Distinct NF-κB Regulation by Shear Stress Through Ras-Dependent IκBα Oscillations: Real-Time Analysis of Flow-Mediated Activation in Live Cells

• Published in: Circulation research 2005-04, Vol.96 (6), p.626-634
• Main Authors: Ganguli, Arunima, Persson, Linda, Palmer, Ian R, Evans, Iona, Yang, Lin, Smallwood, Rod, Black, Richard, Qwarnstrom, Eva E
• Format: Article
• Language: English
• Subjects: Biological and medical sciences; Fundamental and applied biological sciences. Psychology; Vertebrates: cardiovascular system
• ISSN: 0009-7330; 1524-4571
• DOI: 10.1161/01.RES.0000160435.83210.95

NF-κB, a transcription factor central to inflammatory regulation during development of atherosclerosis, is activated by soluble mediators and through biomechanical inputs such as flow-mediated shear- stress. To investigate the molecular mechanisms underlying shear stress mediated signal transduction in vascular cells we have developed a system that applies flow-mediated shear stress in a controlled manner, while inserted in a confocal microscope. In combination with GFP-based methods, this allows continuous monitoring of flow induced signal transduction in live cells and in real time. Flow-mediated shear stress, induced using the system, caused a successive increase in NF-κB–regulated gene activation. Experiments assessing the mechanisms underlying the NF-κB induced activity showed time and flow rate dependent effects on the inhibitor, IκBα, involving nuclear translocation characterized by a biphasic or cyclic pattern. The effect was observed in both endothelial- and smooth muscle cells, demonstrated to impact noncomplexed IκBα, and to involve mechanisms distinct from those mediating cytokine signals. In contrast, effects on the NF-κB subunit relA were similar to those observed during cytokine stimulation. Further experiments showed the flow induced inter-compartmental transport of IκBα to be regulated through the Ras GTP-ase, demonstrating a pronounced reduction in the effects following blocking of Ras activity. These studies show that flow-mediated shear stress, regulated by the Ras GTP-ase, uses distinct mechanisms of NF-κB control at the molecular level. The oscillatory pattern, reflecting inter-compartmental translocation of IκΒα, is likely to have fundamental impact on pathway regulation and on development of shear stress-induced distinct vascular cell phenotypes.