PDX1 regulation of FABP1 and novel target genes in human intestinal epithelial Caco-2 cells

Abstract

The transcription factor pancreatic and duodenal homeobox 1 (PDX1) plays an essential role in pancreatic development and in maintaining proper islet function via target gene regulation. Few intestinal PDX1 targets, however, have been described. We sought to define novel PDX1-regulated intestinal genes. Caco-2 human intestinal epithelial cells were engineered to overexpress PDX1 and gene expression profiles relative to control cells were assessed. Expression of 80 genes significantly increased while that of 49 genes significantly decreased more than 4-fold following PDX1 overexpression in differentiated Caco-2 cells. Analysis of the differentially regulated genes with known functional annotations revealed genes encoding transcription factors, growth factors, kinases, digestive glycosidases, nutrient transporters, nutrient binding proteins, and structural components. The gene for fatty acid binding protein 1, liver, FABP1, is repressed by PDX1 in Caco-2 cells. PDX1 overexpression in Caco-2 cells also results in repression of promoter activity driven by the 0.6 kb FABP1 promoter. PDX1 regulation of promoter activity is consistent with the decrease in FABP1 RNA abundance resulting from PDX1 overexpression and identifies FABP1 as a candidate PDX1 target. PDX1 repression of FABP1, LCT, and SI suggests a role for PDX1 in patterning anterior intestinal development.

Introduction

Development of the gastrointestinal tract is associated with programmed patterning of epithelial gene expression along the gut A-P axis. Global expression profiling using microarray analysis has revealed distinct gene expression patterns with sharp segmental boundaries for multiple intestine-specific genes along the length of the small intestine [1]. The mechanisms regulating spatial restriction of intestine-specific gene expression along the anterior-posterior axis during gastrointestinal development, however, are largely unknown. Transcription factors expressed in specific regions along the A-P gut axis during gastrointestinal organogenesis have been characterized as spatial regulators. An evolutionarily-conserved group of homeodomain-containing genes that regulate endodermal and mesodermal cell fates during gut ontogeny are grouped in chromosomal regions known as the Hox and Parahox clusters, reviewed by Krumlauf [2] and Beck [3]. The spatial expression domains for several Parahox transcription factors expressed in endoderm-derived organs have been mapped along the A-P gut axis [4]. The Parahox gene, PDX1, pancreatic duodenal homeobox 1, PDX1 (also known as IPF-1, IDX-1 and STF-1) is expressed in the developing pancreas and duodenum. PDX1 is required for pancreas development and the maintenance of functional islet ß cells [5], [6], [7], [8], [9]. Specifically, mice null for the PDX1 gene, PDX1^−/−, fail to form a pancreas and die in the neonatal period within a week of birth [6], [7].

In addition to the pancreas, PDX1 is maximally expressed in the most anterior duodenal region of the intestinal tract with decreased expression in the distal small intestine [10]. In neonatal PDX1^−/− null mice, the rostral duodenum shows dilated cystic malformations at the stomach/duodenum junction felt to be abnormal Brunner’s glands and areas of local ectopic GLUT2-positive cuboidal epithelium [7]. Just distal of the abnormal epithelium, the numbers of enteroendocrine cells in the villi are greatly reduced. In further support of a role for PDX1 in patterning intestinal development and cell differentiation, mice with misexpression of PDX1 targeted to the large intestine manifest an altered midgut-hindgut union [11] and immature intestinal epithelial rat IEC-6 cells can differentiate into enteroendocrine cells in response to PDX1 overexpression [12]. PDX1 is a known regulator of a number of genes essential for maintaining pancreatic cell identity and function including insulin [13], glucose transporter 2 [14], glucokinase [15], islet amyloid polypeptide [16], [17], [18] and somatostatin [8], [9]. Both activator [8], [9], [13], [14], [15], [16], [17], [18], [19], [20], [21], [22], [23], [24], [25], [26] and repressor [11], [27], [28], [29], [30], [31] functions have been described for PDX1. Few genes expressed in the intestine, however, have been identified as intestinal targets capable of being regulated by PDX1. In order to obtain further insights into the role of PDX1 in regulating intestinal cell fate determination in epithelial cells, we sought to identify downstream target genes regulated by PDX1. cDNA microarray approaches were used to characterize the effects on gene expression profiles following overexpression of PDX1 in Caco-2 cells, a human adenocarcinoma-derived cell line that mimics a small intestinal enterocyte phenotype with respect to expression of several digestive hydrolases [32].