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

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.

Birt-Hogg-Dube syndrome is a novel ciliopathy.

Birt-Hogg-Dubé (BHD) syndrome is an autosomal dominant disorder where patients are predisposed to kidney cancer, lung and kidney cysts and benign skin tumors. BHD is caused by heterozygous mutations affecting folliculin (FLCN), a conserved protein that is considered a tumor suppressor. Previous research has uncovered multiple roles for FLCN in cellular physiology, yet it remains unclear how these translate to BHD lesions. Since BHD manifests hallmark characteristics of ciliopathies, we speculated that FLCN might also have a ciliary role. Our data indicate that FLCN localizes to motile and non-motile cilia, centrosomes and the mitotic spindle. Alteration of FLCN levels can cause changes to the onset of ciliogenesis, without abrogating it. In three-dimensional culture, abnormal expression of FLCN disrupts polarized growth of kidney cells and deregulates canonical Wnt signalling. Our findings further suggest that BHD-causing FLCN mutants may retain partial functionality. Thus, several BHD symptoms may be due to abnormal levels of FLCN rather than its complete loss and accordingly, we show expression of mutant FLCN in a BHD-associated renal carcinoma. We propose that BHD is a novel ciliopathy, its symptoms at least partly due to abnormal ciliogenesis and canonical Wnt signalling.

Cartilage-like composition of keloid scar extracellular matrix suggests fibroblast mis-differentiation in disease.

Following wound damage to the skin, the scarring spectrum is wide-ranging, from a manageable normal scar through to pathological keloids. The question remains whether these fibrotic lesions represent simply a quantitative extreme, or alternatively, whether they are qualitatively distinct. A three-way comparison of the extracellular matrix (ECM) composition of normal skin, normal scar and keloids was performed using quantitative discovery-based proteomics. This approach identified 40 proteins that were significantly altered in keloids compared to normal scars, and strikingly, 23 keloid-unique proteins. The major alterations in keloids, when functionally grouped, showed many changes in proteins involved in ECM assembly and fibrillogenesis, but also a keloid-associated loss of proteases, and a unique cartilage-like composition, which was also evident histologically. The presence of Aggrecan and Collagen II in keloids suggest greater plasticity and mis-differentiation of the constituent cells. This study characterises the ECM of both scar types to a depth previously underappreciated. This thorough molecular description of keloid lesions relative to normal scars is an essential step towards our understanding of this debilitating clinical problem, and how best to treat it.

Connexins: sensors of epidermal integrity that are therapeutic targets.

Gap junction proteins (connexins) are differentially expressed throughout the multiple layers of the epidermis. A variety of skin conditions arise with aberrant connexin expression or function and suggest that maintaining the epidermal gap junction network has many important roles in preserving epidermal integrity and homeostasis. Mutations in a number of connexins lead to epidermal dysplasias giving rise to a range of dermatological disorders of differing severity. 'Gain of function' mutations reveal connexin-mediated roles in calcium signalling within the epidermis. Connexins are involved in epidermal innate immunity, inflammation control and in wound repair. The therapeutic potential of targeting connexins to improve wound healing responses is now clear. This review discusses the role of connexins in epidermal integrity, and examines the emerging evidence that connexins act as epidermal sensors to a variety of mechanical, temperature, pathogen-induced and chemical stimuli. Connexins thus act as an integral component of the skin's protective barrier.

Porokeratotic eccrine nevus may be caused by somatic connexin26 mutations.

Porokeratotic eccrine ostial and dermal duct nevus, or porokeratotic eccrine nevus (PEN), is a hyperkeratotic epidermal nevus. Several cases of widespread involvement have been reported, including one in association with the keratitis-ichthyosis-deafness (KID) syndrome (OMIM #148210), a rare disorder caused by mutations in the GJB2 gene coding for the gap junction protein connexin26 (Cx26). The molecular cause is, as yet, unknown. We have noted that PEN histopathology is shared by KID. The clinical appearance of PEN can resemble that of KID syndrome. Furthermore, a recent report of cutaneous mosaicism for a GJB2 mutation associated with KID describes linear hyperkeratotic skin lesions that might be consistent with PEN. From this, we hypothesized that PEN might be caused by Cx26 mutations associated with KID or similar gap junction disorders. Thus, we analyzed the GJB2 gene in skin samples from two patients referred with generalized PEN. In both, we found GJB2 mutations in the PEN lesions but not in unaffected skin or peripheral blood. One mutation was already known to cause the KID syndrome, and the other had not been previously associated with skin symptoms. We provide extensive functional data to support its pathogenicity. We conclude that PEN may be caused by mosaic GJB2 mutations.

The anti-arrhythmic peptide AAP10 remodels Cx43 and Cx40 expression and function.

The anti-arrhythmic peptide AAP10 has previously been shown to acutely upregulate electrical cell-to-cell coupling mediated via connexin 43 gap junctions. In the present work, we have further examined the connexin (Cx) specificity and mechanism of action of this peptide in HeLa cells expressing Cx43, Cx40 or Cx26. The ability of cells to transfer the small fluorescent dyes Alexa 488 (MW 570) or Alexa 594 (MW 759), as markers for metabolic coupling mediated via gap junctions, before and after exposure to AAP10 and/or the protein kinase C inhibitor chelerythrine for 5 h was determined by microinjection analysis. Immunofluorescence analysis assessed the effect of AAP10 on the spatial localisation of each Cx sub-type. Cell extracts were isolated for Western blot and reverse transcription polymerase chain reaction analysis at 0, 5, 10, 18 and 24 h following exposure to AAP10 and the relative Cx expression profiles determined. AAP10 enhanced the ability of Cx43 and, to a lesser extent, Cx40 to transfer Alexa 488. It also enhanced the ability of Cx43 to transfer Alexa 594 but not Cx40. Inhibition of protein kinase C blocked this enhanced response in both Cx sub-types. Western blot analysis determined that AAP10 induced Cx40 protein expression over periods of up to 24 h with an associated increase in the localisation of Cx40 at points of cell-to-cell contact following 24-h exposure. Cx43 expression was transiently induced following exposure to the peptide for 5-10 h, with an associated increase in Cx43 at points of cell-to-cell contact, returning to control levels by 18-24 h, via a post-translational mechanism independent of chelerythrine. A transient increase in Cx40 mRNA expression but not Cx43 mRNA expression was also observed. By contrast, AAP10 had no effect on the ability of Cx26 gap junctions to transfer the dyes or on the level of Cx26 expression. We propose that AAP10 is a versatile peptide that remodels metabolic coupling via Cx43 and to a lesser extent Cx40 gap junction channels via an initial protein-kinase-C-dependent pathway modifying local responses at the plasma membrane. This is followed by enhanced Cx43 or Cx40 protein expression.