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P190B RhoGAP Overexpression in the Developing Mammary Epithelium Induces TGFβ-dependent Fibroblast Activation

Rho GTPases mediate stromal-epithelial interactions that are important for mammary epithelial cell (MEC) morphogenesis. Increased extracellular matrix (ECM) deposition and reorganization affect MEC morphogenesis in a Rho GTPase-dependent manner. Although the effects of altered ECM on MEC morphogenes...

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Autores principales: Gillette, Melissa, Bray, Kristi, Blumenthaler, Alisa, Vargo-Gogola, Tracy
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2013
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3661508/
https://www.ncbi.nlm.nih.gov/pubmed/23717689
http://dx.doi.org/10.1371/journal.pone.0065105
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author Gillette, Melissa
Bray, Kristi
Blumenthaler, Alisa
Vargo-Gogola, Tracy
author_facet Gillette, Melissa
Bray, Kristi
Blumenthaler, Alisa
Vargo-Gogola, Tracy
author_sort Gillette, Melissa
collection PubMed
description Rho GTPases mediate stromal-epithelial interactions that are important for mammary epithelial cell (MEC) morphogenesis. Increased extracellular matrix (ECM) deposition and reorganization affect MEC morphogenesis in a Rho GTPase-dependent manner. Although the effects of altered ECM on MEC morphogenesis have been described, how MECs regulate stromal deposition is not well understood. Previously, we showed that p190B RhoGAP overexpression disrupts mammary gland morphogenesis by inducing hyperbranching in association with stromal alterations. We therefore hypothesized that MEC overexpression of p190B regulates paracrine interactions to impact fibroblast activation. Using a combination of in vivo morphometric and immunohistochemical analyses and primary cell culture assays, we found that p190B overexpression in MECs activates fibroblasts leading to increased collagen, fibronectin, and laminin production and elevated expression of the collagen crosslinking enzyme lysyl oxidase. Phosphorylation of the TGF-β effector SMAD2 and expression of the TGF-β target gene αSma were increased in p190B-associated fibroblasts, suggesting that elevated TGF-β signaling promoted fibroblast activation. Mechanical tension and TGF-β cooperate to activate fibroblasts. Interestingly, active TGF-β was elevated in conditioned medium from p190B overexpressing MECs compared to control MECs, and p190B overexpressing MECs exhibited increased contractility in a collagen gel contraction assay. These data suggest that paracrine signaling from the p190B overexpressing MECs may activate TGF-β signaling in adjacent fibroblasts. In support of this, transfer of conditioned medium from p190B overexpressing MECs onto wildtype fibroblasts or co-culture of p190B overexpressing MECs with wildtype fibroblasts increased SMAD2 phosphorylation and mRNA expression of ECM genes in the fibroblasts when compared to fibroblasts treated with control CM or co-cultured with control MECs. The increased ECM gene expression and SMAD2 phosphorylation were blocked by treatment with a TGF-β receptor inhibitor. Taken together, these data suggest that p190B overexpression in the mammary epithelium induces fibroblast activation via elevated TGF-β paracrine signaling.
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spelling pubmed-36615082013-05-28 P190B RhoGAP Overexpression in the Developing Mammary Epithelium Induces TGFβ-dependent Fibroblast Activation Gillette, Melissa Bray, Kristi Blumenthaler, Alisa Vargo-Gogola, Tracy PLoS One Research Article Rho GTPases mediate stromal-epithelial interactions that are important for mammary epithelial cell (MEC) morphogenesis. Increased extracellular matrix (ECM) deposition and reorganization affect MEC morphogenesis in a Rho GTPase-dependent manner. Although the effects of altered ECM on MEC morphogenesis have been described, how MECs regulate stromal deposition is not well understood. Previously, we showed that p190B RhoGAP overexpression disrupts mammary gland morphogenesis by inducing hyperbranching in association with stromal alterations. We therefore hypothesized that MEC overexpression of p190B regulates paracrine interactions to impact fibroblast activation. Using a combination of in vivo morphometric and immunohistochemical analyses and primary cell culture assays, we found that p190B overexpression in MECs activates fibroblasts leading to increased collagen, fibronectin, and laminin production and elevated expression of the collagen crosslinking enzyme lysyl oxidase. Phosphorylation of the TGF-β effector SMAD2 and expression of the TGF-β target gene αSma were increased in p190B-associated fibroblasts, suggesting that elevated TGF-β signaling promoted fibroblast activation. Mechanical tension and TGF-β cooperate to activate fibroblasts. Interestingly, active TGF-β was elevated in conditioned medium from p190B overexpressing MECs compared to control MECs, and p190B overexpressing MECs exhibited increased contractility in a collagen gel contraction assay. These data suggest that paracrine signaling from the p190B overexpressing MECs may activate TGF-β signaling in adjacent fibroblasts. In support of this, transfer of conditioned medium from p190B overexpressing MECs onto wildtype fibroblasts or co-culture of p190B overexpressing MECs with wildtype fibroblasts increased SMAD2 phosphorylation and mRNA expression of ECM genes in the fibroblasts when compared to fibroblasts treated with control CM or co-cultured with control MECs. The increased ECM gene expression and SMAD2 phosphorylation were blocked by treatment with a TGF-β receptor inhibitor. Taken together, these data suggest that p190B overexpression in the mammary epithelium induces fibroblast activation via elevated TGF-β paracrine signaling. Public Library of Science 2013-05-22 /pmc/articles/PMC3661508/ /pubmed/23717689 http://dx.doi.org/10.1371/journal.pone.0065105 Text en © 2013 Gillette et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Gillette, Melissa
Bray, Kristi
Blumenthaler, Alisa
Vargo-Gogola, Tracy
P190B RhoGAP Overexpression in the Developing Mammary Epithelium Induces TGFβ-dependent Fibroblast Activation
title P190B RhoGAP Overexpression in the Developing Mammary Epithelium Induces TGFβ-dependent Fibroblast Activation
title_full P190B RhoGAP Overexpression in the Developing Mammary Epithelium Induces TGFβ-dependent Fibroblast Activation
title_fullStr P190B RhoGAP Overexpression in the Developing Mammary Epithelium Induces TGFβ-dependent Fibroblast Activation
title_full_unstemmed P190B RhoGAP Overexpression in the Developing Mammary Epithelium Induces TGFβ-dependent Fibroblast Activation
title_short P190B RhoGAP Overexpression in the Developing Mammary Epithelium Induces TGFβ-dependent Fibroblast Activation
title_sort p190b rhogap overexpression in the developing mammary epithelium induces tgfβ-dependent fibroblast activation
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3661508/
https://www.ncbi.nlm.nih.gov/pubmed/23717689
http://dx.doi.org/10.1371/journal.pone.0065105
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