A COMPLEX RETINOIC ACID RESPONSE ELEMENT IN THE UNCOUPLING PROTEIN GENE DEFINES A NOVEL ROLE FOR RETINOIDS IN THERMOGENESIS

Retinoids have been implicated in the control of cell proliferation an d differentiation, and in several developmental processes. We report h ere the molecular bases for a metabolic role of RA, by showing that th e expression of the uncoupling protein (UCP), the Bey element in brown adipose tissue (BAT) thermogenesis, is stimulated by retinoic acid (R A). Both all-trans-RA and 9-cis-RA powerfully increase UCP messenger R NA levels in isolated rat brown adipocytes. Transient transfection exp eriments in HIB-1B cells, a BAT-derived cell line, identified the sequ ence -2399/-2490 (called R90) as the RA-responsive sequence in the rat UCP gene. R90 mediated a 20- to 70-fold stimulation of the chloramphe nicol acetyl transferase reporter gene by maximal concentrations of al l-trans-RA or 9-cis-RA. Non-BAT cells were significantly less responsi ve. RE effect was also less when chloramphenicol acetyl transferase ge ne was driven by a heterologous promoter instead of the UCP minimal pr omoter. By footprinting and site-directed mutagenesis, we identified t hree discrete sequences as being essential for the RA response within R90, thus defining the complex RA response element (RARE) of this gene . Critical bases in these sequences are arranged in pairs of putative half-sites. RAR gamma-RXR heterodimers can bind to the R90 as revealed by electrophoretic mobility shift assays using in vitro translated re ceptors, and HIB-1B nuclear extracts with anti-RAR gamma or anti-RXR a ntibodies. The participation of RAR gamma-RXR heterodimers in RA stimu lation is further supported by transient transfection experiments over expressing selected receptors and dose-response analyses of RA isomers and analogues. These results show that retinoids strongly stimulate t he rat UCP gene expression through a complex RARE, composed of three p airs of half-sites, and define a novel role for retinoids in the regul ation of facultative thermogenesis and energy expenditure.