Herbelet Sandrine, De Vlieghere Elly, Gonçalves Amanda, De Paepe Boel, Schmidt Karsten, Nys Eline, Weynants Laurens, Weis Joachim, Van Peer Gert, Vandesompele Jo, Schmidt Jens, De Wever Olivier, De Bleecker Jan L
Department of Neurology, Ghent University and Ghent University Hospital, Ghent, Belgium.
Cancer Research Institute Ghent and Department of Radiation Oncology and Experimental Cancer Research, Ghent University, Ghent, Belgium.
Front Physiol. 2018 Feb 21;9:126. doi: 10.3389/fphys.2018.00126. eCollection 2018.
Regeneration in skeletal muscle relies on regulated myoblast migration and differentiation, in which the transcription factor nuclear factor of activated T-cells 5 (NFAT5) participates. Impaired muscle regeneration and chronic inflammation are prevalent in myositis. Little is known about the impact of inflammation on NFAT5 localization and expression in this group of diseases. The goal of this study was to investigate NFAT5 physiology in unaffected myoblasts exposed to cytokine or hyperosmolar stress and in myositis. NFAT5 intracellular localization and expression were studied using a cell culture model of myositis. Myoblasts were exposed to DMEM solutions enriched with pro-inflammatory cytokines IFN-γ with IL-1β or hyperosmolar DMEM obtained by NaCl supplementation. NFAT5 localization was visualized using immunohistochemistry (IHC) and Western blotting (WB) in fractionated cell lysates. NFAT5 expression was assessed by WB and RT-qPCR. localization and expression of NFAT5 were studied in muscle biopsies of patients diagnosed with polymyositis ( = 6), dermatomyositis ( = 10), inclusion body myositis ( = 11) and were compared to NFAT5 localization and expression in non-myopathic controls ( = 13). Muscle biopsies were studied by means of quantitative IHC and WB of total protein extracts. In unaffected myoblasts, hyperosmolar stress ensues in NFAT5 nuclear translocation and increased NFAT5 mRNA and protein expression. In contrast, pro-inflammatory cytokines did not lead to NFAT5 nuclear translocation nor increased expression. Cytokines IL-1β with IFN-γ induced colocalization of NFAT5 with histone deacetylase 6 (HDAC6), involved in cell motility. In muscle biopsies from dermatomyositis and polymyositis patients, NFAT5 colocalized with HDAC6, while in IBM, this was often absent. Our data suggest impaired NFAT5 localization and expression in unaffected myoblasts in response to inflammation. This disturbed myogenic NFAT5 physiology could possibly explain deleterious effects on muscle regeneration in myositis.
骨骼肌的再生依赖于成肌细胞的迁移和分化的调控，其中活化T细胞核因子5（NFAT5）转录因子参与其中。肌肉再生受损和慢性炎症在肌炎中很常见。关于炎症对这组疾病中NFAT5定位和表达的影响知之甚少。本研究的目的是研究在暴露于细胞因子或高渗应激的未受影响的成肌细胞以及在肌炎中NFAT5的生理学。使用肌炎细胞培养模型研究NFAT5的细胞内定位和表达。将成肌细胞暴露于富含促炎细胞因子IFN-γ和IL-1β的DMEM溶液或通过补充NaCl获得的高渗DMEM中。使用免疫组织化学（IHC）和分级细胞裂解物中的蛋白质印迹（WB）观察NFAT5的定位。通过WB和RT-qPCR评估NFAT5的表达。在诊断为多发性肌炎（n = 6）、皮肌炎（n = 10）、包涵体肌炎（n = 11）的患者的肌肉活检中研究NFAT5的定位和表达，并与非肌病对照（n = 13）中的NFAT5定位和表达进行比较。通过定量IHC和总蛋白提取物的WB研究肌肉活检。在未受影响的成肌细胞中，高渗应激导致NFAT5核转位以及NFAT5 mRNA和蛋白表达增加。相反，促炎细胞因子不会导致NFAT5核转位或表达增加。细胞因子IL-1β与IFN-γ诱导NFAT5与参与细胞运动的组蛋白去乙酰化酶6（HDAC6）共定位。在皮肌炎和多发性肌炎患者的肌肉活检中，NFAT5与HDAC6共定位，而在包涵体肌炎中，这种情况通常不存在。我们的数据表明，在未受影响的成肌细胞中，NFAT5的定位和表达因炎症而受损。这种肌源性NFAT5生理学紊乱可能解释了对肌炎中肌肉再生的有害影响。
