Abstract 1442: Phosphorylation of alpha4 Integrin by PKA is Required for Endothelial Cell Alignment Induced by Shear Stress.
Endothelial cells in the blood vessel intima respond to laminar shear stress by aligning in the direction of flow, a process which is athero-protective. In this study we identified a novel mechanism regulating shear stress-induced cytoskeletal alignment in microvascular endothelial cells. The cytoplasmic tails of alpha4 integrins expressed in these cells become phosphorylated at Ser988 as early as five minutes following exposure to physiological levels of shear stress. This phosphorylation response is enhanced at the downstream edges of cells relative to the source of flow. Downstream alpha4 integrin phosphorylation induced by shear is dependent on activity of protein kinase A, which we previously showed phosphorylates alpha4 integrins at the leading edge of migrating cells. Furthermore, PKA activity is required for alignment of endothelial cells under prolonged shear. Using endothelial cells isolated from alpha4(S988A) knock-in mice, we demonstrate that alignment induced by 20 hours in shear requires alpha4 integrin phosphorylation at Ser988. Lastly, we found that shear-induced polarized activation of the small GTPase Rac1, which is necessary for alignment, requires PKA activity. We propose a model whereby shear-induced PKA-dependent alpha4 integrin phosphorylation at the downstream edge of endothelial cells promotes localized Rac1 activation, which in turn directs cytoskeletal rearrangements leading to alignment in shear. These findings present a unique mechanism of anti-atherogenic endothelial cell function under blood flow and suggest potential therapeutic targets in modulating this response.