Decreased Histone Deacetylase Activity Research Articles

Cryptotanshinone Reverses Corticosteroid Insensitivity by Inhibition of Phosphoinositide-3-Kinase-δ in Chronic Obstructive Pulmonary Disease.

Corticosteroid insensitivity has become a major barrier in the treatment of chronic obstructive pulmonary disease (COPD). It is known that oxidative stress reduces the expression and activity of histone deacetylase (HDAC)-2 by activating phosphoinositide-3-kinase-δ(PI3Kδ)/Akt pathway, which is a common mechanism. The aim of this study was to investigate whether cryptotanshinone (CPT) can improve corticosteroid sensitivity and to investigate the molecular mechanisms by which this occurs. Corticosteroid sensitivity in peripheral blood mononuclear cells (PBMCs) collected from COPD patients, or in human monocytic U937 monocytic cells exposed to cigarette smoke extract (CSE), was quantified as the dexamethasone concentration required to achieve 30% inhibition of tumor necrosis factor-α (TNFα)-induced interleukin 8 (IL-8) production in the presence or absence of cryptotanshinone. PI3K/Akt activity (measured as the relative ratio of phosphorylated Akt at Ser-473 to total Akt) and HDAC2 expression levels were determined by western blotting. HDAC activity was evaluated by a Fluo-Lys HDAC activity assay kit in U937 monocytic cells. Both PBMCs in patients with COPD and U937 cells exposed to CSE were found to be insensitive to dexamethasone, accompanied by increased phosphorylated Akt (pAkt) and decreased HDAC2 protein expression. The pretreatment of cryptotanshinone restored their sensitivity to dexamethasone, and simultaneously downregulated the level of phosphorylated Akt and upregulated the level of HDAC2 protein. Pretreatment with cryptotanshinone or IC87114 reversed the decrease in HDAC activity in CSE-stimulated U937 cells. Cryptotanshinone restores corticosteroid sensitivity induced by oxidative stress via inhibition of PI3Kδ and is a potential treatment for corticosteroid-insensitive diseases such as COPD.

HSV-2 increases replication of HIV in human T cells

Abstract Herpes simplex virus 2 (HSV-2) infection is a major factor driving the global HIV epidemic, leading to increased HIV plasma viral loads (PVLs) and transmission. Previous studies which focused on mucosal responses cannot explain the observed effects of HSV-2 on HIV PVLs. We sought to determine mechanisms by which HSV-2 increases HIV PVLs. CD4 T cells are recruited to sites of HSV-2 lesions and HSV-2 can infect these cells. Phenotyping of CD4 cells infected with HSV-2 in vitro suggested a preference for T follicular helper cells, an HIV latent reservoir. HSV infection of latently infected Jurkat-2D10 cells resulted in a 10-fold increase in HIV ltr expression. After exposing CD4 cells from HIV+ donors to HSV-2, HIV p24 was detected only in cells expressing HSV-2 glycoprotein B (gB), a marker of productive HSV-2 infection. Bulk and single cell RNAseq on FACS-purified, HSV-infected and uninfected CD4 cells demonstrated reduced expression of HIV restriction factors in HSV+ cells; HSV exposed but uninfected cells exhibited higher expression of interferon response genes, potentially explaining their resistance to HIV reactivation. HSV+ cells exhibited increased expression of the lncRNA MALAT1, which may promote HIV reactivation through epigenetic modification, and HSV infection of 2D10 and CD4 cells led to decreased histone deacetylase activity. Transfection of 2D10 cells with HSV protein VP16 resulted in significantly increased HIV ltr and MALAT1 gene expression, and reduced epigenetic silencing marks. HIV proteins were also increased by proteomic analyses. These findings identify several mechanisms by which HSV-2 directly triggers HIV reactivation, such as the up-regulation of the lncRNA MALAT1. Supported by grants from the NIH (R01 AI134367, T32 AI007501, T32 GM007288)

Individual variation in buffalo somatic cell cloning efficiency is related to glycolytic metabolism.

Mammalian individuals differ in their somatic cell cloning efficiency, but the mechanisms leading to this variation is poorly understood. Here we found that high cloning efficiency buffalo fetal fibroblasts (BFFs) displayed robust energy metabolism, looser chromatin structure, high H3K9 acetylation and low heterochromatin protein 1α (HP1α) expression. High cloning efficiency BFFs had more H3K9ac regions near to the upstream of glycolysis genes by ChIP-seq, and involved more openness loci related to glycolysis genes through ATAC-seq. The expression of these glycolysis genes was also found to be higher in high cloning efficiency BFFs by qRT-PCR. Two key enzymes of glycolysis, PDKs and LDH, were confirmed to be associated with histone acetylation and chromatin openness of BFFs. Treatment of low cloning efficiency BFFs with PS48 (activator of PDK1) resulted in an increase in the intracellular lactate production and H3K9 acetylation, decrease in histone deacetylase activity and HP1α expression, less condensed chromatin structure and more cloning embryos developing to blastocysts. These results indicate that the cloning efficiency of buffalo somatic cells is associated with their glycolytic metabolism and chromatin structure, and can be improved by increasing glycolytic metabolism.

Histone deacetylase inhibition by suberoylanilide hydroxamic acid: a therapeutic approach to treat human uterine leiomyoma

To evaluate the effect of inhibition of histone deacetylases (HDACs) by suberoylanilide hydroxamic acid (SAHA) treatment of human uterine leiomyoma primary (HULP) cells invitro on cell proliferation, cell cycle, extracellular matrix (ECM) formation, and transforming growth factor β3 (TGF-β3) signaling. Prospective study comparing uterine leiomyoma (UL) vs. adjacent myometrium (MM) tissue and cells with or without SAHA treatment. Hospital and university laboratories. Women with UL without any hormone treatment. Myomectomy or hysterectomy surgery in women for leiomyoma disease. HDAC activity was assessed by enzyme-linked immunosorbent assay, and gene expression was assessed by quantitative real-time polymerase chain reaction. Effects of SAHA on HULP cells were analyzed by CellTiter(Promega, Madison, Wisconsin), Western blot, and quantitative real-time polymerase chain reaction. The expression of HDAC genes (HDAC1, fold change [FC] = 1.65; HDAC3, FC = 2.08; HDAC6, FC = 2.42) and activity (0.56 vs. 0.10 optical density [OD]/h/mg) was significantly increased in UL vs. MM tissue. SAHA decreased HDAC activity in HULP cells but not in MM cells. Cell viability significantly decreased in HULP cells (81.68% at 5 μM SAHA, 73.46% at 10 μM SAHA), but not in MM cells. Proliferating cell nuclear antigen expression was significantly inhibited in SAHA-treated HULP cells (5 μM SAHA, FC = 0.556; 10 μM SAHA, FC = 0.622). Cell cycle markers, including C-MYC (5 μM SAHA, FC = 0.828) and CCND1 (5 μM SAHA, FC = 0.583; 10 μM SAHA, FC = 0.482), were significantly down-regulated after SAHA treatment. SAHA significantly inhibited ECM protein expression, including FIBRONECTIN (5 μM SAHA, FC = 0.815; 10 μM SAHA, FC = 0.673) and COLLAGEN I (5 μM SAHA, FC = 0.599; 10 μM SAHA, FC = 0.635), in HULP cells. TGFβ3 and MMP9 gene expression was also significantly down-regulated by 10 μM SAHA (TGFβ3, FC = 0.596; MMP9, FC = 0.677). SAHA treatment inhibits cell proliferation, cell cycle, ECM formation, and TGF-β3 signaling in HULP cells, suggesting that histone deacetylation may be useful for treatment of UL.

Histone Deacetylase 5 Is an Early Epigenetic Regulator of Intermittent Hypoxia Induced Sympathetic Nerve Activation and Blood Pressure.

Intermittent hypoxia (IH) is a hallmark manifestation of obstructive sleep apnea (OSA). Long term IH (LT-IH) triggers epigenetic reprogramming of the redox state involving DNA hypermethylation in the carotid body chemo reflex pathway resulting in persistent sympathetic activation and hypertension. Present study examined whether IH also activates epigenetic mechanism(s) other than DNA methylation. Histone modification by lysine acetylation is another major epigenetic mechanism associated with gene regulation. Equilibrium between the activities of histone acetyltransferases (HATs) and histone deacetylases (HDACs) determine the level of lysine acetylation. Here we report that exposure of rat pheochromocytoma (PC)-12 cells to IH in vitro exhibited reduced HDAC enzyme activity due to proteasomal degradation of HDAC3 and HDAC5 proteins. Mechanistic investigations showed that IH-evoked decrease in HDAC activity increases lysine acetylation of α subunit of hypoxia inducible factor (HIF)-1α as well as Histone (H3) protein resulting in increased HIF-1 transcriptional activity. Trichostatin A (TSA), an inhibitor of HDACs, mimicked the effects of IH. Studies on rats treated with 10 days of IH or TSA showed reduced HDAC activity, HDAC5 protein, and increased HIF-1 dependent NADPH oxidase (NOX)-4 transcription in adrenal medullae (AM) resulting in elevated plasma catecholamines and blood pressure. Likewise, heme oxygenase (HO)-2 null mice, which exhibit IH because of high incidence of spontaneous apneas (apnea index 72 ± 1.2 apnea/h), also showed decreased HDAC activity and HDAC5 protein in the AM along with elevated circulating norepinephrine levels. These findings demonstrate that lysine acetylation of histone and non-histone proteins is an early epigenetic mechanism associated with sympathetic nerve activation and hypertension in rodent models of IH.

4-Hexylresorcinol Inhibits Class I Histone Deacetylases in Human Umbilical Cord Endothelial Cells

Histone deacetylases (HDACs) are key enzymes for post-translational modification and influence on various cellular activities. Thus, HDACs are associated with many diseases and their inhibitors have clinical significance. Here, 4-Hexylresorcinol (4HR) was studied as an inhibitor for class I HDACs using the HDAC inhibitor (HDACi) Trichostatin-A as a positive control. The 4HR was administered 1–100 μM to human umbilical endothelial cells (HUVECs) and the HDAC expression and activity were examined. The 4HR decreased the expression level of HDAC1, 3, 4, and 5 in a time and dose-dependent manner. The 4HR also increased acetylated lysine and decreased HDAC activity significantly (p < 0.05). Collectively, 4HR was a new class I HDAC inhibitor that reduced the expression and activity of HDAC in HUVECs.

Exercise enhances the expression of brain-derived neurotrophic factor in the hippocampus accompanied by epigenetic alterations in senescence-accelerated mice prone 8

Exercise increases the expression of brain-derived neurotrophic factor (BDNF) in the hippocampus and beneficially contributes to cognitive function and is accompanied by epigenetic changes. Specifically, the activity levels of histone acetyltransferases (HATs) and histone deacetylases (HDACs) regulate histone acetylation and modulate gene transcription. The objective of the present study was to assess the effect of an exercise regimen over a long period on the expression of BDNF, tropomyosin receptor kinase B (TrkB) and p75, and the activity of HATs and HDACs in the degenerative hippocampus. We used senescence-accelerated mice (SAM), and specifically, 3-month-old SAM resistant 1 (SAMR1) and SAM prone 8 (SAMP8) strains. Mice were distributed into four groups based on accelerated senescence and exercise status. Mice in the exercise groups exercised on a treadmill for 45 min a day, 3 days a week, for 6 months. Exercise significantly increased BDNF expression and decreased the expression of p75 in both SAMR1 and SAMP8. In addition, aging decreased HAT activity, whereas exercise decreased HDAC activity and increased the activity ratio of HAT to HDAC (HAT/HDAC). Therefore, the present study revealed that despite accelerated senescence, exercise up-regulated the expression of BDNF along with decreased the expression of p75, a receptor involved in apoptotic signaling. Furthermore, exercise increased HAT/HDAC, which might beneficially contribute to the transcriptional regulation for degenerative changes in the hippocampus.

Sulforaphane absorption and histone deacetylase activity following single dosing of broccoli sprout supplement in normal dogs.

The role of epigenetic alterations during cancer has gained increasing attention and has resulted in a paradigm shift in our understanding of mechanisms leading to cancer susceptibility. Sulforaphane (SFN) is a naturally occurring isothiocyanate derived from the precursor glucosinolate, glucoraphanin (GFN), which is found in cruciferous vegetables such as broccoli. Sulforaphane has been shown to suppress tumour growth by several mechanisms including inhibiting histone deacetylases. The objective of this study was to provide a detailed analysis of sulforaphane absorption following a single oral dose of a broccoli sprout supplement in normal dogs. A single dose of broccoli sprout supplement (with active myrosinase) was orally administered to 10 healthy adult dogs. Blood and urine samples were collected prior to dosing, and at various time points post‐dosing. Plasma total SFN metabolite levels peaked at 4 h post‐consumption and were cleared by 24 h post‐consumption. Urinary SFN metabolites peaked at 4 h post‐consumption, and remained detectable at 24 and 48 h post‐supplement consumption. A trend for decrease in histone deacetylase activity was observed at 1 h post‐consumption and a significant decrease was observed at 24 h post‐consumption. The data presented herein indicate that oral SFN is absorbed in dogs, SFN metabolites are detectable in plasma and urine post‐dosing, and SFN and its metabolites have some effect on histone deacetylase activity following a single dose.

Anticancer properties of sodium selenite in human glioblastoma cell cluster spheroids

Glioblastoma (GBM) is the most common type of primary tumor of the central nervous system with a poor prognosis, needing the development of new therapeutic drugs. Few studies focused on sodium selenite (SS) effects in cancer cells cultured as multicellular tumor spheroids (MCTS or 3D) closer to in vivo tumor.We investigated SS anticancer effects in three human GBM cell lines cultured in 3D: LN229, U87 (O(6)-methyguanine-DNA-methyltransferase (MGMT) negative) and T98G (MGMT positive). SS absorption was evaluated and the cytotoxicity of SS and temozolomide (TMZ), the standard drug used against GBM, were compared. SS impacts on proliferation, cell death, and invasiveness were evaluated as well as epigenetic modifications by focusing on histone deacetylase (HDAC) activity and dimethyl-histone-3-lysine-9 methylation (H3K9m2), after 24h to 72h SS exposition.SS was absorbed by spheroids and was more cytotoxic than TMZ (i.e., for LN229, the IC50 was 38 fold-more elevated for TMZ than SS, at 72h). SS induced a cell cycle arrest in the S phase and apoptosis via caspase-3. SS decreased carbonic anhydrase-9 (CA9) expression, invasion on a Matrigel matrix and modulated E- and N-Cadherin transcript expressions. SS decreased HDAC activity and modulated H3K9m2 levels.3D model provides a relevant strategy to screen new drugs and SS is a promising drug against GBM that should now be tested in GBM animal models.

Delphinidin induces apoptosis via cleaved HDAC3-mediated p53 acetylation and oligomerization in prostate cancer cells.

Delphinidin is a major anthocyanidin compound found in various fruits. It has anti-inflammatory, anti-oxidant, and various other biological activities. In this study, we identified the epigenetic modulators that mediate the apoptotic effect of delphinidin in human prostate cancer cells. We found that treatment of LNCaP cells (a p53 wild-type, human prostate cancer cell line) with delphinidin increased caspase-3, −7, and −8 activity, whereas it decreased histone deacetylase activity. Among class I HDACs, the activity of HDAC3 was specifically inhibited by delphinidin. Moreover, the induction of apoptosis by delphinidin was dependent on caspase-mediated cleavage of HDAC3, which results in the acetylation and stabilization of p53. We also observed that delphinidin potently upregulated pro-apoptotic genes that are positively regulated by p53, and downregulated various anti-apoptotic genes. Taken together, these results show that delphinidin induces p53-mediated apoptosis by suppressing HDAC activity and activating p53 acetylation in human prostate cancer LNCaP cells. Therefore, delphinidin may be useful in the prevention of prostate cancer.

Chromatin remodeling by curcumin alters endogenous aryl hydrocarbon receptor signaling

The aim of this study was to gain more information about the mechanisms that regulate expression of the aryl hydrocarbon receptor (AHR) target gene CYP1A1. Human hepatoma cells (HepG2 and Huh7) and human immortalized keratinocytes (HaCaT) were treated with different concentrations of the dietary polyphenolic compound curcumin (CUR) alone or in combination with the natural AHR agonist 6-formylindolo[3,2-b]carbazole (FICZ). In an earlier study, we described that CUR can activate the AHR indirectly by inhibiting metabolic clearance of FICZ. Here, we measured cell viability, activation of AHR signaling, oxidative stress and histone modifying activities in response to CUR at concentrations ranging from 0.1 to 50 μM. We observed apparent non-linear responses on cell viability and activation of AHR signaling. The CYP1A1 expression and the CYP1A1 enzyme activity in the presence of CUR reflected the histone acetylation efficiency observed in nuclear extracts. At the lowest concentration, CUR significantly decreased histone deacetylase activity and increased the FICZ-induced CYP1A1 activity. In contrast, at the highest concentration, CUR increased the formation of reactive oxygen species, significantly inhibited histone acetylation, and temporally decreased FICZ-induced CYP1A1 activity. The results suggest that CUR can both increase and decrease the accessibility of DNA and thereby influence transcriptional responses to the ligand-activated AHR. This suggestion was supported by the fact that chromatin remodeling treatments with trichostatin A, p300, or 5-aza-dC increased CYP1A1 transcription. We conclude that the AHR-dependent transcriptional efficiency is modified by factors that influence the cellular redox status and the chromatin structure.

Elevated nuclear sphingoid base-1-phosphates and decreased histone deacetylase activity after fumonisin B1 treatment in mouse embryonic fibroblasts

Fumonisin B1 (FB1) is a mycotoxin produced by a common fungal contaminant of corn. Administration of FB1 to pregnant LM/Bc mice induces exencephaly in embryos, and ingestion of FB1-contaminated food during early pregnancy is associated with increased risk for neural tube defects (NTDs) in humans. FB1 inhibits ceramide synthase enzymes in sphingolipid biosynthesis, causing sphinganine (Sa) and bioactive sphinganine-1-phosphate (Sa1P) accumulation in blood, cells, and tissues. Sphingosine kinases (Sphk) phosphorylate Sa to form Sa1P. Upon activation, Sphk1 associates primarily with the plasma membrane, while Sphk2 is found predominantly in the nucleus. In cells over-expressing Sphk2, accumulation of Sa1P in the nuclear compartment inhibits histone deacetylase (HDAC) activity, causing increased acetylation of histone lysine residues. In this study, FB1 treatment in LM/Bc mouse embryonic fibroblasts (MEFs) resulted in significant accumulation of Sa1P in nuclear extracts relative to cytoplasmic extracts. Elevated nuclear Sa1P corresponded to decreased histone deacetylase (HDAC) activity and increased histone acetylation at H2BK12, H3K9, H3K18, and H3K23. Treatment of LM/Bc MEFs with a selective Sphk1 inhibitor, PF-543, or with ABC294640, a selective Sphk2 inhibitor, significantly reduced nuclear Sa1P accumulation after FB1, although Sa1P levels remained significantly increased relative to basal levels. Concurrent treatment with both PF-543 and ABC294640 prevented nuclear accumulation of Sa1P in response to FB1. Other HDAC inhibitors are known to cause NTDs, so these results suggest that FB1-induced disruption of sphingolipid metabolism leading to nuclear Sa1P accumulation, HDAC inhibition, and histone hyperacetylation is a potential mechanism for FB1-induced NTDs.

Sodium Selenite Decreased HDAC Activity, Cell Proliferation and Induced Apoptosis in Three Human Glioblastoma Cells.

Selenium (Se) is an essential trace element for human health which also has antitumor properties. Little is known about its effects on brain tumor cells (BTC). The aim of this study was to investigate the anticancer effects of sodium selenite (SS) including histone deacetylase (HDAC) activity in three human glioblastoma (GBM) cell lines (LN229, T98G and U87). LN229, T98G and U87 GBM cell lines were treated with variable doses of SS for time varying from 24 to 72h. HDAC activity, cell proliferation, toxicity, cell death process, caspase-3 and MMP2 activities and Se absorption were evaluated. SS modulated all the parameters tested in a dose- and time-dependent manner. We found that SS decreased HDAC activity, blocked cell proliferation and cell cycle at the G2 phase, triggered an apoptotic cell death process caspase-3-dependent and reduced MMP2 activities. All these effects were performed whereas SS was weakly absorbed (<2%). SS decreasing HDAC activity exhibited interesting antitumor properties in GBM cells which may be taken into account in the novel strategies for achieving tumor growth inhibition and cytotoxicity. Epigenetic modifications induced by SS should be evaluated in further studies.

Elevated Nuclear and Cytoplasmic FTY720-Phosphate in Mouse Embryonic Fibroblasts Suggests the Potential for Multiple Mechanisms in FTY720-Induced Neural Tube Defects.

FTY720 (fingolimod) is a U.S. Food and Drug Administration-approved drug to treat relapsing remitting multiple sclerosis. FTY720 treatment in pregnant inbred LM/Bc mice results in approximately 60% of embryos having a neural tube defect (NTD). Sphingosine kinases (Sphk1, Sphk2) phosphorylate FTY720 invivo to form the bioactive metabolite FTY720-1-phosphate (FTY720-P). Cytoplasmic FTY720-P is an agonist for 4 of the 5 sphingosine-1-phosphate (S1P) receptors (S1P1, 3-5) and can also act as a functional antagonist of S1P1, whereas FTY720-P generated in the nucleus inhibits histone deacetylases (HDACs), leading to increased histone acetylation. This study demonstrates that treatment of LM/Bc mouse embryonic fibroblasts (MEFs) with FTY720 results in a significant accumulation of FTY720-P in both the cytoplasmic and nuclear compartments. Elevated nuclear FTY720-P is associated with decreased HDAC activity and increased histone acetylation at H3K18 and H3K23 in LM/Bc MEFs. Treatment of LM/Bc MEFs with FTY720 and a selective Sphk2 inhibitor, ABC294640, significantly reduces the amount of FTY720-P that accumulates in the nucleus. The data provide insight into the relative amounts of FTY720-P generated in the nuclear versus cytoplasmic subcellular compartments after FTY720 treatment and the specific Sphk isoforms involved. The results of this study suggest that FTY720-induced NTDs may involve multiple mechanisms, including: (1) sustained and/or altered S1P receptor activation and signaling by FTY720-P produced in the cytoplasm and (2) HDAC inhibition and histone hyperacetylation by FTY720-P generated in the nucleus that could lead to epigenetic changes in gene regulation.

Haemophilus influenzae induces steroid-resistant inflammatory responses in COPD.

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

Over‐expression of prothymosin‐α antagonizes TGFβ signalling to promote the development of emphysema

Emphysema, a major consequence of chronic obstructive pulmonary disease (COPD), is characterized by the permanent airflow restriction resulting from enlargement of alveolar airspace and loss of lung elasticity. Transforming growth factor-β (TGFβ) signalling regulates the balance of matrix metalloproteinase (MMP)/tissue inhibitor of matrix metalloproteinase (TIMP) to control matrix homeostasis. Patients with COPD have dysregulated TGFβ signalling and reduced histone deacetylase (HDAC) activity through epigenetic up-regulation of histone acetylation in the promoters of pro-inflammatory genes. However, the potential link between decreased HDAC activity and dysregulated TGFβ signalling in emphysema pathogenesis remains to be determined. Prothymosin α (ProT), a highly conserved acidic nuclear protein, plays a role in the acetylation of histone and non-histone proteins. The aim of this study was to test the hypothesis that ProT inhibits TGFβ-Smad signalling through Smad7, thereby contributing to emphysema pathogenesis. We show that ProT enhances Smad7 acetylation by decreasing its association with HDAC and thereby down-regulates TGFβ-Smad signalling. ProT caused an imbalance between MMP and TIMP through acetylated Smad7 in favour of MMP expression. In addition to interfering with R-Smad activation and targeting receptors for degradation in the cytoplasm, acetylated Smad7 potentiated by ProT competitively antagonized binding of the pSmad2/3-Smad4 complex to the TIMP-3 promoter, resulting in reduced TIMP-3 expression. These effects were detected in ProT-over-expressing cells, lungs of ProT transgenic mice displaying an emphysema phenotype and in emphysema patients. Importantly, increased Smad7 and reduced TIMP-3 were found in the lungs of emphysema patients and mice with cigarette smoke extract (CSE)-induced emphysema. Such effects could be abrogated by silencing endogenous ProT expression. Collectively, our results uncover acetylated Smad7 regulated by ProT as an important determinant in dysregulated TGFβ signalling that contributes to emphysema pathogenesis.

Toll-like receptor-3 mediates HIV-1 transactivation via NFκB and JNK pathways and histone acetylation, but prolonged activation suppresses Tat and HIV-1 replication

TLR3 has been implicated in the pathogenesis of several viral infections, including SIV- and HIV-1-induced inflammation and AIDS. However the molecular mechanisms of these TLR3-mediated effects are not known, and it is not known whether HIV interacts with cellular TLR3 to affect disease process. Here we investigate the effects of TLR3 ligands on HIV-1 transactivation using both primary human macrophages and cells containing integrated copies of the HIV-1 promoter. We demonstrate that TLR3 activation induced upregulation of transcription factors such as c-Jun, CCAAT/enhancer-binding protein alpha (CEBPA), signal transducer and activator of transcription (STAT)-1, STAT-2, RELB, and nuclear factor kappa-B1 (NFκB1), most of which are known to regulate the HIV promoter activity. We also demonstrate that TLR3 activation increased HIV-1 transactivation via the c-Jun N-terminal kinase (JNK) and NFκB pathways. This was associated with epigenetic modifications, including decreased histone deacetylase activity, increased histone acetyl transferase (HAT) activity, and increased acetylation of histones H3 and H4 at lysine residues in the nucleosome-0 and nucleosome-1 of the HIV-1 promoter. However, prolonged TLR3 activation decreased HIV-1 transactivation, decreased HAT activity and Tat transcription, and suppressed viral replication. Overall, data suggests that TLR3 can act as viral sensor to mediate viral transactivation, cellular signaling, innate immune response, and inflammation in HIV-infected humans. Our study provides novel insights into the molecular basis for these TLR3-mediated effects.

The mitogen-activated protein kinase CgHog1 is required for iron homeostasis, adherence and virulence in Candida glabrata.

Candida glabrata has emerged as a major fungal pathogen over the last two decades, although our understanding of its survival strategies inside the mammalian host remains rudimentary. An important requirement for survival in vivo is the ability to acquire critical nutrients such as iron from host niches of varied iron content. In the present study, we demonstrate for the first time that C. glabrata cells respond to high external iron levels via activation of two stress-responsive mitogen-activated protein kinases, CgHog1 and CgSlt2, and lack of either kinase results in sensitivity to the high-iron medium. Furthermore, we show that CgHOG1 deletion led to perturbed iron homeostasis (elevated intracellular iron content and high mitochondrial aconitase activity), reduced survival in macrophages and attenuated virulence in the murine model of disseminated candidiasis. Consistently, several genes implicated in iron acquisition and storage displayed deregulated expression in the Cghog1∆ mutant. Genome-wide transcriptional profiling analysis revealed upregulation of genes implicated in DNA repair, RNA processing and autophagy, and downregulation of genes related to cellular respiration and organonitrogen compound metabolism under iron-limiting conditions. In contrast, genes involved in the respiratory electron transport chain were induced under iron-replete conditions. Gene expression microarrays also identified a set of iron-responsive regulon in C. glabrata. Lastly, we present evidence for the iron-regulated expression of the major adhesin-encoding EPA1 gene, decreased histone deacetylase activity in a high-iron environment and increased adherence of iron-surplus-medium-grown C. glabrata cells to epithelial cells. Together, our findings yield novel insights into iron abundance-based regulation of transcriptional and mitogen-activated protein kinase signaling pathways in C. glabrata.

Comparative Study between Valproic Acid Combined with Conventional Chemotherapy Versus Conventional Chemotherapy Alone in Egyptian Acute Myeloid Leukemia Patients

It has been postulated that inhibition of Histone Deacetylase (HDAC) can restore normal acetylation of histone proteins and transcription factors, and to be of benefit in the treatment of cancer. So, this study aimed at assessing the response to histone deacetylase inhibitor valproic acid combined with conventional chemotherapy versus conventional chemotherapy alone in Egyptian Acute Myeloid Leukemia (AML) patients. Thirty newly diagnosed AML patients were divided into 2 groups. Group 1 consisted of 15 AML patients received conventional chemotherapy while group 2 consisted of 15 AML patients received valproic acid 40 mg/kg for 7 days and conventional chemotherapy. Ten healthy persons of matched age and sex were considered group 3 (controls). Serum histone deacetylase activity, vascular endothelial growth factor, basic fibroblast growth factor, tumor necrosis factor α, glutathione S transferase and nuclear factor κB in nuclear extract before and after chemotherapy were measured in all patients and controls. Results revealed better clinical response with no side effects with valproic acid than conventional chemotherapy alone (p=0.021). This was associated with statistically significant decrease in histone deacetylase activity in patients receiving valproic acid compared to the other AML group (p=0.00019). There was significant negative correlation between age and HDAC activity at initial presentation in patients receiving valproic acid (p=0.039) while no significant correlation was detected with the other studied laboratory parameters. Our results revealed that valproic acid in the tested dose was safe and associated with better therapeutic response when used in combination with conventional chemotherapy.

Oxidative stress regulates IL-4 gene expression in mast cells through the reduction of histone deacetylase.

Many proinflammatory cytokines are regulated by the acetylation and deacetylation of the core histone. Since dysregulation of T helper 2 cytokine production is a key in the pathogenesis of allergic diseases, we examined the role of histone deacetylase (HDAC) on interleukin (IL)-4 gene expression in mast cells. We also examined whether oxidative stress has any impact on HDAC activity. In vitro study. Academic research laboratory. An IgE-sensitized mast cell line (RBL-2H3 cells) was treated with varying concentrations of the HDAC inhibitors trichostatin A (TSA) and H2O2 and stimulated with antigens. The amount of IL-4 gene expression was quantified by real-time polymerase chain reaction. Quantitative measurement of IL-4 in the cell supernatant was performed using enzyme-linked immunosorbent assay. Moreover, HDAC activity was measured with the use of a nonisotopic assay that utilized an HDAC Fluorescent Activity Assay Kit. IL-4 mRNA expression was induced by antigens in IgE-sensitized RBL-2H3 cells. Pretreatment with TSA and H2O2 enhanced IL-4 mRNA expression up to 5-fold in a dose-dependent manner. Furthermore, HDAC activity in RBL-2H3 cells was reduced after treatment with H2O2. Our results suggest that oxidative stress may up-regulate IL-4 gene expression in mast cells via a decrease in HDAC activity.