Specialty Areas: Airway Epithelial Cell Biology and Airway Surface Liquid Homeostasis in CF and COPD
It has recently been shown that a critical component of airways innate defense is the thin (7 µm) liquid layer lining airway surfaces, the periciliary liquid (PCL), that provides a low viscosity solution for ciliary beating and acts a lubricant layer for mucus transport. Normal airways appear to be able to sense the PCL volume and adjust ion channel activity accordingly by unknown mechanisms. A proposed model for ASL volume regulation is shown in Figure 1.
Apical membrane ion channel activity controls the amount of salt (and water) on airway surfaces and hence, PCL volume and mucus hydration levels. It has recently been proposed that the initiating event in CF lung disease is depletion of the PCL due to abnormal ion channel activity (i.e. a lack of CFTR), which causes dehydrated mucus to adhere to airway surfaces, preventing it from being cleared (Fig. 2), causing increased bacterial infections.
The long term goal of this laboratory is to understand how homeostasis of PCL volume occurs in airway epithelia under normal and pathophysiological conditions. Currently, research in the Tarran lab is focused on three main areas, listed below, and we utilize cell biological and biochemical techniques coupled with in vivo translational approaches to address these questions:
- Regulation of epithelial cell function by the extracellular environment: We have hypothesized that nucleotides, proteases and other molecules contained in the ASL (ATP for example) can regulate airway ion transport. Fig. 3 depicts cystic fibrosis airway cells that have lost the ability to regulate ASL volume following infection with GFP-labeled viruses that inhibit intracellular Ca2+ signaling by depleting extracellular ATP.
- Gender differences in cystic fibrosis lung disease: Females suffer more severely with cystic fibrosis than their male counterparts, resulting in a significantly shorter lifespan. This gender difference becomes apparent after puberty. Accordingly, we are currently investigating whether estrogens affect Ca2+ signaling and ASL homeostasis. Figure 4 shows an example of simultaneous Fura-2 imaging (as a marker of intracellular Ca2+) and a fusion protein of Estrogen Receptor α (ER α) conjugated to orange fluorescent protein (mOr).
- The effects of cigarette smoke on epithelial airway ion transport: Similar to CF, long term tobacco exposure also results in chronic mucus accumulation. We have found that acute tobacco exposure causes a loss of CFTR from the plasma membrane (Fig. 5) and are currently investigating this phenomenon
- Ca2+ imaging
- Confocal microscopy
- Electrophysiology (In vivo nasal potential difference measurements, microelectrodes & Ussing chambers)
- Fluorescence resonance energy transfer (FRET)
- Mass Spectrometry
- Molecular Biology & Real Time (q)PCR
- Tissue culture
- Western Blot
- Scott DW, Walker MP, Sesma J, Wu B, Stuhlmiller TJ, Sabater JR, Abraham WM, Crowder TM, Christensen DJ, Tarran R. SPX-101 is a novel epithelial sodium channel-targeted therapeutic for cystic fibrosis that restores mucus transport. Am J Respir Crit Care Med. 2017 Sep 15;196(6):734-744. doi: 10.1164/rccm.201612-2445OC. PMID: 28481660.
- Wu T, Huang J, Moore PJ, Little MS, Walton WG, Fellner RC, Alexis NE, Peter Di Y, Redinbo MR, Tilley SL, Tarran R. Identification of BPIFA1/SPLUNC1 as an epithelium-derived smooth muscle relaxing factor. Nat Commun. 2017 Feb 6;8:14118. doi: 10.1038/ncomms14118. PMID: 28165446; PMCID: PMC5303822.
- Rasmussen JE, Sheridan JT, Polk W, Davies CM, Tarran R. Cigarette smoke-induced Ca2+ release leads to cystic fibrosis transmembrane conductance regulator (CFTR) dysfunction. J Biol Chem. 2014 Mar 14;289(11):7671-81. doi: 10.1074/jbc.M113.545137. Epub 2014 Jan 21. PMID: 24448802; PMCID: PMC3953278.
- Garland AL, Walton WG, Coakley RD, Tan CD, Gilmore RC, Hobbs CA, Tripathy A, Clunes LA, Bencharit S, Stutts MJ, Betts L, Redinbo MR, Tarran R. Molecular basis for pH-dependent mucosal dehydration in cystic fibrosis airways. Proc Natl Acad Sci U S A. 2013 Oct 1;110(40):15973-8. doi: 10.1073/pnas.1311999110. Epub 2013 Sep 16. PMID: 24043776; PMCID: PMC3791714.
- Clunes LA, Davies CM, Coakley RD, Aleksandrov AA, Henderson AG, Zeman KL, Worthington EN, Gentzsch M, Kreda SM, Cholon D, Bennett WD, Riordan JR, Boucher RC, Tarran R. Cigarette smoke exposure induces CFTR internalization and insolubility, leading to airway surface liquid dehydration. FASEB J. 2012 Feb;26(2):533-45. doi: 10.1096/fj.11-192377. Epub 2011 Oct 11. PMID: 21990373; PMCID: PMC3290447.
- Garcia-Caballero A, Rasmussen JE, Gaillard E, Watson MJ, Olsen JC, Donaldson SH, Stutts MJ, Tarran R. SPLUNC1 regulates airway surface liquid volume by protecting ENaC from proteolytic cleavage. Proc Natl Acad Sci U S A. 2009 Jul 7;106(27):11412-7. doi: 10.1073/pnas.0903609106. Epub 2009 Jun 18. PMID: 19541605; PMCID: PMC2708735. Erratum in: Proc Natl Acad Sci U S A. 2009 Sep 1;106(35):15091.
- Coakley RD, Sun H, Clunes LA, Rasmussen JE, Stackhouse JR, Okada SF, Fricks I, Young SL, Tarran R. 17beta-Estradiol inhibits Ca2+-dependent homeostasis of airway surface liquid volume in human cystic fibrosis airway epithelia. J Clin Invest. 2008 Dec;118(12):4025-35. doi: 10.1172/JCI33893. Epub 2008 Nov 20. PMID: 19033671; PMCID: PMC2582929.