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Background/Aims: The serum- and glucocorticoid-inducible kinase Sgk1 contributes to cardiac remodeling and development of heart failure, which is paralelled by Sgk1-dependent stimulation of the cardiac Na+/H+ exchanger Nhe1. Glucocorticoids are powerful stimulators of Sgk1 expression and influence cardiac remodeling. The present study thus explored whether the glucocorticoid receptor agonist dexamethasone influenced cardiac Sgk1 expression, as well as activity, expression and phosphorylation at Ser703 of the cardiac Na+/H+ exchanger Nhe1. Methods: Experiments were performed in HL-1 cardiomyocytes and gene targeted mice lacking functional Sgk1 (sgk1-/-) and respective wild type mice (sgk1+/+). Gene expression was determined by quantitative RT-PCR and Nhe1 phosphorylation was determined utilizing a specific antibody against a 14-3-3 binding motif at P-Ser703, which represents a putative phosphorylation site recognition motif for Sgk1 and is involved in Nhe1 activation. Cytosolic pH (pHi) was determined utilizing 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) fluorescence and Nhe activity by the Na+-dependent realkalinization after an ammonium pulse. Results: Treatment of HL-1 cardiomyocytes with dexamethasone was followed by a significant increase in Sgk1 mRNA expression, parallelled by increased Na+/H+ exchanger activity. Furthermore, dexamethasone significantly increased Nhe1 and Spp1 mRNA expression. The effects of dexamethasone were blunted by cotreatment of HL-1 cardiomyocytes with the Sgk1 inhibitor EMD638683. Cotreatment with Nhe1 inhibitor cariporide similarly prevented dexamethasone-stimulated Spp1 mRNA expression. In sgk1+/+ mice, dexamethasone significantly increased cardiac Sgk1 mRNA levels. In sgk1+/+ mice, but not in sgk1-/- mice, dexamethasone significantly increased cardiac Nhe1 mRNA expression and Nhe1 phosphorylation at Ser703. Furthermore, cardiac Spp1, Ctgf, Nppa and Nppb mRNA levels were significantly increased in dexamethasone treated sgk1+/+ mice, effects significantly blunted in sgk1-/- mice. Conclusions: Sgk1 is critically involved in the phosphorylation and activation of the cardiac Na+/H+ exchanger Nhe1. Copyright © 2013 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Chorea-acanthocytosis (ChAc), a lethal disease caused by defective chorein, is characterized by neurodegeneration and erythrocyte acanthocytosis. The functional significance of chorein in other cell types remained ill-defined. The present study revealed chorein expression in blood platelets. As compared to platelets from healthy volunteers, platelets from patients with ChAc displayed a 47% increased globular/filamentous actin ratio, indicating actin depolymerization. Moreover, phosphoinositide-3-kinase subunit p85 phosphorylation, p21 protein-activated kinase (PAK1) phosphorylation, as well as vesicle-associated membrane protein 8 (VAMP8) expression were significantly reduced in platelets from patients with ChAc (by 17, 22, and 39%, respectively) and in megakaryocytic (MEG-01) cells following chorein silencing (by 16, 54, and 11%, respectively). Activation-induced platelet secretion from dense granules (ATP release) and α granules (P-selectin exposure) were significantly less (by 55% after stimulation with 1 µg/ml CRP and by 33% after stimulation with 5 µM TRAP, respectively) in ChAc platelets than in control platelets. Furthermore, platelet aggregation following stimulation with different platelet agonists was significantly impaired. These observations reveal a completely novel function of chorein, i.e., regulation of secretion and aggregation of blood platelets. [ABSTRACT FROM AUTHOR]

Highlights: [•] In blood platelets, thrombin upregulates protein abundance of Ca2+ channel Orai1. [•] The effect is paralleled by transient formation of mRNA from pre-RNA. [•] The effect is blunted by inhibition of translation but not by inhibition of transcription. [•] The effect is blunted by pharmacological inhibition of phosphoinositide-3-kinase. [ABSTRACT FROM AUTHOR]

Abstract: Activation of platelets by thrombin opens pore forming channel protein Orai1 with subsequent store operated Ca2+ entry (SOCE) and Ca2+ dependent platelet granule release, integrin αIIbβ3 activation, adhesion, aggregation and thrombus formation. Orai1 and thus SOCE as well as platelet activation are up-regulated by the serum- and glucocorticoid-inducible kinase-1 (SGK1), which transcriptionally regulates Orai1 expression in megakaryocytes and thus determines Orai1 protein abundance in mature, circulating platelets. As platelets are devoid of nuclei, they are unable to modify protein abundance by regulation of transcription. However, they contain mRNA and thus could express novel protein by stimulation of protein translation. Translation is sensitive to actin polymerization and phosphoinositide-3-kinase (PI3K). Translational regulation of SGK1 expression has never been described before. The present study thus explored whether thrombin regulates SGK1 expression in platelets. As a result, according to RT-PCR mRNA encoding SGK1 is present in circulating platelets and significantly decreased by activation of platelets with thrombin (1U/ml). The protein abundance of SGK1 is significantly enhanced by thrombin treatment, an effect significantly decreased by inhibition of translation with puromycin (100nM) but not by inhibition of transcription with actinomycin (4μg/ml). The increase of SGK1 protein abundance is blunted by inhibition of PI3K with wortmannin (100nM) or LY294002 (25μM), and by disruption of the cytoskeleton with cytochalasin B (1μM). In conclusion, activation of platelets with thrombin stimulates the translation of SGK1. [Copyright &y& Elsevier]