Inhibition of histone deacetylase on ventricular remodeling in infarcted rats

Histone deacetylase (HDAC) determines the acetylation status of histones and, thereby, controls the regulation of gene expression. HDAC inhibitors have been shown to inhibit cardiomyocyte growth in vitro and in vivo. We assessed whether HDAC inhibitors exert a beneficial effect on the remodeling heart in infarcted rats. At 24 h after ligation of the left anterior descending artery, male Wistar rats were randomized to vehicle, HDAC inhibitors [valproic acid (VPA) and tributyrin], an agonist of HDAC (theophylline), VPA + theophylline, or tributyrin + theophylline for 4 wk. Significant ventricular hypertrophy was detected as increased myocyte size at the border zone isolated by enzymatic dissociation after infarction. Cardiomyocyte hypertrophy and collagen formation at the remote region and border zone were significantly attenuated by VPA and tributyrin with a similar potency compared with that induced by the vehicle. Left ventricular shortening fraction was significantly higher in the VPA- and tributyrin-treated groups than in the vehicle-treated group. Increased synthesis of atrial natriuretic peptide mRNA after infarction was confirmed by RT-PCR, consistent with the results of immunohistochemistry and Western blot for acetyl histone H4. The beneficial effects of VPA and tributyrin were abolished by theophylline, implicating HDAC as the relevant target. Inhibition of HDAC by VPA or tributyrin can attenuate ventricular remodeling after infarction. This might provide a worthwhile therapeutic target.     

Ellagic acid inhibits adipocyte differentiation through coactivator-associated arginine methyltransferase 1-mediated chromatin modification

Chromatin remodeling is a key mechanism in adipocyte differentiation. However, it is unknown whether dietary polyphenols are epigenetic effectors for adiposity control. Ellagic acid (EA) is a naturally occurring polyphenol in numerous fruits and vegetables. Recently, EA-containing foods have been reported to reduce adiposity. In the present study, we sought to determine whether EA inhibits adipogenesis by modifying chromatin remodeling in human adipogenic stem cells (hASCs). qPCR microarray of chromatin modification enzymes revealed that 10 μmol/L of EA significantly inhibits histone deacetylase (HDAC)9 down-regulation. In addition, EA was associated with up-regulation of HDAC activity and a marked reduction of histone acetylation levels. However, chemical inhibition of HDAC activity or depletion of HDAC9 by siRNA were not sufficient to reverse the antiadipogenic effects of EA. Intriguingly, EA treatment was also associated with reduced histone 3 arginine 17 methylation levels (H3R17me2), implying the inhibitory role of EA in coactivator-associated arginine methyltransferase 1 (CARM)1 activity during adipogenesis. Boosting CARM1 activity by delivering cell-penetrating peptides of CARM1 not only recovered H3R17me2 but also restored adipogenesis evidenced by H3 acetylation at lysine 9, HDAC9 down-regulation, PPARγ expression and triglyceride accumulation. Taken together, our data suggest that reduced CARM1 activity by EA results in a decrease of H3R17me2 levels, which may interrupt consecutive histone remodeling steps for adipocyte differentiation including histone acetylation and HDAC9 dissociation from chromatin. Our work provides the mechanistic insights into how EA, a polyphenol ubiquitously found in fruits and vegetables, attenuates human adipocyte differentiation by altering chromatin remodeling.     

Protective Effects of Valproic Acid,a Histone Deacetylase Inhibitor,against Hyperoxic Lung Injury in a Neonatal Rat Model

ObjectiveHistone acetylation and deacetylation may play a role in the pathogenesis of inflammatory lung diseases. We evaluated the preventive effect of valproic acid (VPA), a histone deacetylase (HDAC) inhibitor, on neonatal hyperoxic lung injury.MethodsForty newborn rat pups were randomized in normoxia, normoxia+VPA, hyperoxia and hyperoxia+VPA groups. Pups in the normoxia and normoxia+VPA groups were kept in room air and received daily saline and VPA (30 mg/kg) injections, respectively, while those in hyperoxia and hyperoxia+VPA groups were exposed to 95% O₂ and received daily saline and VPA (30 mg/kg) injections for 10 days, respectively. Growth, histopathological, biochemical and molecular biological indicators of lung injury, apoptosis, inflammation, fibrosis and histone acetylation were evaluated.ResultsVPA treatment during hyperoxia significantly improved weight gain, histopathologic grade, radial alveolar count and lamellar body membrane protein expression, while it decreased number of TUNEL(+) cells and active Caspase-3 expression. Expressions of TGFβ3 and phospho-SMAD2 proteins and levels of tissue proinflammatory cytokines as well as lipid peroxidation biomarkers were reduced, while anti-oxidative enzyme activities were enhanced by VPA treatment. VPA administration also reduced HDAC activity while increasing acetylated H3 and H4 protein expressions.ConclusionsThe present study shows for the first time that VPA treatment ameliorates lung damage in a neonatal rat model of hyperoxic lung injury. The preventive effect of VPA involves HDAC inhibition.     

Combination of valproic acid and ATRA restores RARβ2 expression and induces differentiation in cervical cancer through the PI3K/Akt pathway

Epigenetic silencing of the tumor suppressor gene, RARβ2, through histone deacetylation has been established as an important process of cervical carcinogenesis. This pivotal role has led to the suggestion that a combination of retinoids selective for RARβ2 with histone deacetylase (HDAC) inhibitors may have therapeutic potential. Valproic acid (VPA), a HDAC inhibitor, has a critical role in the regulation of gene expression through histone acetylation and causes transformed cells to undergo growth arrest, differentiation, and apoptosis. Therefore, we hypothesized that the combination of VPA and ATRA could restore RARβ2 expression, thus resulting in enhanced anti-neoplastic activity in cervical cancer. Here, we show that VPA combined with ATRA led to hyperacetylation of histone H3 and a significant alteration of gene expression in cervical cancer cells, including RARβ2 gene expression, which was upregulated 50- to 90-fold. The combination therapy effectively inhibited the growth of cervical cancer cells more than the single agent treatment both in vitro and in vivo. The additive effects were associated with a significant upregulation of p21(CIP1) and p53 as well as a pronounced decrease in p-Stat3. Furthermore, the combined treatment led to cell cycle arrest predominantly at the G1 phase, and it preferentially induced cell differentiation rather than apoptosis in cervical cancer cells. The differentiation program was determined by the presence of E-cadherinmediated adhesion and activation of the PI3K/Akt pathway. Taken together, these results provide new insight into the mechanisms of enhanced antitumor activity of the HDAC inhibitor and ATRA regimen, thus offering a new therapeutic strategy for cervical cancer patients.     

Oxidative stress reduces histone deacetylase 2 activity and enhances IL-8 gene expression: role of tyrosine nitration

Oxidative stress is a characteristic of chronic inflammatory diseases. The reactive oxygen intermediate hydrogen peroxide (H(2)O(2)) is an important signaling molecule that modulates gene expression. We have demonstrated that H(2)O(2) significantly enhanced cytokine production in BEAS-2B cells, with a maximal effect at 4h. This did not result from enhanced NF-kappaB activation, but through decreased activity of histone deacetylase (HDAC)2. This results in increased inflammatory gene expression following acetylation of specific histone residues. Decreased HDAC2 activity was associated with tyrosine nitration status. Peroxynitrite and SIN-1, a peroxynitrite generator, were also able to reduce HDAC2 activity via tyrosine nitration. Our data suggest that oxidative stress contributes to worsening inflammation via reduction of HDAC2 activity through HDAC2 nitration. This novel mechanism of inflammation may be important in increasing the severity and chronicity of inflammatory diseases.     

Nuclear histone deacetylases are not required for global histone deacetylation during meiotic maturation in porcine oocytes

Histone acetylation plays an important role in the regulation of chromatin structure and gene function. In mammalian oocytes, histones H3 and H4 are highly acetylated during the germinal vesicle (GV) stage, and global histone deacetylation takes place via a histone deacetylase (HDAC)-dependent mechanism after GV breakdown (GVBD). The presence of HDACs in the GVs of mammalian oocytes in spite of the high acetylation states of nuclear histones indicates that the HDACs in the nucleus are inactive but become activated after GVBD. However, the fluctuation pattern, the localization of HDAC activity during meiotic maturation and, moreover, the responsibility of nuclear HDACs for global histone deacetylation are still unknown. Here, we demonstrated using porcine oocytes that total HDAC activity was maintained throughout meiotic maturation, and high HDAC activity was observed in both the nucleus and the cytoplasm at the GV stage. The experiments with valproic acid (VPA), a specific class I HDAC inhibitor, revealed that the HDACs in GVs were class I, and those in the cytoplasm were other than class I. Interestingly, VPA had no effect on global histone deacetylation after GVBD, indicating that nuclear HDACs were not required for global histone deacetylation. To confirm this possibility, we removed the nuclei from immature oocytes, injected somatic cell nuclei into the enucleated oocytes, and showed that injected somatic cell nuclei were dramatically deacetylated after nuclear envelope breakdown. These results revealed that nuclear contents, including class I HDACs, are not required for the global histone deacetylation during meiosis, and that cytoplasmic HDACs other than class I are responsible for this process.     

Characterization of valproic acid‐initiated homologous recombination

BACKGROUND : Valproic acid (VPA) is a frequently used antiepileptic agent and known teratogen. Previous research suggests that inhibition of histone deacetylases (HDACs) may play a role in VPA‐induced teratogenicity. We have also shown that VPA exposure leads to both an increase in reactive oxygen species (ROS) production and increased frequency of homologous recombination (HR). METHODS : In the present study, we evaluated the role of HDAC inhibition in VPA‐initiated HR to determine if HDAC inhibition could alter repair activity and/or cause DNA double‐strand breaks (DSBs), which would then initiate repair. Histone acetylation status was assessed to determine if VPA exposure led to HDAC inhibition in CHO 33 cells. RESULTS : Our results demonstrate that VPA (5 mM) exposure leads to increased acetylated histone H3 and H4 protein levels after 10 to 24 hr. Secondly, in our recombination assay where an artificial DNA DSB was induced in CHO 33 cells to assess repair activity, VPA exposure did not affect the repair activity of VPA‐initiated HR. Subsequently, to determine if VPA could increase susceptibility to DNA DSBs, the number of γ‐H2AX foci was assessed using immunocytochemistry and results revealed an increase in γ‐H2AX foci after 10‐ to 24‐hr exposure to VPA. CONCLUSIONS : Although we demonstrated the protective effect of polyethylene glycol‐catalase against VPA‐induced HR and the generation of intracellular ROS within 24 hr, we did not observed an increase in DNA oxidation. These studies suggest that HDAC inhibition and ROS signaling may play roles in DNA maintenance and cell‐cycle arrest in initiating DNA damage and repair. Birth Defects Res (Part B) 89:124–132, 2010. © 2010 Wiley‐Liss, Inc.     

Induction of osteogenic differentiation of human mesenchymal stem cells by histone deacetylase inhibitors

Valproic acid (VPA) has been used as an anticonvulsant agent for the treatment of epilepsy, as well as a mood stabilizer for the treatment of bipolar disorder, for several decades. The mechanism of action for these effects remains to be elucidated and is most likely multifactorial. Recently, VPA has been reported to inhibit histone deacetylase (HDAC) and HDAC has been reported to play roles in differentiation of mammalian cells. In this study, the effects of HDAC inhibitors on differentiation and proliferation of human adipose tissue-derived stromal cells (hADSC) and bone marrow stromal cells (hBMSC) were determined. VPA increased osteogenic differentiation in a dose dependent manner. The pretreatment of VPA before induction of differentiation also showed stimulatory effects on osteogenic differentiation of hMSC. Trichostatin A (TSA), another HDAC inhibitor, also increased osteogenic differentiation, whereas valpromide (VPM), a structural analog of VPA which does not possess HDAC inhibitory effects, did not show any effect on osteogenic differentiation on hADSC. RT-PCR and Real-time PCR analysis revealed that VPA treatment increased osterix, osteopontin, BMP-2, and Runx2 expression. The addition of noggin inhibited VPA-induced potentiation of osteogenic differentiation. VPA inhibited proliferation of hADSC and hBMSC. Our results suggest that VPA enhance osteogenic differentiation, probably due to inhibition of HDAC, and could be useful for in vivo bone engineering using hMSC.     

Acetate supplementation increases brain histone acetylation and inhibits histone deacetylase activity and expression

Acetate supplementation increases brain, heart, and liver acetyl-CoA levels and reduces lipopolysaccharide-induced neuroinflammation. Because intracellular acetyl-CoA can be used to alter histone acetylation-state, using Western blot analysis, we measured the temporal effect that acetate supplementation had on brain and liver histone acetylation following a single oral dose of glyceryl triacetate (6 g/kg). In parallel experiments, we measured the effect that acetate supplementation had on histone deacetylase (HDAC) and histone acetyltransferase (HAT) enzymic activities and the expression levels of HDAC class I and II enzymes using Western blot analysis. We found that acetate supplementation increased the acetylation-state of brain histone H4 at lysine 8 at 2 and 4 h, histone H4 at lysine 16 at 4 and 24 h, and histone H3 at lysine 9 at 4 h following treatment. No changes in other forms of brain or liver H3 and H4 acetylation-state were found at any post-treatment times measured. Enzymic HAT and HDAC assays on brain extracts showed that acetate supplementation had no effect on HAT activity, but significantly inhibited by 2-fold HDAC activity at 2 and 4 h post-treatment. Western blot analysis demonstrated that HDAC 2 levels were decreased at 4 h following treatment. Based on these results, we conclude that acetyl-CoA derived from acetate supplementation increases brain histone acetylation-state by reducing HDAC activity and expression.     

Histone Acetylation Regulation in Sleep Deprivation-Induced Spatial Memory Impairment

Sleep disorders negatively affect cognition and health. Recent evidence has indicated that chromatin remodeling via histone acetylation regulates cognitive function. This study aimed to investigate the possible roles of histone acetylation in sleep deprivation (SD)-induced cognitive impairment. Results of the Morris water maze test showed that 3 days of SD can cause spatial memory impairment in Wistar rats. SD can also decrease histone acetylation levels, increase histone deacetylase 2 (HDAC2) expression, and decrease histone acetyltransferase (CBP) expression. Furthermore, SD can reduce H3 and H4 acetylation levels in the promoters of the brain-derived neurotrophic factor (Bdnf) gene and thus significantly downregulate BDNF expression and impair the activity of key BDNF signaling pathways (pCaMKII, pErk2, and pCREB). However, treatment with the HDAC inhibitor trichostatin A attenuated all the negative effects induced by SD. Therefore, BDNF and its histone acetylation regulation may play important roles in SD-induced spatial memory impairment, whereas HDAC inhibition possibly confers protection against SD-induced impairment in spatial memory and hippocampal functions.     

The histone deacetylase inhibitor valproic acid selectively induces proteasomal degradation of HDAC2

Histone-modifying enzymes play essential roles in physiological and aberrant gene regulation. Since histone deacetylases (HDACs) are promising targets of cancer therapy, it is important to understand the mechanisms of HDAC regulation. Selective modulators of HDAC isoenzymes could serve as efficient and well-tolerated drugs. We show that HDAC2 undergoes basal turnover by the ubiquitin-proteasome pathway. Valproic acid (VPA), in addition to selectively inhibiting the catalytic activity of class I HDACs, induces proteasomal degradation of HDAC2, in contrast to other inhibitors such as trichostatin A (TSA). Basal and VPA-induced HDAC2 turnover critically depend on the E2 ubiquitin conjugase Ubc8 and the E3 ubiquitin ligase RLIM. Ubc8 gene expression is induced by both VPA and TSA, whereas only TSA simultaneously reduces RLIM protein levels and therefore fails to induce HDAC2 degradation. Thus, poly-ubiquitination and proteasomal degradation provide an isoenzyme-selective mechanism for downregulation of HDAC2.