Sahu Itishri, Pelzl Lisann, Sukkar Basma, Fakhri Hajar, Al-Maghout Tamer, Cao Hang, Hauser Stefan, Gutti Ravi, Gawaz Meinrad, Lang Florian
Department of Cardiology and Vascular Medicine and Physiology, University of Tübingen, Tübingen, Germany.
Department of Biochemistry, School of Life Sciences, University of Hyderabad, Hyderabad, India.
FASEB J. 2017 Aug;31(8):3439-3448. doi: 10.1096/fj.201601211R. Epub 2017 Apr 26.
The transcription factor nuclear factor of activated T cells 5 (NFAT5) is up-regulated in several clinical disorders, including dehydration. NFAT5-sensitive genes include serum and glucocorticoid-inducible kinase 1 (SGK1). The kinase is a powerful regulator of Orai1, a Ca channel accomplishing store-operated Ca entry (SOCE). Orai1 is stimulated after intracellular store depletion by the Ca sensors stromal interaction molecule 1 (STIM1), or STIM2, or both. In the present study, we explored whether nuclear factor of activated T cell (NFAT)-5 influences Ca signaling in megakaryocytes. To this end, human megakaryocytic (MEG-01) cells were transfected with NFAT5 or with siNFAT5. Platelets and megakaryocytes were isolated from wild-type mice with either access to water or dehydration by 36 h of water deprivation. Transcript levels were determined with quantitative RT-PCR and protein abundance by Western blot analysis and flow cytometry, cytosolic (intracellular) Ca concentration ([Ca]) by fura-2-fluorescence. SOCE was estimated from the increase of [Ca] following readdition of extracellular Ca after store depletion with thapsigargin (1 µM). Platelet degranulation was estimated from P-selectin abundance and integrin activation from αβ integrin abundance determined by flow cytometry. As a result, NFAT5 transfection or exposure to hypertonicity (+40 mM NaCl) of MEG-01 cells increased Orai1, Orai2, STIM1, and STIM2 transcript levels. Orai1 transcript levels were decreased by NFAT5 silencing. NFAT5 transfection and IκB inhibitor BMS 345541 (5 µM) increased SOCE, whereas NFAT5 silencing and SGK1 inhibitor GSK650394 (10 µM) decreased SOCE. In the mice, dehydration increased NFAT5 and Orai1 protein abundance in megakaryocytes and NFAT5, Orai1, and Orai2 abundance in platelets. Dehydration further augmented the degranulation and integrin activation by thrombin and collagen-related peptide. In summary, NFAT5 is a powerful regulator of Orai1-expression and SOCE in megakaryocytes.-Sahu, I., Pelzl, L., Sukkar, B., Fakhri, H., al-Maghout, T., Cao, H., Hauser, S., Gutti, R., Gawaz, M., Lang, F. NFAT5-sensitive Orai1 expression and store-operated Ca entry in megakaryocytes.
转录因子活化T细胞核因子5（NFAT5）在包括脱水在内的多种临床病症中上调。NFAT5敏感基因包括血清和糖皮质激素诱导激酶1（SGK1）。该激酶是Orai1的强大调节因子，Orai1是一种实现储存性钙内流（SOCE）的钙通道。在细胞内储存耗尽后，Orai1会被钙传感器基质相互作用分子1（STIM1）或STIM2或两者刺激。在本研究中，我们探讨了活化T细胞核因子（NFAT）-5是否影响巨核细胞中的钙信号传导。为此，用NFAT5或siNFAT5转染人巨核细胞（MEG-01）。从可自由饮水或通过剥夺水分36小时而脱水的野生型小鼠中分离血小板和巨核细胞。通过定量RT-PCR测定转录水平，通过蛋白质印迹分析和流式细胞术测定蛋白质丰度，通过fura-2荧光测定胞质（细胞内）钙浓度（[Ca]）。在用毒胡萝卜素（1μM）耗尽储存后重新添加细胞外钙后，根据[Ca]的增加来估计SOCE。根据流式细胞术测定的P-选择素丰度估计血小板脱颗粒，并根据αβ整合素丰度估计整合素活化。结果，NFAT5转染或MEG-01细胞暴露于高渗（+40 mM NaCl）会增加Orai_{1}、Orai_{2}、STIM_{1}和STIM_{2}的转录水平。NFAT5沉默会降低Orai_{1}转录水平。NFAT5转染和IκB抑制剂BMS 345541（5μM）增加SOCE，而NFAT5沉默和SGK1抑制剂GSK650394（10μM）降低SOCE。在小鼠中，脱水会增加巨核细胞中NFAT5和Orai_{1}的蛋白质丰度以及血小板中NFAT5、Orai_{1}和Orai_{2}的丰度。脱水进一步增强了凝血酶和胶原相关肽引起的脱颗粒和整合素活化。总之，NFAT5是巨核细胞中Orai_{1}表达和SOCE的强大调节因子。-萨胡，I.，佩尔兹，L.，苏卡尔，B.，法赫里，H.，马古特，T.，曹，H.，豪泽，S.，古蒂，R.，加瓦兹，M.，朗，F.巨核细胞中NFAT5敏感的Orai_{1}表达和储存性钙内流 。
