RhoA GTPase Switch Controls Cx43-Hemichannel Activity through the Contractile System

Raf Ponsaerts,Luc Leybaert,Geert Bultynck,Fréderic Hertens,Jan B Parys,Johan Vereecke,Catheleyne D’Hondt,Bernard Himpens,Anna Maria Delprato

doi:10.1371/journal.pone.0042074

Raf Ponsaerts, Luc Leybaert + Show 7 more

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https://doi.org/10.1371/journal.pone.0042074

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Journal: PLoS ONE	Publication Date: Jul 30, 2012
Citations: 48	License type: CC BY 4.0

Affiliation: Ghent University, KU Leuven

Abstract

ATP-dependent paracrine signaling, mediated via the release of ATP through plasma membrane-embedded hemichannels of the connexin family, coordinates a synchronized response between neighboring cells. Connexin 43 (Cx43) hemichannels that are present in the plasma membrane need to be tightly regulated to ensure cell viability. In monolayers of bovine corneal endothelial cells (BCEC),Cx43-mediated ATP release is strongly inhibited when the cells are treated with inflammatory mediators, in particular thrombin and histamine. In this study we investigated the involvement of RhoA activation in the inhibition of hemichannel-mediated ATP release in BCEC. We found that RhoA activation occurs rapidly and transiently upon thrombin treatment of BCEC. The RhoA activity correlated with the onset of actomyosin contractility that is involved in the inhibition of Cx43 hemichannels. RhoA activation and inhibition of Cx43-hemichannel activity were both prevented by pre-treatment of the cells with C3-toxin as well as knock down of RhoA by siRNA. These findings provide evidence that RhoA activation is a key player in thrombin-induced inhibition of Cx43-hemichannel activity. This study demonstrates that RhoA GTPase activity is involved in the acute inhibition of ATP-dependent paracrine signaling, mediated by Cx43 hemichannels, in response to the inflammatory mediator thrombin. Therefore, RhoA appears to be an important molecular switch that controls Cx43 hemichannel openings and hemichannel-mediated ATP-dependent paracrine intercellular communication under (patho)physiological conditions of stress.

Highlights

Intercellular communication can occur through direct and indirect pathways
We show that RhoA is activated upon exposure of bovine corneal endothelial cells (BCEC) to thrombin and that chemical inhibition of RhoA activity with C3 and genetic inhibition of RhoA expression with siRNA alleviates the inhibition of Connexin 43 (Cx43) hemichannels by increased contractility in BCEC induced by thrombin exposure
Our findings indicate that thrombin activates RhoA in BCEC cells and that C3-treatment prevents the inhibitory effect of thrombin on intercellular communication as assayed by Ca2z-wave propagation, ATP release and Lucifer Yellow (LY)-uptake

Summary

Introduction

Intercellular communication can occur through direct and indirect pathways. Direct signaling is possible via connexin (Cx)based gap junction channels that connect the cytoplasm of adjacent cells. An important indirect manner of intercellular communication is via paracrine signaling. The activity of Cx hemichannels is tightly regulated and characterized by a low open probability under physiological conditions to prevent deleterious events such as excessive loss of metabolites [1]. The latter may occur under pathological conditions like inflammation [2,3], ischemia [4] and oxidative stress [5]

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