Use of cell-based screening to identify small-molecule compounds that modulate claudin-4 expression
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Claudins constitute a family of at least 27 proteins with four transmembrane domains, and play a pivotal role in maintaining tight-junctions seals in diverse epithelial tissues. The expression of claudin-4 often changes in intestinal tissues of inflammatory bowel disease and various human cancers. Therefore, claudin-4 is a promising target for treatment of these diseases. In our previous study, we established a reporter cell line to monitor claudin-4 expression on the basis of a functional claudin-4 promoter. Using this cell line, we have performed a cell-based screen of a library containing 2642 biologically active small-molecule compounds to identify modulators of claudin-4 expression. The screen identified 24 potential modulators of the claudin-4 promoter activity. Fourteen of these compounds (12 of them novel) induced endogenous claudin-4 expression. The identified compounds might serve as lead compounds targeting aberrant gene expression in inflammatory bowel disease.
KeywordsClaudin Inflammatory bowel disorder Reporter assay Screening Small-molecule compounds Tight junction
This work was supported in part by a grant from the Ministry of Health, Labor, and Welfare, Japan, and by the Platform for Drug Discovery, Informatics, and Structural Life Science from the Ministry of Education, Culture, Sports, Science, and Technology, Japan.
Supplementary Figure 1—Screen for small-molecule compounds modulating luciferase activity. Claudin-4 reporter cells were seeded in 96-well plates, treated with 10 μM TSA, apicidin, scriptaid, or SAHA for 24 h, and luciferase activity in cell lysates was measured. Relative luciferase activity is the ratio of luciferase activity in the compound-treated cells to that in the DMSO-treated cells.
Supplementary Figure 2—Dose-dependent effects of the claudin-4 modulator candidates on luciferase activity. Claudin-4 reporter cells were treated with daunorubicin, halcinonide, chetomin, fluvastatin, strophanthidin, trichostatin A (TSA), SAHA, scriptaid, sapintoxin D, nabumetone, or thiabendazole at the indicated concentrations for 24 h. Relative luciferase activity is the ratio of luciferase activity in the compound-treated cells to that in the DMSO-treated cells. The data are means ± S.D. (n = 3).
Supplementary Figure 3—Effects of claudin-4 modulator candidates on cell viability. Claudin-4 reporter cells were seeded in 96-well plates and treated with T-2 toxin, HT-2 toxin, homoharringtonine, 17-AAG, or geldanamycin (10 μM each). After 24 h, the LDH release was determined. Tween20 (0.2%) was used as a positive control. The data are means ± S.D. (n = 3).
Supplementary Figure 4—Effects of claudin-4 modulator candidates (T-2 toxin, HT-2 toxin, Ochratoxin, Homoharringtonine, 17-AAG, and Geldanamycin) on claudin-4 mRNA expression. Claudin-4 reporter cells were treated with T-2 toxin (10 M), HT-2 toxin (10 M), Ochratoxin (10 M), Homoharringtonine (10 M), 17-AAG (10 M), or Geldanamycin (10 M) for 24 h, and claudin-4 mRNA expression was analyzed by qPCR. Claudin-4 mRNA level was normalized to the GAPDH level. The data are means ± S.D. (n = 3).
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