Xenopus cytosolic thyroid hormone-binding protein (xCTBP) is aldehyde dehydrogenase catalyzing the formation of retinoic acid.
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Amino acid sequencing of an internal peptide fragment derived from purified Xenopus cytosolic thyroid hormone-binding protein (xCTBP) demonstrates high similarity to the corresponding sequence of mammalian aldehyde dehydrogenase 1 (ALDH1) (Yamauchi, K., and Tata, J. R. (1994) Eur. J. Biochem. 225, 1105-1112). Here we show that xCTBP was co-purified with ALDH and 3,3',5-triiodo-L-thyronine (T3) binding activities. By photoaffinity labeling with [125I]T3, a T3-binding site in the xCTBP was estimated to reside in amino acid residues 93-114, which is distinct from the active site of the enzyme but present in the NAD+ binding domain. The amino acid sequences deduced from the two isolated xALDH1 cDNAs (xALDH1-I and xALDH1-II) were 94.6% identical to each other and very similar to those of mammalian ALDH1 enzymes. The two recombinant xALDH1 proteins exhibit both T3 binding activity and ALDH activity converting retinal to retinoic acid (RA), which are similar to those of xCTBP. The mRNAs were present abundantly in kidney and intestine of adult female Xenopus. Interestingly, their T3 binding activities were inhibited by NAD+ and NADH but not by NADP+ and NADPH, whereas NAD+ was required for their ALDH activities. Our results demonstrate that xCTBP is identical to ALDH1 and suggest that this protein might modulate RA synthesis and intracellular level of free T3. (+info)
Stimulation of premature retinoic acid synthesis in Xenopus embryos following premature expression of aldehyde dehydrogenase ALDH1.
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In order for nuclear retinoic acid receptors to mediate retinoid signaling, the ligand retinoic acid must first be produced from its vitamin A precursor retinal. Biochemical studies have shown that retinal can be metabolized in vitro to retinoic acid by members of the aldehyde dehydrogenase enzyme family, including ALDH1. Here we describe the first direct evidence that ALDH1 plays a physiological role in retinoic acid synthesis by analysis of retinoid signaling in Xenopus embryos, which have plentiful stores of maternally derived retinal. The Xenopus ALDH1 gene was cloned and shown to be highly conserved with chick and mammalian homologs. Xenopus ALDH1 was not expressed at blastula and gastrula stages, but was expressed at the neurula stage. We used a retinoic acid bioassay to demonstrate that retinoic acid is normally undetectable in embryos from fertilization to the initial gastrula stage, but that a tremendous increase in retinoic acid occurs during neurulation when ALDH1 is first expressed. Overexpression of ALDH1 by injection of Xenopus embryos with mRNAs encoding the mouse, chick or Xenopus ALDH1 homologs induced high levels of retinoic acid detection during the blastula stage. Thus, premature expression of ALDH1 stimulates premature synthesis of retinoic acid. These findings reveal an important conserved role for ALDH1 in retinoic acid synthesis in vivo, and demonstrate that conversion of retinoids from the aldehyde form to the carboxylic acid form is a crucial regulatory step in retinoid signaling. (+info)
Dorsal and ventral retinal territories defined by retinoic acid synthesis, break-down and nuclear receptor expression.
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Determination of the dorso-ventral dimension of the vertebrate retina is known to involve retinoic acid (RA), in that high RA activates expression of a ventral retinaldehyde dehydrogenase and low RA of a dorsal dehydrogenase. Here we show that in the early eye vesicle of the mouse embryo, expression of the dorsal dehydrogenase is preceded by, and transiently overlaps with, the RA-degrading oxidase CYP26. Subsequently in the embryonic retina, CYP26 forms a narrow horizontal boundary between the dorsal and ventral dehydrogenases, creating a trough between very high ventral and moderately high dorsal RA levels. Most of the RA receptors are expressed uniformly throughout the retina except for the RA-sensitive RARbeta, which is down-regulated in the CYP26 stripe. The orphan receptor COUP-TFII, which modulates RA responses, colocalizes with the dorsal dehydrogenase. The organization of the embryonic vertebrate retina into dorsal and ventral territories divided by a horizontal boundary has parallels to the division of the Drosophila eye disc into dorsal, equatorial and ventral zones, indicating that the similarities in eye morphogenesis extend beyond single molecules to topographical patterns. (+info)
Differential distribution of retinoic acid synthesis in the chicken embryo as determined by immunolocalization of the retinoic acid synthetic enzyme, RALDH-2.
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Retinaldehyde dehydrogenase type 2 (RALDH-2) is a major retinoic acid generating enzyme in the early embryo. Here we report the immunolocalization of this enzyme (RALDH-2-IR) in stage 6-29 chicken embryos; we also show that tissues that exhibit strong RALDH-2-IR in the embryo contain RALDH-2 and synthesize retinoic acid. RALDH-2-IR indicates dynamic and discrete patterns of retinoic acid synthesis in the embryo, particularly within the somitic mesoderm, lateral mesoderm, kidney, heart, and spinal motor neurons. Prior to somitogenesis, RALDH-2-IR is present in the paraxial mesoderm with a rostral boundary at the level of the presumptive first somite; as the somites form, they exhibit strong RALDH-2-IR. Cervical presomitic mesoderm exhibits RALDH-2-IR but thoracic presomitic mesoderm does not. Neural crest cells do not express detectable levels of RALDH-2, but migrating crest cells are associated with RALDH-2 expressing mesoderm. The developing limb mesoderm expresses little RALDH-2-IR; however, RALDH-2-IR is strongly expressed in tissues adjacent to the limb. The most lateral, earliest-projecting motor neurons at all levels of the spinal cord exhibit RALDH-2-IR. Subsequently, many additional motor neurons in the brachial and lumbar cord regions express RALDH-2-IR. Motor neuronal expression of RALDH-2-IR is present in the growing axons as they extend to the periphery, indicating a potential role of retinoic acid in nerve influences on peripheral differentiation. With the exception of a transient expression in the facial/vestibulocochlear nucleus, cranial motor neurons do not express detectable levels of RALDH-2-IR. (+info)
Levels of retinoic acid and retinaldehyde dehydrogenase expression in eyes of the Mitf-vit mouse model of retinal degeneration.
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PURPOSE: Several reports have characterized the retinal degeneration observed in the Mitf(vit) mutant mouse. Despite these reports, the factor(s) that may cause or modulate the degeneration still are not well defined; however, it is known that the photoreceptors of Mitf(vit) mice die through an apoptotic mechanism. We reported previously that retinoid metabolism in the RPE of Mitf(vit)++ mice is perturbed. Retinoids regulate genes via the RAR and RXR nuclear receptor pathway that are involved in numerous cellular responses including apoptosis. It is possible that retinoic acid (RA) modulates the retinal degeneration observed in the Mitf(vit) mice. The purpose of this study was to evaluate the levels of RA in whole eyes, as well as its distribution between neural retina and RPE, of the Mitf(vit) mutant mouse model. An additional purpose was to examine the expression of the RA generating enzyme, retinaldehyde dehydrogenase (AHD2), in the eyes of mutant and control mice. METHODS: The distribution of AHD2 in eyes of pre- and postnatal Mitf(vit) and C57BL/6 wild-type mice was determined immunohistochemically. Quantitative and qualitative analyses of RA were performed using reversed-phase high performance liquid chromatography (HPLC). RESULTS: The distribution of AHD2 in ocular tissues was similar between pre- and postnatal Mitf(vit) and C57BL/6 control mice. At postnatal week 10, however, a marked increase in AHD2 immunoreactivity was noted in the central dorsal neural retina of Mitf(vit) mice. No differences in the level of total RA in whole eyes were noted between Mitf(vit) and control mice at early postnatal ages. By 10 weeks of age there was a significant elevation of RA that was localized to the neural retina. CONCLUSIONS: In this study, we show a high level of AHD2 and RA in the neural retina of Mitf(vit) mice relative to control mice. It is possible that this elevation of RAs contributes to the retinal degeneration observed in Mitf(vit) mice either by inducing apoptosis or by enhancing the effect of some other factor(s) involved in the apoptotic pathway. (+info)
Retinoic acid biosynthetic enzyme ALDH1 localizes in a subset of retinoid-dependent tissues during xenopus development.
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Control of retinoic acid synthesis in vertebrate organisms is undoubtedly important for regulating the numerous retinoid signaling events which occur during development. The mechanisms which accomplish this task involve enzymes such as class I aldehyde dehydrogenase (ALDH1), which has recently been found to be conserved from amphibians to mammals and which functions as a retinoic acid biosynthetic enzyme in vivo. Here we have found that Xenopus ALDH1 mRNA and protein is expressed in a subset of retinoid-dependent tissues which develop shortly after neurulation during the tail bud stages. ALDH1 mRNA was first clearly detectable by in situ hybridization in stage 28 tail bud embryos localized in the olfactory placode and pronephros, and at stage 35 mRNA was also detected in the pronephric duct. Antibodies were generated against Xenopus ALDH1, and immunohistochemistry was used to demonstrate that ALDH1 protein accumulates in the olfactory placode, pronephros, and dorsal retina at stage 28, and additionally in the lens placode and pronephric duct at stage 35. Neither ALDH1 mRNA nor protein was detected in the posterior region of Xenopus embryos during the tail bud stages. In contrast to neurula stage embryos in which retinoic acid is distributed in an anteroposterior gradient with the high end posteriorly, we found that tail bud stage embryos have retinoic acid present in significant levels in both the head and trunk regions, but with no detection in the posterior region. These findings are consistent with ALDH1 contributing to retinoic acid synthesis needed for development of certain head structures (olfactory placodes, dorsal retina, lens placode) and certain trunk structures (pronephros and pronephric duct). Dev Dyn 1999;215:264-272. (+info)
Identification of cytosolic aldehyde dehydrogenase 1 from non-small cell lung carcinomas as a flavopiridol-binding protein.
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The synthetic flavone flavopiridol can be cytostatic or cytotoxic to mammalian cells, depending on the concentration of the drug and the duration of exposure. It has been shown to inhibit the cyclin-dependent kinase (CDK) family of cell cycle regulatory enzymes. However, the existence of additional potential targets for drug action remains a matter of interest to define. To identify cellular targets, flavopiridol was immobilized. CDKs, particularly CDK 4, bound weakly to immobilized flavopiridol when ATP was absent but not in its presence. Two proteins with molecular weights of 40 kDa and 120 kDa had high affinities to the immobilized flavopiridol independent of the presence of ATP. They were present in all cell lines analyzed: cervical (HeLa), prostate and non-small cell lung carcinoma (NSCLC) cell lines. A 60-kDa protein, which was present only in NSCLC cells and bound similarly well to immobilized flavopiridol, was identified as cytosolic aldehyde dehydrogenase class 1 (ALDH-1). The level of this protein correlated with the resistance of NSCLC cell lines to cytotoxicity caused by 500 nM flavopiridol but not higher flavopiridol concentrations. Despite binding to ALDH-1, there was no inhibition of dehydrogenase activity by flavopiridol concentrations as high as 20 microM and flavopiridol was not metabolized by ALDH-1. The results suggest that high cellular levels of ALDH-1 may reduce cytotoxicity of flavopiridol and contribute to relative resistance to the drug. This is the first report that flavopiridol binds to proteins other than CDKs. (+info)
Interactions of retinoid binding proteins and enzymes in retinoid metabolism.
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Naturally occurring retinoids (vitamin A or retinol and its active metabolites) are vital for vision, controlling the differentiation program of epithelial cells in the digestive tract and respiratory system, skin, bone, the nervous system, the immune system, and for hematopoiesis. Retinoids are essential for growth, reproduction (conception and embryonic development), and resistance to and recovery from infection. The functions of retinoids in the embryo begin soon after conception and continue throughout the lifespan of all vertebrates. Both naturally occurring and synthetic retinoids are used in the therapy of various skin diseases, especially acne, for augmenting the treatment of diabetes, and as cancer chemopreventive agents. Retinol metabolites serve as ligands that activate specific transcription factors in the superfamily of steroid/retinoid/thyroid/vitamin D/orphan receptors and thereby control gene expression. Additionally, retinoids may also function through non-genomic actions. Various retinoid binding proteins serve as partners in retinoid function. These binding proteins show high specificity and affinity for specific retinoids and seem to control retinoid metabolism in vivo qualitatively and quantitatively by reducing 'free' retinoid concentrations, protecting retinoids from non-specific interactions, and chaperoning access of metabolic enzymes to retinoids. Implementation of the physiological effects of retinoids depends on the spatial-temporal expressions of binding proteins, receptors and metabolic enzymes. This review will discuss current understanding of the enzymes that catalyze retinol and retinoic acid metabolism and their unique and integral relationship to retinoid binding proteins. (+info)