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.
Project description:Eye lenses of various diurnal geckos contain up to 12% iota-crystallin. This protein is related to cellular retinol-binding protein type I (CRBP I) but has 3,4-didehydroretinol, rather than retinol, as a ligand. The 3,4-didehydroretinol gives the lens a yellow color, thus protecting the retina by absorbing short-wave radiation. iota-Crystallin could be either the gecko's housekeeping CRBP I, recruited for an additional function in the lens, or the specialized product of a duplicated CRBP I gene. The finding of the same CRBP I-like sequence in lens and liver cDNA of the gecko Lygodactylus picturatus now supports the former option. Comparison with iota-crystallin of a distantly related gecko, Gonatodes vittatus, and with mammalian CRBP I, suggests that acquiring the additional lens function is associated with increased amino acid changes. Compared with the rat CRBP I structure, the iota-crystallin model shows reduced negative surface charge, which might facilitate the required tight protein packing in the lens. Other changes may provide increased stability, advantageous for a long-living lens protein, without frustrating its role as retinol transporter outside the lens. Despite a number of replacements in the ligand pocket, recombinant iota-crystallin binds 3,4-didehydroretinol and retinol with similar and high affinity (approximately 1.6 nM). Availability of ligand thus determines whether it binds 3,4-didehydroretinol, as in the lens, or retinol, in other tissues. iota-Crystallin presents a striking example of exploiting the potential of an existing gene without prior duplication.
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:Metabolism of vitamin A, all-trans-retinol, leads to the formation of 11-cis-retinaldehyde, the visual chromophore, and all-trans-retinoic acid, which is involved in the regulation of gene expression through the retinoic acid receptor. Enzymes and binding proteins involved in retinoid metabolism are highly conserved across species. We previously described a novel mammalian enzyme that saturates the 13-14 double bond of all-trans-retinol to produce all-trans-13,14-dihydroretinol, which then follows the same metabolic fate as that of all-trans-retinol. Specifically, all-trans-13,14-dihydroretinol is transiently oxidized to all-trans-13,14-dihydroretinoic acid before being oxidized further by Cyp26 enzymes. Here, we report the identification of two putative RetSat homologues in zebrafish, one of which, zebrafish RetSat A (zRetSat A), also had retinol saturase activity, whereas zebrafish RetSat B (zRetSat B) was inactive under similar conditions. Unlike mouse RetSat (mRetSat), zRetSat A had an altered bond specificity saturating either the 13-14 or 7-8 double bonds of all-trans-retinol to produce either all-trans-13,14-dihydroretinol or all-trans-7,8-dihydroretinol, respectively. zRetSat A also saturated the 13-14 or 7-8 double bonds of all-trans-3,4-didehydroretinol (vitamin A2), a second endogenous form of vitamin A in zebrafish. The dual enzymatic activity of zRetSat A displays a newly acquired specificity for the 13-14 double bond retained in higher vertebrates and also the evolutionarily preserved activity of bacterial phytoene desaturases and plant carotenoid isomerases. Expression of zRetSat A was restricted to the liver and intestine of hatchlings and adult zebrafish, whereas zRetSat B was expressed in the same tissues but at earlier developmental stages. Exogenous all-trans-retinol, all-trans-13,14-dihydroretinol, or all-trans-7,8-dihydroretinol led to the strong induction of the expression of the retinoic acid-metabolizing enzyme, Cyp26A1, arguing for an active signaling function of dihydroretinoid metabolites in zebrafish. These findings point to a conserved function but altered specificity of RetSat in vertebrates, leading to the generation of various dihydroretinoid compounds, some of which could have signaling functions.
Project description:Recently, zebrafish and human cytochrome P450 (P450) 27C1 enzymes have been shown to be retinoid 3,4-desaturases. The enzyme is unusual among mammalian P450s in that the predominant oxidation is a desaturation and in that hydroxylation represents only a minor pathway. We show by proteomic analysis that P450 27C1 is localized to human skin, with two proteins of different sizes present, one being a cleavage product of the full-length form. P450 27C1 oxidized all-trans-retinol to 3,4-dehydroretinol, 4-hydroxy (OH) retinol, and 3-OH retinol in a 100:3:2 ratio. Neither 3-OH nor 4-OH retinol was an intermediate in desaturation. No kinetic burst was observed in the steady state; neither the rate of substrate binding nor product release was rate-limiting. Ferric P450 27C1 reduction by adrenodoxin was 3-fold faster in the presence of the substrate and was ?5-fold faster than the overall turnover. Kinetic isotope effects of 1.5-2.3 (on kcat/Km ) were observed with 3,3-, 4,4-, and 3,3,4,4-deuterated retinol. Deuteration at C-4 produced a 4-fold increase in 3-hydroxylation due to metabolic switching, with no observable effect on 4-hydroxylation. Deuteration at C-3 produced a strong kinetic isotope effect for 3-hydroxylation but not 4-hydroxylation. Analysis of the products of deuterated retinol showed a lack of scrambling of a putative allylic radical at C-3 and C-4. We conclude that the most likely catalytic mechanism begins with abstraction of a hydrogen atom from C-4 (or possibly C-3) initiating the desaturation pathway, followed by a sequential abstraction of a hydrogen atom or proton-coupled electron transfer. Adrenodoxin reduction and hydrogen abstraction both contribute to rate limitation.
Project description:Retinoids are known to affect skin cell proliferation and differentiation and are key molecules that target retinoid and retinoic acid receptors (RXRs and RARs), leading to physiological and pharmacologic effects. Our aim was to elucidate the role of the retinol-binding protein receptor STRA6, mediating cellular uptake of retinol, on skin structure and function. Our results indicate that STRA6 is constitutively expressed in human epidermal keratinocytes and dermal fibroblasts and is regulated via RAR/RXR-mediated pathways. HaCaT (Human adult low Calcium high Temperature) cells with stable STRA6 knockdown (STRA6KD) showed increased proliferation. Consistently, human organotypic 3D skin models using stable STRA6KD HaCaT cells showed a significantly thicker epidermis and enhanced expression of activation, differentiation, and proliferation markers. The effects were reversible after treatment with free retinol. Human skin reconstitution employing STRA6KD HaCaT cells leads to massive epithelial thickening under in vivo conditions in SCID mice. We propose that STRA6KD could lead to cellular vitamin A deficiency in keratinocytes. Consequently, STRA6 has a role for regulating retinoid homeostasis and in helping to program signaling that drives proliferation and differentiation of human skin cells. By its influence on hyperproliferation-associated differentiation, STRA6 could also have a role in skin regeneration and could be a target for pharmacological approaches to improve wound healing.
Project description:1. Four major radioactive fractions have been isolated from the livers of vitamin A-deficient rats given [6,7-(14)C(2)]retinoic acid. 2. At least one of these was more potent than retinoic acid and approximately equal to retinol in the growth assay for vitamin A activity. 3. The biologically active material was chromatographically distinct from retinoic acid, retinol and retinal. 4. Alkaline hydrolysis of this material yielded an acidic compound containing all the radioactivity. 5. The methyl ester of the acidic product was unlike the methyl ester of retinoic acid in its chromatographic behaviour. 6. It is suggested that this metabolite may represent the active form of retinol in its growth-supporting role.
Project description:The transport of retinoic acid in plasma was examined in vitamin A-deficient rats maintained on small doses of radioactively labelled retinoic acid. After ultracentrifugation of serum adjusted to density 1.21, most of the radioactivity (83%) was associated with the proteins of density greater than 1.21, and not with the serum lipoproteins. Gel filtration of the labelled serum on Sephadex G-200 showed that the radioactive label was associated with protein in the molecular-weight range of serum albumin. On polyacrylamide-gel electrophoresis almost all of the recovered radioactivity migrated with serum albumin. Similar esults were obtained with serum from a normal control rat given a single oral dose of [(14)C]retinoic acid. These findings indicate that retinoic acid is transported in rat serum bound to serum albumin, and not by retinol-binding protein (the specific transport protein for plasma retinol). Several tissues and the entire remaining carcase of each rat were extracted with ethanol-acetone to determine the tissue distribution of retinoic acid and some of its metabolites. The total recover of radioactive compounds in in the entire body of the rat was about 7-9mug, representing less than 5% or 10% respectively of the total administered label in the two dosage groups studied. The results confirm that retinoic acid is not stored in any tissue. Most of the radioactive material was found in the carcase, rather than in the specific tissues analysed. Two-thirds of the radioactivity in the carcase appeared to represent unchanged retinoic acid. Of the tissues examined, the liver, kidneys and intestine had relatively high concentrations of radioactive compounds, whereas the testes and fat-pads had the lowest concentrations.
Project description:Retinol dehydrogenases catalyze the rate-limiting step in the biosynthesis of retinoic acid, a bioactive lipid molecule that regulates the expression of hundreds of genes by binding to nuclear transcription factors, the retinoic acid receptors. Several enzymes exhibit retinol dehydrogenase activities in vitro; however, their physiological relevance for retinoic acid biosynthesis in vivo remains unclear. Here, we present evidence that two murine epidermal retinol dehydrogenases, short-chain dehydrogenase/reductase family 16C member 5 (SDR16C5) and SDR16C6, contribute to retinoic acid biosynthesis in living cells and are also essential for the oxidation of retinol to retinaldehyde in vivo Mice with targeted knockout of the more catalytically active SDR16C6 enzyme have no obvious phenotype, possibly due to functional redundancy, because Sdr16c5 and Sdr16c6 exhibit an overlapping expression pattern during later developmental stages and in adulthood. Mice that lack both enzymes are viable and fertile but display accelerated hair growth after shaving and also enlarged meibomian glands, consistent with a nearly 80% reduction in the retinol dehydrogenase activities of skin membrane fractions from the Sdr16c5/Sdr16c6 double-knockout mice. The up-regulation of hair-follicle stem cell genes is consistent with reduced retinoic acid signaling in the skin of the double-knockout mice. These results indicate that the retinol dehydrogenase activities of murine SDR16C5 and SDR16C6 enzymes are not critical for survival but are responsible for most of the retinol dehydrogenase activity in skin, essential for the regulation of the hair-follicle cycle, and required for the maintenance of both sebaceous and meibomian glands.
Project description:alpha1-Proteinase inhibitor is a serpin and can inhibit most serine proteinases. The cornea is one of several extrahepatic tissues that synthesizes this inhibitor. In the presence of retinol, corneal alpha1-proteinase inhibitor levels were increased 3.8-fold. The maximal response was achieved 2 h after the addition of retinol (1 microM final concentration) to the culture medium. A similar increase in alpha1-proteinase inhibitor was observed with retinaldehyde (1 nM final concentration). Concentrations of alpha1-proteinase inhibitor in other tested cells (Hep G2, CaCo 2, MCF-7, monocytes and macrophages) remained unchanged in the presence of retinol. Retinoic acid did not affect alpha1-proteinase inhibitor levels in the cornea or the other cells tested. The acute-phase cytokine, interleukin-6, increased alpha1-proteinase inhibitor levels in all tested tissues/cells except the cornea. These results demonstrate that alpha1-proteinase inhibitor levels are controlled differently in the cornea compared with other tissues/cells. alpha1-Proteinase inhibitor is the first protein identified whose levels are regulated by a mechanism supported by retinol and retinaldehyde but not retinoic acid.
Project description:Retinoic acid biosynthesis in vertebrates occurs in two consecutive steps: the oxidation of retinol to retinaldehyde followed by the oxidation of retinaldehyde to retinoic acid. Enzymes of the MDR (medium-chain dehydrogenase/reductase), SDR (short-chain dehydrogenase/reductase) and AKR (aldo-keto reductase) superfamilies have been reported to catalyse the conversion between retinol and retinaldehyde. Estimation of the relative contribution of enzymes of each type was difficult since kinetics were performed with different methodologies, but SDRs would supposedly play a major role because of their low K(m) values, and because they were found to be active with retinol bound to CRBPI (cellular retinol binding protein type I). In the present study we employed detergent-free assays and HPLC-based methodology to characterize side-by-side the retinoid-converting activities of human MDR [ADH (alcohol dehydrogenase) 1B2 and ADH4), SDR (RoDH (retinol dehydrogenase)-4 and RDH11] and AKR (AKR1B1 and AKR1B10) enzymes. Our results demonstrate that none of the enzymes, including the SDR members, are active with CRBPI-bound retinoids, which questions the previously suggested role of CRBPI as a retinol supplier in the retinoic acid synthesis pathway. The members of all three superfamilies exhibit similar and low K(m) values for retinoids (0.12-1.1 microM), whilst they strongly differ in their kcat values, which range from 0.35 min(-1) for AKR1B1 to 302 min(-1) for ADH4. ADHs appear to be more effective retinol dehydrogenases than SDRs because of their higher kcat values, whereas RDH11 and AKR1B10 are efficient retinaldehyde reductases. Cell culture studies support a role for RoDH-4 as a retinol dehydrogenase and for AKR1B1 as a retinaldehyde reductase in vivo.