Stimulation of ORAI1 expression, store-operated Ca2+ entry, and osteogenic signaling by high glucose exposure of human aortic smooth muscle cells.

Abstract

Diabetes and chronic kidney disease (CKD) both trigger vascular osteogenic signaling and calcification leading to early death by cardiovascular events. Osteogenic signaling involves upregulation of the transcription factors CBFA1, MSX2, and SOX9, as well as alkaline phosphatase (ALP), an enzyme fostering calcification by degrading the calcification inhibitor pyrophosphate. In CKD, osteogenic signaling is triggered by hyperphosphatemia, which upregulates the serum and glucocorticoid-inducible kinase SGK1, a strong stimulator of the Ca2+-channel ORAI1. The channel is activated by STIM1 and accomplishes store-operated Ca2+-entry (SOCE). The present study explored whether exposure of human aortic smooth muscle cells (HAoSMCs) to high extracellular glucose concentrations similarly upregulates ORAI1 and/or STIM1 expression, SOCE, and osteogenic signaling. To this end, HAoSMCs were exposed to high extracellular glucose concentrations (15mM, 24h) without or with additional exposure to the phosphate donor ß-glycerophosphate. Transcript levels were estimated using qRT-PCR, protein abundance using Western blotting, ALP activity using a colorimetric assay kit, calcium deposits utilizing Alizarin red staining, 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 (1μM). As a result, glucose enhanced the transcript levels of SGK1 and ORAI1, ORAI2, and STIM2, protein abundance of ORAI1, SOCE, the transcript levels of CBFA1, MSX2, SOX9, and ALPL, as well as calcium deposits. Moreover, glucose significantly augmented the stimulating effect of ß-glycerophosphate on transcript levels of SGK1 and ORAI1, SOCE, the transcript levels of osteogenic markers, as well as calcium deposits. ORAI1 inhibitor MRS1845 (10μM) significantly blunted the glucose-induced upregulation of the CBFA1 and MSX2 transcript levels. In conclusion, the hyperglycemia of diabetes stimulates expression of SGK1 and ORAI1, thus, augmenting store-operated Ca2+-entry and osteogenic signaling in HAoSMCs.