The canonical Wnt/ÌÄå_Ìâå_-catenin signaling is involved in the remodeling of epithelial tissue during early embryonic development. An essential component of the canonical Wnt pathway is ÌÄå_Ìâå_-catenin, whose dual role in the cell allows it to function either as a bridge between cadherins and the actin cytoskeleton at the adherens junctions, or as a transcriptional transactivator of Wnt target genes when bound to lymphoid enhancer factor (LEF)/ T-cell factor (TCF) family of transcription factors. The dual function ÌÄå_Ìâå_-catenin in cell adhesion and transcription is tightly regulated in vertebrates in order to maintain a balanced level of canonical Wnt signaling. Deregulation of this conserved developmental pathway is frequently associated with disease, especially in pre-malignant lesions and metastatic disease. In this research dissertation, a novel role for the ARF6 GTPase in the regulation of Wnt/ÌÄå_Ìâå_-catenin signaling via its effect on the turnover of adhesion molecules in Madin-Darby canine kidney (MDCK) epithelial cells, is demonstrated.
Activation of ARF6 during canonical Wnt signaling led to intracellular accumulation of pools of active ÌÄå_Ìâå_-catenin by promoting E-cadherin endocytosis and triggering an intracellular ERK-CK2 signaling cascade that results in the dissociation of ÌÄå_Ìâå_-catenin from ÌÄå_Ìâå±-catenin complexes. The contribution of membrane bound ÌÄå_Ìâå_-catenin to transcription initiation was based on the presence of N-terminally dephosphorylated ÌÄå_Ìâå_-catenin, which has been shown to mediate ÌÄå_Ìâå_-catenin transactivation. Furthermore, ERK phosphorylates LRP6 to amplify the Wnt transduction pathway.
Wnt/ÌÄå_Ìâå_-catenin signaling also initiated a proliferation response that correlated with the luminal filling of epithelial glandular structures. Sustained proliferation of cells may be facilitated by ARF6-dependent endocytosis of the LRP6 receptor and ERK kinase. Furthermore, we showed that the ERK-CK2 signaling cascade mediates the proliferation response that sustains luminal filling in cysts and acinar structures. Finally, the identification of consensus sites for interaction with transcription factors regulated by Wnt in the ARF6 promoter is indicative of potential transcriptional control by signaling pathways that contribute to epithelial-mesenchymal transitions. In summary, this study identifies ARF6 as a new target of Wnt/ÌÄå_Ìâå_-catenin activation with a therapeutic potential in pre-invasive lesions of glandular epithelial tissue.