Identification of a Functional Hepatocyte Nuclear Factor 4 Binding Site in the Neutral Ceramidase Promoter

The brush border membrane of the differentiated small intestinal epithelial cell is studded with membrane bound hydrolytic ectoenzymes involved in digestion. Previous studies of the regulation of genes encoding brush border enzymes have especially implicated the transcription factors hepatocyte nuclear factor HNF-1 and Cdx2. Recent genome-wide studies have, however, also identified HNF-4 alpha as a transcription factor with a high number of target genes in the differentiated small intestinal epithelial cell. The Asah2 gene encodes neutral ceramidase, which is a hydrolytic brush border enzyme involved in ceramide digestion. It was the purpose of the present work to experimentally verify the functional importance of a HNF-4 alpha binding site predicted by bioinformatic analysis to be present in the Asah2 promoter. Using supershift analysis, HNF-4 alpha overexpression, and HNF-4 alpha knockdown experiments it was confirmed that the predicted HNF-4 alpha binding site identified in the Asah2 promoter is functional. The results support the hypothesis that HNF-4 alpha might be important for intestinal glycolipid metabolism. J. Cell. Biochem. 111: 1330-1336, 2010. (C) 2010 Wiley-Liss, Inc.