Keiko Nohara

Modulation of oestrogen receptor signalling by association with the activated dioxin receptor

Environmental contaminants affect a wide variety of biological events in many species. Dioxins are typical environmental contaminants that exert adverse oestrogen-related effects. Although their anti-oestrogenic actions are well described, dioxins can also induce endometriosis and oestrogen-dependent tumours, implying possible oestrogenic effects. However, the molecular mechanism underlying oestrogen-related actions of dioxins remains largely unknown. A heterodimer of the dioxin receptor (AhR) and Arnt, which are basic helix–loop–helix/PAS-family transcription factors, mediates most of the toxic effects of dioxins. Here we show that the agonist-activated AhR/Arnt heterodimer directly associates with oestrogen receptors ER-α and ER-β. This association results in the recruitment of unliganded ER and the co-activator p300 to oestrogen-responsive gene promoters, leading to activation of transcription and oestrogenic effects. The function of liganded ER is attenuated. Oestrogenic actions of AhR agonists were detected in wild-type ovariectomized mouse uteri, but were absent in AhR-/- or ER-α-/- ovariectomized mice. Our findings suggest a novel mechanism by which ER-mediated oestrogen signalling is modulated by a co-regulatory-like function of activated AhR/Arnt, giving rise to adverse oestrogen-related actions of dioxin-type environmental contaminants.

Dioxin receptor is a ligand-dependent E3 ubiquitin ligase

Fat-soluble ligands, including sex steroid hormones and environmental toxins, activate ligand-dependent DNA-sequence-specific transcriptional factors that transduce signals through target-gene-selective transcriptional regulation. However, the mechanisms of cellular perception of fat-soluble ligand signals through other target-selective systems remain unclear. The ubiquitin–proteasome system regulates selective protein degradation, in which the E3 ubiquitin ligases determine target specificity. Here we characterize a fat-soluble ligand-dependent ubiquitin ligase complex in human cell lines, in which dioxin receptor (AhR) is integrated as a component of a novel cullin 4B ubiquitin ligase complex, CUL4BAhR. Complex assembly and ubiquitin ligase activity of CUL4BAhR in vitro and in vivo are dependent on the AhR ligand. In the CUL4BAhR complex, ligand-activated AhR acts as a substrate-specific adaptor component that targets sex steroid receptors for degradation. Thus, our findings uncover a function for AhR as an atypical component of the ubiquitin ligase complex and demonstrate a non-genomic signalling pathway in which fat-soluble ligands regulate target-protein-selective degradation through a ubiquitin ligase complex.

Aryl hydrocarbon receptor regulates Stat1 activation and participates in the development of Th17 cells

IL-17-producing T helper cells (Th17) have been recently identified as a previously undescribed subset of helper T cells. Here, we demonstrate that aryl hydrocarbon receptor (Ahr) has an important regulatory function in the commitment of Th17 cells. Ahr was robustly induced under Th17-polarizing conditions. Ahr-deficient naïve T cells showed a considerable loss in the ability to differentiate into Th17 cells when induced by TGF-β plus IL-6. We were able to demonstrate that Ahr interacts with Stat1 and Stat5, which negatively regulate Th17 development. Whereas Stat1 activation returned to its basal level in Ahr wild type naïve T cells 24 h after stimulation with TGF-β plus IL-6, Stat1 remained activated in Ahr-deficient naïve T cells after stimulation. These results indicate that Ahr participates in Th17 cell differentiation through regulating Stat1 activation, a finding that constitutes additional mechanisms in the modulation of Th17 cell development.

Aryl hydrocarbon receptor negatively regulates dendritic cell immunogenicity via a kynurenine-dependent mechanism

Although an immunoregulatory role of aryl hydrocarbon receptor (Ahr) has been demonstrated in T cells and macrophages, little is known about its function in dendritic cells (DC). Here, we show that lipopolysaccharide (LPS) and CpG stimulate Ahr expression in bone marrow-derived dendritic cells (BMDC). Furthermore, we found that Ahr is required to induce indoleamine 2,3-dioxygenase (IDO) expression, an immunosuppressive enzyme that catabolizes tryptophan into kynurenine (Kyn) and other metabolites in DC. In the presence of LPS or CpG, Ahr-deficient (Ahr−/−) mature BMDC induced immune responses characterized by reduced Kyn and IL-10 production compared with results observed with tolerogenic mature WT BMDC. In a coculture system with LPS- or CpG-stimulated BMDC and naive T cells, Ahr−/− BMDC inhibited naive T-cell differentiation into regulatory T (Treg) cells, which likely facilitated Th17 cell development and promoted naive T-cell proliferation. Addition of synthetic L-Kyn to the coculture system skewed the differentiation of naive T cells to Treg cells rather than Th17 cells. Taken together, our results demonstrate a previously unknown negatively regulatory role for Ahr in DC-mediated immunogenesis in the presence of LPS or CpG, which, in turn, alters the Kyn-dependent generation of Treg cells and Th17 cells from naive T cells.

A Method for Micro-Determination of Total Microcystin Content in Waterblooms of Cyanobacteria (Blue-Green Algae)

Abstract Microcystins as cyanobacterial (blue-green algal) toxins are found from toxic waterblooms in freshwater lakes and reservoirs for drinking water all over the world. In this paper, we demonstrated a micro-determination method for total microcystin content in waterblooms of toxic cyanobacteria. This method based on a quantitative analysis of 2-methyl-3-methoxy-4-phenylbutyric acid(MMPB) as an oxidation product of microcystins by gas chromatography(GC) with a flame ionization detector or high performance liquid chromatography(HPLC) with a fluorescence monitor. In the case of GC analysis, methyl ester of MMPB in the pmol range of concentration was determined. On the other hand, in the HPLC analysis, MMPB in the fmol range of concentration was determined by labeling with 2-(2,3-naphthalimino)ethyl trifluoromethanesulfonate as a fluorescent reagent. These results pointed out that this micro-analysis is able to be applied to the monitoring of microcystins in drinking water, reservoirs and freshwater lakes.

Aryl hydrocarbon receptor deficiency in T cells suppresses the development of collagen-induced arthritis

The contributions of aryl hydrocarbon receptor (Ahr) to the pathogenesis of rheumatoid arthritis have not been elucidated. Here, we show that Ahr deficiency ameliorated collagen-induced arthritis, a mouse model of RA. Collagen-immunized Ahr KO mice showed decreased serum levels of such proinflammatory cytokines as IL-1β and IL-6. The Th17 and Th1 cell populations in lymph nodes from these mice decreased and increased, respectively, whereas the percentage of regulatory T cells was unchanged. Interestingly, a lack of Ahr specifically in T cells significantly suppressed collagen-induced arthritis development, whereas Ahr deficiency in macrophages had no effect. These finding indicate that the development of experimental autoimmune arthritis depends on the presence of Ahr in T cells, and that Th1/Th17 balance may be particularly important for this process.

Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on T cell-derived cytokine production in ovalbumin (OVA)-immunized C57Bl/6 mice.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is known to suppress both cellular and humoral immunity. Effector T cell-derived type-2 cytokines, including IL-4 and IL-5, play pivotal roles in humoral immunity. Herein, we studied whether TCDD affects type-2 cytokine productions during the immune response. C57Bl/6 mice were intraperitoneally immunized with ovalbumin (OVA) and orally administered 5 or 20 microg TCDD/kg on Day 0, and then challenged with OVA on Day 21. Seven days later (Day 28), antigen-specific antibodies in plasma, and T cell-derived cytokines produced by splenocytes and proliferation of splenocytes upon ex vivo re-stimulation with OVA were investigated. The quantities of IgM class and IgG1 class OVA-specific antibodies in plasma were reduced by 5 or 20 microg TCDD/kg and by 20 microg TCDD/kg, respectively. While thymus weight and cellularity were reduced by 20 microg TCDD/kg, spleen weight and cellularity were not changed by either 5 or 20 microg TCDD/kg. The proportions of B and T cells in the spleen were not affected by TCDD exposure. On the other hand, splenocytes from mice treated with 5 or 20 microg TCDD/kg were shown to produce less IL-4 or IL-5 upon ex vivo re-stimulation with OVA. Production of the T cell growth factor IL-2 was also decreased in splenocytes from TCDD-treated mice. In contrast, the type-1 cytokine IFN-gamma was increased by TCDD. Twenty micrograms of TCDD/kg suppressed OVA- or T cell mitogen (Con A)-stimulated proliferation of splenocytes, but did not affect B cell mitogen (LPS)-stimulated proliferation. These results suggested compromised T cell activation and suppressed type-2 cytokine production by T cells to be involved in the impaired humoral immunity associated with TCDD exposure.

Constitutively Active Aryl Hydrocarbon Receptor Expressed Specifically in T-Lineage Cells Causes Thymus Involution and Suppresses the Immunization-Induced Increase in Splenocytes

The aryl hydrocarbon receptor (AhR) is a transcription factor belonging to the basic helix-loop-helix-PER-ARNT-SIM superfamily. Xenobiotics, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin, bind the receptor and trigger diverse biological reactions. Thymocyte development and T cell-dependent immune reactions are sensitive targets of AhR-dependent 2,3,7,8-tetrachlorodibenzo-p-dioxin toxicity. However, the exact role of the AhR in T cells in animals exposed to exogenous ligands has not been clarified because indirect effects of activated AhR in other cell types cannot be excluded. In this study, we generated transgenic (Tg) mice expressing a constitutively active mutant of AhR under the regulation of a T cell-specific CD2 promoter to examine AhR function in T cells. The mRNAs of the constitutively active mutant of AhR and an AhR-induced gene, CYP1A1, were expressed in the thymus and spleen of the Tg mice. The transgene expression was clearly detected in the thymocytes, CD4, and CD8 T cells, but not in the B cells or thymus stromal cells. These Tg mice had a decreased number of thymocytes and an increased percentage of CD8 single-positive thymocytes, but their splenocytes were much less affected. By contrast, the increase in number of T cells and B cells taking place in the spleen after immunization was significantly suppressed in the Tg mice. These results clearly show that AhR activation in the T-lineage cells is directly involved in thymocyte loss and skewed differentiation. They also indicate that AhR activation in T cells and not in B cells suppresses the immunization-induced increase in both T cells and B cells.

Induction of apoptosis in mouse thymocytes by cadmium

In the thymus apoptosis is an important process in T cell maturation and differentiation. Cadmium (Cd) is an ubiquitous toxic metal that is capable of modulating immune responses. To investigate the induction of apoptosis and immunomodulation by environmental chemicals, we cultured mouse thymocytes with Cd and/or dexamethasone (DEX). DNA fragmentation was analyzed by gel electrophoresis, ELISA and flow cytometry. Treatment with either Cd or DEX induced DNA fragmentation in the thymocytes. Exposure to 10 microM Cd killed thymocytes by apoptosis rather than necrosis. However, no synergistic or additive effect was observed in the induction of apoptosis when DEX was added to the Cd. These results suggest that Cd may modulate the function of the thymus by the induction of apoptosis through mechanisms that differ from those used by DEX.

Effect of environmental pollutants on the production of pro-inflammatory cytokines by normal human dermal keratinocytes.

The effect of the environmental pollutants, diesel exhaust particles (DEP) and formaldehyde (FA), on the production of pro-inflammatory cytokines (interleukin (IL)-1alpha, IL-1beta, tumor necrosis factor (TNF)-alpha and IL-8) by normal human dermal keratinocytes (hKCs) was investigated. Normal hKCs were incubated with various concentrations of DEP (0.4, 0.8, 4, or 20 microg/ml) or FA (0.25, 0.5, 1, or 5 microg/ml), and cytokine production was then determined by enzyme-linked immunosorbent assay (ELISA). DEP (20 microg/ml) induced IL-1beta production without altering cell growth. The increased production of IL-1beta induced by this concentration of DEP was further enhanced by the presence of phorbol 12-myristate 13-acetate (PMA), although PMA alone did not affect the levels of IL-1beta. IL-8 production was also increased by DEP (0.4 and 0.8 microg/ml), which is consistent with the results that these concentrations of DEP increased the number of cells significantly after 72 h incubation. Although FA alone did not stimulate the production of IL-1beta or IL-8 by keratinocytes, FA (0.5 microg/ml and 5 microg/ml) significantly increased IL-8 and IL-1beta production, respectively, in cells stimulated with PMA. IL-1alpha production was not modulated by FA or DEP even in the presence of PMA. TNF-alpha was produced by unstimulated keratinocytes at barely detectable levels after 48 h incubation. Although basal levels of TNF-alpha in the culture supernatants were increased after stimulation with PMA, neither pollutant alone nor combination with PMA affected the levels of TNF-alpha. These in vitro findings suggest that environmental pollutants may act as modulating factors of cutaneous inflammation by affecting the ability of keratinocytes to release pro-inflammatory cytokines.