Stretch of Septic Monolayers Figure 7. Sham and 2CLP monolayers were cyclically stretched to a magnitude of 12% DSA for 60 minutes following a 1-hr incubation with either Jasplakinolide (1 mM, 2CLP) or DMSO (Sham and 2CLP), and permeability to BODIPY-ouabain was analyzed. Stretch resulted in significant permeability increases in DMSO-treated 2CLP monolayers only, compared to unstretched sham and 2CLP monolayers. Treatment with Jasplakinolide prevented all permeability increases. Significance defined as p,0.05 with multiple comparison correction (b = 4), *significantly different than stretched sham (mean 6 SE, N$3 isolations). doi:10.1371/journal.pone.0038748.g007 associated with enhanced MAPk signaling activation in our model, which is counter to our hypothesis of additive activation. Interestingly we did observe MAPk activation following stretch to 25% DSA, however inhibition of MAPk signaling in 2CLP monolayers did not prevent stretch-induced permeability in- creases, suggesting that an alternative mechanism is responsible for the increase in permeability. The tight junction is an apically located protein complex that controls paracellular permeability of epithelial monolayers, and we hypothesized that the expression levels of TJ proteins, which determine how restrictive the tight junctions are to paracellular fluid and ion motion, would change with injurious levels of stretch. However, we observed no significant alterations in protein expression in 2CLP monolayers following stretch to 12% DSA, despite increases in monolayer permeability. Therefore, we conclude that stretch-induced (60 minutes) permeability changes observed in 2CLP monolayers are not due to loss of TJ proteins. The actin cytoskeleton is integral to the formation and maintenance of the tight junction barrier, and depolymerization, or reorganization, of the actin cytoskeleton has been linked to disruption of junctional integrity in epithelial cells [23,33–39]. Therefore we hypothesized that regulation of the actin cytoskel- eton is compromised in 2CLP monolayers, and that cytoskeletal alterations in 2CLP monolayers result in the permeability increases observed following stretch to 12% DSA. Visualization of actin stress fibers demonstrated organizational differences between 2CLP and sham cytoskeletons with and without stretch. We further demonstrate that with low magnitude stretch in 2CLP monolayers, the phosphorylation decrease of MLCK may indicate an alteration in actin contractile force generation during de- formation. MLCK is a Ca2+ dependent signaling molecule that is activated by calmodulin [40]. Phosphorylation of MLCK on serine-1760 inhibits binding of calmodulin to MLCK, preventing its activation [41,42]. Therefore, in 2CLP monolayers, MLCK is potentially more readily activated in response to changes in intracellular Ca2+ due to reduced regulatory signaling. Studies by Turner and colleagues have demonstrated that constitutive MLCK activation increases permeability and destabilizes the junction of non-pulmonary cell lines [43,44]. Consequently it is not unreasonable to speculate that the lower phosphorylation levels of the regulatory site of MLCK in 2CLP monolayers could contribute to altered barrier function following stretch. Furthermore, we observe that the cytoskeletal-tight junction complex is more detergent-soluble in unstretched 2CLP mono- layers, as well as a significant decrease in occludin expression in 2CLP monolayers compared to sham without stretch, as reported previously [15]. Occludin has been shown to regulate the function of tight junctions through its binding with other TJ proteins [45,46]. Occludin has been shown to directly interact with claudins, ZO-1, and actin at its C-terminus, and we speculate that with sepsis, the reduction in occludin expression, even without stretch, leads to a more disorganized and leaky tight junction [47]. Jasplakinolide prevents the depolymerization of actin fibers, and has been shown to reduce stretch-induced permeability increases in healthy epithelial monolayers cyclically stretched to 37% DSA for 1 hour [12,48]. Furthermore, treatment with jasplakinolide has recently been shown by our lab to prevent formation of cortical actin rings in healthy primary epithelial cells cyclically stretched to 25% DSA for 10 minutes [22]. Pretreatment with jasplakinolide prevented significant increases in 2CLP monolayer permeability following low-level mechanical stretch. Future studies will focus on elucidating the relationship between acto-myosin tension and anchoring of the tight junction complex in septic and healthy epithelial monolayers. In conclusion, we have shown that cultured epithelial mono- layers composed of cells isolated from septic animals have a lower threshold for cyclic stretch-induced barrier dysfunction than one composed of cells from control animals The lower threshold is not due to enhanced activation of MAPk signaling pathways as hypothesized, but rather due to alteration in the actin cytoskeleton PLoS ONE | www.plosone.org 8 June 2012 | Volume 7 | Issue 6 | e38748