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

Abstract

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 Ca 2+-channel accomplishing store-operated ca 2+-entry (SOCE). ORAI1 is stimulated following intracellular store depletion by Ca 2+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 Ca 2+-concentration ([Ca 2+ ] i) by Fura-2-fluorescence, and SOCE from increase of [Ca 2+ ] i following re-addition of extracellular ca 2+ 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 storeoperated ca 2+-entry. Compromised renal elimination of phosphate leads in chronic kidney disease (CKD) to increase of phosphate concentration in plasma and tissues which in turn triggers vascular calcification leading to cardiovascular events and the respective increases of morbidity and mortality 1-4. Calcium deposition in the vascular wall involves osteo-/chrondrogenic reprogramming of vascular smooth muscle cells (VSMCs) 5-8. The signaling includes up-regulation of the transcription factor NFAT5 (nuclear factor of activated T cells 5) 9-12. NFAT5 was originally

Effect of MgCl2 and GdCl3 on ORAI1 Expression and Store-Operated Ca2+ Entry in Megakaryocytes

“…As shown in Figure 1 , MgCl 2 treatment significantly upregulated CaSR expression with or without prior exposure to ß-glycerophosphate. In agreement with earlier observations [ 30 ], a 24-h pretreatment of human megakaryocytes with the phosphate donor ß-glycerophosphate upregulated the transcript levels of NFAT5 , SGK1 , ORAI1 , ORAI2 , ORAI3 , STIM1 and STIM2 . All those effects were significantly blunted or virtually abrogated by additional exposure to 1.5 mM MgCl 2 .…”

“…The present study confirms previous observations [ 30 ] demonstrating the upregulation of NFAT5, ORAI1, ORAI2, ORAI3, STIM1 and STIM2 expression as well as SOCE by the phosphate donor ß-glycerophosphate in megakaryocytes.…”

“…Platelets are generated by megakaryocytes, and protein abundance in platelets depends on megakaryocytic transcript and protein expression [ 28 , 29 ]. Megakaryocytes express ORAI1, ORAI2 and ORAI3 [ 30 ], thus accomplishing SOCE [ 31 , 32 , 33 , 34 ]. ORAI expression and SOCE are upregulated by the phosphate donor ß-glycerophosphate [ 30 ].…”

“…Megakaryocytes express ORAI1, ORAI2 and ORAI3 [ 30 ], thus accomplishing SOCE [ 31 , 32 , 33 , 34 ]. ORAI expression and SOCE are upregulated by the phosphate donor ß-glycerophosphate [ 30 ]. The stimulation of vascular calcification by phosphate has been shown to be reversed by Mg 2+ and the calcium-sensing receptor (CaSR) agonist Gd 3+ [ 17 , 35 ].…”

Severe COVID-19 infection is associated with increased antibody-mediated platelet apoptosis

“…Cytosolic Ca 2+ concentration was determine using Fluo-3/AM (Sigma-Aldrich, St. Louis, USA) as previously described 24 , with minor modifications. In brief, PRP were incubated with Fluo3/AM (3 μM) in TRIS (trisaminomethane) buffer (10 mM TRIS, 0.9% NaCl, 1 mM CaCl 2 , pH 7.4) for 30 min* at 37°C.…”