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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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STAT5 Knockdown amplifies pro-inflammatory cytokine-induced hyperpermeability in IEC monolayerHT-29 IEC monolayers on transwell filters, with and without stat5 RNAi, were exposed to IFN-γ (10 ng/ml) induction for 18 h and then TNF-α (10 ng/ml) stimulation for 12 h.A. Paracellular permeability in HT-29 cell monolayers was assessed as FD4 clearance by the apical-to-basolateral flux of FD4 and TEER. The inset indicated STAT5 knockdown in the monolayers. Results were expressed as the mean ± SEM (n = 6).B-D. HT-29 monolayer was interfered with stat5 siRNA, p65 nuclear abundance (B), pMLC levels in TP (C) and cytosolic IκB α and β levels (D) were assessed in the presence and absence of IFN-γ and TNF-α co-stimulation. Signal intensity was determined by densitometry, results were expressed as the mean ± SEM (n = 5).E,F. DNA binding activity in the NF-κB promoter activity (E) or MLCK promoter containing κB binding sites (F) was analysed with nuclear extracts by EMSAs. Cold competition assays were performed by adding unlabeled oligonucleotides in 100-fold molar excess; NF-κB identity was confirmed by specific p65 antibody (2 µg) as indicated, and the arrow represented the shifted bands. The representative result is shown from five repeated experiments.
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fig07: STAT5 Knockdown amplifies pro-inflammatory cytokine-induced hyperpermeability in IEC monolayerHT-29 IEC monolayers on transwell filters, with and without stat5 RNAi, were exposed to IFN-γ (10 ng/ml) induction for 18 h and then TNF-α (10 ng/ml) stimulation for 12 h.A. Paracellular permeability in HT-29 cell monolayers was assessed as FD4 clearance by the apical-to-basolateral flux of FD4 and TEER. The inset indicated STAT5 knockdown in the monolayers. Results were expressed as the mean ± SEM (n = 6).B-D. HT-29 monolayer was interfered with stat5 siRNA, p65 nuclear abundance (B), pMLC levels in TP (C) and cytosolic IκB α and β levels (D) were assessed in the presence and absence of IFN-γ and TNF-α co-stimulation. Signal intensity was determined by densitometry, results were expressed as the mean ± SEM (n = 5).E,F. DNA binding activity in the NF-κB promoter activity (E) or MLCK promoter containing κB binding sites (F) was analysed with nuclear extracts by EMSAs. Cold competition assays were performed by adding unlabeled oligonucleotides in 100-fold molar excess; NF-κB identity was confirmed by specific p65 antibody (2 µg) as indicated, and the arrow represented the shifted bands. The representative result is shown from five repeated experiments.

Mentions: NF-κB activation is sufficient to upregulate transcription of MLCK from the long MLCK promoter, and some evidences suggest that TJPs (TJPs; occludin, claudin-1 and ZO-1) are internalized through an NF-κB-dependent pathway (Graham et al, 2006; Tang et al, 2010). We thus ask whether there is a transcriptional regulation between NF-κB and STAT5 activation in MLCK-mediated IEC barrier function that impacts intestinal immune response to mucosal inflammation. To mimic in vivo inflammatory signalling, we combined IFN-γ (10 ng/ml) with TNF-α (10 ng/ml) to basolaterally stimulate the HT-29 monolayers. Cytokine-induced monolayer hyperpermeability was significantly enhanced by STAT5 knockdown compared to cytokine-treated controls (Fig 7A). We then demonstrated that NF-κB activity and pMLC levels were consistently increased in stat5 knockdown HT-29 IEC cell monolayers under baseline conditions, and were enhanced by IFN-γ and TNF-α co-administration in the presence of stat5 RNAi (Fig 7B and C). Conversely, IκBα, but not IκBβ, was reduced in STAT5 knockdown HT-29 IEC monolayers. This reduction was potentiated by IFN-γ and TNF-α co-administration in the presence of stat5 RNAi (Fig 7D). We next measured NF-κB and MLCK promoter activity by Electrophoretic Mobility Shift Assay (EMSA). Consistently, we found that NF-κB-mediated transcription as well as DNA binding activity on the NF-κB binding element from the MLCK promoter was promoted in IEC monolayers by stat5 RNAi, and was further enhanced in the cytokine-stimulated monolayers with STAT5 knockdown (Fig 7E and F). Taken together, STAT5 modulates NF-κB DNA binding in the MLCK promoter to suppress TNF-α-induced MLCK transcription; whereas STAT5 deficiency in IECs increases NF-κB-activated MLCK to induce a persistent TJ barrier dysfunction, resulting in the impairment of intestinal mucosal wound healing in response to gut injury.


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)

STAT5 Knockdown amplifies pro-inflammatory cytokine-induced hyperpermeability in IEC monolayerHT-29 IEC monolayers on transwell filters, with and without stat5 RNAi, were exposed to IFN-γ (10 ng/ml) induction for 18 h and then TNF-α (10 ng/ml) stimulation for 12 h.A. Paracellular permeability in HT-29 cell monolayers was assessed as FD4 clearance by the apical-to-basolateral flux of FD4 and TEER. The inset indicated STAT5 knockdown in the monolayers. Results were expressed as the mean ± SEM (n = 6).B-D. HT-29 monolayer was interfered with stat5 siRNA, p65 nuclear abundance (B), pMLC levels in TP (C) and cytosolic IκB α and β levels (D) were assessed in the presence and absence of IFN-γ and TNF-α co-stimulation. Signal intensity was determined by densitometry, results were expressed as the mean ± SEM (n = 5).E,F. DNA binding activity in the NF-κB promoter activity (E) or MLCK promoter containing κB binding sites (F) was analysed with nuclear extracts by EMSAs. Cold competition assays were performed by adding unlabeled oligonucleotides in 100-fold molar excess; NF-κB identity was confirmed by specific p65 antibody (2 µg) as indicated, and the arrow represented the shifted bands. The representative result is shown from five repeated experiments.
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fig07: STAT5 Knockdown amplifies pro-inflammatory cytokine-induced hyperpermeability in IEC monolayerHT-29 IEC monolayers on transwell filters, with and without stat5 RNAi, were exposed to IFN-γ (10 ng/ml) induction for 18 h and then TNF-α (10 ng/ml) stimulation for 12 h.A. Paracellular permeability in HT-29 cell monolayers was assessed as FD4 clearance by the apical-to-basolateral flux of FD4 and TEER. The inset indicated STAT5 knockdown in the monolayers. Results were expressed as the mean ± SEM (n = 6).B-D. HT-29 monolayer was interfered with stat5 siRNA, p65 nuclear abundance (B), pMLC levels in TP (C) and cytosolic IκB α and β levels (D) were assessed in the presence and absence of IFN-γ and TNF-α co-stimulation. Signal intensity was determined by densitometry, results were expressed as the mean ± SEM (n = 5).E,F. DNA binding activity in the NF-κB promoter activity (E) or MLCK promoter containing κB binding sites (F) was analysed with nuclear extracts by EMSAs. Cold competition assays were performed by adding unlabeled oligonucleotides in 100-fold molar excess; NF-κB identity was confirmed by specific p65 antibody (2 µg) as indicated, and the arrow represented the shifted bands. The representative result is shown from five repeated experiments.
Mentions: NF-κB activation is sufficient to upregulate transcription of MLCK from the long MLCK promoter, and some evidences suggest that TJPs (TJPs; occludin, claudin-1 and ZO-1) are internalized through an NF-κB-dependent pathway (Graham et al, 2006; Tang et al, 2010). We thus ask whether there is a transcriptional regulation between NF-κB and STAT5 activation in MLCK-mediated IEC barrier function that impacts intestinal immune response to mucosal inflammation. To mimic in vivo inflammatory signalling, we combined IFN-γ (10 ng/ml) with TNF-α (10 ng/ml) to basolaterally stimulate the HT-29 monolayers. Cytokine-induced monolayer hyperpermeability was significantly enhanced by STAT5 knockdown compared to cytokine-treated controls (Fig 7A). We then demonstrated that NF-κB activity and pMLC levels were consistently increased in stat5 knockdown HT-29 IEC cell monolayers under baseline conditions, and were enhanced by IFN-γ and TNF-α co-administration in the presence of stat5 RNAi (Fig 7B and C). Conversely, IκBα, but not IκBβ, was reduced in STAT5 knockdown HT-29 IEC monolayers. This reduction was potentiated by IFN-γ and TNF-α co-administration in the presence of stat5 RNAi (Fig 7D). We next measured NF-κB and MLCK promoter activity by Electrophoretic Mobility Shift Assay (EMSA). Consistently, we found that NF-κB-mediated transcription as well as DNA binding activity on the NF-κB binding element from the MLCK promoter was promoted in IEC monolayers by stat5 RNAi, and was further enhanced in the cytokine-stimulated monolayers with STAT5 knockdown (Fig 7E and F). Taken together, STAT5 modulates NF-κB DNA binding in the MLCK promoter to suppress TNF-α-induced MLCK transcription; whereas STAT5 deficiency in IECs increases NF-κB-activated MLCK to induce a persistent TJ barrier dysfunction, resulting in the impairment of intestinal mucosal wound healing in response to gut injury.

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