Samuel A. Molina

Mix and match: Investigating heteromeric and heterotypic gap junction channels in model systems and native tissues

This review is based in part on a roundtable discussion session: “Physiological roles for heterotypic/heteromeric channels” at the 2013 International Gap Junction Conference (IGJC 2013) in Charleston, South Carolina. It is well recognized that multiple connexins can specifically co‐assemble to form mixed gap junction channels with unique properties as a means to regulate intercellular communication. Compatibility determinants for both heteromeric and heterotypic gap junction channel formation have been identified and associated with specific connexin amino acid motifs. Hetero‐oligomerization is also a regulated process; differences in connexin quality control and monomer stability are likely to play integral roles to control interactions between compatible connexins. Gap junctions in oligodendrocyte:astrocyte communication and in the cardiovascular system have emerged as key systems where heterotypic and heteromeric channels have unique physiologic roles. There are several methodologies to study heteromeric and heterotypic channels that are best applied to either heterologous expression systems, native tissues or both. There remains a need to use and develop different experimental approaches in order to understand the prevalence and roles for mixed gap junction channels in human physiology.

Claudins: Gatekeepers of lung epithelial function.

The lung must maintain a proper barrier between airspaces and fluid filled tissues in order to maintain lung fluid balance. Central to maintaining lung fluid balance are epithelial cells which create a barrier to water and solutes. The barrier function of these cells is mainly provided by tight junction proteins known as claudins. Epithelial barrier function varies depending on the different needs within the segments of the respiratory tree. In the lower airways, fluid is required to maintain mucociliary clearance, whereas in the terminal alveolar airspaces a thin layer of surfactant enriched fluid lowers surface tension to prevent airspace collapse and is critical for gas exchange. As the epithelial cells within the segments of the respiratory tree differ, the composition of claudins found in these epithelial cells is also different. Among these differences is claudin-18 which is uniquely expressed by the alveolar epithelial cells. Other claudins, notably claudin-4 and claudin-7, are more ubiquitously expressed throughout the respiratory epithelium. Claudin-5 is expressed by both pulmonary epithelial and endothelial cells. Based on in vitro and in vivo model systems and histologic analysis of lungs from human patients, roles for specific claudins in maintaining barrier function and protecting the lung from the effects of acute injury and disease are being identified. One surprising finding is that claudin-18 and claudin-4 control lung cell phenotype and inflammation beyond simply maintaining a selective paracellular permeability barrier. This suggests claudins have more nuanced roles for the control of airway and alveolar physiology in the healthy and diseased lung.

Regulation of claudin/zonula occludens-1 complexes by hetero-claudin interactions

Claudins are tetraspan transmembrane tight-junction proteins that regulate epithelial barriers. In the distal airspaces of the lung, alveolar epithelial tight junctions are crucial to regulate airspace fluid. Chronic alcohol abuse weakens alveolar tight junctions, priming the lung for acute respiratory distress syndrome, a frequently lethal condition caused by airspace flooding. Here we demonstrate that in response to alcohol, increased claudin-5 paradoxically accompanies an increase in paracellular leak and rearrangement of alveolar tight junctions. Claudin-5 is necessary and sufficient to diminish alveolar epithelial barrier function by impairing the ability of claudin-18 to interact with a scaffold protein, zonula occludens 1 (ZO-1), demonstrating that one claudin affects the ability of another claudin to interact with the tight-junction scaffold. Critically, a claudin-5 peptide mimetic reverses the deleterious effects of alcohol on alveolar barrier function. Thus, claudin controlled claudin-scaffold protein interactions are a novel target to regulate tight-junction permeability.

Insulin signaling via the PI3-kinase/Akt pathway regulates airway glucose uptake and barrier function in a CFTR-dependent manner

Cystic fibrosis-related diabetes is the most common comorbidity associated with cystic fibrosis (CF) and correlates with increased rates of lung function decline. Because glucose is a nutrient present in the airways of patients with bacterial airway infections and because insulin controls glucose metabolism, the effect of insulin on CF airway epithelia was investigated to determine the role of insulin receptors and glucose transport in regulating glucose availability in the airway. The response to insulin by human airway epithelial cells was characterized by quantitative PCR, immunoblot, immunofluorescence, and glucose uptake assays. Phosphatidylinositol 3-kinase/protein kinase B (Akt) signaling and cystic fibrosis transmembrane conductance regulator (CFTR) activity were analyzed by pharmacological and immunoblot assays. We found that normal human primary airway epithelial cells expressed glucose transporter 4 and that application of insulin stimulated cytochalasin B-inhibitable glucose uptake, consistent with a requirement for glucose transporter translocation. Application of insulin to normal primary human airway epithelial cells promoted airway barrier function as demonstrated by increased transepithelial electrical resistance and decreased paracellular flux of small molecules. This provides the first demonstration that airway cells express insulin-regulated glucose transporters that act in concert with tight junctions to form an airway glucose barrier. However, insulin failed to increase glucose uptake or decrease paracellular flux of small molecules in human airway epithelia expressing F508del-CFTR. Insulin stimulation of Akt1 and Akt2 signaling in CF airway cells was diminished compared with that observed in airway cells expressing wild-type CFTR. These results indicate that the airway glucose barrier is regulated by insulin and is dysfunctional in CF.

Calibrated flux measurements reveal a nanostructure-stimulated transcytotic pathway

Abstract Transport of therapeutic agents across epithelial barriers is an important element in drug delivery. Transepithelial flux is widely used as a measure of transit across an epithelium, however it is most typically employed as a relative as opposed to absolute measure of molecular movement. Here, we have used the calcium switch approach to measure the maximum rate of paracellular flux through unencumbered intercellular junctions as a method to calibrate the flux rates for a series of tracers ranging in 0.6–900 kDa in size across barriers composed of human colon epithelial (Caco‐2) cells. We then examined the effects of nanostructured films (NSFs) on transepithelial transport. Two different NSF patterns were used, Defined Nanostructure (DN) 2 imprinted on polypropylene (PP) and DN3 imprinted on polyether ether ketone (PEEK). NSFs made direct contact with cells and decreased their barrier function, as measured by transepithelial resistance (TER), however cell viability was not affected. When NSF‐induced transepithelial transport of Fab fragment (55 kDa) and IgG (160 kDa) was measured, it was unexpectedly found to be significantly greater than the maximum paracellular rate as predicted using cells cultured in low calcium. These data suggested that NSFs stimulate an active transport pathway, most likely transcytosis, in addition to increasing paracellular flux. Transport of IgG via transcytosis was confirmed by immunofluorescence confocal microscopy, since NSFs induced a significant level of IgG endocytosis by Caco‐2 cells. Thus, NSF‐induced IgG flux was attributable to both transcytosis and the paracellular route. These data provide the first demonstration that transcytosis can be stimulated by NSFs and that this was concurrent with increased paracellular permeability. Moreover, NSFs with distinct architecture paired with specific substrates have the potential to provide an effective means to regulate transepithelial transport in order to optimize drug delivery. Graphical abstract Figure. No caption available. HighlightsContact of epithelial cells with nanostructured surfaces enhances transepithelial flux of soluble tracers.The calcium switch method enables maximum paracellular flux rates across an epithelial monolayer to be measured.Two distinct nanostructures simultaneously stimulate both transcytosis and paracellular diffusion of IgG.

Use of Super-resolution Immunofluorescence Microscopy to Analyze Tight Junction Protein Interactions in situ

Sites of intercellular contact are regulated by structures known as junctions. Among the several classes of intercellular junctions are tight junctions that enable epithelia to form barriers by controlling the permeability of soluble molecules, water and ions across extracellular contacts between cells [1]. Tight junctions are composed of a complex of transmembrane proteins linked to the actin cytoskeleton by cytoplasmic scaffold proteins. Paracellular permeability is directly controlled by transmembrane proteins called claudins [2]. There are about two dozen different human claudin genes; tissue-specific expression of claudins confers distinct permeability to different epithelia. Most claudins directly bind to the scaffold protein zonula occludens-1 (ZO-1) that, in turn, binds to cortical actin which promotes tight junction formation [3]. This is just one of the many protein-protein interactions that are required to form fully functional junctions.

Alcohol and the Alveolar Epithelium

The distal airways are covered with a heterogeneous layer of cells known as the alveolar epithelium. Alveolar epithelial cells provide the major barrier between the airspace and fluid filled tissue compartments. As such, regulation of the alveolar epithelium is critical to maintain a healthy lung and for optimal gas exchange. In this chapter, we discuss functional roles for alveolar epithelial cells with particular emphasis on intercellular junctions and communication. As a thin layer of cells directly exposed to atmospheric oxygen, alveoli are particularly sensitive to oxidant insults. Alcohol significantly diminishes the normal antioxidant reserves of the alveolar epithelium, thereby rendering it sensitized for an exaggerated damage response to acute and chronic injuries. The effects of alcohol on alveolar epithelia are discussed along with open questions and potential therapeutic targets to prevent the pathophysiology of alcoholic lung disease.

Endocannabinoid receptor CB2R is significantly expressed in aspirin-exacerbated respiratory disease: a pilot study: Significant CB2R expression in AERD

The endocannabinoid system represents a highly conserved, innate signaling network with direct and indirect control of eicosanoid‐mediated inflammation. Activation of the type 2 cannabinoid receptor (CB2R) leads to decreased type 2 inflammation and reduced production of arachidonic acid (AA). Given that altered AA metabolism is associated with aspirin‐exacerbated respiratory disease (AERD), we hypothesized that expression of the CB2R gene CNR2 is increased in AERD.

Cystic Fibrosis: An Overview of the Past, Present, and the Future

Cystic fibrosis (CF) is the most common lethal genetic disorder amongst Caucasians with a current life expectancy of about 40 years. A dysfunctional CF transmembrane conductance regulator chloride channel is the single primary cause of this complex systemic disease that negatively affects every secretory organ in the body. In this chapter, we present our current understanding of CF through model systems that shed light on the pathophysiological consequences of the disease. In particular, we discuss its epidemiological and clinical impact with a focus on the identified and exploratory defects within airway epithelial cells. Finally, we discuss therapeutic approaches as they relate to the underlying disease pathophysiology.