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

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.