Wilson disease is caused by accumulation of Cu(2+) in cells, which results in liver cirrhosis and, occasionally, anemia. Here, we show that Cu(2+) triggers hepatocyte apoptosis through activation of acid sphingomyelinase (Asm) and release of ceramide. Genetic deficiency or pharmacological inhibition of Asm prevented Cu(2+)-induced hepatocyte apoptosis and protected rats, genetically prone to develop Wilson disease, from acute hepatocyte death, liver failure and early death. Cu(2+) induced the secretion of activated Asm from leukocytes, leading to ceramide release in and phosphatidylserine exposure on erythrocytes, events also prevented by inhibition of Asm. Phosphatidylserine exposure resulted in immediate clearance of affected erythrocytes from the blood in mice. Accordingly, individuals with Wilson disease showed elevated plasma levels of Asm, and displayed a constitutive increase of ceramide- and phosphatidylserine-positive erythrocytes. Our data suggest a previously unidentified mechanism for liver cirrhosis and anemia in Wilson disease.

Glucocorticoid excess predisposes to the development of diabetes, at least in part through impairment of insulin secretion. The underlying mechanism has remained elusive. We show here that dexamethasone upregulates transcription and expression of the serumand glucocorticoid-inducible kinase 1 (SGK1) in insulinsecreting cells, an effect reversed by mifepristone (RU486), an antagonist of the nuclear glucocorticoid receptor. G lucocorticoids are known to induce diabetes (1-3). In addition to peripheral insulin resistance and increased hepatic glucose production by stimulating gluconeogenesis (4), glucocorticoids interfere with insulin secretion of pancreatic ␤-cells (5-7). Despite extensive (8 -12) studies, the molecular mechanism is still a matter of debate. Increased expression of ␣ 2 -adrenoceptors has been proposed to account for dexamethasone-induced inhibition of insulin secretion (9). Thus, transgenic mice overexpressing glucocorticoid receptors in ␤-cells show 30% more UK14304 binding, a selective adrenoceptor agonist, than wild-type islets (2). These mice are glucose intolerant and have reduced plasma insulin levels. Since pertussis toxin and cAMP overcome dexamethasone inhibition of glucose-induced insulin release, decreased cAMP levels during dexamethasone treatment may be responsible for inhibition of secretion (6,13). Furthermore, dexamethasone was reported to decrease Glut2 protein abundance at the plasma membrane, a change that may contribute to impaired glucose-induced insulin secretion (8). Dexamethasone also downregulates glucokinase mRNA in an insulin-secreting cell line (14). Mifepristone (RU486), a nuclear glucocorticoid receptor antagonist, completely abolished dexamethasone-induced inhibition of insulin secretion (5,6), pointing to the involvement of glucocorticoid-dependent gene expression. Glucocorticoid-sensitive genes include the serum-and glucocorticoid-inducible kinase 1 (SGK1) (rev. in 15). The kinase is expressed in virtually all human tissues tested. Unlike its isoforms SGK2 and SGK3 and the related kinase protein kinase B, SGK1 is under strong transcriptional control of glucocorticoids (15) and mineralocorticoids (16). SGK1 has been shown to regulate a variety of ion channels including K ϩ channels such as voltage-gated K v channels (17).Ion channel activity is in turn decisive for insulin secretion from pancreatic ␤-cells. 4-AP, 4-aminopyridine; GAPDH, glyceraldehyde-3-phsophate dehydrogenase; SGK1, serum-and glucocorticoid-inducible kinase 1; TEA, tetraethylammonium.

Sunitinib, a multikinase inhibitor, stimulates apoptosis and is thus utilized for the treatment of malignancy. Even though lacking mitochondria and nuclei, critical elements in apoptosis of nucleated cells, erythrocytes may undergo eryptosis, an apoptosis-like suicidal death, characterized by cell shrinkage and cell membrane scrambling with phosphatidylserineexposure at the cell surface. Triggers of eryptosis include activation of Ca²⁺ permeable cation channels with subsequent increase of cytosolic Ca²⁺-activity ([Ca²⁺]_i), ceramide formation, ATP-depletion, stimulation of p38 kinase and caspase activation. The present study explored, whether sunitinib stimulates eryptosis. [Ca²⁺]_i was estimated from Fluo-3-fluorescence, cell volume from forward scatter, phosphatidylserine-exposure from annexin-V-binding, hemolysis from hemoglobin release, ceramide abundance from anti-ceramide antibody binding, and cytosolic ATP from luciferin–luciferase activity. A 48 h exposure to sunitinib (10 µM) significantly decreased forward scatter and increased annexin-V-binding, effects paralleled by significant increase of [Ca²⁺]_i. Sunitinib exposure was followed by a slight but significant increase of hemolysis. Sunitinib induced annexin-V-binding was slightly, but significantly blunted by removal of extracellular Ca²⁺, by p38 kinase inhibitor SB203580 (10 µM) and by the pancaspase inhibitor zVAD (10 µM). Sunitinib, however, did not significantly modify cytosolic ATP and ceramide abundance. The present observations reveal that sunitinib is able to trigger suicidal death in erythrocytes even in the absence of nuclei and mitochondria.

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Background/Aims: Anemia, a common condition in the elderly, could result from impaired formation and/or from accelerated loss of circulating erythrocytes. The latter could result from premature suicidal erythrocyte death or eryptosis characterized by phosphatidylserine (PS) exposure at the erythrocyte surface. Triggers of eryptosis include increased cytosolic Ca²⁺-concentration ([Ca²⁺]_i), oxidative stress and ceramide. The present study explored whether eryptosis is altered in elderly individuals and, if so, to identify underlying mechanisms. Methods: Blood was drawn from healthy young (n=11, age 31.3±1.7 years) and elderly (n=16, age 88.6±0.9 years) individuals. PS exposure was estimated from annexin V-binding, cell volume from forward scatter, [Ca²⁺]_i from Fluo3-fluorescence, reactive oxygen species (ROS) from 2',7'dichlorodihydrofluorescein fluorescence, reduced glutathione (GSH) from mercury orange fluorescence and ceramide from FITC-conjugated antibody binding in flow cytometry. Measurements were made in erythrocytes from freshly drawn blood and in erythrocytes exposed in vitro for 24 h to plasma from young or elderly individuals. Results: Elderly individuals suffered from severe anemia (hemoglobin 10.5±0.3 g/100 ml) despite enhanced number of reticulocytes (2.3±0.2%). The percentage of PS-exposing erythrocytes was significantly higher in the elderly (2.5±0.2%) than in the young volunteers (1.3±0.1%). The increase in PS exposure was paralleled by significant increase of ROS and significantly decreased levels of reduced GSH. Erythrocyte [Ca²⁺]_i, and ceramide abundance tended to be higher in the elderly, differences, however, not reaching statistical significance. Conclusions: The anemia of elderly individuals is mainly if not exclusively due to enhanced eryptosis, resulting at least in part from GSH deficiency and increased oxidative stress.

Hexavalent (VI) chromium is a global contaminant with cytotoxic activity. Chromium (VI) induces oxidative stress, inflammation, cell proliferation, malignant transformation and may trigger carcinogenesis and at the same time apoptosis. The toxic effects of chromium (VI) at least partially result from mitochondrial injury and DNA damage. Erythrocytes lack mitochondria and nuclei but may experience an apoptosis-like suicidal cell death, i.e. eryptosis, which is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine exposure at the cell surface. Eryptosis may result from increase of cytosolic Ca(2+) activity, ATP depletion and/or ceramide formation. The present study explored, whether chromium (VI) triggers eryptosis. Fluo-3-fluorescence was employed to determine cytosolic Ca(2+)-concentration, forward scatter to estimate cell volume, binding of fluorescent annexin V to detect phosphatidylserine exposure, hemoglobin concentration in the supernatant to quantify hemolysis, luciferin-luciferase to determine cytosolic ATP concentration and fluorescent anti-ceramide antibodies to uncover ceramide formation. A 48 h exposure to chromium (VI) (≥10 μM) significantly increased cytosolic Ca(2+)-concentration, decreased ATP concentration (20 μM), decreased forward scatter, increased annexin V-binding and increased (albeit to a much smaller extent) hemolysis. Chromium (VI) did not significantly modify ceramide formation. The effect of 20 μM chromium (VI) on annexin V binding was partially reversed in the nominal absence of Ca(2+). The present observations disclose a novel effect of chromium (VI), i.e. Ca(2+) entry and cytosolic ATP depletion in erythrocytes, effects resulting in eryptosis with cell shrinkage and cell membrane scrambling.

Background: Sorafenib (Nexavar^®), a polytyrosine kinase inhibitor, stimulates apoptosis and is thus widely used for chemotherapy in hepatocellular carcinoma (HCC). Hematological side effects of Nexavar^® chemotherapy include anemia. Erythrocytes may undergo apoptosis-like suicidal death or eryptosis, which is characterized by cell shrinkage and phosphatidylserine-exposure at the cell surface. Signaling leading to eryptosis include increase in cytosolic Ca²⁺activity ([Ca²⁺]_i), formation of ceramide, ATP-depletion and oxidative stress. The present study explored, whether sorafenib triggers eryptosis in vitro and in vivo. Methods: [Ca²⁺]_i was estimated from Fluo3-fluorescence, cell volume from forward scatter, phosphatidylserine-exposure from annexin-V-binding, hemolysis from hemoglobin release, ceramide with antibody binding-dependent fluorescence, cytosolic ATP with a luciferin–luciferase-based assay, and oxidative stress from 2’,7’ dichlorodihydrofluorescein diacetate (DCFDA) fluorescence. Results: A 48 h exposure of erythrocytes to sorafenib (≥0.5 µM) significantly increased Fluo 3 fluorescence, decreased forward scatter, increased annexin-V-binding and triggered slight hemolysis (≥5 µM), but did not significantly modify ceramide abundance and cytosolic ATP. Sorafenib treatment significantly enhanced DCFDA-fluorescence and the reducing agents N-acetyl-L-cysteine and tiron significantly blunted sorafenib-induced phosphatidylserine exposure. Nexavar^® chemotherapy in HCC patients significantly enhanced the number of phosphatidylserine-exposing erythrocytes. Conclusions: The present observations disclose novel effects of sorafenib, i.e. stimulation of suicidal erythrocyte death or eryptosis, which may contribute to the pathogenesis of anemia in Nexavar^®-based chemotherapy.

The mitogen- and stress-activated kinase MSK1/2 plays a decisive role in apoptosis. In analogy to apoptosis of nucleated cells, suicidal erythrocyte death called eryptosis is characterized by cell shrinkage and cell membrane scrambling leading to phosphatidylserine (PS) externalization. Here, we explored whether MSK1/2 participates in the regulation of eryptosis. To this end, erythrocytes were isolated from mice lacking functional MSK1/2 (msk−/−) and corresponding wild-type mice (msk+/+). Blood count, hematocrit, hemoglobin concentration and mean erythrocyte volume were similar in both msk−/− and msk+/+ mice, but reticulocyte count was significantly increased in msk−/− mice. Cell membrane PS exposure was similar in untreated msk−/− and msk+/+ erythrocytes, but was enhanced by pathophysiological cell stressors ex vivo such as hyperosmotic shock or energy depletion to significantly higher levels in msk−/− erythrocytes than in msk+/+ erythrocytes. Cell shrinkage following hyperosmotic shock and energy depletion, as well as hemolysis following decrease of extracellular osmolarity was more pronounced in msk−/− erythrocytes. The in vivo clearance of autologously-infused CFSE-labeled erythrocytes from circulating blood was faster in msk−/− mice. The spleens from msk−/− mice contained a significantly greater number of PS-exposing erythrocytes than spleens from msk+/+ mice. The present observations point to accelerated eryptosis and subsequent clearance of erythrocytes leading to enhanced erythrocyte turnover in MSK1/2-deficient mice.