Increased Histone Deacetylase Activity Research Articles

O06 Naïve human keratinocytes initiate a chromatin-protective mechanism in response to ultraviolet radiation through the upregulation of histone deacetylase 5

Abstract Introduction and aims During normal development and throughout life, epigenetic changes arise in response to an individuals’ behaviour and environmental exposures. Histone deacetylases (HDACs) are chromatin-modifying enzymes that regulate gene expression and their activity and expression in primary human keratinocytes alters with increasing age. Solar ultraviolet radiation (UVR) is a major causative factor of skin ageing, but how this factor affects HDAC expression and activity is largely unknown. We aimed to determine how UVR exposure affects HDAC expression and activity in primary human keratinocytes. Methods Normal human epidermal keratinocytes (NHEKs) from juvenile (aged 1–6 years; n = 3), middle-aged (56–61 years; n = 3) and aged (83–85 years; n = 3) donors were exposed to solar-simulated radiation (SSR) and HDAC abundance and activity were assayed. Results Exposure to SSR significantly increased HDAC activity in primary NHEKs irrespective of donor age (P < 0.05). However, Western blotting for HDAC abundance revealed that there was a differential response to SSR exposure across age groups; juvenile NHEKs expressed significantly more HDAC5 in response to SSR (P < 0.05) than cells from middle-aged or aged donors. Furthermore, following SSR exposure of juvenile NHEKs, the cellular localization of HDAC5 was redistributed to the nucleus from the cytoplasm. Dual immunofluorescence staining revealed that those NHEKs with nuclear HDAC5 expression were also positive for p16INK4a, a robust marker of cellular senescence (P < 0.01). Conclusions Our Results suggest that naïve human keratinocytes are responsive to UVR exposure and appear to initiate a chromatin-protective mechanism through the upregulation of HDAC5 to prevent gene expression of damaged DNA.

Abstract 006: Age And Sex Differentially Regulate Epigenetic Mechanisms In The Heart

Understanding epigenetic mechanisms associated with aging might reveal potential biomarkers for aging as well as therapeutic targets for reversing or ameliorating aging pathologies, including cardiovascular disease. There is a sex difference in the age of onset of cardiovascular disease, where females are protected against age related changes, including hypertension and fibrosis, until menopause. We hypothesized that epigenetic mechanisms would be differentially regulated in a sex and age dependent manner, with changes in the older females reflecting the loss of estrogen with aging. To test this hypothesis, we examined heart tissue from 4-month (4M), 12-month (12M), and 24-month (24M)-old male and female C57BL/6JN mice (National Institute of Aging). Surprisingly histone deacetylase (HDAC) activity (ELISA) was significantly higher in all female mice than in age-matched males (4M, female 5.2 ± 0.6 vs male 2.8 vs 0.5; 12M, female 4.2 ± 0.4 vs male 1.3 ± 0.2; 24M, female 8.1 ± 0.6 vs male 2.3 ± 0.4; ng/min/mg protein; p < 0.05). In females age increased HDAC activity (35% increase 24M v 4M; p < 0.05). Western blot analysis revealed that expression levels of profibrotic class I HDACs were significantly higher in 12M and 24M old female mice (HDAC1, 52% and 48%, p < 0.05 and HDAC2, 52% and 79%, p < 0.05), respectively, as compared with age-matched males. However, histone acetyltransferase activity was comparable in all male and female groups as was histone 3 (H3) acetylation at lysine 9 and 27. Levels of circulating histones were determined in the serum. Histones are released in the extracellular space during disease state, act as damage-associated molecular pattern molecules and activate inflammatory pathways. Circulating H3 levels in serum increased significantly with age in both sexes (male 12M, 17.1 ± 1.4; and 24M, 21.4 ± 0.8 vs. 4M, 4.0 ± 0.6; ng/ml, p < 0.05; female 12M, 13.8 ± 2.1 and 24M, 17.4 ± 1.4 vs. 4M, 7.2 ± 1.0 p < 0.05). The data demonstrate the effect of age and sex in heart tissues with aged female mice exhibiting higher levels of profibrotic epigenetic enzymes compared to males. These findings will have important implications in age-related cardiac injury as aging is a primary risk factor for hypertension and related heart disease in men and women.

Disruption of histone acetylation homeostasis triggers cognitive dysfunction in experimental diabetes

Epigenetic mechanisms related to diabetes-afflicted CNS complications are largely unknown. The present study investigated the role of histone acetylation mechanisms triggering cognitive dysfunction in the Type 1 and 2 diabetic mice model. Dynamic changes in diabetic parameters like fasting blood glucose levels, glucose tolerance test, and insulin levels were observed after the induction of diabetes. Cognitive performance was significantly diminished in T1D and T2D mice examined by the Morris water maze, novel object recognition test, and Y Maze as compared to controls. Histone profiling revealed a significant reduction in H3K9/14 and H4K12 acetylation in the cortex and hippocampus of T1D and T2D mice vs Controls. While histone deacetylase (HDAC) activity was significantly elevated in brain regions of T1D and T2D mice, the histone acetyltransferase (HAT) activity remain unchanged. Significantly increased HDAC 2, HDAC 3 protein and mRNA expression observed in T1D and T2D brain regions may corroborate for increased HDAC activity. No significant change was observed in protein and mRNA expression of HDAC 1, 5, 6, and 7 in diabetic brains. Reduced H3K9/14 and H4K12 acetylation paralleled transcriptional repression of memory-related markers BDNF, SYP, and PSD-95 in the cortex and hippocampus of T1D and T2D. Pharmacological inhibition of HDAC activity by Trichostatin A enhanced the cognitive changes observed in T1D and T2D by ameliorating BDNF, SYP, Psd-95. The present study provides a better insight into molecular mechanisms related to diabetes-dependent memory changes that can help to generate new advances for therapeutics to be developed in this area.

Pharmacological inhibition of histone deacetylases ameliorates cognitive impairment after intracerebral hemorrhage with epigenetic alteration in the hippocampus

ObjectivesPost-stroke cognitive impairment (PSCI) interferes with neurorehabilitation in patients with stroke. Epigenetic regulation of the hippocampus has been targeted to ameliorate cognitive function. In particular, the acetylation level of histones is modulated by exercise, a potent therapy for patients with stroke. Materials and MethodsWe examined the effects of exercise and pharmacological inhibition of histone deacetylase (HDAC) using sodium butyrate (NaB) on cognitive function and epigenetic factors in the hippocampus after intracerebral hemorrhage (ICH) to seek beneficial neuronal conditioning against PSCI. Forty rats were randomly assigned to five groups: sham, control, NaB, exercise, and NaB plus exercise groups (n=8 in each group). Except for those in the sham group, all rats underwent stereotaxic ICH surgery with a microinjection of collagenase solution. Intraperitoneal administration of NaB (300 mg/kg) and treadmill exercise (11 m/min for 30 min) were conducted for approximately 4 weeks starting 3 days post-surgery. ResultsICH reduced cognitive function, as detected by the object location test, accompanied by enhanced activity of HDACs. Although exercise did not modulate HDAC activity or cognitive function, repetitive NaB administration increased HDAC activity and ameliorated cognitive impairment induced by ICH. ConclusionsThis study suggests that pharmacological treatment with an HDAC inhibitor could potentially present an enriched epigenetic platform in the hippocampus and ameliorate PSCI for neurorehabilitation following ICH.

Effects of follicle-stimulating hormone, insulin-like growth factor 1, fibroblast growth factor 2, and fibroblast growth factor 9 on sirtuins expression and histone deacetylase activity in bovine granulosa cells

Granulosa cells (GC) are critical regulators of fertility. During the process of ovarian folliculogenesis, these cells undergo profound changes while producing steroid hormones that are important to control follicular growth, oocyte maturation, and ovulation. Sirtuins are enzymes that regulate several biological processes and have been associated with control of GC function. However, how sirtuins are regulated in GC during ovarian folliculogenesis remains to be unveiled. The present study was designed to investigate effects of hormones that control GC proliferation, differentiation, and steroidogenesis on expression of the seven members of the mammalian sirtuins family (SIRT1-7) and on histone deacetylase activity of nuclear sirtuins (SIRT1, 6, and 7) in GC. Bovine granulosa cells were isolated from small antral follicles (1–5 mm) and were treated with or without follicle-stimulating hormone (FSH), insulin-like growth factor 1 (IGF-1), and fibroblast growth factors 2 (FGF2) and 9 (FGF9). Following treatments, cell proliferation was determined via a cell analyzer, estradiol synthesis and histone deacetylase activity were determined via ELISA, and sirtuins mRNA expression was determined via qPCR. Treatments with FSH and IGF-1 stimulated cell proliferation while addition of FGF2 or FGF9 suppressed estradiol production stimulated by FSH plus IGF-1. In terms of treatments that regulated sirtuins expression in GC, fibroblast growth factors were the most impactful: FGF2 alone increased SIRT1 mRNA expression in comparison to several treatments and increased mRNA abundance of SIRT2 and SIRT7 when added to the combination of FSH and IGF-1; the addition of FGF9 to the combination of FSH and IGF-1 increased mRNA expression of SIRT2, SIRT3, SIRT4, SIRT6, and SIRT7 and increased mRNA expression of SIRT5 in comparison to the negative control group that received no treatment. Also, FGF2 alone increased histone deacetylase activity of sirtuins in comparison to all treatments that contained FSH and/or IGF-1. Furthermore, several correlations were observed between treatments and sirtuins expression and activity, between estradiol or GC numbers and sirtuins expression, and between expression of sirtuins. As FGF2 and FGF9 are considered anti-differentiation factors of GC that stimulate GC proliferation while suppressing estradiol production in combination with FSH and IGF-1, data of this study suggest that sirtuins are associated with control of differentiation of bovine GC.

CRISPR Activation/Interference Screen to Identify Genetic Networks in HDAC-Inhibitor-Resistant Cells.

Epigenetic alterations have been identified in various tumor types. In part, these alterations are mediated via increased histone deacetylase activity. Although preclinical results of monotherapies with histone deacetylase inhibitors (HDACi) are promising, success in clinical trials is limited. Reasons for these limitations may be de novo or acquired resistance to HDAC inhibitors that could be overcome with rational combination therapies. This requires knowledge of resistance mechanism along with the involved genetic networks. One way to identify such genetic networks is the implementation of a CRISPR-based technology allowing transcriptional repression (CRISPRi) and activation (CRISPRa) at a genome-wide scale. We describe a simple approach to amplify and validate sgRNA libraries, generate a myeloid progenitor cell line expressing catalytically dead Cas9 (dCas9) fusion proteins with transcriptional effectors to repress or activate genetic regions of interest and demonstrate a complementary genome-wide HDACi resistance screening approach. Furthermore, we present bioinformatics tools for quality control and analysis of the sequencing data.

Preoperative environment enrichment preserved neuroligin 1 expression possibly via epigenetic regulation to reduce postoperative cognitive dysfunction in mice.

AimsPostoperative cognitive dysfunction (POCD) is a common and significant syndrome. Our previous studies have shown that surgery reduces dendritic arborization and spine density and that environment enrichment (EE) reduces POCD. Neuroligin 1 is a postsynaptic protein involved in the formation of postsynaptic protein complex. This study was designed to determine the role of neuroligin 1 in the protection of EE against POCD and the mechanisms for EE to affect neuroligin 1 expression.MethodsEight‐week‐old C57BL/6J male mice with or without EE for 3, 7, or 14 days had right carotid artery exposure under isoflurane anesthesia. An anti‐neuroligin 1 antibody at 1.5 µg/mouse was injected intracerebroventricularly at one and two weeks before the surgery. Mice were subjected to the Barnes maze and fear conditioning tests from one week after the surgery. Cerebral cortex and hippocampus were harvested after surgery.ResultsMice with surgery had poorer performance in the Barnes maze and fear conditioning tests than control mice. EE for 2 weeks, but not EE for 3 or 7 days, improved the performance of surgery mice in these tests. Surgery reduced neuroligin 1 in the hippocampus. Preoperative EE for 2 weeks attenuated this reduction. The anti‐neuroligin 1 antibody worsened the performance of mice with surgery plus EE in the Barnes maze and fear conditioning tests. Surgery increased histone deacetylase activity and decreased the acetylated histone in the hippocampus. EE attenuated these surgery effects.ConclusionOur results suggest that preoperative EE for 2 weeks reduces POCD. This effect may be mediated by preserving neuroligin 1 expression via attenuating surgery‐induced epigenetic dysregulation in the brain.

Class I HDAC modulates angiotensin II-induced fibroblast migration and mitochondrial overactivity.

Inhibition of histone deacetylases (HDACs) attenuates cardiac fibrosis. In this study, we evaluated whether the inhibition of class I HDACs can attenuate angiotensin II (ANG II)-induced fibrogenesis and mitochondrial malfunction through its effects on reactive oxygen species (ROS) and calcium dysregulation in human cardiac fibroblasts (CFs). Seahorse XF24 extracellular flux analyser, fluorescence staining, Western blotting, HDAC activity assays and Transwell migration assay were used to study mitochondrial respiration, adenosine triphosphate (ATP) production, mitochondrial calcium uptake and ROS, HDAC expression and activity and fibroblast activity in CFs without (control) or with ANG II (100nM) and/or MS-275 (HDAC class 1 inhibitor, 10μM) for 24h. ANG II increased HDAC activity without changing protein expression in CFs. Compared with controls, ANG II-treated CFs had greater migration activity, higher ATP production, maximal respiration and spare capacity with higher mitochondrial Ca2+ uptake and ROS generation, which was attenuated by the administration of MS-275. ANG II activated CFs by increasing mitochondrial calcium content and ATP production, which may be caused by increased HDAC activity. Inhibition of HDAC1 attenuated the effects of ANG II by reducing mitochondrial ROS generation and calcium overload. Modulating mitochondrial function by regulation of HDAC may be a novel strategy for controlling CF activity.

Sodium acetate-mediated inhibition of histone deacetylase alleviates hepatic lipid dysregulation and its accompanied injury in streptozotocin-nicotinamide-induced diabetic rats

ObjectiveHepatic lipid dysregulation with consequent lipotoxicity remains critical in the progression of non-alcoholic fatty liver disease, a rising prevalent complication of diabetes mellitus particularly type 2 diabetes. Diabetes-associated hepatic complications are among the leading causes of liver-related morbidity and mortality worldwide. Short chain fatty acids (SCFAs) have been demonstrated to regulate glycemic metabolism but its effect on diabetes-driven hepatic perturbation is unknown. This study is therefore designed to investigate the effect of SCFAs, acetate on diabetes-characterised hepatic lipotoxicity, and plausible involvement of histone deacetylase (HDAC) activity. MethodsAdult male Wistar rats (230−260 g) were allotted into groups (n = 6/group) namely: control (vehicle; p.o.), sodium acetate (SAT)-treated (200 mg/kg), diabetic with/without SAT groups. Diabetes was induced by intraperitoneal injection of streptozotocin 65 mg/kg after a dose of nicotinamide 110 mg/kg. ResultsData from diabetic animals showed increased fasting glycemia and insulinemia, decreased insulin sensitivity and body weight with increased relative hepatic mass. It also revealed increased hepatic lipid, serum/hepatic malondialdehyde, tissue necrosis factor-α, uric acid, aspartate transaminase, alanine aminotransferase and decreased glutathione content with elevated hepatic HDAC. Histologically, the hepatic tissue was characterised with disrupted architecture, inflammation of central vein and foci of periportal and sinusoidal cellular infiltration. However, these alterations were attenuated by sodium acetate. ConclusionThe study demonstrates that diabetes mellitus drives hepatic lipotoxicity, characterised with lipid accumulation, excessive lipid peroxidation, pro-inflammation, depleted glutathione content and accompanied by increased HDAC activity. Besides, the study suggests that acetate ameliorates diabetes-associated hepatic lipotoxicity through HDAC suppression and enhancement of insulin sensitivity.

Aza-PBHA, a potent histone deacetylase inhibitor, inhibits human gastric-cancer cell migration via PKCα-mediated AHR-HDAC interactions

Recently, histone deacetylase inhibitors (HDACi) have become widely used in anti-cancer treatment; however, due to acquired drug resistance and their relatively low specificity, they are largely ineffective against late-stage cancer. Thus, it is critical to elucidate the molecular mechanisms underlying these issues, so as to identify novel therapeutic targets to prevent late-stage cancer progression and resistance acquisition. The present study investigated the Aryl hydrocarbon receptor (AHR), that has been shown to mediate histone acetylation by regulating histone deacetylase (HDAC) activity during HDACi treatment in human gastric-cancer cell lines (i.e. AGS and NCI-N87 cells). The potent HDACi, Aza-PBHA, was thus shown to upregulate AHR expression in both AGS and NCI-N87 cell lines, and to increase histone acetylation levels by facilitating AHR/HDAC interactions. Conversely, AHR knockdown increased HDAC activity. Aza-PBHA also increased PKCα phosphorylation and membrane translocation; however, interestingly, PKCα inhibition reduced the Aza-PBHA-increased AHR and histone acetylation levels, and inhibited the formation of the AHR/HDAC complex, likely upregulating Aza-PBHA-inhibited cell migration. Thus, our results suggest that Aza-PBHA treatment increased AHR levels to suppress HDAC activity, and inhibited cell migration by activating PKCα activation. These findings support the use of drugs to control AHR-related epigenetic regulation as a promising potential method to prevent acquired resistance to cancer treatments.

SUN-151 TGF-BETA1 MODIFIES HISTONE ACETYLATION AND ACETYL-COENZYME A METABOLISM IN RENAL MYOFIBROBLASTS

Histone acetylation is an important modulator of gene expression in fibrosis. This study examined the effect of the pre-eminent fibrogenic cytokine TGF-beta1 on histone 3 (H3) acetylation and its regulatory kinetics in renal myofibroblasts. Primary cultures of fibroblasts propagated from rat kidneys 3-days after ureteric obstruction were treated with 1ng/ml recombinant TGF-beta1 or vehicle for 48 hours. Flow cytometry (FC) was used to compare expression of alpha smooth muscle actin (SMA) and relative enrichment of H3 marks, while whole transcriptome sequencing (RNA-seq), FC and kinetic assays were used to examine the role of enzymes regulating acetylation. The effect of TGF-beta1 on acetyl-CoA (substrate) availability and synthesis were studied kinetically. TGF-beta1 -induced myofibroblast activation (increased SMA expression) (p<0.001 vs control) was accompanied by a net decrease in total H3 acetylation (p<0.001 vs control), although changes in individual marks were variable. This was paralleled by a generalised reduction in histone acetyltransferases (HAT), and divergent changes in histone deacetylase (HDAC) enzymes at both transcript and protein levels. Globally this was manifest in a reduction in total HAT activity (p<0.001) and increase in HDAC activity (p<0.01). TGF-beta1 induced a shift in cellular metabolism from oxidative respiration to aerobic glycolysis resulting in reduced acetyl-CoA. The reduction in total H3 acetylation could be rescued by providing exogenous citrate (p=ns vs control), a source of acetyl-CoA, without ameliorating changes in HAT/HDAC activity. TGF-beta1 produces a metabolic reprogramming in renal fibroblasts, with less H3 acetylation through reduced acetyltransferase activity, increased deacetylation and changes in carbon availability. Our results suggest that acetyl-CoA availability predominates over HAT and HDAC activity as a key determinant of H3 acetylation in response to TGF-beta1

Blocking histone deacetylase activity as a novel target for epithelial barrier defects in patients with allergic rhinitis

A defective epithelial barrier is found in patients with allergic rhinitis (AR) and asthma; however, the underlying mechanisms remain poorly understood. Histone deacetylase (HDAC) activity has been identified as a crucial driver of allergic inflammation and tight junction dysfunction. We investigated whether HDAC activity has been altered in patients with AR and in a mouse model of house dust mite (HDM)-induced allergic asthma and whether it contributed to epithelial barrier dysfunction. Primary nasal epithelial cells of control subjects and patients with AR were cultured at the air-liquid interface to study transepithelial electrical resistance and paracellular flux of fluorescein isothiocyanate-dextran (4kDa) together with mRNA expression and immunofluorescence staining of tight junctions. Air-liquid interface cultures were stimulated with different concentrations of JNJ-26481585, a broad-spectrum HDAC inhibitor. Invivo the effect of JNJ-26481585 on mucosal permeability and tight junction function was evaluated in a mouse model of HDM-induced allergic airway inflammation. General HDAC activity was greater in nasal epithelial cells of patients with AR and correlated inversely with epithelial integrity. Treatment of nasal epithelial cells with JNJ-26481585 restored epithelial integrity by promoting tight junction expression and protein reorganization. HDM-sensitized mice were treated with JNJ-26481585 to demonstrate the invivo role of HDACs. Treated mice did not have allergic airway inflammation and had no bronchial hyperreactivity. Moreover, JNJ-26481585 treatment restored nasal mucosal function by promoting tight junction expression. Our findings identify increased HDAC activity as a potential tissue-injury mechanism responsible for dysregulated epithelial cell repair, leading to defective epithelial barriers in AR. Blocking HDAC activity is a promising novel target for therapeutic intervention in patients with airway diseases.

Linking aberrant chromatin features in chronic lymphocytic leukemia to transcription factor networks

In chronic lymphocytic leukemia (CLL), a diverse set of genetic mutations is embedded in a deregulated epigenetic landscape that drives cancerogenesis. To elucidate the role of aberrant chromatin features, we mapped DNA methylation, seven histone modifications, nucleosome positions, chromatin accessibility, binding of EBF1 and CTCF, as well as the transcriptome of B cells from CLL patients and healthy donors. A globally increased histone deacetylase activity was detected and half of the genome comprised transcriptionally downregulated partially DNA methylated domains demarcated by CTCF. CLL samples displayed a H3K4me3 redistribution and nucleosome gain at promoters as well as changes of enhancer activity and enhancer linkage to target genes. A DNA binding motif analysis identified transcription factors that gained or lost binding in CLL at sites with aberrant chromatin features. These findings were integrated into a gene regulatory enhancer containing network enriched for B‐cell receptor signaling pathway components. Our study predicts novel molecular links to targets of CLL therapies and provides a valuable resource for further studies on the epigenetic contribution to the disease.

Carbocisteine Improves Histone Deacetylase 2 Deacetylation Activity via Regulating Sumoylation of Histone Deacetylase 2 in Human Tracheobronchial Epithelial Cells.

Histone deacetylase (HDAC) 2 plays a vital role in modifying histones to mediate inflammatory responses, while HDAC2 itself is commonly regulated by post-translational modifications. Small ubiquitin-related modifier (SUMO), as an important PTM factor, is involved in the regulation of multiple protein functions. Our previous studies have shown that carbocisteine (S-CMC) reversed cigarette smoke extract (CSE)-induced down-regulation of HDAC2 expression/activity in a thiol/GSH-dependent manner and enhanced sensitivity of steroid therapy. However, the mechanism by which S-CMC regulates HDAC2 is worth further exploring. Our study aimed to investigate the relationships between HDAC2 sumoylation and its deacetylase activity under oxidative stress and the molecular mechanism of S-CMC to regulate HDAC2 activity that mediates inflammatory responses in human bronchial epithelial cells. We found that modification of HDAC2 by SUMO1 and SUMO2/3 occurred in 16HBE cells under physiological conditions, and CSE induced SUMO1 modification of HDAC2 in a dose and time-dependent manner. K462 and K51 of HDAC2 were the two major modification sites of SUMO1, and the K51 site mediated deacetylation activity and function of HDAC2 on histone H4 that regulates IL-8 secretion. S-CMC inhibited CSE-induced SUMO1 modification of HDAC2 in the presence of thiol/GSH, increased HDAC activity, and decreased IL-8 expression. Our study may provide novel mechanistic explanation of S-CMC to ameliorate steroid sensitivity treatment in chronic obstructive pulmonary disease.

The neuroprotective action of 3,3\u2032-diindolylmethane against ischemia involves an inhibition of apoptosis and autophagy that depends on HDAC and AhR/CYP1A1 but not ER\u03b1/CYP19A1 signaling

There are no studies examining the effects of 3,3′-diindolylmethane (DIM) in neuronal cells subjected to ischemia. Little is also known about the roles of apoptosis and autophagy as well as AhR and ERα signaling and HDACs in DIM action. We demonstrated for the first time the strong neuroprotective capacity of DIM in mouse primary hippocampal cell cultures exposed to ischemia at early and later stages of neuronal development. The protective effects of DIM were mediated via inhibition of ischemia-induced apoptosis and autophagy that was accompanied by a decrease in AhR/CYP1A1 signaling and an increase in HDAC activity. DIM decreased the levels of pro-apoptotic factors, i.e., Fas, Caspase-3, and p38 mitogen-activated protein kinase (MAPK). DIM also reduced the protein levels of autophagy-related Beclin-1 (BECN1) and microtubule-associated proteins 1A/1B light chain (LC3), partially reversed the ischemia-induced decrease in Nucleoporin 62 (NUP62) and inhibited autophagosome formation. In addition, DIM completely reversed the ischemia-induced decrease in histone deacetylase (HDAC) activity in hippocampal neurons. Although DIM inhibited AhR/CYP1A1 signaling, it did not influence the protein expression levels of ERα and ERα-regulated CYP19A1 which are known to be controlled by AhR. This study demonstrated for the first time, that the neuroprotective action of 3,3′-diindolylmethane against ischemia involves an inhibition of apoptosis and autophagy and depends on AhR/CYP1A1 signaling and HDAC activity, thus creating the possibility of developing new therapeutic strategies that target neuronal degeneration at specific molecular levels.

TGF-β1 modifies histone acetylation and acetyl-coenzyme A metabolism in renal myofibroblasts.

Histone acetylation is an important modulator of gene expression in fibrosis. This study examined the effect of the pre-eminent fibrogenic cytokine TGF-b1 on histone 3 (H3) acetylation and its regulatory kinetics in renal myofibroblasts. Fibroblasts propagated from rat kidneys after ureteric obstruction were treated with recombinant TGF-b1 or vehicle for 48 hours. TGF-b1 -induced myofibroblast activation was accompanied by a net decrease in total H3 acetylation, although changes in individual marks were variable. This was paralleled by a generalised reduction in histone acetyltransferases (HAT), and divergent changes in histone deacetylase (HDAC) enzymes at both transcript and protein levels. Globally this was manifest in a reduction in total HAT activity and increase in HDAC activity. TGF-b1 induced a shift in cellular metabolism from oxidative respiration to aerobic glycolysis resulting in reduced acetyl-CoA. The reduction in total H3 acetylation could be rescued by providing exogenous citrate or alternative sources of acetyl-CoA, without ameliorating changes in HAT/HDAC activity. In conclusion, TGF-b1 produces a metabolic reprogramming in renal fibroblasts, with less H3 acetylation through reduced acetylation, increased deacetylation and changes in carbon availability. Our results suggest that acetyl-CoA availability predominates over HAT and HDAC activity as a key determinant of H3 acetylation in response to TGF-b1.

Exercise and low-level GABAA receptor inhibition modulate locomotor activity and the expression of BDNF accompanied by changes in epigenetic regulation in the hippocampus

Aerobic exercise is known to increase expression of brain-derived neurotrophic factor (BDNF) in the hippocampus and to improve cognitive function. The inhibition of GABAergic synapses enhances hippocampal plasticity as well as learning and memory. The objective of the present study was to examine the interactive effect of low-level GABAA receptor inhibition and exercise on behavior tests (cognitive function and locomotor activity), expression of BDNF and epigenetic regulations including the activity levels of histone acetyltransferases (HATs) and histone deacetylases (HDACs) in the hippocampus.ICR mice were divided into two groups: those who did not participate in exercise and those who participated in exercise. Each group was subdivided into two other groups: the one who received vehicle and the one who received GABAA receptor antagonist, bicucullin. We administered saline or bicuculline intraperitoneally to the mice at a non-epileptic dose of 0.25 mg/kg, whereas the mice were exercised on a treadmill for approximately 1 h a day, 5 days a week for 4 weeks. Novel-object recognition test and locomotor activity were assessed at a rest day approximately 4 days before the euthanasia. The mice were euthanized 4 h after the last exercise session.Aerobic exercise for 4 weeks increased mRNA and protein expression of BDNF in the hippocampus, accompanied by enhanced HAT activity. Alternatively, bicuculline administration increased HDAC activity in the hippocampus. Furthermore, exercise in the presence of bicuculline administration increased locomotor activity, indicating that exercise combined with low-level GABAA receptor inhibition potentiated the activity of the mice. Altogether, the present study suggested that exercise beneficially contributes to neuroprotection in the hippocampus accompanied by the up-regulation of BDNF expression and epigenetic regulation, whereas the chronic inhibition of GABAA receptor potentiates exercise-induced behavioral activity.

In Vivo 6-([18F]Fluoroacetamido)-1-hexanoicanilide PET Imaging of Altered Histone Deacetylase Activity in Chemotherapy-Induced Neurotoxicity.

Background Histone deacetylases (HDACs) regulate gene expression by changing histone deacetylation status. Neurotoxicity is one of the major side effects of cisplatin, which reacts with deoxyribonucleic acid (DNA) and has excellent antitumor effects. Suberoylanilide hydroxamic acid (SAHA) is an HDAC inhibitor with neuroprotective effects against cisplatin-induced neurotoxicity. Purpose We investigated how cisplatin with and without SAHA pretreatment affects HDAC expression/activity in the brain by using 6-([18F]fluoroacetamido)-1-hexanoicanilide ([18F]FAHA) as a positron emission tomography (PET) imaging agent for HDAC IIa. Materials and Methods [18F]FAHA and [18F]fluoro-2-deoxy-2-D-glucose ([18F]FDG) PET studies were done in 24 mice on 2 consecutive days and again 1 week later. The mice were divided into three groups according to drug administration between the first and second imaging sessions (Group A: cisplatin 2 mg/kg, twice; Group B: cisplatin 4 mg/kg, twice; Group C: cisplatin 4 mg/kg, twice, and SAHA 300 mg/kg pretreatment, 4 times). Results The Ki value of [18F]FAHA was increased and the percentage of injected dose/tissue g (% ID/g) of [18F]FDG was decreased in the brains of animals in Groups A and B. The Ki value of [18F]FAHA and % ID/g of [18F]FDG were not significantly different in Group C. Conclusions [18F]FAHA PET clearly showed increased HDAC activity suggestive of cisplatin neurotoxicity in vivo, which was blocked by SAHA pretreatment.

Abstract P161: Inhibition of Class I HDAC Enhances the Expression of Npr1 and Repairs Renal Pathology in Gene-targeted Haplotype Mice

The objective of the present study was to determine the effect of class I histone deacetylase (HDAC) inhibitor, mocetinostat (MGCD0103) on the expression of Npr1 (coding for natriuretic peptide receptor-A; NPRA) and renal pathology in Npr1 gene-disrupted haplotype (50% of normal) mice. Adult male Npr1 haplotype (1-copy; Npr1 +/- ), wild-type (2-copy; Npr1 +/+ ), and gene-duplicated (3-copy; Npr1 ++/+ ) mice were injected intraperitonealy with MGCD0103 (2 mg/kg) at alternate days for 2-weeks. After MGCD0103 treatment, the renal Npr1 mRNA was increased in Npr1 +/- mice by 7.3-fold (p &lt; 0.01), Npr1 +/+ mice by 4.4-fold (p &lt; 0.05) and Npr1 ++/+ mice by 3.6-fold (p &lt; 0.05) compared with vehicle-treated controls. The MGCD0103 also enhanced renal cGMP levels (pmol/mg protein) in Npr1 +/- (57 ± 7 vs. control, 12 ± 1; p &lt; 0.01), Npr1 +/+ (125 ± 9 vs. control, 66 ± 5; p &lt; 0.01), and Npr1 ++/+ (202 ± 7 vs. control, 127 ± 15; p &lt; 0.05) mice. An increased HDAC activity (ng/min/mg protein) was observed in Npr1 +/- mice (24.4 ± 2.8; p &lt; 0.05); however, Npr1 ++/+ mice showed lower HDAC activity (6.4 ± 0.7; p &lt; 0.05); compared with Npr1 +/+ mice (15.9 ± 1.3). Treatment with MGCD0103 significantly attenuated HDAC activity by almost 50% (15.3 ± 1.2; p &lt; 0.01) in Npr1 haplotype mice. A significant decrease in systolic blood pressure was observed in MGCD0103-treated Npr1 +/- mice (106 ± 0.6 vs. control, 131 ± 1.9, p &lt; 0.001). Significantly lower creatinine clearance (μl/min) was observed in Npr1 +/- (51 ± 11; p &lt; 0.05) vs. Npr1 +/+ mice (130 ± 14.0; p &lt; 0.05) and treatment with MGCD0103 increased creatinine clearance in Npr1 +/- mice (105 ± 13; p &lt; 0.05) vs. controls. Higher urinary albumin to creatinine ratio was detected in Npr1 +/- mice (0.7 ± 0.05) vs. Npr1 +/+ animals (0.5 ± 0.05; p &lt; 0.05) and a complete reversal was observed in drug-treated Npr1 +/- mice (0.4 ± 0.04). The present results provide direct evidence that class I HDAC inhibition upregulates NPRA expression in vivo and repairs the renal pathology in Npr1 +/- haplotype mice. The present findings will have important implications in the treatment and prevention of hypertension and renal pathophysiological conditions.

Regulation of Nitrogen Mustard-Induced Lung Macrophage Activation by Valproic Acid, a Histone Deacetylase Inhibitor.

Nitrogen mustard (NM)-induced lung injury is associated with an accumulation of proinflammatory/cytotoxic M1 and antiinflammatory/wound repair M2 macrophages, which have been implicated in tissue injury and repair. Herein, we analyzed the effects of valproic acid (VPA), a histone deacetylase (HDAC) inhibitor with antiinflammatory and antioxidant activity, on lung macrophages responding to NM. Treatment of rats with NM (0.125 mg/kg, i.t.) resulted in structural alterations in the lung and a macrophage-rich inflammatory cell infiltrate, at 3 d and 7 d. This was accompanied by expression of PCNA, a marker of proliferation, and CYPb5, HO-1, and MnSOD, markers of oxidative stress. Administration of VPA (300 mg/kg/day; i.p.), beginning 30 min after NM, reduced increases in PCNA, CYPb5, HO-1, and MnSOD. This was associated with increases in immature CD11b+CD43+ M1 macrophages in the lung, and decreases in mature CD11b+CD43- M2 macrophages 3 d post NM, suggesting delayed maturation and phenotypic switching. VPA also attenuated NM-induced increases in lung iNOS+ and CCR2+ M1 macrophages, a response correlated with downregulation of NOS2, IL12B, PTGS2, MMP-9, and CCR2 expression. Conversely, numbers of CD68+, CD163+ , and ATR-1α+ M2 macrophages increased after VPA, along with the expression of IL10, ApoE, and ATR-1A. NM exposure resulted in increased HDAC activity and upregulation of HDAC2 and acetylated H3K9 in the lung. Whereas VPA blunted the effects of NM on HDAC2 expression, histone H3K9 acetylation increased. These data suggest that alterations in the balance between histone acetylases and deacetylases contribute to lung macrophage maturation and activation following NM exposure.