Retinoid Response Elements Research Articles

A pleiotropic element in the medium-chain acyl coenzyme A dehydrogenase gene promoter mediates transcriptional regulation by multiple nuclear receptor transcription factors and defines novel receptor-DNA binding motifs.

We previously identified a complex regulatory element in the medium-chain acyl coenzyme A dehydrogenase gene promoter that confers transcriptional regulation by the retinoid receptors RAR and RXR and the orphan nuclear receptor HNF-4. In this study we demonstrate a trans-repressing regulatory function for the orphan receptor COUP-TF at this same nuclear receptor response element (NRRE-1). The transcriptional regulatory properties and receptor binding sequences of each nuclear receptor response element within NRRE-1 are also characterized. NRRE-1 consists of four potential nuclear hormone receptor hexamer binding sites, arranged as [<--1-(n)s-2-->-3-->(n)4<--4], three of which are used in alternative pairwise binding by COUP-TF and HNF-4 homodimers and by RAR-RXR heterodimers, as demonstrated by mobility shift assays and methylation interference analysis. Binding and transactivation studies with mutant NRRE-1 elements confirmed the existence of distinct retinoid, COUP-TF, and HNF-4 response elements that define novel receptor binding motifs: COUP-TF homodimers bound sites 1 and 3 (two hexamer repeat sequences arranged as an everted imperfect repeat separated by 14 bp or ER14), RAR-RXR heterodimers bound sites 1 and 2 (ER8), and HNF-4 homodimers bound sites 2 and 3 (imperfect DR0). Mixing cotransfection experiments demonstrated that the nuclear receptor dimers compete at NRRE-1 to modulate constitutive and ligand-mediated transcriptional activity. These data suggest a mechanism for the transcriptional modulation of genes encoding enzymes involved in cellular metabolism.

A Pleiotropic Element in the Medium-Chain Acyl Coenzyme A Dehydrogenase Gene Promoter Mediates Transcriptional Regulation by Multiple Nuclear Receptor Transcription Factors and Defines Novel Receptor-DNA Binding Motifs

We previously identified a complex regulatory element in the medium-chain acyl coenzyme A dehydrogenase gene promoter that confers transcriptional regulation by the retinoid receptors RAR and RXR and the orphan nuclear receptor HNF-4. In this study we demonstrate a trans-repressing regulatory function for the orphan receptor COUP-TF at this same nuclear receptor response element (NRRE-1). The transcriptional regulatory properties and receptor binding sequences of each nuclear receptor response element within NRRE-1 are also characterized. NRRE-1 consists of four potential nuclear hormone receptor hexamer binding sites, arranged as [&lt;--1-(n)s-2--&gt;-3--&gt;(n)4&lt;--4], three of which are used in alternative pairwise binding by COUP-TF and HNF-4 homodimers and by RAR-RXR heterodimers, as demonstrated by mobility shift assays and methylation interference analysis. Binding and transactivation studies with mutant NRRE-1 elements confirmed the existence of distinct retinoid, COUP-TF, and HNF-4 response elements that define novel receptor binding motifs: COUP-TF homodimers bound sites 1 and 3 (two hexamer repeat sequences arranged as an everted imperfect repeat separated by 14 bp or ER14), RAR-RXR heterodimers bound sites 1 and 2 (ER8), and HNF-4 homodimers bound sites 2 and 3 (imperfect DR0). Mixing cotransfection experiments demonstrated that the nuclear receptor dimers compete at NRRE-1 to modulate constitutive and ligand-mediated transcriptional activity. These data suggest a mechanism for the transcriptional modulation of genes encoding enzymes involved in cellular metabolism.

RZRs, a new family of retinoid-related orphan receptors that function as both monomers and homodimers.

Members of the superfamily of nuclear receptors share the greatest homology in their DNA-binding domains. We have used reverse transcription-polymerase chain reaction and highly degenerate primers based on the amino acid sequence of the zinc finger motif of known nuclear receptors to identify novel members of the family. Starting with rat brain RNA, we have isolated an orphan receptor that we call RZR beta. The sequence of its nearly full-length complementary DNA shows great similarity to RZR alpha, a receptor we recently identified from human umbilical vein endothelial cells. These RZR subtypes represent members of a new family of orphan nuclear receptors that most likely regulate specific gene expression. Sequence comparison with other known nuclear receptors reveals great similarity for both RZR subtypes to retinoic acid and retinoid-X receptors. By Northern blot analyses, we found RZR beta messenger RNA only in brain, whereas RZR alpha is expressed in many tissues. We show here that the RZRs bind as monomers to natural retinoid response elements formed by (A/G)GGTCA half-sites. However, a T-residue in the -1 position of this motif greatly enhances the DNA binding affinity of RZRs, whereas the -2 position has no influence. We show that RZRs can bind as homodimers on response elements formed by palindromes, inverted palindromes, or direct repeats of two TAGGTCA half-sites. Interestingly, these response elements display dramatically reduced affinity for retinoic acid receptor-retinoid-X receptor heterodimers. Thus, the 5'-flanking sequence of hexameric half-sites appears to be crucial to direct the activity of several nuclear receptors. On monomeric as well as dimeric binding sites, RZRs show constitutive transactivational activity that can be enhanced by unidentified components of fetal calf serum.

Presence of a Retinoid Responsive Element in the Promoter Region of the Human Cytochrome P4501A1 Gene

Cytochrome P4501A1 (CYP1A1) plays a key role in the metabolic activation of procarcinogenic compounds, leading to skin carcinogenesis. It is therefore important to determine whether its enzymatic activity is altered by topically administered drugs. We investigated, in cultured normal human keratinocytes (NHK), the effects of retinoic acid (RA) and synthetic analogs on the regulation of the CYP1A1 gene. Using transient transfections and gel shift assays, we demonstrated that the human CYP1A1 gene promoter was differentially regulated by retinoid receptors. We report, for the first time, that a RA responsive element 5′-CTT AGGTCACCAC GGGGCA-3′ (RARE1A1) is present within the promoter region of the CYP1A1 gene.

Down-regulation of retinoic acid receptor activity associated with decreased alpha and gamma isoforms expression in F9 embryonal carcinoma cells differentiated by retinoic acid.

F9 embryonal carcinoma cells differentiate in response to retinoic acid (RA). To investigate the regulation of RA receptors (RARs) expression during this process, cDNA probes specific for the major RAR isoforms were used. In contrast to the level of RAR beta 2 mRNA which was high in cells treated 5 days with RA and below detection in untreated cells, as previously described, the steady state levels of RAR alpha 1, alpha 2, gamma 1, and gamma 2 mRNAs were markedly decreased in the RA-differentiated cells as compared to untreated cells. The down-regulation of the RA-responsive system in differentiated cells was also evident in gel shift assays as a marked decrease in binding capacity to a retinoid acid response element (beta 2RARE), as well as in chloramphenicol acetyltransferase (CAT) assays as a sixfold decrease in RA-mediated transacting activity via this element. The down-regulation of RAR DNA-binding and transacting activity coincided with the burst in tissue plasminogen activator secretion and thus, occurred at the hinge between early and late differentiation. The down-regulation of RA responsiveness may constitute an important event in the transition between early and late differentiation stage in F9 cells.

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.

Retinoid-dependent transcriptional suppression of cytokeratin gene expression in human epidermal squamous cell carcinoma cells

Abstract We have previously demonstrated that cytokeratin levels are coordinately regulated in normal cultured human keratinocytes. In the present study we examine the mechanism of this regulation using human squamous cell carcinoma (SCC) cells. Treatment of SCC-13 cells with 20 or 200 n M trans-retinoic acid results in nearly complete suppression of cytokeratin K5 and K6 expression. This change is accompanied by a simultaneous reduction (> 20-fold) in the level of the mRNAs encoding K5 and K6. Transcriptional analysis indicates that the transcription rate of the K5 and K6 genes drops by approximately four to fivefold in retinoid treated nuclei. Retinol (2000 n M) also promotes this change. In contrast, cytokeratin K19 does not increase in the presence of retinoic acid, thus the normal coordinate regulation of keratin gene expression by retinoids appears to be uncoupled in SCC-13 cells. However, this does not represent a general defect in positive regulation of gene expression by retinoids, since in a transient transfection assay trans-retinoic acid positively regulates a reporter plasmid containing the retinoid response element from the retinoic acid receptor-β gene. The synthetic retinoids Ro 13–6298 (ethyl ester) and its metabolic derivative Ro 13–7410 (free acid) are both active in modulating the differentiation of normal keratinocytes. In contrast, only Ro 13–7410 is active in SCC-13 cells. As Ro 13–6298 binds poorly to the retinoic acid receptors, this suggests that SCC-13 cells, unlike normal keratinocytes, lack the ability to convert Ro 13–6298 to the active Ro 13–7410. These results further suggest that an inability to metabolize retinoid ethyl esters may be a property of transformed epidermal keratinocytes.

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

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.

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

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.

Identification of a novel retinoid-responsive element in the promoter region of the medium chain acyl-coenzyme A dehydrogenase gene.

To study the mechanisms involved in regulation of nuclear genes encoding mitochondrial enzymes in oxidative energy pathways, the promoter region of the medium-chain acyl-CoA dehydrogenase (MCAD) gene was analyzed. A series of hexamer sequences known to bind and confer responsiveness to a subset of members of the nuclear receptor superfamily of transcription factors was identified. Cotransfection of an MCAD promoter-chloramphenicol acetyltransferase (CAT) reporter plasmid with retinoic acid receptor (RAR)alpha, beta, or retinoid X receptor alpha (RXR alpha) resulted in 10-15-fold transcriptional activation in response to retinoic acid. The retinoic acid-induced activation was 3-4-fold higher with RXR alpha than with either RAR alpha or RAR beta. Deletional analysis confirmed that a region between -341 and -308 base pairs upstream of the MCAD gene cap site conferred the RA-responsive transcriptional activation to homologous and heterologous promoters. Gel mobility shift assays demonstrated that the MCAD RARE interacted directly with overexpressed receptors. Mutational analysis of the RARE delineated three hexamer binding sequences with unique orientation and spacing compared to other reported retinoid responsive elements. These results indicate that the MCAD gene promoter region contains a novel regulatory element that interacts with members of the retinoid receptor family, with preferential activation by RXR alpha. This element likely plays a role in the transcriptional regulation of this gene and perhaps others involved in oxidative energy metabolism.

Axis derangement in Xenopus laevis through nocodazole and deuterium oxide treatments : [formula omitted]1 Dept. of Biosciences, South Gujarat Univ., Surat 395007, India. 2 Hubrecht Laboratory, Uppsalalaan 8, 3584 CT Utrecht, Netherlands

The field of vitamin A research has witnessed a remarkable surge in interest since the late 1980's, when the retinoid receptors were discovered and their genes cloned. Heterodimeric interactions between the retinoid X receptors (RXRs α, β, and γ), which bind 9-cis-retinoic acid, and other hormone receptors, including the retinoic acid receptors (RARs α, β, and γ), the thyroid hormone receptor (TR), the vitamin D receptor (VDR), the peroxisomal proliferator activated receptor (PPAR), and others make hormone action dependent on retinoid homeostasis. Retinoid response elements (RAREs) are present in the promoter and/or enhancer regions of several genes, including some of the homeobox genes, which control development and differentiation. The interaction between hormones and retinoids is added additional orders of complexity by the diversity of the RAREs including the spacer length, their 5′ or 3′ position, and their coexistence in composite sequences with other hormone response elements (e.g., an estrogen response element in the lactablbumin gene promoter, see Table 2). Control of normal epithelial differentiation is a fundamental function of retinoids. The histogenesis of squamous metaplasia caused by vitamin A deficiency is a stepwise process, which permits the gradual transition of phenotypes from simple-columnar, typical of the endocervical epithelium, to pseudostratified, to stratified-squamous and, eventually, to keratinizing. Conversely, the maintenance of the squamous keratinizing differentiation in the ectocervix and vagina requires estrogen. In the absence of this hormone, the squamous stratified ectocervical epithelium retrogrades to a simpler, two or three cell layer morphology, with the topmost layer expressing keratin K8, typical of the endocervical epithelium and mucous cells. Retinoid and estrogen receptor transcript expression is governed by dietary retinoid status and by estrogen availability, with squamous cells mostly expressing RARγ and estrogen receptor transcripts, and columnar cells mostly expressing RARβ. RXR transcripts appear mostly expressed in proliferating cells. The relevance of the retinoid receptors to carcinogenesis is highlighted in the work on acute promyelocytic leukemia. This work has demonstrated that the fusion gene PML-RARα, resulting from the t(15;17) chromosomal translocation, is etiologically connected with the disease and with complete remission after oral retinoid administration. Developments in retinoid metabolism, including the cloning of the cytochrome P450RAI and the connection between RA metabolism and cell growth inhibition, have recently taken place. Recent work has also shown that pharmacological dietary retinoic acid specifically inhibits malignant conversion in the mouse two-stage carcinogenesis system. Because RA upregulates retinoid receptor expression, it seems that retinoid receptors function as tumor suppressors. p]This field should serve as a paradigm for things to come for other essential nutrients, and spells out the notion that nutritional sciences are indeed fundamentally important, because they can contribute significantly to our understanding of different diseases and provide effective therapeutic approaches.