SKINCONNECTION · Investigating the Role of Abnormal Hemichannel Activities of Cx26 Mutations on Epidermal Keratinocyte Calcium Homeostasis and Differentiation

Periodic Report Summary 2 - SKINCONNECTION (Investigating the Role of Abnormal Hemichannel Activities of Cx26 Mutations on Epidermal Keratinocyte Calcium Homeostasis and Differentiation)

Skin is the biggest organ of all animal species and it fulfills critical functions for their survival. The epidermis and its appendages surround and protect the body against physical/chemical insults, serves as barrier against pathogenic microorganisms and facilitates the maintenance of the body core temperature by controlling the fluid loss. In order to perform these functions, the epidermis undergoes enormous proliferation and differentiation processes in a very precise manner. Alteration of this balance due to mutations in genes functioning in the epidermis leads to disorders. Some of these diseases have been shown to be linked to mutations in connexin (Cx) genes, which are the subunits of gap junction channels in vertebrates. Although connexins are widely expressed throughout the epidermis, their exact roles in the epidermal homeostasis are not exactly known. For this, characterization of disease causing mutations can be an invaluable tool for understanding connexin function in the skin and the etiology of pathological phenotypes. Mutations in Cx26 are the leading cause of recessive non-syndromic hereditary loss in addition to dominant deafness associated with skin disorders such as keratitis-ichthyosis-deafness (KID) syndrome. In vitro characterization of Cx26 mutations that cause skin disorders revealed that mutant proteins/channels acquires novel properties different than wild-type connexin proteins/channels. For example, KID syndrome associated Cx26 mutations were demonstrated to form active hemichannels that might result in uncontrolled exchange of molecules between the cytosol and the extracellular space, influencing cellular homeostasis. One of the mechanisms that can be altered due to abnormal Cx26 hemichannels might be the calcium (Ca2+) homeostasis in cells. Ca2+ is crucial for the epidermal homeostasis where it plays roles in keratinocyte growth, proliferation, differentiation and the maintenance of the epidermal barrier (stratum corneum) as well as the regulation of the function of junctional molecules. Further, there is a Ca2+ gradient in the epidermis with the highest concentration in the stratum corneum and the lowest concentration in the basal layer. The alteration of this gradient is also associated with different skin disorders as changes in the extracellular calcium concentration manipulate intercellular calcium signals and interfere with the functions of calcium-dependent molecules, affecting the proliferation and differentiation mechanisms of keratinocytes. In this project, recently identified Cx26 mutations associated with KID syndrome, I30N, D50A, D50Y and A88V, was proposed to lead to the formation of aberrant hemichannels that may alter the epidermal Ca2+ balance by releasing metabolites such as ATP into the extracellular space. Therefore, these changes may interfere with keratinocyte proliferation and differentiation processes leading to phenotypes observed in affected individuals. In order to investigate this, the ability of mutant proteins to form abnormal hemichannels was investigated in communication deficient cells. Then, normal human keratinocyte cell lines with mutant Cx26 clones were examined to analyze the effect of mutant proteins/channels on cellular events, changes in metabolite levels in the extracellular environments and Ca2+ metabolism in cells. Furthermore, keratinocytes with mutant Cx26 proteins was proposed to be grown in 3D cultures in order to mimic the normal epidermal organization where effects of abnormal hemichannels on cells and on Ca2+ signals in 3D environment would be characterized. Initially, Cx26 mutations associated with KID syndrome, I30N, D50A, D50Y and A88V, were generated and cloned into sequencing vectors. After the verification of insertion of mutations via sequencing, mutant cDNAs were subcloned into mammalian expression vectors in order to characterize the properties of mutant proteins and channels in gap junctional communication deficient cell lines (i.e. HeLa and N2A). Mutant proteins failed to form gap junction plaques at the cell-to-cell surfaces of adjacent cells unlike Cx26 wild-type (WT) proteins that form gap junction plaques between neighboring cells. Functional studies of channel activities by using fluorescent dye uptake assays showed that cells with mutant channels were able to take more fluorescent dyes from the medium into the cells than cells containing Cx26 WT channels. These suggested that KID syndrome mutations lead to the formation of aberrant hemichannels that are overly active compared to Cx26 WT channels. To determine the effect of mutations on cellular homeostasis in keratinocytes, we transfected HaCaT cells with WT and mutant constructs. The localization of mutant proteins were examined and showed that mutant proteins did not form gap junction plaques between adjacent cells in contrast to Cx26 WT proteins. The failure of mutant proteins to form gap junctional plaques at cell-cell contact sites might be due to problems in biosynthetic pathway. Examination of BiP/GRP78 levels, an endoplasmic reticulum marker, did not suggest any unfolded/misfolded protein response in the ER. However, mutant Cx26 I30N and D50Y proteins were found to co-localize with golgin 97, a Golgi apparatus marker. These results suggest that a fraction of mutant proteins may retain in the Golgi apparatus, potentially interfering with modification and/or oligomerization. In keratinocytes, the effect of mutations on intracellular calcium content was examined where cells with Cx26 I30N and D50Y mutations had elevated intracellular calcium levels compared to WT expressing cells. This alteration in calcium levels could be due to paracrine effect of ATP released into the extracellular space through aberrant hemichannels so we compared ATP levels between Cx26 WT and mutant expressing cells. There was no statistically significant difference in the amount of ATP in the extracellular space between groups, suggesting the involvement of other mechanisms in the elevation of intracellular calcium signals. Other KID syndrome associated Cx26 mutations in transfected cells have shown to result in cell death due to uncontrolled exchange of molecules across the plasma membrane. In a similar manner, Cx26 I30N and D50Y mutations interfered with the cell viability as there was an increase in the number of cells undergoing cell death in cells with mutant proteins compared to Cx26 WT expressing keratinocytes. In this study, we aimed to understand the role of Cx26 in the skin homeostasis by examining the mechanisms leading to epidermal disorders due to Cx26 mutations. As a result we observed the involvement of aberrant hemichannels in the KID syndrome that can interfere with cellular processes such as calcium homeostasis and cell survival mechanisms. Understanding how these changes in keratinocytes result in pathological phenotypes of affected individuals remains to be elucidated and it eventually may help to develop treatment options for the affected individuals.

Data: CORDIS, © European Union