Detrimental ELAVL-1/HuR-dependent GSK3β mRNA stabilization impairs resolution in acute respiratory distress syndrome

<div><p>A hallmark of acute respiratory distress syndrome (ARDS) is accumulation of protein-rich edema in the distal airspaces and its removal is critical for patient survival. Previous studies have shown a detrimental role of Glycogen Synthase Kinase (GSK) 3β during ARDS via inhibition of alveolar epithelial protein transport. We hypothesized that post-transcriptional regulation of GSK3β could play a functional role in ARDS resolution. To address this hypothesis, we performed an <i>in silico</i> analysis to identify regulatory genes whose expression correlation to GSK3β messenger RNA utilizing two lung cancer cell line array datasets. Among potential regulatory partners of GSK3β, these studies identified the RNA-binding protein ELAVL-1/HuR (Embryonic Lethal, Abnormal Vision, Drosophila-Like) as a central component in a likely GSK3β signaling network. ELAVL-1/HuR is a RNA-binding protein that selectively binds to AU-rich elements of mRNA and enhances its stability thereby increasing target gene expression. Subsequent studies with siRNA suppression of ELAVL-1/HuR demonstrated deceased GSK3β mRNA and protein expression and improved clearance of FITC-albumin in A549 cells. Conversely, stabilization of ELAVL-1/HuR with the proteasome inhibitor MG-132 resulted in induction of GSK3β at mRNA and protein level and attenuated FITC-albumin clearance. Utilizing ventilator-induced lung injury or intra-tracheal installation of hydrochloric acid to induce ARDS in mice, we observed increased mRNA and protein expression of ELAVL-1/HuR and GSK3β. Together, our findings indicate a previously unknown interaction between GSK3β and ELAV-1 during ARDS, and suggest the inhibition of the ELAV-1- GSK3β pathways as a novel ARDS treatment approach.</p></div

Suppression of ELAVL-1/HUR leads to down regulation of GSK3β <i>in vitro</i> and subsequently increased of epithelial albumin clearance.

<p><b>(</b>A) ELAVL-1/HUR protein expression and suppression in nuclear cell lysate in cells treated with ELAVL-1/HUR siRNA. (B) mRNA and protein expression of GSK3β in cells transfected with siELLAVL-1. Binding (C) and uptake (D) of FITC labelled albumin All experiments were conducted in A549 cells, n 3–4; data represent the mean ± SEM * p< 0.05, ***p<0.001, ****p<0.0001.</p

Summary- acute lung injury induces ELAVL-1/HuR transcription, which augments GSK3β mRNA mediated impairment of alveolar protein clearance.

<p>Summary- acute lung injury induces ELAVL-1/HuR transcription, which augments GSK3β mRNA mediated impairment of alveolar protein clearance.</p

Stabilization of ELAVL-1/HUR up regulates GSK3β in vitro and attenuates epithelial albumin clearance.

<p>Cells were incubated with 20 μM of the lysosomal inhibitor MG-132 at indicated time points to stabilize ELAV-1. (A) ELAVL-1/HUR protein expression was determined in the nuclear cell lysate via western blot. (B) mRNA and protein expression of GSK3β in cells treated with MG-132. Binding (C) and uptake (D) of FITC labelled albumin All experiments were conducted in A549 cells, n = 4; data represent the mean ± SEM * p< 0.05, **p<0.01, ***p<0.001, n.s. = non significant.</p

<i>In silico</i> identification of ELAVL-1/HUR as a potential regulator of GSK3β.

<p>(A) Gene expression data sets from the Cancer Cell Line Encyclopedia (CCLE) and UT Southwestern Medical Center (UTSW) were analyzed independently to find genes correlated to GSK3β expression. We identified 120 common transcripts with correlation coefficient |r|> 0.55 in both lung cell data sets (B) Gene network analysis based on previously known direct (solid lines) and indirect (dashed lines) biological connections for the identified transcripts with a positive (red) and negative correlation (green) r-value >0.55) to GSK3β expression in two, independent lung cancer cell line datasets.</p

A1R is involved in adenosine-induced VVEC barrier function. Effect of A1R siRNA on CCPA-induced increase in TER in VVEC.

<p>(<b>A, B</b>) VVEC were incubated with A1R specific siRNA or non-specific siRNA for 48 h and then cells were stimulated with CCPA (1 nM) in TER measurement assay. The depletion of A1R mRNA and protein was confirmed by RT-PCR (<b>C</b>) and the Western blot analysis with anti-A1R antibody. (<b>D</b>). Results are presented as mean ± SE from three independent experiments.</p

PI3K/Akt pathway mediates adenosine-induced increase in TER in VVEC.

<p>VVEC-Co (<b>A</b>) and VVEC-Hyp (<b>B</b>) were pre-incubated with LY294002 (5 µM; PI3K inhibitor) or GSK690693 (10 nM; Akt inhibitor) for 30 min and then exposed to adenosine. Barrier function was measured by TER assay. Results were obtained from three independent experiments and are presented as mean ± SE. * p<0.05.</p

Effects of adenosine receptor agonists on the VVEC barrier function.

<p>Activation of A1R improves VVEC barrier function. VVE-Co (<b>A</b>) and VVEC-Hyp (<b>B</b>) were stimulated with various agonists of adenosine receptors (CCPA, 1 nM; CGS21680, 30 nM; BAY 60-5683 10 nM; IB-MECA, 1 nM) and barrier function was analyzed by TER. VVE-Co (<b>C</b>) and VVEC-Hyp (<b>D</b>) were stimulated with adenosine (Ado, 100 μM) with and without A1R specific antagonist (PSB 36, 1 nM, 30 min), and barrier function was analyzed by TER. VVE-Co (E) and VVEC-Hyp (F) were stimulated with A1R specific agonist (CCPA, 1 nM) with and without A1R antagonist (PSB 36, 1 nM, 30 min), and barrier function was analyzed by TER.</p

Schematic representation of the proposed signaling pathway of adenosine-induced enhancement of barrier function in VVEC.

<p>Schematic representation of the proposed signaling pathway of adenosine-induced enhancement of barrier function in VVEC.</p

Adenosine enhances the VVEC barrier function.

<p>VVEC monolayers in ECIS arrays were incubated in serum free medium for 1 h. Adenosine (50–500 µM) was added to VVEC-Co (<b>A</b>) or VVEC-Hyp (<b>B</b>) after a steady baseline was established, and the TER measurements continued for 6 h. Data are representative of multiple independent experiments (minimum of three).</p