AKT induces senescence in primary esophageal epithelial cells but is permissive for differentiation as revealed in organotypic culture.

Epidermal growth factor receptor (EGFR) overexpression and activation is critical in the initiation and progression of cancers, especially those of epithelial origin. EGFR activation is associated with the induction of divergent signal transduction pathways and a gamut of cellular processes; however, the cell-type and tissue-type specificity conferred by certain pathways remains to be elucidated. In the context of the esophageal epithelium, a prototype stratified squamous epithelium, EGFR overexpression is relevant in the earliest events of carcinogenesis as modeled in a three-dimensional organotypic culture system. We demonstrate that the phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway, and not the MEK/MAPK (mitogen-activated protein kinase) pathway, is preferentially activated in EGFR-mediated esophageal epithelial hyperplasia, a premalignant lesion. The hyperplasia was abolished with direct inhibition of PI3K and of AKT but not with inhibition of the MAPK pathway. With the introduction of an inducible AKT vector in both primary and immortalized esophageal epithelial cells, we find that AKT overexpression and activation is permissive for complete epithelial formation in organotypic culture, but imposes a growth constraint in cells grown in monolayer. In organotypic culture, AKT mediates changes related to cell shape and size with an expansion of the differentiated compartment.

Central role of the plakoglobin-binding domain for desmoglein 3 incorporation into desmosomes.

The carboxy-termini of classical cadherins and desmocollins have been shown to play an important role in initiating desmosome assembly. In this study we wanted to determine whether the carboxy- terminal cytoplasmic domains of desmoglein 3 are important for targeting it to the desmosome. By generating stably transfected A431 cell lines with chimeric constructs encoding for the extracellular domain of E-cadherin and the transmembrane and intracellular region of human desmoglein 3, we could show that the cytoplasmic tail is sufficient to target the protein to the desmosome. By generating truncations of the carboxy-terminus we investigated the importance of the various intracellular subdomains. Whereas the construct encoding the intracellular cadherin-type segment domain still allowed its incorporation into the desmosome, further truncation, leaving only the intracellular anchor domain, did not. Deletion of the 87 amino acid long plakoglobin-binding site within the intracellular cadherin-type segment domain demonstrated that this region is essential for targeting desmoglein 3 to the desmosome. Absent the plakoglobin-binding site the chimeric molecule colocalizes with beta-catenin rather than desmoplakin. We conclude that binding of plakoglobin to desmoglein 3 is an important step in desmosome assembly and leads to the incorporation of desmoglein 3 into the desmosome.