scholarly journals An RPE cell line as a useful in vitro model for studying retinoic acid receptor β: expression and affinity

2008 ◽  
Vol 28 (6) ◽  
pp. 327-334 ◽  
Author(s):  
Barbara Pavan ◽  
Alessandro Dalpiaz ◽  
Carla Biondi ◽  
Marzia Nieddu ◽  
Antonella De Luca ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Retinoic Acid Receptor ◽  
Retinal Pigment ◽  
Nuclear Extract ◽  
Retinal Pigment Epithelial Cells ◽  
Receptor Subtypes ◽  
Ligand Interaction ◽  
Acid Receptor ◽  
Nuclear Extracts

Retinoids mediate their biological effect by interacting with specific nuclear receptors. Of the several known RAR (retinoic acid receptor) subtypes, RAR-β is of particular interest, since its expression is silenced in many cancers and it is believed to be a tumour suppressor. Specific ligands of RAR-β can potentially be used in anti-cancer therapy. In the present study, we have investigated the feasibility of using HRPE cells (human retinal pigment epithelial cells) as an experimental model for characterizing RAR-β–ligand interaction. RT–PCR (reverse transcription–PCR) and Western blot analyses show that HRPE cells specifically express only RAR-β and none of the other receptor subtypes. In addition, we show that the expression of RAR-β increases with increasing passage number of the cells. Interestingly, the increase in RAR-β expression is not associated with telomere shortening, a typical biomarker of cellular senescence. In the present study, we also describe a protocol for characterizing RAR-β–ligand interactions using nuclear extract from late passage HRPE cells as a source of endogenous RAR-β. Using [3H]CD367 as the ligand, RAR-β in HRPE cells showed an affinity of 9.6±0.6 nM and a Bmax of 780±14 fmol/mg of protein. We have confirmed the feasibility of using this assay to detect the interaction of ligands with RAR-β by investigating the ability of certain flavonoids to inhibit the binding of [3H]CD367 to nuclear extracts from HRPE cells. The inhibition constant of the flavonoids for RAR-β was between approx. 1–30 μM, showing that the flavonoids interact with RAR-β with low affinity.

scholarly journals Retinoic acid receptor gamma: specific immunodetection and phosphorylation.

1991 ◽  
Vol 115 (2) ◽  
pp. 535-545 ◽  
Author(s):  
C Rochette-Egly ◽  
Y Lutz ◽  
M Saunders ◽  
I Scheuer ◽  
M P Gaub ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Polyclonal Antibodies ◽  
Retinoic Acid Receptor ◽  
Amino Acid Sequences ◽  
Expression Vectors ◽  
Acid Receptor ◽  
F Region ◽  
Nuclear Extracts ◽  
F9 Embryonal Carcinoma Cells ◽  
Human And Mouse

Synthetic peptides corresponding to cDNA-deduced amino acid sequences unique to the human and mouse retinoic acid receptor gamma 1 (hRAR-gamma 1 and mRAR-gamma 1, respectively) were used to generate anti-RAR-gamma 1 antibodies. Four mAbs were selected, which were directed against peptides found in region A1 (Ab1 gamma (A1)), region F (Ab2 gamma (mF) and Ab4 gamma (hF)) and region D2 (Ab5 gamma (D2)). These antibodies specifically immunoprecipitated and recognized by Western blotting RAR-gamma 1 proteins in COS-1 cells transfected with expression vectors containing the RAR-gamma 1 cDNAs. They all reacted with both human and mouse RAR-gamma 1 proteins, except Ab4 gamma (hF) that was specific for hRAR-gamma 1. Rabbit polyclonal antibodies, directed against a peptide from the mRAR-gamma 1 F region were also obtained (RP gamma (mF)) and found to be specific for mouse RAR-gamma 1 protein. Furthermore, in gel retardation/shift assays the antibodies specifically retarded the migration of complexes obtained with a RA response element (RARE). Antibodies raised against regions D2 and F also recognized the RAR-gamma 2 isoform which differs from RAR-gamma 1 only in the A region. On the other hand, antibodies directed against the A1 region of RAR-gamma 1 (Ab1 gamma (A1)) only reacted with the RAR-gamma 1 protein. The antibodies characterized here allowed us to detect the presence of mRAR-gamma 1 and gamma 2 isoforms in mouse embryos and F9 embryonal carcinoma cells nuclear extracts. They were also used to demonstrate that the mRAR-gamma 1 protein can be phosphorylated and that the phosphorylation occurs mainly in the NH2-terminal A/B region.

scholarly journals Inhibitory effect of retinoic acid receptor agonists on in vitro chondrogenic differentiation

2019 ◽  
Vol 95 (2) ◽  
pp. 202-208
Author(s):  
Yusuke Sumitani ◽  
Kenta Uchibe ◽  
Kaya Yoshida ◽  
Yao Weng ◽  
Jiajie Guo ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Chondrogenic Differentiation ◽  
Retinoic Acid Receptor ◽  
Inhibitory Effect ◽  
Acid Receptor ◽  
Receptor Agonists

scholarly journals Effects of Vitamin-A Status on Hamster Tracheal Epithellum in Viva in Vitro

1989 ◽  
Vol 11 (3) ◽  
pp. 1-6 ◽  
Author(s):  
Luigi M. De Luca ◽  
Elizabeth M. McDowell
Keyword(s):  
Retinoic Acid ◽  
Vitamin A ◽  
Retinoic Acid Receptor ◽  
Normal Liver ◽  
Acid Receptor ◽  
New Horizons ◽  
Vitamin A Status ◽  
Essential Nutrient ◽  
Hepatoma Tissue

In this paper we have suggested the new concept of exotrophic cells, i.e. cells that have conditionally escaped the need for an essential nutrient, such as vitamin A. These exotrophs might become fixed by a mutation and eventually contribute to the tumorigenic phenotype. The discovery of the retinoic acid receptor (RAR) has opened up new horizons in the search for the mechanism of action of retinoic acid [17; 18]. It is intriguing that a second retinoic acid receptor, RARE, is abundantly expressed in hepatoma tissue and not in normal liver; Benbrook et al. [191 suggest that the erroneous expression of the RARE might contribute to tumour development in liver. How and whether these findings relate to the vitamin-A-deficient status of hepatoma cells remains to be understood.

scholarly journals Attenuation of Hypertrophy in Human MSCs via Treatment with a Retinoic Acid Receptor Inverse Agonist

2020 ◽  
Vol 21 (4) ◽  
pp. 1444 ◽  
Author(s):  
Moritz Riedl ◽  
Christina Witzmann ◽  
Matthias Koch ◽  
Siegmund Lang ◽  
Maximilian Kerschbaum ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Cell Fate ◽  
Limb Development ◽  
Retinoic Acid Receptor ◽  
Cartilage Regeneration ◽  
Inverse Agonist ◽  
Cartilage Tissue ◽  
Human Mscs ◽  
Acid Receptor

In vitro chondrogenically differentiated mesenchymal stem cells (MSCs) have a tendency to undergo hypertrophy, mirroring the fate of transient “chondrocytes” in the growth plate. As hypertrophy would result in ossification, this fact limits their use in cartilage tissue engineering applications. During limb development, retinoic acid receptor (RAR) signaling exerts an important influence on cell fate of mesenchymal progenitors. While retinoids foster hypertrophy, suppression of RAR signaling seems to be required for chondrogenic differentiation. Therefore, we hypothesized that treatment of chondrogenically differentiating hMSCs with the RAR inverse agonist, BMS204,493 (further named BMS), would attenuate hypertrophy. We induced hypertrophy in chondrogenic precultured MSC pellets by the addition of bone morphogenetic protein 4. Direct activation of the RAR pathway by application of the physiological RAR agonist retinoic acid (RA) further enhanced the hypertrophic phenotype. However, BMS treatment reduced hypertrophic conversion in hMSCs, shown by decreased cell size, number of hypertrophic cells, and collagen type X deposition in histological analyses. BMS effects were dependent on the time point of application and strongest after early treatment during chondrogenic precultivation. The possibility of modifing hypertrophic cartilage via attenuation of RAR signaling by BMS could be helpful in producing stable engineered tissue for cartilage regeneration.

Modulation of Retinoic Acid Receptor Subtypes by 5- and 8-Substituted (Naphthalen-2-yl)-based Arotinoids

ChemMedChem ◽  
2015 ◽  
Vol 10 (8) ◽  
pp. 1378-1391
Author(s):  
Susana Álvarez ◽  
Michele Lieb ◽  
Claudio Martínez ◽  
Harshal Khanwalkar ◽  
Fátima Rodríguez-Barrios ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Retinoic Acid Receptor ◽  
Receptor Subtypes ◽  
Acid Receptor

scholarly journals Ligand-Activated Retinoic Acid Receptor Inhibits AP-1 Transactivation by Disrupting c-Jun/c-Fos Dimerization

1999 ◽  
Vol 13 (2) ◽  
pp. 276-285 ◽  
Author(s):  
Xiao-Feng Zhou ◽  
Xi-Qiang Shen ◽  
Lirim Shemshedini
Keyword(s):  
Retinoic Acid ◽  
Dna Binding ◽  
Transcriptional Activity ◽  
Retinoic Acid Receptor ◽  
Cellular Growth ◽  
Dependent Manner ◽  
Acid Receptor ◽  
Retinoid Receptors ◽  
Two Hybrid

Abstract In the presence of retinoic acid (RA), the retinoid receptors, retinoic acid receptor (RAR) and retinoid X receptor (RXR), are able to up-regulate transcription directly by binding to RA-responsive elements on the promoters of responsive genes. Liganded RARs and RXRs are also capable of down-regulating transcription, but, by contrast, this is an indirect effect, mediated by the interaction of these nuclear receptors not with DNA but the transcription factor activating protein-1 (AP-1). AP-1 is a dimeric complex of the protooncoproteins c-Jun and c-Fos and directly regulates transcription of genes important for cellular growth. Previous in vitro results have suggested that RARs can block AP-1 DNA binding. Using a mammalian two-hybrid system, we report here that human RARα (hRARα) can disrupt in a RA-dependent manner the homo- and heterodimerization properties of c-Jun and c-Fos. This inhibition of dimerization is cell specific, occurring only in those cells that exhibit RA-induced repression of AP-1 transcriptional activity. Furthermore, this mechanism appears to be specific for the RARs, since another potent inhibitor of AP-1 activity, the glucocorticoid receptor, does not affect AP-1 dimerization. Our data argue for a novel mechanism by which RARs can repress AP-1 DNA binding, in which liganded RARs are able to interfere with c-Jun/c-Jun homodimerization and c-Jun/c-Fos heterodimerization and, in this way, may prevent the formation of AP-1 complexes capable of DNA binding.

scholarly journals Pharmacological Activity of Retinoic Acid Receptor Alpha-Selective Antagonists in Vitro and in Vivo

2013 ◽  
Vol 4 (5) ◽  
pp. 446-450 ◽  
Author(s):  
Sanny S. W. Chung ◽  
Rebecca A. D. Cuellar ◽  
Xiangyuan Wang ◽  
Peter R. Reczek ◽  
Gunda I. Georg ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Pharmacological Activity ◽  
Retinoic Acid Receptor ◽  
Retinoic Acid Receptor Alpha ◽  
Acid Receptor ◽  
Receptor Alpha ◽  
Selective Antagonists
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