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Biosynthesis of 3,4-didehydroretinol from retinol by human skin keratinocytes in culture.

ABSTRACT: The uptake and metabolism of radiolabelled retinol was studied in cultivated human skin cells. Normal epidermal keratinocytes in primary culture were able to incorporate unbound [11,12-3H]all-trans-retinol from the growth medium and transform it into 3,4-didehydroretinol (dehydroretinol) in a dose- and time-dependent manner. A total of 23% of the radioactive label became cell-associated during a 48-h incubation period when added at 7 nM to differentiated keratinocytes submerged in serum-containing, high-calcium (1.56 mM) culture medium. At that time point, 25-30% of cell-bound radioactive retinol had been converted into dehydroretinol, with no labelled retinal, dehydroretinal, retinoic acid or dehydroretinoic acid being detected in cells or medium. Thus dehydroretinol, which occurs physiologically in mammalian skin tissue in vivo, was identified as the predominant neutral retinol metabolite in cultured keratinocytes using h.p.l.c. and anhydro-derivatization procedures. At least 94% of the product, along with its precursor, was present in the cells in esterified form, with no traces of the compound being secreted into the cell environment. The rate of formation of dehydroretinol from its precursor was significantly lower in keratinocytes grown in serum-free, low-calcium (0.09 mM) culture medium, and in medium pre-incubated with excess unlabelled substrate. Furthermore, the application of 13-cis-retinoic acid (isotretinoin), a therapeutic retinoid drug known to markedly reduce dehydroretinol levels in human skin, blocked the biosynthesis of this metabolite in cultured keratinocytes. The 3,4-dehydrogenation pathway observed in this study could not be shown to operate to any significant extent in cultures of human epidermal melanocytes or dermal fibroblasts, supporting the hypothesis that keratinocytes represent the principal cell type involved in dehydroretinol formation from retinol in human skin.

SUBMITTER: Rollman O

PROVIDER: S-EPMC1134419 | biostudies-other | 1993 Aug

REPOSITORIES: biostudies-other

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Project description:Cultured keratinocytes offer an attractive model for studying the metabolism of retinol in relation to cell differentiation, since the extent of keratinocyte differentiation can be modulated experimentally. The metabolism of retinol and retinal was studied in cytosol fractions prepared from two distinct keratinocyte populations, differentiating and non-differentiated. The enzymic activities were analysed using physiological concentrations of [3H]retinol and [3H]retinal in the presence of cofactors. The products formed were quantified by h.p.l.c. In the population of differentiating keratinocytes, the formation of retinoic acid from retinol occurred at a rate of 4.49 +/- 0.17 pmol/h per mg of protein, but no such conversion was observed in the population of non-differentiated cells. However, when retinal was used as substrate, retinoic acid was formed in both cell populations, at rates of 14.4 pmol/h per mg of protein in non-differentiated and 51.6 pmol/h per mg of protein in differentiating keratinocytes. Using PAGE/radiobinding assay, we demonstrated that retinoic acid formed from retinol was bound in differentiating keratinocytes to endogenous cellular retinoic acid-binding protein (CRABP). Furthermore, retinal was reduced to retinol in the presence of NADH in both differentiating and non-differentiated keratinocytes at a similar rate (8 pmol/h per mg of protein). Although retinal could not be detected under physiological conditions, it was found in significant amounts at pH 8.5-9, which is optimal for enzymic activity. This indicates that in keratinocytes retinal is an intermediate metabolite in retinoic acid formation from retinol. The enzymes catalysing the conversion of retinol into retinoic acid were found to differ from other alcohol and aldehyde dehydrogenases, since the formation of retinoic acid was not significantly affected by specific inhibitors of alcohol metabolism, such as 4-methylpyrazole and disulfiram. Moreover, the cytosol of non-differentiated keratinocytes did not generate retinoic acid from retinol despite showing alcohol dehydrogenase activity. The results suggest that: (1) retinol metabolism in human keratinocytes is different from that of other alcohols, (2) retinal is an intermediate metabolite in the conversion of retinol into retinoic acid, and (3) differentiating keratinocytes rich in CRABP are probably target cells for retinoic acid action.

Project description:Targets of Retinoic Acid (RA) and 3,4-didehydroretinoic acid (ddRA) were identified in primary human epidermal keratinocytes grown in the presence of atRA or ddRA for 4 and 24 hours. Retinoids (natural forms and synthetic derivatives of vitamin A) are used as therapeutic agents for numerous skin diseases such as keratinization disorders (e.g. ichthyoses) and psoriasis. Two endogenous ligands for retinoic acid receptors exist, retinoic acid (atRA) and 3,4-didehydroretinoic acid (ddRA). In primary human epidermal keratinocytes many transcriptional targets for atRA are known, whereas the targets for ddRA are unknown. In an attempt to determine the targets, we compared the effect of atRA and ddRA on transcriptional profiles in undifferentiated and differentiating human primary keratinocytes. First, as expected, many genes were induced or suppressed in response to keratinocyte differentiation. Furthermore, the two retinoids affected substantially more genes in differentiated keratinocytes (more than 350) than in proliferating keratinocytes (20). In differentiating keratinocytes markers of cornification were suppressed suggesting a de-differentiating effect by the two retinoids. When comparing the expression profile of atRA to that of ddRA, no differently regulated genes were found. The array analysis also found that a minor number of miRNAs and a large number of non-coding transcripts were changed during differentiation and in response to the two retinoids. Furthermore, the expression of all, except one, genes known to cause autosomal recessive congenital ichthyosis (ARCI) were found to be induced by differentiation. These results comprehensively document that atRA and ddRA exert similar transcriptional changes in keratinocytes and also add new insights into the molecular mechanism influenced by retinoids in the epidermis. Furthermore, it suggests which ARCI patients could benefit from therapy with retinoids. Proliferating and differentiated keratinocytes were harvested after 4 and 24 hours after treatment with atRA, ddRA and vehicle for RNA extraction and hybridization on Affymetrix microarrays. Triplicate samples were generated for each time point and each treatment.

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Biosynthesis of 3,4-didehydroretinol from retinol by human skin keratinocytes in culture.

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