Reversal of phosphate-induced ORAI1 expression, store-operated Ca2+ entry and osteogenic signaling by MgCl2 in human aortic smooth muscle cells

Abstract

In chronic kidney disease, renal phosphate retention leads to hyperphosphatemia with subsequent vascular osteogenic signaling and calcification. Osteogenic signaling involves up-regulation of the transcription factors CBFA1, MSX2, and SOX9, as well as alkaline phosphatase (ALP), an enzyme stimulating calcification by degrading the calcification inhibitor pyrophosphate. Stimulation of osteogenic signaling and calcification by phosphate donor β-glycerophosphate in human aortic smooth muscle cells (HAoSMCs) is attenuated by MgCl₂, an effect mimicked by Ca²⁺-sensing receptor agonist GdCl₃. Most recent observations revealed that the effect of β-glycerophosphate on osteogenic signaling requires ORAI1, a Ca²⁺-channel accomplishing store-operated Ca²⁺-entry (SOCE), which is stimulated by Ca²⁺-sensor STIM1. The present study explored whether ORAI1 and/or STIM1 expression and, thus, SOCE and osteogenic signaling in HAoSMCs are sensitive to MgCl₂ and/or GdCl₃. To this end, transcript levels were estimated using q-RT-PCR, protein abundance with western blotting, 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). As a result, 24 h exposure to β-glycerophosphate (2 mM) significantly enhanced transcript levels of ORAI1 and STIM1 as well as SOCE, effects significantly blunted or virtually abrogated by 1.5 mM MgCl₂ and by 50 μM GdCl₃. In conclusion, MgCl₂ and GdCl₃ are powerful inhibitors of ORAI1 and STIM1 expression and store-operated Ca²⁺-entry, effects affecting osteogenic signalling in vascular smooth muscle cells.

Keywords: Ca(2+)-sensing receptor; GdCl(3); HAoSMCs; MgCl(2); ORAI1; Osteogenic signaling; SOCE; STIM1.