Fas-, caspase 8-, and caspase 3-dependent signaling regulates the activity of the aminophospholipid translocase and phosphatidylserine externalization in human erythrocytes.
Apoptosis and erythrocyte senescence share the common feature of exposure of phosphatidylserine (PS) in the outer leaflet of the cells. Western analysis showed that mature red cells contain Fas, FasL, Fas-associated death domain (FADD), caspase 8, and caspase 3. Circulating, aged cells showed colocalization of Fas with the raft marker proteins Galpha(s) and CD59; the existence of Fas-associated FasL, FADD and caspase 8; and caspase 8 and caspase 3 activity. Aged red cells had significantly lower aminophospholipid translocase activity and higher levels of PS externalization in comparison with young cells. In support of our contention that caspases play a functional role in the mature red cell, the oxidatively stressed red cell recapitulated apoptotic events, including translocation of Fas into rafts, formation of a Fas-associated complex, and activation of caspases 8 and 3. These events were independent of calpain but dependent on reactive oxygen species (ROS) as evident from the effects of the ROS scavenger N-acetylcysteine. Caspase activation was associated with loss of aminophospholipid translocase activity and with PS externalization. ROS was not generated by treatment of cells with t-butyl hydroperoxide at 10 degrees C, and Fas did not translocate into rafts. Concomitantly, neither formation of a Fas-associated signaling complex nor caspase activation could be observed, supporting the view that translocation of Fas into rafts was the trigger for the chain of events leading to caspase 3 activation. Our data demonstrate for the first time the novel involvement of Fas/caspase 8/caspase 3-dependent signaling in an enucleated cell leading to PS externalization, a central feature of erythrophagocytosis and erythrocyte biology.
Mandal
Debabrata
D
Department of Chemistry, Bose Institute 93/1 Acharya Prafulla Chandra Road, Kolkata-700009, India.
Mazumder
Arindam
A
Das
Pradeep
P
Kundu
Manikuntala
M
Basu
Joyoti
J
D048868:Adaptor Proteins, Signal Transducing / Q000097:blood; D017209:Apoptosis; D053148:Caspase 3; D053181:Caspase 8; D020169:Caspases / Q000097:blood*; D004905:Erythrocyte Aging / Q000502:physiology; D004912:Erythrocytes / Q000166:cytology / Q000378:metabolism*; D053222:Fas Ligand Protein; D053200:Fas-Associated Death Domain Protein; D006801:Humans; D066298:In Vitro Techniques; D008562:Membrane Glycoproteins / Q000097:blood; D021962:Membrane Microdomains / Q000378:metabolism; D018384:Oxidative Stress; D010587:Phagocytosis; D010718:Phosphatidylserines / Q000097:blood*; D049294:Phospholipid Transfer Proteins / Q000097:blood*; D018124:Receptors, Tumor Necrosis Factor / Q000097:blood*; D015398:Signal Transduction; D048069:Tumor Necrosis Factors / Q000097:blood; D019014:fas Receptor
D048868:Adaptor Proteins, Signal Transducing; C505533:FADD protein, human; C491901:FAS protein, human; C505661:FASLG protein, human; D053222:Fas Ligand Protein; D053200:Fas-Associated Death Domain Protein; D008562:Membrane Glycoproteins; D010718:Phosphatidylserines; D049294:Phospholipid Transfer Proteins; D018124:Receptors, Tumor Necrosis Factor; D048069:Tumor Necrosis Factors; D019014:fas Receptor; C505407:CASP3 protein, human; C505530:CASP8 protein, human; D053148:Caspase 3; D053181:Caspase 8; D020169:Caspases