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(Circulation Research. 2005;96:626.)
© 2005 American Heart Association, Inc.

Cellular Biology

Distinct NF-B Regulation by Shear Stress Through Ras-Dependent IB Oscillations

Real-Time Analysis of Flow-Mediated Activation in Live Cells

Arunima Ganguli^*, Linda Persson^*, Ian R. Palmer^*, Iona Evans, Lin Yang, Rod Smallwood, Richard Black, Eva E. Qwarnstrom

From the Academic Unit of Cell Biology (A.G., L.P., I.R.P., I.E., L.Y., E.E.Q.), School of Medicine and Biomedical Sciences, University of Sheffield; the Division of Clinical Engineering (R.B.), School of Clinical Sciences, Faculty of Medicine, University of Liverpool; and the Department of Computer Science (R.S.), University of Sheffield, UK.

Correspondence to Prof Eva Qwarnstrom, Head Academic Unit of Cell Biology, School of Medicine and Biomedical Sciences, University of Sheffield, Glossop Rd, Sheffield S10 2 JF, UK. E-mail e.qwarnstrom{at}sheffield.ac.uk

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

Key Words: flow-mediated shear stress • IB • NF-B • relA • signal transduction

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