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Journal of Histochemistry and Cytochemistry, Vol. 46, 825-832, July 1998, Copyright © 1998, The Histochemical Society, Inc.

ARTICLE

Localization of Xenobiotic-responsive Element Binding Protein in Rat Hepatocyte Nuclei After Methylcholanthrene Administration as Revealed by In Situ Southwestern Hybridization

Youko Asakaa, Jun Watanabea, and Shinsuke Kanamuraa
a Department of Anatomy, Kansai Medical University, Moriguchi, Osaka, Japan

Correspondence to: Shinsuke Kanamura, Dept. of Anatomy, Kansai Medical Univ., 10-15 Fumizono-cho, Moriguchi, Osaka 570, Japan.

Xenobiotic-responsive element binding protein (XRE-BP), a heterodimer of aryl hydrocarbon receptor (AhR) and its nuclear translocator (Arnt), regulates the transcription of cytochrome P-450 1A1 gene (CYP1A1) through XRE in response to xenobiotic inducers. For a better understanding of the regulatory mechanism of CYP1A1 through XRE, localization of XRE-BP was examined in liver sections or isolated hepatocyte nuclei from control and 3-methylcholanthrene (MC)-treated rats by in situ Southwestern hybridization, using synthetic XRE as a probe, and was observed by confocal laser scanning microscopy and electron microscopy. Gel mobility shift assay and competitive binding assay showed specificity of the synthetic XRE probe. XRE-BP was exclusively localized in hepatocyte nuclei in liver sections from animals 3 hr after MC injection, whereas the protein was absent in hepatocyte cytoplasm in MC-treated animals and in hepatocyte nuclei and cytoplasm in control animals. In isolated hepatocyte nuclei, XRE-BP began to accumulate in the central region between 0.5 and 3 hr, showed a peak between 3 and 6 hr, decreased gradually between 6 and 72 hr, and disappeared at 72 hr after MC injection. The protein was scarce in peripheral and nucleolar regions of the nucleus. Therefore, XRE-BP is formed in the nuclei of hepatocytes after MC stimulation. In addition, XRE-BP was found in isolated hepatocyte nuclei from control animals after preincubation with cytoplasmic lysate from MC-treated animals, although the protein was absent in the nuclei before the preincubation. These findings strongly suggest that AhR translocates from hepatocyte cytoplasm to the nucleus and forms XRE-BP in the nucleus after MC stimulation. (J Histochem Cytochem 46:825–832, 1998)

Key Words: xenobiotic-responsive element, DNA binding protein, in situ Southwestern, hybridization, cytochrome P-450, methylcholanthrene, liver, rat


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[Abstract] [Full Text]



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