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

ABSTRACT: In chronic kidney disease, hyperphosphatemia upregulates the Ca²⁺ channel ORAI and its activating Ca²⁺ sensor STIM in megakaryocytes and platelets. ORAI1 and STIM1 accomplish store-operated Ca²⁺ entry (SOCE) and play a key role in platelet activation. Signaling linking phosphate to upregulation of ORAI1 and STIM1 includes transcription factor NFAT5 and serum and glucocorticoid-inducible kinase SGK1. In vascular smooth muscle cells, the effect of hyperphosphatemia on ORAI1/STIM1 expression and SOCE is suppressed by Mg²⁺ and the calcium-sensing receptor (CaSR) agonist Gd³⁺. The present study explored whether sustained exposure to Mg²⁺ or Gd³⁺ interferes with the phosphate-induced upregulation of NFAT5, SGK1, ORAI1,2,3, STIM1,2 and SOCE in megakaryocytes. To this end, human megakaryocytic Meg-01 cells were treated with 2 mM ß-glycerophosphate for 24 h in the absence and presence of either 1.5 mM MgCl₂ or 50 µM GdCl₃. Transcript levels were estimated utilizing q-RT-PCR, protein abundance by Western blotting, cytosolic Ca²⁺ concentration ([Ca²⁺]_i) by Fura-2 fluorescence and SOCE from the increase in [Ca²⁺]_i following re-addition of extracellular Ca²⁺ after store depletion with thapsigargin (1 µM). As a result, Mg²⁺ and Gd³⁺ upregulated CaSR and blunted or virtually abolished the phosphate-induced upregulation of NFAT5, SGK1, ORAI1,2,3, STIM1,2 and SOCE in megakaryocytes. In conclusion, Mg²⁺ and the CaSR agonist Gd³⁺ interfere with phosphate-induced dysregulation of [Ca²⁺]_i in megakaryocytes.

SUBMITTER: Zhou K 

PROVIDER: S-EPMC8036595 | biostudies-literature |

REPOSITORIES: biostudies-literature