trichostatin-a and Inflammation

Diverse cellular functions including the regulation of inflammatory gene expression, DNA repair and cell proliferation are regulated by epigenetic changes. Transcriptional co-activators possess intrinsic histone acetyltransferase (HAT) activity, and histone acetylation plays a major role in inflammatory gene expression. Other marks such as histone methylation are also associated with gene induction and gene repression. Recent evidence implicates histone acetylation and methylation as being crucial for the development of tolerance in macrophages and CpG methylation for T regulatory cell development and function. The expression of the enzymes that lay down or remove these epigenetic marks have not been well studied in human airways disease, but reduced HDAC2 expression and activity is reported in lung macrophages, biopsies and blood cells from patients with COPD, severe asthma and smoking asthma. In vitro, inhibitors of histone deacetylases (HDAC) often lead to a further induction of inflammatory gene expression. This is not always the case, however, as HATs and HDACs also target non-histone proteins particularly transcription factors to alter their activity. Furthermore, trichostatin A, an HDAC inhibitor, can reduce inflammation in a murine model of allergic asthma. This effect of HDAC inhibitors may be due to their effects on cell death acting through acetylation of non-histone proteins. The role of epigenetic modifications in inflammatory gene expression and in the control of cell function in the airways is becoming clearer. Targeting specific enzymes involved in this process may lead to new therapeutic agents, in particular, in situations where current anti-inflammatory therapies are currently suboptimal.

Topics: Acetylation; Animals; Asthma; DNA Methylation; Enzyme Inhibitors; Epigenesis, Genetic; Gene Expression Regulation; Histone Acetyltransferases; Histone Deacetylase Inhibitors; Histone Deacetylases; Histones; Humans; Hydroxamic Acids; Immune Tolerance; Inflammation; Pulmonary Disease, Chronic Obstructive; Transcriptional Activation

Eosinophils are one of the cells that play a critical role in the pathogenesis of allergic diseases. The increase in the number of eosinophils in such diseases is regulated by interleukin-5 (IL-5). The author have prepared recombinant rat IL-5 using a baculovirus expression system and examined its biological activities in rat eosinophils. It was demonstrated that recombinant rat IL-5 prolongs the survival of mature eosinophils and differentiates immature eosinophils into mature eosinophils, suggesting that rat IL-5 is a factor for eosinophilia in rats. Recombinant rat eosinophil-associated ribonuclease (Ear)-1 and Ear-2 were also prepared. Eosinophil granule proteins are thought to cause tissue damage due to their cytotoxic activity, but using recombinant rat Ear-1 and Ear-2, it was found that rat Ear-1 and Ear-2 have strong RNase A activity and bactericidal activity, suggesting that these proteins play critical roles in host defense. Finally, the important role of acetylation of histones was clarified in the differentiation of HL-60 clone 15 cells into eosinophils using the histone deacetylase inhibitors sodium n-butyrate, apicidin, and trichostatin A. These findings would be useful for further investigations of the role of eosinophils in allergic inflammation.

Topics: Acetylation; Animals; Butyrates; Cell Differentiation; Cell Division; Enzyme Inhibitors; Eosinophil Cationic Protein; Eosinophil Granule Proteins; Eosinophils; Histone Deacetylase Inhibitors; Histones; HL-60 Cells; Humans; Hydroxamic Acids; Hypersensitivity; Inflammation; Interleukin-5; Peptides, Cyclic; Rats; Recombinant Proteins

Chlamydia pneumoniae (Cpn) is a gram-negative intracellular pathogen that causes a variety of pulmonary diseases, and there is growing evidence that it may play a role in Alzheimer's disease (AD) pathogenesis. Cpn can interact functionally with host histones, altering the host's epigenetic regulatory system by introducing bacterial products into the host tissue and inducing a persistent inflammatory response. Because Cpn is difficult to propagate, isolate, and detect, a modified LPS-like neuroinflammation model was established using lyophilized cell free supernatant (CFS) obtained from infected cell cultures, and the effects of CFS were compared to LPS. The neuroprotective effects of Trichostatin A (TSA), givinostat, and RG108, which are effective on epigenetic mechanisms, and the antibiotic rifampin, were studied in this newly introduced model and in the presence of amyloid beta (Aβ) 1-42. The neuroprotective effects of the drugs, as well as the effects of CFS and LPS, were evaluated in Aβ-induced neurotoxicity using a real-time cell analysis system, total ROS, and apoptotic impact. TSA, RG108, givinostat, and rifampin all demonstrated neuroprotective effects in both this novel model and Aβ-induced neurotoxicity. The findings are expected to provide early evidence on neuroprotective actions against Cpn-induced neuroinflammation and Aβ-induced neurotoxicity, which could represent a new treatment option for AD, for which there are currently few treatment options.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Carbamates; Chlamydophila Infections; Chlamydophila pneumoniae; Epigenesis, Genetic; Humans; Hydroxamic Acids; Inflammation; Neuroprotective Agents; Peptide Fragments; Phthalimides; THP-1 Cells; Tryptophan

Pre-exposure to volatile anesthetics inhibits inflammation induced by various stimuli, including surgical procedures and ischemia. We hypothesize that volatile anesthetics may induce anti-inflammatory effects via a mechanism involving regulation of histone deacetylases (HDACs). Pre-exposure of 1.5% isoflurane for 0.5 h induced anti-inflammatory effects [measured by cytokine production of tumor necrosis factor-ɑ, interleukin-8 (IL-8) and IL-1β] in both human THP-1 cells and primary human peripheral blood monocytes stimulated by lipopolysaccharide. In human THP-1 cells, coadministration of the HDAC inhibitor trichostatin A (TSA) blocked the isoflurane-induced anti-inflammatory effects. TSA also blocked isoflurane-upregulated HDAC1-3 expression and isoflurane-reduced nuclear translocation of p65 and p50 subunits of nuclear factor-κB (NF-κB). The ability of isoflurane to reduce NF-κB nuclear translocation and proinflammatory responses in the cell line was blocked by gene silencing of HDAC1 and HDAC2, but not by gene silencing of HDAC3. A coimmunoprecipitation assay demonstrated that the decreased interaction between HDAC1 and HDAC2 through lipopolysaccharide was restored by isoflurane pretreatment. These findings were validated in primary human peripheral blood monocytes  wherein gene silencing of HDAC1 and HDAC2 resulted in increased cytokine production and NF-κB nuclear translocation induced by isoflurane pre-exposure and lipopolysaccharide stimulation. These results indicate that anti-inflammatory effects of the volatile anesthetic isoflurane in human monocytes involve regulation of HDAC1 and HDAC2.

Topics: Anesthetics, Inhalation; Cell Line; Gene Silencing; Histone Deacetylase 1; Histone Deacetylase 2; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Hydroxamic Acids; Inflammation; Interleukin-1beta; Interleukin-8; Isoflurane; Lipopolysaccharides; Monocytes; NF-kappa B; Tumor Necrosis Factor-alpha

Excessive neuroinflammatory responses play important roles in the development of postoperative cognitive dysfunction (POCD). Neurofilaments (NFs) were essential to the structure of axon and nerve conduction; and the abnormal degradation of NFs were always accompanied with degenerative diseases, which were also characterized by excessive neuroinflammatory responses in brain. However, it is still unclear whether the NFs were involved in the POCD. In this study, the LC-MS/MS method was used to explore the neuroinflammatory response and NFs of POCD in aged rats. Moreover, trichostatin A (TSA), an inflammation-related drug, was selected to test whether it could improve the surgery-induced cognitive dysfunction, inflammatory responses and NFs. Evident cognitive dysfunction, excessive microglia activation, neuroinflammatory responses and upregulated NFs in hippocampus were observed in the POCD group. TSA pretreatment could significantly mitigate these changes. The KEGG analysis revealed that nine pathways were enriched in the TSA + surgery group (versus the surgery group). Among them, two signaling pathways were closely related with the changes of NFs proteins. In conclusion, surgery could impair the cognitive function and aggravate neuroinflammation and NFs. The TSA could significantly improve these changes which might be related to the activation of the "focal adhesion" and "ECM-receptor interaction" pathways.

Topics: Animals; Cognitive Dysfunction; Hippocampus; Hydroxamic Acids; Inflammation; Intermediate Filaments; Laparotomy; Microglia; Postoperative Complications; Rats

Class I and II histone deacetylases (HDAC) are considered important regulators of immunity and inflammation. Modulation of HDAC expression and activity is associated with altered inflammatory responses but reports are controversial and the specific impact of single HDACs is not clear. We examined class I and II HDACs in TLR-4 signaling pathways in murine macrophages with a focus on IκB kinase epsilon (IKKε) which has not been investigated in this context before. Therefore, we applied the pan-HDAC inhibitors (HDACi) trichostatin A (TSA) and suberoylanilide hydroxamic acid (SAHA) as well as HDAC-specific siRNA. Administration of HDACi reduced HDAC activity and decreased expression of IKKε although its acetylation was increased. Other pro-inflammatory genes (IL-1β, iNOS, TNFα) also decreased while COX-2 expression increased. HDAC 2, 3 and 4, respectively, might be involved in IKKε and iNOS downregulation with potential participation of NF-κB transcription factor inhibition. Suppression of HDAC 1-3, activation of NF-κB and RNA stabilization mechanisms might contribute to increased COX-2 expression. In conclusion, our results indicate that TSA and SAHA exert a number of histone- and HDAC-independent functions. Furthermore, the data show that different HDAC enzymes fulfill different functions in macrophages and might lead to both pro- and anti-inflammatory effects which have to be considered in therapeutic approaches.

Topics: Animals; Cyclooxygenase 2; Gene Expression Regulation; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Hydroxamic Acids; I-kappa B Kinase; Inflammation; Interleukin-1beta; Mice; Nitric Oxide Synthase Type II; RNA, Small Interfering; Signal Transduction; Toll-Like Receptor 4; Tumor Necrosis Factor-alpha; Vorinostat

Sepsis is a syndrome of life-threatening organ dysfunction caused by dysregulated host responses to infection. Macrophage polarization is a key process involved in the pathogenesis of sepsis. Recent evidence has demonstrated that autophagy participates in the regulation of macrophage polarization in different phases of inflammation. Here, we investigated whether trichostatin A (TSA), a histone deacetylase (HDAC) inhibitor, promotes the macrophage M2 phenotype by enhancing autophagy to counteract excessive inflammation in a cecal ligation and puncture (CLP) mouse model. TSA stimulation increased the proportions of M2 marker (CD206, CD124 and CD23)-labeled RAW264.7 macrophages. Furthermore, with increasing TSA doses, autophagy was enhanced gradually. Interestingly, the autophagy activator rapamycin (Rap), also known as an mTOR inhibitor, unexpectedly decreased the proportions of M2 marker-labeled macrophages. However, TSA treatment reversed the Rap-induced decreases in CD206-labeled macrophages. Next, we stimulated different groups of RAW264.7 cells with the autophagy inhibitors MHY1485 or 3-methyladenine (3-MA). Inhibition of autophagy at any stage in the process suppressed TSA-induced macrophage M2 polarization, but the effect was not associated with mTOR activity. In vivo, TSA administration promoted peritoneal macrophage M2 polarization, increased LC3 II expression, attenuated sepsis-induced organ (lung, liver and kidney) injury, and altered systemic inflammatory cytokine secretion. However, 3-MA abolished the protective effects of TSA in CLP mice and decreased the number of M2 peritoneal macrophages. Therefore, TSA promotes the macrophage M2 phenotype by enhancing autophagy to reduce systemic inflammation and ultimately improves the survival of mice with polymicrobial sepsis.

Topics: Animals; Autophagy; Biomarkers; Cell Line; Cytokines; Disease Models, Animal; Histone Deacetylase Inhibitors; Hydroxamic Acids; Inflammation; Ligation; Lung; Macrophage Activation; Macrophages, Peritoneal; Male; Mice; Mice, Inbred C57BL; Phenotype; Punctures; RAW 264.7 Cells; Sepsis

Inflammation causes proliferation of intestinal smooth muscle cells (ISMC), contributing to a thickened intestinal wall and to stricture formation in Crohn's disease. Proliferation of ISMC in vitro and in vivo caused decreased expression of marker proteins, but the underlying cause is unclear. Since epigenetic change is important in other systems, we used immunocytochemistry, immunoblotting, and quantitative PCR to examine epigenetic modification in cell lines from rat colon at low passage or after extended growth to evaluate phenotype. Exposure to the histone deacetylase (HDAC) inhibitor trichostatin A or the DNA methyltransferase inhibitor 5-azacytidine reversed the characteristic loss of phenotypic markers among high-passage cell lines of ISMC. Expression of smooth muscle actin and smooth muscle protein 22, as well as functional expression of the neurotrophin glial cell line-derived neurotrophic factor, was markedly increased. Increased expression of muscarinic receptor 3 and myosin light chain kinase was correlated with an upregulated response to cholinergic stimulation. In human ISMC (hISMC) lines from the terminal ileum, phenotype was similarly affected by extended proliferation. However, in hISMC from resected Crohn's strictures, we observed a significantly reduced contractile phenotype compared with patient-matched intrinsic controls that was associated with increased patient-specific expression of DNA methyltransferase 1, HDAC2, and HDAC5. Therefore, protracted growth causes epigenetic alterations that account for an altered phenotype of ISMC. A similar process may promote stricture formation in Crohn's disease, where the potential for halting progression, or even reversal, of disease through control of phenotypic modulation may become a novel treatment option.

Topics: Actins; Animals; Azacitidine; Cell Proliferation; Crohn Disease; DNA (Cytosine-5-)-Methyltransferase 1; DNA Methylation; Epigenesis, Genetic; Gene Expression Regulation; Histone Deacetylase 2; Histone Deacetylases; Humans; Hydroxamic Acids; Ileum; Inflammation; Intestinal Mucosa; Intestines; Muscle Contraction; Myocytes, Smooth Muscle; Rats

RGS10 has emerged as a key regulator of proinflammatory cytokine production in microglia, functioning as an important neuroprotective factor. Although RGS10 is normally expressed in microglia at high levels, expression is silenced in vitro following activation of TLR4 receptor. Given the ability of RGS10 to regulate inflammatory signaling, dynamic regulation of RGS10 levels in microglia may be an important mechanism to tune inflammatory responses. The goals of the current study were to confirm that RGS10 is suppressed in an in vivo inflammatory model of microglial activation and to determine the mechanism for activation-dependent silencing of Rgs10 expression in microglia. We demonstrate that endogenous RGS10 is present in spinal cord microglia, and RGS10 protein levels are suppressed in the spinal cord in a nerve injury-induced neuropathic pain mouse model. We show that the histone deacetylase (HDAC) enzyme inhibitor trichostatin A blocks the ability of lipopolysaccharide (LPS) to suppress Rgs10 transcription in BV-2 and primary microglia, demonstrating that HDAC enzymes are required for LPS silencing of Rgs10 Furthermore, we used chromatin immunoprecipitation to demonstrate that H3 histones at the Rgs10 proximal promoter are deacetylated in BV-2 microglia following LPS activation, and HDAC1 association at the Rgs10 promoter is enhanced following LPS stimulation. Finally, we have shown that sphingosine 1-phosphate, an endogenous microglial signaling mediator that inhibits HDAC activity, enhances basal Rgs10 expression in BV-2 microglia, suggesting that Rgs10 expression is dynamically regulated in microglia in response to multiple signals.

Topics: Acetylation; Animals; Azacitidine; Cell Line; Chemokine CXCL2; Disease Models, Animal; Gene Silencing; Histone Deacetylase Inhibitors; Histone Deacetylases; Hydroxamic Acids; Inflammation; Lipopolysaccharides; Lysophospholipids; Methyltransferases; Mice, Inbred C57BL; Microglia; Promoter Regions, Genetic; Receptors, G-Protein-Coupled; RGS Proteins; RNA, Messenger; Signal Transduction; Sphingosine; Transcription, Genetic; Tumor Necrosis Factor-alpha

Histone deacetylase inhibitors (HDACi) are currently used in the routine clinical treatment of cancer. Alongside the antitumor activity of HDACi, increased attention has been paid to their anti‑inflammatory effects. The present study aimed to analyze the inhibitory effects of the HDACi Trichostatin A (TSA), on the release of inflammatory mediators from macrophages differentiated from U‑937 cells. A low dose of TSA (50 nM) was able to effectively decrease the levels of inflammatory cytokines in the cell supernatants, independent of apoptosis. In addition, the potential underlying mechanisms were explored, and TSA was shown to promote, rather than inhibit, the acetylation of histones. Furthermore, the inflammation‑induced enhanced expression of class I HDACs was effectively inhibited by TSA. In addition, TSA enhanced the lipopolysaccharide (LPS)‑induced expression of cyclooxygenase‑2, but suppressed the LPS‑induced expression of chemokine (C‑C motif) ligand 7. The acetylation level of nuclear factor‑κB p65 was decreased by LPS, but increased following treatment with TSA. In conclusion, TSA was able to inhibit inflammation in macrophages. However, whether the mechanism by which TSA inhibits inflammation is through significantly enhancing histone acetylation, in order to selectively suppress the expression of proinflammatory genes, and/or through regulating non‑histone acetylation requires further research.

Topics: Acetylation; Apoptosis; Cell Line, Tumor; Cell Survival; Cytokines; Dose-Response Relationship, Drug; Gene Expression Regulation; Histone Deacetylases; Histones; Humans; Hydroxamic Acids; Inflammation; Macrophages; Tetradecanoylphorbol Acetate; Transcription Factor RelA

T lymphocytes, which are characterized by longevity and immune memory, play an important role in airway inflammation in asthma. Here, we assessed the association between immune memory and histone deacetylation and/or acetylation status.. CD4 + CD45RB(low) cells (memory T (Tm)) obtained from the spleens of asthma mice models were co-cultured with glucocorticoids (GCs), trichostatin A (TSA) or anacardic acid (AA) and adoptively transferred to naïve mice. Interleukin (IL)-4, 5 and 13 and IFN-γ concentrations were measured in culture supernatants and bronchoalveolar lavage fluid (BALF). Histone deacetylase (HDAC) and histone acetyltransferase (HAT) activities and the expression of T-bet, GATA-3, HDACs 1-11 and alveolar eosinophilic inflammation index (AEII) were determined in lung tissues.. Culture supernatants and the BALF showed similar cytokine profiles. AA and GCs significantly inhibited HAT activity (P = 0.002 and P = 0.018), whereas TSA inhibited and GCs promoted HDAC activity (P = 0.004 and P = 0.025). HDACs 7, 9 and 10 were upregulated by AA and GCs (all P < 0.032), while HDAC11 was upregulated by GCs (P = 0.028). GC-induced inhibition of Tm histone acetylation alleviated AEII by downregulating IL-4, 5 and 13, similar to the effect of AA.. Histone hyperacetylation status induced by low expression of HDACs 7, 9 and 10 in allergen-specific Tm cells contributes to eosinophilic airway inflammation. The mechanism by which GCs improve airway inflammation involves the upregulation of HDACs 7, 9, 10 and 11 and especially HDAC-10. The role of individual HDACs and AA as novel therapeutic agents for allergic asthma needs to be explored in the future.

Topics: Acetylation; Allergens; Anacardic Acids; Animals; Asthma; Blotting, Western; Bronchoalveolar Lavage Fluid; Cell Culture Techniques; Cytokines; Disease Models, Animal; Eosinophils; Glucocorticoids; Histone Acetyltransferases; Histone Deacetylases; Histones; Hydroxamic Acids; Inflammation; Lung; Male; Mice; Mice, Inbred BALB C; T-Lymphocytes

To compare the expression of histone deacetylase(HDAC)1-11 of human periodontal ligament stem cells(PDLSC)in normal and inflammatory microenvironments, and to investigate the effect of histone deacetylase inhibitor trichostatin A(TSA)on the osteogenic differentiation potential of PDLSC in inflammatory microenvironment induced by tumor necrosis factor-α(TNF-α)stimulation.. PDLSC were isolated from periodontal ligament tissues obtained from the surgically extracted human teeth and cultured by single-colony selection. The expression of HDAC1-11 in cells with or without TNF-α(10 μg/L)stimulation was evaluated by quantitative real time-PCR(RT-PCR). The effect of TSA on cell proliferation was investigated by methyl thiazolyl tetrazolium(MTT)assay. The influence of TSA on osteogenic differentiation of PDLSC in inflammatory microenvironment with TNF-α stimulation was assessed by alizarin red staining, quantitative RT-PCR and Western blotting, respectively.. The expression of HDAC in PDLSC with TNF-α stimulation was significantly higher than that in normal PDLSC(P<0.05)(except HDAC7, P=0.243). TSA had no significant effect on PDLSC proliferation at the concentration of 50 nmol/L(P=0.232). The alizarin red staining showed that PDLSC in TNF-α group generated less mineralized nodule than the control group, while the cell matrix mineralization in TSA group was improved obviously. TNF-α had an inhibitory effect on the expression of osteogenesis related genes, runt-related transcription factor-2(RUNX2)and alkaline phosphatase(ALP), with relative gene expression ratio(experimental/control)decreased to 0.17 ± 0.02 and 0.32 ± 0.03, while TSA could significantly increase the genes' expression to 0.67±0.03 and 0.89±0.02(P<0.01). Western blotting test showed that in TNF-α group the expression of osteogenesis related proteins was obviously reduced, and compared with the TNF-α group, TSA could significantly promote the expression of proteinsin inflammatory microenvironment.. PDLSC in inflammatory microenvironment by TNF-α stimulation had a higher expression of HDAC than that in normal conditions. TSA, as a histone deacetylase inhibitor, could significantly promote the osteogenic differentiation potential of PDLSC in inflammatory microenvironment by suppressing HDAC.

Topics: Alkaline Phosphatase; Anthraquinones; Cell Differentiation; Cell Proliferation; Core Binding Factor Alpha 1 Subunit; Gene Expression; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Hydroxamic Acids; Inflammation; Osteogenesis; Periodontal Ligament; Staining and Labeling; Stem Cell Niche; Stem Cells; Tumor Necrosis Factor-alpha

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infections in infants and young children. However, the majority of RSV-infected patients only show mild symptoms. Different severities of infection and responses among the RSV-infected population indicate that epigenetic regulation as well as personal genetic background may affect RSV infectivity. Histone deacetylase (HDAC) is an important epigenetic regulator in lung diseases. The present study aimed to explore the possible connection between HDAC expression and RSV-induced lung inflammation. To address this question, RSV-infected airway epithelial cells (BEAS‑2B) were prepared and a mouse model of RSV infection was established, and then treated with various concentrations of HDAC inhibitors (HDACis), namely trichostatin A (TSA) and suberoylanilide hydroxamic acid (SAHA). Viral replication and markers of virus-induced airway inflammation or oxidative stress were assessed. The activation of the nuclear factor-κB (NF-κB), cyclo-oxygenase-2 (COX-2), mitogen-activated protein kinase (MAPK) and signal transducer and activator of transcription 3 (STAT3) signaling pathways was evaluated by western blot analysis. Our results showed that RSV infection in airway epithelial cells (AECs) significantly decreased histone acetylation levels by altering HDAC2 expression. The treatment of RSV-infected AECs with HDACis significantly restricted RSV replication by upregulating the interferon-α (IFN-α) related signaling pathways. The treatment of RSV-infected AECs with HDACis also significantly inhibited RSV-induced pro-inflammatory cytokine release [interleukin (IL)-6 and IL-8] and oxidative stress-related molecule production [malondialdehyde (MDA), and nitrogen monoxide (NO)]. The activation of NF-κB, COX-2, MAPK and Stat3, which orchestrate pro‑inflammatory gene expression and oxidative stress injury, was also significantly inhibited. Our in vivo study using a mouse model of RSV infection validated these results. Treatment with HDACis alleviated airway inflammation and reduced in vivo RSV replication. Our data demonstrated that RSV reduced histone acetylation by enhancing HDAC2 expression. Treatment with HDACis (TSA/SAHA) significantly inhibited RSV replication and decreased RSV-induced airway inflammation and oxidative stress. Therefore, the inhibition of HDACs represents a novel therapeutic approach in modulating RSV-induced lung disease.

Topics: Animals; Blotting, Western; Bronchi; Cell Line; Cell Line, Tumor; Cyclooxygenase 2; Cytokines; Epithelial Cells; Female; Histone Deacetylase 2; Histone Deacetylase Inhibitors; Host-Pathogen Interactions; Humans; Hydroxamic Acids; Inflammation; Mice, Inbred BALB C; Mitogen-Activated Protein Kinases; NF-kappa B; Pneumonia; Respiratory Syncytial Virus Infections; Respiratory Syncytial Viruses; Respiratory System; STAT3 Transcription Factor; Vorinostat

Sex steroid hormones have been reported to induce inflammation causing dysregulation of cytokines in prostate cancer cells. However, the underlying epigenetic mechanism has not well been studied. The objective of this study was to evaluate the effect of sex steroid hormones on epigenetic DNA methylation changes in prostate cancer cells using a signature PCR methylation array panel that correspond to 96 genes with biological function in the human inflammatory and autoimmune signals in prostate cancer. Of the 96-gene panel, 32 genes showed at least 10% differentially methylation level in response to hormonal treatment when compared to untreated cells. Genes that were hypomethylated included CXCL12, CXCL5, CCL25, IL1F8, IL13RAI, STAT5A, CXCR4 and TLR5; and genes that were hypermethylated included ELA2, TOLLIP, LAG3, CD276 and MALT1. Quantitative RT-PCR analysis of select genes represented in a cytokine expression array panel showed inverse association between DNA methylation and gene expression for TOLLIP, CXCL5, CCL18 and IL5 genes and treatment of prostate cancer cells with 5'-aza-2'-deoxycytidine with or without trichostatin A induced up-regulation of TOLLIP expression. Further analysis of relative gene expression of matched prostate cancer tissues when compared to benign tissues from individual patients with prostate cancer showed increased and significant expression for CCL18 (2.6-fold; p<0.001), a modest yet significant increase in IL5 expression (1.17-fold; p=0.015), and a modest increase in CXCL5 expression (1.4-fold; p=0.25). In conclusion, our studies demonstrate that sex steroid hormones can induce aberrant gene expression via differential methylation changes in prostate carcinogenesis.

Topics: Azacitidine; Biomarkers, Tumor; Cell Line, Tumor; Chemokine CXCL5; Chemokines, CC; Cytokines; Decitabine; DNA Methylation; Epigenesis, Genetic; Gene Expression Regulation, Neoplastic; Gonadal Steroid Hormones; Humans; Hydroxamic Acids; Inflammation; Interleukin-5; Male; Polymerase Chain Reaction; Promoter Regions, Genetic; Prostatic Neoplasms; Tissue Array Analysis; Tumor Cells, Cultured

Histone deacetylase inhibitors (HDACi) were recently shown to suppress inflammatory responses in experimental models of autoimmune and inflammatory diseases. In this study, the protective effects of Trichostatin A (TSA), an HDACi, on experimental acute-on-chronic liver failure (ACLF) in rat were explored. An ACLF model was established in rats, and animals were randomly divided into control, model, and TSA-treated groups. The rats in TSA-treated group received TSA (2 mg/kg) at 2 h before induction of ACLF. Samples were obtained at 24 h after ACLF induction. We found that the rats in model group showed severe damage to liver tissue at 24 h after ACLF induction. TSA improved liver injury effectively. Serum tumor necrosis factor-alpha (TNF-α), interferon-γ (IFN-γ), interleukin (IL)-10, and IL-18 levels were significantly increased in model group compared with control group, but TSA reduced serum TNF-α, IFN-γ, IL-10, and IL-18 levels effectively compared with model group. In addition, TSA reduced the total HDAC activity, promoted the acetylation of histone, and decreased the expressions of class I HDAC in liver tissue. TSA also increased the acetylation levels and decreased phosphorylation levels in NF-κB p65. The median survival time of the rats was significantly prolonged in TSA-treated group. To conclude, TSA can inhibit the release of multiple inflammatory cytokines, prolong the survival time, and protect against ACLF in rats. The mechanisms were probably through enhancing the acetylation levels of non-histones rather than histone.

Topics: Acetylation; Acute-On-Chronic Liver Failure; Animals; Galactosamine; Histone Deacetylase Inhibitors; Histone Deacetylases; Histones; Hydroxamic Acids; Inflammation; Interferon-gamma; Interleukin-10; Interleukin-18; Lipopolysaccharides; Liver; Random Allocation; Rats; Rats, Wistar; Serum Albumin; Transcription Factor RelA; Tumor Necrosis Factor-alpha

Our previous study demonstrated that persistent pain can epigenetically suppress the transcription of Gad2 [encoding glutamic acid decarboxylase 65 (GAD65)] and consequently impair the inhibitory function of GABAergic synapses in central pain-modulating neurons. This contributes to the development of persistent pain sensitization. Histone deacetylase (HDAC) inhibitors increased GAD65 activity considerably, restored GABA synaptic function, and rendered sensitized pain behavior less pronounced. However, the molecular mechanisms by which HDAC regulates GABAergic transmission through GAD65 under pain conditions are unknown. This work showed that HDAC inhibitor-induced increases in colocalization of GAD65 and synaptic protein synapsin I on the presynaptic axon terminals of the nucleus raphe magnus (NRM) were blocked by a TrkB receptor antagonist K252a [(9S,10R,12R)-2,3,9,10,11,12-hexahydro-10-hydroxy-9-methyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3-fg:3',2',1'-kl]pyrrolo[3,4-i][1,6]benzodiazocine-10-carboxylic acid methyl ester], indicating that BDNF-TrkB signaling may be required in GAD65 modulation of GABA synaptic function. At the brain-derived neurotrophic factor (BDNF) promoter, HDAC inhibitors induced significant increases in H3 hyperacetylation, consistent with the increase in BDNF mRNA and total proteins. Although exogenous BDNF facilitated GABA miniature inhibitory postsynaptic currents and GAD65 accumulation in NRM neuronal synapses in normal rats, it failed to do so in animals subjected to persistent inflammation. In addition, blockade of the TrkB receptor with K252a has no effect on miniature inhibitory postsynaptic currents and synaptic GAD65 accumulation under normal conditions. In addition, the analgesic effects of HDAC inhibitors on behavior were blocked by NRM infusion of K252a. These findings suggest that BDNF-TrkB signaling is required for drugs that reverse the epigenetic effects of chronic pain at the gene level, such as HDAC inhibitors.

Topics: Analgesics; Animals; Brain-Derived Neurotrophic Factor; Carbazoles; gamma-Aminobutyric Acid; Glutamate Decarboxylase; Histone Deacetylase Inhibitors; Hydroxamic Acids; Indole Alkaloids; Inflammation; Male; Nucleus Raphe Magnus; Pain; Promoter Regions, Genetic; Rats, Wistar; Receptor, trkB; Signal Transduction; Synapses; Synapsins; Vorinostat

Acute lung injury (ALI) during sepsis is characterized by bilateral alveolar infiltrates, lung edema and respiratory failure. Here, we examined the efficacy the DNA methyl transferase (DNMT) inhibitor 5-Aza 2-deoxycytidine (Aza), the histone deacetylase (HDAC) inhibitor Trichostatin A (TSA), as well as the combination therapy of Aza and TSA (Aza+TSA) provides in the protection of ALI. In LPS-induced mouse ALI, post-treatment with a single dose of Aza+TSA showed substantial attenuation of adverse lung histopathological changes and inflammation. Importantly, these protective effects were due to substantial macrophage phenotypic changes observed in LPS-stimulated macrophages treated with Aza+TSA as compared with untreated LPS-induced macrophages or LPS-stimulated macrophages treated with either drug alone. Further, we observed significantly lower levels of pro-inflammatory molecules and higher levels of anti-inflammatory molecules in LPS-induced macrophages treated with Aza+TSA than in LPS-induced macrophages treated with either drug alone. The protection was ascribed to dual effects by an inhibition of MAPK-HuR-TNF and activation of STAT3-Bcl2 pathways. Combinatorial treatment with Aza+TSA reduces inflammation and promotes an anti-inflammatory M2 macrophage phenotype in ALI, and has a therapeutic potential for patients with sepsis.

Topics: Acute Lung Injury; Animals; Azacitidine; Decitabine; Drug Combinations; Endotoxemia; Epigenesis, Genetic; Histone Deacetylases; Humans; Hydroxamic Acids; Inflammation; Lipopolysaccharides; Macrophages; Methyltransferases; Mice; Sepsis; Signal Transduction

Inflammatory chronic pathologies are complex processes characterized by an imbalance between the resolution of the inflammatory phase and the establishment of tissue repair. The main players in these inflammatory pathologies are bone marrow derived monocytes (BMDMs). However, how monocyte differentiation is modulated to give rise to specific macrophage subpopulations (M1 or M2) that may either maintain the chronic inflammatory process or lead to wound healing is still unclear. Considering that inhibitors of Histone Deacetylase (HDAC) have an anti-inflammatory activity, we asked whether this enzyme would play a role on monocyte differentiation into M1 or M2 phenotype and in the cell shape transition that follows. We then induced murine bone marrow progenitors into monocyte/macrophage differentiation pathway using media containing GM-CSF and the HDAC blocker, Trichostatin A (TSA). We found that the pharmacological inhibition of HDAC activity led to a shape transition from the typical macrophage pancake-like shape into an elongated morphology, which was correlated to a mixed M1/M2 profile of cytokine and chemokine secretion. Our results present, for the first time, that HDAC activity acts as a regulator of macrophage differentiation in the absence of lymphocyte stimuli. We propose that HDAC activity down regulates macrophage plasticity favoring the pro-inflammatory phenotype.

Topics: Animals; Anti-Inflammatory Agents; Bone Marrow Cells; Cell Differentiation; Chemokines; Epigenesis, Genetic; Flow Cytometry; Granulocyte-Macrophage Colony-Stimulating Factor; Histone Deacetylase 1; Hydroxamic Acids; Immunohistochemistry; Inflammation; Macrophages; Male; Mice; Mice, Inbred C57BL; Monocytes; Nitric Oxide; Phenotype; Stem Cells

Impairment of tissue fluid homeostasis and migration of inflammatory cells across the vascular endothelial barrier are crucial factors in the pathogenesis of acute lung injury (ALI). The goal for treatment of ALI is to target pathways that lead to profound dysregulation of the lung endothelial barrier. Although studies have shown that chemical epigenetic modifiers can limit lung inflammation in experimental ALI models, studies to date have not examined efficacy of a combination of DNA methyl transferase inhibitor 5-Aza 2-deoxycytidine and histone deacetylase inhibitor trichostatin A (herein referred to as Aza+TSA) after endotoxemia-induced mouse lung injury. We tested the hypothesis that treatment with Aza+TSA after lipopolysaccharide induction of ALI through epigenetic modification of lung endothelial cells prevents inflammatory lung injury. Combinatorial treatment with Aza+TSA mitigated the increased endothelial permeability response after lipopolysaccharide challenge. In addition, we observed reduced lung inflammation and lung injury. Aza+TSA also significantly reduced mortality in the ALI model. The protection was ascribed to inhibition of the eNOS-Cav1-MLC2 signaling pathway and enhanced acetylation of histone markers on the vascular endothelial-cadherin promoter. In summary, these data show for the first time the efficacy of combinatorial Aza+TSA therapy in preventing ALI in lipopolysaccharide-induced endotoxemia and raise the possibility of an essential role of DNA methyl transferase and histone deacetylase in the mechanism of ALI.

Topics: Acetylation; Acute Lung Injury; Animals; Azacitidine; Blotting, Western; Capillary Permeability; Cell Proliferation; Cell Survival; Chromatin Immunoprecipitation; Decitabine; Disease Models, Animal; Drug Therapy, Combination; Endothelial Cells; Endotoxemia; Enzyme Inhibitors; Flow Cytometry; Fluorescent Antibody Technique; Hydroxamic Acids; In Situ Nick-End Labeling; Inflammation; Lung; Male; Methylation; Mice; Mice, Inbred C57BL; Real-Time Polymerase Chain Reaction

The production of inflammatory proteins such as interleukin-6 (IL-6) by preadipocytes and mature adipocytes is closely associated with the impairment of systemic glucose homeostasis. However, precisely how the production is regulated and the roles of histone deacetylases (HDACs) remain largely unknown. The aim of this study was to establish whether HDAC inhibitors affect the expression of inflammatory proteins in pre/mature adipocytes, and, if so, to determine the mechanism involved. Trichostatin A (TSA), an HDAC inhibitor, enhanced lipopolysaccharide (LPS)-induced production of IL-6 in OP9 preadipocytes but not the mature adipocytes. Moreover, TSA also enhanced palmitic acid-induced IL-6 production and the expression of inflammatory genes induced by LPS in preadipocytes. Although TSA did not affect TLR4 mRNA expression or the activation of MAPKs, a reporter gene assay revealed that the LPS-induced increase in nuclear factor κB (NF-κB) activity was enhanced by TSA. Moreover, TSA increased the level of NF-κB p65 acetylation at lysine 310 and duration of its translocation into the nucleus, which leads to enhancement of NF-κB activity and subsequently expression of inflammatory genes. These findings shed new light on the regulatory roles of HDACs in preadipocytes in the production of inflammatory proteins.

Topics: Adipocytes; Animals; Cell Differentiation; Chemokine CCL2; Enzyme-Linked Immunosorbent Assay; Gene Expression Regulation; Glucose; Histone Deacetylase Inhibitors; Histone Deacetylases; Homeostasis; Hydroxamic Acids; Inflammation; Interleukin-6; Lipopolysaccharides; Lysine; Mice; Palmitic Acid; Stromal Cells; Transcription Factor RelA

Histone deacetylase (HDAC) inhibitors are promising new compounds for the therapy of fibrotic diseases. In this study we compared the effect of two HDAC inhibitors, trichostatin A and valproic acid, in an experimental model of kidney fibrosis. In mice, doxorubicin (adriamycin) can cause nephropathy characterized by chronic proteinuria, glomerular damage and interstitial inflammation and fibrosis, as seen in human focal segmental glomerulosclerosis. Two treatment regimens were applied, treatment was either started prior to the doxorubicin insult or delayed until a significant degree of proteinuria and fibrosis was present. Pre-treatment of trichostatin A significantly hampered glomerulosclerosis and tubulointerstitial fibrosis, as did the pre-treatment with valproic acid. In contrast, the development of proteinuria was only completely inhibited in the pre-treated valproic acid group, and not in the pre-treated trichostatin A animals. In the postponed treatment with valproic acid, a complete resolution of established doxorubicin-induced proteinuria was achieved within three days, whereas trichostatin A could not correct proteinuria in such a treatment regimen. However, both postponed regimens have comparable efficacy in maintaining the kidney fibrosis to the level reached at the start of the treatments. Moreover, not only the process of fibrosis, but also renal inflammation was attenuated by both HDAC inhibitors. Our data confirm a role for HDACs in renal fibrogenesis and point towards a therapeutic potential for HDAC inhibitors. The effect on renal disease progression and manifestation can however be different for individual HDAC inhibitors.

Topics: Acetylation; Animals; Doxorubicin; Female; Fibrosis; Glomerulosclerosis, Focal Segmental; Histone Deacetylase Inhibitors; Hydroxamic Acids; Immunohistochemistry; Inflammation; Kidney; Kidney Diseases; Mice; Mice, Inbred BALB C; Microscopy, Electron, Transmission; Neutrophil Infiltration; RNA, Messenger; Valproic Acid

Inflammatory mechanisms contribute substantially to secondary tissue injury after brain ischemia. Regulatory T cells (Tregs) are key endogenous modulators of postischemic neuroinflammation. We investigated the potential of histone deacetylase inhibition (HDACi) to enhance Treg potency for experimental stroke in mice. HDACi using trichostatin A increased the number of Tregs and boosted their immunosuppressive capacity and interleukin (IL)-10 expression. In vivo treatment reduced infarct volumes and behavioral deficits after cortical brain ischemia, attenuated cerebral proinflammatory cytokine expression, and increased numbers of brain-invading Tregs. A similar effect was obtained using tubastatin, a specific inhibitor of HDAC6 and a key HDAC in Foxp3 regulation. The neuroprotective effect of HDACi depended on the presence of Foxp3(+) Tregs, and in vivo and in vitro studies showed that the anti-inflammatory cytokine IL-10 was their main mediator. In summary, modulation of Treg function by HDACi is a novel and potent target to intervene at the center of neuroinflammation. Furthermore, this novel concept of modulating endogenous immune mechanisms might be translated to a broad spectrum of diseases, including primary neuroinflammatory and neurodegenerative disorders.

Topics: Animals; Histone Deacetylase Inhibitors; Hydroxamic Acids; Inflammation; Interleukin-10; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Stroke; T-Lymphocytes, Regulatory

Histone deacetylase (HDAC) inhibitors may offer novel approaches in the treatment of asthma. We postulate that trichostatin A (TSA), a Class 1 and 2 inhibitor of HDAC, inhibits airway hyperresponsiveness in antigen-challenged mice. Mice were sensitized and challenged with Aspergillus fumigatus antigen (AF) and treated with TSA, dexamethasone, or vehicle. Lung resistance (R(L)) and dynamic compliance were measured, and bronchial alveolar lavage fluid (BALF) was analyzed for numbers of leukocytes and concentrations of cytokines. Human precision-cut lung slices (PCLS) were treated with TSA and their agonist-induced bronchoconstriction was measured, and TSA-treated human airway smooth muscle (ASM) cells were evaluated for the agonist-induced activation of Rho and intracellular release of Ca(2+). The activity of HDAC in murine lungs was enhanced by antigen and abrogated by TSA. TSA also inhibited methacholine (Mch)-induced increases in R(L) and decreases in dynamic compliance in naive control mice and in AF-sensitized and -challenged mice. Total cell counts, concentrations of IL-4, and numbers of eosinophils in BALF were unchanged in mice treated with TSA or vehicle, whereas dexamethasone inhibited the numbers of eosinophils in BALF and concentrations of IL-4. TSA inhibited the carbachol-induced contraction of PCLS. Treatment with TSA inhibited the intracellular release of Ca(2+) in ASM cells in response to histamine, without affecting the activation of Rho. The inhibition of HDAC abrogates airway hyperresponsiveness to Mch in both naive and antigen-challenged mice. TSA inhibits the agonist-induced contraction of PCLS and mobilization of Ca(2+) in ASM cells. Thus, HDAC inhibitors demonstrate a mechanism of action distinct from that of anti-inflammatory agents such as steroids, and represent a promising therapeutic agent for airway disease.

Topics: Animals; Asthma; Blotting, Western; Bronchoalveolar Lavage Fluid; Bronchoconstriction; Calcium; Carbachol; Dexamethasone; Electrophoresis, Polyacrylamide Gel; Female; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Inflammation; Interleukin-4; Interleukin-6; Mice; Mice, Inbred C57BL; Models, Biological

Emerging evidence indicates that myocardial inflammation plays a key role in the pathogenesis of cardiac diseases. But the exact mechanisms for this chronic inflammatory disorder have not been elucidated. Glucocorticoids (GCs) are the most effective anti-inflammatory treatments available for many inflammatory diseases. However, it is unknown whether endogenous GCs are able to exert anti-inflammatory effect on myocardial inflammation. In this study, the potential role of endogenous GCs in the regulation of myocardial inflammation was investigated. We showed that the reduction of endogenous GC level by adrenalectomy promoted the production of basal and lipopolysaccharide (LPS)-induced proinflammatory cytokines, which could be partly reversed by supplementing with exogenous physiological level of hydrocortisone. Inhibition of GC receptor (GR) signaling pathway with GR antagonist mifepristone (RU486) or histone deacetylase inhibitor trichostatin A (TSA) also increased the levels of basal and LPS-induced proinflammatory cytokines. Moreover, blockade of GC-GR signaling pathway by adrenalectomy, RU486 or TSA enhanced LPS-induced myocardial nuclear factor-κB activation and histone acetylation but inhibited myocardial histone deacetylase expression and activity. Cardiac function studies demonstrated that blockade of the GC-GR signaling pathway aggravated inflammation-induced cardiac dysfunction. These findings indicate that endogenous GCs are able to inhibit myocardial inflammation induced by LPS. Endogenous GCs represent an important endogenous anti-inflammatory mechanism for myocardium in rats and such mechanism injury may be an important factor for pathogenesis of cardiac diseases.

Topics: Animals; Cytokines; Disease Models, Animal; Glucocorticoids; Histone Deacetylases; Hydrocortisone; Hydroxamic Acids; Inflammation; Lipopolysaccharides; Male; Mifepristone; Myocardium; Rats; Rats, Sprague-Dawley; Receptors, Glucocorticoid; Signal Transduction

We have shown previously that pan-HDAC inhibitors (HDACIs) m-carboxycinnamic acid bis-hydroxamide (CBHA) and trichostatin A (TSA) attenuated cardiac hypertrophy in BALB/c mice by inducing hyper-acetylation of cardiac chromatin that was accompanied by suppression of pro-inflammatory gene networks. However, it was not feasible to determine the precise contribution of the myocytes- and non-myocytes to HDACI-induced gene expression in the intact heart. Therefore, the current study was undertaken with a primary goal of elucidating temporal changes in the transcriptomes of cardiac myocytes exposed to CBHA and TSA.. We incubated H9c2 cardiac myocytes in growth medium containing either of the two HDACIs for 6h and 24h and analyzed changes in gene expression using Illumina microarrays. H9c2 cells exposed to TSA for 6h and 24h led to differential expression of 468 and 231 genes, respectively. In contrast, cardiac myocytes incubated with CBHA for 6h and 24h elicited differential expression of 768 and 999 genes, respectively. We analyzed CBHA- and TSA-induced differentially expressed genes by Ingenuity Pathway (IPA), Kyoto Encyclopedia of Genes and Genomes (KEGG) and Core_TF programs and discovered that CBHA and TSA impinged on several common gene networks. Thus, both HDACIs induced a repertoire of signaling kinases (PTEN-PI3K-AKT and MAPK) and transcription factors (Myc, p53, NFkB and HNF4A) representing canonical TGFβ, TNF-α, IFNγ and IL-6 specific networks. An overrepresentation of E2F, AP2, EGR1 and SP1 specific motifs was also found in the promoters of the differentially expressed genes. Apparently, TSA elicited predominantly TGFβ- and TNF-α-intensive gene networks regardless of the duration of treatment. In contrast, CBHA elicited TNF-α and IFNγ specific networks at 6 h, followed by elicitation of IL-6 and IFNγ-centered gene networks at 24h.. Our data show that both CBHA and TSA induced similar, but not identical, time-dependent, gene networks in H9c2 cardiac myocytes. Initially, both HDACIs impinged on numerous genes associated with adipokine signaling, intracellular metabolism and energetics, and cell cycle. A continued exposure to either CBHA or TSA led to the emergence of a number of apoptosis- and inflammation-specific gene networks that were apparently suppressed by both HDACIs. Based on these data we posit that the anti-inflammatory and anti-proliferative actions of HDACIs are myocyte-intrinsic. These findings advance our understanding of the mechanisms of actions of HDACIs on cardiac myocytes and reveal potential signaling pathways that may be targeted therapeutically.

Topics: Animals; Binding Sites; Cardiomegaly; Cell Line; Cell Proliferation; Cinnamates; Gene Regulatory Networks; Genomics; Histone Deacetylase Inhibitors; Histone Deacetylases; Hydroxamic Acids; Inflammation; Myocytes, Cardiac; Rats; Signal Transduction; Sirtuins; Software; Time Factors; Transcription Factors; Transcriptome

The present study aims to explore the possible mechanisms that trichostatin A (TSA), a histone deacetylases inhibitor (HDACi), affects the inflammatory signaling pathways of lipopolysaccharide/toll-like receptor 4/nuclear factor-κB (LPS/TLR4/NF-κB). Murine macrophage cell line RAW264.7 cells were employed. MTT assay was used to assess cell viability. The contents of TNF-α, IL-1β and IL-6 in culture supernatant were assayed by enzyme-linked immunosorbent assay (ELISA). TLR4 expression and NF-κB/p65 (Lys310) acetylation were examined by Western blotting. DNA binding activity of NF-κB/p65 was detected by using TransAM(TM) NF-κB/p65 activity assay kit. The results showed that, compared with control group, which was treated by DMSO, the cells treated with TSA (20, 40, 80 ng/mL) showed decreased percentages of cell survival (P < 0.05). The contents of TNF-α, IL-1β and IL-6 in culture supernatant were all increased by LPS (100 ng/mL), whereas reduced by 40 ng/mL TSA pretreatment (P < 0.05). TSA pretreatment inhibited LPS-induced up-regulation of TLR4 protein expression. Acetylation of NF-κB/p65(Lys310), which was already increased by LPS, was further enhanced by TSA (P < 0.05). On the contrary, LPS-increased DNA binding activity of NF-κB/p65 was decreased by pretreatment with TSA (P < 0.05). The results suggest that TSA-induced anti-inflammation may be attributed to decreases in the expression of TLR4 and DNA binding activity of NF-κB/p65.

Topics: Acetylation; Animals; Cell Line; Hydroxamic Acids; Inflammation; Interleukin-1beta; Interleukin-6; Lipopolysaccharides; Macrophages; Mice; Signal Transduction; Toll-Like Receptor 4; Transcription Factor RelA; Tumor Necrosis Factor-alpha; Up-Regulation

Adipocyte fatty acid binding protein (A-FABP) present in macrophages has been implicated in the integration of lipid metabolism and inflammatory response, contributing to development of insulin resistance and atherosclerosis.. This study was conducted to test the hypothesis that the role of fatty acids in the inflammatory pathways is mediated through the modulation of A-FABP expression in macrophages.. Murine RAW 264.7 macrophages were treated with inflammatory insults and fatty acids for quantitative real-time PCR and Western blot analysis. The cells were treated with trichostatin A (TSA), a histone deacetylase inhibitor, for elucidating mechanisms for the regulation of A-FABP expression by fatty acids. RNA interference (RNAi) to knock down A-FABP was utilized to assess its role in inflammatory gene expression.. When RAW 264.7 were incubated with lipopolysaccharides (LPS; 100 ng/ml) or 2.5 ng/ml of tumor necrosis factor α for 18 h, A-FABP mRNA and protein levels were drastically increased. Unsaturated fatty acids (100 μmol/l in complexed with BSA) such as palmitoleic acid, oleic acid, linoleic acid, linolenic acid, and eicosapentaenoic acid, significantly repressed the basal as well as LPS-induced A-FABP expression, whereas palmitic acid did not elicit the same effect. TSA increased A-FABP mRNA levels and abolished the repressive effect of linoleic acid on A-FABP expression in unstimulated and LPS-stimulated macrophages. Depletion of A-FABP expression by 70-80% using RNAi markedly decreased cyclooxygenase 2 mRNA abundance and potentiated the repression by linoleic acid.. Unsaturated fatty acids inhibited the basal as well as LPS-induced A-FABP expression. The mechanism may involve histone deacetylation and anti-inflammatory effect of unsaturated fatty acids may be at least in part attributed to their repression of A-FABP expression in RAW 264.7 macrophages.

Topics: Animals; Blotting, Western; Cell Line; Cyclooxygenase 2; Fatty Acid-Binding Proteins; Fatty Acids, Unsaturated; Gene Expression Regulation; Gene Knockdown Techniques; Histone Deacetylase Inhibitors; Histones; Hydroxamic Acids; Inflammation; Lipid Metabolism; Lipopolysaccharides; Macrophages; Mice; Real-Time Polymerase Chain Reaction; RNA Interference; RNA, Messenger; Tumor Necrosis Factor-alpha

Histone deacetylase inhibitors (HDACi) have been shown to exhibit anti-inflammatory activity, but their mechanism of action is poorly understood. Trichostatin A (TSA) and the cyclic tetrapeptide class inhibitor Ky-2 inhibit both lipopolysaccharide-induced tumor necrosis factor-α (TNF-α) production in rats and TNF-α-induced expression of inflammatory genes in HeLa cells. We assessed the molecular mechanism underlying TSA-induced anti-inflammatory activity by genetically dissecting activation of the nuclear factor-κB (NF-κB) pathway following stimulation with TNF-α. Trichostatin A did not inhibit degradation of IκBα, nuclear translocation and DNA binding of NF-κB; also, the drug did not affect transient expression from exogenous κB-reporter plasmids. However, endogenous expression of inflammatory cytokines such as interleukin-8 (IL-8) was greatly reduced, even in the absence of de novo protein synthesis, suggesting that HDACi directly inhibits NF-κB-induced transcription. Indeed, chromatin immunoprecipitation (ChIP) analysis showed that events related to transcriptional activation of the IL-8 gene region in response to TNF-α, including recruitment of RNA polymerase II (Pol II), were compromised in the presence of TSA. These data indicate that HDAC activity is required for the efficient initiation and/or elongation of inflammatory gene transcription mediated by NF-κB.

Topics: Animals; Blotting, Western; Chromatin Immunoprecipitation; Fluorescent Antibody Technique; Gene Expression; HeLa Cells; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Inflammation; NF-kappa B; Rats; Reverse Transcriptase Polymerase Chain Reaction; RNA Polymerase II; Transcription, Genetic

Epigenetic alteration of the genome has been shown to provide palliative effects in mouse models of certain human autoimmune diseases. We have investigated whether chromatin remodeling could provide protection against autoimmune diabetes in NOD mice. Treatment of female mice during the transition from prediabetic to diabetic stage (18-24 weeks of age) with the well-characterized histone deacetylase inhibitor, trichostatin A effectively reduced the incidence of diabetes. However, similar treatment of overtly diabetic mice during the same time period failed to reverse the disease. Protection against diabetes was accompanied by histone hyperacetylation in pancreas and spleen, enhanced frequency of CD4(+) CD62L(+) cells in the spleen, reduction in cellular infiltration of islets, restoration of normoglycemia and glucose-induced insulin release by beta cells. Activation of splenic T lymphocytes derived from protected mice in vitro with pharmacological agents that bypass the antigen receptor or immobilized anti-CD3 antibody resulted in enhanced expression of Ifng mRNA and protein without altering the expression of Il4, Il17, Il18, Inos and Tnfa genes nor the secretion of IL-2, IL-4, IL-17 and TNF-α proteins. Consistently, expression of the transcription factor involved in Ifng transcription, Tbet/Tbx21 but not Gata3 and Rorgt, respectively, required for the transcription of Il4 and Il17, was upregulated in activated splenocytes of protected mice. These results indicate that chromatin remodeling can lead to amelioration of diabetes by using multiple mechanisms including differential gene transcription. Thus, epigenetic modulation could be a novel therapeutic approach to block the transition from benign to frank diabetes.

Topics: Acetylation; Animals; Chromatin Assembly and Disassembly; Diabetes Mellitus, Type 1; Epigenomics; Female; Gene Expression Regulation; Histone Deacetylase Inhibitors; Histones; Hydroxamic Acids; Immune System; Inflammation; Interferon-gamma; Islets of Langerhans; Mice; Mice, Inbred NOD; T-Box Domain Proteins; T-Lymphocyte Subsets; Up-Regulation

We have reported that the nuclear factor-κB (NF-κB) induces chronic inflammation in the adipose tissue of p65 transgenic (Tg) mice, in which the NF-κB subunit p65 (RelA) is overexpressed from the adipocyte protein 2 (aP2) gene promoter. Tg mice suffer a mild lipodystrophy and exhibit deficiency in adipocyte differentiation. To understand molecular mechanism of the defect in adipocytes, we investigated glyceroneogenesis by examining the activity of cytosolic phosphoenolpyruvate carboxykinase (PEPCK) in adipocytes. In aP2-p65 Tg mice, Pepck expression is inhibited at both the mRNA and protein levels in adipose tissue. The mRNA reduction is a consequence of transcriptional inhibition but not alteration in mRNA stability. The Pepck gene promoter is inhibited by NF-κB, which enhances the corepressor activity through activation of histone deacetylase 3 (HDAC3) in the nucleus. HDAC3 suppresses Pepck transcription by inhibiting the transcriptional activators, peroxisome proliferator-activated receptor-γ, and cAMP response element binding protein. The NF-κB activity is abolished by Hdac3 knockdown or inhibition of HDAC3 catalytic activity. In a chromatin immunoprecipitation assay, HDAC3 interacts with peroxisome proliferator-activated receptor-γ and cAMP response element binding protein in the Pepck promoter when NF-κB is activated by TNF-α. These results suggest that HDAC3 mediates NF-κB activity to repress Pepck transcription. This mechanism is responsible for inhibition of glyceroneogenesis in adipocytes, which contributes to lipodystrophy in the aP2-p65 Tg mice.

Topics: 3T3-L1 Cells; Adipocytes; Adipose Tissue; Animals; Blotting, Western; Cyclic AMP Response Element-Binding Protein; HEK293 Cells; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Hydroxamic Acids; Inflammation; Lipodystrophy; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Phosphoenolpyruvate Carboxylase; PPAR gamma; Promoter Regions, Genetic; Protein Binding; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; Transcription Factor RelA; Triglycerides; Tumor Necrosis Factor-alpha

To study usefulness of bone marrow progenitor cells (BPCs) epigenetically altered by chromatin modifying agents in mediating heart repair after myocardial infarction in mice.. We tested the therapeutic efficacy of bone marrow progenitor cells treated with the clinically-used chromatin modifying agents Trichostatin A (TSA, histone deacetylase inhibitor) and 5Aza-2-deoxycytidine (Aza, DNA methylation inhibitor) in a mouse model of acute myocardial infarction (AMI). Treatment of BPCs with Aza and TSA induced expression of pluripotent genes Oct4, Nanog, Sox2, and thereafter culturing these cells in defined cardiac myocyte-conditioned medium resulted in their differentiation into cardiomyocyte progenitors and subsequently into cardiac myocytes. Their transition was deduced by expression of repertoire of markers: Nkx2.5, GATA4, cardiotroponin T, cardiotroponin I, α-sarcomeric actinin, Mef2c and MHC-α. We observed that the modified BPCs had greater AceH3K9 expression and reduced histone deacetylase1 (HDAC1) and lysine-specific demethylase1 (LSD1) expression compared to untreated BPCs, characteristic of epigenetic changes. Intra-myocardial injection of modified BPCs after AMI in mice significantly improved left ventricular function. These changes were ascribed to differentiation of the injected cells into cardiomyocytes and endothelial cells.. Treatment of BPCs with Aza and TSA converts BPCs into multipotent cells, which can then be differentiated into myocyte progenitors. Transplantation of these modified progenitor cells into infarcted mouse hearts improved left ventricular function secondary to differentiation of cells in the niche into myocytes and endothelial cells.

Topics: Animals; Azacitidine; Bone Marrow Cells; Cell Differentiation; Decitabine; Endothelial Cells; Epigenesis, Genetic; Gene Expression Regulation; Heart; Hydroxamic Acids; Inflammation; Male; Mice; Mice, Inbred C57BL; Myocardial Infarction; Myocytes, Cardiac; Neovascularization, Physiologic; Pluripotent Stem Cells; Stem Cell Transplantation; Stem Cells; Ventricular Dysfunction, Left

We investigated the genome-wide consequences of pan-histone deacetylase inhibitors (HDACIs) trichostatin A (TSA) and m-carboxycinnamic acid bis-hydroxamide (CBHA) in the hearts of BALB/c mice eliciting hypertrophy in response to interleukin-18 (IL-18). Both TSA and CBHA profoundly altered cardiac chromatin structure that occurred concomitantly with normalization of IL-18-induced gene expression and amelioration of cardiac hypertrophy. The hearts of mice exposed to IL-18+/-TSA or CBHA elicited distinct gene expression profiles. Of 184 genes that were differentially regulated by IL-18 and TSA, 33 were regulated in an opposite manner. The hearts of mice treated with IL-18 and/or CBHA elicited 147 differentially expressed genes (DEGs), a third of which were oppositely regulated by IL-18 and CBHA. Ingenuity Pathways and Kyoto Encyclopedia of Genes and Genomes analyses of DEGs showed that IL-18 impinged on TNF-α- and IFNγ-specific gene networks relegated to controlling immunity and inflammation, cardiac metabolism and energetics, and cell proliferation and apoptosis. These TNF-α- and IFNγ-specific gene networks, extensively connected with PI3K, MAPK, and NF-κB signaling pathways, were oppositely regulated by IL-18 and pan-HDACIs. Evidently, both TSA and CBHA caused a two- to fourfold induction of phosphatase and tensin homolog expression to counteract IL-18-induced proinflammatory signaling and cardiac hypertrophy.

Topics: Animals; Cardiomegaly; Chromatin Assembly and Disassembly; Cinnamates; Cluster Analysis; Gene Expression Profiling; Gene Expression Regulation; Gene Regulatory Networks; Histone Deacetylase Inhibitors; Histones; Hydroxamic Acids; Inflammation; Interleukin-18; Intracellular Space; Male; Mice; Mice, Inbred BALB C; Myocardium; Oligonucleotide Array Sequence Analysis; Protein Processing, Post-Translational; Reproducibility of Results; Signal Transduction

Decreased neutrophil apoptosis is implicated in persistent inflammation resulting in systemic inflammatory response syndrome and multiple organ dysfunctions syndromes. Short-chain fatty acids (SCFAs) may be a candidate to control neutrophil apoptosis because SCFAs are normally produced in the gut and related products have been approved for human use. We investigated the effects of SCFAs on apoptosis of activated and non-activated neutrophils and their mechanisms.. Purified neutrophils obtained from healthy volunteers were preincubated for 1 h with or without the G-protein receptor (GPR) inhibitor pertussis toxin (100 ng/mL) or U-73122 (50 ng/mL), extracellular signal-related protein kinase inhibitor PD98059 (10 microM), mitogen-activated protein kinase (MAPK) p38 inhibitor SB203580 (25 microM), Jun kinase inhibitor-I (2 microM), caspase-3 and -7 inhibitor Z-VAD-FMK (100 microM), caspase-8 inhibitor Z-IETD-FMK (50 microM), or caspase-9 inhibitor Z-LEHD-FMK (50 microM). The cells were then cultured with or without SCFAs or trichostatin A, a typical histone deacetylase inhibitor, in the presence or absence of lipopolysaccharide (1 microg/mL) or tumor necrosis factor-alpha (100 ng/mL). Neutrophil apoptosis was assessed by annexin V staining using flow cytometry. The GPR-41 and -43 and apoptosis-related proteins (bax, mcl-1, a1) mRNA were measured by quantitative real-time polymerase chain reaction and the expression of acetylated histone H3 was determined by western blot.. The caspase inhibitors inhibited butyrate- and propionate-induced neutrophil apoptosis treated or untreated with lipopolysaccharide or tumor necrosis factor-alpha, whereas GPR and MAPK inhibitors had no effect. The mRNA expressions of GPR-43 and a1 protein were reduced by butyrate and propionate. The expressions of acetylated histone H3 were induced by butyrate and propionate.. These results suggest that butyrate and propionate increase apoptosis of neutrophils irrespective of their activation state, by factors other than GPRs and MAPKs, and their mechanisms likely relate to their histone deacetylase inhibition activity, which may control a1 mRNA expression.

Topics: Apoptosis; Butyrates; Caspase Inhibitors; Enzyme Inhibitors; Estrenes; Histone Deacetylase Inhibitors; Histones; Humans; Hydroxamic Acids; Inflammation; Lipopolysaccharides; Neutrophils; Pertussis Toxin; Propionates; Proteins; Pyrrolidinones; Receptors, G-Protein-Coupled; Reference Values; RNA, Messenger; Tumor Necrosis Factor-alpha

Macrophages contribute significantly to the pathology of many chronic inflammatory diseases, including rheumatoid arthritis (RA), asthma, and chronic obstructive pulmonary disease. Macrophage activation and survival are tightly regulated by reversible acetylation and deacetylation of histones, transcription factors, and structural proteins. Although histone deacetylase (HDAC) inhibitors (HDACis) demonstrate therapeutic effects in animal models of chronic inflammatory disease, depressed macrophage HDAC activity in patients with asthma, chronic obstructive pulmonary disease, or RA may contribute to inflammation in these diseases, potentially contraindicating the therapeutic administration of HDACis. In this study, we directly examined whether HDACis could influence the activation of macrophages derived from the inflamed joints of patients with RA. We found that inhibition of class I/II HDACs or class III sirtuin HDACs potently blocked the production of IL-6 and TNF-alpha by macrophages from healthy donors and patients with RA. Two HDACis, trichostatin A and nicotinamide, selectively induced macrophage apoptosis associated with specific downregulation of the antiapoptotic protein Bfl-1/A1, and inflammatory stimuli enhanced the sensitivity of macrophages to HDACi-induced apoptosis. Importantly, inflammatory and angiogenic cytokine production in intact RA synovial biopsy explants was also suppressed by HDACis. Our study identifies redundant, but essential, roles for class I/II and sirtuin HDACs in promoting inflammation, angiogenesis, and cell survival in RA.

Topics: Adult; Aged; Apoptosis; Arthritis, Rheumatoid; Blotting, Western; Cells, Cultured; Female; Flow Cytometry; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Inflammation; Interleukin-6; Macrophages; Male; Middle Aged; Minor Histocompatibility Antigens; Niacinamide; Proto-Oncogene Proteins c-bcl-2; Reverse Transcriptase Polymerase Chain Reaction; Synovial Membrane; Tumor Necrosis Factor-alpha

Histone deacetylase inhibitors (HDACi) have been proposed as therapies for certain cancers and as an anti-reservoir therapy for HIV+ individuals with highly active anti-retroviral therapy, yet their roles in glial inflammatory and innate antiviral gene expression have not been defined. In this study, we examined the effects of two non-selective HDACi, trichostatin A and valproic acid, on antiviral and cytokine gene expression in primary human microglia and astrocytes stimulated with TLR3 or TLR4 ligand. HDACi potently suppressed the expression of innate antiviral molecules such as IFNβ, interferon-simulated genes, and proteins involved in TLR3/TLR4 signaling. HDACi also suppressed microglial and astrocytic cytokine and chemokine gene expression, but with different effects on different groups of cytokines. These results have important implications for the clinical use of HDACi.

Topics: Astrocytes; Blotting, Western; Cells, Cultured; Cytokines; Enzyme-Linked Immunosorbent Assay; Gene Expression; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Immunity, Innate; Inflammation; Microglia; Oligonucleotide Array Sequence Analysis; Reverse Transcriptase Polymerase Chain Reaction; Valproic Acid

Broad-spectrum inhibitors of HDACs are therapeutic in many inflammatory disease models but exacerbated disease in a mouse model of atherosclerosis. HDAC inhibitors have anti- and proinflammatory effects on macrophages in vitro. We report here that several broad-spectrum HDAC inhibitors, including TSA and SAHA, suppressed the LPS-induced mRNA expression of the proinflammatory mediators Edn-1, Ccl-7/MCP-3, and Il-12p40 but amplified the expression of the proatherogenic factors Cox-2 and Pai-1/serpine1 in primary mouse BMM. Similar effects were also apparent in LPS-stimulated TEPM and HMDM. The pro- and anti-inflammatory effects of TSA were separable over a concentration range, implying that individual HDACs have differential effects on macrophage inflammatory responses. The HDAC1-selective inhibitor, MS-275, retained proinflammatory effects (amplification of LPS-induced expression of Cox-2 and Pai-1 in BMM) but suppressed only some inflammatory responses. In contrast, 17a (a reportedly HDAC6-selective inhibitor) retained anti-inflammatory but not proinflammatory properties. Despite this, HDAC6(-/-) macrophages showed normal LPS-induced expression of HDAC-dependent inflammatory genes, arguing that the anti-inflammatory effects of 17a are not a result of inhibition of HDAC6 alone. Thus, 17a provides a tool to identify individual HDACs with proinflammatory properties.

Topics: Animals; Blotting, Western; Chromatin Immunoprecipitation; Cyclooxygenase 2; Enzyme-Linked Immunosorbent Assay; Histone Deacetylase 6; Histone Deacetylase Inhibitors; Histone Deacetylases; Hydroxamic Acids; Inflammation; Inflammation Mediators; Interleukin-12 Subunit p40; Lipopolysaccharides; Luciferases; Macrophages; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Plasminogen Activator Inhibitor 1; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Toll-Like Receptor 4; Vorinostat

Corneal fibroblasts exhibit different phenotypes in different phases of corneal wound healing. In the inflammatory phase, the cells assume a proinflammatory phenotype and produce large amounts of cytokines and chemokines, but in the proliferative and remodeling phases, they adapt a profibrotic state, differentiate into myofibroblasts and increase extracellular matrix protein synthesis, secretion, and deposition. In the present study, the molecular mechanisms regulating the transition of corneal fibroblasts from the proinflammatory state to the profibrotic state were investigated. Corneal fibroblasts were treated with TGFbeta, a known profibrotic and anti-inflammatory factor in wound healing, in the absence or presence of trichostatin A (TSA), a histone deacetylase (HDAC) inhibitor. The results revealed that TGFbeta induced the profibrotic transition of corneal fibroblasts, including increased extracellular matrix synthesis, morphological changes, and assembly of actin filaments. Meanwhile, proinflammatory gene expressions of corneal fibroblasts were down-regulated with the treatment of TGFbeta, as confirmed by cDNA microarray, real time PCR and ELISA. Moreover, TSA reversed the TGFbeta-mediated transition of corneal fibroblasts from the proinflammatory state to the profibrotic state, as accompanied by histone hyperacetylations. In conclusion, TGFbeta suppressed the production of proinflammatory factors and enhanced the expression of matrix remodeling genes of corneal fibroblasts in the transition from the proinflammatory state to the profibrotic state, and the dual roles of TGFbeta on the phenotype regulations of corneal fibroblasts were mediated by altered histone acetylation.

Topics: Acetylation; Actin Cytoskeleton; Cell Shape; Cell Transdifferentiation; Cells, Cultured; Cornea; Enzyme-Linked Immunosorbent Assay; Extracellular Matrix Proteins; Fibroblasts; Fibrosis; Gene Expression Profiling; Gene Expression Regulation; Histone Acetyltransferases; Histone Deacetylase Inhibitors; Histones; Humans; Hydroxamic Acids; Inflammation; Inflammation Mediators; Oligonucleotide Array Sequence Analysis; Phenotype; Polymerase Chain Reaction; Transforming Growth Factor beta

Clostridium difficile toxin A is known to cause actin disaggregation through the enzymatic inactivation of intracellular Rho proteins. Based on the rapid and severe cell rounding of toxin A-exposed cells, we speculated that toxin A may be involved in post-translational modification of tubulin, leading to microtubule instability. In the current study, we observed that toxin A strongly reduced α-tubulin acetylation in human colonocytes and mouse intestine. Fractionation analysis demonstrated that toxin A-induced α-tubulin deacetylation yielded monomeric tubulin, indicating the presence of microtubule depolymerization. Inhibition of the glucosyltransferase activity against Rho proteins of toxin A by UDP-2',3'-dialdehyde significantly abrogated toxin A-induced α-tubulin deacetylation. In colonocytes treated with trichostatin A (TSA), an inhibitor of the HDAC6 tubulin deacetylase, toxin A-induced α-tubulin deacetylation and loss of tight junction were completely blocked. Administration of TSA also attenuated proinflammatory cytokine production, mucosal damage, and epithelial cell apoptosis in mouse intestine exposed to toxin A. These results suggest that toxin A causes microtubule depolymerization by activation of HDAC6-mediated tubulin deacetylation. Indeed, blockage of HDAC6 by TSA markedly attenuates α-tubulin deacetylation, proinflammatory cytokine production, and mucosal damage in a toxin A-induced mouse enteritis model. Tubulin deacetylation is an important component of the intestinal inflammatory cascade following toxin A-mediated Rho inactivation in vitro and in vivo.

Topics: Acetylation; Acute Disease; Animals; Apoptosis; Bacterial Toxins; Cell Line; Colon; Cytokines; Enteritis; Enterotoxins; Enzyme Activation; Epithelial Cells; Histone Deacetylase 6; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Hydroxamic Acids; Inflammation; Intestinal Mucosa; Mice; Microtubules; Protein Processing, Post-Translational; rho GTP-Binding Proteins; Tubulin; Uridine Diphosphate

Interaction of pathogens with cells of the immune system results in activation of inflammatory gene expression. This response, although vital for immune defence, is frequently deleterious to the host due to the exaggerated production of inflammatory proteins. The scope of inflammatory responses reflects the activation state of signalling proteins upstream of inflammatory genes as well as signal-induced assembly of nuclear chromatin complexes that support mRNA expression. Recognition of post-translationally modified histones by nuclear proteins that initiate mRNA transcription and support mRNA elongation is a critical step in the regulation of gene expression. Here we present a novel pharmacological approach that targets inflammatory gene expression by interfering with the recognition of acetylated histones by the bromodomain and extra terminal domain (BET) family of proteins. We describe a synthetic compound (I-BET) that by 'mimicking' acetylated histones disrupts chromatin complexes responsible for the expression of key inflammatory genes in activated macrophages, and confers protection against lipopolysaccharide-induced endotoxic shock and bacteria-induced sepsis. Our findings suggest that synthetic compounds specifically targeting proteins that recognize post-translationally modified histones can serve as a new generation of immunomodulatory drugs.

Topics: Acetylation; Animals; Anti-Inflammatory Agents; Benzodiazepines; Cells, Cultured; Epigenomics; Gene Expression Regulation; Genome-Wide Association Study; Heterocyclic Compounds, 4 or More Rings; Histone Deacetylase Inhibitors; Hydroxamic Acids; Inflammation; Kaplan-Meier Estimate; Lipopolysaccharides; Macrophages; Mice; Mice, Inbred C57BL; Models, Molecular; Protein Serine-Threonine Kinases; Protein Structure, Tertiary; Salmonella Infections; Salmonella typhimurium; Sepsis; Shock, Septic

Phenotypic alteration of vascular smooth muscle cells (SMC) in response to injury or inflammation is an essential component of vascular disease. Evidence suggests that this process is dependent on epigenetic regulatory processes. P300, a histone acetyltransferase (HAT), activates crucial muscle-specific promoters in terminal (non-SMC) myocyte differentiation, and may be essential to SMC modulation as well.. We performed a subanalysis examining transcriptional time-course microarray data obtained using the A404 model of SMC differentiation. Numerous chromatin remodeling genes (up to 62% of such genes on our array platform) showed significant regulation during differentiation. Members of several chromatin-remodeling families demonstrated involvement, including factors instrumental in histone modification, chromatin assembly-disassembly and DNA silencing, suggesting complex, multi-level systemic epigenetic regulation. Further, trichostatin A, a histone deacetylase inhibitor, accelerated expression of SMC differentiation markers in this model. Ontology analysis indicated a high degree of p300 involvement in SMC differentiation, with 60.7% of the known p300 interactome showing significant expression changes. Knockdown of p300 expression accelerated SMC differentiation in A404 cells and human SMCs, while inhibition of p300 HAT activity blunted SMC differentiation. The results suggest a central but complex role for p300 in SMC phenotypic modulation.. Our results support the hypothesis that chromatin remodeling is important for SMC phenotypic switching, and detail wide-ranging involvement of several epigenetic modification families. Additionally, the transcriptional coactivator p300 may be partially degraded during SMC differentiation, leaving an activated subpopulation with increased HAT activity and SMC differentiation-gene specificity.

Topics: Animals; Cell Differentiation; Chromatin; E1A-Associated p300 Protein; Epigenesis, Genetic; Gene Expression Regulation, Enzymologic; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Inflammation; Mice; Myocytes, Smooth Muscle; Oligonucleotide Array Sequence Analysis; Transcriptional Activation

To examine the effects of a histone deacetylase inhibitor, Trichostatin A (TSA), on the behavior of macrophages and subconjunctival fibroblasts in vitro and on ocular surface inflammation and scarring in vivo using an alkali burn wound healing model.. Effects of TSA on expression of inflammation-related growth factors or collagen I were examined by real-time RT-PCR or immunoassay in mouse macrophages or human subconjunctival fibroblasts. Effects of TSA on trans forming growth factor β (TGFβ)/Smad signaling were evaluated with western blotting and/or immunocytochemistry. Alkali-burn injuries on the eyes of mice were performed with three µl of 0.5 N NaOH under general and topical anesthesia. TSA (600 µg/Kg daily) or vehicle was administered to animals via intraperitoneal (i.p.) injection. Histology and real-time RT-PCR investigations evaluated the effects of TSA on the healing process of the cornea.. TSA inhibited TGFβ 1 and vascular endothelial growth factor (VEGF) expression in macrophages, and TGFβ1 and collagen I in ocular fibroblasts. It elevated the expression of 5'-TG-3'-interacting factor (TGIF) and Smad7 in fibroblasts and blocked nuclear translocation of phospho-Smad2. Real-time PCR and immunocytochemistry studies showed that systemic administration of TSA suppressed the inflammation and fibrotic response in the stroma and accelerated epithelial healing in the alkali-burned mouse cornea.. Systemic administration of TSA reduces inflammatory and fibrotic responses in the alkali-burned mouse ocular surface in vivo. The mechanisms of action involve attenuation of Smad signal in mesenchymal cells and reduction in the activation and recruitment of macrophages. TSA has the potential to treat corneal scarring in vivo.

Topics: Animals; Burns, Chemical; Cell Movement; Cell Proliferation; Cells, Cultured; Conjunctiva; Cytokines; Eye Diseases; Fibroblasts; Fibrosis; Gene Expression Regulation; Humans; Hydroxamic Acids; Inflammation; Macrophages; Mice; Neovascularization, Pathologic; Signal Transduction; Transforming Growth Factor beta; Wound Healing

Macrophage-triggered chronic inflammation and smooth muscle cell-initiated vascular remodeling are two major pathophysiologic events during atherogenesis. Major histocompatibility class II (MHC II) transactivator (CIITA) is a key mediator of these processes through transcriptional regulation of interferon gamma (IFN-gamma) induced MHC II activation and type I collagen repression. Transcriptional activity of CIITA is regulated by multiple post-translational modifications. Here we report that CIITA and histone deacetylase 2 (HDAC2) interact in smooth muscle cells and macrophages as assayed by co-immunoprecipitations. HDAC2 deacetylates CIITA whereas both the HDAC inhibitor trichostatin A (TSA) and over-expression of HDAC2 interfering RNA increase CIITA acetylation. HDAC2 down-regulates CIITA recruitment to target promoters as evidenced by chromatin immunoprecipitation assays, and suppresses MHC II activation and collagen repression mediated by CIITA in luciferase reporter assays. Quantitative PCR reveals that TSA enhances MHC II activation and collagen repression by IFN-gamma. Wild type but not enzyme-deficient HDAC2 promotes the degradation of CIITA protein, whereas TSA and the proteasome inhibitor MG132 restore CIITA activity by stabilizing CIITA protein and increasing its association with target promoters. Furthermore, TSA treatment enhances the association of CIITA with the transcription factor RFX5, which ameliorates the down-regulation of CIITA recruitment to target promoters by HDAC2. In conclusion, our data suggest that HDAC2 antagonizes CIITA activity by committing CIITA to protein degradation and decreasing the interaction of CIITA with RFX5 in a deacetylation-dependent manner. Therefore, modulating CIITA activity by targeting HDAC2 may provide potential anti-atherogenic strategies.

Topics: Acetylation; Animals; Atherosclerosis; Cell Line; Collagen Type I; Cysteine Proteinase Inhibitors; DNA-Binding Proteins; Histocompatibility Antigens Class II; Histone Deacetylase 2; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Hydroxamic Acids; Inflammation; Interferon-gamma; Leupeptins; Macrophages; Mice; Myocytes, Smooth Muscle; Nuclear Proteins; Promoter Regions, Genetic; Protein Processing, Post-Translational; Regulatory Factor X Transcription Factors; Repressor Proteins; Trans-Activators; Transcription Factors; Transcription, Genetic

Topics: Acetylation; Animals; Atherosclerosis; Cell Line; Collagen Type I; Cysteine Proteinase Inhibitors; DNA-Binding Proteins; Histocompatibility Antigens Class II; Histone Deacetylase 2; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Hydroxamic Acids; Inflammation; Interferon-gamma; Leupeptins; Macrophages; Mice; Myocytes, Smooth Muscle; Nuclear Proteins; Promoter Regions, Genetic; Protein Processing, Post-Translational; Regulatory Factor X Transcription Factors; Repressor Proteins; Trans-Activators; Transcription Factors; Transcription, Genetic

The effects of short-chain fatty acids (butyrate, propionate, and acetate) and trichostatin A (TSA), a typical histone deacetylase inhibitor, on tumor necrosis factor (TNF)-alpha secretion and nuclear factor kappaB (NF-kappaB) activation in peripheral blood mononuclear cells induced with lipopolysaccharide were evaluated in relation to prostaglandin E(2) (PGE(2)) secretion. Treatment of cells with butyrate; tributyrin, a prodrug of butyrate; propionate; acetate; and TSA down-regulated TNF-alpha secretion but all up-regulated PGE(2) secretion. Butyrate, propionate, and TSA inhibited NF-kappaB activation. The effects of the cyclooxygenase-nonspecific inhibitor, indomethacin; the cyclooxygenase-2 selective inhibitor, N-[2-(cyclohexyloxy)-4-nitro-phenyl] methanesulfonamide; and the general lipoxygenase inhibitor, nordihydroguaiaretic acid, varied in cells treated with each short-chain fatty acids. N-[2-(cyclohexyloxy)-4-nitro-phenyl] methanesulfonamide inhibited the effect of propionate on TNF-alpha secretion, and nordihydroguaiaretic acid inhibited that of acetate. The results showed that butyrate, propionate, and TSA inhibited TNF-alpha production via PGE(2) secretion and down-regulated NF-kappaB activation by lipopolysaccharide. These data suggest that the mechanism of butyrate and propionate action is through histone deacetylation and acetate through lipoxygenase activation in the regulation of proinflammatory responses in cells.

Topics: Adult; Anti-Inflammatory Agents; Colon; Dinoprostone; Enzyme Inhibitors; Fatty Acids, Volatile; Female; Humans; Hydroxamic Acids; Immunologic Factors; Inflammation; Leukocytes, Mononuclear; Male; NF-kappa B; Prodrugs; Triglycerides; Tumor Necrosis Factor-alpha; Young Adult

Protein acetylation regulates the extent of inflammatory responses and disturbances in protein acetylation have been proposed to play an important role in inflammatory and neurodegenerative diseases. We have recently observed that histone deacetylase inhibitors, such as trichostatin A (TSA) and SAHA, strongly potentiate the LPS induced inflammatory response in several rat and mouse inflammatory models. Our aim here was to characterise pro-inflammatory signaling mediated via increased protein acetylation and protein phosphorylation in microglial N9 cells. First we observed that TSA induced pro-inflammatory response was independent of the different Toll-like receptors activated, since LPS, flagellin and unmethylated CpG oligonucleotides, equally potentiated IL-6 secretion from N9 microglia. Next we compared the protein acetylation induced potentiation to that induced by okadaic acid, a well-known inducer of pro-inflammatory responses. The time scale of the IL-6 responses showed that the effects of okadaic acid were clearly early-response effects appearing as soon as 6h after exposure, whereas TSA evoked a significant inhibition in IL-6 secretion up to 12h but after that it induced an exponential increase in cytokine and nitric oxide production up to 24h. It seems that okadaic acid induces an early moderate response and TSA a late but exponential potentiation of microglial inflammatory responses. The pro-inflammatory responses of TSA and okadaic acid were both dependent on NF-kappaB signaling but independent on the DNA-binding capacity of nuclear NF-kappaB complexes. Interestingly, we observed that the transactivation of the NF-kappaB-Luc reporter gene was clearly activated during TSA induced pro-inflammatory potentiation. Our studies imply that the potentiation of the inflammatory response by increased acetylation is due to the enhancement of transactivation of NF-kappaB driven inflammatory genes. Our studies on signaling pathways revealed that PI3K inhibitors LY294002 and Wortmannin blocked the TSA induced pro-inflammatory response but surprisingly did not affect the okadaic acid induced response. Furthermore, LY294002 did not inhibit DNA-binding activity of NF-kappaB but still inhibited NF-kappaB-Luc reporter gene transactivation. These results indicate that PI 3-kinase regulates the transactivation efficiency of NF-kappaB-dependent transcription rather than transduction of NF-kappaB signaling.

Topics: Acetylation; Animals; Chromones; DNA; Electrophoretic Mobility Shift Assay; Enzyme Inhibitors; Enzyme-Linked Immunosorbent Assay; Hydroxamic Acids; In Vitro Techniques; Inflammation; Inflammation Mediators; L-Lactate Dehydrogenase; Lipopolysaccharides; Mice; Microglia; Morpholines; Nerve Tissue Proteins; NF-kappa B; Nitric Oxide; Okadaic Acid; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Rats; Toll-Like Receptors

Bacterial LPS triggers dramatic changes in gene expression in macrophages. We show here that LPS regulated several members of the histone deacetylase (HDAC) family at the mRNA level in murine bone marrow-derived macrophages (BMM). LPS transiently repressed, then induced a number of HDACs (Hdac-4, 5, 7) in BMM, whereas Hdac-1 mRNA was induced more rapidly. Treatment of BMM with trichostatin A (TSA), an inhibitor of HDACs, enhanced LPS-induced expression of the Cox-2, Cxcl2, and Ifit2 genes. In the case of Cox-2, this effect was also apparent at the promoter level. Overexpression of Hdac-8 in RAW264 murine macrophages blocked the ability of LPS to induce Cox-2 mRNA. Another class of LPS-inducible genes, which included Ccl2, Ccl7, and Edn1, was suppressed by TSA, an effect most likely mediated by PU.1 degradation. Hence, HDACs act as potent and selective negative regulators of proinflammatory gene expression and act to prevent excessive inflammatory responses in macrophages.

Topics: Animals; Bone Marrow Cells; Cell Line; Cloning, Molecular; Cyclooxygenase 2; Enzyme Inhibitors; Gene Expression Regulation, Enzymologic; Histone Deacetylase Inhibitors; Histone Deacetylases; Hydroxamic Acids; Inflammation; Lipopolysaccharides; Macrophages; Mice; Mice, Inbred C57BL; Polymerase Chain Reaction; Promoter Regions, Genetic

STAT1 is a transcription factor that plays a crucial role in signaling by interferons (IFNs). In this study we demonstrated that inhibitors of histone deacetylase (HDAC) activity, butyrate, trichostatin A, and suberoylanilide hydroxamic acid, prevented IFNgamma-induced JAK1 activation, STAT1 phosphorylation, its nuclear translocation, and STAT1-dependent gene activation. Furthermore, we showed that silencing of HDAC1, HDAC2, and HDAC3 through RNA interference markedly decreased IFNgamma-driven gene activation and that overexpression of HDAC1, HDAC2, and HDAC3 enhanced STAT1-dependent transcriptional activity. Our data therefore established the essential role of deacetylase activity in STAT1 signaling. Induction of IRF-1 by IFNgamma requires functional STAT1 signaling and was abrogated by butyrate, trichostatin A, suberoylanilide hydroxamic acid, and STAT1 small interfering RNA. In contrast, silencing of STAT1 did not interfere with IFNgamma-induced expression of STAT2 and caspase-7, and HDAC inhibitors did not preclude IFNgamma-induced expression of STAT1, STAT2, and caspase-7, suggesting that HDAC inhibitors impede the expression of IFNgamma target genes whose expression depends on STAT1 but do not interfere with STAT1-independent signaling by IFNgamma. Finally, we showed that inhibitors of deacetylase activity sensitized colon cancer cells to IFNgamma-induced apoptosis through cooperative negative regulation of Bcl-x expression, demonstrating that interruption of the balance between STAT1-dependent and STAT1-independent signaling significantly alters the biological activity of IFNgamma.

Topics: Active Transport, Cell Nucleus; Apoptosis; bcl-X Protein; Blotting, Western; Butyrates; Cell Line; Cell Line, Tumor; Cell Nucleus; DNA-Binding Proteins; Dose-Response Relationship, Drug; Enzyme Inhibitors; Gene Expression Regulation; Gene Silencing; Genes, Reporter; Histone Deacetylase 1; Histone Deacetylase 2; Histone Deacetylases; Humans; Hydroxamic Acids; Inflammation; Interferon-gamma; Microscopy, Fluorescence; Phosphorylation; Proto-Oncogene Proteins c-bcl-2; Repressor Proteins; RNA Interference; RNA, Small Interfering; Serine; Signal Transduction; STAT1 Transcription Factor; Trans-Activators; Transcriptional Activation; Transfection; Tyrosine; Vorinostat

Chronic obstructive pulmonary disease (COPD) is a common chronic inflammatory disease of the lungs with little or no response to glucocorticoids and a high level of oxidative stress. Histone deacetylase (HDAC) activity is reduced in cells of cigarette smokers, and low concentrations of theophylline can increase HDAC activity. We measured the effect of theophylline on HDAC activity and inflammatory gene expression in alveolar macrophages (AM) from patients with COPD. AM from normal smokers showed a decrease in HDAC activity compared with normal control subjects, and this was further reduced in COPD patients (51% decrease, P < 0.01). COPD AMs also showed increased basal release of IL-8 and TNF-alpha, which was poorly suppressed by dexamethasone. Theophylline induced a sixfold increase in HDAC activity in COPD AM lysates and significantly enhanced dexamethasone suppression of induced IL-8 release, an effect that was blocked by the HDAC inhibitor trichostatin A. Therefore, theophylline might restore steroid responsiveness in COPD patients.

Topics: Aged; Blotting, Western; Bronchodilator Agents; Enzyme-Linked Immunosorbent Assay; Female; Glutathione; Histone Deacetylases; Humans; Hydroxamic Acids; Immunohistochemistry; Inflammation; Interleukin-8; Lipopolysaccharides; Macrophages; Male; Middle Aged; Oxidative Stress; Pulmonary Disease, Chronic Obstructive; Smoking; Steroids; Theophylline; Tumor Necrosis Factor-alpha; U937 Cells

Although p53 is known to have dual functions as a transcriptional activator and repressor, there has not been an example where both p53-activating and -repressing elements reside within one target promoter. Previous work from this laboratory defined two different p53 response elements, termed P1 and P2, located at nucleotide positions -70 and -707, respectively, in the rat bradykinin B2 receptor promoter. In this study, through manipulation of the DNA sequence and context, we demonstrate opposing roles for P1 and P2 as transcriptional activator and repressor, respectively. Deletion of P1 abrogates p53-mediated activation. P1 maintains its role as an activator upon relocation to the P2 site and activates transcription from a heterologous promoter construct. Thus, P1 is a bona fide positive p53-response element. In contrast, deletion of P2 enhances P1-induced activation. P2 represses transcription when substituted for P1 or when relocated midway between P1 and P2. P2-mediated repression is sequence-dependent, because it is reversed to activation when P2 is substituted by the P1 or p53 consensus sequences. Moreover, site-directed mutagenesis that converts P2 to a higher affinity p53-binding site results in transcriptional activation rather than repression. Surprisingly, P2 strongly activates a heterologous promoter. Thus, P2-mediated transcriptional repression is both sequence- and context-dependent. Investigations into the mechanisms of P2-mediated repression indicate that it is trichostatin-insensitive and unaffected by CBP or mutation of the minimal repression C-terminal domain of p53. However, gel shift assays suggest that p53 competes with other transcriptional activators for binding to overlapping binding sequences within the P2 element. In conclusion, this study provides a rare example of a transcription factor having two divergent functional effects that are sequence- and context-dependent. The interplay of P1 and P2 may be important in the regulation of bradykinin B2 receptor gene expression in response to inflammatory stress and during development.

Topics: Animals; Base Sequence; Binding Sites; Cell Nucleus; Chloramphenicol O-Acetyltransferase; Gene Deletion; Gene Expression Regulation; HeLa Cells; Humans; Hydroxamic Acids; Inflammation; Models, Genetic; Molecular Sequence Data; Mutagenesis, Site-Directed; Promoter Regions, Genetic; Protein Binding; Protein Structure, Tertiary; Protein Synthesis Inhibitors; Rats; Receptor, Bradykinin B2; Receptors, Bradykinin; Transcriptional Activation; Transfection; Tumor Suppressor Protein p53

Rheumatoid arthritis (RA) is characterized by progressive destruction of the affected joints. The pathophysiology results from genetic susceptibility and autoimmune phenomena, leading to tissue inflammation and synovial hyperplasia termed pannus, which irreversibly destroys cartilage and bone. The current treatment options, which suppress immune responses or ameliorate inflammation, do not halt the destructive process. We found that the histone deacetylase (HDAC) inhibitors (phenylbutyrate and trichostatin A) causing histone hyperacetylation to modulate multiple gene expression not only induced the expression of p21(Cip1) and p16(INK4) in synovial cells but also inhibited the expression of tumor necrosis factor-alpha in affected tissues in adjuvant arthritis, an animal model of RA. Based on the observations that joint swelling is reduced, subintimal mononuclear cell infiltration is decreased, synovial hyperplasia is inhibited, pannus formation is suppressed, and no cartilage or bone destruction is seen, the HDAC inhibitors may represent a new class of compounds for the treatment of RA by simultaneously, coordinately, synergistically, or epigenetically modulating multiple molecular targets in the pathogenesis of RA.

Topics: Acetylation; Animals; Arthritis, Rheumatoid; Blotting, Western; Cell Division; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Disease Models, Animal; Enzyme Inhibitors; Gene Expression Regulation; Histone Deacetylase Inhibitors; Histones; Hydroxamic Acids; Immunohistochemistry; Inflammation; Leukocytes, Mononuclear; Male; Phenylbutyrates; Rats; Rats, Long-Evans; Time Factors; Tumor Necrosis Factor-alpha