Thyroid hormone and p44/42 MAPK inactivation are important in intestinal differentiation. We demonstrated not only that treatment with p44/42 MAPK inhibitor U0126 in intestinal cell line Caco-2 cells reduced the phosphorylation of serine and threonine residues of TR alpha-1, but also that T-3 and U0126 synergistically induced GLUT5 gene expression. EMSA demonstrated that the binding activity of TR alpha-1-RXR heterodimer on GLUT5-TRE in nuclear proteins of Caco-2 cells was synergistically enhanced by co-incubation in vitro with T-3 and CIAP, which strongly de-phosphorylates proteins. ChIP and transfection assays revealed that co-treatment of T-3 and U0126 induces TR alpha-1-RXR binding to GLUT5-TRE on the human GLUT5 enhancer region, and recruitment of the transcriptional complex in cells. These results suggest that inactivation of p44/42 MAPK enhances T-3-induced GLUT5 gene expression in Caco-2 cells through increasing TR alpha-1 transactivity and binding activity to the GLUT5-TRE, probably due to de-phosphorylation of TR alpha-1. (c) 2007 Elsevier Inc. All rights reserved.