Retinoid receptor-specific agonists regulate bovine in vitro early embryonic development, differentiation and expression of genes related to cell cycle arrest and apoptosis

A major goal in reproductive biotechnology is the identification of pathways that regulate early embryonic development and the allocation of cells to the inner cell mass (ICM) and trophectoderm (TE). Retinoids regulate the development and differentiation of the bovine blastocyst in vitro, although the involvement of the retinoid X receptors (RXRs) remains to be clarified. This paper compares the effect of a synthetic RXR agonist (LG100268; LG) with that of the retinoic acid receptor (RAR) agonist all-trans retinoic acid (ATRA) on blastulation. In vitro-produced morulae were treated for 48 h with LG (0.1 microM, 1 microM and 10 microM), ATRA 0.7 microM, or no additives. Treatment with ATRA did not increase the rate of development; however, the LG 0.1 microM treatment increased both the blastocyst development and hatching rate. Cell numbers increased in the ICM with LG 10 microM, while a dose-dependent reduction was observed in the TE in the presence of LG. Gene expression levels of p53 and p66 did not vary with LG but increased with ATRA. Both LG and ATRA activated bax, a pro-apoptotic gene and H2A.Z, a cell cycle-related gene. The above effects suggest the existence of active p53-dependent and -independent apoptotic pathways for ATRA and LG, respectively, in the bovine embryo. The expression of p53 and H2A.Z showed a strong, positive correlation (r=0.93; p<0.0001) in all experimental groups; both proteins are linked through the cell cycle. Agonists of RXR could be used to control blastocyst development and differentiation.     

Control of retinoic acid receptor heterodimerization by ligand-induced structural transitions. A novel mechanism of action for retinoid antagonists

Heterodimerization of retinoic acid receptors (RARs) with 9-cis-retinoic receptors (RXRs) is a prerequisite for binding of RXR.RAR dimers to DNA and for retinoic acid-induced gene regulation. Whether retinoids control RXR/RAR solution interaction remains a debated question, and we have used in vitro and in vivo protein interaction assays to investigate the role of ligand in modulating RXR/RAR interaction in the absence of DNA. Two-hybrid assay in mammalian cells demonstrated that only RAR agonists were able to increase significantly RAR interaction with RXR, whereas RAR antagonists inhibited RXR binding to RAR. Quantitative glutathione S-transferase pull-down assays established that there was a strict correlation between agonist binding affinity for the RAR monomer and the affinity of RXR for liganded RAR, but RAR antagonists were inactive in inducing RXR recruitment to RAR in vitro. Alteration of coactivator- or corepressor-binding interfaces of RXR or RAR did not alter ligand-enhanced dimerization. In contrast, preventing the formation of a stable holoreceptor structure upon agonist binding strongly altered RXR.RAR dimerization. Finally, we observed that RAR interaction with RXR silenced RXR ligand-dependent activation function. We propose that ligand-controlled dimerization of RAR with RXR is an important step in the RXR.RAR activation process. This interaction is dependent upon adequate remodeling of the AF-2 structure and amenable to pharmacological inhibition by structurally modified retinoids.     

Morphogenetic effects of 9-cis-retinoic acid on the regenerating limbs of the axolotl

9-cis-retinoic acid has recently been found to be a high affinity ligand for the retinoic X receptor (RXR). RXRs are believed to be involved in metabolic activities rather than in morphogenetic ones. Interestingly, RXR has been found to form heterodimers involving other receptors from the steroid family, such as the thyroid hormone receptor, vitamin D receptor or retinoic acid receptors (RARs). In this paper we examined whether or not 9-cis-retinoic acid had any morphogenetic properties on the regenerating axolotl limb. It is shown that 9-cis-retinoic acid proximalized regenerating limbs and was somewhat more potent in this action than all-trans-retinoic acid. Based on these observations, the possible roles of other receptors during pattern formation is discussed. Correspondence to: P.A. Tsonis     

The influence of heterodimer partner ultraspiracle/retinoid X receptor on the function of ecdysone receptor

A pair of nuclear receptors, ecdysone receptor (EcR) and ultraspiracle (USP), heterodimerize and transduce ecdysteroid signals. The EcR and its nonsteroidal ligands are being developed for regulation of transgene expression in humans, animals and plants. In mammalian cells, EcR:USP heterodimers can function in the absence of ligand, but EcR/retinoid X receptor (EcR:RXR) heterodimers require the presence of ligand for activation. The heterodimer partner of EcR can influence ligand sensitivity of EcR so that the EcR/Locusta migratoria RXR (EcR:LmRXR) heterodimers are activated at lower concentrations of ligand when compared with the concentrations of ligand required for the activation of EcR/Homo sapiens RXR (EcR:HsRXR) heterodimers. Analysis of chimeric RXRs containing regions of LmRXR and HsRXR and point mutants of HsRXR showed that the amino acid residues present in helix 9 and in the two loops on either end of helix 9 are responsible for improved activity of LmRXR. The EcR:Lm-HsRXR chimera heterodimer induced reporter genes with nanomolar concentration of ligand compared with the micromolar concentration of ligand required for activating the EcR:HsRXR heterodimer. The EcR:Lm-HsRXR chimera heterodimer, but not the EcR:HsRXR heterodimer, supported ligand-dependent induction of reporter gene in a C57BL/6 mouse model.     

Isolation and characterization of unsaturated fatty acids as natural ligands for the retinoid-X receptor

The retinoid-X receptor (RXR) is a ligand activated nuclear receptor that is the heterodimer partner for many class II nuclear receptors. Previously identified natural ligands for this receptor include 9-cis retinoic acid (9cRA), docosahexaenoic acid, and phytanic acid. Our studies were performed to determine if there are any unidentified, physiologically important RXR ligands. Agonists for RXR were purified from rat heart and testes lipid extracts with the use of a cell-based reporter assay to monitor RXR activation. Purified active fractions contained a variety of unsaturated fatty acids and components were quantified by gas-liquid chromatography of derivatized samples. The corresponding fatty acid standards elicited a similar response in the reporter cell assay. Competition binding analysis revealed that the active fatty acids compete with [3H]9cRA for binding to RXR. Non-esterified fatty acids were analyzed from lipid extracts of isolated heart and testes nuclei and endogenous concentrations were found to be within the range of their determined binding affinities. Our studies reveal tissue dependent profiles of RXR agonists and support the idea of unsaturated fatty acids as physiological ligands of RXR.