ABSTRACT
Estrogen-related receptors (NR3B family) and Nurr1, NGFI-B, and Nor1 (NR4A family) are orphan nuclear receptors lacking identified natural ligands. The mechanisms regulating their transcriptional activities have remained elusive. We have previously observed that the members of NR3B and NR4A families are coexpressed in certain cell types such as osteoblasts and that the ability of Nurr1 to transactivate the osteopontin promoter is repressed by ERRs. We have now studied the cross-talk between NR3B and NR4A receptors. We show that NR3B and NR4A receptors mutually repress each others' transcriptional activity. The repression involves intact DNA-binding domains and dimerization interfaces but does not result from competition for DNA binding or from heterodimerization. The activation functions of NR3B and NR4A receptors are dispensable for the cross-talk. In conclusion, we report that cross-talk between NR3B and NR4A receptors is a mechanism modulating the transcriptional activities of these orphan nuclear receptors.
Subject(s)
Cell Nucleus/metabolism , DNA-Binding Proteins/metabolism , Estrogens/metabolism , Receptors, Cytoplasmic and Nuclear/metabolism , Receptors, N-Methyl-D-Aspartate/metabolism , Receptors, Steroid/metabolism , Transcription Factors/metabolism , Binding, Competitive , Cell Line, Tumor , DNA/metabolism , Dimerization , Gene Expression Regulation , HeLa Cells , Humans , Ligands , Nuclear Receptor Subfamily 4, Group A, Member 1 , Osteopontin/blood , Osteopontin/genetics , Promoter Regions, Genetic , Transcription, GeneticABSTRACT
Estrogen-related receptor gamma (ERRgamma) is an orphan nuclear receptor lacking identified natural ligands. We have addressed the requirements for ERRgamma-mediated gene regulation. ERRgamma transactivates constitutively reporter genes driven by ERR response elements (ERREs) or estrogen response elements (EREs). The activation depends on an intact DNA-binding domain (DBD) and activation function-2 (AF2). ERRgamma-mediated transactivation is further enhanced by peroxisome proliferator-activated receptor coactivator-1. Interestingly, ligand-binding domain (LBD) mutations predicted to either enlarge or diminish the putative ligand-binding pocket have no effect on the transcriptional activity implying that ERRgamma activity does not depend on any ligands. Antiestrogens 4OH-tamoxifen (4OHT) and 4-hydroxytoremifene (4OHtor) inhibit the ability of ERR to transactivate ERRE and ERE reporters. In contrast, ERRgamma activates transcription at AP-1 sites in the presence of 4OHT and 4OHtor. Thus, the transcriptional activity of ERRgamma seems not to require ligand binding but is modulated by binding of certain small synthetic ligands.
Subject(s)
Receptors, Cytoplasmic and Nuclear/chemistry , Receptors, Cytoplasmic and Nuclear/physiology , Receptors, Estrogen/chemistry , Receptors, Estrogen/physiology , Tamoxifen/analogs & derivatives , Transcriptional Activation , Binding Sites , Cell Line , DNA-Binding Proteins , Genes, Reporter , Humans , Luciferases/analysis , Point Mutation , Protein Binding , Protein Structure, Secondary , Receptors, Cytoplasmic and Nuclear/genetics , Receptors, Estrogen/genetics , Tamoxifen/chemistry , Tamoxifen/pharmacology , Toremifene/chemistry , Toremifene/pharmacology , Transcription Factors/physiologyABSTRACT
The orphan nuclear receptor Nurr1 is mainly expressed in the central nervous system but is also detected in certain peripheral tissues such as bone. To elucidate the role of Nurr1 in bone, we examined the ability of Nurr1 to regulate osteopontin (OPN) expression in osteoblastic cell lines. Transfection of Nurr1 in osteoblastic cells increased OPN mRNA expression. A dominant negative Nurr1 variant abolished the ability of PTH to induce OPN expression, suggesting that Nurr1 is involved in mediating the regulation of OPN by PTH. Nurr1 efficiently transactivated a luciferase reporter construct driven by the -857/+191 fragment of the mouse OPN promoter. The activation of the OPN promoter was mediated by the monomeric form of Nurr1, required direct binding of Nurr1 to the OPN promoter, and was dependent on the amino-terminal transactivation function-1. The OPN promoter is also regulated by vitamin D receptor and estrogen-related receptors. We show that Nurr1 and vitamin D activate the OPN promoter in a synergistic fashion, whereas Nurr1-mediated transactivation of the OPN promoter is repressed by estrogen-related receptors. In conclusion, Nurr1 activates the OPN promoter directly in osteoblastic cells, suggesting a role for Nurr1 in the regulation of bone homeostasis.
Subject(s)
DNA-Binding Proteins/metabolism , Gene Expression Regulation , Osteoblasts/metabolism , Sialoglycoproteins/biosynthesis , Sialoglycoproteins/genetics , Transcription Factors/metabolism , 3T3 Cells , Animals , Base Sequence , Bone and Bones/metabolism , Cell Line , Cell Line, Tumor , DNA/metabolism , Genes, Dominant , Genes, Reporter , Humans , Luciferases/metabolism , Mice , Molecular Sequence Data , Mutation , Nuclear Receptor Subfamily 4, Group A, Member 2 , Osteopontin , Plasmids/metabolism , Promoter Regions, Genetic , Protein Binding , RNA/metabolism , RNA, Messenger/metabolism , Receptors, Estrogen/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Time Factors , Transcriptional Activation , TransfectionABSTRACT
Estrogen-related receptor gamma (ERRgamma) is an orphan nuclear receptor lacking identified natural ligands. However, 4-hydroxytamoxifen and diethylstilbestrol were recently shown to bind to and inhibit ERRgamma activity. ERR activates transcription constitutively as a monomer. We show here that ERRgamma forms also dimers via its ligand-binding domain. Homodimerization enhances the transcriptional activity. In contrast, heterodimerization with the related receptor ERRalpha inhibits the activities of both ERRgamma and ERRalpha. The inverse ERRgamma agonist 4OHT further inhibits the activity of the ERRgamma-ERRalpha heterodimer, indicating that 4OHT may modulate ERRalpha signaling via ERRgamma. Receptor dimerization thus modulates the transcriptional activities of ERRs.
