Articles in PresS. Am J Physiol Lung Cell Mol Physiol (January 30, 2015). doi:10.1152/ajplung.00309.2014

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Pulmonary epithelial barrier function- some new players and mechanisms Kieran Brune1, James Frank2, Andreas Schwingshackl3, James Finigan4, Venkataramana K. Sidhaye1 1

Division of Pulmonary and Critical Care Medicine, Johns Hopkins University, Baltimore MD, USA, 2The

Division of Pulmonary and Critical Care Medicine, University of California, San Francisco, San Francisco VA Medical Center, and NCIRE/Veterans Health Research Institute, San Francisco, California USA, 3 Department of Pediatrics, University of Tennessee Health Science Center, 4 Division of Oncology, Cancer Center, National Jewish Health

Correspondence: Venkataramana K. Sidhaye, MD Department of Medicine, Division of Pulmonary and Critical Care Medicine Johns Hopkins Asthma and Allergy Center 4B.64 5501 Hopkins Bayview Circle Baltimore, MD 21224 Tel.: 410-550-4893 Fax: 410-550-2612 E-mail: [email protected]

Running Head: New players modulating the epithelial barrier Abstract: The pulmonary epithelium serves as a barrier to prevent access of the inspired luminal contents to the sub epithelium. In addition, the epithelium dictates the lung’s initial responses to both infectious and non-infectious stimuli. One mechanism that the epithelium does this is by coordinating transport of diffusible molecules across the epithelial barrier, both through the cell and between cells. In this review we will discuss a few emerging paradigms of permeability changes through altered ion transport and paracellular regulation by which the epithelium gates its response to potentially detrimental luminal stimuli. This review is a summary of talks presented during a symposium in Experimental Biology geared towards novel and less recognized methods of epithelial barrier regulation. First, we will discuss mechanisms of dynamic regulation of cell-cell contacts in the context of repetitive exposure to inhaled infectious and non-infectious insults. In the second section, we will briefly discuss mechanisms of transcellular ion homeostasis specifically focused on the role of claudins and paracellular ion channel regulation in chronic barrier dysfunction. In the next section, we will address transcellular ion transport and highlight the role of Trek-1 in epithelial responses to lung injury. In the final section, we will outline the role of epithelial growth receptor (EGFR) in barrier regulation in baseline, acute lung injury and airway disease. We will then end with a summary of mechanisms of epithelial control as well as discuss emerging paradigms of the epithelium role in shifting between a structural element that maintains tight cell-cell adhesion to a cell that initiates and participates in immune responses.

Copyright © 2015 by the American Physiological Society.

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Keywords: Lung, epithelial barrier, permeability, transport, ions Introduction

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The lung epithelium is a mucosal surface composed of ciliated cells, mucous producing

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cells, and undifferentiated basal cells that forms the interface between the lumen and

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the parenchyma from the nasal passage to the alveoli. It is the initial site of contact for

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all inspired substances and therefore acts as both a physical and an immunological

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barrier that protects the sub-epithelial tissue.

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The physical barrier, comprised of the mucocilary apparatus, secreted antimicrobial

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substances, and the intercellular junctions, prevents inhaled toxins and pathogens from

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contacting the sub-epithelial tissue. The mucociliary apparatus traps foreign particles

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and removes them from the respiratory tract. Defects in this apparatus can lead to

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recurrent infections as seen in patients with cystic fibrosis(104), COPD(14, 79), and

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ciliary dyskinesis(18, 19). Secreted antimicrobial substances including enzymes,

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protease inhibitors, and antimicrobial peptides further contribute to the epithelial cell’s

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immune response(134). Epithelial secretions contain enzymes such as lysozyme that

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have antimicrobial effects against both gram positive and gram negative bacteria(40,

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76) as well as protease inhibitors including serine protease inhibitor, secretory leukocyte

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protease inhibitor, and elafin which reduce the effects of proteases that are produced by

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pathogens. In addition, surface antimicrobial peptides like human beta defensins

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further protect against numerous pathogens including bacteria and viruses(116, 176).

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The intercellular junctions form adhesive forces that connect neighboring cells and

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separate the external environment from the sub-epithelial tissue. There are three main

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types of intercellular junctions: the tight junctions, the adherens junctions, and the

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desmosomes(137). The tight junctions are located more apically when compared to the

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other junctions and are multi-protein complexes that consist of transmembrane proteins

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including claudins, occludins, and JAMs as well as scaffolding proteins like the zona

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occludens. These junctions form a continuous circumferential ring around the epithelial

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cells which regulates the paracellular transport of ions and certain molecules(63, 157).

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The adherens junctions, composed of E-cadherin and several proteins from the catenin

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family including alpha catenin, beta catenin, and p120 anchor the actin cytoskeleton and

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mediate cell to cell adhesion(41, 63, 78). Together, the tight and adherens junctions

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form the apicoadherens junctions which separate the apical and basolateral membranes

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and block the movement of integral membrane proteins between surfaces to maintain

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epithelial cell polarity(169). The desmosomes, adhesive junctions located on the lateral

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cell membrane, link to the intermediate filaments and help protect against mechanical

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stress. Inhaled toxins such as cigarette smoke damage these intercellular junctions

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resulting in a leaky, hyper-proliferative, and abnormally differentiated epithelium(22, 68,

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73, 130, 150, 191).

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It is increasingly recognized that the epithelium helps to maintain homeostasis in the

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lung and disruption of the epithelial barrier can lead the development of inflammation

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and diseases such as COPD and acute lung injury (ALI). Moreover, novel studies

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demonstrating that restoration of the epithelial barrier through transfer of mitochondria

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from bone marrow derived stromal cells to epithelial cells can protect against ALI further

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establishes the importance of the epithelial barrier (77). In this review we will further

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discuss how the airway epithelium dynamically regulates its response to luminal stimuli

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by altering ion transport and paracellular permeability

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Section I: The lung epithelium: insights into the role of regulation of paracellular

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permeability in epithelial signaling

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The role of cell-cell adhesion in modulating epithelial signaling pathways

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The integrity of the physical barrier and epithelial cell polarity affect the interaction of

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cytokines and growth factors with their receptors thereby modulating the epithelium’s

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immunological response to environmental stimuli. In this respect, the epithelium also

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acts as an immunological barrier. Subtle changes in paracellular permeability can

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impact ligand interaction with its receptor. Moreover, certain cytokines are prevented

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from interacting with their cytokine receptors due to segregation to different sides of the

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apicoadherens junctional complex. Damage to the epithelium can affect the polarized

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distribution of cell surface receptors allowing for increased receptor-ligand interaction

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and the activation of signaling transduction cascades and secretory activity(73, 171,

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200). One specific example of such regulation is the epidermal growth factor (EGF),

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which regulates migration, proliferation, and differentiation following injury and

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enhances epithelial repair(186). In normal human airways, epidermal growth factor

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receptor (EGFR) expression is limited and isolated to the basolateral membrane (187)

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however, expression is increased in response to cigarette smoke(165, 188), mechanical

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stress(139), and inhaled noxious substances like bleomycin(110). In a homeostatic

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state, the EGF ligand is located on the apical surface and therefore separated from its

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receptor on the basolateral surface. Moreover, the apically located growth factor

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heregulin is separated from its receptor erbB2/3 (also known as HER2/3) which is

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located on the basolateral membrane. These growth factors therefore only have access

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to their receptors when the epithelium is damaged allowing for rapid targeted repair of

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the epithelial injury(200). The binding of heregulin to the erbB2/3 receptor leads to cell

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proliferation, differentiation, and mucous secretion(193, 201, 209) which allows the

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damaged epithelium to protect and repair itself. Therefore although the epithelium

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expresses both the growth factors and their receptors, it can regulate the interaction

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such that the effects occur only at times of injury, and one mechanism by which the

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epithelium does this is by regulating paracellular permeability.

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Dynamic regulation of paracellular permeability

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The intercellular junctions do not statically resist entry to all substances but act as

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dynamic structures that can open or close in response to both physiological and

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pathological stimuli(157, 166). By tightening cell-cell adhesion in response to

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mechanical stimuli such as shear stress or chemical environmental exposures such as

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inhaled particulate matter, the epithelia dynamically regulates receptor-ligand

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interactions (Fig. 1)(171). For example, airflow over the epithelium during breathing

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creates shear stress which can lead to changes in barrier properties. Low,

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physiological levels of shear stress are generated during normal tidal volume breathing

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but can increase by several fold during exercise, coughing, and bronchospasm.

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Exposure to shear stress leads to actin rearrangement and barrier enhancement(173).

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In addition, in response to both physiological levels of shear stress as well as pathologic

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stimuli like particulate matter- pollution formed from the mixture of solid particles and

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liquid droplets found in air- there is increased membrane localization of septin 2 that

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decreases paracellular permeability by altering cortical actin rearrangement(171).

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Septin 2 is a member of the family of GTP binding proteins that function in cytoskeletal

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arrangement and cell polarization. Membrane localization of septin 2 allows for

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increased interaction with actin which regulates the rearrangement of cortical actin and

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enhances barrier function. Physiological levels of shear stress also lead to a selective

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decrease in the abundance of the apical water channel aquaporin 5(172, 173), which

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helps to modulate the paracellular permeability in response to the stress. Aquaporin 5

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stabilizes the microtubules(172) and antagonizes cell to cell adhesion(173) thereby

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affecting paracellular permeability. Thus, the epithelium is able to dynamically regulate

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its response to experienced luminal stimuli.

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In summary, the airway epithelium is the first line of defense and prevents inspired

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substances from contacting the sub-epithelial tissue. The epithelium acts as both a

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physical and immunological barrier that continuously responds to both physiologic and

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pathologic stimuli in the environment and dynamically regulates its barrier in response

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to these stimuli. The apicoadherens junctions separate the apical and basolateral

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membranes and therefore membrane integrity can affect cell signaling and the defenses

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mounted in response to environmental exposures. By tightly coordinating epithelial

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responses to environmental exposures and altering epithelial signaling, secreted

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products, and ion and water flux in response to stimuli, the epithelium is able to provide

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a dynamic barrier at the interface between the lung parenchyma and the luminal

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environment. Noxious stimuli such as chronic tobacco exposure, infections, or

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particulate matter and air pollution, can lead to a breakdown in this regulatory response

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leading to chronic epithelial activation which can further propagate the damage resulting

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in a leaky barrier. In the following sections, we will discuss in more detail how the tight

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junction proteins, ion transport, and EGFR contribute to the dynamic epithelial barrier.

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Section II: The lung epithelium: insights in ion transport across the barrier

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Interactions between tight junctions and ion transporters to maintain lung fluid

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balance

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Control of airspace fluid volume and composition in the alveolus is primarily determined

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by transcellular ion transport, which requires a polarized epithelium with intact tight

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junctions. However, paracellular ion, macromolecule, and water transport through intact

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tight junctions also makes a significant contribution to lung fluid balance (115, 199).

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Furthermore, the effects of both bacterial and viral infections are quite complex, with

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data suggesting that a multitude of ion channels are altered in response to infection,

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and there is potential creation of new channels by a host of viruses, termed viroporins.

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The detailed discussions of these effects are beyond the scope of this review. During

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the resolution of pulmonary edema, clearance of fluid from the airspaces is largely

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dependent on a transepithelial sodium concentration gradient driven by Na+/K+-ATPase.

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Apical sodium conductance through ENaC is central to fluid clearance, but chloride and

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potassium channels also make important contributions (13, 37, 98). Apical-to-

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basolateral chloride transport may be particularly important because the maximal rate of

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sodium and water transport from the airspaces appears to be limited by the concomitant

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transport of chloride in many studies (9, 43, 122). Because experimental data suggest

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that a sizable fraction of transepithelial chloride transport occurs through the

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paracellular route in the alveolar epithelium (87), regulated paracellular ion transport

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could influence alveolar fluid clearance rates by this mechanism. In other words,

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changes in the selectivity and magnitude of paracellular ion conductance may influence

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rates of alveolar fluid clearance, either by limiting sodium leak or increasing chloride

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currents. The mechanisms for the regulation of paracellular transport in the lung and the

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potential interrelationship with transcellular transport is not fully understood, but recent

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work has begun to describe some of the major determinants of paracellular

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permselectivity in the alveolar epithelium. These studies have also provided some

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insight into deeper associations between the paracellular and transcellular routes of

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transport that is the regulation of transcellular transporters by cell junction proteins. One

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area of interest is the potential association between Na+/K+-ATPase and tight junction

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claudins.

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Sodium/Potassium ATPase as a regulator of cell junctions

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Sodium/potassium ATPase is made up of catalytic (α) and regulatory (β) subunits.

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Additional regulatory subunits of the FXYD family, such as the γ subunit of Na+/K+-

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ATPase, may also associate with α/β heterodimers and affect function. Pump activity in

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polarized epithelial cells is essential for lung fluid balance and increased pump activity is

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associated with increased rates of vectorial fluid clearance across the alveolar

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epithelium (4, 107). Recent work has uncovered an additional role for Na+/K+-ATPase in

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tight junction formation and maintenance in epithelia (194). For example, the β subunit

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of Na+/K+-ATPase associates with PDZ domain-containing proteins and localizes to tight

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junctions (109, 141). Although the β1 subunit has been reported to function as a cell

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adhesion molecule, enzymatic pump activity of Na+/K+-ATPase is required for tight

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junction formation in MDCK cells (144). The mechanism for this function appears to be

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through Na+/K+-ATPase-mediated activation of RhoA signaling and cytoskeletal

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remodeling at the periphery of epithelial cells. Na+/K+-ATPase may also be required for

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the maintenance of tight junctions. In monolayers of retinal pigment epithelial cells and

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in pancreatic ductal cell (HPAF-II) monolayers with established tight junctions, inhibition

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of Na+/K+-ATPase with ouabain increased paracellular permeability to ions and

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macromolecules (142, 143). However, the junction promoting effects of Na+/K+-ATPase

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may be context or cell-type specific because ouabain has also been reported to

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increase expression of claudins-4 and -1 in MDCK cells in association with decreased

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paracellular macromolecule permeability (97). Although the mechanisms for the

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regulation of tight junction formation and function by Na+/K+-ATPase are not fully

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understood, it appears that Na+/K+-ATPase can modulate tight junction assembly and

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function.

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Claudins as regulators of paracellular permeability

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Differential expression of the claudin family of tight junction proteins is a fundamental

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regulatory mechanism of paracellular permselectivity. Claudins are transmembrane

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proteins that are necessary and sufficient for tight junction strand formation (51). The

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spatially restricted expression of specific claudins along the course of the nephron

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exemplifies their permselectivity function (7). Moreover, point mutations in certain

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claudins result in clinically important changes in ion transport, and substantial

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experimental evidence supports a direct role for claudins in charge-selective

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paracellular ion conductance (6, 102, 195). Claudins on adjacent cells form paracellular

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pores with an approximately 3 Angstrom diameter that function as relatively high

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conductance, un-gated ion channels (101, 145, 197). Claudins also influence

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paracellular macromolecule transport and individual claudins appear to convey different

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properties to this charge-independent, lower conductance pathway (167, 168). A

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leading hypothesis is that individual claudin strands within a tight junction establish the

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ion pore pathway while the persistence or continuity of claudin strands within the

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membrane influences the macromolecule leak pathway (167). Because tight junctions

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consist of multiple layers of claudin-based strands that undergo continuous turnover,

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both conductance pathways can exist simultaneously. Three claudins, -3, -4, and -18.1,

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are abundantly expressed in the alveolar epithelium, although other claudins are

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expressed at low levels (95).

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Lung-specific claudin-18.1 is a requirement for the alveolar epithelial permeability

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barrier

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Claudin-18.1 is of particular interest because it is the only known lung-specific tight

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junction protein and it is the most abundantly expressed claudin in type 1 alveolar

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epithelial cells (95). The role of claudin-18 in alveolar epithelial barrier function has

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recently been examined in knockout mice. Claudin-18 does not appear to be required

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for prenatal lung development; however, upon conversion to air breathing,

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alveolarization is abnormal in claudin-18 knockout mice(96). Claudin-18 null mice have

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a 3-fold increase in alveolar epithelial permeability to macromolecules. There is a similar

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increase in macromolecule permeability in cultured primary alveolar epithelial cells from

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knockout mice and with siRNA knock down of claudin-18 in wild type cells(96). These

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data indicate that claudin-18 provides a macromolecule permeability barrier in the

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alveolar epithelium and suggest that the loss of this function results in abnormal alveolar

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homeostasis and impaired alveolarization. Interestingly, although macromolecule

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permeability is increased, claudin-18 null mice do not develop pulmonary edema(100).

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The reason for this may be that alveolar epithelial sodium and chloride transport is

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increased. The basal alveolar fluid clearance rate in claudin-18 null mice is

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approximately twice that of wild type littermates. Examination of the expression levels of

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ion transporters in whole lung revealed a 2-fold increase in the β1 subunit of Na+/K+-

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ATPase and increased pump activity, but no change or a slight decrease in the α, β

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and γ subunits of ENaC at the mRNA level. Because over-expression of Na+/K+-ATPase

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results in higher rates of alveolar fluid transport in animal models, the increase in β1

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subunit expression may account for most of the increase in alveolar fluid clearance in

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claudin-18 null mice.

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Chloride transport and alveolar fluid clearance in claudin-18 null mice

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As mentioned above, sodium transport in the lung can be limited by the co-transport of

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chloride, so increases in the levels of chloride transporters might be expected to

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facilitate higher rates of sodium and fluid transport. It is notable that CFTR mRNA

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expression is increased by approximately 4-fold in claudin-18 knockout mice, but

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whether CFTR inhibition decreases alveolar fluid clearance in this context has not been

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reported. In addition to CFTR, several other regulators of chloride transport show

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increased expression in the lungs of claudin-18 null mice, including NKCC1, CLC2, and

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SLC26A9. It is possible that these data point to a larger role for chloride transport in

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alveolar fluid clearance than has been previously appreciated. For example, chloride-

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dependent sodium transport into the airspaces appears to be an important mechanism

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for edema formation during hydrostatic stress (183). In the nephron, chloride-dependent

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sodium absorption is a mechanism for increased fluid retention and hypertension. This

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mechanism depends on the coupling of sodium transport to chloride transport via

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NKCC1. Although NKCC1 transport in a basolateral-to-apical direction is well

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established, transport in an apical-to-basolateral direction (or bi-directional transport) is

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possible (183). Therefore, the observed increase in chloride transporters in claudin-18

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null mice is consistent with compensation of fluid clearance facilitated by maximizing

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chloride-limited sodium transport. Further investigation is required, but increased

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NKCC1 expression in claudin-18 null mice might also support the hypothesis that

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apical-to-basolateral chloride-dependent sodium transport is a mechanism for alveolar

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fluid clearance. In summary, the loss of claudin-18 results in increased paracellular

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permeability in the absence of increased lung water. Higher rates of fluid transport in

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these mice may be due to increased expression of the beta subunit of Na+/K+-ATPase

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along with chloride transporters. The mechanisms for the up-regulation of ion

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transporters in the context of claudin-18 deficiency are not known, but one possibility is

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co-regulation of cell junction constituents and ion transporters.

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Functional implications of increased claudin-4 expression in claudin-18 null mice

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Claudin-4 mRNA and protein levels are increased by 4-fold in claudin-18 knockout mice.

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Claudin-4 plays a distinct role in trans-epithelial ion transport and junction formation and

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may partly compensate for the loss of claudin-18. Claudin-4 acts as a relative sodium

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barrier and is also specifically up-regulated during epithelial repair (72, 196, 207). In

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human and murine lungs, higher claudin-4 expression is associated with higher rates of

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alveolar fluid clearance(82, 152, 207), which is suggestive of a protective or reparative

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role for this claudin. Claudin-4 may also play a unique role in trans-epithelial ion

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transport. For example, in zebra fish, the claudin-4 ortholog, claudin-b, associates with

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Na+/K+-ATPase to effect decreased paracellular sodium transport in low ion

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concentration conditions (92, 93). In mouse studies, the loss of claudin-4 results in

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decreased alveolar fluid clearance and more severe pulmonary edema with injury(82,

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207). These data are consistent with the hypothesis that lung epithelial tight junctions

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containing high levels of claudin-4 support higher rates of alveolar fluid clearance. This

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may be because claudin-4 forms high resistance junctions that limit sodium

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conductance resulting in a larger trans-epithelial sodium gradient. Alternatively, claduin-

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4 may play a role in the regulation of Na+/K+-ATPase, but further studies are needed. In

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claudin-18 null mice, higher levels of claudin-4 may partly explain the observed increase

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in alveolar fluid clearance.

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Functional association between claudins and transcellular ion transport

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Taken together, available data suggest a functional association between tight junction

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claudins and transcellular ion transport. The hypothesis that claudin-4 and Na+/K+-

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ATPase have a cooperative role in trans-epithelial sodium transport in the lung requires

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further study, but it is clear that claudin-4 is necessary for normal barrier function and

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maximal rates of fluid clearance. It is also clear that claudin-18 forms a unique

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permeability barrier in the alveolar epithelium and is a requirement for alveolar

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homeostasis. The loss of claudin-18 results in a compensatory increase in claudin-4

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along with numerous changes in transcellular ion transporters in association with

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increased rates of alveolar fluid clearance. The adaptation to chronic barrier leak in

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claudin-18 null mice may provide novel insights into the mechanisms of alveolar ion and

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water transport regulation in diseases characterized by epithelial barrier dysfunction.

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Section III: The lung epithelium: The role of 2-pore domain potassium channels in

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lung epithelium

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Transcellular ion homeostasis in the lung epithelium

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Maintenance of the integrity and the homeostatic properties of the alveolar capillary

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barrier plays a vital role in pulmonary health and disease(111). By separating the air-

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and blood-filled spaces in the lung, the alveolar epithelium is strategically positioned to

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interact closely with both environments and to regulate the electrolyte and water content

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of the alveolar spaces. In this section we will briefly review (1) the major ion transport

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mechanisms regulating the composition of the alveolar lining fluid (ALF) under

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physiologic conditions, and (2) the role of stretch-activated ion channels in the

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development of hyperoxia (HO) - and mechanical stretch-induced Acute Lung Injury

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(ALI) and Acute Respiratory Distress Syndrome (ARDS), with particular emphasis on

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epithelial barrier function and inflammatory cytokine secretion.

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Maintaining tight control over the intra-alveolar environment requires complex

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synchronization of multiple epithelial ion transport processes, and a dynamic balance

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between continuous secretion and re-absorption of Na+ and Cl- determines the

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composition of the ALF(131). When an increase in alveolar fluid absorption is required,

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as in the immediate postnatal period or in conditions associated with pulmonary edema

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like pneumonia and congestive heart failure(136, 205), Na+ is internalized via apical

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amiloride-sensitive ENaC channels and cyclic nucleotide-gated cation channels (CNG)

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in both alveolar type I (AT I) and type II (AT II) cells(33, 36, 37, 45, 74). Internalized Na+

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is then secreted into the interstitial space via the basolateral Na/K ATPase(4, 31). To

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maintain intracellular electroneutrality, apical Cl- absorption occurs mainly via CFTR

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channels or paracellular pathways(36). In combination, these electrolyte shifts create an

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osmotic gradient for water molecules to move from the alveolar into the interstitial space

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either via aquaporin channels or by diffusion (1, 88, 177, 198).

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In contrast, when increased production of ALF is required, as part of physiologic ALF

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turnover and aging, to maintain surfactant homeostasis, or in response to infectious or

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environmental pollution stimuli(67, 81, 106, 120), Cl- anions are actively secreted into

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the alveolar space mainly via apical CFTR channels, ClC channels, and Ca-dependent

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Cl- channels (35, 50, 69, 108). An active driving force for Cl- secretion is maintained by

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basolateral Na/K/2Cl co-transporters, K-Cl co-transporters, HCO3/Cl exchangers(5, 91,

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151, 175, 190), as well as basolateral inwardly and outwardly rectifying Cl- channels(10,

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69). Na+ ions are thought to follow Cl- ions from the interstitial into the alveolar space

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but the molecular mechanisms underlying Na+ secretion are still not fully understood.

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Similarly, basolateral K+ channels play an important role in maintaining ALF by creating

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a favorable electrochemical gradient for Na+ absorption and Cl- secretion, by restoring

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intracellular electroneutrality, and by stabilizing the resting membrane potential(112). A

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variety of K+ channels have been described in both AT I and AT II cells, including

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voltage-gated (Kv), ATP-dependent (KATP), inwardly rectifying (Kir), Ca2+-activated (KCa)

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K+ channels(8, 57), and small (SK) and large conductance (BK) K+ channels(11, 113).

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In addition to homeostatic processes, K+ channels also appear to be involved in

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epithelial wound healing, cell migration, cell proliferation and cytokine secretion in in

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vitro and in vivo models of ALI/ARDS(8, 149, 161, 162, 192) and large conductance

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BKCa channels may act as oxygen sensors in the lung epithelium(80).

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Further complexity is added to this system when accounting for the intricate interactions

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between these ion channels and transporter proteins. For example, CFTR can

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downregulate ENaC expression(158); inhibition of KATP channels can inhibit ENaC

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currents(90); activation of KATP channels can increase ENaC and CFTR currents(98);

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and inhibition of KCa and Kv channels can inhibit ENaC and CFTR currents(17, 62).

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There are numerous limitations in the study of epithelial ion transport processes

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however, more recent approaches employing siRNA and shRNA knockdown of specific

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ion channels(163), and the broader availability of knockout (KO) mice deficient in

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specific ion channels (32, 124) have significantly facilitated research in this field.

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Nevertheless, conditional KO models for specific epithelial ion channels, where a

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particular ion channel is absent from AT I or AT II cells only, are not always readily

375

available.

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The role of ENaC channels in lung fluid balance

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The ENaC channels are composed of 4 subunits (α,β, γ, and δ) coded for by the

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SCNN1A, SCNN1B, SCNN1G, and theSCNN1D genes respectively. The α subunit is

379

required to form a functional ENaC channel and the β and γ subunits amplify the

380

channel’s activity. In mice, homozygous knockout of the α-ENaC subunit leads to

381

retention of fetal lung liquid, respiratory distress, and ultimately death (74). In humans,

382

mutations in the ENaC subunit genes SCNN1A, SCNN1B, and SCNN1G can lead to

383

autosomal recessive pseudohypoaldosteronism (PHA) characterized by life-long salt

384

wasting, hyperkalemia, and dehydration(25). Children who have PHA type 1 with

385

mutations in the genes encoding the α and β subunit have an increased volume of fluid

386

in the lungs due to problems with fluid absorption (84) leading to recurrent respiratory

387

problems. There are, however, case reports of preterm infants with a single

388

homozygous mutation in the α-ENaC subunit who did not have immediate post natal

389

difficulty with lung fluid clearance indicating that the α-ENaC may not be pivotal in the

390

clearance of lung fluid at early stages of lung maturation(75).

391

TGF-β has been shown to be important in the regulation of ENaC activity which has

392

important implications for lung fluid balance. Peters et al. demonstrated that TGF-β can

393

drive the abnormal internalization of the ENaC complex leading to a reduction in this

394

complex at the lung epithelial cell surface resulting in reduced sodium and fluid

395

absorption (135). This data suggests a unique TGF-β dependent mechanism to

396

regulate ion and fluid transport in the lung and potentially a new pathological

397

mechanism of ALI and ARDS.

398

The role of 2-pore domain potassium channels in ALI and ARDS

399

The role of stretch-activated ion channels (SACs) in the development of hyperoxia (HO)

400

and mechanical stretch-induced ALI/ARDS and the consequences of SAC dysregulation

401

on epithelial barrier function and inflammatory cytokine secretion is of particular

402

interest(181, 182). Both in vitro and in vivo studies have demonstrated that mechanical

403

forces can alter trans-epithelial ion transport, which results in a net decrease in alveolar

404

fluid clearance(50, 146, 204) and is directly associated with an increased length of

405

mechanical ventilation and hospital mortality in patients with ALI/ARDS(70, 181).

406

The term SACs refers to a variety of ion channels, including Na+ channels such as

407

ENaC, Cl- channels such as the ClC family, Ca2+ channels such as the TRPV (transient

408

receptor potential vanilloid) family, and numerous K+ channels including 2-pore domain

409

potassium (K2P) channels, KATP and Kir channels(23, 126). SAC channels are

410

ubiquitously expressed in body tissues including the heart, kidneys, genitourinary tract,

411

gastrointestinal tract, leukocytes and great blood vessels(179, 202, 203) and regulate a

412

variety of cellular functions including the resting membrane potential, cell volume, gene

413

expression, cell differentiation and cell contraction processes(16, 39, 154).

414

The K2P channel family consists of 15 members and is divided into 6 subfamilies:

415

TWIK, THIK, TREK, TASK, TALK, TRESK(156). A unique property of K2P channels is

416

that they can be constitutively open creating so-called “K+ leak currents” that maintain

417

the negative resting membrane potential of a cell (12, 127) and in tissues other than the

418

lung, K2P channels have been shown to act as mechano-sensors and mechano-

419

transducers(212). For this review we will focus specifically the role of TREK-1 in

420

ALI/ARDS.

421

Regulation and function of TREK-1 channels in models of ALI/ARDS

422

TREK-1 expression was first detected in mouse, rat and human AECs(163) but also

423

appears to be expressed in airway epithelial cells(213). Preliminary data shows TREK-

424

1 deficient cultured mouse AECs and human bronchial epithelial cells appear protected

425

from a HO- and TNF-α-induced loss of trans-epithelial electrical resistance suggesting a

426

role for TREK-1 in epithelial barrier (unpublished data). In addition, stimulation of

427

TREK-1 deficient mouse and human epithelial cells with TNF-α decreased IL-6 and

428

RANTES secretion and increased in MCP-1 secretion (Fig. 2). In contrast, KC/IL-8

429

secretion remained unchanged between TREK-1 deficient and control cells. While

430

several TNF-α-activated signaling pathways, including p38 kinase, PKC, and c-Jun N-

431

terminal kinases (JNK1/2/3) were altered in TREK-1 deficient AECs, decreased IL-6

432

secretion from TREK-1 deficient AECs was related to impaired phosphorylation of the

433

PKC isoform θ after TNF-α stimulation, whereas increased MCP-1 secretion appeared

434

unrelated to the increased phosphorylation of JNK1/2/3 in TREK-1 deficient AECs. The

435

specific molecular signaling mechanisms underlying these alterations in cytokine

436

secretion is currently being investigated.

437

Based on in vitro findings, it was anticipated that TREK-1 KO mice would be protected

438

from HO- and mechanical stretch-induced lung injury. However, exposure of TREK-1

439

KO mice to 24 hours HO resulted in increased lung injury as evidenced by gross lung

440

anatomy, H&E staining of lung sections (increased inflammatory cell infiltration and

441

hemorrhage), increased lung injury scoring, decreased lung compliance, and increased

442

macrophage and neutrophil accumulation in the BAL fluid of TREK-1 KO mice(164).

443

Interestingly, exposure of TREK-1 KO mice to HO plus mechanical ventilation resulted

444

in no further injury when compared to HO exposure alone(164).

445

Similar to in vitro studies(163), in vivo models show HO exposure is a weak stimulus for

446

cytokine secretion and only HO plus mechanical ventilation increased IL-6, MCP-1, and

447

TNF-α levels in the BAL fluid of both WT and TREK-1 KO mice. In accordance with in

448

vitro findings, there were decreased amounts of IL-6 in HO plus mechanically ventilated

449

TREK-1 KO mice(161). However, in contrast to in vitro findings(162), MCP-1 levels

450

were decreased in HO plus mechanically ventilated TREK-1 KO mice, whereas TNF-α

451

levels were unchanged between control and TREK-1 KO mice(164). Therefore, in this

452

model alterations in cytokine secretion between TREK-1 KO and WT mice were unlikely

453

the cause for the observed increase in lung injury in the TREK-1 KO mice.

454

Nevertheless, the contributions of cytokines such as IL-6, IL-1β and TNF-α to the

455

development of ALI/ARDS in other animal models and in human ARDS patients are well

456

documented(118, 119). The pro-inflammatory environment associated with the

457

secretion of these mediators promotes neutrophil-, macrophage-, T cell- and epithelium-

458

mediated alveolar injury, causes loss of alveolar barrier function, and decreases

459

alveolar surfactant levels(61, 94, 119, 185, 206). Similar to in vitro studies, the specific

460

molecular mechanisms underlying these alterations in BAL cytokine levels in vivo

461

models remain to be determined. In addition, it will be important to investigate the

462

cellular sources for these inflammatory mediators and quantify their relative

463

contributions. Furthermore, it is of major importance to determine the time course of

464

cytokine secretion in these ARDS models. It is quite possible that although after 24

465

hours HO exposure and 4 hours mechanical ventilation BAL IL-6 and MCP-1 levels

466

were decreased in TREK-1 deficient mice, these cytokines were increased at earlier

467

time points and contributed to the observed lung injury. Of note, while TNF-α is a known

468

pro-inflammatory cytokine in the early stages of ARDS, in chronic pulmonary

469

inflammation it may actually exert anti-inflammatory, beneficial effects and promote

470

physiologic wound healing(117).

471

Interestingly, alveolar barrier function appeared uncompromised in the mouse ARDS

472

model since BAL protein levels were unchanged between control and TREK-1 deficient

473

mice(164). HO alone caused only a minor disruption in alveolar barrier function whereas

474

the combination of HO and mechanical ventilation resulted in significant BAL protein

475

accumulation in both mouse types. The protective effect of TREK-1 deficiency observed

476

in vitro on TNF-α- and HO-induced loss of barrier function was not seen in the whole

477

animal model. Whether a close interaction between TREK-1 and epithelial tight junction

478

proteins, as seen in cultured alveolar epithelial cells (unpublished data), also exists in

479

vivo remains to be confirmed.

480

One explanation for the increased lung damage observed in HO exposed TREK-1 KO

481

mice could be an increase in AT I and AT II cell apoptosis in these animals as

482

evidenced by TUNEL staining and corroborated by increased PARP-1 cleavage, one of

483

the distal signaling molecules of the caspase cascade(164). Another possible

484

explanation could be a decrease in surfactant protein-C detected in TREK-1 deficient

485

mice after prolonged HO exposure, potentially making lungs more vulnerable to HO-

486

and mechanical stretch-induced injury(164). The molecular mechanisms linking TREK-1

487

deficiency to epithelial apoptosis and decreased surfactant production are currently

488

unknown. As described above, we have observed substantial alterations in p38, PKC

489

and JNK phosphorylation in TREK-1 deficient epithelial cells after TNF-α

490

stimulation(161, 162), but whether these kinases are involved in apoptosis or surfactant

491

production needs to be determined. Another question that needs to be addressed in

492

future studies is whether K+ flux through TREK-1 channels is required for the observed

493

in vitro and in vivo effects, or whether TREK-1 could exert K+-independent functions and

494

act as a regulatory molecule rather than an ion channel, as reported for example for

495

CFTR(159).

496

In conclusion, while in vitro epithelial TREK-1 deficiency appeared protective against the

497

inflammatory stimuli responsible for the development of ALI/ARDS, in an in vivo model

498

of ALI/ARDS TREK-1 deficiency resulted in increased lung damage. The mechanisms

499

accounting for these differences are currently unknown. Conditional TREK-1 deficient

500

mice should give us insights into the molecular mechanisms responsible for the

501

observed lung damage and help us resolve the discrepancies between in vitro and in

502

vivo models. Once TREK-1 signaling pathways are delineated, the development of

503

TREK-1 activating or inhibiting agents may steer us towards new therapeutic

504

approaches in ALI/ARDS. As with all known ion channel modulators, drug specificity

505

issues will likely constitute future challenges, especially since K2P channels are quite

506

ubiquitously expressed in the body tissues.

507

Section IV: The lung epithelium: NRG-1-HER2/3 Signaling in Epithelial Barrier

508

regulation

509

HER Family Signaling: Background

510

Receptor tyrosine kinases, such as members of the Human Epidermal Growth Factor

511

Receptor (HER) family, and their ligands serve as important signaling molecules that

512

regulate the epithelial response to extracellular signals and environmental stimuli. HER

513

family signaling has been shown to regulate epithelial migration, growth and

514

differentiation as well as epithelial barrier function in numerous tissue beds including

515

skin (184), intestine (38), cornea (123, 211, 215), and the lung (30). Identification of

516

mutations of HER family genes as pathogenic in certain epithelial cancers (85, 180) has

517

led to the successful deployment of targeted tyrosine kinase inhibitors (TKIs) aimed at

518

inhibiting these receptors, particularly in breast and lung cancer (24, 105). However,

519

recent data demonstrate that these receptors regulate pulmonary epithelial barrier

520

function in non-transformed pulmonary epithelia, in particularly in the context of

521

inflammatory lung injury (46, 47).

522

The HER family is a complex signaling network consisting of 4 transmembrane

523

receptors, HER1 or the epidermal growth factor receptor (EGFR), HER2, HER3 and

524

HER4 and 10 known ligands. The HER family is also referred to as the ErbB family after

525

it was discovered that the avian erytheroblastosis virus gene encoded a mutant form of

526

EGFR/HER1 (64, 65, 138). Members of the HER family are expressed on the

527

basolateral surface of airway and alveolar type I and II epithelial cells. The 4 HER

528

receptors all share a similar structure including an extracellular ligand binding domain, a

529

single transmembrane domain, and a cytoplasmic tyrosine kinase domain (209). HER

530

receptors exist in quiescent cells as single “closed” monomers. Ligand binding induces

531

a conformational change to an “open” configuration allowing for homo or hetero-

532

dimerization leading to phosphorylation of intracellular tyrosine residues and activation

533

of the kinase domain. This allows for docking of cytoplasmic proteins that facilitate

534

initiation of downstream signaling. Certain structural distinctions influence how specific

535

monomers interact to form signaling competent dimer pairs. HER2 has no ligand yet,

536

under basal conditions, exists in a receptive “open” confirmation making it the preferred

537

binding partner of other HER receptors. In contrast, HER3 has at least 4 different

538

ligands yet does not have a functioning kinase and partners with other receptors to

539

influence signaling. Though it does not directly phosphorylate other proteins, HER3

540

interacts with other intra-cellular proteins and signaling pathways such as the PI3 kinase

541

pathway (103). Additionally, in cancer cells, HER3-depedent signaling is a resistance

542

mechanism against HER2-targeted TKI therapy (52). Broadly, ligands are organized

543

depending on which receptors they bind and activate. Epidermal growth factor (EGF),

544

transforming growth factor alpha (TGF-α) and amphiregulin bind EGFR/HER1

545

exclusively while betacellulin, heparin binding EGF (HB-EGF) and epiregulin can bind

546

EGFR/HER1 or HER4. The final group of ligands includes the neuregulins (NRG-1-4).

547

The 4 NRG proteins all share a similar EGF like domain which is sufficient for receptor

548

activation yet differ in tissue distribution structurally which can influence signaling, NRG-

549

1 being the isoform most commonly studied.

550

HER family signaling is a complex, multilayered network influenced by factors such as

551

dimerization partner, cell type (e.g. cancer vs. untransformed) and ligand. (148). For

552

example, activated EGFR phosphorylates αCbl and Grb2 but only when activated in

553

partnership with HER2 as opposed to other HER family members (58, 129).

554

Additionally, while several ligands can bind and activate different HER receptors, each

555

ligand elicits specific intra-cellular responses depending cell type (e.g. transformed vs.

556

non-transformed) and the availability of other HER receptors. In HER2 deficient cells,

557

NRG-1 can induce EGFR/HER3 dimerization and activation but when HER2 is

558

available, NRG-1 preferentially induces HER2/3 heterodimers eliciting specific

559

intracellular signaling patterns (46, 58).

560

Accumulating data suggests that the HER family is important in regulating epithelial

561

barrier function in the lung. Activation of EGFR with TGF-α leads to epithelial

562

proliferation, spreading and motility (99, 153) (86) and enhances wound closure in

563

cultured alveolar epithelial cells (86). Additionally, epithelial injury leads to shedding of

564

HER family ligands and wound closure is delayed after exposure to an EGFR inhibitor.

565

Similarly, NRG-1 shedding and HER2 activation is also observed in a scratch model of

566

wound closure and blockade of HER2 impedes healing following wounding (200).

567

Notably, exogenous NRG-1 increases epithelial permeability similar in a monolayer of

568

epithelial cells (46). These data suggest that HER activation can enhance, improve, or

569

worsen epithelial barrier function, possibly depending on the continuity of the barrier at

570

the time of receptor activation.

571

NRG-1-HER2/3: Regulation of the Pulmonary Epithelial Barrier

572

Recent evidence has identified the NRG-1-HER2/3 system as activated in and a critical

573

modulator of pulmonary epithelial barrier function in acute lung injury (ALI) and the

574

Acute Respiratory Distress Syndrome (ARDS). ALI and ARDS are syndromes marked

575

by a hyper-inflammatory state with an increase in cytokines such as IL-6 and IL-8 that

576

are felt to participate in disease pathogenesis. In particular, the cytokine IL-1β has been

577

implicated as a central mediator of ALI, both in animal models and humans(53, 71, 114,

578

132, 133, 140, 174). IL-1β is increased in the lavage of ARDS patients and exogenous

579

IL-1β leads to increased epithelial permeability in an epithelial monolayer as well as in

580

animal models (89). Therapeutically, strategies to block IL-1β signaling is protective in

581

bleomycin and ventilator-induced models of lung injury (53, 132) (49). Epithelial NRG-1-

582

HER2 signaling has been recently identified as activated by IL-1β and central in the

583

pathogenesis of epithelial injury and permeability induced by IL-1β. In cultured airway

584

and alveolar cells, IL-1β-mediated increases in epithelial permeability are HER2

585

dependent (46). IL-1β induces shedding of NRG-1 and HER2 activation without

586

evidence of EGFR activation. The protease ADAM17 has been identified as the

587

sheddase responsible for NRG-1 shedding. Pharmacologic (inhibitors) or genetic

588

(siRNA) strategies to inhibit ADAM17, NRG-1 or HER2 significantly attenuate IL-1β-

589

mediated changes in epithelial barrier function. Hence, an overall pathway of IL-1β-

590

ADAM17-NRG-1-HER2/3 leading to increased epithelial permeability has emerged as a

591

new important regulator of epithelial barrier function in ALI.

592

NRG-1-HER2/3 signaling also participates in animal models of lung injury. Bleomycin

593

induced ALI is associated with both increased broncho-alveolar lavage NRG-1

594

indicative of surface shedding as well as pulmonary HER2 activation. Pharmacologic

595

ADAM17 inhibition attenuates NRG-1 cleavage, pulmonary HER2 phosphorylation and

596

indices of injury in mice exposed to bleomycin. Bleomycin serves as a model of both

597

acute and chronic, fibrotic lung injury and targeting HER2 is protective in the fibrotic

598

phase as well as the acute phase of lung injury following bleomycin. Use of an inhibitor

599

that prevents HER2 dimerization with HER3 prevented both HER2/3 activation as well

600

as fibrosis at 21 days after bleomycin exposure (44). Similarly, transgenic mice lacking

601

pulmonary epithelial HER3 are protected from bleomycin-induced fibrosis (125). Though

602

in these studies indices of acute injury were not specifically studied, there was

603

suggestion of protection in alveolar leak early following bleomycin. Mechanistically,

604

alterations in alveolar epithelial permeability have been linked to a pro-fibrotic response

605

in the lung (2, 3, 15, 178) possibly providing a link between early acute injury marked by

606

compromised alveolar epithelial barrier function and delayed fibrotic injury.

607

Proteases are critical participants in the HER family signaling as they translate external

608

signals into HER receptor activation through shedding of surface ligands. Numerous

609

agonists which induce epithelia barrier dysfunction do so via protease-mediated HER

610

ligand shedding leading to receptor activation. Examples of this include IL-1β,

611

mechanical stretch, and hypertonic stress. In particular, ADAM17 has been identified as

612

the sheddase responsible for cleaving numerous HER family ligands and has been

613

linked to disease marked by inflammation and increased permeability (21, 27, 29, 55,

614

211). In vitro, blocking ADAM17 reduces IL-1β-induced epithelial permeability through

615

blocking NRG-1 shedding and subsequent HER2 activation. In vivo, intra-tracheal

616

instillation of the ADAM17 inhibitor attenuated bleomycin induced ALI including protein

617

leak. This was associated with decreased NRG-1 lavage levels and HER2 activation.

618

While ADAM17 KO mice display embryonic lethality, mice deficient in the endogenous

619

inhibitor of ADAM17- Tissue Inhibitor of Metalloprotease (TIMP)-3- have delayed

620

resolution of lung injury following bleomycin administration supporting the importance of

621

this sheddase in ALI, though the role of HER ligands and receptors was not specifically

622

investigated.

623

Perhaps the most compelling data indicating that the HER family participates in

624

epithelial barrier disruption in the lung is that patients with ARDS have evidence of

625

active ligand shedding in the lung. TGF-α is elevated in lavage from patients suggesting

626

pathway activation. Similarly, increased NRG-1 lavage levels have been detected from

627

patients with ARDS. As a biomarker, NRG-1 levels were significantly correlated with

628

inflammation as measured by inflammatory cytokines. Notably, NRG-1 elevations were

629

associated with worse outcome as measured by ventilator free days. Plasma NRG-1 is

630

also increased in ARDS patients suggesting that the source of NRG-1 leading to HER2

631

activation could be extra-pulmonary.

632 633

NRG-1-HER2/3 Signaling- Mechanisms Regulating Epithelial Cell-Cell adhesion

634

and Permeability.

635

Knowledge regarding the mechanisms by which HER2 and the other HER receptors

636

influence epithelial permeability is incomplete but several pathways have been outlined.

637

HER proteins modulate cell-cell adhesion proteins of the tight junction and the adherens

638

junction. EGFR alters claudin expression and spatial re-arrangement of claudins and

639

ZO-1 (28, 189). HER2 has also been linked to the tight junction proteins ZO-1 and

640

occludin (54, 56) as well as integrin β4 in regulating cell-cell adhesion (66). Recent data

641

suggest that the adherens junction protein β-catenin is a required intermediary

642

influencing HER2 mediated barrier function in the lung. Phosphorylation at tyrosine Y-

643

654 of β-catenin has been linked to altered barrier function. NRG-1-mediated HER2

644

activation leads to β-catenin Y-654 phosphorylation as well as disruption of β-catenin–

645

E-cadherin interaction at the cell junction in pulmonary epithelial cells (48). This is

646

associated with impaired E-cadherin-mediated cell-cell adhesion. Finally loss of β-

647

catenin via shRNA knock down results in a significant attenuation in NRG-1- HER2/3

648

induced barrier dysfunction. Together, this data suggest that NRG-1-HER3-HER2

649

activation leads to β-catenin tyrosine phosphorylation, disruption of adherens junction,

650

and alteration of the pulmonary epithelial barrier (Fig. 3).

651

Additionally, processes inherent in barrier function such as proliferation and cell

652

migration suggest other pathways by which HER proteins might influence the epithelial

653

barrier. The GTPases Rho and Rac have both been linked to HER family signaling. In a

654

breast cancer epithelial cell line, HER2-dependent Rac activation was required for

655

NRG-1 mediated cell migration (208). Rac activation following NRG-1 is likely via

656

HER2-dependent P-Rex1 activation as down-regulation of P-Rex1 inhibited Rac

657

activation and cell migration following NRG-1 (121). Similarly, EGFR activation leads to

658

Rac activation and Rac-dependent cell migration (34). During epithelial wound closure,

659

Rho activation follows EGFR activation and, in a corneal epithelial wound model, Rho

660

inhibition decreased both cell migration and proliferation resulting in delayed wound

661

closure (210).

662

Proteins involved in formation of tight junctions and adherens junctions are also

663

mobilized after HER family activation. EGFR activation alters expression levels of

664

claudins and the cellular localization of claudins and ZO-1 (28, 189). NRG-1-HER2

665

mediated changes in epithelial permeability are also associated with alterations in

666

cellular localization of the tight junction proteins ZO-1 and occludin (54, 56). Integrin β4,

667

an adhesion molecule which regulates cell-cell junctions (66) and epithelial proliferation

668

(26), amplifies HER2 activation in epithelial cells to activate STAT and disrupt cell-cell-

669

adhesion (60). Previous reports have suggested a structural and functional link between

670

the adherens junction protein β-catenin and HER2. Structurally, HER2 co-localizes to

671

the basolateral plasma membrane via binding to the PDZ domain of erbin (20). Erbin

672

has been identified as a binding partner for the p120 catenin protein p0071 as well as

673

direct binding partner for β-catenin (59, 147). In gastric and breast cancer epithelial

674

cells, TGF-α induces EGFR dependent HER2 activation, β-catenin phosphorylation and

675

β-catenin-HER2 association, though the role of HER2 in β-catenin phosphorylation was

676

not specifically studied (83, 128, 160, 170). Functionally, HER2 and β-catenin have both

677

been linked to breast cancer pathogenesis. In murine breast cancer models utilizing a

678

constitutively active HER2, β-catenin signaling was activated and required for tumor

679

initiation and metastasis (155). Blockade of HER2 using trastuzumab blocked both

680

HER2 activation and β-catenin phosphorylation as well as β-catenin dependent

681

transcription of surviving breast cancer cells (214).

682 683

Conclusion

684

The airway epithelium is a physical and immunological barrier that sits at the interface

685

between the lumen and the lung parenchyma and is the initial site of contact for all

686

inspired substances. It is constantly exposed to both physiological and pathological

687

stimuli and dynamically regulates its barrier. This dynamic regulation occurs via

688

changes in cell-cell adhesion due to altered cytoskeletal and junctional protein

689

arrangement and expression, as well as changes in transcellular and paracellular ion

690

permeability in response to these stimuli to help maintain homeostasis. The ion

691

channels on the apical and basolateral cell surfaces and the junctional complexes,

692

which directly connect adjacent epithelial cells, play a key role in controlling the

693

movement of substances from the lumen into the sub-epithelial space both via

694

paracellular and transcellular transport. The junctions also separate the apical and

695

basolateral cell surface which maintains the specialized function of each cell surface,

696

controls the interaction between various cytokines, growth factors, and their receptors,

697

and modulates the extent and location of inflammatory response to stimuli. Exposure to

698

noxious stimuli such as mechanical stretch/trauma, hyperoxia, infection, cigarette

699

smoke, or particulate matter, damages the epithelial barrier resulting in a loss of

700

compartmentalization and a hyperproliferative, inflammatory state that is seen in many

701

pulmonary diseases.

702

The junction proteins and ion channels interact to maintain lung fluid balance. Initial

703

studies indicate that changes in the levels of the tight junction claudins can alter fluid

704

balance. However, further work needs to be done to characterize the compensatory

705

shifts in junction proteins that occur in response to physiological and pathological stimuli

706

in order to maintain tissue homeostasis.

707

As outlined earlier, stretch activated ion channels play a role in epithelial barrier

708

function. TREK-1 has been linked to the pathogenesis of ALI/ARDS however there are

709

conflicting results in in vitro and in vivo models of ALI/ARDS. Future studies delineating

710

the role of TREK-1 in ALI/ARDS could potentially lead to the development of new

711

targeted therapies for this disease. The ENaC complex is another ion channel shown to

712

be important in lung fluid balance but better defining the role of the individual subunits in

713

fluid balance as well as identifying other pathways that contribute to the trafficking of

714

this complex will be important in further understanding the pathogenesis of diseases

715

such as ARDS.

716

Although it is hypothesized that changes in the physical barrier in response to chronic

717

stimulation by both physiological and pathological stimuli precipitate the immunological

718

changes that lead to diseases such as COPD, ALI, and ARDs, the sequence and time

719

course of these changes is yet to be determined. As discussed in this review, the

720

epithelium dynamically regulates its response to a variety of stimuli to promote repair

721

and regeneration however; we do not understand mechanisms of dysregulation of this

722

dynamic repair process. Future studies should further explore the link between altered

723

transcellular and paracellular transport and the activation of the inflammatory cascades

724

that result in chronic epithelial barrier dysfunction and injury. In addition, much attention

725

has been devoted to pathways that mediate lung inflammation and injury however,

726

pathways that protect against lung injury are not well studied and therefore few have

727

been detailed in the literature. It is critical to note that Islam et al. demonstrated that

728

both epithelial injury and the downstream consequences of disease can be reversed by

729

targeting the epithelium, which was done using mitochondrial transfer (77). However,

730

this raises a future directive to not only identify pathways involved in damage, but to

731

also investigate protective mechanisms. Recently, the apelin-APJ pathway has been

732

shown to be protective against ARDS/ALI but more work needs to be done to identify

733

other pathways that protect against lung inflammation and injury(42). Our future

734

directive is not to simply define and dissect disrupted molecular pathways in the

735

epithelium, but target and manipulate protective mechanisms to alter the course of

736

disease. In conditions such as COPD, where there is a long lead time prior to the onset

737

of disease, there is ample opportunity to prevent the development by manipulating

738

epithelial targets. In conditions such as ARDS, we need to fine tune mechanisms to

739

target the epithelium to ameliorate the severity of disease and amplify repair and

740

resolution.

741

As discussed earlier in this review, the maintenance of ion and fluid homeostasis in the

742

lung is complex and requires the dynamic interaction between numerous ion channels

743

and transporter proteins. Studies have started to explore the connections between

744

paracellular and transcellular transport in maintaining fluid balance and ion

745

homeostasis. However, our knowledge regarding this subject is still in its infancy. We

746

have already identified certain transporters, such as Aquaporin 5, which have roles in

747

dictating both transcellular water transport and paracellular permeability as outlined in

748

Section I. Further defining the contribution of these transporters to the pathogenesis of

749

different pulmonary diseases could potentially lead to the development of novel

750

therapeutics. Indeed, Ivacaftor, a drug approved to treat patients with cystic fibrosis

751

who have specific mutations in the CFTR protein, is one such example, which could

752

have reaches into non-cystic fibrosis lung diseases as well.

753

In summary, the airway epithelium is a dynamic barrier that constantly responds to

754

luminal stimuli and coordinates its response to maintain homeostasis in the lung.

755

Breakdown in this coordinated response can lead to a myriad of lung diseases.

756

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Fig.1. The EGFR is restricted to the basal surface of the airway epithelial cells. This figure serves to highlight how the integrity of the apicoadherens junction can regulate epithelial cell signaling. Under conditions of low paracellular permeability created by the cytoskeleton and cell-cell contacts, there is segregation of EGF ligand and receptor, thereby inhibiting constant cellular signaling. In conditions of increased paracellular permeability, EGF can bind its receptor and activate its pathway where phosphorylated ERK serves as readout of EGFR activation. Thus, the epithelial barrier can modulate epithelial signaling pathways. Fig. 2: We propose that hyperoxia, mechanical stretch and inflammatory cytokines alter the regulation of K2P channel function. This can then impact on cytokine secretion and epithelial barrier function and this dysregulation of these proteins can contribute to the pathogenesis of ALI through downstream. Fig. 3. Cytokine activation can lead to increased free NRG-1. Initial disruption of the epithelial barrier allows for NRG-1-HER3-HER2 activation. The activation of this complex can leads to β-catenin tyrosine phosphorylation, which further propogates disruption of adherens junction, and alteration of the pulmonary epithelial barrier.

Fig. 1

Fig. 2

IL-1β NRG-1 Free NRG-1

Pulmonary Epithelial Cell IL-1R

Occludin ADAM17

ZO-1

Claudin

JAM/CAR HER3

HER2 Epithelial Barrier Disruption

P P

PP P Actin β-catenin

E-cadherin

EGFR

Fig. 3