Individual Subunits of Heterodimers Comprised of Retinoic Acid and Retinoid X Receptors Interact with Their Ligands Independently†

Retinoid receptors, which are members of the nuclear hormone receptor superfamily, act as ligand-dependent transcription factors. They mediate the effects of retinoic acid primarily as heterodimers of retinoic acid receptors (RARs) and retinoid X receptors (RXRs). To analyse their function, xRXR beta synthetic mRNA was injected into Xenopus embryos in combination with normal and mutated xRAR alpha transcripts. Two informative phenotypes are reported here. Firstly, over-expression of xRXR beta with xRAR alpha results in the formation of ectopic primary neurons. Secondly, blocking retinoid signalling with a mutated xRAR alpha results in a lack of primary neurons. These two phenotypes, from contra-acting manipulations, indicate a role for retinoid signalling during neurogenesis.

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Retinoid X receptors stimulate and 9-cis retinoic acid inhibits 1,25-dihydroxyvitamin D3-activated expression of the rat osteocalcin gene

Molecular and Cellular Biology ◽

10.1128/mcb.13.9.5907-5917.1993 ◽

1993 ◽

Vol 13 (9) ◽

pp. 5907-5917

Author(s):

P N MacDonald ◽

D R Dowd ◽

S Nakajima ◽

M A Galligan ◽

M C Reeder ◽

...

Keyword(s):

Gene Expression ◽

Vitamin D ◽

Retinoic Acid ◽

Transcriptional Level ◽

Mobility Shift ◽

Dihydroxyvitamin D3 ◽

Retinoid X Receptors ◽

1,25 Dihydroxyvitamin D3 ◽

Osteocalcin Gene ◽

X Receptors

The vitamin D receptor (VDR) binds the vitamin D-responsive element (VDRE) as a heterodimer with an unidentified receptor auxiliary factor (RAF) present in mammalian cell nuclear extracts. VDR also interacts with the retinoid X receptors (RXRs), implying that RAF may be related to the RXRs. Here we demonstrate that highly purified HeLa cell RAF contained RXR beta immunoreactivity and that both activities copurified and precisely coeluted in high-resolution hydroxylapatite chromatography. Furthermore, an RXR beta-specific antibody disrupted VDR-RAF-VDRE complexes in mobility shift assays. These data strongly indicate that HeLa RAF is highly related to or is identical to RXR beta. Consequently, the effect of the 9-cis retinoic acid ligand for RXRs was examined in 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]-activated gene expression systems. Increasing concentrations of 9-cis retinoic acid (1 nM to 1 microM) markedly reduced 1,25(OH)2D3-dependent accumulation of osteocalcin mRNA in osteoblast-like ROS 17/2.8 cells. All-trans retinoic acid also interfered with vitamin D responsiveness, but it was consistently less potent than the 9-cis isomer. Transient transfection studies revealed that attenuation by 9-cis retinoic acid was at the transcriptional level and was mediated through interactions at the osteocalcin VDRE. Furthermore, overexpression of both RXR beta and RXR alpha augmented 1,25(OH)2D3 responsiveness in transient expression studies. Direct analysis of VDRE binding in mobility shift assays demonstrated that heteromeric interactions between VDR and RXR were enhanced by 1,25(OH)2D3 and were not affected appreciably by 9-cis retinoic acid, except that inhibition was observed at high retinoid concentrations. These data suggest a regulatory mechanism for osteocalcin gene expression that involves 1,25(OH)2D3-induced heterodimerization of VDR and unliganded RXR. 9-cis retinoic acid may attenuate 1,25(OH)2D3 responsiveness by diverting RXRs away from VDR-mediated transcription and towards other RXR-dependent transcriptional pathways.

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Terminal differentiation in keratinocytes involves positive as well as negative regulation by retinoic acid receptors and retinoid X receptors at retinoid response elements

Molecular and Cellular Biology ◽

10.1128/mcb.12.11.4862-4871.1992 ◽

1992 ◽

Vol 12 (11) ◽

pp. 4862-4871

Author(s):

B J Aneskievich ◽

E Fuchs

Keyword(s):

Retinoic Acid ◽

Terminal Differentiation ◽

Cell Types ◽

Epidermal Differentiation ◽

Retinoic Acid Receptors ◽

Keratinocyte Differentiation ◽

Epidermal Keratinocytes ◽

Retinoid X Receptors ◽

Response Elements ◽

X Receptors

Terminal differentiation of epidermal keratinocytes is inhibited by 1 microM retinoic acid, a concentration which induces differentiation in a number of cell types, including F9 teratocarcinoma cells. The molecular basis for these opposing retinoid responses is unknown, although retinoic acid receptors (RARs) and retinoid X receptors (RXRs) have been detected in both cell types. When F9 cells are stably transfected with a truncated RAR alpha lacking the E/F domain necessary for ligand binding and RAR/RXR dimerization, action at retinoid response elements is suppressed and cells produce a retinoic acid-resistant phenotype; i.e., they are blocked in differentiation (A. S. Espeseth, S. P. Murphy, and E. Linney, Genes Dev. 3:1647-1656, 1989). If retinoid receptors influence epidermal differentiation only in a negative fashion, then suppression of transactivation at retinoid response elements would be expected to enhance, rather than block, keratinocyte differentiation. In this study, we show that surprisingly, even though constitutive expression of an analogous truncated RAR gamma in keratinocytes specifically suppressed transactivation at retinoid response elements, keratinocytes were blocked, rather than enhanced, in their ability to undergo morphological and biochemical features of differentiation. These findings demonstrate a direct and hitherto unrecognized role for RARs and RXRs in positively as well as negatively regulating epidermal differentiation. Additionally, our studies extend those of Espeseth et al. (Genes Dev. 3:1647-1656, 1989), indicating a novel RAR function independent of the E/F domain.

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