Cloning of monkey RALDH1 and characterization of retinoid metabolism in monkey kidney proximal tubule cells

Abstract

All-trans and 9-cis retinoic acids function as ligands for retinoic acid receptors (RARs and RXRs), which are ligand-dependent transcription factors and play important roles in development and cellular differentiation. Several retinal dehydrogenases are likely to contribute to the production of all-trans and 9-cis RAs in vivo, but their respective roles in different tissues are still poorly characterized. We have previously characterized and cloned from kidney tissues the rat retinal dehydrogenase type 1 (RALDH1), which oxidizes all-trans and 9-cis retinal with high efficiency but is inactive with 13-cis retinal. Here we have characterized the retinal-oxidizing activity in monkey JTC12 cells, which are derived from kidney proximal tubules. In vitro assay of cell lysates revealed the presence of a NAD ؉ -dependent dehydrogenase that catalyzed the oxidation of all-trans , 9-cis, and 13-cis retinal. Northern blot analysis of JTC12 RNAs and cloning by reverse transcriptionpolymerase chain reaction demonstrated expression of a monkey homolog of RALDH1. Bacterially expressed JTC12 RALDH1 catalyzed conversion of all three retinal isomers, with a higher catalytic efficiency for 9-cis retinal than for alltrans and 13-cis retinal. Accordingly, live JTC12 produced 9-cis retinoic acid more efficiently than all-trans retinoic acid from their respective retinal precursors.Only metabolites corresponding to the same steric conformation were formed from 9-cis or all-trans retinal, indicating a lack of detectable isomerizing activity in JTC12 cells. Retinoids are important regulators of cell growth, differentiation, and embryonic development (1-3). They are also needed for normal vision, reproduction, and immunity (4-6). The biological actions of retinoids are mediated through binding and modulation of retinoic acid receptors (RARs) or retinoid X receptors (RXRs), which function as ligand-dependent transcription factors (7). All-trans retinoic acid (RA) is a natural ligand for RARs, and 9-cis RA binds to both RARs and RXRs (8). The influence of vitamin A (retinol) in the control of gene expression is made possible by enzymes regulating RA synthesis. RA is formed from retinol via a 2-step metabolic pathway that involves oxidation of retinol to retinal and then of retinal to RA (9, 10). Although the metabolic pathways leading to the formation of all-trans and 9-cis RAs are beginning to be elucidated, the enzymes controlling production of these compounds within specific cells and tissues are still poorly characterized. In particular, it is not firmly established whether both all-trans and 9-cis RAs can be produced from the precursor all-trans retinol. Several studies have shown that externally supplied all-trans, 9-cis , and 13-cis RAs are isomerized into cis or trans RAs in cells and tissues and appear to reach equilibrium (11-16). Since no isomerase(s) involved in these processes have been identified, it is generally believed that the interconversion of cis -trans RA occurs in cells through nonenzymatic mechanism(s), alth...

The search for non‐chordate retinoic acid signaling: lessons from chordates

“…In contrast, in vivo data have failed to substantiate almost all claims for a physiological role of 9-cis RA in vertebrate development (Costaridis et al, '96;Mic et al, 2003). In fact, 9-cis RA has been inconspicuous, failing detection in more recent experiments in vertebrates (Costaridis et al, '96;Mic et al, 2003), except when animals or cultured cells are previously treated with 9-cis precursors (e.g., Brodeur et al, 2003). More importantly, there is direct evidence that whereas an RAR-specific agonist can rescue the dramatic RA insufficiency of RALDH2 knockout embryos, an RXR-specific agonist fails to do so .…”

Multiple Retinol and Retinal Dehydrogenases Catalyze All-trans-retinoic Acid Biosynthesis in Astrocytes

“…Error bars, means Ϯ S.E. expression in Escherichia coli, each could occur in different intracellular microenvironments (31,58,59). To test this premise, primary astrocytes were immunostained with Raldh1 or -2 antibodies together with the astrocyte marker GFAP.…”

Estrogen Receptor Alpha Regulates Retinaldehyde Dehydrogenase 1 Expression in Rat Anterior Pituitary Cells

“…RALdh1 is a dehydrogenase that generates RA in Xenopus [24], chicken [25], and mammals including human [6,7,26]. Rat RALdh1 catalyzes the oxidation of all-trans-retinal and 9-cis-retinal to all-trans-RA and 9-cis-RA, respectively [27,28].…”