Abstract
The integrity of cell-cell contacts such as adherens junctions (AJ) and tight junctions (TJ) is essential for the function of epithelia. During carcinogenesis, the increased motility and invasiveness of tumor cells reflect the loss of characteristic epithelial features, including cell adhesion. While beta-catenin, a component of AJ, plays a well characterized dual role in cell adhesion and signal transduction leading to epithelial cell transformation, little is known about possible roles of tight junction components in signaling processes. Here we show that mutants of the TJ protein zonula occludens protein-1 (ZO-1), which encode the PDZ domains (ZO-1 PDZ) but no longer localize at the plasma membrane, induce a dramatic epithelial to mesenchymal transition (EMT) of Madin-Darby canine kidney I (MDCKI) cells. The observed EMT of these MDCK-PDZ cells is characterized by a repression of epithelial marker genes, a restricted differentiation potential and a significantly induced tumorigenicity. Intriguingly, the beta-catenin signaling pathway is activated in the cells expressing the ZO-1 PDZ protein. Ectopic expression of the adenomatous polyposis coli tumor suppressor gene, known to down-regulate activated beta-catenin signaling, reverts the transformed fibroblastoid phenotype of MDCK-PDZ cells. Thus, cytoplasmic localization of the ZO-1 PDZ domains induces an EMT in MDCKI cells, most likely by modulating beta-catenin signaling.
Highlights
The integrity of cell-cell contacts such as adherens junctions (AJ) and tight junctions (TJ) is essential for the function of epithelia
Progression of these areas to carcinomas in situ and malignant tumors depends on construct encoding the PDZ and Src homology 3 (SH3) domains; MDCK-PSG, Madin-Darby canine kidney I (MDCKI) cells expressing a zonula occludens protein-1 (ZO-1) construct encoding the PDZ, SH3, and GUK domains; PDZ, PSD-95/disk large tumor suppressor protein Dlg/ZO-1; SH3, Src homology domain; Tcf/Lef, T cell factor/lymphocyte enhancer factor; Transepithelial electrical resistance (TER), transepithelial electrical resistance; TJ, tight junction; ZO, zonula occludens; PCR, polymerase chain reaction; RT, reverse transcription; DMEM, Dulbecco’s modified Eagle’s medium; PAGE, polyacrylamide gel electrophoresis; bp, base pair(s); BES, 2-[bis(2-hydroxyethyl)amino]ethanesulfonic acid; PBS, phosphate-buffered saline; GST, glutathione S-transferase; MAGUK, membrane-associated guanylate kinase; TOP, multimerized wild-type Lef binding sites; FOP, multimerized mutant Lef binding sites
We show that expression of ZO-1 mutants encoding the N terminus comprising the PDZ domains that localize to the cytosol induces a dramatic epithelial to mesenchymal transition (EMT) in MDCKI cells
Summary
The integrity of cell-cell contacts such as adherens junctions (AJ) and tight junctions (TJ) is essential for the function of epithelia. Mutations in the adenomatous polyposis coli (APC) gene are thought to initiate the process, leading to aberrant crypt foci that develop into areas of benign epithelial hyperplasia or dysplasia and adenomas Progression of these areas to carcinomas in situ and malignant tumors depends on construct encoding the PDZ and SH3 domains; MDCK-PSG, MDCKI cells expressing a ZO-1 construct encoding the PDZ, SH3, and GUK domains; PDZ, PSD-95/disk large tumor suppressor protein Dlg/ZO-1; SH3, Src homology domain; Tcf/Lef, T cell factor/lymphocyte enhancer factor; TER, transepithelial electrical resistance; TJ, tight junction; ZO, zonula occludens; PCR, polymerase chain reaction; RT, reverse transcription; DMEM, Dulbecco’s modified Eagle’s medium; PAGE, polyacrylamide gel electrophoresis; bp, base pair(s); BES, 2-[bis(2-hydroxyethyl)amino]ethanesulfonic acid; PBS, phosphate-buffered saline; GST, glutathione S-transferase; MAGUK, membrane-associated guanylate kinase; TOP, multimerized wild-type Lef binding sites; FOP, multimerized mutant Lef binding sites. This process, known as epithelial to mesenchymal transition (EMT), includes the disruption of apical-basolateral polarity, the disassembly of AJ and TJ, and the ability of the cells to degrade the basement membrane, to migrate, and to form metastases at distant sites
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