Jun-ichi Nishikawa

Structure-dependent activation of peroxisome proliferator-activated receptor (PPAR) γ by organotin compounds

Organotin compounds such as tributyltin (TBT) and triphenyltin (TPT) are frequent environmental contaminants and are suspected of disrupting endocrine function in vertebrates and invertebrates. Previously, we reported that TBT and TPT function as powerful agonists for peroxisome proliferator-activated receptor (PPAR) gamma and stimulate adipocyte differentiation via the PPARgamma signaling pathway. Our current study investigates the structure-dependent binding of butyltin and phenyltin compounds to PPARgamma and their ability to activate the receptor. A Scatchard analysis with purified recombinant PPARgamma demonstrated that [(14)C]TPT binds to PPARgamma with an equilibrium dissociation constant (K(d)) of 66.6+/-5.2 nM, which approximated the 46.2+/-2.5 nM K(d) of a typical PPARgamma agonist, [(3)H]rosiglitazone (Rosi). TBT, TPT, diphenyltin (DPT), and tetrabutyltin (TeBT) blocked the binding of [(3)H]Rosi to PPARgamma in a competitive manner, and all tested organotin compounds except monobutyltin blocked the binding of [(14)C]TPT to PPARgamma in a competitive manner. Unexpectedly, Rosi did not compete at all with [(14)C]TPT for binding to PPARgamma, and contrary to the results of the competition assay, TBT and TeBT, but not dibutyltin, transcriptionally activated a GAL-PPARgamma chimeric receptor. All tested phenyltin compounds transcriptionally activated GAL-PPARgamma with an order of potency of TPT>DPT>monophenyltin. In addition, treatment of human choriocarcinoma cells with TBT, TeBT, and all tested phenyltin compounds stimulated production of human chorionic gonadotropin, which is upregulated by PPARgamma-mediated transcription. Our observations indicate that trialkylated and triphenylated tin compounds are the most potent PPARgamma agonists among the alkylated and phenylated tin compounds, and a phenyl substituent on a tin atom enhances the potency of organotin compounds as a PPARgamma agonist much more than a butyl substituent.

Screening and detection of the in vitro agonistic activity of xenobiotics on the retinoic acid receptor

The retinoic acid receptors (RARs) play key roles in various biological processes in response to endogenous retinoic acids. However, excessive embryonic exposure to specific ligands for each subtype of the RAR was reported to induce specific developmental abnormalities. We measured the RAR agonistic activity of 543 chemicals using an assay system adopting yeast cells transfected with the human RAR gamma and a coactivator. Eighty-five of the 543 chemicals, including 16 organochlorine pesticides, 14 styrene dimers, 9 monoalkylphenols and 6 parabens, exhibited RAR gamma agonistic effects in this assay. In particular, monoalkylphenols having a 6-9 carbon alkyl group para to the phenolic hydroxyl group possessed high affinity for the RAR gamma, and their activities were 1.363-0.446% of that of all-trans RA. para-Alkylphenols chlorinated at the ortho position also were about as active or more active than their unchlorinated analogs. In addition, all tested styrene dimers showed positive effects, and the activity of 1-phenyltetralin, the strongest in this category, was 1.169% that of all-trans RA. A number of chemicals having binding affinity for the RAR gamma were revealed in this study (both newly identified and confirmed), further comprehensive studies of in vitro and in vivo effects via the RARs are required for the reliable risk assessment of chemicals. In vitro receptor binding studies represent an important step in hazard identification and suggest a potential mechanism of action, which can be an important step in risk assessment and in particular for screening studies to identify potential toxicity and inform mechanistic studies.

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.

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.

Effects of coumestrol on lipid and glucose metabolism as a farnesoid X receptor ligand

In the course of an effort to identify novel agonists of the farnesoid X receptor (FXR), coumestrol was determined to be one such ligand. Reporter and in vitro coactivator interaction assays revealed that coumestrol bound and activated FXR. Treatment of Hep G2 cells with coumestrol stimulated the expression of FXR target genes, thereby regulating the expression of target genes of the liver X receptor and hepatocyte nuclear factor-4alpha. Through these actions, coumestrol is expected to exert beneficial effects on lipid and glucose metabolism.

Induction of integrin β3 in PGE2-stimulated adhesion of mastocytoma P-815 cells to the Arg-Gly-Asp-enriched fragment of fibronectin

We previously demonstrated that prostaglandin (PG) E₂ stimulates adhesion of mastocytoma P-815 cells (P-815 cells) to the Arg-Gly-Asp (RGD)-enriched matrix via the PGE₂ receptor subtype EP4 [Hatae N, Kita A, Tanaka S, Sugimoto Y, Ichikawa A. Induction of adherent activity in mastocytoma P-815 cells by the cooperation of two prostaglandin E₂ receptor subtypes, EP3 and EP4. J Biol Chem 2003;278:17977-81]. Here we investigated the role of various integrin subtypes in the induction of adherent activity in PGE(2)-stimulated P-815 cells. FACS analysis showed that P-815 cells express high levels of integrin α4, α5, β1 and β2 subunits and moderate levels of integrin αIIb, αv, β3 and β7 subunits. When treated with PGE₂, the EP4 agonist ONO-AE1-329 or the cell permeable cAMP analogue, 8-Br-cAMP, P-815 cells showed markedly increased cell surface expression of integrin αIIb, αv and β3 subunits, and these expressions were significantly reduced by addition of the protein synthesis inhibitor cycloheximide. Along with increased cell surface expression, mRNA and protein levels of the integrin β3 subunit, but not of integrin αIIb and αv subunits, were simultaneously elevated. On the other hand, adhesion of P-815 cells in response to PGE₂ or 8-Br-cAMP was abolished by antibodies specific for integrin αv and β3 subunits, but not by antibodies for integrin α4, α5, β1, β2 and β7 subunits. Moreover, treatment with tirofiban, an integrin αIIbβ3 antagonist, or eptifibatide, an integrin αvβ3/αIIbβ3 antagonist resulted in a decrease in adhesion of P-815 cells in response to PGE₂ or 8-Br-cAMP. These results suggest that de novo synthesis of the integrin β3 subunit plays a pivotal role in PGE₂-induced adhesion of P-815 cells to the RGD-enriched matrix through EP4-mediated cAMP signaling.

Site-directed mutagenesis of rat thioltransferase: Effects of essential cysteine residues for the protection against oxidative stress

A cDNA of rat liver thioltransferase was cloned and then expressed using pMAL‐c expression vector in Escherichia coli. Recombinant rat liver thioltransferase was expressed as a fusion protein with maltose‐binding protein and then purified by amylose resin column chromatography to be homogeneity on 12.5% SDS‐polyacrylamide gel electrophoretic analysis. The expressed proteins were shown as two bands at around 53 and 41 kDa, suggesting that the high molecular one was a fusion protein of recombinant thioltransferase (11.7 plus 41 kDa) and the other (smaller one) was a maltose‐binding protein (41 kDa). A recombinant thioltransferase catalyzed a thiol/disulfide exchange reaction in the same way as thioltransferases purified from various sources. Compared with wild type, the mutants C23A, C26A, C79A, and C83A showed 0%, 17%, 82%, and 86% in the enzymatic activity, respectively. In addition, wild‐type‐transfected bacteria expressed in bacterial cells showed a strong resistance to H2O2 treatment as well as the case of active mutants (C79A and C83A), but inactive mutants (C23A and C26A) showed no resistance to H2O2 treatment as same as mocktransfection. Thioltransferase can be important for survival of bacterial cells under oxidative stress.

Identification of Candidate Target Cyp Genes for microRNAs Whose Expression Is Altered by PCN and TCPOBOP, Representative Ligands of PXR and CAR

MicroRNAs (miRNAs) are small non-coding RNAs that are involved in mRNA post-transcriptional regulation. The deregulation of miRNAs affects the expression of drug-metabolizing enzymes, drug transporters, and nuclear receptors, all of which are important in regulating drug metabolism. miRNA expression can be altered by several endogenous or exogenous agents, such as steroid hormones, carcinogens, and therapeutic drugs. However, it is unclear whether hepatic miRNA expression is regulated by nuclear receptors, such as pregnane X receptor (PXR) and constitutive androstane receptor (CAR), which are indispensable for the expression of the CYPs. Here we investigated the effects of the mouse PXR and CAR ligands pregnenolone-16α-carbonitrile (PCN) and 1,4-bis[(3,5-dichloropyridin-2-yl)oxy]benzene (TCPOBOP) on hepatic miRNA expression in mice. We found that the expression of 9 miRNAs was increased (>2-fold) and of 4 miRNAs was decreased (>50%) in response to PCN, while TCPOBOP treatment led to the up-regulation of 8 miRNAs and down-regulation of 6 miRNAs. Using several miRNA target prediction algorithms, we found that the predicted target genes included several lesser known Cyp genes (Cyp1a1, Cyp1b1, Cyp2b10, Cyp2c38, Cyp2u1, Cyp4a12a/b, Cyp4v3, Cyp17a1, Cyp39a1, and Cyp51). We analyzed the expression of these genes in response to PCN and TCPOBOP and found changes in their mRNA levels, some of which were negatively correlated with the expression of their corresponding miRNAs, suggesting that miRNAs may play a role in regulating Cyp enzyme expression. Further studies will be required to fully elucidate the miRNA regulatory mechanisms that contribute to modulating CYP expression.