Tsuyoshi Nakanishi

A Mollusk Retinoic Acid Receptor (RAR) Ortholog Sheds Light on the Evolution of Ligand Binding

Nuclear receptors are transcription factors that regulate networks of target genes in response to small molecules. There is a strong bias in our knowledge of these receptors because they were mainly characterized in classical model organisms, mostly vertebrates. Therefore, the evolutionary origins of specific ligand-receptor couples still remain elusive. Here we present the identification and characterization of a retinoic acid receptor (RAR) from the mollusk Nucella lapillus (NlRAR). We show that this receptor specifically binds to DNA response elements organized in direct repeats as a heterodimer with retinoid X receptor. Surprisingly, we also find that NlRAR does not bind all-trans retinoic acid or any other retinoid we tested. Furthermore, NlRAR is unable to activate the transcription of reporter genes in response to stimulation by retinoids and to recruit coactivators in the presence of these compounds. Three-dimensional modeling of the ligand-binding domain of NlRAR reveals an overall structure that is similar to vertebrate RARs. However, in the ligand-binding pocket (LBP) of the mollusk receptor, the alteration of several residues interacting with the ligand has apparently led to an overall decrease in the strength of the interaction with the ligand. Accordingly, mutations of NlRAR at key positions within the LBP generate receptors that are responsive to retinoids. Altogether our data suggest that, in mollusks, RAR has lost its affinity for all-trans retinoic acid, highlighting the evolutionary plasticity of its LBP. When put in an evolutionary context, our results reveal new structural and functional features of nuclear receptors validated by millions of years of evolution that were impossible to reveal in model organisms.

Contamination with retinoic acid receptor agonists in two rivers in the Kinki region of Japan.

This study was conducted to investigate the agonistic activity against human retinoic acid receptor (RAR) alpha in the Lake Biwa-Yodo River and the Ina River in the Kinki region of Japan. To accomplish this, a yeast two-hybrid assay was used to elucidate the spatial and temporal variations and potential sources of RARalpha agonist contamination in the river basins. RARalpha agonistic activity was commonly detected in the surface water samples collected along two rivers at different periods, with maximum all-trans retinoic acid (atRA) equivalents of 47.6 ng-atRA/L and 23.5 ng-atRA/L being observed in Lake Biwa-Yodo River and Ina River, respectively. The results indicated that RARalpha agonists are always present and widespread in the rivers. Comparative investigation of RARalpha and estrogen receptor alpha agonistic activities at 20 stations along each river revealed that the spatial variation pattern of RARalpha agonist contamination was entirely different from that of the estrogenic compound contamination. This suggests that the effluent from municipal wastewater treatment plants, a primary source of estrogenic compounds, seemed not to be the cause of RARalpha agonist contamination in the rivers. Fractionation using high performance liquid chromatography (HPLC) directed by the bioassay found two bioactive fractions from river water samples, suggesting the presence of at least two RARalpha agonists in the rivers. Although a trial conducted to identify RARalpha agonists in the major bioactive fraction was not completed as part of this study, comparison of retention times in HPLC analysis and quantification with liquid chromatography-mass spectrometry analysis revealed that the major causative contaminants responsible for the RARalpha agonistic activity were not RAs (natural RAR ligands) and 4-oxo-RAs, while 4-oxo-RAs were identified as the major RAR agonists in sewage in Beijing, China. These findings suggest that there are unknown RARalpha agonists with high activity in the rivers.

Screening of agonistic activities against four nuclear receptors in wastewater treatment plants in Japan using a yeast two-hybrid assay.

To assess the potential endocrine disruptive effects through multiple nuclear receptors (NRs), especially non-steroidal NRs, in municipal wastewater, we examined the agonistic activities on four NRs (estrogen receptor alpha, thyroid hormone receptor alpha, retinoic acid receptor alpha and retinoid X receptor alpha) of untreated and treated wastewater from municipal wastewater treatment plants (WWTPs) in Japan using a yeast two-hybrid assay. Investigation of the influent and effluent of seven WWTPs revealed that agonistic activities against steroidal and non-steroidal NRs were always detected in the influents and partially remained in the effluents. Further investigation of four WWTPs employing conventional activated sludge, pseudo-anoxic-oxic, anoxic-oxic and anaerobic-anoxic-oxic processes revealed that the ability to reduce the agonistic activity against each of the four NRs varies depending on the treatment process. These results indicated that municipal wastewater in Japan commonly contains endocrine disrupting chemicals that exert agonistic activities on steroidal and non-steroidal NRs, and that some of these chemicals are released into the natural aquatic environment. Although the results obtained in yeast assays suggested that measured levels of non-steroidal NR agonists in the effluent of WWTPs were not likely to cause any biological effect, further study is required to assess their possible risks in detail.

Retinoic acid receptor agonists regulate expression of ATP-binding cassette transporter G1 in macrophages.

ABC transporter G1 (ABCG1) plays a pivotal role in HDL-mediated cholesterol efflux and atherogenesis. We investigated whether, and how, retinoic acid receptors (RARs) regulate ABCG1 expression in macrophages. All-trans retinoic acid (ATRA), an RAR ligand, increased ABCG1 protein levels and apoA-I/HDL-mediated cholesterol efflux from the macrophages. Both ATRA and other RAR agonists, TTNPB and Am580, increased major transcripts driven by promoter B upstream of exon 5, though minor transcripts driven by promoter A upstream of exon 1 were only increased by ATRA. The stimulatory effects of ATRA on ABCG1 expression were completely abolished in the presence of RAR/RXR antagonists but were only partially canceled in the presence of an LXR antagonist. Adenovirus with overexpressed oxysterol sulfotransferase abolished the LXR pathway, as previously reported, and ATRA-responsiveness in ABCA1/ABCG1 expressions were respectively attenuated by 38 and 22% compared to the control virus. Promoter assays revealed that ABCG1 levels were regulated more by promoter B than promoter A, and ATRA activated promoter B in a liver X receptor-responsive element (LXRE)-dependent manner. Further, LXRE-B in intron 7, but not LXRE-A in intron 5, enhanced ATRA responsiveness under overexpression of all RAR isoforms-RARα/β/γ. In contrast, the activation of promoter B by TTNPB depended on LXRE-B and RARα, but not on RARβ/γ. Finally, chromatin immunoprecipitation and gel-shift assays revealed a specific and direct repeat 4-dependent binding of RARα to LXRE-B. In conclusion, RAR ligands increase ABCA1/G1 expression and apoA-I/HDL-mediated cholesterol efflux from macrophages, and modulate ABCG1 promoter activity via LXRE-dependent mechanisms.

Organotin compounds cause structure-dependent induction of progesterone in human choriocarcinoma Jar cells.

Organotin compounds, such as tributyltin (TBT) and triphenyltin (TPT), are typical environmental contaminants and suspected endocrine-disrupting chemicals because they cause masculinization in female mollusks. In addition, previous studies have suggested that the endocrine disruption by organotin compounds leads to activation of peroxisome proliferator-activated receptor (PPAR)γ and retinoid X receptor (RXR). However, whether organotin compounds cause crucial toxicities in human development and reproduction is unclear. We here investigated the structure-dependent effect of 12 tin compounds on mRNA transcription of 3β-hydroxysteroid dehydrogenase type I (3β-HSD I) and progesterone production in human choriocarcinoma Jar cells. TBT, TPT, dibutyltin, monophenyltin, tripropyltin, and tricyclohexyltin enhanced progesterone production in a dose-dependent fashion. Although tetraalkyltin compounds such as tetrabutyltin increased progesterone production, the concentrations necessary for activation were 30-100 times greater than those for trialkyltins. All tested active organotins increased 3β-HSD I mRNA transcription. We further investigated the correlation between the agonistic activity of organotin compounds on PPARγ and their ability to promote progesterone production. Except for DBTCl2, the active organotins significantly induced the transactivation function of PPARγ. In addition, PPARγ knockdown significantly suppressed the induction of mRNA transcription of 3β-HSD I by all active organotins except DBTCl2. These results suggest that some organotin compounds promote progesterone biosynthesis in vitro by inducing 3β-HSD I mRNA transcription via the PPARγ signaling pathway. The placenta represents a potential target organ for these compounds, whose endocrine-disrupting effects might cause local changes in progesterone concentration in pregnant women.

The zinc-sensing transcription factor MTF-1 mediates zinc-induced epigenetic changes in chromatin of the mouse metallothionein-I promoter.

Metallothionein (MT) is a small, cysteine-rich protein active in zinc homeostasis, cadmium detoxification, and protection against reactive oxygen species. Mouse MT-I gene transcription is regulated by metal response element-binding transcription factor-1 (MTF-1), which is recruited to the promoter by zinc. We examined alterations in the chromatin structure of the MT-I promoter associated with enhanced transcriptional activation. MTF-1 proved essential for zinc-induced epigenetic changes in the MT-I promoter. Chromatin immunoprecipitation assays demonstrated that zinc treatment rapidly decreased Lys⁴-trimethylated and Lys⁹-acetylated histone H3 in the promoter and decreased total histone H3 but not histone H3.3. Micrococcal nuclease sensitivity of the MT-I promoter was increased by zinc. Thus, the chromatin structure in the promoter may be locally disrupted by zinc-induced nucleosome removal. Without MTF-1 these changes were not observed, and an MTF-1 deletion mutant recruited to the MT-I promoter by zinc that did not recruit the coactivator p300 or activate MT-I transcription did not affect histone H3 in the MT-I promoter in response to zinc. Interleukin-6, which induces MT-I transcription independently of MTF-1, did not reduce histone H3 levels in the promoter. Rapid disruption of nucleosome structure at the MT-I promoter is mediated by zinc-responsive recruitment of an active MTF-1-coactivator complex.

Role of megalin and the soluble form of its ligand RAP in Cd-metallothionein endocytosis and Cd-metallothionein-induced nephrotoxicity in vivo.

Orally administered Cd is predominantly distributed to the intestine, and the majority of this mucosal Cd is bound to metallothionein (MT). MT attenuates heavy metal-induced cytotoxicity by sequestering these metals and lowering their intracellular concentrations. In addition, MT acts as an extracellular transporter of orally administered Cd to the kidney. Because of its low molecular weight, the Cd-MT complex is freely filtered at the glomerulus, and the filtered Cd-MT is then incorporated into renal proximal tubular cells. Megalin, a multiligand endocytic receptor (also known as low-density lipoprotein receptor-related protein 2 or Lrp2), acts as the receptor for Cd-MT in a renal proximal tubular cell model. Here, we used the soluble form of 39-kDa receptor-associated protein (sRAP; also known as Lrpap1), a ligand of megalin, to inhibit megalin function, and then analyzed the effect of megalin loss on Cd-MT distribution and Cd-MT-induced nephrotoxicity in an animal model. Administration of sRAP to mice caused acute loss of megalin function by removing megalin in the brush border membrane. The pre-injection of sRAP decreased renal Cd content and decreased Cd-MT-induced kidney damage. Our results demonstrate that sRAP reduces Cd-MT-induced kidney toxicity in vivo.

Enhanced in vivo gene transfer into the placenta using RGD fiber-mutant adenovirus vector.

Among viral vectors, the fiber-mutant adenovirus vector carrying the Arg-Gly-Asp (RGD) peptide sequence (Ad-RGD) seems to have potential for both clinical gene therapy and basic research. As a part of a thorough evaluation of Ad-RGD in preclinical studies, we designed an experiment to investigate in detail the distribution of Ad-RGD compared with conventional adenovirus vector (WT-Ad) in pregnant mice. Surprisingly, Ad-RGD had substantial placental tropism, at 10-100 times that of WT-Ad. Transgene expression was sustained for at least 7 days, and Ad-RGD expressing firefly luciferase or red fluorescent protein has so far caused no placental dysfunction leading to fetal death. Ad-RGD showed high levels of transduction efficiency in in vitro-differentiated trophoblast stem cells, in which higher expression of αvβ3 integrin than in undifferentiated cells was observed. Our results suggest that the use of Ad-RGD or another RGD-mediated targeting strategy holds promise for drug delivery to the placenta.

Practical method for PCB degradation using Pd/C–H2–Mg system

Polychlorinated biphenyls (PCBs) were mainly used as lubricants and coolants in electrical equipment. However, their chemical stabilities as well as hydrophobic properties caused persistent environmental pollution and damage to human health based on their bioaccumulative property. PCBs are currently targeted for worldwide elimination and should be disposed by 2028 based on the Stockholm Convention on Persistent Organic Pollutants. The conventional PCB degradation methods require high-heat, high-pressure or/and strongly basic conditions. The development of a safer and more practical method, therefore, is desired. We have reported a catalytic degradation method of PCBs based on a palladium on carbon (Pd/C)-catalyzed dechlorination in the presence of Et(3)N under ambient hydrogen pressure and temperature. In this study, we demonstrate a more practical system using magnesium metal instead of Et(3)N for the dechlorination of a variety of aromatic chlorides. The method was applicable for the complete degradation of a variety of PCB mixtures, such as Aroclor 1242, 1248, 1254 and PCBs removed from a capacitor to produce only biphenyl and magnesium chloride as the maritime component, both of which are less toxic and easily separable. Moreover, the Pd/C could be recovered and reused at least five times without any loss of catalytic activity. The present Pd/C-Mg-H(2) system is a simple, safe, inexpensive, and environmentally-benign degradation method of PCBs.

Structure-Dependent Activity of Phthalate Esters and Phthalate Monoesters Binding to Human Constitutive Androstane Receptor

The present study investigated the human constitutive androstane receptor (CAR) binding activities of 23 phthalate esters and 10 phthalate monoesters using a fast and sensitive human CAR yeast two-hybrid assay. Of 23 phthalate esters, 16 were evaluated as positive, and the 10% relative effective concentrations (REC10) ranged from 0.28 (BBP) to 29.51 μM (DEHP), whereas no obvious binding activities were found for the phthalate esters having alkyl chains more than six carbons in length. Of 10 phthalate monoesters, only monoethyl phthalate (MEP), monoisobutyl phthalate (MIBP), and mono-(2-ethyhexyl) tetrabromophthalate (TBMEHP) elicited human CAR binding activities. The REC10 values of MEP and MIBP were 4.27 and 14.13 μM, respectively, higher than those of their corresponding phthalate esters (1.45 μM for DEP and 0.83 μM for DIBP), whereas TBMEHP (0.66 μM) was much lower than TBHP (>10(2) μM). A molecular docking method was performed to simulate the interaction modes between phthalates and human CAR, and active phthalates were found to lie at almost the same site in the human CAR pocket. The docking results suggest that the strong binding of phthalates to human CAR arises primarily from hydrophobic interactions, π-π interactions, and steric effects and that weak hydrogen bonds and weak halogen bonds greatly contribute to the high binding activity of TBMEHP. In conclusion, the current study clarified that an extensive array of phthalates are activators of human CAR.