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Shear‐Induced Extracellular Regulated Kinase Signaling to eNOS is Increased When Autophagy is Compromised in Endothelial Cells
Author(s) -
Ruan Ting,
Bharath Leena,
Mueller Robert,
Goodrich Rebekah,
Graham Timothy,
Symons J David
Publication year - 2015
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.29.1_supplement.956.4
Subject(s) - enos , autophagy , gene knockdown , small interfering rna , chemistry , extracellular , kinase , nitric oxide , protein kinase a , medicine , microbiology and biotechnology , transfection , endothelium , endocrinology , nitric oxide synthase , biology , apoptosis , biochemistry , gene
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‐eNOS S1177 (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/cm 2 ) increased (p<0.05) autophagy (e.g., ↑LC3‐II:LC3‐1, ↓p62, ↑LC3‐GFP puncta formation), p‐eNOS S1177 , and NO production (assessed via DAF, EPR), but p‐eNOS T495 (a negative eNOS regulatory site) was unchanged. In ECs with >85% knockdown of Atg3 protein expression via siRNA, shear–induced increases in autophagy, p‐eNOS S1177 , and NO generation were prevented, but p‐eNOS T495 increased (p<0.05). As such, shear‐induced increases (p<0.05) in p‐eNOS S1177 : p‐eNOS T495 occur in ECs with intact but not compromised autophagy. Extracellular regulated kinase 1/2 (ERK1/2), which phosphorylates p‐eNOS T495 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‐eNOS S1177 : p‐eNOS T495 were restored. Work in progress is exploring mechanisms whereby autophagy suppression might alter kinase signaling to eNOS.