Volume 118, Issue 6, Pages 1001-1011 (June 2000) Claudins regulate the intestinal barrier in response to immune mediators  Tetsushi Kinugasa, Takanori Sakaguchi, Xuibin Gu, Hans–Christian Reinecker  Gastroenterology  Volume 118, Issue 6, Pages 1001-1011 (June 2000) DOI: 10.1016/S0016-5085(00)70351-9 Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 1 IL-17 enhances the development of the tight junctional barrier of T84 cell monolayers without regulating cell proliferation. (A) Development of TER of T84 cells stimulated with media alone or addition of 100 ng/mL IL-17, assessed as described in Materials and Methods. Data was obtained from triplicate samples. (B) Mannitol flux through T84 cell monolayers with or without stimulation by IL-17. [3H]Mannitol was added apically to each transwell. (C) Proliferation of T84 cells without or in the presence of IL-17 for the indicated time period, assessed in triplicate as described in Materials and Methods. One representative experiment of 3 with similar results for each panel.*P < 0.01 compared with unstimulated cells; Student t test. Gastroenterology 2000 118, 1001-1011DOI: (10.1016/S0016-5085(00)70351-9) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 2 MEK activity is required for basal- and cytokine-induced tight junction assembly in intestinal epithelial cells. (A) Development of TER in T84 cell monolayers in the presence of IL-17 alone, IL-17 and PD98059, PD98095 alone, and without stimulants. TER was determined in triplicates. (B) Proliferation of T84 cells was assessed without or in the presence of IL-17 and/or PD98059 for the indicated time period. One representative experiment of 3 with similar results in both A and B. *P < 0.01 compared with unstimulated cells; Student t test. Gastroenterology 2000 118, 1001-1011DOI: (10.1016/S0016-5085(00)70351-9) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 3 Functional MEK is required for claudin-2 but not claudin-1 protein expression in intestinal epithelial cells. (A) Claudin protein expression was determined by Western blotting after incubation of T84 cells with or without IL-17, the MEK inhibitor PD98059, or the PI3-kinase inhibitor wortmannin in combination or alone for 3 days. The same immunoblot was hybridized with claudin-1 antibodies, stripped, and rehybridized with antibodies specific for claudin-2, as described in Materials and Methods. (B) Regulation of claudin protein expression correlates with MEK-dependent activation of ERK MAPKs. T84 cells were pretreated with PD98059 or wortmannin for 1 hour and stimulated with IL-17 for 15 minutes. Activation and expression of ERK-1 and ERK-2 were assessed as described in Materials and Methods. One experiment of 3 with similar results. (C) Transient ERK-1 and ERK-2 activation in T84 cells induced by IL-17. T84 cells were incubated for the indicated time periods with IL-17, and cell lysates were resolved on 4%-20% SDS-polyacrylamide gel. Activated and total ERK-1 and ERK-2 expressions were determined by immunoblotting as described in Materials and Methods. (D) Immunoblot analysis of IL-17–induced claudin-1 and claudin-2 protein expression in membrane fraction isolated from T84 cells. (E) Densitometric analysis of claudin-1 and claudin-2 protein expression shown in D. One experiment of 3 with similar results. Gastroenterology 2000 118, 1001-1011DOI: (10.1016/S0016-5085(00)70351-9) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 4 MEK mediates basal and IL-17–induced claudin expression in intestinal epithelial cells. (A) Northern blot analysis of claudin mRNA expression in T84 cells stimulated with IL-17 (lanes 1-3), IL-17 and PD98059 (lanes 4-6), or PD98059 (lanes 7-9). The same Northern blot was subsequently hybridized with probes for claudin-1, claudin-2, and GAPDH, as described in Materials and Methods. (B) Claudin mRNA expression was densitometrically analyzed after normalization for GAPDH expression and expressed as mean density per area for claudin-1 (□) and claudin-2 (■). One experiment of 3 with similar results. Gastroenterology 2000 118, 1001-1011DOI: (10.1016/S0016-5085(00)70351-9) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 5 Expression and membrane association of claudin-1 and claudin-2 are differentially regulated by inhibition of MEK activity in intestinal epithelial cells. T84 cells were grown on cover slips and immunofluorescence staining was performed as described in Materials and Methods for the detection of (A-D) claudin-1 or (E-H) claudin-2. Claudin expression was determined in the absence (A and E) or presence of either 100 ng/mL IL-17 (B and F) or 5 μmol/L PD98059 alone (C and G) or in combination (D and H). One representative immunostaining of 3 performed for each group (original magnification 100×). Gastroenterology 2000 118, 1001-1011DOI: (10.1016/S0016-5085(00)70351-9) Copyright © 2000 American Gastroenterological Association Terms and Conditions