Functional inhibition of retinoic acid response by dominant negative retinoic acid receptor mutants.

Abstract

The diverse effects of retinoids on the development, growth, and homeostasis of vertebrate organisms are mediated in part by three distinct isoforms of retinoic acid receptors (RARs). These proteins, which are structurally and functionally closely related to thyroid hormone receptors and the oncogene product v-ErbA, regulate patterns of gene expression in target tissues. One approach to study the distinct effects of retinoic acid in cells is to subvert this activity of endogenous receptors by expression of dominant negative receptor derivatives. We demonstrate here that RARa, RARf8, and RARy can be converted into potent negative transcriptional regulators that block wild-type RAR function. Furthermore, these mutant RARs, but not the wild-type receptors, actively repress the basal transcription level of target promoters. When expressed in transgenic mice, the most potent of these inhibitory receptor mutants is apparently able to disturb developmental processes by inducing a cleft palate in transgenic offspring.Retinoids and, in particular, retinoic acid (RA) are a group of vitamin A-derived substances that exert striking effects on vertebrate development. RA can promote differentiation of a wide variety of cell types in vitro, while in vivo RA has been shown to provide positional information and to influence pattern formation (for review, see refs. 1 and 2). RA is also known to be teratogenic in mammals, causing a range of abnormalities in the face, cleft palate, and malformations of the central nervous system as well as the limbs (3, 4).The identification of three distinct isoforms of RA receptors (RARs; refs. 5-11) as members of the nuclear receptor superfamily provides a model for the molecular mechanism of action of RA. This family of receptors controls cell function by regulating gene expression in target tissues in a ligandinducible fashion. With the exception of the N and C termini, which are almost completely divergent (9), the three RARs are remarkably similar to one another and are structurally and functionally closely related to thyroid hormone receptors (12, 13). Recently, a second RA response pathway has been identified and shown to be mediated by a family of retinoid X receptors (RXRs; refs. 14-17). RXRs differ from RARs in primary sequence and in their response to retinoids (18, 19). They have also been shown to function as coregulators of RARs, thyroid hormone receptors (TRs), and vitamin D3 receptors (VDRs) (16,17,20).We have previously described that the viral homologue of the TR, the v-erbA oncogene product, can function as a potent TR antagonist (21). The constitutive negative phenotype of v-ErbA is due to mutations in the C terminus of the TR (21-23), a region where v-ErbA, the TR, and RARs exhibit 53% amino acid similarity. In the present study, a further characterization of the structure/function relationship of these receptors has allowed us to convert RARa, RAR,3, and RARy into potent negative transcriptional regulators that no longer respond to RA. Furthermore, these mutant RARs ...

Progress toward discerning the genetics of cleft lip

“…Transgenic and knockout mice studies have shown that these genes are important for facial development [36,37]. Various human studies have reported both positive and negative results near the RARA gene.…”

Endogenous retinoid X receptors can function as hormone receptors in pituitary cells.

“…Our results also support the concept that some of the biological effects of RA and 9-cis RA might be mediated solely by RXRs acting as hormone receptors. This is particularly interesting in view of the tissue-specific and developmental regulation of multiple RXR subtypes (17,26,43,56,59) and the recently discovered phenotypic similarities between mice lacking RXRot and those with congenital vitamin A deficiency (57).…”

Retinoid-induced G1 Arrest and Differentiation Activation Are Associated with a Switch to Cyclin-dependent Kinase-activating Kinase Hypophosphorylation of Retinoic Acid Receptor α

“…However, introducing a normal RAR␣ cDNA into these cells restores their differentiating response to . Both the ligand-dependent transcriptional activation function AF-2 (located in the RAR␣ E region) and the ligand-independent transcriptional activation function AF-1 (located in the RAR␣ A/B region) are involved in cell differentiation (33,34). In vitro studies show that CAK phosphorylates RAR␣ at both the A/B and F regions (23).…”