ToxSci Advance Access originally published online on May 28, 2007
Toxicological Sciences 2007 99(1):43-50; doi:10.1093/toxsci/kfm138
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The Putative Tumor Suppressor Tsc-22 is Downregulated Early in Chemically Induced Hepatocarcinogenesis and may be a Suppressor of Gadd45b
* Laboratory of Molecular Carcinogenesis
Toxicology Operations Branch, Environmental Toxicology Program, National Institute of Environmental Health Sciences, National Institute of Health, Research Triangle Park, North Carolina 27709
1 To whom requests for reprints should be addressed at Department of Pathology, Biosafety Research Center, Foods, Drugs and Pesticides, 582-2 Shioshinden, Iwata-shi, Shizuoka 437-1213, Japan. Fax: +81-538-58-1343. E-mail: kotorogzo{at}yahoo.co.jp.
Received February 25, 2007; accepted May 16, 2007
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Tsc-22 is a novel tumor suppressor gene that represents a new class of transcription factors that has transcriptional repressor activity. We found Tsc-22 downregulation in livers from B6C3F1 mice following treatment for 2 weeks with carcinogenic doses of the antianxiety drug oxazepam (2500 ppm) or the peroxisome proliferator Wyeth-14,643 (500 ppm) but not with two other carcinogens such as o-nitrotoluene or methyleugenol or three noncarcinogens including p-nitrotoluene, eugenol, or acetaminophen. The expression of Tsc-22 was also repressed in B6C3F1 mouse liver tumors that were induced by several chemicals from 2-year carcinogenicity studies as well as in spontaneous liver tumors. To identify potential Tsc-22 target genes in mouse liver, we transfected small interference RNA (SiRNA) designed to inhibit Tsc-22 into murine liver BNL-CL.2 cells. We selected two potential transcriptional targets of Tsc-22, growth arrest and DNA damage–inducible gene 45 ß (Gadd45b) and leucine zipper, putative tumor suppressor 2 (Lzts2) to test based on our previous complementary DNA microarray studies, showing that expression of these cancer-associated genes was increased when Tsc-22 was repressed. SiRNA treatment of BNL-CL.2 cells with Tsc-22 oligonucleotides but not nonspecific oligonucleotides decreased RNA and protein expression of Tsc-22 by 80–90%, while expression of Gadd45b gene, but not Lzts2, was increased over time after an initial decrease. Treatment of these cells with oxazepam for 48 h also resulted in decreased Tsc-22 and increased Gadd45b expression. These data provide evidence that Tsc-22 is a suppressor of Gadd45b expression, which may contribute to an early antiapoptotic response.
Key Words: Non-genotoxic; tumor suppressor gene; SiRNA.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To investigate early mechanisms of mouse liver carcinogenesis following exposure to nongenotoxic carcinogens, we used complementary DNA (cDNA) microarray analysis previously to identify 25 or 36 genes with altered expression in livers from B6C3F1 mice treated with oxazepam for 2 weeks or 6 months, respectively (Iida et al., 2003
). One interesting gene that was downregulated two to threefold at both time points was transforming growth factor-ß (TGF-ß)–stimulated clone 22 (Tsc-22). Tsc-22 was first isolated as a TGF-ß–inducible gene in a mouse osteoblast cell line (Shibanuma et al., 1992) and is an alternative spliced transcript of the TGF-ß1–inducible transcript 4 gene (Tgfb1i4, Gene acc. NM_009366
[GenBank]
). Tsc-22 is the only member of a novel family of transcription factors that contains a transcriptional repressor domain and does not have a DNA-binding domain. The mouse Tsc-22 gene product demonstrates strong evolutionary conservation with 98.5% and 100% identity compared to the human and rat proteins, respectively (Hamil and Hall, 1994; Jay et al., 1996). Kester et al. identified a TSC-22 binding protein, THG-1 (also called TILZ2), and they demonstrated that TSC-22 can homodimerize and heterodimerize with THG-1; they also showed transcriptional repressor activity by the Tsc-22–THG-1 complex (Kester et al., 1999).
Human TSC-22 was downregulated in salivary gland tumors as compared with normal salivary gland tissue and upregulated by the anticancer drug vesnarinone in a salivary gland carcinoma cell line (Kawamata et al., 1998; Nakashiro et al., 1998). After the salivary gland cancer cell line TYS received apoptotic stimuli, TSC-22 translocated from the cytoplasm to the nucleus and showed transcription-regulatory activity (Hino et al., 2000). In T47D mammary carcinoma cells TSC-22 was induced by progestin, an anticancer agent (Kester et al., 1997). Because TSC-22 was induced by anticancer agents, it was suggested to have tumor suppressor activity.
Given that Tsc-22 is a putative tumor suppressor and transcriptional repressor, we hypothesized that genes expressing an inverse relationship to Tsc-22 may be targets of Tsc-22 action. From analysis of our previous cDNA microarray results, several genes were upregulated when Tsc-22 was suppressed in mice treated with oxazepam and Wyeth-14,643 for 2 weeks and 6 months, respectively, suggesting that these genes might be targets of Tsc-22. In the present study, expression of two of these genes, growth arrest and DNA damage–inducible 45ß (Gadd45b, genbank no. NM_008655
[GenBank]
), and leucine zipper, putative tumor suppressor 2 (Lzts2, genbank no. NM_145503
[GenBank]
) was measured by real-time and quantitative (RTAQ)-PCR to see if they were upregulated in liver after treatment for 2 weeks by carcinogens other than oxazepam or Wyeth-14,643 or by noncarcinogens in a pattern opposite to Tsc-22 expression. Gadd45b has been associated with both cell growth control and cellular response to DNA damage, demonstrating different cellular responses to different ligands. Gadd45b appears to prevent apoptotic cell death in response to tumor necrosis factor-
(TNF-) (De Smaele et al., 2001), whereas it triggers apoptosis in response to TGF-ß (Yoo et al., 2003). Lzts2, also called Lapser1, is a putative liver tumor suppressor gene that also may play a role in regulation of cell growth (Cabeza-Arvelaiz et al., 2001).
To test the hypothesis that Tsc-22 plays a role in carcinogenesis, we first examined Tsc-22 expression and protein accumulation in liver tissues following treatment with various known carcinogens and noncarcinogens for 2 weeks, in liver tumors induced by different carcinogens, and in murine liver cell lines after treatment with TGF-ß1 or oxazepam. To identify potential Tsc-22 target genes in mouse liver, we utilized small interference RNA (SiRNA) technology to inhibit Tsc-22 expression selectively in murine embryonic liver cell line, BNL-CL.2 and examined the expression of two genes, Gadd45b and Lzts2, identified in microarray experiments as being upregulated when Tsc-22 was repressed.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Animals and experimental design.
Six-week-old male or female B6C3F1 mice were obtained for the study as described previously (Iida et al., 2003
). The mice were exposed for 2 weeks to different compounds, including the following treatment groups: oxazepam, male and female mice, 125 ppm (noncarcinogenic) and 2500 ppm (carcinogenic); o-nitrotoluene, female mice, 1250 ppm (noncarcinogenic) and 5000 ppm (carcinogenic), and male mice, 5000 ppm (100% hemangiosarcomas, but no hepatocellular tumors); p-nitrotoluene, female mice, 5000 ppm (noncarcinogenic); methyleugenol, female mice, 75 mg/kg (carcinogenic); eugenol, female mice, 75 mg/kg (noncarcinogenic); acetaminophen, female mice, 6000 ppm (noncarcinogenic). Beginning on a Wednesday, all animals were dosed in the feed except for the methyleugenol and eugenol groups, which were dosed by gavage daily at 9–10 A.M. Monday to Friday. All mice were killed between 9 and 10 A.M. 2 weeks after the start of the treatment. Age-matched untreated male and female mice were used as controls. All mice received the highest standard of humane care in accordance with protocols approved by the National Institute of Environmental Health Sciences (NIEHS) animal care and use committee and the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals. Frozen liver neoplasms were obtained from the National Toxicology Program (NTP) Archives at NIEHS from previous NTP 2-year carcinogenicity studies to compare changes in gene expression with livers from earlier time points after treatments.
Cell culture.
The murine embryonic hepatocellular carcinoma (HCC) cell line, BNL-1ME A.7R.1 (tumorgenic) and the embryonic murine liver cell line, BNL-CL.2 (nontumorigenic) cells were obtained from the American Type Culture Collection (Rockville, MD) and maintained in Dulbecco's modified Eagle medium (DMEM) supplemented with 1% L-glutamine, antibiotics (1% penicillin G), and 2% heat-inactivated fetal bovine serum (Life Technologies/BRL, Gaithersburg, MD) in a humidified atmosphere of 95% air and 5% CO2 at 37°C. Cells (5 x 105/ml) were seeded in 5 ml of medium in a 60-mm2 sterile tissue culture dishes overnight to achieve 80% confluency. Cells were incubated for 48 h in the absence or presence of oxazepam (Roussel Corp, Englewood Cliffs, NJ) or for 96 h in the absence or presence of TGF-ß1 (Sigma, St Louis, MI) in order to maintain optimal cell growth condition. Cells were harvested after incubation, and RNA and protein were isolated.
RNA extraction and cDNA synthesis.
Total liver RNA from tissues and cells was extracted using TRIzol (Life Technologies, Gaithersburg, MD) according to the manufacturer's recommendations or prepared using an RNeasy Mini kit (Qiagen, Inc., Valencia, CA). Intact 28S and 18S ribosomal RNAs from the extracted total RNA from liver tissues were visualized on a formaldehyde denaturing 1.2% agarose gel. One microgram of total RNA from each sample was subjected to cDNA synthesis using MuLV Reverse Transcriptase (Applied Biosystems, Foster City, CA) according to manufacturer's protocol.
Northern blot analysis and RTAQ-PCR.
For Northern blots, 5 µg of total RNA was loaded on a 1% formaldehyde gel and transferred to a Nylon membrane (LI-COR, Lincoln, NE) overnight. cDNA probes were labeled with [-32P] deoxy-cytidine triphosphate (Amersham, Piscataway, NJ) using a Prime-it RmT Random Primer Labeling Kit (Stratagene, La Jolla, CA). The membrane was hybridized with labeled cDNA probes in ULTRAhyb -OS Buffer (Ambion, Austin, TX) at 42°C overnight and developed. The probe used was a 201-bp fragment containing parts of exon 4 of mouse Tsc-22 cDNA and a 270-bp fragment of mouse ß-actin as a normalization control. RTAQ-PCR was carried out as described previously (Iida et al., 2003). The primers used for Northern blot analysis and RTAQ-PCR are listed in Table 1. ß-Actin expression was quantified to normalize the amount of cDNA in each sample.
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Production of Tsc-22 antibodies.
Polyclonal rabbit anti-mouse antibodies were raised against two Tsc-22 specific peptides, each synthesized as multiple antigen peptides (MAP) conjugated peptides by Invitrogen. The MAP peptides were 8- to 18-kDa molecules consisting of a central core of seven lysine residues with eight identical peptide chains (12–20 residues in length) extending outward from the core with individual peptide subunits attached to the central core via the C-terminal carboxyl groups. The sequences of the peptides, VAMDLGVYQLRHFSI and LSSLLGTENASVRLD, corresponded to the N-terminal region of the predicted amino acid sequence of the Tsc-22 protein. This region was chosen based on its unique sequence identity of the predicted sequence of the TSC-22 transcript and lack of predicted cross-reactivity with the predicted amino acid sequence from the highly related Tilz1b transcript of the Tgfb1i4 gene. Initial priming with 1 mg of peptide in complete Freunds adjuvant were followed by four times monthly boosts with 1 mg of peptide in incomplete Freunds adjuvant. Rabbit serum was prepared from blood drawn 14 days after injection.
Western blot analysis using polymer immunocomplexes.
Cellular proteins were extracted from frozen tissues in radioimmunoprecipitation assay buffer as described previously (Iida et al., 2003). Total protein samples (30–50 µg) were denatured by boiling in Laemmli sample buffer, resolved on sodium dodecyl sulfate–polyacrylamide gel electrophoresis gels (15% Tris–HCl gels, Bio-Rad, Hercules, CA) and transferred to PVDF membranes (Millipore, Bedford, MA). For the Tsc-22 antibody we used the Envision system for Western blot analysis (Fukuda et al., 2000). In brief, 100 ng of affinity-purified anti-Tsc-22 antibody was diluted in 500 µl of PBST-nonfat milk (phosphate-buffered saline with 0.1% Tween 20 and 1% nonfat milk) and mixed with 250 µl of Envision+/HRP, Rabbit consisting of dextran polymer (Dako, Carpinteria, CA) for 1 h at room temperature with gentle shaking. Then, 250 µl of normal rabbit serum was added and mixed with antibody solutions for 1 hr at room temperature with gentle shaking. After adjusting the volume to 5 ml with PBST-nonfat milk, it was applied to blocked membrane with 1% PBST-nonfat milk and incubated for 1 hr at room temperature with gentle shaking. Then the membrane was washed with PBST-nonfat milk at overnight and developed with an ECL kit (Amersham Biosciences Corp.). The NIH Image program was used to measure the mean band densities on the western blots. We also tested an affinity-purified anti-human TSC-22 antibody, which was kindly provided by Dr. Hitoshi Kawamata (Dokkyo university school of medicine, Tochigi, Japan) for confirmation, and similar results were obtained using multiple samples.
SiRNA experiments.
Small interfering RNA duplex oligonucleotides for Tsc-22 (#1: 5'-GCAGATCAAAGAACTAATA-3', #2: 5'-CAATAGCTCTGGTGCAAGT-3', #3: 5'- GGCGATGGATCTAGGAGTT-3', #4: 5'-GCTCAGGATCAACCGCATA-3') were purchased from Dharmacon, Lafayette, CO. Cells were transfected with TransIT-TKO Transfection Reagent (Mirus Corporation, Madison, WI) according to the manufacturer's protocol in the presence of SiRNA. SiRNA against a nonspecific control duplex (cat#D-001206-09-20, Dharmacon) was used as a control. The four specific duplexes were tested separately and in a pool.
SiRNA transfection.
Cultured BNL-CL.2 cells were transfected with SiRNA. In brief, these cells were grown at 37°C in DMEM supplemented with 10% fetal calf serum, 1% penicillin, and 1% L-glutamine. Cells were passaged regularly to maintain exponential growth. The day before transfection, cells were trypsinized, diluted with fresh medium without antibiotics, and transferred to 12-well plates (2.5 x 105 per well). Transient transfection of SiRNAs (100nM final concentration) was performed using TransIT-TKO Transfection Reagent (Mirus Corporation) following the manufacturer's protocol.
Statistical analyses.
The significance of differences in Tsc-22 expression resulting from each chemical treatment was examined by one-way analysis of variance (ANOVA). When ANOVA tests were significant then Dunnett's multiple comparisons were performed between the expression of Tsc-22 and Gadd45b or Lzts2 for specific chemical treatments. Student's t-tests were used to compare the expression each gene (relative to ß-actin) in treated versus untreated liver tissues. The significance level of ANOVA tests was 0.05, Dunnett's tests and Student's t-tests were 0.05 and 0.01 (two-tailed). Statistical analysis was performed using SAS 9.1.3 (SAS Institute, Cary, NC).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In a previous microarray study we examined early expression changes in liver after treatment for 2 weeks or 6 months with carcinogenic doses of oxazepam or Wyeth-14,643 (Iida et al., 2003
). We detected downregulation of a cDNA clone identified as the Tgfb1i4 gene (unpublished data). This clone was found to be a chimera, and its sequence could not be confirmed. However, by utilizing primers to different parts of the Tgfb1i4 gene that encode Q-rich and repressor domains (Fig. 1), we found by RTAQ-PCR that the Tsc-22 transcript was downregulated in these livers (Table 2). In contrast, an alternative transcript, designated Tilz1b was only expressed at a low level in control livers and was not changed in the livers from treated mice (data not shown). Only the Tsc-22 transcript contains the repressor domain, while it lacks the Q-rich domain that contains the DNA-binding region. However, the Tilz1b and Tsc-22 proteins share the leucine zipper domain. We made a probe to the leucine zipper region, and only the 1.8-kb Tsc-22, but not the 5.0-kb Tilz1b transcript, was detected by Northern analysis in mouse livers (Fig. 2a).
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To investigate the role of Tsc-22 in early mouse liver carcinogenesis, we first examined by RTAQ-PCR its expression in livers and in liver tumors following treatment with several known carcinogens and noncarcinogens. Confirming the microarray data, Tsc-22 was downregulated in liver tissues from mice treated with carcinogenic doses of oxazepam (2500 ppm) or Wyeth-14,643 for 2 weeks and 6 months (Table 2). Suppression of Tsc-22 protein in liver following treatment of mice for 2 weeks or 6 months with oxazepam or Wyeth-14,643 was also detected by western blot analysis (example in Fig. 2b). However, Tsc-22 expression by RTAQ-PCR was not changed by treatment for 2 weeks with a noncarcinogenic dose of oxazepam (125 ppm) or with other weakly genotoxic carcinogens such as o-nitrotoluene or methyleugenol or with noncarcinogens including p-nitrotoluene or acetaminophen (Table 2). In contrast, almost all liver tumors examined that developed after treatment with oxazepam, TCDD, methylene chloride, and anthraquinone, and those from untreated mice, exhibited strongly decreased expression of the Tsc-22 gene compared to control liver tissues (Table 3). These data on liver tumors and on liver tissue after 2 weeks of treatment indicate that Tsc-22 downregulation may play a role in all mouse liver carcinogenesis and be an early event in the carcinogenic process following treatment with certain chemicals such as oxazepam or Wyeth-14,643.
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To examine if downregulation of Tsc-22 was a direct effect of oxazepam in hepatocyte-derived cells, we utilized the embryonic murine liver cell line BNL-CL.2 nontumorigenic cells and the murine embryonic hepatocarcinoma cell line BNL-1ME A.7R.1 tumorigenic line. We first showed that Tsc-22 was induced in both cell lines when treated with TGF-ß (Fig. 2a, lanes 11 and 12, and data not shown). We then treated liver BNL-CL.2 cells with 5 or 50 µg of oxazepam to assess its effect on gene expression (Fig. 3). At the higher concentration Tsc-22 expression by RTAQ-PCR was decreased about sevenfold at 48 h relative to the control level, supporting our findings of suppression of Tsc-22 by oxazepam in vivo.
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Since Tsc-22 has transcriptional repressor activity, target genes of Tsc-22 would be induced when Tsc-22 is repressed, such as after oxazepam treatment. Based on cDNA microarray analysis (Iida et al., 2003
), we identified four potential cancer-related genes with increased expression by oxazepam and Wyeth-14,643 at 2 weeks and 6 months when Tsc-22 expression was suppressed. These genes were Gadd45b, Lzts2, Maff, and Igfbp1. Then, expression of these genes in livers after 2 weeks of treatment with other chemicals was measured by RTAQ-PCR to see if there was an inverse pattern compared to Tsc-22 expression (data not shown). Expression of two of those genes, Gadd45b and Lzts2, was altered in a pattern opposite to Tsc-22 expression (Table 2), and thus we considered them to be potential Tsc-22 transcriptional target genes. We also assessed Gadd45b expression in the embryonic murine liver cell line BNL-CL.2, and we found that Gadd45b expression was increased about sixfold after the initial decrease at 1 h (from 0.3- to 1.8-fold in expression over time using actin expression as a control) (Fig. 3). To further investigate whether Gadd45b and Lzts2 were Tsc-22 transcriptional targets in mouse liver, we used the SiRNA technology to knockdown Tsc-22 transcript expression in BNL-CL.2 cells. We tested four individual SiRNA duplexes and a pool of all four duplexes designed to knockdown Tsc-22 specifically. Twenty-four, 48, 72, and 96 h after transfection with specific and nonspecific SiRNA oligos, RNA and protein lysates were harvested and analyzed by RTAQ-PCR and Western blot analysis. We found that Tsc-22 protein was decreased at 24–72 h (Fig. 4a), and RNA expression was suppressed by Tsc-22 duplex No. 2 80–90% at all time points (Fig. 4b). In fact, all of the specific SiRNA oligos elicited downregulation of Tsc-22 (data not shown), while a nonspecific oligo duplex had little to no effect on Tsc-22 protein expression level (Fig. 4a). In addition, the SiRNA oligos did not cause nonspecific downregulation of expression, as demonstrated by the Lamin A/C control (Fig. 4a). We also assessed Gadd45b and Lzts2 RNA gene expression in the Tsc-22 knockdown BNL-CL.2 cells. Gadd45b expression was decreased at 24 h but then significantly increased about fourfold over time to 96 h suggesting that Tsc-22 repression contributed to the activation of Gadd45b by Dunnett's tests (Fig. 4b). In contrast, expression of Lzts2 was not changed in Tsc-22 knockdown cells, indicating that this gene is not likely a target of Tsc-22 transcriptional repression.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Several investigations have reported that human TSC-22 was decreased in certain cancers and may act as a tumor suppressor gene. For example, it has been reported that TSC-22 expression was downregulated in benign and malignant human salivary gland tumors (Nakashiro et al., 1998
). In addition, TSC-22 expression was decreased in astrocytomas of different malignancy grades and in other types of human brain tumors (Shostak et al., 2003). These results suggest that TSC-22 downregulation plays a role in the development of cancer in diverse tissues.
In this study we demonstrated that Tsc-22 was downregulated two to threefold in mouse liver tumors induced by several chemicals and in spontaneous liver tumors. Tsc-22 expression was also repressed as early as 2 weeks in livers from mice following treatment with certain carcinogens, such as oxazepam or Wyeth-14,643, but not with other carcinogens, o-nitrotoluene or methyleugenol, or with several noncarcinogens. In a study of Sprague–Dawley rats treated with genotoxic or nongenotoxic carcinogens for 5 days, reduction in Tsc-22 expression correlated strongly with carcinogenicity in liver (Kramer et al., 2004). Recently, Michel et al. (2005) reported that TSC-22 has the potential to be an acute early molecular marker for nongenotoxic hepatocarcinogenesis in rodents. Thus, our results as well as those of other investigators suggest a role for early downregulation of Tsc-22 in liver and hepatocarcinogenesis in rodents after treatment with several nongenotoxic carcinogens.
The present study also provides evidence that Gadd45b is a potential target of Tsc-22 transcriptional repression. The basis for the hypothesis was the finding of Gadd45b expression in liver in an opposite pattern to Tsc-22 expression following several different chemical treatments in vivo (Iida et al., 2003, 2005). The findings of increases in Gadd45b expression after oxazepam treatment in vitro and after specific Tsc-22 knockdown by SiRNA oligonucleotides supported the hypothesis. Because Tsc-22 does not have a classical DNA-binding domain, we did not expect to find any Tsc-22 elements in the promoter region of Gadd45b. It has been suggested that Tsc-22 may act by dimerizing through its leucine zipper domain with different partners in repression or activation complexes (Kester et al., 1999). Thus, the loss of Tsc-22 expression in liver, such as after oxazepam treatment, would allow another transcription factor to activate target genes, such as Gadd45b.
One known binding partner for TSC-22 is THG-1, and this heterodimer has been shown to have transcriptional repressor activity (Kester et al., 1999). In our previous microarray experiment (Iida et al., 2005) and in RTAQ-PCR analyses, we found no change in Thg-1 expression levels after in vivo treatment with any of the carcinogens or noncarcinogens. Based on expression pattern analysis following the microarray experiment, one candidate for a transcriptional activation factor that may interact directly or indirectly with Tsc-22 is TEA domain family member 2 (Tead2) that was found to have an opposite expression pattern to Tsc-22 in liver following treatment of mice with the different carcinogens and noncarcinogens (data not shown). In a National Center for Biotechnology Information BLAST search to find sequences producing significant alignments between Tead2 and Tsc-22 or Thg-1 proteins, a region of Thg-1 from aa321 to aa387 was identified with an expected alignment score with Tead2 of 0.002. Significant alignment was not found for Tead2 and Tsc-22. Further study is needed to identify the Tsc-22 transcriptional complex, but the use of microarray pattern analysis may help identify potential members.
It is unclear why Gadd45b expression decreased at 24 and 48 h after Tsc-22 SiRNA treatment. However, this was consistent with an immediate decrease in Gadd45b expression after oxazepam treatment. The increase after that initial decrease was sixfold from 1 to 48 h after oxazepam treatment and about fourfold from 24 to 96 h after SiRNA treatment. It is likely that expression of Gadd45b is controlled by a complex with different transcriptional elements, one of which may be Tsc-22. The BNL.CL.2 cells may provide a good model for further study of the relationship between Tsc-22 and Gadd45b, the functions of these genes, and the nature of the Tsc-22 transcriptional complex.
In a previous study to examine early expression changes in mouse liver carcinogenesis, we hypothesized that the shift in balance between cell death and survival is likely to play an important initial role (Iida et al., 2003). Inhibition of apoptosis is an important mechanism of tumor induction by nongenotoxic hepatocarcinogens (Christensen et al., 1998; Roberts, 1996; Schwarz et al., 1995). Increased expression of Tsc-22 has been associated with increases in apoptosis (Ohta et al., 1997). Gadd45b has been reported to play both a pro- and an antiapoptotic role in carcinogenesis (De Smaele et al., 2001; Yoo et al., 2003). Induction of Gadd45b may induce apoptosis after treatment with TGF-ß or block apoptosis after treatment with TNF- (De Smaele et al., 2001). A recent study demonstrated that Gadd45b directly blocks TNF-–mediated phosphorylation of MKK7 and JNK kinase–associated apoptosis (Papa et al., 2004). Preliminary findings in our laboratory found no change in frequency of apoptotic hepatocytes following treatment of mice with oxazepam for 2 weeks compared to 3% in liver cells from untreated mice (unpublished data). Moreover, in our previous study we found that TNF-–induced protein 2 (Tnfaip2) was also induced in liver after 2 weeks treatment with oxazepam (Iida et al., 2003), suggesting that oxazepam may cause a similar antiapoptotic effect as TNF-. Thus, in our present study the repression of Tsc-22 and strong activation of Gadd45b in liver after treatment with oxazepam suggests a critical role in blocking apoptosis and enhancing cell proliferation early in the carcinogenic process.
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NOTES |
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2 Present address: Department of Pathology, Biosafety Research Center, Foods, Drugs and Pesticides, Shizuoka 437-1213, Japan.
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ACKNOWLEDGMENTS |
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We thank Masaya Suzuki for his excellent statistical analysis. We thank Dr Hitoshi Kawamata for kindly providing anti-human TSC-22 antibody for comparison with our antibody and Drs Robert Sills, Jeanelle Martinez and Eiji Maki for their helpful comments about the manuscript. This research was supported by the Intramural Research Program of the NIEHS, NIH.
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