Enterocyte STAT5 promotes mucosal wound healing via suppression of myosin light chain kinase-mediated loss of barrier function and inflammation.
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.
Affiliation: Division of Gastroenterology, Hepatology and Nutrition, Cincinnati Children's Hospital Medical Center (CCHMC), Cincinnati, OH, USA.Show MeSH
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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.
Affiliation: Division of Gastroenterology, Hepatology and Nutrition, Cincinnati Children's Hospital Medical Center (CCHMC), Cincinnati, OH, USA.