Toshiharu Iwasaki

Identification of the functional domain of thyroid hormone receptor responsible for polychlorinated biphenyl-mediated suppression of its action in vitro.

Background Polychlorinated biphenyls (PCBs), polychlorinated dibenzo-p-dioxins, and poly-chlorinated dibenzofurans adversely affect the health of humans and various animals. Such effects might be partially exerted through the thyroid hormone (TH) system. We previously reported that one of the hydroxylated PCB congeners suppresses TH receptor (TR)-mediated transcription by dissociating TR from the TH response element (TRE). However, the binding site of PCB within TR has not yet been identified. Objectives We aimed to identify the functional TR domain responsible for the PCB-mediated suppression of TR action by comparing the magnitude of suppression using several representative PCB/dioxin congeners. Materials and methods We generated chimeric receptors by combining TR and glucocorticoid receptor (GR) and determined receptor-mediated transcription using transient transfection-based reporter gene assays, and TR-TRE binding using electrophoretic mobility shift assays. Results Although several PCB congeners, including the hydroxylated forms, suppressed TR-mediated transcription to various degrees, 2,3,7,8-tetrachlorodibenzo-p-dioxin did not alter TR action, but 2,3,4,7,8-pentachlorodibenzofuran weakly suppressed it. The magnitude of suppression correlated with that of TR–TRE dissociation. The suppression by PCB congeners was evident from experiments using chimeric receptors containing a TR DNA-binding domain (DBD) but not a GR-DBD. Conclusions Several nondioxin-like PCB congeners and hydroxylated PCB compounds suppress TR action by dissociating TR from TRE through interaction with TR-DBD.

Hydroxylated PCB induces Ca2+ oscillations and alterations of membrane potential in cultured cortical cells

Polychlorinated biphenyls (PCBs) are known as environmental pollutants that may cause adverse health effects. Although some congeners have been shown to affect brain development or function, the molecular mechanisms mediating their toxicity are not yet fully understood. Since signal transduction via intracellular Ca2+ is crucial for neuronal development and plasticity, we investigated the effect of PCBs on Ca2+ homeostasis and membrane potential in cultured mouse cortical cells. Acute exposure to hydroxylated PCB 106 [4(OH)‐2′,3,3′,4′,5′‐pentachlorobiphenyl, OH‐PCB 106, 0.1u2009μm] caused recurring Ca2+ oscillations that were classified into three prototypes. Although extracellular Ca2+ deprivation significantly reduced the oscillations, 54% of the cells still showed different patterns of oscillations or gradual increase in the intracellular Ca2+ concentration, indicating possible involvement of multiple Ca2+ channels in a cell‐specific manner. Such a possibility was further confirmed by differential responses to several channel/receptor blockers, including nifedipine, ryanodine, xestospongine and tetrodotoxin. Although all chemicals had partial inhibition action in different subsets of neurons, nifedipine blocked the OH‐PCB 106 action in the largest subpopulation of cells and with the greatest magnitude. Ryanodine also blocked the action with a similar magnitude, but in a smaller subpopulation of cells. Moreover, OH‐PCB 106 induced depolarization of the plasma membrane in all the recorded cells. Taken together, our results indicate that OH‐PCB 106 alters membrane potential as well as Ca2+ dynamics in part by inducing extracellular influx and/or intracellular release of Ca2+. These mechanisms may be responsible for their neurotoxicity. Copyright

1,2,5,6,9,10-αHexabromocyclododecane (HBCD) Impairs Thyroid Hormone-Induced Dendrite Arborization of Purkinje Cells and Suppresses Thyroid Hormone Receptor-Mediated Transcription

Abstract1,2,5,6,9,10-αHexabromocyclododecane (HBCD) is a nonaromatic, brominated cyclic alkane used as an additive flame retardant. It bioaccumulates, persists in the environment, and has been detected in humans and wildlife. Its developmental neurotoxicity is of great concern. We investigated the effect of HBCD on thyroid hormone (TH) receptor (TR)-mediated transcription using transient transfection-based reporter gene assays and found that a low-dose (10−10xa0M) HBCD suppressed TR-mediated transcription. We further examined the effect of HBCD on interaction of TR with TH response element (TRE) and found a partial dissociation of TR from TRE. HBCD did not dissociate steroid receptor coactivator-1 from TR in the presence of TH; neither did it recruit corepressors (N-CoR and SMRT) to TR in the absence of TH. Furthermore, low-dose HBCD (10−10xa0M) significantly suppressed TH-induced dendrite arborization of Purkinje cells in primary cerebellar culture derived from newborn rat. These results show that low-dose HBCD can potentially disrupt TR-mediated transactivation and impairs Purkinje cell dendritogenesis, suggesting that HBCD can interfere with TH action in target organs, including the developing brain.

Ligand-induced downregulation of TrkA is partly regulated through ubiquitination by Cbl

c‐Cbl physically interacts with TrkA by anti bait coimmunoprecipitation (View interaction)

Brain-derived neurotrophic factor (BDNF) ameliorates the suppression of thyroid hormone-induced granule cell neurite extension by hexabromocyclododecane (HBCD)

Thyroid hormone (TH) plays an essential role in growth and differentiation of the central nervous system. Deficiency of TH during perinatal period results in abnormal brain development known as cretinism in human. We recently reported that an environmental chemical 1,2,5,6,9,10-α-hexabromocyclododecane (HBCD) suppressed TH receptor (TR)-mediated transcription. To examine the effect of HBCD on cerebellar granule cells, we used purified rat cerebellar granule cells in reaggregate culture. Low dose HBCD (10(-10)M) significantly suppressed TH-induced neurite extension of granule cell aggregate. To clarify further the mechanisms of such suppression, we added brain-derived neurotrophic factor (BDNF) into culture medium, since BDNF plays a critical role in promoting granule cell development and is regulated by TH. BDNF completely rescued HBCD-induced suppression of granule cell neurite extension in the presence of T3. These results indicate that HBCD may disrupt TH-mediated brain development at least in part due to a disruption of the T3 stimulated increase in BDNF and BDNF may possess ability to ameliorate the effect of HBCD in granule cells.

Suppression of thyroid hormone receptor-mediated transcription and disruption of thyroid hormone-induced cerebellar morphogenesis by the polybrominated biphenyl mixture, BP-6.

Polybrominated biphenyls (PBBs) are polyhalogenated, bioaccumulative flame retardant chemicals, which have been used in a variety of consumer and household products. They were accidentally introduced into the food chain in Michigan in 1973 and have remained a source of health concern. Studies have shown that exposure to PBB may cause adverse neurotoxic effects. We therefore examined the effects of BP-6, a PBB mixture, on thyroid hormone (TH) receptor (TR)-mediated transcription, on TH-induced Purkinje cell dendritogenesis, and on TH-induced cerebellar granule cell neurite extension. Our study shows that BP-6 suppressed TR-mediated transcription in CV-1 cells. Mammalian two-hybrid studies revealed that BP-6 did not inhibit coactivator binding to TR nor did it recruit corepressors to TR. Further examination using the liquid chemiluminescent DNA pull down assay revealed partial dissociation of TR from TH response element (TRE). In primary rat cerebellar culture, BP-6 significantly suppressed TH-induced dendrite arborization of Purkinje cells, and in reaggregate rat granule cell culture, impaired TH-induced neurite extension of granule cells. Taken together, our results indicate that BP-6 may disrupt TH homeostasis and consequently impair normal neuronal development.

Liquid chemiluminescent DNA pull-down assay to measure nuclear receptor-DNA binding in solution

Electrophoretic mobility shift assays (EMSAs) are commonly used to investigate protein-DNA binding in vitro. However, EMSA can generate considerable amounts of undesirable waste, particularly when toxic compounds are examined. We therefore developed a novel in vitro protein-DNA binding assay called liquid chemiluminescent DNA pull-down assay, which is based on solution hybridization between digoxigenin-labeled DNA and glutathione S-transferase (GST)-fused DNA binding protein bound to glutathione-Sepharose beads.

Retinoic Acid Receptor–Related Orphan Receptor Alpha–Enhanced Thyroid Hormone Receptor–Mediated Transcription Requires Its Ligand Binding Domain Which Is Not, by Itself, Sufficient: Possible Direct Interaction of Two Receptors

BACKGROUNDnNatural mutant staggerer (sg) mice harbor a mutated retinoic acid receptor-related orphan receptor alpha (RORalpha). A genetic deletion corresponding to the ligand-binding domain (LBD) of RORalpha results in aberrant cerebellar development in the sg mice. These mice show similar neurotrophin expression to that seen in perinatal hypothyroid animals. RORalpha augments thyroid hormone receptor (TR)-mediated transcription, which may be partly responsible for the similar cerebellar abnormalities between sg and hypothyroid animals. The objective of this study is to examine further the mechanisms of augmentation of TR action by RORalpha. We examined whether TR directly binds to ROR and which regions of TR or ROR are required for the TR-ROR interaction.nnnMETHODSnA transient transfection-based reporter gene assay was performed to measure the activity of TR-mediated transcription in CV-1 cells. To examine TR-RORalpha binding mammalian two-hybrid and glutathione-S-transferase (GST) pull-down assays were carried out.nnnRESULTSnAlthough full-length RORalpha augmented TRalpha1- or beta1-mediated transcription, such augmentation was not observed with sg-type mutant RORalpha (RORsg) that contained the RORalpha N-terminal and DNA-binding domain (DBD) and a part of the LBD. On the other hand, the transcription of Gal4-DBD-fused TRbeta1-LBD was suppressed by RORalpha, indicating that RORalpha does not interact with TR-LBD. Full-length TRbeta1 bound to RORalpha or RORsg in GST pull-down assays; however, RORalpha-LBD did not bind to TRalpha1 or beta1.nnnCONCLUSIONnThe full-length forms of both RORalpha and TR are essential for the augmentation of TR-mediated transcription by RORalpha.

Augmentation of estrogen receptor-mediated transcription by steroid and xenobiotic receptor

The estrogen receptor (ER) is a key regulator of proliferation and differentiation in breast cancer cells. In the present study, the effect of steroid and xenobiotic receptor (SXR) on 17β-estradiol (E2)-induced transcription through ERα was studied. SXR augmented ER-mediated transcription in the presence of E2 in MCF-7 breast cancer-derived cells and CV-1 fibroblast-derived cells. On the other hand, SXR alone did not affect the estrogen response element (ERE)-containing promoter activity in CV-1 cells. SXR did not directly bind to ERα or ERE inxa0vitro, indicating that SXR may affect ER-mediated transcription by altering cofactor binding to ER. Although SXR did not alter the binding between ERα and p300/CBP interacting protein (p/CIP), it decreased the binding of a specific corepressor, silencing mediator of retinoid and thyroid hormone receptors (SMRT) to liganded ERα as assessed by mammalian two-hybrid, glutathione S-transferase pull-down, immunoprecipitation and newly developed Liquid Chemiluminescent DNA Pull-Down Assays. These results indicate that SXR augmented ER-mediated transcription by dissociating SMRT from ERα. Thus, the expression of SXR in breast cancer cells may alter the ER signaling, which may play crucial role for growth and differentiation of breast cancer cells.