E-cadherin Differentially Regulates the Assembly of Connexin43 and Connexin32 into Gap Junctions in Human Squamous Carcinoma Cells

Souvik Chakraborty,Shalini Mitra,Matthias M Falk,Steve H Caplan,Margaret J Wheelock,Keith R Johnson,Parmender P Mehta

doi:10.1074/jbc.m109.053348

Souvik Chakraborty, Shalini Mitra + Show 5 more

Open Access

https://doi.org/10.1074/jbc.m109.053348

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Abstract

It is as yet unknown how the assembly of connexins (Cx) into gap junctions (GJ) is initiated upon cell-cell contact. We investigated whether the trafficking and assembly of Cx43 and Cx32 into GJs were contingent upon cell-cell adhesion mediated by E-cadherin. We also examined the role of the carboxyl termini of these Cxs in initiating the formation of GJs. Using cadherin and Cx-null cells, and by introducing Cx43 and Cx32, either alone or in combination with E-cadherin, our studies demonstrated that E-cadherin-mediated cell-cell adhesion was neither essential nor sufficient to initiate GJ assembly de novo in A431D human squamous carcinoma cells. However, E-cadherin facilitated the growth and assembly of preformed GJs composed of Cx43, although the growth of cells on Transwell filters was required to initiate the assembly of Cx32. Our results also documented that the carboxyl termini of both Cxs were required in this cell type to initiate the formation of GJs de novo. Our findings also showed that GJ puncta composed of Cx43 co-localized extensively with ZO-1 and actin fibers at cell peripheries and that ZO-1 knockdown attenuated Cx43 assembly. These findings suggest that the assembly of Cx43 and Cx32 into GJs is differentially modulated by E-cadherin-mediated cell-cell adhesion and that direct or indirect cross-talk between carboxyl tails of Cxs and actin cytoskeleton via ZO-1 may regulate GJ assembly and growth.

Highlights

Because some Cxs are expressed in a tissue-specific manner [18], and have divergent carboxyl termini that might interact with the cadherins directly or indirectly to regulate gap junctions (GJ) assembly [2, 18, 21, 22], we assessed the role of these termini in initiating the formation of GJs in the presence and absence of E-cadherin
Using cadherin- and Cx-null A431D cells, derived from human squamous carcinoma cell line A431(19, 20), and by introducing Cx43, a ubiquitously expressed Cx [22], and Cx32, a Cx preferentially expressed by the well differentiated and polarized cells [23], either alone or in combination with E-cadherin, we show that E-cadherin-mediated cell-cell adhesion facilitates the growth and assembly of only preformed GJs but is not sufficient to trigger the assembly of GJs de novo
Our data imply that the assembly of different Cxs into GJs is governed in a complex manner by E-cadherin, which in turn may depend on the motifs in the Cxs themselves and additional proteins that permit or impede docking of connexons and clustering of gap junctional channels

Summary

Introduction

We sought to investigate if cell-cell adhesion mediated by E-cadherin expression was sufficient by itself to induce the assembly of Cxs into GJs or whether additional events were required. Because both cadherins and Cxs belong to a large family of related proteins, and most cells in vivo and in vitro are likely to express more than one family member [2, 10, 18], we thought to delineate these mechanisms in a cell culture model. Our data imply that the assembly of different Cxs into GJs is governed in a complex manner by E-cadherin, which in turn may depend on the motifs in the Cxs themselves and additional proteins that permit or impede docking of connexons and clustering of gap junctional channels

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