Connexin43 levels are increased in mouse neural crest cells exposed to homocysteine

BACKGROUND: Elevated homocysteine levels during embryonic development can result in neural tube and cardiovascular defects. The mechanisms that underlie the toxic effect of homocysteine are largely unknown. METHODS: We cultured mouse neural tube explants to study the effects of homocysteine on the migratory behavior of neural crest cells and on the levels of the gap junction protein Connexin43 (C x 43) and the actin- and C x 43-interacting protein ZO-1. RESULTS: Homocysteine exposure resulted in a significantly augmented maximal migration distance (MMD). The level of C x 43 immunolabeling was 2 times higher in the cytoplasm and cell protrusions of neural crest cells in homocysteine-treated cultures than in control cultures. Furthermore, colocalization of C x 43 and ZO-1 was increased in neural crest cell protrusions by this treatment. CONCLUSION: Increased C x 43 levels were previously shown to result in abnormal embryonic development. Our data raises the hypothesis that the embryotoxic effects of homocysteine may be mediated in part by its effects on C x 43 expression level and gap junction function in neural crest cells.     

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 carboxy‐terminal tail of connexin43 gap junction protein is sufficient to mediate cytoskeleton changes in human glioma cells

Connexin43 (Cx43) is a ubiquitously expressed member of the gap junction protein family that mediates gap junction intercellular communication (GJIC) by allowing exchange of cytosolic materials. Previous studies have used Cx43 truncated at the cytoplasmic tail (C‐tail) to demonstrate that the C‐tail is essential to regulate cell growth and motility. Therefore, the aim of our study was to delineate the respective role of the truncated Cx43 and the C‐tail in mediating Cx43‐dependent signaling. A truncated Cx43 expressing the channel part of the protein (TrCx43, amino acid 1–242) and a construct encompassing only the C‐tail from amino acid 243 (243Cx43) were transduced into LN18 human glioma cells. Our results showed that the ability of Cx43 to suppress growth was independent of GJIC as assessed by dye transfer, but was dependent on the presence of a rigid extracellular matrix. We further demonstrated that the C‐tail alone is sufficient to promote motility. Surprisingly, Cx43 is also able to increase migration in the absence of the C‐tail, suggesting the presence of at least two distinct signaling mechanisms utilized by Cx43 to affect motility. Finally, we used time‐lapse imaging to examine the behavior of migrating cells and it was apparent that the C‐tail was associated with a lamellipodia‐based migration not observed in either mock or TrCx43 expressing LN18 cells. Our study shows for the first time that a free C‐tail is sufficient to induce Cx43‐dependent changes in cell morphology and that Cx43 signaling is linked to the regulation of the actin cytoskeleton. J. Cell. Biochem. 110: 589–597, 2010. © 2010 Wiley‐Liss, Inc.     

Interaction of arsenic with gap junction protein connexin 43 alters gap junctional intercellular communication

Chronic exposure to Arsenic pollution in ground water is one of the largest environmental health disasters in the world. The toxicity of trivalent Arsenicals primarily happens due to its interaction with sulfhydryl groups in proteins. Arsenic binding to the protein can change the conformation of the protein and alter its interactions with other proteins leading to tissue damage. Therefore, much importance has been given to the studies of Arsenic bound proteins, for the purpose of understanding the origins of toxicity and to explore therapeutics. Here we study the dynamic effect of Arsenic on Connexin 43 (Cx43), a protein that forms the gap junctions, whose alteration deeply perturbs the cell-to-cell communication vital for maintaining tissue homeostasis. In silico molecular modelling and in vitro studies comparing Arsenic treated and untreated conditions show distinct results. Gap junction communication is severely disrupted by Arsenic due to reduced availability of unaltered Cx43 in the membrane bound form. In silico and Inductively Coupled Plasma Mass Spectrometry studies revealed the interaction of Arsenic to the Cx43 preferably occurs through surface exposed cysteines, thereby capping the thiol groups that form disulfide bonds in the tertiary structure. This leads to disruption of Cx43 oligomerization, and altered Cx43 is incompetent for transportation to the membrane surface, often forming aggregates primarily localizing in the endoplasmic reticulum. Loss of functional Cx43 on the cell surface have a deleterious effect on cellular homeostasis leading to selective vulnerability to cell death and tissue damage.     

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.     

Correlation between expression of cadherin and gap junctional communication in human lung carcinoma cells

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骨组织细胞Cx43形成的间隙连接及半通道研究进展

连接子蛋43（connexin 43,Cx43）是骨组织中主要的间隙连接（gap junction）蛋白和半通道（hemichannel）蛋白,由Cx43形成的间隙连接及半通道实现了骨组织细胞间的直接通讯。连接子蛋白对骨组织的正常发育、骨重建过程的建立与平衡是非常重要的。目前研究指出,Cx43不仅参与了骨组织的力学响应过程,也参与了二磷酸盐、甲状旁腺激素等药物对骨重建的调节过程。该文以骨组织细胞内信号传递途径的关键分子Cx43为对象,就其目前的研究现状作一综述。     

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.     

Modulation of mouse neural crest cell motility by N-cadherin and connexin 43 gap junctions.

Connexin 43 (Cx43alpha1) gap junction has been shown to have an essential role in mediating functional coupling of neural crest cells and in modulating neural crest cell migration. Here, we showed that N-cadherin and wnt1 are required for efficient dye coupling but not for the expression of Cx43alpha1 gap junctions in neural crest cells. Cell motility was found to be altered in the N-cadherin-deficient neural crest cells, but the alterations were different from that elicited by Cx43alpha1 deficiency. In contrast, wnt1-deficient neural crest cells showed no discernible change in cell motility. These observations suggest that dye coupling may not be a good measure of gap junction communication relevant to motility. Alternatively, Cx43alpha1 may serve a novel function in motility. We observed that p120 catenin (p120ctn), an Armadillo protein known to modulate cell motility, is colocalized not only with N-cadherin but also with Cx43alpha1. Moreover, the subcellular distribution of p120ctn was altered with N-cadherin or Cx43alpha1 deficiency. Based on these findings, we propose a model in which Cx43alpha1 and N-cadherin may modulate neural crest cell motility by engaging in a dynamic cross-talk with the cell's locomotory apparatus through p120ctn signaling.     

Allocation of epiblast cells to germ layer derivatives during mouse gastrulation as studied with a retroviral vector

The embryonic ectoderm, or epiblast, is the source of the three primary germ layers that form during gastrulation in the mouse embryo. Previous studies have investigated the fate of epiblast cells in early gastrulation stages using clonal analysis of cell lineage and in late gastrulation stages using transplantation of labeled grafts. In this study, we studied the fate of late gastrulation stage epiblast using a clonal analysis based on a retroviral vector encoding the Escherichia coli lacZ gene. We found that by reducing the volume of viral suspension injected into each embryo, it was possible to achieve single infectious events. Our analysis of 20 embryos singly infected at the late streak stage and 21 at the head fold stage revealed clonal descendants in only a single germ layer in each embryo. These results indicate that allocation of epiblast progenitors to a single germ layer fate has occurred by late gastrulation in mouse embryos. © 1995 Wiley-Liss, Inc.     

2-Methoxyethanol inhibits gap junctional communication in rat myometrial myocytes

The glycol ethers 2-methoxyethanol (2-ME) and 2-ethoxyethanol (2-EE) prolong gestation in rodents. Because gap junctions in the myometrium likely facilitate parturition, the present study examined inhibition of gap junctional communication by 2-ME and 2-EE in myometrial smooth-muscle cell cultures. To measure gap junctional communication, the fluorescent dye Lucifer yellow was injected into cultured cells and the transfer of the dye to adjacent cells was scored with epifluorescence microscopy. The data are presented as the percentage of cells adjacent to the microinjected cell that exhibited dye following microinjection. A 30 min treatment with 32 or 63 mmol/L 2-ME decreased dye transfer to 71% and 63%, respectively (p0.05; control 90%). Similarly, 2-EE inhibited dye transfer, although myometrial cells were less sensitive to 2-EE compared to 2-ME. Dye transfer returned to control levels after 2 h in the continued presence of 2-ME. The primary metabolite of 2-ME, methoxyacetic acid (MAA), had no effect on dye transfer at concentrations equimolar to 2-ME. Because 2-ME and 2-EE inhibited gap junctional communication only at high concentrations and because the inhibition reversed in the continued presence of the compounds, it is suggested that glycol ethers delay parturition by a mechanism independent of a direct action on myometrial gap junctions.     

Gene transfer and expression in mouse preimplantation embryos by recombinant adenovirus vector

Replication-defective recombinant adenovirus, Adex4SRLacZL, was used as a vector for transferring exogenous genes in mouse zona pellucida-free eggs at the pronuclear stage. The vector contained the E. coli LacZ reporter gene under the control of the SRα promoter (SV40 early promoter-fused HTLV-I LTR), and the expression of the reporter gene was examined during preimplantation development in culture. Histochemical staining of the embryos for β-galactosidase activity showed that the exogenous LacZ gene as expressed in 98% of the embryos at the morula-blastocyst stages. As in the microinjection method, the exogenous genes could be pursued from the 2-cell stage. Neither apparent morphological changes nor cytotoxic effects were observed. Both the percentages of embryos expressing reporter genes and the rate of development to the blastocyst stage were higher in the adenovirus vector-treated embryos than in the microinjected ones. These results suggest that the adenovirus vector system is a useful tool in investigating the genetic control of early mammalian development. © 1995 wiley-Liss, Inc.     

Dose-dependent differential upregulation of CCN1/Cyr61 and CCN3/NOV by the gap junction protein Connexin43 in glioma cells

Gap junctions form channels that allow exchange of materials between cells and are composed of transmembrane protein subunits called connexins. While connexins are believed to mediate cellular signaling by permitting intercellular communication to occur, there is also increasing evidence that suggest connexins may mediate growth control via a junction-independent mechanism. Connexin43 (Cx43) is the most abundant gap junction protein found in astrocytes, and gliomas exhibit reduced Cx43 expression. We have previously observed that restoration of Cx43 levels in glioma cells led to increased expression of CCN3 (NOV) proteins. We now report that overexpression of Cx43 in C6-glioma cells (C6-Cx43) also upregulates the expression of CCN1 (Cyr61). Both CCN1 and CCN3 belong to the Cyr61/Connective tissue growth factor/Nephroblastoma-overexpressed (CCN) family of secretory proteins. The CCN proteins are tightly associated with the extracellular matrix and have important roles in cell proliferation and migration. CCN1 promotes growth in glioma cells, as shown by the increased proliferation rate of CCN1-overexpressing C6 cells. In addition to its effect on cell growth, CCN1 also increased the motility of glioma cells in the presence of extracellular substrates such as fibronectin. Gliomas expressing high levels of Cx43 preferentially upregulated CCN3 which resulted in reduced growth rate. CCN3 could also be observed in Cx43 gap junction plaques in confluent C6-Cx43H culture at the stationary phase of their growth. Our results suggest that the dissimilar growth characteristics between high and low Cx43 expressors may be due to differential regulation of CCN3 by varying levels of Cx43.     

Regulated expression of neurofibromin in migrating neural crest cells of avian embryos

Neurofibromatosis type 1 (NF1) is a common human genetic disease involving various neural crest (NC)-derived cell types, in particular, Schwann cells and melanocytes. The gene responsible for NF1 encodes the protein neurofibromin, which contains a domain with amino acid sequence homology to the ras-guanosine triphosphatase activating protein, suggesting that neurofibromin may play a role in intracellular signaling pathways regulating cellular proliferation or differentiation, or both. To determine whether neurofibromin plays a role in NC cell development, we used antibodies raised against human neurofibromin fusion proteins in western blot and immunocytochemical studies of early avian embryos. These antibodies specifically recognized the 235 kD chicken neurofibromin protein, which was expressed in migrating trunk and cranial NC cells of early embryos (E1.5 to E2), as well as in endothelial and smooth muscle cells of blood vessels and in a subpopulation of non-NC-derived cells in the dermamyotome. At slightly later stages (E3 to E5), neurofibromin immunostaining was observed in various NC derivatives, including dorsal root ganglia and peripheral nerves, as well as non-NC-derived cell types, including heart, skeletal muscle, and kidney. At still later stages (E7 to E9), neurofibromin immunoreactivity was found in almost all tissues in vivo. To determine whether the levels of neurofibromin changed during melanocyte and Schwann cell development, tissue culture experiments were performed. Cultured NC cells were found to express neurofibromin at early time points in culture, but the levels of immunoreactivity decreased as the cells underwent pigmentation. Schwann cells, on the other hand, continued to express neurofibromin in culture. These data suggest, therefore, that neurofibromin may play a role in the development of both NC cells and a variety of non-NC-derived tissues. © 1995 John Wiley & Sons, Inc.     

Radiation-induced bystander effects in malignant trophoblast cells are independent from gap junctional communication

It is controversially discussed that irradiation induces bystander effects via gap junction channels and/or diffusible cellular factors such as nitric oxide or cytokines excreted from the cells into the environment. But up to now the molecular mechanism leading to a bystander response is not well understood. To discriminate between both mechanisms of bystander response, (i) mediated by gap junctional communication and/or (ii) mediated by diffusible molecules, we used non-communicating Jeg3 malignant trophoblast cells transfected with inducible gap junction proteins, connexin43 and connexin26, respectively, based on the Tet-On system. We co-cultivated X-ray irradiated and non-irradiated bystander Jeg3 cells for 4 h, separated both cell populations by flow cytometry and evaluated the expression of activated p53 by Western blot analysis. The experimental design was proven with communicating versus non-communicating Jeg3 cells. Interestingly, our results revealed a bystander effect which was independent from gap junctional communication properties and the connexin isoform expressed. Therefore, it seems more likely that the bystander effect is not mediated via gap junction channels but rather by paracrine mechanisms via excreted molecules in Jeg3 cells.     

Oligodendroglial and pan‐neural crest expression of Cre recombinase directed by Sox10 enhancer

Utilizing a recently identified Sox10 distal enhancer directing Cre expression, we report S4F:Cre, a transgenic mouse line capable of inducing recombination in oligodendroglia and all examined neural crest derived tissues. Assayed using R26R:LacZ reporter mice expression was detected in neural crest derived tissues including the forming facial skeleton, dorsal root ganglia, sympathetic ganglia, enteric nervous system, aortae, and melanoblasts, consistent with Sox10 expression. LacZ reporter expression was also detected in non‐neural crest derived tissues including the oligodendrocytes and the ventral neural tube. This line provides appreciable differences in Cre expression pattern from other transgenic mouse lines that mark neural crest populations, including additional populations defined by the expression of other SoxE proteins. The S4F:Cre transgenic line will thus serve as a powerful tool for lineage tracing, gene function characterization, and genome manipulation in these populations. genesis 47:765–770, 2009. © 2009 Wiley‐Liss, Inc.