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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 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.
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pone-0026352-g006: 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.

Mentions: The control of tight junction dynamics that maintain IEC barrier function is not entirely understood, but MLCK plays a key role in the reduction of TJ integrity in response to TNF in IECs [45]. To assess whether the drop in TER associated with TNFAIP3 deficiency involved dysregulation of the MLCK signaling pathway, we treated cells with membrane-permeant inhibitor of myosin light chain kinase (PIK), a highly specific inhibitor of MLCK activity in IEC [46]. PIK did not directly inhibit TNFAIP3′s deubiquitinating activity and did not significantly change the TER of untreated cells (Figures S5, S6). As before, TNF induced a rapid decrease in TER in scrambled shRNA-expressing cells, and this decrease was more pronounced in TNFAIP3 shRNA-expressing cells. Inhibition of MLCK by PIK completely prevented the rapid decrease in TER in both scrambled and TNFAIP3 deficient cells (Figure 6A). This suggests that TNFAIP3 regulates barrier function by acting at some point in the TNF/MLCK/TJ pathway. Phosphorylation of myosin light chain (MLC) is a standard indicator of MLCK activity [10]. We therefore assessed phospho-MLC levels in TNF-treated cells to determine whether TNFAIP3 inhibits TNF-induced MLCK activation. Cells constitutively expressing TNFAIP3 did not display altered phosphorylation of MLC in response to TNF treatment (Figure 6B). These data suggest that TNFAIP3 regulates MLCK-mediated barrier function through a mechanism that does not involve inhibition of MLCK activity itself.


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 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.
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pone-0026352-g006: 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.
Mentions: The control of tight junction dynamics that maintain IEC barrier function is not entirely understood, but MLCK plays a key role in the reduction of TJ integrity in response to TNF in IECs [45]. To assess whether the drop in TER associated with TNFAIP3 deficiency involved dysregulation of the MLCK signaling pathway, we treated cells with membrane-permeant inhibitor of myosin light chain kinase (PIK), a highly specific inhibitor of MLCK activity in IEC [46]. PIK did not directly inhibit TNFAIP3′s deubiquitinating activity and did not significantly change the TER of untreated cells (Figures S5, S6). As before, TNF induced a rapid decrease in TER in scrambled shRNA-expressing cells, and this decrease was more pronounced in TNFAIP3 shRNA-expressing cells. Inhibition of MLCK by PIK completely prevented the rapid decrease in TER in both scrambled and TNFAIP3 deficient cells (Figure 6A). This suggests that TNFAIP3 regulates barrier function by acting at some point in the TNF/MLCK/TJ pathway. Phosphorylation of myosin light chain (MLC) is a standard indicator of MLCK activity [10]. We therefore assessed phospho-MLC levels in TNF-treated cells to determine whether TNFAIP3 inhibits TNF-induced MLCK activation. Cells constitutively expressing TNFAIP3 did not display altered phosphorylation of MLC in response to TNF treatment (Figure 6B). These data suggest that TNFAIP3 regulates MLCK-mediated barrier function through a mechanism that does not involve inhibition of MLCK activity itself.

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