Basma Sukkar

Impairment of renal phosphate elimination in chronic kidney disease (CKD) leads to enhanced plasma and tissue phosphate concentration, which in turn up-regulates transcription factor NFAT5 and serum & glucocorticoid-inducible kinase SGK1. The kinase upregulates ORAI1, a Ca²⁺-channel accomplishing store-operated Ca²⁺-entry (SOCE). ORAI1 is stimulated following intracellular store depletion by Ca²⁺-sensors STIM1 and/or STIM2. In megakaryocytes and blood platelets SOCE and thus ORAI1 are powerful regulators of activity. The present study explored whether the phosphate-donor ß-glycerophosphate augments NFAT5, ORAI1,2,3 and/or STIM1,2 expressions and thus SOCE in megakaryocytes. Human megakaryocytic Meg01cells were exposed to 2 mM of phosphate-donor ß-glycerophosphate for 24 hours. Platelets were isolated from blood samples of patients with impaired kidney function or control volunteers. Transcript levels were estimated utilizing q-RT-PCR, cytosolic Ca²⁺-concentration ([Ca²⁺]_i) by Fura-2-fluorescence, and SOCE from increase of [Ca²⁺]_i following re-addition of extracellular Ca²⁺ after store depletion with thapsigargin (1 µM). NFAT5 and ORAI1 protein abundance was estimated with Western blots. As a result, ß-glycerophosphate increased NFAT5, ORAI1/2/3, STIM1/2 transcript levels, as well as SOCE. Transcript levels of NFAT5, SGK1, ORAI1/2/3, and STIM1/2 as well as NFAT5 and ORAI1 protein abundance were significantly higher in platelets isolated from patients with impaired kidney function than in platelets from control volunteers. In conclusion, phosphate-donor ß-glycerophosphate triggers a signaling cascade of NFAT5/SGK1/ORAI/STIM, thus up-regulating store-operated Ca²⁺-entry.

Compromised renal phosphate elimination in chronic kidney disease (CKD) leads to hyperphosphatemia, which in turn triggers osteo-/chondrogenic signaling in

The transcription factor p53 suppresses tumor growth by inducing nucleated cell apoptosis and cycle arrest. Because of its influence on primitive erythroid cell differentiation and survival, p53 is an important determinant of erythropoiesis. However, the impact of p53 on the fate of erythrocytes, cells lacking nucleus and mitochondria, during their post-maturation phase in the circulation remained elusive. Erythrocyte survival may be compromised by suicidal erythrocyte death or eryptosis, which is hallmarked by phosphatidylserine translocation and stimulated by increase of cytosolic Ca²⁺ concentration. Here, we comparatively examined erythrocyte homeostasis in p53-mutant mice (Trp53^tm1Tyj/J) and in corresponding WT mice (C57BL/6J) by analyzing eryptosis and erythropoiesis. To this end, spontaneous cell membrane phosphatidylserine exposure and cytosolic Ca²⁺ concentration were higher in erythrocytes drawn from Trp53^tm1Tyj/J mice than from WT mice. Eryptosis induced by glucose deprivation, a pathophysiological cell stressor, was slightly, but significantly more prominent in erythrocytes drawn from Trp53^tm1Tyj/J mice as compared to WT mice. The loss of erythrocytes by eryptosis was fully compensated by enhanced erythropoiesis in Trp53^tm1Tyj/J mice, as reflected by increased reticulocytosis and abundance of erythroid precursor cells in the bone marrow. Accordingly, erythrocyte number, packed cell volume and hemoglobin were similar in Trp53^tm1Tyj/J and WT mice. Taken together, functional p53 deficiency enhances the turnover of circulating erythrocytes by parallel increase of eryptosis and stimulated compensatory erythropoiesis.

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.

Background: The widely expressed protein chorein fosters activation of the phosphoinositide 3 kinase (PI3K) pathway thus supporting cell survival. Loss of function mutations of the chorein encoding gene VPS13A (vacuolar protein sorting-associated protein 13A) causes chorea-acanthocytosis (ChAc), a neurodegenerative disorder paralleled by deformations of erythrocytes. In mice, genetic knockout of chorein leads to enhanced neuronal apoptosis. PI3K dependent signalling upregulates Orai1, a pore forming channel protein accomplishing store operated Ca²⁺ entry (SOCE). Increased Orai1 expression and SOCE have been shown to confer survival of tumor cells. SOCE could be up-regulated by lithium. The present study explored, whether SOCE and/or apoptosis are altered in ChAc fibroblasts and could be modified by lithium treatment. Methods: Fibroblasts were isolated from ChAc patients and age-matched healthy volunteers. Cytosolic Ca²⁺ activity ([Ca²⁺]_i) was estimated from Fura-2-fluorescence, SOCE from increase of [Ca²⁺]_i following Ca²⁺ re-addition after Ca²⁺-store depletion with sarcoendoplasmatic Ca²⁺-ATPase (SERCA) inhibitor thapsigargin (1 µM), and apoptosis from annexin-V/propidium iodide staining quantified in flow cytometry. Results: SOCE was significantly smaller in ChAc fibroblasts than in control fibroblasts. Lithium (2 mM, 24 hours) significantly increased and Orai1 blocker 2-Aminoethoxydiphenyl Borate (2-APB, 50 µM, 24 hours) significantly decreased SOCE. Annexin-V-binding and propidium iodide staining were significantly higher in ChAc fibroblasts than in control fibroblasts. In ChAc fibroblasts annexin-V-binding and propidium iodide staining were significantly decreased by lithium treatment, significantly increased by 2-APB and virtually lithium insensitive in the presence of 2-APB. Conclusions: In ChAc fibroblasts, downregulation of SOCE contributes to enhanced susceptibility to apoptosis. Both, decreased SOCE and enhanced apoptosis of ChAc fibroblasts can be reversed by lithium treatment.

The transcription factor nuclear factor of activated T cells 5 (NFAT5) is up‐regulated in several clinical disorders, including dehydration. NFAT5‐sensitive genes include serum and glucocorticoid‐inducible kinase 1 (SGK1). The kinase is a powerful regulator of Orai1, a Ca ²⁺ channel accomplishing store‐operated Ca ²⁺ entry (SOCE). Orai1 is stimulated after intracellular store depletion by the Ca ²⁺ sensors stromal interaction molecule 1 (STIM1), or STIM2, or both. In the present study, we explored whether nuclear factor of activated T cell (NFAT)‐5 influences Ca ²⁺ signaling in megakaryocytes. To this end, human megakaryocytic (MEG‐01) cells were transfected with NFAT5 or with siNFAT5. Platelets and megakaryocytes were isolated from wild‐type mice with either access to water ad libitum or dehydration by 36 h of water deprivation. Transcript levels were determined with quantitative RT‐PCR and protein abundance by Western blot analysis and flow cytometry, cytosolic (intracellular) Ca ²⁺ concentration ([Ca ²⁺ ] _i ) by fura‐2‐fluorescence. SOCE was estimated from the increase of [Ca ²⁺ ] _i following readdition of extracellular Ca ²⁺ after store depletion with thapsigargin (1 mM). Platelet degranulation was estimated from P‐selectin abundance and integrin activation from α _IIb b ₃ integrin abundance determined by flow cytometry. As a result, NFAT5 transfection or exposure to hypertonicity (+40 mM NaCl) of MEG‐01 cells increased Orai1, Orai2, STIM1, and STIM2 transcript levels. Orai1 transcript levels were decreased by NFAT5 silencing. NFAT5 transfection and IkB inhibitor BMS 345541 (5 mM) increased SOCE, whereas NFAT5 silencing and SGK1 inhibitor GSK650394 (10 mM) decreased SOCE. In the mice, dehydration increased NFAT5 and Orai1 protein abundance in megakaryocytes and NFAT5, Orai1, and Orai2 abundance in platelets. Dehydration further augmented the degranulation and integrin activation by thrombin and collagen‐related peptide. In summary, NFAT5 is a powerful regulator of Orai1‐expression and SOCE in megakaryocytes.—Sahu, I., Pelzl, L., Sukkar, B., Fakhri, H., al‐Maghout, T., Cao, H., Hauser, S., Gutti, R., Gawaz, M., Lang, F. NFAT5‐sensitive Orai1 expression and store‐operated Ca ²⁺ entry in megakaryocytes. FASEB J . 31, 3439–3448 (2017). www.fasebj.org

Chorea-Acanthocytosis (ChAc), a neurodegenerative disorder, results from loss-of-function-mutations of chorein-encoding gene VPS13A. In tumour cells chorein up-regulates ORAI1, a Ca²⁺-channel accomplishing store operated Ca²⁺-entry (SOCE) upon stimulation by STIM1. Furthermore SOCE could be up-regulated by lithium. The present study explored whether SOCE impacts on neuron apoptosis. 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, as well as apoptosis from annexin-V-binding and propidium-iodide uptake determined by flow cytometry. As a result, ORAI1 and STIM1 transcript levels and protein abundance and SOCE were significantly smaller and the percentage apoptotic cells significantly higher in ChAc neurons than in control neurons. Lithium treatment (2 mM, 24 hours) increased significantly ORAI1 and STIM1 transcript levels and protein abundance, an effect reversed by inhibition of Serum & Glucocorticoid inducible Kinase 1. ORAI1 blocker 2-APB (50 µM, 24 hours) significantly decreased SOCE, markedly increased apoptosis and abrogated the anti-apoptotic effect of lithium. In conclusion, enhanced neuronal apoptosis in ChAc at least partially results from decreased ORAI1 expression and SOCE, which could be reversed by lithium treatment.