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

Abstract

Background: Impaired renal elimination of phosphate in chronic kidney disease (CKD) contributes to hyperphosphatemia, which in turn upregulates the expression of nuclear factor of activated T cells 5 (NFAT5) and serum and glucocorticoid-inducible kinase SGK1 (SGK1) in megakaryocytes and platelets. SGK1 is a potent stimulator of ORAI1, a Ca2+-channel activated by Ca2+ sensor stromal interaction molecule 1 (STIM1) following the internal Ca2+ store depletion. Both ORAI1 and STIM1 are considered major components of store-operated Ca2+ entry (SOCE) and play a crucial role in platelet activation, thus accounting for the high risk for cardiovascular events in CKD patients. In vascular smooth muscle cells, sustained exposure to MgCl2 and the Ca2+-sensing receptor (CaSR) agonist GdCl3 significantly attenuated the stimulatory effect of phosphate on ORAI1/STIM1 expression as well as SOCE activity. Our present study investigated whether phosphate-triggered upregulation of NFAT5, SGK1, ORAI1/2/3, STIM1/2 and SOCE is similarly sensitive to MgCl2 or GdCl3 in megakaryocytes. Methods: Human megakaryocytic cells (Meg-01) were exposed to 1.5 mM MgCl2 or 50 µM GdCl3 for 24 h without or with 2 mM β-glycerophosphate treatment. Transcript and protein abundance were evaluated utilizing qPCR and immunoblotting, respectively. Cytosolic Ca2+ activity was estimated by ratiometric Ca2+ imaging with Fura-2 fluorescence. SOCE activity was determined from the peak and slope of the increase in the 340/380 nm ratio, following extracellular Ca2+ re-addition after thapsigargin-evoked store depletion. Results: 1.5 mM MgCl2 and 50 µM GdCl3 upregulated CaSR expression and effectively reversed the phosphate-stimulated SOCE enhancement via inhibiting the signalling cascade of NFAT5/SGK1/ORAIs/STIMs. Conclusions: MgCl2 and the CaSR agonist GdCl3 are powerful regulators of ORAI1/STIM1 expression and SOCE, involved in phosphate-mediated Ca2+ signalling in megakaryocytes.