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Martin Petkovich - One of the best experts on this subject based on the ideXlab platform.
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the retinoic acid metabolizing enzyme CYP26B1 regulates cd4 t cell differentiation and function
PLOS ONE, 2013Co-Authors: Alistair Chenery, Martin Petkovich, Michael T Underhill, Kyle Burrows, Frann Antignano, Colby ZaphAbstract:The vitamin A metabolite retinoic acid (RA) has potent immunomodulatory properties that affect T cell differentiation, migration and function. However, the precise role of RA metabolism in T cells remains unclear. Catabolism of RA is mediated by the Cyp26 family of cytochrome P450 oxidases. We examined the role of CYP26B1, the T cell-specific family member, in CD4+ T cells. Mice with a conditional knockout of CYP26B1 in T cells (CYP26B1−/− mice) displayed normal lymphoid development but showed an increased sensitivity to serum retinoids, which led to increased differentiation under both inducible regulatory T (iTreg) cell- and TH17 cell-polarizing conditions in vitro. Further, CYP26B1 expression was differentially regulated in iTreg and TH17 cells. Transfer of naive CYP26B1−/− CD4+ T cells into Rag1−/− mice resulted in significantly reduced disease in a model of T cell-dependent colitis. Our results show that T cell-specific expression of CYP26B1 is required for the development of T cell-mediated colitis and may be applicable to the development of therapeutics that target CYP26B1 for the treatment of inflammatory bowel disease.
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functional properties and substrate characterization of human cyp26a1 CYP26B1 and cyp26c1 expressed by recombinant baculovirus in insect cells
Journal of Pharmacological and Toxicological Methods, 2011Co-Authors: Christian F Helvig, Glenville Jones, Mohammed Taimi, Don Cameron, Martin PetkovichAbstract:Abstract Introduction The cytochrome P450 CYP26 family of retinoic acid (RA) metabolizing enzymes, comprising CYP26A1, CYP26B1, and CYP26C1 is critical for establishing patterns of RA distribution during embryonic development and retinoid homeostasis in the adult. All three members of this family can metabolize all trans -RA. CYP26C1 has also been shown to efficiently metabolize the 9 -cis isomer of RA. Methods We have co-expressed each of the CYP26 enzymes along with the NADPH-cytochrome P450 oxidoreductase using a baculovirus/Sf9 insect cell expression system to determine the enzymatic activities of these enzymes in cell free preparations and have established an in vitro binding assay to permit comparison of binding affinities of the three CYP26 enzymes. Results We demonstrated that the expressed enzymes can efficiently coordinate heme, as verified by spectral-difference analysis. All CYP26s efficiently metabolized all -trans -RA to polar aqueous-soluble metabolites, and in competition experiments exhibited IC 50 values of 16, 27, and 15 nM for CYP26A1, B1, and C1 respectively for all- trans -RA. Furthermore, this metabolism was blocked with the CYP inhibitor ketoconazole. CYP26C1 metabolism of all trans -RA could also be effectively competed with 9- cis RA, with IC 50 of 62 nM, and was sensitive to ketoconazole inhibition. Discussion CYP26 enzymes are functionally expressed in microsomal fractions of insect cells and stably bind radiolabeled RA isomers with affinities respecting their substrate specificities. We demonstrated that compared to CYP26A and CYP26B, only CYP26C1 was able to bind with high affinity to 9- cis- RA. These assays will be useful for the screening of synthetic substrates and inhibitors of CYP26 enzymes and may be applicable to other cytochrome P450s and their respective substrates.
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genetic deletion of CYP26B1 negatively impacts limb skeletogenesis by inhibiting chondrogenesis
Journal of Cell Science, 2011Co-Authors: Helen J Dranse, Martin Petkovich, Arthur V Sampaio, Michael T UnderhillAbstract:CYP26B1 , a retinoic acid (RA)-metabolising enzyme, is expressed in the developing limb bud, and CYP26B1 −/− mice present with severe limb defects. These malformations might be attributable to an RA-induced patterning defect; however, recent reports suggest that RA is dispensable for limb patterning. In this study, we examined the role of endogenous retinoid signalling in skeletogenesis using CYP26B1 −/− mice and transgenic mice in which CYP26B1 is conditionally deleted under control of the Prrx1 promoter beginning at ~E9.5 ( Prrx1Cre + /CYP26B1 fl/fl ). We found that the limb phenotype in Prrx1Cre + /CYP26B1 fl/fl mice was less severe than that observed in CYP26B1 −/− animals and that a change in retinoid signalling contributed to the difference in phenotypes. We systematically examined the role of endogenous RA signalling in chondrogenesis and found that CYP26B1 −/− cells and limb mesenchymal cells treated with a CYP inhibitor, are maintained in a pre-chondrogenic state, exhibit reduced chondroblast differentiation and have modestly accelerated chondrocyte hypertrophy. Furthermore, CYP26B1 −/− mesenchyme exhibited an increase in expression of genes in a closely related tendogenic lineage, indicating that retinoid signals in the limb interfere with differentiation and maintain progenitor status. Together, these findings support an important function for RA in regulating the behaviour of mesenchymal progenitors, and their subsequent differentiation and maturation.
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analysis of CYP26B1 rarg compound null mice reveals two genetically separable effects of retinoic acid on limb outgrowth
Developmental Biology, 2010Co-Authors: Tracie Pennimpede, Don Cameron, Glenn Maclean, Martin PetkovichAbstract:abstract Article history:Received for publication 26 September 2009Revised 17 December 2009Accepted 18 December 2009Available online 4 January 2010Keywords:CYP26B1RARγLimb developmentProximo-distal patterningRetinoic acidTeratogenesisMouse The role of retinoic acid (RA) in limb development is unclear, although it has been suggested to be aproximalizing factor which plays a morphogenetic role in pattern formation. Exogenous RA produces ateratogenic effect on limb morphology; similarly, changes in the endogenous distribution of RA followinggenetic ablation of the RA-metabolizing enzyme, CYP26B1, result in phocomelia accompanied by changes inexpression of proximo-distal (P–D) patterning genes, increased cell death, and delayed chondrocytematuration. Here we show that disruption of RA receptor (RAR) gamma in a CYP26B1 −/ background is ableto partially rescue limb skeletal morphology without restoring normal expression of proximo-distalpatterning genes. We further show that embryos deficient in CYP26B1 exhibit early localized domains ofmesenchymal cell death, which are reduced in compound-null animals. This model reveals two geneticallyseparable effects of RA in the limb: an apoptotic effect mediated by RARγ in the presence of ectopic RA, and aP–D patterning defect which is uncovered following the loss of both CYP26B1 and RARγ. These data providegenetic evidence to clarify the roles of both RA and CYP26B1 in limb outgrowth and proximo-distalpatterning.© 2009 Elsevier Inc. All rights reserved.
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CYP26B1 expression in murine sertoli cells is required to maintain male germ cells in an undifferentiated state during embryogenesis
PLOS ONE, 2009Co-Authors: Glenn Maclean, Don Cameron, Margaret Clagettdame, Martin PetkovichAbstract:In mammals, germ cells within the developing gonad follow a sexually dimorphic pathway. Germ cells in the murine ovary enter meiotic prophase during embryogenesis, whereas germ cells in the embryonic testis arrest in G0 of mitotic cell cycle and do not enter meiosis until after birth. In mice, retinoic acid (RA) signaling has been implicated in controlling entry into meiosis in germ cells, as meiosis in male embryonic germ cells is blocked by the activity of a RA-catabolizing enzyme, CYP26B1. However, the mechanisms regulating mitotic arrest in male germ cells are not well understood. CYP26B1 expression in the testes begins in somatic cells at embryonic day (E) 11.5, prior to mitotic arrest, and persists throughout fetal development. Here, we show that Sertoli cell-specific loss of CYP26B1 activity between E15.5 and E16.5, several days after germ cell sex determination, causes male germ cells to exit from G0, re-enter the mitotic cell cycle and initiate meiotic prophase. These results suggest that male germ cells retain the developmental potential to differentiate in meiosis until at least at E15.5. CYP26B1 in Sertoli cells acts as a masculinizing factor to arrest male germ cells in the G0 phase of the cell cycle and prevents them from entering meiosis, and thus is essential for the maintenance of the undifferentiated state of male germ cells during embryonic development.
Leif A. Eriksson - One of the best experts on this subject based on the ideXlab platform.
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homology models of human all trans retinoic acid metabolizing enzymes CYP26B1 and CYP26B1 spliced variant
Journal of Chemical Information and Modeling, 2012Co-Authors: Patricia Saenzmendez, Åke Strid, Allan Sirsjö, Ali Ateia Elmabsout, Helena Savenstrand, Mohamed Khalid Alhaj Awadalla, Leif A. ErikssonAbstract:Homology models of CYP26B1 (cytochrome P450RAI2) and CYP26B1 spliced variant were derived using the crystal structure of cyanobacterial CYP120A1 as template for the model building. The quality of the homology models generated were carefully evaluated, and the natural substrate all-trans-retinoic acid (atRA), several tetralone-derived retinoic acid metabolizing blocking agents (RAMBAs), and a well-known potent inhibitor of CYP26B1 (R115866) were docked into the homology model of full-length cytochrome P450 26B1. The results show that in the model of the full-length CYP26B1, the protein is capable of distinguishing between the natural substrate (atRA), R115866, and the tetralone derivatives. The spliced variant of CYP26B1 model displays a reduced affinity for atRA compared to the full-length enzyme, in accordance with recently described experimental information.
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cloning and functional studies of a splice variant of CYP26B1 a cellular storage protein for all trans retinoic acid
in Vivo, 2010Co-Authors: Ali Ateia Elmabsout, Magnus Karlsson, Åke Strid, Leif A. Eriksson, Olesya Krivospitskaya, Helena Savenstrand, Ashok Kumar Kumawat, Torma Hans, Allan SirsjöAbstract:BackgroundAll-trans retinoic acid (atRA) plays an essential role in the regulation of gene expression, cell growth and differentiation and is also important for normal cardiovascular development but may in turn be involved in cardiovascular diseases, i.e. atherosclerosis and restenosis. The cellular atRA levels are under strict control involving several cytochromes P450 isoforms (CYPs). CYP26 may be the most important regulator of atRA catabolism in vascular cells. The present study describes the molecular cloning, characterization and function of atRA-induced expression of a spliced variant of the CYP26B1 gene.Methodology/Principal FindingsThe coding region of the spliced CYP26B1 lacking exon 2 was amplified from cDNA synthesized from atRA-treated human aortic smooth muscle cells and sequenced. Both the spliced variant and full length CYP26B1 was found to be expressed in cultured human endothelial and smooth muscle cells, and in normal and atherosclerotic vessel. atRA induced both variants of CYP26B1 in cultured vascular cells. Furthermore, the levels of spliced mRNA transcript were 4.5 times higher in the atherosclerotic lesion compared to normal arteries and the expression in the lesions was increased 20-fold upon atRA treatment. The spliced CYP26B1 still has the capability to degrade atRA, but at an initial rate one-third that of the corresponding full length enzyme. Transfection of COS-1 and THP-1 cells with the CYP26B1 spliced variant indicated either an increase or a decrease in the catabolism of atRA, probably depending on the expression of other atRA catabolizing enzymes in the cells.Conclusions/SignificanceVascular cells express the spliced variant of CYP26B1 lacking exon 2 and it is also increased in atherosclerotic lesions. The spliced variant displays a slower and reduced degradation of atRA as compared to the full-length enzyme. Further studies are needed, however, to clarify the substrate specificity and role of the CYP26B1 splice variant in health and disease.
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Homology Models and Molecular Modeling of Human Retinoic Acid Metabolizing Enzymes Cytochrome P450 26A1 (CYP26A1) and P450 26B1 (CYP26B1).
Journal of chemical theory and computation, 2008Co-Authors: Magnus Karlsson, Åke Strid, Allan Sirsjö, Leif A. ErikssonAbstract:Homology models of cytochrome P450 26A1 and cytochrome P450 26B1 were constructed using the crystal structures of human, CYP2C8, CYP2C9, and CYP3A4 as templates for the model building. The homology models generated were investigated for their docking capacities against the natural substrate all-trans-retinoic acid (atRA), five different tetralone-derived retinoic acid metabolizing blocking agents (RAMBAs), and R115866. Interaction energies (IE) and linear interaction energies (LIE) were calculated for all inhibitors in both homology models after molecular dynamics (MD) simulation of the enzyme-ligand complexes. The results revealed that the homologues had the capacity to distinguish between strong and weak inhibitors. Important residues in the active site were identified from the CYP26A1/B1-atRA complexes. Residues involved in hydrophobic interactions with atRA were Pro113, Phe222, Phe299, Val370, Pro371, and Phe374 in CYP26A1 and Leu88, Pro118, Phe222, Phe295, Ile368, and Tyr272 in CYP26B1. Hydrogen bonding interactions were observed between the atRA carboxylate group and Arg 90 in CYP26A1 and with Arg76, Arg95, and Ser369 in CYP26B1.
Jing-pian Peng - One of the best experts on this subject based on the ideXlab platform.
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retinoic acid metabolizing enzyme cyp26a1 is implicated in rat embryo implantation
Human Reproduction, 2010Co-Authors: Hong-fei Xia, Ying Yang, Jing Sun, Jing-pian PengAbstract:background: The retinoic acid metabolizing enzyme Cyp26a1 plays a pivotal role in vertebrate embryo development. Cyp26a1 was characterized previously as a differentially expressed gene in peri-implantation rat uteri via suppressive subtracted hybridization analysis. However, the role of Cyp26a1 in rat embryo implantation remained elusive. methods: The expression of Cyp26a1 in the uteri of early pregnancy, pseudopregnancy and artificial decidualization was detected by northern blotting, real time-PCR, in situ hybridization, western blotting and immunofluorescent staining. The effect of Cyp26a1 on apoptosis of endometrial stromal cells (ESCs) isolated from rat uteri was determined by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) and Hoechst staining. Apoptosis-related proteins in ESCs were detected by western blotting. results: Cyp26a1 showed distinctive expression patterns in embryos and uteri during the peri-implantation period, with a remarkable increase (P , 0.01 versus Days 4–5) in mRNA and protein in the implantation phase (Days 5.5–6.5 of pregnancy). CYP26A1 was specifically localized in glandular epithelium, luminal epithelium and decidua basalis. The level of CYP26A1 protein was significantly increased in uteri of artificial decidualization (P , 0.01 versus control). Forced Cyp26a1 overexpression significantly reduced the sensitivity of ESCs to etoposideinduced apoptosis, with reductions in p53 (P , 0.01) and Fas (P , 0.05) proteins versus control, while in contrast, FasL (P , 0.01) and proliferating cell nuclear antigen (P , 0.05) proteins increased. conclusions: Cyp26a1 is spatiotemporally expressed in the uterus during embryo implantation and decidualization. Overexpression of Cyp26a1 attenuates the process of uterine stromal cell apoptosis, probably via down-regulating the expression of p53 and FasL.
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retinoic acid metabolizing enzyme cytochrome p450 26a1 cyp26a1 is essential for implantation functional study of its role in early pregnancy
Journal of Cellular Physiology, 2010Co-Authors: Bingchen Han, Ying Yang, Hong-fei Xia, Jing Sun, Jing-pian PengAbstract:Vitamin A (VA) is required for normal fetal development and successful pregnancy. Excessive VA intake during pregnancy may lead to adverse maternal and fetal effects. Cytochrome P450 26A1 (cyp26a1), a retinoic acid (RA)-metabolizing enzyme, is involved in VA metabolism. It has been shown that cyp26a1 is expressed in female reproductive tract, especially in uterus. In order to investigate the role of cyp26a1 during pregnancy, we constructed a recombinant plasmid DNA vaccine encoding cyp26a1 protein and immunized mice with the plasmid. Compared to control groups, the pregnancy rate of the cyp26a1 plasmid-immunized mice were significantly decreased (P < 0.01). Further results showed that both cyp26a1 mRNA and protein were specifically induced in the uterus during implantation period and localized in the uterine luminal epithelium. Importantly, the number of implantation sites was also significantly reduced (P < 0.05) after the uterine injection of cyp26a1-specific antisense oligos or anti-cyp26a1 antibody on day 3 of pregnancy. Accordingly, the expression of RA-related cellular retinoic acid binding protein 1 and tissue transglutaminase was markedly increased (P < 0.05) in the uterine luminal epithelium after intrauterine injection treatments. These data demonstrate that uterine cyp26a1 activity is important for the maintenance of pregnancy, especially during the process of blastocyst implantation. J. Cell. Physiol. 223: 471–479, 2010. © 2010 Wiley-Liss, Inc.
Nina Isoherranen - One of the best experts on this subject based on the ideXlab platform.
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Scaling in vitro activity of CYP3A7 suggests human fetal livers do not clear retinoic acid entering from maternal circulation
Nature Publishing Group, 2019Co-Authors: Ariel R Topletz, Guo Zhong, Nina IsoherranenAbstract:Abstract All-trans-retinoic acid (atRA), the active metabolite of vitamin A, is a critical signaling molecule during embryonic and fetal development and is necessary for maternal health. Fetal exposure to endogenous atRA is tightly regulated during gestation in a tissue specific manner and maternal exposure to exogenous retinoids during pregnancy is teratogenic. The clearance of atRA is primarily mediated by the cytochrome P450 (CYP) 26 enzymes, which play an essential role in controlling retinoid gradients during organogenesis. We hypothesized that CYP26 enzymes in the human fetal liver also function as a protective barrier to prevent maternal atRA reaching fetal circulation. Using human fetal liver tissue, we found that the mRNA of CYP26A1 and CYP26B1 enzymes is expressed in the human fetal liver. However, based on inhibition studies, metabolite profiles and correlation of atRA metabolism with testosterone hydroxylation, clearance of atRA in the fetal livers was mediated by CYP3A7. Based on in vitro-to-in vivo scaling, atRA clearance in the fetal liver was quantitatively minimal, thus providing an insufficient maternal-fetal barrier for atRA exposure
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inhibition of the all trans retinoic acid atra hydroxylases cyp26a1 and CYP26B1 results in dynamic tissue specific changes in endogenous atra signaling
Drug Metabolism and Disposition, 2017Co-Authors: Faith Stevison, Sasmita Tripathy, Cathryn A Hogarth, Travis Kent, Nina IsoherranenAbstract:All-trans retinoic acid (atRA), the active metabolite of vitamin A, is a ligand for several nuclear receptors and acts as a critical regulator of many physiologic processes. The cytochrome P450 family 26 (CYP26) enzymes are responsible for atRA clearance, and are potential drug targets to increase concentrations of endogenous atRA in a tissue-specific manner. Talarozole is a potent inhibitor of CYP26A1 and CYP26B1, and has shown some success in clinical trials. However, it is not known what magnitude of change is needed in tissue atRA concentrations to promote atRA signaling changes. The aim of this study was to quantify the increase in endogenous atRA concentrations necessary to alter atRA signaling in target organs, and to establish the relationship between CYP26 inhibition and altered atRA concentrations in tissues. Following a single 2.5-mg/kg dose of talarozole to mice, atRA concentrations increased up to 5.7-, 2.7-, and 2.5-fold in serum, liver, and testis, respectively, resulting in induction of Cyp26a1 in the liver and testis and Rar β and Pgc 1β in liver. The increase in atRA concentrations was well predicted from talarozole pharmacokinetics and in vitro data of CYP26 inhibition. After multiple doses of talarozole, a significant increase in atRA concentrations was observed in serum but not in liver or testis. This lack of increase in atRA concentrations correlated with an increase in CYP26A1 expression in the liver. The increased atRA concentrations in serum without a change in liver suggest that CYP26B1 in extrahepatic sites plays a key role in regulating systemic atRA exposure.
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direct protein protein interactions and substrate channeling between cellular retinoic acid binding proteins and CYP26B1
FEBS Letters, 2016Co-Authors: Cara H Nelson, Alex Zelter, Justin D Lutz, Chi Chi Peng, Catherine K Yeung, Nina IsoherranenAbstract:Cellular retinoic acid binding proteins (CRABPs) bind all-trans-retinoic acid (atRA) tightly. This study aimed to determine whether atRA is channeled directly to cytochrome P450 (CYP) CYP26B1 by CRABPs, and whether CRABPs interact directly with CYP26B1. atRA bound to CRABPs (holo-CRABP) was efficiently metabolized by CYP26B1. Isotope dilution experiments showed that delivery of atRA to CYP26B1 in solution was similar with or without CRABP. Holo-CRABPs had higher affinity for CYP26B1 than free atRA, but both apo-CRABPs inhibited the formation of 4-OH-RA by CYP26B1. Similar protein-protein interactions between soluble binding proteins and CYPs may be important for other lipophilic CYP substrates.
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identification of tazarotenic acid as the first xenobiotic substrate of human retinoic acid hydroxylase cyp26a1 and CYP26B1
Journal of Pharmacology and Experimental Therapeutics, 2016Co-Authors: Robert S Foti, Alex Zelter, Nina Isoherranen, Leslie J Dickmann, Brian Buttrick, Philippe Diaz, Dominique DouguetAbstract:Cytochrome P450 (CYP) 26A1 and 26B1 are heme-containing enzymes responsible for metabolizing all-trans retinoic acid (at-RA). No crystal structures have been solved, and therefore homology models that provide structural information are extremely valuable for the development of inhibitors of cytochrome P450 family 26 (CYP26). The objectives of this study were to use homology models of CYP26A1 and CYP26B1 to characterize substrate binding characteristics, to compare structural aspects of their active sites, and to support the role of CYP26 in the metabolism of xenobiotics. Each model was verified by dockingat-RA in the active site and comparing the results to known metabolic profiles ofat-RA. The models were then used to predict the metabolic sites of tazarotenic acid with results verified by in vitro metabolite identification experiments. The CYP26A1 and CYP26B1 homology models predicted that the benzothiopyranyl moiety of tazarotenic acid would be oriented toward the heme of each enzyme and suggested that tazarotenic acid would be a substrate of CYP26A1 and CYP26B1. Metabolite identification experiments indicated that CYP26A1 and CYP26B1 oxidatively metabolized tazarotenic acid on the predicted moiety, with in vitro rates of metabolite formation by CYP26A1 and CYP26B1 being the highest across a panel of enzymes. Molecular analysis of the active sites estimated the active-site volumes of CYP26A1 and CYP26B1 to be 918 A(3)and 977 A(3), respectively. Overall, the homology models presented herein describe the enzyme characteristics leading to the metabolism of tazarotenic acid by CYP26A1 and CYP26B1 and support a potential role for the CYP26 enzymes in the metabolism of xenobiotics.
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induction of cyp26a1 by metabolites of retinoic acid evidence that cyp26a1 is an important enzyme in the elimination of active retinoids
Molecular Pharmacology, 2015Co-Authors: Ariel R Topletz, Wendel L Nelson, Robert S Foti, Sasmita Tripathy, Jakob A Shimshoni, Nina IsoherranenAbstract:All-trans-retinoic acid (atRA), the active metabolite of vitamin A, induces gene transcription via binding to nuclear retinoic acid receptors (RARs). The primary hydroxylated metabolites formed from atRA by CYP26A1, and the subsequent metabolite 4-oxo-atRA, bind to RARs and potentially have biologic activity. Hence, CYP26A1, the main atRA hydroxylase, may function either to deplete bioactive retinoids or to form active metabolites. This study aimed to determine the role of CYP26A1 in modulating RAR activation via formation and elimination of active retinoids. After treatment of HepG2 cells with atRA, (4S)-OH-atRA, (4R)-OH-atRA, 4-oxo-atRA, and 18-OH-atRA, mRNAs of CYP26A1 and RARβ were increased 300- to 3000-fold, with 4-oxo-atRA and atRA being the most potent inducers. However, >60% of the 4-OH-atRA enantiomers were converted to 4-oxo-atRA in the first 12 hours of treatment, suggesting that the activity of the 4-OH-atRA was due to 4-oxo-atRA. In human hepatocytes, atRA, 4-OH-atRA, and 4-oxo-atRA induced CYP26A1 and 4-oxo-atRA formation was observed from 4-OH-atRA. In HepG2 cells, 4-oxo-atRA formation was observed even in the absence of CYP26A1 activity and this formation was not inhibited by ketoconazole. In human liver microsomes, 4-oxo-atRA formation was supported by NAD+, suggesting that 4-oxo-atRA formation is mediated by a microsomal alcohol dehydrogenase. Although 4-oxo-atRA was not formed by CYP26A1, it was depleted by CYP26A1 (Km = 63 nM and intrinsic clearance = 90 μl/min per pmol). Similarly, CYP26A1 depleted 18-OH-atRA and the 4-OH-atRA enantiomers. These data support the role of CYP26A1 to clear bioactive retinoids, and suggest that the enzyme forming active 4-oxo-atRA may be important in modulating retinoid action.
Allan Sirsjö - One of the best experts on this subject based on the ideXlab platform.
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homology models of human all trans retinoic acid metabolizing enzymes CYP26B1 and CYP26B1 spliced variant
Journal of Chemical Information and Modeling, 2012Co-Authors: Patricia Saenzmendez, Åke Strid, Allan Sirsjö, Ali Ateia Elmabsout, Helena Savenstrand, Mohamed Khalid Alhaj Awadalla, Leif A. ErikssonAbstract:Homology models of CYP26B1 (cytochrome P450RAI2) and CYP26B1 spliced variant were derived using the crystal structure of cyanobacterial CYP120A1 as template for the model building. The quality of the homology models generated were carefully evaluated, and the natural substrate all-trans-retinoic acid (atRA), several tetralone-derived retinoic acid metabolizing blocking agents (RAMBAs), and a well-known potent inhibitor of CYP26B1 (R115866) were docked into the homology model of full-length cytochrome P450 26B1. The results show that in the model of the full-length CYP26B1, the protein is capable of distinguishing between the natural substrate (atRA), R115866, and the tetralone derivatives. The spliced variant of CYP26B1 model displays a reduced affinity for atRA compared to the full-length enzyme, in accordance with recently described experimental information.
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CYP26B1 plays a major role in the regulation of all trans retinoic acid metabolism and signaling in human aortic smooth muscle cells
Journal of Vascular Research, 2011Co-Authors: Pauline Ocaya, Ali Ateia Elmabsout, Andreas C Gidlof, Peder S Olofsson, Hans Torma, Allan SirsjöAbstract:Aim: The cytochrome P450 enzymes of the CYP26 family are involved in the catabolism of the biologically active retinoid all-trans-retinoic acid (atRA). Since it is possible that an increased local CYP26 activity would reduce the effects of retinoids in vascular injury, we investigated the role of CYP26 in the regulation of atRA levels in human aortic smooth muscle cells (AOSMCs). Methods: The expression of CYP26 was investigated in cultured AOSMCs using real-time PCR. The metabolism of atRA was analyzed by high-performance liquid chromatography, and the inhibitor R115866 or small interfering RNA (siRNA) was used to suppress CYP26 activity/expression. Results: AOSMCs expressed CYP26B1 constitutively and atRA exposure augmented CYP26B1 mRNA levels. Silencing of the CYP26B1 gene expression or reduction of CYP26B1 enzymatic activity by using siRNA or the inhibitor R115866, respectively, increased atRA-mediated signaling and resulted in decreased cell proliferation. The CYP26 inhibitor also induced expression of atRA-responsive genes. Therefore, atRA-induced CYP26 expression accelerated atRA inactivation in AOSMCs, giving rise to an atRA-CYP26 feedback loop. Inhibition of this loop with a CYP26 inhibitor increased retinoid signaling. Conclusion: The results suggest that CYP26 inhibitors may be a therapeutic alternative to exogenous retinoid administration.
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cloning and functional studies of a splice variant of CYP26B1 a cellular storage protein for all trans retinoic acid
in Vivo, 2010Co-Authors: Ali Ateia Elmabsout, Magnus Karlsson, Åke Strid, Leif A. Eriksson, Olesya Krivospitskaya, Helena Savenstrand, Ashok Kumar Kumawat, Torma Hans, Allan SirsjöAbstract:BackgroundAll-trans retinoic acid (atRA) plays an essential role in the regulation of gene expression, cell growth and differentiation and is also important for normal cardiovascular development but may in turn be involved in cardiovascular diseases, i.e. atherosclerosis and restenosis. The cellular atRA levels are under strict control involving several cytochromes P450 isoforms (CYPs). CYP26 may be the most important regulator of atRA catabolism in vascular cells. The present study describes the molecular cloning, characterization and function of atRA-induced expression of a spliced variant of the CYP26B1 gene.Methodology/Principal FindingsThe coding region of the spliced CYP26B1 lacking exon 2 was amplified from cDNA synthesized from atRA-treated human aortic smooth muscle cells and sequenced. Both the spliced variant and full length CYP26B1 was found to be expressed in cultured human endothelial and smooth muscle cells, and in normal and atherosclerotic vessel. atRA induced both variants of CYP26B1 in cultured vascular cells. Furthermore, the levels of spliced mRNA transcript were 4.5 times higher in the atherosclerotic lesion compared to normal arteries and the expression in the lesions was increased 20-fold upon atRA treatment. The spliced CYP26B1 still has the capability to degrade atRA, but at an initial rate one-third that of the corresponding full length enzyme. Transfection of COS-1 and THP-1 cells with the CYP26B1 spliced variant indicated either an increase or a decrease in the catabolism of atRA, probably depending on the expression of other atRA catabolizing enzymes in the cells.Conclusions/SignificanceVascular cells express the spliced variant of CYP26B1 lacking exon 2 and it is also increased in atherosclerotic lesions. The spliced variant displays a slower and reduced degradation of atRA as compared to the full-length enzyme. Further studies are needed, however, to clarify the substrate specificity and role of the CYP26B1 splice variant in health and disease.
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Homology Models and Molecular Modeling of Human Retinoic Acid Metabolizing Enzymes Cytochrome P450 26A1 (CYP26A1) and P450 26B1 (CYP26B1).
Journal of chemical theory and computation, 2008Co-Authors: Magnus Karlsson, Åke Strid, Allan Sirsjö, Leif A. ErikssonAbstract:Homology models of cytochrome P450 26A1 and cytochrome P450 26B1 were constructed using the crystal structures of human, CYP2C8, CYP2C9, and CYP3A4 as templates for the model building. The homology models generated were investigated for their docking capacities against the natural substrate all-trans-retinoic acid (atRA), five different tetralone-derived retinoic acid metabolizing blocking agents (RAMBAs), and R115866. Interaction energies (IE) and linear interaction energies (LIE) were calculated for all inhibitors in both homology models after molecular dynamics (MD) simulation of the enzyme-ligand complexes. The results revealed that the homologues had the capacity to distinguish between strong and weak inhibitors. Important residues in the active site were identified from the CYP26A1/B1-atRA complexes. Residues involved in hydrophobic interactions with atRA were Pro113, Phe222, Phe299, Val370, Pro371, and Phe374 in CYP26A1 and Leu88, Pro118, Phe222, Phe295, Ile368, and Tyr272 in CYP26B1. Hydrogen bonding interactions were observed between the atRA carboxylate group and Arg 90 in CYP26A1 and with Arg76, Arg95, and Ser369 in CYP26B1.
