Silencing of CREB Inhibits HDAC2/TLR4/NF-κB Cascade to Relieve Severe Acute Pancreatitis-Induced Myocardial Injury.

The purpose of the present study is to investigate the role of CREB in cardiomyocytes proliferation in regulation of HDAC2-dependent TLR4/NF-κB pathway in severe acute pancreatitis (SAP)-induced myocardial injury. The SAP rat model was developed by injecting sodium touracholate into SD rats and then infected with lentivirus vectors expressing sh-CREB in the presence/absence of LPS. The pathological alterations of rat pancreatic and cardiac tissues were observed by HE staining. TUNEL assay was used to study apoptosis of cardiomyocytes. Next, the loss- and gain-function assay was conducted in LPS-induced myocardial injury cardiomyocytes to define the roles of CREB, HDAC2, and TLR4 in cardiomyocyte proliferation, apoptosis, inflammation, and myocardial injury in vitro. ChIP assay was used to study the enrichment of CREB bound to HDAC2 promoter. RT-qPCR and Western blot analysis were used to detect the expressions of related mRNA and proteins in the NF-κB pathway, respectively. CREB was found to be overexpressed in both SAP tissues and cells. CREB directly bound to the promoter of HDAC2 and activated its expression. Overexpressed CREB or HDAC2 inhibited proliferation and promoted apoptosis of cardiomyocytes. Suppression of CREB inhibited the HDAC2/TLR4/NF-κB cascade to promote proliferation and inhibit apoptosis of cardiomyocytes. The in vitro results were validated in vivo experiments. Coherently, suppression of CREB can inhibit HDAC2/TLR4/NF-κB cascade to promote cardiomyocyte proliferation, thus ameliorating SAP-induced myocardial injury.

Endothelial-Specific Overexpression of Histone Deacetylase 2 Protects Mice against Endothelial Dysfunction and Atherosclerosis.

BACKGROUND/AIMS: We recently described a novel regulatory role for histone deacetylase 2 (HDAC2) in protecting endothelial cells from oxidized low-density lipoprotein (OxLDL)-induced injury. In this study, we examined the effects of endothelial-specific HDAC2 overexpression on endothelial-dependent vasorelaxation and atherogenesis in vivo. METHODS: Endothelial-specific HDAC2-overexpressing transgenic mice (HDAC2-Tg) were generated under control of the Tie2 promoter. An atherosclerosis model was produced by injecting HDAC2-Tg and wild-type (WT) mice with adeno-associated virus encoding a PCSK9 gain-of-function mutant under control of a liver-specific promoter and feeding them a high-fat diet for 12 weeks. Aortic stiffness in vivo was determined by measuring pulse wave velocity. Wire myography was used to measure endothelium dependent (acetylcholine) and independent (sodium nitroprusside) relaxation in isolated mice aortas. Atherosclerotic plaque burden in aortas was determined by Oil Red O staining and protein expression was determined by western blotting. RESULTS: At baseline, HDAC2-Tg mice had normal mean arterial blood pressure (MAP) and body weight, but pulse wave velocity (PWV), an inverse measure of vascular health and stiffness, was decreased, suggesting that their vessels were more compliant. Moreover, basal nitric oxide production was enhanced in the vessels of HDAC2-Tg mice as compared to that in WT controls, although no significant differences in acetylcholine (endothelial component)- or sodium nitroprusside (non-endothelial component)-mediated relaxation were observed. However, after exposure to OxLDL, aortas from HDAC2-Tg mice exhibited greater acetylcholine-induced relaxation than did those from WT mice. Thus, endothelial-specific vasodilator production was enhanced despite oxidative injury. Atherosclerosis induction in WT mice led to a significant increase in PWV, but in HDAC2-Tg mice, PWV and MAP remained unchanged. Further, aortic rings from HDAC2-Tg exhibited better endothelial-dependent vascular relaxation than did those from WT mice, but not when treated with nitric oxide synthase inhibitor L-NAME. Finally, plaque burden, determined by Oil red O staining, was significantly increased in WT, but not HDAC2-Tg mice, subjected to the atherogenic model. Deletion of endothelial HDAC2 led to impaired endothelial cell-dependent vascular relaxation and increased PWV, compared with those in littermate controls. CONCLUSION: HDAC2 protects against endothelial dysfunction and atherogenesis induced by oxidized lipids. Hence, overexpression or activation of HDAC2 represents a novel therapy for endothelial dysfunction and atherosclerosis. HDAC2-Tg mice provide an opportunity to determine the role of endothelial HDAC2 in vascular endothelial homeostasis.

NF-κB p65-dependent transcriptional regulation of histone deacetylase 2 contributes to the chronic constriction injury-induced neuropathic pain via the microRNA-183/TXNIP/NLRP3 axis.

BACKGROUND: Neuropathic pain is related to the sustained activation of neuroglial cells and the production of proinflammatory cytokines in the spinal dorsal horn. However, the clinical efficacy of currently available treatments is very limited. The transcription factor nuclear factor κB (NF-κB) is a ubiquitously expressed protein family and considered to be crucial in autoimmunity. Thus, our study aimed to examine the influence of NF-κB p65 in chronic constriction injury (CCI)-induced neuropathic pain as well as its underlying mechanism. METHODS: A rat model of neuropathic pain was established by CCI induction followed by isolation of microglial cells. The binding of NF-κB p65 to HDAC2, of miR-183 to TXNIP, and of TXNIP to NLRP3 was investigated. Expression of miR-183, NF-κB p65, HDAC2, TXNIP, and NLRP3 was determined with their functions in CCI rats and microglial cells analyzed by gain- and loss-of-function experiments. RESULTS: NF-κB p65 and HDAC2 were upregulated while miR-183 was downregulated in the dorsal horn of the CCI rat spinal cord. NF-κB p65 was bound to the HDAC2 promoter and then increased its expression. HDAC2 reduced miR-183 expression by deacetylation of histone H4. Additionally, miR-183 negatively regulated TXNIP. Mechanistically, NF-κB p65 downregulated the miR-183 expression via the upregulation of HDAC2 and further induced inflammatory response by activating the TXNIP-NLRP3 inflammasome axis, thus aggravating the neuropathic pain in CCI rats and microglial cells. CONCLUSION: These results revealed a novel transcriptional mechanism of interplay between NF-κB and HDAC2 focusing on neuropathic pain via the miR-183/TXNIP/NLRP3 axis.

L1CAM Beneficially Inhibits Histone Deacetylase 2 Expression under Conditions of Alzheimer's Disease.

BACKGROUND: Cognitive capacities in Alzheimer's Disease (AD) are impaired by an epigenetic blockade mediated by histone deacetylase 2 (HDAC2), which prevents the transcription of genes that are important for synaptic plasticity. OBJECTIVE: Investigation of the functional relationship between cell adhesion molecule L1 and HDAC2 in AD. METHODS: Cultures of dissociated cortical and hippocampal neurons from wild-type or L1-deficient mice were treated with Aß1-42 for 24 h. After removal of Aß1-42 cells were treated with the recombinant L1 extracellular domain (rL1) for 24 h followed by immunohistochemistry, western blotting, and reverse transcription PCR to evaluate the interaction between L1 and HDAC2. RESULTS: Aß and HDAC2 protein levels were increased in APPSWE/L1+/- mutant brains compared to APPSWE mutant brains. Administration of the recombinant extracellular domain of L1 to cultured cortical and hippocampal neurons reduced HDAC2 mRNA and protein levels. In parallel, reduced phosphorylation levels of glucocorticoid receptor 1 (GR1), which is implicated in regulating HDAC2 levels, was observed in response to L1 administration. Application of a glucocorticoid receptor inhibitor reduced Aß-induced GR1 phosphorylation and prevented the increase in HDAC2 levels. HDAC2 protein levels were increased in cultured cortical neurons from L1-deficient mice. This change could be reversed by the administration of the recombinant extracellular domain of L1. CONCLUSION: Our results suggest that some functionally interdependent activities of L1 and HDAC2 contribute to ameliorating the phenotype of AD by GR1 dephosphorylation, which leads to reduced HDAC2 expression. The combined findings encourage further investigations on the beneficial effects of L1 in the treatment of AD.

HDAC2 hyperexpression alters hippocampal neuronal transcription and microglial activity in neuroinflammation-induced cognitive dysfunction.

BACKGROUND: Inflammation can induce cognitive dysfunction in patients who undergo surgery. Previous studies have demonstrated that both acute peripheral inflammation and anaesthetic insults, especially isoflurane (ISO), are risk factors for memory impairment. Few studies are currently investigating the role of ISO under acute peri-inflammatory conditions, and it is difficult to predict whether ISO can aggravate inflammation-induced cognitive deficits. HDACs, which are essential for learning, participate in the deacetylation of lysine residues and the regulation of gene transcription. However, the cell-specific mechanism of HDACs in inflammation-induced cognitive impairment remains unknown. METHODS: Three-month-old C57BL/6 mice were treated with single versus combined exposure to LPS injected intraperitoneally (i.p.) to simulate acute abdominal inflammation and isoflurane to investigate the role of anaesthesia and acute peripheral inflammation in cognitive impairment. Behavioural tests, Western blotting, ELISA, immunofluorescence, qRT-PCR, and ChIP assays were performed to detect memory, the expressions of inflammatory cytokines, HDAC2, BDNF, c-Fos, acetyl-H3, microglial activity, Bdnf mRNA, c-fos mRNA, and Bdnf and c-fos transcription in the hippocampus. RESULTS: LPS, but not isoflurane, induced neuroinflammation-induced memory impairment and reduced histone acetylation by upregulating histone deacetylase 2 (HDAC2) in dorsal hippocampal CaMKII+ neurons. The hyperexpression of HDAC2 in neurons was mediated by the activation of microglia. The decreased level of histone acetylation suppressed the transcription of Bdnf and c-fos and the expressions of BDNF and c-Fos, which subsequently impaired memory. The adeno-associated virus ShHdac2, which suppresses Hdac2 after injection into the dorsal hippocampus, reversed microglial activation, hippocampal glutamatergic BDNF and c-Fos expressions, and memory deficits. CONCLUSIONS: Reversing HDAC2 in hippocampal CaMKII+ neurons exert a neuroprotective effect against neuroinflammation-induced memory deficits.

Klf4 Promotes Dentinogenesis and Odontoblastic Differentiation via Modulation of TGF-ß Signaling Pathway and Interaction With Histone Acetylation.

Transcription factors bind to cell-specific cis-regulatory elements, such as enhancers and promoters, to initiate much of the gene expression program of different biological process. Odontoblast differentiation is a necessary step for tooth formation and is also governed by a complex gene regulatory network. Our previous in vitro experiments showed that Krüppel-like factor 4 (KLF4) can promote odontoblastic differentiation of both mouse dental papillary cells (mDPCs) and human dental pulp cells; however, its mechanism remains unclear. We first used Wnt1-Cre; KLF4fx/fx (Klf4 cKO) mice to examine the role of KLF4 during odontoblast differentiation in vivo and demonstrated significantly impaired dentin mineralization and enlarged pulp/root canals. Additionally, combinatory analysis using RNA-seq and ATAC-seq revealed genomewide direct regulatory targets of KLF4 in mouse odontoblasts. We found that KLF4 can directly activate the TGF-ß signaling pathway at the beginning of odontoblast differentiation with Runx2 as a cofactor. Furthermore, we found that KLF4 can directly upregulate the expression levels of Dmp1 and Sp7, which are markers of odontoblastic differentiation, through binding to their promoters. Interestingly, as a transcription factor, KLF4 can also recruit histone acetylase as a regulatory companion to the downstream target genes to positively or negatively regulate transcription. To further investigate other regulatory companions of KLF4, we chose histone acetylase HDAC3 and P300. Immunoprecipitation demonstrated that KLF4 interacted with P300 and HDAC3. Next, ChIP analysis detected P300 and HDAC3 enrichment on the promoter region of KLF4 target genes Dmp1 and Sp7. HDAC3 mainly interacted with KLF4 on day 0 of odontoblastic induction, whereas P300 interacted on day 7 of induction. These temporal-specific interactions regulated Dmp1 and Sp7 transcription, thus regulating dentinogenesis. Taken together, these results demonstrated that KLF4 regulates Dmp1 and Sp7 transcription via the modulation of histone acetylation and is vital to dentinogenesis. © 2019 American Society for Bone and Mineral Research.

Theophylline inhibits cigarette smoke-induced inflammation in skeletal muscle by upregulating HDAC2 expression and decreasing NF-κB activation.

Inflammation is associated with skeletal muscle dysfunction and atrophy in patients with chronic obstructive pulmonary disease (COPD). Theophylline has an anti-inflammatory role in COPD. However, the effects of theophylline on inflammation in skeletal muscle in COPD have rarely been reported. The aims of this study were to explore whether theophylline has an anti-inflammatory effect on skeletal muscle in a mouse model of emphysema and to investigate the molecular mechanism underlying this effect. In mice, cigarette smoke (CS) exposure for 28 wk resulted in atrophy of the gastrocnemius muscle. Histone deacetylase 2 (HDAC2) and nuclear factor-κBp65 (NF-κBp65) mRNA and protein levels were significantly decreased and increased, respectively, in gastrocnemius muscle. This effect was revered by aminophylline. The exposure of murine skeletal muscle C2C12 cells to CS extract (CSE) significantly increased IL-8 and TNF-α levels as well as NF-κBp65 mRNA and protein levels and NF-κBp65 activity. This effect was reversed by theophylline. HDAC2 knockdown enhanced the activity of NF-κBp65 and increased IL-8 and TNF-α levels in C2C12 cells. CSE significantly increased the interaction of HDAC2 with NF-κBp65 in C2C12 cells. These data suggest that theophylline has an anti-inflammatory effect on skeletal muscle in a mouse model of emphysema by upregulating HDAC2 expression and decreasing NF-κBp65 activation.

Histone deacetylases mediate GABAA receptor expression, physiology, and behavioral maladaptations in rat models of alcohol dependence.

Alcohol use disorders are chronic debilitating diseases characterized by severe withdrawal symptoms that contribute to morbidity and relapse. GABAA receptor (GABAAR) adaptations have long been implicated in the chronic effects of alcohol and contribute to many withdrawal symptoms associated with alcohol dependence. In rodents, GABAAR hypofunction results from decreases in Gabra1 expression, although the underlying mechanism controlling Gabra1 expression after chronic ethanol exposure is still unknown. We found that chronic ethanol exposure using either ethanol gavage or two-bottle choice voluntary access paradigms decreased Gabra1 expression and increased Hdac2 and Hdac3 expression. Administration of the HDAC inhibitor trichostatin A (TSA) after chronic ethanol exposure prevents the decrease in Gabra1 expression and function as well as the increase in Hdac2 and Hdac3 expression in both the cortex and the medial prefrontal cortex (mPFC). Chronic ethanol exposure and withdrawal, but not acute ethanol exposure or acute withdrawal, cause a selective upregulation of HDAC2 and HDAC3 associated with the Gabra1 promoter that accompanies a decrease in H3 acetylation of the Gabra1 promoter and the reduction in GABAAR α1 subunit expression. TSA administration prevented each of these molecular events as well as behavioral manifestations of ethanol dependence, including tolerance to zolpidem-induced loss of righting reflex, reduced open-arm time in the elevated plus maze, reduced center-time and locomotor activity in the open-field assay, and TSA reduced voluntary ethanol consumption. The results show how chronic ethanol exposure regulates the highly prominent GABAAR α1 subunit by an epigenetic mechanism that represents a potential treatment modality for alcohol dependence.

Clinicopathological features and prediction values of HDAC1, HDAC2, HDAC3, and HDAC11 in classical Hodgkin lymphoma.

Histone deacetylases (HDACs) are involved in multiple physical and pathological processes in classical Hodgkin lymphoma (cHL). The prognostic value of HDACs in cHL patients has not been discussed. The aim of the current study is to investigate the HDAC1, HDAC2, HDAC3, and HDAC11 expressions, and to evaluate the correlation of HDAC1, HDAC2, HDAC3, and HDAC11 expressions with the survival rate in cHL patients. We retrospectively analyzed clinicopathological data of 28 patients who were diagnosed with cHL between August 2002 and March 2010. Immunohistochemistry was used to detect the expression of HDAC1, HDAC2, HDAC3, and HDAC11 in these patients. The results showed that HDAC1, HDAC3, and HDAC11 were expressed at a higher level in Hodgkin Reed-Sternberg cells, whereas HDAC2 was expressed at a lower level in Hodgkin Reed-Sternberg cells. The expression of HDAC2 had a relationship with pathological type (P=0.012). There was also a correlation between the expression of HDAC11 and the erythrocyte sedimentation rate (P=0.054). Other clinicopathological parameters had no significant correlation with the expression of HDAC1, HDAC2, HDAC3, and HDAC11 in terms of survival (P>0.05). The 10-year total survival rate by Cox multivariate analysis, after taking into account all clinical and pathologic factors, showed that bulky disease retained significance (P=0.028). Higher expression of HDAC1 predicted shorter progression-free survival and overall survival (OS) in cHL patients (P<0.05, in both cases), and higher expression of HDAC11 might be correlated with lower OS (P=0.05). The study showed that the expressions of HDAC2 and HDAC11 have a particular relationship with the pathologic subtype. Increased expression of HDAC1 was correlated negatively with progression-free survival and OS, and increased expression of HDAC11 had a borderline relationship with the OS rate in patients with cHL.

Histone Deacetylase 2 Inhibition Attenuates Downregulation of Hippocampal Plasticity Gene Expression during Aging.

The brain undergoes several anatomical, biochemical, and molecular changes during aging, which subsequently result in downregulation of synaptic plasticity genes and decline of memory. However, the regulation of these genes during aging is not clearly understood. Previously, we reported that the expression of histone deacetylase (HDAC)2 was upregulated in the hippocampus of old mice and negatively correlated with the decline in recognition memory. As HDAC2 regulates key synaptic plasticity neuronal immediate early genes (IEGs), we have examined their expression and epigenetic regulation. We noted that the expression of neuronal IEGs decreased both at mRNA and protein level in the hippocampus of old mice. To explore the underlying regulation, we analyzed the binding of HDAC2 and level of histone acetylation at the promoter of neuronal IEGs. While the binding of HDAC2 was higher, H3K9 and H3K14 acetylation level was lower at the promoter of these genes in old as compared to young and adult mice. Further, we inhibited HDAC2 non-specifically by sodium butyrate and specifically by antisense oligonucleotide to recover epigenetic modification, expression of neuronal IEGs, and memory in old mice. Inhibition of HDAC2 increased histone H3K9 and H3K14 acetylation level at the promoter of neuronal IEGs, their expression, and recognition memory in old mice as compared to control. Thus, inhibition of HDAC2 can be used as a therapeutic target to recover decline in memory due to aging and associated neurological disorders.

TRPV1 Regulates Stress Responses through HDAC2.

Stress causes changes in neurotransmission in the brain, thereby influencing stress-induced behaviors. However, it is unclear how neurotransmission systems orchestrate stress responses at the molecular and cellular levels. Transient receptor potential vanilloid 1 (TRPV1), a non-selective cation channel involved mainly in pain sensation, affects mood and neuroplasticity in the brain, where its role is poorly understood. Here, we show that Trpv1-deficient (Trpv1-/-) mice are more stress resilient than control mice after chronic unpredictable stress. We also found that glucocorticoid receptor (GR)-mediated histone deacetylase 2 (HDAC) 2 expression and activity are reduced in the Trpv1-/- mice and that HDAC2-regulated, cell-cycle- and neuroplasticity-related molecules are altered. Hippocampal knockdown of TRPV1 had similar effects, and its behavioral effects were blocked by HDAC2 overexpression. Collectively, our findings indicate that HDAC2 is a molecular link between TRPV1 activity and stress responses.

An Acute Methamphetamine Injection Downregulates the Expression of Several Histone Deacetylases (HDACs) in the Mouse Nucleus Accumbens: Potential Regulatory Role of HDAC2 Expression.

Methamphetamine (METH) administration alters gene expression in the nucleus accumbens (NAc). We recently demonstrated that an acute METH injection produced prolonged increases in the expression of immediate early genes in the NAc of HDAC2-deficient mice, suggesting that HDAC2 might be an important regulator of gene expression in the rodent brain. Here, we tested the possibility that HDAC2 deletion might also impact METH-induced changes in the expression of various HDAC classes in the NAc. Wild-type (WT) and HDAC2 knockout (KO) mice were given a METH (20 mg/kg) injection, and NAc tissue was collected at 1, 2, and 8 h post treatment. We found that METH decreased HDAC3, HDAC4, HDAC7, HDAC8, and HDAC11 mRNA expression but increased HDAC6 mRNA levels in the NAc of WT mice. In contrast, the METH injection increased HDAC3, HDAC4, HDAC7, HDAC8, and HDAC11 mRNA levels in HDAC2KO mice. These observations suggest that METH may induce large-scale transcriptional changes in the NAc by regulating the expression of several HDACs, in part, via HDAC2-dependent mechanisms since some of the HDACs showed differential responses between the two genotypes. Our findings further implicate HDACs as potential novel therapeutic targets for neurotoxic complications associated with the abuse of certain psychostimulants.

A Novel Role of Silibinin as a Putative Epigenetic Modulator in Human Prostate Carcinoma.

Silibinin, extracted from milk thistle (Silybum marianum L.), has exhibited considerable preclinical activity against prostate carcinoma. Its antitumor and chemopreventive activities have been associated with diverse effects on cell cycle, apoptosis, and receptor-dependent mitogenic signaling pathways. Here we hypothesized that silibinin's pleiotropic effects may reflect its interference with epigenetic mechanisms in human prostate cancer cells. More specifically, we have demonstrated that silibinin reduces gene expression levels of the Polycomb Repressive Complex 2 (PRC2) members Enhancer of Zeste Homolog 2 (EZH2), Suppressor of Zeste Homolog 12 (SUZ12), and Embryonic Ectoderm Development (EED) in DU145 and PC3 human prostate cancer cells, as evidenced by Real Time Polymerase Chain Reaction (RT-PCR). Furthermore immunoblot and immunofluorescence analysis revealed that silibinin-mediated reduction of EZH2 levels was accompanied by an increase in trimethylation of histone H3 on lysine (Κ)-27 residue (H3K27me3) levels and that such response was, in part, dependent on decreased expression levels of phosphorylated Akt (ser473) (pAkt) and phosphorylated EZH2 (ser21) (pEZH2). Additionally silibinin exerted other epigenetic effects involving an increase in total DNA methyltransferase (DNMT) activity while it decreased histone deacetylases 1-2 (HDACs1-2) expression levels. We conclude that silibinin induces epigenetic alterations in human prostate cancer cells, suggesting that subsequent disruptions of central processes in chromatin conformation may account for some of its diverse anticancer effects.

Neuronal gene repression in Niemann-Pick type C models is mediated by the c-Abl/HDAC2 signaling pathway.

BACKGROUND: Niemann-Pick type C (NPC) disease is a fatal neurodegenerative disorder characterized by the accumulation of free cholesterol in lysosomes. There are currently no effective FDA-approved treatments for NPC, although in the last years the inhibition of histone deacetylases (HDACs) has emerged as a potential treatment for this disease. However, the molecular mechanisms that deregulate HDAC activity in NPC disease are unknown. Previously our group had shown that the proapoptotic tyrosine kinase c-Abl signaling is activated in NPC neurons. Here, we demonstrate that c-Abl activity increases HDAC2 levels inducing neuronal gene repression of key synaptic genes in NPC models. RESULTS: Our data show that: i) HDAC2 levels and activity are increased in NPC neuronal models and in Npc1(-/-) mice; ii) inhibition of c-Abl or c-Abl deficiency prevents the increase of HDAC2 protein levels and activity in NPC neuronal models; iii) c-Abl inhibition decreases the levels of HDAC2 tyrosine phosphorylation; iv) treatment with methyl-ß-cyclodextrin and vitamin E decreases the activation of the c-Abl/HDAC2 pathway in NPC neurons; v) in vivo treatment with two c-Abl inhibitors prevents the increase of HDAC2 protein levels in the brain of Npc1(-/-) mice; and vi) c-Abl inhibition prevents HDAC2 recruitment to the promoter of neuronal genes, triggering an increase in their expression. CONCLUSION: Our data show the involvement of the c-Abl/HDAC2 signaling pathway in the regulation of neuronal gene expression in NPC neuronal models. Thus, inhibition of c-Abl could be a pharmacological target for preventing the deleterious effects of increased HDAC2 levels in NPC disease.

Histone Deacetylase 2 in Sudden Sensorineural Hearing Loss Patients in Response to Intratympanic Methylprednisolone Perfusion.

OBJECTIVE: To evaluate the expression of histone deacetylase 2 (HDAC2) in peripheral blood mononuclear cells (PBMCs) from patients with sudden sensorineural hearing loss (SSNHL) who were refractory to systemic glucocorticoid treatment and to identify the relationship between the level of HDAC2 and glucocorticoid insensitivity. STUDY DESIGN: Prospective clinical study. SETTING: This study was conducted in Nanjing Drum Tower Hospital, Nanjing University Medical School. SUBJECTS AND METHODS: PBMCs were collected from 42 refractory SSNHL patients. After a 10-day intratympanic methylprednisolone perfusion (IMP) and systemic Ginkgo biloba extract treatment, the SSNHL patients were divided into 2 groups according to their hearing recovery after IMP (IMP sensitive and insensitive). Real-time polymerase chain reaction and HDAC2 protein assays were used to detect the relative expression levels of HDAC2 in PBMCs. The HDAC2 mRNA expression and protein levels in PBMCs collected from 17 volunteers were used as normal HDAC2 reference levels. RESULTS: Compared with normal reference levels, HDAC2 protein levels were significantly reduced, while the HDAC2 mRNA expression was much higher in all refractory SSNHL patients before IMP. HDAC2 mRNA expression and HDAC2 protein levels were significantly elevated in the IMP-sensitive group, while no change was observed in the IMP-insensitive group after IMP plus systemic antioxidant treatment. CONCLUSIONS: Reduced HDAC2 protein levels may be 1 of the mechanistic underpinnings of corticosteroid insensitivity in refractory SSNHL patients. IMP can increase HDAC2 protein levels and the expression of HDAC2 mRNA in IMP-sensitive patients. HDAC2 protein levels might be regulated through posttranslational modifications.

Assessment of Tissue Level of Histone Deactylase-2 (HDAC-2) in Patients With Mycosis Fungoides.

BACKGROUND: Histone deactylases (HDAC) have a role in the pathogenesis of mycosis fungoides (MF) through their actions on different apoptosis pathways. OBJECTIVE: To assess the possible role played by HDAC-2 in MF by estimating the tissue expression of HDAC2 mRNA in different stages of MF. METHODS: This study included 28 MF patients and 30 controls. The HDAC-2 levels were detected by real-time polymerase chain reaction (PCR). Correlations of HDAC-2 levels with clinical presentation and different stages of MF were analyzed. RESULTS: Mean HDAC-2 level was significantly higher in patients (P < .001) than in controls. HDAC-2 highest mean value was significantly detected in patients with stage IIb, and the lowest mean value was detected in patients with stage Ia (P < .001). CONCLUSION: Up-regulation of tissue HDAC-2 in MF patients might develop a new approach in the understanding of the pathogenesis of MF. Histone deactylases are important targets for molecular cancer therapeutics.

Valproic acid (VPA) reduces sensorimotor gating deficits and HDAC2 overexpression in the MAM animal model of schizophrenia.

BACKGROUND: Evidence indicates that the disruption of epigenetic processes might play an important role in the development of schizophrenia symptoms. The present study investigated the role of histone acetylation in the development of sensorimotor gating deficits in a neurodevelopmental model of schizophrenia based on prenatal administration of methylazoxymethanol (MAM) at embryonic day 17. METHODS: Valproic acid (VPA), an inhibitor of class I histone deacetylases, was administered (250 mg/kg, twice a day for 7 consecutive days) in early adolescence (23rd-29th day) or early adulthood (63rd-69th day) to rats. The effect of VPA treatment on the sensorimotor gating deficits induced by prenatal MAM administration was analyzed in adult rats at postnatal day 70 (P70). In addition, the effects of VPA administration (at the same doses) on MAM-induced changes in the levels of histone H3 acetylation at lysine 9 (H3K9ac) and histone deacetylase 2 (HDAC2) in the medial prefrontal cortex (mPFC) were determined at P70 using Western blot. RESULTS: VPA administration in either adolescence or early adulthood prevented the sensorimotor gating deficits induced by MAM. However, VPA administration in early adolescence or early adulthood did not alter H3K9ac levels induced by MAM. In contrast, VPA administration in either adolescence or adulthood prevented the increase in HDAC2 level evoked by MAM. CONCLUSIONS: Prenatal MAM administration impaired histone acetylation in the mPFC, which might be involved in the development of some of the neurobehavioral deficits (i.e., sensorimotor gating deficits) associated with schizophrenia. Blockade of HDAC2 might prevent the disruption of sensorimotor gating in adulthood.

Aberrant expression and activity of histone deacetylases in sporadic idiopathic pulmonary fibrosis.

BACKGROUND: Activation and differentiation of fibroblasts into contractile protein-expressing myofibroblasts and their acquired apoptosis-resistant phenotype are critical factors towards the development of idiopathic pulmonary fibrosis (IPF), a fatal disease characterised by distorted pulmonary structure and excessive extracellular matrix (ECM) deposition. The molecular mechanisms underlying these processes in IPF remain incompletely understood. We investigated the possible implication of aberrant overexpression and activity of histone deacetylases (HDACs) in IPF. METHODS: We analysed lung tissues from patients with sporadic IPF (n=26) and non-diseased control lungs (n=16) for expression of class I and II HDACs. Primary IPF fibroblasts were treated with HDAC inhibitors (HDACi) LBH589 or valproic acid (VPA). RESULTS: Compared to control lungs, protein levels of class I (HDAC1, HDAC2, HDAC3, HDAC8) and class II HDACs (HDAC4, HDAC 5, HDAC 7, HDAC 9) were significantly elevated in IPF lungs. Using immunohistochemistry, strong induction of nearly all HDAC enzymes was observed in myofibroblasts of fibroblast foci and in abnormal bronchiolar basal cells at sites of aberrant re-epithelialisation in IPF lungs, but not in controls. Treatment of primary IPF fibroblasts with the pan-HDACi LBH589 resulted in significantly reduced expression of genes associated with ECM synthesis, proliferation and cell survival, as well as in suppression of HDAC7, and was paralleled by induction of endoplasmic reticulum stress and apoptosis. The profibrotic and apoptosis-resistant phenotype of IPF fibroblasts was also partly attenuated by the class I HDACi VPA. CONCLUSIONS: Aberrant overexpression of HDACs in basal cells of IPF lungs may contribute to the bronchiolisation process in this disease. Similarly, generation and apoptosis resistance of IPF fibroblasts are mediated by enhanced activity of HDAC enzymes. Therefore, pan-HDAC inhibition by LBH589 may present a novel therapeutic option for patients with IPF.

Loss of the deubiquitylase BAP1 alters class I histone deacetylase expression and sensitivity of mesothelioma cells to HDAC inhibitors.

Histone deacetylases are important targets for cancer therapeutics, but their regulation is poorly understood. Our data show coordinated transcription of HDAC1 and HDAC2 in lung cancer cell lines, but suggest HDAC2 protein expression is cell-context specific. Through an unbiased siRNA screen we found that BRCA1-associated protein 1 (BAP1) regulates their expression, with HDAC2 reduced and HDAC1 increased in BAP1 depleted cells. BAP1 loss-of-function is increasingly reported in cancers including thoracic malignancies, with frequent mutation in malignant pleural mesothelioma. Endogenous HDAC2 directly correlates with BAP1 across a panel of lung cancer cell lines, and is downregulated in mesothelioma cell lines with genetic BAP1 inactivation. We find that BAP1 regulates HDAC2 by increasing transcript abundance, rather than opposing its ubiquitylation. Importantly, although total cellular HDAC activity is unaffected by transient depletion of HDAC2 or of BAP1 due to HDAC1 compensation, this isoenzyme imbalance sensitizes MSTO-211H cells to HDAC inhibitors. However, other established mesothelioma cell lines with low endogenous HDAC2 have adapted to become more resistant to HDAC inhibition. Our work establishes a mechanism by which BAP1 loss alters sensitivity of cancer cells to HDAC inhibitors. Assessment of BAP1 and HDAC expression may ultimately help identify patients likely to respond to HDAC inhibitors.

Post-occlusion administration of sodium butyrate attenuates cognitive impairment in a rat model of chronic cerebral hypoperfusion.

Chronic cerebral hypoperfusion (CCH) has been commonly associated with Alzheimer's disease and other types of dementia, but therapies that can improve cerebral blood flow displayed little effect on impaired cognition. Epigenetic intervention with histone deacetylase inhibitors, such as sodium butyrate (SB), on the other hand has been shown to improve cognition in several animal models of dementia. To investigate the effect of SB on cognitive impairment induced by CCH in rats, adult male SD rats were given intraperitoneal injections of SB at a daily dose of 840mg/kg for 4weeks, from the 29th day after permanent occlusion of bilateral common carotid arteries (2VO). Learning and memory were assessed by Morris water maze and novel object recognition. Following behavioral tests, western blotting of histone acetylation, of transcription factors, of neuronal/synaptic proteins, were performed using rat hippocampus and cortex. The data showed that SB treatment alleviated hippocampal dependent spatial learning disability in 2VO rats, and altered HDAC1/2 mRNA level, histone H4 acetylation and Nrf2 transcriptional activation in rat hippocampus. Accordingly, cognition-protective effect of SB appeared to be partially mediated by enhancing histone acetylation and hence by facilitating the transcription of Nrf2 downstream genes in the hippocampus. Thus, SB might be considered for putative treatment for CCH-related cognitive impairment.