Cell-cell contact formation governs Ca2+ signaling by TRPC4 in the vascular endothelium: evidence for a regulatory TRPC4-beta-catenin interaction.
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.
Graziani
Annarita
A
From the Institute of Pharmaceutical Sciences-Pharmacology and Toxicology, University of Graz, Universitätsplatz 2, 8010 Graz, Austria.
Poteser
Michael
M
From the Institute of Pharmaceutical Sciences-Pharmacology and Toxicology, University of Graz, Universitätsplatz 2, 8010 Graz, Austria.
Heupel
Wolfgang-Moritz
WM
the Institute of Anatomy and Cell Biology, University of Würzburg, Koellikerstrasse 6, 97070 Würzburg, Germany.
Schleifer
Hannes
H
From the Institute of Pharmaceutical Sciences-Pharmacology and Toxicology, University of Graz, Universitätsplatz 2, 8010 Graz, Austria.
Krenn
Martin
M
From the Institute of Pharmaceutical Sciences-Pharmacology and Toxicology, University of Graz, Universitätsplatz 2, 8010 Graz, Austria.
Drenckhahn
Detlev
D
the Institute of Anatomy and Cell Biology, University of Würzburg, Koellikerstrasse 6, 97070 Würzburg, Germany.
Romanin
Christoph
C
the Institute of Biophysics, University of Linz, A-4040 Linz, Austria, and.
Baumgartner
Werner
W
the Department of Cellular Neurobionics, Institute of Biology II, RWTH-Aachen University, Kopernikusstrasse 16, 52056 Aachen, Germany.
Groschner
Klaus
K
From the Institute of Pharmaceutical Sciences-Pharmacology and Toxicology, University of Graz, Universitätsplatz 2, 8010 Graz, Austria. Electronic address: klaus.groschner@uni-graz.at.
D015703:Antigens, CD / Q000378:metabolism; D015153:Blotting, Western; D015820:Cadherins / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D002448:Cell Adhesion / Q000187:drug effects; D002450:Cell Communication / Q000502:physiology*; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D049109:Cell Proliferation / Q000187:drug effects; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D004730:Endothelium, Vascular / Q000378:metabolism*; D004815:Epidermal Growth Factor / Q000494:pharmacology; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D047468:Immunoprecipitation; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence; D011485:Protein Binding; D034622:RNA Interference; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D050052:TRPC Cation Channels / Q000235:genetics / Q000378:metabolism*; D051176:beta Catenin / Q000378:metabolism; D000099152:Cadherin 5
D015703:Antigens, CD; D015820:Cadherins; D002118:Calcium; D004815:Epidermal Growth Factor; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D050052:TRPC Cation Channels; D051176:beta Catenin; D000099152:Cadherin 5; C489571:TRPC4 ion channel