Regulation of α-antitrypsin gene expression in human intestinal epithelial cell line Caco-2 by HNF-1α and HNF-4

There is still relatively limited information about mechanisms of gene expression in enterocytes and mechanisms by which gene expression is regulated during enterocyte differentiation. Using the human intestinal epithelial cell line Caco-2, which spontaneously differentiates from a cryptlike to a villouslike enterocyte, we have previously shown that there is a marked increase in transcription of the well-characterized alpha(1)-antitrypsin (alpha(1)-AT) gene during enterocyte differentiation. In this study we examined the possibility of identifying the cis-acting elements and trans-acting DNA-binding proteins responsible for expression of the alpha(1)-AT gene in Caco-2 cells during differentiation. Footprint analysis and electrophoretic mobility shift assays showed that hepatocyte nuclear factor-lot (HNF-1 alpha), HNF-1 beta, and HNF-4 from nuclear extracts of Caco-2 cells specifically bound to two regions in the proximal promoter of the alpha(1)-AT gene. Cotransfection studies showed that HNF-1 alpha and HNF-4 had a synergistic effect on alpha(1)-AT gene expression. RNA blot analysis showed that HNF-1 alpha and HNF-4 mRNA levels and electrophoretic mobility shift assays showed that HNF-1 alpha binding activity increase coordinately with alpha(1)-AT mRNA levels during differentiation of Caco-2 cells. Finally, overexpression of antisense ribozymes for HNF-1 alpha in Caco-2 cells resulted in a selective decrease in endogenous alpha(1)-AT gene expression. Together, these results provide evidence that HNF-1 alpha and HNF-4 play a role in the mechanism by which the alpha(1)-AT gene is upregulated during enterocyte differentiation in the model Caco-2 cell system.