Amsler Lab Research

Leak Pathway Regulation

We are investigating the effect of hydrogen peroxide treatment, as a model of ischemia/reperfusion injury, on the paracellular permeability of large compounds across sheets of a kidney epithelial cell line, MDCK Type II, that forms tight junctions and behaves in culture like a normal reabsorptive epithelium.

Flux Assay

MDCK cells are grown on permeable membrane filters. Cells are pretreated for 1 hour with low concentrations of hydrogen peroxide (~50 microM). A large fluorescent molecule is added to the solution bathing the basolateral surface of the cells (see figure). Samples of the solution bathing the apical surface of the cells are taken periodically. Fluorescence is measured. The amount of the fluorescent molecule that has entered the apical fluid compartment is calculated as a function of time to determine the rate of movement of the fluorescent molecule through the Leak Pathway. We have shown that the Leak Pathway is the only pathway by which these fluorescent molecules cross the MDCK cell epithelium.

Role of Occludin in Mediating the Effect of Hydrogen Peroxide on Leak Pathway Permeability

We showed that treatment of MDCK cells with hydrogen peroxide at non-lethal concentrations increased Leak Pathway permeability in a concentration-dependent manner (www.ncbi.nlm.nih.gov/pmc/articles/PMC4725295/pdf/nihms-751910.pdf) . In contrast, it had no effect on Pore Pathway permeability. The ability of hydrogen peroxide to increase Leak Pathway permeability was negatively correlated with the cellular content of occludin protein (see figure). Hydrogen peroxide treatment did not affect the cellular content of occludin protein but did alter its subcellular localization modestly. Previous studies demonstrated that some tight junction proteins move into and out of the tight junction region (see, e.g., Shen et al., J Cell Biol. 2008 May 19;181(4):683-95. doi: 10.1083/jcb.200711165). We showed that treatment with hydrogen peroxide slowed the movement of occludin into the tight junction region. These results suggest occludin protein content and/or dynamics contribute to modulation of Leak Pathway permeability by hydrogen peroxide.

Role of ERK 1/2 in Mediating the Effect of Hydrogen Peroxide on Leak Pathway Permeability

The effect of manipulations on cellular processes is typically mediated by their effect on the activities of intracellular enzymes, called signaling proteins. We screened the effect of hydrogen peroxide treatment on multiple signaling proteins and showed that it stimulated the phosphorylation of the signaling protein Extracellular signal-Regulated Kinase 1/2 (ERK 1/2), a marker for its enzyme activity (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6171043/). Inhibitors of ERK 1/2 activation inhibited the ability of hydrogen peroxide to increase Leak Pathway permeability. As shown above, MDCK cells in which occludin protein content was decreased by siRNA knockdown exhibited an enhanced response of Leak Pathway permeability to hydrogen peroxide treatment. In contrast, siRNA knockdown of ZO-1 protein content dramatically diminished the ability of hydrogen peroxide to increase Leak Pathway permeability (see figure). Interestingly, complete knockout of the expression of a protein, TOCA-1, that organizes the actin into a branching F-actin network similarly decreased the ability of hydrogen peroxide to increase Leak Pathway permeability. ZO-1 binds to TOCA-1 and targets it to the tight junction region. Both ZO-1 knockdown MDCK cells and TOCA-1 knockout MDCK cells exhibited an altered response to blebbistatin, a myosin II ATPase inhibitor which blocks actomyosin contraction. These results support the hypothesis that coupling of the tight junction to the F-actin cytoskeleton via ZO-1 plays an important role in regulating Leak Pathway permeability.

The Future

While we have learned much about Leak Pathway regulation, many questions remain. Since ERK 1/2 is a protein kinase, i.e., it phosphorylates proteins, this suggests that Leak Pathway permeability is regulated through the phosphorylation of one or more cell proteins. What are these proteins and how does their phosphorylation alter their behavior? What exactly are the roles of the different tight junction proteins in mediating the Leak Pathway? How is the actomyosin network involved in this regulation? Studies have implicated Myosin Light Chain Kinase (MLCK) in regulation of paracellular permeability (see e.g., Yu et al., Proc Natl Acad Sci U S A. 2010 May 4;107(18):8237-41. doi: 10.1073/pnas.0908869107). It is currently unclear if MLCK is involved in the hydrogen peroxide-induced increase in Leak Pathway permeability in the MDCK cells. These are just some of the questions we are continuing to pursue. Through these studies, we hope to determine how Leak Pathway permeability is regulated under normal conditions and how this regulation is disrupted in disease states.

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