acetylation in prostate cancer cells. HDAC6 over-expression was able to reverse SFN-induced cyotoxicity. In PrEC cells, SFN caused only a transient reduction in HDAC activity with no change in any other endpoints tested. The differences in sensitivity to SFN in PrEC and PC3 are likely not due to differences in SFN metabolism or differences in phase 2 enzyme induction. SFN exerts differential effects on cell proliferation, HDAC activity and downstream targets in normal and cancer cells.

(-)-Epigallocatechin-3-gallate reactivates silenced tumor suppressor genes, Cip1/p21 and p16INK4a, by reducing DNA methylation and increasing histones acetylation in human skin cancer cells

The anti-skin carcinogenic effects of green tea catechins have been studied extensively in vitro and in vivo models but the precise epigenetic molecular mechanisms are still unclear. Accumulating data suggest that dietary phytochemicals may alter cancer risk by modifications of epigenetic processes in the cells. The present study was designed to investigate whether tea catechins, particularly (-)-epigallocatechin-3-gallate (EGCG), would modify epigenetic events to regulate DNA methylation-silenced tumor suppressor genes in skin cancer cells. DNA methylation, histone modifications and tumor suppressor gene expressions were studied in detail using human epidermoid carcinoma A431 cells as an in vitro model after EGCG treatment using cytostaining, western blotting, dot blot analysis, real-time polymerase chain reaction and enzymatic activity assays. Our study shows that EGCG treatment decreased global DNA methylation levels in A431 cells in a dose-dependent manner. EGCG decreased the levels of 5-methylcytosine, DNA methyltransferase (DNMT) activity, messenger RNA (mRNA) and protein levels of DNMT1, DNMT3a and DNMT3b. EGCG decreased histone deacetylase activity and increased levels of acetylated lysine 9 and 14 on histone H3 (H3-Lys 9 and 14) and acetylated lysine 5, 12 and 16 on histone H4 but decreased levels of methylated H3-Lys 9. Additionally, EGCG treatment resulted in re-expression of the mRNA and proteins of silenced tumor suppressor genes, p16INK4a and Cip1/p21. Together, our study provides new insight into the epigenetic mechanism of action of EGCG that may contribute to the chemoprevention of skin cancer and may have important implications for epigenetic therapy.

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.

Differential effects of sulforaphane on histone deacetylases, cell cycle arrest and apoptosis in normal and prostate cancer cells

Epidemiological data indicate that a diet high in cruciferous vegetables may lower the overall risk of prostate cancer. The ability of sulforaphane (SFN), an isothiocyanate derived from cruciferous vegetables, to inhibit histone deacetylases (HDAC) may be one mechanism by which it acts as a chemoprevention agent. Interestingly, the antiproliferative effects of pharmacological HDAC inhibitors are specific to cancer cells. The objective of this study was to compare the effects of SFN on HDAC inhibition and downstream molecular targets in both normal (PrEC) and cancerous (PC3) prostate epithelial cells. We hypothesize that SFN will act similar to pharmacological HDAC inhibitors and selectively target cancer cells. In PC3, SFN decreased HDAC activity, decreased HDACs 3, 6, and 8, increased acetylated histone H3 at the promoter for p21, induced p21 expression and increased tubulin acetylation. These data suggest that SFN targets both Class I and II HDACs in prostate cancer cells. In PrEC, SFN caused only a transient reduction in HDAC activity with no change in any other endpoints tested. SFN also induced selective cell cycle arrest and apoptosis in PC3 but not PrEC. We conclude that SFN exerts differential effects on HDAC activity and downstream targets in normal and cancer cells. Together, these data highlight the potential of SFN, a dietary HDAC inhibitor as an effective chemoprevention agent. Funded by NIHCA122906.Grant Funding Source: NIHCA122906