| Fluid shear stress, one of the hemodynamic forces acting on the vessel wall, regulates vascular structure and function. Endothelium, forming the inner lining of all blood vessels, is constantly exposed to hemodynamic forces. Flow alters endothelial cell (EC) functions such as orientation in the vessel wall, distribution of cytoskeletal molecules, and gene expression for several proteins, including platelet-derived growth factor A- and B-chain, tissue plasminogen activator, and endothelial nitric oxide synthase (eNOS). The changes of cellular physiology and gene expression are mediated by flow-induced activation of mechanosensitive pathways including receptor tyrosine kinases (RTKs, such as VEGFR), members of mitogen-activated protein kinase (MAPK) family (such as ERK1/2, P38, and BMK1/ERK5), and non-RTKs (including FAK, PYK2, and Src). However, proximal mechanisms by which ECs respond to flow causing downstream signaling remain poorly defined. We proposed that the early signaling events (e.g. ROS, calcium, and phosphoylation of PECAM1) are required for the activation of protein kinases in flow signaling.; First I demonstrated that PYK2 is a proximal target of flow-derived ROS and intracellular calcium and is also an upstream kinase that phosphorylates Cas. To understand the mechanisms that regulate BMK1 function we used a yeast two-hybrid system to find BMK1 associated proteins including beta-catenin. However, beta-catenin did not regulate flow-induced BMK1 activation and BMK1 did not stabilize beta-catenin in the cytosol. Later, by using a proteomics method and siRNA technique, I investigated the functions of membrane adhesion molecules and RTKs in response to flow. As such, I tested the hypothesis that membrane molecules (PECAM1 or Tie2) in signaling modules are important for MAPK and Akt/eNOS activation by flow. We found that PECAM1/Tie2-SHP2 complexes participate in the activation of ERK1/2, Akt, and eNOS by flow. The PECAM1/SHP2 complex is important for flow-induced BMK1 activation, but not Tie2. These results suggest that ROS, calcium, membrane adhesion molecules, and RTKs are required for flow-induced activation of protein kinases and eNOS. |
