Sphingosine-1-Phosphate Is a Novel Regulator of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Activity

RESEARCH ARTICLE Sphingosine-1-Phosphate Is a Novel Regulator of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Activity Firhan A. Malik1☯, Anja Meissner1,2☯, Illya Semenkov1, Steven Molinski3,4, Stan Pasyk3,4, Saumel Ahmadi1,4, Hai H. Bui5, Christine E. Bear1,3,4, Darcy Lidington1,6‡, SteffenSebastian Bolz1,6,7‡* 1 Department of Physiology, University of Toronto, Medical Science Building, 1 King’s College Circle, Toronto, M5S 1A8 Canada, 2 Department of Brain Ischemia and Neurodegeneration, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Rosello 161, 6th floor, 08036 Barcelona, Spain, 3 Department of Biochemistry, University of Toronto, Medical Science Building, 1 King’s College Circle, Toronto, M5S 1A8 Canada, 4 Programme in Molecular Structure and Function in the Research Institute, The Hospital for Sick Children, 555 University Avenue, Toronto, MG5 1X8 Canada, 5 Lilly Research Laboratories, Indianapolis, Indiana 46285, United States of America, 6 Toronto Centre for Microvascular Medicine, University of Toronto and The Li Ka Shing Knowledge Institute at St. Michael’s Hospital, 209 Victoria Street, Toronto, M5B 1T8 Canada, 7 Heart & Stroke / Richard Lewar Centre of Excellence for Cardiovascular Research, University of Toronto, 50 College Street, Toronto, M5S 3E2 Canada OPEN ACCESS Citation: Malik FA, Meissner A, Semenkov I, Molinski S, Pasyk S, Ahmadi S, et al. (2015) Sphingosine-1Phosphate Is a Novel Regulator of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Activity. PLoS ONE 10(6): e0130313. doi:10.1371/ journal.pone.0130313 Academic Editor: Michael B. Butterworth, University of Pittsburgh, School of Medicine, UNITED STATES Received: August 13, 2014 Accepted: May 18, 2015 Published: June 16, 2015 Copyright: © 2015 Malik et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: Operating Funding: Heart and Stroke Foundation of Ontario Grant in Aid G13-0002610 (Operating Grant to Steffen-Sebastian Bolz). University of Toronto Research Funding (Operations Funding provided to Steffen-Sebastian Bolz). Stipend Support: Heart and Stroke Foundation of Ontario Career Investigator Award CI-7432 (Stipend Support to Steffen-Sebastian Bolz). This work is also supported by HSFO New Investigator (SSB, NIA6581) and the Natural Sciences and Engineering ☯ These authors contributed equally to this work. ‡ These senior authors contributed equally to this work. * Abstract The cystic fibrosis transmembrane conductance regulator (CFTR) attenuates sphingosine1-phosphate (S1P) signaling in resistance arteries and has emerged as a prominent regulator of myogenic vasoconstriction. This investigation demonstrates that S1P inhibits CFTR activity via adenosine monophosphate-activated kinase (AMPK), establishing a potential feedback link. In Baby Hamster Kidney (BHK) cells expressing wild-type human CFTR, S1P (1μmol/L) attenuates forskolin-stimulated, CFTR-dependent iodide efflux. S1P’s inhibitory effect is rapid (within 30 seconds), transient and correlates with CFTR serine residue 737 (S737) phosphorylation. Both S1P receptor antagonism (4μmol/L VPC 23019) and AMPK inhibition (80μmol/L Compound C or AMPK siRNA) attenuate S1P-stimluated (i) AMPK phosphorylation, (ii) CFTR S737 phosphorylation and (iii) CFTR activity inhibition. In BHK cells expressing the ΔF508 CFTR mutant (CFTRΔF508), the most common mutation causing cystic fibrosis, both S1P receptor antagonism and AMPK inhibition enhance CFTR activity, without instigating discernable correction. In summary, we demonstrate that S1P/ AMPK signaling transiently attenuates CFTR activity. Since our previous work positions CFTR as a negative S1P signaling regulator, this signaling link may positively reinforce S1P signals. This discovery has clinical ramifications for the treatment of disease states associated with enhanced S1P signaling and/or deficient CFTR activity (e.g. cystic fibrosis, heart failure). S1P receptor/AMPK inhibition could synergistically enhance the efficacy of therapeutic strategies aiming to correct aberrant CFTR trafficking. PLOS ONE | DOI:10.1371/journal.pone.0130313 June 16, 2015 1 / 15 S1P Regulates CFTR via AMPK Research Council of Canada PGS-D Doctoral Scholarship (Stipend Support to Firhan A Malik). The funders had no role in study design, data collection and analysis, decision to publish or the preparation of the publication. Eli Lilly and Company provided support in the form of salaries for authors [HBH], but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section. Competing Interests: Dr. Hai H. Bui is employed by Eli Lilly and Company (Lilly Research Laboratories, Indianapolis, IN). This affiliation does not alter the authors' adherence to PLOS ONE policies on sharing data and materials. All other authors confirm that they have no competing interests to declare. Introduction Sphingosine-1-phosphate (S1P) is a key endogenous regulator of resistance artery myogenic vasoconstriction [1–3]. Pressure elevation stimulates sphingosine kinase 1 (Sphk1) and hence, S1P production in microvascular smooth muscle cells (VSMCs) [2], which subsequently activates an array of pro-constrictive signaling cascades [4]. Specifically, S1P signaling concurrently activates myosin light chain kinase and inhibits myosin light chain phosphatase, thereby driving substantial myosin light chain phosphorylation and consequently, potent vasoconstriction. To finely tune S1P signaling, a robust degradation mechanism counterbalances endogenous S1P production. This mechanism depends on two key elements: first, the cystic fibrosis transmembrane conductance regulator (CFTR) transports extracellular S1P across the plasma membrane, thereby sequestering it from its receptors; the internalized S1P is then degraded by the intracellular S1P phosphohydrolase 1 (SPP1) [5,6]. Our previous work in resistance arteries has characterized Sphk1 and CFTR/SPP1 as the principal counteracting signaling elements within a signaling framework that precisely controls S1P bioavailability and consequently, its pro-constrictive actions. In principle, inversely regulating this signaling tandem (i.e., decreasing S1P degradation when production increases and vice versa) would enable more efficient control of S1P signal onset, amplitude and duration. However, while we have defined a rapid mechanism that enhances S1P synthesis in response to transmural pressure elevation [2], a similarly rapid mechanism that depresses S1P degradation (i.e., regulates CFTR/SPP1) h (...truncated)