Accelerated apoptotic death and in vivo turnover of erythrocytes in mice lacking functional mitogen- and stress-activated kinase MSK1/2

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.

In the present study, we explored whether MSK1/2 influences the survival of erythrocytes in response to pathophysiological cell stressors such as hyperosmotic shock and energy depletion. To this end, the eryptotic phenotype was characterized in mice lacking functional MSK1/2 (msk −/− ) and their corresponding wild type mice (msk +/+ ).
Expression of MSK1 and MSK2 in human and murine erythrocytes. Immunoblotting was employed to test whether MSK1 and/or MSK2 are expressed in erythrocytes. To this end, erythrocytes from humans or from mice were isolated and purified. Equal amounts of protein lysates were made and immunoblotting was performed. GAPDH served as a loading control. Expression of MSK1 and MSK2 was determined in lysates from murine whole blood and from purified murine erythrocytes. As illustrated in Fig. 1, the incubation with MSK1 and MSK2 specific antibodies both yielded a band of 90 (MSK1) and 86 (MSK2) kDa in murine and human erythrocytes, respectively.
Increased vulnerability of msk −/− erythrocytes to energy-sensitive eryptosis. Additional experiments were performed in the presence and absence of glucose, as energy depletion is known to  foster eryptosis 38 . As shown in Fig. 5, annexin V-binding reflecting phosphatidylserine exposure at the erythrocyte surface was significantly increased by 12 h glucose depletion, an effect significantly higher in msk −/− than in msk +/+ erythrocytes. Furthermore, as shown in Fig. 6, forward scatter was significantly reduced by energy depletion in erythrocytes from both msk −/− and msk +/+ mice. This effect tended to be larger in msk −/− than in msk +/+ erythrocytes, an effect, however, not reaching statistical significance (Fig. 6).
Enhanced in vivo clearance and entrapment of eryptotic erythrocytes in the spleens of msk −/− mice. Eryptotic erythrocytes are rapidly cleared from circulating blood 36 . Thus, additional experiments were performed to disclose a possible effect of MSK1/2 deficiency on erythrocyte clearance.
To determine the life span of circulating erythrocytes, blood was drawn from msk −/− and msk +/+ mice and erythrocytes were labelled with CFSE and injected autologously in the mice of the respective genotype. As shown in Fig. 7A, within 4 and 5 days CFSE-labeled msk −/− erythrocytes disappeared from circulating blood of msk −/− mice more rapidly than CFSE-labeled msk +/+ erythrocytes from circulating blood of msk +/+ mice. Thus, the life span of msk −/− erythrocytes in msk −/− mice was significantly shorter than the life span of msk +/+ erythrocytes in msk +/+ mice. The labelled erythrocytes were mainly trapped in the spleen. The ratio of spleen weight to body weight was slightly but significantly larger in msk −/− mice as compared to msk +/+ mice (Fig. 7B). The number of fluorescent annexin V-binding and thus phosphatidylserine-exposing erythrocytes as visualized by fluorescence confocal microscopy was again higher in the spleens from msk −/− mice than in the spleens from msk +/+ mice reflecting enhanced trapping of eryptotic erythrocytes in msk −/− mice (Fig. 7C,D).

Discussion
According to the present observations, a lack of MSK1/2 enhances the susceptibility of erythrocytes to undergo suicidal erythrocyte death or eryptosis following pathophysiological cell stressors such as hyperosmotic shock and energy depletion. The MSK1/2-deficient (msk −/− ) mice did not exhibit overt anemia but showed marked increase in erythrocyte turnover that contributes to a mild increase in splenic mass. Moreover, the erythrocytes from msk −/− mice are more sensitive than erythrocytes from msk +/+ mice to triggers of eryptosis, including hyperosmotic shock and energy depletion. On the other hand, MSK1/2 deficiency decreases the resistance against hemolysis following decrease of extracellular Hyperosmotic shock and energy deletion trigger eryptosis only in a subset of the erythrocyte population, indicating that the circulating erythrocytes are not uniformly sensitive to those triggers of eryptosis. As a matter of fact, the susceptibility of circulating erythrocytes towards triggers of eryptosis increases with erythrocyte age 39,40 . On the other hand, evidence has been reported that newly formed erythrocytes are highly susceptible to suicidal death, a phenomenon called neocytolysis [41][42][43] . Along those lines, considerable diversity of lysophosphatidic acid (LPA) induced Ca 2+ influx and phospatidylserine translocation was observed in seemingly morphologically homogeneous erythrocyte populations 44 . The Ca 2+ response to LPA was virtually lacking in reticulocytes and still highly variable in old erythrocytes 44 .
Collectively, the present observations highlight the significance of MSK1/2 for erythrocyte survival. Phosphatidylserine-exposing cells are bound to macrophages 45 , engulfed and degraded 46 , and thus rapidly cleared from circulating blood 36,37 . Along those lines, msk −/− erythrocytes are cleared more rapidly from the circulation. The accelerated erythrocyte death and clearance from circulating blood is outweighed by compensatory increase of erythropoiesis in msk −/− mice, which is reflected by increased numbers of circulating reticulocytes in those mice.
Mechanistically, exposure of erythrocytes to hypertonic extracellular environment in vitro simulates the osmotic conditions encountered in the kidney medulla. Under pathological conditions such as acute renal failure, erythrocytes are trapped in the kidney medulla, thus predisposing erythrocytes to eryptosis 33 . It is, therefore, tempting to speculate that MSK1/2 influences erythrocyte survival and its ramifications in systemic conditions such as renal failure. The MSK1/2 upstream molecule p38 MAPK orchestrates adaptation to hypertonicity in mammalian cells 47,48 . In nucleated cells, hypertonic shock modulates cAMP response element-binding protein via activation of MSK1-dependent signaling 49 . In erythrocytes, a similar parallel can be drawn as hyperosmotic shock elicits phosphorylation of p38 MAPK that regulates the eryptosis machinery 50 . The msk −/− erythrocytes have further an enhanced sensitivity to the eryptotic effect of cellular energy deprivation, another powerful stimulator of eryptosis 38 . Signaling involved in the regulation of eryptosis following cellular energy depletion includes protein kinase C, AMP activate kinase and Janus kinase 3 19 .
According to the present data MSK1/2 contributes to both osmo-and energy-sensitive regulation of erythrocyte survival. Without stimulation of eryptosis, the percentage of eryptotic cells is similar in msk −/− mice and in msk +/+ mice. The susceptibility of the erythrocytes from msk −/− mice to eryptosis is, however, apparent following osmotic shock and energy depletion. Eryptosis is enhanced by erythrocyte age, a wide variety of anemia-causing xenobiotics and endogenous substances 19 and several clinical disorders, including iron deficiency, phosphate depletion, hepatic failure, dehydration, fever, Hemolytic Uremic Syndrome, end stage renal disease, sepsis, malaria, malignancy and Wilson's disease 19,51 . Eryptosis may further influence erythrocyte storage for transfusion 52 . MSK1 deficiency may enhance the susceptibility to the eryptotic effect of those xenobiotics, endogenous substances and clinical disorders. In view of the accelerated clearance of erythrocytes and a mild splenomegaly in msk −/− mice, triggers of eryptosis are apparently operative in the blood of those mice.
In conclusion, lack of MSK1/2 leads to enhanced susceptibility to suicidal erythrocyte death or eryptosis following osmotic shock and energy depletion leading to accelerated splenic trapping of circulating erythrocytes.

Materials and Methods
Human erythrocytes. Highly purified erythrocyte concentrates were provided by the blood bank of the University of Tübingen. The erythrocyte concentrates were virtually free of white blood cells and contained less than 1% platelets. The Committee approving the experiments, in name, is the ethics committee of the University of Tübingen, given report number: 184/2003V. Informed consent was obtained from all subjects.
Mice. Experiments were performed in 9-to 16-wk-old MSK1/2-deficient mice (msk −/− ) as well as sex-and age matched wild-type mice (msk +/+ ) which were fed a control diet (C1314; Altromin, Heidenau, Germany) and had access to drinking water ad libitum. The msk −/− mice have been described previously 15,18 . The animals were maintained under specific pathogen-free conditions and all experiments described in the methods were carried out in accordance with the approved guidelines (American Physiological Society as well as the German law and the EU Animals Scientific Procedures Act for the welfare of animals) and were approved by local authorities of the state of Baden-Württemberg. Blood count and isolation of murine erythrocytes. For all experiments except for the blood count, heparin blood was retrieved from the retrobulbar plexus of mice 62 . For the blood count, EDTA blood was analyzed using an electronic hematology particle counter (type MDM 905 from Medical Diagnostics Marx; Butzbach, Germany) equipped with a photometric unit for haemoglobin determination. To obtain pure erythrocytes, murine erythrocytes were separated utilizing Ficoll (Biochrom AG, Germany) and washed twice with Ringer solution containing (in mM): 125 NaCl, 5 KCl, 1 MgSO 4 , and 32 HEPES/NaOH (pH 7.4), 5 glucose, and 1 CaCl 2 .
Reticulocyte count. For determination of the reticulocyte count EDTA-whole blood (5 μ l) was added to 1 ml Retic-COUNT (Thiazole orange) reagent from Becton Dickinson. Samples were stained for 30 min at room temperature, and flow cytometry was performed according to the manufacturer's instructions. Forward scatter (FSC), side scatter (SSC), and Thiazole orange-fluorescence intensity (in FL-1) of the blood cells were determined. The number of Retic-COUNT positive reticulocytes was expressed as the percentage of the total gated erythrocyte populations. Gating of erythrocytes was achieved by analysis of FSC vs. SSC dot plots using CellQuest software.
Determination of the osmotic resistance. For measurement of osmotic resistance 2 μ l erythrocyte pellets were exposed in a 96 well plate for 2 min to phosphate-buffered saline (PBS) solutions (in mM: 1.05 KH 2 PO 4 , 2.97 Na 2 HPO 4 , 155.2 NaCl) of decreasing osmolarity as prepared by mixing a PBS solution with a defined volume of distilled water. After centrifugation (500 g for 5 min), the Hb concentration of the supernatants was determined photometrically (at 405 nm).

Measurement of the in vivo clearance of fluorescence-labeled erythrocytes. The in vivo
clearance of fluorescence-labeled erythrocytes was determined as described previously 63 . Briefly, erythrocytes (obtained from 200 μ l blood) were fluorescence-labeled by staining the cells with 5 μ M carboxyfluorescein-diacetate-succinimidyl-ester (CFSE) (Molecular Probes, Leiden, Netherlands) in PBS and incubated for 30 min at 37 °C. After washing twice in PBS containing 10% FCS the pellet was resuspended in Ringer solution (37 °C), and 100 μ l of the CFSE-labelled erythrocytes (50% hematocrit) were injected into the tail vein of the recipient mouse. As indicated, blood was retrieved from the tail veins of the mice, and CFSE-dependent fluorescence intensity of the erythrocytes was measured as described above. The percentage of CFSE-positive erythrocytes was calculated in % of the total labelled fraction determined 10 min after injection.
Confocal microscopy. For the detection of annexin V-binding and CFSE-dependent fluorescence of erythrocytes in the spleen, the spleens of msk −/− and msk +/+ mice were homogenized mechanically in 1 ml cold PBS. The suspension was then centrifuged at 500 g for 10 min at 4 °C. The cell pellet was resuspended in 200 μ l cold PBS. Five μ l of Annexin V-APC (BD, Heidelberg, Germany) were added, and incubation was carried out for 20 min at 37 °C protected from light. Then, the suspension was transferred onto a glass slide and mounted with Prolong ® Gold antifade reagent (Invitrogen). Images were taken on a Zeiss LSM 5 EXCITER Confocal Laser Scanning Microscope (Carl Zeiss MicroImaging GmbH, Germany) with a water immersion Plan-Neofluar 63/1.3 NA DIC.
Statistics. Data are expressed as arithmetic means ± SEM, and statistical analysis was made using ANOVA or t-test, as appropriate. n denotes the number of different erythrocyte specimens studied.