ARTICLE |
Correspondence to: Shinsuke Kanamura, Dept. of Anatomy, Kansai Medical Univ., 10-15 Fumizono-cho, Moriguchi, Osaka 570, Japan.
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Summary |
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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 46:825832, 1998)
Key Words: xenobiotic-responsive element, DNA binding protein, in situ Southwestern, hybridization, cytochrome P-450, methylcholanthrene, liver, rat
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Introduction |
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Xenobiotic-responsive element (XRE) is an enhancer that drives expression of cytochrome P-450 1A1 (P-450 1A1) in response to xenobiotic inducers such as 3-methylcholanthrene (MC) and dioxin (
Recently,
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Materials and Methods |
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A total of 76 male SpragueDawley rats, 1012 weeks old, were used. The animals were fed standard laboratory chow and water ad libitum. The animals received a single IP injection of MC (Sigma, St Louis, MO; 25 mg/kg body weight) dissolved in corn oil or corn oil alone (control). The animal experiments were performed according to the guidelines of Kansai Medical University.
Detection of XRE-BP in Sections
Three hours after MC or corn oil injection, livers of five MC-treated and five control animals were briefly perfused via the portal vein with cold saline and with cold 0.1 M phosphate buffer (pH 7.4) containing 4% paraformaldehyde for 10 min under sodium pentobarbital anesthesia. Small blocks cut from the left lobe of perfusion-fixed livers were further fixed in the same fixative at 4C for 1 hr and washed in 0.1 M phosphate buffer containing 8% sucrose (pH 7.4) at 4C for 1 hr. Frozen sections 6 µm thick were cut from the blocks and attached to aminopropyltriethoxysilane (APS)- coated glass slides. The sections were preincubated with 5 x sodium citratesodium chloride solution (SSC) containing 1% blocking reagent attached to a DIG Detection System Kit (Boehringer Mannheim; Mannheim, Germany) at 30C for 3 hr and then hybridized with oligonucleotide probe labeled with digoxigenin (DIG) by 3'-end-labeling (100 µl/slide). A double-stranded XRE probe prepared from equimolaramounts of (+) strand (XRE sequence) and (-) strand (complementary to XRE sequence) was used in this experiment. The hybridization medium contained the blocking reagent, 5 x SSC, and 300 pmol/ml of one of the labeled probes. The sequence of (+) strand probe was 5'-CCT CCA GGC TCT TCT CAC GCA ACT CCG GGG CAC-3'. This corresponds to an XRE sequence at the 5'-flanking region of CYP1A1 (-1029 to -997) (
Detection of XRE-BP in Isolated Hepatocyte Nuclei
Three hours after MC or corn oil injection, livers of five MC-treated and 5 control animals were perfused with cold saline via the portal vein for 3 min under anesthesia and homogenized separately with 2 volumes of 0.05 M Tris-HCl buffer containing 25 mM KCl, 5 mM MgCl2, and 0.25 M sucrose (pH 7.5). The resulting homogenates were subjected to sucrose gradient centrifugation according to the method of
To examine accumulation and withdrawal of XRE-BP in hepatocyte nuclei, the nuclei were isolated from animals 0 (within 1 min after the injection), 0.25, 0.5, 1, 3, 6, 9, 12, 24, 36, 48, or 72 hr after injection of MC (three animals/time point), smeared onto glass slides, fixed, hybridized with DIG-labeled double-stranded probe (300 pmol/ml), and processed for CLSM as above.
For electron microscopy, the fixed nuclei on slides were immersed in methanol containing 0.3% (v/v) hydrogen peroxide for 30 min at 20C to block endogenous peroxidase activity, washed well with PBS, preincubated with the blocking reagent, and incubated in the hybridization medium containing DIG-labeled double-stranded probe (300 pmol/ml) at 30C for 15 hr as above. The slides were processed for immunohistochemical detection of DIG using horseradish peroxidase (HRP)-labeled anti-DIG antibody (Boehringer Mannheim), and stained with PBS containing diaminobenzidine (10 mg/ml) and 0.03% (v/v) hydrogen peroxide. The slides were washed with PBS and immersed in 1% (v/v) osmium tetroxide for 1 min at room temperature. The nuclei thus stained were dehydrated and embedded in Spurr's resin. After polymerization, the glass slide was removed by heating from the nuclei embedded in the Spurr block. Unstained thin sections were cut from the block and examined in a JEM 100-S electron microscope (JEOL; Tokyo, Japan).
Biochemical Methods
Livers of five MC-treated and five control animals were perfused under anesthesia with cold saline for 3 min via the portal vein 3 hr after the injection. The perfused livers were homogenized separately with 9 volumes of 1 mM Tris-HCl buffer containing 1 mM EDTA and 0.25 M sucrose (pH 7.4) at 4C. A portion of each homogenate (2 ml) was pooled and the remaining homogenates were centrifuged at 1000 x g for 15 min at 4C. The resulting precipitates containing hepatocyte nuclei were washed with the buffer by centrifugation at 1000 x g for 15 min at 4C and suspended in the buffer (nuclear fractions). The supernatants from the first centrifugation were centrifuged at 1000 x g for 15 min at 4C to remove contaminated nuclei and pooled (cytoplasmic fractions). After measurement of protein content by the method of
For gel mobility shift assay, aliquots of the lysates (10 µl) were incubated with nonlabeled (+) strand probe, (-) strand probe, or double-stranded probe (100 pmol) at 30C for 30 min and subjected to polyacrylamide gel electrophoresis. The resulting gels were stained with ethidium bromide and viewed under UV irradiation.
For dot Southwestern analysis, aliquots of the lysates (1 µl) were immobilized on nitrocellulose filters, incubated with DIG-labeled double-stranded probe (100 pmol) at 30C for 30 min, processed for immunohistochemical detection of DIG using the HRP-labeled anti-DIG antibody, and stained with diaminobenzidine and hydrogen peroxide as above. For dot Western analysis, the membranes were stained by the indirect immunoperoxidase method as previously described (
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Results |
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Gel Mobility Shift Assay
When cell lysates or nuclear lysates from MC-treated animals were subjected to gel mobility shift assay, a labeled protein band was found at about 200 kD for (+) strand and double-stranded probe (Figure 1). The value is identical with that of XRE-BP (
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Localization of XRE-BP in Sections
Fluorescence due to DIG-labeled double-stranded probe was strong in hepatocyte nuclei but was absent in hepatocyte cytoplasm and nonparenchymal cells in sections from animals 3 hr after MC injection (Figure 2A and Figure 2C). The intensity of fluorescence in the nuclei appeared uniform within the liver lobule. When sections from MC-treated or control animals were incubated with DIG-labeled double-stranded probe in the presence of a 100-fold molar excess of unlabeled probe, no fluorescence was observed (Figure 2B and Figure 2D). Fluorescence was negative in sections from control animals (Figure 2E and Figure 2F).
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Localization of XRE-BP in Isolated Hepatocyte Nuclei
In isolated hepatocyte nuclei from animals 3 hr after injection of MC, fluorescence due to DIG-labeled double-stranded or (+) strand probe was strong in the central region of the nuclei except for a few dotted nonfluorescent portions with a diameter of 23 µm within the central region, whereas it was weak along the periphery of the nuclei (Figure 3AD). Fluorescence due to DIG-labeled (-) strand probe was negative in the nuclei from MC-treated animals (Figure 3E). Binding of DIG-labeled (+) strand or double-stranded probe to the nuclei from MC-treated animals was inhibited by an excess amount of unlabeled double-stranded or (+) strand probe. Fluorescence was absent in the nuclei from control animals when the nuclei were incubated with any of the three labeled probes (Figure 3FH).
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By electron microscopy, electron-dense reaction product was abundant in the nucleoplasm of central region of isolated hepatocyte nuclei from MC-treated animals (Figure 4A and Figure 4B). The reaction product appeared to be associated with eu- (active) chromatin. However, the product was scarce in nucleoli and nucleoplasm around the nucleoli (nucleolar region) within the central region. Therefore, the nonfluorescent portions observed in the central region by CLSM represent the nucleolar region. In addition, the product was scarce in the nucleoplasm along the periphery of the nuclei beneath the nuclear membrane (peripheral region). The binding of DIG-labeled double-stranded probe to the nuclei from MC-treated animals was inhibited by excess amount of unlabeled probe (Figure 4C). In the nuclei from control animals, the reaction product was scarce in all regions of the nucleus.
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Accumulation and Withdrawal of XRE-BP in Isolated Hepatocyte Nuclei
No fluorescence due to XRE-BP was found in hepatocyte nuclei 0 (within 1 min) or 0.25 hr (15 min) after MC injection (Figure 5AC). Fluorescence appeared and became strong in the central region of nuclei between 0.5 and 3 hr, and remained unchanged between 3 and 6 hr after the injection (Figure 5DF). The fluorescence gradually diminished between 6 and 72 hr, and disappeared 72 hr after the injection (Figure 5GI). In the peripheral or nucleolar region of the nucleus, weak fluorescence was occasionally seen between 1 and 24 hr after the injection.
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XRE-BP in Hepatocyte Nuclei from Control Animals Preincubated with Cytoplasmic Lysate from MC-treated or Control Animals
Isolated hepatocyte nuclei from control animals showed no or very weak fluorescence before preincubation with cytoplasmic lysate from MC-treated or control animals (Figure 6A) or after preincubation with the lysate from control animals (Figure 6B). Strong fluorescence was found in isolated hepatocyte nuclei from control animals after preincubation with cytoplasmic lysate from MC-treated animals (Figure 6C) or in the nuclei from MC-treated animals after preincubation with cytoplasmic lysate from control animals (Figure 6D).
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Dot Southwestern Analysis of XRE-BP and Dot Western Analysis of AhR in Nuclear or Cytoplasmic Lysates from Control or MC-treated Animals
When nuclear or cytoplasmic lysates from livers of control or MC-treated animals were subjected to dot Southwestern analysis, reaction was strong in nuclear lysates from MC-treated animals but very weak in cytoplasmic lysates from MC-treated animals and in nuclear and cytoplasmic lysates from control animals (Figure 7A). When the lysates from livers of control or MC-treated animals were subjected to dot Western blot analysis, immunostaining for AhR was strong in nuclear or cytoplasmic lysates from livers of MC-treated animals and cytoplasmic lysates from livers of control animals, whereas nuclear lysates from control animals showed very weak reaction (Figure 7B). Similar results were obtained when nuclear or cytoplasmic lysates from the muscle, intestine, or kidney were analyzed by Western blot analysis (Figure 7C).
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Discussion |
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As shown in the present study, fluorescence due to DIG-labeled double-stranded XRE probe was observed preferentially in hepatocyte nuclei in sections from MC-treated animals. No fluorescence was found in hepatocyte cytoplasm in sections from MC-treated animals or in hepatocyte nuclei and cytoplasm in sections from control animals. Binding of the labeled probe was inhibited by an excess amount of unlabeled probe. Furthermore, gel mobility shift assay showed that the probe bound specifically to a single protein band at about 200 kD, which is identical to the molecular weight of XRE-BP (
The mechanism for regulating the expression of CYP1A1 through XRE has been postulated as follows (
After dioxin stimulation, AhR associated with heat shock protein 90 (HSP90) in the cytoplasm of Hepa-1 cells releases HSP90 and may associate with dioxin (
AhR and Arnt contain basic helixloophelix (bHLH) motifs near their amino termini (
XRE-BP appeared in the central region in isolated hepatocyte nuclei 0.5 hr after injection of MC; it takes about 30 min to form XRE-BP in vivo after the injection. Subsequent accumulation of XRE-BP in the central region of nuclei, found between 0.5 and 3 hr after the injection, was prominent. However, fluorescence due to XRE-BP was weak in the peripheral and nucleolar regions of the nuclei from MC-treated animals. Thereafter, the protein decreased gradually in the nuclei between 6 and 72 hr after MC injection. XRE-BP is therefore formed primarily in the central region of hepatocyte nuclei after MC stimulation. The findings also suggest that dissociation or degradation of XRE-BP occurs in the central region of the nuclei. The decrease in XRE-BP in isolated nuclei from MC-treated animals is consistent with observations of the lability of the liganded AhR in the nucleus of Hepa-1 cells (
However, it is unclear whether the amount of XRE-BP accumulated in the nuclei relates directly to the transcriptional activity of CYP1A1. The expression of P-450 1A1 protein is shown to be more prominent in centrilobular hepatocytes than periportal hepatocytes in adult rats after MC injection (
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Acknowledgments |
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Supported in part by grants from the Japanese Ministry of Education, Science and Culture (06670041 and 09470005).
Received for publication September 16, 1997; accepted March 2, 1998.
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