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Abstract: Gαi2, a heterotrimeric G‐protein subunit, regulates various cell functions including ion channel activity, cell differentiation, proliferation and apoptosis. Platelet‐expressed Gαi2 is decisive for the extent of tissue injury following ischemia/reperfusion. However, it is not known whether Gαi2 plays a role in the regulation of platelet apoptosis, which is characterized by caspase activation, cell shrinkage and cell membrane scrambling with phosphatidylserine (PS) translocation to the platelet surface. Stimulators of platelet apoptosis include thrombin and collagen‐related peptide (CoRP), which are further known to enhance degranulation and activation of αIIbβ3‐integrin and caspases. Using FACS analysis, we examined the impact of agonist treatment on activation and apoptosis in platelets drawn from mice lacking Gαi2 and their wild‐type (WT) littermates. As a result, treatment with either thrombin (0.01 U/mL) or CoRP (2 μg/mL or 5 μg/mL) significantly upregulated PS‐exposure and significantly decreased forward scatter, reflecting cell size, in both genotypes. Exposure to CoRP triggered a significant increase in active caspase 3, ceramide formation, surface P‐selectin, and αIIbβ3‐integrin activation. These molecular alterations were significantly less pronounced in Gαi2‐deficient platelets as compared to WT platelets. In conclusion, our data highlight a previously unreported role of Gαi2 signaling in governing platelet activation and apoptosis. [ABSTRACT FROM AUTHOR]

The transcription factor nuclear factor of activated T cells 5 (NFAT5) is up-regulated in several clinical disorders, including dehydration. NFAT5-sensitive genes include serum and glucocorticoid-inducible kinase 1 (SGK1). The kinase is a powerful regulator of Orai1, a Ca2+ channel accomplishing store-operated Ca2+ entry (SOCE). Orai1 is stimulated after intracellular store depletion by the Ca2+ sensors stromal interaction molecule 1 (STIM1), or STIM2, or both. In the present study, we explored whether nuclear factor of activated T cell (NFAT)-5 influences Ca2+ signaling in megakaryocytes. To this end, human megakaryocytic (MEG-01) cells were transfected with NFAT5 or with siNFAT5. Platelets and megakaryocytes were isolated from wild-type mice with either access to water ad libitum or dehydration by 36 h of water deprivation. Transcript levels were determined with quantitative RT-PCR and protein abundance by Western blot analysis and flow cytometry, cytosolic (intracellular) Ca2+ concentration ([Ca2+]i) by fura-2-fluorescence. SOCE was estimated from the increase of [Ca2+]i following readdition of extracellular Ca2+ after store depletion with thapsigargin (1 µM). Platelet degranulation was estimated from P-selectin abundance and integrin activation from aIIbß3 integrin abundance determined by flow cytometry. As a result, NFAT5 transfection or exposure to hypertonicity (+40 mM NaCl) of MEG-01 cells increased Orai1, Orai2, STIM1, and STIM2 transcript levels. Orai1 transcript levels were decreased by NFAT5 silencing. NFAT5 transfection and IκB inhibitor BMS 345541 (5 µM) increased SOCE, whereas NFAT5 silencing and SGK1 inhibitor GSK650394 (10 µM) decreased SOCE. In the mice, dehydration increased NFAT5 and Orai1 protein abundance in megakaryocytes and NFAT5, Orai1, and Orai2 abundance in platelets. Dehydration further augmented the degranulation and integrin activation by thrombin and collagen-related peptide. In summary, NFAT5 is a powerful regulator of Orai1-expression and SOCE in megakaryocytes. [ABSTRACT FROM AUTHOR]

Background: The anion exchanger pendrin (SLC26A4) is required for proper development of the inner ear, and contributes to iodide organification in thyroid glands as well as anion transport in various epithelia, such as airways and renal tubules. SLC26A4 deficiency leads to Pendred syndrome, which is characterized by hearing loss with enlarged vestibular aqueducts and variable hypothyroidism and goiter. Pendrin expression in kidney, heart, lung and thyroid is up-regulated by the mineralocorticoid deoxycorticosterone (DOCA). Platelets express anion exchangers but virtually nothing is known about the molecular identity and regulation of those carriers. Other carriers such as the Na+/H+ exchanger are regulated by the mineralocorticoid-sensitive serum and glucocorticoid inducible kinase SGK1. Methods: The present study utilized i) quantitative reverse transcription polymerase chain reaction (RT-qPCR) to quantify the transcript levels of Slc26a4 as compared to Gapdh and ii) western blotting to assess Slc26a4 protein abundance in murine platelets from gene-targeted mice lacking Sgk1 (sgk1-/-) and respective wild type animals (sgk1+/+) treated without or with a subcutaneous injection of 2.5 mg DOCA for 3 h, or in sgk1+/+ platelets with or without in vitro treatment for 1 h with 10 µg/ml DOCA. Results: Slc26a4 was expressed in platelets, and in vitro DOCA treatment increased Slc26a4 mRNA levels in platelets isolated from sgk1+/+ mice. Moreover, in vivo DOCA treatment significantly up-regulated Slc26a4 mRNA levels in platelets isolated from sgk1+/+ but not sgk1-/- mice. An increase in Sgk1 mRNA levels paralleled that of Slc26a4 mRNA levels in platelets of sgk1+/+ mice. In addition, DOCA treatment further increased Slc26a4 protein abundance in platelets isolated from sgk1+/+ mice. Conclusions: Pendrin is expressed in platelets and is presumably regulated by SGK1 and mineralocorticoids. © 2014 S. Karger AG, Basel [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]