Effects of neuroinflammation on glia-glia gap junctional intercellular communication: a perspective

Gap junctions serve as intercellular conduits that allow for the direct transfer of small molecular weight molecules (up to 1 kDa) including ions involved in cellular excitability, metabolic precursors, and second messengers. The observation of extensive intercellular coupling and large numbers of gap junctions in the central nervous system (CNS) suggests a syncytium-like organization of glial compartments. Inflammation is a hallmark of various CNS diseases such as bacterial and viral infections, multiple sclerosis, Alzheimer's disease, and cerebral ischemia. A general consequence of brain inflammation is reactive gliosis typified by astrocyte hypertrophy and proliferation of astrocytes and microglia. Changes in gap junction intercellular communication as reflected by alterations in dye coupling and connexin expression have been associated with numerous CNS inflammatory diseases, which may have dramatic implications on the survival of neuronal and glial populations in the context of neuroinflammation. A review of the effects of inflammatory products on glia-glia gap junctional communication and glial glutamate release is presented. In addition, the hypothesis of a "syncytial switch" based upon differential regulation of gap junction expression in astrocytes and microglia during normal CNS homeostasis and neuroinflammation is proposed.     

Staphylococcus aureus-derived peptidoglycan induces Cx43 expression and functional gap junction intercellular communication in microglia

Gap junctions serve as intercellular conduits that allow the exchange of small molecular weight molecules (up to 1 kDa) including ions, metabolic precursors and second messengers. Microglia are capable of recognizing peptidoglycan (PGN) derived from the outer cell wall of Staphylococcus aureus, a prevalent CNS pathogen, and respond with the robust elaboration of numerous pro-inflammatory mediators. Based on recent reports demonstrating the ability of tumor necrosis factor-alpha and interferon-gamma to induce gap junction coupling in macrophages and microglia, it is possible that pro-inflammatory mediators released from PGN-activated microglia are capable of inducing microglial gap junction communication. In this study, we examined the effects of S. aureus-derived PGN on Cx43, the major connexin in microglial gap junction channels, and functional gap junction communication using single-cell microinjections of Lucifer yellow (LY). Exposure of primary mouse microglia to PGN led to a significant increase in Cx43 mRNA and protein expression. LY microinjection studies revealed that PGN-treated microglia were functionally coupled via gap junctions, the specificity of which was confirmed by the reversal of activation-induced dye coupling by the gap junction blocker 18-alpha-glycyrrhetinic acid. In contrast to PGN-activated microglia, unstimulated cells consistently failed to exhibit LY dye coupling. These results indicate that PGN stimulation can induce the formation of a functional microglial syncytium, suggesting that these cells may be capable of influencing neuro-inflammatory responses in the context of CNS bacterial infections through gap junction intercellular communication.     

Endocytosis and post-endocytic sorting of connexins

The connexins constitute a family of integral membrane proteins that form intercellular channels, enabling adjacent cells in solid tissues to directly exchange ions and small molecules. These channels assemble into distinct plasma membrane domains known as gap junctions. Gap junction intercellular communication plays critical roles in numerous cellular processes, including control of cell growth and differentiation, maintenance of tissue homeostasis and embryonic development. Gap junctions are dynamic plasma membrane domains, and there is increasing evidence that modulation of endocytosis and post-endocytic trafficking of connexins are important mechanisms for regulating the level of functional gap junctions at the plasma membrane. The emerging picture is that multiple pathways exist for endocytosis and sorting of connexins to lysosomes, and that these pathways are differentially regulated in response to physiological and pathophysiological stimuli. Recent studies suggest that endocytosis and lysosomal degradation of connexins is controlled by a complex interplay between phosphorylation and ubiquitination. This review summarizes recent progress in understanding the molecular mechanisms involved in endocytosis and post-endocytic sorting of connexins, and the relevance of these processes to the regulation of gap junction intercellular communication under normal and pathophysiological conditions. This article is part of a Special Issue entitled: The Communicating junctions, composition, structure and characteristics.     

Gap junctions and connexin-mediated communication in the immune system

Gap junctions and connexins are present in the immune system. In haematopoiesis, connexin 43, the most widely distributed gap junction protein, appears to be a key player in the development of progenitor cells and their communication with stromal cells. Connexin 43 is expressed by macrophages, neutrophils and mast cells. Lymphocytes also express connexin 43, and inhibition of gap junction channels in these cells by using highly specific connexin mimetic reagents has profound effects on immunoglobulin secretion and synthesis of cytokines. Lymphocytes and leukocytes also communicate directly in vitro with endothelial cells via gap junctions. Connexins are implicated in inflammatory reactions in a range of tissues. Their involvement in atherosclerotic plaque formation in the vascular system is also a current growth point in research, and could lead to the development of therapeutic interventions.     

Molecular and morphological approach to study the innexin gap junctions in Rhynchosciara americana

Gap junctions mediate communication between adjacent cells and are fundamental to the development and homeostasis in multicellular organisms. In invertebrates, gap junctions are formed by transmembrane proteins called innexins. Gap junctions allow the passage of small molecules through an intercellular channel, between a cell and another adjacent cell. The dipteran Rhynchosciara americana has contributed to studying the biology of invertebrates and the study of the interaction and regulation of genes during biological development. Therefore, this paper aimed to study the R. americana innexin-2 by molecular characterization, analysis of the expression profile and cellular localization. The molecular characterization results confirm that the message is from a gap junction protein and analysis of the expression and cellular localization profile shows that innexin-2 can participate in many physiological processes during the development of R. americana.     

Mix and match: Investigating heteromeric and heterotypic gap junction channels in model systems and native tissues

This review is based in part on a roundtable discussion session: “Physiological roles for heterotypic/heteromeric channels” at the 2013 International Gap Junction Conference (IGJC 2013) in Charleston, South Carolina. It is well recognized that multiple connexins can specifically co-assemble to form mixed gap junction channels with unique properties as a means to regulate intercellular communication. Compatibility determinants for both heteromeric and heterotypic gap junction channel formation have been identified and associated with specific connexin amino acid motifs. Hetero-oligomerization is also a regulated process; differences in connexin quality control and monomer stability are likely to play integral roles to control interactions between compatible connexins. Gap junctions in oligodendrocyte:astrocyte communication and in the cardiovascular system have emerged as key systems where heterotypic and heteromeric channels have unique physiologic roles. There are several methodologies to study heteromeric and heterotypic channels that are best applied to either heterologous expression systems, native tissues or both. There remains a need to use and develop different experimental approaches in order to understand the prevalence and roles for mixed gap junction channels in human physiology.     

Regulation of gap junctions by phosphorylation of connexins

Gap junctions are a unique type of intercellular junction found in most animal cell types. Gap junctions permit the intercellular passage of small molecules and have been implicated in diverse biological processes, such as development, cellular metabolism, and cellular growth control. In vertebrates, gap junctions are composed of proteins from the "connexin" gene family. The majority of connexins are modified posttranslationally by phosphorylation, primarily on serine amino acids; however, phosphotyrosine has also been detected in connexin from cells coexpressing nonreceptor tyrosine protein kinases. Connexins are targeted by numerous protein kinases, of which some have been identified: protein kinase C, mitogen-activated protein kinase, and the v-Src tyrosine protein kinase. Phosphorylation has been implicated in the regulation of a broad variety of connexin processes, such as the trafficking, assembly/disassembly, degradation, as well as the gating of gap junction channels. This review examines the consequences of connexin phosphorylation for the regulation of gap junctional communication.     

Gap junctions and cancer: implications and perspectives

Gap junctions are made of intercellular channels which permit the diffusion from cytoplasm to cytoplasm of small hydrophilic molecules (<1,200 Da) such as ions, sugars, amino acids, nucleotides, second messengers (calcium, inositol triphosphate, etc.). Since their discovery in the early sixties, several groups have described the loss of their function in cancer cells. The accumulation of such data led to the hypothesis that gap junctions are involved in the carcinogenesis process. This assumption has been confirmed by data establishing that gap junctional intercellular communication is inhibited by most of the tumor promoters and that the restoration of such a communication, by transfection of cDNAs encoding gap junction proteins (connexins), inhibits the aberrant growth rates of tumorigenic cells. Despite these important informations, several fundamental questions remain still open. First, we do not know how gap junctions mediate such a tumor suppressor effect and whether it may depend either on the cell type or on the connexin type. Moreover, most of the data concerning a possible involvement of gap junctions in carcinogenesis have been obtained from in vitro and animal models. The very few results which have been currently collected from human tumors are not sufficient to have a clear idea concerning the real involvement of gap junctions in sporadic human cancers. These points as well as other unresolved questions about the role of gap junctional intercellular communication in carcinogenesis are mentioned. To bring some answers, some prospects are proposed with the objective to use gap junctions for increasing the effect of anticancer therapies.     

Two Classes of Gap Junction Channels Mediate Soma-Germline Interactions Essential for Germline Proliferation and Gametogenesis in Caenorhabditis elegans

In all animals examined, somatic cells of the gonad control multiple biological processes essential for germline development. Gap junction channels, composed of connexins in vertebrates and innexins in invertebrates, permit direct intercellular communication between cells and frequently form between somatic gonadal cells and germ cells. Gap junctions comprise hexameric hemichannels in apposing cells that dock to form channels for the exchange of small molecules. Here we report essential roles for two classes of gap junction channels, composed of five innexin proteins, in supporting the proliferation of germline stem cells and gametogenesis in the nematode Caenorhabditis elegans. Transmission electron microscopy of freeze-fracture replicas and fluorescence microscopy show that gap junctions between somatic cells and germ cells are more extensive than previously appreciated and are found throughout the gonad. One class of gap junctions, composed of INX-8 and INX-9 in the soma and INX-14 and INX-21 in the germ line, is required for the proliferation and differentiation of germline stem cells. Genetic epistasis experiments establish a role for these gap junction channels in germline proliferation independent of the glp-1/Notch pathway. A second class of gap junctions, composed of somatic INX-8 and INX-9 and germline INX-14 and INX-22, is required for the negative regulation of oocyte meiotic maturation. Rescue of gap junction channel formation in the stem cell niche rescues germline proliferation and uncovers a later channel requirement for embryonic viability. This analysis reveals gap junctions as a central organizing feature of many soma–germline interactions in C. elegans.     

Gap junction gene and protein families: Connexins,innexins, and pannexins

Gap junction channels facilitate the intercellular exchange of ions and small molecules. While this process is critical to all multicellular organisms, the proteins that form gap junction channels are not conserved. Vertebrate gap junctions are formed by connexins, while invertebrate gap junctions are formed by innexins. Interestingly, vertebrates and lower chordates contain innexin homologs, the pannexins, which also form channels, but rarely (if ever) make intercellular channels. While the connexin and the innexin/pannexin polypeptides do not share significant sequence similarity, all three of these protein families share a similar membrane topology and some similarities in quaternary structure. This article is part of a Special Issue entitled: Gap Junction Proteins edited by Jean Claude Herve.     

Acute loss of Cell–Cell Communication Caused by G Protein–coupled Receptors: A Critical Role for c-Src

Gap junctions mediate cell–cell communication in almost all tissues, but little is known about their regulation by physiological stimuli. Using a novel single-electrode technique, together with dye coupling studies, we show that in cells expressing gap junction protein connexin43, cell–cell communication is rapidly disrupted by G protein–coupled receptor agonists, notably lysophosphatidic acid, thrombin, and neuropeptides. In the continuous presence of agonist, junctional communication fully recovers within 1–2 h of receptor stimulation. In contrast, a desensitization-defective G protein–coupled receptor mediates prolonged uncoupling, indicating that recovery of communication is controlled, at least in part, by receptor desensitization. Agonist-induced gap junction closure consistently follows inositol lipid breakdown and membrane depolarization and coincides with Rho-mediated cytoskeletal remodeling. However, we find that gap junction closure is independent of Ca²⁺, protein kinase C, mitogen-activated protein kinase, or membrane potential, and requires neither Rho nor Ras activation. Gap junction closure is prevented by tyrphostins, by dominant-negative c-Src, and in Src-deficient cells. Thus, G protein–coupled receptors use a Src tyrosine kinase pathway to transiently inhibit connexin43-based cell–cell communication.     

Retinoids regulate the formation and degradation of gap junctions in androgen-responsive human prostate cancer cells

The retinoids, the natural or synthetic derivatives of Vitamin A (retinol), are essential for the normal development of prostate and have been shown to modulate prostate cancer progression in vivo as well as to modulate growth of several prostate cancer cell lines. 9-cis-retinoic acid and all-trans-retinoic acid are the two most important metabolites of retinol. Gap junctions, formed of proteins called connexins, are ensembles of intercellular channels that permit the exchange of small growth regulatory molecules between adjoining cells. Gap junctional communication is instrumental in the control of cell growth. We examined the effect of 9-cis-retinoic acid and all-trans retinoic acid on the formation and degradation of gap junctions as well as on junctional communication in an androgen-responsive prostate cancer cell line, LNCaP, which expressed retrovirally introduced connexin32, a connexin expressed by the luminal cells and well-differentiated cells of prostate tumors. Our results showed that 9-cis-retinoic acid and all-trans retinoic acid enhanced the assembly of connexin32 into gap junctions. Our results further showed that 9-cis-retinoic acid and all-trans-retinoic acid prevented androgen-regulated degradation of gap junctions, post-translationally, independent of androgen receptor mediated signaling. Finally, our findings showed that formation of gap junctions sensitized connexin32-expressing LNCaP cells to the growth modifying effects of 9-cis-retinoic acid, all-trans-retinoic acid and androgens. Thus, the effects of retinoids and androgens on growth and the formation and degradation of gap junctions and their function might be related to their ability to modulate prostate growth and cancer.     

Formation of the gap junction nexus: binding partners for connexins.

Gap junctions are the morphological correlates of direct cell-cell communication and are formed of hexameric assemblies of gap junction proteins (connexins) into hemichannels (or connexons) provided by each coupled cell. Gap junction channels formed by each of the connexin subtypes (of which there are as many as 20) display different properties, which have been attributed to differences in amino acid sequences of gating domains of the connexins. Recent studies additionally indicate that connexin proteins interact with other cellular components to form a protein complex termed the Nexus. This review summarizes current knowledge regarding the protein-protein interactions involving of connexin proteins and proposes hypothesized functions for these interactions.     

Dynamic connexin43 expression and gap junctional communication during endoderm differentiation of F9 embryonal carcinoma cells

Gap junctional communication permits the direct intercellular exchange of small molecules and ions. In vertebrates, gap junctions are formed by the conjunction of two connexons, each consisting of a hexamer of connexin proteins, and are either established or degraded depending on the nature of the tissue formed. Gap junction function has been implicated in both directing developmental cell fate decisions and in tissue homeostasis/metabolite exchange. In mouse development, formation of the extra embryonal parietal endoderm from visceral endoderm is the first epithelial-mesenchyme transition to occur. This transition can be mimicked in vitro, by F9 embryonal carcinoma (EC) cells treated with retinoic acid, to form (epithelial) primitive or visceral endoderm, and then with parathyroid hormone-related peptide (PTHrP) to induce the transition to (mesenchymal) parietal endoderm. Here, we demonstrate that connexin43 mRNA and protein expression levels, protein phosphorylation and subcellular localization are dynamically regulated during F9 EC cell differentiation. Dye injection showed that this complex regulation of connexin43 is correlated with functional gap junctional communication. Similar patterns of connexin43 expression, localization and communication were found in visceral and parietal endoderm isolated ex vivo from mouse embryos at day 8.5 of gestation. However, in F9 cells this tightly regulated gap junctional communication does not appear to be required for the differentiation process as such.     

The supramolecular architecture of junctional microdomains in native lens membranes

Gap junctions formed by connexons and thin junctions formed by lens-specific aquaporin 0 (AQP0) mediate the tight packing of fibre cells necessary for lens transparency. Gap junctions conduct water, ions and metabolites between cells, whereas junctional AQP0 seems to be involved in cell adhesion. High-resolution atomic force microscopy (AFM) showed the supramolecular organization of these proteins in native lens core membranes, in which AQP0 forms two-dimensional arrays that are surrounded by densely packed gap junction channels. These junctional microdomains simultaneously provide adhesion and communication between fibre cells. The AFM topographs also showed that the extracellular loops of AQP0 in junctional microdomains adopt a conformation that closely resembles the structure of junctional AQP0, in which the water pore is thought to be closed. Finally, time-lapse AFM imaging provided insights into AQP0 array formation. This first high-resolution view of a multicomponent eukaryotic membrane shows how membrane proteins self-assemble into functional microdomains.     

Sharing signals: connecting lung epithelial cells with gap junction channels

Gap junction channels enable the direct flow of signaling molecules and metabolites between cells. Alveolar epithelial cells show great variability in the expression of gap junction proteins (connexins) as a function of cell phenotype and cell state. Differential connexin expression and control by alveolar epithelial cells have the potential to enable these cells to regulate the extent of intercellular coupling in response to cell stress and to regulate surfactant secretion. However, defining the precise signals transmitted through gap junction channels and the cross talk between gap junctions and other signaling pathways has proven difficult. Insights from what is known about roles for gap junctions in other systems in the context of the connexin expression pattern by lung cells can be used to predict potential roles for gap junctional communication between alveolar epithelial cells.     

Downregulation of Connexin 32 Attenuates Hypoxia/Reoxygenation Injury in Liver Cells

Gap junction intercellular communication is involved in ischemia–reperfusion (IR) injury of organs. Connexins are proteins that are critical to the function of gap junctions. To clarify the role of gap junctions in IR injury in liver cells, the function of gap junctions was modulated in an in vitro hypoxia/reoxygenation (H/R) model. BRL‐3A rat liver cells, endogenously expressing connexins Cx32 and Cx43, were used to model the process of hepatic IR injury. Suppression of gap junction activity was achieved genetically, using Cx32‐specific small interfering RNA (siRNA), or chemically, with pharmacological inhibitors, oleamide, and 18‐α‐GA. BRL‐3A cells subjected to H/R exhibited reduced cell survival and pathologies indicative of IR injury. Cx32‐specific siRNA, oleamide, and 18‐α‐GA, respectively, decreased gap junction permeability, as assessed by the parachute assay. Pretreatment with Cx32‐specific siRNA increased cell survival. Pretreatment with oleamide or 18‐α‐GA did not improve cell survival. Modulating gap junction by Cx32 gene silencing protected BRL‐3A liver cells from H/R.     

Enhanced gap junction formation with LDL and apolipoprotein B.

Gap junctions are plasma membrane specializations involved in direct cell-cell communication. Intercellular communication is dependent upon the assembly of gap junction structures and would be influenced by agents which alter the assembly process. We investigated the effects of low density lipoprotein (LDL) on gap junction assembly between cultured Novikoff cells using quantitative dye transfer and freeze-fracture electron microscopic methods. We observed a concentration-dependent increase in dye transfer (maximum effect at 2.5 micrograms/ml) and a sixfold increase in the number of aggregated gap junction particles per cell. Immunoblots of Novikoff cells probed with anti-connexin43 antibody revealed no detectable increase in gap junction protein (connexin) levels. The influence of the different components of LDL on junction formation was also examined. First, we treated cells with cholesterol (0-150 microM) in serum-free BSA media and observed a decrease in junction assembly. Second, we added apolipoprotein-B (apo-B) in phosphatidyl choline vesicles to the cells and observed a concentration-dependent increase in dye transfer (maximum effect at 2.5 micrograms protein/ml) and a fivefold increase in the number of aggregated gap junction particles per cell. The addition of phosphatidyl choline vesicles without apo-B had no effect on gap junction formation. Thus, we demonstrated that gap junction assembly can be modulated by LDL and apo-B treatments.     

Molecular organization of gap junction membrane channels

Gap junctions regulate a variety of cell functions by creating a conduit between two apposing tissue cells. Gap junctions are unique among membrane channels. Not only do the constituent membrane channels span two cell membranes, but the intercellular channels pack into discrete cell-cell contact areas formingin vivo closely packed arrays. Gap junction membrane channels can be isolated either as two-dimensional crystals, individual intercellular channels, or individual hemichannels. The family of gap junction proteins, the connexins, create a family of gap junctions channels and structures. Each channel has distinct physiological properties but a similar overall structure. This review focuses on three aspects of gap junction structure: (1) the molecular structure of the gap junction membrane channel and hemichannel, (2) the packing of the intercellular channels into arrays, and (3) the ways that different connexins can combine into gap junction channel structures with distinct physiological properties. The physiological implications of the different structural forms are discussed.