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We undertook a study of molecular interference of nuclear orphan receptors. Nuclear receptor response element-1 (NRRE-1) from the human medium-chain acyl coenzyme A dehydrogenase (MCAD) gene promoter was shown to contain three hexamer elements (site 1 through 3) that are known to interact with a number of nuclear receptors including chicken ovalbumin upstream promoter transcription factor (COUP-TF) and estrogen-related receptor alpha (ERRalpha). We demonstrated that the peroxisome proliferator-activated receptor alpha/9-cis-retinoic acid receptor alpha (PPARalpha/RXRalpha) heterodimer complex can also bind to the two hexamer repeat sequences (between site 1 and site 3) arranged as an everted imperfect repeat separated by 14 bp (ER14). Mutations of the putative core elements have shown that these three sites are differentially involved in ERRalpha and PPARalpha/RXRalpha binding. Homodimer of ERRalpha was shown to interact between site 1 and site 3 (ER14). To date, no nuclear receptor is known to bind to response elements over such long intervals. Interestingly, site 1 was shown to be essential for ERRalpha binding while site 3 supports its binding only in the presence of site 1. Furthermore, it was shown that the binding profile of ERRalpha and PPARalpha/RXRalpha are competitive rather than making a high order complex within NRRE-1. At the cellular level, transcriptional activation driven by the PPARalpha/RXRalpha complex was counteracted by the expression of ERRalpha in HeLa cells. These results suggest that ERRalpha and PPARalpha/RXRalpha could interfere with each other's function through binding to similar DNA elements, thereby finetuning the transcriptional outcome of the target gene. Our findings suggest a mechanism whereby multiple nuclear receptors can activate or repress DNA binding or transcription via a single pleiotropic regulatory element.

The effects of two vitamin D analogues, EB1089 and CB1093, on insulin-like growth factor binding protein (IGFBP) expression have been examined in MCF-7 and Hs578T human breast cancer cell lines. Both vitamin D analogues inhibited IGF-1 stimulated growth of MCF-7 cells and enhanced the production of IGFBP-3 as determined by Western-ligand blotting. Recombinant human IGFBP-3 inhibited the growth of MCF-7 cells over the concentration range 1-235 ng/ml. Hs578T cells were unresponsive to the mitogenic effects of IGF-1 but growth was inhibited by the two vitamin D analogues. Treatment of Hs578T cells with EB1089 and CB1093 (10 nM) as well as 100 nM 9-cis retinoic acid (9-cis RA) or all-trans retinoic acid (ATRA) was associated with increased accumulation of IGFBP-3 in conditioned medium. Furthermore, cotreatment of Hs578T cells with EB1089 and 9-cis RA led to augmented effects on both inhibition of cell growth and IGFBP-3 accumulation in conditioned medium as assessed by Western ligand blotting and radioimmunoassay. These findings suggest a role for IGFBP-3 in the growth inhibitory effects of vitamin D analogues.

Thyroid hormone receptors (TRs) often modulate transcriptional activity of target genes by heterodimerization with the 9-cis retinoic acid receptor (RXR). On positive thyroid response elements (TREs), RXR favors binding of the TR-RXR complex to DNA and stimulates transcription. RXR action on negative TREs is unclear. Furthermore, the single half-site configuration of many negative TREs does not favor the binding of a classic TR-RXR heterodimer. In a comparative study using CV-1 cells (relatively RXR- and TR-deficient) and JEG-3 cells (relatively TR-deficient), we demonstrate the importance of RXR in the negative transcriptional regulation of genes of the hypothalamo-pituitary axis by tri-iodothyronine. While RXR has variable effects on ligand-independent activation produced by TRs, it was required for efficient ligand-dependent repression of the TRH gene for TRalpha1 and TRbeta1 and of the TSH genes by all TRs. Using different RXR constructs we also observed the importance of the C-terminus of RXR but not of the N-terminus nor the DNA-binding domain, in the potentiation of negative regulation. We thus suggest that, with regard to negative regulation of the TRH and TSH genes by thyroid hormones, RXR behaves more like a cofactor than a classic heterodimerization partner.

Estrogen synthesis in adipose tissue is associated with the development of breast cancer. Tumors are preferentially found in breast quadrants with strongest expression of the cytochrome P450 aromatase (encoded by the gene CYP19A1). Several promoters regulated by various hormonal factors drive aromatase expression in human breast adipose fibroblasts (BAFs). As adipose tissue is a major source of retinoids, in this study, we investigated their role in the regulation of aromatase expression. The retinoids all-trans-retinoic acid (at-RA) and 9-cis-RA induce aromatase activity in human BAFs. In BAFs, at-RA induces aromatase gene expression via promoter I.4. In 3T3-L1 cells, both retinoids specifically drive luciferase reporter gene expression under the control of aromatase promoter I.4, whereas other promoters active in human adipose tissue are insensitive. Activation by retinoids depends on a 467 bp fragment (−256/+211) of promoter I.4 containing four putative retinoic acid response elements (RAREs). Site-directed mutagenesis revealed that only RARE2 (+91/+105) mediates the retinoid-dependent induction of reporter gene activity. In 3T3-L1 preadipocytes and human BAFs, RA receptor α (RARα (RARA)) expression is predominant, whereas RARβ (RARB) or RARγ (RARG) expression is low. Electrophoretic mobility shift assays with nuclear extracts obtained from human BAFs and 3T3-L1 cells identified a specific RARE2-binding complex. Retinoids enhanced complex formation, whereas pre-incubation with anti-RARα antibodies prohibited the binding of RARα to RARE2. Chromatin immunoprecipitation showed RA-dependent binding of RARα to the RARE2-containing promoter region in vivo. Furthermore, we provide evidence that RARE2 is also necessary for the basal activation of promoter I.4 in these cells. Taken together, these findings indicate a novel retinoid-dependent mechanism of aromatase activity induction in adipose tissue.