Expression Levels Of Histone Deacetylases Research Articles

Abstract 5395: The influence of hyperthermia on epigenetic signaling in dysbiotic tumor vasculature

Abstract The overall use of antibiotics has increased significantly in recent years. Although they resolve infections, oral antibiotics (ABX) severely alter the gut microbiota, disrupting commensal bacteria crucial for maintenance of various homeostatic processes. ABX-induced microbial imbalance, or dysbiosis, has systemic and long-lasting deleterious effects on the host. For example, we and others have shown that distal, non-GI tract solid tumors show accelerated progression during dysbiosis due to vascular immune suppression. Localized hyperthermia has been increasingly recognized to elicit various cellular responses, yet the epigenetic effects of hyperthermia on the tumor vasculature during ABX-induced dysbiosis are unknown. The balance of histone acetylation and deacetylation plays a critical role in the regulation of gene expression and thereby cellular processes. Therefore, we assessed which histone deacetylases (HDACs) are expressed and their abundance in endothelial cells of various sources with or without dysbiotic stress and/or hyperthermia to understand the utility of thermal medicine to promote normal functioning of the vasculature in dysbiotic patients. We found that HDAC expression levels differed among endothelial origins and that hyperthermia (60 min of either 41.5 °C or 43 °C) influenced the HDAC levels differentially. Especially HDAC 2, 3 and 8 were inhibited by hyperthermia. Additionally, we found that the vasculature receptors for endogenous HDAC inhibitors, GPR41, GPR43, and GPR109a are less abundant intrinsically on dysbiotic than orthobiotic tumor endothelial cells and that hyperthermia increased this expression. Although further studies are warranted, these results demonstrate that hyperthermia exerts epigenetic effects and is capable of overcoming the dysbiosis-induced phenotype. Since anti-cancer treatment with HDAC inhibitors has shown promise in the clinic but is hindered by systemic toxicities, localized hyperthermia may become a viable alternative in the future. Citation Format: Hailey Kristian, Samir Jenkins, Robert Griffin, Ruud Petrus Dings. The influence of hyperthermia on epigenetic signaling in dysbiotic tumor vasculature [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5395.

Icariside II protects dopaminergic neurons from 1‑methyl‑4‑phenylpyridinium‑induced neurotoxicity by downregulating HDAC2 to restore mitochondrial function.

Parkinson's disease (PD) is the second most common neurodegenerative disease after Alzheimer's disease (AD). Icariside II (ICS II) is known to confer notable therapeutic effects against a variety of neurodegenerative diseases, such as AD. Therefore, the present study aimed to evaluate the possible effects of ICS II on 1-methyl-4-phenylpyridinium (MPP+)-induced SK-N-SH cell injury, in addition to understanding the underlying mechanism of action. The MPP+-induced SK-N-SH cell model was used to simulate PD in vitro. The viability and mitochondrial membrane potential of SK-N-SH cells were detected by MTT assay and JC-1 staining, respectively. Lactate dehydrogenase (LDH) release, ATP levels and complex I activity in treated SK-N-SH cells were measured using LDH activity, ATP and Complex I assay kits, respectively. The protein expression levels of histone deacetylase 2 (HDAC2) and γ-H2A histone family member X and the copy number of mitochondrial DNA were measured by western blotting or reverse transcription-quantitative PCR, respectively. Autodock 4.2 was used to predict the molecular docking site of ICS II on HDAC2. The results of the present study demonstrated that ICS II mitigated SK-N-SH cytotoxicity induced by MPP+. Specifically, ICS II alleviated DNA damage and restored mitochondrial function in SK-N-SH cells treated with MPP+. In addition, ICS II reduced the HDAC2 protein expression levels in MPP+-induced SK-N-SH cells. However, overexpression of HDAC2 reversed the protective effects of ICS II on DNA damage and mitochondrial dysfunction in MPP+-induced SK-N-SH cells. In conclusion, the results of the present study suggest that ICS II can protect dopaminergic neurons from MPP+-induced neurotoxicity by downregulating HDAC2 expression to restore mitochondrial function.

Caffeic acid improves intestinal barrier functions by regulating colonic bacteria and tight junction protein expression and alleviating inflammation in weaning piglets

The experiment investigated the effect of caffeic acid on bacteria, short-chain fatty acids (SCFA), and the expression of tight junction protein and inflammation related genes in the colon of weaning piglets. Thirty-six weaning piglets were allocated to three treatment groups, which were fed with a basal diet, a basal diet supplemented with 250 mg/kg or 500 mg/kg caffeic acid for 28 days. The results showed that caffeic acid treatment increased the contents of acetate acid, propionate acid and total SCFA. Moreover, real-time quantitative PCR showed that the number of Bifidobacterium (p < 0.05) and Lactobacillus (p < 0.05) were increased and the number of Escherichia coli (p < 0.05) was decreased by caffeic acid in colonic mucosa. Real-time quantitative PCR also showed that the mRNA levels of zonula occludens-1 (p < 0.01), claudin-1 (p < 0.01), occludin (p < 0.01), mucin 1 (MUC1) (p < 0.01), MUC2 (p < 0.01), interleukin 4 (IL-4) (p < 0.01) and IL-10 (p < 0.05) were increased, while the mRNA expression levels of histone deacetylases (p < 0.01), IL-1 (p < 0.01), IL-6 (p < 0.01) and tumor necrosis factor-α (TNF-α) (p < 0.01) were decreased, by caffeic acid in colonic mucosa. These results suggested that caffeic acid could improve intestinal barrier function in weaned pigs, which might be mediated by regulating colonic bacteria and tight junction protein expression and alleviating inflammation.

EGCG prevents pressure overload‑induced myocardial remodeling by downregulating overexpression of HDAC5 in mice.

Myocardial remodeling is a complex pathological process and its mechanism is unclear. The present study investigated whether epigallocatechin gallate (EGCG) prevents myocardial remodeling by regulating histone acetylation and explored the mechanisms underlying this effect in the heart of a mouse model of transverse aortic constriction (TAC). A TAC mouse model was created by partial thoracic aortic banding (TAB). Subsequently, TAC mice were injected with EGCG at a dose of 50 mg/kg/day for 12 weeks. The hearts of mice were collected for analysis 4, 8 and 12 weeks after TAC. Histopathological changes in the heart were observed by hematoxylin and eosin, Masson trichrome and wheat germ agglutinin staining. Protein expression levels were investigated using western blotting. Cardiac function of mice was detected by echocardiography. The level of histone acetylated lysine 27 on histone H3 (H3K27ac) first increased and then decreased in the hearts of mice at 4, 8 and 12 weeks after TAC. The expression levels of two genes associated with pathological myocardial remodeling, atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP), also increased initially but then decreased. The expression levels of histone deacetylase 5 (HDAC5) gradually increased in the hearts of mice at 4, 8 and 12 weeks after TAC. Furthermore, EGCG increased acetylation of H3K27ac by inhibiting HDAC5 in the heart of TAC mice treated with EGCG for 12 weeks. EGCG normalized the transcriptional activity of heart nuclear transcription factor myocyte enhancer factor 2A in TAC mice treated for 12 weeks. The low expression levels of myocardial remodeling-associated genes (ANP and BNP) were reversed by EGCG treatment for 12 weeks in TAC mice. In addition, EGCG reversed cardiac enlargement and improved cardiac function and survival in TAC mice when treated with EGCG for 12 weeks. Modification of the HDAC5-mediated imbalance in histone H3K27ac served a key role in pathological myocardial remodeling. The present results show that EGCG prevented and delayed myocardial remodeling in TAC mice by inhibiting HDAC5.

Roles of HDAC2, eIF5, and eIF6 in Lung Cancer Tumorigenesis

The expression levels of histone deacetylase 2 (HDAC2), eukaryotic initiation factor 5 (eIF5), and eukaryotic initiation factor 6 (eIF6), and relationship between HDAC2 and eIF5 or eIF6 in lung cancer tissues were investigated, in order to charify the relationship between HDAC2 and the prognosis of lung cancer patients and its influence on the expression of eIF5 and eIF6. The expression of HDAC2, eIF5, and eIF6 in lung cancer tissues was detected by quantitative reverse transcription polymerase chain reaction. The expression correlation between HDAC2 and eIF5 or eIF6 was tested using a t test. The correlation between HDAC2 and eIF5 or eIF6 was analyzed using the TCGA database. The identified cells were constructed with small interfering siRNA and HDAC2 overexpression plasmid. The proliferation and migration ability of the identified cells was investigated by CCK8 and Transwell assays, respectively. HDAC2, eIF5, and eIF6 were overexpressed in lung cancer tissues, and HDAC2 expression level was negatively correlated with the prognosis of lung cancer patients. HDAC2 expression level was positively correlated with eIF5 and eIF6 expression levels. HDAC2 could regulate the expression of eIF5 and eIF6. The regulation of proliferation and invasion of lung cancer cells by HDAC2 depended on eIF5 and eIF6. HDAC2, eIF5, and eIF6 were closely related with lung cancer tumorigenesis, which might be potential biological markers and therapeutic targets for lung cancer.

Expression and prognostic analyses of HDACs in human gastric cancer based on bioinformatic analysis.

Gastric cancer (GC) is a common cancerous tumor, and is the third leading cause of cancer mortality worldwide. Although comprehensive therapies of GC have been widely used in clinical set ups, advanced gastric cancer carries is characterized by poor prognosis, probably due to lack of effective prognostic biomarkers. Mammalian histone deacetylase family, histone deacetylases (HDACs), play significant roles in initiation and progression of tumors. Aberrant expression of HDACs is reported in many cancer types including gastric cancer, and may serve as candidate biomarkers or therapeutic targets for GC patients.Gene Expression Profiling Interactive Analysis was used to explore mRNA levels of HDACs in GC. Kaplan–Meier plotter was used to determine the prognostic value of HDACs mRNA expression in GC. Genomic profiles including mutations of HDACs were retrieved from cBioPortal webserver. A protein–protein interaction network was constructed using STRING database. GeneMANIA was used to retrieve additional genes or proteins related to HDACs. R software was used for functional enrichment analyses.Analysis of mRNA levels of HDAC1/2/4/8/9 showed that they were upregulated in GC tissues, whereas HDAC6/10 was downregulated in GC tissues. Aberrant expression of HDAC1/3/4/5/6/7/8/10/11 was all correlated with prognosis in GC. In addition, expression levels of HDACs were correlated with different Lauren classifications, and clinical stages, lymph node status, treatment, and human epidermal growth factor receptor 2 status in GC.The findings of this study showed that HDAC members are potential biomarkers for diagnosis or prognosis of gastric cancer. However, further studies should be conducted to validate these findings.

Dynamic Regulation of Histone Acetylation By Nuclear Proteolysis Controls Cell Cycle Gene Expression and Chromosome Integrity in Multiple Myeloma

Transcription factors are generally short-lived proteins that undergo active turnover. The dynamic interaction of transcription factors and co-regulators with promoters and enhancers allows cells to continuously adjust gene expression. Whereas the composition and binding of transcription factors at genomic sites is the focus of a widespread research effort, relatively little is known about how these complexes are being removed by the ubiquitin-proteasome system (UPS).Multiple myeloma (MM), the second most common hematopoietic malignancy, has become a model disease for drugs that interfere with the UPS through either blocking or facilitating protein elimination. The proteasome inhibitor Bortezomib, for instance, is used as first-line treatment in myeloma; yet, the process by which myeloma cells are killed by this drug is ill-defined. Since transcription factors are prime targets of proteasomal degradation, our research is focused on defining how proteolysis regulates transcriptional dynamics in this disease and determining the therapeutic relevance for treatment with proteasome inhibitors.Following proteasome inhibition in multiple myeloma cell lines, we performed chromatin-immunoprecipitation for histone H3 acetylation (K27) and multiple histone deacetylases and used next generation sequencing (ChIP-seq) to identify unique gene clusters that are actively regulated by the proteasome and quantify epigenetic changes in dependence of protein turnover. Our findings reveal that cell cycle genes, particularly subsets of genes involved in centromere formation and sister chromatid segregation during mitosis, are associated with nuclear protein turnover and are transcriptionally repressed by proteasome inhibition. Notably, proteasome inhibition increased the recruitment of specific histone deacetylases (HDACs) to the promoters of genes involved in centromere formation. We are investigating the mechanisms for UPS regulation of HDAC abundance at specific genomic locations. Moreover, data analysis of a panel of multiple myeloma patients shows that the expression levels of HDACs correlate with patient survival, specifically when treated with proteasome inhibitors. We are currently exploring how histone modifications and proteasome activity crosstalk in a therapeutically relevant manner in multiple myeloma.Finally, to determine whether epigenetic regulation of centromere stability might play a role in sensitivity or the development of resistance to proteasome inhibitors, we established and optimized long-term treatment of multiple myeloma cell lines with low doses of proteasome inhibitors. Remarkably, we observed reduced immunofluorescence staining for centromeric protein A (CENP-A) in multiple myeloma cells receiving drugs over an extended period, suggesting a loss of centromere integrity.This research project will contribute to our understanding of epigenetic and transcriptional dynamics in MM. With our focus on the continuously changing abundance of transcription factors and co-regulators at promoters of cell cycle genes, we seek to unlock new pathways for molecular therapy, as well as identify more specific targets for treatment compared to blunt proteasome inhibition. Elucidating the mechanisms of action and resistance to proteasome inhibition will help advance future MM therapy. DisclosuresLulla:ASH Scholar Award: Research Funding; Leukemia Texas Research grant: Research Funding; Junior Faculty sees funding-Baylor College of Medicine: Research Funding; Lymphoma SPORE: Research Funding; ASBMT Young Investigator Award: Research Funding; Leukemia Texas: Membership on an entity's Board of Directors or advisory committees. Catic:Cancer Prevention and Research Institute of Texas: Research Funding; Ted Nash Long Life Foundation: Research Funding.

H3K14 hyperacetylation‑mediated c‑Myc binding to the miR‑30a‑5p gene promoter under hypoxia postconditioning protects senescent cardiomyocytes from hypoxia/reoxygenation injury.

Our previous study reported that microRNA (miR)-30a-5p upregulation under hypoxia postconditioning (HPostC) exert a protective effect on aged H9C2 cells against hypoxia/reoxygenation injury via DNA methyltransferase 3B-induced DNA hypomethylation at the miR-30a-5p gene promoter. This suggests that miR-30a-5p may be a potential preventative and therapeutic target for ischemic heart disease in aged myocardium. The present study aimed to investigate the underlying mechanisms of miR-30a-5p transcription in aged myocardium in ischemic heart disease. Cardiomyocytes were treated with 8 mg/ml D-galactose for 9 days, and then exposed to hypoxic conditions. Cell viability was determined using a cell viability assay. Expression levels of histone deacetylase 2 (HDAC2), LC3B-II/I, beclin-1 and p62 were detected via reverse transcription-quantitative PCR and western blotting. Chromatin immunoprecipitation-PCR and luciferase reporter assays were performed to evaluate the effect of c-Myc binding and activity on the miR-30a-5p promoter in senescent cardiomyocytes following HPostC. It was found that HPostC enhanced the acetylation levels of H3K14 at the miR-30a-5p gene promoter in senescent cardiomyocytes, which attributed to the decreased expression of HDAC2. In addition, c-Myc could positively regulate miR-30a-5p transcription to inhibit senescent cardiomyocyte autophagy. Mechanically, it was observed that increased H3K14 acetylation level exposed to romidepsin facilitated c-Myc binding to the miR-30a-5p gene promoter region, which led to the increased transcription of miR-30a-5p. Taken together, these results demonstrated that HDAC2-mediated H3K14 hyperacetylation promoted c-Myc binding to the miR-30a-5p gene promoter, which contributed to HPostC senescent cardioprotection.

Anti-inflammatory Effects of Proanthocyanidin-rich Cranberry Extract through the Suppression of NF-kB Pathway and Histone Acetylase in RAW 264.7 and Mouse Bone Marrow-derived Macrophages

Obesity-mediated chronic inflammation promotes the progression of obesity to metabolic anti-inflammatory effect of cranberries by decreasing plasma inflammatory cytokines. However, its specific mechanisms of action remain unclear. The nuclear factor-κB (NF-κB) pathway in macrophages plays a critical role in regulating the expression of many inflammatory genes, and histone acetylation has been identified as a key epigenetic modification for the NF-κB p65-mediated inflammatory responses. The objective of the study was to investigate if proanthocyanidin (PAC)-rich cranberry extract (CBE) suppresses histone acetylation and NF-κB p65 activation in RAW 264.7 macrophages and mouse bone marrow-derived macrophages (BMDMs). Treatment with 5% and 15% PAC-containing CBEs markedly suppressed the expression of pro-inflammatory mediators (iNos, Cox-2, Tnfα, Mcp-1 and Il-6) in both RAW 264.7 macrophages and BMDMs stimulated with lipopolysaccharides (LPS). CBE significantly reduced LPS-induced phosphorylation of p65 in both cell types without changing total p65 expression levels. Moreover, 15% PAC-CBE increased the expression levels of histone deacetylase 3 (HDAC3) with a concomitant decrease in histone H4 acetylation levels. These results suggest that CBE increases HDAC3 protein expression with the subsequent inhibition of p65 phosphorylation to mediate anti-inflammatory effects in macrophages. Cranberries may serve as a dietary agent to attenuate chronic inflammation in patients with obesity and related complications.

Isoflurane preconditioning effects on brain damage induced by electromagnetic pulse radiation through epigenetic modification of BDNF gene transcription.

The effects of electromagnetic pulse (EMP) radiation on cognitive impairment have attracted much attention, but the mechanism is still unclear. Regulation of brain-derived neurotrophic factor (BDNF) gene expression has been found to promote memory formation and neuronal survival. Isoflurane preconditioning (IP) was reported to have a neuroprotective effect. In this study, we verified the protective effect of IP against brain injury induced by EMP exposure and examined the relation of this effect with BDNF gene regulation. Twenty-four hours before EMP exposure, rats were pretreated with 2% inhaled isoflurane for 30 minutes. At 24 hours after EMP injury, the Morris water maze test was carried out. Meanwhile, the other rats were executed and their brain tissues were used for Nissl staining, qRT-PCR, western blot and chromatin immunoprecipitation. The Morris water maze results showed that 2% IP improved the spatial learning and memory ability of the rats. The Nissl staining results showed 2% of IP alleviated neuronal damage. Also, we detected the mRNA and protein expression of BDNF, and 2% IP significantly increased the expression of BDNF. We also found the expression level of histone deacetylase 2 (HDAC2) was increased and that EMP exposure significantly decreased H3 acetylation, while 2% IP reversed these phenomena, individually, BDNF transcription was activated, and neurogenesis after EMP exposure was alleviated. Our results suggested that 2% of IP alleviates cognitive impairment induced by EMP exposure in rats. Also, the sustained elevated level of BDNF gene transcription may be an essential mechanism for stimulating neurogenesis because of the increased level of HDAC2-dependent H3 acetylation.

In vivo human brain expression of histone deacetylases in bipolar disorder

The etiology of bipolar disorder (BD) is unknown and the neurobiological underpinnings are not fully understood. Both genetic and environmental factors contribute to the risk of BD, which may be linked through epigenetic mechanisms, including those regulated by histone deacetylase (HDAC) enzymes. This study measures in vivo HDAC expression in individuals with BD for the first time using the HDAC-specific radiotracer [11C]Martinostat. Eleven participants with BD and 11 age- and sex-matched control participants (CON) completed a simultaneous magnetic resonance – positron emission tomography (MR-PET) scan with [11C]Martinostat. Lower [11C]Martinostat uptake was found in the right amygdala of BD compared to CON. We assessed uptake in the dorsolateral prefrontal cortex (DLPFC) to compare previous findings of lower uptake in the DLPFC in schizophrenia and found no group differences in BD. Exploratory whole-brain voxelwise analysis showed lower [11C]Martinostat uptake in the bilateral thalamus, orbitofrontal cortex, right hippocampus, and right amygdala in BD compared to CON. Furthermore, regional [11C]Martinostat uptake was associated with emotion regulation in BD in fronto-limbic areas, which aligns with findings from previous structural, functional, and molecular neuroimaging studies in BD. Regional [11C]Martinostat uptake was associated with attention in BD in fronto-parietal and temporal regions. These findings indicate a potential role of HDACs in BD pathophysiology. In particular, HDAC expression levels may modulate attention and emotion regulation, which represent two core clinical features of BD.

Sustained increased CaMKII phosphorylation is involved in the impaired regression of isoproterenol-induced cardiac hypertrophy in rats

To understand the mechanism underlying the regression of cardiac hypertrophy, we investigated the pathological changes after isoproterenol (ISO) withdrawal in ISO-induced cardiomyopathy models in rats and neonatal cardiomyocytes. Cardiac hypertrophy was induced in rats by two weeks of ISO administration; however, the hypertrophy did not regress after three weeks of natural maintenance after ISO administration was withdrawn (ISO-wdr group). The remaining hypertrophy in the ISO-wdr group was accompanied by a sustained increase in the level of phosphorylated Ca2+/calmodulin-dependent protein kinase II (p-CaMKII). Additionally, the increased expression levels of histone deacetylase 4 (HDAC4) and the CaV1.2 channel and amounts of CaMKII bound with HDAC4 and CaV1.2 were not recovered in the ISO-wdr group. The results in cardiomyocyte models were similar to those seen in rat models. Losartan, metoprolol or amlodipine neither ameliorated the increase in atrial natriuretic peptide nor inhibited the increase in p-CaMKII and bound CaMKII. In contrast, autocamtide-2-related inhibitor peptide, a CaMKII inhibitor, reduced these increases. This study investigated the phosphorylation status of CaMKII after hypertrophic stimulus was withdrawn for the first time and proposed that CaMKII as well as its complexes with CaV1.2 could be potential targets to achieve effective regression of cardiac hypertrophy.

Synergetic responses of intestinal microbiota and epithelium to dietary inulin supplementation in pigs.

Inulin is a soluble dietary fiber that has been implicated in regulating the intestinal health. Here, we describe a synergetic response of intestinal microbiota and epithelial functions to increased intake of inulin in a porcine model. Twenty growing-pigs were randomly allocated to two groups (n = 10) and fed with a basal diet (BD) or BD containing 0.5% inulin (INU) for 21 days. We show that INU supplementation not only elevated villus height and the abundance of zonula occludens-1 (ZO-1), but also increased acetate and butyrate concentrations in cecum (P < 0.05). Moreover, INU decreased IL-6 and TNFα secretion, and reduced intestinal epithelial cell apoptosis in ileum and cecum (P < 0.05). Interestingly, we observed an elevated 16S rRNA gene copies in cecum after INU ingestion (P < 0.05). INU had no influence on overall diversity, but acutely altered the abundance of specific bacteria. INU decreased the abundance of phylum Proteobacteria in ileum, but increased the phylum Bacteroidetes in the ileum and cecum (P < 0.05). INU significantly elevated the Lactobacillus spp. and Bacteroides spp. in the ileum and cecum, respectively. Importantly, INU elevated the expression levels of GPR43, GLP-2, and ZO-1, but decreased the expression levels of histone deacetylase 1 (HDAC1) and TNFα in the ileum and cecum mucosa (P < 0.05). Moreover, INU also elevated the expression levels of GPR109A and angiopoietin-4 (ANG-4) in the cecum mucosa (P < 0.05). This study indicated how the intestinal microbiome and epithelium adapt to inulin ingestion, and furthered our understanding of the mechanisms behind the dietary fiber-modulated intestinal microbiota and health.

ATP depletion induced cochlear hair cells death through histone deacetylation in vitro

Previous studies have shown histone modifications being present in cochlear hair cells in animal models of hearing loss. Our past studies have shown that ATP depletion, histone deacetylase (HDAC) upregulation, and histone deacetylation occur in cochlea after noise exposure, and these are linked to hair cell death. Whether ATP depletion correlates with the expression level of HDACs and acetylation of histones is still unknown. In this study, we investigated the changes in the expression of HDACs and the level of histone acetylation in cochlear hair cells using an ATP-depleted explant culture of mouse organ of Corti. We found that the expression of HDAC3 and HDAC6 increased and hair cells were lost after oligomycin A (OA) treatment. Meanwhile, the acetylation level of histone H2B reduced. However, when oligomycin was combined with an HDAC inhibitor, trichostatin A (TSA), the acetylation level of histone H3 was restored. Moreover, combined treatment of oligomycin and TSA or sodium butyrate (NaB) attenuated oligomycin-induced cochlear hair cell loss. In conclusion, our results indicated that ATP depletion led to histone deacetylation and eventually resulted in hair cell death.

MicroRNA-489 suppresses isoproterenol-induced cardiac fibrosis by downregulating histone deacetylase 2.

Cardiac fibrosis is a hallmark of cardiovascular diseases. Several studies have indicated that microRNAs (miRs) are associated with the development of cardiac fibrosis. However, to date, the underlying molecular mechanisms of miR-489 in cardiac fibrosis have not been studied. The present study investigated the biological function of miR-489 in isoproterenol (ISO)-induced cardiac fibrosis. It was observed that miR-489 was downregulated in the heart tissue and cardiac fibroblasts (CFs) obtained from rats with ISO-induced cardiac fibrosis, as compared with the levels in the control group. By contrast, the expression levels of histone deacetylase 2 (HDAC2), collagen I (Col1A1) and α-smooth muscle actin (α-SMA) were increased in the heart tissue and CFs obtained from ISO-treated rats compared with the control group. Furthermore, ISO-treated CFs were transfected with a miR-489 mimic, which resulted in decreased viability and differentiation of CFs compared with the control group. Bioinformatics analysis and a dual-luciferase reporter assay further revealed that HDAC2 is a downstream target of miR-489. Subsequently, a loss-of-function experiment demonstrated that depletion of HDAC2 decreased the expression levels of Col1A1 and α-SMA in CFs. Taken together, the results obtained in the present study revealed that the miR-489/HDAC2 signaling pathway may serve as a novel regulatory mechanism in ISO-induced cardiac fibrosis and may increase the understanding on cardiac fibrosis.

Amelioration of clinical course and demyelination in the cuprizone mouse model in relation to ketogenic diet.

Ketogenic diet (KD) is defined as a high-fat, low-carbohydrate diet with appropriate amounts of protein, which has broad neuroprotective effects. However, the mechanisms of ameliorating the demyelination and of the neuroprotective effects of KD have not yet been completely elucidated. Therefore, the present study investigated the protection mechanism of KD treatment in the cuprizone (bis-cyclohexanone oxalydihydrazone, CPZ)-induced demyelination mice model, with special emphasis on neuroinflammation. After the KD treatment, an increased ketone body level in the blood of mice was detected, and a significant increase in the distance traveled within the central area was observed in the open field test, which reflected the increased exploration and decreased anxiety of mice that received CPZ. The results of Luxol fast blue and myelin basic protein (MBP) immunohistochemistry staining for the evaluation of the myelin content within the corpus callosum revealed a noticeable increase in the number of myelinated fibers and myelin score after KD administration in these animals. Concomitant, the protein expressions of glial fibrillary acidic protein (GFAP, an astrocyte marker), ionized calcium-binding adaptor molecule 1 (Iba-1, a microglial marker), CD68 (an activated microglia marker) and CD16/32 (a M1 microglial marker) were down-regulated, while the expression of oligodendrocyte lineage transcription factor 2 (OLIG2, an oligodendrocyte precursor cells marker) was up-regulated by the KD treatment. In addition, the KD treatment not only reduced the level of the C-X-C motif chemokine 10 (CXCL10), which is correlated to the recruitment of activated microglia, but also inhibited the production of proinflammatory cytokines, including interleukin 1β (IL-1β) and tumor necrosis factor-α (TNF-α), which are closely correlated to the M1 phenotype microglia. It is noteworthy, that the expression levels of histone deacetylase 3 (HADC3) and nod-like receptor pyrin domain containing 3 (NLRP3) significantly decreased after KD administration. In conclusion, these data demonstrate that KD decreased the reactive astrocytes and activated the microglia in the corpus callosum, and that KD inhibited the HADC3 and NLRP3 inflammasome signaling pathway in CPZ-treated mice. This suggests that the inhibition of the HADC3 and NLRP3 signaling pathway may be a novel mechanism by which KD exerts its protective actions for the treatment of demyelinating diseases.

Aberrant histone deacetylase 1 expression upregulates vimentin expression via an NF‑κB‑dependent pathway in hepatocellular carcinoma

Aberrantly elevated expression levels of histone deacetylase 1 (HDAC1) and vimentin are closely associated with disease progression in hepatocellular carcinoma (HCC). It was previously demonstrated that knocking down expression of HDAC1 resulted in a concurrent decrease in the expression levels of vimentin. However, a causal link between these two proteins has not yet been demonstrated, to the best of our knowledge. In the present study, the association between HDAC1 and vimentin was investigated using an HDAC1 overexpression platform. HDAC1 and vimentin were significantly increased in HCC cells, and HDAC1 overexpression enhanced vimentin mRNA and protein expression levels in an HDAC1 dose-dependent manner. Subsequently, truncation and mutation of a vimentin promoter demonstrated that HDAC1-induced vimentin expression was dependent on a nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) binding site in the vimentin promoter sequence. Furthermore, HDAC1 induced vimentin expression by promoting NF-κB translocation between the cytoplasm and the nucleus, as opposed to modulating the total expression level of vimentin directly. The data in the present study demonstrated that HDAC1 is overexpressed in HCC and that HDAC1 may upregulate vimentin expression through the NF-κB signaling pathway, thus demonstrating a causal link between HDAC1 and vimentin in HCC, and may provide valuable information in understanding the pathogenesis of HCC.

Histone deacetylase activity is required for Botrylloides leachii whole-body regeneration.

The colonial tunicate Botrylloides leachii is exceptional at regenerating from a piece of vascular tunic after loss of all adults from the colony. Previous transcriptome analyses indicate a brief period of healing before regeneration of a new adult (zooid) in as little as 8-10 days. However, there is little understanding of how the resulting changes to gene expression, required to drive regeneration, are initiated and how the overall process is regulated. Rapid changes to transcription often occur in response to chromatin changes, mediated by histone modifications such as histone acetylation. Here, we investigated a group of key epigenetic modifiers, histone deacetylases (HDAC), which are known to play an important role in many biological processes such as development, healing and regeneration. Through our transcriptome data, we identified and quantified the expression levels of HDAC and histone acetyltransferase enzymes during whole-body regeneration (WBR). To determine whether HDAC activity is required for WBR, we inhibited its action using valproic acid and trichostatin A. HDAC inhibition prevented the final morphological changes normally associated with WBR and resulted in aberrant gene expression. Botrylloides leachii genes including Slit2, TGF-β, Piwi and Fzd4 all showed altered mRNA levels upon HDAC inhibition in comparison with the control samples. Additionally, atypical expression of Bl_Piwi was found in immunocytes upon HDAC inhibition. Together, these results show that HDAC function, specifically HDAC I/IIa class enzymes, are vital for B. leachii to undergo WBR successfully.

The increase of interfollicular epidermal stem cells and regulation of aryl hydrocarbon receptor and its repressors in the skin through hydroporation with anti-aging cocktail.

Hydroporation is a procedure that involves a subsonic flow of air and microdroplets into the skin. We previously reported that hydroporation treatment with a cocktail solution containing copper-glycyl-L-histidyl-L-lysyl, oligo hyaluronic acid, rhodiola extract, tranexamic acid, and β-glucan yielded positive effects on skin aging. The aim of this study was to evaluate the effects of hydroporation with anti-aging cocktail on interfollicular epidermal stem cells (IFESCs) and expression of aryl hydrocarbon receptor (AhR)/AhR repressor (AhRR) in the skin. Skin samples from six volunteers who were treated with hydroporation were analyzed via confocal microscopic examination. Markers for dermal matrix (procollagen type I and fibrillin-1) and basement membrane (type IV collagen and integrin α6) were increased after treatment. Moreover, there was a significant increase in the expression level of histone deacetylase 1-positive/p63-negative basal cells, which we previously reported as interfollicular epidermal stem cells. The expression level of AhR was significantly decreased, whereas that of AhRR was increased. This indicates an alteration in the interaction between the skin and environment posttreatment. Anti-aging hydroporation treatment recovered the stem cell potential of basal cells. Moreover, this treatment decreased AhR and increased AhRR in the skin, which may protect the skin from the harmful environment.

PO-365 Isothiocyanates as potent epigenetic regulators in human malignant melanoma chemoprevention

IntroductionGlucosinolates (GLs) are phytochemicals abundant in cruciferous vegetables, which are hydrolysed, by myrosinase, to a range of isothiocyanates (ITCs). These molecules are biologically active metabolites capable of mediating a plurality of anticancer effects including cell cycle arrest, inhibition of proliferation and apoptotic induction. Their wide range of biological properties may be reflected by their ability to interfere with the epigenetic machinery at both DNA and histone levels. The aim of the current study is to investigate how ITCs interact with the epigenetic pathway(s) response in order to induce apoptosis by modifying the expression of key apoptotic genes in an in vitro model of human malignant melanoma.Material and methodsOur in vitro human malignant melanoma model consists of (i) immortalised normal keratinocyte (HaCaT) cells; (ii) malignant melanoma (A375) cells and epidermoid carcinoma (A431) cells subjected to the following ITCs: R, S-Sulforaphane (SFN), Phenethyl Isothiocyanate (PEITC), Benzyl Isothiocyanate (BITC), Allyl Isothiocyanate (AITC) and Iberin (IBN) over different concentrations and time points of exposure. Apoptotic induction was confirmed by TaqMan qPCR gene expression profiling arrays while involvement of the epigenetic machinery was assessed by western immunoblotting for determining protein expression levels of histone deacetylases (HDACs), histone acetyltransferases (HATs) and various other histone modification tags.Results and discussionsOur results showed that all ITCs were capable of inducing apoptosis as evident by the differential expression of key target genes in a manner where PEITC and IBN were involved primarily in up-regulation compared to SFN and AITC both of which were involved in down-regulation of the majority of these apoptotic genes. Finally, differences in HDAC and HAT protein expression levels, among ITC treatments, were evident in addition to their differential compartmentalization between nucleus and cytosol.ConclusionOverall our results suggest an ITC-dependent cytotoxicity effect which is mediated via apoptotic induction and is underlined, at least partially, by epigenetic pathway(s) response mechanism(s). Our data support the notion that ITCs may be promising candidates in the context of epigenetic therapy for the treatment of human malignant melanoma.