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 threedimensional 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. Epidermal growth factor receptor (EGFR)1 is a transmembrane protein receptor with tyrosine kinase activity that triggers numerous signaling pathways (1-3). Activation of the EGFR tyrosine kinase results in the generation of a number of intracellular signals, which culminate in not only cell proliferation but also other processes that are crucial to cancer progression, including angiogenesis, metastatic spread, and the inhibition of apoptosis. These events are mediated through various downstream targets of EGFR (e.g. the serine/threonine kinase Raf and mitogen-activated protein/extracellular signalregulated kinase 1/2). In addition, Ras activation by EGFR is required for a vast array of cellular functions, foremost of which is the regulation of cellular proliferation. Activation of EGFR also results in the activation of the lipid kinase phosphatidylinositol 3-kinase, generating the second messenger phosphatidylinositol 3,4,5-trisphosphate, which in turn activates Akt. We have previously demonstrated that there is differential activation of the Akt isoforms by EGFR in esophageal cancer cells (4). Apart from the mitogen-activated protein kinase and phosphatidylinositol 3-kinase pathways, EGFR also activates other pathways such as phospholipase-C and its downstream protein kinase cascades, small GTPases such as Rho, and multiple signal transducer and activator of transcription (STAT) isoforms.EGFR activation is not only important in normal cellular processes, but it is frequently altered or overexpressed in many malignancies, especially those of squamous cell origin. Mechanisms that mediate E...

The epidermal growth factor receptor (EGFR) activates several signaling cascades in response to epidermal growth factor stimulation. One of these signaling events involves tyrosine phosphorylation of signal transducer and activator of transcription (STAT), whereas another involves activation of the phosphatidylinositol 3-OH kinase pathway. Two possibilities for STAT activation exist: a janus kinase (JAK)-dependent and a JAK-independent mechanism. Herein, we demonstrate that EGFR overexpression in primary esophageal keratinocytes activates STAT in a JAK-dependent fashion with the functional consequence of enhanced cell migration, which can be abolished by use of a JAK-specific inhibitor, AG-490. We determined the mechanisms underlying the signal transduction pathway responsible for increased cell migration. Stimulation of EGFR induces Tyr701 phosphorylation of STAT1 and initiates complex formation of STAT1 and STAT3 with JAK1 and JAK2. Thereafter, the STATs translocate to the nucleus within 15 min. In addition, we found that activation of this signaling pathway results in matrix metalloproteinase-1 (MMP-1) activity. By contrast, Akt activation does not impact the EGFR-STATs-JAKs complex formation and nuclear translocation of the STATs with subsequent MMP-1 activity, although Akt activation may contribute to cell migration through an independent mechanism. Taken together, we find that the recruitment of the STAT-JAK complex by EGFR is responsible for keratinocyte migration that, in turn, might be mediated by MMP-1 activation.

The exact mechanisms of the development of pancreatic fibrosis are still unknown. To clarify the relationship between pancreatic fibrosis and free radicals, the effect of the administration of a superoxide dismutase (SOD) inhibitor, diethyldithiocarbamate (DDC), on pancreatic fibrosis in rats was studied. A single intraperitoneal injection of 500 mg/kg of DDC significantly reduced SOD activity and significantly increased lipid peroxidation products in the pancreas, showing no histologic changes of inflammation or necrosis. Repeated administration of 500 mg/kg DDC, twice a week, caused inter- and intralobular fibrosis with atrophy of acinar cells in the pancreas for at least 2 weeks without fibrosis of the liver and kidney. Administration of allopurinol showed preventive effects against DDC-induced pancreatic fibrosis. In conclusion, repeated administration of DDC, which caused pancreatic fibrosis, is a new experimental model of pancreatic fibrosis from the viewpoint of oxidative stress.

Heparanase is an endo-beta-glucuronidase that specifically cleaves heparan sulfate (HS) chains. Heparanase is involved in the process of metastasis and angiogenesis through the degradation of HS chains of the extracellular matrix and cell surface. Recently, we demonstrated that heparanase was localized in the cell nucleus of normal esophageal epithelium and esophageal cancer, and that its expression was correlated with cell differentiation. However, the nuclear function of heparanase remains unknown. To elucidate the role of heparanase in esophageal epithelial differentiation, primary human esophageal cells were grown in monolayer as well as organotypic cultures, and cell differentiation was induced. Expression of heparanase, HS, involucrin, and p27 was determined by immunostaining and Western blotting. SF4, a novel pharmacological inhibitor, was used to specifically inhibit heparanase activity. Upon esophageal cell differentiation, heparanase was translocated from the cytoplasm to the nucleus. Such translocation of heparanase appeared to be associated with the degradation of HS chains in the nucleus and changes in the expression of keratinocyte differentiation markers such as p27 and involucrin, whose induction was inhibited by SF4. Furthermore, these in vitro observations agreed with the expression pattern of heparanase, HS, involucrin, cytokeratin 13, and p27 in normal esophageal epithelium. Nuclear translocation of heparanase and its catalytic cleavage of HS may play a critical role in the differentiation of esophageal epithelial cells. Our study provides a novel insight into the role of heparanase in an essential differentiation process.