Neutral ceramidase gene: role in regulating ceramide-induced apoptosis Moonsuk S. Choi a,1 , Mary A. Anderson a,1,2 , Zhongjian Zhang a , Drazen B. Zimonjic b , Nicolae Popescu b , Anil B. Mukherjee a, * a Section on Developmental Genetics, Heritable Disorders Branch, The National Institute of Child Health and Human Development, The National Institutes of Health, Room 9S241, Building 10, Bethesda, MD 20892-1830, USA b The National Cancer Institute, The National Institutes of Health, Bethesda, MD 20892-1830, USA Received 19 February 2003; received in revised form 24 April 2003; accepted 22 May 2003 Received by F. Salvatore Abstract The sphingolipid, ceramide, is a natural dietary constituent and a potent mediator of apoptosis. If left undegraded, it may induce apoptosis and cause disruption of cellular integrity. A potential mechanism to prevent ceramide-induced apoptosis in various organs may involve ceramidases that facilitate the degradation of ceramide. In this study, we first isolated and characterized the murine neutral ceramidase (N- CDase) gene, mapped its chromosomal location and determined its developmental and organ-specific expression. Then we used cultured mesangial cells as our in vitro model and mouse gastrointestinal (GI) tract as the in vivo model to determine the effects of an inhibitor of N- CDase, D-erythro-MAPP, to delineate whether N-CDase plays a role in preventing ceramide-induced apoptosis. Our results show that: (i) the structure of the murine neutral ceramidase gene is virtually identical to that of the human gene; (ii) it is localized on chromosome 19 at bands C2 – C3 that is syntenic to human chromosome 10q24 – 26; (iii) N-CDase expression is developmentally regulated and it is expressed at high levels in cultured mesangial cells and in specific regions of the mouse small intestine; (iv) inhibition of N-CDase by D-erythro-MAPP leads to increased ceramide levels and consequent apoptosis in cultured mesangial cells; (v) mice treated with D-erythro-MAPP alone also caused apoptosis in the small intestine; and (vi) mice treated with D-erythro-MAPP prior to feeding C2 ceramide manifest markedly elevated levels of apoptosis in the GI tract raising the possibility that neutral ceramidase plays a detoxifying role against inadvertent stimulation of ceramide- induced apoptosis in organs that come in contact with this sphingolipid. We propose that N-CDase is an essential component of an innate detoxifying mechanism to prevent ceramide-induced apoptosis. D 2003 Elsevier B.V. All rights reserved. Keywords: Neutral/alkaline ceramidase; Ceramide; Apoptosis; Gastrointentinal 1. Introduction The sphingomyelin (SM) cycle is a major signal trans- duction pathway that mediates the action of a large variety of cytokines, stress ligands, and growth factors responsible for the onset of important cellular responses (reviewed in Hannun and Luberto, 2000). This pathway is initiated by the activation of sphingomyelinase (SMase). SMase catalyzes the hydrolysis of SM generating ceramide and phosphocho- line. Ceramide is a major intracellular messenger that mediates cellular responses in apoptosis (reviewed in Cuvil- lier, 2002), cell cycle arrest (Dbaibo et al., 1995), and differentiation (Okazaki et al., 1989; Dobrowsky et al., 1994). Cellular ceramide can be further metabolized by ceramidases that hydrolyze the N-acyl linkage between a fatty acid and sphingosine. Sphingosine can then be further metabolized to sphingosine-1-phosphate, which regulates cell proliferation by inhibiting caspases (reviewed in Cuvil- lier, 2002). Moreover, ceramide and its metabolites, sphin- gosine and sphingosine-1-phosphate, play integral roles in cellular signaling (Hannun, 1996). In view of the critical 0378-1119/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0378-1119(03)00721-2 Abbreviations: C2-cer, C2-ceramide; DMEM, Dulbecco’s modified Eagle’s medium; D-erythro-MAPP, D-erythro-2-(N-myristoylamino)-1-phe- nyl-1-propanol; FISH, fluorescence in situ hybridization; FITC, fluorescein isothiocyanate; GI tract, gastrointestinal tract; N-CDase, neutral/alkaline ceramidase; PI, propidium iodide; PCR, polymerase chain reaction; RT- PCR, reverse transcription-PCR; SM, sphingomyelin; SMase, sphingo- myelinase; TUNEL, terminal deoxynucleotide transferase (TdT) mediated dUTP digoxigenin nick and labeling. * Corresponding author. Tel.: +1-301-496-7213; fax: +1-301-402- 6632. E-mail address: mukherja@exchange.nih.gov (A.B. Mukherjee). 1 Equally contributed. 2 Present address: Digene, 1201 Clopper Road, Gaithersburg, MD 20878, USA. www.elsevier.com/locate/gene Gene 315 (2003) 113 – 122