Abstract
ORAI and stromal interaction molecule (STIM) are store-operated channel molecules that play essential roles in human physiology through a coupling mechanism of internal Ca(2+) store to Ca(2+) influx. However, the roles of ORAI and STIM in vascular endothelial cells under diabetic conditions remain unknown. Here, we investigated expression and signalling pathways of ORAI and STIM regulated by high glucose or hyperglycaemia using in vitro cell models, in vivo diabetic mice and tissues from patients. We found that ORAI1-3 and STIM1-2 were ubiquitously expressed in human vasculatures. Their expression was upregulated by chronic treatment with high glucose (HG, 25mM D-glucose), which was accompanied by enhanced store-operated Ca(2+) influx in vascular endothelial cells. The increased expression was also observed in the aortae from genetically modified Akita diabetic mice (C57BL/6-Ins2(Akita)/J) and streptozocin-induced diabetic mice, and aortae from diabetic patients. HG-induced upregulation of ORAI and STIM genes was prevented by the calcineurin inhibitor cyclosporin A and NFATc3 siRNA. Additionally, in vivo treatment with the nuclear factor of activated T cells (NFAT) inhibitor A-285222 prevented the gene upregulation in Akita mice. However, HG had no direct effects on ORAI1-3 currents and the channel activation process through cytosolic STIM1 movement in the cells co-expressing STIM1-EYFP/ORAIs. We concluded that upregulation of STIM/ORAI through Ca(2+)-calcineurin-NFAT pathway is a novel mechanism causing abnormal Ca(2+) homeostasis and endothelial dysfunction under hyperglycaemia. ORAI1-3 and STIM1-2 are ubiquitously expressed in vasculatures and upregulated by high glucose. Increased expression is confirmed in Akita (Ins2(Akita)/J) and STZ diabetic mice and patients. Upregulation mechanism is mediated by Ca(2+)/calcineurin/NFATc3 signalling. High glucose has no direct effects on ORAI1-3 channel activity and channel activation process.
Highlights
Cardiovascular mortality has decreased over the last 40 years in Western countries; patients with diabetes still have high mortality and show a two- to four-fold increased risk of cardiovascular events over those without diabetes [1]
Using real-time PCR, we found that the mRNA levels of ORAI1, 2, 3 and STIM1, 2 were significantly upregulated in the endothelial cells incubated with high glucose (HG) (25 mM) for 72 h (Fig. 1E)
In this study, we have shown that store-operated Ca2+ entry in vascular endothelial cells and smooth muscle cells is enhanced by chronic treatment with HG
Summary
Cardiovascular mortality has decreased over the last 40 years in Western countries; patients with diabetes still have high mortality and show a two- to four-fold increased risk of cardiovascular events over those without diabetes [1]. Ca2+ signalling is critical in regulating endothelial function Many inflammatory mediators, such as thrombin, histamine and cytokines, increase intracellular Ca2+ concentration ([Ca2+]i) through activation of G protein-coupled receptors or receptor tyrosine kinases [3, 4]. ORAIs mediate the highly Ca2+-selective and inward rectifying Ca2+ release-activated Ca2+ current (ICRAC) [5, 6], while TRPCs mediate the non-selective Ca2+-permeable cationic current with outward rectification [7, 8] These channels are associated with the Ca2+ sensor protein STIM1 (stromal interaction molecule 1) in the endoplasmic reticulum (ER), which senses the depleted Ca2+ store and couples to the SOCs in the plasma membrane through subplasmalemmal translocation and clustering [9,10,11,12,13]
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