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Apoptotic Sphingolipid Signaling by Ceramides in Lung Endothelial Cells

¹ Division of Pulmonary and Critical Care Medicine, ⁴ Division of Clinical Pharmacology, and ⁷ Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland; ² Division of Pulmonary, Allergy, Critical Care and Occupational Medicine, Indiana University, Indianapolis, Indiana; ³ Section of Pulmonary and Critical Care Medicine, Yale University School of Medicine, New Haven, Connecticut; ⁵ Division of Pulmonary and Critical Care Medicine, University of Chicago, Chicago, Illinois; and ⁶ Division of Molecular Genetics, Mount Sinai School of Medicine, New York, New York

Correspondence and requests for reprints should be addressed to Irina Petrache, M.D., Indiana University, Division of Pulmonary, Allergy, Critical Care and Occupational Medicine, Van Nuys Medical Science Building, 635 Barnhill Drive, MS224, Indianapolis, IN 46202-5120. E-mail: ipetrach{at}iupui.edu

The de novo pathway of ceramide synthesis has been implicated in the pathogenesis of excessive lung apoptosis and murine emphysema. Intracellular and paracellular-generated ceramides may trigger apoptosis and propagate the death signals to neighboring cells, respectively. In this study we compared the sphingolipid signaling pathways triggered by the paracellular- versus intracellular-generated ceramides as they induce lung endothelial cell apoptosis, a process important in emphysema development. Intermediate–chain length (C_8:0) extracellular ceramides, used as a surrogate of paracellular ceramides, triggered caspase-3 activation in primary mouse lung endothelial cells, similar to TNF-–generated endogenous ceramides. Inhibitory siRNA against serine palmitoyl transferase subunit 1 but not acid sphingomyelinase inhibited both C_8:0 ceramide– and TNF- (plus cycloheximide)–induced apoptosis, consistent with the requirement for activation of the de novo pathway of sphingolipid synthesis. Tandem mass spectrometry analysis detected increases in both relative and absolute levels of C_16:0 ceramide in response to C_8:0 and TNF- treatments. These results implicate the de novo pathway of ceramide synthesis in the apoptotic effects of both paracellular ceramides and TNF-–stimulated intracellular ceramides in primary lung endothelial cells. The serine palmitoyl synthase-regulated ceramides synthesis may contribute to the amplification of pulmonary vascular injury induced by excessive ceramides.

Key Words: apoptosis • lung • cytokines • signaling • sphingolipids

CLINICAL RELEVANCE Prevention of lung microvascular endothelial apoptosis modulated by serine palmitoyl transferase might evolve as a therapeutic strategy in emphysema.