Cell-cell contact formation governs Ca2+ signaling by TRPC4 in the vascular endothelium: evidence for a regulatory TRPC4-beta-catenin interaction

J Biol Chem. 2010 Feb 5;285(6):4213-4223. doi: 10.1074/jbc.M109.060301. Epub 2009 Dec 8.

Abstract

TRPC4 is well recognized as a prominent cation channel in the vascular endothelium, but its contribution to agonist-induced endothelial Ca(2+) entry is still a matter of controversy. Here we report that the cellular targeting and Ca(2+) signaling function of TRPC4 is determined by the state of cell-cell adhesions during endothelial phenotype transitions. TRPC4 surface expression in human microvascular endothelial cells (HMEC-1) increased with the formation of cell-cell contacts. Epidermal growth factor recruited TRPC4 into the plasma membrane of proliferating cells but initiated retrieval of TRPC4 from the plasma membrane in quiescent, barrier-forming cells. Epidermal growth factor-induced Ca(2+) entry was strongly promoted by the formation of cell-cell contacts, and both siRNA and dominant negative knockdown experiments revealed that TRPC4 mediates stimulated Ca(2+) entry exclusively in proliferating clusters that form immature cell-cell contacts. TRPC4 co-precipitated with the junctional proteins beta-catenin and VE-cadherin. Analysis of cellular localization of fluorescent fusion proteins provided further evidence for recruitment of TRPC4 into junctional complexes. Analysis of TRPC4 function in the HEK293 expression system identified beta-catenin as a signaling molecule that enables cell-cell contact-dependent promotion of TRPC4 function. Our results place TRPC4 as a Ca(2+) entry channel that is regulated by cell-cell contact formation and interaction with beta-catenin. TRPC4 is suggested to serve stimulated Ca(2+) entry in a specific endothelial state during the transition from a proliferating to a quiescent phenotype. Thus, TRPC4 may adopt divergent, as yet unappreciated functions in endothelial Ca(2+) homeostasis and emerges as a potential key player in endothelial phenotype switching and tuning of cellular growth factor signaling.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antigens, CD / metabolism
  • Blotting, Western
  • Cadherins / metabolism
  • Calcium / metabolism*
  • Cell Adhesion / drug effects
  • Cell Communication / physiology*
  • Cell Line
  • Cell Membrane / drug effects
  • Cell Membrane / metabolism
  • Cell Proliferation / drug effects
  • Endothelial Cells / cytology
  • Endothelial Cells / drug effects
  • Endothelial Cells / metabolism
  • Endothelium, Vascular / metabolism*
  • Epidermal Growth Factor / pharmacology
  • Fluorescence Resonance Energy Transfer
  • Humans
  • Immunoprecipitation
  • Luminescent Proteins / genetics
  • Luminescent Proteins / metabolism
  • Microscopy, Fluorescence
  • Protein Binding
  • RNA Interference
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Signal Transduction*
  • TRPC Cation Channels / genetics
  • TRPC Cation Channels / metabolism*
  • beta Catenin / metabolism

Substances

  • Antigens, CD
  • Cadherins
  • Luminescent Proteins
  • Recombinant Fusion Proteins
  • TRPC Cation Channels
  • TRPC4 ion channel
  • beta Catenin
  • cadherin 5
  • Epidermal Growth Factor
  • Calcium