P059 Human alpha-Defensin 6 regulated by both Atoh1 and beta-catenin might be the pathogenesis of Japanese Crohn's Disease
R. Hayashi*1, K. Tsuchiya2, S. Hibiya3, K. Fukushima3, N. Horita3, E. Okada3, A. Araki3, K. Ohtsuka3, M. Watanabe3
1Hiroshima University Hospital, Gastroenterology and Metabolism, Hiroshima, Japan, 2Tokyo Medical and Dental University, Advanced Therapeutics for Gastrointestinal Diseases, Tokyo, Japan, 3Tokyo Medical and Dental University, Gastroenterology and Hepatology, Tokyo, Japan
Antimicrobial mucosal barrier dysfunction, including the reduction of Human alpha-Defensin (HD) 5, is one of the most crucial pathogenesis of Crohn's disease (CD). Human Paneth cells produce two α-defensin peptides, which called HD5 and HD6. Recently, it has been reported that HD6 promotes mucosal innate immunity through self-assembled peptide "nanonets" whereas HD5 has the antimicrobial activity. The transcriptional regulation of HD6 has not been elucidated. Moreover the association of HD6 expression with CD also remains unknown.
We therefore aimed to elucidate the transcriptional regulation of HD6 and the pathogenesis of CD by HD6 expression.
To investigate the regulation of HD6 expression, We transgened Atoh1 into colon cancer cell line; SW480 by lentivirus infection. The expression of HD6 was assessed by quantitative RT-PCR. The transcriptional activity of HD6 promoter was assessed by the luciferase reporter assay. For the analysis of the HD6 expression in CD, non-inflamed jejunum biopsy specimens of 15 CD patients and 9 healthy controls using double balloon endoscopy (DBE) were assessed.
HD6 was significantly increased by Atoh1 expression in SW480 whereas other antimicrobial peptides such as HD5, Lysozyme and phospholipase A2 were not changed. Atoh1 also enhanced the transcriptional activity of HD6 promoter. We found that HD6 promoter within 200-bp from ATG contains a transcription factor (TCF) binding site and four E-box binding site. The deletion of each binding sites revealed that not only TCF4/β-catenin protein complex but also Atoh1 is indispensable for HD6 expression. Moreover, ChIP assay showed that Atoh1 directly binds to the promoter region of HD6.
Finally, we assessed the HD6 expression in non-inflamed mucosa CD. The microarray using mapping biopsy of entire small intestine in 4 CD patients showed that almost inflammatory related genes were not shown in jejunum compared with 4 non-IBD healthy controls, suggesting that the pathogenesis before the onset of CD might remain in jejunum. HD6 positive Paneth cells of CD patients were significantly lower than that of healthy controls. Moreover, immunostaining showed that β-catenin and Atoh1 are co-expressed in the nuclei of HD6 positive cells, whereas β-catenin is not expressed in the nuclei of HD6 negative crypts.
Both TCF4/β-catenin protein and Atoh1 are essential to express HD6 in different from HD5. The decrease of HD6 in small intestine might cause mucosal barrier dysfunction suggesting that HD6 might be one of the pathogenesis of CD.