Ligand-dependent synergy of thyroid hormone and retinoid X receptors.

The binding of thyroid hormone receptors to DNA is enhanced by heterodimerization with nuclear proteins. One such heterodimerization partner has recently been characterized as the retinoid X receptor. 9-cis-Retinoic acid has been identified as a natural ligand for retinoid X receptors, suggesting a potential receptor-mediated interaction between thyroid hormone and 9-cis-retinoic acid in the regulation of thyroid hormone-responsive genes. A transient cotransfection assay was used to test for such an interaction. When a complex thyroid hormone response element composed of both direct and inverted repeat hexamers was tested, these two ligands activated gene expression synergistically. In contrast, when the response element consisted only of directly repeated hexamers, unliganded retinoid X receptors enhanced thyroid hormone responsiveness, but 9-cis-retinoic acid induced no additional activation. The results suggest a unique mechanism to achieve differential suggest a unique mechanism to achieve differential thyroid hormone sensitivity of thyroid hormone-responsive genes within a cell. Genes with appropriate response elements will show amplification of the thyroid hormone response by 9-cis-retinoic acid in the presence of retinoid X receptors; other thyroid hormone-responsive genes will be influenced by retinoid X receptors, but not 9-cis-retinoic acid.     

Mutations in retinoid X receptor that impair heterodimerization with specific nuclear hormone receptor

Retinoid X receptor (RXR) serves as a promiscuous heterodimerization partner for many nuclear receptors through the identity box, a 40-amino acid subregion within the ligand binding domain. In this study, we randomly mutated two specific residues within the human RXRalpha identity box region previously identified as important determinants in heterodimerization (i.e. Ala(416) and Arg(421)). Interestingly, most of these mutants still retained wild type interactions with thyroid hormone receptor (TR), retinoic acid receptor, peroxisome proliferator-activated receptor alpha, small heterodimer partner, and constitutive androstane receptor. However, RXR-A416D and R421L were specifically impaired for interactions with TR, whereas RXR-A416K lost both TR and retinoic acid receptor interactions. Accordingly, RXR-A416D did not support T3 transactivation in mammalian cells, whereas RXR-A416K was not supportive of transactivation by retinoids or T3. These results provide a basis upon which to further design mutant RXRs highly selective in heterodimerization, potentially useful tools to probe nuclear receptor function in vivo.     

RXR alpha, a promiscuous partner of retinoic acid and thyroid hormone receptors.

Retinoic acid receptor (RAR), thyroid hormone receptor (T3R) and vitamin D3 receptor (VD3R) differ from steroid hormone receptors in that they bind and transactivate through responsive elements organized as direct rather than inverted repeats. We now show that recombinant RAR and T3R are monomers in solution and cannot form stable homodimeric complexes on their responsive elements. Stable binding of the receptors to their responsive elements requires heterodimerization with a nuclear factor. This auxiliary factor is tightly associated with RAR and T3R in the absence of DNA and co-purifies with both receptors. As demonstrated by extensive purification, the same auxiliary factor is required for stable DNA binding of RAR as for that of T3R; the factor also facilitates the formation of a stable VD3R-DNA complex. The auxiliary factor is identical to the retinoid X receptor alpha (RXR alpha) by biochemical and functional criteria. The identification of RXR alpha as a dimerization partner for the RARs, T3Rs and VD3R has important implications as to the function of these receptors and their ligands in development, homeostasis and neoplasia.     

Anti-tumor effects of all-trans retinoic acid are enhanced by genistein

The effects of all-trans retinoic acid (ATRA) on cancer are complex. ATRA has anti-cancer effects as it promotes cancer cell differentiation. However, ATRA also up-regulates expression of vascular endothelial growth factor (VEGF) in cancer cells, which leads to angiogenesis and can, thus, facilitate cancer growth. Genistein, a crucial non-nutrient component in soybean, exhibits anti-cancer effects by inhibiting protein tyrosine kinase that is involved in up-regulation of VEGF. We hypothesized that genistein, applied simultaneously with ATRA, would counter its undesired angiogenic effects and, thus, enhance the anti-cancer effects of ATRA. The purpose of this study was to document potential synergistic effects of genistein and ATRA in A549 lung adenocarcinoma cells. We further explored the role of genistein on countering the ATRA-induced VEGF expression. We demonstrate that genistein enhances the ATRA-induced growth inhibition of A549 cells by promoting apoptosis. Further, the combined use of ATRA and genistein leads to cancer cell arrest in G0/G1 and G2/M cell cycle phases. Finally, expression of VEGF (both mRNA and protein) was diminished in A549 cells exposed to both ATRA and genistein. In conclusion, our results demonstrate that genistein effectively enhances anti-cancer effects of ATRA, particularly, by countering the ATRA-induced up-regulation of VEGF. Our study provides an experimental basis for combined use of ATRA and genistein in the treatment of lung cancer.     

Retinoic acid receptor: a simulation analysis of retinoic acid binding and the resulting conformational changes.

The binding/escape mechanism of all- trans retinoic acid with respect to the ligand-binding domain of the nuclear receptor RARgamma has been studied by molecular dynamic simulations. The entry/exit channel was shown to be on the side of the activation helix by the use of multiple copy dynamics. Three independent minimum energy paths from the liganded structure to a model for the unliganded structure were calculated with the conjugate peak refinement method. Ligand escape takes place in the early steps of the transition during rearrangement of the binding pocket; the latter involves inward motion of the beta-sheet and outward motions of the Omega-loop and helix H6. The correlated rearrangements involved in the escape phase are similar and occur in the same order for the different paths. After the escape phase, the conformational changes affect primarily the C-terminal helices H11-H12 and the Omega-loop. The three paths are significantly different for this reorganization phase and reveal a multiplicity of possibilities, in agreement with the idea that the apo state is structurally less constrained. The present calculations extend the crystallographic results, confirming the "mouse trap" mechanism and stressing the importance of the helix H3 conformation and of the contacts between the Omega-loop and helices H11 and H6. They are in good agreement with known mutants and point to other functionally important residues, especially in helices H3 and H11, suggesting mutations that may affect the ligand-binding function and the associated conformational changes.     

Ligand-dependent and -independent transactivation by thyroid hormone receptor beta 2 is determined by the structure of the hormone response element.

Chicken thyroid hormone receptor beta 2 (cTR beta 2) is likely to serve specific functions in gene regulation since it possesses a unique N-terminal domain and is expressed in very few tissues. We demonstrate here that TR beta 2 exhibits distinct transactivation properties which are dependent on the availability of ligand and on the structure of the hormone response element. First, a strong ligand-independent transactivation was observed with hormone response elements composed of direct repeats and everted repeats. Second, TR beta 2 was induced by triiodothyronine to transactivate more efficiently than TR beta 0 on palindromic and everted-repeat types of hormone response elements. However, coexpression of the retinoid X receptor reduced the strong transactivation by TR beta 2 but not by TR beta 0 via palindromic response elements, suggesting that TR beta 2 can transactivate as a homodimer. Finally, the N terminus of TR beta 2 contains two distinct transactivation regions rich in tyrosines, which are essential for transactivation. Our results thus show that the activity of the novel transactivating region of TR beta 2 is dependent on the organization of the half-sites in the response element.