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First published online 26 March 2003
doi: 10.1242/jcs.00389

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Regulation of tight junctions during the epithelium-mesenchyme transition: direct repression of the gene expression of claudins/occludin by Snail

Junichi Ikenouchi^*, Miho Matsuda^*, Mikio Furuse and Shoichiro Tsukita

Department of Cell Biology, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan

View larger version (82K): [in a new window]   Fig. 1. Establishment of Eph4 and CSG1 stable transfectants expressing mouse Snail. Mouse Snail cDNA was isolated and introduced into mouse epithelial cell lines Eph4 and CSG1. Phase contrast images revealed the in vitro Snail-induced EMT in Eph4 and CSG1 cells (A). Both Eph4-Mock and CSG1-Mock cells exhibited a typical cobblestone-like appearance. By contrast, when Snail was overexpressed, these cells acquired a more fibroblastic phenotype (Eph4- and CSG1-mSnail). Northern blotting confirmed the absence and presence of Snail mRNA in parental and transfectants, respectively (B). As a control, the mRNA levels of GAPDH are shown. Bars, 60 µm.

View larger version (82K): [in a new window]   Fig. 2. Behavior of AJ and TJ constituents in Snail-overexpressed epithelial cells. (A) Immunoblotting of Eph4 cells, two independent Eph4-mSnail clones and NIH/3T3 cells with antibodies specific for AJ and TJ components. In Eph4-mSnail cells, E-cadherin, claudin-3 and occludin became undetectable at the protein level. By contrast, the expression levels of p120 and ZO-1 (E-cadherin- and claudin/occludin-binding undercoat protein, respectively) did not appear to be altered significantly by the Snail overexpression, although p120 showed significant mobility shifts for unknown reasons. The expression pattern of AJ and TJ components in Eph4-mSnail cells is very similar to that in NIH/3T3 fibroblasts. Cytokeratin-18 (CK18) is an epithelial marker. (B) Immunofluorescence microscopy of Eph4 and Eph4-mSnail cells with antibodies specific for AJ and TJ components. In Eph4-mSnail cells, E-cadherin as well as claudin-3/occludin became undetectable not only in the cell-cell contact regions but also in the cytoplasm. By contrast, Snail appeared to translocate ZO-1 and p120 from the junctional regions to the cytoplasm. Bars, 30 µm.

View larger version (53K): [in a new window]   Fig. 3. The mRNA levels of AJ and TJ constituents in epithelial cells overexpressing Snail. (A) Northern blotting of Eph4, Eph4-mSnail and NIH/3T3 cells. Snail completely shut down the transcription of E-cadherin, claudin genes and occludin, but not of ZO-1 or cytokeratin-18. As a control, the GAPDH gene was detected in equal amounts in all samples. (B) Comparison of the mRNA levels of E-cadherin, claudin-4, claudin-7, occludin, Snail and ZO-1 between mouse epithelial cells (Eph4, MTD-1A and CSG1) and fibroblasts (NIH/3T3 and L).

View larger version (13K): [in a new window]   Fig. 4. Schematic representation of the promoter region of human E-cadherin, mouse claudin-3, claudin-4 and claudin-7, and human occludin. The putative transcription start point for each claudin promoter was estimated according to the expressed sequence tag database. Open box, E-box; +1, putative transcription start point; ORF, open reading frame. For the occludin gene, two possible transcription start points have been suggested (Mankertz et al., 2000).

View larger version (22K): [in a new window]   Fig. 5. Snail-induced repression of the promoter activities of claudin genes and occludin. Luciferase reporter constructs carrying mouse claudin-3, claudin-4 or claudin-7 promoter were transfected into Eph4 epithelial cells or NIH/3T3 fibroblasts singly or together with the Snail expression vector. In Eph4 cells, the claudin promoters induced a three- to tenfold increase in relative luciferase activity above that observed in NIH/3T3 cells, indicating that the claudin promoters are activated in an epithelium-specific manner (A). When mouse Snail was coexpressed in Eph4 cells, the activities of claudin promoters were remarkably repressed (B), and the repression of claudin-7 promoter was depended on the dose of Snail (C). The Snail mutant lacking its N-terminal SNAG domain showed no repressor activity for the claudin-7 promoter (D). Similarly, luciferase reporter construct carrying human occludin promoter was transfected into human HT29 epithelial cells (or Eph4 cells) singly or together with the Snail expression vector. The human occludin promoter was repressed by Snail in both HT29 cells (E) and Eph4 cells (data not shown). All results correspond to the average of three independent experiments.

View larger version (29K): [in a new window]   Fig. 6. Impairment of Snail-induced repression of the claudin-7 promoter by mutations of the E-boxes. (A) A short fragment of the mouse claudin-7 promoter region (–110 to +190; Fig. 4). This short fragment includes five E-boxes (E1-E5) and showed the epithelium-specific promoter activity (data not shown). Double-stranded oligonucleotides corresponding to the E4-containing sequence (underlined sequence) were used in the electrophoretic mobility shift/oligonucleotide precipitation assays in Fig. 7. (B,C) Mutational analyses. The core sequence, 5'-CA(G/C)(G/C)TG-3', of E-boxes (E1E5) was mutated to 5'-AA(G/C)(G/C)TA-3' in various combinations (shadowed boxes). Luciferase reporter constructs carrying wild-type or these mutated claudin-7 promoters were transfected into Eph4 cells together with a mouse Snail expression vector (mSnail) or an empty vector (pCAG). (B) Luciferase activity found in cells co-transfected with a wild-type reporter construct and pCAG empty vector was defined as 1.0. (C) The same set of data expressed as the Snail-induced repression ratio (+mSnail/+pCAG in B) for individual reporter constructs. As the number of mutated E-boxes increased, the claudin-7 promoter became less sensitive to Snail. For no known reason, even when all five E-boxes were mutagenized, the Snail-induced repression ratio did not reach 1.0. The arrowhead shows the putative transcription start point. All results correspond to the average of three independent experiments.

View larger version (56K): [in a new window]   Fig. 7. Direct binding of Snail to the E-box in claudin-7 and occludin promoters. (A) Electrophoretic mobility shift assay for the interaction of Snail with an E-box in the claudin-7 promoter. 32P-labeled double-stranded oligonucleotides corresponding to the sequence containing one of the E-boxes of the claudin-7 promoter (E4 in Fig. 6) formed a DNA-protein complex with the in vitro translated/HA-tagged mouse Snail (HA-mSnail) (arrowhead in lane 2), but not with the in vitro translated luciferase (control; lane 1). This complex formation was not observed when mutated oligonucleotides were used (lane 3). This complex formation was affected by an increased amount of unlabeled wild-type oligonucleotides (arrowhead in lanes 4-6), but not by that of unlabeled mutated oligonucleotides (arrowhead in lanes 7-9). The DNA-protein complex band shifted upwards when incubated with anti-HA pAb (arrow in lane 11) but not when incubated with anti-GFP pAb (control, lane 10). (B) Electrophoretic mobility shift assay for the interaction of Snail with an E-box in the occludin promoter. Results were very similar to those shown in A. (C) Biotinylated oligonucleotide precipitation assay. The nuclear extract was prepared from 293 cells transfected with an HA-Snail expression vector (HA-Snail) or an empty vector (con). This extract was incubated with biotin-labeled double-stranded wild-type (wt) or mutated (mut) oligonucleotides corresponding to the sequence containing the E-box of the claudin-7 promoter (E4 in Fig. 6). The oligonucleotides were then recovered using streptavidin-conjugated agarose beads; bound HA-Snail was detected by immunoblotting with anti-HA mAb. HA-Snail bound specifically to the wild type, not mutated, oligonucleotides (lanes 1-3). A similar specific binding was detected when the oligonucleotides of the E-box sequence in the occludin promoter were used (lanes 4-6).