Vasopressin-stimulated ORAI1 expression and store-operated Ca2+ entry in aortic smooth muscle cells

Abstract

Vascular calcification may result from stimulation of osteogenic signalling with upregulation of the transcription factors CBFA1, MSX2 and SOX9, as well as alkaline phosphatase (ALPL), which degrades and thus inactivates the calcification inhibitor pyrophosphate. Osteogenic signalling further involves upregulation of the Ca²⁺-channel ORAI1. The channel is activated by STIM1 and then accomplishes store-operated Ca²⁺ entry. ORAI1 and STIM1 are upregulated by the serum & glucocorticoid inducible kinase 1 (SGK1) which is critically important for osteogenic signalling. Stimulators of vascular calcification include vasopressin. The present study explored whether exposure of human aortic smooth muscle cells (HAoSMCs) to vasopressin upregulates ORAI1 and/or STIM1 expression, store-operated Ca²⁺ entry and osteogenic signalling. To this end, HAoSMCs were exposed to vasopressin (100 nM, 24 h) without or with additional exposure to ORAI1 blocker MRS1845 (10 μM) or SGK1 inhibitor GSK-650394 (1 μM). Transcript levels were measured using q-RT-PCR, cytosolic Ca²⁺-concentration ([Ca²⁺]_i) by Fura-2-fluorescence, and store-operated Ca²⁺ entry from increase of [Ca²⁺]_i following re-addition of extracellular Ca²⁺ after store depletion with thapsigargin (1 μM). As a result, vasopressin enhanced the transcript levels of ORAI1 and STIM1, store-operated Ca²⁺ entry, as well as the transcript levels of CBFA1, MSX2, SOX9 and ALPL. The effect of vasopressin on store-operated Ca²⁺ entry as well as on transcript levels of CBFA1, MSX2, SOX9 and ALPL was virtually abrogated by MRS1845 and GSK-650394. In conclusion, vasopressin stimulates expression of ORAI1/STIM1, thus augmenting store-operated Ca²⁺ entry and osteogenic signalling. In HAoSMCs, vasopressin (VP) upregulates Ca²⁺ channel ORAI1 and its activator STIM1. VP upregulates store-operated Ca²⁺ entry (SOCE) and osteogenic signalling (OS). VP-induced SOCE, OS and Ca²⁺-deposition are disrupted by ORAI1 inhibitor MRS1845. VP-induced SOCE, OS and Ca²⁺-deposition are disrupted by SGK1 blocker GSK-650394. KEY MESSAGES: • In HAoSMCs, vasopressin (VP) upregulates Ca²⁺ channel ORAI1 and its activator STIM1. • VP upregulates store-operated Ca²⁺ entry (SOCE) and osteogenic signalling (OS). • VP-induced SOCE, OS and Ca²⁺-deposition are disrupted by ORAI1 inhibitor MRS1845. • VP-induced SOCE, OS and Ca²⁺-deposition are disrupted by SGK1 blocker GSK-650394.

Keywords: Alkaline phosphatase; HAoSMCs; ORAI1; Osteogenic signalling; STIM1; Store-operated Ca2+ entry.