Al-Maghout Tamer, Pelzl Lisann, Sahu Itishri, Sukkar Basma, Hosseinzadeh Zohreh, Gutti Ravi, Laufer Stefan, Voelkl Jakob, Pieske Burkert, Gawaz Meinrad, Lang Florian
Department of Internal Medicine III, University of Tuebingen, Tuebingen, Germany.
Department of Biochemistry, School of Life Sciences, University of Hyderabad, Hyderabad, India.
Cell Physiol Biochem. 2017;42(6):2169-2181. doi: 10.1159/000479992. Epub 2017 Aug 15.
TGFβ1, a decisive regulator of megakaryocyte maturation and platelet formation, has previously been shown to up-regulate both, store operated Ca2+ entry (SOCE) and Ca2+ extrusion by Na+/Ca2+ exchange. The growth factor thus augments the increase of cytosolic Ca2+ activity ([Ca2+]i) following release of Ca2+ from intracellular stores and accelerates the subsequent decline of [Ca2+]i. The effect on SOCE is dependent on a signaling cascade including p38 kinase, serum & glucocorticoid inducible kinase SGK1, and nuclear factor NFκB. The specific Na+/Ca2+ exchanger isoforms involved and the signalling regulating the Na+/Ca2+ exchangers remained, however elusive. The present study explored, whether TGFβ1 influences the expression and function of K+ insensitive (NCX) and K+ sensitive (NCKX) Na+/Ca2+ exchangers, and aimed to shed light on the signalling involved.
In human megakaryocytic cells (MEG01) RT-PCR was performed to quantify NCX/NCKX isoform transcript levels, [Ca2+]i was determined by Fura-2 fluorescence, and Na+/Ca2+ exchanger activity was estimated from the increase of [Ca2+]i following switch from an extracellular solution with 130 or 90 mM Na+ and 0 mM Ca2+ to an extracellular solution with 0 Na+ and 2 mM Ca2+. K+ concentration was 0 mM for analysis of NCX and 40 mM for analysis of NCKX.
TGFβ1 (60 ng/ml, 24 h) significantly increased the transcript levels of NCX1, NCKX1, NCKX2 and NCKX5. Moreover, TGFβ1 (60 ng/ml, 24 h) significantly increased the activity of both, NCX and NCKX. The effect of TGFβ1 on NCX and NCKX transcript levels and activity was significantly blunted by p38 kinase inhibitor Skepinone-L (1 µM), the effect on NCX and NCKX activity further by SGK1 inhibitor GSK-650394 (10 µM) and NFκB inhibitor Wogonin (100 µM).
TGFβ1 markedly up-regulates transcription of NCX1, NCKX1, NCKX2, and NCKX5 and thus Na+/Ca2+ exchanger activity, an effect requiring p38 kinase, SGK1 and NFκB.
转化生长因子β1（TGFβ1）是巨核细胞成熟和血小板形成的决定性调节因子，此前已表明它能上调储存性钙内流（SOCE）以及通过钠/钙交换进行的钙外排。因此，这种生长因子会增强细胞内钙库释放钙后胞质钙活性（[Ca2+]i）的增加，并加速随后[Ca2+]i的下降。对SOCE的影响依赖于包括p38激酶、血清及糖皮质激素诱导激酶SGK1和核因子NFκB在内的信号级联反应。然而，所涉及的具体钠/钙交换异构体以及调节钠/钙交换体的信号仍不清楚。本研究探讨了TGFβ1是否影响钾离子不敏感型（NCX）和钾离子敏感型（NCKX）钠/钙交换体的表达和功能，并旨在阐明其中涉及的信号传导。
在人巨核细胞（MEG01）中进行逆转录聚合酶链反应（RT-PCR）以定量NCX/NCKX异构体转录水平，通过Fura-2荧光测定[Ca2+]i，并根据从含130或90 mM钠和0 mM钙的细胞外溶液切换到含0钠和2 mM钙的细胞外溶液后[Ca2+]i的增加来估计钠/钙交换体活性。分析NCX时钾离子浓度为0 mM，分析NCKX时钾离子浓度为40 mM。
TGFβ1（60 ng/ml，24小时）显著增加了NCX1、NCKX1、NCKX2和NCKX5的转录水平。此外，TGFβ1（60 ng/ml，24小时）显著增加了NCX和NCKX的活性。p38激酶抑制剂Skepinone-L（1 μM）显著减弱了TGFβ1对NCX和NCKX转录水平及活性的影响，SGK1抑制剂GSK-650394（10 μM）和NFκB抑制剂汉黄芩素（100 μM）进一步减弱了对NCX和NCKX活性的影响。
TGFβ1显著上调NCX1、NCKX1、NCKX2和NCKX5的转录，从而增加钠/钙交换体活性，这一效应需要p38激酶、SGK1和NFκB。
