Distinct NF-κB Regulation by Shear Stress Through Ras-Dependent IκBα Oscillations

Abstract

NF-kappaB, 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-kappaB-regulated gene activation. Experiments assessing the mechanisms underlying the NF-kappaB induced activity showed time and flow rate dependent effects on the inhibitor, IkappaBalpha, 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 IkappaBalpha, and to involve mechanisms distinct from those mediating cytokine signals. In contrast, effects on the NF-kappaB subunit relA were similar to those observed during cytokine stimulation. Further experiments showed the flow induced inter-compartmental transport of IkappaBalpha 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-kappaB control at the molecular level. The oscillatory pattern, reflecting inter-compartmental translocation of IkappaBetaalpha, is likely to have fundamental impact on pathway regulation and on development of shear stress-induced distinct vascular cell phenotypes.