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• W1972319607 abstract "Retinol and retinyl esters are converted with time to slowly increasing amounts of all-trans retinoic acid (RA) in cultured human keratinocytes. Exogenous RA has been shown to limit retinol oxidation and to increase retinol esterification. Because significant amounts of retinol are present in biologic systems, we examined whether RA and all-trans-retinoyl-β-D-glucuronide (RAG) interact with retinol in exhibiting their activities on HaCaT keratinocytes maintained in a retinoid-free culture system. RA was more potent than RAG and retinol in inducing ultrastructural changes attributed to retinoids, inhibiting cell proliferation as well as enhancing keratin 19 expression. In addition, retinoids were able to induce cellular retinoic acid-binding protein II mRNA levels in the cultures, whereas early RA and late RAG activity was detected. The described biologic effects of RA and RAG were diminished by simultaneous cell exposure to retinol. HaCaT cells quickly metabolized retinol to retinyl esters and consequently to low amounts of RA. RA treatment led to an early high peak of cellular RA followed by reduction to trace amounts. Treatment with RAG resulted in constantly high cellular RAG and low RA levels. Under the combined RA and retinol treatment retinyl esters were increased and RA was reduced in HaCaT cells, whereas extracellular RA levels were similar to those obtained by RA alone. On the other hand, the combination of RAG and retinol resulted in higher extracellular RAG, similar cellular RAG, and lower cellular RA levels than those obtained by RAG alone without any change in retinyl esters. This study demonstrates that retinoid signaling by RA and RAG is attenuated by simultaneous exposure of HaCaT keratinocytes in vitro to retinol. The presence of retinol in the medium alters the rate of RA or RAG metabolism and thus cellular RA concentrations. The intensity of retinoid signal is probably dependent on cellular RA levels. The resulting “antagonism” among retinoids is consistent with the presence of an autoregulatory mechanism in human keratinocytes offering protection against excessive accumulation of cellular RA. Retinol and retinyl esters are converted with time to slowly increasing amounts of all-trans retinoic acid (RA) in cultured human keratinocytes. Exogenous RA has been shown to limit retinol oxidation and to increase retinol esterification. Because significant amounts of retinol are present in biologic systems, we examined whether RA and all-trans-retinoyl-β-D-glucuronide (RAG) interact with retinol in exhibiting their activities on HaCaT keratinocytes maintained in a retinoid-free culture system. RA was more potent than RAG and retinol in inducing ultrastructural changes attributed to retinoids, inhibiting cell proliferation as well as enhancing keratin 19 expression. In addition, retinoids were able to induce cellular retinoic acid-binding protein II mRNA levels in the cultures, whereas early RA and late RAG activity was detected. The described biologic effects of RA and RAG were diminished by simultaneous cell exposure to retinol. HaCaT cells quickly metabolized retinol to retinyl esters and consequently to low amounts of RA. RA treatment led to an early high peak of cellular RA followed by reduction to trace amounts. Treatment with RAG resulted in constantly high cellular RAG and low RA levels. Under the combined RA and retinol treatment retinyl esters were increased and RA was reduced in HaCaT cells, whereas extracellular RA levels were similar to those obtained by RA alone. On the other hand, the combination of RAG and retinol resulted in higher extracellular RAG, similar cellular RAG, and lower cellular RA levels than those obtained by RAG alone without any change in retinyl esters. This study demonstrates that retinoid signaling by RA and RAG is attenuated by simultaneous exposure of HaCaT keratinocytes in vitro to retinol. The presence of retinol in the medium alters the rate of RA or RAG metabolism and thus cellular RA concentrations. The intensity of retinoid signal is probably dependent on cellular RA levels. The resulting “antagonism” among retinoids is consistent with the presence of an autoregulatory mechanism in human keratinocytes offering protection against excessive accumulation of cellular RA. cellular retinoic acid-binding protein II 50% inhibitory concentration all-trans retinoic acid all-trans retinoyl-β-D-glucuronide Human skin requires retinol for regulation of keratinocyte growth and differentiation (Fuchs, 1990Fuchs E. Epidermal keratinization: the bare essentials.J Cell Biol. 1990; 111: 2807-2814Crossref PubMed Scopus (589) Google Scholar;Darmon, 1991Darmon M. Retinoic acid in skin and epithelia.Sem Dev Biol. 1991; 2: 219-228Google Scholar). The biologic activity of retinol is considered to be predominantly exhibited by its natural oxidation product, all-trans retinoic acid (RA) (Kurlandsky et al., 1994Kurlandsky S.B. Xiao J.-H. Duell E.A. Voorhees J.J. Fisher G.J. Biological activity of all-trans-retinol requires metabolic conversion to all-trans-retinoic acid and is mediated through activation of nuclear receptors in human keratinocytes.J Biol Chem. 1994; 269: 32821-32827Abstract Full Text PDF PubMed Google Scholar). Excess retinol is mainly handled by human and mouse epidermis in vivo by ester formation (Kang et al., 1995Kang S. Duell E.A. Fisher G.J. et al.Application of retinol to human skin in vivo induces epidermal hyperplasia and cellular retinoid binding proteins characteristic of retinoic acid but without measurable retinoic acid levels or irritation.J Invest Dermatol. 1995; 105: 549-556Crossref PubMed Scopus (264) Google Scholar;Sass et al., 1996Sass J.O. Didierjean L. Carraux P. Plum C. Nau H. Saurat J.-H. Metabolism of topical retinaldehyde and retinol by mouse skin in vivo: predominant formation of retinyl esters and identification of 14-hydroxy-4, 14-retro-retinol.Exp Dermatol. 1996; 5: 267-271Crossref PubMed Scopus (17) Google Scholar). On the other hand, synthetic RA and other retinoids have been successfully used for treatment of various epidermal disorders (Zouboulis and Orfanos, 1995Zouboulis Ch.C. Orfanos C.E. Retinoide – Zwölf Jahre Wirksamkeit und Verträglichkeit der systemischen Therapie.in: Tebbe B. Geordt S. Orfanos C.E. Dermatologie Heutiger Stand. Thieme, Stuttgart, New York1995: 301-308Google Scholar;Orfanos et al., 1997Orfanos C.E. Zouboulis Ch.C. Almond-Roesler B. Geilen C.C. Current use and future potential role of retinoids in dermatology.Drugs. 1997; 53: 358-388Crossref PubMed Scopus (306) Google Scholar). RA binds to and induces cellular retinoic acid-binding protein II (CRABP II) as well as binds to and activates nuclear retinoic acid receptors (Giguère, 1994Giguère V. Retinoic acid receptors and cellular retinoid binding proteins: complex interplay in retinoid signaling.Endocrine Rev. 1994; 15: 61-79Crossref PubMed Google Scholar;Ross and Gardner, 1994Ross A.C. Gardner E.M. The function of vitamin A in cellular growth and differentiation, and its roles during pregnancy and lactation.Adv Exp Med Biol. 1994; 352: 187-200Crossref PubMed Scopus (91) Google Scholar). Most actions of RA are now recognized to be mediated through activation of retinoic acid receptors. Epidermal keratinocytes in vivo probably regulate their RA levels by induction of RA 4-hydroxylase (Duell et al., 1992Duell E.A. Åström A. Griffiths C.E.M. Chambon P. Voorhees J.J. Human skin levels of retinoic acid and cytochrome P-450-derived 4-hydroxyretinoic acid after topical application of retinoic acid in vivo compared to concentrations required to stimulate retinoic acid receptor-mediated transcription.In Vitro. J Clin Invest. 1992; 90: 1269-1274Crossref PubMed Scopus (109) Google Scholar). RA inactivation by 4-hydroxylation prevents RA accumulation in the epidermis during application of retinoids to human skin and leads to cellular RA concentrations of 20 nM or lower (Kang et al., 1995Kang S. Duell E.A. Fisher G.J. et al.Application of retinol to human skin in vivo induces epidermal hyperplasia and cellular retinoid binding proteins characteristic of retinoic acid but without measurable retinoic acid levels or irritation.J Invest Dermatol. 1995; 105: 549-556Crossref PubMed Scopus (264) Google Scholar;Duell et al., 1996Duell E.A. Kang S. Voorhees J.J. Retinoic acid isomers applied to human skin in vivo each induce a 4-hydroxylase that inactivates only trans retinoic acid.J Invest Dermatol. 1996; 106: 316-320Crossref PubMed Scopus (48) Google Scholar), which seem to be optimal for a normal pattern of keratinocyte differentiation (Asselineau et al., 1989Asselineau D. Bernard B.A. Bailly C. Darmon M. Retinoid acid improves epidermal morphogenesis.Dev Biol. 1989; 133: 322-335Crossref PubMed Scopus (191) Google Scholar). Recent work showed that retinol, retinaldehyde, 9-cis retinoic acid, and 13-cis retinoic acid are not able to regulate their own hydroxylation and that only RA induces its own inactivation by RA 4-hydroxylase (Duell et al., 1996Duell E.A. Kang S. Voorhees J.J. Retinoic acid isomers applied to human skin in vivo each induce a 4-hydroxylase that inactivates only trans retinoic acid.J Invest Dermatol. 1996; 106: 316-320Crossref PubMed Scopus (48) Google Scholar). In contrast to the findings on human skin in vivo, RA metabolism in human keratinocytes maintained in serum-containing cultures is robust and is not further induced by the addition of exogenous RA (Randolph and Simon, 1997Randolph R.K. Simon M. Metabolism of all-trans-retinoic acid by cultured human epidermal keratinocytes.J Lipid Res. 1997; 38: 1374-1383Abstract Full Text PDF PubMed Google Scholar). This extensive RA metabolism proceeds without detectable levels of 4-hydroxy RA. It is still unknown whether this metabolic pathway, i.e., multiple sequential enzymatic reactions, involves 4-hydroxylase activity, but retinol and retinyl esters can be converted with time to slowly increasing amounts of RA (Randolph and Simon, 1993Randolph R.K. Simon M. Characterization of retinol metabolism in cultured human epidermal keratinocytes.J Biol Chem. 1993; 268: 9198-9205Abstract Full Text PDF PubMed Google Scholar;Kurlandsky et al., 1994Kurlandsky S.B. Xiao J.-H. Duell E.A. Voorhees J.J. Fisher G.J. Biological activity of all-trans-retinol requires metabolic conversion to all-trans-retinoic acid and is mediated through activation of nuclear receptors in human keratinocytes.J Biol Chem. 1994; 269: 32821-32827Abstract Full Text PDF PubMed Google Scholar). In addition, significant amounts of retinol are present in biologic systems (Giguère, 1994Giguère V. Retinoic acid receptors and cellular retinoid binding proteins: complex interplay in retinoid signaling.Endocrine Rev. 1994; 15: 61-79Crossref PubMed Google Scholar). Therefore, the effects of exogenous and endogenous RA on human keratinocytes in vitro can be hardly discriminated in common culture systems. In this study we developed a simple retinoid-free culture system for epithelial cells to examine whether RA and all-trans retinoyl-β-D-glucuronide (RAG), a natural RA derivative (Dunagin et al., 1965Dunagin Jr, P.E. Meadows Jr, E.H. Olson J.A. Retinoyl beta-glucuronic acid: a major metabolite of vitamin A in rat bile.Science. 1965; 148: 86-87Crossref PubMed Scopus (56) Google Scholar), may interact with retinol in exhibiting their activities on human keratinocytes in vitro. Bovine serum albumin (BSA) was shown to be a key molecule in this model. The spontaneously immortalized, nontumorigenic human HaCaT keratinocytes were used instead of normal human keratinocytes in our studies because HaCaT cells (i) could be maintained in a defined retinoid-free culture system described below providing reproducible results, and (ii) present a similar differentiation pattern to normal keratinocytes (Schürer et al., 1993Schürer N. Kohne A. Schliep V. Barlag K. Goerz G. Lipid composition and synthesis of HaCaT cells, an immortalized human keratinocyte line, in comparison with normal human adult keratinocytes.Exp Dermatol. 1993; 2: 179-185Crossref PubMed Scopus (64) Google Scholar) and have been successfully used in several pharmacologic studies in vitro (Jeffes et al., 1995Jeffes 3rd, E.W. McCullough J.L. Pittelkow M.R. et al.Methotrexate therapy of psoriasis: differential sensitivity of proliferating lymphoid and epithelial cells to the cytotoxic and growth-inhibitory effects of methotrexate.J Invest Dermatol. 1995; 104: 183-188Crossref PubMed Scopus (118) Google Scholar;Lehmann, 1997Lehmann B. HaCaT cell line as a model system for vitamin D3 metabolism in human skin.J Invest Dermatol. 1997; 108: 78-82Abstract Full Text PDF PubMed Scopus (75) Google Scholar;Stein et al., 1997Stein M. Bernd A. Ramirez-Bosca A. Kippenberger S. Holzmann H. Measurement of anti-inflammatory effects of glucocorticoids on human keratinocytes in vitro. Comparison of normal human keratinocytes with the keratinocyte cell line HaCaT.Arzneimittelforschung. 1997; 47: 1266-1270PubMed Google Scholar). We found that the biologic effects of RA and of RAG on human keratinocytes in vitro are diminished by simultaneous exposure of the cells to retinol. Enhanced or decreased activity of retinoids was associated with increased or reduced cellular RA, respectively. Moreover, we demonstrated that retinoids can induce CRABP II mRNA in retinoid-free cultured human keratinocytes. The spontaneously immortalized, nontumorigenic human keratinocyte line HaCaT was kindly provided by Prof. N.E. Fusenig and Dr. D. Breitkreutz (German Cancer Research Center, Heidelberg, Germany) (Boukamp et al., 1988Boukamp P. Petrussevska R.T. Breitkreutz D. Hornung J. Markham A. Fusenig N.E. Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line.J Cell Biol. 1988; 106: 761-778Crossref PubMed Scopus (3464) Google Scholar) and was cultured in Dulbecco’s modified Eagle’s (DME)/Ham’s F 12 medium (1:1; Gibco, Berlin, Germany) supplemented with 10% fetal calf serum, 1.4 mM L-glutamine, 100 IU penicillin per ml, and 100 μg streptomycin per ml (all Seromed, Berlin, Germany) with 5% CO2 at 37°C. For all studies passages between 50 and 100 were used. Before performing the experiments, HaCaT cells have been accustomed to diminished fetal calf serum concentrations and have been finally subcultivated five times in serum-free DME/Ham’s F 12 medium only supplemented with L-glutamine and antibiotics. BSA (Boehringer, Mannheim, Germany) was dissolved in serum-free DME/Ham’s F 12 medium to give concentrations from 0.16 to 40 mg per ml. Retinol (Sigma, Deisenhofen, Germany) was dissolved in dimethyl sulfoxide (DMSO; Merck, Darmstadt, Germany) and subsequently in serum-free DME/Ham’s F 12 medium with or without BSA (1 mg per ml), giving final concentrations from 10–13 to 10–5 M retinol and 0.2% DMSO. RA (gift of Hoffmann-La Roche, Basel, Switzerland) and RAG were dissolved in DMSO and subsequently in serum-free DME/Ham’s F 12 medium supplemented with 1 mg BSA per ml, giving final retinoid concentrations from 10–12 to 10–6 M and 0.2% DMSO. RAG was synthesized following a modification (Foerster et al., 1996Foerster M. Sass J.O. Rühl R. Nau H. Comparative studies on effects of all-trans-retinoic acid and all-trans-retinoyl-β-D-glucuronide on the development of foetal mouse thymus in an organ culture system..Toxicol in Vitro. 1996; 10: 7-15Crossref PubMed Scopus (11) Google Scholar) of the method described byBarua and Olson, 1989Barua A.B. Olson J.A. Chemical synthesis of all-trans-[11–3H]retinoyl-β-glucuronide and its metabolites in rats.In Vivo. Biochem J. 1989; 263: 403-409Crossref PubMed Scopus (35) Google Scholar. All chemicals for RAG synthesis were a gift of BASF AG (Ludwigshafen, Germany). 0.2% DMSO alone served as control. Retinoids were handled under dim amber light. The morphology of HaCaT cells was evaluated by inverted photomicroscopy and electron microscopy. For transmission electron microscopic examination, HaCaT cell cultures were grown to confluence on plastic Lux Thermanox cover slips (Miles Laboratories, Naperville, IL) and were processed using standard techniques (Detmar et al., 1989Detmar M. Mayer-da-Silva A. Stadler R. Orfanos C.E. Effects of azelaic acid on proliferation and ultrastructure of mouse keratinocytes in vitro.J Invest Dermatol. 1989; 93: 70-74Abstract Full Text PDF PubMed Google Scholar). HaCaT cells were maintained in 96 well culture plates (Falcon, Jersey, NJ). Cell proliferation was assessed by the 4-methylumbelliferyl heptanoate fluorescence assay and measured automatically, as described before (Stadler et al., 1989Stadler R. Detmar M. Stephanek K. Bangemann C. Orfanos C.E. A rapid fluorometric assay for the determination of keratinocyte proliferation in vitro.J Invest Dermatol. 1989; 93: 532-534Abstract Full Text PDF PubMed Google Scholar;Zouboulis et al., 1991Zouboulis Ch.C. Garbe C. Krasagakis K. Krüger S. Orfanos C.E. A fluorometric rapid microassay to identify anti-proliferative compounds for human melanoma cells.In Vitro. Melanoma Res. 1991; 1: 91-95Crossref PubMed Scopus (36) Google Scholar). On the day of evaluation culture medium was removed, the cells were washed twice with phosphate-buffered saline without Ca2+ and Mg2+ (pH 7.2, Seromed), and 100 μl of a 100 μg 4-methylumbelliferyl heptanoate (Serva, Heidelberg, Germany) per ml solution in phosphate-buffered saline were added to each well. The plates were then incubated for 30 min at 37°C and their fluorescence was read on a Titertek Fluoroscan II (Flow, Meckenheim, Germany). The results are given as absolute fluorescence units using 355 nm excitation and 460 nm emission filters. Retinol and its metabolites were analyzed in HaCaT cells and in collected supernatants by a reverse-phase HPLC method according toCollins et al., 1992Collins M.D. Eckhoff C. Chahoud I. Bochert G. Nau H. 4-Methylpyrazole partially ameliorated the teratogenicity of retinol and reduced the metabolic formation of all-trans-retinoic acid in the mouse.Arch Toxicol. 1992; 66: 652-659Crossref PubMed Scopus (92) Google Scholar, which provides a single run analysis of both polar retinoids, i.e., RA, RAG, and their isomers, and nonpolar retinoids, i.e., retinol and retinyl esters. Sample enrichment by solid phase extraction preceded HPLC analysis with the exception that butylated hydroxytoluene was omitted. If below 100 mg, the weight of cell samples was adjusted to this value by addition of water. One hundred milligrams cell preparation were homogenized in 300 μl isopropanol (Merck), using a Branson B-12 sonifier (Danbury, CT). In contrast to the described method, only 300 μl of the tissue supernatant were diluted 3-fold with 2% ammonium acetate (Merck) solution and submitted to solid phase extraction. Adjustment of the eluent pH was not necessary for our samples and the internal standard was omitted as done byEckhoff et al., 1990Eckhoff C. Wittfoht W. Nau H. Slikker Jr, W. Characterization of oxidized and glucouronidated metabolites of retinol in monkey plasma by thermospray liquid chromatography/mass spectrometry.Biomed Environ Mass Spectrom. 1990; 19: 428-433Crossref PubMed Scopus (34) Google Scholar. In all experiments, retinoids were identified by comparison with retention times and ratios of absorbance units at the two detection wavelengths (340/356 nm) of reference compounds, which were determined simultaneously with a Shimadzu SPD-10A detector (Kyoto, Japan). Multilinear calibration was performed by analysis of solutions of 5% (wt/vol) BSA in phosphate-buffered saline (Merck), which had been spiked with known amounts of retinoids. Polar reference retinoids were a gift of Hoffmann-La Roche. Retinyl palmitate was purchased from Sigma. Synthesis of retinyl esters was previously described byEckhoff et al., 1989Eckhoff C.H. Löfberg B. Chahoud J. Bochert G. Nau H. Transplacental pharmacokinetics and teratogenicity of a single dose of retinol (vitamin A) during organogenesis in the mouse.Toxicol Letters. 1989; 48: 171-184Crossref PubMed Scopus (49) Google Scholar. Total RNA was isolated from subconfluent HaCaT cells using a commercial kit (RNAzol; WAK-Chemie, Bad Homburg, Germany) and stored at –70°C until use. RNA concentrations were determined by absorbance at 260 nm and verified by nondenaturing agarose gel electrophoresis and ethidium bromide staining (Thompson et al., 1985Thompson C.B. Challoner P.B. Neiman P.E. Groudine M. Levels of c-myc oncogene mRNA are invariant throughout the cell cycle.Nature. 1985; 314: 363-366Crossref PubMed Scopus (247) Google Scholar). Thirty micrograms of total RNA were size-fractionated by electrophoresis in 1% formaldehyde-agarose gels containing 0.2 μg ethidium bromide per ml and transferred to derivatized nylon membranes (GeneScreenPlus; NEN, Dreieich, Germany) (Elder et al., 1990Elder J.T. Tavakkol A. Klein S.B. Zeigler M.E. Wicha M. Voorhees J.J. Protooncogene expression in normal and psoriatic skin.J Invest Dermatol. 1990; 94: 19-25Abstract Full Text PDF PubMed Google Scholar). Complete transfer of RNA was documented by examining the gels under ultraviolet light. The blots were sequentially hybridized against CRABP II and β-actin cDNA probes labeled to a specific activity of 3 × 109 cpm DNA per μg with 32P-dCTP (Amersham, Braunschweig, Germany) by the random priming technique (Feinberg and Vogelstein, 1983Feinberg A.P. Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity.Arch Biochem. 1983; 132: 6-13Crossref Scopus (16654) Google Scholar) at a concentration of 4 × 105 cpm hybridization buffer per ml. The CRABP II probe was a purified insert fragment from human CRABP II cDNA (Åström et al., 1991Åström A. Tavakkol A. Pettersson U. Cromie M. Elder J.T. Voorhees J.J. Molecular cloning of two human cellular retinoic acid-binding proteins (CRABP). Retinoic acid-induced expression of CRABP II but not CRABP-I in adult human skin in vivo and in skin fibroblasts in vitro.J Biol Chem. 1991; 266: 17662-17666Abstract Full Text PDF PubMed Google Scholar), which was kindly provided by Dr. A. Tavakkol (Colgate-Palmolive, Piscataway, NJ). The β-actin probe was a gift of Dr. J. Eberle of our Department of Dermatology. The prehybridization and hybridization procedures are described elsewhere (Åström et al., 1991Åström A. Tavakkol A. Pettersson U. Cromie M. Elder J.T. Voorhees J.J. Molecular cloning of two human cellular retinoic acid-binding proteins (CRABP). Retinoic acid-induced expression of CRABP II but not CRABP-I in adult human skin in vivo and in skin fibroblasts in vitro.J Biol Chem. 1991; 266: 17662-17666Abstract Full Text PDF PubMed Google Scholar). Northern blots were visualized by screen-enhanced autoradiography performed by exposing Kodak X-OMAT AR films (Kodak, Rochester, NJ) to the blots in Kodak X-Omatik Regular cassettes in the presence of intensifying screens (Kodak) at –70°C for 24 h. The films were developed in a Kodak M6B RP X-Omat processor. HaCaT cells were grown to confluency in 100 mm culture dishes (Falcon) prior to treatment with the test substances. The cells were then manually scrapped from the culture dishes, harvested into centrifuge tubes, homogenized, and extracted as described (Mischke and Wild, 1987Mischke D. Wild G. Polymorphic keratins in human epidermis.J Invest Dermatol. 1987; 88: 191-197Abstract Full Text PDF PubMed Google Scholar). The resulting pellet, highly enriched in cytoskeletal proteins, was solubilized in sample buffer. One-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) according toLaemmli, 1970Laemmli U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature. 1970; 27: 680-685Crossref Scopus (207012) Google Scholar was performed as described (Mischke and Wild, 1987Mischke D. Wild G. Polymorphic keratins in human epidermis.J Invest Dermatol. 1987; 88: 191-197Abstract Full Text PDF PubMed Google Scholar). Gels were stained in 0.2% Coomassie blue (Serva-Blue R; Serva). For quantitation of mRNA and keratin levels a computed-assisted image analysis system (Bio-Profil, Vilber Lourmat, Marne La Vallée, France) was used. In northern analysis, the intensity of CRABP II mRNA levels was normalized to β-actin, which was used as control gene, by dividing the signal intensity obtained for CRABP II with that of β-actin. The results of the western analysis obtained by SDS-PAGE were presented as keratin 19/total protein volume, because keratin 19 is known to be upregulated by retinoids (Korge et al., 1990Korge B. Stadler R. Mischke D. Effect of retinoids on hyperproliferation-associated keratins K6 and K16 in cultured human keratinocytes: a quantitative analysis.J Invest Dermatol. 1990; 95: 450-455Abstract Full Text PDF PubMed Google Scholar). The results are presented as mean values, error bars represent SD. In proliferation experiments, each mean value represents six or 10 samples. In northern analysis and in HPLC analysis of retinoid levels in medium, each mean value represents three samples. In western analysis and HPLC studies of cellular retinoids three samples were pooled to give a single value. Statistical comparisons were performed by Student’s t test using a two-tailed hypothesis. Differences were defined as statistically significant at p < 0.05. Assessment of the influence of the seeding density (125–3000 cells per well in a 96 well culture plate) on HaCaT cell proliferation revealed that 2000 cells per well produced an optimum exponential growth for cells grown in medium containing 10% fetal calf serum, 0.8–1 mg BSA per ml, and in serum-free medium (Figure 1a ). In addition, the decreased rate of HaCaT cell proliferation in serum-free medium (26% of the proliferation rate obtained in medium containing 10% fetal calf serum at 96 h) was almost normalized by 0.31–5 mg BSA per ml (Figure 1b). HaCaT cells that became spindle-shaped under serum-free conditions reestablished their epithelial morphology in BSA-supplemented medium with or without retinol, but not in only retinol-supplemented medium (Figure 2 ). Under serum-free conditions retinol was inactive on HaCaT cell proliferation at concentrations of 10–13–10–7 M, only inhibited cell proliferation at 10–6 M, and was toxic for HaCaT cells at 10–5 M (Figure 3 ). In contrast, a dose-dependent inhibition of HaCaT cell proliferation was detected at concentrations of 10–9–10–5 M [14%–82% at 96 h, 50% inhibitory concentration (IC50) ≈ 10–7 M] when retinol has been added in BSA (1 mg per ml)-supplemented medium.Figure 2Morphology of HaCaT cells maintained at different culture conditions. (a) HaCaT cells cultured in serum-free medium exhibited a mostly spindle-shaped morphology. Epithelial morphology was reestablished into 48 h after addition of (b) 10% fetal calf serum, (c) 1 mg BSA per ml, (d) 1 mg BSA per ml + retinol 10–8 M, but not after addition of (e) retinol 10–8 M. Scale bar: 400 μm.View Large Image Figure ViewerDownload (PPT)Figure 3Proliferation of HaCaT cells for 96 h in serum-free medium supplemented with different concentrations of retinol and combined retinol and 1 mg BSA per ml. Medium was renewed every 2 d. Retinol (ROL) added in serum-free medium was inactive at concentrations of 10–13–10–7 M, only inhibited cell proliferation at 10–6 M, and was toxic at 10–5 M. Retinol added in BSA-supplemented medium exhibited a dose-dependent inhibition of HaCaT cell proliferation at concentrations of 10–9–10–5 M (14%–82% at 96 h). The results are presented as mean values of six samples, error bars represent SD. Con., serum-free control; BSA, BSA-supplemented control; dAFU, difference of the AFU values between 96 and 0 h, representing proliferating cells. **p < 0.01;***p < 0.001.View Large Image Figure ViewerDownload (PPT) Confluent HaCaT cell cultures treated for 7 d with retinol 10–8 M, RA 10–10 M, and RAG 10–8 M exhibited shortening and rarefication of desmosomes, formation of numerous microvilli on both the free and the internal cell surfaces, widening of the intercellular spaces and formation of structures reminiscent of canaliculi, when compared with the untreated controls (Figure 4 ). Under RA 10–8 M the observed retinoid effects were magnified: cell membrane retraction and loss of microvilli, remnants of desmosome plaques, and loss of cell-to-cell contact were apparent. The activity of retinoids on the proliferation of HaCaT cells was investigated by two experimental protocols: (i) application of a single retinoid concentration (10–7 M) for increasing duration without medium and retinoid renewal, and (ii) continuous application of retinoids in increasing concentrations with renewal of medium and retinoids every 48 h. After application of retinol for 6, 12, 24, 48, 72, 96, and 120 h without renewal, no change of cell proliferation was found during a period of 120 h (Figure 5 ). In contrast, short treatment with RA over 6 h inhibited cell proliferation for at least 120 h (82% ± 3% inhibition, p < 0.001) in a similar magnitude with RA treatment for 120 h. Combined RA/retinol treatment did not influence the anti-proliferative RA activity (83% ± 2% inhibition). On the other hand, RAG required 120 h to exhibit maximum inhibition of cell proliferation (68% ± 2%), whereas simultaneous RAG/retinol treatment decreased RAG effectivity by 13% (p < 0.001). Under continuous treatment and retinoid renewal every 48 h, RA and RAG showed a narrow concentration range where they developed anti-proliferative effects. RA IC50 was 10–9.5 M and RAG IC50 about 10–8.5 M at 120 h. Simultaneous RA/retinol (10–6 M) and RAG/retinol (10–6 M) treatment exhibited weaker anti-proliferative effects than RA and RAG alone, respectively (RA/ROL IC50 = 10–8 M, p < 0.001; RAG/ROL IC50 > 10–8 M, p < 0." @default.
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• W1972319607 date "1999-02-01" @default.
• W1972319607 modified "2023-10-10" @default.
• W1972319607 title "Retinoid Signaling by all-trans Retinoic Acid and all-trans Retinoyl-β-D-Glucuronide Is Attenuated by Simultaneous Exposure of Human Keratinocytes to Retinol" @default.
• W1972319607 cites W10463414 @default.
• W1972319607 cites W14103738 @default.
• W1972319607 cites W1535301107 @default.
• W1972319607 cites W1592146973 @default.
• W1972319607 cites W1603565203 @default.
• W1972319607 cites W1604444075 @default.
• W1972319607 cites W1967440349 @default.
• W1972319607 cites W1969934702 @default.
• W1972319607 cites W1981602259 @default.
• W1972319607 cites W1983851236 @default.
• W1972319607 cites W1984919477 @default.
• W1972319607 cites W1993899202 @default.
• W1972319607 cites W1995087043 @default.
• W1972319607 cites W1997191582 @default.
• W1972319607 cites W2003262612 @default.
• W1972319607 cites W2005529170 @default.
• W1972319607 cites W2010579819 @default.
• W1972319607 cites W2014315882 @default.
• W1972319607 cites W2017282278 @default.
• W1972319607 cites W2017451939 @default.
• W1972319607 cites W2019736888 @default.
• W1972319607 cites W2024445459 @default.
• W1972319607 cites W2024576761 @default.
• W1972319607 cites W2025236508 @default.
• W1972319607 cites W2025922732 @default.
• W1972319607 cites W2030888198 @default.
• W1972319607 cites W2035105655 @default.
• W1972319607 cites W2035943027 @default.
• W1972319607 cites W2038435300 @default.
• W1972319607 cites W2040507798 @default.
• W1972319607 cites W2041460044 @default.
• W1972319607 cites W2041465863 @default.
• W1972319607 cites W2049038526 @default.
• W1972319607 cites W2051747151 @default.
• W1972319607 cites W2052955310 @default.
• W1972319607 cites W2058497139 @default.
• W1972319607 cites W2061643706 @default.
• W1972319607 cites W2065693137 @default.
• W1972319607 cites W2066851739 @default.
• W1972319607 cites W2067990211 @default.
• W1972319607 cites W2076235025 @default.
• W1972319607 cites W2079122253 @default.
• W1972319607 cites W2081287406 @default.
• W1972319607 cites W2085312769 @default.
• W1972319607 cites W2085776873 @default.
• W1972319607 cites W2086437094 @default.
• W1972319607 cites W2090537316 @default.
• W1972319607 cites W2100837269 @default.
• W1972319607 cites W2143464311 @default.
• W1972319607 cites W2184392777 @default.
• W1972319607 cites W2317386814 @default.
• W1972319607 cites W2339097139 @default.
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