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Pangala V. Bhat - One of the best experts on this subject based on the ideXlab platform.
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kinetic characterization of recombinant mouse Retinal Dehydrogenase types 3 and 4 for Retinal substrates
Biochimica et Biophysica Acta, 2009Co-Authors: Aurelia Sima, Sylvie Mader, Maxime Parisotto, Pangala V. BhatAbstract:Abstract Background Retinal Dehydrogenases (RALDHs) catalyze the dehydrogenation of Retinal into retinoic acids (RAs), which are required for embryogenesis and tissue differentiation. This study sought to determine the detailed kinetic properties of 2 mouse RALDHs, namely RALDH3 and 4, for Retinal isomer substrates, to better define their specificities in RA isomer synthesis. Methods RALDH3 and 4 were expressed in Escherichia coli as His-tagged proteins and affinity-purified. Enzyme kinetics were performed with Retinal isomer substrates. The enzymatic products were analyzed by high pressure liquid chromatography. Results RALDH3 oxidized all-trans Retinal with high catalytic efficiency (Vmax/Km = 77.9) but did not show activity for either 9-cis or 13-cis Retinal substrates. On the other hand, RALDH4 was inactive for all-trans Retinal substrate, exhibited high activity for 9-cis Retinal oxidation (Vmax/Km = 27.4), and oxidized 13-cis Retinal with lower catalytic efficiency (Vmax/Km = 8.24). β-ionone, a potent inhibitor of RALDH4 activity, suppressed 9-cis and 13-cis Retinal oxidation competitively with inhibition constants of 0.60 and 0.32, respectively, but had no effect on RALDH3 activity. The divalent cation MgCl2 activated 13-cis Retinal oxidation by RALDH4 by 3-fold, did not significantly influence 9-cis Retinal oxidation, and slightly activated RALDH3 activity. Conclusions These data extend the kinetic characterization of RALDH3 and 4, providing their specificities for Retinal isomer substrates. General significance The kinetic characterization of RALDHs should give useful information in determining amino acid residues that are involved in the specificity for Retinal isomers and on the role of these enzymes in the synthesis of RAs in specific tissues.
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Kinetic characterization of recombinant mouse Retinal Dehydrogenase types 3 and 4 for Retinal substrates.
Biochimica et biophysica acta, 2009Co-Authors: Aurelia Sima, Sylvie Mader, Maxime Parisotto, Pangala V. BhatAbstract:Retinal Dehydrogenases (RALDHs) catalyze the dehydrogenation of Retinal into retinoic acids (RAs), which are required for embryogenesis and tissue differentiation. This study sought to determine the detailed kinetic properties of 2 mouse RALDHs, namely RALDH3 and 4, for Retinal isomer substrates, to better define their specificities in RA isomer synthesis. RALDH3 and 4 were expressed in Escherichia coli as His-tagged proteins and affinity-purified. Enzyme kinetics were performed with Retinal isomer substrates. The enzymatic products were analyzed by high pressure liquid chromatography. RALDH3 oxidized all-trans Retinal with high catalytic efficiency (Vmax/Km=77.9) but did not show activity for either 9-cis or 13-cis Retinal substrates. On the other hand, RALDH4 was inactive for all-trans Retinal substrate, exhibited high activity for 9-cis Retinal oxidation (Vmax/Km=27.4), and oxidized 13-cis Retinal with lower catalytic efficiency (Vmax/Km=8.24). beta-ionone, a potent inhibitor of RALDH4 activity, suppressed 9-cis and 13-cis Retinal oxidation competitively with inhibition constants of 0.60 and 0.32, respectively, but had no effect on RALDH3 activity. The divalent cation MgCl2 activated 13-cis Retinal oxidation by RALDH4 by 3-fold, did not significantly influence 9-cis Retinal oxidation, and slightly activated RALDH3 activity. These data extend the kinetic characterization of RALDH3 and 4, providing their specificities for Retinal isomer substrates. The kinetic characterization of RALDHs should give useful information in determining amino acid residues that are involved in the specificity for Retinal isomers and on the role of these enzymes in the synthesis of RAs in specific tissues.
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Isomer-specific retinoic acid biosynthesis in HeLa cells expressing recombinant class I aldehyde Dehydrogenases.
Biochimica et biophysica acta, 2007Co-Authors: Hélène Brodeur, Sylvie Mader, Maxime Parisotto, Samuel Chagnon, Pangala V. BhatAbstract:Retinal Dehydrogenase type 1 (RALDH1) catalyzes the oxidation of all-trans and 9-cis Retinal to the respective retinoic acids (RAs), whereas another member of the aldehyde Dehydrogenase family, the phenobarbital-induced aldehyde Dehydrogenase (PB-ALDH), is very poorly active. We have previously generated chimeras between these two enzymes that displayed selectivity for Retinal isomers in crude bacterial extracts. To examine whether the selectivity of the recombinant enzymes is retained in intact cells, we first assessed whether retinoid-isomerizing activity is present in cultured eukaryotic cells. Our results demonstrate that the only RA isomers detected in RALDH1-expressing or non-expressing cells corresponded to the same steric conformation as the supplied retinoids, indicating a lack of measurable 9-cis/all-trans retinoid-isomerizing activity. Finally, HeLa cells transfected with RALDH1 derivatives that were Retinal isomer-selective in vitro produced only the corresponding RA isomers, establishing these enzymes as useful tools to assess the respective roles of the two RA isomers in vivo.
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Kinetic properties of chimeric class I aldehyde Dehydrogenases for Retinal isomers
Biochemistry and cell biology = Biochimie et biologie cellulaire, 2006Co-Authors: Hélène Brodeur, Sylvie Mader, Maxime Parisotto, Samuel Chagnon, Pangala V. BhatAbstract:Retinal Dehydrogenase type 1 (RALDH1) catalyzes the oxidation of all-trans and 9-cis Retinal to the respective retinoic acids (RAs), whereas another member of the aldehyde Dehydrogenase (ALDH) family, the phenobarbital-induced aldehyde Dehydrogenase (PB-ALDH), is very poorly active. We have previously generated chimeras between these 2 enzymes that displayed selectivity for Retinal isomers in crude bacterial extracts. Here we have characterized the kinetic properties of the corresponding purified recombinant proteins. The all-trans selective chimera RALDH-131 converted all-trans Retinal to all-trans RA with 2.9-fold lower efficiency than the wild-type RALDH1 and had only residual activity with 9-cis Retinal. The converse chimera PB-131 was specific for 9-cis Retinal, with no residual activity for all-trans Retinal. MgCl2 inhibited the activities of RALDH1 and PB-131, but not of RALDH-131, suggesting that amino acids 132-510 in RALDH are necessary for inhibition by MgCl2. These data demonstrate that the chimeric enzymes act as Retinal isomer-selective ALDHs, and suggest that these enzymes may be useful to study the roles of cis RA isomers in embryogenesis and differentiation in vivo.
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Enzymatic characterization of recombinant mouse Retinal Dehydrogenase type 1
Biochemical pharmacology, 2003Co-Authors: Isabelle Gagnon, Gregg Duester, Pangala V. BhatAbstract:Retinal Dehydrogenases (RALDHs) convert Retinal into retinoic acids (RAs), which are important signaling molecules in embryogenesis and tissue differentiation. We expressed mouse RALDH type 1 (mRALDH1) in Escherichia coli and studied the kinetic properties of the recombinant enzyme for Retinal substrates. Purified recombinant mRALDH1 catalyzed the oxidation of all-trans and 9-cis Retinal but not 13-cis Retinal, and exhibited two pH optimums, 7.8 and 9.4, for all-trans and 9-cis Retinal substrates, respectively. The K(m) for all-trans Retinal (11.6 micro M) was 3-fold higher than for 9-cis Retinal (3.59 micro M). However, the conversion efficiencies of either all-trans or 9-cis Retinal to the respective RAs were similar. MgCl(2) inhibited the oxidation of both all-trans and 9-cis Retinal. Chloral hydrate and acetaldehyde competitively suppressed all-trans Retinal oxidation with inhibition constants (K(i)) of 4.99 and 49.4 micro M, respectively. Retinol, on the other hand, blocked the reaction uncompetitively. These data extend the kinetic characterization of mRALDH1, provide insight into the possible role of this enzyme in the biogenesis of RAs, and should give useful information on the determination of amino acid residues that play crucial roles in the catalysis of all-trans and 9-cis Retinal.
Joseph L. Napoli - One of the best experts on this subject based on the ideXlab platform.
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Mouse Retinal Dehydrogenase 4 (RALDH4), molecular cloning, cellular expression, and activity in 9-cis-retinoic acid biosynthesis in intact cells.
The Journal of biological chemistry, 2003Co-Authors: Min Lin, Min Zhang, Michael Abraham, Susan M. Smith, Joseph L. NapoliAbstract:This study describes cDNA cloning and characterization of mouse RALDH4. The 2.3-kb cDNA encodes an aldehyde Dehydrogenase of 487 amino acid residues, about two-orders of magnitude more active in vitro with 9-cis-Retinal than with all-trans-Retinal. RALDH4 recognizes as substrate 9-cis-Retinal generated in transfected cells by the short-chain Dehydrogenases CRAD1, CRAD3, or RDH1, to reconstitute a path of 9-cis-retinoic acid biosynthesis in situ. Northern blot analysis showed expression of RALDH4 mRNA in adult mouse liver and kidney. In situ hybridization revealed expression of RALDH4 in liver on embryo day 14.5, in adult hepatocytes, and kidney cortex. Immunohistochemistry confirmed RALDH4 expression in hepatocytes and showed that hepatocytes also express RALDH1, RALDH2, and RALDH3. Kidney expresses the RALDH4 protein primarily in the proximal and distal convoluted tubules of the cortex but not in the glomeruli or the medulla. Kidney expresses RALDH2 in the proximal convoluted tubules of the cortex but not in the distal convoluted tubules or glomeruli. Kidney expresses RALDH1 and RALDH2 in the medulla. The enzymatic characteristics of RALDH4, its expression in fetal liver, and its unique expression pattern in adult kidney compared with RALDH1, -2, and -3 suggest that it could meet specific needs for 9-cis-retinoic acid biosynthesis.
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Molecular characterization of a mouse short chain Dehydrogenase/reductase active with all-trans-retinol in intact cells, mRDH1.
The Journal of biological chemistry, 2001Co-Authors: Min Zhang, Susan M. Smith, Weiguo Chen, Joseph L. NapoliAbstract:Abstract Metabolic activation of retinol (vitamin A) via sequential actions of retinol and Retinal Dehydrogenases produces the active metabolite all-trans-retinoic acid. This work reports cDNA cloning, enzymatic characterization, function in a reconstituted path of all-trans-retinoic acid biosynthesis in cell culture, and mRNA expression patterns in adult tissues and embryos of a mouse retinol Dehydrogenase, RDH1. RDH1 represents a new member of the short chain Dehydrogenase/reductase superfamily that differs from other mouse RDH in relative activity with all-trans and cis-retinols. RDH1 has a multifunctional catalytic nature, as do other short chain Dehydrogenase/reductases. In addition to retinol Dehydrogenase activity, RDH1 has strong 3α-hydroxy and weak 17β-hydroxy steroid Dehydrogenase activities. RDH1 has widespread and intense mRNA expression in tissues of embryonic and adult mice. The mouse embryo expresses RDH1 as early as 7.0 days post-coitus, and expression is especially intense within the neural tube, gut, and neural crest at embryo day 10.5. Cells cotransfected with RDH1 and any one of three Retinal Dehydrogenase isozymes synthesize all-trans-retinoic acid from retinol, demonstrating that RDH1contributes to a path of all-trans-retinoic acid biosynthesis in intact cells. These characteristics are consistent with RDH1 functioning in a path of all-trans-retinoic acid biosynthesis starting early during embryogenesis.
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Analysis of mouse Retinal Dehydrogenase type 2 promoter and expression.
Genomics, 2001Co-Authors: Xianshu Wang, Zuzana Sperkova, Joseph L. NapoliAbstract:The mouse RALDH2 gene spans >50 kb, has a structure similar to that of human class 1 aldehyde Dehydrogenase genes, and localizes to the central region of chromosome 9 by single-strand polymorphism analysis. Expression of mouse RALDH2 was detected in testis, lung, brain, and heart (Northern blot) and in liver and kidney (RNase protection assays). Expression was not detected by RNase protection assay in testis of vitamin A-deficient rats, and all-trans-retinoic acid dosing did not increase expression in vitamin A-deficient rat testis. A 2.3-kb section of the gene 5' to the transcription start site included neither retinoic acid nor retinoid X response elements, but included TATA and CCAAT motifs and AP, AHR, CREB, ER, Ets, and SREBP sites. The promoter initiated transcription of a luciferase reporter in human embryonic kidney cells (EBNA) and mouse Leydig- (TM3) and Sertoli-derived (TM4) cell lines, but neither all-trans-retinoic acid nor 9-cis-retinoic acid affected reporter transcription. These data suggest that relatively weak RALDH2 expression in vitamin A-deficient testis reflects vastly decreased numbers of germ cells, the major site of expression.
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Cellular expression of Retinal Dehydrogenase types 1 and 2: effects of vitamin A status on testis mRNA.
Journal of cellular physiology, 2001Co-Authors: Yan Zhai, Zuzanna Sperkova, Joseph L. NapoliAbstract:We examined expression of Retinal Dehydrogenase (RALDH) types 1 and 2 in liver and lung, and the effect of vitamin A status on testis expression by in situ hybridization. Liver expressed RALDH1 and RALDH2 only in stellate cells and hepatocytes, respectively. Lung expressed RALDH1 and RALDH2 throughout the epithelia of the airways, from the principal bronchi to the respiratory bronchiole. Vitamin A-sufficient rats expressed RALDH1 in spermatocytes, with less intense expression in spermatogonia and spermatids, and expressed RALDH2 in interstitial cells, spermatogonia, and spermatocytes. Neither Sertoli nor peritubular cells showed detectable RALDH1 or RALDH2 mRNA. Vitamin A deficiency produced a sevenfold increase in RALDH1 and a 70-fold decrease in RALDH2 mRNA in testis. In each case, the net change reflected extensive loss of germ cells, increased intensity of expression in residual germ cells, and expression in Sertoli and peritubular cells. Low-dose RA relatively early during vitamin A depletion supported spermatogenesis and affected expression of both RALDHs, but did not reinstate “vitamin A normal” expression patterns. These results show that: RALDH1 and RALDH2 have distinct mRNA expression patterns in multiple cell types in three vitamin A target tissues; RALDH expression occurs in cell types that express cellular retinol-binding protein and retinol Dehydrogenase isozymes (except stellate cells, for which retinol Dehydrogenase expression remains unknown); vitamin A deficiency and RA supplementation affects the loci and intensity of RALDH mRNAs in testis; and low-dose RA does not substitute completely for retinol. Overall, these data provide insight into the unique functions of RALDH1 and RALDH2 in retinoid metabolism. J. Cell. Physiol. 186:220–232, 2001. © 2001 Wiley-Liss, Inc.
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cdna cloning and expression of a human aldehyde Dehydrogenase aldh active with 9 cis Retinal and identification of a rat ortholog aldh12
Journal of Biological Chemistry, 2000Co-Authors: Min Lin, Joseph L. NapoliAbstract:This report describes the isolation of a heretofore uncharacterized aldehyde Dehydrogenase (ALDH) with Retinal Dehydrogenase activity from rat kidney and the cloning and expression of a cDNA that encodes its human ortholog, the previously unknown ALDH12. The human ALDH12 cDNA predicts a 487-residue protein with the 23 invariant amino acids, four conserved regions, cofactor binding motif (G209 XGX3G), and active site cysteine residue (Cys287) that typify members of the ALDH superfamily. ALDH12 seems at least as efficient (Vm/Km) in converting 9-cis-Retinal into the retinoid X receptor ligand 9-cis-retinoic acid as two previously identified ALDHs with 9-cis-Retinal Dehydrogenase activity, rat Retinal Dehydrogenase (RALDH) 1 and RALDH2. ALDH12, however, has ∼40-fold higher activity with 9-cis- Retinal than with all-trans-Retinal, whereas RALDH1 and RALDH2 have equivalent and ∼4-fold less efficiencies for 9-cis-Retinal versusall-trans-Retinal, respectively. Therefore, ALDH12 is the first known ALDH to show a preference for 9-cis-Retinal relative to all-trans-Retinal. Evidence consistent with the possibility that ALDH12 could function in a pathway of 9-cis-retinoic acid biosynthesis in vivoincludes biosynthesis of 9-cis-retinoic acid from 9-cis-retinol in cells co-transfected with cDNAs encoding ALDH12 and the 9-cis-retinol/androgen Dehydrogenase, cis-retinoid/androgen Dehydrogenase type 1. Intense ALDH12 mRNA expression in adult and fetal liver and kidney, two organs that reportedly have relatively high concentrations of 9-cis-retinol, reinforces this notion.
David E. Ong - One of the best experts on this subject based on the ideXlab platform.
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Immunolocalization of Enzymes, Binding Proteins, and Receptors Sufficient for Retinoic Acid Synthesis and Signaling During the Hair Cycle
The Journal of investigative dermatology, 2007Co-Authors: Helen B. Everts, John P. Sundberg, Lloyd E. King, David E. OngAbstract:Retinoic acid (RA) is essential for maintenance of most epithelial tissues. One RA biosynthesis pathway consists of cellular retinol-binding protein (Crbp), retinol Dehydrogenase (Dhrs9/eRoldh), Retinal Dehydrogenase 1-3 (Aldh1a1-3), and cellular RA-binding protein 2 (Crabp2). Previously, we localized Aldh1a2 and Aldh1a3 to both epithelial and mesenchymal cells within the hair follicle throughout the hair cycle. This study expands that observation by examining the complete pathway of RA biosynthesis and signaling via RA receptors α , β , and γ by immunohistochemistry in C57BL/6J mice wax-stripped to initiate and synchronize the cycle. This pathway of RA biosynthesis and signaling localized to the majority of layers of the hair follicle, sebaceous gland, and interfollicular epidermis in a hair cycle-dependent manner, suggesting that RA biosynthesis within the hair follicle is regulated in both a spatial and temporal manner. This localization pattern also revealed insights into epithelial–mesenchymal interactions and differentiation state differences within the RA biosynthesis and signaling pathway, as well as novel observations on nuclear versus cytoplasmic localization of Crabp2 and RA receptors. This complex pattern of RA biosynthesis and signaling identified by immunolocalization suggests that endogenous RA regulates specific aspects of hair follicle growth, differentiation, and cycling.
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Immunolocalization of retinoic acid biosynthesis systems in selected sites in rat.
Experimental cell research, 2005Co-Authors: Helen B. Everts, John P. Sundberg, David E. OngAbstract:Vitamin A deficiency leads to focal metaplasia of numerous epithelial tissues with altered differentiation from columnar (in general) to stratified squamous cells. This process can be reversed with vitamin A repletion. Previously, we described a system of retinoic acid (RA) synthesis in the cycling rat uterus consisting of cellular retinol binding protein (Crbp), epithelial retinol Dehydrogenase (eRoldh), Retinal Dehydrogenase 2 (Aldh1a2), and cellular retinoic acid binding protein type II (Crabp2). Western blot analysis, RT-PCR, and immunohistochemistry were performed to test whether this retinoic acid synthesis system was also present in other vitamin A sensitive tissues. We found that combinations of Crbp, eRoldh, Aldh1a2 or Aldh1a3, and Crabp2 were present in all vitamin A sensitive tissues examined. In the ureter, while eRoldh was present, another short chain alcohol Dehydrogenase reductase (possibly Roldh 1, 2, or 3) was in higher concentration in the transitional epithelia. In several tissues, Crbp, Aldh1a2, and/or Aldh1a3 localized to mesenchyme and/or epithelial cells, while eRoldh and Crabp2 were expressed only in epithelial cells. This suggests that mesenchymal-epithelial interactions may be as important in the adult as they are during development and that local synthesis of RA is important in maintenance of these tissues.
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Estrogen Directly Induces Expression of Retinoic Acid Biosynthetic Enzymes, Compartmentalized between the Epithelium and Underlying Stromal Cells in Rat Uterus
Endocrinology, 2004Co-Authors: Bharati Kakkad, David E. OngAbstract:Estrogen (E2) has been shown to induce the biosynthesis of retinoic acid (RA) in rat uterus. Here we examined whether E2 could directly induce the enzymes involved in this process by using the ovariectomized rat. A retinol Dehydrogenase that we have previously described, eRolDH, and the Retinal Dehydrogenase, RalDH II, were found to have markedly increased uterine mRNA levels withi n4ho f E 2 administration, independent of the prior administration of puromycin. eRolDH and RalDH II and their mRNAs were also increased in uteri of rats during estrus. This indicated that RA biosynthesis in rat uterus is directly controlled by E2 and provides a direct link between the action of a steroid hormone and retinoid action. We also examined the cell-specific localization of RalDH II by immunohistochemistry. The enzyme was observed in the stromal compartment, particularly in cells close to the uterine lumenal epithelium. eRolDH was observed only in the lining epithelial cells. Taken together with the previous observations of cellular retinol-binding protein and cellular retinoic acid-binding protein, type two also being expressed in the lumenal epithelium, we propose that RA production is compartmentalized, with retinol oxidation occurring in the lumenal epithelium and subsequent oxidation of Retinal to RA occurring in the underlying stromal cells. (Endocrinology 145: 4756 – 4762, 2004)
Makoto Iwata - One of the best experts on this subject based on the ideXlab platform.
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Retinoic acid and GM-CSF coordinately induce Retinal Dehydrogenase 2 (RALDH2) expression through cooperation between the RAR/RXR complex and Sp1 in dendritic cells.
PloS one, 2014Co-Authors: Yoshiharu Ohoka, Aya Yokota-nakatsuma, Naoko Maeda, Hajime Takeuchi, Makoto IwataAbstract:Retinoic acid (RA)-producing dendritic cells (DCs) play critical roles in gut immunity. Retinal Dehydrogenase 2 (RALDH2) encoded by Aldh1a2 is a key enzyme for generating RA in DCs. Granulocyte-macrophage colony-stimulating factor (GM-CSF) potently induces RALDH2 expression in DCs in an RA-dependent manner, and RA alone weakly induces the expression. However, how GM-CSF and RA induce RALDH2 expression remains unclear. Here, we show that GM-CSF-induced activation of the transcription factor Sp1 and RA-dependent signaling via the RA receptor (RAR)/retinoid X receptor (RXR) complex contribute to Aldh1a2 expression. The RAR antagonist LE540 and the Sp1 inhibitor mithramycin A inhibited GM-CSF-induced Aldh1a2 expression in fms-related tyrosine kinase 3 ligand-generated bone marrow-derived DCs (BM-DCs). ERK and p38 MAPK inhibitors suppressed GM-CSF-induced nuclear translocation of Sp1 and Aldh1a2 expression. Sp1 and the RARα/RXRα complex bound to GC-rich Sp1-binding sites and an RA response element (RARE) half-site, respectively, near the TATA box in the mouse Aldh1a2 promoter. The DNA sequences around these sites were highly conserved among different species. In the presence of RA, ectopic expression of RARα/RXRα and Sp1 synergistically enhanced Aldh1a2 promoter-reporter activity. GM-CSF did not significantly induce Aldh1a2 expression in plasmacytoid DCs, peritoneal macrophages, or T cells, and the Aldh1a2 promoter in these cells was mostly unmethylated. These results suggest that GM-CSF/RA-induced RALDH2 expression in DCs requires cooperative binding of Sp1 and the RAR/RXR complex to the Aldh1a2 promoter, and can be regulated by a DNA methylation-independent mechanism.
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retinoic acid and gm csf coordinately induce Retinal Dehydrogenase 2 raldh2 expression through cooperation between the rar rxr complex and sp1 in dendritic cells
PLOS ONE, 2014Co-Authors: Yoshiharu Ohoka, Naoko Maeda, Hajime Takeuchi, Aya Yokotanakatsuma, Makoto IwataAbstract:Retinoic acid (RA)-producing dendritic cells (DCs) play critical roles in gut immunity. Retinal Dehydrogenase 2 (RALDH2) encoded by Aldh1a2 is a key enzyme for generating RA in DCs. Granulocyte–macrophage colony-stimulating factor (GM-CSF) potently induces RALDH2 expression in DCs in an RA-dependent manner, and RA alone weakly induces the expression. However, how GM-CSF and RA induce RALDH2 expression remains unclear. Here, we show that GM-CSF-induced activation of the transcription factor Sp1 and RA-dependent signaling via the RA receptor (RAR)/retinoid X receptor (RXR) complex contribute to Aldh1a2 expression. The RAR antagonist LE540 and the Sp1 inhibitor mithramycin A inhibited GM-CSF-induced Aldh1a2 expression in fms-related tyrosine kinase 3 ligand-generated bone marrow-derived DCs (BM-DCs). ERK and p38 MAPK inhibitors suppressed GM-CSF-induced nuclear translocation of Sp1 and Aldh1a2 expression. Sp1 and the RARα/RXRα complex bound to GC-rich Sp1-binding sites and an RA response element (RARE) half-site, respectively, near the TATA box in the mouse Aldh1a2 promoter. The DNA sequences around these sites were highly conserved among different species. In the presence of RA, ectopic expression of RARα/RXRα and Sp1 synergistically enhanced Aldh1a2 promoter-reporter activity. GM-CSF did not significantly induce Aldh1a2 expression in plasmacytoid DCs, peritoneal macrophages, or T cells, and the Aldh1a2 promoter in these cells was mostly unmethylated. These results suggest that GM-CSF/RA-induced RALDH2 expression in DCs requires cooperative binding of Sp1 and the RAR/RXR complex to the Aldh1a2 promoter, and can be regulated by a DNA methylation-independent mechanism.
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Retinoic acid production by intestinal dendritic cells.
Vitamins and hormones, 2011Co-Authors: Makoto Iwata, Aya YokotaAbstract:Subpopulations of dendritic cells (DCs) in the small intestine and its related lymphoid organs can produce retinoic acid (RA) from vitamin A (retinol). Through the RA production, these DCs play a pivotal role in imprinting lymphocytes with gut-homing specificity, and contribute to the development of immune tolerance by enhancing the differentiation of Foxp3(+) regulatory T cells and inhibiting that of inflammatory Th17 cells. The RA-producing capacity in these DCs mostly depends on the expression of Retinal Dehydrogenase 2 (RALDH2, ALDH1A2). It is likely that the RALDH2 expression is induced in DCs by the microenvironmental factors in the small intestine and its related lymphoid organs. The major factor responsible for the RALDH2 expression appears to be GM-CSF. RA itself is essential for the GM-CSF-induced RALDH2 expression. IL-4 and IL-13 also enhance RALDH2 expression, but are dispensable. Toll-like receptor-mediated signals can also enhance the GM-CSF-induced RALDH2 expression in immature DCs.
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Retinoic Acid Imprints Gut-Homing Specificity on T Cells
Immunity, 2004Co-Authors: Makoto Iwata, Asami Hirakiyama, Yuko Eshima, Hiroyuki Kagechika, Chieko Kato, Si-young SongAbstract:For a preferential homing of T cells to the gut, expression of the integrin alpha4beta7 and the chemokine receptor CCR9 is essential and is induced by antigenic stimulation with dendritic cells from the gut-associated lymphoid organs. Here, we show that the vitamin A (retinol) metabolite, retinoic acid, enhances the expression of alpha4beta7 and CCR9 on T cells upon activation and imprints them with the gut tropism. Dendritic cells from the gut-associated lymphoid organs produced retinoic acid from retinol. The enhanced alpha4beta7 expression on T cells by antigenic stimulation with these dendritic cells was suppressed by the Retinal Dehydrogenase inhibitor citral and the retinoic acid receptor antagonist LE135. Accordingly, vitamin A deficiency caused a reduction in alpha4beta7(+) memory/activated T cells in lymphoid organs and a depletion of T cells from the intestinal lamina propria. These findings revealed a novel role for retinoic acid in the imprinting of gut-homing specificity on T cells.
Helen B. Everts - One of the best experts on this subject based on the ideXlab platform.
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Dietary Vitamin A Dose Dependently Regulates BMP4 and WNT7A in Hair Follicles
The FASEB Journal, 2017Co-Authors: Helen B. Everts, Judy Reu, John P. Sundberg, Liye SuoAbstract:Hair cycles through a growing phase (anagen), apoptosis phase (categen), and resting phase (telogen). Telogen is further divided into refractory and competent telogen based on bone morphogenetic protein 4 (BMP4) expression. BMP4 inhibits anagen induction by directly inhibiting wingless-related MMTV integration site 7A (WNT7A) and other WNTs. Transient activation of WNT signaling in hair follicle stem cells triggers anagen induction; while sustained WNT signaling activation in hair follicle stem cells leads to skin cancer. Previously, we localized a complete set of proteins involved in retinoic acid (RA) synthesis and signaling to the hair follicle, whose expression changed throughout the hair cycle. We also found that dietary vitamin A altered the hair cycle in a dose dependent manner. The purpose of this study was to further define these hair cycle changes. We initially examined localization of RA synthesis proteins Retinal Dehydrogenase 2 (ALDH1A2), cellular RA binding protein 2 (CRABP2), RA degradation...
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Immunolocalization of Enzymes, Binding Proteins, and Receptors Sufficient for Retinoic Acid Synthesis and Signaling During the Hair Cycle
The Journal of investigative dermatology, 2007Co-Authors: Helen B. Everts, John P. Sundberg, Lloyd E. King, David E. OngAbstract:Retinoic acid (RA) is essential for maintenance of most epithelial tissues. One RA biosynthesis pathway consists of cellular retinol-binding protein (Crbp), retinol Dehydrogenase (Dhrs9/eRoldh), Retinal Dehydrogenase 1-3 (Aldh1a1-3), and cellular RA-binding protein 2 (Crabp2). Previously, we localized Aldh1a2 and Aldh1a3 to both epithelial and mesenchymal cells within the hair follicle throughout the hair cycle. This study expands that observation by examining the complete pathway of RA biosynthesis and signaling via RA receptors α , β , and γ by immunohistochemistry in C57BL/6J mice wax-stripped to initiate and synchronize the cycle. This pathway of RA biosynthesis and signaling localized to the majority of layers of the hair follicle, sebaceous gland, and interfollicular epidermis in a hair cycle-dependent manner, suggesting that RA biosynthesis within the hair follicle is regulated in both a spatial and temporal manner. This localization pattern also revealed insights into epithelial–mesenchymal interactions and differentiation state differences within the RA biosynthesis and signaling pathway, as well as novel observations on nuclear versus cytoplasmic localization of Crabp2 and RA receptors. This complex pattern of RA biosynthesis and signaling identified by immunolocalization suggests that endogenous RA regulates specific aspects of hair follicle growth, differentiation, and cycling.
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Immunolocalization of retinoic acid biosynthesis systems in selected sites in rat.
Experimental cell research, 2005Co-Authors: Helen B. Everts, John P. Sundberg, David E. OngAbstract:Vitamin A deficiency leads to focal metaplasia of numerous epithelial tissues with altered differentiation from columnar (in general) to stratified squamous cells. This process can be reversed with vitamin A repletion. Previously, we described a system of retinoic acid (RA) synthesis in the cycling rat uterus consisting of cellular retinol binding protein (Crbp), epithelial retinol Dehydrogenase (eRoldh), Retinal Dehydrogenase 2 (Aldh1a2), and cellular retinoic acid binding protein type II (Crabp2). Western blot analysis, RT-PCR, and immunohistochemistry were performed to test whether this retinoic acid synthesis system was also present in other vitamin A sensitive tissues. We found that combinations of Crbp, eRoldh, Aldh1a2 or Aldh1a3, and Crabp2 were present in all vitamin A sensitive tissues examined. In the ureter, while eRoldh was present, another short chain alcohol Dehydrogenase reductase (possibly Roldh 1, 2, or 3) was in higher concentration in the transitional epithelia. In several tissues, Crbp, Aldh1a2, and/or Aldh1a3 localized to mesenchyme and/or epithelial cells, while eRoldh and Crabp2 were expressed only in epithelial cells. This suggests that mesenchymal-epithelial interactions may be as important in the adult as they are during development and that local synthesis of RA is important in maintenance of these tissues.