IntroductionPlatelet adhesion, activation, and aggregation are essential for primary hemostasis at sites of vascular injury but are also critically important for the development of acute thrombotic occlusion at regions of atherosclerotic plaque rupture, the major pathophysiologic mechanism underlying myocardial infarction and ischemic stroke. 1 Platelet activation is triggered by various agonists, including subendothelial collagen, ADP released from activated platelets, thrombin generated by the coagulation cascade, or the collagen receptor glycoprotein VI (GPVI)-specific agonists convulxin (CVX) and collagen-related peptide (CRP). 2 The agonists lead to platelet granule release, integrin ␣ IIb ␤ 3 activation, phosphatidylserine exposure, aggregation, and thrombus formation. 2 All those platelet responses depend on an increase of cytosolic Ca 2ϩ concentration ([Ca 2ϩ ] i ), 3,4 which is accomplished by inositol-1,4,5-triphosphatemediated Ca 2ϩ release from intracellular stores triggering subsequent stimulation of store-operated Ca 2ϩ entry (SOCE) across the plasma membrane. 5 Two key players in platelet SOCE have recently been identified: The 4-transmembrane-spanning poreforming calcium release-activated channel moiety Orai1, which mediates entry of extracellular Ca 2ϩ , and stromal interaction molecule 1 (STIM1), an Orai1 regulating Ca 2ϩ sensor expressed predominantly in the endoplasmic reticulum. [6][7][8] Regulators of Orai1 in other cell types include receptor for activated protein kinase C-1, 9 reactive oxygen species, 10 and lipid rafts. 11 However, regulation of Orai1 in platelets is poorly understood. Platelet activation has been shown to be regulated in vitro and in vivo by the PI3K/Akt signaling cascade. 12,13 Interference with PI3K signaling has previously been shown to compromise Ca 2ϩ influx into platelets. 14,15 Signaling molecules regulated by PI3K signaling include the serum-and glucocorticoid-inducible kinase 1 (SGK1), a kinase belonging to the AGC family of serine/threonine protein kinases. 16,17 SGK1 has originally been cloned as a glucocorticoidsensitive gene but later shown to be regulated by a variety of hormones and other triggers, including thrombin, growth factors IGF-1 and TGF-␤, oxidative stress, and ischemia. 17 SGK1 has previously been reported to regulate a wide variety of carriers and ion channels, including the epithelial Ca 2ϩ channels TRPV5 and TRPV6. 17 Most recently, SGK1 has been shown to be critically important for the Ca 2ϩ entry into mast cells after activation of the IgE receptor, 18 an effect mediated by regulation of Orai1. 19 Furthermore, SGK1 participates in the regulation of renal tubular Na ϩ reabsorption, salt appetite, and thus blood pressure. 17 A gain-of-function SGK1 gene variant, the combined presence of single nucleotide polymorphism in intron 6 (rs1743966) and in exon 8 (rs1057293), is associated with enhanced blood pressure. 20 Submitted June 9, 2011; accepted August 28, 2011. Prepublished online as Blood First Edition paper, October 26, 2011; DOI 10.1182...

Ca(2+) signaling includes store-operated Ca(2+) entry (SOCE) following depletion of endoplasmic reticulum (ER) Ca(2+) stores. On store depletion, the ER Ca(2+) sensor STIM1 activates Orai1, the pore-forming unit of Ca(2+)-release-activated Ca(2+) (CRAC) channels. Here, we show that Orai1 is regulated by serum- and glucocorticoid-inducible kinase 1 (SGK1), a growth factor-regulated kinase. Membrane Orai1 protein abundance, I(CRAC), and SOCE in human embryonic kidney (HEK293) cells stably expressing Orai1 and transfected with STIM1 were each significantly enhanced by coexpression of constitutively active (S422D)SGK1 (by+81, +378, and+136%, respectively) but not by inactive (K127N)SGK1. Coexpression of the ubiquitin ligase Nedd4-2, an established negatively regulated SGK1 target, down-regulated SOCE (by -48%) and I(CRAC) (by -60%), an effect reversed by expression of (S422D)SGK1 (by +175 and +173%, respectively). Orai1 protein abundance and SOCE were significantly lower in mast cells from SGK1-knockout (sgk1(-/-)) mice (by -37% and -52%, respectively) than in mast cells from wild-type (sgk1(+/+)) littermates. Activation of SOCE by sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase-inhibitor thapsigargin (2 μM) stimulated migration, an effect significantly higher (by +306%) in (S422D)SGK1-expressing than in (K127N)SGK1-expressing HEK293 cells, and also significantly higher (by +108%) in sgk1(+/+) than in sgk1(-/-) mast cells. SGK1 is thus a novel key player in the regulation of SOCE.

Alterations of cytosolic Ca 2ϩ activity participate in the regulation of a wide variety of cellular functions including excitation-contraction coupling, exocytosis, migration, cell proliferation, and cell death (1-4). Cytosolic Ca 2ϩ is increased by release of Ca 2ϩ from intracellular stores and/or Ca 2ϩ entry across the cell membrane (5). Ca 2ϩ release from intracellular stores results in the stimulation of Ca 2ϩ release-activated Ca 2ϩ channel (CRAC) 2 (6, 7), which consists of the pore forming units Orai1, -2, and/or -3 (8 -10) and the endoplasmic reticulum-located regulatory subunit STIM1 or -2 (11-13). The stimulation of the channel leads to the inward current I CRAC and the store-operated Ca 2ϩ entry (SOCE). Recent observations uncovered the powerful stimulation of I CRAC and SOCE by the serum and glucocorticoid-inducible kinase SGK1 (14), a kinase stimulated by growth factors and involved in stress response (15) and regulation of cell survival (16). SGK1 is partially effective through phosphorylation of the ubiquitin ligase Nedd4-2 (neuronal precursor cells expressed developmentally down-regulated). Nedd4-2 ubiquitinates Orai1, thus preparing the channel protein for degradation (14). The effect of Nedd4-2 on Orai1 parallels that of Nedd4-2 on the epithelial Na ϩ channel ENaC (16, 17). The phosphorylation of Nedd4-2 leads to binding of the ubiquitin ligase to the protein 14-3-3, which prevents the interaction with the channel protein (18). Accordingly, SGK1 enhances Orai1 protein abundance in the cell membrane (14). STIM is similarly regulated by ubiquitination (19). However, the effect of SGK1 on Orai1 protein abundance is only in part explained by Nedd4-2-dependent protein degradation. Therefore, further experiments were performed to explore whether SGK1, in addition, stimulates Orai1 and/or STIM1 expression. As a matter of fact, RT-PCR revealed an increase of Orai1 and STIM1 transcript levels after expression of constitutively active SGK1. Thus, further experiments were performed to uncover the transcription factor involved. Previously, SGK1 has been shown to foster nuclear translocation and activation of nuclear factor B (NF-B) (20 -22). Accordingly, this study explored the putative involvement of NF-B subunits p65 (RELA), p50 (NFKB1), and p52 (NFKB2) in the regulation of Orai1 and STIM1 expression. EXPERIMENTAL PROCEDURES

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