Connexin-built channels as modulators of lung inflammation

Thesis

Reference

Connexin-built channels as modulators of lung inflammation

RICHANI SARIEDDINE, Maya

Abstract

L'épithélium respiratoire est exposé au milieu extérieur étant ainsi en contact avec de nombreux pathogènes. Suite à une infection, des cellules inflammatoires comme le neutrophile transmigrent vers le tissue pulmonaire en traversant la barrière alvéolo-capillaire.

Parmi les différents types d'interactions cellulaires, la communication intercellulaire directe via les jonctions communicantes pourrait participer aux réponses inflammatoires du poumon. Les jonctions communicantes, formées par l'apposition de deux hémicanaux membranaires, permettent le passage d'ions et de petites molécules entre les cellules en contact. Chaque hémicanal est constitué par l'association de six connexines. Les hémicanaux peuvent former des jonctions communicantes ou peuvent rester libres à la membrane des cellules. Peu est connu sur l'implication des connexines dans le recrutement des leucocytes, leur adhésion et leur transmigration à travers la barrière alvéolo-capillaire. L'objectif de mon travail de thèse est de déterminer le rôle des connexines et des jonctions communicantes dans les interactions cellulaires entre le [...]

RICHANI SARIEDDINE, Maya. Connexin-built channels as modulators of lung inflammation. Thèse de doctorat : Univ. Genève, 2010, no. Sc. 4174

URN : urn:nbn:ch:unige-54078

DOI : 10.13097/archive-ouverte/unige:5407

Available at:

http://archive-ouverte.unige.ch/unige:5407

UNIVERSITÉ DE GENÈVE

Département de Zoologie et Biologie Animale FACULTÉ DES SCIENCES Prof. Jean-Louis Bény

Département de l’Enfant et de l’Adolescent FACULTÉ DE MÉDECINE Dr. Marc Chanson

“Connexin-built Channels as Modulators of Lung Inflammation”

THÈSE

présentée à la Faculté des sciences de l’Université de Genève pour obtenir le grade de Docteur ès sciences, mention biologie

par

Maya RICHANI SARIEDDINE

de Chwaifat (LIBAN)

Thèse N° 4174

Genève

Centre d’Impression de l’Université de Genève 2010

ACKNOWLEDGEMENTS

I would like to thank Dr. Marc Chanson, my thesis supervisor, for giving me the opportunity to work in his group, for his guidance and support through the years of my thesis.

I am also thankful to Prof. Claire-Anne Siegrist for being my thesis co-director, to Prof.

Werner Schlegel and Prof. Constance Barazzone for being my mentors and to Prof. Brenda Kwak and her team for showing me support and encouragements during my thesis.

I am grateful to Prof. Jean-Louis Bény, Prof. Beat Imhof and Prof. Lhousseine Touqui for accepting to be members of my thesis committee.

I finally want to thank my colleagues, Ludwig Scheckenback, Isabelle Scerri, Tecla Dudez, Bernard Foglia, Marc Bacchetta, Sophie Crespin, Davide Losa and Sandrine Jaccard for their helpful discussions and advice. I also thank them for contributing with their work and expertise to the completion of my thesis.

DEDICATION

I dedicate my PhD thesis to my parents, Mona and Ziad Richani, and to my grandparents, Effat and Anis Halabi. I would like to thank them for their love and support. I am also grateful to my husband, Rabih Sarieddine, my brother, Nagib Richani and my loving uncles, Adonis and Rabih Halabi for always encouraging me to go further. I finally want to mention my son, Rayan Sarieddine, who came into my life and fulfilled it with joy during the last year of my thesis.

CONTENTS

Pages

ABBREVIATIONS

RÉSUMÉ

SUMMARY

I. INTRODUCTION

1. The lung respiratory epithelium

2. Defense mechanisms in the lung

A. Maintenance of lung sterility 14

B. Pathogen sensing 15

C. The toll-like receptors’ signaling cascade 16

D. Leukocyte recruitment 16

3. Inflammation in respiratory diseases

A. Common lung diseases 21

B. Cellular orchestration during immuno-inflammatory pathologies 22

4. Gap junctions, connexins and intercellular communication

A. Discovery of gap junctions 24

B. Structure of gap junctions 25

C. Structural diversity of connexin genes 27

D. Topology of connexins 29

E. Assembly and degradation of gap junctions 30

F. Regulation of gap junction channels 34

G. Function of gap junctions 35

5. Gap junctions and inflammation

A. Modulation of gap junctions in inflammatory conditions 37 B. Disease-like phenotypes in genetically modified animals for connexins 38

C. Connexins in the immune system 39

D. Intercellular communication in the lung 43

II. AIM OF THE PROJECT

III. CHAPTER 1: “Cx43 modulates neutrophil recruitment to the lung”

IV. CHAPTER 2: “Contribution of Cx43 and Cx37 expressed in neutrophils to the lung inflammatory response”

60

V. CHAPTER 3: “Endothelial-specific deletion of Cx40 promotes atherosclerosis by increasing CD73-dependent leukocyte adhesion”

75

VI. GENERAL DISCUSSION AND CONCLUDING REMARKS

VII. REFERENCES

ABBREVIATIONS

5'-ecto-nucleotidase CD73

Acute lung injury ALI

Acute respiratory distress syndrome ARDS

Adenosine ADO

Alveolar type I pneumocytes ATI

Alveolar type II pneumocytes ATII

Amino terminus of connexins NT

Bronchoalveolar lavage BAL

Carboxyl terminus of connexins CT

Cell adhesion molecule CAM

Chronic obstructive pulmonary disease COPD

Connexins Cx

Cx43 blocking peptide ⁴³Gap26

Cystic fibrosis CF

Cystic fibrosis transmembrane conductance regulator CFTR Endothelial cell-selective adhesion molecule ESAM

ER-associated degradation ERAD

Extracellular loops of connexins E1, E2

Gap junctional intercellular communication GJIC Hydrophobic transmembrane domains of connexins M1-M4

Inositol-triphosphate IP3

Interleukin IL-

Junctional adhesion molecules A, B and C JAM-A, JAM-B and JAM-C

Krebs-Ringer-Bicarbonate KRB

Lipopolysaccharide LPS

Lucifer Yellow LY

Metalloproteinase MMP

Non phosphorylated Cx43 NP Cx43

Nuclear factor-B NF-κB

Platelet-activating factors PAF

Polymorphomononuclear cell PMN

Pseudomonas aeruginosa P. aeruginosa

Endoplasmic reticulum ER

Surfactant proteins A-D SP A-D

Toll-like receptor TLR

Trans-Golgi network TGN

Tumour necrosis factor-α TNF-α

RÉSUMÉ

Hémicanaux et Jonctions Communicantes : Des Modulateurs de l’Inflammation Pulmonaire

L’épithélium respiratoire est exposé au milieu extérieur étant ainsi en contact avec de nombreux pathogènes. Le poumon doit lutter contre ces corps étrangers en développant un système de défense adéquat afin de maintenir la stérilité pulmonaire. Suite à une infection, des cellules inflammatoires comme le neutrophile transmigrent vers le tissue pulmonaire en traversant la barrière alvéolo-capillaire jusqu’au site d’inflammation. Dans le poumon inflammé, des mécanismes de défense sont mis en place par une coordination cellulaire.

Parmi les différents types d’interactions cellulaires, la communication intercellulaire directe via les jonctions communicantes (“Gap Junctional Intercellular Communication”, GJIC) pourrait participer aux réponses inflammatoires du poumon. Les jonctions communicantes sont formées par l’apposition de deux hémicanaux membranaires qui permettent le passage d’ions et de petites molécules entre les cellules en contact. Chaque hémicanal est constitué par l’association de six connexines (Cx). Les hémicanaux peuvent former des jonctions communicantes ou peuvent rester libres à la membrane des cellules, participant également au relâchement de molécules vers le milieu extracellulaire.

La partie respiratoire du poumon comprend des cellules épithéliales alvéolaires de type I et II entourées d’un réseau de cellules endothéliales. Différentes connexines sont exprimées par ces cellules parmi lesquelles la Cx37, la Cx40 et la Cx43 sont les plus étudiées.

En particulier, la Cx43 s’est avérée être impliquée dans la transmission de messages pro- inflammatoires au niveau des cellules endothéliales du poumon par l’intermédiaire d’une

cette connexine à l’intensité de la réponse inflammatoire du poumon in vivo n’est toujours pas établie. De plus, peu est connu sur l’implication des connexines dans le recrutement des leucocytes, leur adhésion et leur transmigration à travers la barrière alvéolo-capillaire.

L’objectif de mon travail de thèse est de déterminer le rôle des connexines et des jonctions communicantes dans les interactions cellulaires entre le neutrophile et la barrière alvéolo- capillaire en utilisant des approches in vivo et in vitro.

Dans le premier chapitre de ma thèse, j’ai étudié la contribution de la Cx43 dans le recrutement du neutrophile suite à une injection intratrachéale (IT) de lipopolysaccharide (LPS), provenant de Pseudomonas aeruginosa, dans des modèles de souris génétiquement modifiées pour la Cx43. La communication intercellulaire directe peut être réduite en diminuant l’expression de la Cx43 ou augmentée en tronquant le domaine C-terminal de la Cx43. Ainsi, j’ai comparé la réponse inflammatoire dans des souris hétérozygotes pour la Cx43 (Cx43^+/-) et pour la forme tronquée non réglable de la Cx43 (Cx43^K258stop/-). Dans les poumons de souris Cx43^K258stop/-, une réponse inflammatoire exagérée a été mise en évidence contrairement aux souris Cx43^+/- où l’inflammation pulmonaires était significativement atténuée. De plus, j’ai pu montrer que les médiateurs de l’inflammation ont induit l’expression de la Cx43 dans les septa alvéolaires de souris sauvages. L’importance de la Cx43 sur l’adhérence du neutrophile a été également évaluée in vitro en bloquant la conductivité des canaux Cx43 grâce à un peptide spécifique (⁴³Gap26). En effet, l’adhérence du neutrophile sur des cellules endothéliales et alvéolaires épithéliales murines traitées avec ce peptide a significativement diminué. Le peptide a pu être utilisé in vivo par injection IT dans des poumons de souris inflammés, diminuant ainsi l’inflammation pulmonaire de 65%. La Cx43 joue donc un rôle de médiateur pro-inflammatoire et pourrait représenter une cible pharmacologique pour contrôler le recrutement des leucocytes durant les maladies inflammatoires pulmonaires.

Parmi les différents mécanismes qui régulent l’adhérence du neutrophile, la libération de nucléotides durant l’inflammation semble avoir un lien avec les connexines. Les neutrophiles secréteraient de l’ATP par l’intermédiaire des hémicanaux. Dans le second chapitre de ma thèse, j’ai évalué l’implication de la Cx43 du neutrophile lors de son interaction avec l’endothélium. J’ai analysé l’adhérence de ces leucocytes sur des lignés de cellules endothéliales en utilisant des neutrophiles circulants de souris Cx43^+/- et de souris Cx43^K258stop/-. L’adhésion de neutrophiles traités au ⁴³Gap26 pour fermer les hémicanaux de surface a également été étudiée. En parallèle aux tests d’adhésion, la fonction de phagocytose du neutrophile a été évaluée. Les résultats montrent que la capacité des neutrophiles à phagocyter et leur capacité à adhérer ne sont pas altérées suite à la modification génétique ou à l’inhibition pharmacologique de la Cx43 exprimée par ces neutrophiles. Par contre, j’ai trouvé que l’adhérence des neutrophiles provenant de souris knock-out pour la Cx37 était significativement augmentée. La Cx37 semble ainsi être impliquée dans l’interaction entre le neutrophile et la cellule endothéliale. Cette implication a été confirmée in vivo en évaluant la transmigration grâce à une technique de transfert adoptif où des neutrophiles fluorescents de souris génétiquement modifiées ont été injectés dans une souris sauvage receveuse ayant subit une injection IT de LPS. Nos résultats suggèrent que les interactions entre le neutrophile et l’endothélium n’impliquent pas l’expression de la Cx43 dans le neutrophile. Ils impliqueraient plutôt l’expression de la Cx37 qui semble être importante pour la régulation de l’adhésion et de la transmigration du neutrophile, éventuellement à travers un relâchement d’ATP.

Suite à son relâchement, l’ATP est hydrolysée en AMP puis convertie en adénosine par la 5'-ecto-nucléotidase (CD73) à la surface des cellules endothéliales. L’adénosine active les récepteurs A2B qui vont engendrer une activité anti-inflammatoire et diminuer ainsi l’adhérence des leucocytes à l’endothélium. Dans le troisième chapitre, j’ai travaillé sur des souris pour lesquelles la Cx40 a été spécifiquement invalidée dans l’endothélium (souris

Cx40del). D’une manière intéressante, ces souris présentent une diminution de l’expression du CD73 au niveau de l’endothélium pulmonaire et une augmentation du recrutement des neutrophiles vers l’espace bronchoalvéolaire dans la phase initiale de la réponse inflammatoire. De plus, l’inhibition de la Cx40 endothéliale par une approche in vitro a diminué l’expression et l’activité du CD73 tout en augmentant l’adhérence des neutrophiles dans des lignées de cellules endothéliales. J’ai également développé un système de co-culture pour démontrer la contribution de la GJIC via la Cx40 dans la propagation intercellulaire de signaux anti-inflammatoires affectant l’adhérence du neutrophile.

L’ensemble des résultats de ma thèse montrent des rôles opposés pour la Cx43 et la Cx40 dans la propagation de signaux endothéliaux durant l’inflammation pulmonaire. La Cx43 est un médiateur pro-inflammatoire alors que la Cx40 endothéliale serait impliquée dans des évènements anti-inflammatoires en diminuant l’adhésion des leucocytes circulants à la surface des vaisseaux. La Cx37 dans le neutrophile contribuerait à ces évènements. En effet, une hypothèse est que l’ATP relâchée par les hémicanaux Cx37 des neutrophiles servirait de substrats aux ecto-enzymes à la surface des cellules endothéliales, générant ainsi un signal anti-adhésif. La propagation de ce signal impliquerait la GJIC via la Cx40 endothéliale. En présence d’un stimulus pro-inflammatoire, l’induction de la Cx43 dans les septa alvéolaires favoriserait alors la propagation de vagues calciques, assurant l’adhésion et la transmigration des neutrophiles. En résumé, je propose l’hypothèse selon laquelle la communication cellulaire orchestrée par différentes connexines crée un équilibre entre les mécanismes pro- et anti- adhésifs nécessaires à la régulation du recrutement des neutrophiles durant la réponse inflammatoire pulmonaire.

SUMMARY

Connexin-built Channels as Modulators of Lung Inflammation

The lung respiratory epithelium has a large area of exposure to the external environment and is continuously subjected to foreign bodies. Although several mechanisms including cough reflex and mucociliary clearance participate in the evacuation of inhaled harmful particles, the lung still has to develop an adequate and orchestrated host defense system to maintain the sterility of the pulmonary tissue. Upon infection, inflammatory cells such as neutrophils transmigrate across the lung endothelial and alveolar epithelial barriers to sites of inflammation, where defense mechanisms work in concert via cellular coordination. These cell interactions involve in part gap junctional intercellular communication (GJIC) and an emerging field of research is focusing on the implication of gap junctions during lung inflammatory responses. Gap junctions consist of connexin (Cx) hemichannels that dock to each other on neighboring cells, hence allowing for ionic and molecular exchange. Connexin hemichannels can also stay free at the membrane and may participate in the release of molecules to the extracellular milieu.

The respiratory portion of the lungs consists of alveolar type I and II epithelial cells surrounded by a capillary endothelial network. Many connexins are expressed in these cells, among which Cx37, Cx40, and Cx43 are mostly documented. Interestingly, the latter connexin has been shown to be implicated in the spread of proinflammatory responses in the lung capillary bed via calcium waves occurring through gap junctions. However, whether this connexin contributes to the intensity of lung inflammation in vivo is not known. In addition, the implication of connexins in leukocyte recruitment, adhesion and transmigration across the

of my thesis is to determine the role of connexin-built channels in the cellular interactions occurring between neutrophils and the lung endothelial and alveolar epithelial cells using in vivo and in vitro approaches.

In the first chapter of my thesis, I studied the contribution of Cx43 to neutrophils’

recruitment upon intratracheal (IT) instillation of Pseudomonas aeruginosa lipopolysaccharide (LPS) in genetically modified mouse models for Cx43. GJIC can be reduced by decreasing Cx43 expression or increased by truncating Cx43 C-terminal domain leading to a long lasting channel opening. We therefore compared lung inflammation in heterozygous mice for Cx43 (Cx43^+/-) and in mice harboring the truncated non regulatable Cx43 allele (Cx43^K258stop/-). Interestingly, increasing Cx43-mediated GJIC in Cx43K258stop/-

mice led to an exaggerated lung inflammation whereas decreasing Cx43 expression in Cx43^+/- mice attenuated their inflammatory response. In addition, lung inflammation in wild-type mice was associated with the induction of Cx43 in alveolar septa by the action of inflammatory mediators. The importance of Cx43 on neutrophils’ adhesion was evaluated in vitro by decreasing Cx43-mediated GJIC using a specific Cx43 blocking peptide (⁴³Gap26).

The adhesion of neutrophils decreased on mouse endothelial and epithelial cell lines treated with ⁴³Gap26. The effect of the peptide was finally evaluated in vivo by IT instillation in inflamed lungs. I observed a protection of 65% against lung inflammation. Cx43 therefore acts as a pro-inflammatory mediator and may represent a pharmacological target to control leukocyte recruitment in lung diseases.

Among the mechanisms regulating neutrophil adhesion to the endothelium, nucleotide liberation at sites of inflammation has recently received increasing attention regarding its relation to connexins. It was previously reported that neutrophils may release ATP via connexin hemichannels. In the second chapter, I evaluated if leukocyte-endothelial cell interactions are related to Cx43 expression in neutrophils. I monitored the adhesion of these

leukocytes on endothelial cell lines by using circulating neutrophils from Cx43^+/- mice and Cx43^K258stop/- mice. The adhesion of neutrophils pretreated with the ⁴³Gap26 to close Cx43 hemichannels at the leukocyte surface was also tested. In parallel to adhesion tests, the phagocytic functional property of neutrophils was also studied. The results show that phagocytosis and adhesion of neutrophils were not modified upon genetic modulation or pharmacological inhibition of Cx43. In contrast, I found that adhesion of neutrophils from Cx37 knock-out mice was significantly increased suggesting an implication for this connexin in the neutrophil-endothelial cell interactions. These results were confirmed in vivo by evaluating transmigration across the lung endothelial and alveolar epithelial barriers via an adoptive-transfer technique. Thus, fluorescent neutrophils from genetically modified mice were injected retro-orbitally in a surrogate wild-type mouse subjected to IT instillation of LPS. Our observations show that neutrophil-endothelial cell interactions do not involve Cx43 expression in neutrophils. Interestingly, I found that Cx37 in neutrophils seems to be crucial for regulating their adhesion and transmigration, possibly through ATP release.

Upon its release, ATP is hydrolyzed to AMP which will be further converted to adenosine at endothelial cells’ surface by the 5'-ecto-nucleotidase (CD73). Adenosine activates A2B receptors which are known to mediate anti-inflammatory events by decreasing leukocyte adhesion to the endothelium. In the third chapter, I studied mice with endothelial specific deletion of Cx40 (Cx40del mice). Interestingly, Cx40del mice showed a decreased expression of CD73 in the lung capillary endothelium and an early increased neutrophil recruitment form the microcirculation in response to LPS IT instillation. Moreover, knock- down of endothelial Cx40 using in vitro approaches decreased CD73 expression and activity while increasing neutrophil adhesion to a mouse endothelial cell line. I further developed a co-culture system to demonstrate the contribution of Cx40-mediated GJIC in the propagation of anti-inflammatory signals affecting neutrophil adhesion.

Altogether, the results presented in my thesis argue for opposite roles for Cx43 and Cx40 in endothelial signal propagation during lung inflammation. Cx43 is a pro-inflammatory mediator, in contrast to endothelial Cx40 which appeared to participate in anti-inflammatory events by decreasing the adhesion of circulating leukocytes to the endothelium. Cx37 expression in neutrophils contributes to these events. Indeed, ATP may be released via Cx37 hemichannels and used as a substrate for ecto-enzymes at the surface of endothelial cells, thus creating an anti-adhesive signal. The propagation of this signal involves endothelial Cx40- mediated GJIC. In the presence of a pro-inflammatory stimulus, Cx43 induction in alveolar septa favors the propagation of calcium waves, thus ensuring leukocyte adhesion and transmigration. In summary, I propose the hypothesis that cellular communication orchestrated by different connexins creates a balance between pro- and anti- inflammatory events to regulate neutrophils’ recruitment to the alveolar space during lung inflammation.

I. INTRODUCTION

1. The lung respiratory epithelium

The respiratory epithelium is positioned at the interface between the body and the environment and has a large area of exposure to the external milieu. The mature respiratory system consists of two main portions with distinct functions: the air-conducting portion and the respiratory portion (Figure 1). The air-conducting portion extends from the nasal cavities to the bronchioles thus providing a passage for inhaled and exhaled air in and out of the respiratory portion. The human conducting airways are lined by a tall pseudostratified epithelium that includes several cell types, among which basal, ciliated and secretory cells are the most abundant. This structure is referred to as the airway epithelium. Submucosal seromucus glands are additional structures of the airway epithelium and they are mostly found in the air-conducting portion.

Fig.1: Schematic representation of human lungs from the air-conducting to the respiratory

The respiratory portion in lungs is the site for gas exchange between blood and air. It is composed of the respiratory bronchioles, alveolar ducts and alveoli, which are surrounded by an important capillary network essential for gas exchange. These structures, referred to as the alveolar epithelium, are lined by a thin epithelium composed of alveolar type I (ATI) and type II (ATII) pneumocytes. The large flat ATI cells mediate gas exchange while the cuboidal ATII cells, progenitors of ATI pneumocytes, can synthesize, store, secrete and recycle pulmonary surfactants, which allow for effective gas exchange (Koval, 2002; Sugahara et al., 2006; Mason, 2006; Andreeva, Kutuzov and Voyno-Yasenetskaya, 2007).

The interalveolar septa are very thin layers of tissue that constitute the interface between air and blood (Figure 2). These layers are so thin that they cannot be resolved into their constituents by light microscopy. Electron microscopy showed that interalveolar septa are consistently composed of two layers: a capillary endothelium and an epithelium, separated by a very narrow interstitial space (Figure 2 A).

Fig.2: A, Scanning electron microscope view of lung alveoli, magnified at Å-750. The arrow points to the narrow interstitial space separating the alveolar endothelium and epithelim. Al, Alveolus. Adapted from Albertine, Williams and Hyde, 2000. B, Schematic view of alveoli and interalveolar septum. Modified from “http://www.netterimages.com/image/14561.htm”.

Nearly half the contact surface between capillary endothelium and alveolar epithelium is formed exclusively by the fused basement membranes of these two cell layers. In some parts, the basement membranes are separated and the intercalated space between them contains thin elastic fibers and small bundles of collagen fibrils (Figure 2) (Weibel, 1973).

The highly differentiated respiratory epithelium represents an enormous and delicate interface with the external environment because it is continuously exposed to antigens and potentially pathogenic microbes throughout respiration. In the air-conducting portion, the epithelium is covered by the airway surface liquid, which includes overlying mucous layer onto which inhaled particles are trapped and evacuated through mucociliary clearance.

However, for some harmful antigens, an immediate and intense defense action is also required to eliminate lung invaders as early as possible in order to preserve the function of the lung as a vital organ for gas exchange. Hence, lung epithelium not only conditions the incoming air moisture but orchestrates, as well, the pulmonary defense system essential to maintain the sterility of pulmonary tissues (Diamond, Legarda and Ryan, 2000; Holgate et al., 2000).

2. Defense mechanisms in the lung

The body is continuously exposed to pollutants, dust, irritant, allergens as well as to microbes and harmful pathogens including bacteria and viruses. To face all these insults, the body has to defend itself by developing an adequate immune system. Immunity involves innate and adaptive systems that share components and act in concert to provide defense. The innate immunity constitutes a rapid response in a non-specific manner that will initiate inflammation and mobilize the adaptive immunity which is antigen-specific. An adequate orchestration of the immune response at sites of injury is essential to provide a balance between inflammatory and anti-inflammatory events in order to ensure tissue homeostasis.

A. Maintenance of lung sterility

Homeostasis in pulmonary tissues is maintained by the concerted effects of mechanical, innate, and adaptive host defense systems that will recognize and localize pathogens. Upon recognition, pathogens are killed and removed in order to maintain lung sterility. In some lung diseases, failure in these mechanisms allows local or systemic infection and destruction of lung tissues. Several mechanisms in the lungs such as cough reflex and mucociliary clearance complement the innate and adaptive responses (Figure 3). In addition, the production of antimicrobial polypeptides by epithelial cells contributes as well to the clearance of the invading pathogens (Tiddens, Silverman and Bush, 2000; Travis, Singh and Wlesh, 2001;

Knowles and Boucher, 2002; Whitsett, 2002; Strieter, Belperio and Keane, 2003).

Fig.3: Schematic view of the airway epithelium with its resident and recruited leukocytes. The host defense mechanism of the respiratory epithelium involves mucociliary clearance (removal of pathogens by cough and beating cilia), the production by epithelial cells of substances with antimicrobial activities (including mucins, defensins, nitric oxide and surfactants), and the release of inflammatory mediators (cytokines) by epithelial cells and macrophages. Macrophages, B and T lymphocytes, neutrophils and epithelial cells represent cellular components of the innate and adaptive immune systems. Modified from Bals, Weiner

The lung epithelium will detect foreign bodies and will act accordingly by secreting a variety of substances such as mucins, defensins, nitric oxide and surfactants that will prevent pulmonary edema and nonspecifically shield the respiratory tract from microbial attack (Figure 3) (Oliveira et al., 2007). Surfactant lies on the surface of alveoli and contains four surfactant proteins (SP A-D). SP-A and SP-D are hydrophilic calcium-dependent lectins that participate in host defense by acting as antimicrobial substances with the ability to agglutinate a variety of fungi, bacteria, and viruses, hence facilitating their clearance (Mason, 2006;

Kuroki, Takahashi and Nishitani, 2007).

B. Pathogen sensing

Pulmonary innate immunity is mediated by airway and alveolar epithelial cells as well as resident leukocytes such as alveolar macrophages, and recruited lymphocytes and neutrophils (Figure 3) (Moldoveanu et al., 2009). The airway epithelium and alveolar macrophages are the first sites of contact with inhaled microbes. They are equipped with sensing receptors including toll-like receptors (TLRs) that allow them to detect bacterial exposure and respond accordingly by increasing their defense system (Basu and Fenton, 2004; Guillot et al., 2004;

Hoth et al., 2009). Toll-like receptors, involved in the recognition of various pathogen- associated molecular patterns (PAMPs), represent a conserved family of receptors linking innate and adaptive immunity (Takeda, Kaisho and Akira, 2003; Chaudhuri et al., 2005;

Akira, Uematsu and Takeuchi, 2006; Chaudhuri et al., 2007; Chignard et al., 2007; Si-Tahar, Touqui and Chignard, 2009). The TLR family distinguishes 10 types of receptors, each designated for the recognition of a specific category of invaders. In particular, TLR3, TLR7 and TLR8 are implicated in viral detection, TLR2 senses gram-positive bacteria and peptidoglycan while TLR4 recognizes endotoxins and lipopolysaccharide (LPS) from gram- negative bacteria such as Pseudomonas aeruginosa (P. aeruginosa) (Schurr et al., 2005;

Baumgarten et al, 2006; Menendez and Torres, 2007; Wang, Kurt-Jones and Finberg, 2007).

The latter is a cytotoxic strain that can invade the respiratory system in some lung diseases. In this case, LPS activates a TLR4 signaling system in order to clear the bacteria and prevent lethal lung injury and bacteremia (Faure et al., 2004; Yamada et al., 2008).

C. The toll-like receptors’ signaling cascade

Ligand–TLR interactions during lung infection will trigger the binding of at least one adaptor molecule, MyD88, to the intracellular domain of the TLR. This is the first step of a signaling cascade that leads to the activation of various nuclear factors, including nuclear factor-B (NF-κB), that will stimulate and regulate the expression of multiple genes of inflammatory mediators and adhesion molecules (Pahl, 1999; Ciesielski et al., 2002; Li and Verma, 2002).

Indeed, NF-B will induce the production of a large array of cytokines and chemokines essential for the initiation and orchestration of the subsequent innate and adaptive immune response (Adler et al., 1994; Strieter, Belperio and Keane, 2002; Chignard et al., 2007). In particular, activated alveolar macrophages, epithelial cells and endothelial cells will produce tumour necrosis factor-α (TNF-α), interleukins (IL-)/chemokines (IL-1β, IL-6, IL-8, IL-10, IL-12), and granulocyte/macrophage colony stimulating factor (GM-CSF), as well as platelet- activating factors (PAF) that will act on leukocytes and stimulate their transmigration into the site of inflammation (Strieter, Belperio and Keane, 2002; Bals and Hiemstra, 2004;

Hippenstiel et al., 2006).

D. Leukocyte recruitment

Leukocyte activation and extravasation from the bloodstream into the sites of injury in lungs is essential to provide a protection against many bacterial pathogens. Leukocyte emigration relies on many sequential steps during which leukocytes will tether and roll on the endothelial

surface in response to selectins. Activated leukocytes will tightly adhere to the endothelium; a step triggered by chemokine signals and controlled by specific cell adhesion molecules (CAMs) and leukocyte integrins (Figure 4). Signaling molecules mediate the cross- talk between the CAM-integrin complex in order to determine the tightness of adhesion upon which leukocytes continue to transmigrate or return back to the blood stream (Ley et al., 2007).

Fig.4:Leukocyte extravasation from bloodstream into tissue occurs in four main steps. First leukocytes tether and roll on the endothelial surface in response to selectins. They are then activated in response to chemokines and arrested in response to integrin and cell adhesion molecules to firmly adhere on the vascular surface and transmigrate into the injured tissue via diapedesis. Modified from “http://www.mpi-muenster.mpg.de/nvz/Abb1e.jpg”

Leukocytes that are primed for transmigration undergo diapedesis, which is a step that consists on the passage of recruited cells across the inter-endothelial barrier. Leukocyte transendothelial migration is mediated by junctional adhesion molecules (JAM-A, JAM‑B, JAM‑C) as well as endothelial cell-selective adhesion molecules (ESAMs). Interestingly, JAMs mediate leukocyte transmigration in a stimulus specific manner in response to interleukins whereas ESAMs influence transmigration in a leukocyte-specific manner

favouring neutrophil rather than lymphocyte T transmigration (Vestweber, 2002; Muller, 2003; Ley, 2007). In the context of lung injury, leukocyte recruitment involves cell to cell contacts not only between inflammatory and alveolar endothelial cells, but also between leukocytes and alveolar epithelial cells, which results in leukocyte transmigration across the thin endothelial and epithelial barriers that separate the bloodstream from the pulmonary air space (Hogg and Doerschuk, 1995; Doerschuk et al., 1999; Wagner and Roth, 2000; Mizgerd, 2002; Craig et al., 2009).

The first blood cells to appear into the alveolar space after lung injury or infection are polymorphomononuclear cells (PMNs) among which neutrophils are the most abundant.

Neutrophils will adhere to the injured capillary endothelium and marginate through the interstitium into the alveolus. Inflamed alveoli are filled with protein-rich edema fluid responsible for surfactant inactivation, which will hamper in turn the correct restoration of the lungs’ vascular permeability during inflammation (Figure 5) (Lewis and Jobe, 1993; Greene et al., 1999; Ware and Matthay, 2000). Neutrophil transmigration is followed by that of monocytes, which will differentiate into macrophages. In the air space, alveolar macrophages secrete cytokines/chemokines, including TNF-, IL-1, IL-6, IL-8 and IL-10, that will act locally to modulate chemotaxis and activate neutrophils. Interleukin-8 is a chemotactic agent for circulating leukocytes while TNF- increases the expression of cell adhesion molecules on lung capillary endothelial cells to enhance leukocyte adhesion during their recruitment.

Moreover, activated neutrophils can release proinflammatory molecules including oxidants, leukotrienes, PAF and proteases such as elastase, which will induce IL-8 production in epithelial cells; they also produce IL-8, providing therefore a positive feedback for enhancing their own recruitment to sites of injury (Wardlaw, 1990; Takahashi et al., 1993; Strieter, Belperio and Keane, 2002; Strieter, Belperio and Keane, 2003). Finally the immune response is completed with the immigration of lymphocytes. The production of cytokines and of a

variety of growth factors such as IL-1and IL-1 by damaged tissue also triggers the migration of resident cells and the restoration of tissue integrity by the production of a new extracellular matrix by fibroblasts (Figure 5) (Ware and Matthay, 2000).

Fig.5: The normal alveolus (left-hand side) and the injured alveolus in the acute phase of immuno-inflammatory pathologies such as acute lung injury and acute respiratory distress syndrome (right-hand side). Ware and Matthay, 2000.

The initiation, maintenance and resolution of pulmonary innate responses therefore

alveolar macrophages, endothelial cells and fibroblasts are altogether involved in the initiation and regulation of air space inflammation through a variety of cytokines and chemokines which contribute in turn to the recognition of pathogens, the recruitment of neutrophils and mononuclear cells and the removal of the invading micro-organism (Strieter, Belperio and Keane, 2002; Strieter, Belperio and Keane, 2003). Initiation and amplification of immune responses are usually followed by a phase of resolution (Gordon, 2007). Neutrophils undergo surface changes to become apoptotic when infected with bacterial residues, which allow them to be recognized and ingested by phagocytic macrophages. Moreover, the regulation of cytokine production by the macrophage inhibitory factor (MIF), for instance, is important to maintain the balance between proinflammatory and anti-inflammatory mediators, which is essential for an adequate resolution of inflammatory responses (Figure 5). Several endogenous inhibitors of proinflammatory cytokines have also been described in the broncho- alveolar space including IL-1 receptor antagonist and soluble TNF- receptor, which are known to regulate the release of inflammatory mediators (Donnelly et al., 1997; Pittet et al., 1997; Matthay, 1999). Altogether, apoptosis, adequate removal of inflammatory cells by macrophages at sites of infection as well as suppression of inflammatory mediator production therefore appear to be essential for the resolution of inflammatory responses (Fadok et al., 1998; Ware and Matthay, 2000).

3. Inflammation in respiratory diseases

It sometimes happens that resolution of inflammation does not properly occur. In such cases, the incomplete resolution of inflammatory responses results in excessive tissue damage and a massive invasion of leukocytes with decreased apoptotic signals, leading to acute and chronic conditions (Matute-Bello et al., 1997).

A. Common lung diseases

Lung homeostasis is crucial for providing protection against excessive leukocyte recruitment during inflammation. Acute lung injury (ALI) is a condition characterized by an exaggerated tissue inflammation and a massive invasion of leukocytes to the broncho-alveolar space in response to micro-organisms. Acute lung injury develops rapidly across the lungs and may lead to irreversible bronchial and alveolar epithelial cell damage as well as to the formation of protein-rich hyaline membranes in alveoli (Figure 5) (Ware and Matthay, 2000; MacCallum and Evans, 2005). The illness syndrome of ALI consists of hypoxemic respiratory failure with bilateral pulmonary infiltrates, leading to high mortality in both children and adults. The severity of the airway inflammation can be related to the severity of the disease. However, despite recent advances regarding pathophysiology, treatment and long-term outcome of ALI, its incidence and outcome are not predictable (Thomsen and Morris, 1995; Ware and Matthay, 2000; Rubenfeld et al., 2005). Acute lung injury is encountered in pneumonia and acute respiratory distress syndrome (ARDS), which is characterized by a more severe hypoxemia (Markowicz et al., 2000). In some life-threatening bacterial lung diseases, ALI is dominated by the excessive infiltration of neutrophils to overcome the abnormal bacterial colonization. When the resolution of ALI is incomplete, a chronic lung inflammation might take over with a persisting spread of infection and a delayed apoptosis and phagocytosis of immune cells, which lead to a prolonged inflammatory cell infiltration in lungs (Moldoveanu et al., 2009). Chronic airway inflammation is characteristic in asthma and in chronic obstructive pulmonary disease (COPD), and can lead to chronic bronchitis with airflow obstruction, mucus hyper secretion and interstitial pulmonary fibrosis. Cystic fibrosis (CF) disease, caused by a mutation in the gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR), is another exemple to illustrate chronic lung inflammation, dominated however by neutrophil recruitment to the airways (Boucher, 2004).

B. Cellular orchestration during immuno-inflammatory pathologies

Many studies regarding the extended spread of inflammation in ALI, ARDS and COPD are shedding the light on the importance of cellular coordination to overcome lung infiltrates. The mechanisms underlying lung inflammation and leukocyte accumulation during lower respiratory tract bacterial infection are learned from experimental animal models, particularly in mice. Inoculation of murine airways with LPS from gram-negative bacteria (P. aeruginosa, Escherichia coli, etc…) leads to a range of inflammatory responses including a persistent release of chemokines and a time-dependent neutrophil influx, which is considered as an index for the development and outcome of ALI and ARDS (Reutershan et al., 2005). Alveolar macrophages along with endothelial and epithelial cells play a critical role in recognizing the inoculated LPS via TLR4 and initiating in turn neutrophil recruitment from the vascular space to the airspace (Figure 6) (Skerrett et al., 2004; Mizgerd and Skerrett, 2008; Craig et al., 2009). Although TLR4 expression on endothelial and epithelial cells is essential for sequestration of neutrophils in the lung after systemic exposure to LPS, TLR4 expression on hematopoietic cells, such as macrophages and neutrophils, has also been shown to be important. Indeed, TLR4 on these cells was up-regulated by LPS and appeared to be critical to the movement of neutrophils from the vascular space to the airspace after LPS inhalation (Figure 5 and 6) (Koay et al., 2002; Andonegui et al., 2003; Hollingsworth et al., 2005;

Janardhan et al., 2006). TLR4 on alveolar endothelial cells, alveolar epithelial cells and hematopoietic cells in the lungs therefore seems to orchestrate the cooperation of inflammatory cells in their recognition of the invading pathogen and the initiation of the host inflammatory response with the goal of restoration of tissue integrity (Martin, 2000; Barton and Medzhitov, 2003; Takeda, Kaisho and Akira, 2003; Hollingsworth et al., 2005).

Fig.6: LPS initiates a TLR4 signaling that will activate alveolar macrophages and alveolar epithelial and endothelial cells, which in turn will induce a systemic release of cytokines such as TNF-, IL-6, IL-8 and other proinflammatory molecules that will recruit neutrophils to the bronchoalveolar space in lungs. The red arrow represents neutrophil transmigration across the endo-alveolar barrier in lungs.

Cooperation of the cells involved in the immune system not only relies on the TLR4- induced secretion of inflammatory mediators but has also been shown to rely on direct cellular interactions. Interestingly, the exchange of some signaling molecules between endothelial cells and leukocytes during their extravasations occurred via intercellular communication and appeared to control tight adhesions, mediated by integrins and their ligands, between leukocytes and endothelial cells. Moreover, diapedesis, which also involves cell to cell contacts, was also shown to involve gap junctions (Sáez et al., 2000a; Wong, Christen and Kwak, 2004). Gap junctions are intercellular channels that permit the cytoplasmic exchange of signals between neighbouring cells. This process is referred to as gap junctional intercellular communication (GJIC). The next part of this introduction will be dedicated to gap junction channels, where their structure and functional properties as well as

4. Gap junctions, connexins and intercellular communication

A. Discovery of gap junctions

Cross-talk between cells can be accomplished by paracrine or autocrine mechanisms implicating the release of molecules into the extracellular environment, such as neurotransmitters, hormones, and growth factors. An alternative mechanism for cellular communication is the direct transfer of molecules within cells without reaching the extracellular environment. This intercellular communication is mediated by a cluster of channels, known as gap junctions, forming low resistance pathways that allow the passage of ions, small metabolites, siRNA and linear peptides between neighboring cells (Goodenough et al., 1996; Mese, Richard and White, 2007; Harris and Darren, 2009). Gap junctional intercellular communication relies on the existence of intercellular protein channels that span the lipid bilayers of adjacent cells. The first indication for the existence of a low-resistance pathway was proposed in 1952 by Weidmann, who worked with strips of myocardium and found that the space constant for the spread of currents extends beyond the expected value for a single Purkinjie fiber (Weidmann, 1952). Stronger evidence supporting Weidmann hypothesis was provided by the discovery of electrical transmission at the giant crayfish motor synapses (Furshpan and Potter, 1959). Later, this type of pathway was demonstrated in other excitable tissues and in nonexcitable cell types (Loewenstein and Kanno, 1964; Barr, Dewey and Berger, 1965; Orkand, Nicholls and Kuffler, 1966; Potter, Furshpan and Lennox, 1966; Bennett, 1966; Revel, Yee, and Hudspeth, 1971; Harris, Spray and Bennett, 1981).

Today, it is accepted that cells of most tissues can communicate with their neighbors via intercellular pathways. The structure responsible for this type of intercellular communication was termed “gap junctions” by Revel in 1967 (Revel and Karnovsky, 1967). Gap junctions therefore allow for the direct exchange of information between cells and offer a third pathway for intercellular communication with the synapse and the neuromuscular junction (Wells and

Bonetta, 2005). In vertebrates, only few cell types do not form gap junctions in their fully differentiated state, including red blood cells, spermatozoa and skeletal muscle. Nevertheless, the progenitors of these cells do express gap junctions (Rosendaal et al., 1994; Constantin, Cronier and Raymond, 1997; Proulx, Merrifield and Naus, 1997; Mok, Yeung and Luk, 1999).

B. Structure of gap junctions

Gap junctions consist of aggregates of transmembrane hemichannels (or connexons), that dock to similar connexons on neighboring cells (Figure 7). This interaction produces a gap junction channel consisting of a protein-lined conduit directly connecting adjacent cells.

Three-dimensional structure of a recombinant gap junction channel revealed that opposing connexons are staggered by 30° and packed in the intercellular gap, resulting in a tight seal between the two hemichannels (Perkins, Goodenough and Sosinsky, 1998; Unger et al., 1999).

The wall of each connexon is formed of six multi-gene protein subunits, named connexins (Cx), creating a 2nm-diameter aqueous pore that allows diffusion of molecules of about 1 kDa between the cytoplasm of neighboring cells (Figure 7) (Goodenough, Goliger and Paul, 1996; Kumar and Gilula, 1996; Spray, 1996; Harris, 2001; Evans and Martin, 2002;

Maeda et al., 2009). Gap junction channels possess distinctive permeabilities for various signaling molecules, which depend on the connexin member(s) that form them (Nicholson, 2000; Harris, 2001).

Fig.7: The transmembrane core proteins of gap junctions are the connexins. Six identical or distinct connexins assemble to form connexons. Connexons then dock with their counterparts in the neighboring cell to form intercellular channels (Goodenough and Paul, 2003).

The assembly of six identical connexins into a connexon forms a homomeric hemichannel, while the assembly of different connexins forms a heteromeric hemichannel (White and Bruzzone, 1996; He et al., 1999; Moreno et al., 2004). Interestingly, the oligomerization of connexin molecules into a connexon, made of single or multiple connexin types appears to be based on specific signals located within the connexin polypeptides (White and Bruzzone, 1996). Connexons can then stay free at the cell membrane or they can form gap junctions by docking with another hemichannel at sites of cell to cell contact. Two identical docking connexons can therefore form a homotypic channel; while heterotypic channel can be generated by two connexons having different connexin isotypes (Figure 8)

(Sáez et al., 2003). The mechanisms that regulate connexin hetero-oligomerization remain however unknown.

Fig.8: Schematic drawing showing possible arrangements of connexons to form heterotypic and heteromeric gap junction channels. Connexons consisting of six connexin subunits (red and blue) may be homomeric (identical subunits) or heteromeric (more than one connexin isotype), and when associated end to end, form membrane channels that may be homotypic or heterotypic (adapted from Kumar and Gilula, 1996).

C. Structural diversity of connexin genes

The commonly used nomenclature distinguishes connexins by their molecular mass deduced from their respective cDNAs. Thus, 37, 40 and 43 kDa connexin proteins are referred to as Cx37, Cx40 and Cx43, respectively; Cx43 is by far the most well known connexin among gap junction proteins. The alternative gene nomenclature of connexins is based on their evolution regarding their sequence homologies. This nomenclature uses the letters Gja to Gjd and GJA to GJD for mice and human genes, respectively, followed by a consecutive number assigned to each connexin (Söhl and Willecke, 2003). For instance, the GJA1 gene corresponds to human Cx43 while the Gja5 gene corresponds to mouse Cx40.

Most cells and tissues express more than one connexin isotype. To date, 20 connexin genes have been identified in the mouse genome and 21 in the human genome (Sohl and Willecke, 2003). The available information indicates that many connexin genes have a similar organization, and virtually all have only single copies in the haploid genome (Willecke et al., 2002). Most of the connexin genes share a common structure: a first exon containing the 5’

untranslated region (5’-UTR) followed by an intron of varying length, and a second exon containing the remaining 5’-UTR, the complete coding region, and the 3’-UTR. Exceptions to this gene structure are the Cx32 and Cx45 genes that contain three exons, and the Cx36 gene which shows a coding region located on its two exons interrupted by an intron (Figure 9 A) (Willecke et al., 2002; Sáez et al., 2003). The sequence similarities between connexins and their similar gene structures suggest that they have arisen by gene duplication (Figure 9 B).

Fig.9: A, Structures of connexin genes. Top: common organized structure for connexin genes. Bottom:

in some connexin genes (e.g., Cx36), the coding region is interrupted by an intron. Untranslated and translated regions are depicted as boxes in dark gray and light gray, respectively. The genomic DNA sequences not present in the mRNA are represented by the black lines.

B, Phylogenetic tree for the connexin family (http://workbench.sdsc.edu).

All sequences used for the aligment are from human origin with the exception of rat Cx33. Adapted from Sáez et al., 2003.

D. Topology of connexins

Connexins are transmembrane proteins that have a common topology with four alpha-helical hydrophobic transmembrane domains (M1-M4), two extracellular loops (E1 and E2), a cytoplasmic loop, and an amino and carboxyl terminus (NT and CT, respectively) located on the cytoplasmic membrane (Figure 10) (Yeager and Harris, 2007).

Fig.10: Model showing membrane topology for the connexin polypeptide. M1–M4 represent the four transmembrane domains, E1 and E2 the two intracellular loops; the amino (N) and carboxy (C) termini are located intracellularly (Kumar and Gilula, 1996).

The four transmembrane spanning regions contain many identical residues or conservative substitutions among the different connexins. In addition, the two extracellular loops E1 and E2 contain about 31 and 34 amino acids, respectively, and are covalently connected by disulphide bonds. Interestingly, the end-to-end binding between two connexons occurs via non-covalent interactions between the extracellular loops, each of which contains three conserved cysteine residues that constitute intramolecular disulfide bridges to create a tight seal for prohibiting the exchange of substances between the channel lumen and the extracellular milieu (Unger et al., 1999). Finally, while the NT domain has been shown to be involved in gating polarity, the second extracellular loop (E2) of connexins appeared to be

important in determining which connexins can interact to form heterotypic channels (Verselis, Ginter and Bargiello, 1994; White et al., 1994; White et al., 1995; Purnick et al., 2000).

In contrast to the transmembrane domains, the cytoplasmic loop and the CT domain vary extensively in length and amino acid composition, being unique to each connexin.

Indeed, the CT tail of connexins is a major determinant of the connexin molecular mass and is thought to play key regulatory roles since it contains many sites subjected to phosphorylation, as well as internalization motifs and other sites implicated in the interaction of connexins with other proteins. In particular, the C-terminus of Cx43 has around 150 amino acids and contains multiple sites for protein kinase C (PKC), PKA, PKG and Src kinase phosphorylation.

Connexin 43 CT also interacts with proteins present in other cell junctions and with cytoskeletal components (Thomas et al., 2002; Giepmans, 2004; Herve et al., 2004; Solan and Lampe, 2009). Homozygous mice that are generated to lack the CT of Cx43 die shortly after birth due to a defective epidermal barrier, and removal of the Cx43 CT tail may also lead to prevention of normal cell growth (Moorby, 2000; Maass et al., 2004). Finally, recent in vivo evidence in heterozygous mice for the CT of Cx43 show that the CT domain is involved in regulating the localization, number and size of Cx43 cardiac gap junction plaques while protein interactions or posttranslational modifications taking place within the Cx43 CT appear not to be required for the assembly of functional gap junctions in this tissue (Maass et al., 2007).

E. Assembly and degradation of gap junctions

Connexins’ mRNA leaves the nucleus and heads toward the rough endoplasmic reticulum (ER) where it is translated. Newly synthesized connexins will fold then assemble with other connexins to form hemichannels. The location at which connexins oligomerize into connexons is connexin-type dependent. For instance, Cx32 assembles in the ER or in the

ER/Golgi intermediate compartment while Cx43 assembles in the trans-Golgi network (TGN) (Musil and Goodenough, 1993; Rahman, Carlile and Evans, 1993; Martin et al., 2001; Das Sarma, Wang and Koval, 2002). Moreover, in studies performed in liver, Cx26 oligomers are detected in the ER/Golgi intermediate compartment while oligomers containing both Cx26 and Cx32 are preferentially detected in a Golgi membrane fraction (Figure 11) (Diez, Ahmad and Evans, 1999). This might have implications for the formation of heteromeric connexons (Laird, 2006).

Fig.11: Diagram illustrating the pathways from transcription to degradation of connexins.

TGN, trans-Golgi network; ERAD, ER-associated-degradation. (Laird, 2006).

Although some connexin oligomers such as Cx26 are directly integrated into the plasma membranes after translation, most of the newly synthesized and assembled connexins, including Cx43 and to a minor extent Cx26, follow the exocytic/secretory pathway which transports them in vesicles to the plasma membrane along microtubules (Figure 11) (George, Kendall and Evans, 1999; Martin et al., 2001; Ahmad and Evans, 2002). Indeed, it is believed that microtubule assembly promotes the delivery of connexin hemichannels from the cell interior directly to the cell-cell border by targeting gap junctions to tight or adherens junctions, which favours the clustering of gap junctions and the formation of gap junction’s plaques (Figure 12) (Shaw et al., 2007). In this way, connexons are inserted into the plasma membrane near tight or adherens junctions, at regions of cadherin/catenin-mediated cell adhesion, where gap junction plaques formation can take place as shown from the colocalization of connexins with E-cadehrin or catenin during plaque assembly in regenerating liver (Jongen et al., 1991; Fujimoto et al., 1997; Shaw et al., 2007).

Although tight and adherens junctions are thought to mediate the assembly of gap junction plaques, gap junctions have also been shown to mediate, reciprocally, the assembly of tight and adherens junctions, suggesting a close association and a significant interplay between gap, tight and adherens junctions. Such association probably involves tight junctions associated proteins such as ZO-1 and ZO-2 motifs capable of binding to components in all three junctional complexes (Figure 12) (Jongen et al., 1991; Meyer et al., 1992; Itoh et al., 1993; Duffy, Delmar and Spray, 2002; Thomas et al., 2002). The growth of gap junction plaques occurs by incorporation of additional hemichannels to the plasma membrane followed by their assembly at the periphery of existing gap junctions (Lauf et al., 2002; Gaietta et al., 2002). However, how connexins are brought together to assemble into connexons, how these connexons are incorporated into the plasma membrane and how gap junctional channels aggregate and form junctional plaques is not yet resolved (Sáez et al., 2003).

Fig.12: Arrangement of gap, tight and junctional complexes (Laird, 2006).

Many morphological and biochemical approaches have recently been applied to elucidate the mechanisms of connexins and gap junction degradation (Beyer and Berthoud, 2002). Connexins can be mono- or poly-ubiquitinated, depending on which they can be targeted for ER-associated degradation (ERAD) or tagged for internalization and lysosomal degradation (Figure 11) (Laird, 2006). Further electron microscopy studies have suggested that the ubiquitinated gap junctions may undergo endosomal internalization of entire junctions forming an “annular ring”. This ring interacts with actin filaments and moves toward to proteasomal and lysosmal multivesicular bodies, or autophagosomal compartments where its protein components are degraded (Figure 11) (Larsen and Tung, 1978; Murray et al., 1981;

Mazet, Wittenberg and Spray, 1985; Severs et al., 1989; Vaughan and Lasater, 1992; Jordan et al., 2001; Beyer and Berthoud, 2002; Gilleron et al., 2009). However, while some studies focused on the importance of the ubiquitin-proteasome pathway in Cx43 gap junction’s degradation, others showed that older Cx43 are removed from the center of gap junction

al., 2002). Connexin43 gap junction plaques as well as Cx43 polypeptides exhibit rapid turnover rates but the preference of one pathway of degradation over the other seems to depend on the cell type and on the CT tail of connexins, and within the same cell type, the chosen pathway for connexin degradation might also depend on the metabolic/pathological state of the cell (Sáez et al., 2003; Laird, 2006). Signals and motifs located on the CT domain of Cx43 appeared to be implicated in the fate of connexins by regulating their sorting to the lysosomal pathway or to the ubiquitin-proteasome degradation system. In particular, a tyrosine-based sorting signal has been shown to control Cx43 turnover by targeting the protein for lysosmal degradation, hence playing a key role in regulating the process of connexin endocytosis and internalisation via endosomal/lysosmal compartment, thereby regulating the strength of GJIC (Thomas et al., 2003). Interestingly, the loss of Cx43 CT domain in transgenic mice resulted in an increased cellular communication and increased expression of Cx43 proteins in the heart, clearly demonstrating the importance of the CT domain regarding connexin expression, turnover, function and regulation at the junctional membrane (Maass et al., 2007).

F. Regulation of gap junction channels

Both homotypic and heterotypic gap junction channels display different biophysical properties. Their selective permeability gating can vary over a wide range. Hence, some connexin combination can produce intermediate or new conductive or gating properties that could modify the property of gap junction channels thereby influencing the communication between cells (White and Bruzzone, 1996; Ramanan et al., 1999; Banach and Weingart, 2000;

Valiunas et al., 2001; Goldberg, Valiunas and Brink, 2004; Moreno et al., 2004). The extent of GJIC can be evaluated by the use of specific molecular fluorescent tracers, such as the negatively charged Lucifer Yellow (LY) or calcein dies, and the positively charged

neurobiotin tracer. Interestingly, these tracers clearly demonstrate the different permeability of each connexin for cations or anions since LY, for instance, could pass through Cx32, Cx40 and Cx43 channels but could not be transmitted to neighbouring cells via Cx30 and Cx46 gap junction channels (Nicholson, 2000; Harris, 2001).

Gating regulation of gap junctions is not only determined by their connexin composition but also by multiple other factors such as intracellular calcium and proton concentrations, transjunctional membrane potential (the difference of membrane potentials between cells) and protein phosphorylation (Lampe and Lau, 2000; Harris, 2001; Ripps, Qian and Zakevicius, 2002; Stout and Charles, 2003; Basilio et al., 2004; Sáez et al., 2005; Spray, Ye and Ransom, 2006; Moreno and Lau 2007). Connexins sense intracellular changes via their CT tail; it was shown for example that gap junction channels from truncated Cx43 are less sensitive to intracellular acidification than normal Cx43 channels are (Liu et al., 1993).

The CT domain of connexins not only responds to stimuli but also interacts with other domains of the Cx43 protein to serve as a gating particle in a “ball and chain” model (Delmar et al., 2004). Differential phosphorlyation of connexin’s CT via protein kinases and phosphatases might affect in some cases the gating of gap junction channels, hence affecting GJIC (Moreno, Fishman and Spray, 1992; Chanson et al., 1993; Kwak et al., 1995; Cottrell et al., 2003; Lampe and Lau, 2004). For instance, ser-368 in Cx43 appears to be an important phosphorylation site that underlies cellular uncoupling in fibroblasts (Liu and Johnson, 1999;

Lampe et al., 2000). Interestingly, the pattern of Cx43 phosphorylation and its functional consequences appeared to be cell type dependent (Sáez et al., 1998; Lampe and Lau, 2000).

G. Function of gap junctions

Gap junctions have many physiological roles and numerous processes are driven by GJIC. For instance, GJIC are implicated in the rapid transmission of action potentials in heart and in

neuronal tissue via electrical synapses (Kirchhoff et al., 1998; Simon, Goodenough and Paul, 1998). Gap junctions ensure electrotonic signals’ transmission, which is essential to coordinate contraction of cardiac and smooth muscles. Moreover, metabolites and nutrients, such as nucleotides and glucose, can diffuse through gap junction channels, a process that depends on the channel type (Goldberg, Lampe and Nicholson, 1999; Fanchaouy et al., 2005;

Eltzschig et al., 2006). For instance, Cx32 channels are more permeable to adenosine than Cx43 channels, while the latter allows more readily the passage of ATP as compared to other connexin channels (Goldberg, Moreno and Lampe, 2002). Gap junctions also allow the diffusion of second messengers, such as calcium, inositol-triphosphate (IP3) and cyclic nucleotides which might be involved in the induction of apoptosis, gene transcription, embryogenesis and growth control (Sáez et al., 1989; Lecanda et al., 2000; Niessen et al., 2000; Plum et al., 2000; White, 2002; Alexander and Goldberg, 2003; Stains et al., 2003;

Michon et al., 2005). Gap junctions are therefore involved in tissue homeostasis, which depends not simply on intercellular communication but also on the specific pattern of connexin gene expression.

Connexins in one tissue can be differentially regulated by hormones, growth factors and proinflammatory mediators, thus ensuring a fine-tuned regulation of GJIC (Stagg and Fletcher, 1990; Harris, 2001; Evans and Martin, 2002; Thomas et al., 2002). Altered pattern of connexin expression and/or GJIC regulation would lead to changes in cell coupling thereby leading to tissue dysfunctions and pathological disease conditions. Indeed, critical functions for gap junction channels have been elucidated in mice with distinct phenotypes due to targeted deletions of connexins and in human by the discovery of disease-causing mutations in connexin genes (White and Paul, 1999; Willecke et al., 2002). Mutations in connexins or defective production of gap junctions in humans are associated to deafness, Charcot-Marie- Tooth X-linked neuropathy, cataractogenesis and a variety of skin disorders (Thomas and