RESUMO
BACKGROUND/AIMS: Inflammation is the body's natural response to stress in the broadest sense. The regulatory mechanisms that control this process, some of which are still unclear, are needed to balance the immune response, but also when insufficient, can cause immunodeficiency resulting in infection, cancer, neurodegeneration or other serious disorders. In this study, we focused on defining the role of lysine-specific demethylase 1 (LSD1), an enzyme involved in modulating the methylation state of lysine, including histone and non-histone proteins, in shaping the inflammatory profile of endothelial cells. METHODS: To determine the role of LSD1 in the inflammatory response of ECs, cells were stimulated with lipopolysaccharide (100 ng/ml LPS) in the presence and absence of an LSD1 inhibitor (2-PCPA). A transcription model of LSD1 deficient cells (HMEC-1 LSD1 KD) obtained by lentiviral shRNA transduction was also used. The indicated cellular models were analyzed by gene profiling, monitoring of p65 shuttling by Western blotting and immunofluorescence staining. Also chromatin immunoprecipitation (ChIP) was performed to identify the interactions between selected: IL-6/p65 and LSD1. RESULTS: Analysis of both experimental models revealed an altered inflammatory response following both LSD1 inhibition and LSD1 silencing. We observed decreased U-937 monocytes recruitment to LPS-activated endothelial cells and decreased extracellular secretion of many proinflammatory cytokines, also confirmed at the transcript level by RT-qPCR. Monitoring of the LPS-induced p65 translocation revealed inhibition of the NF-kB subunit in LSD1 KD vs nonT as well as due to pretreatment of 2-PCPA cells. Gene profiling performed with RNA microarrays confirmed the obtained biochemical data at the transcript level. CONCLUSION: In conclusion, the conducted studies showed a proinflammatory profile of LSD1 activity in endothelial cells, revealed by the inhibition of the enzyme activity and confirmed at the transcriptional level by the inhibition of its expression. Although we found significant changes in the modification of interactions between monocytes and endothelial cells as well as in cytokine/chemokine release and expression that were consistent with the altered NF-κB-p65 translocation into the nucleus, we did not identify a direct interaction between LSD1 and the transcription factor. Our finding may have important implications for prevention of cardiovascular diseases at their first stage - activation of the endothelium as well as for tumor cell biology, providing evidence for the use of LSD1 inhibitors to reduce the inflammatory response, which enhances tumor tissue remodeling, angiogenesis and metastasis.
Assuntos
Células Endoteliais/metabolismo , Histona Desmetilases/metabolismo , Inflamação/metabolismo , Linhagem Celular , Histona Desmetilases/genética , Humanos , Inflamação/genética , NF-kappa B/metabolismo , Interferência de RNA , Transdução de SinaisRESUMO
Epigenetic mechanisms, including histone post-translational modifications, are central regulators of cell cycle control. The euchromatic G9a histone methyltransferase (G9a HMT) is a key enzyme catalyzing histone H3 methylation on lysines 9 and 27, and its dysregulation has been linked to uncontrolled proliferation of tumor cells. Here, we have investigated the effect of G9a HMT silencing on cell proliferation of microvascular endothelial cells, a process necessary to sustain tumor growth through the formation of the vascular capillary network. Inhibition of G9a HMT activity in human microvascular endothelial cells (HMEC-1) was performed either pharmacologically, by treatment of cells with BIX-01294 or chaetocin, or transcriptionally, using shRNA. Cell viability and proliferation were examined using the resazurin reduction assay, flow cytometry and immunostaining of phosphorylated checkpoint kinase 1 (pSer317Chk1). Expression of cell cycle- and redox homeostasis-related genes was determined by quantitative PCR. Reactive oxygen species production was measured by oxidation of the fluorescent probe 2',7'-dichlorodihydrofluorescein diacetate and the cell's total antioxidant capacity by using the ABTS assay. Inhibition of G9a HMT activity by BIX-01294 treatment or by shRNA attenuated the proliferation of HMEC-1, nuclear localization of phosphorylated Chk1, and induced cell cycle arrest in G1 phase. Transcriptional analysis demonstrated increased gene expression of the cyclin-dependent kinase (CDK) inhibitor p21, and also of Rb1, in BIX-01294 treated cells. Decreased proliferation rate was accompanied by enhanced antioxidant potential of HMEC-1 cells, as demonstrated by reduced production of reactive oxygen species, increased total antioxidant capacity and expression of the antioxidant enzymes catalase and superoxide dismutase 1. Collectively, our results demonstrate of the central role of G9a HMT in the promotion of endothelial cells proliferation, and suggest that endothelial G9a HMT may be a target in the treatment of vascular proliferative disorders and tumor neovascularization.
Assuntos
Proliferação de Células/fisiologia , Células Endoteliais/fisiologia , Antígenos de Histocompatibilidade/fisiologia , Histona-Lisina N-Metiltransferase/fisiologia , Microvasos/citologia , Azepinas/farmacologia , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Antígenos de Histocompatibilidade/genética , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Histona-Lisina N-Metiltransferase/genética , Homeostase , Humanos , Oxirredução , Quinazolinas/farmacologia , RNA Interferente Pequeno/genéticaRESUMO
Novel approaches to cancer chemotherapy employ metabolic differences between normal and tumor cells, including the high dependence of cancer cells on glycolysis ("Warburg effect"). 3-Bromopyruvate (3-BP), inhibitor of glycolysis, belongs to anticancer drugs basing on this principle. 3-BP was tested for its capacity to kill human non-invasive MCF-7 and invasive MDA-MB-231 breast cancer cells. We found that 3-BP was more toxic for MDA-MB-231 cells than for MCF-7 cells. In both cell lines, a statistically significant decrease of ATP and glutathione was observed in a time- and 3-BP concentration-dependent manner. Transient increases in the level of reactive oxygen species and reactive oxygen species was observed, more pronounced in MCF-7 cells, followed by a decreasing tendency. Activities of glutathione peroxidase, glutathione reductase (GR) and glutathione S-transferase (GST) decreased in 3-BP treated MDA-MB-231 cells. For MCF-7 cells decreases of GR and GST activities were noted only at the highest concentration of 3-BP.These results point to induction of oxidative stress by 3-BP via depletion of antioxidants and inactivation of antioxidant enzymes, more pronounced in MDA-MB-231 cells, more sensitive to 3-BP.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Piruvatos/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Relação Dose-Resposta a Droga , Feminino , Glutationa Redutase/metabolismo , Glutationa Transferase/metabolismo , Humanos , Células MCF-7 , Invasividade Neoplásica , Proteínas de Neoplasias/metabolismo , Oxirredução/efeitos dos fármacosRESUMO
Ketone bodies (KBs), comprising ß-hydroxybutyrate, acetoacetate and acetone, are a set of fuel molecules serving as an alternative energy source to glucose. KBs are mainly produced by the liver from fatty acids during periods of fasting, and prolonged or intense physical activity. In diabetes, mainly type-1, ketoacidosis is the pathological response to glucose malabsorption. Endogenous production of ketone bodies is promoted by consumption of a ketogenic diet (KD), a diet virtually devoid of carbohydrates. Despite its recently widespread use, the systemic impact of KD is only partially understood, and ranges from physiologically beneficial outcomes in particular circumstances to potentially harmful effects. Here, we firstly review ketone body metabolism and molecular signaling, to then link the understanding of ketone bodies' biochemistry to controversies regarding their putative or proven medical benefits. We overview the physiological consequences of ketone bodies' consumption, focusing on (i) KB-induced histone post-translational modifications, particularly ß-hydroxybutyrylation and acetylation, which appears to be the core epigenetic mechanisms of activity of ß-hydroxybutyrate to modulate inflammation; (ii) inflammatory responses to a KD; (iii) proven benefits of the KD in the context of neuronal disease and cancer; and (iv) consequences of the KD's application on cardiovascular health and on physical performance.
Assuntos
Diabetes Mellitus Tipo 1 , Dieta Cetogênica , Epigênese Genética , Neoplasias , Doenças do Sistema Nervoso , Ácido 3-Hidroxibutírico/metabolismo , Acetoacetatos/metabolismo , Animais , Diabetes Mellitus Tipo 1/dietoterapia , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/patologia , Epigenômica , Humanos , Corpos Cetônicos/genética , Corpos Cetônicos/metabolismo , Cetose/dietoterapia , Cetose/genética , Cetose/metabolismo , Cetose/patologia , Metabolômica , Neoplasias/dietoterapia , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Doenças do Sistema Nervoso/dietoterapia , Doenças do Sistema Nervoso/genética , Doenças do Sistema Nervoso/metabolismo , Doenças do Sistema Nervoso/patologiaRESUMO
Butyrate and R-ß-hydroxybutyrate are two related short chain fatty acids naturally found in mammals. Butyrate, produced by enteric butyric bacteria, is present at millimolar concentrations in the gastrointestinal tract and at lower levels in blood; R-ß-hydroxybutyrate, the main ketone body, produced by the liver during fasting can reach millimolar concentrations in the circulation. Both molecules have been shown to be histone deacetylase (HDAC) inhibitors, and their administration has been associated to an improved metabolic profile and better cellular oxidative status, with butyrate inducing PGC1α and fatty acid oxidation and R-ß-hydroxybutyrate upregulating oxidative stress resistance factors FOXO3A and MT2 in mouse kidney. Because of the chemical and functional similarity between the two molecules, we compared here their impact on multiple cell types, evaluating i) histone acetylation and hydroxybutyrylation levels by immunoblotting, ii) transcriptional regulation of metabolic and inflammatory genes by quantitative PCR and iii) cytokine secretion profiles using proteome profiling array analysis. We confirm that butyrate is a strong HDAC inhibitor, a characteristic we could not identify in R-ß-hydroxybutyrate in vivo nor in vitro. Butyrate had an extensive impact on gene transcription in rat myotubes, upregulating PGC1α, CPT1b, mitochondrial sirtuins (SIRT3-5), and the mitochondrial anti-oxidative genes SOD2 and catalase. In endothelial cells, butyrate suppressed gene expression and LPS-induced secretion of several pro-inflammatory genes, while R-ß-hydroxybutyrate acted as a slightly pro-inflammatory molecule. Our observations indicate that butyrate induces transcriptional changes to a higher extent than R-ß-hydroxybutyrate in rat myotubes and endothelial cells, in keep with its HDAC inhibitory activity. Also, in contrast with previous reports, R-ß-hydroxybutyrate, while inducing histone ß-hydroxybutyrylation, did not display a readily detectable HDAC inhibitor activity and exerted a slight pro-inflammatory action on endothelial cells.
Assuntos
Anti-Inflamatórios/farmacologia , Butiratos/farmacologia , Inibidores de Histona Desacetilases/farmacologia , Inflamação/tratamento farmacológico , Acetilação/efeitos dos fármacos , Animais , Células Endoteliais/efeitos dos fármacos , Proteína Forkhead Box O3/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Histona Desacetilases/efeitos dos fármacos , Humanos , Hidroxibutiratos/farmacologia , Metalotioneína/genética , Camundongos , Estresse Oxidativo/efeitos dos fármacos , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Ratos , Transcrição Gênica/efeitos dos fármacosRESUMO
: The methylation of histone lysine residues modifies chromatin conformation and regulates the expression of genes implicated in cell metabolism. Lysine-specific demethylase 1 (LSD1) is a flavin-dependent monoamine oxidase that can demethylate mono- and dimethylated histone lysines 4 and 9 (H3K4 and H3K9). The removal of methyl groups from the lysine residues of histone and non-histone proteins was found to be an important regulatory factor of cell proliferation. However, its role has not been fully elucidated. In this study, we assessed LSD1-mediated cell cycle progression using a human endothelial cell model. The short hairpin RNA knockdown of LSD1 inhibits the G2/M phase of cell cycle progression by checkpoint kinase 1 (Chk1) phosphorylation (S137). We observed elevated DNA damage, which was consistent with the increased detection of double-strand breaks as well as purines and pyrimidines oxidation, which accompanied the activation of ATR/ATRIP signaling by H2AXS139 phosphorylation. The irreversible pharmacological inhibition of LSD1 by 2-phenylcyclopropylamine (2-PCPA) inactivated its enzymatic activity, causing significant changes in heterochromatin and euchromatin conformation assessed by chromatin assembly factor 1 subunit A (CAF1A) and heterochromatin protein 1 isoform α and γ (HP1α/γ) immunofluorescence analysis. We conclude that the knockdown of LSD1 in endothelial cells leads to increased HP1-positive chromatin, the stimulation of DNA repair processes, and the dysregulation of proliferation machinery.
Assuntos
Quinase 1 do Ponto de Checagem/metabolismo , Cromatina/metabolismo , Células Endoteliais , Histona Desmetilases/fisiologia , Linhagem Celular , Homólogo 5 da Proteína Cromobox , Proteínas Cromossômicas não Histona/metabolismo , Reparo do DNA , Desmetilação , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Pontos de Checagem da Fase G2 do Ciclo Celular/fisiologia , Inativação Gênica , Histona Desmetilases/genética , Humanos , Pontos de Checagem da Fase M do Ciclo Celular/fisiologia , Fosforilação , Processamento de Proteína Pós-TraducionalRESUMO
Rather than being a passive barrier between circulating blood and smooth muscle cells and the underlying tissues, the endothelium is a fundamental functional component of the vasculature, and could be viewed as the largest human endocrine gland/organ, secreting multiple pro-/antiangiogenic factors, cytokines and low-molecular-weight mediators controlling the vascular tone. The location of endothelium, at the interface between the circulation and the tissues, makes this epithelial layer particularly exposed to physical and chemical cues coming from the bloodstream. In response to such stimuli, the endothelium modulates its morphology and functions to maintain vascular homeostasis. Dietary components significantly affect the proper functioning of the endothelium. High-calories and high-fat western diets, in the long term, cause endothelial dysfunction, which is a major contributor to the development of the metabolic syndrome and its pathological consequences, including atherosclerosis, diabetes, and hypertension. On the contrary, plant-derived antioxidant molecules and polyphenols have been shown to exert beneficial effects on endothelial function. Extensive research in the last decade has clearly shown the close relationship between food intake, dietary habits, and gene expression, which is driven by the action of macro- and micronutrients on chromatin regulation. Nutrient-induced chromatin epigenetic modifications via DNA methylation and histone post-translational modifications, especially in the context of the western diet, significantly contribute to the dysregulation of endothelial functioning. Here, we review the current understanding on how dietary components (macronutrients, antioxidants), acting on epigenetic mechanisms, regulate endothelial physiology, and physiopathology. © 2016 BioFactors, 43(1):5-16, 2017.
Assuntos
Endotélio Vascular/fisiologia , Epigênese Genética , Animais , Antioxidantes/farmacologia , Dieta , Células Endoteliais/fisiologia , Endotélio Vascular/citologia , Expressão Gênica , HumanosRESUMO
Posttranslational modifications of histone tails can alter chromatin structure and regulate gene transcription. While recent studies implicate the lysine/arginine protein methyltransferases in the regulation of genes for endothelial metabolism, the role of AMI-1 and AMI-5 compounds in angiogenesis remains unknown. Here, we show that global inhibition of arginine and lysine histone methyltransferases (HMTs) by AMI-5 induced an angiostatic profile in human microvascular endothelial cells and human umbilical vein endothelial cells. Based on FACS analysis, we found that inhibition of HMTs significantly affects proliferation of endothelial cells, by suppressing cell cycle progression in the G0/G1 phase. Immunofluorescent studies of the endothelial cells replication pattern by 5-ethynyl-2'-deoxyuridine incorporation disclosed that AMI-5, and the arginine methyltransferase inhibitor AMI-1, induced heterochromatin formation and a number of nuclear abnormalities, such as formation of micronuclei (MNs) and nucleoplasmic bridges (NPBs), which are markers of chromosomal instability. In addition to the modification of the cell cycle machinery in response to AMIs treatment, also endothelial cells migration and capillary-like tube formation processes were significantly inhibited, implicating a stimulatory role of HMTs in angiogenesis.