Localization of xenobiotic-responsive element binding protein in rat hepatocyte nuclei after methylcholanthrene administration as revealed by in situ Southwestern hybridization.
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.
Asaka
Y
Y
Department of Anatomy, Kansai Medical University, Moriguchi, Osaka, Japan.
Watanabe
J
J
Kanamura
S
S
D000818:Animals; D015139:Blotting, Southern; D015153:Blotting, Western; D002467:Cell Nucleus / Q000737:chemistry* / Q000187:drug effects / Q000648:ultrastructure; D004268:DNA-Binding Proteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D017403:In Situ Hybridization; D008099:Liver / Q000737:chemistry* / Q000187:drug effects / Q000648:ultrastructure; D008297:Male; D008748:Methylcholanthrene / Q000494:pharmacology; D008854:Microscopy, Electron; D051381:Rats; D017207:Rats, Sprague-Dawley
D004268:DNA-Binding Proteins; D008748:Methylcholanthrene