基于糖皮质激素受体 GR 和 NFAT5 描述一种可能解释糖皮质激素在杜氏肌营养不良成纤维细胞中抗增殖作用的新机制。
Description of a Novel Mechanism Possibly Explaining the Antiproliferative Properties of Glucocorticoids in Duchenne Muscular Dystrophy Fibroblasts Based on Glucocorticoid Receptor GR and NFAT5.
机构信息
Department of Neurology, Ghent University Hospital, Ghent University, C. Heymanslaan 10, 9000 Ghent, Belgium.
Neuromuscular Reference Center, Ghent University Hospital, C. Heymanslaan 10, 9000 Ghent, Belgium.
出版信息
Int J Mol Sci. 2020 Dec 3;21(23):9225. doi: 10.3390/ijms21239225.
Glucocorticoids are drugs of choice in Duchenne muscular dystrophy (DMD), prolonging patients' ambulation. Their mode of action at the protein level is not completely understood. In DMD, muscle tissue is replaced by fibrotic tissue produced by fibroblasts, reducing mobility. Nuclear factor of activated T-cells 5 (NFAT5) is involved in fibroblast proliferation. By treating one DMD fibroblast cell culture and one of unaffected skeletal muscle fibroblasts with methylprednisolone (MP) or hydrocortisone (HC) for 24 h or 12 d, the antiproliferative properties of glucocorticoids could be unraveled. NFAT5 localization and expression was explored by immunocytochemistry (ICC), Western blotting (WB) and RT-qPCR. NFAT5 and glucocorticoid receptor (GR) colocalization was measured by ImageJ. GR siRNA was used, evaluating GR's influence on NFAT5 expression during MP and HC treatment. Cell proliferation was monitored by IncuCyte ZOOM. In DMD fibroblasts, treatment with MP for 24 h induced dots (ICC) positive for NFAT5 and colocalizing with GR. After 12 d of MP or HC in DMD fibroblasts, NFAT5 expression was decreased (RT-qPCR and WB) and growth arrest was observed (Incucyte ZOOM), whereas NFAT5 expression and cell growth remained unchanged in unaffected skeletal muscle fibroblasts. This study may help understand the antiproliferative properties of glucocorticoids in DMD fibroblasts.
糖皮质激素是杜氏肌营养不良症 (DMD) 的首选药物，可延长患者的活动能力。但其在蛋白质水平的作用机制尚不完全清楚。在 DMD 中，肌肉组织被成纤维细胞产生的纤维组织所取代，从而降低了运动能力。活化 T 细胞核因子 5（NFAT5）参与成纤维细胞增殖。通过用甲泼尼龙（MP）或氢化可的松（HC）处理一个 DMD 成纤维细胞培养物和一个未受影响的骨骼肌成纤维细胞 24 小时或 12 天，可揭示糖皮质激素的抗增殖特性。通过免疫细胞化学（ICC）、Western blot（WB）和 RT-qPCR 探索 NFAT5 的定位和表达。使用 ImageJ 测量 NFAT5 和糖皮质激素受体（GR）的共定位。GR siRNA 被用来评估 GR 在 MP 和 HC 处理期间对 NFAT5 表达的影响。通过 IncuCyte ZOOM 监测细胞增殖。在 DMD 成纤维细胞中，MP 处理 24 小时诱导 NFAT5 阳性斑点（ICC），并与 GR 共定位。在 DMD 成纤维细胞中用 MP 或 HC 处理 12 天后，NFAT5 表达减少（RT-qPCR 和 WB）并观察到生长停滞（Incucyte ZOOM），而未受影响的骨骼肌成纤维细胞中 NFAT5 表达和细胞生长保持不变。这项研究可能有助于理解糖皮质激素在 DMD 成纤维细胞中的抗增殖特性。
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