Rights statement: This is the peer reviewed version of the following article: Easton, JA, Albuloushi, AK, Kamps, MAF, et al. A rare missense mutation in GJB3 (Cx31G45E) is associated with a unique cellular phenotype resulting in necrotic cell death. Experimental Dermatology. doi: 10.1111/exd.13542 which has been published in final form at https://onlinelibrary.wiley.com/doi/abs/10.1111/exd.13542 This article may be used for non-commercial purposes in accordance With Wiley Terms and Conditions for self-archiving.
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Research output: Contribution to Journal/Magazine › Journal article › peer-review
Research output: Contribution to Journal/Magazine › Journal article › peer-review
}
TY - JOUR
T1 - A rare missense mutation in GJB3 (Cx31G45E) is associated with a unique cellular phenotype resulting in necrotic cell death
AU - Easton, Jennifer A.
AU - Albuloushi, Ahmad K.
AU - Kamps, Miriam A.F.
AU - Brouns, Gladys H.M.R.
AU - Broers, Jos L.V.
AU - Coull, Barry J.
AU - Oji, Vincent
AU - van Geel, Michel
AU - van Steensel, Maurice A.M.
AU - Martin, Patricia E.
N1 - This is the peer reviewed version of the following article: Easton, JA, Albuloushi, AK, Kamps, MAF, et al. A rare missense mutation in GJB3 (Cx31G45E) is associated with a unique cellular phenotype resulting in necrotic cell death. Experimental Dermatology. doi: 10.1111/exd.13542 which has been published in final form at https://onlinelibrary.wiley.com/doi/abs/10.1111/exd.13542 This article may be used for non-commercial purposes in accordance With Wiley Terms and Conditions for self-archiving.
PY - 2019/10/1
Y1 - 2019/10/1
N2 - Erythrokeratodermia variabilis et progressiva (EKV-P) is caused by mutations in either the GJB3 (Cx31) or GJB4 genes (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 and HaCaT cells, a model human keratinocyte cell line. Cx31WT-GFP localised to the plasma membrane, but expression of Cx31G45E-GFP caused vacuolar expansion of the endoplasmic reticulum (ER), the mutant protein accumulated within the ER membrane and disassembly of the microtubular network occurred. No ER stress responses were evoked. Cx31WT-myc-myc-6xHis and Cx31G45E-GFP co-immunoprecipitated, indicative of heteromeric interaction, but co-expression with Cx31WT-mCherry, Cx26 or Cx30.3 did not mitigate the phenotype. Cx31 and Cx31G45E both co-immunoprecipitated with Cx43, indicating the ability to form heteromeric connexons. WT-Cx31 and Cx43 assembled into large gap junction plaques at points of cell-to-cell contact; Cx31G45E restricted the ability of Cx43 to reach the plasma membrane in both HaCaT cells and HeLa cells stably expressing Cx43 where the proteins strongly co-localised with the vacolourised ER. Cell viability assays identified an increase in cell death in cells expressing Cx31G45E-GFP, which FACS analysis determined was necrotic. Blocking connexin channel function with 18α-glycyrrhetinic acid did not completely rescue necrosis or prevent propidium iodide uptake, suggesting that expression of Cx31G45E-GFP damages the cellular membrane independent of its channel function. Our data suggest that entrapment of Cx43 and necrotic cell death in the epidermis could underlie the EKV skin phenotype.
AB - Erythrokeratodermia variabilis et progressiva (EKV-P) is caused by mutations in either the GJB3 (Cx31) or GJB4 genes (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 and HaCaT cells, a model human keratinocyte cell line. Cx31WT-GFP localised to the plasma membrane, but expression of Cx31G45E-GFP caused vacuolar expansion of the endoplasmic reticulum (ER), the mutant protein accumulated within the ER membrane and disassembly of the microtubular network occurred. No ER stress responses were evoked. Cx31WT-myc-myc-6xHis and Cx31G45E-GFP co-immunoprecipitated, indicative of heteromeric interaction, but co-expression with Cx31WT-mCherry, Cx26 or Cx30.3 did not mitigate the phenotype. Cx31 and Cx31G45E both co-immunoprecipitated with Cx43, indicating the ability to form heteromeric connexons. WT-Cx31 and Cx43 assembled into large gap junction plaques at points of cell-to-cell contact; Cx31G45E restricted the ability of Cx43 to reach the plasma membrane in both HaCaT cells and HeLa cells stably expressing Cx43 where the proteins strongly co-localised with the vacolourised ER. Cell viability assays identified an increase in cell death in cells expressing Cx31G45E-GFP, which FACS analysis determined was necrotic. Blocking connexin channel function with 18α-glycyrrhetinic acid did not completely rescue necrosis or prevent propidium iodide uptake, suggesting that expression of Cx31G45E-GFP damages the cellular membrane independent of its channel function. Our data suggest that entrapment of Cx43 and necrotic cell death in the epidermis could underlie the EKV skin phenotype.
KW - Cell death
KW - Connexin
KW - EKV-P
KW - Heteromeric connexins
U2 - 10.1111/exd.13542
DO - 10.1111/exd.13542
M3 - Journal article
AN - SCOPUS:85046656514
VL - 28
SP - 1106
EP - 1113
JO - Experimental Dermatology
JF - Experimental Dermatology
SN - 0906-6705
IS - 10
ER -