Flow activates ERK1/2 and endothelial nitric oxide synthase via a pathway involving PECAM1, SHP2, and Tie2.
Blood flow modulates endothelial cell (EC) functions through specific signaling events. Previous data show that flow stimulates SHP2 translocation to cell membranes and binding to phosphotyrosine proteins. Flow-induced ERK1/2 phosphorylation depends on SHP2 phosphatase activity and SHP2 binding to phospho-PECAM1 (platelet endothelial adhesion molecule 1), suggesting that SHP2 forms a signaling module with PECAM1. We hypothesized that flow induces assembly of the multi-protein complexes with SHP2 that are required for downstream signaling. ECs were exposed to flow for 10 min, and endogenous SHP2 was immunoprecipitated. SHP2-associated proteins were analyzed by SDS-PAGE and identified by mass spectrometry. Tie2 and several known SHP2-binding proteins were identified in flow-induced SHP2 complexes. Flow significantly increased tyrosine phosphorylation of both Tie2 and PECAM1 and their association with SHP2. To evaluate their functional roles, ECs were treated with Tie2 or PECAM1 small interfering RNA (siRNA). Tie2 and PECAM1 expression decreased >80% after siRNA treatment, and flow-stimulated phosphorylation of ERK1/2, Akt, and endothelial nitric oxide synthase was significantly inhibited by Tie2 and PECAM1 siRNA. Tie2 phosphorylation by flow was significantly inhibited by PECAM1 siRNA treatment. These results establish Tie2 transactivation via PECAM1 as an early event in flow-mediated mechanotransduction and suggest an important role for a PECAM1-SHP2-Tie2 pathway in flow-mediated signal transduction.
Cardiovascular Research Institute and Department of Medicine, University of Rochester, New York 14642, USA.
Lung-Kuo
LK
Tai
Qinlei
Q
Zheng
Shi
S
Pan
Zheng-Gen
ZG
Jin
Bradford C
BC
Berk
D000818:Animals; D001775:Blood Circulation / Q000502:physiology*; D002417:Cattle; D002478:Cells, Cultured; D004789:Enzyme Activation; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000502:physiology*; D019950:Mitogen-Activated Protein Kinase 1 / Q000502:physiology*; D048052:Mitogen-Activated Protein Kinase 3 / Q000502:physiology*; D019001:Nitric Oxide Synthase / Q000502:physiology*; D052250:Nitric Oxide Synthase Type III; D010766:Phosphorylation; D019408:Platelet Endothelial Cell Adhesion Molecule-1 / Q000502:physiology*; D017346:Protein Serine-Threonine Kinases / Q000502:physiology; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; D017027:Protein Tyrosine Phosphatases / Q000502:physiology*; D011518:Proto-Oncogene Proteins / Q000502:physiology; D051057:Proto-Oncogene Proteins c-akt; D042787:Receptor, TIE-2 / Q000502:physiology*; D015398:Signal Transduction / Q000502:physiology*; D015533:Transcriptional Activation
D047908:Intracellular Signaling Peptides and Proteins; D019408:Platelet Endothelial Cell Adhesion Molecule-1; D011518:Proto-Oncogene Proteins; C496314:NOS3 protein, human; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; D042787:Receptor, TIE-2; C494918:AKT1 protein, human; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; C516724:PTPN11 protein, human; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; D017027:Protein Tyrosine Phosphatases