Gap junctional communication is required to maintain mouse cortical neural progenitor cells in a proliferative state

The mechanisms that determine whether neural stem cells remain in a proliferative state or differentiate into neurons or glia are largely unknown. Here we establish a pivotal role for gap junction-mediated intercellular communication in determining the proliferation and survival of mouse neural progenitor cells (NPCs). When cultured in the presence of basic fibroblast growth factor (bFGF), NPCs express the gap junction protein connexin 43 and are dye-coupled. Upon withdrawal of bFGF, levels of connexin 43 and dye coupling decrease, and the cells cease proliferating and differentiate into neurons; the induction of gap junctions by bFGF is mediated by p42/p44 mitogen-activated protein kinases. Inhibition of gap junctions abolishes the ability of bFGF to maintain NPCs in a proliferative state resulting in cell differentiation or cell death, while overexpression of connexin 43 promotes NPC self-renewal in the absence of bFGF. In addition to promoting their proliferation, gap junctions are required for the survival of NPCs. Gap junctional communication is therefore both necessary and sufficient to maintain NPCs in a self-renewing state.     

Bone morphogenetic protein-2 modulation of chondrogenic differentiation in vitro involves gap junction-mediated intercellular communication

Undifferentiated mesenchymal cells in the limb bud integrate a complex array of local and systemic signals during the process of cell condensation and chondrogenic differentiation. To address the relationship between bone morphogenetic protein (BMP) signaling and gap junction-mediated intercellular communication, we examined the effects of BMP-2 and a gap junction blocker 18 alpha glycyrrhetinic acid (18alpha-GCA) on mesenchymal cell condensation and chondrogenic differentiation in an in vitro chondrogenic model. We find that connexin43 protein expression significantly correlates with early mesenchymal cellular condensation and chondrogenesis in high-density limb bud cell culture. The level of connexin43 mRNA is maximally upregulated 48 h after treatment with recombinant human BMP-2 with corresponding changes in protein expression. Inhibition of gap junction-mediated intercellular communication with 2.5 microM 18alpha-GCA decreases chondrogenic differentiation by 50% at 96 h without effects on housekeeping genes. Exposure to 18alpha-GCA for only the first 24-48 h after plating does not affect condensation or later chondrogenic differentiation suggesting that gap junction-mediated intercellular communication is not critical for the initial phase of condensation but is important for the onset of differentiation. 18alpha-GCA can also block the chondrogenic effects of BMP-2 without effects on cell number or connexin43 expression. These observations demonstrate 18alpha-GCA-sensitive regulation of intercellular communication in limb mesenchymal cells undergoing chondrogenic differentiation and suggest that BMP-2 induced chondrogenic differentiation may be mediated in part through the modulation of connexin43 expression and gap junction-mediated intercellular communication.     

Gap junction blockage interferes with neuronal and astroglial differentiation of mouse P19 embryonal carcinoma cells

During embryonic development, cells not only increase in number, they also undergo specialization and differentiate into diverse cell types that are organized into different tissues and organs. Nervous system development, for example, involves a complex series of events such as neuronal and astroglial differentiation that are coordinated among adjacent cells. The organization of growth and differentiation may be mediated, at least partly, by exchange of small ions and molecules via intercellular gap junction channels. These structures are mode of connexons (hemichannels), which are hexameric assemblies of the gap junction proteins, connexins. We investigated the role of intercellular communication in neuronal and astroglial differentiation by using a gap junction blocking agent, carbenoxolone (CBX), in comparison to its inactive (control) analog, glycyrrhizic acid (GZA). We used the mouse P19 embryonal carcinoma cell line, which differentiates into neurons and astrocytes upon retinoic acid (RA) induction. Our results show that both GZA- and CBX-treated cells express alpha 1 connexin (connexin43). The level of alpha 1 connexin decreases upon RA induction. CBX treated cells show significant reduction in both neuronal (5-fold) and astrocytic (13-fold) differentiation compared with those of control. These results clearly indicate that the blockage of gap junction-mediated intercellular communication interferes with differentiation of P19 cells into neurons and astrocytes.     

Gap junctions are required for the propagation of spreading depression

Spreading depression (SD) is a slowly propagating depression of cerebral neuronal activity and transmembrance ionic gradients, that arises in response to a variety of noxious stimuli. SD bears a strong resemblance to gap junction-mediated calcium waves among cultured astrocytes. Here, we show that gap junction-mediated intercellular diffusion is necessary for the generation of SD. Waves of SD in the isolated chicken retina were blocked by five different inhibitors of gap junctional coupling, which was assessed by the intercellular transit of Lucifer Yellow (LY). Each of these gap junction blockers inhibited both the migration of SD and the diffusion of LY in a dose-dependent manner. In contrast, glutamate-evoked calcium influx into retinal cells was not affected by these compounds. The results indicate that intercellular coupling through gap junctions is required for SD. Gap junction-mediated communication might therefore constitute an important mechanism in both normative and pathological brain function. © 1995 John Wiley & Sons, Inc.     

Effect of thioridazine on gap junction intercellular communication in connexin 43-expressing cells

Propagation of electrical activity between myocytes in the heart requires gap junction channels, which contribute to coordinated conduction of the heartbeat. Some antipsychotic drugs, such as thioridazine and its active metabolite, mesoridazine, have known cardiac conduction side-effects, which have resulted in fatal or nearly fatal clinical consequences in patients. The physiological mechanisms responsible for these cardiac side-effects are unknown. We tested the effect of thioridazine and mesoridazine on gap junction-mediated intercellular communication between cells that express the major cardiac gap junction subtype connexin 43. Micromolar concentrations of thioridazine and mesoridazine inhibited gap junction-mediated intercellular communication between WB-F344 epithelial cells in a dose-dependent manner, as measured by fluorescent dye transfer. Kinetic analyses demonstrated that inhibition by 10 μmol/L thioridazine occurred within 5 min, achieved its maximal effect within 1 h, and was maintained for at least 24 h. Inhibition was reversible within 1 h upon removal of the drug. Western blot analysis of connexin 43 in a membrane-enriched fraction of WB-F344 cells treated with thioridazine revealed decreased amounts of unphosphorylated connexin 43, and appearance of a phosphorylated connexin 43 band that co-migrated with a “hyperphosphorylated” connexin 43 band present in TPA-inhibited cells. When tested for its effects on cardiomyocytes isolated from neonatal rats, thioridazine decreased fluorescent dye transfer between colonies of beating myocytes. Microinjection of individual cells with fluorescent dye also showed inhibition of dye transfer in thioridazine-treated cells compared to vehicle-treated cells. In addition, thioridazine, like TPA, inhibited rhythmic beating of myocytes within 15 min of application. In light of the fact that the thioridazine and mesoridazine concentrations used in these experiments are in the range of those used clinically in patients, our results suggest that inhibition of gap junction intercellular communication may be one factor contributing to the cardiac side-effects observed in some patients taking these medications.     

Cytosolic stress reduces degradation of connexin43 internalized from the cell surface and enhances gap junction formation and function

The protein constituents of gap junctions, connexins, have a rapid basal rate of degradation even after transport to the cell surface. We have used cell surface biotinylation to label gap junction-unassembled plasma membrane pools of connexin43 (Cx43) and show that their degradation is inhibited by mild hyperthermia, oxidative stress, and proteasome inhibitors. Cytosolic stress does not perturb endocytosis of biotinylated Cx43, but instead it seems to interfere with its targeting and/or transport to the lysosome, possibly by increasing the level of unfolded protein in the cytosol. This allows more Cx43 molecules to recycle to the cell surface, where they are assembled into long-lived, functional gap junctions in otherwise gap junction assembly-inefficient cells. Cytosolic stress also slowed degradation of biotinylated Cx43 in gap junction assembly-efficient normal rat kidney fibroblasts, and reduced the rate at which gap junctions disappeared from cell interfaces under conditions that blocked transport of nascent connexin molecules to the plasma membrane. These data demonstrate that degradation from the cell surface can be down-regulated by physiologically relevant forms of stress. For connexins, this may serve to enhance or preserve gap junction-mediated intercellular communication even under conditions in which protein synthesis and/or intracellular transport are compromised.     

Shapes of astrocyte networks in the juvenile brain

The high level of intercellular communication mediated by gap junctions between astrocytes indicates that, besides individual astrocytic domains, a second level of organization might exist for these glial cells as they form communicating networks. Therefore,the contribution of astrocytes to brain function should also be considered to result from coordinated groups of cells. To evaluate the shape and extent of these networks we have studied the expression of connexin 43, a major gap junction protein in astrocytes, and the intercellular diffusion of gap junction tracers in two structures of the developing brain, the hippocampus and the cerebral cortex. We report that the shape of astrocytic networks depends on their location within neuronal compartments ina defined brain structure. Interestingly, not all astrocytes are coupled, which indicates that connections within these networks are restricted. As gap junctional communication in astrocytes is reported to contribute to several glial functions, differences in the shape of astrocytic networks might have consequences on neuronal activity and survival.     

SRC utilizes Cas to block gap junctional communication mediated by connexin43

The Src tyrosine kinase phosphorylates Cas (Crk-associated substrate) to confer anchorage independence and invasive growth potential to transformed cells. Gap junctional communication is often lower between aggressive tumor cells compared with normal or benign precursors. The gap junction protein connexin43 (Cx43) is a tumor suppressor that can inhibit tumor cell growth. Src can phosphorylate Cx43 to block gap junctional communication between transformed cells. However, mechanisms by which this event actually closes intercellular channels have not been clearly defined. Here, we report that Src and Cas associate with each other at intercellular junctions. In addition, Cas is required for Src to reduce dye transfer and electrical coupling between cells expressing Cx43. Thus, Src utilizes Cas to inhibit gap junctional communication mediated by Cx43. This finding introduces a novel role of the Cas focal adhesion linker protein in the gap junction complex. This observation may help explain how gap junctional communication can be suppressed between malignant and metastatic tumor cells.     

The enhancement of glucose uptake caused by the collapse of gap junction communication is due to an increase in astrocyte proliferation

We have previously shown that several gap junction uncouplers increase the uptake of glucose in astrocytes. The aim of the present work was to study whether the increase in glucose uptake was a consequence of the inhibition of gap junction communication and the purpose of this effect. Our results show that alpha-glycyrrhetinic acid and endothelin-1 increase the uptake of glucose in highly, but not in poorly, coupled astrocytes. This effect depended on connexin 43 levels and was abolished when the inhibition of gap junction communication was prevented by tolbutamide or ouabain. The inhibition of gap junctions increased the rate of glucose incorporation into DNA and RNA, which was inhibited by treatment with dehydroepiandrosterone, an inhibitor of glucose-6-phosphate dehydrogenase, the regulatory enzyme of the pentose phosphate pathway. The inhibition of gap junctions significantly increased astrocyte proliferation, which was counteracted by tolbutamide. These effects were not observed in poorly coupled astrocytes expressing low levels of connexin 43. The increase in astrocyte proliferation caused by gap junction inhibition was prevented when either glucose uptake or the pentose phosphate pathway were inhibited. We conclude that the inhibition of gap junction communication induces astrocyte proliferation, resulting in an enhancement of glucose uptake and its utilization through the pentose phosphate pathway to provide ribose-5-phosphate for the synthesis of nucleic acids.     

Degradation of Connexins from the Plasma Membrane Is Regulated by Inhibitors of Protein Synthesis

Little is known about the mechanism and regulation of connexin turnover from the plasma membrane. We have used a combination of cell surface biotinylation, immunofluorescence microscopy, and scrape-load dye transfer assays to investigate the effect of the protein synthesis inhibitor cycloheximide on connexin43 and connexin32 after their transport to the plasmalemma. The results obtained demonstrate that cycloheximide inhibits the turnover of connexins from the surface of both gap junction assembly-deficient and -efficient cells. Moreover, cell surface connexin saved from destruction by cycloheximide can assemble into long-lived, functional gap junctional plaques. These findings support the concept that downregulation of connexin degradation from the plasma membrane can serve as a mechanism to enhance gap junction-mediated intercellular communication.     

Degradation of connexins from the plasma membrane is regulated by inhibitors of protein synthesis

Little is known about the mechanism and regulation of connexin turnover from the plasma membrane. We have used a combination of cell surface biotinylation, immunofluorescence microscopy, and scrape-load dye transfer assays to investigate the effect of the protein synthesis inhibitor cycloheximide on connexin43 and connexin32 after their transport to the plasmalemma. The results obtained demonstrate that cycloheximide inhibits the turnover of connexins from the surface of both gap junction assembly-deficient and -efficient cells. Moreover, cell surface connexin saved from destruction by cycloheximide can assemble into long-lived, functional gap junctional plaques. These findings support the concept that downregulation of connexin degradation from the plasma membrane can serve as a mechanism to enhance gap junction-mediated intercellular communication.     

Immunoglobulin and cytokine expression in mixed lymphocyte cultures is reduced by disruption of gap junction intercellular communication.

Connexins (Cx), the protein subunits assembled into gap junction intercellular communication channels, are expressed in primary lymphoid organs and by circulating leukocytes. Human tonsil-derived T and B lymphocytes express Cx40 and 43; circulating human T, B, and NK lymphocytes express Cx43 and directly transfer between each other a low molecular dye indicative that functional gap junctions exist. We now identify specific properties in the immune system underwritten by gap junctions. Mixed lymphocytes cultured in the presence of two reagents with independent inhibitory action on gap junction communication, a connexin mimetic peptide and 18-alpha-glycyrrhetinic acid, markedly reduced the secretion of IgM, IgG, and IgA. The secretion of these immunoglobulins by purified B cells was also reduced by the two classes of gap junction inhibitors. Complex temporal inhibitory effects on the expression of mRNA encoding interleukins, especially IL-10, were also observed. The results indicate that intercellular signaling across gap junctions is an important component of the mechanisms underlying metabolic cooperation in the immune system.     

Effects of mechanical strain on the function of Gap junctions in osteocytes are mediated through the prostaglandin EP2 receptor

Osteocytes embedded in the matrix of bone are thought to be mechanosensory cells that translate mechanical strain into biochemical signals that regulate bone modeling and remodeling. We have shown previously that fluid flow shear stress dramatically induces prostaglandin release and COX-2 mRNA expression in osteocyte-like MLO-Y4 cells, and that prostaglandin E2 (PGE2) released by these cells functions in an autocrine manner to regulate gap junction function and connexin 43 (Cx43) expression. Here we show that fluid flow regulates gap junctions through the PGE2 receptor EP2 activation of cAMP-dependent protein kinase A (PKA) signaling. The expression of the EP2 receptor, but not the subtypes EP1,EP3, and EP4, increased in response to fluid flow. Application of PGE2 or conditioned medium from fluid flow-treated cells to non-stressed MLO-Y4 cells increased expression of the EP2 receptor. The EP2 receptor antagonist, AH6809, suppressed the stimulatory effects of PGE2 and fluid flow-conditioned medium on the expression of the EP2 receptor, on Cx43 protein expression, and on gap junction-mediated intercellular coupling. In contrast, the EP2 receptor agonist butaprost, not the E1/E3 receptor agonist sulprostone, stimulated the expression of Cx43 and gap junction function. Fluid flow conditioned medium and PGE2 stimulated cAMP production and PKA activity suggesting that PGE2 released by mechanically stimulated cells is responsible for the activation of cAMP and PKA. The adenylate cyclase activators, forskolin and 8-bromo-cAMP, enhanced intercellular connectivity, the number of functional gap junctions, and Cx43 protein expression, whereas the PKA inhibitor, H89, inhibited the stimulatory effect of PGE2 on gap junctions. These studies suggest that the EP2 receptor mediates the effects of autocrine PGE2 on the osteocyte gap junction in response to fluid flow-induced shear stress. These data support the hypothesis that the EP2 receptor, cAMP, and PKA are critical components of the signaling cascade between mechanical strain and gap junction-mediated communication between osteocytes.     

The role of gap junction membrane channels in development

In most developmental systems, gap junction-mediated cell-cell communication (GJC) can be detected from very early stages of embryogenesis. This usually results in the entire embryo becoming linked as a syncytium. However, as development progresses, GJC becomes restricted at discrete boundaries, leading to the subdivision of the embryo into communication compartment domains. Analysis of gap junction gene expression suggests that this functional subdivision of GJC may be mediated by the differential expression of the connexin gene family. The temporal-spatial pattern of connexin gene expression during mouse embryogenesis is highly suggestive of a role for gap junctions in inductive interactions, being regionally restricted in distinct developmentally significant domains. Using reverse genetic approaches to manipulate connexin gene function, direct evidence has been obtained for the connexin 43 (Cx43) gap junction gene playing a role in mammalian development. The challenges in the future are the identification of the target cell populations and the cell signaling processes in which Cx43-mediated cell-cell interactions are critically required in mammalian development. Our preliminary observations suggest that neural crest cells may be one such cell population.     

The functional expression of connexin 43 in articular chondrocytes is increased by interleukin 1beta: evidence for a Ca2+-dependent mechanism

Cell-to-cell interactions and gap junctions-dependent communication are crucially involved in chondrogenic differentiation, while in adult articular cartilage direct intercellular communication occurs mainly among chondrocytes facing the outer cartilage layer. Chondrocytes extracted from adult articular cartilage and grown in primary culture express connexin 43 and form functional gap junctions capable of sustaining the propagation of intercellular Ca2+ waves. Degradation of articular cartilage is a characteristic feature of arthritic diseases and is associated to increased levels of interleukin-1 (IL-1) in the synovial fluid. We have examined the effects of IL-1 on gap junctional communication in cultured rabbit articular chondrocytes. Incubation with IL-1 potentiated the transmission of intercellular Ca2+ waves and the intercellular transfer of Lucifer yellow. The stimulatory effect was accompanied by a dose-dependent increase in the expression of connexin 43 and by an enhanced connexin 43 immunostaining at sites of cell-to-cell contact. IL-1 stimulation induced a dose-dependent increase of cytosolic Ca2+ and activates protein tyrosine phosphorylation. IL-1-dependent up-regulation of connexin 43 could be prevented by intracellular Ca2+ chelation, but not by inhibitors of protein tyrosine kinases, suggesting a crucial role of cytosolic Ca2+ in regulating the expression of connexin 43. IL-1 is one of the most potent cytokines that promotes cartilage catabolism: its modulation of intercellular communication represents a novel mechanism by which proinflammatory mediators regulate the activity of cartilage cells.     

Gap junctions in human synovial cells and tissue

Our objective was to establish the existence of intercellular communication through gap junctions in synovial lining cells and in primary and passaged cultures of human synovial cells. Communication between cells was assessed using the nystatin perforated-patch method, fluorescent dye transfer, immunochemistry, transmission electron microscopy, and immunoblotting. Functional gap junctions were observed in primary and passaged cultures and were based on measurements of the transient current response to a step voltage. The average resistance between cells in small aggregates was 300 +/- 150 MOmega. Gap junctions were also observed between synovial lining cells in tissue explants; the size of the cell network in synovial tissue was estimated to be greater than 40 cells. Intercellular communication between cultured cells and between synovial lining cells was confirmed by dye injection. Punctate fluorescent regions were seen along intercellular contacts between cultured cells and in synovial membranes in cells and tissue immunostained for connexin43. The presence of the protein was verified in immunoblots. Regular 2-nm intermembrane gap separations characteristic of gap junctions were seen in transmission electron micrographs of synovial biopsies. The results showed that formation of gap-junction channels capable of mediating ionic and molecular communication was a regular feature of synovial cells, both in tissue and in cultured cells. The gap junctions contained connexin43 protein and perhaps other proteins. The physiological purpose of gap junctions in synovial cells is unknown, but it is reasonable to anticipate that intercellular communication serves some presently unrecognized function.     

Connexin make-up of endothelial gap junctions in the rat pulmonary artery as revealed by immunoconfocal microscopy and triple-label immunogold electron microscopy.

Integration of vascular endothelial function relies on multiple signaling mechanisms, including direct cell-cell communication through gap junctions. Gap junction proteins expressed in the endothelium include connexin37, connexin40, and connexin43. To investigate whether individual endothelial cells in vivo express all three connexin types and, if so, whether multiple connexins are assembled into the same gap junction plaque, we used affinity-purified connexin-specific antibodies raised in three different species to permit multiple-label immunoconfocal and immunoelectron microscopy in the rat main pulmonary artery. Immunoconfocal microscopy showed a high incidence of co-localization between connexin43 and connexin40, but lower incidences of co-localization between connexin37 and connexin40 or connexin43. Immunoelectron microscopy revealed that 83% of gap junction profiles contained all three connexins, with the proportion of connexin40 labeling being significantly higher than that of connexin37 or connexin43. The presence of three different connexin types of distinct properties in vitro provides potential for complex regulation and functional differentiation of endothelial intercellular communication properties in vivo.     

Selective permeability of different connexin channels to the second messenger cyclic AMP

Gap junctions are intercellular conduits that are formed in vertebrates by connexin proteins and allow diffusion exchange of intracellular ions and small molecules. At least 20 different connexin genes in the human and mouse genome are cell-type specifically expressed with overlapping expression patterns. A possible explanation for this diversity could be different permeability of biologically important molecules, such as second messenger molecules. We have recently demonstrated that cyclic nucleotide-gated channels can be used to quantify gap junction-mediated diffusion of cyclic AMP. Using this method we have compared the relative permeability of gap junction channels composed of connexin 26, 32, 36, 43, 45, or 47 proteins toward the second messenger cAMP. Here we show that cAMP permeates through the investigated connexin channels with up to 30-fold different efficacy. Our results suggest that intercellular cAMP signaling in different cell types can be affected by the connexin expression pattern.