Shear Stress Regulates Endothelial Nitric Oxide Synthase Expression Through c-Src by Divergent Signaling Pathways

• Published in: Circulation research 2001-11, Vol.89 (11), p.1073-1080
• Main Authors: Davis, Michael E, Cai, Hua, Drummond, Grant R, Harrison, David G
• Format: Article
• Language: English
• Subjects: Animals; Biological and medical sciences; Blood vessels and receptors; Cattle; Cells, Cultured; Endothelium, Vascular - metabolism; Enzyme Inhibitors - pharmacology; Fundamental and applied biological sciences. Psychology; Kinetics; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 1 - metabolism; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinase Kinases - antagonists & inhibitors; Mitogen-Activated Protein Kinases - metabolism; Nitric Oxide Synthase - biosynthesis; Nitric Oxide Synthase - genetics; Nitric Oxide Synthase Type III; Proto-Oncogene Proteins c-raf - metabolism; Proto-Oncogene Proteins p21(ras) - physiology; Proto-Oncogene Proteins pp60(c-src) - antagonists & inhibitors; Proto-Oncogene Proteins pp60(c-src) - physiology; RNA Stability; RNA, Messenger - biosynthesis; Stress, Mechanical; Transcriptional Activation; Vertebrates: cardiovascular system
• ISSN: 0009-7330; 1524-4571
• DOI: 10.1161/hh2301.100806

In this study, we defined the signaling cascade responsible for increased eNOS mRNA expression in response to laminar shear stress. This pathway depends on the tyrosine kinase c-Src because shear induction of eNOS mRNA is blocked by the c-Src inhibitors PP1 and PP2, as well as an adenovirus encoding kinase inactive c-Src. After activation of c-Src, this pathway diverges. One arm is responsible for the short-term (6 hour) increase in eNOS mRNA. This involves a transient, 1-hour increase in eNOS transcription, as detected by nuclear run-on, that is dependent on activation of Ras and is blocked by adenoviral infection with dominant negative Ras. Downstream of Ras, MEK1/2 and ERK1/2 are important in this pathway, as 2 inhibitors of MEK1/2, PD98059 and UO126, completely prevented this early increase in eNOS mRNA. ERK1/2 was rapidly phosphorylated in response to shear, and this was prevented by c-Src and Ras inhibition. Further, Raf is phosphorylated in response to shear stress, and this is prevented by c-Src inhibition, suggesting that Raf may transduce the signal between Ras and ERK1/2. The second arm of the pathway linking activation of c-Src to eNOS expression involves stabilization of eNOS mRNA by shear stress. This response to shear is completely abrogated by the c-Src inhibitor PP1 but not altered by Ras or MEK1/2 inhibition. Thus, c-Src plays a central role in modulation of eNOS expression in response to shear stress via divergent pathways involving a short-term increase in eNOS transcription and a longer-term stabilization of eNOS mRNA.