Department of Pharmacology, Experimental Therapy & Toxicology, Eberhard Karls University, 72074 Tübingen, Germany.
Institute of Preventive Veterinary Medicine, Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu 611130, China.
Int J Mol Sci. 2021 Mar 24;22(7):3292. doi: 10.3390/ijms22073292.
In chronic kidney disease, hyperphosphatemia upregulates the Ca channel ORAI and its activating Ca sensor STIM in megakaryocytes and platelets. ORAI1 and STIM1 accomplish store-operated Ca entry (SOCE) and play a key role in platelet activation. Signaling linking phosphate to upregulation of ORAI1 and STIM1 includes transcription factor NFAT5 and serum and glucocorticoid-inducible kinase SGK1. In vascular smooth muscle cells, the effect of hyperphosphatemia on ORAI1/STIM1 expression and SOCE is suppressed by Mg and the calcium-sensing receptor (CaSR) agonist Gd. The present study explored whether sustained exposure to Mg or Gd interferes with the phosphate-induced upregulation of NFAT5, SGK1, ORAI1,2,3, STIM1,2 and SOCE in megakaryocytes. To this end, human megakaryocytic Meg-01 cells were treated with 2 mM ß-glycerophosphate for 24 h in the absence and presence of either 1.5 mM MgCl or 50 µM GdCl. Transcript levels were estimated utilizing q-RT-PCR, protein abundance by Western blotting, cytosolic Ca concentration ([Ca]) by Fura-2 fluorescence and SOCE from the increase in [Ca] following re-addition of extracellular Ca after store depletion with thapsigargin (1 µM). As a result, Mg and Gd upregulated CaSR and blunted or virtually abolished the phosphate-induced upregulation of NFAT5, SGK1, ORAI1,2,3, STIM1,2 and SOCE in megakaryocytes. In conclusion, Mg and the CaSR agonist Gd interfere with phosphate-induced dysregulation of [Ca] in megakaryocytes.
在慢性肾脏病中，高磷血症上调巨核细胞和血小板中的钙通道 ORAI 和其激活钙传感器 STIM。ORAI1 和 STIM1 完成钙库操纵性钙内流 (SOCE)，并在血小板激活中发挥关键作用。将磷酸盐与 ORAI1 和 STIM1 的上调相关联的信号包括转录因子 NFAT5 和血清和糖皮质激素诱导激酶 SGK1。在血管平滑肌细胞中，高磷血症对 ORAI1/STIM1 表达和 SOCE 的影响被镁和钙敏感受体 (CaSR) 激动剂 Gd 抑制。本研究探讨了持续暴露于镁或 Gd 是否会干扰磷酸盐诱导的 NFAT5、SGK1、ORAI1、2、3、STIM1、2 和 SOCE 在巨核细胞中的上调。为此，在不存在或存在 1.5 mM MgCl 或 50 µM GdCl 的情况下，用 2 mM β-甘油磷酸处理人巨核细胞 Meg-01 细胞 24 小时。利用 q-RT-PCR 估计转录物水平，通过 Western blot 测定蛋白丰度，通过 Fura-2 荧光测定细胞浆 Ca 浓度 ([Ca])，并通过在钙库耗竭后（1 µM 他普西龙）再添加细胞外 Ca 以增加 [Ca] 来测定 SOCE。结果，镁和 Gd 上调了 CaSR，并减弱或几乎消除了磷酸盐诱导的 NFAT5、SGK1、ORAI1、2、3、STIM1、2 和 SOCE 在巨核细胞中的上调。总之，镁和 CaSR 激动剂 Gd 干扰了巨核细胞中磷酸盐诱导的 Ca 失调。
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