Role of Connexin 43 in Endothelial Cell-Induced Mural Cell Differentiation

Persistent Link:
http://hdl.handle.net/10150/306774
Title:
Role of Connexin 43 in Endothelial Cell-Induced Mural Cell Differentiation
Author:
Angelov, Stoyan N.
Issue Date:
2013
Publisher:
The University of Arizona.
Rights:
Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.
Abstract:
Objective: Endothelial cell (EC)-induced mesenchymal cell (MC) differentiation toward a mural cell phenotype requires transforming growth factor beta (TGF-β), cell contact and connexin 43 (Cx43)- or Cx45- heterocellular gap junction intercellular communication (GJIC). However, the identity of the communicated signal, the features of Cx43 required, and the possible regulatory mechanisms have not been elucidated and were investigated herein. Methods & Results: To determine whether channel functionality and the major regulatory domain (the carboxyl terminus, CT) of connexin Cx43 are necessary to support EC-induced differentiation, Cx43 deficient MCs (incapable of undergoing EC-induced mural cell differentiation without re-expression of Cx43 or Cx45) were transduced with wild-type (Cx43wt), channel dead, or truncated (Cx43tr-residues 258-382 deleted) versions of Cx43 and their ability to support EC-induced differentiation was assessed. Our data indicate that both channel functionality and presence of the CT domain are both necessary for EC-induced mural cell differentiation. Moreover, expression of Cx40 did not restore ability of MCs to undergo EC-induced mural cell differentiation, despite supporting GJIC. To determine whether (and which) specific regulatory sites in the carboxyl terminus are necessary for EC-induced mural cell differentiation, constructs of Cx43 with serine to alanine substitutions at the mitogen activated protein kinase (MAPK) or protein kinase C (PKC) target sites were introduced into Cx43 deficient MCs and their ability to undergo EC-induced differentiation was tested. The data indicated that the MAPK targeted serines (S255,279,2982) are necessary, while the PKC targeted serine (S368) is dispensable, for this process. To determine whether calcium ions might be the messengers communicated between ECs and MCs, we investigated whether elevation in EC free intracellular calcium concentration (with ionomycin treatment) can replace Cx43-mediated GJIC, activate TGF-β and induce differentiation. Conclusions: Channel functionality, CT domain and the MAPK target sites in Cx43 are all necessary, and neither alone is sufficient, for Cx43-mediated, EC-induced mural cell differentiation. Unlike Cx43, Cx40 is not capable of supporting EC-induced differentiation, despite supporting GJIC. Calcium is unlikely to be the messenger critical to TGF-β activation during EC-induced differentiation, but similar signaling pathways can be initiated. Taken together, these data support a role for connexins in EC-induced differentiation that is complex and goes beyond that of a simple conduit.
Type:
text; Electronic Dissertation
Keywords:
Endothelial Cell; Mural cell differentiation; TGF Beta; Physiological Sciences; Connexin 43
Degree Name:
Ph.D.
Degree Level:
doctoral
Degree Program:
Graduate College; Physiological Sciences
Degree Grantor:
University of Arizona
Advisor:
Burt, Janis M.

Full metadata record

DC FieldValue Language
dc.language.isoen_USen_US
dc.titleRole of Connexin 43 in Endothelial Cell-Induced Mural Cell Differentiationen_US
dc.creatorAngelov, Stoyan N.en_US
dc.contributor.authorAngelov, Stoyan N.en_US
dc.date.issued2013-
dc.publisherThe University of Arizona.en_US
dc.rightsCopyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.en_US
dc.description.abstractObjective: Endothelial cell (EC)-induced mesenchymal cell (MC) differentiation toward a mural cell phenotype requires transforming growth factor beta (TGF-β), cell contact and connexin 43 (Cx43)- or Cx45- heterocellular gap junction intercellular communication (GJIC). However, the identity of the communicated signal, the features of Cx43 required, and the possible regulatory mechanisms have not been elucidated and were investigated herein. Methods & Results: To determine whether channel functionality and the major regulatory domain (the carboxyl terminus, CT) of connexin Cx43 are necessary to support EC-induced differentiation, Cx43 deficient MCs (incapable of undergoing EC-induced mural cell differentiation without re-expression of Cx43 or Cx45) were transduced with wild-type (Cx43wt), channel dead, or truncated (Cx43tr-residues 258-382 deleted) versions of Cx43 and their ability to support EC-induced differentiation was assessed. Our data indicate that both channel functionality and presence of the CT domain are both necessary for EC-induced mural cell differentiation. Moreover, expression of Cx40 did not restore ability of MCs to undergo EC-induced mural cell differentiation, despite supporting GJIC. To determine whether (and which) specific regulatory sites in the carboxyl terminus are necessary for EC-induced mural cell differentiation, constructs of Cx43 with serine to alanine substitutions at the mitogen activated protein kinase (MAPK) or protein kinase C (PKC) target sites were introduced into Cx43 deficient MCs and their ability to undergo EC-induced differentiation was tested. The data indicated that the MAPK targeted serines (S255,279,2982) are necessary, while the PKC targeted serine (S368) is dispensable, for this process. To determine whether calcium ions might be the messengers communicated between ECs and MCs, we investigated whether elevation in EC free intracellular calcium concentration (with ionomycin treatment) can replace Cx43-mediated GJIC, activate TGF-β and induce differentiation. Conclusions: Channel functionality, CT domain and the MAPK target sites in Cx43 are all necessary, and neither alone is sufficient, for Cx43-mediated, EC-induced mural cell differentiation. Unlike Cx43, Cx40 is not capable of supporting EC-induced differentiation, despite supporting GJIC. Calcium is unlikely to be the messenger critical to TGF-β activation during EC-induced differentiation, but similar signaling pathways can be initiated. Taken together, these data support a role for connexins in EC-induced differentiation that is complex and goes beyond that of a simple conduit.en_US
dc.typetexten_US
dc.typeElectronic Dissertationen_US
dc.subjectEndothelial Cellen_US
dc.subjectMural cell differentiationen_US
dc.subjectTGF Betaen_US
dc.subjectPhysiological Sciencesen_US
dc.subjectConnexin 43en_US
thesis.degree.namePh.D.en_US
thesis.degree.leveldoctoralen_US
thesis.degree.disciplineGraduate Collegeen_US
thesis.degree.disciplinePhysiological Sciencesen_US
thesis.degree.grantorUniversity of Arizonaen_US
dc.contributor.advisorBurt, Janis M.en_US
dc.contributor.committeememberBurt, Janis M.en_US
dc.contributor.committeememberSimon, Alexander M.en_US
dc.contributor.committeememberBoitano, Scotten_US
All Items in UA Campus Repository are protected by copyright, with all rights reserved, unless otherwise indicated.