期刊
JOURNAL OF CELLULAR AND MOLECULAR MEDICINE
卷 12, 期 4, 页码 1331-1346出版社
WILEY
DOI: 10.1111/j.1582-4934.2008.00302.x
关键词
tumour necrosis factor-alpha; NF-kappa B subunit; intestinal permeability; tight junction; myosin light chain kinase
资金
- NIDDK NIH HHS [R0 1-DK-64165-01, R01 DK064165] Funding Source: Medline
The patients with Crohn's disease (CD) have a 'leaky gut' manifested by an increase in intestinal epithelial tight junction (TJ) permeability. Tumour necrosis factor-alpha (TNF-alpha) is a proto-typical pro-inflammatory cytokine that plays a central role in intestinal inflammation of CD. An important pro-inflammatory action of TNF-alpha is to cause a functional opening of intestinal TJ barrier. Previous studies have shown that TNF-alpha increase in TJ permeability was regulated by an increase in myosin light chain kinase (MLCK) gene activity and protein expression. The major aim of this study was to elucidate the cellular and molecular mechanisms that mediate basal and TNF-alpha-induced increase in MLCK gene activity. By progressive 5' deletion, minimal MLCK promoter was localized between -313 to +118 on MLCK promoter. A p53 binding site located within minimal promoter region was identified as an essential determinant for basal promoter activity. A 4 bp start site and a 5 bp downstream promoter element were required for MLCK gene activity. TNF-alpha-induced increase in MLCK promoter activity was mediated by NF-kappa B activation. There were eight kappa B binding sites on MLCK promoter. The NF-kappa B1 site at +48 to +57 mediated TNF-alpha-induced increase in MLCK promoter activity. The NF-kappa B2 site at -325 to -316 had a repressive role on promoter activity. The opposite effects on promoter activity were due to differences in the NF-kappa B dimer type binding to the kappa B sites. p50/p65 dimer preferentially binds to the NF-kappa B1 site and up-regulates promoter activity; while p50/p50 dimer preferentially binds to the NF-kappa B2 site and down-regulates promoter activity. In conclusion, we have identified the minimal MLCK promoter region, essential molecular determinants and molecular mechanisms that mediate basal and TNF-alpha-induced modulation of MLCK promoter activity in Caco-2 intestinal epithelial cells. These studies provide novel insight into the cellular and molecular mechanisms that regulate basal and TNF-alpha-induced modulation of MLCK gene activity.
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