Activation of Retinoic Acid Receptor-dependent Transcription by All-trans-retinoic Acid Metabolites and Isomers

• Published in: The Journal of biological chemistry 2002-08, Vol.277 (35), p.31491-31498
• Main Authors: Idres, Nadia, Marill, Julie, Flexor, Maria A, Chabot, Guy G
• Format: Article
• Language: English
• Subjects: Animals; Binding, Competitive; Chlorocebus aethiops; COS Cells; Genetic Vectors; Isomerism; Kinetics; Molecular Structure; Receptors, Retinoic Acid - genetics; Receptors, Retinoic Acid - metabolism; Retinoic Acid Receptor alpha; Retinoic Acid Receptor gamma; Structure-Activity Relationship; Transcription, Genetic - drug effects; Transcriptional Activation; Transfection; Tretinoin - analogs & derivatives; Tretinoin - pharmacology
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M205016200

We have shown that four metabolites of all- trans -retinoic acid (ATRA) (4-oxo-, 4-OH-, 18-OH-, and 5,6-epoxy-RA) can induce maturation of NB4 promyelocytic leukemia cells (Idres, N., Benoit, G., Flexor, M. A., Lanotte, M., and Chabot, G. G. (2001) Cancer Res. 61, 700–705). To better understand the mechanism of action of ATRA metabolites and isomers, we assessed their binding to retinoic acid receptors (RARs) and activation of RAR-mediated transcription via a retinoic acid response element (RARE). Competition binding experiments with tritiated ATRA showed that all metabolites could bind to RARs with variable affinity. For transactivation studies, COS-7 cells were cotransfected with RARα, β, or γ expression vectors and the reporter plasmid RARE-tk-Luc, and the retinoid concentrations for half-maximal luciferase activity (EC 50 ) were determined. All retinoids tested could activate the three RAR isotypes. The lowest EC 50 value for RARα was with 9- cis -RA (13 n m ), followed by 4-oxo-RA (33 n m ), 5,6-epoxy-RA (77 n m ), 13- cis -RA (124 n m ), 18-OH-RA (162 n m ), ATRA (169 n m ), and 4-OH-RA (791 n m ). For RARβ, the EC 50 values increased as follows: 4-oxo-RA (8 n m ), ATRA (9 n m ), 18-OH-RA (14 n m ), 5,6-epoxy-RA (35 n m ), 13- cis -RA (47 n m ), 4-OH-RA (64 n m ), and 9- cis -RA (173 n m ). For RARγ the EC 50 values were: ATRA (2 n m ), 5,6-epoxy-RA (4 n m ), 18-OH-RA (14 n m ), 13- cis -RA (36 n m ), 9- cis -RA (58 n m ), 4-oxo-RA (89 n m ), and 4-OH-RA (94 n m ). By comparing the -fold induction of luciferase activity, all retinoids tested were equipotent at transactivating RARE-tk-Luc whatever the RAR considered. However, the best induction of the transcription was obtained for RARα, which was 5-fold higher than for RARβ and 10-fold higher than for RARγ. In conclusion, these data show that ATRA metabolites can bind to and activate the three RARs with variable relative affinity but with similar efficacy. These results suggest that ATRA metabolites may activate several signaling pathways and probably play an important role in cellular physiology and cancer therapy.