Epidermal Growth Factor Receptor Mediates Increased Cell Proliferation, Migration, and Aggregation in Esophageal Keratinocytes in Vitro and in Vivo

Claudia D Andl,Takaaki Mizushima,Hiroshi Nakagawa,Kenji Oyama,Hideki Harada,Katerina Chruma,Meenhard Herlyn,Anil K Rustgi

doi:10.1074/jbc.m209148200

Abstract

Epidermal growth factor receptor (EGFR) overexpression is observed in a number of malignancies, especially those of esophageal squamous cell origin. However, little is known about the biological functions of EGFR in primary esophageal squamous epithelial cells. Using newly established primary human esophageal squamous epithelial cells as a platform, we overexpressed EGFR through retroviral transduction and established novel three-dimensional organotypic cultures. Additionally, EGFR was targeted in a cell type- and tissue-specific fashion to the esophageal epithelium in transgenic mice. EGFR overexpression in primary esophageal keratinocytes resulted in the biochemical activation of Akt and STAT pathways and induced enhanced cell migration and cell aggregation. When established in organotypic culture, EGFR-overexpressing cells had evidence of epithelial cell hyperproliferation and hyperplasia. These effects were also observed in EGFR-overexpressing transgenic mice and the esophageal cell lines established thereof. In particular, EGFR-induced effects upon aggregation appear to be mediated through the relocalization of p120 from the cytoplasm to the membrane and increased interaction with E-cadherin. EGFR modulates cell migration through the up-regulation of matrix metalloproteinase 1. Taken together, the functional effects of EGFR overexpression help to explain its role in the initiating steps of esophageal squamous carcinogenesis.

Highlights

Epidermal growth factor receptor (EGFR)1 is a transmembrane protein receptor with tyrosine kinase activity that triggers numerous signaling pathways [1,2,3]
EGFR Overexpression and Activation by Retroviral Transduction in Normal Human Esophageal Epithelial Cells—Human primary esophageal cells, designated as EPC1 and EPC2, were established and had morphologic, cytogenetic, and biochemical properties of normal cells as illustrated by normal diploid status, expression of cytokeratins 5 and 14 found in basal cells, absence of p53 mutation, and the ability to be differentiated in a postconfluent state or with high calcium concentration (1.0 –1.2 mM)
Parental cells as well as green fluorescent protein (GFP) and EGFR-EPC2 cells were grown on a collagen gel containing human skin fibroblasts submerged in the tissue culture medium for 4 days followed by cultivation at the air-liquid interface for 7 days to induce terminal differentiation

Summary

EXPERIMENTAL PROCEDURES

Cell Lines—Primary esophageal keratinocytes, designated as EPC1 and EPC2, from normal human esophagus were established. The growth curves of EPC cells and those transduced with green fluorescent protein (GFP) or EGFR (see below) were generated by plating cells in six-well plates (1.0 ϫ 104 cells/plate) and grown in KSFM supplemented with 40 ␮g/ml bovine pituitary extract. Immunoprecipitation—Preconfluent cells starved in KBM medium and stimulated with 10 ng/ml EGF for 15 min as well as unstimulated cells were washed with phosphate-buffered saline without calcium and magnesium (PBS) and incubated with 700 ␮l of lysis buffer (1% Triton X-100, 1% Nonidet P-40, 50 mM Tris, pH 8, and proteinase inhibitors 2 ␮g/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride, 10 mM NaF, 2 mM Na3VO4, 5 mM sodium pyrophosphate; less stringent buffer does not contain Nonidet P-40) for 30 min on ice. After washing with PBS, objects were incubated with Texas Red- conjugated secondary antibody (Molecular Probes, Inc., Eugene, OR) or fluorescein isothiocyanate-conjugated secondary antibody (Roche Molecular Biochemicals) for 1 h

EGFR Induces Cell Migration and Cell Adhesion

RESULTS

DISCUSSION