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TNFAIP3 maintains intestinal barrier function and supports epithelial cell tight junctions.

Kolodziej LE, Lodolce JP, Chang JE, Schneider JR, Grimm WA, Bartulis SJ, Zhu X, Messer JS, Murphy SF, Reddy N, Turner JR, Boone DL - PLoS ONE (2011)

Bottom Line: In cultured human intestinal epithelial cell lines, TNFAIP3 expression regulated both TNF-induced and myosin light chain kinase-regulated tight junction dynamics but did not affect myosin light chain kinase activity.We also found that TNFAIP3 deubiquitinates polyubiquitinated occludin.These in vivo and in vitro studies support the role of TNFAIP3 in promoting intestinal epithelial barrier integrity and demonstrate its novel ability to maintain intestinal homeostasis through tight junction protein regulation.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of Chicago, Chicago, Illinois, United States of America.

ABSTRACT
Tight junctions between intestinal epithelial cells mediate the permeability of the intestinal barrier, and loss of intestinal barrier function mediated by TNF signaling is associated with the inflammatory pathophysiology observed in Crohn's disease and celiac disease. Thus, factors that modulate intestinal epithelial cell response to TNF may be critical for the maintenance of barrier function. TNF alpha-induced protein 3 (TNFAIP3) is a cytosolic protein that acts in a negative feedback loop to regulate cell signaling induced by Toll-like receptor ligands and TNF, suggesting that TNFAIP3 may play a role in regulating the intestinal barrier. To investigate the specific role of TNFAIP3 in intestinal barrier function we assessed barrier permeability in TNFAIP3(-/-) mice and LPS-treated villin-TNFAIP3 transgenic mice. TNFAIP3(-/-) mice had greater intestinal permeability compared to wild-type littermates, while villin-TNFAIP3 transgenic mice were protected from increases in permeability seen within LPS-treated wild-type littermates, indicating that barrier permeability is controlled by TNFAIP3. In cultured human intestinal epithelial cell lines, TNFAIP3 expression regulated both TNF-induced and myosin light chain kinase-regulated tight junction dynamics but did not affect myosin light chain kinase activity. Immunohistochemistry of mouse intestine revealed that TNFAIP3 expression inhibits LPS-induced loss of the tight junction protein occludin from the apical border of the intestinal epithelium. We also found that TNFAIP3 deubiquitinates polyubiquitinated occludin. These in vivo and in vitro studies support the role of TNFAIP3 in promoting intestinal epithelial barrier integrity and demonstrate its novel ability to maintain intestinal homeostasis through tight junction protein regulation.

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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.
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pone-0026352-g007: 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.

Mentions: The ability of TNFAIP3 to regulate TJ dynamics, but not MLCK activity, suggests that TNFAIP3 might act on the TJ itself. The enzymatic activity of TNFAIP3 may directly or indirectly alter the ubiquitination of TJ proteins, such as occludin. Ubiquitination of occludin mediates its endocytosis, thus increasing permeability of the tight junction [13]. To determine whether TNFAIP3 associates with occludin in cells, we immunoprecipitated endogenous TNFAIP3 and tested for the presence of associated occludin. TNFAIP3 and occludin were associated in unstimulated cells and this association was diminished 10 minutes after stimulation with TNF. Thus, TNFAIP3 and occludin associate in cells in a manner that is regulated by TNF stimulation (Figure 7A). To determine whether TNFAIP3 can deubiquitinate occludin, we incubated recombinant N-terminal TNFAIP3 with ubiquitinated occludin in vitro. We found that TNFAIP3 was able to decrease the total ubiquitination of occludin (Figure 7B). In addition we transfected epitope-tagged forms of TNFAIP3, occludin, and K48R-ubiquitin into HEK 293T cells. The mutation in ubiquitin from a lysine to arginine at position 48 prevents degradative polyubiquitin chains from forming on substrates; thus, the Myc-epitope tag will mark only mono, K63-linked and other non-K48-linked polyubiquitin chains. The introduction of an increasing amount of TNFAIP3 into cells resulted in decreased ubiquitination of co-transfected occludin (Figure 7B). These results indicate that TNFAIP3 can alter the ubiquitination of occludin, and are consistent with the retention of occludin at the apical membrane in the intestines of LPS-treated villin-TNFAIP3 mice.


TNFAIP3 maintains intestinal barrier function and supports epithelial cell tight junctions.

Kolodziej LE, Lodolce JP, Chang JE, Schneider JR, Grimm WA, Bartulis SJ, Zhu X, Messer JS, Murphy SF, Reddy N, Turner JR, Boone DL - PLoS ONE (2011)

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.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3198775&req=5

pone-0026352-g007: 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.
Mentions: The ability of TNFAIP3 to regulate TJ dynamics, but not MLCK activity, suggests that TNFAIP3 might act on the TJ itself. The enzymatic activity of TNFAIP3 may directly or indirectly alter the ubiquitination of TJ proteins, such as occludin. Ubiquitination of occludin mediates its endocytosis, thus increasing permeability of the tight junction [13]. To determine whether TNFAIP3 associates with occludin in cells, we immunoprecipitated endogenous TNFAIP3 and tested for the presence of associated occludin. TNFAIP3 and occludin were associated in unstimulated cells and this association was diminished 10 minutes after stimulation with TNF. Thus, TNFAIP3 and occludin associate in cells in a manner that is regulated by TNF stimulation (Figure 7A). To determine whether TNFAIP3 can deubiquitinate occludin, we incubated recombinant N-terminal TNFAIP3 with ubiquitinated occludin in vitro. We found that TNFAIP3 was able to decrease the total ubiquitination of occludin (Figure 7B). In addition we transfected epitope-tagged forms of TNFAIP3, occludin, and K48R-ubiquitin into HEK 293T cells. The mutation in ubiquitin from a lysine to arginine at position 48 prevents degradative polyubiquitin chains from forming on substrates; thus, the Myc-epitope tag will mark only mono, K63-linked and other non-K48-linked polyubiquitin chains. The introduction of an increasing amount of TNFAIP3 into cells resulted in decreased ubiquitination of co-transfected occludin (Figure 7B). These results indicate that TNFAIP3 can alter the ubiquitination of occludin, and are consistent with the retention of occludin at the apical membrane in the intestines of LPS-treated villin-TNFAIP3 mice.

Bottom Line: In cultured human intestinal epithelial cell lines, TNFAIP3 expression regulated both TNF-induced and myosin light chain kinase-regulated tight junction dynamics but did not affect myosin light chain kinase activity.We also found that TNFAIP3 deubiquitinates polyubiquitinated occludin.These in vivo and in vitro studies support the role of TNFAIP3 in promoting intestinal epithelial barrier integrity and demonstrate its novel ability to maintain intestinal homeostasis through tight junction protein regulation.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of Chicago, Chicago, Illinois, United States of America.

ABSTRACT
Tight junctions between intestinal epithelial cells mediate the permeability of the intestinal barrier, and loss of intestinal barrier function mediated by TNF signaling is associated with the inflammatory pathophysiology observed in Crohn's disease and celiac disease. Thus, factors that modulate intestinal epithelial cell response to TNF may be critical for the maintenance of barrier function. TNF alpha-induced protein 3 (TNFAIP3) is a cytosolic protein that acts in a negative feedback loop to regulate cell signaling induced by Toll-like receptor ligands and TNF, suggesting that TNFAIP3 may play a role in regulating the intestinal barrier. To investigate the specific role of TNFAIP3 in intestinal barrier function we assessed barrier permeability in TNFAIP3(-/-) mice and LPS-treated villin-TNFAIP3 transgenic mice. TNFAIP3(-/-) mice had greater intestinal permeability compared to wild-type littermates, while villin-TNFAIP3 transgenic mice were protected from increases in permeability seen within LPS-treated wild-type littermates, indicating that barrier permeability is controlled by TNFAIP3. In cultured human intestinal epithelial cell lines, TNFAIP3 expression regulated both TNF-induced and myosin light chain kinase-regulated tight junction dynamics but did not affect myosin light chain kinase activity. Immunohistochemistry of mouse intestine revealed that TNFAIP3 expression inhibits LPS-induced loss of the tight junction protein occludin from the apical border of the intestinal epithelium. We also found that TNFAIP3 deubiquitinates polyubiquitinated occludin. These in vivo and in vitro studies support the role of TNFAIP3 in promoting intestinal epithelial barrier integrity and demonstrate its novel ability to maintain intestinal homeostasis through tight junction protein regulation.

Show MeSH
Related in: MedlinePlus