Mechanistic effect of the human GJB6 gene and its mutations in HaCaT cell proliferation and apoptosis

Lu, Yuting; Zhang, Ruili; Wang, Zhenying; Zhou, Shuhua; Song, Yun‐Chun; Chen, Lamei; Chen, Nan; Liu, Wenmin; Ji, C Y; Wu, Wenxian; Zhang, Li

doi:10.1590/1414-431x20187560

Cited by 7 publications

(9 citation statements)

References 17 publications

(20 reference statements)

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“…1). There was also a distinct lack of gap junction plaques between cells expressing mutant Cx30-A88V, indicating that HEI-OC1 cells are also susceptible to Cx30-A88V-induced cytotoxicity, as seen previously in HeLa, REK and HaCaT cells (Berger et al, 2014;Essenfelder et al, 2004;Lu et al, 2018). To further elucidate the subcellular localization of the Cx30-A88V mutant in the limited number of cells that survived, we immunostained for markers of intracellular compartments and found that a substantial amount of Cx30-A88V co-localized with GM130, a marker for the Golgi apparatus ( Fig.…”

Section: Resultssupporting

confidence: 72%

“…The Cx30-A88V mutant is cytotoxic to HEI-OC1 cells Our lab and others have shown that the Cx30-A88V mutant is cytotoxic when overexpressed in HeLa and REK cell lines, and that co-expression of Cx26 is unable to rescue this effect (Berger et al, 2014;Essenfelder et al, 2004;Lu et al, 2018). To determine whether cochlear cells, which would normally express Cx30 in vivo, are particularly resistant to the cytotoxic effects of the mutant, Cx30…”

Section: Resultsmentioning

confidence: 99%

“…Although the Cx30-A88V mutant causes an ectodermal dysplasia known as Clouston syndrome, it has not been reported to cause hearing loss when patients have a normal complement of Cx26 (Jan et al, 2004;Sugiura et al, 2013). This is surprising for two reasons: (1) we and others have shown that the Cx30-A88V mutant is highly toxic to cells when overexpressed in an in vitro system (Berger et al, 2014;Essenfelder et al, 2004;Lu et al, 2018), and (2) Cx30 is highly abundant in the inner ear (Ahmad et al, 2003;Forge et al, 2003b;Kikuchi et al, 1995;Lautermann et al, 1998), which suggests that any perturbations in its normal function could have negative consequences in the exquisitely sensitive sensory epithelium (Johnson et al, 2017;Sun et al, 2005;Zhang et al, 2005). In the present study, we aimed to provide an improved understanding of the role Cx30 plays in supporting the cochlear cells that are essential for hearing across a normal lifespan.…”

Section: Discussionmentioning

confidence: 99%

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The connexin30 A88V mutant reduces cochlear gap junction expression and confers long-term protection against hearing loss

Kelly

Abitbol

Hulme

et al. 2018

Journal of Cell Science

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Mutations in the genes that encode the gap junction proteins connexin 26 (Cx26, encoded by GJB2) and Cx30 (GJB6) are the leading cause of hereditary hearing loss. That said, the Cx30 p.Ala88Val (A88V) mutant causes Clouston syndrome, but not hearing loss. Here, we report that the Cx30-A88V mutant, despite being toxic to inner ear-derived HEI-OC1 cells, conferred remarkable long-term protection against age-related high frequency hearing loss in Cx30 A88V/A88V mice. During early development, there were no overt structural differences in the cochlea between genotypes, including a normal complement of hair cells; however, the supporting cell Cx30 gap junction plaques in mutant mice were reduced in size. In adulthood, Cx30 A88V/A88V mutant mice had a reduction of cochlear Cx30 mRNA and protein, yet a full complement of hair cells. Conversely, the age-related high frequency hearing loss in Cx30 +/+ and Cx30 +/A88V mice was due to extensive loss of outer hair cells. Our data suggest that the Cx30-A88V mutant confers long-term hearing protection and prevention of hair cell death, possibly via a feedback mechanism that leads to the reduction of total Cx30 gap junction expression in the cochlea.

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Section: Resultssupporting

confidence: 72%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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The connexin30 A88V mutant reduces cochlear gap junction expression and confers long-term protection against hearing loss

Kelly

Abitbol

Hulme

et al. 2018

Journal of Cell Science

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“…In both HeLa cells and rat epidermal keratinocytes (REK cells), expression of Cx30 p.A88V correlated with cell death due to potent activation of cleaved caspase-3 [15] . Likewise HaCaT cells, a human-derived keratinocyte cell line [16] , became apoptotic via activation of caspase-3, -8, -9, and PARA following expression of Cx30 p.G11R or Cx30 p.A88V [17] .…”

Section: Introductionmentioning

confidence: 99%

A potent antagonist antibody targeting connexin hemichannels alleviates Clouston syndrome symptoms in mutant mice

et al. 2020

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Background Numerous currently incurable human diseases have been causally linked to mutations in connexin (Cx) genes. In several instances, pathological mutations generate abnormally active Cx hemichannels, referred to also as “leaky” hemichannels. The goal of this study was to assay the in vivo efficacy of a potent antagonist antibody targeting Cx hemichannels. Methods We employed the antibody to treat Cx30 A88V/A88V adult mutant mice, the only available animal model of Clouston syndrome, a rare orphan disease caused by Cx30 p.A88V leaky hemichannels. To gain mechanistic insight into antibody action, we also performed patch clamp recordings, Ca 2+ imaging and ATP release assay in vitro . Findings Two weeks of antibody treatment sufficed to repress cell hyperproliferation in skin and reduce hypertrophic sebaceous glands (SGs) to wild type (wt) levels. These effects were obtained whether mutant mice were treated topically, by application of an antibody cream formulation, or systemically, by intraperitoneal antibody injection. Experiments with mouse primary keratinocytes and HaCaT cells revealed the antibody blocked Ca 2+ influx and diminished ATP release through leaky Cx30 p.A88V hemichannels. Interpretation Our results show anti-Cx antibody treatment was effective in vivo and sufficient to counteract the effects of pathological connexin expression in Cx30 A88V/A88V mice. In vitro experiments suggest antibodies gained control over leaky hemichannels and contributed to restoring epidermal homeostasis. Therefore, regulating cell physiology by antibodies targeting the extracellular domain of Cxs may enforce an entirely new therapeutic strategy. These findings support the further development of antibodies as drugs to address unmet medical needs for Cx-related diseases. Fund Fondazione Telethon, GGP19148; University of Padova, SID/BIRD187130; Consiglio Nazionale delle Ricerche, DSB.AD008.370.003\TERABIO-IBCN; National Science Foundation of China, 31770776; Science and Technology Commission of Shanghai Municipality, 16DZ1910200.

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“…Interestingly, these tissues engage in CSF production and circulation ( Brinker et al, 2014 ), which might explain, in part, the hydrocephalus phenotype seen in aging Cx30 A88V/A88V mice. Using cellular expression models, our group and others have previously shown that the Cx30-A88V mutant is cytotoxic when ectopically expressed in HeLa cells and keratinocytes ( Berger et al, 2014 ; Essenfelder et al, 2004 ; Lu et al, 2018 ), most probably owing to its assembly into leaky hemichannels ( Essenfelder et al, 2004 ; Kuang et al, 2020 ). This raises the possibility that the homozygous expression of the A88V mutant is causing ependymal cells to malfunction.…”

Section: Discussionmentioning

confidence: 99%

Mutant Cx30-A88V mice exhibit hydrocephaly and sex-dependent behavioral abnormalities, implicating a functional role for Cx30 in the brain

Novielli‐Kuntz

Press

Barr

et al. 2021

Disease Models &Amp; Mechanisms

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Connexin 30 (Cx30; also known as Gjb6 when referring to the mouse gene) is expressed in ependymal cells of the brain ventricles, in leptomeningeal cells and in astrocytes rich in connexin 43 (Cx43), leading us to question whether patients harboring GJB6 mutations exhibit any brain anomalies. Here, we used mice harboring the human disease-associated A88V Cx30 mutation to address this gap in knowledge. Brain Cx30 levels were lower in male and female Cx30A88V/A88V mice compared with Cx30A88V/+ and Cx30+/+ mice, whereas Cx43 levels were lower only in female Cx30 mutant mice. Characterization of brain morphology revealed a disrupted ependymal cell layer, significant hydrocephalus and enlarged ventricles in 3- to 6-month-old adult male and female Cx30A88V/A88V mice compared with Cx30A88V/+ or Cx30+/+ sex-matched littermate mice. To determine the functional significance of these molecular and morphological changes, we investigated a number of behavioral activities in these mice. Interestingly, only female Cx30A88V/A88V mice exhibited abnormal behavior compared with all other groups. Cx30A88V/A88V female mice demonstrated increased locomotor and exploratory activity in both the open field and the elevated plus maze. They also exhibited dramatically reduced ability to learn the location of the escape platform during Morris water maze training, although they were able to swim as well as other genotypes. Our findings suggest that the homozygous A88V mutation in Cx30 causes major morphological changes in the brain of aging mice, possibly attributable to an abnormal ependymal cell layer. Remarkably, these changes had a more pronounced consequence for cognitive function in female mice, which is likely to be linked to the dysregulation of both Cx30 and Cx43 levels in the brain.

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Mechanistic effect of the human GJB6 gene and its mutations in HaCaT cell proliferation and apoptosis

Cited by 7 publications

References 17 publications

The connexin30 A88V mutant reduces cochlear gap junction expression and confers long-term protection against hearing loss

The connexin30 A88V mutant reduces cochlear gap junction expression and confers long-term protection against hearing loss

A potent antagonist antibody targeting connexin hemichannels alleviates Clouston syndrome symptoms in mutant mice

Mutant Cx30-A88V mice exhibit hydrocephaly and sex-dependent behavioral abnormalities, implicating a functional role for Cx30 in the brain

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