Interplay between Cystic Fibrosis Transmembrane Regulator and Gap Junction Channels Made of Connexins 45, 40, 32 and 50 Expressed in Oocytes

The cystic fibrosis transmembrane regulator (CFTR) is a Cl⁻ channel known to influence other channels, including connexin (Cx) channels. To study the functional interaction between CFTR and gap junction channels, we coexpressed in Xenopus oocytes CFTR and either Cx45, Cx40, Cx32 or Cx50 and monitored junctional conductance (G _j) and its sensitivity to transjunctional voltage (V _j) by the dual voltage-clamp method. Application of forskolin induced a Cl⁻ current; increased G _j approximately 750%, 560%, 64% and 8% in Cx45, Cx40, Cx32 and Cx50, respectively; and decreased sensitivity to V _j gating, monitored by a change in the ratio between G _j steady state and G _j peak (G _jSS/G _jPK) at the pulse. In oocyte pairs expressing just Cx45 in one oocyte (#1) and both Cx45 and CFTR in the other (#2), with negative pulses applied to oocyte #1 forskolin application still increased G _j and decreased the sensitivity to V _j gating, indicating that CFTR activation is effective even when it affects only one of the two hemichannels and that the G _j and V _j changes are not artifacts of decreased membrane resistance in the pulsed oocyte. COOH-terminus truncation reduced the forskolin effect on Cx40 (Cx40TR) but not on Cx32 (Cx32TR) channels. The data suggest a cross-talk between CFTR and a variety of gap junction channels. Cytoskeletal scaffolding proteins and/or other intermediate cytoplasmic proteins are likely to play a role in CFTR-Cx interaction.     

Is Intercellular Communication Via Gap Junctions Required for Myoblast Fusion?

Fusion of myoblasts to form syncitial muscle cells results from a complex series of sequential events including cell alignment, cell adhesion and cell communication. The aim of the present investigation was to assess whether intercellular communication through gap junctions would be required for subsequent membrane fusion. The presence of the gap junction protein connexin 43 at areas of contact between prefusing rat L6 myoblasts was established by immunofluorescent staining. These myoblasts were dye-coupled, as demonstrated by the use of the scrape-loading/dye transfer technique. L6 myoblast dye coupling was reversibly blocked by heptanol in short term experiments as well as after chronic treatment. After a single addition of 3.5 mM heptanol, gap junctions remained blocked for up to 8 hours, then this inhibitory effect decreased gradually, likely because the alcohol was evaporated. Changing heptanol solutions every 8 hours during the time course of L6 differentiation resulted in a lasting drastic inhibition of myoblast fusion. We further investigated the effect of heptanol and of other uncoupling agents on the differentiation of primary cultures of embryonic chicken myoblasts. These cells are transiently coupled by gap junctions before myoblast fusion and prolonged application of heptanol, octanol and 18-β-glycyrrhetinic acid also inhibited their fusion. The effect of heptanol and octanol was neither due to a cytotoxic effect nor to a modification of cell proliferation. Moreover, heptanol treatment did not alter myoblast alignment and adhesion. Taken together these observations suggest that intercellular communication might be a necessary step for myoblast fusion.     

Tetracysteine Genetic Tags Complexed with Biarsenical Ligands as a Tool for Investigating Gap Junction Structure and Dynamics

Gap junctions (GJ) are defined as contact regions between two adjacent cells containing tens to thousands of closely packed membrane channels. Cells dynamically modulate communication through GJ by regulating the synthesis, transport and turnover of these channels. Previously, we engineered a recombinant connexin43 (Cx43) by genetically appending a small tetracysteine peptide motif containing the sequence -Cys-Cys-Xaa-Xaa-Cys-Cys- to the carboxy terminus of Cx43 (Cx43-TC) (3). Cx43-TC was stably expressed in HeLa cells and was specifically labeled by exposing the cells to membrane-permeant non-fluorescent ligands, such as FlAsH (a fluorescein derivative) and ReAsH (a resorufin derivative). Direct correlation of live cell images with high resolution EM detection was possible because bound ReAsH not only becomes fluorescent, but can also be used to initiate the photoconversion of diaminobenzidine (DAB) that causes the localized polymerization of an insoluble osmiophilic precipitate then visible by EM. Cx43-TC GJ's could be labeled with ReAsH and photooxidized to give selectively stained channels. Here, how the development of these tetracysteine tags complexed with appropriate ligands are useful for experiments spanning resolution ranges from light microscopy to electron tomography to molecular purification and detection is described.     

Differentiating connexin hemichannels and pannexin channels in cellular ATP release

Adenosine triphosphate (ATP) plays a fundamental role in cellular communication, with its extracellular accumulation triggering purinergic signaling cascades in a diversity of cell types. While the roles for purinergic signaling in health and disease have been well established, identification and differentiation of the specific mechanisms controlling cellular ATP release is less well understood. Multiple mechanisms have been proposed to regulate ATP release with connexin (Cx) hemichannels and pannexin (Panx) channels receiving major focus. However, segregating the specific roles of Panxs and Cxs in ATP release in a plethora of physiological and pathological contexts has remained enigmatic. This multifaceted problem has arisen from the selectivity of pharmacological inhibitors for Panxs and Cxs, methodological differences in assessing Panx and Cx function and the potential compensation by other isoforms in gene silencing and genetic knockout models. Consequently, there remains a void in the current understanding of specific contributions of Panxs and Cxs in releasing ATP during homeostasis and disease. Differentiating the distinct signaling pathways that regulate these two channels will advance our current knowledge of cellular communication and aid in the development of novel rationally-designed drugs for modulation of Panx and Cx activity, respectively.     

The Clouston syndrome mutation connexin30 A88V leads to hyperproliferation of sebaceous glands and hearing impairments in mice

Distinct mutations in the gap junction protein connexin30 (Cx30) can cause the ectodermal dysplasia Clouston syndrome in humans. We have generated a new mouse line expressing the Clouston syndrome mutation Cx30A88V under the control of the endogenous Cx30 promoter. Our results show that the mutated Cx30A88V protein is incorporated in gap junctional plaques of the epidermis. Homozygous Cx30A88V mice reveal hyperproliferative and enlarged sebaceous glands as well as a mild palmoplantar hyperkeratosis. Additionally, homozygous mutant mice show an altered hearing profile compared to control mice. We conclude that the Cx30A88V mutation triggers hyperproliferation in the skin and changes the cochlear homeostasis in mice.     

CTGF facilitates cell‐cell communication in chondrocytes via PI3K/Akt signalling pathway

PurposesGap junction intercellular communication (GJIC) is essential for articular cartilage to respond appropriately to physical or biological stimuli and maintain homeostasis. Connective tissue growth factor (CTGF), identified as an endochondral ossification genetic factor, plays a vital role in cell proliferation, migration and adhesion. However, how CTGF regulates GJIC in chondrocytes is still unknown. This study aims to explore the effects of CTGF on GJIC in chondrocytes and its potential biomechanism.Materials and methodsqPCR was performed to determine the expression of gene profile in the CCN family in chondrocytes. After CTGF treatment, CCK‐8 assay and scratch assay were performed to explore cell proliferation and migration. A scrape loading/dye transfer assay was adopted to visualize GJIC in living chondrocytes. Western blot analysis was done to detect the expression of Cx43 and PI3K/Akt signalling. Immunofluorescence staining was used to show protein distribution. siRNA targeting CTGF was used to detect the influence on cell‐cell communication.ResultsThe CTGF (CCN2) was shown to be the highest expressed member of the CCN family in chondrocytes. CTGF facilitated functional gap junction intercellular communication in chondrocytes through up‐regulation of Cx43 expressions. CTGF activated PI3K/Akt signalling to promote Akt phosphorylation and translocation. Suppressing CTGF also reduced the expression of Cx43. The inhibition of PI3K/Akt signalling decreased the expressions of Cx43 and thus impaired gap junction intercellular communication enhanced by CTGF.ConclusionsFor the first time, we provide evidence to show CTGF facilitates cell communication in chondrocytes via PI3K/Akt signalling pathway.     

Cardiac to cancer: Connecting connexins to clinical opportunity

Gap junctions and their connexin components are indispensable in mediating the cellular coordination required for tissue and organ homeostasis. The critical nature of their existence mandates a connection to disease while at the same time offering therapeutic potential. Therapeutic intervention may be offered through the pharmacological and molecular disruption of the pathways involved in connexin biosynthesis, gap junction assembly, stabilization, or degradation. Chemical inhibitors aimed at closing connexin channels, peptide mimetics corresponding to short connexin sequences, and gene therapy approaches have been incredibly useful molecular tools in deciphering the complexities associated with connexin biology. Recently, therapeutic potential in targeting connexins has evolved from basic research in cell-based models to clinical opportunity in the form of human trials. Clinical promise is particularly evident with regards to targeting connexin43 in the context of wound healing. The following review is aimed at highlighting novel advances where the pharmacological manipulation of connexin biology has proven beneficial in animals or humans.     

A strategy for the suppression of tumorigenesis induced by biomaterials: Restoration of transformed phenotype of polyetherurethane-induced tumor cells by Cx43 transfection

Biomaterials such as polyetherurethans (PEUs) are the scaffolding, which is indispensable for the development of the bio-artificial organs. However, PEUs can induce tumors in subcutaneous implantation sites in rat. We have shown that the different inhibitory potential of gap junctional intercellular communication (GJIC) on the surface of the biomaterials, including PEUs, is a key step in determining the tumorigenic potential. Here we show that suppression of a gap junctional protein connexin 43 (Cx43) plays an important role in in vivo tumorigenesis induced by PEUs for the first time and that Cx43 transfection may be an effective strategy for preventing tumorigenesis induced by biomaterials. Rat tumor cell line U41 is derived from tumors in the subcutaneous implantation of PEU films. The GJIC and the expression of Cx43 were suppressed in U41. The restoration of normal phenotype, such as reduction of growth rate, recovery of contact inhibition and loss of colony formation ability in soft agar, was achieved by Cx43 transfection. These results strongly suggest that suppression of Cx43 expression plays an important role in the development of rat malignant fibrous histiocytoma (MFHC) caused by PEUs and that Cx43 transfection is effective for prevention of tumorigenesis induced by PEUs. This revised version was published online in August 2006 with corrections to the Cover Date.