Recent research have emphasized the role of the dioxin receptor (AhR) in maintaining cell morphology adhesion and migration. in both normal and transformed epithelial cells. and (10). In additional cell types such as primary mouse endothelial cells (11) and CD4?CD8? thymocytes (4 12 AhR activation promoted cell migration to newly formed blood vessels and to the spleen respectively. The fact that AhR depletion increased primary keratinocytes migration and improved wound healing led us to suggest that AhR could be involved in the epithelial-to-mesenchymal transition (EMT). EMT is a phenotypic switch that permanently or transiently converts epithelial cells into motile mesenchymal-like cells. During this process epithelial cells suffer a spectrum of changes that affect their adhesion to neighboring cells and to the substratum their migration and their normal functioning (13). EMT is essential during embryonic development and in tissue repair although a large body of evidence indicates that it also contributes to pathology (13-15). Because EMT enables epithelial cells with migration and invasion capabilities it is generally accepted that it contributes to the early stages of tumor metastasis (15 16 Among the EMT features that are conserved in most epithelial cell types are the repression of the adherents junctions protein E-cadherin (E-Cad) the up-regulation of mesenchymal markers vimentin fibronectin and N-cadherin (N-Cad) and the change toward a mesenchymal-like morphology (13 17 18 Several transcription factors promote EMT through the down-regulation of E-Cad (13 15 and a central role has been given to members of the Snail family of proteins (Snail and modulate common as well as Spautin-1 specific gene regulatory pathways that likely differentiate their contribution to cancer progression and dissemination (22). An additional inducer of EMT is the extracellular cytokine transforming growth factor β (TGFβ) which can be produced and secreted by tumor cells or by the stroma. TGFβ induces EMT and cancer metastasis (23-25) possibly by promoting the early dissolution of the tight junctions that interconnect epithelial cells (26 27 AhR is functionally related to TGFβ in different cell types and and (10). Interestingly TGFβ exerts cell type-specific effects on AhR by inhibiting receptor expression and activation in A549 lung cancer cells while enhancing receptor function in HepG2 hepatoma cells (32 33 Thus it is likely that AhR and TGFβ could cross-talk during EMT. In this study we have investigated the role of AhR in EMT under both basal and TGFβ-induced conditions with the aim to determine whether or not AhR expression restrains the acquisition of a migratory EMT phenotype in epithelial cells. Thus we have used primary keratinocytes from (Santa Cruz); N-cadherin (Invitrogen); fibronectin (Chemicon) vimentin α-smooth muscle actin and β-actin (Sigma). The AhR agonist 6-formylindolo[3 Spautin-1 2 (FICZ) Spautin-1 was from Enzo and the AhR antagonist α-naphthoflavone (α-naph) was from Sigma. The pharmacological inhibitor of the TGFβ pathway SB431542 was from Selleckchem. Rhodamine-phalloidin was from Invitrogen. Cdx1 Matrigel solution was from BD Biosciences. TaqDNA polymerase was from Ecogen. iScript reverse transcription supermix and SYBR Spautin-1 Green master mix were obtained from Bio-Rad. Small hairpin RNA was from Sigma. Small interfering RNA for AhR and scrambled siRNA were synthesized by Dharmacon. The constitutively active form of the AhR (CA-AhR) was produced from the wild type mouse receptor by deleting the minimal PAS-B motif (amino acids 288-421) without altering the N-terminal half of the binding domain (PAS-A). This constitutively active receptor heterodimerizes with ARNT and has intrinsic transcriptional activity in a ligand-independent manner (34). Recombinant human TGFβ (Sigma) was added to the cultures at 10 ng/ml (primary keratinocytes and HaCaT cells) or 5 ng/ml (NMuMG cells). Control cultures were treated with the same volume of solvent (PBS). Retroviral Transduction NMuMG cells were stably transduced with expression vectors containing a small hairpin RNA for AhR (sh-AhR) or a constitutively active form of the protein (CA-AhR) as described (Stanford University Medical Center). In brief constructs LMP-sh-AhR pBABE-CA-AhR or the empty vectors pBABE+LMP were transfected by calcium phosphate precipitation in Phoenix cells and virus production was allowed for 48 h. NMuMG cells were exposed overnight to the viral supernatants and 48 h later selection was began with 1.