GJB4 variants linked to skin disease exhibit a trafficking deficiency en route to gap junction formation that can be restored by co-expression of select connexins

Abstract

Epidermal keratinocytes are enriched with at least nine connexins that are key regulators of epidermal homeostasis. The role of Cx30.3 in keratinocytes and epidermal health became evident when fourteen autosomal dominant mutations in the Cx30.3-encoding GJB4 gene were linked to a rare and incurable skin disorder called erythrokeratodermia variabilis et progressiva (EKVP). While these variants are linked to EKVP, they remain largely uncharacterized hindering therapeutic options. In this study, we characterize the expression and functional status of three EKVP-linked Cx30.3 mutants (G12D, T85P, and F189Y) in tissue-relevant and differentiation-competent rat epidermal keratinocytes. We found that GFP-tagged Cx30.3 mutants were non-functional likely due to their impaired trafficking and primary entrapment within the endoplasmic reticulum (ER). However, all mutants failed to increase BiP/GRP78 levels suggesting they were not inducing an unfolded protein response. FLAG-tagged Cx30.3 mutants were also trafficking impaired yet occasionally exhibited some capacity to assemble into gap junctions. The pathological impact of these mutants may extend beyond their trafficking deficiencies as keratinocytes expressing FLAG-tagged Cx30.3 mutants exhibited increased propidium iodide uptake in the absence of divalent cations. Attempts to rescue the delivery of trafficking impaired GFP-tagged Cx30.3 mutants into gap junctions by chemical chaperone treatment were ineffective. However, co-expression of wild type Cx30.3 greatly enhanced the assembly of Cx30.3 mutants into gap junctions, although endogenous levels of Cx30.3 do not appear to prevent the skin pathology found in patients harboring these autosomal dominant mutations. In addition, a spectrum of connexin isoforms (Cx26, Cx30, and Cx43) exhibited the differential ability to trans-dominantly rescue the assembly of GFP-tagged Cx30.3 mutants into gap junctions suggesting a broad range of connexins found in keratinocytes may favourably interact with Cx30.3 mutants. We conclude that selective upregulation of compatible wild type connexins in keratinocytes may have potential therapeutic value in rescuing epidermal defects invoked by Cx30.3 EKVP-linked mutants.

Connexins in epidermal health and diseases: insights into their mutations, implications, and therapeutic solutions

“…This mutation significantly reduced Cx30.3 expression in the patient's epidermis, and consistent with this, the mutant protein displayed reduced expression and a more diffuse distribution compared to the membrane-localized wild-type Cx30.3 in HeLa cells (Zhang et al, 2022). Similarly, Cx30.3-G12D, Cx30.3-T85P, and Cx30.3-F189Y mutant proteins remained within the ER and exhibited impaired transport to the plasma membrane in rat epidermal keratinocytes (Lucaciu et al, 2023). Interestingly, these mutant protein-expressing cells also exhibited increased propidium iodide uptake, potentially indicating the presence of active hemichannels.…”

“…Notably, co-expression of either wild-type Cx30.3 or other skin connexins (Cx26, Cx30, and Cx43) with Cx30.3 mutants facilitated the incorporation of mutant proteins into GJ at the plasma membrane. This finding suggests that upregulation of wild-type connexins in the epidermis may hold therapeutic potential for Cx30.3-related EKVP (Lucaciu et al, 2023).…”

“…Transitioning to the granular layer, Cx30.3, Cx31, and Cx43 are expressed at high levels, whereas Cx26, Cx31.1, Cx40, and Cx45 are found at lower levels (Figure 3) (Martin et al, 2014;Zhang and Cui, 2017). Beyond their specific distribution in the epidermis, there is a link between various skin disorders and mutations in Cx26, Cx30, Cx30.3, Cx31, and Cx43, suggesting their substantial roles in establishing and maintaining the integrity of the epidermal barrier (García-Vega et al, 2019;Lucaciu et al, 2023).…”

Cx31.1 can selectively intermix with co-expressed connexins to facilitate its assembly into gap junctions

“…Cx43 interacting with β-connexins is not without precedent, as Cx30.3 and mutant Cx26 (H73R or S183F) have been found to bind Cx43 in Cos7 cells and Xenopus oocytes, respectively ( Okamoto et al, 2020 ; Shuja et al, 2016 ). Furthermore, we have recently shown that Cx43 can intermix into the same gap junctions with Cx30.3 and Cx30.3 variants (G12D, T85P and F189Y) in keratinocytes ( Lucaciu et al, 2023a ). Next, we investigated Cx26, Cx30.3 and Cx31 as potential connexins that could rescue the cell surface delivery of Cx31.1 as they are likely to be expressed within the same keratinocyte and epidermal strata in a healthy epidermis ( Aasen and Kelsell, 2009 ).…”

“…This raises questions regarding whether these selective interactions with Cx31.1 have pathological implications. Notably, various gain- and/or loss-of-function mutations in five connexins (Cx26, Cx30, Cx30.3, Cx31 and Cx43) have been linked to a variety of congenital skin diseases ( Scott et al, 2012 ; Sellitto et al, 2021 ; Lucaciu et al, 2023a ). As such, it is possible that mutations within these distinct connexin isoforms alter interactions with Cx31.1, which might explain the clinical variability of disease presentation.…”