Distinct NF-kappaB regulation by shear stress through Ras-dependent IkappaBalpha oscillations: real-time analysis of flow-mediated activation in live cells.
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.
Ganguli
Arunima
A
Academic Unit of Cell Biology, School of Medicine and Biomedical Sciences, University of Sheffield, UK.
Persson
Linda
L
Palmer
Ian R
IR
Evans
Iona
I
Yang
Lin
L
Smallwood
Rod
R
Black
Richard
R
Qwarnstrom
Eva E
EE
D000818:Animals; D002478:Cells, Cultured / Q000378:metabolism; D003199:Computer Systems; D031541:Fluorescence Resonance Energy Transfer; D017930:Genes, Reporter; D011905:Genes, ras; D000882:Haplorhini; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D020672:I-kappa B Proteins / Q000502:physiology*; D016209:Interleukin-8 / Q000235:genetics; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics; D018613:Microscopy, Confocal; D032389:Myocytes, Smooth Muscle / Q000378:metabolism; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000502:physiology*; D010641:Phenotype; D011401:Promoter Regions, Genetic; D021381:Protein Transport; D016283:Proto-Oncogene Proteins p21(ras) / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000502:physiology; D012212:Rheology; D015398:Signal Transduction / Q000502:physiology; D013314:Stress, Mechanical; D051996:Transcription Factor RelA; D014158:Transcription, Genetic; D014162:Transfection
D020672:I-kappa B Proteins; D016209:Interleukin-8; D008164:Luminescent Proteins; D016328:NF-kappa B; C000606204:NFKBIA protein, human; D011993:Recombinant Fusion Proteins; D051996:Transcription Factor RelA; D000072000:NF-KappaB Inhibitor alpha; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras)