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To examine factors that regulate ceramide production during keratinization of the human stratum corneum (SC), we developed a reconstructed human epidermal keratinization model in which a fresh layer of SC is newly formed within 1 week. Addition of the UDP-glucose: ceramide glucosyltransferase inhibitor 1-phenyl-2-decanoylamino-3-morpholino-1-propanol significantly diminished SC ceramide levels (expressed as µg/mg protein) with decreased glucosylceramide levels. Desipramine hydrochloride, an inhibitor of sphingomyelinase, also significantly reduced SC ceramide levels. Similarly, conduritol B epoxide, an inhibitor of β-glucocerebrosidase, significantly down-regulated SC ceramide levels and significantly increased glucosylceramide levels. These results indicate the reliability of this model to elucidate ceramide synthesis regulating factors. Using this model, we assessed the effects of the inflammatory cytokine interleukin-1α (IL-1α), several bioactive sphingolipids and all-trans retinoic acid (RA) on ceramide levels in the SC. Whereas treatment with IL-1α (at 10 nM) significantly down-regulated ceramide levels, treatment with sphingosylphosphorylcholine (at 50 µM) or sphingosine-1-phosphate (at 10 or 20 µM) distinctly up-regulated ceramide levels. Interestingly, RA (at low as 10 nM) significantly up-regulated ceramide levels without affecting the formation of the SC or levels of keratinization-related proteins in the epidermis. The increased levels of ceramide were accompanied by a significantly increased secretion of granulocyte-macrophage colony-stimulating factor as well as by a significantly down-regulated expression of acid-ceramidase at both the gene and protein levels. Taken together, our results underscore the superiority of this reconstructed human epidermal keratinization model to analyze factors that regulate ceramide synthesis, especially in human SC.
School of Bioscience and Biotechnology, Tokyo University of Technology, 1404-1 Katakura, Hachioji, Tokyo, 192-0982, Japan.
This article was published in the following journal.
Name: Archives of dermatological research
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