A novel role for reactive oxygen species in the regulation of RhoA; implications for endothelial permeability and leukocyte transmigration

Amir Aghajanian: A novel role for reactive oxygen species in the regulation of RhoA; implications for endothelial permeability and leukocyte transmigration. The endothelial lining of the vasculature plays a critical role in regulating the passage of fluid, macromolecules, and cells between the blood and surrounding tissues. Vascular permeability is tightly regulated and is modulated during both physiological and pathological situations. Our laboratory is interested in the mechanisms which regulate vascular permeability and the transmigration of leukocytes during inflammation. The migration of leukocytes across the endothelial barrier is called " leukocyte transendothelial migration " (TEM). Deciphering the mechanisms which regulate TEM is important to understanding and managing inflammatory diseases. My interest in TEM is focused on pathways which involve small GTPases. My specific interest is in the role of Rho GTPases and their regulation by reactive oxygen species (ROS) during leukocyte TEM. Although ROS have been largely seen as mediators of oxidative damage, more recently, ROS have been recognized as necessary components of cell signaling pathways. Importantly, ROS have been shown to play an important role in regulating vascular permeability and TEM. iv In this dissertation, I show that physiological levels of ROS can directly activate RhoA in cells. In vitro studies had previously identified two critical cysteine residues in the nucleotide binding pocket of RhoA that are oxidatively modified by ROS. My work showed that this oxidative regulation of RhoA can occur in a cellular context. Before these studies, the regulation of small GTPases had almost exclusively focused on regulatory proteins. Importantly, my work identifies direct oxidative modification as a novel way to regulate RhoA activity. I extended this work by investigating the regulation of RhoA by ROS in the context of leukocyte TEM. Based on these findings and my previous work, I hypothesized that leukocyte adhesion to endothelial cells initiates ROS generation and the direct activation of RhoA to promote leukocyte TEM. In preliminary studies, I found that crosslinking of a cell adhesion molecule (ICAM-1) stimulates RhoA and Rac1 activation. In addition the activation of RhoA appears to be dependent on ROS, as seen by studies of redox-insensitive mutants. The work presented in this dissertation lays the framework for future studies on the role of ROS during leukocyte adhesion and TEM.