With this proposal, we will test the overall hypothesis that regulation of cAMP levels in microdomains of signaling, rather than on the global scale, determines the function of the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) in health and disease. The CFTR is a cAMP-stimulated anion channel that is critical for ion- and water homeostasis across many epithelia including in the airways and the gastrointestinal tract. CFTR hypofunction, as a result of mutations in the CFTR gene in Cystic Fibrosis (CF), or smoke exposure in Chronic Obstructive Pulmonary Disease (COPD), results in reduced levels of this channel in the apical membrane of epithelia which causes abnormal fluid and electrolyte transport. It is well established that CFTR function is stimulated by PKA phosphorylation. However, stimulating CFTR-dependent ion transport in CF model systems by increasing global cellular cAMP to supra-physiological levels with a combination of adenylyl cyclase activators and broad-spectrum PDE inhibitors has been met with mixed results and successes in the past. With this research proposal, we wish to test two novel approaches to overcome this limitation.
Specific Aim 1 of this proposal will test the hypothesis that specific isoforms of cyclic nucleotide phosphodiesterases (PDEs), the enzymes that degrade and inactivate cAMP, are physically tethered to signaling complexes involving the CFTR and control the activity of this channel in compartmentalized microdomains of cAMP signaling. We wish to test the idea that inactivation of this localized pool of PDE activity or its displacement from CFTR signaling complexes provides a safer and more effective approach to stimulate CFTR function compared to increasing global cAMP levels, as the latter approach induces significant cellular feedback responses and undesirable side effects that ultimately limit its efficacy. The human genome encodes for 21 PDE genes, which are likely expressed as more than 100 protein variants. Inhibitors of type 4 PDEs (PDE4s) have been shown to stimulate CFTR in immortalized epithelial cell lines. We have now generated preliminary data showing that PDE4 isoforms are the major regulators of CFTR activity in primary lung epithelial cells and are physically tethered to this channel suggesting that these are the PDEs that control cAMP in CFTR microdomains.
In Specific Aim 1 of this proposal, we will characterize the interaction of CFTR with individual PDE4 isoforms and test the effect of selective inactivation of these PDE4s on CFTR function in non-CF and CF cells and tissues.
In Specific Aim 2 of this proposal, we will generate novel FRET-based biosensors that can measure cAMP levels and PKA activity in the immediate vicinity of the CFTR. This will allow us to trace the pools of cAMP that determine CFTR function, identify the key regulators in this compartment, and explore possible differences in cAMP signaling events between CF and non-CF epithelia.

Public Health Relevance

Abnormal expression and function of the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) is central to several diseases with high public health relevance including cystic fibrosis (CF). The identification of specific cyclic nucleotide phosphodiesterase 4 (PDE4) isoforms as regulators of CFTR activity will open new opportunities to target PDE4s as a therapeutic approach in CF. This proposal aims to validate the idea that targeting microdomains of cAMP signaling, rather than global cAMP levels, is an effective therapeutic approach, which will advance the development of more selective and, thus, safer drugs.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Exploratory/Developmental Grants (R21)
Project #
1R21HL107960-01A1
Application #
8243487
Study Section
Lung Cellular, Molecular, and Immunobiology Study Section (LCMI)
Program Officer
Banks-Schlegel, Susan P
Project Start
2012-03-01
Project End
2014-02-28
Budget Start
2012-03-01
Budget End
2013-02-28
Support Year
1
Fiscal Year
2012
Total Cost
$193,125
Indirect Cost
$68,125
Name
University of California San Francisco
Department
Obstetrics & Gynecology
Type
Schools of Medicine
DUNS #
094878337
City
San Francisco
State
CA
Country
United States
Zip Code
94143
Blanchard, Elise; Zlock, Lorna; Lao, Anna et al. (2014) Anchored PDE4 regulates chloride conductance in wild-type and ýýF508-CFTR human airway epithelia. FASEB J 28:791-801
Haj Slimane, Zeineb; Bedioune, Ibrahim; Lechêne, Patrick et al. (2014) Control of cytoplasmic and nuclear protein kinase A by phosphodiesterases and phosphatases in cardiac myocytes. Cardiovasc Res 102:97-106
Mika, Delphine; Richter, Wito; Westenbroek, Ruth E et al. (2014) PDE4B mediates local feedback regulation of ??-adrenergic cAMP signaling in a sarcolemmal compartment of cardiac myocytes. J Cell Sci 127:1033-42
Cazabat, Laure; Ragazzon, Bruno; Varin, Audrey et al. (2014) Inactivation of the Carney complex gene 1 (PRKAR1A) alters spatiotemporal regulation of cAMP and cAMP-dependent protein kinase: a study using genetically encoded FRET-based reporters. Hum Mol Genet 23:1163-74
Xie, Moses; Blackman, Brigitte; Scheitrum, Colleen et al. (2014) The upstream conserved regions (UCRs) mediate homo- and hetero-oligomerization of type 4 cyclic nucleotide phosphodiesterases (PDE4s). Biochem J 459:539-50
Jung, Eun Suk; Park, Joonhee; Gee, Heon Yung et al. (2014) Shank2 mutant mice display a hypersecretory response to cholera toxin. J Physiol 592:1809-21
Conti, Marco; Mika, Delphine; Richter, Wito (2014) Cyclic AMP compartments and signaling specificity: role of cyclic nucleotide phosphodiesterases. J Gen Physiol 143:29-38
Richter, Wito; Menniti, Frank S; Zhang, Han-Ting et al. (2013) PDE4 as a target for cognition enhancement. Expert Opin Ther Targets 17:1011-27
Richter, Wito; Mika, Delphine; Blanchard, Elise et al. (2013) *1-adrenergic receptor antagonists signal via PDE4 translocation. EMBO Rep 14:276-83