Endothelin-1 Decreases Gap Junctional Intercellular Communication by Inducing Phosphorylation of Connexin 43 in Human Ovarian Carcinoma Cells

Pier Giorgio Natali,Laura Rosanò,Maria Rita Nicotra,Valeriana Di Castro,Francesca Spinella,Anna Bagnato

doi:10.1074/jbc.m304785200

Abstract

Endothelin-1 (ET-1) is overexpressed in ovarian carcinoma and acts as an autocrine factor selectively through the ETA receptor (ETAR) to promote tumor cell proliferation, survival, neovascularization, and invasiveness. Loss of gap junctional intercellular communication (GJIC) is critical for tumor progression by allowing the cells to escape growth control. Exposure of HEY and OVCA 433 ovarian carcinoma cell lines to ET-1 led to a 50-75% inhibition in intercellular communication and to a decrease in the connexin 43 (Cx43)-based gap junction plaques. To investigate the phosphorylation state of Cx43, ovarian carcinoma cell lysates were immunoprecipitated and transient tyrosine phosphorylation of Cx43 was detected in ET-1-treated cells. BQ 123, a selective ETAR antagonist, blocked the ET-1-induced Cx43 phosphorylation and cellular uncoupling. Gap junction closure was prevented by tyrphostin 25 and by the selective c-Src inhibitor, PP2. Furthermore, the increased Cx43 tyrosine phosphorylation was correlated with ET-1-induced increase of c-Src activity, and PP2 suppressed the ET-1-induced Cx43 tyrosine phosphorylation, indicating that inhibition of Cx43-based GJIC is mainly mediated by the Src tyrosine kinase pathway. In vivo, the inhibition of human ovarian tumor growth in nude mice induced by the potent ETAR antagonist, ABT-627, was associated with a reduction of Cx43 phosphorylation. These findings indicate that the signaling mechanisms involved in GJIC disruption on ovarian carcinoma cells depend on ETAR activation, which leads to the Cx43 tyrosine phosphorylation mediated by c-Src, suggesting that ETAR blockade may contribute to the control of ovarian carcinoma growth and progression also by preventing the loss of GJIC.

Highlights

Endothelin-1 (ET-1) is overexpressed in ovarian carcinoma and acts as an autocrine factor selectively through the ETA receptor (ETAR) to promote tumor cell proliferation, survival, neovascularization, and invasiveness
These findings indicate that the signaling mechanisms involved in gap junctional intercellular communication (GJIC) disruption on ovarian carcinoma cells depend on ETAR activation, which leads to the connexin 43 (Cx43) tyrosine phosphorylation mediated by c-Src, suggesting that ETAR blockade may contribute to the control of ovarian carcinoma growth and progression by preventing the loss of GJIC
Using two ovarian carcinoma cell lines, HEY and OVCA 433, in which the ET-1/ ETAR autocrine pathway is biologically active, we report here for the first time in tumor cells that 1) ET-1 significantly decreases intercellular communication and Cx43 expression at the cell surface; 2) these activities require ETAR activation; and 3) Cx43 phosphorylation is mainly mediated by the Src tyrosine kinase pathway

Summary

Introduction

Endothelin-1 (ET-1) is overexpressed in ovarian carcinoma and acts as an autocrine factor selectively through the ETA receptor (ETAR) to promote tumor cell proliferation, survival, neovascularization, and invasiveness. The inhibition of human ovarian tumor growth in nude mice induced by the potent ETAR antagonist, ABT-627, was associated with a reduction of Cx43 phosphorylation These findings indicate that the signaling mechanisms involved in GJIC disruption on ovarian carcinoma cells depend on ETAR activation, which leads to the Cx43 tyrosine phosphorylation mediated by c-Src, suggesting that ETAR blockade may contribute to the control of ovarian carcinoma growth and progression by preventing the loss of GJIC. ET-1 acts an autocrine factor selectively through ETAR [2] Ligand binding to this receptor results in activation of a pertussis toxin-insensitive G protein that stimulates phospholipase C activity and increases intracellular Ca2ϩ levels, activation of protein kinase C, mitogen-activated protein kinase, and p125 focal adhesion kinase phosphorylation [11]. GJs are composed of transmembrane channel proteins, termed connexins (Cxs), that directly connect the cytoplasm of two adjacent cells, allowing the cells to review and shape the functional state of their neighbors by exchanging signaling molecules, a process called gap junctional intercellular communication (GJIC) [22]

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