Transforming growth factor-␤ (TGF-␤) and interferon-␥ (IFN-␥Physical interaction between Smad3 and YB-1 was demonstrated by immunoprecipitation-Western blot analyses, and electrophoretic mobility shift assays using the recombinant Smad3 and YB-1 proteins indicated that YB-1 forms a complex with Smad3 bound to the Smadbinding element. Glutathione S-transferase pull-down assays showed that YB-1 binds to the MH1 domain of Smad3, whereas the central and carboxyl-terminal regions of YB-1 were required for its interaction with Smad3. YB-1 also interferes with the Smad3-p300 interaction by its preferential binding to p300. Altogether, the results provide a novel insight into the mechanism by which IFN-␥/YB-1 counteracts TGF-␤/Smad3. They also indicate that IFN-␥/YB-1 inhibits COL1A2 transcription by dual actions: via the IFN-␥ response element and through a cross-talk with the TGF-␤/Smad signaling pathway.

We have demonstrated previously that a proximal element within the human ␣2(I) collagen gene (COL1A2) promoter mediates transcriptional repression by interferon-␥ (IFN-␥), and designated this region the IFN-␥ response element (IgRE). Screening of a human fibroblast cDNA expression library with a radiolabeled IgRE probe exclusively yielded clones with a sequence identical to that of the transcription factor YB-1. Electrophoretic mobility shift assays (EMSA) using various IgRE-derived oligonucleotide probes containing serial two-base mutations showed that YB-1 protein was preferentially bound to the pyrimidine-rich sequence within the IgRE. This region is located immediately downstream of and partly overlaps the previously reported Sp1/Sp3 binding site. Overexpression of YB-1 in human dermal fibroblasts decreased steady state levels of COL1A2 mRNA and repressed COL1A2 promoter activity in a dose-dependent manner. This inhibitory effect of YB-1 on COL1A2 expression was abolished by mutations of the IgRE shown to prevent YB-1 binding in EMSA. In addition, these mutations also abolished the inhibitory effect of IFN-␥, suggesting that YB-1 mediates the inhibitory action of IFN-␥ on COL1A2 promoter through its binding to the IgRE. Also, overexpression of a deletion mutant YB-1, which lacks the carboxyl-terminal domain, abrogated the repression of COL1A2 transcription by IFN-␥. A functional correlation between IFN-␥ and YB-1 was further supported by luciferase assays using four tandem repeats of the Y-box consensus oligonucleotide linked to a minimal promoter. EMSA and Western blot analysis using cytoplasmic and nuclear proteins implied that IFN-␥ promotes the nuclear translocation of YB-1. Direct evidence for the nuclear translocation of YB-1 by IFN-␥ was further provided by using a YB-1-green fluorescent protein expression plasmid transfected into human fibroblasts. Altogether, this study represents the definitive identification of the transcription factor responsible for IFN-␥-elicited inhibition of COL1A2 expression, namely YB-1.

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The metabolic fate of pyriproxyfen [4-phenoxyphenyl (RS)-2-(2-pyridyloxy]propyl ether, Sumilarv) was examined in rats and mice given single oral doses of [pyridyl-2,6-14C]-or \phenoxyphenyl-14C]pyriproxyfen at doses of 2 and 1000 mg/kg. The carbon-14 was excreted almost completely into urine and feces within 7 days after dosing and fecal excretion of carbon-14 predominated in both animals. Excretion of carbon-14 into feces and urine was, respectively, 84-97% and 4-12% of the dose in rats and 64-91% and 9-38% in mice. Major metabolic reactions of pyriproxyfen were (1) hydroxylation at the 4-position of the terminal phenyl ring, (2) hydroxylation at the 2-position of the terminal phenyl ring, (3) hydroxylation at the 5-position of the pyridyl ring, (4) dephenylation, (5) cleavage of ether linkages, and (6) conjugation of the resultant phenols with sulfuric acid or glucuronic acid. Although there was generally no marked difference in the metabolic profile of pyriproxyfen between the two species, significant sex-related differences were found in metabolic reactions 1, 3, and 6 in the rat but not in the mouse.

Estrogenic and antiestrogenic activity of pyrethroid insecticides (d-trans-allethrin, cypermethrin, empenthrin, fenvalerate, imiprothrin, permethrin, d-phenothrin and prallethrin) was evaluated using a suite of three in vitro assays based on classic human estrogen receptor alpha (hER alpha)-mediated mechanisms. A mammalian cell-based luciferase reporter gene assay was developed for examining effects on hER alpha-mediated gene activation. hER alpha-independent effects on the gene activation were examined using control cells with constitutive luciferase activation by a herpes simplex virus thymidine kinase (HSV-TK) promoter for determining appropriate dose levels of test chemicals. Moreover, the test chemical-dependent interaction between hER alpha and a coactivator (transcriptional intermediary factor 2: TIF2) was analyzed by a yeast two-hybrid method, competitive binding to hER alpha being assayed by a fluorescence polarization method. Significant (p < 0.05) positive effects of estrogenic substances (E2/estradiol, diethylstilbestrol, and p-nonylphenol) were detected in all assays. An antiestrogen, 4-hydroxytamoxifen, significantly inhibited E2-mediated transactivation and interaction between hER alpha and TIF2 through hER alpha binding (p < 0.05). However, none of the pyrethroids tested showed significant (p < 0.05) estrogenic or antiestrogenic effects (100 nM-10 microM), indicating that they do not impact on the classic hER alpha-mediated activation pathway in vitro.