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Molecular mechanism of tumor necrosis factor-alpha modulation of intestinal epithelial tight junction barrier.
Am J Physiol Gastrointest Liver Physiol. 2006 Mar; 290(3):G496-504.AJ

Abstract

A TNF-alpha-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-alpha-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-alpha 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-alpha-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-alpha regulation of intestinal TJ barrier, we identified and cloned for the first time a functionally active MLCK promoter region. TNF-alpha 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-alpha-induced increase in MLCK transcription corresponded to a sequential increase in MLCK protein expression, MLCK activity, and Caco-2 TJ permeability. The TNF-alpha-induced increase in MLCK promoter activity was mediated by NF-kappaB activation, and the inhibition of NF-kappaB activation prevented the TNF-alpha-induced increase in promoter activity and the subsequent increase in MLCK protein expression and Caco-2 TJ permeability. The TNF-alpha-induced activation of MLCK promoter was mediated by binding of the activated NF-kappaB p50/p65 dimer to the downstream kappaB binding site (-84 to -75) on the MLCK promoter region; deletion of the kappaB binding site prevented the TNF-alpha increase in promoter activity. Additionally, siRNA silencing of NF-kappaB p65 also prevented the TNF-alpha increase in MLCK promoter activity. In conclusion, our findings indicated that the TNF-alpha-induced increase in intestinal epithelial TJ permeability was mediated by NF-kappaB p50/p65 binding and activation of the MLCK promoter. NF-kappaB 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.

Authors+Show Affiliations

Department of Internal Medicine-Gastroenterology, MSC10 5550, University of New Mexico, Albuquerque, NM 87131-0001, USA.No affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, N.I.H., Extramural
Research Support, U.S. Gov't, Non-P.H.S.

Language

eng

PubMed ID

16474009

Citation

Ye, Dongmei, et al. "Molecular Mechanism of Tumor Necrosis Factor-alpha Modulation of Intestinal Epithelial Tight Junction Barrier." American Journal of Physiology. Gastrointestinal and Liver Physiology, vol. 290, no. 3, 2006, pp. G496-504.
Ye D, Ma I, Ma TY. Molecular mechanism of tumor necrosis factor-alpha modulation of intestinal epithelial tight junction barrier. Am J Physiol Gastrointest Liver Physiol. 2006;290(3):G496-504.
Ye, D., Ma, I., & Ma, T. Y. (2006). Molecular mechanism of tumor necrosis factor-alpha modulation of intestinal epithelial tight junction barrier. American Journal of Physiology. Gastrointestinal and Liver Physiology, 290(3), G496-504.
Ye D, Ma I, Ma TY. Molecular Mechanism of Tumor Necrosis Factor-alpha Modulation of Intestinal Epithelial Tight Junction Barrier. Am J Physiol Gastrointest Liver Physiol. 2006;290(3):G496-504. PubMed PMID: 16474009.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Molecular mechanism of tumor necrosis factor-alpha modulation of intestinal epithelial tight junction barrier. AU - Ye,Dongmei, AU - Ma,Iris, AU - Ma,Thomas Y, PY - 2006/2/14/pubmed PY - 2006/3/17/medline PY - 2006/2/14/entrez SP - G496 EP - 504 JF - American journal of physiology. Gastrointestinal and liver physiology JO - Am J Physiol Gastrointest Liver Physiol VL - 290 IS - 3 N2 - A TNF-alpha-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-alpha-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-alpha 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-alpha-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-alpha regulation of intestinal TJ barrier, we identified and cloned for the first time a functionally active MLCK promoter region. TNF-alpha 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-alpha-induced increase in MLCK transcription corresponded to a sequential increase in MLCK protein expression, MLCK activity, and Caco-2 TJ permeability. The TNF-alpha-induced increase in MLCK promoter activity was mediated by NF-kappaB activation, and the inhibition of NF-kappaB activation prevented the TNF-alpha-induced increase in promoter activity and the subsequent increase in MLCK protein expression and Caco-2 TJ permeability. The TNF-alpha-induced activation of MLCK promoter was mediated by binding of the activated NF-kappaB p50/p65 dimer to the downstream kappaB binding site (-84 to -75) on the MLCK promoter region; deletion of the kappaB binding site prevented the TNF-alpha increase in promoter activity. Additionally, siRNA silencing of NF-kappaB p65 also prevented the TNF-alpha increase in MLCK promoter activity. In conclusion, our findings indicated that the TNF-alpha-induced increase in intestinal epithelial TJ permeability was mediated by NF-kappaB p50/p65 binding and activation of the MLCK promoter. NF-kappaB 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. SN - 0193-1857 UR - https://www.unboundmedicine.com/medline/citation/16474009/Molecular_mechanism_of_tumor_necrosis_factor_alpha_modulation_of_intestinal_epithelial_tight_junction_barrier_ L2 - https://journals.physiology.org/doi/10.1152/ajpgi.00318.2005?url_ver=Z39.88-2003&rfr_id=ori:rid:crossref.org&rfr_dat=cr_pub=pubmed DB - PRIME DP - Unbound Medicine ER -