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

Sonidegib, a Novel Inhibitor of Suicidal Erythrocyte Death

Background/Aims: The Hedgehog pathway disrupting drug sonidegib is used in the treatment of basal cell carcinoma. Side effects of sonidegib include anemia, which could result either from impaired erythropoiesis or from loss of erythrocytes e.g. due to suicidal erythrocyte death or eryptosis, which is characterized by cell membrane scrambling with phosphatidylserine translocation to the cell surface and by cell shrinkage. Eryptosis is stimulated by cell stress, including energy depletion, hyperosmotic shock, oxidative stress and excessive increase of cytosolic Ca2+ activity ([Ca2+]i). The present study explored, whether sonidegib exerts an effect on eryptosis. Methods: Human erythrocytes have been treated with energy depletion (glucose withdrawal for 48 hours), hyperosmotic shock (addition of 550 mM sucrose for 6 hours), oxidative stress (addition of 0.3 mM tert-butylhydroperoxide [tBOOH] for 50 min) or Ca2+ ionophore ionomycin (1 µM for 60 min) in absence and presence of sonidegib (2-6 µg/ ml). After treatment flow cytometry was employed to quantify phosphatidylserine exposure at the cell surface from annexin-V-binding, and cell volume from forward scatter. Hemolysis was estimated from the hemoglobin concentration in the supernatant. Results: In the absence of cell stress exposure to sonidegib did not significantly modify annexin-V-binding or forward scatter, but triggered hemolysis. Energy depletion, hyperosmotic shock, oxidative stress and ionomycin, all markedly and significantly increased the percentage of annexin-V-binding erythrocytes, and decreased the forward scatter. Sonidegib significantly blunted the effect of energy depletion, hyperosmotic shock, and oxidative stress, but not of ionomycin on annexin-V-binding. Sonidegib further significantly blunted the effect of energy depletion, but not of hyperosmotic shock, oxidative stress, and ionomycin on forward scatter. Conclusions: Sonidegib is a novel inhibitor of erythrocyte cell membrane scrambling following energy depletion, hyperosmotic shock and oxidative stress

Inhibition of Suicidal Erythrocyte Death by Volasertib

Background/Aims: The Polo-like kinase 1 (Plk1) inhibitor volasertib is used in the treatment of malignancy. Volasertib is partially effective by triggering suicidal death or apoptosis of tumor cells. Similar to apoptosis of nucleated cells, erythrocytes may enter suicidal cell death or eryptosis, which is characterized by cell membrane scrambling with phosphatidylserine translocation to the cell surface and by cell shrinkage. Stimulators of eryptosis include energy depletion, hyperosmotic shock, oxidative stress and excessive increase of cytosolic Ca2+ activity ([Ca2+]i). The present study explored, whether volasertib impacts on eryptosis. Methods: Human erythrocytes have been exposed to energy depletion (glucose withdrawal for 48 hours), hyperosmotic shock (addition of 550 mM sucrose for 6 hours), oxidative stress (addition of 0.3 mM tert-butylhydroperoxide [tBOOH] for 50 min) or Ca2+ ionophore ionomycin (1 µM for 60 min) in absence and presence of volasertib (0.5-1.5 µg/ml) and flow cytometry was employed to quantify phosphatidylserine exposure at the cell surface from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3 fluorescence, reactive oxygen species from 2’,7’-dichlorodihydrofluorescein diacetate (DCFDA) fluorescence and ceramide abundance utilizing antibodies. For comparison, annexin-V-binding and forward scatter were determined following a 48 hours exposure of human leukemic K562 cells in RPMI-1640 medium to volasertib. Results: Treatment with volasertib alone did not significantly modify annexin-V-binding or forward scatter in mature erythrocytes. Energy depletion, hyperosmotic shock, oxidative stress and ionomycin, all markedly and significantly increased the percentage of annexin-V-binding erythrocytes, and decreased the forward scatter. Volasertib significantly blunted the effect of energy depletion and hyperosmotic shock, but not of oxidative stress and ionomycin on annexin-V-binding. Volasertib did not significantly influence the effect of any maneuver on forward scatter. In K562 cells, volasertib enhanced annexin-V-binding and decreased the forward scatter. Conclusions: Volasertib is a novel inhibitor of erythrocyte cell membrane scrambling following energy depletion and hyperosmotic shock, effects contrasting the stimulation of K562 cell apoptosis