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Intestinal alkaline phosphatase (IAP) is an enterocyte differentiation marker that functions to limit fat absorption. Zinc finger binding protein-89 (ZBP-89) is a Kruppel-type transcription factor that appears to promote a differentiated phenotype in the intestinal epithelium. The purpose of this study was to investigate the regulation of IAP gene expression by ZBP-89. RT-PCR, quantitative real-time RT-PCR, Western blot analyses, and reporter assays were used to determine the regulation of IAP by ZBP-89 in HT-29 and Caco-2 colon cancer cells. ZBP-89 knockdown was achieved by specific short interfering (si)RNA. EMSA and chromatin immunoprecipitation (CHIP) were performed to examine the binding of ZBP-89 to the IAP promoter. The results of RT-PCR, quantitative real-time PCR, and Western blot analyses showed that ZBP-89 was expressed at low levels in Caco-2 and HT-29 cells, whereas IAP was minimally expressed and absent in these cells, respectively. Transfection with ZBP-89 expression plamid increased IAP mRNA and protein levels in both cell lines, whereas knockdown of endogenous ZBP-89 by siRNA reduced basal levels of IAP gene expression in Caco-2 cells. IAP-luciferase reporter assays, EMSA, and ChIP established that ZBP-89 activated the IAP gene through a response element (ZBP-89 response element: 5'-CCTCCTCCC-3') located between -1018 and -1010 bp upstream of the AUG start codon. We conclude that ZBP-89 is a direct transcriptional activator of the enterocyte differentiation marker IAP. These findings are consistent with the role that this transcription factor is thought to play as a tumor suppressor and suggests its possible function in the physiology of fat absorption. carcinogenesis; development; enterocyte differentiation; eukaryotic promoter; transient transfection

We have examined the role that the caudal-related homeobox transcription factors Cdx1 and Cdx2 play in activating the enterocyte differentiation marker gene intestinal alkaline phosphatase (IAP). Human colon cancer Caco-2 cells were transiently transfected with Cdx1 and/or Cdx2, and semiquantitative RT-PCR was used to study the effects on IAP mRNA expression. Transfections with a variety of IAP-luciferase reporter constructs were used to identify a Cdx response element located within the human IAP gene promoter. Protein-DNA interactions were examined by EMSA. Results showed that Cdx1 markedly induced IAP mRNA expression, whereas Cdx2 did not, and, in fact, inhibited the Cdx1 effects. Functional analysis revealed that Cdx1 transactivates (fourfold, P < 0.05) the IAP promoter through a novel Cdx response element (GTTTAGA) located between -2369 and -2375 upstream of the translational start site. EMSA showed that both Cdx1 and Cdx2 could bind to the cis element, but in cotransfection experiments, Cdx2 inhibited the Cdx1 effects by ~50%. Thus we have identified a previously unrecognized interaction between two important gut transcription factors, Cdx1 and Cdx2, in the context of IAP gene regulation. Cdx1 activates the IAP gene via a novel cis element, whereas Cdx2 inhibits the Cdx1 effects. cell cycle; enterocyte differentiation; gene regulation; gut development; homeostasis; intestinal alkaline phosphatase; tumorigenesis