Cystic Fibrosis (CF) is a life-limiting systemic inherited illness with a tremendous financial burden. Relentlessly progressive pulmonary disease is still the primary cause of the morbidity and mortality in CF. The cystic fibrosis transmembrane regulator (CFTR) is a cAMP-regulated chloride channel that is highly expressed in fetal airway epithelia. However, CFTR function is not required until after birth when it maintains periciliary fluid balance and sustains effective mucociliary clearance. After birth, and in the absence of CFTR-mediated chloride secretion, the epithelial sodium channel, ENaC, is unregulated and drives excessive sodium and fluid absorption. This dehydrates the airways and increases the viscosity of airways secretions, thereby impairing bacterial clearance. We have proposed that alternative chloride channels can be exploited to bypass the CFTR defect. We will test the hypothesis that over-expression and activation of the ClC-2 channel in the postnatal period will rescue the CF murine lung by restoring chloride secretion and down-regulation of ENaC. Our long term goal is to discover the technology and determine the mechanism that will allow compensation of the mutant CFTR with an endogenous gene product, ClC-2.
Aim 1 : To determine the sequence of protein interactions by which ClC2 is targeted to the plasma membrane. The hypothesis is that HSP90 complexes regulate apical plasma membrane expression of ClC-2. We demonstrate that HSP90 interacts with ClC-2 and we have developed the technology to identify ClC-2 and its binding partners on 2D gels followed by MALDI-TOF. The goal is to discover the factors that maximize surface ClC-2 expression and function.
Aim 2 : To determine whether ClC-2 and ENaC interact through Cl- current to modulate ion transport in the CF mouse lung. The hypothesis is that chloride conductance through ClC-2 will inhibit ENaC function. We predict that in CF mice, sustained activation of ClC-2 will decrease resting potential difference and the amiloride inhibited fraction of PD.
Aim 3 : To determine whether ClC-2 function can regulate the inflammatory response to infection or LPS challenge. The hypothesis is that stimulating ClC-2 expression and ClC-2 mediated chloride transport will restore normal levels of inflammatory responses in CF mice. These studies will produce the following novel contributions: 1) demonstration that an alternative chloride channel can bypass the CFTR defect in an animal model of CF: 2) dissemination and sharing of a novel epithelial-specific, doxycycline-regulated TET-On human ClC-2 mouse model;3) assessment of two novel ClC-2 activators, from Sucampo Pharmaceuticals, for CF lung disease;and 4) techniques to induce and assess CFTR-mediated airways inflammation in CF mice.

Public Health Relevance

We propose to test the hypothesis that over-expression and activation of pH- and voltage activated ClC-2 channels will rescue the cystic fibrosis mouse. Critical protein:protein interactions will be interrogated during trafficking of ClC-2 to apical membranes of airway epithelial cells. The product of this work will be a method of stimulating chloride transport to compensate for mutant CFTR, reduce sodium absorption and relieve airway inflammation in vivo.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
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Special Emphasis Panel (ZRG1-RES-C (02))
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Banks-Schlegel, Susan P
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Johns Hopkins University
Schools of Medicine
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Tran, Ian; Ji, Changhoon; Ni, Inzer et al. (2015) Role of Cigarette Smoke-Induced Aggresome Formation in Chronic Obstructive Pulmonary Disease-Emphysema Pathogenesis. Am J Respir Cell Mol Biol 53:159-73
Lee, Seakwoo; Henderson, Mark J; Schiffhauer, Eric et al. (2014) Interference with ubiquitination in CFTR modifies stability of core glycosylated and cell surface pools. Mol Cell Biol 34:2554-65
Schiffhauer, Eric S; Vij, Neeraj; Kovbasnjuk, Olga et al. (2013) Dual activation of CFTR and CLCN2 by lubiprostone in murine nasal epithelia. Am J Physiol Lung Cell Mol Physiol 304:L324-31
Henderson, Mark J; Singh, Om V; Zeitlin, Pamela L (2010) Applications of proteomic technologies for understanding the premature proteolysis of CFTR. Expert Rev Proteomics 7:473-86
Henderson, Mark J; Vij, Neeraj; Zeitlin, Pamela L (2010) Ubiquitin C-terminal hydrolase-L1 protects cystic fibrosis transmembrane conductance regulator from early stages of proteasomal degradation. J Biol Chem 285:11314-25
Vij, Neeraj; Mazur, Steven; Zeitlin, Pamela L (2009) CFTR is a negative regulator of NFkappaB mediated innate immune response. PLoS One 4:e4664
MacDonald, Kelvin D; McKenzie, Karen R; Henderson, Mark J et al. (2008) Lubiprostone activates non-CFTR-dependent respiratory epithelial chloride secretion in cystic fibrosis mice. Am J Physiol Lung Cell Mol Physiol 295:L933-40
Vij, Neeraj; Amoako, Martha O; Mazur, Steven et al. (2008) CHOP transcription factor mediates IL-8 signaling in cystic fibrosis bronchial epithelial cells. Am J Respir Cell Mol Biol 38:176-84
Zeitlin, Pamela L (2008) Cystic fibrosis and estrogens: a perfect storm. J Clin Invest 118:3841-4
Singh, Om V; Pollard, Harvey B; Zeitlin, Pamela L (2008) Chemical rescue of deltaF508-CFTR mimics genetic repair in cystic fibrosis bronchial epithelial cells. Mol Cell Proteomics 7:1099-110

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