BRIEF REPORT |
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 |
---|
![]() ![]() ![]() ![]() |
---|
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 51:973976, 2003)
Key Words: gastrointestinal tract, in situ hybridization, mucosal immunity, VDUP1, TBP-2
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() |
---|
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) (-rays, and anti-cancer agents (
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 20462470 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 5267 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% paraformaldehydePBS 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 (
|
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 (
Redox regulation is deeply involved in biologically important phenomena such as differentiation and apoptosis (
![]() |
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 |
---|
![]() ![]() ![]() ![]() |
---|
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]
FenoglioPreiser CM, Noffsinger AE, Stemmermann GN, Lantz PE, Listrom MB, Rilke FO (1999) Gastrointestinal Pathology: an Atlas and Text. Philadelphia, LippincottRaven 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
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
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 differentiationa matter of choice. Science 294:2115-2116
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]