Emerging functions and clinical prospects of connexins and pannexins in melanoma.

Cellular communication through gap junctions and hemichannels formed by connexins and through channels made by pannexins allows for metabolic cooperation and control of cellular activity and signalling. These channel proteins have been described to be tumour suppressors that regulate features such as cell death, proliferation and differentiation. However, they display cancer type-dependent and stage-dependent functions and may facilitate tumour progression through junctional and non-junctional pathways. The accumulated knowledge and emerging strategies to target connexins and pannexins are providing novel clinical opportunities for the treatment of cancer. Here, we provide an updated overview of the role of connexins and pannexins in malignant melanoma. We discuss how targeting of these channel proteins may be used to potentiate antitumour effects in therapeutic settings, including through improved immune-mediated tumour elimination.

Gadolinium Chloride Restores the Function of the Gap Junctional Intercellular Communication between Hepatocytes in a Liver Injury.

BACKGROUND: Gadolinium chloride (GdCl3) has been reported to attenuate liver injury caused by a variety of toxicants. Gap junctional intercellular communication (GJIC) is thought to be essential in controlling liver homeostasis and pathology. Here we evaluate the effects of GdCl3 on functional GJIC and connexin expression in mouse models and primary hepatocytes. METHODS: Mice were administered GdCl3 intraperitoneally the day before a carbon tetrachloride (CCl4) injection or bile duct ligation (BDL) operation. Primary hepatocytes were treated with CCl4 or lipopolysaccharides (LPS), with or without GdCl3. A scrape loading/dye transfer assay was performed to assess the GJIC function. The expression of connexins was examined by real-time reverse transcription polymerase chain reaction (RT-PCR), western blot and immunofluorescent staining. RESULTS: CCl4 treatment or the BDL operation led to the dysfunction of GJIC and a down-regulation of Cx32 and Cx26 in injured liver. GdCl3 administration restored GJIC function between hepatocytes by facilitating the transfer of fluorescent dye from one cell into adjacent cells via GJIC, and markedly prevented the decrease of Cx32 and Cx26 in injured liver. In primary hepatocytes, CCl4 or LPS treatment induced an obvious decline of Cx32 and Cx26, whereas GdCl3 pretreatment prevented the down-regulation of connexins. In vivo GdCl3 protected hepatocytes and attenuated the liver inflammation and fibrosis in liver injury mouse models. CONCLUSION: GdCl3 administration protects functional GJIC between hepatocytes, and prevents the decrease of connexin proteins at mRNA and protein levels during liver injury, leading to the alleviation of chronic liver injury.

Pannexin 1 Channels Contribute to IL-1ß Expression via NLRP3/Caspase-1 Inflammasome in Aspergillus Fumigatus Keratitis.

Purpose: Pannexin 1 channels are deemed to play important roles in inflammation. However, there is limited information regarding their roles in fungal infection diseases, especially fungal keratitis. This study aimed to investigate the role of pannexin 1 channels in Aspergillus fumigatus (A. fumigatus) keratitis. Materials and Methods: Mouse models or immortalized human corneal epithelial cells (HCECs) were infected with or without A. fumigatus for given time. The expression of pannexin 1 channels was tested by qPCR, western blot and immunofluorescence staining. Mice of A. fumigatus keratitis were pretreated with carbenoxolone (CBX) or 2'(3')-O-(4-Benzoylbenzoyl) adenosine-5'-triphosphate (BzATP) to block or activate the opening of pannexin 1 channels respectively. The clinical score was recorded. Cornea tissues were examined for the downstream signals of pannexin 1 channels, including NLRP3, Caspase-1 and IL-1ß, and myeloperoxidase (MPO) by PCR and ELISA. Data were analyzed with commercial data analysis software and a P < 0.05 was considered to be statistically significant. Results: Upon A. fumigatus infection, pannexin 1 expression increased at both the mRNA and the protein levels in mice corneas (P< 0.05, n = 3). Immunofluorescence indicated that pannexin 1 channels were mainly located in the corneal epithelial layer, and they were upregulated after A. fumigatus infection. In vitro, the same tendency was found at the mRNA and the protein levels in HCECs (P< 0.05, n = 8). In mouse model, blockage of pannexin 1 channels by CBX caused more severely keratitis. The downstream signals of pannexin 1 channels (NLRP3/Caspase-1/IL-1ß) and MPO were down-regulated. Whereas activation the opening of pannexin 1 channels by BzATP reduced corneal infection with increased expression of Caspase-1 and IL-1ß. Conclusions: Pannexin 1 channels play important roles in the regulation of progression and leucocytes aggregation during corneal A. fumigatus infection via the NLRP3/Caspase-1/IL-ß pathway.

Involvement of Connexin Hemichannels in the Inflammatory Response of Chronic Diseases.

Over the last decade it has become evident that under normal conditions connexin hemichannels are either not expressed (e.g., skeletal muscle) or are expressed in very low numbers with low open probability in various mammalian tissues (e.g., liver and central nervous system (CNS)).[...].

Connexin Hemichannels in Astrocytes: An Assessment of Controversies Regarding Their Functional Characteristics.

Astrocytes in the mammalian central nervous system are interconnected by gap junctions made from connexins of the subtypes Cx30 and Cx43. These proteins may exist as hemichannels in the plasma membrane in the absence of a 'docked' counterpart on the neighboring cell. A variety of stimuli are reported to open the hemichannels and thereby create a permeation pathway through the plasma membrane. Cx30 and Cx43 have, in their hemichannel configuration, been proposed to act as ion channels and membrane pathways for different molecules, such as fluorescent dyes, ATP, prostaglandins, and glutamate. Published studies about astrocyte hemichannel behavior, however, have been highly variable and/or contradictory. The field of connexin hemichannel research has been complicated by great variability in the experimental preparations employed, a lack of highly specific pharmacological inhibitors and by confounding changes associated with genetically modified animal models. This review attempts to critically assess the gating, inhibition and permeability of astrocytic connexin hemichannels and proposes that connexins in their hemichannel configuration act as gated pores with isoform-specific permeant selectivity. We expect that some, or all, of the controversies discussed here will be resolved by future research and sincerely hope that this review serves to motivate such clarifying investigations.

Rational design of new NO and redox sensitivity into connexin26 hemichannels.

CO2 directly opens hemichannels of connexin26 (Cx26) by carbamylating K125, thereby allowing salt bridge formation with R104 of the neighbouring subunit in the connexin hexamer. The formation of the inter-subunit carbamate bridges within the hexameric hemichannel traps it in the open state. Here, we use insights derived from this model to test whether the range of agonists capable of opening Cx26 can be extended by promoting the formation of analogous inter-subunit bridges via different mechanisms. The mutation K125C gives potential for nitrosylation on Cys125 and formation of an SNO bridge to R104 of the neighbouring subunit. Unlike wild-type Cx26 hemichannels, which are insensitive to NO and NO2 (-), hemichannels comprising Cx26(K125C) can be opened by NO2 (-) and NO donors. However, NO2 (-) was unable to modulate the doubly mutated (K125C, R104A) hemichannels, indicating that an inter-subunit bridge between C125 and R104 is required for the opening action of NO2 (-). In a further test, we introduced two mutations into Cx26, K125C and R104C, to allow disulfide bridge formation across the inter-subunit boundary. These doubly mutated hemichannels open in response to changes in intracellular redox potential.

Valproic acid enhances anti-tumor effect of mesenchymal stem cell mediated HSV-TK gene therapy in intracranial glioma.

Suicide gene therapy of glioma based on herpes simplex virus type I thymidine kinase (HSV-TK) and prodrug ganciclovir (GCV) suffers from the lack of efficacy in clinical trials, which is mostly due to low transduction efficacy and absence of bystander effect in tumor cells. Recently, stem cells as cellular delivery vehicles of prodrug converting gene has emerged as a new treatment strategy for malignant glioma. In this study, we evaluated the anti-glioma effect of suicide gene therapy using human bone marrow mesenchymal stem cells expressing HSV-TK (MSCs-TK) combined with valproic acid (VPA), which can upregulate the gap junction proteins and may enhance the bystander effect of suicide gene therapy. Expression of HSV-TK in MSCs was confirmed by RT-PCR analysis and the sensitivity of MSCs-TK to GCV was assessed. A bystander effect was observed in co-cultures of MSCs-TK and U87 glioma cells by GCV in a dose-dependent manner. VPA induced the expression of the gap junction proteins connexin (Cx) 43 and 26 in glioma cell and thereby enhanced the bystander effect in co-culture experiment. The enhanced bystander effect was inhibited by the gap junction inhibitor 18-ß-glycyrrhetinic acid (18-GA). Moreover, the combined treatment with VPA and MSCs-TK synergistically enhanced apoptosis in glioma cells by caspase activation. In vivo efficacy experiments showed that combination treatment of MSCs-TK and VPA significantly inhibited tumor growth and prolonged the survival of glioma-bearing mice compared with single-treatment groups. In addition, TUNEL staining also demonstrated a significant increase in the number of apoptotic cells in the combination treated group compared with single-treatment groups. Taken together, these results provide the rational for designing novel experimental protocols to increase bystander killing effect against intracranial gliomas using MSCs-TK and VPA.

P2X7 receptor agonists pre- and postcondition the heart against ischemia-reperfusion injury by opening pannexin-1/P2X7 channels.

Protection of the heart from ischemia-reperfusion injury can be achieved by ischemic preconditioning and ischemic postconditioning. Previous studies revealed that a complex of pannexin-1 with the P2X(7) receptor forms a channel during ischemic preconditioning and ischemic postconditioning that results in the release of endogenous cardioprotectants. ATP binds to P2X(7) receptors, inducing the formation of a channel in association with pannexin-1. We hypothesized that this channel would provide a pathway for the release of these same cardioprotectants. Preconditioning-isolated perfused rat hearts with 0.4 µM ATP preceding 40 min of ischemia minimized infarct size upon subsequent reperfusion (5% of risk area) and resulted in >80% recovery of left ventricular developed pressure. Postconditioning with ATP after ischemia during reperfusion was also protective (6% infarct and 72% recovery of left ventricular developed pressure). Antagonists of both pannexin-1 (carbenoxolone and mefloquine) and P2X(7) receptors (brilliant blue G and A438079) blocked ATP pre- and postconditioning, indicating that ATP protection was elicited via the opening of a pannexin-1/P2X(7) channel. An antagonist of binding of the endogenous cardioprotectant sphingosine 1-phosphate to its G protein-coupled receptor diminished protection by ATP, which is also consistent with an ATP-dependent release of cardioprotectants. Suramin, an antagonist of binding of ATP (and ADP) to P2Y receptors, was without effect on ATP protection. Benzoyl benzoyl-ATP, a more specific P2X(7) agonist, was also a potent pre- and postconditioning agent and sensitive to blockade by pannexin-1/P2X(7) channel antagonists. The data point out for the first time the potential of P2X(7) agonists as cardioprotectants.

Gap junctions and connexins as therapeutic targets in cancer.

IMPORTANCE OF THE FIELD: Connexins (Cxs) and gap junctional intercellular communications (GJICs) play roles in cancer development, growth and metastasis. Experimental studies suggest that targeting Cxs may be a novel technique, either to inhibit tumor cell growth directly or to sensitize to various therapies. AREAS COVERED IN THIS REVIEW: A brief introduction to the role of Cxs in cancer. The focus is mainly on data available in the literature regarding therapeutic aspects. WHAT THE READER WILL GAIN: This article reviews the various strategies that take advantage of gap junctions and connexins to eliminate cancer cells, including use of the bystander effect (BE) in gene therapy, the effect of connexins on chemosensitization, the role of apoptotic processes and interactions with the microenvironment. Attempts to restore connexin expression at the transcriptional and post-transcriptional levels are described, as well as promising strategies recently explored. The potential and limitations of the approaches are discussed. TAKE HOME MESSAGE: Connexins have multiple facets, singly, in hemichannel complexes, in gap junctions or interacting with different proteins. The regulation of their expression is not fully resolved and selective manipulation of Cxs expression is therefore a challenge. Although the therapeutic potential of connexins is undeniable, more effort is needed to study the regulation and functions of these proteins.