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Journal of Cell Science, Vol 113, Issue 11 2085-2090, Copyright © 2000 by Company of Biologists
JOURNAL ARTICLES |
J Mankertz, S Tavalali, H Schmitz, A Mankertz, EO Riecken, M Fromm and JD Schulzke
Medizinische Klinik I Gastroenterologie und Infektiologie , Universitatsklinikum Benjamin Franklin, Freie Universitat Berlin, Hindenburgdamm 30, Germany. mankertz@medizin.fu-berlin.de.
The 65 kDa protein occludin is a membrane-spanning part of the epithelial tight junction, which is the main barrier of the paracellular pathway. The function of occludin as part of tight junctions is still poorly understood and even less is known about the regulatory mechanisms that influence occludin gene expression. This study aimed to identify the sequences essential in cis for genomic regulation of tight junction formation and to investigate their funcional role in cytokine-dependent tight junction regulation. Using genome walking cloning of occludin-specific human genomic DNA sequences, a 1853 bp DNA fragment containing the transcription start point of occludin cDNA sequences was amplified and sequenced. Subcloning of this fragment in front of the luciferase reporter gene revealed strong expression of enzymatic activity after transfection of the human intestinal cell line HT-29/B6. With subsequent deletions of parts of the promoter fragment, its size was reduced to 280 bp that are necessary and sufficient to mediate promoter activity. Tumor necrosis factor alpha and another cytokine involved in inflammation, interferon gamma, reduced transepithelial resistance in HT-29/B6 cells, which was preceded by a decrease in occludin mRNA expression as revealed by northern blot analysis. Tumor necrosis factor alpha and interferon gamma diminished occludin promoter activity alone and even synergistically, suggesting a genomic regulation of alterations of the paracellular barrier. In conclusion, proinflammatory cytokines such as tumor necrosis factor alpha and interferon gamma can downregulate the expression of the transmembrane tight junction strand protein occludin, paralleling the barrier disturbance detected electrophysiologically. This could be an important mechanism in gastrointestinal diseases accompanied by barrier defects, for example inflammatory bowel diseases.
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