Thrombospondin-1/CD47 signaling modulates transmembrane cation conductance, survival, and deformability of human red blood cells.

BACKGROUND: Thrombospondin-1 (TSP-1), a Ca2+-binding trimeric glycoprotein secreted by multiple cell types, has been implicated in the pathophysiology of several clinical conditions. Signaling involving TSP-1, through its cognate receptor CD47, orchestrates a wide array of cellular functions including cytoskeletal organization, migration, cell-cell interaction, cell proliferation, autophagy, and apoptosis. In the present study, we investigated the impact of TSP-1/CD47 signaling on Ca2+ dynamics, survival, and deformability of human red blood cells (RBCs). METHODS: Whole-cell patch-clamp was employed to examine transmembrane cation conductance. RBC intracellular Ca2+ levels and multiple indices of RBC cell death were determined using cytofluorometry analysis. RBC morphology and microvesiculation were examined using imaging flow cytometry. RBC deformability was measured using laser-assisted optical rotational cell analyzer. RESULTS: Exposure of RBCs to recombinant human TSP-1 significantly increased RBC intracellular Ca2+ levels. As judged by electrophysiology experiments, TSP-1 treatment elicited an amiloride-sensitive inward current alluding to a possible Ca2+ influx via non-selective cation channels. Exogenous TSP-1 promoted microparticle shedding as well as enhancing Ca2+- and nitric oxide-mediated RBC cell death. Monoclonal (mouse IgG1) antibody-mediated CD47 ligation using 1F7 recapitulated the cell death-inducing effects of TSP-1. Furthermore, TSP-1 treatment altered RBC cell shape and stiffness (maximum elongation index). CONCLUSIONS: Taken together, our data unravel a new role for TSP-1/CD47 signaling in mediating Ca2+ influx into RBCs, a mechanism potentially contributing to their dysfunction in a variety of systemic diseases. Video abstract.

Inhibition of suicidal erythrocyte death by pyrogallol.

Pyrogallol, a polyphenolic component of Acacia nilotica has previously been reported to induce apoptosis of diverse cell types. Pyrogallol is in part effective by influencing gene expression and by interference with mitochondrial function. Despite lack of nuclei and mitochondria, erythrocytes may undergo eryptosis, a suicidal death apparent from phosphatidylserine translocation to the erythrocyte surface and cell shrinkage. Eryptosis is triggered by glucose depletion, by oxidation, by hyperosmotic cell shrinkage and by excessive Ca2+ entry. As enhanced eryptosis is a common cause of anemia, uncovering inhibitors and stimulators of eryptosis may, both, be of clinical interest. Here we tested, whether eryptosis of human erythrocytes is modified by pyrogallol. Utilizing flow cytometry, phosphatidylserine abundance at the cell surface was estimated from annexin-V-binding and cell volume from forward scatter. Prior to determinations erythrocytes were incubated with or without glucose, without or with added oxidant tert-butyl-hydroperoxide (t-BOOH, 0.5 mM), without or with added hyperosmotic sucrose (550 mM) or without or with added Ca2+ ionophore ionomycin (1 µM). Treatment of erythrocytes with pyrogallol (2-8 µM) was without significant effect on annexin-V-binding and forward scatter. Glucose deprivation, t-BOOH, sucrose and ionomycin, each, triggered annexin-V-binding and decreased forward scatter. Pyrogallol significantly blunted the effects on annexin-V-binding but not on forward scatter. Pyrogallol thus blunts phosphatidylserine translocation in erythrocytes exposed to glucose depletion, oxidative stress, hyperosmotic shock and excessive Ca2+ entry.

Genetic deficiency of the tumor suppressor protein p53 influences erythrocyte survival.

The transcription factor p53 suppresses tumor growth by inducing nucleated cell apoptosis and cycle arrest. Because of its influence on primitive erythroid cell differentiation and survival, p53 is an important determinant of erythropoiesis. However, the impact of p53 on the fate of erythrocytes, cells lacking nucleus and mitochondria, during their post-maturation phase in the circulation remained elusive. Erythrocyte survival may be compromised by suicidal erythrocyte death or eryptosis, which is hallmarked by phosphatidylserine translocation and stimulated by increase of cytosolic Ca2+ concentration. Here, we comparatively examined erythrocyte homeostasis in p53-mutant mice (Trp53tm1Tyj/J) and in corresponding WT mice (C57BL/6J) by analyzing eryptosis and erythropoiesis. To this end, spontaneous cell membrane phosphatidylserine exposure and cytosolic Ca2+ concentration were higher in erythrocytes drawn from Trp53tm1Tyj/J mice than from WT mice. Eryptosis induced by glucose deprivation, a pathophysiological cell stressor, was slightly, but significantly more prominent in erythrocytes drawn from Trp53tm1Tyj/J mice as compared to WT mice. The loss of erythrocytes by eryptosis was fully compensated by enhanced erythropoiesis in Trp53tm1Tyj/J mice, as reflected by increased reticulocytosis and abundance of erythroid precursor cells in the bone marrow. Accordingly, erythrocyte number, packed cell volume and hemoglobin were similar in Trp53tm1Tyj/J and WT mice. Taken together, functional p53 deficiency enhances the turnover of circulating erythrocytes by parallel increase of eryptosis and stimulated compensatory erythropoiesis.

Inhibition of Lithium Sensitive Orai1/ STIM1 Expression and Store Operated Ca2+ Entry in Chorea-Acanthocytosis Neurons by NF-κB Inhibitor Wogonin.

BACKGROUND/AIMS: The neurodegenerative disease Chorea-Acanthocytosis (ChAc) is caused by loss-of-function-mutations of the chorein-encoding gene VPS13A. In ChAc neurons transcript levels and protein abundance of Ca2+ release activated channel moiety (CRAC) Orai1 as well as its regulator STIM1/2 are decreased, resulting in blunted store operated Ca2+-entry (SOCE) and enhanced suicidal cell death. SOCE is up-regulated and cell death decreased by lithium. The effects of lithium are paralleled by upregulation of serum & glucocorticoid inducible kinase SGK1 and abrogated by pharmacological SGK1 inhibition. In other cell types SGK1 has been shown to be partially effective by upregulation of NFκB, a transcription factor stimulating the expression of Orai1 and STIM. The present study explored whether pharmacological inhibition of NFκB interferes with Orai1/STIM1/2 expression and SOCE and their upregulation by lithium in ChAc neurons. METHODS: Cortical neurons were differentiated from induced pluripotent stem cells generated from fibroblasts of ChAc patients and healthy volunteers. Orai1 and STIM1 transcript levels and protein abundance were estimated from qRT-PCR and Western blotting, respectively, cytosolic Ca2+-activity ([Ca2+]i) from Fura-2-fluorescence, SOCE from increase of [Ca2+]i following Ca2+ re-addition after Ca2+-store depletion with sarco-endoplasmatic Ca2+-ATPase inhibitor thapsigargin (1µM), as well as CRAC current utilizing whole cell patch clamp recording. RESULTS: Orai1 and STIM1 transcript levels and protein abundance as well as SOCE and CRAC current were significantly enhanced by lithium treatment (2 mM, 24 hours). These effects were reversed by NFκB inhibitor wogonin (50 µM). CONCLUSION: The stimulation of expression and function of Orai1/STIM1/2 by lithium in ChAc neurons are disrupted by pharmacological NFκB inhibition.

Inhibitory Effect of Afatinib on Platelet Activation and Apoptosis.

BACKGROUND/AIMS: The epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor afatinib is used for the treatment of several malignancies. Afatinib is at least partially effective by triggering apoptosis of tumor cells. Platelets may similarly undergo apoptosis, which is characterized by caspase 3 activation, cell shrinkage and phosphatidylserine translocation. However, an effect of afatinib on platelets has never been reported. The present study explored whether treatment of platelets with afatinib modifies platelet activation and apoptosis in the absence and presence of platelet activators thrombin or collagen related peptide (CRP). METHODS: Platelets isolated from wild-type mice were exposed for 30 minutes to afatinib (18 µg/ml) without or with subsequent treatment with thrombin (0.005 U/ml or 0.01 U/ml) or CRP (2 µg/ml or 5 µg/ml). Flow cytometry was employed to estimate Orai1 abundance at the platelet surface with specific antibodies, cytosolic Ca2+-activity ([Ca2+]i) from Fluo-3 fluorescence, platelet degranulation from P-selectin abundance, integrin activation from αIIbß3 integrin abundance, caspase activity utilizing an Active Caspase-3 Staining kit, phosphatidylserine abundance from annexin-V-binding, platelet volume from forward scatter and aggregation utilizing staining with CD9-APC and CD9-PE. RESULTS: In the absence of thrombin and CRP, the administration of afatinib (18 µg/ml) slightly, but significantly, increased [Ca2+]i and annexin-V-binding, but did not significantly modify Orai1 abundance, P-selectin abundance, activated αIIbß3 integrin, cell volume, caspase activity and aggregation. Exposure of platelets to 0.005 U/ml or 0.01 U/ml thrombin or 2 µg/ml or 5 µg/ ml CRP was followed by a significant increase of Orai1 abundance, increase of [Ca2+]i, P-selectin abundance, αIIbß3 integrin activity, annexin-V-binding, caspase activity, and aggregation, as well as a significant decrease of forward scatter, all effects significantly blunted (thrombin) or virtually abolished (CRP) by afatinib. CONCLUSIONS: Afatinib is a powerful inhibitor of platelet activation, platelet apoptosis and platelet aggregation.

Ceranib-2-induced suicidal erythrocyte death.

Ceramide is known to trigger apoptosis of nucleated cells and eryptosis of erythrocytes. Eryptosis is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Besides ceramide, stimulators of eryptosis include increase of cytosolic Ca(2+) -activity ([Ca(2+) ]i ) and oxidative stress. Ceramide is degraded by acid ceramidase and inhibition of the enzyme similarly triggers apoptosis. The present study explored, whether ceramidase inhibitor Ceranib-2 induces eryptosis. Flow cytometry was employed to quantify phosphatidylserine-exposure at the cell surface from annexin-V-binding, cell volume from forward scatter, [Ca(2+) ]i from Fluo3-fluorescence, reactive oxygen species (ROS) from DCF dependent fluorescence, and ceramide abundance utilizing specific antibodies. Hemolysis was estimated from hemoglobin concentration in the supernatant. A 48 h exposure of human erythrocytes to Ceranib-2 significantly increased the percentage of annexin-V-binding cells (≥50 µM) and the percentage of hemolytic cells (≥10 µM) without significantly modifying forward scatter. Ceranib-2 significantly increased Fluo3-fluorescence, DCF fluorescence and ceramide abundance. The effect of Ceranib-2 on annexin-V-binding was not significantly blunted by removal of extracellular Ca(2+) . Ceranib-2 triggers phospholipid scrambling of the erythrocyte cell membrane, an effect at least in part due to increase of ceramide abundance and induction of oxidative stress, but not dependent on Ca(2+) entry. Copyright © 2016 John Wiley & Sons, Ltd.

Beta-Glycerophosphate-Induced ORAI1 Expression and Store Operated Ca2+ Entry in Megakaryocytes.

Impairment of renal phosphate elimination in chronic kidney disease (CKD) leads to enhanced plasma and tissue phosphate concentration, which in turn up-regulates transcription factor NFAT5 and serum & glucocorticoid-inducible kinase SGK1. The kinase upregulates ORAI1, a Ca2+-channel accomplishing store-operated Ca2+-entry (SOCE). ORAI1 is stimulated following intracellular store depletion by Ca2+-sensors STIM1 and/or STIM2. In megakaryocytes and blood platelets SOCE and thus ORAI1 are powerful regulators of activity. The present study explored whether the phosphate-donor ß-glycerophosphate augments NFAT5, ORAI1,2,3 and/or STIM1,2 expressions and thus SOCE in megakaryocytes. Human megakaryocytic Meg01cells were exposed to 2 mM of phosphate-donor ß-glycerophosphate for 24 hours. Platelets were isolated from blood samples of patients with impaired kidney function or control volunteers. Transcript levels were estimated utilizing q-RT-PCR, cytosolic Ca2+-concentration ([Ca2+]i) by Fura-2-fluorescence, and SOCE from increase of [Ca2+]i following re-addition of extracellular Ca2+ after store depletion with thapsigargin (1 µM). NFAT5 and ORAI1 protein abundance was estimated with Western blots. As a result, ß-glycerophosphate increased NFAT5, ORAI1/2/3, STIM1/2 transcript levels, as well as SOCE. Transcript levels of NFAT5, SGK1, ORAI1/2/3, and STIM1/2 as well as NFAT5 and ORAI1 protein abundance were significantly higher in platelets isolated from patients with impaired kidney function than in platelets from control volunteers. In conclusion, phosphate-donor ß-glycerophosphate triggers a signaling cascade of NFAT5/SGK1/ORAI/STIM, thus up-regulating store-operated Ca2+-entry.

Oxidative stress, eryptosis and anemia: a pivotal mechanistic nexus in systemic diseases.

The average lifespan of circulating erythrocytes usually exceeds hundred days. Prior to that, however, erythrocytes may be exposed to oxidative stress in the circulation which could cause injury and trigger their suicidal death or eryptosis. Oxidative stress activates Ca2+ -permeable nonselective cation channels in the cell membrane, thus, stimulating Ca2+ entry and subsequent cell membrane scrambling resulting in phosphatidylserine exposure and activation of Ca2+ -sensitive K+ channels leading to K+ exit, hyperpolarization, Cl- exit, and ultimately cell shrinkage due to loss of KCl and osmotically driven water. While the mechanistic link between oxidative stress and anemia remains ill-defined, several diseases such as diabetes, hepatic failure, malignancy, chronic kidney disease and inflammation have been identified to display both increased oxidative stress as well as eryptosis. Recent compelling evidence suggests that oxidative stress is an important perpetrator in accelerating erythrocyte loss in different systemic conditions and an underlying mechanism for anemia associated with these pathological states. In the present review, we discuss the role of oxidative stress in reducing erythrocyte survival and provide novel insights into the possible use of antioxidants as putative antieryptotic and antianemic agents in a variety of systemic diseases.

Correction to: Triggering of eryptosis, the suicidal erythrocyte death, by phenoxodiol.

The original version of this article contains several mistakes due to the missed corrections.

Triggering of eryptosis, the suicidal erythrocyte death, by phenoxodiol.

Phenoxodiol is used for the treatment of malignancy. The substance is effective by triggering suicidal tumor cell death or apoptosis. At least in theory, phenoxodiol could similarly stimulate suicidal erythrocyte death or eryptosis. Eryptosis is characterized by cell shrinkage and breakdown of cell membrane asymmetry with phosphatidylserine translocation to the erythrocyte surface. Signaling of eryptosis includes increase of cytosolic Ca2+ activity ([Ca2+]i), formation of reactive oxygen species (ROS), and increase of ceramide abundance at the cell surface. The present study explored whether phenoxodiol induces eryptosis and whether it modifies Ca2+ entry, ROS, and ceramide. Using flow cytometry, phosphatidylserine exposure at the cell surface was quantified from annexin V binding, cell volume from forward scatter, [Ca2+]i from Fluo3 fluorescence, ROS from DCFDA-dependent fluorescence, and ceramide abundance utilizing specific antibodies. A 48-h exposure of human erythrocytes to phenoxodiol (100 µg/ml [416 µM]) significantly increased the percentage of annexin V binding cells, significantly decreased average forward scatter and Fluo3 fluorescence and significantly increased ceramide abundance, but did not significantly modify DCFDA fluorescence. The effect of phenoxodiol on annexin V binding tended to decrease following removal of extracellular Ca2+, an effect, however, not reaching statistical significance. In conclusion, phenoxodiol triggers eryptosis, an effect paralleled by increase of ceramide abundance.