ZO-1 protein is required for hydrogen peroxide to increase MDCK cell paracellular permeability in an ERK 1/2-dependent manner.

Hydrogen peroxide (H2O2) increases paracellular permeability of Madin-Darby canine kidney (MDCK) cells, but the mechanism mediating this effect remains unclear. Treatment of MDCK cells with H2O2 activated ERK 1/2. Inhibition of ERK 1/2 activation blocked the ability of H2O2 to increase paracellular permeability. Knockdown of zonula occludens-1 (ZO-1) protein but not occludin eliminated the ability of H2O2 to increase paracellular permeability. H2O2 treatment did not, however, affect the total cell content or contents of the Triton X-100-soluble and -insoluble fractions for occludin, ZO-1, or ZO-2. H2O2 treatment decreased the number of F-actin stress fibers in the basal portion of the cells. Similar to wild-type MDCK cells, H2O2 increased ERK 1/2 activation in ZO-1 knockdown and occludin knockdown cells. Inhibition of ERK 1/2 activation blocked the increase in paracellular permeability in occludin knockdown cells. ZO-1 knockdown cell paracellular permeability was regulated by PP1, an src inhibitor, indicating that the loss of response to H2O2 was not a general loss of the ability to regulate the paracellular barrier. Inhibition of myosin ATPase activity with blebbistatin increased paracellular permeability in ZO-1 knockdown cells but not in wild-type MDCK cells. H2O2 treatment sensitized wild-type MDCK cells to inhibition of myosin ATPase. Knockdown of TOCA-1 protein, which promotes formation of local branched actin networks, reproduced the effects of ZO-1 protein knockdown. These results demonstrate that H2O2 increases MDCK cell paracellular permeability through activation of ERK 1/2. This H2O2 action requires ZO-1 protein and TOCA-1 protein, suggesting involvement of the actin cytoskeleton.

Tiled microarray identification of novel viral transcript structures and distinct transcriptional profiles during two modes of productive murine gammaherpesvirus 68 infection.

We applied a custom tiled microarray to examine murine gammaherpesvirus 68 (MHV68) polyadenylated transcript expression in a time course of de novo infection of fibroblast cells and following phorbol ester-mediated reactivation from a latently infected B cell line. During de novo infection, all open reading frames (ORFs) were transcribed and clustered into four major temporal groups that were overlapping yet distinct from clusters based on the phorbol ester-stimulated B cell reactivation time course. High-density transcript analysis at 2-h intervals during de novo infection mapped gene boundaries with a 20-nucleotide resolution, including a previously undefined ORF73 transcript and the MHV68 ORF63 homolog of Kaposi's sarcoma-associated herpesvirus vNLRP1. ORF6 transcript initiation was mapped by tiled array and confirmed by 5' rapid amplification of cDNA ends. The ∼1.3-kb region upstream of ORF6 was responsive to lytic infection and MHV68 RTA, identifying a novel RTA-responsive promoter. Transcription in intergenic regions consistent with the previously defined expressed genomic regions was detected during both types of productive infection. We conclude that the MHV68 transcriptome is dynamic and distinct during de novo fibroblast infection and upon phorbol ester-stimulated B cell reactivation, highlighting the need to evaluate further transcript structure and the context-dependent molecular events that govern viral gene expression during chronic infection.