Journal of Histochemistry and Cytochemistry, Vol. 51, 973-976, July 2003, Copyright © 2003, The Histochemical Society, Inc.


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Localization of Thioredoxin-interacting Protein (TXNIP) mRNA in Epithelium of Human Gastrointestinal Tract

Yasuo Takahashia, Yukimoto Ishiib, Akiko Murataa, Toshihito Nagataa, and Satoshi Asaia
a Division of Genetic and Genomic Medicine, Medical Research Center, Nihon University School of Medicine, Tokyo, Japan
b 3rd Department of Surgery, Nihon University School of Medicine, Tokyo, Japan

Correspondence to: Yasuo Takahashi, Div. of Genetic and Genomic Medicine, Medical Research Center, Nihon U. School of Medicine, 30-1 Oyaguchi Kamimachi, Itabashi-ku, Tokyo 173-8610, Japan. E-mail: yasuot@med.nihon-u.ac.jp


  Summary
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Summary
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Literature Cited

Thioredoxin-interacting protein (TXNIP) is a negative regulator of thioredoxin. However, its role in the gastrointestinal (GI) epithelium is as yet unknown. Using in situ hybridization, we demonstrated that mRNA of TXNIP was differentially expressed in the epithelium of the human GI tract. TXNIP transcript was especially prominent in terminal differentiated cells. TXNIP was also highly expressed in lymphocytes in the lymphoid follicles. Our results suggest a new potential role of TXNIP in the differentiation of epithelial cells and in mucosal immunity of the GI tract. (J Histochem Cytochem 51:973–976, 2003)

Key Words: gastrointestinal tract, in situ hybridization, mucosal immunity, VDUP1, TBP-2


  Introduction
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Summary
Introduction
Literature Cited

Thioredoxin-interacting protein (TXNIP), also known as VDUP1 (vitamin D3 upregulated protein 1) or TBP-2 (thioredoxin-binding protein-2), is a negative regulator of thioredoxin (TRX) (Nishiyama et al. 1999 ), an intracellular thiol reductant, and an important regulator of redox balance (Yodoi et al. 2002 ). Recent studies have gradually clarified the involvement of TXNIP in biologically important cellular events such as differentiation and apoptosis. The TXNIP gene was originally reported to be upregulated in HL60 cells by vitamin D3 treatment (Chen and DeLuca 1994 ), which can induce differentiation in some hematopoietic cells. Moreover, TXNIP is induced by a variety of stresses, including H2O2, heat shock, UV, {gamma}-rays, and anti-cancer agents (Junn et al. 2000 ; Takahashi et al. 2002 ), which are well known to act as a common mediator of apoptosis in many types of cells. Finally, TXNIP functions as an oxidative stress mediator, inducing apoptosis by inhibiting TRX activity via the interaction between TRX and proliferation-associated gene (PAG) or between TRX and apoptosis signal-regulating kinase 1 (ASK-1) (Junn et al. 2000 ). Another possible role of TXNIP in an anti-tumor effect has been suggested in rat mammary tumors and human gastrointestinal (GI) cancers (Yang et al. 1998 ; Ikarashi et al. 2002 ). However, the nature of TXNIP in the GI epithelium is as yet unknown. The GI epithelium maintains homeostasis by a balance among proliferation, differentiation, and apoptosis, all of which are affected by the cell's position in the epithelium (Augenlicht et al. 1999 ; Fenoglio-Preiser et al. 1999 ; Clatworthy and Subramanian 2001 ; van den Brink et al. 2001 ). Therefore, it is of interest to know whether TXNIP expression is influenced by the cell's position in the epithelium, e.g., along the crypt-to-surface axis or the gland-to-pit axis.

To examine the localization of TXNIP mRNA expression along the human GI tract, we used in situ hybridization (ISH) analysis. Digoxigenin (DIG)-labeled riboprobes (sense and antisense) for TXNIP (424 bp, nucleotides 2046–2470 of the TXNIP mRNA sequence; GenBank XM_002093) were transcribed from PCR-generated DNA templates. A consensus T7 RNA polymerase-binding sequence was incorporated in the primer sets. The antisense TXNIP probe was transcribed from a DNA template generated with forward primer (5'-CAATGGAGAGAGCTTTCCCTG-3') and reverse primer (5'-GGCCAGTGAATTGTAA-TACGACTCACTATAGGGAGGCGGCAGCAGCAA-CCCTTTCACA-3'). For the sense TXNIP probe, forward primer (5'-GGCCAGTGAATTGTAATACGA-CTCACTATAGGGAGGCGGCAATGGAGAGAGCT-TTCCCTG-3') and reverse primer (5'-CAGCAGCA-ACCCTTTCACA-3') were used. The authenticity of each PCR product was confirmed by electrophoresis and by direct sequencing. After purification of the PCR products, in vitro transcription was carried out using a DIG RNA labeling kit (Roche Diagnostics; Basel, Switzerland).

Tissue samples (three from stomach and three from large bowel) for ISH analysis were obtained from six patients (age 52–67 years) who had undergone surgical resection of different areas of the GI tract. Normal tissue adjacent to the resected pathological tissue was also analyzed in all cases, with verification by histopathological assessment. Tissue samples were washed with PBS and immediately embedded in OCT compound in hexane and dry ice, and stored at -80C until sectioning.

Ten-µm-thick sections were cut (-15C; HM560M, Microm, Walldorf, Germany) and mounted on silanized slides (Matsunami; Osaka, Japan). All sections were fixed in 4% paraformaldehyde–PBS for 15 min, incubated twice in PBS containing 0.1% active DEPC for 15 min, and equilibrated in 5 x SSC for 15 min. Hybridization was performed in hybridization buffer (50% formamide, 5 x SSC, 5 x Denhardt's, 500 µg/ml salmon sperm DNA, 250 µg/ml tRNA, 1 mM DTT) containing antisense or sense riboprobes at 500 ng/ml in a humid chamber for 20 hr at 58C. After hybridization the sections were washed in 2 x SCC for 30 min at room temperature (RT), then in 2 x SCC and 0.1 x SCC for 1 hr at 65C, and finally incubated in TBST buffer (125 mM Tris, 150 mM NaCl, 2 mM KCl, 0.1% Tween-20, pH 8.0) for 15 min at RT. For digoxigenin detection, the sections were blocked with DAKO Protein Block Serum-Free (DAKO; Kyoto, Japan) for 20 min and then incubated with alkaline phosphatase-conjugated anti-digoxgenin antibody (Roche Diagnostics) diluted 1:500 in blocking buffer (Roche Diagnostics) for 1 hr at RT. After washes in TBST with 2 mM levamisole, the signal was visualized by overnight incubation with BM purple substrate (Roche Diagnostics). Finally, the sections were counterstained with nuclear fast red and mounted in NEW MX (Matsunami).

In this study, ISH analysis demonstrated that TXNIP mRNA was differentially expressed in the epithelium of colon and stomach. TXNIP transcript was prominent in the upper portion of the colon crypts, especially at the flat surface (Fig 1A and Fig 1G). On the contrary, TXNIP transcript was prominent in the lower portion of the gastric glands and was also present in the superficial epithelial cells of the gastric pits and the flat surface (Fig 1B and Fig 1C). These discrepancies may come from differences in the cells' function and the mechanism of cell differentiation between colon and gastric epithelium. The differentiated epithelial cells in the colon are replaced every few days by progenitor stem cells that proliferate in the base of crypts. They move up towards the luminal surface as they differentiate, and then undergo apoptosis after reaching the top of the colonic crypt (Augenlicht et al. 1999 ; Clatworthy and Subramanian 2001 ). On the other hand, in the gastric epithelium, progenitor stem cells proliferate in the mucous neck region to replace all of the specialized cells lining the gastric glands, gastric pits, and luminal surface. From this area the foveolar cells migrate upward and are exfoliated at the luminal surface. Other cell lineages migrate downward and differentiate into parietal, chief, and endocrine cells (Fenoglio-Preiser et al. 1999 ). These mature glandular cells are thought to die by a process of apoptosis at the fundus of the gastric glands (Fenoglio-Preiser et al. 1999 ). Although these cell types, which were positive for TXNIP in the present study, varied along the GI tract, they were all terminal differentiated cells leading to apoptosis. These results suggest that TXNIP may play a role in epithelial differentiation in the GI tract.



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Figure 1. Localization of TXNIP mRNA in colonic (A,D,G–I) and gastric epithelium (B,C,E,F). Antisense hybridization (A–C,G–I); sense hybridization (D–F). TXNIP transcript is abundant at the flat surface of the colon crypts (A,G). TXNIP mRNA signal is observed in the lower portion of the gastric glands (B) and in the superficial epithelial cells at the flat surface (C). Sense probe hybridization produced no signal over background (D–F). TXNIP-positive cells are present in a lymphoid follicle (H) and in the lamina propria (I). Bars: A,B,D,E,H,I = 10 µm; C,F,G = 30 µm.

It is also of interest to know whether TXNIP is expressed in lymphocytes in the GI epithelium, because TXNIP was reported to be upregulated in human myeloid leukemic HL60 cells in response to vitamin D3, suggesting its possible role in differentiation of some hematopoietic cells (Chen and DeLuca 1994 ). The present study demonstrated that lymphocytes in lymphoid follicles expressed TXNIP abundantly (Fig 1H). Interestingly, TXNIP transcript was predominant in the parafollicular region of the lymphoid follicles, which is rich in T-cells, but was not present in the central germinal center, containing primarily B-cells and macrophages. Colon follicles, as well as Peyer's patches in the small intestine, are the major inductive sites of mucosal immunity in the GI tract, where there are two important immune responses. One is B-cell differentiation into plasma cells to produce antigen-specific immunoglobulins. The other is T-cell activation and differentiation into functionally distinct subsets, Th1 and Th2, which can secrete various cytokines (Neurath et al. 2002 ). These mature lymphocytes then migrate to effector sites, such as the lamina propria (Fenoglio-Preiser et al. 1999 ). In this study, sparse cells expressing the TXNIP transcript were present in the lamina propria of the colon and gastric epithelium (Fig 1I). These cells included not only lymphocytes but also large cells that were inconsistent with mature lymphocytes, suggesting the involvement of other cell types in the lamina propria in the synthesis of TXNIP. Our observations, together with previous reports, suggest that TXNIP plays a role in mucosal immunity in the GI tract via differentiation of some hematopoietic cells.

Redox regulation is deeply involved in biologically important phenomena such as differentiation and apoptosis (Yodoi et al. 2002 ). Because TRX is one of the major components of the thiol-reducing system and is important in regulating the redox balance (Yodoi et al. 2002 ), the TRX–TXNIP interaction may also be important as a redox regulatory mechanism in cellular processes. Our study, which demonstrated that TXNIP transcript was abundant in differentiated epithelial cells and in lymphocytes in the lymphoid follicles, suggests that redox regulation may be involved in epithelial differentiation and in mucosal immunity of the GI tract via the TRX–TXNIP interaction. Although the number of specimens was small, our study provides new insights into the molecular mechanisms underlying homeostasis of the GI epithelium.


  Acknowledgments

Supported in part by a Nihon University Research Grant for 2002 and by a Grant-in Aid from the Japanese Ministry of Education, Culture, Sports, Science and Technology to promote advanced scientific research, awarded to Nihon University.

Received for publication October 21, 2002; accepted February 5, 2003.


  Literature Cited
Top
Summary
Introduction
Literature Cited

Augenlicht L, Velcich A, Mariadason J, Bordonaro M, Heerdt B (1999) Colonic cell proliferation, differentiation, and apoptosis. Adv Exp Med Biol 470:15-22[Medline]

Chen KS, DeLuca HF (1994) Isolation and characterization of a novel cDNA from HL-60 cells treated with 1,25-dihydroxyvitamin D-3. Biochim Biophys Acta 1219:26-32[Medline]

Clatworthy JP, Subramanian V (2001) Stem cells and the regulation of proliferation, differentiation and patterning in the intestinal epithelium: emerging insights from gene expression patterns, transgenic and gene ablation studies. Mech Dev 101:3-9[Medline]

Fenoglio–Preiser CM, Noffsinger AE, Stemmermann GN, Lantz PE, Listrom MB, Rilke FO (1999) Gastrointestinal Pathology: an Atlas and Text. Philadelphia, Lippincott–Raven Publishers

Ikarashi M, Takahashi Y, Nagata T, Ishii Y, Ishikawa K, Asai S (2002) Vitamin D3 up-regulated protein 1 (VDUP1) expression in gastrointestinal cancer and its relation to stage of disease. Anticancer Res 22:4045-4048[Medline]

Junn E, Han SH, Im JY, Yang Y, Cho EW, Um HD, Kim DK et al. (2000) Vitamin D3 up-regulated protein 1 mediates oxidative stress via suppressing the thioredoxin function. J Immunol 164:6287-6295[Abstract/Free Full Text]

Neurath MF, Finotto SG, Limcher LH (2002) The role of Th1/Th2 polarization in mucosal immunity. Nature Med 8:567-573[Medline]

Nishiyama A, Matsui M, Iwata S, Hirota K, Masutani H, Nakamura H, Takagi Y et al. (1999) Identification of thioredoxin-binding protein-2/vitamin D(3) up-regulated protein 1 as a negative regulator of thioredoxin function and expression. J Biol Chem 274:21645-21650[Abstract/Free Full Text]

Takahashi Y, Nagata T, Ishii Y, Ikarashi M, Ishikawa K, Asai S (2002) Up-regulation of vitamin D3 up-regulated protein 1 gene in response to 5-fluorouracil in colon carcinoma SW620. Oncol Rep 9:75-79[Medline]

van den Brink GR, de Santa Barbara P, Roberts DJ (2001) Development, epithelial cell differentiation—a matter of choice. Science 294:2115-2116[Free Full Text]

Yang X, Young LH, Voigt JM (1998) Expression of a vitamin D-regulated gene (VDUP-1) in untreated- and MNU-treated rat mammary tissue. Breast Cancer Res Treat 48:33-44[Medline]

Yodoi J, Nakamura H, Masutani H (2002) Redox regulation of stress signals: possible roles of dendritic stellate TRX producer cells (DST cell types). Biol Chem 383:585-590[Medline]