Togo Ikuta

Association of CYP1B1 genetic polymorphism with incidence to breast and lung cancer

Cytochrome P450 1B1 (CYP1B1) participates in the metabolic activation of a number of procarcinogens including benzo[a]pyrene and the hydroxylation of 17beta-estradiol at the C-4 position. In this study, we investigated the association between CYP1B1 genetic polymorphism and breast or lung cancer incidence. The Ala-Ser polymorphism at codon 119 in presumed substrate recognition site 1 was significantly associated with the incidence of breast or squamous cell carcinoma of the lung. On the other hand, Leu-Val polymorphism at codon 432 did not show any association to the cancers. An allele containing both Ala and Leu simultaneously, comprised 75% of alleles among 315 Japanese healthy controls, was significantly inversely associated with breast cancer incidence. When expressed in a recombinant system, this CYP1B1 cDNA showed the lowest 17beta-estradiol 4-hydroxylase activity among four different variant forms of CYP1B1. Thus, inter-individual differences in activation of procarcinogens or metabolism of oestrogen originating from genetic polymorphisms of the human CYP1B1 gene may contribute to the susceptibility of human cancers.

Aryl hydrocarbon receptor suppresses intestinal carcinogenesis in ApcMin/+ mice with natural ligands

Intestinal cancer is one of the most common human cancers. Aberrant activation of the canonical Wnt signaling cascade, for example, caused by adenomatous polyposis coli (APC) gene mutations, leads to increased stabilization and accumulation of β-catenin, resulting in initiation of intestinal carcinogenesis. The aryl hydrocarbon receptor (AhR) has dual roles in regulating intracellular protein levels both as a ligand-activated transcription factor and as a ligand-dependent E3 ubiquitin ligase. Here, we show that the AhR E3 ubiquitin ligase has a role in suppression of intestinal carcinogenesis by a previously undescribed ligand-dependent β-catenin degradation pathway that is independent of and parallel to the APC system. This function of AhR is activated by both xenobiotics and natural AhR ligands, such as indole derivatives that are converted from dietary tryptophan and glucosinolates by intestinal microbes, and suppresses intestinal tumor development in ApcMin/+ mice. These findings suggest that chemoprevention with naturally-occurring and chemically-designed AhR ligands can be used to successfully prevent intestinal cancers.

A Nuclear Localization Signal of Human Aryl Hydrocarbon Receptor Nuclear Translocator/Hypoxia-inducible Factor 1β Is a Novel Bipartite Type Recognized by the Two Components of Nuclear Pore-targeting Complex

Aryl hydrocarbon receptor nuclear translocator (ARNT) is a component of the transcription factors, aryl hydrocarbon receptor (AhR) and hypoxia-inducible factor 1, which transactivate their target genes, such as CYP1A1 and erythropoietin, in response to xenobiotic aromatic hydrocarbons and to low O2concentration, respectively. Since ARNT was isolated as a factor required for the nuclear translocation of AhR from the cytoplasm in response to xenobiotics, the subcellular localization of ARNT has been of great interest. In this investigation, we analyzed the subcellular distribution of ARNT using transient expression of a fusion gene with β-galactosidase and microinjection of recombinant proteins containing various fragments of ARNT in the linker region of glutathioneS-transferase/green fluorescent protein. We found a clear nuclear localization of ARNT in the absence of exogenous ligands to AhR, and identified the nuclear localization signal (NLS) of amino acid residues 39–61. The characterized NLS consists of 23 amino acids, and can be classified as a novel variant of the bipartite type on the basis of having two separate regions responsible for efficient nuclear translocation activity, but considerable deviation of the sequence from the consensus of the classical bipartite type NLSs. Like the well characterized NLS of the SV40 T-antigen, this variant bipartite type of ARNT NLS was also mediated by the two components of nuclear pore targeting complex, PTAC58 and PTAC97, to target to the nuclear rim in an in vitro nuclear transport assay.

Hypersensitivity of Aryl Hydrocarbon Receptor-Deficient Mice to Lipopolysaccharide-Induced Septic Shock

ABSTRACT Aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor, is known to mediate a wide variety of pharmacological and toxicological effects caused by polycyclic aromatic hydrocarbons. Recent studies have revealed that AhR is involved in the normal development and homeostasis of many organs. Here, we demonstrate that AhR knockout (AhR KO) mice are hypersensitive to lipopolysaccharide (LPS)-induced septic shock, mainly due to the dysfunction of their macrophages. In response to LPS, bone marrow-derived macrophages (BMDM) of AhR KO mice secreted an enhanced amount of interleukin-1β (IL-1β). Since the enhanced IL-1β secretion was suppressed by supplementing Plasminogen activator inhibitor-2 (Pai-2) expression through transduction with Pai-2-expressing adenoviruses, reduced Pai-2 expression could be a cause of the increased IL-1β secretion by AhR KO mouse BMDM. Analysis of gene expression revealed that AhR directly regulates the expression of Pai-2 through a mechanism involving NF-κB but not AhR nuclear translocator (Arnt), in an LPS-dependent manner. Together with the result that administration of the AhR ligand 3-methylcholanthrene partially protected mice with wild-type AhR from endotoxin-induced death, these results raise the possibility that an appropriate AhR ligand may be useful for treating patients with inflammatory disorders.

AhR protein trafficking and function in the skin.

Because aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor, its nuclear translocation in response to ligands may be directly linked to transcriptional activation of target genes. We have investigated the biological significance of AhR from the perspective of its subcellular localization and revealed that AhR possesses a functional nuclear localization signal (NLS) as well as a nuclear export signal (NES) which controls the distribution of AhR between the cytoplasm and nucleus. The intracellular localization of AhR is regulated by phosphorylation of amino acid residues in the vicinity of the NLS and NES. In cell culture systems, cell density affects not only its intracellular distribution of AhR, but also its transactivation activity of the target genes such as transcriptional repressor Slug, which is important for the induction of epithelial-mesenchymal transitions. These effects of AhR observed in cultured cells are proposed to be reflected on the in vivo response such as morphogenesis and tumor formation. This review summarizes recent work on the control mechanism of AhR localization and progress in understanding the physiological role of AhR in the skin. We propose that AhR is involved in normal skin formation during fetal development as well as in pathological states such as epidermal wound healing and skin carcinogenesis.

B lymphocyte-induced maturation protein 1 is a novel target gene of aryl hydrocarbon receptor

BACKGROUND The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor. When environmental pollutants, including chemical carcinogens, bind to AhR, the receptor translocates to nucleus and transcriptionally activates target genes including drug metabolizing enzymes such as P450s. Recent studies have shown that AhR mediates various responses, including cellular growth, differentiation, immune system and development. OBJECTIVE In this study, we investigated the physiological function of AhR in skin. METHODS Distribution of AhR in murine skin was examined by immunohistochemistry. Expression of a target gene which is transcriptionally activated by AhR is analysed by RT-PCR. RESULTS We found that AhR co-localizes with the transcriptional repressor B lymphocyte maturation protein 1 (Blimp1) in sebaceous gland. In this report, we show that expression of Blimp1 is induced by treatment with AhR ligands, such as methylcolanthrene (MC) in sebocyte and keratinocyte cell lines. Exposure to ultraviolet B, which has been reported to generate AhR ligand intracellularly, also increased Blimp1 mRNA. This ligand-dependent induction of Blimp1 requires the expression of both AhR and ARNT, since transfection of siRNA specific to either AhR or ARNT significantly reduced Blimp1 mRNA in response to MC. Analysis using kinase inhibitors revealed that ligand-dependent induction of Blimp1, but not that of CYP1A1, is inhibited by staurosporine. TPA, a potent activator of protein kinase C, increased Blimp1 mRNA but not CYP1A1. CONCLUSION These data indicate that Blimp1 is a novel AhR-target gene in epidermal keratinocyte and sebocyte.

Tissue distribution of aryl hydrocarbon receptor in the intestine: Implication of putative roles in tumor suppression

Intestinal homeostasis is maintained by complex interactions between intestinal microorganisms and the gut immune system. Dysregulation of gut immunity may lead to inflammatory disorders and tumorigenesis. We previously have shown the tumor suppressive effects of aryl hydrocarbon receptor (AhR) in intestinal carcinogenesis. In the present study, we investigated AhR distribution in the mouse and human intestine by histochemical analysis. In the normal intestine, AhR was mainly localized in the stroma containing immune cells in the lamina propria and lymphoid follicles. On the other hand, in the tumor tissue from human colon cancer and that developed in Apc(Min/+)mice, AhR expression was elevated. AhR immunostaining was found in both stromal and tumor cells. Although AhR was localized in the cytoplasm of tumor cells in most cases, nuclear AhR was also observed in some. AhR knockdown using siRNA resulted in significant promotion of cell growth in colon cancer cell lines. Furthermore, AhR activation by AhR ligands supplemented in culture medium suppressed cell growth. Our study results suggest that tumor suppressive roles of AhR are estimated in two distinct ways: in normal tissue, AhR is associated with tumor prevention by regulating gut immunity, whereas in tumor cells, it is involved in growth suppression.

Acetylation of nuclear localization signal controls importin-mediated nuclear transport of Ku70

Ku70 participates in various intra-and extra-nucleic processes. For multifunctional control, machinery that precisely regulates the intracellular localization of Ku70 is essential. Recently, it was reported that acetylation of Ku70 regulates its function. Here, we demonstrate that specific lysine residues in Ku70 that are targets of acetylation are critical for regulating nuclear transport in vivo. Ku70-GFP fusion proteins transiently expressed in cultured cells localized in the nucleus, whereas mimicking acetylation of K553 or K556 in the Ku70 nuclear localization signal (NLS) by substituting these lysine residues with glutamine markedly decreased the nuclear localization of Ku70. Moreover, the Ku70-importin interaction was suppressed in the K553Q and K556Q mutants. Theoretical estimations indicated that the binding energy between the Ku70 NLS and importin-α decreases with acetylation of lysine residues in the Ku70 NLS, similar to the case when these lysine residues are substituted with glutamine. These results suggest that acetylation of specific lysine residues in the Ku70 NLS is a key switch that controls the localization of Ku70 by modulating interactions between Ku70 and nuclear transport factors.

Aryl Hydrocarbon Receptor Suppresses Cecal Carcinogenesis

The aryl hydrocarbon receptor (AhR) has dual roles in regulating intracellular protein levels as both a ligand-activated transcription factor and a ligand-dependent E3 ubiquitin ligase. We showed that the AhR E3 ubiquitin ligase has a role in the suppression of intestinal carcinogenesis by a previously undescribed ligand-dependent β-catenin degradation pathway. This function of AhR is activated by both xenobiotics and natural AhR ligands, such as indole derivatives that are converted from dietary tryptophan and glucosinolates by intestinal microbes, and which suppresses intestinal tumor development in Apc Min/+ mice. To elucidate whether the tumors develop autonomously in AhR −/− mice as a result of impaired β-catenin degradation or in association with accelerated inflammation, we performed two kinds of experiments using germ-free (GF) AhR −/− mice or compound mutant mice lacking genes for AhR and adaptor apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), which play an essential role in caspase-1 activation in inflammasomes. Both GF AhR −/− and AhR −/− •ASC −/− mice showed considerably reduced tumor development compared with that in AhR −/− mice, albeit in a “cancer-prone” state with aberrant β-catenin accumulation. These results revealed an important role of the bacteria-triggered or ASC-mediated inflammation signaling pathway in mouse intestinal tumorigenesis. Furthermore, they also suggest a possible chemical therapeutic intervention that involves AhR ligands and inhibitors of the inflammation pathway.