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

Abstract

Transforming Growth Factor β1 (TGFβ1) plays an important role in the maturation of megakaryocyte and formation of platelets. TGFβ1 can up-regulate Ca2+ entry through store operated Ca2+ entry (SOCE) and on the contrary, it can up-regulate Ca2+ exclusion by upregulating the activity of Na+/Ca2+ exchangers. TGFβ1 first enhances the increase of intracellular Ca2+ triggered by the release of Ca2+ from intracellular stores, then it enhances the subsequent decline of [Ca2+]i. The mechanism of action, by which TGFβ1 up-regulates SOCE, is based on a signalling pathway requires the activation of p38 MAP Kinase, Serum & Glucocorticoid inducible Kinase (SGK1), and Nuclear Factor κB (NFκB). On the other hand, the mechanism of action, by which TGFβ1 upregulates Na+/Ca2+ exchangers remained unidentified, as well as the specific Na+/Ca2+ exchanger isoforms involved in the process of up-regulation. The present study aimed to identify, whether TGFβ1 influences the expression and activity of K+-independent (NCX) and K+-dependent (NCKX) Na+/Ca2+ exchangers, and aimed also to explore the signalling involved. Methods: In human megakaryocytic cells (MEG01), Fura-2 fluorescence was utilized to observe cytosolic Ca2+ activity [Ca2+]i. The activity of Na+/Ca2+ exchanger was studied by observing the rise in [Ca2+]i resulting from changing the extracellular solution from a solution with 0 mM Ca2+ and 130 mM Na+ to a solution with 2 mM Ca2+ and 0 Na+. For analysis of NCX, the concentration of K+ was 0 mM. For analysis of NCKX, the concentration of K+ was 40 mM. In order to quantify transcription levels of NCX/NCKX isoform, RT-PCR was applied. Results: TGFβ1 (60 ng/ml, 24 h) was found to increase significantly the transcription levels of certain isoforms of NCX/NCKX including: NCX1, NCKX1, NCKX2 and NCKX5. Additionally, the activity of NCX and NCKX was shown to be increased significantly in the presence of TGFβ1 (60 ng/ml, 24 h). Skepinone-L (1 μM), a p38 MAP Kinase inhibitor, caused a significant downregulation of the effect of TGFβ1 on both transcription levels and activity of NCX and NCKX. GSK-650394 (10 μM), an inhibitor of SGK1, and Wogonin (100 μM), and inhibitor of NFκB, caused a significant downregulation of the effect of TGFβ1 on the activity of NCX and NCKX. Conclusions: P38 MAP Kinase, SGK1 and NFκB are involved in the signaling pathway by which TGFβ1 increases the activity of Na+/Ca2+ exchanger and the transcription levels of NCX1, NCKX1, NCKX2, and NCKX5.