Shear‐Induced Extracellular Regulated Kinase Signaling to eNOS is Increased When Autophagy is Compromised in Endothelial Cells

Abstract

Frictional forces (i.e., “shear stress”) exerted by flowing blood along the surface of the endothelium are potent stimuli for endothelial nitric oxide (NO) synthase (eNOS) phosphorylation and NO production. Recently we reported that shear‐induced increases in p‐eNOSS1177 (a positive eNOS regulatory site) and NO generation are prevented when the process of autophagy is disrupted in ECs via knockdown of AuTophaGy‐related protein 3 (Atg3) using siRNA. We sought to determine whether shear‐induced kinase stimulation / inhibition of eNOS is altered when autophagy is compromised in ECs. In ECs transfected with scrambled siRNA, shear stress (3 h x 20 dyn/cm2) increased (p<0.05) autophagy (e.g., ↑LC3‐II:LC3‐1, ↓p62, ↑LC3‐GFP puncta formation), p‐eNOSS1177, and NO production (assessed via DAF, EPR), but p‐eNOST495 (a negative eNOS regulatory site) was unchanged. In ECs with >85% knockdown of Atg3 protein expression via siRNA, shear–induced increases in autophagy, p‐eNOSS1177, and NO generation were prevented, but p‐eNOST495 increased (p<0.05). As such, shear‐induced increases (p<0.05) in p‐eNOSS1177 : p‐eNOST495 occur in ECs with intact but not compromised autophagy. Extracellular regulated kinase 1/2 (ERK1/2), which phosphorylates p‐eNOST495 in ECs, increased (p<0.05) in response to shear stress only after Atg3 siRNA. Interestingly, when ERK1/2 activity was inhibited (FR180204) after Atg3 siRNA, shear‐induced increases p‐eNOSS1177 : p‐eNOST495 were restored. Work in progress is exploring mechanisms whereby autophagy suppression might alter kinase signaling to eNOS.