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

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 [H-3] 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 alpha(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, Z., R. Tandon, J. Ziembicki, J. Nagano, K. M. Hujer, R. T. Miller, and C. Huang. Role of ceramide in Ca2+-sensing receptor-induced apoptosis.