Abstract
Retinoic acid is generated by a two-step mechanism. First, retinol is converted into retinal by a retinol dehydrogenase, and, subsequently, retinoic acid is formed by a retinal dehydrogenase. In vitro, several enzymes are suggested to act in this metabolic pathway. However, little is known regarding their capacity to contribute to retinoic acid biosynthesis in vivo. We have developed a versatile cell reporter system to analyze the role of several of these enzymes in 9-cis-retinoic acid biosynthesis in vivo. Using a Gal4-retinoid X receptor fusion protein-based luciferase reporter assay, the formation of 9-cis-retinoic acid from 9-cis-retinol was measured in cells transfected with expression plasmids encoding different combinations of retinol and retinal dehydrogenases. The results suggested that efficient formation of 9-cis-retinoic acid required co-expression of retinol and retinal dehydrogenases. Interestingly, the cytosolic alcohol dehydrogenase 4 failed to efficiently catalyze 9-cis-retinol oxidation. A structure-activity analysis showed that mutants of two retinol dehydrogenases, devoid of the carboxyl-terminal cytoplasmic tails, displayed greatly reduced enzymatic activities in vivo, but were active in vitro. The cytoplasmic tails mediate efficient endoplasmic reticulum localization of the enzymes, suggesting that the unique milieu in the endoplasmic reticulum compartment is necessary for in vivo activity of microsomal retinol dehydrogenases.
Highlights
Retinol and its derivatives are essential dietary compounds needed in a variety of physiological processes, e.g. embryonic development, reproduction, cell differentiation, postnatal growth, maintenance of the immune system, and vision (1–3)
A Reporter Assay Able to Measure the in Vivo Enzymatic Conversion of 9cROL to 9-cis-retinoic acid (9cRA) in Transfected Cells—Three different cell lines, JEG-3 cells, COS-1 cells, and 293 cells, were separately transfected with expression plasmids encoding the cis-retinol-specific dehydrogenase CRAD1 and the retinal dehydrogenase Raldh2 in an attempt to reconstruct a metabolic pathway able to generate 9cRA from 9cROL
The results showed that all three cell lines efficiently converted 9cROL to 9cRA when co-expressing CRAD1 and Raldh2 (Fig. 1A)
Summary
9cRA, 9-cis-retinoic acid; 9cROL, 9-cisretinol; 9cRAL, 9-cis-retinal; atRA, all-trans-retinoic acid; atROL, alltrans-retinol; atRAL, all-trans-retinal; CRAD, cis-retinol/androgen dehydrogenase; ER, endoplasmic reticulum; RA, retinoic acid; RAR, retinoic acid receptor; RDH, retinol dehydrogenase; RT, reverse transcription; PCR, polymerase chain reaction; RXR, retinoid X receptor; Raldh, retinal dehydrogenase; ADH, medium chain alcohol dehydrogenase; PAGE, polyacrylamide gel electrophoresis; HPLC, high performance liquid chromatography; CHO, Chinese hamster ovary. Two classes of RDHs have been implicated in oxidation of the different stereo isomers of retinol, i.e. microsomal members of the short chain alcohol dehydrogenase/reductases, and the cytosolic medium chain alcohol dehydrogenases (ADHs). Genetic evidence for a role of the microsomal RDHs in ocular retinoid metabolism comes from the identification of mutations in RDH5 in patients suffering from fundus albipunctatus (19, 20) These patients have a reduced rate of synthesis of 11-cis-retinal resulting in accumulation of white spots in the retina and stationary night blindness. Studies on mice carrying target deletions in the genes encoding ADH1 and ADH4 do not show any obvious phenotypes related to altered metabolism of endogenous retinoids (22, 23) Instead, these data provide compelling evidence suggesting that ADH1 and ADH4 may not be critically required in generation of RA in vivo. We provide evidence suggesting that the unique milieu in the ER is necessary for efficient generation of 9cRA in vivo
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
