tretinoin and 3-4-didehydroretinoic-acid

Topics: Animals; Morphogenesis; Tretinoin; Vitamin A

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 (>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.

Topics: Adult; Cell Differentiation; Cell Proliferation; Cells, Cultured; Chromosome Disorders; Female; Genes, Recessive; Humans; Ichthyosiform Erythroderma, Congenital; Keratinocytes; Microarray Analysis; Primary Cell Culture; RNA, Messenger; Transcriptional Activation; Transcriptome; Tretinoin

All-trans 3,4-didehydroretinoic acid (at-ddRA) has been identified as a biologically important retinoid in avian, but not mammalian, embryonic development. In this report, we show that at-ddRA, like all-trans retinoic acid (atRA), supports the survival and differentiation of sympathetic neurons of the embryonic chick. Furthermore, the expression of the retinoid-responsive gene RARbeta2 is increased in neurons exposed to either at-ddRA or atRA. The mechanism whereby at-ddRA exerts its effects in chick neurons may involve binding to and activation of nuclear retinoid receptors. For this reason, the binding of recombinant chick RARbeta2 to at-ddRA and to receptor-specific DNA response elements was examined and compared with the binding characteristics of recombinant murine RARbeta2. The chick RARbeta2, like the mammalian RAR, binds to [3H]atRA with high affinity (Kd=0.7-2 nM). Furthermore, both chick and murine RARbeta2 bind equally well to at-ddRA, atRA, and 9-cis RA, but neither receptor shows appreciable binding to 13-cis RA. The chick RARbeta2 recognizes previously described retinoic acid response elements of mammalian gene promoters and, like mammalian RARbeta2, shows enhanced binding in the presence of RXR. This study provides evidence that at-ddRA, like atRA, supports neuronal development in the chick by its interaction with nuclear retinoid receptors.

Topics: Animals; Baculoviridae; Base Sequence; Cell Differentiation; Cell Survival; Chick Embryo; Dose-Response Relationship, Drug; Ganglia, Sympathetic; Ligands; Mice; Molecular Sequence Data; Neurites; Neurons; Receptors, Retinoic Acid; Recombinant Proteins; RNA, Messenger; Species Specificity; Tretinoin

Retinoic acid (RA) is present in the chick limb bud, and excess RA induces limb duplications. Here, we have investigated the role of endogenous RA during chick limb development by preventing the synthesis of RA and testing the effect on various genes expressed during limb initiation and outgrowth.. We demonstrate that the stage 20/21 limb bud synthesizes didehydroretinoic acid (ddRA), and that the posterior half of the limb bud synthesizes ddRA at a higher rate than the anterior half. Disulphiram inhibits this synthesis at micromolar concentrations. Administering disulphiram to embryos prior to limb bud outgrowth (stages 12-18) abolishes outgrowth, and no limb develops in the majority of cases. Disulphiram treatment also prevents the expression of Sonic hedgehog (Shh), but the expression of the fibroblast growth factor-8 gene (Fgf-8) appears as normal in the ectoderm over the prospective limb bud. The application of a bead soaked in RA can rescue Shh expression. Disulphiram treatment of later limb buds (stages 20-23) similarly down-regulates Shh, and also Fgf-4, expression, whereas the expression of Fgf-8, as at earlier stages, is initially unaffected. Again, RA can rescue the expression of Shh in these limb buds.. RA, in conjunction with Fgf-8, may be needed for the induction of the chick limb bud and the induction of Shh and Fgf-4 expression. The expression of Shh and Fgf-4 remains dependent upon the continued synthesis of RA within the limb bud. Didehydroretinoic acid is the major active retinoid in the stage 20 chick limb bud.

Topics: Animals; Chick Embryo; Disulfiram; Extremities; Gene Expression Regulation, Developmental; Tretinoin

This study demonstrates early embryonic metabolism of exogenous all-trans-3,4-didehydroretinol (vitamin A2) to all-trans-3,4-didehydroretinal and to all-trans-3,4-didehydroretinoic acid in Xenopus embryos during neurulation. The latter metabolite was recently shown to bind with high affinity and to activate various retinoic acid receptors. Embryos treated with all-trans-3,4-didehydroretinol during early or late gastrulation exhibited abnormalities along the anteroposterior axis. The abnormalities were primarily in the posterior regions of the embryo, with only minor defects anteriorally. Eye malformations, typical for early exposure to 9-cis- and all-trans-retinols and retinals (companion paper), were not observed. We also present evidence that all-trans-3,4-didehydroretinoic acid is present endogenously during early neurulation and is evenly distributed along the anteroposterior axis. After treatment with all-trans-3,4-didehydroretinol, embryonic levels of all-trans-3,4-didehydroretinoic acid exceeded endogenous levels of this metabolite during early and late neurulation. We hypothesize that the dysmorphogenic effects produced by treatment of Xenopus embryos with the alcohol precursor, all-trans-3,4-didehydroretinol, are the result of its embryonic conversion to its corresponding acid ligand.

Topics: Abnormalities, Drug-Induced; Animals; Chromatography, High Pressure Liquid; Embryo, Nonmammalian; Female; Phenotype; Teratogens; Tretinoin; Vitamin A; Xenopus laevis

Retinol metabolism was examined in cultured human epidermal keratinocytes incubated with medium containing physiological concentrations of bovine serum albumin-free fatty acid complexes and [3H]retinol. Unsaturated 16- and 18-carbon fatty acids: (1) promoted up to a 50% increase in the steady state total cell retinoid mass which was accounted for as retinyl ester corresponding to the added fatty acid, (2) decreased the rate of endogenous retinyl ester utilization up to 80%, (3) decreased the steady state cellular concentrations of retinol, 3,4-didehydroretinol and their respective carboxylic acids up to 80%, and (4) did not alter the rate of retinoic acid metabolism. Increasing the medium retinol concentration augmented the fatty acid-stimulated increase in esters and blunted the extent of decrease in alcohols and acids. Saturated fatty acids with 14, 16, 17, and 18 carbons also reduced the rate of retinyl ester utilization and the cellular concentrations of the retinoid alcohols and acids but had little to no effect on retinyl ester synthesis. The data demonstrate that exogenous fatty acids possess the capacity to regulate the cellular concentrations of substrate retinoid alcohols, active retinoid acids, and the overall rate of retinol metabolism via substrate-mediated stimulation of retinol esterification and decreasing retinyl ester utilization. Fatty acids thus possess the potential to play a physiological role in modulating retinoid signaling in keratinocytes by regulating cellular concentrations of active retinoids.

Topics: Cells, Cultured; Culture Media; Esterification; Fatty Acids; Humans; Keratinocytes; Oleic Acid; Oleic Acids; Retinoids; Stereoisomerism; Structure-Activity Relationship; Tretinoin; Vitamin A

To determine the prevalence of subclinical vitamin A deficiency (vitamin A inadequacy) in Indonesian pregnant women as assessed by the modified relative dose response test.. Cross-sectional study of the vitamin A statuses of pregnant (second trimester) women randomly selected from ten different villages.. West Java, Indonesia.. A group of 144 pregnant women recruited from the local health posts.. Modified relative dose response (MRDR) test, serum retinol determination and gynecological examinations.. The mean (s.d.) MRDR ratio was 0.039 +/- 0.031. Of the women tested, the vitamin A status of 17% was provisionally classified as being marginal (subclinically deficient) (MRDR ratio > or = 0.060), of 35% as being uncertain (MRDR ratio between 0.030 and 0.060) and of 48% as being satisfactory (MRDR ratio < or = 0.030).. If the vitamin A statuses of the 'uncertain' group are also deemed to be unsatisfactory, approximately half of the pregnant Indonesian women tested could benefit from an increased intake of vitamin A.

Topics: Adolescent; Adult; Cross-Sectional Studies; Dose-Response Relationship, Drug; Female; Humans; Indonesia; Nutrition Surveys; Nutritional Status; Pregnancy; Pregnancy Complications; Prevalence; Tretinoin; Vitamin A; Vitamin A Deficiency

The biologic activities of retinoic acid and 3,4-didehydroretinoic acid, two endogenous vitamin A derivatives in various tissues, were compared to their affinities for the nuclear retinoic acid receptors and their ability to induce transcriptional activation. Both retinoids were equipotent inducers of differentiation of F9 teratocarcinoma cells. In a morphologic assay, using reconstructed skin, retinoic acid and 3,4-didehydroretinoic acid inhibited keratinization at a concentration of 100 nM. In cultured keratinocytes, a 50% inhibition of the production of the keratinocyte transglutaminase enzyme was achieved with about 20 nM for both retinoids. The in vitro binding to the nuclear retinoic acid receptors alpha, beta, and gamma showed that retinoic acid and 3,4-didehydroretinoic acid had almost equal affinities for the receptors with Kds ranging from 3 to 47 nM. The transcriptional activation resulting from the addition of the two retinoids to cells co-transfected with alpha, beta, or gamma retinoic acid receptor expression vectors and a retinoic acid responsive element linked to the chloramphenicol acetyltransferase reporter gene was similar. Finally, it was demonstrated that retinoic acid did not metabolize to 3,4-didehydroretinoic acid, and a slow conversion of 3,4-didehydroretinoic acid into retinoic acid was not sufficient to explain the biologic effects produced by the former compound. In conclusion, the present study demonstrates that retinoic acid and 3,4-didehydroretinoic acid have the same activity in several different test systems, but their metabolism differs depending on the cell type used.

Topics: Cells, Cultured; Culture Media, Serum-Free; Epidermis; Humans; Keratinocytes; Keratins; Morphogenesis; Receptors, Retinoic Acid; Transcription, Genetic; Transcriptional Activation; Transglutaminases; Tretinoin

Topical all-trans retinoic acid (RA) modulates growth and differentiation of skin and is used in the treatment of various dermatological disorders. RA is metabolized to 4-hydroxy RA, 4-oxo RA and 5,6-epoxy RA, which are believed to be markedly less active than RA. 3,4-didehydroretinoic acid (ddRA) is a metabolite of 3,4-didehydroretinol which is present in skin. ddRA is biologically active and acts as a morphogen. We have determined the relative biological activity of ddRA, 4-hydroxy RA, 4-oxo RA and 5,6-epoxy RA as assessed by three retinoid responsive systems relevant to skin. RA, ddRA, 4-hydroxy RA, 4-oxo RA and 5,6-epoxy RA (10-100 nM) reduced epidermal transglutaminase activity in human keratinocytes to similar extents, and inhibited alpha-melanocyte-stimulating hormone-isobutylmethylxanthine-inducible tyrosinase activity in Cloudman S-91 mouse melanoma cells by 67, 39, 48, 51 and 19%, respectively, at 100 nM. Daily topical application of the retinoids to hairless mouse skin for 4 days resulted in dose-dependent changes in epidermal thickness and global histological score. The relative potencies of RA, ddRA, 4-hydroxy RA, 4-oxo RA and 5,6-epoxy RA, as calculated by parallel line assay, were 1.0, 0.60, 0.34, 0.29 and 0.18, respectively, for epidermal hyperplasia and 1.0, 0.78, 0.23, 0.14 and 0.08, respectively, for global histological score. Interestingly, the compounds exhibited a similar rank order of potency with respect to induction of cellular retinoic acid binding protein-II mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adult; Animals; Carrier Proteins; Cells, Cultured; Epidermis; Humans; Keratinocytes; Male; Melanoma, Experimental; Mice; Mice, Hairless; Monophenol Monooxygenase; Receptors, Retinoic Acid; RNA, Messenger; Skin; Transglutaminases; Tretinoin; Tumor Cells, Cultured

The expression of mRNA for retinoic acid receptor beta (RAR-beta) was induced by all trans-retinoic acid in murine S91 melanoma cells. The induction of RAR-beta was dose-dependent, rapid and insensitive to cycloheximide. Both 13-cis-retinoic acid and 3,4-didehydro-all trans-retinoic acid also induced expression of RAR-beta but were only effective at concentrations 100-fold greater than all trans-retinoic acid. The expression of RAR-alpha and RAR-gamma was unaffected by retinoic acid.

Topics: Animals; Carrier Proteins; Cell Line; Cycloheximide; Gene Expression; Kinetics; Melanoma, Experimental; Mice; Receptors, Retinoic Acid; RNA, Messenger; Stereoisomerism; Tretinoin

The modified relative-dose-response (MRDR) assay has been validated in rats as a function of vitamin A status and tested in a group of American children. In this study the MRDR assay was applied to West Javan children who are at risk of being vitamin A deficient. Of 86 children enrolled, 75 were tested. In a time-course study involving 22 children aged 3.7-5.3 y, blood samples were taken at different times after doses of 0.35 mumol 3,4-didehydroretinyl acetate/kg body wt. Generally, the ratio of dehydroretinol to retinol (DR-R ratio) peaked between 4 and 8 h. Thereafter, in a survey of 53 children aged 0.6-4.8 y, single blood samples were drawn 5 h after the dehydroretinyl acetate dose. The DR-R ratio ranged from 0.0028 to 0.169. With a DR-R ratio of 0.03 as the cutoff value, 62% of the children were judged to be of marginal vitamin A status.

Topics: Anthropometry; Child, Preschool; Dose-Response Relationship, Drug; Humans; Indonesia; Infant; Nutritional Status; Time Factors; Tretinoin; Vitamin A; Vitamin A Deficiency

The relative-dose-response (RDR) assay first proposed by Underwood has proved to be a useful indicator of marginal vitamin A status. We suggested that 3,4-didehydroretinol might be used in a simpler assay that requires only one blood sample for analysis. In the present study 24 healthy children aged 3.7-7.1 y were given 100 micrograms 3,4-didehydroretinyl acetate/kg body wt in corn oil, followed by ice cream (90 mL). A blood sample was taken 2, 4, 5, 6, 8, 10, or 24 h after the dose. The mean ratio of dehydroretinol to retinaol (DR-R ratio) plateaued at approximately 0.02 in the plasma between 4 and 10 h. Only three children showed ratios greater than 0.03. Upon reinvestigation, the DR-R ratio remained greater than 0.03 in one child at 5 h. After vitamin A treatment the ratio decreased to 0.019. Thus, a tentative DR-R cutoff ratio for a satisfactory vitamin A status in healthy American children is 0.03.

Topics: Child; Child, Preschool; Dose-Response Relationship, Drug; Female; Humans; Iowa; Male; Nutritional Status; Reference Values; Tretinoin; Vitamin A

There is increasing evidence that retinoic acid is a morphogen involved in vertebrate development. This evidence comes in part from studies of the chick wing bud, in which local application of all-trans-retinoic acid results in a duplication of the digit pattern along the anteroposterior axis. Retinoic acid may be only one of several morphogenetic signalling compounds required for limb pattern formation. To identify novel morphogenetically active compounds, fractionated extracts of whole chick embryos were tested for their ability to induce digit pattern duplications. We describe here the isolation of a new activity present in the limb bud, which we have identified as all-trans-3,4-didehydroretinoic acid. The 3,4-didehydroretinoic acid is generated in situ from retinol through a 3,4-didehydroretinol intermediate. We show that 3,4-didehydroretinoic acid and retinoic acid are equipotent in evoking digit duplications. These findings suggest that there are at least two endogenous retinoids with morphogenetic properties in the chick limb.

Topics: Animals; Chick Embryo; Chromatography, High Pressure Liquid; Gas Chromatography-Mass Spectrometry; Morphogenesis; Retinoids; Tretinoin; Wings, Animal