Connexin and pannexin channels in cancer

Communication among cells via direct cell-cell contact by connexin gap junctions, or between cell and extracellular environment via pannexin channels or connexin hemichannels, is a key factor in cell function and tissue homeostasis. Upon malignant transformation in different cancer types, the dysregulation of these connexin and pannexin channels and their effect in cellular communication, can either enhance or suppress tumorigenesis and metastasis. In this review, we will highlight the latest reports on the role of the well characterized connexin family and its ability to form gap junctions and hemichannels in cancer. We will also introduce the more recently discovered family of pannexin channels and our current knowledge about their involvement in cancer progression.     

Connexin and pannexin channels in cancer

Communication among cells via direct cell-cell contact by connexin gap junctions, or between cell and extracellular environment via pannexin channels or connexin hemichannels, is a key factor in cell function and tissue homeostasis. Upon malignant transformation in different cancer types, the dysregulation of these connexin and pannexin channels and their effect in cellular communication, can either enhance or suppress tumorigenesis and metastasis. In this review, we will highlight the latest reports on the role of the well characterized connexin family and its ability to form gap junctions and hemichannels in cancer. We will also introduce the more recently discovered family of pannexin channels and our current knowledge about their involvement in cancer progression.

     

Connexin and pannexin hemichannels of neurons and astrocytes

Hemichannels are large pore ion channels that in the traditional view are formed when half a gap connexin junction opens to the extracellular space. It is now evident that other ion channel families, including the newly discovered pannexin family can form channels with all the nascent properties of hemichannels. This suggests that hemichannels should now be defined to include members of non-connexin families. Several connexin, and two pannexins are expressed in neurons and astrocytes where they may function in release of ATP and glutamate. Additionally, pannexin-1 appears to play a role in neuronal death. Hemichannels form a novel and unique class of ion channels that likely have diverse physiological and pathophysiological roles in the nervous system.     

Connexin and pannexin hemichannels of neurons and astrocytes

Hemichannels are large pore ion channels that in the traditional view are formed when half a gap connexin junction opens to the extracellular space. It is now evident that other ion channel families, including the newly discovered pannexin family can form channels with all the nascent properties of hemichannels. This suggests that hemichannels should now be defined to include members of non-connexin families. Several connexin, and two pannexins are expressed in neurons and astrocytes where they may function in release of ATP and glutamate. Additionally, pannexin-1 appears to play a role in neuronal death. Hemichannels form a novel and unique class of ion channels that likely have diverse physiological and pathophysiological roles in the nervous system.     

Connexin and pannexin as modulators of myocardial injury

Multicellular organisms have developed a variety of mechanisms that allow communication between their cells. Whereas some of these systems, as neurotransmission or hormones, make possible communication between remote areas, direct cell-to-cell communication through specific membrane channels keep in contact neighboring cells. Direct communication between the cytoplasm of adjacent cells is achieved in vertebrates by membrane channels formed by connexins. However, in addition to allowing exchange of ions and small metabolites between the cytoplasms of adjacent cells, connexin channels also communicate the cytosol with the extracellular space, thus enabling a completely different communication system, involving activation of extracellular receptors. Recently, the demonstration of connexin at the inner mitochondrial membrane of cardiomyocytes, probably forming hemichannels, has enlarged the list of actions of connexins. Some of these mechanisms are also shared by a different family of proteins, termed pannexins. Importantly, these systems allow not only communication between healthy cells, but also play an important role during different types of injury. The aim of this review is to discuss the role played by both connexin hemichannels and pannexin channels in cell communication and injury. This article is part of a Special Issue entitled: The Communicating junctions, composition, structure and characteristics.     

Plant polyphenols in cell-cell interaction and communication

Plant polyphenols including flavonoids and tannins are important constituent of our everyday diet and medical herbals. It is broadly accepted that polyphenols may protect us from toxins, carcinogens and pollutants though the mechanisms of the polyphenols action is still not clear. Here we discuss the ability of polyphenols and especially gallate rich compounds like tannins and catechin gallates to interact with proteins and lipids, establish binding between adjacent bilayer surfaces and initiate membrane aggregation. This phenomena discovered in model experiments could also influence lateral segregation and compartmentalization of cell surface compounds and assist the cell-cell interaction and signal transduction. The involvement of plant polyphenols in communication between cells could be an important factor responsible for anticarcinogenic, vascular and cardioprotective activity of these compounds and speculated to be implicated in the evolution of human brain and intelligence.Key words: human evolution, polyphenols, flavonoids, tannins, membranes, lipid rafts, bilayer structure, cell communicationPlant polyphenols including flavonoids and tannins are present on our daily diet. The opinion on their influence on the human health is contradictory and ranges widely from positive to skeptic and even to alarming.^1–3 Obviously the processes of their functioning in our body should be studied in more details.The polyphenols are know not only as patent antioxidants but also as cell metabolism regulators.⁴ The biological functioning of these compounds begins from their interaction with the cell surface and penetration through the plasma membrane into the cytoplasm. They can influence various physical properties of membrane lipids including diffusion, melting temperature, detergent solubility, osmotic stability, permeability to water soluble compounds^5–7 and general parameters of lipid packing and intrinsic bilayer curvature related to membrane interaction and fusion. Their penetration through the lipid bilayer inversive correlates with the number of hydroxyl groups and accordingly with the hydrophobicity of molecules.The background of polyphenols functioning is attributed to the ability of these compounds to interact with proteins, lipids and polynucleotides through electrostatic, hydrophobic, and even covalent binding.^6–9 The polyphenolic compounds are generally not very active chemically and only electrostatic and hydrophobic forces are responsible for their interaction within cells. However, after oxidation of catechol and gallate¹⁰ moieties to quinones (Fig. 1) in presence of peroxidases, polyphenol oxidases, alkaline pH, or transition metals they could be involved in reactions with free nucleophilic functional groups such as sulfhydryl, amine, amide, indole and imidazole substituents;¹¹ and result in covalent binding with different residues including lysine, methionine, cysteine and tryptophane.^8,12 We expect that terminal amino groups of phosphatidylethanolamines or sulfolipids of biological membranes can also participate in covalent binding with quinones. The consequence of chemical derivatization of proteins may lead to changes in a-helix and random coiled constituents and be accompanied by modulation of protein physical properties and functioning.⁸Open in a separate windowFigure 1A simplified scheme of catechol and gallate moieties oxidation to quinones and subsequent quinones reaction with terminal amino- and sulfa-groups of proteins and lipids (R₂ or R₃). The catechol and gallate moieties are attached to a larger molecule of polyphenol (R₁). For details see reviews.^8,12Among the polyphenolic compounds, tannins express the strongest influence on the lipid bilayer properties. The influence depends on the presence of numerous gallic moieties in the tannin molecule.¹³ Tannins are able to bind on the membrane surface, establish bridges between apposing bilayers and initiate their interaction and adhesion.¹⁴ The process is accompanied by reduction of the membrane dipole potential, lipid interdigitation and the decrease of interbilayer spacing from 15 Å to 5 Å. According to authors¹³ this happens because tannins (1) are amphipathic and partition into the bilayers interfacial region, (2) are long enough to span the interbilayer space, (3) contain several gallic acids distributed so that they can partition simultaneously into apposing bilayers, and (4) have sufficient gallic acid residues to interact with all lipid headgroups and cover the bilayer surface. The electrostatic interaction between the π electrons in the phenol ring and -N⁺(CH₃)₃ groups of phosphatidylcholines is also should be considered.It is necessary to mention that besides tannins the gallate moiety is present also in some antimalarial agents as rufigallol and exifone.¹⁵ The tea catechin gallate and a number of related compounds including gallocatechin gallate, epicatechin gallate, epigallocatechin gallate contain the gallic moiety and was found to influence the membrane fluidity.¹⁶ Some of the tea gallates reveal anticancer activity correlated with membrane rigidifying effect.¹⁷ Not long ago novel black tea pigments rich with gallate moieties and produced by oxidation of tea epagallocatechin-3-o-gallate were discovered (Fig. 2).¹⁸ The appearance of this kind of pigments in food is very typical example of enzymatic and thermal browning process accompanying various traditional preparation of beverages (tea, coffee, cacao, beer and so on) and meal (roasted or baked vegetables and meet).¹¹Open in a separate windowFigure 2Tannin (A) and a fragment of polymeric chain of gallate rich black tea pigment (B). The triplets of neighboring hydroxyl groups of gallic acid are emphasized by a larger font.According to our hypotheses (Fig. 3) the polyphenols rich with gallate moieties may attach to the cell surface, change physical properties of lipids, initiate lateral segregation and formation of lipid and protein clusters, and finally, initiate binding of neighbouring cells. The lateral segregation of the bilayer compounds is known as a physical background of membranes compartmentalization and lipid rafts formation.^19,20 This process is relevant for cell-cell communication, signalling and metabolism regulation; and, thus, responsible for numerous medically recognizable processes.²¹ It is known that lipid rafts are enriched with cholesterol and sphingolipids that serve as a platform for gathering of signalling and trafficking proteins.²² Tannins and other gallate rich molecules may specifically interact with -N⁺(CH₃)₃ groups of sphingomyelin present in the outer leaflet of plasma membrane. High reactivity of gallates towards nucleophilic moieties of proteins especially in presence of peroxidases may facilitate formation of covalent bridges between adjacent cells.Open in a separate windowFigure 3Polyphenols rich of gallate moieties can interact with the cell surface (A). Then gallates may initiate the phospholipid rigidifying process (physically modified lipids are emphasized by shape and color) (B) and lead to aggregation of some proteins and lipids in clusters (D). Polyphenol molecules could serve as bridges between surfaces of two neighbor cells and initiate cells binding and formation of similar clusters in the membrane of the opposite cell.Plant polyphenols as catechins of tea^23,24 are involved in cell-cell interactions responsible for their anticarcinogenic activity by upregulating the receptor and signaling cascades of communication between cells. Well known cardiovascular protective effect of plant polyphenols can also be explained by influence of these compounds on interaction between endothelial cells.²⁵The cell-cell communication and signal transduction is very important also for functioning of neural networks. In human evolution the plant polyphenol consumption would be considerable improved after invention of the fire cooking. It was supposed that the thermal treatment of meat and vegetables, mostly wild tubers of antic savanna, was an important factor of the brain evolution.²⁶ The increase of protein, lipid and carbohydrates availability contributes the most noticeable gain in human intellectual development.²⁷ The role of polyphenols in neuronal communication and brain evolution is still has to be uncovered. Flavonoids are known to protect the brain from oxidation stress and prevent the aging and numerous pathological processes including neuronal damage during Alzheimer''s disease, Parkinson''s disease, amyotrophic lateral sclerosis.^28,29 It seems the mechanism of polyphenols action on the brain functioning could not be restricted by prevention against oxidation stress because these compounds could also participate in the intracellular signal transduction cascades and modulation of brain functioning.³⁰ This may be explained by interactions of polyphenols with specific proteins and plasma membrane compartments responsible for signal transduction as well as for memory formation and storage.     

Direct cell-cell communication in the blood-forming system

In mammals, bone marrow is the principal tissue where blood is formed during adult life. Paracrine factors are generally considered to control this process but there is considerable evidence that gap junctions are present in haemopoietic tissues. Gap junctions have been implicated in developmental and patterning roles, and we set out to characterize the cells which are coupled, and to provide evidence for their role(s) in blood cell formation. Direct cell-cell communication, shown by dye-transfer, occurs between haemopoietic cells and certain stromal cells. In culture these stromal cells form a mat in which they retain their dye-coupling properties. Freeze-fracture electron microscopy confirms that this coupling is via gap junctions. When haemopoietic cells are cultured on top of these mats dye spreads upwards from the stromal cells into the haemopoietic cells above. Experiments in which haemopoietic cells were cultured alone, with stromal cell conditioned medium, or in direct contact with stromal cell underlays, were therefore carried out. The results of these experiments provide evidence that gap junctional communication may be playing a vital role in maintaining populations of precursor cells which would otherwise differentiate into end cells, leading to the ultimate demise of the system.     

Exosomes: New players in cell-cell communication

Exosomes are small membrane vesicles of endosomal origin, which are secreted from a variety of cell types. During the 1980s exosomes were first described as organelles to remove cell debris and unwanted molecules. The discovery that exosomes contain proteins, messenger and microRNAs suggests a role as mediators in cell-to-cell communication. Exosomes can be transported between different cells and influence physiological pathways in the recipient cells. In the present review, we will summarize the biological function of exosomes and their involvement in physiological and pathological processes. Moreover, the potential clinical application of exosomes as biomarkers and therapeutic tools will be discussed.     

Diverse subcellular distribution profiles of pannexin 1 and pannexin 3

Pannexins have been proposed to play a role in gap junctional intercellular communication and as single-membrane channels, although many of their molecular characteristics differ from connexins. Localization of untagged Panx1 and Panx3 exogenously expressed in five cultured cell lines revealed a cell surface distribution profile with limited evidence of cell surface clustering and variable levels of intracellular pools. However, N-glycosylation-defective mutants of pannexins exhibited a more prominent intracellular distribution with decreased cell surface labeling, suggesting an important role for pannexin glycosylation in trafficking. Similar to wild-type pannexins, the glycosylation-defective mutants failed to noticeably transfer microinjected fluorescent dyes to neighboring cells, suggesting that few, or no functional intercellular channels were formed. Finally, varied distribution patterns of endogenous Panx1 and Panx3 were observed in cells of osteoblast origin and Madin-Darby canine kidney cells. Collectively, diverse expression and distribution profiles of Panx1 and Panx3 suggest that they may have multiple cellular functions.     

Cytonemes and tunneling nanotubules in cell-cell communication and viral pathogenesis

Cells use a variety of intercellular structures, including gap junctions and synapses, for cell-cell communication. Here, we present recent advances in the understanding of thin membrane bridges that function in cell-cell signaling and intercellular transport. Cytonemes or filopodial bridges connect neighboring cells via mechanisms of adhesion, which enable ligand-receptor-mediated transfer of surface-associated cargoes from cell to cell. By contrast, tunneling nanotubes establish tubular conduits between cells that provide for the exchange of both cell-surface molecules and cytoplasmic content. We propose models for the biogenesis of both types of membrane bridges and describe how viruses use these structures for the purpose of cell-to-cell spread.     

Programming microbial population dynamics by engineered cell-cell communication

A major aim of synthetic biology is to program novel cellular behavior using engineered gene circuits. Early endeavors focused on building simple circuits that fulfill simple functions, such as logic gates, bistable toggle switches, and oscillators. These gene circuits have primarily focused on single-cell behaviors since they operate intracellularly. Thus, they are often susceptible to cell-cell variations due to stochastic gene expression. Cell-cell communication offers an efficient strategy to coordinate cellular behavior at the population level. To this end, we review recent advances in engineering cell-cell communication to achieve reliable population dynamics, spanning from communication within single species to multispecies, from one-way sender-receiver communication to two-way communication in synthetic microbial ecosystems. These engineered systems serve as well-defined model systems to better understand design principles of their naturally occurring counterparts and to facilitate novel biotechnology applications.     

Molecules mediating cell-ECM and cell-cell communication in human heart valves

The specific phenotype of different tissues depends on the interactions of cells with neighboring cells and the surrounding extracellular matrix, which is mediated by cell adhesion receptors including integrins, immunoglobulin family members, syndecans, and selectins. The aim of this study was to investigate the adhesion profile of native human valve interstitial cells (ICs) in situ and in vitro by analyzing these adhesion receptors. Flow cytometry and immunocytochemistry was used to quantify the expression of the specific receptors on ICs cultured from all human cardiac valves, and immunohistochemistry were used to profile their distribution pattern in valve tissue sections. The valve leaflets and cultured ICs from all valves expressed alpha1, alpha2, alpha3, alpha4, and alpha5 integrins to varying degrees and percentages with very little expression of alpha6 and alphaV. Valve leaflet ICs from all valves, expressed predominantly beta1 integrin but no beta3 or beta4 integrin. Syndecan-1 and Syndecan-4 were not detected. Intercellular adhesion molecule-1 was weakly detected, whereas vascular adhesion molecule-1 was barely detectable and E-selectin was not detected. This study has delineated the identity of some of the integrins synthesized and expressed by human valve ICs and the specificity of adhesion molecules with which the valve ICs interact with the extracellular matrix and mediate intercellular interactions. This pattern of expression of cell surface adhesion molecules may be considered as a basis for a fingerprint on which to base future cell alternatives and would provide useful information for valve tissue engineering.     

Detection of cell-cell fusion mediated by Ebola virus glycoproteins

Ebola viruses (EboV) are enveloped RNA viruses infecting cells by a pH-dependent process mediated by viral glycoproteins (GP) involving endocytosis of virions and their routing into acidic endosomes. As with well-characterized pH-dependent viral entry proteins, in particular influenza virus hemagglutinin, it is thought that EboV GP require activation by low pH in order to mediate fusion of the viral envelope with the membrane of endosomes. However, it has not yet been possible to confirm the direct role of EboV GP in membrane fusion and the requirement for low-pH activation. It was in particular not possible to induce formation of syncytia by exposing cells expressing EboV GP to acidic medium. Here, we have used an assay based on the induction of a beta-galactosidase (lacZ) reporter gene in target cells to detect cytoplasmic exchanges, indicating membrane fusion, with cells expressing EboV GP (Zaire species). Acidic activation of GP-expressing cells was required for efficient fusion with target cells. The direct role of EboV GP in this process is indicated by its inhibition by anti-GP antibodies and by the lack of activity of mutant GP normally expressed at the cell surface but defective for virus entry. Fusion was not observed when target cells underwent acidic treatment, for example, when they were placed in coculture with GP-expressing cells before the activation step. This unexpected feature, possibly related to the nature of the EboV receptor, could explain the impossibility of inducing formation of syncytia among GP-expressing cells.     

Learning the language of cell-cell communication through connexin channels

A report on the Ninth International Gap Junction Conference, Honolulu, USA, 4-9 August 2001.     

A bacterial cell-cell communication signal with cross-kingdom structural analogues

Extracellular signals are the key components of microbial cell-cell communication systems. This report identified a diffusible signal factor (DSF), which regulates virulence in Xanthomonas campestris pv. campestris, as cis-11-methyl-2-dodecenoic acid, an alpha,beta unsaturated fatty acid. Analysis of DSF derivatives established the double bond at the alpha,beta positions as the most important structural feature for DSF biological activity. A range of bacterial pathogens, including several Mycobacterium species, also displayed DSF-like activity. Furthermore, DSF is structurally and functionally related to farnesoic acid (FA), which regulates morphological transition and virulence by Candida albicans, a fungal pathogen. Similar to FA, which is also an alpha,beta unsaturated fatty acid, DSF inhibits the dimorphic transition of C. albicans at a physiologically relevant concentration. We conclude that alpha,beta unsaturated fatty acids represent a new class of extracellular signals for bacterial and fungal cell-cell communications. As prokaryote-eukaryote interactions are ubiquitous, such cross-kingdom conservation in cell-cell communication systems might have significant ecological and economic importance.     

Feline immunodeficiency virus decreases cell-cell communication and mitochondrial membrane potential.

The in vitro effects of viral replication on mitochondrial membrane potential (MMP) and gap junctional intercellular communication (GJIC) were evaluated as two parameters of potential cellular injury. Two distinct cell types were infected with the Petaluma strain of feline immunodeficiency virus (FIV). Primary astroglia supported acute FIV infection, resulting in syncytia within 3 days of infection, whereas immortalized Crandell feline kidney (CRFK) cells of epithelial origin supported persistent FIV infection in the absence of an obvious cytopathic effect. An examination of cells under conditions that included an infection rate of more than 90% for either population revealed that the astroglia produced about four times more virus than the CRFK cells. The mitochondrial uptake of the cationic fluorescent dye rhodamine 123 in infected astroglia was less than 45% of that of normal control cells, whereas the MMP of the CRFK cells, which produced about one-fourth as much virus, was 80.8% of that of the normal cells. Cell-cell communication between adjacent cells was determined by the recovery of fluorescence following photobleaching of a single cell. In spite of the lower level of innate cell-cell communication among cultured CRFK cells than among astroglia, viral replication resulted in a 30% decrease in the GJIC of both astroglia and CRFK cells. These studies indicate that cell injury, as defined by an inhibition of MMP and GJIC, can occur as a result of persistent and acute infection with the Petaluma strain of FIV.