Abstract
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.
Highlights
UV irradiation represents a major oxidative stressor for mammalian skin
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 required for sphingomyelin synthesis occur(s) in UVB-irradiated cells
We report here a mechanism(s) that accounts for the apparent failure to up-regulate SM production following UVB irradiation that appears linked to the subsequent UVBinduced apoptosis
Summary
Materials—Cer and GlcCer were purchased from Sigma and Matreya (Pleasant Gap, PA), respectively. UVB Irradiation—UVB irradiation was performed as described previously [16, 32]. Cells were irradiated with UVB at 4 °C in PBS followed by incubation with or without 1 M HPA-12 at 37 °C for 30 min. Either UVB-treated or sham-treated purified recombinant CERT protein (50 g) [25] in 0.5 ml of Tris/HCl buffer, pH 7.4, was added to the C5-DMB-Cer filmcoated tubes and incubated for various times at 37 °C. Protein Identification by In-gel Digestion and Liquid Chromatography/Electrospray Ionization-Tandem Mass Spectrometry (LC/ESI-MS/MS) Analysis—Fractions containing immunoprecipitated CERT from UVB- or sham-irradiated cells were analyzed by SDS-PAGE as above. Lipid Synthesis—To examine de novo sphingolipid synthesis following UVB irradiation, cells were cultured with [3H]serine (1.5 Ci/ml), [9,10-3H]palmitic acid (1.5 Ci/ml), or [3H]galactose (1.5 Ci/ml) for the final 6 or 8 h. Details are described under “Experimental Procedures.” Values represent mean Ϯ S.D. from three experiments
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