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 sexual 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 receptor, PPAR). To test this hypothesis, transfection assays in CV-1 cells 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 the presence of 9-cis RA. Furthermore, mobility shift assays demonstrated 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 were similar to residues required for ER binding. Moreover, RXR domain-deleted constructs in transfection assays showed that activation required RXR since an RXR delta AF-2 mutant completely abrogated reporter activity. Oligoprecipitation binding studies with biotinylated ERE and (35)S-labeled in vitro translated RXR constructs confirmed binding of delta AF-2 RXR mutant to the ERE in the presence of baculovirus-expressed PPAR. Finally, in situ hybridization confirmed RXR and PPAR mRNA expression in estrogen responsive tissues. Collectively, these data suggest that RXR and PPAR are present in reproductive tissues, are capable of activating estrogen responsive genes and suggest that the mechanism of activation may involve direct binding of the receptors to estrogen response elements.
Nuñez
S B
SB
Unit on the Molecular Mechanisms of Reproduction, Developmental Endocrinology Branch, National Institutes of Health, Bethesda, MD 20892, USA.
Medin
J A
JA
Braissant
O
O
Kemp
L
L
Wahli
W
W
Ozato
K
K
Segars
J H
JH
D018929:Cell Culture Techniques; D004967:Estrogens / Q000235:genetics* / Q000378:metabolism; D005786:Gene Expression Regulation; D005809:Genes, Regulator / Q000502:physiology; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000235:genetics; D017404:In Situ Hybridization, Fluorescence; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism*; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics / Q000502:physiology*; D011960:Receptors, Estrogen / Q000378:metabolism; D018168:Receptors, Retinoic Acid / Q000235:genetics / Q000502:physiology*; D047488:Retinoid X Receptors; D014157:Transcription Factors / Q000235:genetics / Q000502:physiology*; D014162:Transfection
D004967:Estrogens; D012333:RNA, Messenger; D018160:Receptors, Cytoplasmic and Nuclear; D011960:Receptors, Estrogen; D018168:Receptors, Retinoic Acid; D047488:Retinoid X Receptors; D014157:Transcription Factors; D008156:Luciferases