Thyroid hormone positively regulates the enterocyte differentiation marker intestinal alkaline phosphatase gene via an atypical response element

Thyroid hormone (T₃) is a critical regulator of intestinal epithelial development and homeostasis, but its mechanism of action within the gut is not well understood. We have examined the molecular mechanisms underlying the T₃ activation of the enterocyte differentiation marker intestinal alkaline phosphatase (IAP) gene. RT-PCR and Western blotting showed that thyroid hormone receptors TRα1 and TRβ1 were expressed in human colorectal adenocarcinoma Caco-2 cells. Northern blotting detected expression of two IAP transcripts, which were increased approximately 3-fold in response to T₃. Transient transfection studies with luciferase reporter plasmids carrying various internal and 5′ deletion mutations of the IAP promoter localized a putative thyroid hormone response element (TRE) to a region approximately 620 nucleotides upstream (-620) of the ATG start codon. EMSAs using TRα1-retinoid X receptor a (RXRα) on sequential 5′ and 3′ single nucleotide deletions defined the TRE between -632 and -612 (5′-TT-GAACTCAgccTGAGGTTAC-3′). Compared with the consensus TRE, the IAP-TRE is novel in that it contains an everted repeat of two nonamers (not hexamers) separated by three nucleotides. Neither TRα1 nor RXRα binds to the IAP-TRE; however, TRβ1 binds to this TRE with minimal affinity. In the presence of TR and RXRα, only the TR-RXRα heterodimer binds to the IAP-TRE. Mutagenesis of either nonamer abolishes the biological activity of IAP promoter. We have thus identified a novel response element that appears to mediate the T₃-induced activation of the enterocyte differentiation marker, intestinal alkaline phosphatase.