Increased Histone Deacetylase Activity Research Articles

Epigenetic mechanisms and alcohol use disorders: a potential therapeutic target

Alcohol use disorder is a devastating illness with a profound health impact, and its development is dependent on both genetic and environmental factors. This disease occurs over time and requires changes in brain gene expression. There is converging evidence suggesting that the epigenetic processes may play a role in the alcohol-induced gene regulations and behavior such as the intervention of DNA methylation and histone acetylation. Histone acetylation, like histone methylation, is a highly dynamic process regulated by two classes of enzymes: histone acetyltransferases and histone deacetylases (HDACs). To date, 18 human HDAC isoforms have been characterized, and based on their sequence homologies and cofactor dependencies, they have been phylogenetically categorized into 4 main classes: classes I, II (a and b), III, and IV. In the brain, expression of the different classes of HDACs varies between cell types and also in their subcellular localization (nucleus and/or cytosol). Furthermore, we recently showed that a single ethanol exposure inhibits HDAC activity and increases both H3 and H4 histone acetylation within the amygdala of rats. In the brain of alcoholic patients, ethanol has been shown to induce histone-related and DNA methylation epigenetic changes in several reward regions involved in reward processes such as hippocampus, prefrontal cortex, and amygdala. We recently demonstrated alteration of histone H3 acetylation levels in several brain regions from the reward circuit of rats made dependent to alcohol after chronic and intermittent exposure to ethanol vapor. In neuronal cell line culture, ethanol was shown to induce HDAC expression. In mouse and rat brain, numerous studies reported epigenetic alterations following ethanol exposure. We also demonstrated that both the expression of genes and the activity of enzymes involved in epigenetic mechanisms are changed after repeated administrations of ethanol in mice sensitized to the motor stimulant effect of ethanol (a model of drug-induced neuroplasticity). Numerous studies have shown that HDAC inhibitors are able to counter ethanol-induced behaviors and the ethanol-induced changes in the levels of HDAC and/or levels of acetylated HDAC. For example, trichostatin A (TSA) treatment caused the reversal of ethanol-induced tolerance, anxiety, and ethanol drinking by inhibiting HDAC activity, thereby increasing histone acetylation in the amygdala of rats. Another study demonstrated that TSA prevented the development of ethanol withdrawal induced anxiety in rats by rescuing deficits in histone acetylation induced by increased HDAC activity in the amygdala. We have demonstrated that treatment with the HDAC inhibitor sodium butyrate blocks both the development and the expression of ethanol-induced behavioral sensitization in mice. In this context, converging evidence indicates that HDAC inhibitors could be useful in counteracting ethanol-induced gene regulations via epigenetic mechanisms, that is, HDAC inhibitors could affect different acetylation sites and may also alter the expression of different genes that could in turn counteract the effect of ethanol. Recent work in rodents has shown that systemic administration of pan HDAC class I and II inhibitors, TSA and N-hydroxy-N-phenyl-octanediamide [SuberoylAnilide Hydroxamic Acid] (SAHA), and of the more selective inhibitor (mainly HDAC1 and HDAC9) MS-275, decrease binge-like alcohol drinking in mice. SAHA selectively reduced ethanol operant self-administration and seeking in rats. Our previous study revealed that MS-275 strongly decreased operant ethanol self-administration in alcohol-dependent rats when administered 30 minutes before the session at the second day of injection. We also demonstrated that intra-cerebro-ventricular infusion of MS-275 increases acetylation of Histone 4 within the nucleus accumbens and the dorsolateral striatum, associated to a decrease in ethanol self-administration by about 75%. MS-275 also diminished both the motivation to consume ethanol (25% decrease), relapse (by about 50%) and postponed reacquisition after abstinence. Both literature and several of our studies strongly support the potential therapeutic interest of targeting epigenetic mechanisms in excessive alcohol drinking and strengthen theinterest of focusing on specific isoforms of histone deacetylases.

Histone Deacetylase Inhibition Restores Retinal Pigment Epithelium Function in Hyperglycemia.

In diabetic individuals, macular edema is a major cause of vision loss. This condition is refractory to insulin therapy and has been attributed to metabolic memory. The retinal pigment epithelium (RPE) is central to maintaining fluid balance in the retina, and this function is compromised by the activation of advanced glycation end-product receptors (RAGE). Here we provide evidence that acute administration of the RAGE agonist, glycated-albumin (gAlb) or vascular endothelial growth factor (VEGF), increased histone deacetylase (HDAC) activity in RPE cells. The administration of the class I/II HDAC inhibitor, trichostatin-A (TSA), suppressed gAlb-induced reductions in RPE transepithelial resistance (in vitro) and fluid transport (in vivo). Systemic TSA also restored normal RPE fluid transport in rats with subchronic hyperglycemia. Both gAlb and VEGF increased HDAC activity and reduced acetyl-α-tubulin levels. Tubastatin-A, a relatively specific antagonist of HDAC6, inhibited gAlb-induced changes in RPE cell resistance. These data are consistent with the idea that RPE dysfunction following exposure to gAlb, VEGF, or hyperglycemia is associated with increased HDAC6 activity and decreased acetyl-α-tubulin. Therefore, we propose inhibiting HDAC6 in the RPE as a potential therapy for preserving normal fluid homeostasis in the hyperglycemic retina.

Increased histone deacetylase activity in peripheral blood mononuclear cells of patients with schizophrenia

This study investigates the association between histone deacetylase (HDAC) activity in human peripheral blood mononuclear cells (PBMCs) and schizophrenia. Data were derived from a case-control association study of 19 unrelated adult patients with schizophrenia and 21 matched healthy controls. HDAC activity was measured with a HDAC activity colorimetric assay kit. Our findings suggest that HDAC activity in PBMCs is higher in patients with schizophrenia than in healthy people.

A Role for Nuclear Actin in HDAC 1 and 2 Regulation.

Class I histone deacetylases (HDACs) are known to remove acetyl groups from histone tails. This liberates positive charges on the histone tail and allows for tighter winding of DNA, preventing transcription factor binding and gene activation. Although the functions of HDAC proteins are becoming apparent both biochemically and clinically, how this class of proteins is regulated remains poorly understood. We identified a novel interaction between nuclear actin and HDAC 1 and HDAC 2. Nuclear actin has been previously shown to interact with a growing list of nuclear proteins including chromatin remodeling complexes, transcription factors and RNA polymerases. We find that monomeric actin is able to bind the class I HDAC complex. Furthermore, increasing the concentration of actin in HeLa nuclear extracts was able to suppress overall HDAC function. Conversely, polymerizing nuclear actin increased HDAC activity and decreased histone acetylation. Moreover, the interaction between class I HDACs and nuclear actin was found to be activity dependent. Together, our data suggest nuclear actin is able to regulate HDAC 1 and 2 activity.

Sodium butyrate reduces insulin-resistance, fat accumulation and dyslipidemia in type-2 diabetic rat: A comparative study with metformin

Recent evidences highlighted that histone deacetylases (HDACs) can deacetylate the histone, various transcription factors and regulatory proteins, which directly or indirectly affect glucose metabolism. The present study aimed to evaluate the comparative effects of sodium butyrate (NaB) and metformin on the glucose homeostasis, insulin-resistance, fat accumulation and dyslipidemia in type-2 diabetic rat. Diabetes was developed in Sprague-Dawley rats by the combination of high-fat diet (HFD) and low dose streptozotocin (STZ, 35 mg/kg). NaB at the doses of 200 and 400 mg/kg twice daily as well as metformin (as a positive control) 150 mg/kg twice daily for 10 consecutive weeks were administered by i.p. and oral route, respectively. NaB treatment significantly reduced the plasma glucose, HbA1c, insulin-resistance, dyslipidemia and gluconeogenesis, which are comparable to metformin treatment. Further, NaB treatment ameliorated the micro- and macro-vesicular steatosis in liver and fat deposition in brown adipose tissue, white adipose tissue (adipocytes hypertrophy) as well as pancreatic beta-cell damage. In the present study, both NaB and metformin inhibited the diabetes-associated increased HDACs activity, thereby increased the acetylation of histone H3 in liver. The present findings demonstrated that NaB and metformin reduced insulin-resistance, dyslipidemia, fat accumulation and gluconeogenesis thereby improved the glucose homeostasis in rat. Thus, NaB might be a promising molecule for the prevention and treatment of type-2 diabetes and dyslipidemia.

Montelukast versus Dexamethasone Treatment in a Guinea Pig Model of Chronic Pulmonary Neutrophilic Inflammation

Airway inflammation in chronic obstructive pulmonary disease (COPD) is refractory to corticosteroids and hence COPD treatment is hindered and insufficient. This study assessed the effects of oral treatment with Montelukast (10 and 30 mg/kg) or dexamethasone (20 mg/kg) for 20 days on COPD model induced by chronic exposure to lipopolysaccharide (LPS). Six groups of male guinea pigs were studied. Group 1: naïve group, group 2: exposed to saline nebulization. Groups 3, 4, 5, and 6: exposed to 9 nebulizations of LPS (30 μg/ml) for 1 hour, 48 hours apart with or without treatment with Montelukast or dexamethasone. Airway hyperreactivity (AHR) to methacholine (MCh), histopathological study and bronchoalveolar lavage fluid (BALF) as well as lung tissue analyses were performed 48 hours after the final exposure to LPS (day 20). LPS-induced pulmonary dysfunction was associated with increased neutrophil count, leukotriene (LT) B4, and tumor necrosis factor (TNF)-α in BALF. Moreover, there was an increase in malondialdehyde (MDA) level and a decrease in histone deacetylases(HDAC) activity in the lung tissue. Both Montelukast (10 or 30 mg /kg) and dexamethasone significantly reduced neutrophil count in BALF and inflammatory cells in lung parenchyma as well as TNF-α, and MDA levels. However, dexamethasone was more effective (p < 0.05). Montelukast, at a dose of 30 mg /kg, significantly reduced specific airway resistance after the 9th LPS exposure, attenuated AHR to MCh, decreased LTB4 and increased HDAC activity in comparison to dexamethasone. These results suggest that treatment with Montelukast can be useful in chronic airway inflammatory diseases including COPD poorly responsive to glucocorticoids.

Haemophilus influenzae induces steroid-resistant inflammatory responses in COPD.

BackgroundChronic obstructive pulmonary disease (COPD) is an inflammatory disorder partially resistant to glucocorticoids. A reduced histone deacetylase (HDAC) activity has been proposed to explain this resistance. Haemophilus influenzae frequently colonizes the airways of COPD patients, where it enhances inflammation. The effects of Haemophilus influenzae on HDAC activity have not been investigated before.MethodsThe effects of the presence or absence of Haemophilus influenzae ex-vivo and in vitro were studied. To this end, we determined: (1) cytokine release in alveolar macrophages (AM) from 7 patients with COPD, 5 healthy smokers, 6 healthy non-smokers and (2) HDAC activity, nuclear factor kappa B (NF-κB) activation in a macrophage-like cell line (PMA-transformed U937 cells) co-cultured with epithelial cells. Experiments were repeated with dexamethasone (1 μM) and/or the HDAC enhancer theophylline (10 μM).ResultsHaemophilus influenzae induced a steroid-resistant inflammatory response in AM from COPD and controls and decreased HDAC activity, activated NF-κB and induced the secretion of several cytokines (IL-6, IL-8, IL-1β, IL-10 and TNF-α) (p < 0.001 for all comparisons) in the macrophage-like cell line. Dexamethasone reduced NF-κB activation but it did not modify HDAC activity. The addition of theophylline to dexamethasone increased HDAC activity and suppressed cytokine release completely, without modifying NF-κB activation.ConclusionsThese results indicate that Haemophilus influenzae reduces HDAC activity and induces a NF-κB mediated inflammatory response that is only partially suppressed by glucocorticoids irrespective of having COPD. Yet, the latter can be fully restored by targeting HDAC activity.Electronic supplementary materialThe online version of this article (doi:10.1186/s12890-015-0155-3) contains supplementary material, which is available to authorized users.

Significance of P53 and high mobility group box 1 protein in different levels of liver fibrosis in chronic hepatitis B

To investigate the role of P53 and high mobility group protein 1 (HMGB1) protein expression in liver fibrosis stages in chronic hepatitis B patients. According to the pathological grades, 103 patients were divided into 3 groups: no fibrosis group (n=18), low fibrosis group (n=49) or high fibrosis group (n=36). Serum HMGB1 levels were determined and receiver operating characteristic (ROC) curve was made based on the HMGB1 level and liver fibrosis score. Liver fibrosis model was developed by CCl4 in 60 male SD rats, which were sacrificed 6 or 12 weeks later. The degree of fibrosis was examined by Masson staining; HMGB1 and P53 protein expression were analyzed by Western blot; histone deacetylase (HDAC) activity, TNF-α, IL-1β and IL-6 levels in serum were measured. The serum levels of HMGB1 level in low and high fibrosis groups were significantly higher than that in no fibrosis group (P<0.01, respectively). ROC curve showed that serum HMGB1 in the diagnosis of hepatic fibrosis with cut off at 74 pg/mL, specificity at 65% and sensitivity at 87%. Compared with the control group, HMGB1 expression in both low and high fibrosis group was decreased in nucleus but was increased in cytoplasm, accompanied by the elevated P53 expression, increased HDAC activity and inflammatory cytokine levels (all P<0.01, respectively). P53 and HMGB1 expression was significantly increased in chronic hepatitis B patients with liver fibrosis; serum HMGB1 level was positively correlated with the degree of liver cirrhosis and HMGB1 could be used as a sensitive and specific index for liver fibrosis prognosis.

Increased Histone Deacetylase Activity Involved in the Suppressed Invasion of Cancer Cells Survived from ALA-Mediated Photodynamic Treatment.

Previously, we have found that cancer cells survived from 5-Aminolevulinic acid-mediated photodynamic therapy (ALA-PDT) have abnormal mitochondrial function and suppressed cellular invasiveness. Here we report that both the mRNA expression level and enzymatic activity of histone deacetylase (HDAC) were elevated in the PDT-derived variants with dysfunctional mitochondria. The activated HDAC deacetylated histone H3 and further resulted in the reduced migration and invasion, which correlated with the reduced expression of the invasion-related genes, matrix metalloproteinase 9 (MMP9), paternally expressed gene 1 (PEG1), and miR-355, the intronic miRNA. Using chromatin immunoprecipitation, we further demonstrate the reduced amount of acetylated histone H3 on the promoter regions of MMP9 and PEG1, supporting the down-regulation of these two genes in PDT-derived variants. These results indicate that HDAC activation induced by mitochondrial dysfunction could modulate the cellular invasiveness and its related gene expression. This argument was further verified in the 51-10 cybrid cells with the 4977 bp mtDNA deletion and A375 ρ0 cells with depleted mitochondria. These results indicate that mitochondrial dysfunction might suppress tumor invasion through modulating histone acetylation.

Abstract P047: Epigenetic Mechanisms of Sodium Butyrate- and Retinoic Acid-dependent Attenuation of Renal Fibrosis and Inflammation in Npr1 Gene-targeted Mice

The objective of the present study was to determine the combined effect of sodium butyrate (NaBu), a histone deacetylase (HDAC) inhibitor and all-trans retinoic acid (ATRA) on attenuation of renal fibrosis and inflammation in Npr1 (coding for natriuretic peptide receptor-A) gene-disrupted mutant mice. Adult (18-20 week old) male Npr1 gene-disrupted heterozygous (1-copy; Npr1+/-) wild-type (2-copy; Npr1+/+), and gene-duplicated (3-copy; Npr1++/+) mice were treated by injecting ATRA-NaBu hybrid drug (1.0 mg/kg/day) intraperitoneally for 2-weeks. A marked attenuation in tubulo-interstitial fibrosis was observed in Npr1+/- mice after treatment with ATRA-NaBu (50%, p &lt; 0.001). Western blot analyses exhibited the reduction in renal expression of collagen type I alpha 2 and transforming growth factor-beta (55%, p &lt; 0.001, 67%, p &lt; 0.001, respectively) in ATRA-NaBu-treated Npr1+/- mice compared with vehicle-treated mice. A significant decrease in systolic blood pressure was observed in ATRA-NaBu-treated Npr1+/- mice (treated 110.3 ± 2.6 vs. Npr1 +/- control mice, 126.3 ± 2.7, p &lt; 0.01). The ATRA-NaBu also enhanced plasma cGMP levels (pmol/ml) in Npr1+/- (treated, 22.7 ± 3.3 vs. control, 6.2 ± 1.6; p &lt; 0.05), Npr1+/+ (treated, 50.9 ± 3.5 vs. control, 20.8 ± 3.6; p &lt; 0.05), and Npr1++/+ (treated, 71.4 ± 6.4 vs. control, 34.5 ± 2.9; p &lt; 0.05) mice. Treatment with ATRA-NaBu significantly lowered renal levels (pg/mg protein) of monocyte chemoattractant protein-1 (MCP-1) (treated, 109.5 ± 9.9 vs. control, 24.3 ± 1.7; p &lt; 0.01) in Npr1+/- mice compared with vehicle-treated mice. Western blot analyses confirmed the reduction in renal expression of MCP-1 (72%, p &lt; 0.001) in ATRA-NaBu-treated Npr1+/- mice compared with control mice. Moreover, the increased HDAC activity in Npr1+/- mice was significantly reduced by ATRA-NaBu treatment compared with untreated Npr1+/- control mice. The present results provide direct evidence that ATRA-NaBu acts as a potent antifibrotic agent and repairs the renal pathology in Npr1+/- mice, which will have important implications in prevention of hypertension-related renal pathophysiological conditions.

Cigarette smoke extracts inhibit TLR ligand-induced inflammation via inactivation of NF-κB and activation of histone deacetylase in macrophages (INM7P.353)

Abstract Cigarette smoke (CS) is the major risk factor of chronic obstructive pulmonary disease (COPD). CS has been reported to inhibit Toll-like receptor (TLR) function and phagocytosis in macrophages. Moreover, alveolar macrophages in COPD have impaired responsiveness to TLR stimulation. However, the molecular mechanism of CS-mediated impairments of macrophage immunity is not fully defined. We investigated the inhibitory mechanism of cigarette smoke extracts (CSE) in macrophages. Lipopolysaccharide (LPS, TLR4 ligand), Pam3CSK4 (TLR2 ligand), and CpG-oligodeoxynucleotides (TLR9 ligand) induced IL-6, TNF-α, and IL-1β production in RAW264.7 macrophage cells, which was significantly suppressed by CSE co-treatment. CSE did not block LPS-induced IκBα degradation, MAP kinase activation, and nuclear translocation of NF-κB subunit. However, it markedly suppressed mRNA expression of IL-6, TNF-α, and IL-1β, which implies that CSE regulate inflammatory cytokine production at the transcriptional level. Therefore, we next investigated the activity and modification of transcription factor, NF-κB, and histone deacetylase (HDAC). CSE significantly suppressed basal and LPS-induced NF-κB activity and reduced the level of acetyl-NF-κB p65. CSE increased HDAC activity and decreased the level of acetyl- and phospho-Histone H3 (Lys9/Ser10) expression. Together, these results suggest that CSE-mediated impairments of macrophage immunity are mediated by inhibition of NF-κB activation and activation of HDAC.

Acetylation preserves retinal ganglion cell structure and function in a chronic model of ocular hypertension.

The current studies investigate if the histone deacetylase (HDAC) inhibitor, valproic acid (VPA), can limit retinal ganglion cell (RGC) degeneration in an ocular-hypertensive rat model. Intraocular pressure (IOP) was elevated unilaterally in Brown Norway rats by hypertonic saline injection. Rats received either vehicle or VPA (100 mg/kg) treatment for 28 days. Retinal ganglion cell function and number were assessed by pattern electroretinogram (pERG) and retrograde FluoroGold labeling. Western blotting and a fluorescence assay were used for determination of histone H3 acetylation and HDAC activity, respectively, at 3-day, 1-week, and 2-week time points. Hypertonic saline injections increased IOPs by 7 to 14 mm Hg. In vehicle-treated animals, ocular hypertension resulted in a 29.1% and 39.4% decrease in pERG amplitudes at 2 and 4 weeks, respectively, and a 42.9% decrease in mean RGC density at 4 weeks. In comparison, VPA treatment yielded significant amplitude preservation at 2 and 4 weeks and showed significant RGC density preservation at 4 weeks. No significant difference in RGC densities or IOPs was measured between control eyes of vehicle- and VPA-treated rats. In ocular-hypertensive eyes, class I HDAC activity was significantly elevated within 1 week (13.3 ± 2.2%) and histone H3 acetylation was significantly reduced within 2 weeks following the induction of ocular hypertension. Increase in HDAC activity is a relatively early retinal event induced by elevated IOP, and suppressing HDAC activity can protect RGCs from ocular-hypertensive stress. Together these data provide a basis for developing HDAC inhibitors for the treatment of optic neuropathies.

Acetylation: A lysine modification with neuroprotective effects in ischemic retinal degeneration

Neuroretinal ischemic injury contributes to several degenerative diseases in the eye and the resulting pathogenic processes involving a series of necrotic and apoptotic events. This study investigates the time and extent of changes in acetylation, and whether this influences function and survival of neuroretinal cells following injury. Studies evaluated the time course of changes in histone deacetylase (HDAC) activity, histone-H3 acetylation and caspase-3 activation levels as well as retinal morphology and function (electroretinography) following ischemia. In addition, the effect of two HDAC inhibitors, trichostatin-A and valproic acid were also investigated. In normal eyes, retinal ischemia produced a significant increase in HDAC activity within 2 h that was followed by a corresponding significant decrease in protein acetylation by 4 h. Activated caspase-3 levels were significantly elevated by 24 h. Treatment with HDAC inhibitors blocked the early decrease in protein acetylation and activation of caspase-3. Retinal immunohistochemistry demonstrated that systemic administration of trichostatin-A or valproic acid, resulted in hyperacetylation of all retinal layers after systemic treatment. In addition, HDAC inhibitors provided a significant functional and structural neuroprotection at seven days following injury relative to vehicle-treated eyes. These results provide evidence that increases in HDAC activity is an early event following retinal ischemia, and are accompanied by corresponding decreases in acetylation in advance of caspase-3 activation. In addition to preserving acetylation status, the administration of HDAC inhibitors suppressed caspase activation and provided structural and functional neuroprotection in model of ischemic retinal injury. Taken together these data provide evidence that decrease in retinal acetylation status is a central event in ischemic retinal injury, and the hyperacetylation induced by HDAC inhibition can provide acute neuroprotection.

Histone deacetylase activity and brain-derived neurotrophic factor (BDNF) levels in a pharmacological model of mania

In the present study, we aimed to examine the effects of repeated D-amphetamine (AMPH) exposure, a well-accepted animal model of acute mania in bipolar disorder (BD), and histone deacetylase (HDAC) inhibitors on locomotor behavior and HDAC activity in the prefrontal cortex (PFC) and peripheral blood mononuclear cells (PBMCs) of rats. Moreover, we aimed to assess brain-derived neurotrophic factor (BDNF) protein and mRNA levels in these samples. We treated adult male Wistar rats with 2 mg/kg AMPH or saline intraperitoneally for 14 days. Between the 8th and 14th days, rats also received 47.5 mg/kg lithium (Li), 200 mg/kg sodium valproate (VPT), 2 mg/kg sodium butyrate (SB), or saline. We evaluated locomotor activity in the open-field task and assessed HDAC activity in the PFC and PBMCs, and BDNF levels in the PFC and plasma. AMPH significantly increased locomotor activity, which was reversed by all drugs. This hyperactivity was associated with increased HDAC activity in the PFC, which was partially reversed by Li, VPT, and SB. No differences were found in BDNF levels. Repeated AMPH administration increases HDAC activity in the PFC without altering BDNF levels. The partial reversal of HDAC increase by Li, VPT, and SB may account for their ability to reverse AMPH-induced hyperactivity.

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Histone deacetylase induces accelerated maturation in Xenopus laevis oocytes

In oocyte maturation in Xenopus laevis, nuclear material induces rapid maturation and is required for entry into meiosis II. Nuclear material contains a large number of RNAs and proteins, including histone deacetylase (HDAC); however, it is not known which materials induce accelerated maturation. The HDAC activity modifies transcription rate and is required for normal meiosis; however, its function in oocyte maturation is still unclear. We investigated the function of HDAC activity, which is localized in the nuclear material, in the regulation of the speed of oocyte maturation. Inhibition of HDAC activity with trichostatin A (TSA) induced hyperacetylation of histone H3 and prolonged oocyte maturation. In contrast, increase in HDAC activity with an injection of FLAG-tagged maternal histone deacetylase (HDACm-FLAG) mRNA induced deacetylation of histone H3 and reduced the duration of oocyte maturation. Cdc2 kinase, Cdc25C or mitogen-activated protein kinase (MAPK), which are key regulators of the meiosis, were activated coincidently with maturation progression. In oocytes, the mRNA level of Cdc25C, an activator of Cdc2, was increased by HDACm-FLAG mRNA-injection; in contrast, the mRNA level of Cdc2 inhibitor Wee1 was increased by TSA treatment. These results suggest that HDAC activity is involved in the control of maturation speed through the regulation of mRNA levels of cell cycle regulators. Thus, HDACm is a candidate for the nuclear material component that induces rapid maturation in Xenopus oocytes.

Transcriptional repression of E-cadherin by human papillomavirus type 16 E6.

There is increasing evidence supporting DNA virus regulation of the cell adhesion and tumour suppressor protein, E-cadherin. We previously reported that loss of E-cadherin in human papillomavirus (HPV) type 16-infected epidermis is contributed to by the major viral proto-oncogene E6 and is associated with reduced Langerhans cells density, potentially regulating the immune response. The focus of this study is determining how the HPV16 E6 protein mediates E-cadherin repression. We found that the E-cadherin promoter is repressed in cells expressing E6, resulting in fewer E-cadherin transcripts. On exploring the mechanism for this, repression by increased histone deacetylase activity or by increased binding of trans-repressors to the E-cadherin promoter Epal element was discounted. In contrast, DNA methyltransferase (DNMT) activity was increased in E6 expressing cells. Upon inhibiting DNMT activity using 5-Aza-2′-deoxycytidine, E-cadherin transcription was restored in the presence of HPV16 E6. The E-cadherin promoter was not directly methylated, however a mutational analysis showed general promoter repression and reduced binding of the transactivators Sp1 and AML1 and the repressor Slug. Expression of E7 with E6 resulted in a further reduction in surface E-cadherin levels. This is the first report of HPV16 E6-mediated transcriptional repression of this adhesion molecule and tumour suppressor protein.

Transforming growth factor β1 inhibits bone morphogenic protein (BMP)-2 and BMP-7 signaling via upregulation of Ski-related novel protein N (SnoN): possible mechanism for the failure of BMP therapy?

BackgroundBone morphogenic proteins (BMPs) play a key role in bone formation. Consequently, it was expected that topical application of recombinant human (rh)BMP-2 and rhBMP-7 would improve the healing of complex fractures. However, up to 36% of fracture patients do not respond to this therapy. There are hints that a systemic increase in transforming growth factor β1 (TGFβ1) interferes with beneficial BMP effects. Therefore, in the present work we investigated the influence of rhTGFβ1 on rhBMP signaling in primary human osteoblasts, with the aim of more specifically delineating the underlying regulatory mechanisms.MethodsBMP signaling was detected by adenoviral Smad-binding-element-reporter assays. Gene expression was determined by reverse transcription polymerase chain reaction (RT-PCR) and confirmed at the protein level by western blot. Histone deacetylase (HDAC) activity was determined using a test kit. Data sets were compared by one-way analysis of variance.ResultsOur findings showed that Smad1/5/8-mediated rhBMP-2 and rhBMP-7 signaling is completely blocked by rhTGFβ1. We then investigated expression levels of genes involved in BMP signaling and regulation (for example, Smad1/5/8, TGFβ receptors type I and II, noggin, sclerostin, BMP and activin receptor membrane bound inhibitor (BAMBI), v-ski sarcoma viral oncogene homolog (Ski), Ski-related novel protein N (SnoN) and Smad ubiquitination regulatory factors (Smurfs)) and confirmed the expression of regulated genes at the protein level. Smad7 and SnoN were significantly induced by rhTGFβ1 treatment while expression of Smad1, Smad6, TGFβRII and activin receptor-like kinase 1 (Alk1) was reduced. Elevated SnoN expression was accompanied by increased HDAC activity. Addition of an HDAC inhibitor, namely valproic acid, fully abolished the inhibitory effect of rhTGFβ1 on rhBMP-2 and rhBMP-7 signaling.ConclusionsrhTGFβ1 effectively blocks rhBMP signaling in osteoblasts. As possible mechanism, we postulate an induction of SnoN that increases HDAC activity and thereby reduces the expression of factors required for efficient BMP signaling. Thus, inhibition of HDAC activity may support bone healing during rhBMP therapy in patients with elevated TGFβ serum levels.

Histone Deacetylation Inhibition in Pulmonary Hypertension

Background— Epigenetic programming, dynamically regulated by histone acetylation, is a key mechanism regulating cell proliferation and survival. Little is known about the contribution of histone deacetylase (HDAC) activity to the development of pulmonary arterial hypertension, a condition characterized by profound structural remodeling of pulmonary arteries and arterioles. Methods and Results— HDAC1 and HDAC5 protein levels were elevated in lungs from human idiopathic pulmonary arterial hypertension and in lungs and right ventricles from rats exposed to hypoxia. Immunohistochemistry localized increased expression to remodeled vessels in the lung. Both valproic acid, a class I HDAC inhibitor, and suberoylanilide hydroxamic acid (vorinostat), an inhibitor of class I, II, and IV HDACs, mitigated the development of and reduced established hypoxia-induced pulmonary hypertension in the rat. Both valproic acid and suberoylanilide hydroxamic acid inhibited the imprinted highly proliferative phenotype of fibroblasts and R-cells from pulmonary hypertensive bovine vessels and platelet-derived growth factor–stimulated growth of human vascular smooth muscle cells in culture. Exposure to valproic acid and suberoylanilide hydroxamic acid was associated with increased levels of p21 and FOXO3 and reduced expression of survivin. The significantly higher levels of expression of cKIT, monocyte chemoattractant protein-1, interleukin-6, stromal-derived factor-1, platelet-derived growth factor-b, and S100A4 in R-cells were downregulated by valproic acid and suberoylanilide hydroxamic acid treatment. Conclusions— Increased HDAC activity contributes to the vascular pathology of pulmonary hypertension. The effectiveness of HDAC inhibitors, valproic acid, and suberoylanilide hydroxamic acid, in models of pulmonary arterial hypertension supports a therapeutic strategy based on HDAC inhibition in pulmonary arterial hypertension.

Abstract 3599: Expression and function of HFE in mammary gland iron homeostasis and its relevance to breast cancer

Abstract Emerging evidences on the role of iron-regulatory proteins in tumor growth provide new insights into the role of iron in cancer. Iron, though essential for normal cellular function, is toxic if present in excess. Hemochromatosis is an autosomal recessive disease of iron overload linked to mutations in HFE gene [Histocompatability leukocyte antigen class I-like protein involved in iron (FE) homeostasis]. HFE interacts with transferrin receptors, TfR1 and TfR2, and inhibits cellular iron uptake. The prevalence of the disease is very high with homozygosity incidence of ∼1 in 300 and heterozygosity incidence of ∼1 in 10. The disease manifests with liver cirrhosis/cancer, cardiomyopathy, diabetes, and arthritis. Several studies have shown increased prevalence of HFE mutations in breast cancer patients across all races. There is also evidence of increased levels of iron in breast cancer tissue. Although excessive iron and oxidative stress have been postulated to play a role in breast cancer, the expression/function of HFE in mammary gland iron homeostasis and how mutations in HFE affect breast cancer progression have not been studied directly. Here we demonstrate the expression of HFE mRNA in mouse mammary gland and primary mammary epithelial cells. The mRNA was absent in HFE-/- mice. Immunohistochemistry showed localization of HFE protein in luminal and myoepithelial cells. Similar staining was also seen in primary mammary epithelial cells from wild type mice but not from HFE-/- mice. We then examined the physiological role of HFE in mammary gland. Ferritin is a cytoplasmic protein involved in iron storage, and increase in intracellular iron levels results in increased level of ferritin. Recently, ferritin has also been shown to promote angiogenesis. Western blot and immunohistochemical analysis revealed that mammary tissue from HFE-/- mice had increased ferritin levels in an age-dependent manner. Thus, for the first time we show that HFE-/- mice have increased iron accumulation in the mammary gland. Immunostaining with antibody against 4-hydroxynonenal, one of the byproducts of lipid peroxidation, showed that HFE-/- mammary tissue have oxidative stress mediated by iron accumulation. MTT assay and thymidine incorporation showed that primary mammary epithelial cells from HFE-/- mice proliferate faster than the cells from HFE+/+ mice. Increase in histone deacetylase (HDAC) activity has been implicated in many cancers. We found that mammary tissue from HFE-/- mice has increased HDAC activity compared to wild type tissue. This shows that functional loss of HFE in mammary tissue can also promote cell cycle progression via epigenetic mechanisms. Based on these data, we hypothesize that tissue iron is an important modifier of breast cancer progression and that mutations in HFE (heterozygosity as well as homozygosity) may enhance the incidence and/or progression of breast 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 3599. doi:1538-7445.AM2012-3599