Retinoid X receptor (RXR)

UVI3003 suppresses the activity of Xenopus laevis and human RXRα with an IC50 of 0.22 and 0.24 μM, respectively. UVI3003 is nearly ineffective against hPPARγ and mPPARγ, but fully activates xPPARγ with an EC50 of 12.6 μM [1]. The growth rate of EECD34 cells generated from either extraocular muscle (EOM) or lung epithelium (LEG) was not affected by UVI 3003 (10 μM). Desmin expression and EECD34 cell fusion were shown to differ by 65.4% in response to UVI 3003 [2].

Multiple malformations are induced by UVI3003 in Xenopus tropicalis embryos[1]
Exposure to UVI3003 during different developmental windows induced obviously developmental delay and multiple malformations (Fig. 1). The most common phenotypes were reduced forehead, turbid eye lens and narrow fin in UVI3003 treatment groups. Proctodaeum elongation was observed in all NF10-19 treatment groups and in the NF19-25 high dose group, while enlarged proctodaeum phenotype occurred in late exposure windows. The teratogenic ability of UVI3003 was weak in NF 10-25 stages, while it was significantly increased in NF 25-39 and then decreased in NF 39-43. The body length of embryos decreased significantly in all NF 31-36 NF36-39 treatment groups and in the NF39-41middle and high dose groups (Supplementary Fig. 3).

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PPARγ was down-regulated by UVI3003 in Xenopus tropicalis embryos[1]
The expression of mRNAs encoding RXRs and their heterodimeric partners RARs, PPARs and TRs were evaluated after 7 different UVI3003-exposure windows. We found RARβ was down-regulated in early exposure periods (Fig. 2B2), whereas RXRs, TRα and TRβ were affected after late embryogenesis treatment (Fig. 2A, D). The expression of PPARγ was clearly decreased during all the treatment periods (Fig. 2C3). We tested the effects of TPT treatment during the most sensitive exposure window and found that PPARγ was also down-regulated at high dose (Fig. 3). Thus, our results showed that PPARγ was down-regulated by UVI3003 and TPT in X. tropicalis embryos.

Luciferase reporter assay using in vitro model (Cos 7 cells)[1]
pCMX-GAL4 plasmid fusion constructs of nuclear receptor ligand binding domains GAL4-human RXRα (Perlmann et al., 1996), -Xenopus laevis RXRα (Blumberg et al., 1992), -human PPARγ (Greene et al., 1994), -mouse PPARγ (Kliewer et al., 1994) were previously described (Chamorro-García et al., 2012). We isolated Xenopus laevis PPARγ from a cDNA library by PCR and cloned it into pCMX-GAL4 expression vector, its cloning primers are listed in Supplementary Table 2. One microgram pCMX-GAL4 effector plasmid was co-transfected with 5 μg pCMX-β-galactosidase transfection control, 5 μg tk-(MH100)4-luciferase reporter and 14 μg pUC19 carrier plasmid (per 96-well plate) into Cos7 cells using calcium phosphate-mediated transient transfection (Sambrook and Russell, 2005). UVI3003 was added in 3-fold serial dilutions from 10−5 and 10−4 M for RXRα antagonism and PPARγ activation assays, respectively. TPT was serially diluted 10-fold or 3-fold from 10−6 M for RXRα and PPARγ activation assays. The control compounds HX531 (RXR antagonist), IRX4204 (formerly designated AGN194204 and NRX194204, RXR agonist) and ROSI (rosiglitazone, PPARγ agonist) were tested from 10−5 M in 10-fold serial dilutions (Kanayasu-Toyoda et al., 2005; Vuligonda et al., 1996). All transfections were performed in triplicate and reproduced in multiple experiments.

Proliferation experiments.[2]
Sorted EECD34 cells were plated onto Matrigel coated dishes 8-well Permanox chamber slides or 48-well plates at a density of 10,000 cells/cm2, or 15 well μ-Slide Angiogenesis plates at a density of 7680 cells/cm2. Proliferation media was changed every other day until cells reached the appropriate density. At ~ 30–40% confluence cells were treated with vehicle (ethanol), all-trans retinoic acid (10−6M), the RAR inverse agonist BMS493 (10−5 M), or the RXR antagonist UVI3003 (10−5M) for 24 h in proliferation media with a final concentration of ethanol at 0.1% for all treatments. At the end of the 24-h treatment cell proliferation rates were assessed.

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Fusion experiments.[2]
At ~ 70–80% confluence cells were treated with vehicle (ethanol), all-trans retinoic acid (10−6 M), BMS493 (10−5M), or UVI3003 (10−5 M) for 72 h in fusion media (DMEM, 5% horse serum, 1% penicillin/streptomycin) with the final concentration of ethanol at 0.1% for all treatments. Fusion media containing vehicle, retinoic acid, BMS493, or UVI 3003 were replaced after 48 h. At the end of the 72-h treatment cell fusion was assessed.

Exposure experiments using Xenopus tropicalis embryos[1]
Xenopus tropicalis adults were maintained according to previous methods (Yu et al., 2011). Breeding was induced by subcutaneous injection of human chorionic gonadotrophin (hCG) as described (Yu et al., 2011; Hu et al., 2015a). The exposure experiments were conducted following the Frog Embryo Teratogenesis Assay (FETAX) protocol (Fort and Paul, 2002) with some modifications. Briefly, approximately 12 h after the second injection of hCG, adults were removed from their tanks, and embryos were harvested without removing the jelly coats (Supplementary Fig. 2). UVI3003 was dissolved in DMSO and then diluted into FETAX medium. Four replicate dishes (n = 4) were used in each control or treatment group of 20 embryos for morphological observations and real-time quantitative PCR analysis.[1]
The EC50 of UVI3003 is 0.5 μM after 48 hrs treatment from NF10 in X. tropicalis embryos (Zhu et al., 2014). In this study, we chose 1, 1.5, 2 μM of UVI3003 to treat embryos in short exposure windows (6-8.5 hrs) from gastrulation (Nieuwkoop and Faber stage 10) to larval stage (NF43). 10 embryos were collected immediately after the exposure windows ended for real-time quantitative PCR analysis; the other 10 embryos were rinsed with FETAX medium three times and maintained at 26 ± 0.5°C in the dark for later morphological analysis. All exposure experiments ended when the control embryos reached NF43. To minimize biological variation, embryos for each exposure window were chosen from one pair of frogs.

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Luciferase reporter assay using in vivo model (Xenopus laevis embryos)[1]
Xenopus laevis eggs were fertilized in vitro as described previously (Janesick et al., 2012), and embryos were staged according to Nieuwkoopand Faber (Nieuwkoop and Faber, 1956). Embryos were microinjected at the 2- or 4-cell stage with 50 pg/embryo pCMX-GAL4-xPPARγ mRNAor β-galactosidase (control) mRNA together with 50 pg/embryo tk-(MH100)4-luciferase reporter DNA. Microinjected embryos were treated at stage 8 with the following chemicals (in 0.1× MBS): UVI3003 (1, 5, 10 μM), TPT (0.01, 0.05, 0.1 μM), TBT (RXR and PPARγ agonist, 0.05 μM) or vehicle (0.1% DMSO). For each treatment, 25 embryos were treated in glass 60-mm Petri dishes containing 10 mL of MBS + chemical, and two replicate dishes were used for each concentration. Treated embryos were separated into five-embryo aliquots at neural stage for luciferase assays (Janesick et al., 2012, 2014). Each group of five embryos was considered one biological replicate.

[1]. The unexpected teratogenicity of RXR antagonist UVI3003 via activation of PPARγ in Xenopus tropicalis. Toxicol Appl Pharmacol. 2017 Jan 1;314:91-97.

[2]. Effects of retinoic acid signaling on extraocular muscle myogenic precursor cells in vitro. Exp Cell Res. 2017 Dec 1;361(1):101-111.

The RXR agonist (triphenyltin, TPT) and the RXR antagonist (UVI3003) both show teratogenicity and, unexpectedly, induce similar malformations in Xenopus tropicalis embryos. In the present study, we exposed X. tropicalis embryos to UVI3003 in seven specific developmental windows and identified changes in gene expression. We further measured the ability of UVI3003 to activate Xenopus RXRα (xRXRα) and PPARγ (xPPARγ) in vitro and in vivo. We found that UVI3003 activated xPPARγ either in Cos7 cells (in vitro) or Xenopus embryos (in vivo). UVI3003 did not significantly activate human or mouse PPARγ in vitro; therefore, the activation of Xenopus PPARγ by UVI3003 is novel. The ability of UVI3003 to activate xPPARγ explains why UVI3003 and TPT yield similar phenotypes in Xenopus embryos. Our results indicate that activating PPARγ leads to teratogenic effects in Xenopus embryos. More generally, we infer that chemicals known to specifically modulate mammalian nuclear hormone receptors cannot be assumed to have the same activity in non-mammalian species, such as Xenopus. Rather they must be tested for activity and specificity on receptors of the species in question to avoid making inappropriate conclusions.[1]

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One major difference between limb and extraocular muscles (EOM) is the presence of an enriched population of Pitx2-positive myogenic precursor cells in EOM compared to limb muscle. We hypothesize that retinoic acid regulates Pitx2 expression in EOM myogenic precursor cells and that its effects would differ in leg muscle. The two muscle groups expressed differential retinoic acid receptor (RAR) and retinoid X receptor (RXR) levels. RXR co-localized with the Pitx2-positive cells but not with those expressing Pax7. EOM-derived and LEG-derived EECD34 cells were treated with vehicle, retinoic acid, the RXR agonist bexarotene, the RAR inverse agonist BMS493, or the RXR antagonist UVI 3003. In vitro, fewer EOM-derived EECD34 cells expressed desmin and fused, while more LEG-derived cells expressed desmin and fused when treated with retinoic acid compared to vehicle. Both EOM and LEG-derived EECD34 cells exposed to retinoic acid showed a higher percentage of cells expressing Pitx2 compared to vehicle, supporting the hypothesis that retinoic acid plays a role in maintaining Pitx2 expression. We hypothesize that retinoic acid signaling aids in the maintenance of large numbers of undifferentiated myogenic precursor cells in the EOM, which would be required to maintain EOM normalcy throughout a lifetime of myonuclear turnover.[2]

C28H36O4

436.5830

436.261

C, 77.03; H, 8.31; O, 14.66

847239-17-2

847239-17-2;

44566108

White to off-white solid powder

7.075

2

4

8

32

652

0

CCCCCOC1=CC2=C(C=C1C3=C(C=CC(=C3)/C=C/C(=O)O)O)C(CCC2(C)C)(C)C

APJSHECCIRQQDV-ZRDIBKRKSA-N

InChI=1S/C28H36O4/c1-6-7-8-15-32-25-18-23-22(27(2,3)13-14-28(23,4)5)17-21(25)20-16-19(9-11-24(20)29)10-12-26(30)31/h9-12,16-18,29H,6-8,13-15H2,1-5H3,(H,30,31)/b12-10+

(E)-3-[4-hydroxy-3-(5,5,8,8-tetramethyl-3-pentoxy-6,7-dihydronaphthalen-2-yl)phenyl]prop-2-enoic acid

UVI 3003; 847239-17-2; UVI3003; UVI-3003; (2E)-3-{4-hydroxy-3-[5,5,8,8-tetramethyl-3-(pentyloxy)-5,6,7,8-tetrahydronaphthalen-2-yl]phenyl}prop-2-enoic acid; compound 10e [PMID: 19408900]; compound 10e (PMID: 19408900); (E)-3-[4-hydroxy-3-(5,5,8,8-tetramethyl-3-pentoxy-6,7-dihydronaphthalen-2-yl)phenyl]prop-2-enoic acid;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~100 mg/mL (~229.05 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.73 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (5.73 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)