An extensive body of work has demonstrated that atherosclerotic plaques form preferentially at sites of disturbed blood flow encountered at vessel bifurcations, resulting in part from the loss of anti-inflammatory, antithrombotic and antioxidative effects that occur in straight vessel segments due to laminar pulsatile flow1. The production of nitric oxide (NO) by endothelial nitric oxide synthase (eNOS) is a central mechanism of these protective effects2. Transcriptional and posttranslational regulation of eNOS combine to augment NO production in response to flow-induced mechanical stimulation of the apical endothelial cell surface3. AMP-activated protein kinase (AMPK) and the deacetylase sirtuin 1 (SIRT1) are key mediators of multiple vascular endothelial processes. AMPK mediates metformin-induced protection from oxidative stress4 and regulates coronary hemodynamics5 and arteriogenesis6, while SIRT1 suppresses endothelial senescence produced by disturbed flow7. Laminar flow increases the activity of both AMPK and SIRT1, which then act synergistically on eNOS via phosphorylation by AMPK priming and subsequent deacetylation by SIRT1 to increase NO bioavailability8.
In the current issue of ATVB, Shentu and colleagues now substantially advance our understanding of this important process by demonstrating that AMPK and SIRT1 similarly work together to co-regulate function of the actin-binding protein cortactin, leading to altered peripheral cytoskeletal and membrane structures in vascular endothelial cells that in turn contribute to eNOS activation and decreased atherosclerosis9. Cortactin is a multi-domain cytoskeletal scaffold protein that was first identified more than 20 years ago as a novel F-actin binding protein and substrate of v-Src10. The gene encoding cortactin, EMS1, was initially detected as highly overexpressed in human carcinomas11 but subsequently recognized as ubiquitously expressed in almost all tissues and is especially abundant in the endothelium. Cortactin is an ideal candidate to integrate signal transduction and cytoskeletal structural changes because of its the ability to bind actin, interact with a variety of cytoskeletal effector proteins including Arp 2/3, N-WASP and MLCK, and serve as a target for key regulatory kinases, including Src, ERK and c-Abl12, 13. The peripheral cellular distribution of cortactin and its association with cortical actin were early observations, are implicit in its very name, and further strengthen the idea that cortactin is an important link between extracellular stimuli, cytoskeletal organization and membrane dynamics. Over the last two decades, critical roles for cortactin have been described in cell migration, tumor invasion, host-pathogen interactions, endocytosis, vesicle trafficking, intercellular junctional assembly, leukocyte diapedesis and endothelial barrier function12, 14–16. These processes are essential components of vascular endothelial function and pathology, and it is not surprising that this tissue has served as the backdrop for much of the work highlighting the diverse structural and functional roles of cortactin. As noted above, mechanisms by which blood flow induces atherosclerosis or atheroprotection are intriguing areas of investigation requiring the endothelial cell to translate properties of the local environment, especially flow, into dynamic alterations of the cytoskeleton, cell membrane and biochemical signaling17, 18. The present study identifying cortactin as a key player in atheroprotective eNOS regulation sheds new light on how laminar pulsatile flow mediates atheroprotection and serves to expand the influential role and importance of this protein in endothelial biology.
The authors hypothesized that pulsatile flow leads to concerted regulation of cortactin by AMPK/SIRT1 similar to that observed in eNOS as cortactin contains consensus sequences for both proteins. After confirming specific AMPK phosphorylation of cortactin at Thr-401 in proteomic experiments, they subsequently demonstrate AMPK priming of cortactin for SIRT1 deacetylation in cultured endothelial cells subjected to pulsatile shear stress (PS). Next, employing gene knockdown and both gain and loss of function mutants in an elegant series of experiments, Shentu and colleagues determined that activated cortactin plays a direct role in eNOS activation in response to PS. Mechanistically this occurs via cortactin translocation to the periphery in a close association with F-actin, which subsequently facilitates the transfer of eNOS from lipid to nonlipid raft domains, eNOS phosphorylation, and generation of atheroprotective NO. Finally, in vivo studies in cortactin +/− mice revealed impaired NO production and flow-induced vasodilation associated with reduced caveolae and phosphorylated eNOS at the cellular level. This endothelial dysfunction was associated with an increase in atherosclerotic plaques in ApoE −/− cortactin +/− animals as compared to ApoE −/− cortactin +/+. Taken together these data provide convincing evidence that cytoskeletal and membrane dynamics as regulated by the AMPK/SIRT1/cortactin pathway are key determinants of atheroprotection induced by blood flow patterns.
Cortactin is a rich target for investigation into the coordination of numerous signaling pathways terminating in altered peripheral cytoskeletal and membrane dynamics, qualities vital to endothelial migration, barrier integrity and interaction with the local physical and cellular environment. Interestingly, differential patterns of cortactin phosphorylation lead to markedly different outcomes in actin structure and function. Serine and tyrosine phosphorylation by Src and ERK affect cortactin’s interaction with effectors of actin polymerization and tensile force generation15, 19, 20, with Src tyrosine phosphorylation of cortactin also associated with focal adhesion disassembly and turnover21. While Thr-401 phosphorylation by Akt has been implicated in cancer cell migration and invasion22, the current study adds to this complexity with phosphorylation at this same Thr-401 site by AMPK significantly impacting cortactin-F-actin association and regulation of membrane associated caveolae. The specific alterations in cytoskeletal structure and dynamics responsible for these varied effects remain unknown and are intriguing areas for further study.
The vascular endothelium is a varied and intensely regulated tissue with the ability to adapt its structure and function specifically to the requirements of each organ system and physiologic variable. Exciting work such as the current study by Shentu and colleagues continues to reveal the complex biochemistry underlying such changes at the cellular level. Cortactin represents a prominent cog in this larger wheel. A more thorough understanding of this fascinating protein appears destined to expand the discourse in new and exciting directions which will advance our knowledge of vascular biology and disease, including these newly identified insights into atheroprotective mechanisms mediated by the AMPK/SIRT1 signaling axis.
Acknowledgments
None
Sources of Funding: None
Footnotes
Disclosure: None
References
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