Partial ATP depletion induces Fas- and caspase-mediated apoptosis in MDCK cells.
Brief periods of in vitro hypoxia/ischemia induce apoptosis of cultured renal epithelial cells, but the underlying mechanisms remain unknown. We show that partial ATP depletion (approximately 10-65% of control) results in a duration-dependent induction of apoptosis in Madin-Darby canine kidney (MDCK) cells, as evidenced by internucleosomal DNA cleavage (DNA laddering and in situ nick end labeling), morphological changes (cell shrinkage), and plasma membrane alterations (externalization of phosphatidylserine). The ATP-depleted cells display a significant upregulation of Fas, Fas ligand, and the Fas-associating protein with death domain (FADD). Exogenous application of stimulatory Fas monoclonal antibodies also induces apoptosis in nonischemic MDCK cells, indicating that they retain Fas-dependent pathways of programmed cell death. Furthermore, cleavage of poly(ADP)ribose polymerase (PARP) is evident after ATP depletion, indicating activation of caspases. Indeed, the apoptotic cells display a significant increase in caspase-8 (FLICE) activity. Finally, apoptosis induced by ATP depletion is ameliorated by pretreatment with inhibitors of caspase-8 (IETD), caspase-1 (YVAD), or caspase-3 (DEVD) but is not affected by inhibitors of serine proteases (TPCK). Our results indicate that partial ATP depletion of MDCK cells results in apoptosis and that Fas- and caspase-mediated pathways may play a critical role.
Feldenberg
L R
LR
Division of Pediatric Nephrology, Yale University School of Medicine, New Haven, Connecticut 06520, USA.
Thevananther
S
S
del Rio
M
M
de Leon
M
M
Devarajan
P
P
D048868:Adaptor Proteins, Signal Transducing; D000255:Adenosine Triphosphate / Q000172:deficiency*; D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D002352:Carrier Proteins / Q000378:metabolism; D053181:Caspase 8; D053453:Caspase 9; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism / Q000502:physiology*; D002460:Cell Line; D004285:Dogs; D004791:Enzyme Inhibitors / Q000494:pharmacology; D053222:Fas Ligand Protein; D053200:Fas-Associated Death Domain Protein; D007425:Intracellular Membranes / Q000378:metabolism; D007668:Kidney / Q000166:cytology / Q000378:metabolism / Q000502:physiology*; D008562:Membrane Glycoproteins / Q000378:metabolism; D011065:Poly(ADP-ribose) Polymerases / Q000737:chemistry; D013997:Time Factors; D019014:fas Receptor / Q000502:physiology*
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D061945:Caspase Inhibitors; D004791:Enzyme Inhibitors; D053222:Fas Ligand Protein; D053200:Fas-Associated Death Domain Protein; D008562:Membrane Glycoproteins; D019014:fas Receptor; D000255:Adenosine Triphosphate; D011065:Poly(ADP-ribose) Polymerases; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases