RETINOID-X-RECEPTOR AND PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR ACTIVATE AN ESTROGEN-RESPONSIVE GENE INDEPENDENT OF THE ESTROGEN-RECEPTOR

Estrogen receptors regulate transcription of genes essential for sexua l development and reproductive function. Since the retinoid X receptor (RXR) is able to modulate estrogen responsive genes and both 9-cis RA and fatty acids influenced development of estrogen responsive tumors, we hypothesized that estrogen responsive genes might be modulated by RXR and the fatty acid receptor (peroxisome proliferator-activated rec eptor, PPAR). To test this hypothesis, transfection assays in CV-1 cel ls were performed with an estrogen response element (ERE) coupled to a luciferase reporter construct. Addition of expression vectors for RXR and PPAR resulted in an 11-fold increase in luciferase activity in th e presence of 9-cis RA. Furthermore, mobility shift assays demonstrate d binding of RXR and PPAR to the vitellogenin A2-ERE and an ERE in the oxytocin promoter. Methylation interference assays demonstrated that specific guanine residues required for RXR/PPAR binding to the ERE wer e similar to residues required for ER binding. Moreover, RXR domain-de leted constructs in transfection assays showed that activation require d RXR since an RXR Delta AF-2 mutant completely abrogated reporter act ivity. Oligoprecipitation binding studies with biotinylated ERE and S- 35-labeled in vitro translated RXR constructs confirmed binding of Del ta AF-2 RXR mutant to the ERE in the presence of baculovirus-expressed PPAR. Finally, in situ hybridization confirmed RXR and PPAR mRNA expr ession in estrogen responsive tissues. Collectively, these data sugges t that RXR and PPAR are present in reproductive tissues, are capable o f activating estrogen responsive genes and suggest that the mechanism of activation may involve direct binding of the receptors to estrogen response elements. (C) 1997 Elsevier Science Ireland Ltd.