Roles of aberrant hemichannel activities due to mutant connexin26 in the pathogenesis of KID syndrome

T Taki,K Sugiura,T Takeichi,M Akiyama

doi:10.1038/s41598-018-30757-3

T Taki, K Sugiura + Show 2 more

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https://doi.org/10.1038/s41598-018-30757-3

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Journal: Scientific Reports	Publication Date: Aug 27, 2018
Citations: 14	License type: open-access

Affiliation: Nagoya University, Fujita Health University

Abstract

Germline missense mutations in GJB2 encoding connexin (Cx) 26 have been found in keratitis, ichthyosis and deafness (KID) syndrome. We explored the effects of three mouse Cx26 mutants (Cx26-G12R, -G45E and -D50N) corresponding to KID syndrome-causative human mutants on hemichannel activities leading to cell death and the expression of immune response-associated genes. We analyzed the 3D images of cells expressing wild-type (WT) or mutant Cx26 molecules to demonstrate clearly the intracellular localization of Cx26 mutants and hemichannel formation. High extracellular Ca2+ conditions lead to the closure of gap junction hemichannels in Cx26-G12R or Cx26-G45E expressing cells, resulting in prohibition of the Cx26 mutant-induced cell death. Fluorescent dye uptake assays revealed that cells with Cx26-D50N had aberrantly high hemichannel activities, which were abolished by a hemichannel blocker, carbenoxolone and 18α-Glycyrrhetinic acid. These results further support the idea that abnormal hemichannel activities play important roles in the pathogenesis of KID syndrome. Furthermore, we revealed that the expressions of IL15, CCL5, IL1A, IL23R and TLR5 are down-regulated in keratinocytes expressing Cx26-D50N, suggesting that immune deficiency in KID syndrome expressing Cx26-D50N might be associated not only with skin barrier defects, but also with the down-regulated expression of immune response-related genes.

Highlights

Keratitis, ichthyosis and deafness (KID) syndrome is named for its clinical triad of erythrokeratoderma, vascularizing keratitis and bilateral sensorineural hearing loss
To examine the effects of the KID syndrome-associated Gjb[2] mutations Cx26-G12R and -G45E on the intracellular localization of Cx26, HeLa cells lacking endogenous gap junctions were transiently transfected with pIRES2-AcGFP1 Cx26-WT, -G12R or -G45E-FLAG constructs
wheat germ agglutinin (WGA) cannot bind to the gap junction plaque. These findings clearly indicate that Cx26-WT and Cx26-D50N expressed in the HeLa cells were localized to the plasma membrane, but not to the Golgi apparatus, and that WGA was unable to access the gap junction plaques consisting of Cx26-WT and Cx26-D50N

Summary

Introduction

Ichthyosis and deafness (KID) syndrome is named for its clinical triad of erythrokeratoderma, vascularizing keratitis and bilateral sensorineural hearing loss. Cxs are membrane proteins that are primarily involved in intercellular communication They are synthesized in the endoplasmic reticulum (ER)-Golgi network, and six Cx molecules are oligomerized to form a connexon (hemichannel), which docks at cell–cell contact points to form a gap junction intercellular channel that allows exchanges of electrical signals and biochemically important molecules between neighboring cells. Various experiments have shown that KID syndrome-causative GJB2 mutations result in the formation of Cx26 hemichannels with aberrant activity[15,16,17,18,19,20,21,22,23]. The present study characterizes the effects of three KID syndrome-causative Gjb[2] mutations (Cx26-G12R, -G45E and -D50N) on hemichannel activities, cell death and immune responses of the cells with information on the Ca2+ concentrations for each experiment. We used 18α-Glycyrrhetinic acid (AGA) as an additional hemichannel blocker

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