Figure 1.
Stretch activation of MAPk signaling.
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.
Figure 2.
SP600125 inhibits JNK phosphorylation.
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.
Figure 3.
U0126 inhibits ERK phosphorylation.
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.
Figure 4.
MAPk inhibition prevents stretch induced permeability increases.
Permeability of monolayers treated with 35 µM of SP600125 or 20 µM of U0126 following 0, 10, or 60 minutes of stretch. Permeability of all monolayers stretched for 10 or 60 minutes significantly increased compared to unstretched monolayers (*). 60 minutes of stretch resulted in further increases above 10 minute levels in DMSO monolayers (#). Treatment with either SP600125 or U0126 prevented these further increases, and permeability values were significantly lower then in DMSO monolayers stretched for 60 minutes (-). Significance is defined as p<0.05.
Figure 5.
JNK inhibition prevents loss of occludin.
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).
Figure 6.
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).