MAPk Activation Modulates Permeability of Isolated Rat Alveolar Epithelial Cell Monolayers Following Cyclic Stretch

We cultured (5 days) rat alveolar epithelial cells to investigate the role of mitogen-activated protein kinase (MAPk) signaling in ventilator induced epithelial barrier dysfunction. Cells were stretched to a magnitude of 12% or 37% change in surface area at a rate of 0.25 Hz with and without pretreatment with either the JNK inhibitor SP600125 or the ERK inhibitor U0126. Following stretch (0, 10, 30, or 60 min), MAPk phosphorylation was examined, monolayer permeability to the uncharged tracer carboxyfluorescein measured (0, 10, 60 min of stretch), and occludin expression determined (0 and 60 min of stretch). Stretch to 12%, previously shown not to increase monolayer permeability, did not alter phosphorylation of any MAPk or occludin expression at any time point. Following stretch to 37%, phosphorylation of JNK, ERK, and p38 was significantly higher by 10 minutes than in unstretched monolayers. Phosphorylation of JNK and p38 subsided as stretch continued, and by 30 minutes returned to unstretched levels. Phosphorylation of ERK remained significantly elevated compared to unstretched levels at all stretch durations. Epithelial permeability increased significantly by 10 minutes of stretch compared to unstretched controls, with further significant increases by 60 minutes. Inhibition with U0126 and SP600125 prevented stretch-induced phosphorylation increases of ERK and JNK, respectively, however neither prevented increases in permeability following 10 minutes. Separately, inhibition of JNK or ERK prevented subsequent additional permeability increases as stretch continued to 60 minute time points. Inhibition of JNK, not ERK, prevented loss of occludin, and minimized loss of cell-cell contact following 60 minutes of stretch. These data suggest that stretch-induced JNK signaling modulates epithelial permeability through regulation tight junction protein expression, and is a potential target for clinical treatments during mechanical ventilation

SP600125 inhibits JNK phosphorylation.

<p>Phosphorylation levels of JNK, ERK, and p38 MAPk following treatment with the inhibitor SP600125 (20 or 35 µM) or the control DMSO and 10 minutes of cyclic stretch at a rate of 0.25 Hz to a peak magnitude of 37% ΔSA normalized to stretched DMSO controls. Phosphorylation levels of all MAPks significantly increased above unstretched levels in DMSO treated wells. Both concentrations of SP600125 eliminated significant increases in JNK above unstretched levels. Stretch with a treatment of 35 µM of SP600125 resulted in significantly increased ERK phosphorylation compared to DMSO treated stretched wells. Significance is defined as p<0.05, and is depicted with (—) compared to unstretched, and (δ, lower) (*, higher) compared to DMSO treated. Representative western blots of phosphorylated and total MAPks with each treatment are also presented.</p

Stretch activation of MAPk signaling.

<p>Phosphorylation levels of JNK, ERK, and p38 MAPk following 10, 30, and 60 minutes of cyclic stretch at a rate of 0.25 Hz to a peak magnitude of 37% ΔSA normalized to unstretched controls. Phosphorylation levels of all MAPks significantly increased above unstretched levels (value of 1) following 10 minutes of stretch (*). Following 30 or 60 minutes of stretch, only ERK remained significantly elevated. JNK phosphorylation was significantly greater following 10 minutes than following 60 minutes (#). At the 10 minute time point, phosphorylation of JNK and ERK was significantly higher than phosphorylation of p38. Significance is defined as p<0.05. Representative western blots of phosphorylated and total MAPks following stretch to either 12% or 37% ΔSA are also shown. No change in phosphorylated protein normalized to total protein was observed at any stretch duration with a stretch magnitude of 12% ΔSA.</p

U0126 inhibits ERK phosphorylation.

<p>Phosphorylation levels of JNK, ERK, and p38 MAPk following treatment with the inhibitor U0126 (10 or 20 µM) or the control U0124 (20 µM) and 10 minutes of cyclic stretch at a rate of 0.25 Hz to a peak magnitude of 37% ΔSA normalized to stretched U0124 controls. Phosphorylation levels of all MAPks significantly increased above unstretched levels in U0124 treated wells. Following treatment with 10 or 20 µM of U0126 and 10 minutes of stretch, ERK phosphorylation was significantly reduced compared with U0124 treatment and stretch. Only treatment with 20 µM of U0126 eliminated significant increases in ERK above unstretched levels. Significance is defined as p<0.05, and is depicted with (—) compared to unstretched, and (δ) compared to U0124 treated. Representative western blots of phosphorylated and total MAPks with each treatment are also presented.</p

JNK inhibition prevents loss of occludin.

<p>Occludin concentration in cell monolayers significantly drops following 60 minutes of stretch (0.25 Hz, 37% ΔSA) compared to unstretched monolayers (*, p<0.05). Treatment with the JNK inhibitor SP600125 blocked occludin decreases following 60 minutes of stretch, however ERK inhibition with U0126 did not. (N≥3, μ ± SE).</p

Occludin localization.

<p>Images of epithelial monolayers, stained with occludin antibodies, either treated with DMSO control (A and C) or SP600125 (B and D). Unstretched monolayers (A and B), exhibit strong staining of Occludin at the cell-cell junction. Following stretch, junctional staining is significantly reduced, and large holes form in the monolayer. SP600125 treatment reduces these alterations following stretch, however hole formation is still evident. (Scale bar 100 µm).</p