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Human RARa Reporter Assay Kit
Product Description and Product Data
This is an all-inclusive cell-based luciferase reporter assay kit targeting the Human Retinoic Acid Receptor alpha (RARa). INDIGO’s RAR Alpha reporter assay utilizes proprietary mammalian cells that have been engineered to provide constitutive expression of the RAR Alpha. In addition to RAR Alpha Reporter Cells, this kit provides two optimized media for use during cell culture and in diluting the user’s test samples, a reference agonist, Luciferase Detection Reagent, and a cell culture-ready assay plate. The principal application of this assay is in the screening of test samples to quantify any functional activity, either agonist or antagonist, that they may exert against human RAR Alpha. This kit provides researchers with clear, reproducible results, exceptional cell viability post-thaw, and consistent results lot to lot. Kits must be stored at -80C. Do not store in liquid nitrogen. Note: reporter cells cannot be refrozen or maintained in extended culture.
Features
Clear, Reproducible Results
- All-Inclusive Assay Systems
- Exceptional Cell Viability Post-Thaw
- Consistent Results Lot to Lot
Product Specifications
| Target Type | Nuclear Hormone Receptor | ||
| Species | Human | ||
| Receptor Form | Hybrid | ||
| Assay Mode | Agonist, Antagonist | ||
| Kit Components |
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| Shelf Life | 6 months | ||
| Orthologs Available | Yes | ||
| Shipping Requirements | Dry Ice | ||
| Storage temperature | -80C |
Data
Target Background
Retinoic acid receptors (RARs) are nuclear hormone receptors of the NRB1 class, which function as heterodimers with retinoid X receptors (RXRs). There are three distinct RAR subtypes: RAR alpha, RAR beta, and RAR gamma. RAR alpha is present in most tissue types, whereas RAR beta and RAR gamma expression is more selective. RXR-RAR heterodimers act as ligand-dependent transcriptional regulators by binding to the specific retinoic acid response element (RARE) found in the promoter regions of target genes. In the absence of an RAR agonist, RXR-RAR recruits co-repressor proteins such as NCoR and associated factors such as histone deacetylase to maintain a condensed chromatin structure. RAR agonist binding stimulates co-repressor release and co-activator complexes, such as histone acetyltransferase, are recruited to activate transcription. RARs transduce retinoid signals in vivo, which mediates proper embryogenesis, differentiation and growth arrest. Specifically, RXRalpha-RARgamma heterodimers are necessary for growth arrest and viseral and primitive endodermal differentiation, whereas RXRalpha-RARalpha is required for cAMP-dependent parietal endodermal differentiation. In vitro it has been difficult to elucidate the roles of individual subtypes as functional RAR knockouts generate artificial redundancies that are thought not to exist under normal conditions.
INDIGO’s Human Retinoic Acid Receptor, Alpha (RARα) Reporter Assay System utilizes proprietary mammalian cells engineered to provide constitutive, high-level expression of human RARα (NR1B1), a ligand-dependent transcription factor. Because these cells incorporate a responsive luciferase reporter gene, quantifying expressed luciferase activity provides a sensitive surrogate measure of RARα activity in treated cells.
The primary application of this reporter assay system is in the screening of test samples to quantify functional activity, either agonist or antagonist, that they may exert against Human RARα.
Citations
Retinoic acid (RA, 1), an oxidized form of vitamin A, binds to retinoic acid receptors (RAR) and retinoid X receptors (RXR) to regulate gene expression and has important functions such as cell proliferation and differentiation. Synthetic ligands regarding RAR and RXR have been devised for the treatment of various diseases, particularly promyelocytic leukemia, but their side effects have led to the development of new, less toxic therapeutic agents. Fenretinide (4-HPR, 2), an aminophenol derivative of RA, exhibits potent antiproliferative activity without binding to RAR/RXR, but its clinical trial was discontinued due to side effects of impaired dark adaptation. Assuming that the cyclohexene ring of 4-HPR is the cause of the side effects, methylaminophenol was discovered through structure–activity relationship research, and p-dodecylaminophenol (p-DDAP, 3), which has no side effects or toxicity and is effective against a wide range of cancers, was developed. Therefore, we thought that introducing the motif carboxylic acid found in retinoids, could potentially enhance the anti-proliferative effects. Introducing chain terminal carboxylic functionality into potent p-alkylaminophenols significantly attenuated antiproliferative potencies, while a similar structural modification of weakly potent p-acylaminophenols enhanced growth inhibitory potencies. However, conversion of the carboxylic acid moieties to their methyl esters completely abolished the cell growth inhibitory effects of both series. Insertion of a carboxylic acid moiety, which is important for binding to RA receptors, abolishes the action of p-alkylaminophenols, but enhances the action of p-acylaminophenols. This suggests that the amido functionality may be important for the growth inhibitory effects of the carboxylic acids.
Tris(1,3-dichloro-2-propyl) phosphate (TDCPP) is an organophosphate flame retardant. The primary TDCPP metabolite, bis(1,3-dichloro-2-propyl) phosphate (BDCPP), is detectable in the urine of over 90 % of Americans. Epidemiological studies show sex-specific associations between urinary BDCPP levels and metabolic syndrome, which is an established risk factor for type 2 diabetes, heart disease, and stroke. We used a mouse model to determine whether TDCPP exposure disrupts glucose homeostasis. Six-week old male and female C57BL/6J mice were given ad libitum access to diets containing vehicle (0.1 % DMSO) and TDCPP resulting in the following treatment groups: 0 mg/kg/day, 0.02 mg/kg/day, 1 mg/kg/day, or 100 mg/kg/day. After being on the experimental diet for five weeks without interruption, body composition was analyzed, glucose and insulin tolerance tests were performed, and fasting glucose and insulin levels were quantified. TDCPP at 100 mg/kg/day caused male sex-specific adiposity, fasting hyperglycemia, and insulin resistance. TDCPP-induced modulation of nuclear receptor activation was investigated using an in vitro screen to identify potential mechanisms of metabolic disruption. TDCPP activated farnesoid X receptor (FXR) and pregnane X receptor (PXR), and inhibited the androgen receptor (AR). PXR target genes, but not FXR target genes, were upregulated in livers from mice exposed to 100 mg TDCPP/kg/day. Interestingly, PXR target genes were differentially expressed in livers from both males and females. It remains to be determined whether TDCPP-induced metabolic disruption occurs via modulation of nuclear receptor activity. Taken together, these studies build upon the association of TDCPP exposure and metabolic syndrome in humans by identifying sex-specific effects of TDCPP on glucose homeostasis in mice.
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