Decreased ceramide transport protein (CERT) function alters sphingomyelin production following UVB irradiation.
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 (C(5)-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.
Charruyer
Alexandra
A
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, USA.
Bell
Sean M
SM
Kawano
Miyuki
M
Douangpanya
Sounthala
S
Yen
Ten-Yang
TY
Macher
Bruce A
BA
Kumagai
Keigo
K
Hanada
Kentaro
K
Holleran
Walter M
WM
Uchida
Yoshikazu
Y
D000818:Animals; D017209:Apoptosis; D045744:Cell Line, Tumor; D019281:Dimerization; D006367:HeLa Cells; D006801:Humans; D015603:Keratinocytes / Q000378:metabolism; D051379:Mice; D008954:Models, Biological; D009569:Nitric Oxide / Q000378:metabolism; D018384:Oxidative Stress; D017346:Protein Serine-Threonine Kinases / Q000096:biosynthesis*; D013109:Sphingomyelins / Q000378:metabolism*; D014162:Transfection; D014466:Ultraviolet Rays
D013109:Sphingomyelins; D009569:Nitric Oxide; C119090:CERT1 protein, human; D017346:Protein Serine-Threonine Kinases