The Gap Junction Inhibitor Octanol Decreases Proliferation and Increases Glial Differentiation of Postnatal Neural Progenitor Cells.

Neural precursor cells (NPCs) that persist in the postnatal/adult subventricular zone (SVZ) express connexins that form hemichannels and gap junctions. Gap junctional communication plays a role in NPC proliferation and differentiation during development, but its relevance on postnatal age remains to be elucidated. In this work we aimed to evaluate the effect of the blockade of gap junctional communication on proliferation and cell fate of NPCs obtained from the SVZ of postnatal rats. NPCs were isolated and expanded in culture as neurospheres. Electron microscopy revealed the existence of gap junctions among neurosphere cells. Treatment of cultures with octanol, a broad-spectrum gap junction blocker, or with Gap27, a specific blocker for gap junctions formed by connexin43, produced a significant decrease in bromodeoxyuridine incorporation. Octanol treatment also exerted a dose-dependent antiproliferative effect on glioblastoma cells. To analyze possible actions on NPC fate, cells were seeded in the absence of mitogens. Treatment with octanol led to an increase in the percentage of astrocytes and oligodendrocyte precursors, whereas the percentage of neurons remained unchanged. Gap27 treatment, in contrast, did not modify the differentiation pattern of SVZ NPCs. Our results indicate that general blockade of gap junctions with octanol induces significant effects on the behavior of postnatal SVZ NPCs, by reducing proliferation and promoting glial differentiation.

BIGH3 mediates apoptosis and gap junction failure in osteocytes during renal cell carcinoma bone metastasis progression.

Renal cell carcinoma (RCC) bone metastatis progression is driven by crosstalk between tumor cells and the bone microenvironment, which includes osteoblasts, osteoclasts, and osteocytes. RCC bone metastases (RCCBM) are predominantly osteolytic and resistant to antiresorptive therapy. The molecular mechanisms underlying pathologic osteolysis and disruption of bone homeostasis remain incompletely understood. We previously reported that BIGH3/TGFBI (transforming growth factor-beta-induced protein ig-h3, shortened to BIGH3 henceforth) secreted by colonizing RCC cells drives osteolysis by inhibiting osteoblast differentiation, impairing healing of osteolytic lesions, which is reversible with osteoanabolic agents. Here, we report that BIGH3 induces osteocyte apoptosis in both human RCCBM tissue specimens and in a preclinical mouse model. We also demonstrate that BIGH3 reduces Cx43 expression, blocking gap junction (GJ) function and osteocyte network communication. BIGH3-mediated GJ inhibition is blocked by the lysosomal inhibitor hydroxychloroquine (HCQ), but not osteoanabolic agents. Our results broaden the understanding of pathologic osteolysis in RCCBM and indicate that targeting the BIGH3 mechanism could be a combinational strategy for the treatment of RCCBM-induced bone disease that overcomes the limited efficacy of antiresorptives that target osteoclasts.

New Approach to the Cryopreservation of GV Oocytes and Cumulus Cells through the Lens of Preserving the Intercellular Gap Junctions Based on the Bovine Model.

Differences in structural and functional properties between oocytes and cumulus cells (CCs) may cause low vitrification efficiency for cumulus-oocyte complexes (COCs). We have suggested that the disconnection of CCs and oocytes in order to further cryopreservation in various ways will positively affect the viability after thawing, while further co-culture in vitro will contribute to the restoration of lost intercellular gap junctions. This study aimed to determine the optimal method of cryopreservation of the suspension of CCs to mature GV oocytes in vitro and to determine the level of mRNA expression of the genes (GJA1, GJA4; BCL2, BAX) and gene-specific epigenetic marks (DNMT3A) after cryopreservation and in vitro maturation (IVM) in various culture systems. We have shown that the slow freezing of CCs in microstraws preserved the largest number of viable cells with intact DNA compared with the methods of vitrification and slow freezing in microdroplets. Cryopreservation caused the upregulation of the genes Cx37 and Cx43 in the oocytes to restore gap junctions between cells. In conclusion, the presence of CCs in the co-culture system during IVM of oocytes played an important role in the regulation of the expression of the intercellular proteins Cx37 and Cx43, apoptotic changes, and oocyte methylation. Slow freezing in microstraws was considered to be an optimal method for cryopreservation of CCs.

Spatiotemporal heterogeneity of LMOD1 expression summarizes two modes of cell communication in colorectal cancer.

Cellular communication (CC) influences tumor development by mediating intercellular junctions between cells. However, the role and underlying mechanisms of CC in malignant transformation remain unknown. Here, we investigated the spatiotemporal heterogeneity of CC molecular expression during malignant transformation. It was found that although both tight junctions (TJs) and gap junctions (GJs) were involved in maintaining the tumor microenvironment (TME), they exhibited opposite characteristics. Mechanistically, for epithelial cells (parenchymal component), the expression of TJ molecules consistently decreased during normal-cancer transformation and is a potential oncogenic factor. For fibroblasts (mesenchymal component), the expression of GJs consistently increased during normal-cancer transformation and is a potential oncogenic factor. In addition, the molecular profiles of TJs and GJs were used to stratify colorectal cancer (CRC) patients, where subtypes characterized by high GJ levels and low TJ levels exhibited enhanced mesenchymal signals. Importantly, we propose that leiomodin 1 (LMOD1) is biphasic, with features of both TJs and GJs. LMOD1 not only promotes the activation of cancer-associated fibroblasts (CAFs) but also inhibits the Epithelial-mesenchymal transition (EMT) program in cancer cells. In conclusion, these findings demonstrate the molecular heterogeneity of CC and provide new insights into further understanding of TME heterogeneity.

Structures of wild-type and a constitutively closed mutant of connexin26 shed light on channel regulation by CO2.

Connexins allow intercellular communication by forming gap junction channels (GJCs) between juxtaposed cells. Connexin26 (Cx26) can be regulated directly by CO2. This is proposed to be mediated through carbamylation of K125. We show that mutating K125 to glutamate, mimicking the negative charge of carbamylation, causes Cx26 GJCs to be constitutively closed. Through cryo-EM we observe that the K125E mutation pushes a conformational equilibrium towards the channel having a constricted pore entrance, similar to effects seen on raising the partial pressure of CO2. In previous structures of connexins, the cytoplasmic loop, important in regulation and where K125 is located, is disordered. Through further cryo-EM studies we trap distinct states of Cx26 and observe density for the cytoplasmic loop. The interplay between the position of this loop, the conformations of the transmembrane helices and the position of the N-terminal helix, which controls the aperture to the pore, provides a mechanism for regulation.

Calcium Regulation of Connexin Hemichannels.

Connexin hemichannels (HCs) expressed at the plasma membrane of mammalian cells are of paramount importance for intercellular communication. In physiological conditions, HCs can form gap junction (GJ) channels, providing a direct diffusive path between neighbouring cells. In addition, unpaired HCs provide conduits for the exchange of solutes between the cytoplasm and the extracellular milieu, including messenger molecules involved in paracrine signalling. The synergistic action of membrane potential and Ca2+ ions controls the gating of the large and relatively unselective pore of connexin HCs. The four orders of magnitude difference in gating sensitivity to the extracellular ([Ca2+]e) and the cytosolic ([Ca2+]c) Ca2+ concentrations suggests that at least two different Ca2+ sensors may exist. While [Ca2+]e acts as a spatial modulator of the HC opening, which is most likely dependent on the cell layer, compartment, and organ, [Ca2+]c triggers HC opening and the release of extracellular bursts of messenger molecules. Such molecules include ATP, cAMP, glutamate, NAD+, glutathione, D-serine, and prostaglandins. Lost or abnormal HC regulation by Ca2+ has been associated with several diseases, including deafness, keratitis ichthyosis, palmoplantar keratoderma, Charcot-Marie-Tooth neuropathy, oculodentodigital dysplasia, and congenital cataracts. The fact that both an increased and a decreased Ca2+ sensitivity has been linked to pathological conditions suggests that Ca2+ in healthy cells finely tunes the normal HC function. Overall, further investigation is needed to clarify the structural and chemical modifications of connexin HCs during [Ca2+]e and [Ca2+]c variations. A molecular model that accounts for changes in both Ca2+ and the transmembrane voltage will undoubtedly enhance our interpretation of the experimental results and pave the way for developing therapeutic compounds targeting specific HC dysfunctions.

Perspective and Therapeutic Potential of the Noncoding RNA-Connexin Axis.

Noncoding RNAs (ncRNAs) are a class of nucleotide sequences that cannot be translated into peptides. ncRNAs can function post-transcriptionally by splicing complementary sequences of mRNAs or other ncRNAs or by directly engaging in protein interactions. Over the past few decades, the pervasiveness of ncRNAs in cell physiology and their pivotal roles in various diseases have been identified. One target regulated by ncRNAs is connexin (Cx), a protein that forms gap junctions and hemichannels and facilitates intercellular molecule exchange. The aberrant expression and misdistribution of connexins have been implicated in central nervous system diseases, cardiovascular diseases, bone diseases, and cancer. Current databases and technologies have enabled researchers to identify the direct or indirect relationships between ncRNAs and connexins, thereby elucidating their correlation with diseases. In this review, we selected the literature published in the past five years concerning disorders regulated by ncRNAs via corresponding connexins. Among it, microRNAs that regulate the expression of Cx43 play a crucial role in disease development and are predominantly reviewed. The distinctive perspective of the ncRNA-Cx axis interprets pathology in an epigenetic manner and is expected to motivate research for the development of biomarkers and therapeutics.

WNK kinase, ion channels and arachidonic acid metabolites choreographically execute endothelium-dependent vasodilation.

The smooth muscle-walled blood vessels control blood pressure. The vessel lumen is lined by an endothelial cell (ECs) layer, interconnected to the surrounding smooth muscle cells (SMCs) by myoendothelial gap junctions. Gap junctions also maintain homo-cellular ECs-ECs and SMCs-SMCs connections. This gap junction network nearly equalises both cells' membrane potential and cytosolic ionic composition, whether in resting or stimulated conditions. When acetylcholine (ACh) activates ECs M3 receptors, a complex signalling cascade involving second messengers and ion channels is triggered to induce vasodilation.

Gap junctions in Turing-type periodic feather pattern formation.

Periodic patterning requires coordinated cell-cell interactions at the tissue level. Turing showed, using mathematical modeling, how spatial patterns could arise from the reactions of a diffusive activator-inhibitor pair in an initially homogeneous 2D field. Most activators and inhibitors studied in biological systems are proteins, and the roles of cell-cell interaction, ions, bioelectricity, etc. are only now being identified. Gap junctions (GJs) mediate direct exchanges of ions or small molecules between cells, enabling rapid long-distance communications in a cell collective. They are therefore good candidates for propagating nonprotein-based patterning signals that may act according to the Turing principles. Here, we explore the possible roles of GJs in Turing-type patterning using feather pattern formation as a model. We found 7 of the 12 investigated GJ isoforms are highly dynamically expressed in the developing chicken skin. In ovo functional perturbations of the GJ isoform, connexin 30, by siRNA and the dominant-negative mutant applied before placode development led to disrupted primary feather bud formation. Interestingly, inhibition of gap junctional intercellular communication (GJIC) in the ex vivo skin explant culture allowed the sequential emergence of new feather buds at specific spatial locations relative to the existing primary buds. The results suggest that GJIC may facilitate the propagation of long-distance inhibitory signals. Thus, inhibition of GJs may stimulate Turing-type periodic feather pattern formation during chick skin development, and the removal of GJ activity would enable the emergence of new feather buds if the local environment were competent and the threshold to form buds was reached. We further propose Turing-based computational simulations that can predict the sequential appearance of these ectopic buds. Our models demonstrate how a Turing activator-inhibitor system can continue to generate patterns in the competent morphogenetic field when the level of intercellular communication at the tissue scale is modulated.

Genetically engineered human embryonic kidney cells as a novel vehicle for dual patch clamp study of human gap junction channels.

Mutations in more than half of human connexin genes encoding gap junction (GJ) subunits have been linked to inherited human diseases. Functional studies of human GJ channels are essential for revealing mechanistic insights into the etiology of disease-linked connexin mutants. However, the commonly used Xenopus oocytes, N2A, HeLa, and other model cells for recombinant expression of human connexins have different and significant limitations. Here we developed a human cell line (HEK293) with each of the endogenous connexins (Cx43 and Cx45) knocked out using the CRISPR-Cas9 system. Double knockout HEK293 cells showed no background GJ coupling, were easily transfected with several human connexin genes (such as those encoding Cx46, Cx50, Cx37, Cx45, Cx26, and Cx36) which successfully formed functional GJs and were readily accessible for dual patch clamp analysis. Single knockout Cx43 or Cx45 HEK cell lines could also be used to characterize human GJ channels formed by Cx45 or Cx43, respectively, with an expression level suitable for studying macroscopic and single channel GJ channel properties. A cardiac arrhythmia linked Cx45 mutant R184G failed to form functional GJs in DKO HEK293 cells with impaired localizations. These genetically engineered HEK293 cells are well suited for patch clamp study of human GJ channels.

Astroglial gap junctions strengthen hippocampal network activity by sustaining afterhyperpolarization via KCNQ channels.

Throughout the brain, astrocytes form networks mediated by gap junction channels that promote the activity of neuronal ensembles. Although their inputs on neuronal information processing are well established, how molecular gap junction channels shape neuronal network patterns remains unclear. Here, using astroglial connexin-deficient mice, in which astrocytes are disconnected and neuronal bursting patterns are abnormal, we show that astrocyte networks strengthen bursting activity via dynamic regulation of extracellular potassium levels, independently of glutamate homeostasis or metabolic support. Using a facilitation-depression model, we identify neuronal afterhyperpolarization as the key parameter underlying bursting pattern regulation by extracellular potassium in mice with disconnected astrocytes. We confirm this prediction experimentally and reveal that astroglial network control of extracellular potassium sustains neuronal afterhyperpolarization via KCNQ voltage-gated K+ channels. Altogether, these data delineate how astroglial gap junctions mechanistically strengthen neuronal population bursts and point to approaches for controlling aberrant activity in neurological diseases.

Connexin channels and hemichannels are modulated differently by charge reversal at residues forming the intracellular pocket.

BACKGROUND: Members of the ß-subfamily of connexins contain an intracellular pocket surrounded by amino acid residues from the four transmembrane helices. The presence of this pocket has not previously been investigated in members of the α-, γ-, δ-, and ε-subfamilies. We studied connexin50 (Cx50) as a representative of the α-subfamily, because its structure has been determined and mutations of Cx50 are among the most common genetic causes of congenital cataracts. METHODS: To investigate the presence and function of the intracellular pocket in Cx50 we used molecular dynamics simulation, site-directed mutagenesis, gap junction tracer intercellular transfer, and hemichannel activity detected by electrophysiology and by permeation of charged molecules. RESULTS: Employing molecular dynamics, we determined the presence of the intracellular pocket in Cx50 hemichannels and identified the amino acids participating in its formation. We utilized site-directed mutagenesis to alter a salt-bridge interaction that supports the intracellular pocket and occurs between two residues highly conserved in the connexin family, R33 and E162. Substitution of opposite charges at either position decreased formation of gap junctional plaques and cell-cell communication and modestly reduced hemichannel currents. Simultaneous charge reversal at these positions produced plaque-forming non-functional gap junction channels with highly active hemichannels. CONCLUSIONS: These results show that interactions within the intracellular pocket influence both gap junction channel and hemichannel functions. Disruption of these interactions may be responsible for diseases associated with mutations at these positions.

Electro-Metabolic Coupling of Cumulus-Oocyte Complex.

Oocyte-cumulus cell interaction is essential for oocyte maturation and competence. The bidirectional crosstalk network mediated by gap junctions is fundamental for the metabolic cooperation between these cells. As cumulus cells exhibit a more glycolytic phenotype, they can provide metabolic substrates that the oocyte can use to produce ATP via oxidative phosphorylation. The impairment of mitochondrial activity plays a crucial role in ovarian aging and, thus, in fertility, determining the success or failure of assisted reproductive techniques. This review aims to deepen the knowledge about the electro-metabolic coupling of the cumulus-oocyte complex and to hypothesize a putative role of potassium channel modulators in order to improve fertility, promote intracellular Ca2+ influx, and increase the mitochondrial biogenesis and resulting ATP levels in cumulus cells.

The Role of the Gap Junction Protein Connexin in Adrenal Gland Tumorigenesis.

Gap junctions (GJs) are important in the regulation of cell growth, morphology, differentiation and migration. However, recently, more attention has been paid to their role in the pathogenesis of different diseases as well as tumorigenesis, invasion and metastases. The expression pattern and possible role of connexins (Cxs), as major GJ proteins, under both physiological and pathological conditions in the adrenal gland, were evaluated in this review. The databases Web of Science, PubMed and Scopus were searched. Studies were evaluated if they provided data regarding the connexin expression pattern in the adrenal gland, despite current knowledge of this topic not being widely investigated. Connexin expression in the adrenal gland differs according to different parts of the gland and depends on ACTH release. Cx43 is the most studied connexin expressed in the adrenal gland cortex. In addition, Cx26, Cx32 and Cx50 were also investigated in the human adrenal gland. Cx50 as the most widespread connexin, along with Cx26, Cx29, Cx32, Cx36 and Cx43, has been expressed in the adrenal medulla with distinct cellular distribution. Considerable effort has recently been directed toward connexins as therapeutically targeted molecules. At present, there exist several viable strategies in the development of potential connexin-based therapeutics. The differential and hormone-dependent distribution of gap junctions within adrenal glands, the relatively large gap junction within this gland and the increase in the gap junction size and number following hormonal treatment would indicate that gap junctions play a pivotal role in cell functioning in the adrenal gland.

Neuroprotective gap-junction-mediated bystander transformations in the adult zebrafish spinal cord after injury.

The adult zebrafish spinal cord displays an impressive innate ability to regenerate after traumatic insults, yet the underlying adaptive cellular mechanisms remain elusive. Here, we show that while the cellular and tissue responses after injury are largely conserved among vertebrates, the large-size fast spinal zebrafish motoneurons are remarkably resilient by remaining viable and functional. We also reveal the dynamic changes in motoneuron glutamatergic input, excitability, and calcium signaling, and we underscore the critical role of calretinin (CR) in binding and buffering the intracellular calcium after injury. Importantly, we demonstrate the presence and the dynamics of a neuron-to-neuron bystander neuroprotective biochemical cooperation mediated through gap junction channels. Our findings support a model in which the intimate and dynamic interplay between glutamate signaling, calcium buffering, gap junction channels, and intercellular cooperation upholds cell survival and promotes the initiation of regeneration.

Downregulated expression of lactate dehydrogenase in adult oligodendrocytes and its implication for the transfer of glycolysis products to axons.

Oligodendrocytes and astrocytes are metabolically coupled to neuronal compartments. Pyruvate and lactate can shuttle between glial cells and axons via monocarboxylate transporters. However, lactate can only be synthesized or used in metabolic reactions with the help of lactate dehydrogenase (LDH), a tetramer of LDHA and LDHB subunits in varying compositions. Here we show that mice with a cell type-specific disruption of both Ldha and Ldhb genes in oligodendrocytes lack a pathological phenotype that would be indicative of oligodendroglial dysfunctions or lack of axonal metabolic support. Indeed, when combining immunohistochemical, electron microscopical, and in situ hybridization analyses in adult mice, we found that the vast majority of mature oligodendrocytes lack detectable expression of LDH. Even in neurodegenerative disease models and in mice under metabolic stress LDH was not increased. In contrast, at early development and in the remyelinating brain, LDHA was readily detectable in immature oligodendrocytes. Interestingly, by immunoelectron microscopy LDHA was particularly enriched at gap junctions formed between adjacent astrocytes and at junctions between astrocytes and oligodendrocytes. Our data suggest that oligodendrocytes metabolize lactate during development and remyelination. In contrast, for metabolic support of axons mature oligodendrocytes may export their own glycolysis products as pyruvate rather than lactate. Lacking LDH, these oligodendrocytes can also "funnel" lactate through their "myelinic" channels between gap junction-coupled astrocytes and axons without metabolizing it. We suggest a working model, in which the unequal cellular distribution of LDH in white matter tracts facilitates a rapid and efficient transport of glycolysis products among glial and axonal compartments.

Connexin-36-positive gap junctions in ventral tegmental area GABA neurons sustain opiate dependence.

Drug dependence is characterized by a switch in motivation wherein a positively reinforcing substance can become negatively reinforcing. Put differently, drug use can transform from a form of pleasure-seeking to a form of relief-seeking. Ventral tegmental area (VTA) GABA neurons form an anatomical point of divergence between two double dissociable pathways that have been shown to be functionally implicated and necessary for these respective motivations to seek drugs. The tegmental pedunculopontine nucleus (TPP) is necessary for opiate conditioned place preferences (CPP) in previously drug-naïve rats and mice, whereas dopaminergic (DA) transmission in the nucleus accumbens (NAc) is necessary for opiate CPP in opiate-dependent and withdrawn (ODW) rats and mice. Here, we show that this switch in functional anatomy is contingent upon the gap junction-forming protein, connexin-36 (Cx36), in VTA GABA neurons. Intra-VTA infusions of the Cx36 blocker, mefloquine, in ODW rats resulted in a reversion to a drug-naïve-like state wherein the TPP was necessary for opiate CPP and where opiate withdrawal aversions were lost. Consistent with these data, conditional knockout mice lacking Cx36 in GABA neurons (GAD65-Cre;Cx36 fl(CFP)/fl(CFP)) exhibited a perpetual drug-naïve-like state wherein opiate CPP was always DA independent, and opiate withdrawal aversions were absent even in mice subjected to an opiate dependence and withdrawal induction protocol. Further, viral-mediated rescue of Cx36 in VTA GABA neurons was sufficient to restore their susceptibility to an ODW state wherein opiate CPP was DA dependent. Our findings reveal a functional role for VTA gap junctions that has eluded prevailing circuit models of addiction.

Metformin suppresses NFE2L1 pathway activation to inhibit gap junction beta protein expression in NSCLC.

OBJECTIVE: Non-small-cell lung cancer (NSCLC) is a deadly form of cancer that exhibits extensive intercellular communication which contributed to chemoradiotherapy resistance. Recent evidence suggests that arrange of key proteins are involved in lung cancer progression, including gap junction proteins (GJPs). METHODS AND RESULTS: In this study, we examined the expression patterns of GJPs in NSCLC, uncovering that both gap junction protein, beta 2 (GJB2) and gap junction protein, beta 2 (GJB3) are increased in LUAD and LUSC. We observed a correlation between the upregulation of GJB2, GJB3 in clinical samples and a worse prognosis in patients with NSCLC. By examining the mechanics, we additionally discovered that nuclear factor erythroid-2-related factor 1 (NFE2L1) had the capability to enhance the expression of connexin26 and connexin 31 in the NSCLC cell line A549. In addition, the use of metformin was discovered to cause significant downregulation of gap junction protein, betas (GJBs) by limiting the presence of NFE2L1 in the cytoplasm. CONCLUSION: This emphasizes the potential of targeting GJBs as a viable treatment approach for NSCLC patients receiving metformin.

Expression, Purification, and Nanodisc Reconstitution of Connexin-43 Hemichannels for Structural Characterization by Cryo-Electron Microscopy.

Connexins are polytopic domain membrane proteins that form hexameric hemichannels (HCs) which can assemble into gap junction channels (GJCs) at the interface of two neighboring cells. The HCs may be involved in ion and small-molecule transport across the cellular plasma membrane in response to various stimuli. Despite their importance, relatively few structures of connexin HCs are available to date, compared to the structures of the GJCs. Here, we describe a protocol for expression, purification, and nanodisc reconstitution of connexin-43 (Cx43) HCs, which we have recently structurally characterized using cryo-EM analysis. Application of similar protocols to other connexin family members will lead to breakthroughs in the understanding of the structure and function of connexin HCs.