Abstract
Expression of the cellular adhesion protein N-cadherin is a critical event during epithelial-mesenchymal transition (EMT). The SMAD4 protein has been identified as a mediator of transforming growth factor-β (TGF-β) superfamily signaling, which regulates EMT, but the mechanisms linking TGF-β signaling to N-cadherin expression remain unclear. When the TGF-β pathway is activated, SMAD proteins, including the common mediator SMAD4, are subsequently translocated into the nucleus, where they influence gene transcription via SMAD binding elements (SBEs). Here we describe a mechanism for control of CDH2, the gene encoding N-cadherin, through the canonical TGFβ–SMAD4 pathway. We first identified four previously undescribed SBEs within the CDH2 promoter. Using telomerase immortalized human pancreatic ductal epithelium, we found that TGF-β stimulation prompted specific SMAD4 binding to all four SBEs. Luciferase reporter and SMAD4-knockdown experiments demonstrated that specific SMAD4 binding to the SBE located at −3790 bp to −3795 bp within the promoter region of CDH2 was necessary for TGF-β-stimulated transcription. Expression of N-cadherin on the surface of epithelial cells facilitates motility and invasion, and we demonstrated that knockdown of SMAD4 causes decreased N-cadherin expression, which results in diminished migration and invasion of human pancreatic ductal epithelial cells. Similar reduction of cell motility was produced after CDH2 knockdown. Together, these findings suggest that SMAD4 is critical for the TGF-β-driven upregulation of N-cadherin and the resultant invasive phenotype of human pancreatic ductal epithelial cells during EMT.
Highlights
The transition of epithelial cells to a mesenchymal phenotype (EMT) is a fundamental characteristic of carcinoma cells [1]
As transforming growth factor-b (TGF-b) induces EMT and the TGF-b signaling pathway is transduced by SMAD4, our study has focused on how the SMAD4 restrains N-cadherin expression in human pancreatic ductal epithelium, we hypothesize that SMAD4, through the binding of SMAD binding elements (SBEs), regulates N-cadherin expression and cell invasion and migration
These results indicated that knock down of SMAD4 in Human pancreatic nestin-expressing (HPNE) cells down-regulated TGF-b-driven EMT, it suggested a reversal of this process, known as mesenchymal-epithelial transition (MET) [14,31]
Summary
The transition of epithelial cells to a mesenchymal phenotype (EMT) is a fundamental characteristic of carcinoma cells [1]. A lineage tracing study using genetically engineered mouse models of pancreatic adenocarcinoma demonstrated that EMT of pancreatic epithelial cells leads to their migration into surrounding stroma and entry into the bloodstream. These events were observed before the formation of a solid tumor in the animals [2]. The animal lineage tracing study found that inflammation in the form of pancreatitis increased EMT and subsequent dissemination into the bloodstream [2] Observations in both mouse models and patients identify inflammation-related EMT of pancreatic epithelial cells as an outcome-determining event in pancreatic cancer
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