Disruption of aryl hydrocarbon receptor (AhR) induces regression of the seminal vesicle in aged male mice.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates diverse dioxin toxicities. Despite mediating the adverse effects, the AhR gene is conserved among animal species, suggesting important physiological functions for AhR. In fact, a recent study revealed that AhR has an intrinsic function in female reproduction, though its role in male reproduction is largely unknown. In this study, we show age-dependent regression of the seminal vesicles, probably together with the coagulating gland, in AhR(-/-) male mice. Knockout mice had abnormal vaginal plugs, low sperm counts in the epididymis, and low fertility. Moreover, serum testosterone concentrations and expression of steroidogenic 3betahydroxysteroiddehydrogenase (3betaHsd) and steroidogenic acute regulatory protein (StAR) in testicular Leydig cells were decreased in AhR(-/-) males. Taken together, our results suggest that impaired testosterone synthesis in aged mice induces regression of seminal vesicles and the coagulating glands. Such tissue disappearance likely resulted in abnormal vaginal plug formation, and eventually in low fertility. Together with previous findings demonstrating AhR function in female reproduction, AhR has essential functions in animal reproduction in both sexes.

Molecular mechanisms of AhR functions in the regulation of cytochrome P450 genes.

AhR, a ligand-activated transcription factor, mediates xenobiotic signaling to enhance the expression of target genes, including drug-metabolizing cytochrome P450s. The recent development of several new techniques, including chromatin immunoprecipitation and RNA interference, has expanded and deepened our knowledge of AhR function in the xenobiotic signal transduction. In this review, we briefly summarize our current understanding of the activation and inactivation of AhR activities and discuss the future directions of AhR research.

Parathyroid hormone (PTH) down-regulates PTH/PTH-related protein receptor gene expression in UMR-106 osteoblast-like cells via a 3',5'-cyclic adenosine monophosphate-dependent, protein kinase A-independent pathway.

Parathyroid hormone (PTH) regulates osteoblast function via a G protein-linked PTH/PTH-related protein (PTHrP) receptor. We have studied the mechanisms of PTH/PTHrP receptor gene repression by PTH in UMR-106 osteoblast-like cells. Inhibition of PTH/PTHrP receptor mRNA expression by rat (r) PTH(1-34) and Insulin-like growth factor-I (IGF-I) at 10(-7)M was significant at 1 h and 3 h, and maximal at 2 h and 6 h. A maximal decrease in receptor mRNA abundance by rPTH(1-34) and IGF-I was maintained for 24 h. Inhibition of receptor gene expression by rPTH(1-34) was mimicked in UMR-106 cells by the addition of forskolin (an adenylyl cyclase activator), or 8-(4-chlorophenylthio)-adenine 3',5'-cyclic monophosphate (8-pCPTcAMP; a cAMP analogue). Although H89, a selective protein kinase A (PKA) inhibitor, completely inhibited PKA activity stimulated by rPTH(1-34), forskolin or 8-pCPTcAMP, suppression of PTH/PTHrP receptor mRNA synthesis induced by these substances in UMR-106 cells was not affected by H89. In primary osteoblast cultures, rPTH(1-34) inhibited synthesis of PTH/PTHrP receptor mRNA irrespective of H89. The down-regulation effect of rPTH(1-34) was also unaltered by PD98059 (an extracellularly regulated kinase 1/2 mitogen-activated protein kinase pathway inhibitor). Pretreatment with cycloheximide, a protein synthesis inhibitor, did not alter the inhibition of PTH/PTHrP receptor mRNA expression by rPTH(1-34), indicating that receptor mRNA suppression does not require new protein synthesis. Transcriptional activation of PTH/PTHrP receptor gene promoter (U3P or U4P)-luciferase constructs was decreased by rPTH(1-34), forskolin and 8-pCPTcAMP irrespective of H89. Thus, PTH transcriptionally down-regulates PTH/PTHrP receptor gene expression in osteoblast-like cells via a cAMP-dependent, PKA-independent pathway.

Expression of human and mouse genes encoding polkappa: testis-specific developmental regulation and AhR-dependent inducible transcription.

BACKGROUND: Human polkappa is a newly identified low-fidelity DNA polymerase. While the enzyme bypasses an abasic site and acetylaminofluorene-adduct in an error-prone manner, it bypasses benzo[a]pyrene-N2-dG lesions in a mostly error-free manner by incorporating predominantly dC opposite the bulky lesions. Benzo[a]pyrene (B[a]P) is activated through intracellular process mediated by the arylhydrocarbon receptor (AhR, also called the dioxin receptor), which is a ligand-activated transcription factor with high affinities for aromatic compounds such as B[a]P and dioxin. RESULTS: We examined promoter structures of the human POLK and mouse Polk genes to study how their expressions are regulated. The mouse Polk gene is developmentally regulated in testis and utilizes two transcription start sites during spermatogenesis, while it utilizes only one site in tissues other than testis. Both of the mouse Polk and the human POLK genes have two AhR-binding sites in the promoter regions and the expression of the mouse Polk gene is indeed enhanced upon AhR-activation. CONCLUSIONS: The AhR activation increases expression of the mouse Polk gene and probably the human POLK gene, the product of which bypasses benzo[a]pyrene-N2-dG lesions in a mostly accurate manner. Thus, polkappa seems to function to reduce mutagenesis at benzo[a]pyrene-adducts, although it may also have a role related to spermatogenesis.

Dioxin induces a novel nuclear factor, DIF-3, that is implicated in spermatogenesis.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD; dioxin), a member of a class of environmental pollutants represented by polychlorinated dibenzo-p-dioxins and dibenzofurans, is one of the most toxic artificial compounds ever developed. In this study, we identified a novel TCDD target gene, DIF-3 (dioxin inducible factor-3), by cDNA representational difference analysis. DIF-3 protein is a nuclear factor and possesses a zinc-finger motif at its N-terminus. High DIF-3 mRNA expression in the testes was demonstrated by Northern blot analysis and abundant DIF-3 protein was detected during spermatogenesis. Thus, these results suggest that DIF-3 may be a target gene mediating the reproductive toxicity induced by TCDD.

Parathyroid hormone (PTH) suppresses rat PTH/PTH-related protein receptor gene promoter.

Parathyroid hormone (PTH) regulates osteoblasts via a G protein-linked PTH/PTH-related protein (PTHrP) receptor. PTH effects on PTH/PTHrP receptor gene expression were studied in UMR 106 osteoblast-like cells. In heterogeneous nuclear RNA and Northern analysis, PTH suppressed PTH/PTHrP receptor transcription. We cloned the 7-kb promoter region of the rat PTH/PTHrP receptor gene and transiently transfected chimeric deletion constructs containing the 5'-flanking region and the luciferase gene into UMR 106 cells. In transfected cells the minimal region for basal promoter activity was between positions -128 and +103. The 5'-flanking region of exon U1 contained several putative-binding sites for Sp1 and the myc-associated zinc finger protein (MAZ). The minimal PTH-suppressive region (PTHSR) was between +1 and +25 in exon U1, but the 5'-flanking region or Sp1 and MAZ-binding sites also were required for PTH-mediated repression. By gel mobility shift assay PTH markedly decreased binding of PTHSR-protein complex in UMR 106 cells. The mutation experiments showed that the most critical sequence for the repression of PTH was 5'-GGGGGAGGGGAG-3' (+1 to +12) of PTHSR. This represents the first characterization of a PTH-suppressive region of the PTH/PTHrP receptor gene in rat.

Dioxin suppresses the checkpoint protein, MAD2, by an aryl hydrocarbon receptor-independent pathway.

The compound 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has been shown recently to be carcinogenic, but little is currently known about the molecular mechanism of TCDD affecting cell proliferation and carcinogenesis. In this report, we demonstrate that TCDD suppresses the expression of the checkpoint protein, Mad2. Suppression of Mad2 was also observed in aryl hydrocarbon receptor-deficient mouse embryonic fibroblasts, suggesting that TCDD suppresses Mad2 by a novel TCDD receptor signaling mechanism. In addition, HeLa cells treated with TCDD failed to arrest in mitosis after nocodazole treatment. The Mad2 protein plays a significant role in accurate chromosome segregation in mitotic cells. Our data suggest that TCDD may increase chromosomal instability through the suppression of Mad2 expression.

Budd-Chiari syndrome and extrahepatic portal obstruction associated with congenital antithrombin III deficiency.

We report a patient with Budd-Chiari syndrome (BCS) and extrahepatic portal obstruction (EHO) associated with congenital antithrombin (AT) III deficiency. A 35-year-old man was admitted to Nishi Kobe Medical Center for evaluation of abnormal intrahepatic veins. By various imaging modalities, BCS and EHO were diagnosed. Laboratory data revealed parallel decreases in activity and antigen concentration of AT III despite normal liver function. Taken together, the etiology of both BCS and EHO was considered to be thrombosis, associated with congenital AT III deficiency. Two years after beginning warfarin therapy, the patient has no symptoms and his liver function remains normal. Anticoagulant therapy is considered useful for preventing progression of the disease.

Structure and expression of the Ah receptor repressor gene.

The aryl hydrocarbon receptor (AhR) repressor (AhRR) gene has been isolated and characterized from a mouse genomic library. The gene is distributed as 11 exons in a total length of about 60 kilobase pairs. Fluorescence in situ hybridization analysis has shown that the AhRR gene is located at mouse chromosome 13C2, at rat chromosome 1p11.2, and at human chromosome 5p15.3. The AhRR gene has a TATA-less promoter and several transcription start sites. In addition, putative regulatory DNA sequences such as xenobiotic responsive element (XRE), GC box, and NF-kappaB-binding sites have been identified in the 5'-upstream region of the AhRR gene. Transient transfection analyses of HeLa cells with reporter genes that contain deletions and point mutations in the AhRR promoter revealed that all three XREs mediated the inducible expression of the AhRR gene by 3-methylcholanthrene treatment, and furthermore, GC box sequences were indispensable for a high level of inducible expression and for constitutive expression. Moreover, by using gel mobility shift assays we were able to show that the AhR/Arnt heterodimer binds to the XREs with very low affinity, which is due to three varied nucleotides outside the XRE core sequence. We have also shown that Sp1 and Sp3 can bind to the GC boxes. Finally, both transient transfection analysis and gel mobility shift assay revealed that the AhRR gene is up-regulated by a p65/p50 heterodimer that binds to the NF-kappaB site when the cells has been exposed to 12-O-tetradecanoylphorbol-13-acetate, and this inducible expression was further enhanced by cotreatment of 12-O-tetradecanoylphorbol-13-acetate and 3-methylcholanthrene.

Human arylhydrocarbon receptor repressor (AHRR) gene: genomic structure and analysis of polymorphism in endometriosis.

The diversity of biological effects resulting from exposure to dioxin may reflect the ability of this environmental pollutant to alter gene expression by binding to the arylhydrocarbon receptor (AHR) gene and related genes. AHR function may be regulated by structural variations in AHR itself, in the AHR repressor (AHRR), in the AHR nuclear translocator (ARNT), or in AHR target molecules such as cytochrome P-4501A1 (CYP1A1) and glutathione S-transferase. Analysis of the genomic organization of AHRR revealed an open reading frame consisting of a 2094-bp mRNA encoded by ten exons. We found one novel polymorphism, a substitution of Ala by Pro at codon 185 (GCC to CCC), in exon 5 of the AHRR gene; among 108 healthy unrelated Japanese women, genotypes Ala/Ala, Ala/Pro, and Pro/Pro were represented, respectively, by 20 (18.5%), 49 (45.4%), and 39 (36.1%) individuals. We did not detect previously published polymorphisms of ARNT (D511N) or the CYP1A1 promoter (G-469A and C-459T) in our subjects, suggesting that these polymorphisms are rare in the Japanese population. No association was found between uterine endometriosis and any polymorphisms in the AHRR, AHR, ARNT, or CYP1A1 genes analyzed in the present study.

A case of localized primary sclerosing cholangitis mimicking intrahepatic bile duct cancer.

A 75-year-old woman with chronic hepatitis was regularly followed-up in our hospital. A computed tomography (CT) scan revealed an obviously enlarged intrahepatic bile duct in the posterior branch of the left lateral segment. Percutaneous cholangiography revealed an enlarged posterior branch of the left lateral segment and a narrow stenotic region at the root of this branch. We diagnosed her as having intrahepatic bile duct cancer, and a left lateral segmentectomy of the liver was performed. However, microscopic examination of the resected specimens revealed peribiliary fibrosis in the stenotic bile duct and other areas of the intrahepatic bile duct with no malignant cells. Thus, the final diagnosis was made to be primary sclerosing cholangitis. We must consider primary sclerosing cholangitis in the differential diagnosis of localized stenosis of the intrahepatic bile duct.

Vascularity of advanced gastric carcinoma: evaluation by using power Doppler imaging.

BACKGROUND: We investigated the vascularity of advanced gastric adenocarcinomas by using percutaneous power Doppler imaging. METHODS: Seventeen patients with gastric cancer and 10 without a gastric tumor, but with a slightly thick gastric wall in the B-mode ultrasound, were investigated with the use of power Doppler imaging. The color signals of the gastric lesion were graded as follows: 1, no color signals or the same as the surroundings; 2, color signals were slightly increasing; and 3, color signals were obviously increasing. RESULTS: The color signals of three patients were graded 1, those of eight patients were graded 2 and those of six patients were graded 3 in the gastric cancer group. The color signals of all 10 patients without a gastric tumor were grade 1. This difference was statistically significant (P = 0.0002). CONCLUSIONS: Power Doppler imaging showed vascularity of gastric cancer increasing in the majority of patients (14 of 17: 82%). Thus, power Doppler imaging might be a good screening examination method for gastric cancer.

Effect of angiotensin-converting enzyme inhibitor on cardiopulmonary baroreflex sensitivity in patients with acute myocardial infarction.

We evaluated the effect of angiotensin-converting enzyme inhibition (quinapril) on cardiopulmonary baroreflex sensitivity in 30 patients with uncomplicated myocardial infarction (quinapril group, 15 patients; placebo group, 15 patients) at 5 and 10 days after the onset of myocardial infarction. This study indicates that quinapril improved cardiopulmonary baroreflex and thus reduced sympathetic outflow in patients with acute myocardial infarction.

Transactivation mechanisms of mouse clock transcription factors, mClock and mArnt3.

BACKGROUND: The Arnt3 (also termed as BMAL1 or MOP3)/Clock heterodimer is a positive regulator of circadian rhythm and activates the transcription of target genes such as per1 and vasopressin. RESULTS: We investigated the transcriptional mechanism of mArnt3/mClock heterodimer. While mClock did not possess any distinct activation domain, mArnt3 contained a transcriptional activation domain at the most C-terminal end, the activity of which was not expressed, even in the one hybrid system, until it was bound by mClock. It has been suggested that mClock plays a regulatory or structural role in exerting a transcription enhancing effect of the mArnt3/mClock heterodimer. Deletion proceeding from amino acids 559-492 of mClock markedly reduced the transactivation activity of mArnt3/mClock heterodimer, in consistence with the results of the Clock-delta 19 mutant. Yeast and mammalian two-hybrid systems revealed that CBP and p300 interacted with mArnt3 via the CREB binding domain. The In vivo interaction between mArnt3 and CBP was confirmed by the GST pull down assay. CONCLUSION: Taken together, these results suggest that the mArnt3/mClock heterodimer exerted its transactivation activity via CBP or p300 interacting with mArnt3 in the heterodimer with mClock playing a structural or regulatory role in the transactivation process.

Aortic root dilatation among young competitive athletes: echocardiographic screening of 1929 athletes between 15 and 34 years of age.

BACKGROUND: Aortic dilatation can be lethal for young competitive athletes. The prevalence among athletes is not known, however, and thus a reasonable approach to early recognition remains uncertain. METHODS AND RESULTS: Echocardiograms of 1929 normotensive athletes 15 to 34 years of age were analyzed. Five (0.26%) athletes had aortic dilatation; 4 of the 5 played basketball. This made the prevalence of aortic dilatation 0.96% (4 of 415) among basketball and volleyball players, who represented a population of especially tall athletes. Tallness aside, only 2 of the 5 athletes had features of Marfan syndrome. Among the athletes without aortic dilatation, the relation between body surface area and aortic root dimension was nonlinear and best described with a quadratic regression model. Athletes with aortic dilatation fell well outside the 95% confidence interval. CONCLUSION: Because a higher incidence of aortic dilatation is to be anticipated among very tall athletes, inclusion of echocardiography in screening before participation in certain sports should be considered.

Endoscopic resection of small inflammatory fibroid polyp of the colon.

Inflammatory fibroid polyp (IFP) is a solitary intestinal lesion of unknown etiology. Although IFP is benign, laparotomy for the resection of colonic IFP is performed in most cases because the polyp is usually large. We report a successful endoscopic resection of cecal IFP. It is considered that colonic IFP should be resected endoscopically if the polyp is small and is located submucosally.