Roles of aberrant hemichannel activities due to mutant connexin26 in the pathogenesis of KID syndrome

Germline missense mutations in GJB2 encoding connexin (Cx) 26 have been found in keratitis, ichthyosis and deafness (KID) syndrome. We explored the effects of three mouse Cx26 mutants (Cx26-G12R, -G45E and -D50N) corresponding to KID syndrome-causative human mutants on hemichannel activities leading to cell death and the expression of immune response-associated genes. We analyzed the 3D images of cells expressing wild-type (WT) or mutant Cx26 molecules to demonstrate clearly the intracellular localization of Cx26 mutants and hemichannel formation. High extracellular Ca2+ conditions lead to the closure of gap junction hemichannels in Cx26-G12R or Cx26-G45E expressing cells, resulting in prohibition of the Cx26 mutant-induced cell death. Fluorescent dye uptake assays revealed that cells with Cx26-D50N had aberrantly high hemichannel activities, which were abolished by a hemichannel blocker, carbenoxolone and 18α-Glycyrrhetinic acid. These results further support the idea that abnormal hemichannel activities play important roles in the pathogenesis of KID syndrome. Furthermore, we revealed that the expressions of IL15, CCL5, IL1A, IL23R and TLR5 are down-regulated in keratinocytes expressing Cx26-D50N, suggesting that immune deficiency in KID syndrome expressing Cx26-D50N might be associated not only with skin barrier defects, but also with the down-regulated expression of immune response-related genes.


Intracellular localization of Cx26-WT and Cx26-D50N mutant proteins.
We produced HeLa cells transiently transfected with pIRES2-AcGFP1 Cx26-WT constructs (pIRES2-AcGFP1 Gjb2 WT-FLAG constructs) and HeLa cells transiently transfected with Cx26-D50N-FLAG constructs (pIRES2-AcGFP1 Gjb2 c.148 G > A-FLAG constructs). We incubated the cells in DMEM + FBS, which contained 1.9 mM Ca 2+ , at transfection. Unlike the cells transfected with Gjb2 c.34 G > C (Cx26-G12R) or c.134 G > A (Cx26-G45E), the HeLa cells with Gjb2 WT (Cx26-WT) or c.148 G > A (Cx26-D50N) were able to proliferate even after transfection under the condition of 1.9 mM Ca 2+ concentration. Immunofluorescent staining with ant-FLAG antibody (Cx26-FLAG staining) demonstrated that cells expressing Cx26-WT or Cx26-D50N were able to synthesize Cx26 proteins (Fig. 1, Supplementary Videos S1-S4). Further, Cx26 proteins were localized to the plasma membrane, and gap junction plaques were formed at the cell-to-cell contact zones between adjacent cells (Fig. 1, blue arrows, Supplementary Videos S1-S4). To clearly demonstrate the intracellular location of Cx26-WT and Cx26-D50N proteins, co-labeling with an anti-Cx26-FLAG antibody and an antibody to TGN46, which is a marker for the trans-Golgi network (TGN), or double staining with an anti-Cx26-FLAG antibody and wheat germ agglutinin (WGA) for plasma membrane staining were performed ( Fig. 1 Supplementary Videos S1-S4). There was no overlap of Cx26 and TGN46 signals in cells expressing Cx26-WT or -D50N (Fig. 1A,B, blue arrowheads, Supplementary Videos S1 and S2). Furthermore, Cx26-FLAG and WGA co-staining showed that Cx26-FLAG staining overlapped with WGA staining in the cell membrane area without cell-to-cell contact and verified that Cx26-WT and -D50N were localized to the plasma membrane under the condition of 1.9 mM Ca 2+ concentration (Fig. 1C,D, purple arrowheads, Supplementary Videos S3 and S4). In contrast, gap junction plaques showed no overlap of Cx26-FLAG with WGA ( Fig. 1C,D, blue arrows, Supplementary Videos S3 and S4). WGA labels glycoproteins or glycolipids on the outer surface of the cell membrane 26 . Completely built gap junction plaques consist of a compact assembly of connexin molecules lacking surface glycoproteins and glycolipids. Thus, WGA cannot bind to the gap junction plaque. These findings clearly indicate that Cx26-WT and Cx26-D50N expressed in the HeLa cells were localized to the plasma membrane, but not to the Golgi apparatus, and that WGA was unable to access the gap junction plaques consisting of Cx26-WT and Cx26-D50N.
High extracellular Ca 2+ concentration rescues cells producing Cx26-G12R mutants and those producing Cx26-G45E mutants. We tested whether cell death due to Cx26-G12R mutants or Cx26-G45E mutants could be abolished by increased extracellular Ca 2+ concentration (3.8 mM). The cell death induced by the Gjb2 mutation Cx26-G12R and that induced by the Gjb2 mutation Cx26-G45E were prohibited by the high extracellular Ca 2+ concentration (3.8 mM). Cells transfected with Cx26-G12R or Cx26-G45E mutant DNA constructs showed no overlap of Cx26-FLAG and TGN46 ( Fig. 2A,B, blue arrowheads, Supplementary Videos S5 and S6) under the condition of high extracellular Ca 2+ concentration. Under the high extracellular Ca 2+ condition, HeLa cells with Cx26-G12R or Cx26-G45E also formed gap junction plaques without WGA co-staining between adjacent cells (Fig. 2, blue arrows, Supplementary Videos S5-S8). Hemichannels co-stained with WGA were also detected (Fig. 2C,D, purple arrowheads, Supplementary Videos S7 and S8). These results suggest that both Cx26-G12R-expressing cells and Cx26-G45E-expressing cells were rescued by the high extracellular Ca 2+ concentration and that the Cx26 proteins formed gap junction plaques and hemichannels in the rescued cells, similarly to gap junction plaques and hemichannels in the cells transfected with Cx26-WT or Cx26-D50N mutants. Consequently, cells carrying Cx26-G12R or Cx26-G45E were judged to be insufficiently healthy for further mutant protein localization studies and for gene expression profiling under the condition of physiological Ca 2+ concentration.
Altered activities of Cx26-D50N hemichannels. We determined the effects of Cx26-D50N in mammalian cell lines with a fluorescent dye uptake assay using neurobiotin (NB) (Fig. 3). Under Ca 2+ -free conditions, the mean fluorescent intensity of NB in cells with Cx26-WT or Cx26-D50N hemichannels was three times as high as that in negative control cells (p < 0.01 (1.2 mM), NB uptake was almost the same in cells with Cx26-WT as in the negative control cells, but NB uptake by Cx26-D50N-expressing cells was about 1.7 times as large as those by cells with Cx26-WT and negative control cells (p < 0.01) (Fig. 3A). NB dye uptakes by the cells with Cx26-WT or Cx26-D50N under the condition of 1.2 mM Ca 2+ concentration were less than those under the Ca 2+ -free conditions (p < 0.01). These results suggest that both normal hemichannels of Cx26-WT and aberrant hemichannels of Cx26-D50N had similar activities under the Ca 2+ -free conditions, but that aberrant hemichannel activities were aggravated under the condition of physiological Ca 2+ concentration.
To further verify the formation of abnormal hemichannels in the plasma membrane, dye uptake studies were performed with the presence of hemichannel blockers, CBX and AGA (Fig. 3B,C). Under the Ca 2+ -free condition, the treatment of cells with 100 μM CBX or AGA for 20 min reduced the levels of dye uptake in both cells producing Cx26-WT and Cx26-D50N compared to their counterparts without CBX or AGA treatment, respectively (p < 0.01) (Fig. 3B). Under the condition of physiological Ca 2+ concentration (1.2 mM), treatments of cells with 100 μM CBX or AGA for 20 min resulted in a 40% reduction in the levels of dye uptake (mean fluorescent intensities) in Cx26-D50N-expressing cells compared to their counterparts without CBX or AGA treatment, respectively (p < 0.01) (Fig. 3C). In contrast, cells producing Cx26-WT with or without hemichannel blocker treatment showed almost the same levels of dye uptake (mean fluorescent intensities). These findings suggest that the increase in the uptake of NB into cells was mediated by aberrant hemichannels consisting of Cx26-D50N.

Cx26-D50N mutant down-regulates expression of genes involved in immune responses by keratinocytes. To identify down-regulated genes in the HaCaT cells (human skin keratinocytes) producing
Cx26-D50N, we analyzed data from gene expression profiling using the Clariom S array. Using a minimum fold change of 2.5, we selected 69 down-regulated genes. We evaluated these genes by using the functional annotation chart of DAVID Bioinformatics Resources 6.8 (https://david.ncifcrf.gov/). We initially chose 6 terms involved in immunological processes: "immunity", "positive regulation of T cell proliferation", "inflammatory response", "innate immune response", "TNF signaling pathway" and "defense response to bacterium". Under these terms, we found 16 genes associated with immune function (IL23R, FCN1, CLEC4E, IFNL1, IFNL3, BIRC3, IL1A, IL15, USP41, CD180, TAPBPL, HMHB1, CCL5, TLR5, FCAMR and USP41) out of the 69 differentially expressed genes (16/69 genes, 23%). Next, we evaluated these genes by using the functional annotation clustering of DAVID Bioinformatics Resources 6.8. We adopted an enrichment score of more than 1.98 and an adjusted p-value of less than 0.05 to select these down-regulated genes, and we chose 9/16 genes involved in immunological processes. Among these genes, we selected a total of 5 genes (IL15, CCL5, IL1A, IL23R, and TLR5) that have been reported in numerous papers in the past. We confirmed the down-regulation of mRNA expression of those genes by real-time PCR (Fig. 4) and concluded that Cx26-D50N expression resulted in the down-regulated mRNA expression of IL15, CCL5, IL1A, IL23R and TLR5.

Discussion
Elucidation of the effects of KID syndrome-causative Cx26 mutations might provide clues on how Cx26 functions in normal epidermal homeostasis and on the pathomechanisms of disease phenotypes in KID syndrome patients. In the present study, we examined how three Gjb2 mutants (Cx26-G12R, -G45E and -D50N) causative of KID syndrome affected the formation and function of the hemichannels and gap junctions in transfected HeLa cells. In addition, we analyzed the cells by 3D imaging. In the 3D images, we are able to evaluate the molecular localization sites more accurately, although in 2D images we sometimes misunderstood the colocalization sites of target molecules.
GJB2 mutations causative of KID syndrome have been shown to induce elevated hemichannel activities 22,27,28 . In the present study, the Gjb2 mutations Cx26-G12R and Cx26-G45E led to increased hemichannel activity and induced cell death. The Gjb2 mutation Cx26-D50N induced high hemichannel activity, but showed no cellular lethality. The cell death induced by the Gjb2 mutation Cx26-G12R and that induced by the Gjb2 mutation Cx26-G45E were rescued by the addition of extracellular Ca 2+ . It was previously shown that cells with GJB2 mutations are rescued from cell death by the introduction of Ca 2+ to the extracellular media during incubation, although cells carrying different GJB2 mutations showed different reactions to high extracellular Ca 2+ levels 28 . It is known that elevated extracellular Ca 2+ drives the hemichannels into their closed state 24 . The high extracellular Ca 2+ condition prohibited cell death induced by the GJB2 mutant Cx26-G12R and that induced by the GJB2 mutant Cx26-G45E 25,27,28 .
Regarding the hemichannel current, the cells carrying the GJB2 mutation Cx26-G12R responded more quickly and completely to the high Ca 2+ concentration switch than did the cells with the GJB2 mutation Cx26-D50N 28 . Elevation of the external Ca 2+ concentration reduced the amplitude of hemichannel currents by shifting the voltage activation curve of the channels to more positive potentials 29 . This indicates that different activation voltages resulting from the mutations might cause higher hemichannel activity leading to cellular lethality. The Cx26 mutant Cx26-D50N was previously reported to induce elevated membrane currents in Xenopus oocytes as measured electrophysiologically 30 . Cells expressing the Cx26-D50N showed abnormal hemichannel activity and increased cell death in the absence of elevated extracellular Ca 2+28 . Recently, several studies have used highly sophisticated methodologies to investigate the calcium gating of Cx26-D50N hemichannels. Lopez et al. [30][31][32] reported that extracellular Ca 2+ destabilizes the open state of human Cx26 hemichannels by disrupting a salt bridge interaction between residues D50 and K61 located close to the extracellular entrance of the pore of the adjacent Cx26-WT-expressing cells. Purple signals (purple arrowheads) represent the co-localization of Cx26-WT and WGA, which are putative hemichannels. (D) Gjb2 c.148 G > A-transfected cells were doublestained for rhodamine-labeled WGA (red) and Cx26-D50N-FLAG (blue). Gap junction formation (blue) is indicated with blue arrows. Purple signals (purple arrowheads) indicate the co-localization of Cx26-D50N and WGA, suggesting hemichannel formation in the cell surface. Scale bars: 20 μm.  hemichannels. This open-state destabilization is thought to facilitate hemichannel closure 30,31 . A highly conserved electrostatic network located at the extracellular entrance of the pore is involved in the gating of hemichannels of all connexin types. Extracellular Ca 2+ disrupts the open-channel form of this network, resulting in a closed conformation 32 . Another study by Helmuth et al. 33 revealed that Q48 and D50 tightly interact and that disruption of this interaction activates hemichannel gating along the voltage axis. Further shifts in gating activation by extracellular Ca 2+ persist even in the absence of the Q48-D50 interaction, but the shifts require an Asp (D) or Glu (E) at position 50 33 . This fact suggests an independent electrostatic mechanism of gate activation that significantly involves position 50. The difference of Cx26-D50N from Cx26-G12R and Cx26-G45E in terms of their in vivo and in vitro effects may due to the unique characteristics of D50. In the present study, hemichannel activity in Cx26-D50N was less responsive to changes in the extracellular Ca 2+ concentration. Summarizing the results of hemichannel activities in the present study, we clearly demonstrated the formation of aberrant hemichannels by the KID syndrome-causative Gjb2 mutations Cx26-G12R, -G45E and -D50N. The present results further support the idea that abnormal hemichannel activities play an important role(s) in the pathogenesis of KID syndrome.
Hearing impairment-associated Cx26 mutants are roughly classified into two categories. Cx26 mutants in the first category are still localized to the cell membrane and form non-functional gap junctions. Cx26 mutants in the second category accumulate in the cytoplasm due to the abnormal trafficking 25 . The Cx26 mutants in this category localize within subcellular structures, including the ER, the ER-Golgi intermediate compartment and the Golgi apparatus 34-37 . In the present study, it was difficult to clarify the relationship between the Cx26 mutants and the trans-Golgi network. However, in the 3D images, none of the Cx26 mutants showed overlapping localization with TGN46 protein signals under the condition of high extracellular Ca 2+ levels. This indicates that the Gjb2 mutants Cx26-G12R, -G45E and -D50N can be classified into the first category. Most of the mutations associated with KID syndrome have shown to cause aberrant hemichannel activities, leading to the altered regulation of molecular exchanges through the plasma membrane 22,25,27,28 . As is true for the many characterized cell lines from KID syndrome patients 16,25 , in the present study, cells with Cx26 constructs with the mutation Cx26-D50N showed increased uptake of NB fluorescent dye, and the increased uptake was abolished by the hemichannel blockers CBX and AGA. This suggests that aberrant hemichannel activities are involved in the pathomechanisms of KID syndrome phenotypes due to the Cx26-D50N mutation.
Patients with KID syndrome are at high risk for neoplastic complications or cutaneous infections such as those of Candida albicans and Trichophyton rubrum 6 . Although the genetic defect of KID syndrome has been identified, the mechanisms behind the high incidence of neoplastic complications and fungal infections in KID syndrome are poorly understood. Increased risk of malignant tumors and chronic infections in KID syndrome is likely attributable to impaired epithelial barrier function or defective immune function 38 . Regarding immune function in KID syndrome patients, some studies have reported it to be normal 39,40 , but others have showed immune dysregulation 41 or have suggested primary systemic immunodeficiency 3,42 . In the present study, we determined that human keratinocytes expressing the Cx26-D50N mutant show down-regulated expression of IL15, CCL5, IL1A, IL23R and TLR5, compared with keratinocytes expressing Cx26-WT. Interleukin-15 (IL-15) exhibits biological activities similar to those of interleukin-2 and enhances the proliferation of CD8 + cytotoxic T cells and natural killer cells, which in turn eliminate tumor cells [43][44][45][46][47][48][49] . Chemokine C-C motif ligand 5 (CCL5) plays an important role in recruiting various leukocytes, including T cells, macrophages, eosinophils, and basophils, to inflammatory sites. In collaboration with certain cytokines released from T cells, CCL5 also activates natural killer cells to C-C chemokine-activated killer cell and induces their proliferation 50 . Interleukin-1α (IL1α) is produced by cells of various types, including activated macrophages, keratinocytes, stimulated B lymphocytes, and fibroblasts, and it is a potent mediator of inflammation and immune reactions 51 . In humans, high levels of Interleukin-23 receptor (IL-23R) expression are detected on activated/memory T cells, NK cells, macrophages, dendritic cells and monocytes. IL-23 and Interleukin-12 (IL-12) share a common subunit in their receptor complex. The receptor complex is a heterodimer made up of 2 subunits, IL-23R and IL-12β1 52 . Stimulation of the receptor complex activates janus activating kinase 2 (JAK2) and tyrosine kinase 2 (TYK2), resulting in phosphorylation of the receptor complex, and the formation of docking sites for signal transducers and activators of transcription (STATs) 1, 3, 4 and 5. The STATs are subsequently dimerized, phosphorylated and translocated into the nucleus, activating target genes 53 . Importantly, the phosphorylation of STAT4 is essential for increasing interferon (IFN) γ production and the subsequent differentiation of Th1 cells, whereas STAT3 is important for the development of interleukin 17-producing helper T cells (Th17 cells) 54,55 . Overall, this process orchestrates the cytokine cascade, activating the necessary immune cells involved in the eradication of any pathogenic/antigenic challenge.
Toll-like receptors (TLRs) provide efficient and immediate immune responses to bacterial, fungal, and viral infections by recognizing diverse molecules released from them 56 . One study reports that an Asian KID syndrome patient with fungal infection expressed only a lower level of TLR2 mRNA 57 . The present results clearly indicate that keratinocytes expressing the Cx26-D50N mutant show down-regulated expression of important molecules involved in the epidermal immune responses including IL-15, CCL5, IL-1α, IL-23 receptor and TLR5. We speculate that the down-regulation might cause immunodeficiency in the epidermis of KID syndrome due to the Cx26-D50N mutant, resulting in the malignant tumors and chronic infections seen in the patients.
Observations of 2-dimensional plane images are insufficient to clarify the intracellular localization of Cx26 proteins and the formation sites of hemichannels. In the present study, we analyzed the 3D images of cells expressing WT and mutant Cx26 molecules and were able to demonstrate clearly the intracellular localization of Cx26 proteins and hemichannel formation sites. In conclusion, we clearly demonstrated that aberrant hemichannels form due to the KID syndrome-causative Gjb2 mutations Cx26-G12R, -G45E and -D50N. Our findings further support the idea that abnormal hemichannel activities are involved in the pathogenesis of KID syndrome. In addition, we revealed that the expressions of IL15, CCL5, IL1A, IL23R and TLR5 are down-regulated in keratinocytes expressing the Cx26-D50N mutation. These findings suggest that the immune deficiency of KID syndrome caused by Cx26-D50N mutation owes not only to skin barrier dysfunction, but also to the down-regulated expression of immune response-related genes in the keratinocytes. TRITC-conjugated swine polyclonal anti-rabbit immunoglobulins antibody (1:1000) (Dako Cytomation, Glostrup, Denmark)) were applied for 45 min at 37 °C in the dark. After two more washings with PBS, coverslips were mounted with VECTORSHIELD mounting medium (Vector laboratories, California, USA) on glass slides. For co-labeling for rhodamine-labeled WGA and Cx26-FLAG, after blocking with 10% BSA, rhodamine-labeled WGA (5 mg/ml, Vector laboratories, California, USA) diluted 1:500 was applied to cells for 30 min at 37 °C. After two washings with PBS, the cells were incubated with primary antibodies (M2 monoclonal mouse antibody against FLAG (1:1000)) overnight at RT. After two more washings with PBS, secondary antibodies (goat polyclonal anti-mouse IgG antibody, Alexa Flour 350 (1:1000) (Invitrogen, Massachusetts, USA)) were applied for 45 min at 37 °C in the dark. After two more washings with PBS, coverslips were mounted with VECTORSHIELD mounting medium on glass slides.
Dye uptake assays. HeLa cells (2.0 × 10 4 ) were grown over glass coverslips in 8-well glass slides for dye uptake assays with an NB tracer (NB, FW 322.8, Vector Laboratories). At 48 h post-transfection (Ca 2+ concentration, 1.9 mM), the cells were washed with PBS and incubated with Ca 2+ -free medium or a medium with a physiological calcium concentration (1.2 mM Ca 2+ ) for 20 min at 37 °C. The cells were then incubated with 0.5 mg/ ml NB in the same Ca 2+ concentration medium for 20 min at 37 °C 22 . Next, the cells were washed with DMEM containing 4.0 mM CaCl 2 twice for 10 min and PBS once. The cells were fixed with 4% PFA for 15 min at room temperature. After being washed twice with PBS, the fixed cells were permeabilized with 0.25% Triton-X 100 for 30 min, washed twice with PBS, and blocked with 10% BSA for 30 min at 37 °C. Following the two washings with PBS, the cells were incubated with primary antibodies (mouse monoclonal anti-FLAGR M2 antibody (1:1000)) overnight at RT. After two more washings with PBS, TRITC-conjugated rabbit polyclonal anti-mouse immunoglobulins antibody (1:1000) and Alaxa Flour 350-conjugated Streptavidin (1:200) (Invitrogen, Massachusetts, USA) were applied for 45 min at 37 °C in the dark. After two more washings with PBS, images were acquired using the A1R confocal laser scanning microscope system with fixed exposure times. For treatments with carbenoxolone (CBX, Sigma-Aldrich, St Louis, USA) or 18α-Glycyrrhetinic acid (AGA, Sigma-Aldrich), the cells were initially incubated with calcium-free medium containing 100 μM CBX or AGA for 20 min, and then NB in the same medium was applied to the cells as described above.
Image analysis for signal intensity determination was performed with ImageJ software (NIH, Bethesda, USA). We selected 2 high-power view fields under the microscope. During image analysis, after the background in merged images of red and green channels was subtracted, the same parameters were applied to threshold the images for the measurement of blue signal intensities of fluorescence only in the GFP-positive cells 16 . Each experiment was performed at least twice.

Transfection experiments targeting HaCaT cells for microarray analysis.
HaCaT cells were transfected using the Amaxa Cell Line Nucleofector Kit V (Lonza, Cologne, Germany) according to the manufacturer's instructions. The cells were seeded on the plate and the medium was changed 3 h after seeding. At 8 h of incubation, the cells were subjected to microarray analysis and quantitative real-time reverse transcription (qRT)-PCR assays.
Microarray analysis. We isolated total RNA from the transfected HaCaT cells using the RNeasy Mini Kit (Qiagen, Hiden Germany). Whole-genome expression profiling was performed using Clariom S Array (Affymetrix, Santa Clara, California, USA). Differentially expressed genes were defined as those showing a 2.5-fold or greater change in expression between the cells transfected with Gjb2 WT constructs (Cx26-WT) and those transfected with the c.134 G > A constructs (Cx26-D50N) using the Affymetrix Transcriptome Analysis Console Ver 3.1.0.5 (Affymetrix). The list of down-regulated genes was analyzed using gene-set enrichment analyses from DAVID Bioinformatics Resources 6.8 (https://david.ncifcrf.gov/) in order to identify the functions of these genes. We used an enrichment score of more than 1.9 and an adjusted p-value of less than 0.05 to select these down-regulated genes.
qRT-PCR. We isolated total RNA from the transfected HaCaT cells using the RNeasy Mini Kit (Qiagen). We reverse-transcribed 250 ng total RNA using the Prime Script RT Reagent Kit (Takara, Shiga, Japan) according to the manufacturer's instructions. The recovered cDNA was diluted 10-fold with DW for qRT-PCR. mRNA expression levels were measured by qRT-PCR using the Light Cycler System (Roche, Basel, Switzerland). The PCRs were set up in microcapillary tubes filled with 10 μL of reaction agents including 2.5 μL of diluted cDNA solution, and the PCR program was set according to the manufacturer's instructions. The primers and probes used for qRT-PCR are listed in Supplementary Table 1. Each experiment was performed at least three times.

Statistical analyses.
All results are expressed as mean (± standard deviation). In the dye uptake assay, we selected 2 high-power view fields under the microscope without any random sampling method and each experiment was performed at least twice. In the qRT-PCR analysis, each experiment was performed at least three times. Analysis of dye uptake samples and qRT-PCR samples was done by Student's t-test. Statistical significance was shown as *p < 0.01, † p < 0.05.