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Enterocyte STAT5 promotes mucosal wound healing via suppression of myosin light chain kinase-mediated loss of barrier function and inflammation.

Gilbert S, Zhang R, Denson L, Moriggl R, Steinbrecher K, Shroyer N, Lin J, Han X - EMBO Mol Med (2012)

Bottom Line: Consistently, knockdown of stat5 in IEC monolayers led to increased NF-κB DNA binding to MLCK promoter, myosin light chain phosphorylation and tight junction (TJ) permeability, which were potentiated by administration of tumour necrosis factor-α (TNF-α), and prevented by concurrent NF-κB knockdown.Collectively, enterocyte STAT5 signalling protects against TJ barrier dysfunction and promotes intestinal mucosal wound healing via an interaction with NF-κB to suppress MLCK.Targeting IEC STAT5 signalling may be a novel therapeutic approach for treating intestinal barrier dysfunction in IBD.

View Article: PubMed Central - PubMed

Affiliation: Division of Gastroenterology, Hepatology and Nutrition, Cincinnati Children's Hospital Medical Center (CCHMC), Cincinnati, OH, USA.

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Knockdown of RelA/p65 in IEC monolayers rescues hyperpermeability induced by interfering with stat5 expressionHT-29 IEC monolayers, grown on Transwell filters, were interfered with RelA/p65 and/or stat5a and b (stat5) siRNA and exposed to IFN-γ (10 ng/ml) induction for 18 h and then stimulation with TNF-α (10 ng/ml) for 12 h. Para-cellular permeability in monolayers was assessed as FD-4 clearance and TEER.Total proteins (TP) were extracted, P-p65, p65, pMLC, MLC and Bax were determined by WB. Results were expressed as the mean ± SEM (n = 6). ∧p < 0.01 versus Controls (transfection reagent only), *p < 0.05 versus cytokine-treated controls, &p < 0.05 versus STAT5 knockdown monolayer. #p < 0.01 versus cytokine-treated STAT5 knockdown monolayer.STAT5 signalling in the IEC barrier function and mucosal injury. STAT5⊣ MLCK → ZOs feedback mechanism stabilizes IEC TJ barrier function under basal conditions (1); STAT5⊣ NFκB → MLCK negative feedback loop regulates IEC barrier function under inflammatory conditions (2) and mucosal immune responses to gut injury (3). MA, membrane-associated TJPs.
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fig09: Knockdown of RelA/p65 in IEC monolayers rescues hyperpermeability induced by interfering with stat5 expressionHT-29 IEC monolayers, grown on Transwell filters, were interfered with RelA/p65 and/or stat5a and b (stat5) siRNA and exposed to IFN-γ (10 ng/ml) induction for 18 h and then stimulation with TNF-α (10 ng/ml) for 12 h. Para-cellular permeability in monolayers was assessed as FD-4 clearance and TEER.Total proteins (TP) were extracted, P-p65, p65, pMLC, MLC and Bax were determined by WB. Results were expressed as the mean ± SEM (n = 6). ∧p < 0.01 versus Controls (transfection reagent only), *p < 0.05 versus cytokine-treated controls, &p < 0.05 versus STAT5 knockdown monolayer. #p < 0.01 versus cytokine-treated STAT5 knockdown monolayer.STAT5 signalling in the IEC barrier function and mucosal injury. STAT5⊣ MLCK → ZOs feedback mechanism stabilizes IEC TJ barrier function under basal conditions (1); STAT5⊣ NFκB → MLCK negative feedback loop regulates IEC barrier function under inflammatory conditions (2) and mucosal immune responses to gut injury (3). MA, membrane-associated TJPs.

Mentions: To further demonstrate that STAT5 regulates NF-κB activity in IECs, further modulates MLC phosphorylation, we performed a simultaneous knockdown of STAT5 and RelA/p65 in HT-29 monolayers. We observed that paracellular permeability was increased in STAT5 knockdown monolayers, but not in HT-29 monolayers following RelA/p65 knock down (Fig 9A). Monolayer permeability induced by STAT5 knockdown was further potentiated by IFN-γ (10 ng/ml) and TNF-α (10 ng/ml) co-administration, while RelA/p65 knockdown promoted resistance of proinflammatory cytokine-induced hyperpermeability in monolayers (Fig 9A). Surprisingly, STAT5 and RelA/p65 double knockdown rescued the hyperpermeability that was induced by pro-inflammatory cytokines in single STAT5 knockdown monolayers (Fig 9A). Thus, the reduction of NF-κB may prevent hyperpermeability caused by the loss of STAT5 in IEC monolayers. Furthermore, we consistently found that STAT5 knockdown increased the levels of pMLC and potentiated cytokine-induced pMLC, which was restored by concurrent RelA/p65 knockdown (Fig 9B). Knockdown of STAT5 and basolateral stimulation with cytokines, however, did not increase the expression of the pro-apoptotic protein Bax (Fig 9B). Taken together, STAT5 in enterocytes may negatively regulate NF-κB activation of MLCK and facilitates the protection of intestinal hyperpermeability induced by pro-inflammatory cytokines (STAT5⊣ NF-κB → MLCK; Fig 9C).


Enterocyte STAT5 promotes mucosal wound healing via suppression of myosin light chain kinase-mediated loss of barrier function and inflammation.

Gilbert S, Zhang R, Denson L, Moriggl R, Steinbrecher K, Shroyer N, Lin J, Han X - EMBO Mol Med (2012)

Knockdown of RelA/p65 in IEC monolayers rescues hyperpermeability induced by interfering with stat5 expressionHT-29 IEC monolayers, grown on Transwell filters, were interfered with RelA/p65 and/or stat5a and b (stat5) siRNA and exposed to IFN-γ (10 ng/ml) induction for 18 h and then stimulation with TNF-α (10 ng/ml) for 12 h. Para-cellular permeability in monolayers was assessed as FD-4 clearance and TEER.Total proteins (TP) were extracted, P-p65, p65, pMLC, MLC and Bax were determined by WB. Results were expressed as the mean ± SEM (n = 6). ∧p < 0.01 versus Controls (transfection reagent only), *p < 0.05 versus cytokine-treated controls, &p < 0.05 versus STAT5 knockdown monolayer. #p < 0.01 versus cytokine-treated STAT5 knockdown monolayer.STAT5 signalling in the IEC barrier function and mucosal injury. STAT5⊣ MLCK → ZOs feedback mechanism stabilizes IEC TJ barrier function under basal conditions (1); STAT5⊣ NFκB → MLCK negative feedback loop regulates IEC barrier function under inflammatory conditions (2) and mucosal immune responses to gut injury (3). MA, membrane-associated TJPs.
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fig09: Knockdown of RelA/p65 in IEC monolayers rescues hyperpermeability induced by interfering with stat5 expressionHT-29 IEC monolayers, grown on Transwell filters, were interfered with RelA/p65 and/or stat5a and b (stat5) siRNA and exposed to IFN-γ (10 ng/ml) induction for 18 h and then stimulation with TNF-α (10 ng/ml) for 12 h. Para-cellular permeability in monolayers was assessed as FD-4 clearance and TEER.Total proteins (TP) were extracted, P-p65, p65, pMLC, MLC and Bax were determined by WB. Results were expressed as the mean ± SEM (n = 6). ∧p < 0.01 versus Controls (transfection reagent only), *p < 0.05 versus cytokine-treated controls, &p < 0.05 versus STAT5 knockdown monolayer. #p < 0.01 versus cytokine-treated STAT5 knockdown monolayer.STAT5 signalling in the IEC barrier function and mucosal injury. STAT5⊣ MLCK → ZOs feedback mechanism stabilizes IEC TJ barrier function under basal conditions (1); STAT5⊣ NFκB → MLCK negative feedback loop regulates IEC barrier function under inflammatory conditions (2) and mucosal immune responses to gut injury (3). MA, membrane-associated TJPs.
Mentions: To further demonstrate that STAT5 regulates NF-κB activity in IECs, further modulates MLC phosphorylation, we performed a simultaneous knockdown of STAT5 and RelA/p65 in HT-29 monolayers. We observed that paracellular permeability was increased in STAT5 knockdown monolayers, but not in HT-29 monolayers following RelA/p65 knock down (Fig 9A). Monolayer permeability induced by STAT5 knockdown was further potentiated by IFN-γ (10 ng/ml) and TNF-α (10 ng/ml) co-administration, while RelA/p65 knockdown promoted resistance of proinflammatory cytokine-induced hyperpermeability in monolayers (Fig 9A). Surprisingly, STAT5 and RelA/p65 double knockdown rescued the hyperpermeability that was induced by pro-inflammatory cytokines in single STAT5 knockdown monolayers (Fig 9A). Thus, the reduction of NF-κB may prevent hyperpermeability caused by the loss of STAT5 in IEC monolayers. Furthermore, we consistently found that STAT5 knockdown increased the levels of pMLC and potentiated cytokine-induced pMLC, which was restored by concurrent RelA/p65 knockdown (Fig 9B). Knockdown of STAT5 and basolateral stimulation with cytokines, however, did not increase the expression of the pro-apoptotic protein Bax (Fig 9B). Taken together, STAT5 in enterocytes may negatively regulate NF-κB activation of MLCK and facilitates the protection of intestinal hyperpermeability induced by pro-inflammatory cytokines (STAT5⊣ NF-κB → MLCK; Fig 9C).

Bottom Line: Consistently, knockdown of stat5 in IEC monolayers led to increased NF-κB DNA binding to MLCK promoter, myosin light chain phosphorylation and tight junction (TJ) permeability, which were potentiated by administration of tumour necrosis factor-α (TNF-α), and prevented by concurrent NF-κB knockdown.Collectively, enterocyte STAT5 signalling protects against TJ barrier dysfunction and promotes intestinal mucosal wound healing via an interaction with NF-κB to suppress MLCK.Targeting IEC STAT5 signalling may be a novel therapeutic approach for treating intestinal barrier dysfunction in IBD.

View Article: PubMed Central - PubMed

Affiliation: Division of Gastroenterology, Hepatology and Nutrition, Cincinnati Children's Hospital Medical Center (CCHMC), Cincinnati, OH, USA.

Show MeSH
Related in: MedlinePlus