Published online before print February 24, 2005, doi: 10.1161/01.RES.0000160435.83210.95
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Submitted on August 17, 2004
Revised on February 10, 2005
Accepted on February 11, 2005
Distinct NF-
B Regulation by Shear Stress Through Ras-Dependent IB
Oscillations. Real-Time Analysis of Flow-Mediated Activation in Live Cells
Arunima Ganguli ;
From the Academy 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 Department of Biomedical Engineering (R.B.), University of Liverpool; and the Department of Computer Science (R.S.), University of Sheffield, UK.
* To whom correspondence should be addressed. 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 caused a successive induction of NF-B mediated 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 • I
B •
NF-B •
relA •
signal transduction
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