The epidermis expresses a number of connexin (Cx) proteins that are
The epidermis expresses a number of connexin (Cx) proteins that are implicated in gap junction-mediated cell communication. associated with deafness neuropathy and skin disease indicating that Cx31 has a role in both epidermal differentiation plus auditory and neuronal function (13). Multiple autosomal dominant [including (R42P)Cx31 (C86S)Cx31 and (G12D)Cx31 (14 15 and recessive [(L34P)Cx31 (16)] Cx31 mutations are associated with the skin disease erythrokeratoderma variabilis (EKV) a disorder characterized by hyperkeratotic plaques with fixed and transient erythematous patches (17). A dominant neuropathy mutation (66delD)Cx31 has also been identified in a family with sensorineural hearing loss and peripheral neuropathy (12) and other mutations in Cx31 are associated with non-syndromic hearing loss (18-20). Previous investigations involving microscopy of EGFP-tagged Cxs expressed have shown that the EKV mutants and the neuropathy mutant have impaired trafficking to the plasma membrane with a predominantly cytoplasmic localization in contrast to the wild-type which forms aggregates at the plasma membrane and gap junction-like plaques (21). Unlike wild-type or (66delD)Cx31 the expression of the EKV mutants are associated with elevated levels of cell death (21-23) through a mechanism which remains to be elucidated. Basal activity of Cx hemichannels is low with the majority remaining closed most of the time (24) but opening may be induced by stimuli including low extracellular calcium (4 25 26 Open hemichannels can release molecules such as ATP glutamate and NAD+ and lead to uptake of others (reviewed in 1). Decreased cell viability caused by the expression of some Cx26 and Cx30 disease-associated mutants has been attributed to the presence of hemichannels at the plasma membrane which are ‘leaky’ when cells are incubated in physiological levels of extracellular calcium with rescue of the Cilomilast phenotype occurring under high levels of calcium (3 27 28 It is suggested that this abnormal hemichannel activity can contribute to the disease phenotype (29). In Cilomilast contrast data from another study investigating Cx26 skin and deafness mutants suggest that aberrant hemichannels are not the Cilomilast major mechanism of cell death for these mutants (30). In this investigation high levels of cell death were still observed with (R42P)Cx31 (C86S)Cx31 and (G12D)Cx31 when incubated in high extracellular calcium indicating that hemichannel-mediated cell death is not the major mechanism for these mutants. We describe a novel association of the expression of EKV-associated mutants with upregulation of components of the unfolded protein response (UPR) decreases cell viability the mechanism of which was unknown (21 23 However the neuropathy-associated (66delD)Cx31 also mistraffics but does not increase cell death (21 23 Our data confirm and extend this observation. Confocal microscopy in three different cell lines revealed that the skin disease mutants are characterized by bright cytoplasmic aggregates (>3 μm in diameter) and the neuropathy Rabbit polyclonal to TranscriptionfactorSp1. mutant by smaller punctate structures (<2 μm in diameter) whereas subcellular fractionation demonstrates that both classes are membrane bound. This led us to hypothesise that this difference in intracellular localization could give Cilomilast an indication as to why the two classes of mutants cause different diseases and cell phenotypes. The G12S and E208K Cx32 mutants both linked to X-linked Charcot-Marie-Tooth disease (CMTX) have been found to colocalize with the Golgi apparatus (32) and the ER (33) respectively. We have excluded similar backing-up of mutant Cx31 protein in a range of intermediate transport organelles as well as accumulation in the lysosome. The skin disease mutants were detected in the proteasome known to be involved in the degradation steps of the turn-over cycle of Cx protein (6 34 In addition we observed an upregulation of proteasome markers as well as proteins containing the ER resident motif KDEL and the ER chaperone BiP/GRP78. Aberrant hemichannels are not the main mechanism of EKV-associated mutant cell death ATP has been hypothesised as a paracrine signalling molecule (1-3). Furthermore it has been suggested that deregulated release of ATP and other molecules could disrupt paracrine signalling within the skin affecting keratinocyte differentiation and leading to hyperproliferation (3 27 The cell.