Hydrogen peroxide-induced cytoskeletal rearrangement in cultured pulmonary endothelial cells

Abstract

Although the signaling pathways leading to hydrogen peroxide (H2O2)-induced endothelial monolayer permeability remain ambiguous, cytoskeletal proteins are known to be essential for maintaining endothelial integrity and regulating solute flux through the monolayer. We have recently demonstrated that thrombin-induced actin reorganization in bovine pulmonary artery endothelial cells (BPAEC) requires activation of both myosin light chain kinase (MLCK) and protein kinase C (PKC). Therefore, the present study was designed to investigate the effects of H2O2 on actin reorganization in BPAEC. H2O2 initiated sustained recruitment of actin to the cytoskeleton and transient myosin recruitment in a time- and concentration-dependent manner. The H2O2-induced actin recruitment was significantly inhibited by the calmodulin antagonists, W7 and TFP, but not by the MLCK inhibitor, KT5926, nor the PKC inhibitors, H7 and calphostin C. H2O2 also caused actin filament rearrangement in BPAEC with disruption of the dense peripheral bands and formation of stress fibers. These alterations occurred prior to actin translocation to the cytoskeleton and are prevented by inhibition of either MLCK or PKC. High concentrations of H2O2 transiently attenuated PKC activity but slightly increased the phosphorylation of the prominent PKC substrate and actin-binding protein, myristoylated alanine-rich C kinase substrate (MARCKS), by 5 min. However, MARCKS phosphorylation was reduced to below basal levels by 30 min. On the other hand, H2O2 induced a time- and dose-dependent phosphorylation of myosin light chains which was eliminated by both MLCK and PKC inhibitors. These data suggest that MLCK contributes to H2O2-induced myosin light chain phosphorylation and actin rearrangement and that PKC may play a permissive role. Neither of these enzymes appears to be involved in the H2O2-induced recruitment of actin to the cytoskeleton.

Reactive Oxygen Species in Inflammation and Tissue Injury

“…Permeability increasing mediators such as thrombin, histamine, and H 2 O 2 enhance MLC phosphorylation, which precedes the onset of endothelial contraction (Fig. 10) (303,387,470). The myosin phosphorylation in the endothelial cells is regulated by two enzymes: nonmuscle MLCK and myosinassociated protein phosphatase.…”

“…Activated MLCK directly phosphorylates MLC at Thr-18 and Ser-19 site, which induced isometric tension development and actin polymerization in endothelial cells (136). Zhao et al (470) reported that H 2 O 2 also induced MLC phosphorylation in endothelial cells. Though the inhibition of MLCK blocked H 2 O 2 -induced MLC phosphorylation, yet it did not affect H 2 O 2 -induced actin and myosin assembly.…”

“…Oxidant-mediated activation of PKC also occurred through phosphorylation of distinct residues of different PKC isoforms (224). Several lines of evidence suggest that PKC is likely to play a permissive role in H 2 O 2 -mediated endothelial permeability and lung edema (200,389,470 …”

Hydrogen Peroxide Potentiates Volume-sensitive Excitatory Amino Acid Release via a Mechanism Involving Ca2+/Calmodulin-dependent Protein Kinase II

“…Two Ca 2ϩ /calmodulin-dependent enzymes known to be activated by H 2 O 2 , the myosin light chain kinase (MLCK), and CaMKII, have also been found to regulate VRACs (20,27,49,50). We therefore explored whether changes in intracellular Ca 2ϩ and the activation of Ca 2ϩ /calmodulin-dependent enzymes are involved in the H 2 O 2 effect.…”

Vascular Endothelial Growth Factor Causes Translocation of p47 to Membrane Ruffles through WAVE1

“…High levels of exogenous oxidants increase recruitment of actin to the cytoskeleton, and conversely actin cytoskeletal disruption precipitates oxidant-dependent NF-␤ activation (8,9). Oxidant production is also most prominent in actively migrating endothelial cells at the margin of a monolayer wound and, in particular, is concentrated in membrane ruffles of these highly motile cells (10).…”