STAT6 Induces MLCK1-Dependent Epithelial Tight Junction Dysfunction and Promotes Intestinal Inflammation and Tumorigenesis

TNF-α plays a central role in the intestinal inflammation of various inflammatory disorders including Crohn's disease (CD). TNF-α-induced increase in intestinal epithelial tight junction (TJ) permeability has been proposed as one of the proinflammatory mechanisms contributing to the intestinal inflammation. The intracellular mechanisms involved in the TNF-α-induced increase in intestinal TJ permeability remain unclear. The purpose of this study was to investigate the possibility that the TNF-α-induced increase in intestinal epithelial TJ permeability was regulated by myosin light-chain kinase (MLCK) protein expression, using an in vitro intestinal epithelial model system consisting of the filter-grown Caco-2 intestinal epithelial monolayers. TNF-α (10 ng/ml) produced a time-dependent increase in Caco-2 MLCK expression. The TNF-α increase in MLCK protein expression paralleled the increase in Caco-2 TJ permeability, and the inhibition of the TNF-α-induced MLCK expression (by cycloheximide) prevented the increase in Caco-2 TJ permeability, suggesting that MLCK expression may be required for the increase in Caco-2 TJ permeability. The TNF-α increase in MLCK protein expression was preceded by an increase in MLCK mRNA expression but not an alteration in MLCK protein degradation. Actinomycin-D prevented the TNF-α increase in MLCK mRNA expression and the subsequent increase in MLCK protein expression and Caco-2 TJ permeability, suggesting that the increase in MLCK mRNA transcription led to the increase in MLCK expression. The TNF-α increase in MLCK protein expression was also associated with an increase in Caco-2 MLCK activity. The cycloheximide inhibition of MLCK protein expression prevented the TNF-α increase in MLCK activity and Caco-2 TJ permeability. Moreover, inhibitors of MLCK, Mg2+-myosin ATPase, and metabolic energy prevented the TNF-α increase in Caco-2 TJ permeability, suggesting that the increase in MLCK activity was required for the TNF-α-induced opening of the Caco-2 TJ barrier. In conclusion, our results indicate for the first time that 1) the TNF-α increase in Caco-2 TJ permeability was mediated by an increase in MLCK protein expression, 2) the increase in MLCK protein expression was regulated by an increase in MLCK mRNA transcription, and 3) the increase in Caco-2 TJ permeability required MLCK protein expression-dependent increase in MLCK activity.

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Molecular mechanism of tumor necrosis factor-α modulation of intestinal epithelial tight junction barrier

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00318.2005 ◽

2006 ◽

Vol 290 (3) ◽

pp. G496-G504 ◽

Cited By ~ 214

Author(s):

Dongmei Ye ◽

Iris Ma ◽

Thomas Y. Ma

Keyword(s):

Protein Expression ◽

Tight Junction ◽

Binding Site ◽

Promoter Region ◽

Promoter Activity ◽

Molecular Mechanisms ◽

Intestinal Inflammation ◽

Intestinal Epithelial ◽

Tnf Α ◽

Epithelial Tight Junction

A TNF-α-induced increase in intestinal epithelial tight junction (TJ) permeability has been proposed to be an important proinflammatory mechanism contributing to intestinal inflammation in Crohn's disease and other inflammatory conditions. Previous studies from our laboratory suggested that the TNF-α-induced increase in intestinal TJ permeability was mediated by an increase in myosin light chain kinase (MLCK) protein expression. However, the molecular mechanisms that mediate the TNF-α increase in intestinal TJ permeability and MLCK protein expression remain unknown. The purpose of this study was to delineate the intracellular and molecular mechanisms that mediate the TNF-α-induced increase in intestinal TJ permeability; using an in vitro intestinal epithelial model system consisting of filter-grown Caco-2 intestinal epithelial monolayers. To examine the molecular mechanisms involved in the TNF-α regulation of intestinal TJ barrier, we identified and cloned for the first time a functionally active MLCK promoter region. TNF-α treatment of filter-grown Caco-2 monolayers transfected with plasmid vector containing the MLCK promoter region produced an increase in MLCK promoter activity and MLCK transcription. The TNF-α-induced increase in MLCK transcription corresponded to a sequential increase in MLCK protein expression, MLCK activity, and Caco-2 TJ permeability. The TNF-α-induced increase in MLCK promoter activity was mediated by NF-κB activation, and the inhibition of NF-κB activation prevented the TNF-α-induced increase in promoter activity and the subsequent increase in MLCK protein expression and Caco-2 TJ permeability. The TNF-α-induced activation of MLCK promoter was mediated by binding of the activated NF-κB p50/p65 dimer to the downstream κB binding site (−84 to −75) on the MLCK promoter region; deletion of the κB binding site prevented the TNF-α increase in promoter activity. Additionally, siRNA silencing of NF-κB p65 also prevented the TNF-α increase in MLCK promoter activity. In conclusion, our findings indicated that the TNF-α-induced increase in intestinal epithelial TJ permeability was mediated by NF-κB p50/p65 binding and activation of the MLCK promoter. NF-κB p50/p65 activation of the MLCK promoter then leads to a stepwise increase in MLCK transcription, expression and activity, and MLCK-mediated opening of the intestinal TJ barrier.

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Non-hematopoietic STAT6 induces epithelial tight junction dysfunction and promotes intestinal inflammation and tumorigenesis

Mucosal Immunology ◽

10.1038/s41385-019-0204-y ◽

2019 ◽

Vol 12 (6) ◽

pp. 1304-1315 ◽

Cited By ~ 2

Author(s):

Yuli Lin ◽

Bingji Li ◽

Xuguang Yang ◽

Ting Liu ◽

Tiancong Shi ◽

...

Keyword(s):

Colorectal Cancer ◽

Tight Junction ◽

Intestinal Inflammation ◽

Clinical Stage ◽

Tumor Formation ◽

Lymphoid Cells ◽

Gut Permeability ◽

Direct Role ◽

Epithelial Tight Junction

Abstract Enhanced gut permeability due to dysregulated epithelial tight junction is often associated with inflammatory bowel diseases (IBD), which have a greater risk for developing colorectal cancer. STAT6 activation was detected in inflamed colonic epithelium of active IBD patients, suggesting a role of epithelial STAT6 in colitis development. Here, we demonstrated that non-hematopoietic STAT6, but not hematopoietic STAT6, triggered DSS-induced colitis and subsequent tumorigenesis. This could be due to the enhancing-effect of STAT6 on gut permeability and microbiota translocation via interruption of epithelial tight junction integrity. Mechanistically, long-myosin light-chain kinase (MLCK1) was identified as a target of STAT6, leading to epithelial tight junction dysfunction and microbiota-driven colitis. Furthermore, neutralization of IL-13, which was primarily derived from type 2 innate lymphoid cells (ILC2) in a microbiota-dependent way, inhibited epithelial STAT6 activation and improved gut permeability and DSS-induced colitis. Importantly, pharmacological inhibition of STAT6 reduces murine intestinal tumor formation, and tumoral p-STAT6 levels positively correlated to the clinical stage and poor prognosis of human colorectal cancer. Thus, our study reveals a direct role of STAT6 in the disruption of epithelial tight junction integrity and colitis development, and suggests STAT6 as a potential therapeutic and prophylactic target for IBD and colitis-associated cancer.

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Lactobacillus acidophilus Induces a Strain-specific and Toll-Like Receptor 2–Dependent Enhancement of Intestinal Epithelial Tight Junction Barrier and Protection Against Intestinal Inflammation

American Journal Of Pathology ◽

10.1016/j.ajpath.2021.02.003 ◽

2021 ◽

Vol 191 (5) ◽

pp. 872-884 ◽

Cited By ~ 2

Author(s):

Rana Al-Sadi ◽

Prashant Nighot ◽

Meghali Nighot ◽

Mohammad Haque ◽

Manmeet Rawat ◽

...

Keyword(s):

Tight Junction ◽

Lactobacillus Acidophilus ◽

Intestinal Inflammation ◽

Intestinal Epithelial ◽

Toll Like Receptor ◽

Epithelial Tight Junction ◽

Toll Like Receptor 2

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IL-1β and the Intestinal Epithelial Tight Junction Barrier

Frontiers in Immunology ◽

10.3389/fimmu.2021.767456 ◽

2021 ◽

Vol 12 ◽

Author(s):

Lauren W. Kaminsky ◽

Rana Al-Sadi ◽

Thomas Y. Ma

Keyword(s):

Tight Junction ◽

Intestinal Inflammation ◽

Inflammatory Diseases ◽

Gene Activation ◽

Nuclear Factor Κb ◽

Systemic Circulation ◽

Intestinal Lumen ◽

Intestinal Epithelial ◽

Kinase Gene ◽

Epithelial Tight Junction

The intestinal epithelial tight junction (TJ) barrier controls the paracellular permeation of contents from the intestinal lumen into the intestinal tissue and systemic circulation. A defective intestinal TJ barrier has been implicated as an important pathogenic factor in inflammatory diseases of the gut including Crohn’s disease, ulcerative colitis, necrotizing enterocolitis, and celiac disease. Previous studies have shown that pro-inflammatory cytokines, which are produced during intestinal inflammation, including interleukin-1β (IL-1β), tumor necrosis factor-α, and interferon-γ, have important intestinal TJ barrier-modulating actions. Recent studies have shown that the IL-1β-induced increase in intestinal TJ permeability is an important contributing factor of intestinal inflammation. The IL-1β-induced increase in intestinal TJ permeability is mediated by regulatory signaling pathways and activation of nuclear transcription factor nuclear factor-κB, myosin light chain kinase gene activation, and post-transcriptional occludin gene modulation by microRNA and contributes to the intestinal inflammatory process. In this review, the regulatory role of IL-1β on intestinal TJ barrier, the intracellular mechanisms that mediate the IL-1β modulation of intestinal TJ permeability, and the potential therapeutic targeting of the TJ barrier are discussed.

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