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

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

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

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

Inhibition of Lithium Sensitive Orai1/ STIM1 Expression and Store Operated Ca2+ Entry in Chorea-Acanthocytosis Neurons by NF-κB Inhibitor Wogonin

Background/Aims: The neurodegenerative disease Chorea-Acanthocytosis (ChAc) is caused by loss-of-function-mutations of the chorein-encoding gene VPS13A. In ChAc neurons transcript levels and protein abundance of Ca2+ release activated channel moiety (CRAC) Orai1 as well as its regulator STIM1/2 are decreased, resulting in blunted store operated Ca2+-entry (SOCE) and enhanced suicidal cell death. SOCE is up-regulated and cell death decreased by lithium. The effects of lithium are paralleled by upregulation of serum & glucocorticoid inducible kinase SGK1 and abrogated by pharmacological SGK1 inhibition. In other cell types SGK1 has been shown to be partially effective by upregulation of NFκB, a transcription factor stimulating the expression of Orai1 and STIM. The present study explored whether pharmacological inhibition of NFκB interferes with Orai1/STIM1/2 expression and SOCE and their upregulation by lithium in ChAc neurons. Methods: Cortical neurons were differentiated from induced pluripotent stem cells generated from fibroblasts of ChAc patients and healthy volunteers. Orai1 and STIM1 transcript levels and protein abundance were estimated from qRT-PCR and Western blotting, respectively, cytosolic Ca2+-activity ([Ca2+]i) from Fura-2-fluorescence, SOCE from increase of [Ca2+]i following Ca2+ re-addition after Ca2+-store depletion with sarco-endoplasmatic Ca2+-ATPase inhibitor thapsigargin (1µM), as well as CRAC current utilizing whole cell patch clamp recording. Results: Orai1 and STIM1 transcript levels and protein abundance as well as SOCE and CRAC current were significantly enhanced by lithium treatment (2 mM, 24 hours). These effects were reversed by NFκB inhibitor wogonin (50 µM). Conclusion: The stimulation of expression and function of Orai1/STIM1/2 by lithium in ChAc neurons are disrupted by pharmacological NFκB inhibition

Impact of Na+/Ca2+ Exchangers on Therapy Resistance of Ovary Carcinoma Cells

Background/Aims: According to previous observations, enhanced store-operated Ca2+-entry (SOCE) accomplished by the pore forming ion channel unit Orai1 and its regulator STIM1 contribute to therapy resistance of ovary carcinoma cells. Ca2+ signaling is further shaped by Ca2+ extrusion through K+-independent (NCX) and/or K+-dependent (NCKX) Na+/Ca2+-exchangers. The present study thus explored whether therapy resistance is further paralleled by altered expression and/or function of Na+/Ca2+-exchangers. Methods: In therapy resistant (A2780cis) and therapy sensitive (A2780sens) ovary carcinoma cells transcript levels were estimated from RT-PCR, cytosolic Ca2+-activity ([Ca2+]i) from Fura-2-fluorescence, Na+/Ca2+-exchanger activity from the increase of [Ca2+]i (Δ[Ca2+]i) and from whole cell current (Ica) following abrupt replacement of Na+ containing (130 mM) and Ca2+ free extracellular perfusate by Na+ free and Ca2+ containing (2 mM) extracellular perfusate, as well as cell death from PI -staining in flow cytometry. Results: The transcript levels of NCX3, NCKX4, NCKX5, and NCKX6, slope and peak of Δ[Ca2+]i as well as Ica were significantly higher in therapy resistant than in therapy sensitive ovary carcinoma cells. The Na+/Ca2+-exchanger inhibitor KB-R7943 (10 µM) significantly blunted Δ[Ca2+]i and significantly augmented the cisplatin-induced cell death of therapy resistant ovary carcinoma cells without significantly modifying cisplatin-induced cell death of therapy sensitive ovary carcinoma cells. Conclusion: Enhanced Na+/Ca2+-exchanger activity may contribute to the therapy sensitivity of ovary carcinoma cells