Lipopolysaccharide induces lung fibroblast proliferation through Toll-like receptor 4 signaling and the phosphoinositide3-kinase-Akt pathway

Pulmonary fibrosis is characterized by lung fibroblast proliferation and collagen secretion. In lipopolysaccharide (LPS)-induced acute lung injury (ALI), aberrant proliferation of lung fibroblasts is initiated in early disease stages, but the underlying mechanism remains unknown. In this study, we knocked down Toll-like receptor 4 (TLR4) expression in cultured mouse lung fibroblasts using TLR4-siRNA-lentivirus in order to investigate the effects of LPS challenge on lung fibroblast proliferation, phosphoinositide3-kinase (PI3K)-Akt pathway activation, and phosphatase and tensin homolog (PTEN) expression. Lung fibroblast proliferation, detected by BrdU assay, was unaffected by 1 mug/mL LPS challenge up to 24 hours, but at 72 hours, cell proliferation increased significantly. This proliferation was inhibited by siRNA-mediated TLR4 knockdown or treatment with the PI3K inhibitor, Ly294002. In addition, siRNA-mediated knockdown of TLR4 inhibited the LPS-induced up-regulation of TLR4, down-regulation of PTEN, and activation of the PI3K-Akt pathway (overexpression of phospho-Akt) at 72 hours, as detected by real-time PCR and Western blot analysis. Treatment with the PTEN inhibitor, bpV(phen), led to activation of the PI3K-Akt pathway. Neither the baseline expression nor LPS-induced down-regulation of PTEN in lung fibroblasts was influenced by PI3K activation state. PTEN inhibition was sufficient to exert the LPS effect on lung fibroblast proliferation, and PI3K-Akt pathway inhibition could reverse this process. Collectively, these results indicate that LPS can promote lung fibroblast proliferation via a TLR4 signaling mechanism that involves PTEN expression down-regulation and PI3K-Akt pathway activation. Moreover, PI3K-Akt pathway activation is a downstream effect of PTEN inhibition and plays a critical role in lung fibroblast proliferation. This mechanism could contribute to, and possibly accelerate, pulmonary fibrosis in the early stages of ALI/ARDS.     

Position effect variegation and epigenetic modification of a transgene in a pig model

Sequences proximal to transgene integration sites are able to regulate transgene expression, resulting in complex position effect variegation. Position effect variegation can cause differences in epigenetic modifications, such as DNA methylation and histone acetylation. However, it is not known which factor, position effect or epigenetic modification, plays a more important role in the regulation of transgene expression. We analyzed transgene expression patterns and epigenetic modifications of transgenic pigs expressing green fluorescent protein, driven by the cytomegalovirus (CMV) promoter. DNA hypermethylation and loss of acetylation of specific histone H3 and H4 lysines, except H4K16 acetylation in the CMV promoter, were consistent with a low level of transgene expression. Moreover, the degree of DNA methylation and histone H3/H4 acetylation in the promoter region depended on the integration site; consequently, position effect variegation caused variations in epigenetic modifications. The transgenic pig fibroblast cell lines were treated with DNA methyltransferase inhibitor 5-Aza-2'-deoxycytidine and/or histone deacetylase inhibitor trichostatin A. Transgene expression was promoted by reversing the DNA hypermethylation and histone hypoacetylation status. The differences in DNA methylation and histone acetylation in the CMV promoter region in these cell lines were not significant; however, significant differences in transgene expression were detected, demonstrating that variegation of transgene expression is affected by the integration site. We conclude that in this pig model, position effect variegation affects transgene expression.     

Acetate reduces microglia inflammatory signaling in vitro

Acetate supplementation increases brain acetyl‐CoA and histone acetylation and reduces lipopolysaccharide (LPS)‐induced neuroglial activation and interleukin (IL)‐1β expression in vivo. To determine how acetate imparts these properties, we tested the hypothesis that acetate metabolism reduces inflammatory signaling in microglia. To test this, we measured the effect acetate treatment had on cytokine expression, mitogen‐activated protein kinase (MAPK) signaling, histone H3 at lysine 9 acetylation, and alterations of nuclear factor‐kappa B (NF‐κB) in primary and BV‐2 cultured microglia. We found that treatment induced H3K9 hyperacetylation and reversed LPS‐induced H3K9 hypoacetylation similar to that found in vivo. LPS also increased IL‐1β, IL‐6, and tumor necrosis factor‐alpha (TNF‐α) mRNA and protein, whereas treatment returned the protein to control levels and only partially attenuated IL‐6 mRNA. In contrast, treatment increased mRNA levels of transforming growth factor‐β1 (TGF‐β1) and both IL‐4 mRNA and protein. LPS increased p38 MAPK and JNK phosphorylation at 4 and 2–4 h, respectively, whereas treatment reduced p38 MAPK and JNK phosphorylation only at 2 h. In addition, treatment reversed the LPS‐induced elevation of NF‐κB p65 protein and phosphorylation at serine 468 and induced acetylation at lysine 310. These data suggest that acetate metabolism reduces inflammatory signaling and alters histone and non‐histone protein acetylation.     

SIRT1‐mediated epigenetic downregulation of plasminogen activator inhibitor‐1 prevents vascular endothelial replicative senescence

The inactivation of plasminogen activator inhibitor‐1 (PAI‐1) has been shown to exert beneficial effects in age‐related vascular diseases. Limited information is available on the molecular mechanisms regarding the negatively regulated expression of PAI‐1 in the vascular system. In this study, we observed an inverse correlation between SIRT1, a class III histone deacetylase, and PAI‐1 expression in human atherosclerotic plaques and the aortas of old mice, suggesting that internal negative regulation exists between SIRT1 and PAI‐1. SIRT1 overexpression reversed the increased PAI‐1 expression in senescent human umbilical vein endothelial cells (HUVECs) and aortas of old mice, accompanied by decreased SA‐β‐gal activity in vitro and improved endothelial function and reduced arterial stiffness in vivo. Moreover, the SIRT1‐mediated inhibition of PAI‐1 expression exerted an antisenescence effect in HUVECs. Furthermore, we demonstrated that SIRT1 is able to bind to the PAI‐1 promoter, resulting in a decrease in the acetylation of histone H4 lysine 16 (H4K16) on the PAI‐1 promoter region. Thus, our findings suggest that the SIRT1‐mediated epigenetic inhibition of PAI‐1 expression exerts a protective effect in vascular endothelial senescence.     

Histone Deacetylase Inhibition Impairs Normal Intestinal Cell Proliferation and Promotes Specific Gene Expression

Mechanisms that maintain proliferation and delay cell differentiation in the intestinal crypt are not yet fully understood. We have previously shown the implication of histone methylation in the regulation of enterocytic differentiation. In this study, we investigated the role of histone deacetylation as an important epigenetic mechanism that controls proliferation and differentiation of intestinal cells using the histone deacetylase inhibitor suberanilohydroxamic acid (SAHA) on the proliferation and differentiation of human and mouse intestinal cells. Treatment of newly confluent Caco‐2/15 cells with SAHA resulted in growth arrest, increased histone acetylation and up‐regulation of the expression of intestine‐specific genes such as those encoding sucrase‐isomaltase, villin and the ion exchanger SLC26A3. Although SAHA has been recently used in clinical trials for cancer treatment, its effect on normal intestinal cells has not been documented. Analyses of small and large intestines of mice treated with SAHA revealed a repression of crypt cell proliferation and a higher expression of sucrase‐isomaltase in both segments compared to control mice. Expression of SLC26A3 was also significantly up‐regulated in the colons of mice after SAHA administration. Finally, SAHA was also found to strongly inhibit normal human intestinal crypt cell proliferation in vitro. These results demonstrate the important implication of epigenetic mechanisms such as histone acetylation/deacetylation in the regulation of normal intestinal cell fate and proliferation. J. Cell. Biochem. 116: 2695–2708, 2015. © 2015 The Authors. Journal of Cellular Biochemistry published by Wiley Periodicals, Inc.     

Adrenaline stimulates the proliferation and migration of mesenchymal stem cells towards the LPS‐induced lung injury

Bone marrow‐derived mesenchymal stem cells (BMSCs) could modulate inflammation in experimental lung injury. On the other hand, adrenergic receptor agonists could increase DNA synthesis of stem cells. Therefore, we investigated the therapeutic role of adrenaline‐stimulated BMSCs on lipopolysaccharide (LPS)‐induced lung injury. BMSCs were cultured with adrenergic receptor agonists or antagonists. Suspensions of lung cells or sliced lung tissue from animals with or without LPS‐induced injury were co‐cultured with BMSCs. LPS‐stimulated alveolar macrophages were co‐cultured with BMSCs (with adrenaline stimulation or not) in Transwell for 6 hrs. A preliminary animal experiment was conducted to validate the findings in ex vivo study. We found that adrenaline at 10 μM enhanced proliferation of BMSCs through both α‐ and β‐adrenergic receptors. Adrenaline promoted the migration of BMSCs towards LPS‐injured lung cells or lung tissue. Adrenaline‐stimulated BMSCs decreased the inflammation of LPS‐stimulated macrophages, probably through the expression and secretion of several paracrine factors. Adrenaline reduced the extent of injury in LPS‐injured rats. Our data indicate that adrenaline‐stimulated BMSCs might contribute to the prevention from acute lung injury through the activation of adrenergic receptors, promotion of proliferation and migration towards injured lung, and modulation of inflammation.     

Epigenetic characterization of the PBEF and TIMP-2 genes in the developing placentae of normal mice

Reprogramming errors, which appear frequently in cloned animals, are reflected by aberrant gene expression. We previously reported the aberrant expression of TIMP-2 and PBEF in cloned placenta and differential expression of PBEF genes during pregnancy. To examine the epigenetic modifications that regulate dynamic gene expression in developing placentae, we herein analyzed the mRNA and protein expression levels of PBEF and TIMP-2 in the placentae of normal mice during pregnancy and then examined potential correlations with epigenetic modifications. DNA methylation pattern analysis revealed no difference, but ChIP assays using antibodies against H3-K9/K14 and H4-K5 histone acetylation revealed that the H3-K9/K14 acetylation levels, but not the H4-K5 acetylation levels, of the TIMP-2 and PBEF loci were significantly correlated with their gene expression levels during placentation in normal mice. These results suggest that epigenetic changes may regulate gene expression level in the developing placentae of normal mice and that inappropriate epigenetic reprogramming might be one cause of the abnormal placentae seen in cloned animals.     

Simulated microgravity‐induced epigenetic changes in human lymphocytes

Real space flight and modeled microgravity conditions result in changes in the expression of genes that control important cellular functions. However, the mechanisms for microgravity‐induced gene expression changes are not clear. The epigenetic changes of DNA methylation and chromatin histones modifications are known to regulate gene expression. The objectives of this study were to investigate whether simulated microgravity alters (a) the DNA methylation and histone acetylation, and (b) the expression of DNMT1, DNMT3a, DNMT3b, and HDAC1 genes that regulate epigenetic events. To achieve these objectives, human T‐lymphocyte cells were grown in a rotary cell culture system (RCCS) that simulates microgravity, and in parallel under normal gravitational conditions as control. The microgravity‐induced DNA methylation changes were detected by methylation sensitive‐random amplified polymorphic DNA (MS‐RAPD) analysis of genomic DNA. The gene expression was measured by Quantitative Real‐time PCR. The expression of DNMT1, DNMT3a, and DNMT3b was found to be increased at 72 h, and decreased at 7 days in microgravity exposed cells. The MS‐RAPD analysis revealed that simulated microgravity exposure results in DNA hypomethylation and mutational changes. Gene expression analysis revealed microgravity exposure time‐dependent decreased expression of HDAC1. Decreased expression of HDAC1 should result in increased level of acetylated histone H3, however a decreased level of acetylated H3 was observed in microgravity condition, indicating thereby that other HDACs may be involved in regulation of H3 deacetylation. The findings of this study suggest that epigenetic events could be one of the mechanistic bases for microgravity‐induced gene expression changes and associated adverse health effects. J. Cell. Biochem. 111: 123–129, 2010. © 2010 Wiley‐Liss, Inc.     

乙酰转移酶MYST家族的生物学功能

组蛋白乙酰化是表观遗传修饰的重要方式,主要受到组蛋白乙酰转移酶（histone acetyltransferases, HATs)和组蛋白去乙酰化酶（histone deacetylase, HDACs）催化. MYST是人类HATs的4大家族之一,包括MOF(males absent on the first),TIP60 (tat interacting protein 60 kD),结合ORC1的组蛋白乙酰转移酶（histone acetyltransferase binding to ORC1, HBO1),单核细胞白血病锌指蛋白(monocytic leukemia zinc finger protein, MOZ)和MOZ相关蛋白（MOZ related factor, MORF）等,均具有典型的MYST结构域.MYST介导的乙酰化是重要的翻译后修饰,其催化底物包括组蛋白和非组蛋白,如组蛋白H3, H4, H2A, H2A突变体,以及许多参与DNA代谢、细胞增殖和发育调控的蛋白因子. MYST蛋白家族参与许多细胞的生理过程,本文主要综述其在调节基因转录、DNA损伤修复和肿瘤发生发展等方面的生物学功能.