Retinoic acid receptor/retinoid X receptor heterodimers can be activated through both subunits providing a basis for synergistic transactivation and cellular differentiation

The receptor for 9-cis-retinoic acid, retinoid X receptor (RXR), forms heterodimers with several nuclear receptors, including the receptor for all-trans-retinoic acid, RAR. Previous studies have shown that retinoic acid receptor can be activated in RAR/RXR heterodimers, whereas RXR is believed to be a silent co-factor. In this report we show that efficient growth arrest and differentiation of the human monocytic cell line U-937 require activation of both RAR and RXR. Also, we demonstrate that the allosteric inhibition of RXR is not obligatory and that RXR can be activated in the RAR/RXR heterodimer in the presence of RAR ligands. Remarkably, RXR inhibition by RAR can also be relieved by an RAR antagonist. Moreover, the dose response of RXR agonists differ between RXR homodimers and RAR/RXR heterodimers, indicating that these complexes are pharmacologically distinct. Finally, the AF2 activation domain of both subunits contribute to activation even if only one of the receptors is associated with ligand. Our data emphasize the importance of signaling through both subunits of a heterodimer in the physiological response to retinoids and show that the activity of RXR is dependent on both the identity and the ligand binding state of its partner.     

Negative feedback control of the retinoid-retinoic acid/retinoid X receptor pathway by the human TR4 orphan receptor, a member of the steroid receptor superfamily

Amino acid sequence analysis indicates that the human TR4 orphan receptor (TR4) is a member of the estrogen/thyroid receptor subfamily of the steroid/thyroid receptor superfamily and recognizes the AGGTCA direct repeat (DR) of the hormone response element. Here we demonstrate using the electrophoretic mobility shift assay that TR4 binds specifically to DR with a spacing of 1 and 5 base pairs (DR1 and DR5), which are the response elements for retinoic acid receptor (RAR) and retinoid X receptor (RXR), respectively. A reporter gene assay using chloramphenicol acetyltransferase demonstrated that TR4 repressed RA-induced transactivation in a TR4 dose-dependent manner. Inhibition of the retinoid signal pathway also occurs through natural response elements found in CRBPII and RARbeta genes. Our data suggest that the mechanism of repression may not involve the formation of functionally inactive heterodimers between TR4 and RAR or RXR. Instead, we show that TR4 may compete for hormone response elements with RAR and RXR due to its higher binding affinity. Furthermore, treatment of F9 murine teratocarcinoma (F9) cells with 10(-6) M all-trans-retinoic acid increased TR4 mRNA levels, and this change was accompanied by an increased amount of endogenous TR4 protein that can bind to RXRE in electrophoretic mobility shift assay. Our data therefore strongly suggest that the retinoid signal pathway can be regulated by TR4 in a negative feedback control mechanism, which may restrict retinoic acid signaling to certain elements in a cell-specific fashion.     

Interaction between retinoic acid and vitamin D signaling pathways

The nuclear signaling pathways for retinoids and vitamin D differ in the specificity of the respective receptors for response elements. Two pathways for the action of both retinoic acid receptors (RARs) and vitamin D receptors (VDRs) have been identified, one being retinoid X receptor (RXR)-dependent and the other being RXR-independent. Moreover, RXRs were found to function as homodimers. In several steps we converted the retinoid specific response element of the human retinoic acid receptor beta promoter into the vitamin D/retinoic acid response element of the human osteocalcin promoter. We found that VDR homodimers only bind to the motif RGGTGA. The extended osteocalcin element also contains an imperfect direct repeat based on the motif RGGTGA spaced by three nucleotides, which is bound by RXR homodimers and activated by 9-cis-retinoic acid. The responsiveness of the osteocalcin element to all-trans-retinoic acid is mediated neither by RAR homodimers nor by RAR-RXR heterodimers. However, a VDR-RAR heterodimer binds to the osteocalcin response element and mediates activation by all-trans-retinoic acid. This heterodimer also binds to pure retinoid response elements, but it does not mediate activation by vitamin D alone. In combination with all-trans-retinoic acid, however, vitamin D enhances VDR-RAR heterodimer-mediated gene expression. This finding suggests a direct interaction between nuclear signaling by retinoic acid and vitamin D increasing the combinatorial possibilities for gene regulation by the nuclear receptors involved.     

Genetic control of the development by retinoic acid

Two families of nuclear receptors for retinoic acid (RA) have been characterized. Members of the RAR family (types alpha, beta and gamma and their isoforms alpha 1, alpha 2, beta 1 to beta 4, and gamma 1 and gamma 2) are activated by most physiologically occurring retinoids (all-trans RA, 9-cis RA, 4oxo RA and 3,4 dihyroRA). In contrast, members of the RXR family (types alpha, beta and gamma and their isoforms) are activated by 9cis-RA only. In addition to the multiplicity of receptors, the complexity of retinoid signalling is further increased by the fact that, at least in vitro, RARs bind to their cognate response elements as heterodimers with RXRs. Moreover, RXRs can also bind, in vitro, to some DNA elements as homodimers, and are heterodimeric partners for other nuclear receptors, including TRs, VDR, PPARs and a number of orphan nuclear receptors. To evaluate the functions of the different RARs and RXRs types and isoforms, we have generated null mutant mice by targeted gene disruption in ES cells. As to the functions of RARs, we found that RAR alpha 1 and RAR gamma 2 null mutant mice are apparently normal. Mice deficient in RAR alpha or RAR gamma (i.e., all alpha or gamma isoforms disrupted) show aspects of the post-natal vitamin A deficiency (VAD) syndrome which can be cured or prevented by RA, including post-natal lethality, poor weight gain and male sterility. RAR beta 2 (and RAR beta) null mutants display a retrolenticular membrane which represents the most frequent defect of the fetal VAD syndrome. That these abnormalities were restricted to a small subset of the tissues normally expressing these receptors suggested that some degree of functional redundancy should exist in the RAR family. To test this hypothesis we then generated RAR double null mutants. RAR alpha beta, RAR alpha gamma and RAR beta gamma compound mutants exhibit all the malformations of the fetal VAD syndrome, thus demonstrating that RA is the vitamin A derivative which plays a crucial role at many different stages and in different structures during organogenesis. Interestingly, almost all the structures derived from mesenchymal neural crests cells (NCC) are affected in RAR compound mutants. As to the functions of RXRs, RXR gamma null mutants are viable, fertile and morphologically normal. In contrast, RXR alpha null fetuses display a thin ventricular wall and die in utero from cardiac failure. A myocardial hypoplasia has also been observed in some RAR compound mutants as well as in VAD fetuses. Thus, RXR alpha seems to act as an inhibitor of ventricular cardiocyte differentiation and/or as a positive regulator of their proliferation, and these functions might involve heterodimerization with RARs and activation by RA. RXR beta null mutants are viable but the males are sterile, most probably because of an abnormal lipid metabolism in the Sertoli cells. New abnormalities, absent in RXR alpha mutants, are generated in RXR alpha/RAR (alpha, beta or gamma) compound mutants. All these abnormalities are also seen in RAR double mutants as well as in VAD fetuses. In contrast, such manifestations of synergism are not observed between the RXR beta or RXR gamma and the RAR (alpha, beta or gamma) null mutations. These data strongly support the conclusion that RXR alpha/RAR heterodimers represent the main functional units of the RA signalling pathway during embryonic development. Moreover, since RXR gamma-/-/RXR beta-/-/RXR alpha +/-mutants are viable, a single allele of RXR alpha can perform most of the developmental RXR functions.