Volume 137, Issue 6, Pages 2030-2040.e5 (December 2009) Protection of Epithelial Barrier Function by the Crohn's Disease Associated Gene Protein Tyrosine Phosphatase N2  Michael Scharl, Gisela Paul, Achim Weber, Barbara C. Jung, Michael J. Docherty, Martin Hausmann, Gerhard Rogler, Kim E. Barrett, Declan F. McCole  Gastroenterology  Volume 137, Issue 6, Pages 2030-2040.e5 (December 2009) DOI: 10.1053/j.gastro.2009.07.078 Copyright © 2009 AGA Institute Terms and Conditions

Figure 1 Elevated PTPN2 activity parallels decreased STAT phosphorylation in IFN-γ-treated T84 cells. (A) PTPN2 was immunoprecipitated from whole cell lysates. Blots were probed for PTPN2 to show equivalent protein loading. The graph demonstrates IFN-γ-stimulated PTPN2 activity (n = 4). (B) Representative Western blots show phosphorylated cytoplasmic (Tyr701, above) and total STAT1 (below) after IFN-γ treatment, followed by densitometric analysis (n = 5). (C) Western blots and densitometry showing phosphorylated nuclear (above) and total STAT1 (below) in IFN-γ-treated cells (n = 5). (D) Phosphorylated cytoplasmic (Tyr705) and total STAT3 in IFN-γ-treated T84 cells shown by representative Western blots and densitometry (n = 4). Data in B, C, and D are shown as a percentage of the respective control. Asterisks indicate significant differences compared to the respective control (*P < .05, **P < .01, ***P < .001). #P < .05; ###P < .001 vs 24-hour IFN-γ treatment. Gastroenterology 2009 137, 2030-2040.e5DOI: (10.1053/j.gastro.2009.07.078) Copyright © 2009 AGA Institute Terms and Conditions

Figure 2 IFN-γ increases PTPN2 levels in T84 cells and human colonic biopsy specimens. (A) IFN-γ-stimulated PTPN2 mRNA expression normalized to GAPDH (n = 3, performed in triplicate). (B) Cytoplasmic PTPN2 protein in IFN-γ-treated T84 cells shown by representative Western blots. Lamin A/C was used throughout as a loading control. The relative protein level was assessed by densitometry (n = 4). (C) Nuclear PTPN2 protein in IFN-γ-treated T84 cells is demonstrated by representative Western blots and densitometry (n = 4). Data are expressed as a percentage of the respective control. (D) PTPN2 mRNA in human terminal ileum and colon biopsy specimens from CD patients with active disease (n = 9) and in remission (n = 7) as well as from control subjects (n = 9). PTPN2 mRNA expression was normalized to the housekeeping gene GAPDH. Measurements were performed in triplicate. Asterisks denote significant differences from the respective control (*P < .05, **P < .01, ***P < .001). Gastroenterology 2009 137, 2030-2040.e5DOI: (10.1053/j.gastro.2009.07.078) Copyright © 2009 AGA Institute Terms and Conditions

Figure 3 PTPN2 knockdown enhances IFN-γ-stimulated STAT1 and 3 phosphorylation. T84 cells were transfected with either nonspecific or PTPN2 siRNA and treated with IFN-γ (24 hours). (A) Representative Western blots for PTPN2 and lamin A/C followed by densitometric analysis (n = 3). (B and C) Representative Western blots and densitometry showing STAT1 and 3 phosphorylation and expression (n = 3). Figure parts A–C represent analyses of whole cell lysates. Asterisks indicate significant differences vs the respective control (*P < .05, **P < .01, ***P < .001). ###P < .001 vs 24-hour IFN-γ treatment of control siRNA-transfected cells. (D) Representative Western blots showing PTPN2 and lamin A/C, STAT1 and 3 expression,and phosphorylation in cytoplasmic and nuclear lysates (n = 2). Gastroenterology 2009 137, 2030-2040.e5DOI: (10.1053/j.gastro.2009.07.078) Copyright © 2009 AGA Institute Terms and Conditions

Figure 4 AMPK regulation of IFNγ-induced STAT1 phosphorylation in T84 cells. (A) Western blots and densitometric analysis of cytoplasmic (n = 4) and (B) nuclear PTPN2 (n = 3) after treatment with IFNγ and/or CC (50 μM, bilaterally). (C) Western blot and densitometric analysis of cytoplasmic (n = 4) and (D) nuclear (n = 4) phosphorylated and total STAT1 in response to IFNγ (72h) and/or CC. Black dashes in (c) to (f) indicate that the gel has been cropped at this position. Asterisks indicate significant differences vs. the respective control (*P < .05, **P < .01, ***P < .001). #P < .05 vs 72h IFNγ. Gastroenterology 2009 137, 2030-2040.e5DOI: (10.1053/j.gastro.2009.07.078) Copyright © 2009 AGA Institute Terms and Conditions

Figure 5 Effect of AMPK knockdown on PTPN2 distribution. (A) Confocal microscopy shows PTPN2 (green) in T84 cells. Nuclear staining is blue. Each panel shows a representative image for 1 experiment. Three experiments were performed for each condition. PTPN2 was equally distributed between the nucleus (arrow) and cytoplasm in control cells. Cytoplasmic PTPN2 accumulates (arrow) in response to IFN-γ treatment (72 hours). Compound C had no effect on PTPN2 distribution vs control cells. However, CC prevented the IFN-γ-stimulated nuclear exit of PTPN2 (arrows). (B) Representative Western blots show AMPKα1 levels in 24-hour IFN-γ-treated T84 cells transfected with either control or AMPKα1 siRNA and densitometric analysis (n = 4). (C) PTPN2 and lamin A/C expression (n = 4). (D) Representative Western blots and densitometric analysis of phosphorylated and total STAT1 (n = 4). Asterisks indicate significant differences vs the respective control (**P < .01, ***P < .001). #P < .05 vs 72-hour IFN-γ treatment of control siRNA cells. Gastroenterology 2009 137, 2030-2040.e5DOI: (10.1053/j.gastro.2009.07.078) Copyright © 2009 AGA Institute Terms and Conditions

Figure 6 FITC-dextran flux, TER, and claudin-2 protein in PTPN2-deficient T84 cells. (A and B) Western blots showing decreased PTPN2 but not lamin A/C protein in PTPN2-deficient T84 cells after IFN-γ (72 hours) treatment. (A) FITC-dextran flux (n = 3); (B) TER (n = 3); (C) Western blot and densitometric analysis of claudin-2 expression (n = 3); and (D) expression of the tight junction proteins claudin-4, occludin, and ZO-1 (n = 3); in control or PTPN2 siRNA-transfected cells in response to IFN-γ. Asterisks indicate significant differences vs the respective control (*P < .05, ***P < .001). #P < .05, ###P < .001 vs 72-hour IFN-γ treatment of control siRNA cells. Gastroenterology 2009 137, 2030-2040.e5DOI: (10.1053/j.gastro.2009.07.078) Copyright © 2009 AGA Institute Terms and Conditions

Supplementary Figure 1 Dose-response curve of PTPN2 protein by 24-hour IFN-γ treatment in whole cell lysates. T84 cells were either left untreated or treated with IFN-γ in concentrations of 10, 100, 1000, and 3000 U/mL for 24 hours. Representative Western blots demonstrate the protein levels of PTPN2 and of the loading control, the nuclear envelope protein lamin A/C, in T84 cells. The graph shows the densitometric analysis of 2 samples per condition in arbitrary units. Gastroenterology 2009 137, 2030-2040.e5DOI: (10.1053/j.gastro.2009.07.078) Copyright © 2009 AGA Institute Terms and Conditions

Supplementary Figure 2 mRNA levels of IFN-γ in human biopsy specimens from CD and control patients. Relative IFN-γ mRNA in human terminal ileum and colon biopsy specimens from CD patients with active disease (n = 9) and in remission (n = 7) as well as from control subjects (n = 9). IFN-γ mRNA expression was normalized to the housekeeping gene GAPDH. Measurements were performed in triplicates. Gastroenterology 2009 137, 2030-2040.e5DOI: (10.1053/j.gastro.2009.07.078) Copyright © 2009 AGA Institute Terms and Conditions

Supplementary Figure 3 Protein expression of the nuclear envelope protein lamin A/C in T84 cells. The Western blot shows the relative protein expression of lamin A/C in separately collected whole cell, cytoplasmic, and nuclear lysates of T84 cells. Two samples were analyzed for each cell lysis method. The graph shows the densitometric analysis of the Western blot in arbitrary units. Gastroenterology 2009 137, 2030-2040.e5DOI: (10.1053/j.gastro.2009.07.078) Copyright © 2009 AGA Institute Terms and Conditions

Supplementary Figure 4 PTPN2 knockdown enhances IFN-γ-stimulated STAT1 phosphorylation and potentiates IFN-γ-induced transepithelial permeability in HT29cl.19a IEC. HT29cl.19a cells were transfected with either nonspecific siRNA or PTPN2 siRNA and treated with IFN-γ. (A) Representative Western blots show the protein level of PTPN2 and lamin A/C in whole cell lysates of 24-hour IFN-γ-treated HT29cl.19a cells (n = 4). (B) Representative Western blots showing STAT1 phosphorylation and expression in whole cell lysates of 24-hour IFN-γ-treated HT29cl.19a cells (n = 4). (C) FITC-dextran flux across 72-hour IFN-γ-treated HT29cl.19a monolayers transfected with either control or PTPN2 siRNA (n = 4). Asterisks indicate significant differences vs the respective control (*P < .05, ***P < .001). ##P < .01 vs 72-hour IFN-γ treatment of control siRNA transfected cells. Gastroenterology 2009 137, 2030-2040.e5DOI: (10.1053/j.gastro.2009.07.078) Copyright © 2009 AGA Institute Terms and Conditions

Supplementary Figure 5 Representative histologic H&E stainings of intestinal biopsy specimens derived from control patients, patients with CD in remission, and patients with active CD. (A) Colonic biopsy specimen from a healthy control person and (B) from a patient with Crohn's disease in remission reveal no signs of significant inflammation. (C) In contrast, colonic biopsy specimen from a patient with clinically active Crohn's disease demonstrates mucosal thickening and pronounced immune cell infiltration compared with control (A) and CD in remission biopsy specimens (B). H&E staining; scale bar, 200 μm. Gastroenterology 2009 137, 2030-2040.e5DOI: (10.1053/j.gastro.2009.07.078) Copyright © 2009 AGA Institute Terms and Conditions

Supplementary Figure 6 The AMPK activator AICAR (1 mmol/L) affects neither PTPN2 expression nor STAT1 phosphorylation in T84 cells. (A) Representative Western blots show the phosphorylation of AMPK in response to IFN-γ treatment for 72 hours (n = 3). (B) Expression of PTPN2 and the loading control lamin A/C is shown by blots representative of 3 similar experiments, where T84 cells were treated with IFN-γ, AICAR, or the combination thereof. (C) STAT1 phosphorylation and expression in T84 cells treated with IFN-γ, AICAR, or the combination thereof. The Western blots are representative of 3 similar experiments. Gastroenterology 2009 137, 2030-2040.e5DOI: (10.1053/j.gastro.2009.07.078) Copyright © 2009 AGA Institute Terms and Conditions

Supplementary Figure 7 Effect of IFN-γ treatment on PTPN2 distribution in T84 IEC. Confocal microscopy shows PTPN2 (green) in T84 cells. Nuclear staining is blue. Each panel shows a representative image for 1 experiment. Three experiments were performed with similar results for each condition. PTPN2 is equally distributed between the nucleus (arrow) and cytoplasm in control cells. A similar result was obtained in 24-hour IFN-γ-treated cells (arrow), indicating that IFN-γ does not affect PTPN2 localization by treatment for 24 hours. However, in response to 72-hour IFN-γ treatment (shown previously in Figure 5), PTPN2 appears to be less prominent in the nucleus and shows a more intense accumulation (arrow) in the cytoplasm. Gastroenterology 2009 137, 2030-2040.e5DOI: (10.1053/j.gastro.2009.07.078) Copyright © 2009 AGA Institute Terms and Conditions

Supplementary Figure 8 The claudin-2 promoter region contains a putative STAT binding motif. Claudin-2 promoter region is shown represented as described by Sakaguchi et al.39 To identify a putative STAT1/STAT3 binding sequence in the claudin-2 promoter region,39,40 we screened the claudin-2 promoter region39 for such a motif. All STATs recognize the same palindromic sequence—TTCNNNGAA (where N is a variable spacer). By blast search, we identified such a palindromic motif 261 to 253 bp upstream of the transcriptional start point in the claudin-2 promoter. The identified sequence, 5′-TTCCCGGAA-3′, matches the criteria for a possible STAT1 binding motif40 and matches exactly with the STAT3 binding motif in the gene encoding human p21WAF.40 Putative binding motifs are underlined. Gastroenterology 2009 137, 2030-2040.e5DOI: (10.1053/j.gastro.2009.07.078) Copyright © 2009 AGA Institute Terms and Conditions