Functional evidence for retinoid X receptor (RXR) as a nonsilent partner in the thyroid hormone receptor/RXR heterodimer

Many members of the thyroid hormone/retinoid receptor subfamily (type II nuclear receptors) function as heterodimers with the retinoid X receptor (RXR). In heterodimers which are referred to as permissive, such as peroxisome proliferator activated receptor/RXR, both partners can bind cognate ligands and elicit ligand-dependent transactivation. In contrast, the thyroid hormone receptor (TR)/RXR heterodimer is believed to be nonpermissive, where RXR is thought to be incapable of ligand binding and is often referred to as a silent partner. In this report, we used a sensitive derepression assay system that we developed previously to reexamine the TR/RXR interrelationship. We provide functional evidence suggesting that in a TR/RXR heterodimer, the RXR component can bind its ligand in vivo. Ligand binding by RXR does not appear to directly activate the TR/RXR heterodimer; instead, it leads to a (at least transient or dynamic) dissociation of a cellular inhibitor(s)/corepressor(s) from its TR partner and thus may serve to modulate unliganded TR-mediated repression and/or liganded TR-mediated activation. Our results argue against the current silent-partner model for RXR in the TR/RXR heterodimer and reveal an unexpected aspect of cross regulation between TR and RXR.     

Purification, cloning, and RXR identity of the HeLa cell factor with which RAR or TR heterodimerizes to bind target sequences efficiently.

We have purified and cloned a HeLa cell nuclear protein that strongly stimulates binding of retinoic acid and thyroid hormone receptors (RARs and TRs) to response elements. The purified protein is a human retinoid X receptor beta (hRXR beta). Three murine members of the RXR family (mRXR alpha, beta, and gamma) have also been cloned, and their interactions with RARs and TRs have been investigated. Under conditions where RAR, RXR, and TR bound poorly as homodimers to various response elements, strongly cooperative RAR-RXR and TR-RXR binding was observed. The binding efficiency was dependent on the sequence, relative orientation, and spacing of the repeated motifs of response elements. We show also that unstable RAR-RXR heterodimers were formed in solution, and that C-terminal sequences and the DNA-binding domains of both receptors were required for efficient formation of stable heterodimers on response elements. These findings suggest a convergence of the signaling pathways of some members of the nuclear receptor superfamily.     

Retinoid X receptors and retinoid response in neuroblastoma cells

Retinoic acid (RA) modulates differentiation and apoptosis of neural cells via RA receptors (RARs) and retinoid X receptors (RXRs). Neuroblastoma cells are potentially useful models for elucidating the molecular mechanisms of RA in neural cells, and responses to different isomers of RA have been interpreted in terms of differential homo- and heterodimerization of RXRs. The aim of this study was to identify the RXR types expressed in neuroblast and substrate-adherent neuroblastoma cells, and to study the participation of these RXRs in RAR heterodimers. RXRbeta was the predominant RXR type in N-type SH SY 5Y cells and S-type SH EP cells. Gel shift and supershift assays demonstrated that RARbeta and RARgamma predominantly heterodimerize with RXRbeta. In SH SY 5Y cells, RARgamma/RXRbeta was the predominant heterodimer binding to the DR5 RARE in the absence of 9-cis RA (9C), whereas the balance shifted in favor of RARbeta/RXRbeta in the presence of ligand. There was a marked difference between the N- and S-type neuroblastoma cells in retinoid receptor-DNA interactions, and this may underlie the differential effects of retinoids in these neuroblastoma cell types. There was no evidence to indicate that 9C functions via RXR homodimers in either SH SY 5Y or SH EP neuroblastoma cells. The results of this study suggest that interactions between retinoid receptors and other nuclear proteins may be critical determinants of retinoid responses in neural cells.     

Thyroid hormone alters the DNA binding properties of chicken thyroid hormone receptors alpha and beta.

The effects of thyroid hormone agonists on thyroid hormone receptor (TR)/DNA complex formation was investigated to elucidate the mechanism by which TRs transactivate genes in response to ligand. The data, obtained from gel shift experiments, indicate that thyroid hormones alter the conformation of TRs bound to DNA, irrespective of if the element is occupied by monomeric TR, homodimeric TR/TR, or heterodimeric complexes with the retinoid receptors RAR or RXR. Furthermore, triiodo-thyronine (T3) prevents 2 TR molecules from binding to oligonucleotides containing direct repeats or inverted palindromes of the consensus AGGTCA motif, an effect that was not detected with palindromic elements. Heterodimers bound to direct repeats were less affected: RXR/TR were fully and RAR/TR complexes partially resistant to thyroid hormone. The data suggest that a ligand-induced conformational change in TR prevents double TR occupancy of a response element containing 2 direct repeats of the consensus binding motif, possibly by steric hindrance, whereas such an event does not prevent TR/RXR heterodimers from binding to DNA. Finally, our data show that a monomeric, liganded TR bound preferentially to the second half site in a AGGTCActcaAGGTCA element, and therefore indicate that nucleotides adjacent to the consensus half site contribute to binding specificity.     

The role of the retinoid receptor,RAR/RXR heterodimer,in liver physiology

Activated by retinoids, metabolites of vitamin A, the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs) play important roles in a wide variety of biological processes, including embryo development, homeostasis, cell proliferation, differentiation and death. In this review, we summarized the functional roles of nuclear receptor RAR/RXR heterodimers in liver physiology. Specifically, RAR/RXR modulate the synthesis and metabolism of lipids and bile acids in hepatocytes, regulate cholesterol transport in macrophages, and repress fibrogenesis in hepatic stellate cells. We have also listed the specific genes that carry these functions and how RAR/RXR regulate their expression in liver cells, providing a mechanistic view of their roles in liver physiology. Meanwhile, we pointed out many questions regarding the detailed signaling of RAR/RXR in regulating the expression of liver genes, and hope future studies will address these issues.     

Novel roles of retinoid X receptor (RXR) and RXR ligand in dynamically modulating the activity of the thyroid hormone receptor/RXR heterodimer

Many members of the type II nuclear receptor subfamily function as heterodimers with the retinoid X receptor (RXR). A permissive heterodimer (e.g. peroxisome proliferator-activated receptor/RXR) allows for ligand binding by both partners of the receptor complex. In contrast, RXR has been thought to be incapable of ligand binding in a nonpermissive heterodimer, such as that of thyroid hormone receptor (TR)/RXR, where it has been referred to as a silent partner. However, we recently presented functional evidence suggesting that RXR in the TR/RXR heterodimer can bind its natural ligand 9-cis-RA in cells. Here we extended our study of the interrelationship of TR and RXR. We examined the potential modulatory effect of RXR and its ligand on the activity of TR, primarily using a Gal4-TR chimera. This study led to several novel and unexpected findings: 1) heterodimerization of apo-RXRalpha (in the absence of 9-cis-RA) with Gal4-TR inhibits T3-mediated transactivation; 2) the inhibition of Gal4-TR activity by RXRalpha is further enhanced by 9-cis-RA; 3) two different RXR subtypes (alpha and beta) differentially modulate the activity of Gal4-TR; 4) the N-terminal A/B domains of RXR alpha and beta are largely responsible for their differential modulation of TR activity; and 5) the RXR ligand 9-cis-RA appears to differentially affect T3-mediated transactivation from the Gal4-TR/RXRalpha (which is inhibited by 9-cis-RA) and TRE-bound TR/RXRalpha (which is further activated by 9-cis-RA) heterodimers. Taken together, these results further support our recent proposal that the RXR component in a TR/RXR heterodimer is not silent and, more importantly, reveal novel aspects of regulation of the activity of the TR/RXR heterodimer by RXR and RXR ligand.