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

The cystic fibrosis transmembrane conductance regulator (CFTR) attenuates sphingosine-1-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

S1P mediates its effects via the S1P₁ receptor subtype.

<p>In BHK cells stably expressing CFTR^wt, the chemical S1P_<b>1</b> / S1P_<b>3</b> receptor antagonist VPC23019 (VPC; 4μmol/L, 30min pre-treatment) attenuates S1P (1μmol/L)-stimulated (A) AMPK phosphorylation (n = 8) and abolishes (B) CFTR S737 phosphorylation (n = 9) and (C) iodide efflux inhibition (n = 6). The S1P_<b>1</b> receptor-specific agonist SEW-2871 (SEW; 1μmol/L) increases both (D) AMPK phosphorylation (n = 6), (E) CFTR phosphorylation (n = 5). (F) SEW co-stimulation attenuates iodide efflux to a similar extent as S1P (n = 6 for control and SEW; n = 3 for S1P). * denotes P<0.05 relative to the control; In <i>Panels A-C</i>, + denotes P<0.05 for VPC+S1P, relative to S1P. All iodide efflux data was collected from cell monolayers.</p

S1P attenuates CFTR-dependent iodide efflux via phosphorylation of serine 737.

<p>(A) Shown is a representative western blot demonstrating that 1μmol/L FITC-S1P significantly increases AMPK phosphorylation (control n = 4; FITC-S1P n = 6). The extent of AMPK phosphorylation is qualitatively similar to that elicited by unlabeled S1P (n = 2). (B) Shown is a representative tracing of iodide efflux in BHK cells stably transfected with wild-type CFTR (CFTR^wt). Forskolin (FSK; 20μmol/L) stimulates robust CFTR-dependent iodide efflux; more iodide is released in the vehicle control (4% BSA), compared to cells treated with 1μmol/L S1P. Permeablization with 0.1% Triton X-100 (TX-100) confirms that iodide loading was similar. (C) S1P (1μmol/L) attenuates iodide efflux in temperature-rescued (27°C for 24h) BHK cells stably expressing the ΔF508 CFTR mutant (CFTR^ΔF508; n = 6). (D) S1P maximally inhibits iodide efflux at a 200μmol/L concentration in BHK cells stably expressing CFTR^wt (n = 7). (E) Western blots utilizing an antibody that exclusively binds non-phosphorylated CFTR serine residue 737 (S737) indicate that CFTR^wt is rapidly (within 30 seconds) phosphorylated in response to 1μmol/L S1P (n = 6). The response is transient in nature, with phosphorylation significantly reduced after 5 minutes (n = 6) and fully reversed after 60 minutes (n = 5). FSK (20μmol/L; 5 minutes) also stimulates robust CFTR S737 phosphorylation. (F) S1P fails to inhibit iodide efflux in naïve BHK cells transiently transfected with a plasmid encoding a mutated CFTR containing a serine-to-alanine substitution at amino acid 737 (CFTR^S737A). * denotes P<0.05 relative to the control; in <i>Panel E</i>, + denotes a significant difference between both the control and 0.5 minute S1P treatment groups. <i>Panels B</i> and <i>D</i> display iodide efflux data collected from cell suspensions (i.e., “real time” measurements); <i>Panels C</i> and <i>F</i> display iodide efflux data collected from cell monolayers.</p

S1P/AMPK signaling functionally inhibits CFTR^ΔF508 activity.

<p>(A) AMPK phosphorylation is higher in mesenteric arteries isolated from mice carrying the ΔF508 CFTR mutation, relative to wild-type littermates (n = 4). (B) Targeted, siRNA-mediated AMPK inhibition (n = 6) augments iodide efflux in BHK cells expressing the ΔF508 CFTR mutant (CFTR^ΔF508), relative to the scrambled siRNA control (n = 6). (C) Both S1P_<b>1</b> / S1P_<b>3</b> receptor antagonism (4μmol/L VPC23019 for 24 hours; VPC; con n = 11, VPC n = 7) and pharmacological AMPK inhibition (1μmol/L Compound C for 24 hours; CC; n = 6) improve iodide efflux in BHK cells expressing CFTR^ΔF508. Western blots (inset above) show no change in CFTR^ΔF508 electrophoretic mobility following VPC and CC treatment (n = 3); the corrector VX-809 (3μmol/L for 24 hours; “+ve”; n = 3) acts as a positive control. (D) AMPK inhibition (20μmol/L Compound C) increases FITC-S1P uptake in BHK cells expressing CFTR^ΔF508 (con n = 10, CC n = 5); western blots (inset above) show no change in CFTR^ΔF508 electrophoretic mobility following treatment (n = 3). * denotes P<0.05 relative to the control. All iodide efflux data was collected from cell monolayers.</p

S1P inhibits CFTR by an AMPK-dependent mechanism.

<p>In BHK cells stably expressing CFTR^wt, the chemical AMPK inhibitor compound C (CC; 80μmol/L, 30min pre-treatment) abolishes S1P (1μmol/L)-stimulated (A) AMPK phosphorylation (n = 6), (B) CFTR S737 phosphorylation (n = 6) and (C) attenuates iodide efflux (n = 5–6). (D) Transfecting siRNA targeting the AMPK α1 subunit effectively reduced AMPK α1 protein expression in BHK cells stably expressing CFTR^wt; control siRNA had no effect on AMPK α1 expression (n = 12). (E) As expected, S1P stimulated CFTR S737 phosphorylation (n = 5–6) and attenuated FSK-stimulated iodide efflux (n = 6) in BHK cells expressing CFTR^wt. (F) In contrast, S1P had no effect on CFTR S737 phosphorylation or iodide efflux in BHK cells expressing CFTR^wt following treatment with AMPK-targeting siRNA (n = 5–6). * denotes P<0.05 relative to the control. <i>Panel C</i> displays iodide efflux data collected from cell suspensions (i.e., “real time” measurements); all other iodide efflux data were collected from cell monolayers.</p

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

The cystic fibrosis transmembrane conductance regulator (CFTR) attenuates sphingosine- 1-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