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In Vitro Analysis of PDZ-dependent CFTR Macromolecular Signaling Complexes
10:05

In Vitro Analysis of PDZ-dependent CFTR Macromolecular Signaling Complexes

Published on: August 13, 2012

Deregulated expression and function of CFTR and Cl- secretion after activation of the Ras and Src/PyMT pathways in

S E Davenport1, M Mergey, G Cherqui

  • 1Cystic Fibrosis/Pulmonary Research and Treatment Center, Department of Medicine, University of North Carolina at Chapel Hill 27599-7248, USA.

Biochemical and Biophysical Research Communications
|December 13, 1996
PubMed

Insights

Activated Ras and Src/PyMT pathways up-regulate PKC alpha, inhibiting cystic fibrosis transmembrane conductance regulator (CFTR) expression and chloride secretion in colon cells. This suggests CFTR is essential for colonic ion transport.

Area of Science:

  • Cell biology
  • Molecular biology
  • Gastroenterology

Background:

  • The cystic fibrosis transmembrane conductance regulator (CFTR) is crucial for chloride secretion in epithelial cells.
  • Ras and Src/PyMT signaling pathways are implicated in various cellular processes, including proliferation and differentiation.
  • Dysregulation of ion channels can lead to gastrointestinal disorders.

Purpose of the Study:

  • To investigate the impact of activated Ras and Src/Polyoma Middle T (PyMT) signaling pathways on CFTR expression and function in human colonic Caco-2 cells.
  • To determine the role of Protein Kinase C (PKC) in mediating these effects.
  • To elucidate the mechanisms of chloride secretion in Caco-2 cells.

Main Methods:

  • Utilized Caco-2 cell lines transfected with control vectors (Caco-2-H) or PyMT (Caco-2-MT).
  • Measured short circuit current (Isc) in response to forskolin and ATP to assess CFTR-mediated and calcium-mediated chloride secretion.
  • Analyzed PKC activity and CFTR mRNA expression following phorbol 12-myristate 13-acetate (PMA) treatment.

Main Results:

  • Caco-2-H cells exhibited forskolin- and ATP-stimulated Isc, indicating functional CFTR and calcium-activated chloride channels.
  • Caco-2-MT cells, with high PKC levels, showed no sustained Isc response to forskolin or ATP.
  • PMA treatment down-regulated PKC and restored CFTR mRNA but not functional CFTR in Caco-2-MT cells.

Conclusions:

  • Stable upregulation of PKC alpha, driven by Ras or Src/PyMT pathways, leads to the absence of CFTR expression and chloride secretion.
  • Chloride secretion in Caco-2 cells is primarily mediated by CFTR, with no functional alternate calcium-activated chloride channel.
  • These findings highlight a critical link between specific signaling pathways, PKC activity, and CFTR regulation in the colon.

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