Diversity in connexin biology.

Over 35 years ago the cell biology community was introduced to connexins as the subunit employed to assemble semicrystalline clusters of intercellular channels that had been well described morphologically as gap junctions. The decade that followed would see knowledge of the unexpectedly large 21-member human connexin family grow to reflect unique and overlapping expression patterns in all organ systems. While connexin biology initially focused on their role in constructing highly regulated intercellular channels, this was destined to change as discoveries revealed that connexin hemichannels at the cell surface had novel roles in many cell types, especially when considering connexin pathologies. Acceptance of connexins as having bifunctional channel properties was initially met with some resistance, which has given way in recent years to the premise that connexins have multifunctional properties. Depending on the connexin isoform and cell of origin, connexins have wide-ranging half-lives that vary from a couple of hours to the life expectancy of the cell. Diversity in connexin channel characteristics and molecular properties were further revealed by X-ray crystallography and single-particle cryo-EM. New avenues have seen connexins or connexin fragments playing roles in cell adhesion, tunneling nanotubes, extracellular vesicles, mitochondrial membranes, transcription regulation, and in other emerging cellular functions. These discoveries were largely linked to Cx43, which is prominent in most human organs. Here, we will review the evolution of knowledge on connexin expression in human adults and more recent evidence linking connexins to a highly diverse array of cellular functions.

Degradation of cochlear Connexin26 accelerate the development of age-related hearing loss.

The GJB2 gene, encoding Connexin26 (Cx26), is one of the most common causes of inherited deafness. Clinically, mutations in GJB2 cause congenital deafness or late-onset progressive hearing loss. Recently, it has been reported that Cx26 haploid deficiency accelerates the development of age-related hearing loss (ARHL). However, the roles of cochlear Cx26 in the hearing function of aged animals remain unclear. In this study, we revealed that the Cx26 expression was significantly reduced in the cochleae of aged mice, and further explored the underlying molecular mechanism for Cx26 degradation. Immunofluorescence co-localization results showed that Cx26 was internalized and degraded by lysosomes, which might be one of the important ways for Cx26 degradation in the cochlea of aged mice. Currently, whether the degradation of Cx26 in the cochlea leads directly to ARHL, as well as the mechanism of Cx26 degradation-related hearing loss are still unclear. To address these questions, we generated mice with Cx26 knockout in the adult cochlea as a model for the natural degradation of Cx26. Auditory brainstem response (ABR) results showed that Cx26 knockout mice exhibited high-frequency hearing loss, which gradually progressed over time. Pathological examination also revealed the degeneration of hair cells and spiral ganglions, which is similar to the phenotype of ARHL. In summary, our findings suggest that degradation of Cx26 in the cochlea accelerates the occurrence of ARHL, which may be a novel mechanism of ARHL.

Cytomembrane Trafficking Pathways of Connexin 26, 30, and 43.

The connexin gene family is the most prevalent gene that contributes to hearing loss. Connexins 26 and 30, encoded by GJB2 and GJB6, respectively, are the most abundantly expressed connexins in the inner ear. Connexin 43, which is encoded by GJA1, appears to be widely expressed in various organs, including the heart, skin, the brain, and the inner ear. The mutations that arise in GJB2, GJB6, and GJA1 can all result in comprehensive or non-comprehensive genetic deafness in newborns. As it is predicted that connexins include at least 20 isoforms in humans, the biosynthesis, structural composition, and degradation of connexins must be precisely regulated so that the gap junctions can properly operate. Certain mutations result in connexins possessing a faulty subcellular localization, failing to transport to the cell membrane and preventing gap junction formation, ultimately leading to connexin dysfunction and hearing loss. In this review, we provide a discussion of the transport models for connexin 43, connexins 30 and 26, mutations affecting trafficking pathways of these connexins, the existing controversies in the trafficking pathways of connexins, and the molecules involved in connexin trafficking and their functions. This review can contribute to a new way of understanding the etiological principles of connexin mutations and finding therapeutic strategies for hereditary deafness.

[Methylselenocysteine Promotes Etoposide Cytotoxicity by Enhancing Homotypic Gap Junctions Composed of Connexin 26].

Objective: To investigate the effect of methylselenocysteine (MSC) on the function of homotypic gap junction (GJ) composed of connexin (Cx) 26 and its regulation of chemotherapeutic drug cytotoxicity. Methods: The Tet-on HeLa cells transfected with and stably expressing Cx26 were used as the tool cells. Effects of MSC on cell growth, GJ function, and Cx26 protein expression were examined by MTT method, parachute assay, and Western blot analysis, respectively. The cytotoxicity of chemotherapeutic drugs was determined by standard colony-forming assay, and the relationship between MSC's effect on cytotoxicity of these chemotherapeutic drugs and its regulation of GJ was further analyzed. Results: In Tet-on HeLa cells, doxycycline (Dox) can induce the expression of Cx26, which could then form functional GJs. Within a concentration range of 50 µmol/L, MSC had no significant effect on HeLa cell growth. Non-toxic concentrations of MSC can enhance GJs in a concentration-dependent manner and exert its effect at the nanomolar level. This effect was associated with an induction of Cx26 protein expression by MSC. Among the three common chemotherapeutic agents with different mechanisms of action, etoposide (Eto) presented cytotoxicity differences between HeLa cells cultured at low density (nonconfluent, no GJ formed) and high density (confluent, GJ formed). What's more, the inhibitory effect of Eto combined with MSC on HeLa cell colony formation was stronger than that of Eto alone, and this effect occurred only in HeLa cells with GJ formation. Conclusion: MSC can potentiate the cytotoxicity of Eto by enhancing the GJs composed of Cx26, indicating that combined strategy of selenide and chemotherapy shows potential value in the treatment of malignant tumors.

Solute exchange through gap junctions lessens the adverse effects of inactivating mutations in metabolite-handling genes.

Growth of cancer cells in vitro can be attenuated by genetically inactivating selected metabolic pathways. However, loss-of-function mutations in metabolic pathways are not negatively selected in human cancers, indicating that these genes are not essential in vivo. We hypothesize that spontaneous mutations in 'metabolic genes' will not necessarily produce functional defects because mutation-bearing cells may be rescued by metabolite exchange with neighboring wild-type cells via gap junctions. Using fluorescent substances to probe intercellular diffusion, we show that colorectal cancer (CRC) cells are coupled by gap junctions assembled from connexins, particularly Cx26. Cells with genetically inactivated components of pH regulation (SLC9A1), glycolysis (ALDOA), or mitochondrial respiration (NDUFS1) could be rescued through access to functional proteins in co-cultured wild-type cells. The effect of diffusive coupling was also observed in co-culture xenografts. Rescue was largely dependent on solute exchange via Cx26 channels, a uniformly and constitutively expressed isoform in CRCs. Due to diffusive coupling, the emergent phenotype is less heterogenous than its genotype, and thus an individual cell should not be considered as the unit under selection, at least for metabolite-handling processes. Our findings can explain why certain loss-of-function mutations in genes ascribed as 'essential' do not influence the growth of human cancers.

3D Chromatin Organization Involving MEIS1 Factor in the cis-Regulatory Landscape of GJB2.

The human genome is covered by 8% of candidate cis-regulatory elements. The identification of distal acting regulatory elements and an understanding of their action are crucial to determining their key role in gene expression. Disruptions of such regulatory elements and/or chromatin conformation are likely to play a critical role in human genetic diseases. Non-syndromic hearing loss (i.e., DFNB1) is mostly due to GJB2 (Gap Junction Beta 2) variations and DFNB1 large deletions. Although several GJB2 cis-regulatory elements (CREs) have been described, GJB2 gene regulation remains not well understood. We investigated the endogenous effect of these CREs with CRISPR (clustered regularly interspaced short palindromic repeats) disruptions and observed GJB2 expression. To decipher the GJB2 regulatory landscape, we used the 4C-seq technique and defined new chromatin contacts inside the DFNB1 locus, which permit DNA loops and long-range regulation. Moreover, through ChIP-PCR, we determined the involvement of the MEIS1 transcription factor in GJB2 expression. Taken together, the results of our study enable us to describe the 3D DFNB1 regulatory landscape.

The Expression of Connexin 37, 40, 43, 45 and Pannexin 1 in the Early Human Retina and Choroid Development and Tumorigenesis.

The expression pattern of Connexins (Cx) 37, 40, 43, 45 and Pannexin 1 (Pnx1) was analyzed immunohistochemically, as well as semi-quantitatively and quantitatively in histological sections of developing 8th- to 12th-week human eyes and postnatal healthy eye, in retinoblastoma and different uveal melanomas. Expressions of both Cx37 and Cx43 increased during development but diminished in the postnatal period, being higher in the retina than in the choroid. Cx37 was highly expressed in the choroid of retinoblastoma, and Cx43 in epitheloid melanoma, while they were both increasingly expressed in mixoid melanoma. In contrast, mild retinal Cx40 expression during development increased to strong in postnatal period, while it was significantly higher in the choroid of mixoid melanoma. Cx45 showed significantly higher expression in the developing retina compared to other samples, while it became low postnatally and in all types of melanoma. Pnx1 was increasingly expressed in developing choroid but became lower in the postnatal eye. It was strongly expressed in epithelial and spindle melanoma, and particularly in retinoblastoma. Our results indicate importance of Cx37 and Cx40 expression in normal and pathological vascularization, and Cx43 expression in inflammatory response. Whereas Cx45 is involved in early stages of eye development, Pnx1might influence cell metabolism. Additionally, Cx43 might be a potential biomarker of tumor prognosis.

The protective effects of systemic dexamethasone on sensory epithelial damage and hearing loss in targeted Cx26-null mice.

Mutations in the GJB2 gene (encoding Connexin26(Cx26)) are the most common cause of hereditary deafness, accounting for about a quarter of all cases. Sensory epithelial damage is considered to be one of the main causes of deafness caused by GJB2 gene mutation. Dexamethasone (DEX) is widely used in the treatment of a variety of inner ear diseases including sudden sensorineural hearing loss (SSNHL), noise-induced hearing loss (NIHL), and deafness caused by ototoxic drugs. Whether DEX has a direct therapeutic effect on hereditary deafness, especially GJB2-related deafness, remains unclear. In this study, we revealed that DEX can effectively prevent hair cell death caused by oxidative stress in cochlear explants. Additionally, two distinct Cx26-null mouse models were established to investigate whether systemic administration of DEX alleviate the cochlear sensory epithelial injury or deafness in these models. In a specific longitudinally Cx26-null model that does not cause deafness, systemic administration of DEX prevents the degeneration of outer hair cells (OHCs) induced by Cx26 knockout. Similarly, in a targeted-Deiter's cells (DCs) Cx26-null mouse model that causes deafness, treatment with DEX can almost completely prevent OHCs loss and alleviates auditory threshold shifts at some frequencies. Additionally, we observed that DEX inhibited the recruitment of CD45-positive cells in the targeted-DCs Cx26-null mice. Taken together, our results suggest that the protective effect of dexamethasone on cochlear sensory epithelial damage and partially rescue auditory function may be related to the regulation of inner ear immune response in Cx26 deficiency mouse models.

Inducible knockout of syncytin-a leads to poor placental glucose transport in mice.

INTRODUCTION: Cell-cell fusion of cytotrophoblasts into the syncytiotrophoblast layer is a key process in placental development. Syncytin, an endogenous retroviral envelope protein, is expressed in placental trophoblasts and specifically mediates syncytiotrophoblast layer formation. Syncytin deficiency has been observed in fetal growth-restricted placentas. Abnormal fetal growth, especially fetal growth restriction, is associated with the decreased expression of glucose transporters. Here, we aimed to determine the role of syncytin in fetal growth restriction in placental glucose transport capacity. METHODS: To better explore the function of syncytin in fetal growth-restricted placenta, we generated an inducible knockout mouse model of syncytin-a gene. The expression levels of glucose transporters in BeWo cells were measured before and after HERV-W knockdown. RESULTS: Syncytin-A disruption was associated with significant abnormalities in placental and fetal development in mice. Syncytin-A destruction causes extensive abnormalities in the maternal-fetal exchange structures in the labyrinth, including an extremely reduced number and dramatically irregular distribution of fetal vessels. Moreover, glucose transporter 1, glucose transporters 3, and connexin 26 expression levels decreased after E14.5. Consistently, low glucose transporter 1, glucose transporter 3, and connexin 26 levels were observed in HERV-W-silenced BeWo cells. DISCUSSION: Syncytin-A is crucial for both syncytiotrophoblast layer development and morphogenesis, suggesting that syncytin-A disruption leads to fetal growth restriction associated with abnormalities in the maternal-fetal exchange barrier and decreased glucose transport.

Cellular mechanisms of connexin-based inherited diseases.

The 21-member connexin gene family exhibits distinct tissue expression patterns that can cause a diverse array of over 30 inherited connexin-linked diseases ranging from deafness to skin defects and blindness. Intriguingly, germline mutations can cause disease in one tissue while other tissues that abundantly express the mutant connexin remain disease free, highlighting the importance of the cellular context of mutant expression. Modeling connexin pathologies in genetically modified mice and tissue-relevant cells has informed extensively on no less than a dozen gain- and loss-of-function mechanisms that underpin disease. This review focuses on how a deeper molecular understanding of the over 930 mutations in 11 connexin-encoding genes is foundational for creating a framework for therapeutic interventions.

Generation of hiPSC line UMi030-A from an individual with the hearing loss-related GJB2 mutation c.109G > A.

Genetic variants in the GJB2 gene which encodes for the Connexin 26 protein account for âˆ¼ 60% of cases of genetic hearing loss. A novel hiPSC line was generated from an individual with the hearing loss-related variant c.109G > A in GJB2 leading to the p.V37I alteration in the Connexin26 protein. These cells will help to delineate the role of GJB2 in hearing loss pathogenesis and serve as a platform for drug discovery and development.

Connexin hemichannel inhibition ameliorates epidermal pathology in a mouse model of keratitis ichthyosis deafness syndrome.

Mutations in five different genes encoding connexin channels cause eleven clinically defined human skin diseases. Keratitis ichthyosis deafness (KID) syndrome is caused by point mutations in the GJB2 gene encoding Connexin 26 (Cx26) which result in aberrant activation of connexin hemichannels. KID syndrome has no cure and is associated with bilateral hearing loss, blinding keratitis, palmoplantar keratoderma, ichthyosiform erythroderma and a high incidence of childhood mortality. Here, we have tested whether a topically applied hemichhanel inhibitor (flufenamic acid, FFA) could ameliorate the skin pathology associated with KID syndrome in a transgenic mouse model expressing the lethal Cx26-G45E mutation. We found that FFA blocked the hemichannel activity of Cx26-G45E in vitro, and substantially reduced epidermal pathology in vivo, compared to untreated, or vehicle treated control animals. FFA did not reduce the expression of mutant connexin hemichannel protein, and cessation of FFA treatment allowed disease progression to continue. These results suggested that aberrant hemichannel activity is a major driver of skin disease in KID syndrome, and that the inhibition of mutant hemichannel activity could provide an attractive target to develop novel therapeutic interventions to treat this incurable disease.

Expression and Functionality of Connexin-Based Channels in Human Liver Cancer Cell Lines.

Liver cancer cell lines are frequently used in vitro tools to test candidate anti-cancer agents as well as to elucidate mechanisms of liver carcinogenesis. Among such mechanisms is cellular communication mediated by connexin-based gap junctions. The present study investigated changes in connexin expression and gap junction functionality in liver cancer in vitro. For this purpose, seven human liver cancer cell lines, as well as primary human hepatocytes, were subjected to connexin and gap junction analysis at the transcriptional, translational and activity level. Real-time quantitative reverse transcription polymerase chain reaction analysis showed enhanced expression of connexin43 in the majority of liver cancer cell lines at the expense of connexin32 and connexin26. Some of these changes were paralleled at the protein level, as evidenced by immunoblot analysis and in situ immunocytochemistry. Gap junctional intercellular communication, assessed by the scrape loading/dye transfer assay, was generally low in all liver cancer cell lines. Collectively, these results provide a full scenario of modifications in hepatocyte connexin production and gap junction activity in cultured liver cancer cell lines. The findings may be valuable for the selection of neoplastic hepatocytes for future mechanistic investigation and testing of anti-cancer drugs that target connexins and their channels.

Subcellular Localization of Connexin 26 in Cardiomyocytes and in Cardiomyocyte-Derived Extracellular Vesicles.

Connexins (Cxs) are a family of membrane-spanning proteins, expressed in vertebrates and named according to their molecular weight. They are involved in tissue homeostasis, and they function by acting at several communication levels. Cardiac Cxs are responsible for regular heart function and, among them, Cx26 and Cx43 are widely expressed throughout the heart. Cx26 is present in vessels, as well as in cardiomyocytes, and its localization is scattered all over the cell aside from at the intercalated discs as is the case for the other cardiac Cxs. However, having been found in cardiomyocytes only recently, both its subcellular localization and its functional characterization in cardiomyocytes remain poorly understood. Therefore, in this study we aimed to obtain further data on the localization of Cx26 at the subcellular level. Our TEM immunogold analyses were performed on rat heart ventricles and differentiated H9c2 cardiac cell sections as well as on differentiated H9c2 derived extracellular vesicles. The results confirmed the absence of Cx26 at intercalated discs and showed the presence of Cx26 at the level of different subcellular compartments. The peculiar localization at the level of extracellular vesicles suggested a specific role for cardiac Cx26 in inter-cellular communication in an independent gap junction manner.

Connexin-Based Channel Activity Is Not Specifically Altered by Hepatocarcinogenic Chemicals.

Connexin-based channels play key roles in cellular communication and can be affected by deleterious chemicals. In this study, the effects of various genotoxic carcinogenic compounds, non-genotoxic carcinogenic compounds and non-carcinogenic compounds on the expression and functionality of connexin-based channels, both gap junctions and connexin hemichannels, were investigated in human hepatoma HepaRG cell cultures. Expression of connexin26, connexin32, and connexin43 was evaluated by means of real-time reverse transcription quantitative polymerase chain reaction analysis, immunoblot analysis and in situ immunostaining. Gap junction functionality was assessed via a scrape loading/dye transfer assay. Opening of connexin hemichannels was monitored by measuring extracellular release of adenosine triphosphate. It was found that both genotoxic and non-genotoxic carcinogenic compounds negatively affect connexin32 expression. However, no specific effects related to chemical type were observed at gap junction or connexin hemichannel functionality level.

PUPIL enables mapping and stamping of transient electrical connectivity in developing nervous systems.

Currently, many genetic methods are available for mapping chemical connectivity, but analogous methods for electrical synapses are lacking. Here, we present pupylation-based interaction labeling (PUPIL), a genetically encoded system for noninvasively mapping and stamping transient electrical synapses in the mouse brain. Upon fusion of connexin 26 (CX26) with the ligase PafA, pupylation yields tag puncta following conjugation of its substrate, a biotin- or fluorescent-protein-tagged PupE, to the neighboring proteins of electrical synapses containing CX26-PafA. Tag puncta are validated to correlate well with functional electrical synapses in immature neurons. Furthermore, puncta are retained in mature neurons when electrical synapses mostly disappear-suggesting successful stamping. We use PUPIL to uncover spatial subcellular localizations of electrical synapses and approach their physiological functions during development. Thus, PUPIL is a powerful tool for probing electrical connectivity patterns in complex nervous systems and has great potential for transient receptors and ion channels as well.

Dynamic Spatiotemporal Expression Changes in Connexins of the Developing Primate's Cochlea.

Connexins are gap junction components that are essential for acquiring normal hearing ability. Up to 50% of congenital, autosomal-recessive, non-syndromic deafness can be attributed to variants in GJB2, the gene that encodes connexin 26. Gene therapies modifying the expression of connexins are a feasible treatment option for some patients with genetic hearing losses. However, the expression patterns of these proteins in the human fetus are not fully understood due to ethical concerns. Recently, the common marmoset was used as a primate animal model for the human fetus. In this study, we examined the expression patterns of connexin 26 and connexin 30 in the developing cochlea of this primate. Primate-specific spatiotemporal expression changes were revealed, which suggest the existence of primate-specific control of connexin expression patterns and specific functions of these gap junction proteins. Moreover, our results indicate that treatments for connexin-related hearing loss established in rodent models may not be appropriate for human patients, underscoring the importance of testing these treatments in primate models before applying them in human clinical trials.

Transcriptomic Profile Reveals Deregulation of Hearing-Loss Related Genes in Vestibular Schwannoma Cells Following Electromagnetic Field Exposure.

Hearing loss (HL) is the most common sensory disorder in the world population. One common cause of HL is the presence of vestibular schwannoma (VS), a benign tumor of the VIII cranial nerve, arising from Schwann cell (SC) transformation. In the last decade, the increasing incidence of VS has been correlated to electromagnetic field (EMF) exposure, which might be considered a pathogenic cause of VS development and HL. Here, we explore the molecular mechanisms underlying the biologic changes of human SCs and/or their oncogenic transformation following EMF exposure. Through NGS technology and RNA-Seq transcriptomic analysis, we investigated the genomic profile and the differential display of HL-related genes after chronic EMF. We found that chronic EMF exposure modified the cell proliferation, in parallel with intracellular signaling and metabolic pathways changes, mostly related to translation and mitochondrial activities. Importantly, the expression of HL-related genes such as NEFL, TPRN, OTOGL, GJB2, and REST appeared to be deregulated in chronic EMF exposure. In conclusion, we suggest that, at a preclinical stage, EMF exposure might promote the transformation of VS cells and contribute to HL.

Gap Junction-Mediated Intercellular Communication of cAMP Prevents CDDP-Induced Ototoxicity via cAMP/PKA/CREB Pathway.

In the cochlea, non-sensory supporting cells are directly connected to adjacent supporting cells via gap junctions that allow the exchange of small molecules. We have previously shown that the pharmacological regulation of gap junctions alleviates cisplatin (CDDP)-induced ototoxicity in animal models. In this study, we aimed to identify specific small molecules that pass through gap junctions in the process of CDDP-induced auditory cell death and suggest new mechanisms to prevent hearing loss. We found that the cyclic adenosine monophosphate (cAMP) inducer forskolin (FSK) significantly attenuated CDDP-induced auditory cell death in vitro and ex vivo. The activation of cAMP/PKA/CREB signaling was observed in organ of Corti primary cells treated with FSK, especially in supporting cells. Co-treatment with gap junction enhancers such as all-trans retinoic acid (ATRA) and quinoline showed potentiating effects with FSK on cell survival via activation of cAMP/PKA/CREB. In vivo, the combination of FSK and ATRA was more effective for preventing ototoxicity compared to either single treatment. Our study provides the new insight that gap junction-mediated intercellular communication of cAMP may prevent CDDP-induced ototoxicity.

Connexin 43 and Connexin 26 Involvement in the Ponatinib-Induced Cardiomyopathy: Sex-Related Differences in a Murine Model.

Cardiac connexins (Cxs) are proteins responsible for proper heart function. They form gap junctions that mediate electrical and chemical signalling throughout the cardiac system, and thus enable a synchronized contraction. Connexins can also individually participate in many signal transduction pathways, interacting with intracellular proteins at various cellular compartments. Altered connexin expression and localization have been described in diseased myocardium and the aim of this study is to assess the involvement of Cx43, Cx26, and some related molecules in ponatinib-induced cardiac toxicity. Ponatinib is a new multi-tyrosine kinase inhibitor that has been successfully used against human malignancies, but its cardiotoxicity remains worrisome. Therefore, understanding its signaling mechanism is important to adopt potential anti cardiac damage strategies. Our experiments were performed on hearts from male and female mice treated with ponatinib and with ponatinib plus siRNA-Notch1 by using immunofluorescence, Western blotting, and proteomic analyses. The altered cardiac function and the change in Cxs expression observed in mice after ponatinib treatment, were results dependent on the Notch1 pathway and sex. Females showed a lower susceptibility to ponatinib than males. The downmodulation of cardiac Cx43, Cx26 and miR-122, high pS368-Cx43 phosphorylation, cell viability and survival activation could represent some of the female adaptative/compensatory reactions to ponatinib cardiotoxicity.