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Role of mural Cx43 in vascular interactions and its regulation by breast tumor cells

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abstract
On normal blood vessels, vascular mural cells (pericytes and smooth muscle cells) serve to stabilize the vessel and render the endothelium quiescent. Tumor vessels, in contrast, exhibit abnormal association between mural cells and endothelial cells which allows the endothelium to be angiogenic. The mechanism(s) by which tumors prevent mural-cell mediated vessel stabilization have not been well elucidated. We demonstrate that heterotypic connexin 43 (Cx43)-dependent gap junction activity is required for mural cell-induced quiescence of endothelial cells, and that long term exposure of mural cells to media conditioned by aggressive MDA-MB-231 breast tumor cells downregulates Cx43 mRNA and protein to release endothelial cells from mural cell-mediated growth inhibition. We also report that Ang-Tie signaling is involved in tumor induced Cx43 loss and release of endothelial proliferation. Since Cx43 function and half-life can also be modulated by phosphorylation, we investigated the role of phosphorylation in tumor-induced regulation of Cx43 function. In the present study, we show that short-term exposure of mural cells to tumor conditioned media (CM) activates PKC signaling in mural cells, resulting in inactivating phosphorylation of Cx43 at the PKC site S368 that is both rapid and sustained. Although tumor CM activates several of the PKC isoforms expressed by vSMC, using a panel of pharmacological inhibitors, we have identified PKC as the isoform partially responsible for inhibitory phosphorylation at S368. We confirmed that the mechanisms by which tumors inactivate Cx43, by mRNA downregulation and by phosphorylation are distinct, as PKC inhibition prevents S368 phosphorylation but does not protect against Cx43 loss following long-term tumor CM treatment. Similarly, tumor mural CM activates mural cell MAPKs to induce phosphorylation at Cx43 S255 and S279/282 while MAPK inhibitor UO126 prevents target site phosphorylation, it does not prevent Cx43 protein decrease. Importantly, using the GAP26 peptide we demonstrate that inhibition of gap junction function without protein loss is sufficient to prevent mural cell-mediated endothelial growth inhibition. These data suggest that tumors can override mural cell inhibition of endothelial proliferation to promote angiogenesis in two distinct ways: 1) rapid inactivation of intact Cx43 protein by phosphorylation, and 2) long-term mRNA-mediated loss of Cx43 protein. Strategies to prevent vascular Cx43 loss or inactivation may therefore provide a novel means to inhibit angiogenesis or stabilize tumor vasculature for therapeutic gain.
subject
Angiogenesis
breast tumor
Connexin 43
endothelial proliferation
Gap junction
vascular normalization
contributor
Choudhary, Mayur (author)
Metheny-Barlow, Linda J (committee chair)
Christ, George J (committee member)
Seals, Darren F (committee member)
Kridel, Steven J (committee member)
Soker, Shay (committee member)
date
2012-09-05T08:35:22Z (accessioned)
2014-09-05T08:30:08Z (available)
2012 (issued)
degree
Cancer Biology (discipline)
embargo
2014-09-05 (terms)
identifier
http://hdl.handle.net/10339/37447 (uri)
language
en (iso)
publisher
Wake Forest University
title
Role of mural Cx43 in vascular interactions and its regulation by breast tumor cells
type
Dissertation

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