Beta-Glycerophosphate-Induced ORAI1 Expression and Store Operated Ca2+ Entry in Megakaryocytes

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.

cloned as tonicity responsive enhancer binding protein (TonEBP) stimulated by hyperosmotic cell shrinkage 13,14 and subsequently been shown to be enhanced in several disorders, such as diabetes 15 , inflammation 16 and CKD 12 .
Platelets are released into the blood stream by megakaryocytes, which differentiate from hematopoietic progenitor cells in the bone marrow 26,27 . Megakaryocytes reorganize their cytoplasm into long proplatelet extensions that release platelets into the circulation 27 . Consequently, the proteins expressed in megakaryocytes are expected to be transferred into circulating blood platelets 27 . Recent observations revealed a powerful SGK1 dependent stimulation of ORAI1 expression by NFAT5 overexpression in megakaryocytes 28 .
In view of those observations we hypothesized that the phosphate-donor ß-glycerophosphate mimicking enhanced extracellular phosphate concentration may up-regulate the expression of NFAT5 in megakaryocytes, and that NFAT5 enhances the expression of SGK1, ORAI1 and STIM1 and/or STIM2. Considering that the abundance of the respective proteins in circulating platelets is a function of protein synthesis in megakaryocytes, enhanced extracellular phosphate may up-regulate the expression of NFAT5, SGK1, ORAI1 and STIM1 and/or STIM2 in circulating blood platelets. To the best of our knowledge, however, nothing is hitherto known on the contribution of NFAT5 to the regulation of megakaryocyte or platelet function in CKD patients.
The present study thus explored whether NFAT5, SGK1, ORAI1, ORAI2, ORAI3 STIM1 and/or STIM2 expression in megakaryocytes is sensitive to phosphate-donor ß-glycerophosphate and altered in patients with CKD incl. dialysis-dependency.

Results
As the transcription factor NFAT5 is known to upregulate expression of SGK1 17 which in turn activates transcription factor NFkB with upregulation of ORAI and STIM isoform expression 18 , the present study explored whether the phosphate donor ß-glycerophosphate modifies the transcript levels of the transcription factor nuclear factor of activated T cells 5 (NFAT5), of the NFAT5-regulated serum & glucocorticoid inducible kinase 1 (SGK1), of the SGK1-sensitive Ca 2+ release activated ion channels ORAI1, ORAI2 and ORAI3 as well as of the ORAI activating Ca 2+ sensor isoforms STIM1 and STIM2. As illustrated in Fig. 1, 24 hours exposure of human megakaryocytes to 2 mM ß-glycerophosphate was followed by a significant increase in the transcript levels of NFAT5, SGK1, ORAI1, ORAI2, ORAI3, STIM1, and STIM2 ( Fig. 1A-G). As illustrated in Fig. 1H, exposure to 2 mM ß-glycerophosphate further increases the transcription of fibroblast growth factor 23 (FGF23), a gene sensitive to ORAI1-dependent Ca 2+ entry into UMR106 bone cells 29 .
In order to test, whether the observed upregulation of NFAT5, SGK1, ORAI1, ORAI2, STIM1 and STIM2 by ß-glycerophosphate in megakaryocytes leads to the respective alterations of transcript levels in circulating blood platelets, the cells were isolated from patients with impaired kidney function and control volunteers with normal kidney function. Supplementary Fig. 2 displays creatinine plasma levels, glomerular filtration rate (GFR), as well as numbers of leukocytes and platelets in blood from control volunteers and patients with impaired kidney function. As illustrated in Fig. 3, the transcript levels of NFAT5, SGK1, ORAI1, ORAI2, and STIM2 were significantly higher in blood platelets from patients with impaired kidney function than in platelets from control volunteers. As illustrated in Fig. 4, NFAT5, SGK1, ORAI isoforms and STIM isoforms transcript levels were negatively correlated with GFR, i.e. a decline of GFR was associated with enhanced transcript levels of NFAT5, SGK1, ORAI1, ORAI2, STIM1 and STIM2.
Western blotting was employed in order to test, whether the enhanced NFAT5 and ORAI1 transcript levels in patients with impaired kidney function are paralleled by the respective alterations of protein abundance. As illustrated in Fig. 5, both, NFAT5 and ORAI1 protein abundance was significantly higher in platelets from patients with impaired kidney function than in platelets from control volunteers.

Discussion
The present study discloses a novel effect of the phosphate donor ß-glycerophosphate in megakaryocytes, i.e. the upregulation of ORAI1, ORAI2, ORAI3, STIM1 and STIM2 expression. ORAI1 is a Ca 2+ channel 22 shown to accomplish store operated Ca 2+ entry (SOCE) in multiple cell types 30 including platelets 20 and megakaryocytes 22 . To the best of our knowledge, an effect of phosphate or of phosphate donor ß-glycerophosphate on ORAI, STIM and their isoforms has never been shown before in megakaryocytes or platelets.
The effect of phosphate-donor ß-glycerophosphate is disrupted by pharmacological inhibition of SGK1 and is thus presumably due to upregulation of SGK1 by NFAT5. NFAT5 has previously been shown to increase the expression of SGK1 17 , which is known to trigger the degradation of the inhibitor protein IκBα thus allowing nuclear translocation of the transcription factor NFκB 18 . Genes up-regulated by NFκB include ORAI1 18 .
In view of the upregulation of NFAT5, SGK1, ORAI1, STIM1 and STIM2 expression in megakaryocytes by the phosphate donor ß-glycerophosphate, the transcript levels of NFAT5, SGK1, ORAI1, ORAI2, ORAI3, STIM1 and STIM2 were expected to be enhanced in platelets from patients with impaired kidney function. As a matter of fact, the transcript levels of each, NFAT5, SGK1, ORAI1, ORAI2, STIM1and STIM2 were significantly higher in platelets isolated from patients with impaired kidney function than in platelets isolated from control volunteers. As shown for NFAT5 and ORAI1, the increase of transcript levels is paralleled by the respective increase of protein abundance. NFAT5 expression has been shown to be upregulated by patients with advanced CKD incl. dialysis-dependency in other cell types 12 , but not in megakaryocytes or blood platelets.
The upregulation of ORAI1 and STIM2 in platelets of CKD patients is expected to sensitize the platelets for activators 31 . The presently observed stimulation of ORAI1 and STIM2 could thus contribute to the known high risk of cardiac infarction and stroke in CKD patients 32,33 . Excessive activation of blood platelets is well known www.nature.com/scientificreports www.nature.com/scientificreports/ to enhance the risk of cardiac infarction and stroke 34 . Additional effort is needed to define the contribution of phosphate-sensitive ORAI1 and STIM2 expression in blood platelets to the enhanced cardiovascular risk in patients with advanced CKD.
As shown for other cell types, NFAT5 is up-regulated in further clinical disorders, such as dehydration 11 , diabetes mellitus 15 and inflammatory disease 16 . In those conditions the enhanced NFAT5 expression may lead to stimulation of SGK1 expression with subsequent upregulation of ORAI1, STIM1 and STIM2 in megakaryocytes and sensitization of blood platelets to activating stimuli. The signalling may again involve SGK1-sensitive degradation of the inhibitor protein IκBα thus allowing nuclear translocation of the transcription factor NFκB.
Genes previously shown to be upregulated by NFAT5, SGK1, ORAI1, ORAI2, STIM1 and STIM2 in UMR106 cells include FGF23 29 . Here we demonstrate that the phosphate donor ß-glycerophosphate upregulates FGF23 in megakaryocytes. In view of the putative influence of SOCE on FGF23 transcription in UMR106 cells 29 , it is tempting to speculate that the observed signaling contributes to the phosphate-induced FGF23 release from bone. Whether ORAI1 expression and function is sensitive to phosphate in UMR106 cells remains, however, to be shown.
In conclusion, the phosphate donor ß-glycerophosphate stimulates the expression of NFAT5, SGK1, ORAI and STIM isoforms and thus store operated Ca 2+ entry (SOCE) into megakaryocytes (Fig. 6). Accordingly, NFAT5, SGK1, ORAI and STIM isoform expression is enhanced in platelets of patients with impaired kidney function and could thus contribute to the enhanced cardiovascular risk in those patients.

Materials and Methods
Patients and volunteers. 11   Quantitative PCR. To determine transcript levels of NFAT5, ORAI1, ORAI2, ORAI3, STIM1, STIM2 and FGF23 total RNA was extracted according to manufacturer's instructions with TriFast (Peqlab, Erlangen, Germany) [60][61][62][63][64] . DNAse digestion was performed to avoid DNA contamination and was followed by reverse transcription using random hexamers (Promega, Manheim, Germany) and GoScript ™ reverse transcription system (Promega, Manheim, Germany). Real-time polymerase chain reaction (RT-PCR) amplification of the respective genes were set up in a total volume of 20 µl using 40 ng of cDNA, 500 nM forward and reverse primer and 2x    Western blotting. Protein abundance of NFAT5, ORAI1, and GAPDH was determined by Western blotting [60][61][62][63][64] . After isolation of platelets from control donors and patients with impaired kidney function, platelets were centrifuged for 5 minutes at 600 g and 4 °C. The pellet was washed with ice cold PBS and suspended in 50 μl ice-cold RIPA lysis buffer (Cell Signalling Technology, USA) containing Protease Inhibitor Cocktail (Sigma-Aldrich, Taufkirchen, Germany). After centrifugation (20,000 g, 4 °C for 20 minutes) the supernatant was taken to determine protein concentration using the Bradford assay (BioRad, München, Germany). For Western blotting 50 µg of protein were electro-transferred onto a nitrocellulose membrane after electrophoresis using 12% SDS-PAGE and blocked with 5% milk in TBST at room temperature for 1 h. The membranes were incubated with primary anti-NFAT5 antibody (1:1000, Novus Biologicals), anti-ORAI1 antibody (1:1000, Proteintech, Chicago, USA) and anti-GAPDH antibody (1:1000, Cell Signaling, Danvers, USA) at 4 °C overnight. After washing (TBST), the blots were incubated with secondary anti-rabbit antibody conjugated with horseradish peroxidase (1:1000, Cell Signaling, Danvers, USA) for 1 h at room temperature. Protein bands were detected after additional washes (TBST) with an ECL detection reagent (Amersham, Freiburg, Germany). For densitometry image analysis, western blots were scanned and analyzed by ImageJ software (NIH, USA), and the results are shown as the ratio of total protein to GAPDH normalized to the control group. Protein-Marker VI (Peqlab, Erlangen, Germany) was used as reference to assign the right protein size.
Ethical permission. The study was approved by the ethics committee of the University of Tuebingen (270/2011BO1) and has been executed in accordance with the Declaration of Helsinki. Both, volunteers and patients provided informed written consent. The data have been presented at a conference 66 .