Regulation of alpha1-antitrypsin gene expression in human intestinal epithelial cell line caco-2 by HNF-1alpha 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 alpha1-antitrypsin (alpha1-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 alpha1-AT gene in Caco-2 cells during differentiation. Footprint analysis and electrophoretic mobility shift assays showed that hepatocyte nuclear factor-1alpha (HNF-1alpha), HNF-1beta, and HNF-4 from nuclear extracts of Caco-2 cells specifically bound to two regions in the proximal promoter of the alpha1-AT gene. Cotransfection studies showed that HNF-1alpha and HNF-4 had a synergistic effect on alpha1-AT gene expression. RNA blot analysis showed that HNF-1alpha and HNF-4 mRNA levels and electrophoretic mobility shift assays showed that HNF-1alpha binding activity increase coordinately with alpha1-AT mRNA levels during differentiation of Caco-2 cells. Finally, overexpression of antisense ribozymes for HNF-1alpha in Caco-2 cells resulted in a selective decrease in endogenous alpha1-AT gene expression. Together, these results provide evidence that HNF-1alpha and HNF-4 play a role in the mechanism by which the alpha1-AT gene is upregulated during enterocyte differentiation in the model Caco-2 cell system.