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Results for “Retinoic Acid Receptor alpha”

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At least 19 recordsLinked to original sources
PubMed2005

Expression of estrogen receptor alpha, retinoic acid receptor alpha and cellular retinoic acid binding protein II genes is coordinately regulated in human breast cancer cells.

Human breast cancer cell lines expressing the estrogen receptor alpha (ERalpha), all-trans-retinoic acid (ATRA) receptor alpha (RARalpha) and cellular retinoic acid binding protein II (CRABPII) genes are sensitive to ATRA-mediated growth inhibition. To study the relationship among ERalpha, RARalpha and CRABPII expression, the protein levels of each member were compared in five breast cancer cell lines (T47D, MCF-7, ZR-75-1, Hs587 T and MDA-MB-231 cells) and two immortalized nontumorigenic breast epithelial cell lines (MTSV1.7 and MCF-10A). ERalpha, RARalpha and CRABPII proteins were detected in T47D, MCF-7 and ZR-75-1 cells but not in other tested cell lines. RARalpha and CRABPII proteins were either reduced or undetectable in T47D/C4:2W and MCF-7/ADR cells with lost expression of ERalpha. Estradiol increased and anti-estrogens (tamoxifen and ICI 164,384) downregulated the expression of both RARalpha and CRABPII proteins in T47D and MCF-7 cells. RARalpha antagonist Ro-41-5253 inhibited CRABPII expression, but not RARalpha expression in estradiol-treated T47D and MCF-7 cells. Suppression of ERalpha by small interfering RNA (siRNA) reduced RARalpha and CRABPII gene expression and siRNA suppression of RARalpha reduced CRABPII expression while having no effect on ERalpha in T47D cells. Transient transfection of either RARalpha or ERalpha expression vectors increased CRABPII expression in MDA-MB-231 cells but only RARalpha, not ERalpha, activated hCRABPII promoter reporter. These results indicate that there is a gene activation pathway in which ERalpha drives RARalpha transcription and RARalpha drives CRABPII transcription in ERalpha-positive human breast cancer cells.

Animals↗
PubMed2000

Critical role of tyrosine 277 in the ligand-binding and transactivating properties of retinoic acid receptor alpha.

Retinoic acid receptors specifically bind all-trans-retinoic acid (RA) and function as RA-inducible transcriptional regulatory factors. Binding of RA to RARalpha, beta, and gamma is sensitive to nitration with tetranitromethane, a tyrosine-specific modifying reagent. To identify tyrosine residue(s) that are important for RA binding, we carried out chemical modification experiments with purified RARalpha ligand-binding domain (RARalpha-LBD) subjected to partial acid hydrolysis and selective proteolysis. The chemically modified peptides containing each of the three Tyr residues present in the RARalpha-LBD sequence were then analyzed and identified by high-performance liquid chromatography coupled to electrospray ionization mass spectrometry (HPLC/ESI-MS). We found that RA binding to RARalpha-LBD protected Tyr(277)-containing peptides from nitration. Protection of Tyr(277) could result either from direct masking by the bound ligand or from ligand-induced changes in receptor conformation and tyrosine accessibility. The role of Tyr residues was further documented by site directed mutagenesis using three site-specific RARalpha mutants: Y208A, Y277A, and Y362A. The affinity for RA of these mutant receptors was in the range of that of the wild-type protein, except for the Y277A receptor mutant, which displays a 15-20-fold reduction in affinity and transactivation activity for RA. Whereas mutation of Tyr(277) into alanine had a variable effect on different agonists and antagonists binding, it caused a dramatic decrease of retinoid-dependent transactivation activity. This later effect was also observed with mutation of Tyr(277) into phenylalanine. It is unlikely that major conformational changes are responsible for the lower affinity of RA binding and RA-dependent transactivation since these mutants displayed wild-type dimerization and DNA-binding activities. Limited proteolysis revealed that upon ligand binding, the Y277A mutant induced a conformational change slightly different from that obtained with the wild-type protein. These data could suggest that Tyr(277) play a critical role in the ligand-induced conformational changes required for the activation of RARalpha.

Alanine↗
PubMed1995

Separation of transactivation and AP1 antagonism functions of retinoic acid receptor alpha.

Retinoic acid receptors (RARs) regulate gene expression either by directly binding to the RAR-responsive elements or by antagonizing the action of c-Jun/c-Fos (AP1). AP1 is involved in the expression of metalloproteases, cytokines and other factors which play critical roles in the turnover of extracellular matrix, inflammation and hyperproliferation in diseases such as psoriasis, rheumatoid arthritis and in tumor metastases. We demonstrate here that synthetic retinoids inhibit 12-O-tetradecanoylphorbol-14-acetate-induced transcription from the stromelysin AP1 motif through RAR alpha, -beta, and -gamma. Interestingly, these diaryl acetylenic retinoids, which are potent agonists only for RAR beta and RAR gamma, but not for RAR alpha, in transactivation assays, are able to inhibit AP1-dependent gene expression through RAR alpha. Thus these analogs can differentially affect the transactivation and AP1 antagonistic functions of RAR alpha. These results demonstrate that the transactivation and AP1 antagonistic functions are separable, and it should be possible to develop retinoids that are completely specific for AP1 antagonism through all RARs. Furthermore, using an RAR-selective ligand, we also demonstrate the separation of ligand binding and AP1 antagonism functions of RARs.

Base Sequence↗
PubMed1996

Location of two photoaffinity-labeled sites on the ligand-binding domain of retinoic acid receptor alpha.

Retinoic acid receptors (RARs) consist of six domain structures. The C-terminal region (D/E/F-domains) is involved in ligand binding, dimerization, and ligand-dependent transactivation. Structural information about RARs is required for understanding its complex function. A photoreactive retinoid denoted as ADAM-3, which was designed as the result of comparison of two fluorescent retinoids (DAM-3 and DAM-15), was synthesized and used for photoaffinity labeling of recombinant protein MBP-RAR alpha/E. The photoaffinity-labeled site was determined by an endoprotease combination method which utilizes four endoproteinases in a two-phase digestion procedure. Two major labeled fragments were detected in each digestion, and the results of two-phase digestion allowed identification of the labeled residues as being located within residues 492-510 and 585-594, which correspond to 288-306 and 381-390 in human RAR alpha, respectively.

Affinity Labels↗
PubMed1996

E3, a hematopoietic-specific transcript directly regulated by the retinoic acid receptor alpha.

Retinoic acid (RA)-induced maturation mediated by the retinoic acid receptor alpha (RAR alpha) has been implicated in myeloid development. We have used differential hybridization analysis of a cDNA library constructed from the murine RA-inducible MPRO promyelocyte cell line to identify immediate-early genes induced by RA during granulocytic differentiation. E3, one of nine sequences identified, was upregulated in an immediate-early manner, with transcript levels peaking after 60 minutes exposure to RA. E3 transcripts were RA-inducible in HL60 cells, but not in an RA-resistant subclone, HL60R, that harbors a mutated RAR alpha gene. However, when HL60R cells were transduced with a functional copy of the RAR alpha gene, RA induced a 10-fold increase in E3 mRNA levels. E3 transcripts are present in the myeloid, B-lymphoid, and erythroid lineages, absent in nonhematopoietic cells, and encode a highly hydrophobic, potentially phosphorylated polypeptide of unknown function with significant homology to a putative protein expressed in myeloid cells. The murine E3 promoter harbors a single bipartite retinoic acid response element which in transient transfection assays conferred RA sensitivity. These results indicate that E3 is a hematopoietic-specific gene that is an immediate target for the activated RAR alpha during myelopoiesis.

Amino Acid Sequence↗
PubMed2005

c-Jun N-terminal kinase contributes to aberrant retinoid signaling in lung cancer cells by phosphorylating and inducing proteasomal degradation of retinoic acid receptor alpha.

Retinoic acid (RA) is the ligand for nuclear RA receptors (RARs and RXRs) and is crucial for normal epithelial cell growth and differentiation. During malignant transformation, human bronchial epithelial cells acquire a block in retinoid signaling caused in part by a transcriptional defect in RARs. Here, we show that activation of c-Jun N-terminal kinase (JNK) contributes to RAR dysfunction by phosphorylating RARalpha and inducing degradation through the ubiquitin-proteasomal pathway. Analysis of RARalpha mutants and phosphopeptide mapping revealed that RARalpha residues Thr181, Ser445, and Ser461 are phosphorylated by JNK. Mutation of these residues to alanines prevented efficient ubiquitination of RARalpha and increased the stability of the protein. We investigated the importance of RARalpha phosphorylation by JNK as a mediator of retinoid resistance in lung cancer. Mice that develop lung cancer from activation of a latent K-ras oncogene had high intratumoral JNK activity and low RARalpha levels and were resistant to treatment with an RAR ligand. JNK inhibition in a human lung cancer cell line enhanced RARalpha levels, ligand-induced activity of RXR-RAR dimers, and growth inhibition by RA. These findings point to JNK as a key mediator of aberrant retinoid signaling in lung cancer cells.

3T3 Cells↗
PubMed1996

The AF-2 region of the retinoic acid receptor alpha mediates retinoic acid inhibition of estrogen receptor function in breast cancer cells.

The growth of estrogen receptor (ER)-positive breast cancer cells is inhibited by all-trans-retinoic acid (RA). In the present study, estrogen (E2) induction of pS2 mRNA levels was significantly reduced within 6 h following cotreatment with RA. In transient transfection experiments, RA repressed transactivation from a vitellogenin E2-responsive element by approximately 50% and wild-type RA receptor alpha (RARalpha) or RARbeta enhanced this inhibition. Transfection of truncated RARalpha mutants terminating before or at amino acid 412 markedly decreased RA inhibition of E2-induced reporter gene activity. Expression of RARs with deletions of amino acids 413 and 414 in the transactivation-2 (AF-2) domain also reduced RA inhibition, while deletions and point mutations beyond amino acid 414 behaved like the wild-type RARalpha. RA-treated MCF-7 cells transfected with an RARalpha AF-2 region mutant were twice as sensitive to growth inhibition as untransfected and vector-transfected control cells. Thus, the AF-2 domain in the C terminus of the RARalpha mediates RA inhibition of ER-induced transcription in breast cancer cells. In addition, transcriptional interference between RARs and ERs may contribute to RA inhibition of ER-positive breast cancer cell growth.

Base Sequence↗
PubMed1997

Differential role of homologous positively charged amino acid residues for ligand binding in retinoic acid receptor alpha compared with retinoic acid receptor beta.

The diverse biological actions of retinoic acid (RA) are mediated by retinoic acid receptors (RARs) and retinoid X receptors. Although it has been suggested that the ligand binding domains (LBDs) of RARs share the same novel folding pattern, many RAR subtype-specific agonists and antagonists have been synthesized demonstrating that the LBD of each RAR subtype has unique features. We have examined the role of several positively charged amino acid residues located in the LBD of RARalpha in RA binding. These results are compared with previously published data for the homologous mutations in RARbeta. Lys227 of RARalpha does not appear to be important for RA binding or RA-dependent transactivation, whereas the homologous residue in RARbeta, Lys220, plays an important synergistic role with Arg269 in these two activities. In addition, Arg276 of RARalpha, like its homologous residue Arg269 of RARbeta, was found to play an important role in the binding of RA most likely by interacting with the carboxylate group of RA. However, the orientation of and electronic environment associated with Arg276 in RARalpha appears to be different from that of Arg269 in RARbeta, thus contributing to the uniqueness of the ligand binding pocket of each receptor.

Amino Acids, Diamino↗
PubMed2006

Regulation of retinoic acid receptors alpha, beta and retinoid X receptor alpha after sciatic nerve injury.

Cell culture experiments indicated that activation of the retinoic acid signaling system is involved in axonal regeneration. This hypothesis was tested with sciatic nerve injury in the rat. Since the effect of retinoic acid is mediated via retinoic acid receptors and retinoid X receptors, we investigated mRNA and protein expression of these receptors during injury-induced degeneration and regeneration. Seven days after crush injury, transcript concentrations of all retinoic acid receptors and of retinoid X receptor alpha were significantly higher than in non-lesioned nerves. Protein levels of retinoic acid receptor alpha, retinoic acid receptor beta and retinoid X receptor alpha were upregulated 4, 7 and 14 days after injury. In degenerating nerves a significant increase of retinoic acid receptor alpha was detected 7 and 14 days, and of retinoic acid receptor beta 14 and 21 days after complete transection. Immunohistochemical staining of retinoid receptors revealed their expression in Schwann cells and macrophages. In addition, we observed that retinoic acid receptor alpha and retinoid X receptor alpha appeared in the cell nuclei of macrophages during the lesion-induced inflammatory reaction, and that retinoid X receptor alpha-staining co-localized with some regenerating axons. Experiments with Schwann cell primary cultures revealed an effect of retinoic acid on the expression of the neuregulin receptor ErbB3, suggesting that one function of retinoic acid consists in the regulation of neuroglial interactions after peripheral nerve injury.

Animals↗
PubMed1995

Characterization of cDNAs encoding two chick retinoic acid receptor alpha isoforms and distribution of retinoic acid receptor alpha, beta and gamma transcripts during chick skin development.

The amino acid sequence of the retinoic acid receptors alpha, beta and gamma (RAR alpha, beta and gamma) can be divided into six functional domains (A-F), different isoforms arising from the presence of different A domains by differential splicing. In order to address the respective roles of the different RARs during skin morphogenesis in birds, cDNAs encoding two chick RAR alpha isoforms (alpha1 and alpha2) have been isolated. While the A1 and B-F domains of the RAR alpha are highly conserved across species, the chick A2 domain contains 50% specific amino acids. The three RAR alpha, beta and gamma genes display specific patterns of expression during chick skin morphogenesis. As in mouse, RAR alpha and gamma transcripts are present in both the dermis and epidermis during the first stages of skin appendage formation. Furthermore, Northern blot analysis suggests that different RAR alpha and gamma isoforms could be successively required during feather formation. The RAR gamma gene, continuously expressed in the epidermal cells in both chick and mouse, is thus likely to play a similar role in skin development in these two species. However, RAR alpha transcripts, only transiently detected during mouse skin development, still accumulate in epidermis during the later stages of chick skin differentiation. Furthermore, RAR beta transcripts, never detected during normal development in mouse skin, are actually present at the early stages of chick skin morphogenesis. Thus, our results suggest that the role of the three RAR in skin development has not been strictly conserved in the different classes of vertebrates.

Amino Acid Sequence↗
PubMed1996

Dominant negative mutant of retinoic acid receptor alpha inhibits retinoic acid-induced P19 cell differentiation by binding to DNA.

Retinoic acid (RA) is a potent inducer of P19 cell differentiation. RA activity is thought to be mediated by nuclear RA receptors (RARs), transcription factors whose activity is dependent on RA. There are three RARs called alpha, beta, and gamma. We created truncated versions of the three RARs and compared their activities as inhibitors of RA-mediated gene transcription and of P19 cell differentiation. Only mutants of the RAR alpha were inhibitory in these assays. A mutant of RAR alpha carrying a 10-amino-acid insert was able to heterodimerize with RXRbeta or with the normal RAR alpha and the inhibitory activity of this mutant was dependent on an intact DNA binding domain. We conclude that dominant negative mutants of RAR alpha act by heterodimerizing with RXRs or RARs and binding to RA response elements on DNA, thereby preventing binding of the normal receptors to those sites.

Animals↗
PubMed1994

Binding of 9-cis-retinoic acid and all-trans-retinoic acid to retinoic acid receptors alpha, beta, and gamma. Retinoic acid receptor gamma binds all-trans-retinoic acid preferentially over 9-cis-retinoic acid.

Both 9-cis-retinoic acid (RA) and all-trans-RA (t-RA) compete for [3H]9-cis-RA binding to RA receptors (RAR alpha, beta, and gamma) in nucleosol fractions from transiently transfected COS-1 cells with IC50 values of approximately 12 and 5 nM, respectively. Curiously, 9-cis-RA competes for [3H]t-RA binding to mouse RAR alpha, beta, and gamma with IC50 values of 31, 8, and 60 nM, respectively, while t-RA itself does not exhibit such differential competition (IC50 values for RARs, 5 nM). A similar pattern is observed with human retinoic acid receptors (RARs). Differential binding of 9-cis-RA to the RAR beta and gamma receptors is also found following in vitro transcription and translation of these receptors. Displacement assays demonstrate that t-RA exhibits similar off-rates for RAR alpha, beta, and gamma. However, 9-cis-RA is 6-fold more rapidly displaced from RAR gamma than from RAR beta. When RAR-transfected COS-1 cells are incubated with [3H]t-RA, [3H]-9-cis-RA or various mixtures of these two radioligands, high performance liquid chromatography analysis demonstrates that the ligands bound in nucleosol fractions from RAR beta-transfected cells reflect the isomer content of the media. However, in identical whole cell assays, nucleosol fractions from RAR gamma-transfected cells preferentially bind t-RA over 9-cis-RA, consistent with the in vitro data. These binding kinetics in vitro and in whole cells suggest that there could be differences in the interactions of the receptor subtypes with the endogenous retinoic acids under physiologic conditions.

Animals↗
PubMed1997

Expression of retinoid-X receptors (-alpha,-beta,-gamma) and retinoic acid receptors (-alpha,-beta,-gamma) in normal human skin: an immunohistological evaluation.

Increasing evidence suggests that the retinoid-X receptors (RXR-alpha,-beta,-gamma) play a crucial role in regulating the transcriptional activity of several steroid hormone receptors, including the receptors for retinoic acid (RAR-alpha,-beta,-gamma), 1,25-dihydroxyvitamin D3 and thyroid hormone. We investigated the localization of the different types of RXR-alpha,-beta,-gamma and RAR-alpha,-beta,-gamma proteins in frozen sections of normal human skin (n = 12) in situ, applying recently raised corresponding specific monoclonal antibodies and an immunohistochemical technique that we established for the detection of these nuclear receptors. Our findings indicate that RXR-alpha,-beta,-gamma and RAR-alpha,-beta,-gamma proteins can be detected by immunohistochemistry in normal human skin. In contrast to RXR-alpha,-beta,-gamma as well as RAR-alpha and RAR-gamma proteins that were consistently detected in cell layers of the viable epidermis, RAR-beta was only focally demonstrated in single epidermal cells in three out of 12 biopsies analysed. Immunohistochemical labelling of RAR-alpha,-beta,-gamma and RXR-alpha,-beta,-gamma proteins in epidermal nuclei was also pronounced in the stratum granulosum, suggesting a function of RXR and RAR proteins in the transition from proliferation to differatiation in epidermal keratinocytes. Expression of RXRs and RARs in hair follicles, sebaceous glands and endothelial cell points to a biological function from these nuclear receptors to hair growth as well as to the physiology of sebaceous glands and endothelial cells.

Humans↗
PubMed1999

Overexpression of wild-type retinoic acid receptor alpha (RARalpha) recapitulates retinoic acid-sensitive transformation of primary myeloid progenitors by acute promyelocytic leukemia RARalpha-fusion genes.

Retinoic acid receptor alpha (RARalpha) is the target of several chromosomal translocations associated with acute promyelocytic leukemias (APLs). These rearrangements fuse RARalpha to different partner genes creating the chimeric proteins: PML-RARalpha, PLZF-RARalpha, and NPM-RARalpha. Although the vast majority of APLs respond to retinoic acid therapy, those associated with PLZF-RARalpha are resistant. We have used retroviruses to express PML-RARalpha, PLZF-RARalpha, NPM-RARalpha, RARalpha403 (a dominant negative mutant of RARalpha), and wild-type RARalpha in murine bone marrow progenitors and found that all of these constructs blocked differentiation and led to the immortalization of myeloid progenitors. This cellular transformation is specific to an alteration of the RARalpha pathway because overexpression of RARbeta, RARgamma, or RXRalpha did not result in similar growth perturbations. Pharmacological doses of RA induced differentiation and inhibited proliferation of cells transformed with either of the APL fusion genes, including PLZF-RARalpha, whereas physiological retinoic acid concentrations were sufficient to reverse the phenotype of cells transformed with wild-type RARalpha. The cellular responses to retinoic acid were accompanied by a sharp decrease in the amount of the RARalpha-fusion proteins expressed in the cells. Our findings suggest that the oncogenicity of RARalpha-fusion proteins results from their nature to behave as unliganded RARalpha in the presence of physiological concentrations of retinoic acid.

Animals↗
PubMed1993

Co-amplification of erbB2, topoisomerase II alpha and retinoic acid receptor alpha genes in breast cancer and allelic loss at topoisomerase I on chromosome 20.

The DNA topoisomerase enzymes are targets for the cytotoxic effects of a number of anticancer agents termed topoisomerase inhibitors. We have analysed breast cancer biopsy specimens for genetic alterations at and around topoisomerase loci in order to obtain molecular insight into factors which may determine how tumours respond to chemotherapy. We show that of 50 tumours examined, the topoisomerase II alpha locus is co-amplified in 3 cases out of 6 with erbB2 amplification and that amplification can be accompanied by high expression of topoisomerase II alpha. In our attempts to distinguish amplification from aneuploidy and define the limits of amplification, we also observed co-amplification of the retinoic acid-alpha receptor with erbB2 and topoisomerase II alpha in the same three samples. At the topoisomerase I locus on chromosome 20, we observed allelic loss in two out of 17 samples. Genetics abberations at topoisomerase loci, therefore, appear to be relatively common in breast cancer.

Aged↗
PubMed1993

Expression of topoisomerase II alpha and beta in an adenocarcinoma cell line carrying amplified topoisomerase II alpha and retinoic acid receptor alpha genes.

Human topoisomerase II enzymes are targets for a number of widely used anticancer agents. We have analysed a lung adenocarcinoma cell line CALU3, which has co-amplified topoisomerase II alpha and ERBB2 sequences, for the structure of the amplicon and for expression of both topoisomerase II alpha and beta. The region of chromosome 17q amplified in CALU3 also includes the retinoic acid receptor alpha locus and is therefore similar to the amplicon observed in breast cancers carrying amplified topoisomerase II alpha and retinoic acid receptor sequences. The use of fluorescence in situ hybridisation localises the amplified topoisomerase II alpha sequences to a cluster on one chromosome with single copies localised to others. CALU3 express high levels of topoisomerase II alpha is determined by Western blot, immunofluorescence and enzyme activity. The enzyme activity extracted from CALU3 is sensitive to inhibition by the topoisomerase II poison etoposide. Topoisomerase II beta expression was observed in three lung cancer cell lines including CALU3 and was confined to the nucleoli. Thus, the CALU3 cell line is an ideal model to study the amplification and expression of topoisomerase II alpha in adenocarcinomas.

Blotting, Western↗
PubMed1993

Induction of HOX-2 genes in P19 embryocarcinoma cells is dependent on retinoic acid receptor alpha.

In vertebrate embryos retinoic acid can act as a teratogen and may have a natural function as a morphogen. In both cases the mechanism by which retinoic acid exerts its effect may involve alteration of the level of HOX gene transcription. We have investigated the role of retinoic acid receptor alpha (RAR alpha) in retinoic acid-dependent induction of HOX-2 gene expression in embryocarcinoma (EC) cells by using the P19 RAC65 EC cell line, which is retinoic acid-resistant due to the expression of a dominant negative RAR alpha. No induction of HOX-2 genes by retinoic acid was seen, while in P19 RAC65 EC cells stably transfected with human RAR alpha under the control of a constitutive promoter HOX-2 gene induction is restored. This shows that RAR alpha is involved in HOX gene induction by retinoic acid.

Animals↗
PubMed1996

Allelic fusion of DNA topoisomerase II alpha and retinoic acid receptor alpha genes in adriamycin-resistant p388 murine leukemia revealed by fluorescence in situ hybridization.

Fluorescence in situ hybridization (FISH) analysis of metaphase and decondensed free chromatin fibers from Adriamycin (ADR)-sensitive and ADR-resistant murine cells demonstrated a close juxtaposition of topoisomerase II alpha (Top2a) and retinoic acid receptor alpha (Rara) genes in adjacent chromatin in the drug-resistant cells, and a close but separate genetic proximity in normal murine chromatin. This provides physical evidence that the chromosome 11 allelic rearrangement resulting in a chimeric truncated Top2a/Rara transcript in the ADR-resistant cells is due to a novel fusion of the Topo2a and Rara genes. This is the first description of a Rara gene disruption in cells selected for antineoplastic drug resistance.

Alleles↗