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

There is still relatively limited information about mechanisms of gene expr ession in enterocytes and mechanisms by which gene expression is regulated during enterocyte differentiation. Using the human intestinal epithelial ce ll line Caco-2, which spontaneously differentiates from a cryptlike to a vi llouslike enterocyte, we have previously shown that there is a marked incre ase in transcription of the well-characterized alpha(1)-antitrypsin (alpha( 1)-AT) gene during enterocyte differentiation. In this study we examined th e possibility of identifying the cis-acting elements and trans-acting DNA-b inding proteins responsible for expression of the alpha(1)-AT gene in Caco- 2 cells during differentiation. Footprint analysis and electrophoretic mobi lity 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 bo und to two regions in the proximal promoter of the alpha(1)-AT gene. Cotran sfection 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 l evels during differentiation of Caco-2 cells. Finally, overexpression of an tisense ribozymes for HNF-1 alpha in Caco-2 cells resulted in a selective d ecrease 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 differentiatio n in the model Caco-2 cell system.