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Cytokine Regulation of Gap Junction Connectivity

2001; Elsevier BV; Volume: 158; Issue: 5 Linguagem: Inglês

10.1016/s0002-9440(10)64110-7

1525-2191

Celia F. Brosnan, Eliana Scemes, David C. Spray,

Hearing, Cochlea, Tinnitus, Genetics

In this issue, Chanson et al1Chanson M Berclaz P-Y Scerri I Dudez T Wernke-Dollries K Pizurki L Pavirani A Fiedler MA Suter S Regulation of gap junctional communication by a pro-inflammatory cytokine in cystic fibrosis transmembrane conductance regulator-expressing but not cystic fibrosis airway cells.Am J Pathol. 2001; 158: 1775-1784Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar present data that document the failure of airway cells expressing the mutant form of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) gene to down-regulate gap junction connectivity after treatment with the cytokine tumor necrosis factor-α (TNF-α). However, this response could be restored by transfecting in the wild-type CFTR gene, suggesting that a functional CFTR gene is necessary for regulation of gap junctional communication by TNF-α. The authors postulate that the persistence of gap-junction connectivity in the inflamed CF airway epithelium permits the intercellular diffusion of signaling molecules that serve to activate neighboring cells, thus contributing to the excessive inflammatory response characteristic of the CF airway epithelium. Although these conclusions are based only on estimation of gap-junction connectivity using transfer of the gap-junction permeant dye Lucifer Yellow, the data fit well with several studies that have documented regulation of gap junction connectivity in a number of different cell types by proinflammatory cytokines such as TNF-α and interleukin-1β (IL-1β).2Chandross KJ Spray DC Cohen RI Kumar NM Kremer M Dermietzel R Kessler JA TNF alpha inhibits Schwann cell proliferation, connexin46 expression, and gap junctional communication.Mol Cell Neurosci. 1996; 7: 479-500Crossref PubMed Scopus (63) Google Scholar, 3Chandross KJ Kessler JA Cohen RI Simburger E Spray DC Bieri P Dermietzel R Altered connexin expression after peripheral nerve injury.Mol Cell Neurosci. 1996; 7: 501-518Crossref PubMed Scopus (95) Google Scholar, 4Hu J Cotgreave IA Differential regulation of gap junctions by proinflammatory mediators in vitro.J Clin Invest. 1997; 99: 2312-2316Crossref PubMed Scopus (73) Google Scholar, 5van Rijen HV van Kempen MJ Postma S Jongsma HJ Tumour necrosis factor alpha alters the expression of connexin43, connexin40, and connexin37 in human umbilical vein endothelial cells.Cytokine. 1998; 10: 258-264Crossref PubMed Scopus (125) Google Scholar, 6Gingalewski C Wang K Clemens MG De Maio A Posttranscriptional regulation of connexin 32 expression in liver during acute inflammation.J Cell Physiol. 1996; 166: 461-467Crossref PubMed Scopus (59) Google Scholar, 7Fernandez-Cobo M Gingalewski C Drujan D De Maio A Downregulation of connexin 43 gene expression in rat heart during inflammation: the role of tumour necrosis factor.Cytokine. 1999; 11: 216-224Crossref PubMed Scopus (109) Google Scholar, 8John GR Scemes E Suadicani SO Liu JS Charles PC Lee SC Spray DC Brosnan CF IL-1beta differentially regulates calcium wave propagation between primary human fetal astrocytes via pathways involving P2 receptors and gap junction channels.Proc Natl Acad Sci USA. 1999; 96: 11613-11618Crossref PubMed Scopus (171) Google Scholar These interesting observations raise the questions of how inflammatory cytokines might regulate gap junction connectivity and in what way expression of the mutant CFTR gene could influence this response. Alterations in gap junction expression have been noted in a number of different disease conditions with an underlying inflammatory process. So, for example, in the liver, a rapid loss of Cx32 mRNA and protein was observed after the induction of an acute inflammatory state by injection of endotoxin.6Gingalewski C Wang K Clemens MG De Maio A Posttranscriptional regulation of connexin 32 expression in liver during acute inflammation.J Cell Physiol. 1996; 166: 461-467Crossref PubMed Scopus (59) Google Scholar, 9Temme A Ott T Haberberger T Traub O Willecke K Acute-phase response and circadian expression of connexin26 are not altered in connexin32-deficient mouse liver.Cell Tissue Res. 2000; 300: 111-117Crossref PubMed Scopus (25) Google Scholar Ischemia, which is known to up-regulate the expression of cytokines such as IL-1β and TNF-α in many tissues, has also been associated with down-regulation of gap junctions composed of Cx32 in the liver10Gingalewski C De Maio A Differential decrease in connexin 32 expression in ischemic and nonischemic regions of rat liver during ischemia/reperfusion.J Cell Physiol. 1997; 171: 20-27Crossref PubMed Scopus (22) Google Scholar and Cx43 in the heart.11Green CR Severs NJ Robert Feulgen Prize Lecture: distribution and role of gap junctions in normal myocardium and human ischaemic heart disease.Histochemistry. 1993; 99: 105-120Crossref PubMed Scopus (55) Google Scholar A more direct role for TNF-α in mediating down-regulation of Cx43 in the heart after administration of bacterial endotoxin has been demonstrated by analysis of the response of the Cx43 promoter to this cytokine.7Fernandez-Cobo M Gingalewski C Drujan D De Maio A Downregulation of connexin 43 gene expression in rat heart during inflammation: the role of tumour necrosis factor.Cytokine. 1999; 11: 216-224Crossref PubMed Scopus (109) Google Scholar However, injection of endotoxin into the kidneys or lungs resulted in increased expression of Cx43 at these sites.12Fernandez-Cobo M Gingalewski C De Maio A Expression of the connexin 43 gene is increased in the kidneys and the lungs of rats injected with bacterial lipopolysaccharide.Shock. 1998; 10: 97-102Crossref PubMed Scopus (44) Google Scholar Although these data appear to be contradictory, it is possible that the influx of inflammatory cells could contribute to this response, since Cx43 has been detected in activated leukocytes.13Jara PI Boric MP Saez JC Leukocytes express connexin 43 after activation with lipopolysaccharide and appear to form gap junctions with endothelial cells after ischemia-reperfusion.Proc Natl Acad Sci USA. 1995; 92: 7011-7015Crossref PubMed Scopus (130) Google Scholar However, whether gap junctions form in macrophages remains controversial, and our own experiments failed to detect the presence of functional gap junctions or hemichannels in these cells.14Alves LA Coutinho-Silva R Persechini PM Spray DC Savino W Campos de Carvalho AC Are there functional gap junctions or junctional hemichannels in macrophages?.Blood. 1996; 88: 328-334PubMed Google Scholar More recently, we have examined the expression of Cx43 in reactive astrocytes associated with MS lesions. These lesions are known to contain high levels of IL-1β, a cytokine that down-regulates gap junction connectivity, as well as Cx43 mRNA and protein in human astrocytes in vitro.8John GR Scemes E Suadicani SO Liu JS Charles PC Lee SC Spray DC Brosnan CF IL-1beta differentially regulates calcium wave propagation between primary human fetal astrocytes via pathways involving P2 receptors and gap junction channels.Proc Natl Acad Sci USA. 1999; 96: 11613-11618Crossref PubMed Scopus (171) Google Scholar By immunohistochemistry, the data show a marked loss of Cx43 immunoreactivity within the center of active and chronic-active MS lesions, with normal expression of Cx43 immunoreactivity in the adjacent normal-appearing white matter (G. R. John, manuscript in preparation). Taken together, these data support the conclusion that gap junction expression may indeed be altered at sites of inflammation in vivo and could thus contribute to the pathogenic state. Gap junctions are considered to form the molecular link for coordinated long-distance signaling among individual members of various syncytia. The most widely studied of these coordinated responses is the propagation of calcium waves after stimulation of any one cell within the network. Gap junction-mediated intercellular communication can potentially be life-saving or catastrophic for survival of cell populations challenged with injury or cell death to their members. The potential benefit of intercellular coupling is illustrated by the phenomenon of metabolic cooperation, in which it has been demonstrated that cocultures of cells, each deficient in thymidine or purine nucleotide synthesis, resulted in cell populations with growth rates equivalent to those of wild-type cells.15Pitts JD The discovery of metabolic co-operation.Bioessays. 1998; 20: 1047-1051Crossref PubMed Scopus (30) Google Scholar The rescue of cell populations that would otherwise be destined to die has been termed the "kiss of life" or the "Good Samaritan effect." In inflammatory conditions, the closure of gap junction channels could have several distinct advantages for the host tissue. It could serve to restrict the passage of activatory molecules to neighboring cells, thereby containing the spread of the inflammatory response, as suggested by Chanson et al.1Chanson M Berclaz P-Y Scerri I Dudez T Wernke-Dollries K Pizurki L Pavirani A Fiedler MA Suter S Regulation of gap junctional communication by a pro-inflammatory cytokine in cystic fibrosis transmembrane conductance regulator-expressing but not cystic fibrosis airway cells.Am J Pathol. 2001; 158: 1775-1784Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar Closing of gap junction connections could also restrict the spread of apoptotic signals to adjacent cells within the network; consistent with this are studies that have shown a reduction of infarct size in both the heart and the brain when coupling of cells is reduced.16Taimor G Cardiac gap junctions: good or bad?.Cardiovasc Res. 2000; 48: 8-10Crossref PubMed Scopus (5) Google Scholar, 17Rawanduzy A Hansen A Hansen TW Nedergaard M Effective reduction of infarct volume by gap junction blockade in a rodent model of stroke.J Neurosurg. 1997; 87: 916-920Crossref PubMed Scopus (180) Google Scholar Alternatively, intercellular communication may be a bad thing, if highly toxic molecules produced in one cell are small enough to penetrate the channels from one cell to another. Such an effect was originally shown for transfer of toxic nucleotides by Fugimoto et al18Fujimoto WY Subak-Sharpe JH Seegmiller JE Hypoxanthine-guanine phosphoribosyltransferase deficiency: chemical agents selective for mutant or normal cultured fibroblasts in mixed and heterozygote cultures.Proc Natl Acad Sci USA. 1971; 68: 1516-1519Crossref PubMed Scopus (62) Google Scholar and termed the "kiss of death"; more recently, the same phenomenon has been termed the "bystander effect" and used to increase the extent of tumor cell killing with gancyclovir.19Bi WL Parysek LM Warnick R Stambrook PJ In vitro evidence that metabolic cooperation is responsible for the bystander effect observed with HSV tk retroviral gene therapy.Hum Gene Ther. 1993; 4: 725-731Crossref PubMed Scopus (456) Google Scholar, 20Andrade-Rozental AF Rozental R Hopperstad MG Wu JK Vrionis FD Spray DC Gap junctions: the "kiss of death" and the "kiss of life".Brain Res Brain Res Rev. 2000; 32: 308-315Crossref PubMed Scopus (125) Google Scholar, 21Mesnil M Yamasaki H Bystander effect in herpes simplex virus-thymidine kinase/ganciclovir cancer gene therapy: role of gap-junctional intercellular communication.Cancer Res. 2000; 60: 3989-3999PubMed Google Scholar Moreover, loss of the ability to communicate with adjacent cells could also prove detrimental to the normal coordinated functioning of affected tissues, as well as significantly impede the formation of an organized response to insult. This could be particularly important in the induction of an appropriate protective response against noxious substances and/or infectious agents. Recent studies of calcium wave formation, however, have suggested that cells that function as a coordinated syncytium use two distinct pathways to mediate this response: an intercellular and an extracellular pathway, with gap junctions composing the intercellular pathway and signaling via P2 receptors the extracellular pathway.22Scemes E Suadicani SO Spray DC Intercellular calcium wave communication via gap junction dependent and independent mechanisms.Curr Top Membr. 2000; 49: 145-173Crossref Scopus (9) Google Scholar Extracellular nucleotides function as ligands for P2 receptors, which fall into two major classes: metabotropic P2Y receptors and ionotropic P2X receptors. Activation of metabotropic P2Y receptors leading to the release of Ca2+ from intracellular stores is generally considered to be the extracellular mechanism involved in the generation of calcium waves in non-excitable cells. A particularly striking observation that has emanated from studies of cells in which gap junction connectivity is lost either by cytokine-induced down-regulation8John GR Scemes E Suadicani SO Liu JS Charles PC Lee SC Spray DC Brosnan CF IL-1beta differentially regulates calcium wave propagation between primary human fetal astrocytes via pathways involving P2 receptors and gap junction channels.Proc Natl Acad Sci USA. 1999; 96: 11613-11618Crossref PubMed Scopus (171) Google Scholar or through gene-targeting techniques23Scemes E Suadicani SO Spray DC Intercellular communication in spinal cord astrocytes: fine tuning between gap junctions and P2 nucleotide receptors in calcium wave propagation.J Neurosci. 2000; 20: 1435-1445Crossref PubMed Google Scholar is that this can be shown to result in the enhancement of signaling via the extracellular pathway. Furthermore, in both the Cx43 knockout mouse and in IL-1-treated human astrocytes, this enhanced P2Y receptor signaling was associated with a functional switch in the P2Y nucleotide receptor subtype from one dominated by a response to ATP (P2Y1-like) to one in which ATP and UTP were equipotent (P2Y2-like).8John GR Scemes E Suadicani SO Liu JS Charles PC Lee SC Spray DC Brosnan CF IL-1beta differentially regulates calcium wave propagation between primary human fetal astrocytes via pathways involving P2 receptors and gap junction channels.Proc Natl Acad Sci USA. 1999; 96: 11613-11618Crossref PubMed Scopus (171) Google Scholar, 23Scemes E Suadicani SO Spray DC Intercellular communication in spinal cord astrocytes: fine tuning between gap junctions and P2 nucleotide receptors in calcium wave propagation.J Neurosci. 2000; 20: 1435-1445Crossref PubMed Google Scholar This functional switch in P2Y receptor expression was further supported by reverse transcription-polymerase chain reaction evidence of enhanced P2Y2 expression in the IL-1β-treated cells.8John GR Scemes E Suadicani SO Liu JS Charles PC Lee SC Spray DC Brosnan CF IL-1beta differentially regulates calcium wave propagation between primary human fetal astrocytes via pathways involving P2 receptors and gap junction channels.Proc Natl Acad Sci USA. 1999; 96: 11613-11618Crossref PubMed Scopus (171) Google Scholar These data show that communication within the astrocytic syncytium is sustained by a finely tuned interaction between gap junction-dependent and -independent mechanisms, such that a reduction of gap junction-mediated intercellular communication in Cx43 knockout mice is compensated for by an increased autocrine communication. Such an interplay between gap junctions and paracrine/autocrine signaling provides a high degree of plasticity for intercellular communication between astrocytes. Whether a similar maintenance of syncytial activity will be found in other cell systems, as well as for responses other than calcium wave formation, will be an interesting question to address. What is the possible relevance of an interplay between gap junctions and P2 receptor signaling for inflammatory gene expression in the inflamed airway epithelium? Firstly, we have been able to show that in astrocytes signaling via P2 receptors intersects with components of the cytokine signaling cascade, modulating the nature of the inflammatory genes activated in these cells by cytokines such as IL-1β and TNF-α.24John GR Liu JS John GR Sikora A Lee SC Brosnan CF Modulation of IL-1beta and tumor necrosis factor alpha signaling by P2 purinergic receptors in human fetal astrocytes.J Neurosci. 2000; 20: 5292-5299PubMed Google Scholar, 25John GR, Simpson JE, Woodroofe MN, Lee SC, Brosnan CF: Extracellular nucleotides differentially regulate IL-1beta signaling in primary human astrocytes: implications for inflammatory gene expression. J Neurosci (in press)Google Scholar These data fit well with accumulating evidence that in cells of both myeloid and non-myeloid origin, agonists or antagonists of P2Y and P2X receptors modulate the inflammatory cascade. So, for example, it has been shown that in human macrophages, ATP provides a second stimulus required for the processing and secretion of lipopolysaccharide-induced IL-1.26Griffiths RJ Stam EJ Downs JT Otterness IG ATP induces the release of IL-1 from LPS-primed cells in vivo.J Immunol. 1995; 154: 2821-2828PubMed Google Scholar, 27Ferrari D Chiozzi P Falzoni S Dal Susino M Melchiorri L Baricordi OR Di Virgilio F Extracellular ATP triggers IL-1 beta release by activating the purinergic P2Z receptor of human macrophages.J Immunol. 1997; 159: 1451-1458PubMed Google Scholar In these studies, the activation of the P2X7receptor has been implicated, which results in the formation of a large transmembrane pore that allows the bidirectional passage of molecules up to 900 Da.28Di Virgilio FF Chiozzi P Ferrari D Falzoni S Sanz JM Morelli A Torboli M Bolognesi G Baricordi OR Nucleotide receptors: an emerging family of regulatory molecules in blood cells.Blood. 2001; 97: 587-600Crossref PubMed Scopus (626) Google Scholar Additional data supporting a role for this receptor in lipopolysaccharide-activated cytokine production have recently been generated in mice in which the P2X7 gene has been inactivated.29Solle M Labasi J Perregaux DG Stam E Petrushova N Koller BH Griffiths RJ Gabel CA Altered cytokine production in mice lacking P2X(7) receptors.J Biol Chem. 2001; 276: 125-132Crossref PubMed Scopus (794) Google Scholar ATP has also been found to modulate the generation of reactive oxygen intermediates30Schmid-Antomarchi H Schmid-Alliana A Romey G Ventura MA Breittmayer V Millet MA Husson H Moghrabi B Lazdunski M Rossi B Extracellular ATP and UTP control the generation of reactive oxygen intermediates in human macrophages through the opening of a charybdotoxin-sensitive Ca2+-dependent K+ channel.J Immunol. 1997; 159: 6209-6215PubMed Google Scholar, 31Sikora A Liu J Brosnan C Buell G Chessel I Bloom BR Cutting edge: purinergic signaling regulates radical-mediated bacterial killing mechanisms in macrophages through a P2X7-independent mechanism.J Immunol. 1999; 163: 558-561PubMed Google Scholar and to regulate lipopolysaccharide-induced nitric oxide synthase II and TNF expression both in vivo and in vitro30Schmid-Antomarchi H Schmid-Alliana A Romey G Ventura MA Breittmayer V Millet MA Husson H Moghrabi B Lazdunski M Rossi B Extracellular ATP and UTP control the generation of reactive oxygen intermediates in human macrophages through the opening of a charybdotoxin-sensitive Ca2+-dependent K+ channel.J Immunol. 1997; 159: 6209-6215PubMed Google Scholar, 31Sikora A Liu J Brosnan C Buell G Chessel I Bloom BR Cutting edge: purinergic signaling regulates radical-mediated bacterial killing mechanisms in macrophages through a P2X7-independent mechanism.J Immunol. 1999; 163: 558-561PubMed Google Scholar, 32Tonetti M Sturla L Giovine M Benatti U De Flora A Extracellular ATP enhances mRNA levels of nitric oxide synthase and TNF-alpha in lipopolysaccharide-treated RAW 264.7 murine macrophages.Biochem Biophys Res Commun. 1995; 214: 125-130Crossref PubMed Scopus (62) Google Scholar, 33Denlinger LC Fisette PL Garis KA Kwon G Vazquez-Torres A Simon AD Nguyen B Proctor RA Bertics PJ Corbett JA Regulation of inducible nitric oxide synthase expression by macrophage purinoreceptors and calcium.J Biol Chem. 1996; 271: 337-342Crossref PubMed Scopus (154) Google Scholar, 34Hu Y Fisette PL Denlinger LC Guadarrama AG Sommer JA Proctor RA Bertics PJ Purinergic receptor modulation of lipopolysaccharide signaling and inducible nitric-oxide synthase expression in RAW 264.7 macrophages.J Biol Chem. 1998; 273: 27170-27175Crossref PubMed Scopus (125) Google Scholar, 35Greenberg SS Zhao X Wang JF Hua L Ouyang J cAMP and purinergic P2y receptors upregulate and enhance inducible NO synthase mRNA and protein in vivo.Am J Physiol. 1997; 273: L967-L979PubMed Google Scholar in macrophages, as well as IL-1-induced cytokine and chemokine expression in astrocytes.24John GR Liu JS John GR Sikora A Lee SC Brosnan CF Modulation of IL-1beta and tumor necrosis factor alpha signaling by P2 purinergic receptors in human fetal astrocytes.J Neurosci. 2000; 20: 5292-5299PubMed Google Scholar, 25John GR, Simpson JE, Woodroofe MN, Lee SC, Brosnan CF: Extracellular nucleotides differentially regulate IL-1beta signaling in primary human astrocytes: implications for inflammatory gene expression. J Neurosci (in press)Google Scholar The different specificities for endogenous agonists displayed by P2 receptors suggests that the composition and concentration of nucleotide triphosphates, diphosphates, and monophosphates, as well as nucleosides, in the extracellular space provides the cell with important information concerning changes in the extracellular environment and, in the case of ciliated epithelium, may help coordinate cell activity.36Morales B Barrera N Uribe P Mora C Villalon M Functional cross talk after activation of P2 and P1 receptors in oviductal ciliated cells.Am J Physiol. 2000; 279: C658-C669Google Scholar Furthermore, changes in the expression of these receptors may alter sensitivity to these events. High levels of extracellular nucleotides are released from sources such as platelets, activated leukocytes, and damaged or dying cells, in a number of injurious conditions. This has led to the hypothesis that autocrine/paracrine activation of P2 receptors permits different cell types to communicate with each other and with the extracellular environment through the release and sensing of nucleotides such as ATP and to use this information to fine-tune the response to the extent and nature of the injury.24John GR Liu JS John GR Sikora A Lee SC Brosnan CF Modulation of IL-1beta and tumor necrosis factor alpha signaling by P2 purinergic receptors in human fetal astrocytes.J Neurosci. 2000; 20: 5292-5299PubMed Google Scholar, 25John GR, Simpson JE, Woodroofe MN, Lee SC, Brosnan CF: Extracellular nucleotides differentially regulate IL-1beta signaling in primary human astrocytes: implications for inflammatory gene expression. J Neurosci (in press)Google Scholar Secondly, in recent years it has been proposed that extracellular nucleotide activation of P2 receptors may be useful in the symptomatic therapy of CF. In particular, UTP acting on a P2Y2 receptor has been shown to activate alternative Cl− conductances primarily via the phospholipase C/inositol 1,4,5-triphosphate/intracellular Ca2+ signaling pathway, leading to restoration of salt and water secretion via the activation of a [Ca2+]i-mediated anion conductance.37Jiang c Finkbeiner WE Widdicombe JH McCray PB Miller SS Altered fluid transport across airway epithelium in cystic fibrosis.Science. 1993; 262: 424-427Crossref PubMed Scopus (189) Google Scholar, 38Knowles MR Clarke LL Boucher RC Activation by extracellular nucleotides of chloride secretion in the airway epithelia of patients with cystic fibrosis.N Engl J Med. 1991; 325: 533-538Crossref PubMed Scopus (425) Google Scholar Additional studies now support the conclusion that activation of P2Y2 receptors may also restore a portion of electrogenic bicarbonate secretion in CF airways expressing the [Ca2+]i-mediated anion conductance pathway, which may help to normalize transluminal pH across CF epithelia.39Clarke LL Harline MC Gawenis LR Walker NM Turner JT Weisman GA Extracellular UTP stimulates electrogenic bicarbonate secretion across CFTR knockout gallbladder epithelium.Am J Physiol Gastrointest Liver Physiol. 2000; 279: G132-G138PubMed Google Scholar Support for the concept that the P2Y2 receptor is the dominant P2Y purinoceptor that regulates airway epithelial ion transport has been provided by studies in mice with targeted gene deletions of the P2Y2 receptor.40Cressman VL Lazarowski E Homolya L Boucher RC Koller BH Grubb BR Effect of loss of P2Y(2) receptor gene expression on nucleotide regulation of murine epithelial Cl(−) transport.J Biol Chem. 1999; 274: 26461-26468Crossref PubMed Scopus (165) Google Scholar Thus, if indeed the expression of Cx43 and the P2Y2 receptor show a similar inverse relationship in other cell types, then the failure to down-regulate gap junctions in response to TNF-α in airway epithelial cells expressing the mutant form of the CFTR gene would block the shift from P2Y1 to P2Y2 expression, rendering the cells less sensitive to the potentially beneficial effects of P2Y2 receptor agonists. The mechanism by which Cx43 and P2 receptor expression is linked is unknown. One possible way this might occur would involve the different selective diffusion of signaling molecules through gap junction channels formed of different connexins. Such a mechanism recently was proposed for osteoblastic cell lines in which transcriptional activities of osteoblast-specific promoters were modulated in opposite directions by overexpressing either Cx43 or Cx45.41Lecanda F Towler DA Ziambaras K Cheng SL Koval M Steinberg TH Civitelli R Gap junctional communication modulates gene expression in osteoblastic cells.Mol Biol Cell. 1998; 9: 2249-2258Crossref PubMed Scopus (232) Google Scholar Alternatively, the recent recognition of protein-protein interactions involving connexins allows the possibility that expression of Cx43 might recruit a specific type of P2 receptor to the membrane preferentially (as occurs via the direct binding of ZO-142Giepmans BN Moolenaar WH The gap junction protein connexin43 interacts with the second PDZ domain of the zona occludens-1 protein.Curr Biol. 1998; 8: 931-934Abstract Full Text Full Text PDF PubMed Scopus (480) Google Scholar, 43Toyofuku T Yabuki M Otsu K Kuzuya T Tada M Hori M Functional role of c-Src in gap junctions of the cardiomyopathic heart.Circ Res. 1999; 85: 672-681Crossref PubMed Scopus (67) Google Scholar) or might selectively affect P2 receptor gene expression directly or via a binding protein. Intercellular communication can be regarded as a collective process involving gap junctions, membrane receptors, and other membrane and cytosolic proteins whose activities are coordinated. As for other specialized membrane domains, such as caveolae and synapses, the integral membrane components of gap junctions appear to be linked into a macromolecular complex, the Nexus.44Spray DC Duffy HS Scemes E Gap junctions in glia. Types, role, and plasticity.Adv Exp Med Biol. 1999; 468: 339-359Crossref PubMed Google Scholar Because cytoplasmic domains differ greatly among the 16 known connexins, it seems likely that Nexus components may also vary, and binding affinities within the Nexus containing an individual connexin may be altered by such factors as cytosolic pH, phosphorylation, and binding of other components. For the Cx43 Nexus, binding sites include src homology (SH) and PSD, disks large, zonula occludens (PDZ) binding domains on the Cx43 molecule, and most probably other domains as well. Connexin43 (Cx43) is a tetraspan membrane protein with cytoplasmic amino and carboxyl termini (NT and CT) and a cytoplasmic loop (CL) connecting the second and third transmembrane domains.45Yeager M Structure of cardiac gap junction intercellular channels.J Struct Biol. 1998; 121: 231-245Crossref PubMed Scopus (98) Google Scholar Protein-protein interactions between CT and CL are hypothesized to be responsible for channel closure by acidification,46Ek-Vitorin JF Calero G Morley GE Coombs W Taffet SM Delmar M pH regulation of connexin43: molecular analysis of the gating particle.Biophys J. 1996; 71: 1273-1284Abstract Full Text PDF PubMed Scopus (145) Google Scholar and the ∼16-kd carboxyl terminus contains multiple phosphorylation sites, WSH2/SH3 binding sites, and a single distal PDZ recognition motif. Interactions of Cx43 with proteins containing PDZ, SH2, and SH3 domains is thus hypothesized to serve as a scaffold on which to assemble components of the intercellular signaling pathway into the multiprotein Nexus complex, which couples their activity to downstream signaling molecules. PDZ domains are approximately 90 amino acid modules, which mediate protein-protein interactions by binding to the last 3 or 4 amino acids of the C-termini of their target proteins. The specificities of PDZ domain-containing proteins are quite diverse, with the minimal recognition motif being a hydrophobic residue at the carboxyl terminus.47Songyang Z Fanning AS Fu C Xu J Marfatia SM Chishti AH Crompton A Chan AC Anderson JM Cantley LC Recognition of unique carboxyl-terminal motifs by distinct PDZ domains.Science. 1997; 275: 73-77Crossref PubMed Scopus (1232) Google Scholar In the case of Cx43, the second PDZ domain of the tight junction-associated protein zonula occludens (ZO)-1 has been shown to interact with the most distal four amino acids (DLEI42Giepmans BN Moolenaar WH The gap junction protein connexin43 interacts with the second PDZ domain of the zona occludens-1 protein.Curr Biol. 1998; 8: 931-934Abstract Full Text Full Text PDF PubMed Scopus (480) Google Scholar, 43Toyofuku T Yabuki M Otsu K Kuzuya T Tada M Hori M Functional role of c-Src in gap junctions of the cardiomyopathic heart.Circ Res. 1999; 85: 672-681Crossref PubMed Scopus (67) Google Scholar), and such interaction has been implicated in targeting Cx43 to cell-cell interfaces.43Toyofuku T Yabuki M Otsu K Kuzuya T Tada M Hori M Functional role of c-Src in gap junctions of the cardiomyopathic heart.Circ Res. 1999; 85: 672-681Crossref PubMed Scopus (67) Google Scholar The effectors possessing association motifs, known as src homology 2 (SH2) and src homology 3 (SH3) domains, are protein modules of about 100 and 50 amino acids, respectively.48Pawson T Scott JD Signaling through scaffold, anchoring, and adaptor proteins.Science. 1997; 278: 2075-2080Crossref PubMed Scopus (1916) Google Scholar, 49Cohen NA Brenman JE Snyder SH Bredt DS Binding of the inward rectifier K+ channel Kir 2.3 to PSD-95 is regulated by protein kinase A phosphorylation.Neuron. 1996; 17: 759-767Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar SH2 domains bind to proteins containing phosphotyrosine regions and thus regulate signal transduction events involving tyrosine phosphorylation.50Mayer BJ Hanafusa H Mutagenic analysis of the v-crk oncogene: requirement for SH2 and SH3 domains and correlation between increased cellular phosphotyrosine and transformation.J Virol. 1990; 64: 3581-3589Crossref PubMed Google Scholar, 51Matsuda M Mayer BJ Fukui Y Hanafusa H Binding of transforming protein, P47gag-crk, to a broad range of phosphotyrosine-containing proteins.Science. 1990; 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276: 1780-1788Crossref PubMed Scopus (186) Google Scholar One of the most fundamental questions raised by the report of Chanson et al1Chanson M Berclaz P-Y Scerri I Dudez T Wernke-Dollries K Pizurki L Pavirani A Fiedler MA Suter S Regulation of gap junctional communication by a pro-inflammatory cytokine in cystic fibrosis transmembrane conductance regulator-expressing but not cystic fibrosis airway cells.Am J Pathol. 2001; 158: 1775-1784Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar concerns the mechanism by which Lucifer Yellow dye spread is reduced in non-CF cells treated with TNF-α. Reduction in intercellular communication was detected within 2 minutes after TNF-α addition and reached a plateau at 20 minutes, coinciding with the plateau of induction of NF-κB translocation to the nucleus. The rapidity of the uncoupling would appear to constrain the probable mechanisms to those modulating activity of channels already present in the junctional membrane, rather than acting on connexin synthesis or degradation, which is consistent with unpublished results cited in Chanson et al1Chanson M Berclaz P-Y Scerri I Dudez T Wernke-Dollries K Pizurki L Pavirani A Fiedler MA Suter S Regulation of gap junctional communication by a pro-inflammatory cytokine in cystic fibrosis transmembrane conductance regulator-expressing but not cystic fibrosis airway cells.Am J Pathol. 2001; 158: 1775-1784Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar that Cx43 expression is not compromised by treatment with TNF-α for as long as 90 minutes. Plausible candidates for transduction of the Lucifer Yellow uncoupling would include direct closure of the gap junction channels by cytoplasmic factors such as low pH or lipophilic products of phospholipase activation. It is also possible that kinase activation by TNF-α triggers a transduction cascade that closes the intercellular channels either as a direct result of conformational change in Cx43 or as a consequence of altering the affinities of Cx43 to its binding partners within the Nexus. Although the concept that ion channels (such as connexins, P2Y receptors, and CFTR) may interact with each other as well as with other proteins (such as ZO-1 and other PDZ and/or SH domain proteins) is relatively new, the implications of this concept for intercellular signaling under physiological and pathological conditions are profound. As efforts to understand the interactions highlighted by Chanson et al1Chanson M Berclaz P-Y Scerri I Dudez T Wernke-Dollries K Pizurki L Pavirani A Fiedler MA Suter S Regulation of gap junctional communication by a pro-inflammatory cytokine in cystic fibrosis transmembrane conductance regulator-expressing but not cystic fibrosis airway cells.Am J Pathol. 2001; 158: 1775-1784Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar proceed, we anticipate that the links between cytokines and the channels that mediate intercellular signaling (especially gap junctions and P2 receptors) will become clearer and will point the way toward therapeutic intervention for the regulation of inflammation in a number of different cell types and tissue pathologies.