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PubMed1995

Retinoids, interferon alpha, 1,25-dihydroxyvitamin D3 and their combination inhibit angiogenesis induced by non-HPV-harboring tumor cell lines. RAR alpha mediates the antiangiogenic effect of retinoids.

Retinoids combined with interferon alpha-2a (IFN alpha) or 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] have shown marked synergistic inhibitory effects on angiogenesis induced by tumor cell lines harboring DNA of oncogenic human papillomaviruses (HPV) type 16 or 18. This report demonstrates comparable effects of these compounds on angiogenesis induced by non-HPV-bearing transformed cell lines, including breast carcinoma (T47D) and vulval carcinoma (A431) cell lines. Systemic treatment of mice with all-trans retinoic acid, 13-cis retinoic acid, 9-cis retinoic acid, IFN alpha or 1,25(OH)2D3 significantly decreased tumor cell-induced angiogenesis (TIA). In vitro pretreatment of T47D and A431 cells with these compounds also led to inhibition of their angiogenic capability when tested in the TIA assay. The inhibitory effects of retinoids could be counteracted by a selective antagonist of the nuclear retinoic acid receptor RAR alpha, suggesting a RAR alpha mediated mechanism of angiogenesis inhibition. The antiangiogenic effect of retinoids could be significantly enhanced by combination with IFN alpha or 1,25(OH)2D3. The results provide a further basis for the use of combinations of retinoids with IFN alpha or 1,25(OH)2D3 in the treatment of angiogenesis-dependent malignancies.

Animals↗

PubMed1990

Site of conversion of endogenous all-trans-retinoids to 11-cis-retinoids in the bovine eye.

By use of a new high-resolution high-pressure liquid chromatographic method for the separation of isomeric forms of retinol, retinal, retinyl ester and retinal oxime, various retinoids were analyzed in separated retinal pigment epithelial tissue or neural retinal tissue from fresh bleached bovine eyes after incubation in the dark at either 30 or 4 degrees C for 90 min. 11-cis-Retinoids significantly increased during incubation at 30 degrees C, relative to those at 4 degrees C, in the retinal pigment epithelium, but not in the retina. The major forms of vitamin A in incubated retinal pigment epithelium and neural retina were retinyl esters (70%) and all-trans-retinol (69%), respectively. Thus, in keeping with observations on the isomerization of radioactive retinol in homogenates of eye tissues, the retinal pigment epithelium seems to be the primary site of 11-cis-retinoid formation from endogenous all-trans-retinoids in the bovine eye.

Animals↗

PubMed2005

Biochemical properties of purified human retinol dehydrogenase 12 (RDH12): catalytic efficiency toward retinoids and C9 aldehydes and effects of cellular retinol-binding protein type I (CRBPI) and cellular retinaldehyde-binding protein (CRALBP) on the oxidation and reduction of retinoids.

Retinol dehydrogenase 12 (RDH12) is a novel member of the short-chain dehydrogenase/reductase superfamily of proteins that was recently linked to Leber's congenital amaurosis 3 (LCA). We report the first biochemical characterization of purified human RDH12 and analysis of its expression in human tissues. RDH12 exhibits approximately 2000-fold lower K(m) values for NADP(+) and NADPH than for NAD(+) and NADH and recognizes both retinoids and lipid peroxidation products (C(9) aldehydes) as substrates. The k(cat) values of RDH12 for retinaldehydes and C(9) aldehydes are similar, but the K(m) values are, in general, lower for retinoids. The enzyme exhibits the highest catalytic efficiency for all-trans-retinal (k(cat)/K(m) approximately 900 min(-)(1) microM(-)(1)), followed by 11-cis-retinal (450 min(-)(1) mM(-)(1)) and 9-cis-retinal (100 min(-)(1) mM(-)(1)). Analysis of RDH12 activity toward retinoids in the presence of cellular retinol-binding protein (CRBP) type I or cellular retinaldehyde-binding protein (CRALBP) suggests that RDH12 utilizes the unbound forms of all-trans- and 11-cis-retinoids. As a result, the widely expressed CRBPI, which binds all-trans-retinol with much higher affinity than all-trans-retinaldehyde, restricts the oxidation of all-trans-retinol by RDH12, but has little effect on the reduction of all-trans-retinaldehyde, and CRALBP inhibits the reduction of 11-cis-retinal stronger than the oxidation of 11-cis-retinol, in accord with its higher affinity for 11-cis-retinal. Together, the tissue distribution of RDH12 and its catalytic properties suggest that, in most tissues, RDH12 primarily contributes to the reduction of all-trans-retinaldehyde; however, at saturating concentrations of peroxidic aldehydes in the cells undergoing oxidative stress, for example, photoreceptors, RDH12 might also play a role in detoxification of lipid peroxidation products.

Alcohol Oxidoreductases↗

PubMed1997

Enhancement of Calu-1 human lung carcinoma cell growth in serum-free medium by retinoids: dependence on AP-1 activation, but not on retinoid response element activation.

Many lung cancer cell lines are resistant to the growth inhibitory effects of retinoids. However, some small-cell lung cancer cell lines were inhibited by all trans-retinoic acid (ATRA) in serum-free medium. We compared the responses of seven non-small cell lung cancer (NSCLC) cell lines to ATRA in serum-free medium and in medium supplemented with delipidized serum. Whereas the growth of four cell lines was inhibited more in serum-free medium, the growth of the Calu-1 cell line was stimulated by ATRA in a dose-dependent fashion with a maximum at 10(-8) M. Delipidized serum (>2.5%) but not bovine serum albumin (0.15%) suppressed growth stimulation by ATRA. Transcripts of RA receptors RARalpha and RARgamma but not of RARbeta were detected in Calu-1 cells. Receptor expression, the formation of a complex among receptors and a RA-responsive element (RARE), and the transcriptional activation RARE were not suppressed by serum. Natural retinoids and synthetic receptor class- or subtype-selective retinoid agonists, which activated RARs and RXRs for gene transcription from a RARE, and a RAR antagonist (CD2366), which was unable to do so, stimulated the growth of Calu-1 cells in serum-free medium but not in serum-containing medium. Both ATRA and CD2366 enhanced the transcriptional activation of an Activator Protein-1 (AP-1)-luciferase reporter construct in serum-free medium but not in delipidized serum. Transcriptional activation of the RARE by ATRA occurred both in the presence or absence of delipidized serum. These results demonstrate that retinoid-induced growth stimulation of Calu-1 cells is associated with enhanced AP-1 transactivation but not with RARE transactivation.

Antineoplastic Agents↗

PubMed1993

Correlations of RAR isoforms and cellular retinoid-binding proteins mRNA levels with retinoid-induced teratogenesis.

Retinoic acid (RA) plays an important role in normal embryogenesis; however, excessive doses are teratogenic. At present, the molecular mechanisms responsible for these effects of RA are not well understood. The action of retinoids are believed to be mediated by two classes of proteins, nuclear receptors (retinoic acid receptors [RARs] and retinoid X receptors [RXRs]) and small cellular retinol-binding and retinoic acid-binding proteins (CRBP-I, CRBP-II, CRABP-I and CRABP-II). Teratogenic doses of RA increase the level of RAR-beta 2 mRNA, RAR-alpha 2 mRNA, CRBP-I mRNA and CRABP-II mRNA in mouse conceptuses and embryos. The elevation in the level of only RAR-beta 2 mRNA correlates with the target tissues, as well as developmental stages that are sensitive to the teratogenic effects of RA. In addition, we have screened a few other natural and synthetic retinoids with similar results. These results are consistent with the possibility that RAR-beta 2 may mediate at least some of the effects of retinoids during abnormal development.

Abnormalities, Drug-Induced↗

PubMed1984

Topical and systemic effects of retinoids on horn-filled utriculus size in the rhino mouse. A model to quantify "antikeratinizing" effects of retinoids.

A method was developed to quantify the "antikeratinizing" effects of various retinoids (all-trans-retinoic acid, 13-cis-retinoic acid, motretinide, etretinate) in rhino mouse skin, which contains many keratinized pilosebaceous structures or horn-filled utriculi. Mean utriculus diameters in whole mount epidermis were determined and dose-response relationships were constructed after topical or oral administration of test retinoids. All-trans-retinoic acid was 3.7x, 12.5x, and 50x more potent than 13-cis-retinoic acid, etretinate, and motretinide, respectively, after topical administration. Administered orally, all-trans-retinoic acid was 2.3x more potent than 13-cis-retinoic acid. At 5 mg/kg, each retinoid produced a significant reduction in utriculus size. The rhino mouse model represents a novel and useful assay to quantify antikeratinizing activity and potency differences of biologically active retinoids.

Administration, Oral↗

PubMed1992

Effects of topical retinoids on cytoskeletal proteins: implications for retinoid effects on epidermal differentiation.

In vivo effects of retinoids on epidermal differentiation were investigated by analyzing cytoskeletal proteins in rhino mice treated topically with all-trans-retinoic acid (RA) and other retinoids (13-cis-retinoic acid, etretinate, TTNPB). Non-disulfide-linked cytoskeletal proteins, including keratins from the epidermal "living layers," were first selectively extracted using 9.5 M urea; subsequently, keratins of the stratum corneum were isolated using 9.5 M urea plus a reducing agent. Gel electrophoresis and immunoblot analysis showed that urea extracts of epidermis from vehicle-treated skin were composed predominantly of four major keratins (analogous to human epidermal keratins K1, K5, K10, and K14), and the keratin filament-associated protein filaggrin. In contrast, extracts of epidermis from retinoid-treated skin contained additional keratins (K6, K16, and K17) and almost no detectable filaggrin. Furthermore, similar analysis of stratum corneum keratins demonstrated that extracts from RA-treated skin did not contain the partially proteolyzed keratins typically observed in stratum corneum extracts of control animals. Hyperplasia-inducing agents (salicylic acid, croton oil) caused an increase in keratins K6, K16, and K17, but they did not effect filaggrin or alter proteolysis of stratum corneum keratins. The result that RA induced expression of keratins K6, K16, and K17, as commonly expressed in hyperproliferative epidermis, is consistent with the notion that retinoids increase epidermal cell proliferation in the basal and/or lower spinous layers. The findings that topical RA decreased filaggrin expression and reduced proteolysis of stratum corneum keratins, despite increased size and number of granular cells and the presence of an anucleate stratum corneum, suggest that topical RA may also modulate a later stage of epidermal differentiation involved in stratum corneum formation.

Administration, Topical↗

PubMed2003

Constituents within pulp mill effluent deplete retinoid stores in white sucker and bind to rainbow trout retinoic acid receptors and retinoid X receptors.

Wild female and male white sucker (Catostomus commersoni) inhabiting an area receiving pulp mill effluent had reduced hepatic levels of retinol, didehydroretinol, retinyl esters, and didehydroretinyl esters, while vitamin E levels were unaffected. This disruption of the retinoid system led us to test methanol and dichloromethane extracts from the effluent of 11 pulp mills from across Canada for their ability to bind to rainbow trout (Oncorhynchus mykiss) retinoic acid receptors (RARs) from the gill and retinoid X receptors (RXRs) from the liver. Concentrated extracts of the final effluent from 6 of the 11 pulp mills were able to displace greater than 25% of the receptor-bound [3H]all-trans retinoic acid (RA) or [3H]9-cis RA from trout RARs and RXRs, respectively. The ability of the extracts to displace retinoic acid did not appear to be linked to the pulping or treatment processes. Moreover, extracts with the greatest activity came from thermomechanical mills, suggesting the compounds may originate from the wood furnish. In addition, extracts prepared from wood furnish (wood chips: white spruce [50%], lodgepole pine [47%], and balsam fir [3%]) from one mill were able to displace [3H]RA from the RARs and RXRs. The 4-hydroxy RA, a metabolite of RA that has been shown to be generated in greater quantities in fish exposed to P450-inducing xenobiotics, was able to displace [3H]all-trans RA from trout RARs as effectively as unlabeled all-trans RA. These results suggest that pulp mill effluent may impact the retinoid system of fish at multiple sites, either by decreasing hepatic retinoid stores or through contributing additional ligands (from the wood furnish) that can bind to RA receptors.

Animals↗

PubMed2004

Retinoids and retinoid receptors in the control of energy balance: novel pharmacological strategies in obesity and diabetes.

Obesity and type II diabetes are closely related metabolic diseases with an increasing incidence worldwide. No clear-cut pharmacological treatment for these complex metabolic disturbances is available despite current efforts. New directions and perspectives for the pharmacological or nutritional treatment of these diseases should be defined. In recent years, a growing body of evidence shows that retinoids and retinoic acid receptors are involved in the control of biological aspects (e.g. adiposity and energy expenditure mechanisms), which offers great potential for research on the treatment of obesity and type II diabetes. All-trans retinoic acid is known to inhibit adipocyte differentiation, whereas, molecules activating the retinoid X-receptor (rexinoids) promote the differentiation of adipocytes. Treatment with rexinoids ameliorates glycemic control in rodent models of type II diabetes and obesity, although other findings indicate similar positive effects by inhibiting the receptor. Moreover, natural products of dietary origin, such as phytanic acid can activate RXR and thus, trigger adipose cell differentiation. Finally, the activation of retinoic acid receptors or retinoid X receptors has been reported to induce the gene expression of uncoupling proteins, which are mitochondrial proteins involved in the regulation of energy expenditure and fatty acid metabolism. Further research is required to exploit the capacities of the retinoid-dependent pathways of regulation of adiposity, insulin sensitivity and energy expenditure for drug development in metabolic disturbances.

Animals↗

PubMed2002

The retinoid X receptor-selective retinoid, LGD1069, prevents the development of estrogen receptor-negative mammary tumors in transgenic mice.

Despite the effectiveness of the selective estrogen receptor (ER) modulators in preventing ER-positive breast cancer, chemopreventive agents still need to be developed for the prevention of ER-negative breast cancers. The naturally occurring retinoids are promising agents for the prevention of human cancers but are too toxic for long-term chronic use. We previously demonstrated that the chemopreventive effects of the retinoids could be separated from the toxicity by using an RXR-selective retinoid, LGD1069. The studies described here demonstrate that LGD1069 effectively suppresses ER-negative tumor development in mouse mammary tumor virus-erbB2 transgenic mice with minimal toxicity. These studies suggest that receptor-selective retinoids are promising agents for the prevention of breast cancer and that they may be particularly useful in preventing ER-negative breast cancer.

Animals↗

PubMed2002

The expression of cellular retinoid binding proteins in non-APL leukemic cells and its association with retinoid sensitivity.

Retinoic acid (RA) has important effects on cell differentiation and cell growth and on normal embryonic development. Intracellular retinoid signaling induced by endogenous or exogenous RA is regulated by retinoid binding proteins such as CRBPI, CRABPI and CRABPII and there are data suggesting that the expression of these proteins can influence the sensitivity to the growth inhibitory effects of ATRA. In this study, we investigated the basal and ATRA-induced expression of CRBPI and CRABPI and II in leukemic cell lines and in cells from patients with acute myeloid leukemia (AML). CRBPI as well as CRABPI and II were expressed in all tested cell lines and in leukemic cells from all 18 AML-patients. CRABPII mRNA expression was more abundant than CRBPI and CRABPI in both the cell lines and the patient cells but the levels compared the house keeping gene was lower in the patient cells. In all cell lines and in 69% of the patient samples, ATRA did upregulate CRABPII whereas CRBPI exhibited a varying response and CRABPI was more commonly downregulated. The sensitivity to the growth inhibitory effects of ATRA did not correlate with the basal expression of any of these proteins. However, ATRA-induced upregulation of CRABPII did significantly correlate with the ATRA sensitivity (p < 0.005) as well as with ATRA-induced upregulation of the retinoid receptor RARbeta (p < 0.05). We conclude that the retinoid binding proteins CRBPI and CRABPI and II are expressed in myeloid leukemic cells of non-M3 type but that the level of expression does not affect ATRA sensitivity.

Gene Expression↗

PubMed1982

Identification of the endogenous retinoids associated with three cellular retinoid-binding proteins from bovine retina and retinal pigment epithelium.

The endogenous retinoids associated with three cellular retinoid-binding proteins from bovine retina and retinal pigment epithelium have been identified by their spectral characteristics and their co-migration with authentic retinoids on high performance liquid chromatography. All-trans-retinol is the only retinoid associated with the cellular retinol-binding protein from retina and retinal pigment epithelium. The saturation of the binding site with native ligand is 0.95 +/- 0.05 mol of retinoid/mol of protein for cellular retinol-binding protein purified from frozen and fresh retina and retinal pigment epithelium. All-trans-retinoic acid has been identified as the endogenous ligand associated with the cellular retinoic acid-binding protein of cattle retina. The saturation of the binding site of cellular retinoic acid-binding protein from frozen and fresh retina (the protein is absent in extracts of retinal pigment epithelium) is about 0.2 mol of retinoid/mol of protein, probably a minimum value due to losses of the ligand. Retinoic acid has not been detected previously in retina. Cellular retinaldehyde-binding protein from retina purifies with two endogenous ligands which cochromatograph on high performance liquid chromatography with 11-cis-retinaldehyde and 11-cis-retinol and occur in a ratio of approximately 3:1, respectively. The binding site of cellular retinaldehyde-binding protein from frozen retina is nearly fully occupied with these two ligands (saturation greater than 0.9 mol of retinoids/mol of protein). In contrast, purified cellular retinaldehyde-binding protein from retinal pigment epithelium carries only 11-cis-retinaldehyde as an endogenous ligand. The saturation of the binding site with this ligand is greater than 0.95.

Animals↗

PubMed2006

Expression of the human ABCC6 gene is induced by retinoids through the retinoid X receptor.

Mutations in the human ABCC6 gene are responsible for the disease pseudoxanthoma elasticum, although the physiological function or substrate of the gene product (an ABC transporter known also as MRP6) is not known. We found that the expression of this gene in cells of hepatic origin (where this gene is predominantly expressed in the body) is significantly upregulated by retinoids, acting as agonists of the retinoid X receptor (RXR) rather than the retinoid A receptor (RAR). The direct involvement of this nuclear receptor in the transcriptional regulation of ABCC6 gene expression was confirmed by transient transfection and chromatin immunoprecipitation assays. This constitutes the first direct proof of previously suggested involvement of nuclear hormone receptors in ABCC6 gene expression and the first identification of a transcription factor which may be relevant to regulation of ABCC6 level in tissues and in some PXE patients.

Animals↗

PubMed1994

Synthesis and structure-activity relationships of novel retinoid X receptor-selective retinoids.

Two series of potent retinoid X receptor (RXR)-selective compounds were designed and synthesized based upon recent observation that (E)-4-[2-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-2-naphthalenyl)-1- propenyl]benzoic acid (TTNBP) binds and transactivates only the retinoic acid receptor (RAR) subtypes whereas (E)-4-[2-(3,5,5,8,8-pentamethyl-5,6,7,8- tetrahydro-2-naphthalenyl)-1-propenyl]benzoic acid (3-methyl TTNPB) binds and transactivates both the RAR and RXR subfamilies. Addition of functional groups such as methyl, chloro, bromo, or ethyl to the 3 position of the tetrahydronaphthalene moiety of 4-[(5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-2-naphthyl)carbonyl]benzoic acid (5a) and 4-[1-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-2- naphthyl)ethenyl]benzoic acid (6a) results in compounds which elicit potent and selective activation of the RXR class. Such RXR-selective compounds offer pharmacological tools for elucidating the biological role of the individual retinoid receptors with which they interact. Activation profiles in cotransfection and competitive binding assays as well as molecular modeling calculations demonstrate critical structural determinants that confer selectivity for members of the RXR subfamily. The most potent compound of these series, 4-[1-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphthyl)ethenyl]ben zoi c acid (6b), is the first RXR-selective retinoid (designated as LGD1069) to enter clinical trials for cancer indications.

Benzoates↗

PubMed1997

Regulation of retinoidal actions by diazepinylbenzoic acids. Retinoid synergists which activate the RXR-RAR heterodimers.

In human HL-60 promyelocytic leukemia cells, diazepinylbenzoic acid derivatives can exhibit either antagonistic or synergistic effects on the differentiation-inducing activities of natural or synthetic retinoids, the activity depending largely on the nature of the substituents on the diazepine ring. Thus, a benzolog of the retinoid antagonist LE135 (6), 4-(13H-10,11,12,13-tetrahydro-10, 10,13,13,15-pentamethyldinaphtho[2,3-b][1,2-e]diazepin-7-yl) benzoic acid (LE540, 17), exhibits a 1 order of magnitude higher antagonistic potential than the parental LE135 (6). In contrast, 4-[5H-2,3-(2,5-dimethyl-2,5-hexano)-5-methyldibenzo[b,e] [1,4]diazepin-11-yl]-benzoic acid (HX600, 7), a structural isomer of the antagonistic LE135 (6), enhanced HL-60 cell differentiation induced by RAR agonists, such as Am80 (2). This synergistic effect was further increased for a thiazepine, HX630 (29), and an azepine derivative, HX640 (30); both synergized with Am80 (2) more potently than HX600 (7). Notably, the negative and positive effects of the azepine derivatives on retinoidal actions can be related to their RAR-antagonistic and RXR-agonistic properties, respectively, in the context of the RAR-RXR heterodimer.

Azepines↗

PubMed2003

Retinoid activation of retinoic acid receptor but not retinoid X receptor is sufficient to rescue lethal defect in retinoic acid synthesis.

Two isomers of retinoic acid (RA) may be necessary as ligands for retinoid signaling: all-trans-RA for RA receptors (RARs) and 9-cis-RA for retinoid X receptors (RXRs). This was explored by using retinaldehyde dehydrogenase (Raldh)2-/- mouse embryos lacking mesodermal RA synthesis that display early growth arrest unless rescued by all-trans-RA administration. Because isomerization of all-trans-RA to 9-cis-RA can occur, it is unclear whether both ligands are needed for rescue. We show here that an RAR-specific ligand can rescue Raldh2-/- embryos as efficiently as all-trans-RA, whereas an RXR-specific ligand has no effect. Further, whereas all-trans-RA was detected in embryos, 9-cis-RA was undetectable unless a supraphysiological dose of all-trans-RA was administered, revealing that 9-cis-RA is of pharmacological but not physiological significance. Because 9-cis-RA is undetectable and unnecessary for Raldh2-/- rescue, and others have shown that 4-oxo-RA is unnecessary for mouse development, all-trans-RA emerges as the only ligand clearly necessary for retinoid receptor signaling.

Aldehyde Oxidoreductases↗

PubMed1995

Efficient inhibition of activation-induced Fas ligand up-regulation and T cell apoptosis by retinoids requires occupancy of both retinoid X receptors and retinoic acid receptors.

Two retinoic acid (RA) receptors, retinoic acid receptors (RARs) and retinoid X receptors (RXRs), have been identified. All-trans-RA and its 9-cis-isomer are ligands for RARs, but only 9-cis-RA binds RXRs with high affinity. Activation-induced T cell hybridoma death is mediated via the engagement of Fas by activation-up-regulated Fas ligand, and RA prevents this type of apoptosis by inhibiting the induction of Fas ligand expression. To investigate the mechanism of RA action, T hybridoma cells were transfected with cDNA encoding RXR beta or dominant-negative RXR beta. Cells that overexpressed RXR beta were more sensitive to 9-cis-RA rescue from activation-induced death than cells transfected with vector alone. In contrast, cells expressing the dominant-negative RXR beta could not be rescued from death with 9-cis-RA. In wild type cells, an RAR-selective synthetic retinoid had little effect on activation-induced apoptosis, while an RXR-selective agonist prevented apoptosis but only at concentrations about approximately 10-fold greater than that required for 9-cis-RA. Simultaneous addition of the RAR- and RXR-selective retinoids completely prevented activation-induced apoptosis at concentrations where either alone had relatively little protective effect. The same hierarchy of efficacy was found for activation-induced Fas ligand expression. These data demonstrate that binding of both RARs and RXRs is required for efficient inhibition of activation-induced Fas ligand upregulation and T cell apoptosis by retinoic acid.

Animals↗

PubMed1997

Retinoid X receptor (RXR) within the RXR-retinoic acid receptor heterodimer binds its ligand and enhances retinoid-dependent gene expression.

Retinoic acid receptor (RAR) and retinoid X receptor (RXR) form heterodimers and regulate retinoid-mediated gene expression. We studied binding of RXR- and RAR-selective ligands to the RXR-RAR heterodimer and subsequent transcription. In limited proteolysis analyses, both RXR and RAR in the heterodimer bound their respective ligands and underwent a conformational change in the presence of a retinoic acid-responsive element. In reporter analyses, the RAR ligand (but not the RXR ligand), when added singly, activated transcription, but coaddition of the two ligands led to synergistic activation of transcription. This activation required the AF-2 domain of both RXR and RAR. Genomic footprinting analysis was performed with P19 embryonal carcinoma cells, in which transcription of the RARbeta gene is induced upon retinoid addition. Paralleling the reporter activation data, only the RAR ligand induced in vivo occupancy of the RARbeta2 promoter when added singly. However, at suboptimal concentrations of RAR ligand, coaddition of the RXR ligand increased the stability of promoter occupancy. Thus, liganded RXR and RAR both participate in transcription. Finally, when these ligands were tested for teratogenic effects on zebra fish and Xenopus embryos, we found that coadministration of the RXR and RAR ligands caused more severe abnormalities in these embryos than either ligand alone, providing biological support for the synergistic action of the two ligands.

Animals↗