Role of ceramide in Ca2+-sensing receptor-induced apoptosis.
Increased extracellular Ca(2+) ([Ca(2+)](o)) can damage tissues, but the molecular mechanisms by which this occurs are poorly defined. Using HEK 293 cell lines that stably overexpress the Ca(2+)-sensing receptor (CaR), a G protein-coupled receptor, we demonstrate that activation of the CaR leads to apoptosis, which was determined by nuclear condensation, DNA fragmentation, caspase-3 activation, and increased cytosolic cytochrome c. This CaR-induced apoptotic pathway is initiated by CaR-induced accumulation of ceramide which plays an important role in inducing cell death signals by distinct G protein-independent signaling pathways. Pretreatment of wild-type CaR-expressing cells with pertussis toxin inhibited CaR-induced [(3)H]ceramide formation, c-Jun phosphorylation, and caspase-3 activation. The ceramide accumulation, c-Jun phosphorylation, and caspase-3 activation by the CaR can be abolished by sphingomyelinase and ceramide synthase inhibitors in different time frames. Cells that express a nonfunctional mutant CaR that were exposed to the same levels of [Ca(2+)](o) showed no evidence of activation of the apoptotic pathway. In conclusion, we report the involvement of the CaR in stimulating programmed cell death via a pathway involving GTP binding protein alpha subunit (Galpha(i))-dependent ceramide accumulation, activation of stress-activated protein kinase/c-Jun N-terminal kinase, c-Jun phosphorylation, caspase-3 activation, and DNA cleavage.
Wu
Zhenzhen
Z
Department of Medicine, Case Western Reserve University, Cleveland, OH, USA.
Tandon
Rajnish
R
Ziembicki
Jenny
J
Nagano
Junko
J
Hujer
Kristine M
KM
Miller
R Tyler
RT
Huang
Chunfa
C
D017209:Apoptosis / Q000187:drug effects; D002118:Calcium / Q000494:pharmacology; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002460:Cell Line; D002518:Ceramides / Q000096:biosynthesis / Q000502:physiology*; D045304:Cytochromes c / Q000378:metabolism; D004789:Enzyme Activation; D006801:Humans; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism; D048670:MAP Kinase Kinase 4 / Q000378:metabolism; D048055:Mitogen-Activated Protein Kinase 8 / Q000378:metabolism; D037342:Pertussis Toxin / Q000494:pharmacology; D044169:Receptors, Calcium-Sensing / Q000502:physiology*; D015290:Second Messenger Systems / Q000502:physiology*
D002518:Ceramides; D044169:Receptors, Calcium-Sensing; D045304:Cytochromes c; D037342:Pertussis Toxin; D048031:JNK Mitogen-Activated Protein Kinases; D048055:Mitogen-Activated Protein Kinase 8; D048670:MAP Kinase Kinase 4; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; D002118:Calcium