The orphan nuclear hormone receptor estrogen-related receptor A (ERRA, NR3B1) is a constitutive transcription factor that is structurally and functionally related to the classic estrogen receptors. ERRA can recognize both the estrogen response element and its own binding site (ERRE) in either dimeric or monomeric forms. ERRA is also a phosphoprotein whose expression in human breast tumors correlates with that of the receptor tyrosine kinase ErbB2, suggesting that its transcriptional activity could be regulated by signaling cascades. Here, we investigated growth factor regulation of ERRA function and found that it is phosphorylated in MCF-7 breast cancer cells in response to epidermal growth factor (EGF), an event that enhances its DNA binding. Interestingly, treatment with alkaline phosphatase shifts ERRA from a dimeric to a monomeric DNA-binding factor, and only the dimeric form interacts with the coactivator PGC-1A. In vitro, the DNA-binding domain of ERRA is selectively phosphorylated by protein kinase CD (PKCD), which increases its DNA-binding activity, whereas expression of constitutively active PKCD enhances TFF1 promoter activity via the ERRE. However, whereas treatment of MCF-7 cells with the phorbol ester phorbol-12-myristate 13-acetate also enhances ERRA activation of the TFF1 promoter reporter, it does not affect ERRA activity on its own promoter. In agreement, chromatin immunoprecipitation analysis shows that ERRA and RNA polymerase II are preferentially recruited to the TFF1 promoter after EGF treatment, whereas recruitment of these factors to its own promoter is not affected. These results reveal a mechanism through which growth factor signaling can selectively activate ERRA target genes in breast cancer cells. (Cancer Res 2005; 65(14): 6120-29)

The orphan nuclear receptor estrogen-related receptor alpha (ERRalpha, NR3B1) is a constitutively active transcription factor that controls multiple processes, most notably mitochondrial function. ERRalpha preferentially binds to a nine-nucleotide extended half-site sequence TNAAGGTCA, referred to as the ERRE, as either a monomer or a dimer, although how the mode of DNA binding is dictated remains to be determined. Here, we used variants of the extended half-site sequence and selective DNA binding domain mutants of ERRalpha to investigate the effects of ERRE sequence specificity on ERRalpha DNA binding mode, transactivation and interaction with the coactivator protein peroxisome proliferator-activated receptor gamma coactivator 1alpha (PGC-1alpha). We found that the base at the N position of the TNAAGGTCA sequence dictated ERRalpha binding preference as a monomer or dimer. In addition, we demonstrated that the threonine residue at position 124 (Thr(124)) was a determinant of ERRalpha DNA-dependent dimerization. Transfection experiments also indicated that substituting a thymidine for a cytosine at the N position in the ERRE of the native ERRalpha target promoter trefoil factor 1 (TFF1) considerably diminished the transcriptional response of the ERRalpha/PGC-1alpha complex. These results suggest that a single nucleotide in an ERRalpha binding site can determine specific configuration to the receptor and productive interaction with the coactivator PGC-1alpha.

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The vitamin D receptor (VDR) is a ligand-dependent transcription factor that heterodimerizes with retinoid X receptor (RXR) and interacts with the basal transcription machinery and transcriptional cofactors to regulate target gene activity. The p160 coactivator GRIP1 and the distinct coregulator Ski-interacting protein (SKIP)/NCoA-62 synergistically enhance ligand-dependent VDR transcriptional activity. Both coregulators bind directly to and form a ternary complex with VDR, with GRIP1 contacting the activation function-2 (AF-2) domain and SKIP/NCoA-62 interacting through an AF-2 independent interface. It was previously reported that SKIP/NCoA-62 interaction with VDR was independent of the heterodimerization interface (specifically, helices H10/H11). In contrast, the present study defines specific residues within a conserved and surface-exposed region of VDR helix H10 that are required for interaction with SKIP/NCoA-62 and for full ligand-dependent transactivation activity. SKIP/NCoA-62, the basal transcription factor TFIIB, and RXR all interacted with VDR helix H10 mutants at reduced levels compared with wild type in the absence of ligand and exhibited different degrees of increased interaction upon ligand addition. Thus, SKIP/ NCoA-62 interacts with VDR at a highly conserved region not previously associated with coregulator binding to regulate transactivation by a molecular mechanism distinct from that of p160 coactivators. The vitamin D receptor (VDR)1 is a ligand-dependent transcription factor important in the regulation of calcium homeostasis, development, cell growth, and differentiation. VDR belongs to the nuclear receptor superfamily, members of which share a common modular structure including a highly conserved DNA binding domain (DBD) and a conserved ligand binding domain (LBD). The LBD has a predominantly ␣-helical structure with an activation function-2 (AF-2) domain in the COOH-terminal helix (H12). The LBD is the main site of VDR interaction with its heterodimer partner retinoid X receptor (RXR) and basal transcription factors, and the AF-2 domain in combination with the hydrophobic cleft forms an interaction surface for transcriptional corepressors and coactivators (1, 2). In the absence of its ligand 1,25-dihydroxyvitamin D 3

These data show that AA and TZD have synergistic rather than simple additive effects on secretion of HMW adiponectin from human adipocytes and raise the possibility that differences in AA levels may contribute to the variability in adiponectin multimer profiles and efficacy of TZD in humans. Our results also provide a rationale for longitudinal clinical trials investigating the effects of AA supplementation with or without TZD on adiponectin and metabolic profiles.