Abstract
Although inherited GJA1 (encoding Cx43) gene mutations most often lead to oculodentodigital dysplasia and related disorders, four variants have been linked to erythrokeratodermia variabilis et progressiva (EKVP), a skin disorder characterized by erythematous and hyperkeratotic lesions. While two autosomal-dominant EKVP-linked GJA1 mutations have been shown to lead to augmented hemichannels, the consequence(s) of keratinocytes harboring a de novo P283L variant alone or in combination with a de novo T290N variant remain unknown. Interestingly, these variants reside within or adjacent to a carboxy terminus polypeptide motif that has been shown to be important in regulating the internalization and degradation of Cx43. Cx43-rich rat epidermal keratinocytes (REKs) or Cx43-ablated REKs engineered to express fluorescent protein-tagged P283L and/or T290N variants formed prototypical gap junctions at cell–cell interfaces similar to wildtype Cx43. Dye coupling and dye uptake studies further revealed that each variant or a combination of both variants formed functional gap junction channels, with no evidence of augmented hemichannel function or induction of cell death. Tracking the fate of EKVP-associated variants in the presence of the protein secretion blocker brefeldin A, or an inhibitor of protein synthesis cycloheximide, revealed that P283L or the combination of P283L and T290N variants either significantly extended Cx43 residency on the cell surface of keratinocytes or delayed its degradation. However, caution is needed in concluding that this modest change in the Cx43 life cycle is sufficient to cause EKVP, or whether an additional underlying mechanism or another unidentified gene mutation is contributing to the pathogenesis found in patients. This question will be resolved if further patients are identified where whole exome sequencing reveals a Cx43 P283L variant alone or, in combination with a T290N variant, co-segregates with EKVP across several family generations.
Highlights
Connexins (Cx) are a family of hemichannel and intercellular gap junction channelforming proteins that enable cells to communicate with the extracellular milieu and adjoined cells, respectively
We have previously shown that the CRISPR/Cas9 ablation of Cx43 significantly reduces the ability of rat epidermal keratinocytes (REKs) to pass dye [40]
It is apparent that Cx43 plays a pivotal role in the epidermis, as highlighted by a fatal defect in barrier function in genetically-modified mice harboring a C-terminal truncated mutant of Cx43 [49]
Summary
Connexins (Cx) are a family of hemichannel and intercellular gap junction channelforming proteins that enable cells to communicate with the extracellular milieu and adjoined cells, respectively. As keratinocytes of the human epidermis progress through a terminal differentiation program called keratinization, they modulate the expression of at least eight connexin isoforms, namely Cx26, Cx30, Cx30.3, Cx31, Cx32, Cx40, Cx43, and Cx45 [3,4,5,6]. Mutations in the genes that encode five of these keratinocyte connexins are clinically linked to multiple cutaneous disorders with varying degrees of severity [7] One of these disorders is erythrokeratodermia variabilis et progressiva (EKVP), a rare hereditary skin condition characterized by distinct and overlapping erythema and hyperkeratosis that are usually present at birth or develop during infancy, and worsen with age [8]. EKVP is genetically heterogenous with linkages to seven genes that include three connexin-encoding genes: GJB3
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