A reconstructed human epidermal keratinization culture model to characterize ceramide metabolism in the stratum corneum

Abstract

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.

Th1 cytokines accentuate but Th2 cytokines attenuate ceramide production in the stratum corneum of human epidermal equivalents: An implication for the disrupted barrier mechanism in atopic dermatitis

“…The most striking discrepancy occurs for the expression of aSMase following treatment with IL-4 where the protein and the enzymatic activity levels are significantly up-regulated despite the fact that aSMase gene expression is significantly down-regulated by IL-4. Although the discrepancy between the gene expression and the protein/ activity levels for the mechanism(s) involved in the downregulation of ceramide/protein level in the SC by IL-4 remains to be clarified, the reason why the distinctly increased levels of protein and enzymatic activity does not necessarily contribute to the increase in ceramide levels in the SC seems to be as follows: The inhibition or down-regulation of ceramide synthetic enzymes, such as SMase and BGDase, directly results in the diminished level of ceramides in the SC, leaving sphingomyelin and glucosylceramide as reaction residues in the SC, as seen in Niemann-Pick disease [31] and in Gaucher disease [32], respectively, or an in vivo inhibition study using a BGDase inhibitor [33] and in our study using human epidermal equivalents [23]. The stimulation of those activities does not necessarily reflect the increased level of ceramides in the SC because those enzyme activities occur at levels sufficient to completely degrade sphingomyelin and glucosylceramide without leaving their residues in the SC under the normal physiological conditions of keratinization.…”

“…Comparison of the lipid composition by thin layer chromatography (TLC) shows that the pattern of lipids obtained from the whole SC layers of the 3-dimensional epidermal model is almost identical to that from the 3 SC layers of normal human forearm skin [23], indicating the suitability of the 3-dimensional epidermal model. Our recent study using inhibitors of ceramide-producing enzymes in the same epidermal model indicated that the major metabolic pathways leading to ceramide synthesis through the interface between the SC and the granular layer occur in the 3-dimensional epidermal models in a pattern similar to that seen in vivo [23]. Here we report that the Th2-type of inflammation evoked in AD skin is one of the essential factors in down-regulating the level of ceramides in the SC.…”

“…To precisely elucidate the effects of the Th1/Th2 cytokine balance on ceramide levels in the SC as well as on the expression of ceramide metabolic enzymes (CME) at the gene, protein and enzymatic activity levels in the epidermis, we developed an advanced system in which 3-dimensional epidermal sheets (consisting of multi-layered foreskin-derived human keratinocytes) without the SC layers under pre-keratinizing conditions were cultured for 1 week to generate complete SC layers, which could then be separated and ceramides extracted and quantified as per SC protein [23]. Comparison of the lipid composition by thin layer chromatography (TLC) shows that the pattern of lipids obtained from the whole SC layers of the 3-dimensional epidermal model is almost identical to that from the 3 SC layers of normal human forearm skin [23], indicating the suitability of the 3-dimensional epidermal model.…”

Disruption and Destabilization of Meibomian Lipid Films Caused by Increasing Amounts of Ceramides and Cholesterol

“…31 The epidermis, especially the stratified squamous layer, is producing and metabolizing Cer actively. 32 In those tissues where keratinization occurs (such as stratum corneum), Cer form massive intra-and extracellular lamellar membranes and bodies that, together with keratin, FC, and free fatty acids, create a water-permeability barrier. It seems plausible that the excessive amounts of Cer overproduced by abnormal epithelial cells could mix with normal meibomian gland secretions and change its lipid balance in the process that can be exacerbated by the diminishing capacity of acinar cells to produce normal lipids.…”

Current Understanding of Skin Biology Pertinent to Skin Penetration: Skin Biochemistry

“…In the conversion of lamellar granule-associated acylglucosylceramides to covalently bound ω-hydroxyceramides on the outer surface of the cornified envelope, 3 types of enzymes have been implicated - glucocerebrosidase [71,72,73], lipoxygenases [43] and transglutaminase [44]. How are the actions of these enzymes coordinated and regulated?…”