Figure 1.
TNFAIP3 supports occludin localization at intestinal tight junctions.
Immunohistochemistry for occludin (green), actin (red) and DNA (blue) is shown in intestines of untreated wild type (WT) and TNFAIP3−/− mice. A lack of typical perijunctional occludin localization can be observed at the apical surface of epithelial cells in the TNFAIP3 deficient mice.
Figure 2.
TNFAIP3 is required to maintain intestinal barrier function.
Explanted intestinal loops from untreated wild type (WT; triangles) and TNFAIP3 deficient mice (TNFAIP3−/−; circles) were examined for loss of barrier function. The flux of FITC-dextran over time out of the loops was measured in relative fluorescent units (RFU) per cm of intestinal tissue. Increased FITC-dextran flux indicates decreased barrier function. (***p<0.001)
Figure 3.
Intestinal epithelial cell-specific expression of TNFAIP3 protects against LPS-induced loss of barrier function.
Explanted intestinal loops from WT (triangles) or villin-TNFAIP3 transgenic mice (squares) were assayed for barrier function beginning 45 minutes after injection of LPS (0.1 mg/mouse, ∼5 mg/kg i.p.; filled symbols) or in untreated animals (open symbols). The flux of FITC-dextran over time out of the loops was measured in relative fluorescent units (RFU) per cm of intestinal tissue. Increased FITC-dextran flux indicates decreased barrier function. (*p<0.05, ***p<0.001; WT untreated vs. WT LPS treated).
Figure 4.
Intestinal epithelial cell-specific expression of TNFAIP3 protects against LPS-induced disruption of tight junctions.
Immunohistochemistry showing occludin (green), actin (red) and DNA (blue) localization in the intestinal epithelium of untreated (upper two panels) or LPS-treated (lower four panels; 0.1 mg/mouse, ∼5 mg/kg i.p.) mice is displayed. (Upper four panels 40X objective lens; lower two panels 100X objective lens).
Figure 5.
TNFAIP3 regulates TNF-induced tight junction dynamics in intestinal epithelial cells.
Transepithelial electrical resistance (TER) was measured serially in cells grown in semi-permeable supports untreated (open symbols) or treated with TNF (10 ng/ml; closed symbols) for the indicated times. Values represent percentage of initial resistance measurements (% Init Ω). Pools of cells were stably transduced with (A) GFP alone (GFP control; triangles) or GFP and TNFAIP3 (TNFAIP3 +++; squares); or (B) scrambled shRNA (control shRNA; triangles) or TNFAIP3 shRNA (circles). (**p<0.01, ***p<0.001; TNF-treated GFP controls vs. untreated GFP controls (A) or TNF-treated control shRNA/TNF-treated TNFAIP3 shRNA vs. their respective untreated lines (B)).
Figure 6.
TNFAIP3 regulates an MLCK-dependent signal, but does not control MLCK activity.
(A) TER measurements of stably transduced pools of cells expressing scrambled shRNA (control shRNA; triangles) or TNFAIP3 shRNA (circles) were treated with TNF alone (10 ng/ml; open symbols) or TNF plus the cell permeant MLCK inhibitor PIK (200 µM; closed symbols). Values represent percentage of initial resistance measurements (% Init Ω). (***p<0.001; TNF vs. TNF+PIK treated) (B) Immunoblot of phospho-MLC levels using whole cell lysates from GFP transduced (WT), or GFP together with TNFAIP3 transduced (TNFAIP3 +++) cells treated with TNF (10 ng/ml) for the indicated times minutes.
Figure 7.
TNFAIP3 associates with and deubiquitinates occludin.
(A) Immunoblot of occludin in TNFAIP3 immunoprecipitates from HCT116 cells with and without TNF stimulation. (B) Immunoblot for ubiquitin (upper panel) or occludin (center panel) from an in vitro DUB assay with non-K48-linked polyubiquitinated occludin and increasing amounts of recombinant N-terminal (DUB domain only, residues 1–371) TNFAIP3 (0, 5, 10, 20 µM) or isopeptidase T as a positive control (0.5 µM). Ubiquitinated occludin immunoprecipitated from transfected HEK 293T cells was mixed with enzyme for 24 hours and the reaction products were resolved by SDS-PAGE and analyzed for ubiquitination. The lower panel shows an immunoblot from the reaction supernatant (Sup) indicating the relative amount of TNFAIP3 present at the end of the assay. (C) Immunoblot from an in vivo DUB assay showing the ubiquitination of occludin in cells co-transfected with plasmids that express K48R-ubiquitin (0.5 µg), occludin (3 µg), and increasing amounts of full-length TNFAIP3 (0, 1, 5, 10 µg). The upper two panels are immunoblots of lysates immunoprecipitated with antibody against the V5-epitope tag (IP: V5-occludin), and the lower two panels are immunoblots of whole cell lysates (Pre-IP). In panels B and C, the inset numbers in white are the densitometry of the ubiquitin in each lane.