A rare missense mutation in GJB3 (Cx31G45E) is associated with a unique cellular phenotype resulting in necrotic cell death

J.A. Easton, A.K. Alboulshi, M.A.F. Kamps, G.H. Brouns, M.R. Broers, B.J. Coull, V. Oji, M. van Geel, M.A.M. van Steensel, Patricia Martin

Research output: Contribution to journalArticle

4 Downloads (Pure)

Abstract

Erythrokeratodermia variabilis et progressiva (EKV-P) is caused by mutations in either theGJB3 (Cx31) or GJB4 gene (Cx30.3). We identified a rare GJB3 missense mutation,c.134G>A (p.G45E), in two unrelated patients and investigated its cellular characteristics.Expression of Cx31G45E-GFP caused previously undescribed changes within HeLa cells andHaCaT cells, a model human keratinocyte cell line. Cx31WT-GFP localised to the plasmamembrane, but expression of Cx31G45E-GFP caused vacuolar expansion of the endoplasmicreticulum (ER), the mutant protein accumulated within the ER membrane and disassembly ofthe microtubular network occurred. No ER stress responses were evoked. Cx31WT-myc-myc-6xHis and Cx31G45E-GFP co-immunoprecipitated, indicative of heteromeric interaction, butco-expression with Cx31WT-mCherry, Cx26 or Cx30.3 did not mitigate the phenotype. Cx31and Cx31G45E both co-immunoprecipated with Cx43, indicating the ability to formheteromeric connexons. WT-Cx31 and Cx43 assembled into large gap junction plaques atpoints of cell to cell contact, Cx31G45E restricted the ability of Cx43 to reach the plasmamembrane in both HaCaT cells and HeLa cells stably expressing Cx43 where the proteinsstrongly co-localised with the vacolourised ER. Cell viability assays identified an increase incell death in cells expressing Cx31G45E-GFP, which FACS analysis determined wasnecrotic. Blocking connexin channel function with 18a-Glycyrrhetinic acid did notcompletely rescue necrosis or prevent propidium iodide uptake, suggesting that expression ofCx31G45E-GFP damages the cellular membrane independent of its channel function. Ourdata suggests that entrapment of Cx43 and necrotic cell death in the epidermis could underliethe EKV skin phenotype.
Original languageEnglish
JournalExperimental Dermatology
DOIs
Publication statusPublished - 23 Mar 2018

Fingerprint

Connexin 43
Cell death
Missense Mutation
Cell Death
Phenotype
Cells
HeLa Cells
Erythrokeratodermia Variabilis
Glycyrrhetinic Acid
Membranes
Connexins
Propidium
Gap Junctions
Mutant Proteins
Keratinocytes
Epidermis
Assays
Cell Survival
Skin
Necrosis

Keywords

  • cell death
  • cell viability

Cite this

Easton, J.A. ; Alboulshi, A.K. ; Kamps, M.A.F. ; Brouns, G.H. ; Broers, M.R. ; Coull, B.J. ; Oji, V. ; Geel, M. van ; Steensel, M.A.M. van ; Martin, Patricia. / A rare missense mutation in GJB3 (Cx31G45E) is associated with a unique cellular phenotype resulting in necrotic cell death. In: Experimental Dermatology. 2018.
@article{78f0c6709e4a41c6b7be8d13e05e9d35,
title = "A rare missense mutation in GJB3 (Cx31G45E) is associated with a unique cellular phenotype resulting in necrotic cell death",
abstract = "Erythrokeratodermia variabilis et progressiva (EKV-P) is caused by mutations in either theGJB3 (Cx31) or GJB4 gene (Cx30.3). We identified a rare GJB3 missense mutation,c.134G>A (p.G45E), in two unrelated patients and investigated its cellular characteristics.Expression of Cx31G45E-GFP caused previously undescribed changes within HeLa cells andHaCaT cells, a model human keratinocyte cell line. Cx31WT-GFP localised to the plasmamembrane, but expression of Cx31G45E-GFP caused vacuolar expansion of the endoplasmicreticulum (ER), the mutant protein accumulated within the ER membrane and disassembly ofthe microtubular network occurred. No ER stress responses were evoked. Cx31WT-myc-myc-6xHis and Cx31G45E-GFP co-immunoprecipitated, indicative of heteromeric interaction, butco-expression with Cx31WT-mCherry, Cx26 or Cx30.3 did not mitigate the phenotype. Cx31and Cx31G45E both co-immunoprecipated with Cx43, indicating the ability to formheteromeric connexons. WT-Cx31 and Cx43 assembled into large gap junction plaques atpoints of cell to cell contact, Cx31G45E restricted the ability of Cx43 to reach the plasmamembrane in both HaCaT cells and HeLa cells stably expressing Cx43 where the proteinsstrongly co-localised with the vacolourised ER. Cell viability assays identified an increase incell death in cells expressing Cx31G45E-GFP, which FACS analysis determined wasnecrotic. Blocking connexin channel function with 18a-Glycyrrhetinic acid did notcompletely rescue necrosis or prevent propidium iodide uptake, suggesting that expression ofCx31G45E-GFP damages the cellular membrane independent of its channel function. Ourdata suggests that entrapment of Cx43 and necrotic cell death in the epidermis could underliethe EKV skin phenotype.",
keywords = "cell death, cell viability",
author = "J.A. Easton and A.K. Alboulshi and M.A.F. Kamps and G.H. Brouns and M.R. Broers and B.J. Coull and V. Oji and Geel, {M. van} and Steensel, {M.A.M. van} and Patricia Martin",
note = "Acceptance in SAN AAM: 12m embargo",
year = "2018",
month = "3",
day = "23",
doi = "10.1111/exd.13542",
language = "English",
journal = "Experimental Dermatology",
issn = "0906-6705",
publisher = "Wiley-Blackwell Publishing Ltd",

}

A rare missense mutation in GJB3 (Cx31G45E) is associated with a unique cellular phenotype resulting in necrotic cell death. / Easton, J.A.; Alboulshi, A.K.; Kamps, M.A.F.; Brouns, G.H.; Broers, M.R. ; Coull, B.J.; Oji, V.; Geel, M. van; Steensel, M.A.M. van; Martin, Patricia.

In: Experimental Dermatology, 23.03.2018.

Research output: Contribution to journalArticle

TY - JOUR

T1 - A rare missense mutation in GJB3 (Cx31G45E) is associated with a unique cellular phenotype resulting in necrotic cell death

AU - Easton, J.A.

AU - Alboulshi, A.K.

AU - Kamps, M.A.F.

AU - Brouns, G.H.

AU - Broers, M.R.

AU - Coull, B.J.

AU - Oji, V.

AU - Geel, M. van

AU - Steensel, M.A.M. van

AU - Martin, Patricia

N1 - Acceptance in SAN AAM: 12m embargo

PY - 2018/3/23

Y1 - 2018/3/23

N2 - Erythrokeratodermia variabilis et progressiva (EKV-P) is caused by mutations in either theGJB3 (Cx31) or GJB4 gene (Cx30.3). We identified a rare GJB3 missense mutation,c.134G>A (p.G45E), in two unrelated patients and investigated its cellular characteristics.Expression of Cx31G45E-GFP caused previously undescribed changes within HeLa cells andHaCaT cells, a model human keratinocyte cell line. Cx31WT-GFP localised to the plasmamembrane, but expression of Cx31G45E-GFP caused vacuolar expansion of the endoplasmicreticulum (ER), the mutant protein accumulated within the ER membrane and disassembly ofthe microtubular network occurred. No ER stress responses were evoked. Cx31WT-myc-myc-6xHis and Cx31G45E-GFP co-immunoprecipitated, indicative of heteromeric interaction, butco-expression with Cx31WT-mCherry, Cx26 or Cx30.3 did not mitigate the phenotype. Cx31and Cx31G45E both co-immunoprecipated with Cx43, indicating the ability to formheteromeric connexons. WT-Cx31 and Cx43 assembled into large gap junction plaques atpoints of cell to cell contact, Cx31G45E restricted the ability of Cx43 to reach the plasmamembrane in both HaCaT cells and HeLa cells stably expressing Cx43 where the proteinsstrongly co-localised with the vacolourised ER. Cell viability assays identified an increase incell death in cells expressing Cx31G45E-GFP, which FACS analysis determined wasnecrotic. Blocking connexin channel function with 18a-Glycyrrhetinic acid did notcompletely rescue necrosis or prevent propidium iodide uptake, suggesting that expression ofCx31G45E-GFP damages the cellular membrane independent of its channel function. Ourdata suggests that entrapment of Cx43 and necrotic cell death in the epidermis could underliethe EKV skin phenotype.

AB - Erythrokeratodermia variabilis et progressiva (EKV-P) is caused by mutations in either theGJB3 (Cx31) or GJB4 gene (Cx30.3). We identified a rare GJB3 missense mutation,c.134G>A (p.G45E), in two unrelated patients and investigated its cellular characteristics.Expression of Cx31G45E-GFP caused previously undescribed changes within HeLa cells andHaCaT cells, a model human keratinocyte cell line. Cx31WT-GFP localised to the plasmamembrane, but expression of Cx31G45E-GFP caused vacuolar expansion of the endoplasmicreticulum (ER), the mutant protein accumulated within the ER membrane and disassembly ofthe microtubular network occurred. No ER stress responses were evoked. Cx31WT-myc-myc-6xHis and Cx31G45E-GFP co-immunoprecipitated, indicative of heteromeric interaction, butco-expression with Cx31WT-mCherry, Cx26 or Cx30.3 did not mitigate the phenotype. Cx31and Cx31G45E both co-immunoprecipated with Cx43, indicating the ability to formheteromeric connexons. WT-Cx31 and Cx43 assembled into large gap junction plaques atpoints of cell to cell contact, Cx31G45E restricted the ability of Cx43 to reach the plasmamembrane in both HaCaT cells and HeLa cells stably expressing Cx43 where the proteinsstrongly co-localised with the vacolourised ER. Cell viability assays identified an increase incell death in cells expressing Cx31G45E-GFP, which FACS analysis determined wasnecrotic. Blocking connexin channel function with 18a-Glycyrrhetinic acid did notcompletely rescue necrosis or prevent propidium iodide uptake, suggesting that expression ofCx31G45E-GFP damages the cellular membrane independent of its channel function. Ourdata suggests that entrapment of Cx43 and necrotic cell death in the epidermis could underliethe EKV skin phenotype.

KW - cell death

KW - cell viability

U2 - 10.1111/exd.13542

DO - 10.1111/exd.13542

M3 - Article

JO - Experimental Dermatology

JF - Experimental Dermatology

SN - 0906-6705

ER -