SMAD4 regulates cell motility through transcription of N-cadherin in human pancreatic ductal epithelium

PLoS One. 2014 Sep 29;9(9):e107948. doi: 10.1371/journal.pone.0107948. eCollection 2014.

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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Base Sequence
  • Cadherins / genetics*
  • Cell Line, Transformed
  • Cell Movement / physiology*
  • DNA Primers
  • Electrophoretic Mobility Shift Assay
  • Epithelial-Mesenchymal Transition
  • Epithelium / metabolism
  • Humans
  • Pancreatic Ducts / metabolism*
  • Real-Time Polymerase Chain Reaction
  • Smad4 Protein / genetics
  • Smad4 Protein / physiology*
  • Transcription, Genetic / physiology*

Substances

  • Cadherins
  • DNA Primers
  • SMAD4 protein, human
  • Smad4 Protein