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• W2001238939 abstract "Tissue levels of all-trans retinoic acid (RA) are maintained through coordinated regulation of biosynthesis and breakdown. The major pathway for all-trans RA inactivation is initiated by 4-hydroxylation. A novel cytochrome P-450 (CYP26) that catalyzes 4-hydroxylation of all-trans RA has recently been cloned. We have investigated regulation and properties of RA 4-hydroxylase in immortalized human keratinocyte HaCaT cells. In the absence of added retinoid, RA 4-hydroxylase (CYP26) mRNA and protein were minimally detected. Addition of all-trans RA rapidly induced RA 4-hydroxylase mRNA (within 2 h) and activity (within 6 h). Induction of both mRNA and activity was transient, returning to baseline within 48 h, and completely dependent on mRNA synthesis (i.e., blocked by actinomycin D). The synthetic retinoid CD367, which specifically activates nuclear RA receptors, also rapidly induced RA 4-hydroxylase activity. This induction, however, unlike that of all-trans RA, was long-lived (>48 h). This difference was attributable to lack of metabolic inactivation of CD367 in HaCaT cells. CD2665, which inhibits RA receptor-dependent gene transcription, blocked retinoid induction of RA 4-hydroxylase, indicating that it is mediated by RA receptors. Addition of excess unlabeled substrates specific for 10 distinct mammalian P-450 subfamilies did not compete with all-trans RA for RA 4-hydroxylase activity. RA 4-hydroxylase did not hydroxylate 9-cis RA or 13-cis RA. Inhibition of RA 4-hydroxylase activity by ketoconazole potentiated activation of RA receptors by all-trans RA. In summary, RA 4-hydroxylase is a unique, highly specific cytochrome P-450 isoenzyme, whose expression is regulated by its natural substrate, all-trans RA, through activation of RA receptors. RA 4-hydroxylase functions to limit the levels, and thereby the biologic activity of all-trans RA in HaCaT cells. Tissue levels of all-trans retinoic acid (RA) are maintained through coordinated regulation of biosynthesis and breakdown. The major pathway for all-trans RA inactivation is initiated by 4-hydroxylation. A novel cytochrome P-450 (CYP26) that catalyzes 4-hydroxylation of all-trans RA has recently been cloned. We have investigated regulation and properties of RA 4-hydroxylase in immortalized human keratinocyte HaCaT cells. In the absence of added retinoid, RA 4-hydroxylase (CYP26) mRNA and protein were minimally detected. Addition of all-trans RA rapidly induced RA 4-hydroxylase mRNA (within 2 h) and activity (within 6 h). Induction of both mRNA and activity was transient, returning to baseline within 48 h, and completely dependent on mRNA synthesis (i.e., blocked by actinomycin D). The synthetic retinoid CD367, which specifically activates nuclear RA receptors, also rapidly induced RA 4-hydroxylase activity. This induction, however, unlike that of all-trans RA, was long-lived (>48 h). This difference was attributable to lack of metabolic inactivation of CD367 in HaCaT cells. CD2665, which inhibits RA receptor-dependent gene transcription, blocked retinoid induction of RA 4-hydroxylase, indicating that it is mediated by RA receptors. Addition of excess unlabeled substrates specific for 10 distinct mammalian P-450 subfamilies did not compete with all-trans RA for RA 4-hydroxylase activity. RA 4-hydroxylase did not hydroxylate 9-cis RA or 13-cis RA. Inhibition of RA 4-hydroxylase activity by ketoconazole potentiated activation of RA receptors by all-trans RA. In summary, RA 4-hydroxylase is a unique, highly specific cytochrome P-450 isoenzyme, whose expression is regulated by its natural substrate, all-trans RA, through activation of RA receptors. RA 4-hydroxylase functions to limit the levels, and thereby the biologic activity of all-trans RA in HaCaT cells. retinoic acid All-trans retinol and its metabolite all-trans retinoic acid (RA) are critical for a number of biologic processes, including cell growth and differentiation, embryogenesis, vision, spermatogenesis, bone formation, hematopoiesis, and programmed cell death (Lotan, 1980Lotan R. Effects of vitamin A and its analogs (retinoids) on normal and neoplastic cells.Biochim Biophys Acta. 1980; 605: 33-91Crossref PubMed Scopus (1041) Google Scholar;Maden, 1994Maden M. Vitamin A in embryonic development.Nurtr Rev. 1994; 52: S3-S12Crossref Scopus (45) Google Scholar;Gudas, 1994Gudas L.J. Retinoids and vertebrate development.J Biol Chem. 1994; 269: 15399-15402Abstract Full Text PDF PubMed Google Scholar). The importance of RA for epithelial cell proliferation and differentiation (Roberts and Sporn, 1984Roberts A.B. Sporn M.B. Cellular biology and biochemistry of the retinoids.in: Sporn MB Roberts AB Goodman DS The Retinoids. 2. Academic Press, New York1984Crossref Google Scholar;Dolle et al., 1990Dolle P. Ruberte E. Leroy P. Morriss-Kay G. Chambon P. Retinoic acid receptors and cellular retinoid binding proteins. I. A systematic study of their differential pattern of transcription during mouse organogenesis.Development. 1990; 110: 1133-1151Crossref PubMed Google Scholar;Lotan, 1994Lotan R. Suppression of squamous cell carcinoma growth and differentiation by retinoids.Cancer Res. 1994; 54: 1987-1990PubMed Google Scholar), repair of photodamaged skin (Weiss et al., 1988Weiss J.S. Ellis C.N. Headington J.T. Tincoff T. Hamilton T.A. Voorhees J.J. Topical tretinoin improves photodamaged skin: a double blind vehicle-controlled study.Jama. 1988; 259: 527-532Crossref PubMed Scopus (459) Google Scholar;Weinstein et al., 1991Weinstein G.D. Nigra T.P. Pochi P.E. et al.Topical tretinoin for treatment of photodamaged skin. A multicenter study.Arch Dermatol. 1991; 127: 659-665Crossref PubMed Scopus (257) Google Scholar), and suppression of malignant transformation (Sporn and Roberts, 1983Sporn M.B. Roberts A.B. Role of retinoids in differentiation and carcinogenesis.Cancer Res. 1983; 43: 3034-3040PubMed Google Scholar;Moon and Mehta, 1990Moon R.C. Mehta R.G. Retinoid inhibition of experimental carcinogenesis.in: Dawson MI Okamura WH Chemistry and Biology of Synthetic Retinoids. CRC Press, Boca Raton1990Google Scholar;Smith et al., 1992Smith M.A. Parkinson D.R. Cheson B.D. Friedman M.A. Retinoic acid in cancer therapy.J Clin Oncol. 1992; 10: 839-864Crossref PubMed Scopus (528) Google Scholar) has fueled the development of retinoid-based therapeutics. All-trans RA and 13-cis RA have been used to treat acne and psoriasis, and, more recently, certain cancers (Sporn and Roberts, 1983Sporn M.B. Roberts A.B. Role of retinoids in differentiation and carcinogenesis.Cancer Res. 1983; 43: 3034-3040PubMed Google Scholar;Orfanos et al., 1987Orfanos C.E. Ehlert R. Gollnick H. The Retinoids A review of their clinical pharmacology and therapeutic use.Drugs. 1987; 34: 459-503Crossref PubMed Scopus (244) Google Scholar;Smith et al., 1992Smith M.A. Parkinson D.R. Cheson B.D. Friedman M.A. Retinoic acid in cancer therapy.J Clin Oncol. 1992; 10: 839-864Crossref PubMed Scopus (528) Google Scholar). RA has also been used to treat acute promyelocytic leukemia. Whereas patients afflicted with acute promyelocytic leukemia exhibit complete remission of symptoms after treatment with all-trans RA in vivo (Huang et al., 1988Huang M.E. Ye Y.C. Chen S.R. et al.Use of all-trans retinoic acid in the treatment of acute promyelocytic leukemia.Blood. 1988; 72: 567-572Crossref PubMed Google Scholar;Norum, 1993Norum K.R. Acute myeloid leukemia and retinoids.Eur J Clin Nutr. 1993; 47: 77-87PubMed Google Scholar), continuous treatment results in the development of progressive resistance to therapy. Relapse may be related to increased metabolism of RA (Muindi et al., 1992Muindi J. Frankel S.R. Miller W.H. et al.Continuous treatment with all-trans retinoic acid causes a progressive reduction in plasma drug concentrations: implications for relapse and retinoid “resistance” in patients with acute promyelocytic leukemia.Blood. 1992; 79: 299-303PubMed Google Scholar;Kizaki et al., 1996Kizaki M. Ueno H. Yamazoe Y. et al.Mechanisms of retinoid resistance in leukemic cells: possible role of cytochrome P-450 and P-glycoprotein.Blood. 1996; 87: 725-733PubMed Google Scholar;White et al., 1996White J.A. Guo Y.D. Baetz K. et al.Identification of the retinoic acid-inducible all-trans-retinoic acid 4-hydroxylase.J Biol Chem. 1996; 271: 29922-29927Crossref PubMed Scopus (316) Google Scholar) as recent research indicates that topical application of all-trans RA to human or rodent skin increases all-trans RA metabolism (Van Wauwe et al., 1988Van Wauwe J.P. Coene M.C. Goossens J. Van Nijen G. Cools W. Lauwers W. Ketoconazole inhibits the in vitro and in vivo metabolism of all-trans-retinoic acid.J Pharmacol Exp Therapeut. 1988; 245: 718-722PubMed Google Scholar;Vanden Bossche et al., 1988Vanden Bossche V. Willemsens G. Janssen P.A. Cytochrome P-450-dependent metabolism of retinoic acid in rat skin microsomes: inhibition by ketoconazole.Skin Pharmacol. 1988; 1: 176-185Crossref PubMed Scopus (47) Google Scholar;Duell et al., 1992Duell E.A. Astrom 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). Cellular all-trans RA levels are stringently regulated through a balance of uptake, synthesis, and catabolism. In human skin, the primary metabolite of all-trans RA is 4-hydroxy all-trans RA, which is further metabolized to 4-oxo all-trans RA (Duell et al., 1992Duell E.A. Astrom 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). 4-hydroxylation of RA appears to be primarily catalyzed by a novel member of the cytochrome P-450 family (CYP26), which has recently been cloned (Ray et al., 1997Ray W.J. Bain G. Yao M. Gottlieb D.I. CYP26, a novel mammalian cytochrome P450, is induced by reinoic acid and defines a new family.J Biol Chem. 1997; 272: 18702-18708Crossref PubMed Scopus (269) Google Scholar;White et al., 1997White J.A. Beckett-Jones B. Guo Y.D. Dilworth F.J. Bonasoro J. Jones G. Petkovich M. cDNA cloning of human retinoic acid-metabolizing enzyme (hP450RAI) identifies a novel family of cytochromes P450 (CYP26).J Biol Chem. 1997; 272: 18538-18541Crossref PubMed Scopus (337) Google Scholar). This enzyme is expressed in many tissues, including kidney, lung, liver (White et al., 1997White J.A. Beckett-Jones B. Guo Y.D. Dilworth F.J. Bonasoro J. Jones G. Petkovich M. cDNA cloning of human retinoic acid-metabolizing enzyme (hP450RAI) identifies a novel family of cytochromes P450 (CYP26).J Biol Chem. 1997; 272: 18538-18541Crossref PubMed Scopus (337) Google Scholar), and skin (Wang and Fisher, unpublished). Whether there exist other specific RA-inducible RA 4-hydroxylase gene products in addition to CYP26 remains to be determined. We have previously described induction of a highly specific RA 4-hydroxylase activity by all-trans RA in human skin in vivo (Duell et al., 1996Duell E.A. Kang S. Voorhees J.J. Retinoic acid isomers applied to human skin in vivo induce a 4-hydroxylase that inactivates only all-trans retinoic acid.J Invest Dermatol. 1996; 106: 316-320Abstract Full Text PDF PubMed Scopus (48) Google Scholar). The objective of this study was to investigate the mechanism of all-trans RA induction of RA 4-hydroxylase activity in cultured human keratinocytes. For these studies, we utilized the immortalized human keratinocyte HaCaT cell line (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 keratinoctye cell line.J Cell Biol. 1988; 106: 761-771Crossref PubMed Scopus (3464) Google Scholar), because early passage human keratinocytes give variable results depending upon whether they are cultured under serum-free conditions or in serum-containing medium. Early passage human keratinocytes do not express all-trans RA inducible RA 4-hydroxylase activity under serum-free conditions (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 retinoid receptors in human keratinocytes.J Biol Chem. 1994; 269: 32821-32827Abstract Full Text PDF PubMed Google Scholar). Human keratinocytes cultured in serum-containing medium express a substantial RA metabolic pathway, the detectable products of which are more polar than 4-hydroxy and 4-oxo RA; carbon four metabolites are not detected in these cells (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). In serum-containing cultures, the very active RA metabolic pathway may obscure further detection of RA metabolism induced by exogenous RA. HaCaT cells were generously provided by Dr. N.E. Fusenig (German Cancer Research Center, Heidelberg, Germany). Dulbecco’s modified Eagle’s medium (DMEM), heat-inactivated fetal bovine serum, Dulbecco’s phosphate-buffered saline (PBS), and trypsin 2.5% were obtained from Gibco-BRL (Grand Island, NY). All-trans RA, 13-cis RA, cycloheximide, Actinomycin D, tolbutamide, phenylmethylsulfonyl fluoride, aprotinin, leupeptin, NADPH, glycerol, and butylated hydroxytoluene were obtained from Sigma (St. Louis, MO). [3H]All-trans RA was purchased from DuPont NEN (Boston, MA). Extracti-Gel D and Coomassie Plus Protein Assay reagent were from Pierce (Rockford, IL). RA metabolite standards, 4-hydroxy all-trans RA, 4-oxo all-trans RA, 18-hydroxy all-trans RA, and 5,6-epoxy all-trans RA were gifts from Drs. M. Rosenberger and P.F. Sorter of Hoffmann-La Roche (Nutley, NJ). 9-cis RA, [3H]9-cis RA, and [3H]13-cis RA were gifts from Drs. J. Grippo, A. Levin, and P.F. Sorter of Hoffmann-La Roche. Dr. Brahm Shroot of CIRD (Galderma, France) generously provided CD367 [4-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-anthracenyl) benzoic acid] and CD2665 [4-(6-methoxyethoxymethoxy-7-adamantyl-2-naphthyl) benzoic acid]. Caffeine and α-naphthoflavone were purchased from Aldrich (Milwaukee, WI). Liarozole fumarate and ketoconazole were kindly provided by Gert Cauwenburgh of Janssen Research Foundation (Turnhoutseweg, Belgium). Cyclosporine A and s-mephenytoin were gifts from Novartis (Basel, Switzerland). High performance liquid chromatography grade solvents were obtained from Burdick and Jackson Laboratories (Romulus, MI). Spherisorb ODS-1 columns were obtained from Phase Separations (Norwalk, CT). HaCaT cells were cultured in DMEM containing 10% fetal bovine serum. Cells were treated at confluency with retinoids (1000-fold concentrated stock solutions in ethanol) or vehicle under reduced lighting. At appropriate times after addition of retinoids, cells were washed twice with PBS, treated with 0.025% trypsin for 15 min at 37°C, and harvested. Cells were washed three times with PBS, and immediately used for assays, or suspended in a storage medium (PBS, 50% glycerol, 1 mM phenylmethylsulfonyl fluoride, 1 μg leupeptin per ml, and 0.04 units aprotinin per ml) at a cell density of 108 cells per ml, snap-frozen in liquid nitrogen, and stored at –80°C. HaCaT cells (5–15 mg whole cell protein per ml) were suspended in ice-cold homogenization medium [10 mM Tris-HCl (pH 7.4), 1 mM ethylenediamine tetraacetic acid, 0.1 M sucrose, 1 mM phenylmethylsulfonyl fluoride, 0.1 μg leupeptin per ml, and 0.04 units aprotinin per ml] and subjected to 20 strokes in a Dounce homogenizer (40 ml), followed by sonication (30 pulses using a microprobe sonicator) at 0–4°C. The homogenate was centrifuged at 10,000 ×g for 10 min over a sucrose cushion (0.5 M sucrose in homogenization buffer). The pellet was homogenized and centrifuged as above. Pooled supernatants were then centrifuged at 10,000 ×g for 15 min. The supernatant was centrifuged at 100,000 ×g for 60 min. The resulting microsomal pellet was suspended in storage medium (≈10 mg microsomal protein per ml storage medium), snap-frozen in liquid nitrogen, and stored at –80°C. Protein was measured with the Coomassie Plus Protein Assay reagent in a total volume of 2 ml with bovine serum albumin as standard (Bradford, 1976Bradford M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.Anal Biochem. 1976; 72: 248-254Crossref PubMed Scopus (215608) Google Scholar). Whole HaCaT cells (0.1–0.25 mg whole cell protein) or microsomal preparations (0.1 mg microsomal protein) were incubated for 30 min at 37°C with 60 nM [3H]all-trans RA, [3H]9-cis RA, or [3H]13-cis RA, and 2 mM NADPH in reaction buffer [0.1 M Tris-HCl (pH 7.4), 20 mM sodium phosphate buffer (pH 7.4), 5 mM MgCl2, 0.15 M KCl, and 10% glycerol] in a volume of 300 μl. In some experiments, microsomes were incubated with 60 nM [3H]all-trans RA in the presence of 1000 nM unlabeled all-trans RA, 9-cis RA, or 13-cis RA. Reactions were halted by addition of 600 μl chloroform:methanol (2:1, vol/vol), vortexed, and centrifuged at 500 ×g for 10 min. The lower organic fraction containing retinoids was collected, evaporated with nitrogen in the dark, resuspended in methanol, and analyzed using reverse-phase high performance liquid chromatography connected to a flow-through scintillation counter (Duell et al., 1992Duell E.A. Astrom 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 4-hyroxylase activity was calculated as the sum of 4-hydroxy RA plus 4-oxo RA, which is formed from 4-hydroxy RA. 18-hydroxy RA was also detected and was ≈50% of 4-hydroxy RA. HaCaT cells were plated at 50% confluency in 35 mm × 6 well tissue culture plates in DMEM medium containing 10% charcoal-stripped bovine calf serum. Cells were transfected overnight with 1 μg each of βRARE3-tk-CAT (Xiao et al., 1995Xiao J.H. Durand B. Chambon P. Voorhees J.J. Endogenous retinoic acid receptor (RAR) -retinoid X receptor (RXR) heterodimers are the major functional forms regulating retinoid-responsive elements in adult human keratinocytes.J Biol Chem. 1995; 270: 3001-3011Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar) and pSV-β-galactosidase plasmid DNA by the calcium phosphate method (Cullen, 1987Cullen B.R. Use of eucaryotic expression technology in the functional analysis of cloned genes.Meth Enzymol. 1987; 152: 684-704Crossref PubMed Scopus (662) Google Scholar). βRARE3-tk-CAT contains three copies of the RA response element from the mouse RAR-β2 promoter, the thymidine kinase minimal promoter, which regulates 5′ expression of CAT. pSV-β-galactosidase is a constitutively active expression vector for the enzyme β-galactosidase. Following transfection, cells were washed twice with DMEM, replenished with complete DMEM-10% charcoal-striped bovine calf serum, and where indicated pretreated for 60 min with ketoconazole and/or CD-2665. All-trans RA was added for 24 h. Cells were washed twice with PBS, lyzed by freeze-thawing at –80°C in 0.2 ml of 0.1 M Tris buffer (pH 8.0) containing 0.1% Triton X-100. Cell lysates were centrifuged at 14,000 ×g for 10 min. Cell supernatants were assayed for CAT (Neumann et al., 1987Neumann J.R. Morency C.A. Russian K.O. A novel rapid assay for chloramphenicol acetyltransferase gene expression.Biotechniques. 1987; 5: 444-447Google Scholar) and β-galactosidase activity (Miller, 1972Miller J.H. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, New York1972Google Scholar), and CAT activity was normalized to β-galactosidase activity. All transfection experiments were performed in triplicate wells. Total RNA was isolated from HaCaT cells by the guanidinium phenol extraction method (Fisher et al., 1995Fisher G.J. Reddy A.P. Datta S.C. Kang S. Yi J.Y. Chambon P. Voorhees J.J. All-trans retinoic acid induces cellular retinol-binding protein in human skin.In Vivo. J Invest Dermatol. 1995; 105: 80-86Crossref PubMed Scopus (36) Google Scholar). RA 4-hydroxylase (hP450RAI;White et al., 1997White J.A. Beckett-Jones B. Guo Y.D. Dilworth F.J. Bonasoro J. Jones G. Petkovich M. cDNA cloning of human retinoic acid-metabolizing enzyme (hP450RAI) identifies a novel family of cytochromes P450 (CYP26).J Biol Chem. 1997; 272: 18538-18541Crossref PubMed Scopus (337) Google Scholar) and 36B4 (a ribosomal protein used as an internal control;Masiakowski et al., 1982Masiakowski P. Breathnach R. Bloch J. Gannon F. Krust A. Chambon P. Cloning of cDNA sequences of hormone-regulated genes from the MCF-7 human breast cancer cell line.Nucl Acids Res. 1982; 10: 7895-7903Crossref PubMed Scopus (702) Google Scholar) mRNA were determined by northern analysis, using random-primed 32P-labeled cDNA probes, as previously described (Fisher et al., 1995Fisher G.J. Reddy A.P. Datta S.C. Kang S. Yi J.Y. Chambon P. Voorhees J.J. All-trans retinoic acid induces cellular retinol-binding protein in human skin.In Vivo. J Invest Dermatol. 1995; 105: 80-86Crossref PubMed Scopus (36) Google Scholar). Filters were washed, visualized, and quantitated by PhosphoImager. Control cells and cells treated with CD367 were incubated separately with [3H]all-trans RA, and reverse phase HPLC was used to separate resulting retinoids. Chromatograms show a difference in RA metabolites for untreated control cells (Figure 1a) versus cells treated with CD367 (Figure 1b). Only CD367-treated cells produced 4-hydroxyl RA, 18-hydroxyl RA, and 4-oxo RA. In the absence of added retinoids, RA 4-hydroxylase activity was not detectable in HaCaT cells. Addition of all-trans RA, or the nuclear RA receptor-specific ligand CD367 (Fisher et al., 1994Fisher G.J. Talwar H.S. Xiao J.H. et al.Immunological identification and functional quantitation of retinoic acid and retinoid X receptor proteins in human skin.J Biol Chem. 1994; 269: 20629-20635Abstract Full Text PDF PubMed Google Scholar), resulted in induction of RA 4-hydroxylase activity (Figure 1c). CD367 was 6-fold more potent than all-trans RA in inducing RA 4-hydroxylase activity. Fractionation of HaCaT cells revealed that retinoid-inducible RA 4-hydroxylase activity was localized in the microsomal (high-speed pellet) fraction (Figure 1c). The percentage of total cellular RA 4-hydroxylase activity recovered in the microsomal fraction was 50%. The substrate dependence of all-trans RA-induced microsomal RA 4-hydroxylase activity is illustrated in Figure 1(d). Analysis of the data using a Lineweaver–Burk plot (Figure 1d, inset) indicated that the concentration of all-trans RA required for half-maximal activity (Km) was 37 ± 9 nM, and that the maximum rate of reaction (Vmax) was 205 ± 5 pg per min per mg protein (Figure 1d). The time course of induction of RA 4-hydroxylase mRNA and activity by all-trans RA is shown in Figure 2(a). RA 4-hydroxylase mRNA was induced within 2 h, and reached a maximal level within 8 h after addition of all-trans RA. Two RA 4-hydroxylase transcripts were detected, a major band of 1.9 kb and a minor band of 2.3 kb. Induction of RA 4-hydroxylase activity followed induction of its mRNA, first becoming detectable 4 h after addition of all-trans RA. Activity continued to increase, reaching a maximal level 24 h after addition of all-trans RA. Induction of RA 4-hydroxylase activity by all-trans RA was relatively transient, however, returning to near-baseline levels by 48 h (data not shown). This disappearance of RA 4-hydroxylase activity coincided with catabolism of all-trans RA, and was substantially accelerated by removal of all-trans RA from the culture medium. Replacement of the culture media 20 h after addition of all-trans RA, with fresh media lacking exogenous all-trans RA, resulted in complete loss of RA 4-hydroxylase activity within 6 h (data not shown). These data indicate that the continued presence of all-trans RA is required to maintain RA 4-hydroxylase activity, and that the half-life of RA 4-hydroxylase activity is relatively short (less than 6 h) in HaCaT cells. In contrast, RA 4-hydroxylase activity remained maximally elevated for at least 48 h, following induction by CD367. In addition, RA 4-hydroxylase activity remained fully induced for at least 24 h following removal of CD367 from the culture media (data not shown). This long-lived induction of RA 4-hydroxylase activity was likely attributable to the lack of catabolism of CD367 by HaCaT cells. Unlike all-trans RA, which was rapidly catabolized by HaCaT cells, CD367 was metabolically inert (data not shown). A recent report showed that induced RA 4-hydroxylase mRNA levels remained elevated for up to 96 h in the murine F9 teratocarcinoma cell line (Abu-Abed et al., 1998Abu-Abed S.S. Beckett B.R. Chiba H. et al.Mouse P450 RAI (CYP26) expression and retinoic acid inducible retinoic acid metabolism in F9 cells are regulated by retinoic acid γ receptor and retinoid X receptor α.J Biol Chem. 1998; 273: 2409-2415Crossref PubMed Scopus (156) Google Scholar). This difference in expression of RA 4-hydroxylase mRNA after induction may be due to lower RA 4-hydroxylase-mediated catabolism of RA by F9 cells relative to HaCaT cells. Lower catabolism in F9 cells would allow sustained elevation of cellular RA, thus maintaining induced levels of RA 4-hydroxylase activity, as we observed in CD367-treated HaCaT cells. Alternatively, differences in the stability of RA 4-hydroxylase mRNA may exist between F9 and HaCaT cells. The rapid induction of RA 4-hydroxylase mRNA by all-trans RA (Figure 2a) suggests that RA 4-hydroxylase is transcriptionally regulated. We therefore examined the effect of transcription and translation inhibitors on RA 4-hydroxylase induction. Inhibition of transcription by actinomycin D completely blocked induction of RA 4-hydroxylase mRNA and activity (Figure 2b). Inhibition of translation by cycloheximide caused super-induction of RA 4-hydroxylase mRNA, and complete inhibition of induction of RA 4-hydroxylase activity (Figure 2b). Super-induction of inducible transcripts by cycloheximide is commonly observed, and results from increased mRNA stability (Edwards and Mahadevan, 1992Edwards D.R. Mahadevan L.C. Protein synthesis inhibitors differentially superinduce c-fos and c-jun by three distinct mechanisms: lack of evidence for labile repressors.Embo J. 1992; 11: 2415-2424Crossref PubMed Scopus (278) Google Scholar;Schuetz et al., 1995Schuetz J.D. Strom S.C. Schuetz E.G. Induction of P-glycoprotein mRNA by protein synthesis inhibition is not controlled by a transcriptional repressor protein in rat and human liver cells.J Cell Physiol. 1995; 165: 261-272Crossref PubMed Scopus (17) Google Scholar). These data demonstrate that all-trans RA induction of RA 4-hydroxylase is dependent on mRNA and protein synthesis. The above data demonstrate that all-trans RA induction of RA 4-hydroxylase is transcriptionally regulated. The biologic effects of all-trans RA are mediated by nuclear RA receptors (Fisher and Voorhees, 1996Fisher G.J. Voorhees J.J. Molecular mechanisms of retinoid actions in skin.Faseb. 1996; 10: 1002-1013PubMed Google Scholar). These receptors are activated through direct binding of all-trans RA and function to regulate transcription of specific target genes. We therefore investigated the involvement of nuclear RA receptors in induction of RA 4-hydroxylase gene expression by all-trans RA. To do this, we utilized the nuclear RA receptor antagonist CD2665 (Sun et al., 1997Sun S.Y. Yue P. Dawson M.I. et al.Differential effects of synthetic nuclear retinoid receptor-selective retinoids on the growth of human non-small cell lung carcinoma cells.Cancer Res. 1997; 57: 4931-4939PubMed Google Scholar). This synthetic retinoid effectively inhibited all-trans RA induction of nuclear RA receptor-dependent reporter gene expression in HaCaT cells (Figure 3a). CD2665 also inhibited all-trans RA induction of RA 4-hydroxylase activity, in a concentration-dependent manner (Figure 3b). At a ratio of 1:100 (all-trans RA:CD2665) inhibition was nearly complete. These data indicate that all-trans RA induction of RA 4-hydroxylase is mediated by nuclear RA receptors. Recent studies with transformed cell lines have also provided evidence for involvement of nuclear RA receptors in induction of all-trans RA metabolism (van der Leede et al., 1997van der Leede B.M. van der Brink C.E. Pijnappel W.W.M. Sonneveld E. van der Saag P.T. van der Burg B. Autoinduction of retinoic acid metabolism to polar derivatives with decreased biological activity in retinoic acid-sensitive, but not in retinoic acid-resistant human breast cancer cells.J Biol Chem. 1997; 272: 17921-17928Crossref PubMed Scopus (57) Google Scholar;Isogai et al., 1997Isogai M. Chiantore M.V. Haque M. De Scita G. Luca L.M. Expression of a dominant -negative retinoic acid receptor construct reduces retinoic acid metabolism and retinoic acid-induced inhibtion of NIH-3T3 cell growth.Cancer Res. 1997; 57: 4460-4464PubMed Google Scholar;Abu-Abed et al., 1998Abu-Abed S.S. Beckett B.R. Chiba H. et al.Mouse P450 RAI (CYP26) expression and retinoic acid inducible retinoic acid metabolism in F9 cells are regulated by retinoic acid γ receptor and retinoid X receptor α.J Biol Chem. 1998; 273: 2409-2415Crossref PubMed Scopus (156) Google Scholar). Recent studies employing molecular cloning techniques have demonstrated that RA 4-hydroxylase is a member of the cytochrome P-450 supergene family (White et al., 1996White J.A. Guo Y.D. Baetz K. et al.Identification of the retinoic acid-inducible all-trans-retinoic acid 4-hydroxylase.J Biol Chem. 1996; 271: 29922-29927Crossref PubMed Scopus (316) Google Scholar, White et al., 1997White J.A. Beckett-Jones B. Guo Y.D. Dilworth F.J. Bonasoro J. Jones G. Petkovich M. cDNA cloning of human retinoic acid-metabolizing enzyme (hP450RAI) identifies a novel family of cytochromes P450 (CYP26).J Biol Chem. 1997; 272: 18538-18541Crossref PubMed Scopus (337) Google Scholar;Ray et al., 1997Ray W.J. Bain G. Yao M. Gottlieb D.I. CYP26, a novel mammalian cytochrome P450, is induced by reinoic acid and defines a new family.J Biol Chem. 1997; 272: 18702-18708Crossref PubMed Scopus (269) Google Scholar). This multigene family encodes a large number of P-450 isoenzymes that have varying degrees of substrate specificity, and act on a wide range of different substrates (Coon et al., 1996Coon M.J. Vaz A.D. Bestervelt L.L. Cytochrome P-450, 2: peroxidative reactions of diversozymes.Faseb. 1996; 10: 428-434PubMed Google Scholar). To examine the substrate specificity of RA 4-hydroxylase in HaCaT cells, we determined the effect of a series of known substrates for cytochrome P-450 subfamilies on RA 4-hydroxylase activity. Each substrate was added separately at 2-fold, 20-fold, and 100-fold higher concentrations than [3H]all-trans RA in the RA 4-hydroxylase assay. The 11 compounds utilized, and the cytochrome P-450 subfamilies for which they are substrates, are listed in Table I. Any compound tested that was a substrate for, or interacted with, RA 4-hydroxylase would be expected to alter the amount of [3H]4-hydroxy RA product generated in the assay; however, none of the compounds examined had any significant effect on RA 4-hydroxylase activity (data not shown). This finding is consistent with molecular cloning data indicating that RA 4-hydroxylase is unique, and that it defines a new cytochrome P-450 subfamily (White et al., 1996White J.A. Guo Y.D. Baetz K. et al.Identification of the retinoic acid-inducible all-trans-retinoic acid 4-hydroxylase.J Biol Chem. 1996; 271: 29922-29927Crossref PubMed Scopus (316) Google Scholar, White et al., 1997White J.A. Beckett-Jones B. Guo Y.D. Dilworth F.J. Bonasoro J. Jones G. Petkovich M. cDNA cloning of human retinoic acid-metabolizing enzyme (hP450RAI) identifies a novel family of cytochromes P450 (CYP26).J Biol Chem. 1997; 272: 18538-18541Crossref PubMed Scopus (337) Google Scholar;Ray et al., 1997Ray W.J. Bain G. Yao M. Gottlieb D.I. CYP26, a novel mammalian cytochrome P450, is induced by reinoic acid and defines a new family.J Biol Chem. 1997; 272: 18702-18708Crossref PubMed Scopus (269) Google Scholar).Table ICytochrome P-450 substrates tested for their effect on HaCaT cell RA 4-hydroxylase activitySubstrateP-450 subfamilyα-naphthoflavoneCYPIA1CaffeineCYPIA2AcetanilideCYPIA2TolbutamideCYPIICs-mephenytoinCYPIICBenzphetamineCYPIIC8TestosteroneCYPIIDp-nitrophenolCYPIIE1ErythromycinCYPIIIA, IV, VCyclosporineA CYPIIIA, IV, VLauric acidCYPIVA Open table in a new tab We next examined the specificity of RA 4-hydroxylase towards the three naturally occurring isomers of RA: all-trans RA, 9-cis RA, and 13-cis RA. The amount of 4-hydroxy RA formed by HaCaT cell microsomes incubated with [3H]9-cis RA and [3H]13-cis RA was 1% and 5%, respectively, of that formed from all-trans RA (Figure 4). Because 9-cis RA and 13-cis RA spontaneously isomerize to all-trans RA (Duell et al., 1996Duell E.A. Kang S. Voorhees J.J. Retinoic acid isomers applied to human skin in vivo induce a 4-hydroxylase that inactivates only all-trans retinoic acid.J Invest Dermatol. 1996; 106: 316-320Abstract Full Text PDF PubMed Scopus (48) Google Scholar;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), it is likely that the low levels of [3H]4-hydroxy RA formed in incubations containing [3H]9-cis RA or [3H]13-cis RA were generated from [3H]all-trans RA. Our preparations of 9-cis and 13-cis RA contained 4% and 18% all-trans RA, respectively. Addition of excess unlabeled all-trans RA to incubations containing either [3H]9-cis RA or [3H]13-cis RA substantially reduced the amount of [3H]4-hydroxy RA product formed (Figure 4), supporting this supposition.White et al., 1997White J.A. Beckett-Jones B. Guo Y.D. Dilworth F.J. Bonasoro J. Jones G. Petkovich M. cDNA cloning of human retinoic acid-metabolizing enzyme (hP450RAI) identifies a novel family of cytochromes P450 (CYP26).J Biol Chem. 1997; 272: 18538-18541Crossref PubMed Scopus (337) Google Scholar have reported that 9-cis RA served as a good substrate for RA 4-hydroxylase; however, the degree of isomerization of the 9-cis RA used as substrate to all-trans RA was not assessed. Taken together, the above data indicate that microsomal RA 4-hydroxylase activity in HaCaT cells is highly specific for the all-trans isomer of RA.Krekels et al., 1997Krekels MDWG Verhoeven A. Van Dun J. et al.Induction of the oxidative catabolism of retinoic acid in MCF-7 cells.Br J Cancer. 1997; 75: 1098-1104Crossref PubMed Scopus (16) Google Scholar have also reported that all-trans RA is the preferred substrate for RA 4-hydroxylase in MCF-7 breast cancer cells. Finally, we examined the effect of RA 4-hydroxylase on nuclear RA receptor-dependent gene expression. For these functional studies we utilized ketoconazole, which completely inhibits RA 4-hydroxylase activity in HaCaT cells when used at concentrations in excess of 10 μM (Figure 5a). HaCaT cells were transfected with a nuclear RA receptor-dependent reporter gene, and then treated with either all-trans RA (0.1 μM) alone, or in combination with ketoconazole (50 μM). Reporter gene expression was nearly 4-fold greater in the presence of all-trans RA plus ketoconazole, compared with all-trans RA alone (Figure 5b). Activation of the reporter gene by either all-trans RA alone or all-trans RA plus ketoconazole, was inhibited by 90% by the nuclear RA receptor antagonist CD2665, confirming that reporter gene activity was dependent on nuclear RA receptors. These data demonstrate that by conversion of all-trans RA to 4-hydroxy RA, RA 4-hydroxylase functions to limit induction of nuclear RA receptor-regulated gene expression in HaCaT cells. Previous studies have demonstrated that all-trans RA induces its own catabolism to 4-hydroxy RA in human skin (Duell et al., 1996Duell E.A. Kang S. Voorhees J.J. Retinoic acid isomers applied to human skin in vivo induce a 4-hydroxylase that inactivates only all-trans retinoic acid.J Invest Dermatol. 1996; 106: 316-320Abstract Full Text PDF PubMed Scopus (48) Google Scholar) and other organs and cells (Ray et al., 1997Ray W.J. Bain G. Yao M. Gottlieb D.I. CYP26, a novel mammalian cytochrome P450, is induced by reinoic acid and defines a new family.J Biol Chem. 1997; 272: 18702-18708Crossref PubMed Scopus (269) Google Scholar;White et al., 1997White J.A. Beckett-Jones B. Guo Y.D. Dilworth F.J. Bonasoro J. Jones G. Petkovich M. cDNA cloning of human retinoic acid-metabolizing enzyme (hP450RAI) identifies a novel family of cytochromes P450 (CYP26).J Biol Chem. 1997; 272: 18538-18541Crossref PubMed Scopus (337) Google Scholar). We have utilized human keratinocyte HaCaT cells to investigate the mechanism of this induction. The data presented above demonstrate that induction of RA 4-hydroxylase is dependent on nuclear RA receptor-mediated gene transcription. This conclusion is in accord with a recent study demonstrating that disruption of RAR-γ and RAR-α gene expression by homologous recombination causes loss of RA induction of RA 4-hydroxylase in murine F9 cells (Abu-Abed et al., 1998Abu-Abed S.S. Beckett B.R. Chiba H. et al.Mouse P450 RAI (CYP26) expression and retinoic acid inducible retinoic acid metabolism in F9 cells are regulated by retinoic acid γ receptor and retinoid X receptor α.J Biol Chem. 1998; 273: 2409-2415Crossref PubMed Scopus (156) Google Scholar). These findings strongly suggest that the RA 4-hydroxylase gene contains a RA response element that is directly regulated by nuclear RA receptors. Cloning and characterization of the RA 4-hydroxylase gene promoter will reveal whether this hypothesis is correct. 4-hydroxy RA and its metabolites are less potent than all-trans RA in activating nuclear RA receptors (Siegenthaler et al., 1990Siegenthaler G. Saurat J.H. Ponec M. Retinol and retinal metabolism. Relationship to the state of differentiation of cultured human keratinocytes.Biochem J. 1990; 268: 371-378Crossref PubMed Scopus (100) Google Scholar;Reddy et al., 1992Reddy A.P. Chen J.Y. Zacharewski T. Gronemeyer H. Voorhees J.J. Fisher G.J. Characterization and purification of human retinoic acid receptorγ-1 overexpressed in the baculovirus-insect cell system.Biochem J. 1992; 287: 833-840Crossref PubMed Scopus (12) Google Scholar), and in eliciting biologic responses (Reynolds et al., 1993Reynolds N.J. Fisher G.J. Griffiths C.E.M. et al.Retinoic acid metabolites exhibit biological activity in human keratinocytes, mouse melanoma cells and hairless mouse skin.In Vivo. J Pharmacol Exp Therapeut. 1993; 266: 1636-1642PubMed Google Scholar). Therefore, by inducing RA 4-hydroxylase activity, all-trans RA limits its own actions in skin. This negative autoregulatory mechanism operates simultaneously with, and continually controls, the pharmacologic actions of all-trans RA. The actions of all-trans retinol are similarly controlled, because it is converted to all-trans RA. In contrast, most synthetic retinoids are not subject to the natural restraining mechanism provided by RA 4-hydroxylase, due to the stringent substrate specificity of the enzyme. This fact raises the possibility that synthetic retinoids may need to have the equivalent of a built-in, self-adjusting brake in order to be safe and effective. A braking mechanism may be designed by exploiting alternative routes of metabolic disposal, and/or activating RA receptors in a manner that is qualitatively distinct from activation by all-trans RA. Design and development of such synthetic retinoids represents a significant challenge. Supported in part by the Babcock Endowment for Dermatological Research and a grant from the Johnson & Johnson Corporation." @default.
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• W2001238939 title "Retinoic Acid Receptors Regulate Expression of Retinoic Acid 4-Hydroxylase that Specifically Inactivates All-Trans Retinoic Acid in Human Keratinocyte HaCaT Cells" @default.
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