PGE2 in the Regulation of Programmed Erythrocyte Death
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Cell Death and Differentiation (2005) 12, 415–428 & 2005 Nature Publishing Group All rights reserved 1350-9047/05 $30.00 www.nature.com/cdd PGE2 in the regulation of programmed erythrocyte death PA Lang1, DS Kempe1, S Myssina1, V Tanneur1, C Birka1, phosphate-buffered saline; FITC, fluorescein isothiocyanate; S Laufer2, F Lang*,1, T Wieder1 and SM Huber1 WGA, Triticum vulgare lectin; FSC, forward scatter; SSC, sideward scatter; PMSF, phenylmethylsulfonyl fluoride; SDS, 1 Department of Physiology, University of Tu¨bingen, Germany sodium dodecylsulfate; BSA, bovine serum albumin; SDS-PAGE, 2 Institute of Pharmacy, University of Tu¨bingen, Germany sodiumdodecylsulfate polyacrylamide gel electrophoresis; ECL, * Corresponding author: F Lang, Physiologisches Institut der Universita¨t enhanced chemiluminescence Tu¨bingen, Gmelinstr. 5, D-72076 Tu¨bingen, Germany. Tel: þ 49 7071 29 72194; Fax: þ 49 7071 29 5618; E-mail: fl[email protected] Received 05.4.04; revised 15.11.04; accepted 23.11.04; published online 04.3.05 Edited by M Piacentini Introduction Until recently, erythrocytes have been considered unable Abstract to undergo apoptosis, as they lack mitochondria and nuclei, key organelles in the apoptotic machinery of nucleated Hyperosmotic shock, energy depletion, or removal of 1 extracellular ClÀ activates Ca2 þ -permeable cation channels cells. However, most recent observations revealed that treatment of erythrocytes with the Ca2 þ -ionophore ionomycin in erythrocyte membranes. Subsequent Ca2 þ entry induces leads to cell shrinkage, cell membrane blebbing and erythrocyte shrinkage and exposure of phosphatidylserine annexin binding,2–7 all typical features of apoptosis in (PS) at the erythrocyte surface. PS-exposing cells are other cell types.1,8 The breakdown of phosphatidylserine engulfed by macrophages. The present study explored the asymmetry results from the activation of a scramblase À signalling involved. Hyperosmotic shock and Cl removal which is activated by increase of cytosolic Ca2 þ activity.9,10 triggered the release of prostaglandin E2 (PGE2). In whole-cell As macrophages are equipped with receptors specific recording, activation of the cation channels by ClÀ removal for phosphatidylserine,11–13 erythrocytes exposing phospha- was abolished by the cyclooxygenase inhibitor diclophenac. tidylserine at their surface will be rapidly recognized, 14–16 In FACS analysis, phospholipase-A inhibitors quinacrine and engulfed and degraded. Thus, an increase of cytosolic 2 2 þ palmitoyltrifluoromethyl-ketone, and cyclooxygenase inhibi- Ca activity could trigger apoptotic death and clearance tors acetylsalicylic acid and diclophenac, blunted the of erythrocytes. Ca2 þ entry further activates Ca2 þ -sensitive K þ chan- increase of PS exposure following ClÀ removal. PGE (but 2 nels,17,18 leading to or augmenting cell shrinkage.19 Ca2 þ not thromboxane) induced cation channel activation, increase 2 þ 2 þ may enter erythrocytes through Ca -permeable cation in cytosolic Ca concentration, cell shrinkage, PS exposure, channels,20,21 which are activated following removal of calpain activation, and ankyrin-R degradation. The latter was external ClÀ, osmotic shock by increase of extracellular attenuated by calpain inhibitors-I/II, while PGE2-induced PS osmolarity, oxidative stress by addition of t-butylhydroper- exposure was not. In conclusion, hyperosmotic shock or ClÀ oxide (t-BHP) and energy depletion by removal of extracellular 5,6 removal stimulates erythrocyte PS exposure through PGE2 glucose. Activation of those channels triggers breakdown of formation and subsequent activation of Ca2+-permeable phosphatidylserine asymmetry and subsequent erythrocyte 2 þ 5,6 cation channels. death through increased Ca leakage into the cell. Accordingly, erythrocyte death induced by oxidative Cell Death and Differentiation (2005) 12, 415–428. À doi:10.1038/sj.cdd.4401561 stress, energy depletion or removal of external Cl was abrogated and hyperosmotic shock-induced erythrocyte Published online 4 March 2005 death was blunted when the cells are suspended in medium with low free Ca2 þ concentrations.5,6 The signalling pathways Keywords: cell volume; annexin; osmotic cell shrinkage; glu- leading to the activation of the cation channels remained, cose depletion; calcium however, elusive. Subnanomolar concentrations of prostaglandin E2 (PGE2) 22 Abbreviations: PGE2, prostaglandin E2; PS, phosphatidylser- reportedly lead to activation of erythrocyte cation channels, ine; FACS, fluorescence activated cell sorting; t-BHP, t-butylhy- increase in cytosolic Ca2 þ concentration,23 Gardos K þ droxyperoxide; PLA2, phospholipase A2; COX, cyclooxygenase; channel activation, erythrocyte shrinkage and increased PACOCF3, palmitoyltrifluoromethyl ketone; AACOCF3, arachi- erythrocyte filterability.24,25 Thus, the present experiments donyltrifluoromethyl ketone; NMDG, N-methyl-D-glucamine; have been performed to explore the involvement of phospho- EIPA, ethylisopropylamiloride; MAP kinases, mitogen-activated lipase A2 (PLA2) and cyclooxygenase (COX) in the mecha- protein kinase; HEPES, 32-N-2-hydroethylpiperazine-N-2-etha- nisms linking osmotic cell shrinkage, removal of ClÀ, and nesulfonic acid; EGTA, ethylene glycol-bis (b-aminoethyl energy depletion to the activation of the cation channels. It is ether)-N,N,N0,N0-tetraacetic acid; EDTA, ethylenediamine - anticipated that this signalling may be important for properties N,N,N0,N0-tetraacetic acid; DMSO, dimethyl sulfoxide; PBS, and survival of circulating erythrocytes. Erythrocyte cation channel activation PA Lang et al 416 Results acetylsalicylic acid (50 mM; Figure 2e) and diclofenac (10 mM; Figure 2f). Moreover, palmitoyltrifluoromethyl ketone (PA- As disclosed by FACS analysis, some 10% of the erythrocytes COCF3; 4 mM), an inhibitor of the Ca2 þ -independent PLA ,27 7 2 (13 2%; n ¼ 8) incubated in isotonic NaCl Ringer exhibited blocked about 50% of the ClÀ removal-triggered annexin reduced wheat germ lectin (agglutinin) binding (Figure 1a, b) binding (Figure 2g), while arachidonyltrifluoromethyl ketone as a measure of progressive erythrocyte aging.26 Among (AACOCF3; 8 mM), an inhibitor of the cytosolic PLA2, had no those cells, a significantly higher percentage of erythrocytes effect (percentages of annexin-binding cells under control and bound annexin (3.570.5%; n ¼ 8) as compared to (younger) ClÀ-free conditions were 871 and 3177% in the presence of cells with high lectin binding (1.370.2%; n ¼ 8; Figure 1c–e). AACOCF3 and 672 and 3076% in its absence, respectively; These data point to the presence of distinct erythrocyte means7S.E., n ¼ 4). Higher concentrations of AACOCF3 subpopulations which differ in their susceptibility to pro- themselves induced annexin binding of human erythrocytes grammed cell death. An increase of extracellular osmolarity to (data not shown). 850 mOsm by addition of 550 mM sucrose to NaCl Ringer was Similar to its effect on phosphatidylserine exposure upon followed by a sharp increase of the percentage of annexin- hyperosmotic shock or ClÀ removal, quinacrine (25 mM) binding erythrocytes (Figure 2a, left and middle and blunted the annexin binding induced by energy depletion Figure 2b). This increase was significantly blunted in the (Figure 2h). In sharp contrast, ionomycin (1 mM)-triggered presence of the non-specific PLA2 inhibitor quinacrine (25 mM; annexin binding was not sensitive to quinacrine (25 mM; Figure 2a, right, and Figure 2b). The inhibitory effect of Figure 2i). Taken together, the experimental evidence quinacrine on hyperosmotic shock-induced annexin binding strongly suggests the involvement of Ca2 þ -independent was reversed following coincubation with arachidonic acid PLA2 and COX in programmed erythrocyte death triggered (1 mM, Figure 2c). Addition of arachidonic acid (1 mM) alone by osmotic shock or ClÀ removal. did not induce significant annexin binding in isotonic extra- Both experimental manoeuvres, hyperosmotic shock and À cellular fluid (Figure 2c) and did not significantly enhance the Cl removal, indeed stimulated erythrocyte PGE2 release as increase of annexin binding following hyperosmotic shock assessed by competitive ELISA. Hypertonic shock (Figure 3a) À (Figure 2c), suggesting that hyperosmotic shock interferes or Cl -removal (Figure 3b, c) significantly increased the PGE2 with the signalling cascade downstream from PLA2. concentration in the supernatant of erythrocytes within 1 h Replacement of ClÀ by gluconate similarly triggered (850 mOsm) and 3 h (ClÀ-free) of incubation. Under both annexin binding (Figure 2d–g). The percentage of annexin conditions, the PGE2 concentrations in the supernatant binding cells upon 48 h extracellular ClÀ removal was variable remained two- to four-fold enhanced throughout the 6 h of between the individual experiments (5–57% annexin binding) observation (Figure 3a, b). The Ca2 þ ionophore ionomycin À and averaged to 2573% (n ¼ 30). Cl -removal-induced (1 mM for 1 h) did not stimulate erythrocyte PGE2 release annexin binding was again sensitive to quinacrine (25 mM; (Figure 3d). Furthermore, hypertonic shock-stimulated Figure 2d) and inhibited by the cyclooxygenase blockers PGE2 release (850 mOsm for 1 h) was not dependent on a 1000 b 200 e R1 * 4 500 100 13.00 cell counts side sca tter 3 0 0 0 500 1000 100 101 102 103 104 2 forward scatter FL1-H c 4 10 d 4 lectin-FITC+ lectin-FITC 10 1 3 annexinV-Alexa 10 3 10 annexin-binding cells [%] 0.00 0.05 0.34 0.45 0 102 102 FL3-H FL3-H 101 101 positive 15.90 84.05 12.66 86.55 negative 100