Respiratory Research Group, Faculty of Pharmacy, University of Sydney, Sydney, Australia.
Eur Respir J. 2011 Dec;38(6):1444-52. doi: 10.1183/09031936.00129610. Epub 2011 Jun 30.
Airway smooth muscle cells (ASMCs) secrete eotaxin and RANTES (regulated on activation, normal T-cell expressed and secreted) in response to tumour necrosis factor (TNF)-α, which is inhibited by the nuclear factor (NF)-κB inhibitor dimethylfumarate (DMF). NF-κB/IκB (inhibitor of NF-κB) glutathionylation and changes in chromatin remodelling can inhibit NF-κB activity. In this study, we determined whether NF-κB/IκB glutathionylation and reduced histone H3 phosphorylation might underlie the inhibitory effect of DMF on NF-κB activity, and eotaxin and RANTES secretion. Primary human ASMCs were treated with DMF, diamide and/or glutathione (GSH) ethylester (OEt) prior to TNF-α stimulation and were subsequently analysed by ELISA, electrophoretic mobility shift assay, immunofluorescence, co-immunoprecipitation or immunoblotting. DMF reduced intracellular GSH and induced IκBα glutathionylation (IκBα-SSG), which inhibited IκBα degradation, NF-κB p65 nuclear entry and NF-κB/DNA binding. In addition, DMF inhibited the phosphorylation of histone H3, which was possibly mediated by the inhibitory effect of DMF on mitogen- and stress-activated protein kinase (MSK)-1. However, p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase MAPK and MAPK phosphatase-1, upstream of MSK-1, were not inhibited by DMF. Importantly, DMF-mediated effects on NF-κB, histone H3, eotaxin and RANTES were reversed by addition of GSH-OEt. Our data suggest that DMF inhibits NF-κB-dependent eotaxin and RANTES secretion by reduction of GSH with subsequent induction of IκBα-SSG and inhibition of histone H3 phosphorylation. Our findings offer new potential drug targets to reduce airway inflammation in asthma.
气道平滑肌细胞（ASMCs）在肿瘤坏死因子（TNF）-α的刺激下会分泌嗜酸性粒细胞趋化因子和 RANTES（活化调节正常 T 细胞表达和分泌），而核因子（NF）-κB 抑制剂二甲基富马酸（DMF）可抑制其分泌。NF-κB/IκB（NF-κB 抑制剂）谷胱甘肽化和染色质重塑的变化可抑制 NF-κB 活性。在这项研究中，我们确定了 NF-κB/IκB 谷胱甘肽化和组蛋白 H3 磷酸化的减少是否是 DMF 抑制 NF-κB 活性以及嗜酸性粒细胞趋化因子和 RANTES 分泌的基础。用 DMF、二酰胺和/或谷胱甘肽乙酯（OEt）处理原代人 ASMCs ，在 TNF-α刺激之前，通过 ELISA、电泳迁移率变动分析、免疫荧光、共免疫沉淀或免疫印迹法进行分析。DMF 降低了细胞内 GSH 的水平，并诱导了 IκBα谷胱甘肽化（IκBα-SSG），抑制了 IκBα的降解、NF-κB p65 核内进入和 NF-κB/DNA 结合。此外，DMF 抑制了组蛋白 H3 的磷酸化，这可能是由 DMF 对有丝分裂原和应激激活蛋白激酶（MSK）-1 的抑制作用介导的。然而，DMF 并没有抑制 MSK-1 上游的丝裂原激活蛋白激酶（MAPK）p38、细胞外信号调节激酶 MAPK 和 MAPK 磷酸酶-1。重要的是，GSH-OEt 的添加逆转了 DMF 对 NF-κB、组蛋白 H3、嗜酸性粒细胞趋化因子和 RANTES 的介导作用。我们的数据表明，DMF 通过降低 GSH 水平，随后诱导 IκBα-SSG 并抑制组蛋白 H3 磷酸化，从而抑制 NF-κB 依赖性嗜酸性粒细胞趋化因子和 RANTES 的分泌。我们的发现为减少哮喘中的气道炎症提供了新的潜在药物靶点。
