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Journal of Lipid Research, Vol. 46, 1396-1404, July 2005
Copyright © 2005 by American Society for Biochemistry and Molecular Biology

Role of ceramide in Ca²⁺-sensing receptor-induced apoptosis

Zhenzhen Wu^*,, Rajnish Tandon^,, Jenny Ziembicki^,, Junko Nagano^*,, Kristine M. Hujer^*,, R. Tyler Miller^*, and Chunfa Huang^1,*,

^* Departments of Medicine, Case Western Reserve University, Cleveland, OH
Surgery, Case Western Reserve University, Cleveland, OH
Louis Stokes VA Medical Center, Cleveland, OH

Published, JLR Papers in Press, April 1, 2005. DOI 10.1194/jlr.M500071-JLR200

¹ To whom correspondence should be addressed. e-mail: cxh87{at}po.cwru.edu

Increased extracellular Ca²⁺ ([Ca²⁺]_o) can damage tissues, but the molecular mechanisms by which this occurs are poorly defined. Using HEK 293 cell lines that stably overexpress the Ca²⁺-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 [³H]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 [Ca²⁺]_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 (G_i)-dependent ceramide accumulation, activation of stress-activated protein kinase/c-Jun N-terminal kinase, c-Jun phosphorylation, caspase-3 activation, and DNA cleavage.

Abbreviations: [Ca²⁺]_i, intracellular Ca²⁺; [Ca²⁺]_o, extracellular Ca²⁺; CaR, Ca²⁺-sensing receptor; ERK, extracellular signal-regulated kinase; GPCR, G protein-coupled receptor; MAPK, mitogen-activated protein kinase; SAPK/JNK, stress-activated protein kinase/c-Jun N-terminal kinase; SEK1, stress-activated protein kinase/extracellular signal-regulated kinase kinase 1

Supplementary key words stress-activated protein kinase/c-Jun N-terminal kinase • HEK 293 cell • G protein-coupled receptor

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