Requirement for RAR-mediated gene repression in skeletal progenitor differentiation

Chondrogenesis is a multistep process culminating in the establishment of a precisely patterned template for bone formation. Previously, we identified a loss in retinoid receptor–mediated signaling as being necessary and sufficient for expression of the chondroblast phenotype (Weston et al., 2000. J. Cell Biol. 148:679–690). Here we demonstrate a close association between retinoic acid receptor (RAR) activity and the transcriptional activity of Sox9, a transcription factor required for cartilage formation. Specifically, inhibition of RAR-mediated signaling in primary cultures of mouse limb mesenchyme results in increased Sox9 expression and activity. This induction is attenuated by the histone deacetylase inhibitor, trichostatin A, and by coexpression of a dominant negative nuclear receptor corepressor-1, indicating an unexpected requirement for RAR-mediated repression in skeletal progenitor differentiation

Active repression of RAR signaling is required for head formation

The retinoic acid receptors (RARs) recruit coactivator and corepressor proteins to activate or repress the transcription of target genes depending on the presence of retinoic acid (RA). Despite a detailed molecular understanding of how corepressor complexes function, there is no in vivo evidence to support a necessary function for RAR-mediated repression. Signaling through RARs is required for patterning along the anteroposterior (A-P) axis, particularly in the hindbrain and posterior, although the absence of RA is required for correct anterior patterning. Because RARs and corepressors are present in regions in which RA is absent, we hypothesized that repression mediated through unliganded RARs might be important for anterior patterning. To test this hypothesis, specific reagents were used that either reduce or augment RAR-mediated repression. Derepression of RAR signaling by expressing a dominant-negative corepressor resulted in embryos that exhibited phenotypes similar to those treated by RA. Anterior structures such as forebrain and cement gland were greatly reduced, as was the expression of molecular markers. Enhancement of target gene repression using an RAR inverse agonist resulted in up-regulation of anterior neural markers and expansion of anterior structures. Morpholino antisense oligonucleotide-mediated RARα loss-of-function phenocopied the effects of RA treatment and dominant-negative corepressor expression. Microinjection of wild-type or dominant-negative RARα rescued the morpholino phenotype, confirming that RAR is functioning anteriorly as a transcriptional repressor. Lastly, increasing RAR-mediated repression potentiated head-inducing activity of the growth factor inhibitor cerberus, whereas releasing RAR-mediated repression blocked cerberus from inducing ectopic heads. We conclude that RAR-mediated repression of target genes is critical for head formation. This requirement establishes an important biological role for active repression of target genes by nuclear hormone receptors and illustrates a novel function for RARs during vertebrate development

Activation by retinoids of the uncoupling protein UCP1

8 páginas, 5 figuras, 1 tabla -- PAGS nros. 157-164The uncoupling protein from brown adipose tissue (UCP1) is a transporter that catalyzes a regulated discharged of the mitochondrial proton gradient. The proton conductance in UCP1 is inhibited by nucleotides and activated by fatty acids. We have recently shown that all-trans-retinoic acid (ATRA) is a high-affinity activator of UCP1. In the present report, we have set to analyze the structural requirements for the ligands that activate UCP1 and particularly the specificity for different retinoids. For this purpose, we have developed a new protocol to determine the activity of UCP1 in respiring yeast mitochondria that can be adapted for high-throughput screenings. Our results evidence differences between the structural requirements for the activation by fatty acids and retinoids. Thus, although all active retinoids must possess a carboxylate, the introduction of additional polar groups renders them inactive. The linear and rigid structure of these molecules suggests the existence of a long hydrophobic binding pocket. We postulate that the access to the retinoid binding site must occur from the lipid bilayer and this could be at the interface between two transmembrane α-helicesThis work is supported by Allergan Sales Inc. and a project grant from the Spanish Ministry of Science and Technology (BIO2002-00142) to E.R. P.T. was supported by AllerganPeer reviewe

Dorsoventral patterning of the Xenopus eye: A collaboration of retinoid, Hedgehog and FGF receptor signaling

In the developing spinal cord and telencephalon, ventral patterning involves the interplay of Hedgehog (Hh), Retinoic Acid (RA) and Fibroblast Growth Factor (FGF) signaling. In the eye, ventral specification involves Hh signaling, but the roles of RA and FGF signaling are less clear. By overexpression assays in Xenopus embryos, we found that both RA and FGF receptor (FGFR) signaling ventralize the eye, by expanding optic stalk and ventral retina, and repressing dorsal retina character. Co-overexpression experiments show that RA and FGFR can collaborate with Hh signaling and reinforce its ventralizing activity. In loss-of-function experiments, a strong eye dorsalization was observed after triple inhibition of Hh, RA and FGFR signaling, while weaker effects were obtained by inhibiting only one or two of these pathways. These results suggest that the ventral regionalization of the eye is specified by interactions of Hh, RA and FGFR signaling. We argue that similar mechanisms might control ventral neural patterning throughout the central nervous system