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J. Biol. Chem., Vol. 283, Issue 24, 16682-16692, June 13, 2008

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Decreased Ceramide Transport Protein (CERT) Function Alters Sphingomyelin Production following UVB Irradiation^*

Alexandra Charruyer¹, Sean M. Bell¹, Miyuki Kawano, Sounthala Douangpanya, Ten-Yang Yen^¶, Bruce A. Macher^¶, Keigo Kumagai, Kentaro Hanada, Walter M. Holleran^||, and Yoshikazu Uchida²

From the Department of Dermatology, School of Medicine, University of California, Northern California Institute for Research and Education, and Veterans Affairs Medical Center, San Francisco, California 94121, ^||Department of Pharmaceutical Chemistry, School of Pharmacy, University of California, San Francisco, California 94143, Department of Biochemistry and Cell Biology, National Institute of Infectious Diseases, Tokyo 162-8640, Japan, and ^¶Department of Chemistry and Biochemistry, San Francisco State University, San Francisco, California 94132

Increased cellular ceramide accounts in part for UVB irradiation-induced apoptosis in cultured human keratinocytes with concurrent increased glucosylceramide but not sphingomyelin generation in these cells. Given that conversion of ceramide to non-apoptotic metabolites such as sphingomyelin and glucosylceramide protects cells from ceramide-induced apoptosis, we hypothesized that failed up-regulation of sphingomyelin generation contributes to ceramide accumulation following UVB irradiation. Because both sphingomyelin synthase and glucosylceramide synthase activities were significantly decreased in UVB-irradiated keratinocytes, we investigated whether alteration(s) in the function of ceramide transport protein (or CERT) required for sphingomyelin synthesis occur(s) in UVB-irradiated cells. Fluorescently labeled N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-D-erythro-sphingosine (C₅-DMB-ceramide) relocation to the Golgi was diminished after irradiation, consistent with decreased CERT function, whereas the CERT inhibitor N-(3-hydroxy-1-hydroxymethyl-3-phenylpropyl)dodecanamide (1R,3R isomer) (HPA-12) produced an equivalent effect. UVB irradiation also induced the rapid formation of a stable CERT homotrimer complex in keratinocytes as determined by Western immunoblot and mass spectrometry analyses, a finding replicated in HeLa, HEK293T, and HaCaT cells and in murine epidermis. Ceramide binding activity was decreased in recombinant CERT proteins containing the UVB-induced homotrimer. The middle region domain of the CERT protein was required for the homotrimer formation, whereas neither the pleckstrin homology (Golgi-binding) nor the START (ceramide-binding) domains were involved. Finally like UVB-treated keratinocytes, HPA-12 blockade of CERT function increased keratinocyte apoptosis, decreased sphingomyelin synthesis, and led to accumulation of ceramide. Thus, UVB-induced CERT homotrimer formation accounts, at least in part, for apoptosis and failed up-regulation of sphingomyelin synthesis following UVB irradiation, revealing that inactive CERT can attenuate a key metabolic protective mechanism against ceramide-induced apoptosis in keratinocytes.

Received for publication, January 30, 2008 , and in revised form, March 27, 2008.

^* This work was supported, in whole or in part, by National Institutes of Health Grants AR 051077 (to Y. U.), PO1-AR39448 (to W. H.), and P20 MD000262 (to B. A. M.). This work was also supported by a Research Evaluation and Allocation Committee award from the University of California, San Francisco (to Y. U.) and National Science Foundation Grant CHEM-0619163 (to T.-Y. Y. and B. A. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Both authors contributed equally to this work.

² To whom correspondence should be addressed: Dermatology Service and Research Unit (190), Veterans Affairs Medical Center, 4150 Clement St., San Francisco, CA 94121. Tel.: 415-750-2091; Fax: 415-751-3927; E-mail: uchiday{at}derm.ucsf.edu.

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