P38 Kinase, SGK1 and NF-κB Dependent Up-Regulation of Na+/Ca2+ Exchanger Expression and Activity Following TGFß1 Treatment of Megakaryocytes

Abstract

Background: 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. Methods: 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. Results: 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). Conclusions: 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.