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 and differentiation, and in several developmental processes. We report here the molecular bases for a metabolic role of RA, by showing that the expression of the uncoupling protein (UCP), the Bey element in brown adipose tissue (BAT) thermogenesis, is stimulated by retinoic acid (RA). Both all-trans-RA and 9-cis-RA powerfully increase UCP messenger RNA levels in isolated rat brown adipocytes. Transient transfection experiments in HIB-1B cells, a BAT-derived cell line, identified the sequence -2399/-2490 (called R90) as the RA-responsive sequence in the rat UCP gene. R90 mediated a 20- to 70-fold stimulation of the chloramphenicol acetyl transferase reporter gene by maximal concentrations of all-trans-RA or 9-cis-RA. Non-BAT cells were significantly less responsive. RE effect was also less when chloramphenicol acetyl transferase gene was driven by a heterologous promoter instead of the UCP minimal promoter. By footprinting and site-directed mutagenesis, we identified three 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 receptors, and HIB-1B nuclear extracts with anti-RAR gamma or anti-RXR antibodies. The participation of RAR gamma-RXR heterodimers in RA stimulation is further supported by transient transfection experiments overexpressing selected receptors and dose-response analyses of RA isomers and analogues. These results show that retinoids strongly stimulate the rat UCP gene expression through a complex RARE, composed of three pairs of half-sites, and define a novel role for retinoids in the regulation of facultative thermogenesis and energy expenditure.