School of Bioscience and Biotechnology, Tokyo University of Technology, 1404-1 Katakura, Hachioji, Tokyo, 192-0982, Japan.
Arch Dermatol Res. 2012 Sep;304(7):563-77. doi: 10.1007/s00403-012-1232-6. Epub 2012 Mar 27.
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.
为了研究调节人角质层（SC）角蛋白形成过程中神经酰胺产生的因素，我们开发了一种新的重组人表皮角质化模型，其中新的 SC 层在 1 周内形成。UDP-葡萄糖：神经酰胺葡萄糖基转移酶抑制剂 1-苯-2-癸酰氨基-3-吗啉-1-丙烷显著降低了 SC 神经酰胺水平（以每毫克蛋白表示），同时降低了葡萄糖神经酰胺水平。鞘氨醇酶抑制剂盐酸去甲丙咪嗪也显著降低了 SC 神经酰胺水平。同样，β-葡萄糖脑苷脂酶抑制剂 conduritol B 环氧化物也显著下调了 SC 神经酰胺水平，并显著增加了葡萄糖神经酰胺水平。这些结果表明该模型可用于阐明神经酰胺合成调节因子。使用该模型，我们评估了炎症细胞因子白细胞介素-1α（IL-1α）、几种生物活性神经鞘脂和全反式视黄酸（RA）对 SC 中神经酰胺水平的影响。虽然用 10 nM 的 IL-1α处理可显著下调神经酰胺水平，但用 50 µM 的鞘氨醇磷酸胆碱或 10 或 20 µM 的 1-磷酸鞘氨醇处理则明显上调神经酰胺水平。有趣的是，低浓度（10 nM）的 RA 可显著上调神经酰胺水平，而不影响表皮 SC 的形成或角蛋白化相关蛋白的水平。神经酰胺水平的增加伴随着粒细胞-巨噬细胞集落刺激因子的分泌显著增加，以及酸神经酰胺酶的表达在基因和蛋白水平均显著下调。总之，我们的结果强调了这种重组人表皮角质化模型在分析调节神经酰胺合成的因素方面的优越性，特别是在人 SC 中。
