Distinct NF-kappaB regulation by shear stress through Ras-dependent IkappaBalpha oscillations: real-time analysis of flow-mediated activation in live cells.
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.
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ABSTRACT: When bone is mechanically loaded fluid shear stress (FSS) is generated as a result of the movement of interstitial fluid across the membranes of osteoblasts and osteocytes. This external mechanical loading stimulates changes in the activity of cytoplasmic signaling molecules and alters gene expression in bone cells. This process, referred to as mechanotransduction, is vital for maintaining bone health in vivo by regulating the balance between bone formation and bone resorption. This current study focuses on the role of focal adhesions, sites of integrin-mediated cellular attachment to the extracellular matrix, and their proposed function as mechanosensors in bone cells. We examined the role of a key component of focal adhesions and of mechanotransduction, focal adhesion kinase (FAK) in regulation of FSS- and tumor necrosis factor-alpha (TNF-alpha)-induced activation of nuclear factor-kappa B (NF-kappaB) signaling in osteoblasts. Immortalized FAK(+/+) and FAK(-)(/)(-) osteoblasts were exposed to periods of oscillatory fluid shear stress (OFF) and NF-kappaB activation was analyzed. We determined that FAK is required for OFF-induced nuclear translocation and activation of NF-kappaB in osteoblasts. In addition we found that OFF-induced phosphorylation of the IkappaB kinases (IKKalpha/beta) in both FAK(+/+) and FAK(-/-) osteoblasts, but only FAK(+/+) osteoblasts demonstrated the resulting degradation of NF-kappaB inhibitors IkappaBalpha and IkappaBbeta. OFF did not induce the degradation of IkappaBepsilon or the processing of p105 in either FAK(+/+) and FAK(-/-) osteoblasts. To compare the role of FAK in mediating OFF-induced mechanotransduction to the well characterized activation of NF-kappaB by inflammatory cytokines, we exposed FAK(+/+) and FAK(-/-) osteoblasts to TNF-alpha. Interestingly, FAK was not required for TNF-alpha induced NF-kappaB activation in osteoblasts. In addition we determined that TNF-alpha treatment did not induce the degradation of IkappaBbeta as did OFF. These data indicate a novel relationship between FAK and NF-kappaB activation in osteoblast mechanotransduction and demonstrates that the mechanism of FSS-induced NF-kappaB activation in osteoblasts differs from the well characterized TNF-alpha-induced activation.Bone 03/2010; 47(1):74-82. · 4.46 Impact Factor
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ABSTRACT: Endothelial cells are known to respond to hemodynamic forces. Their phenotype has been suggested to differ between atheroprone and atheroprotective regions of the vasculature, which are characterized by the local hemodynamic environment. Once an atherosclerotic plaque has formed in a vessel, the obstruction creates complex spatial gradients in wall shear stress. Endothelial cell response to wall shear stress may be linked to the stability of coronary plaques. Unfortunately, in vitro studies of the endothelial cell involvement in plaque stability have been limited by unrealistic and simplified geometries, which cannot reproduce accurately the hemodynamics created by a coronary stenosis. Hence, in an attempt to better replicate the spatial wall shear stress gradient patterns in an atherosclerotic region, a three dimensional asymmetric stenosis model was created. Human abdominal aortic endothelial cells were exposed to steady flow (Re=50, 100, and 200 and tau=4.5 dyn/cm(2), 9 dyn/cm(2), and 18 dyn/cm(2)) in idealized 50% asymmetric stenosis and straight/tubular in vitro models. Local morphological changes that occur due to magnitude, duration, and spatial gradients were quantified to identify differences in cell response. In the one dimensional flow regions, where flow is fully developed and uniform wall shear stress is observed, cells aligned in flow direction and had a spindlelike shape when compared with static controls. Morphological changes were progressive and a function of time and magnitude in these regions. Cells were more randomly oriented and had a more cobblestone shape in regions of spatial wall shear stress gradients. These regions were present, both proximal and distal, at the stenosis and on the wall opposite to the stenosis. The response of endothelial cells to spatial wall shear stress gradients both in regions of acceleration and deceleration and without flow recirculation has not been previously reported. This study shows the dependence of endothelial cell morphology on spatial wall shear stress gradients and demonstrates that care must be taken to account for altered phenotype due to geometric features. These results may help explain plaque stability, as cells in shoulder regions near an atherosclerotic plaque had a cobblestone morphology indicating that they may be more permeable to subendothelial transport and express prothrombotic factors, which would increase the risk of atherothrombosis.Journal of Biomechanical Engineering 08/2010; 132(8):081013. · 1.52 Impact Factor
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ABSTRACT: Src kinase, the first tyrosine kinase discovered, has been shown to play critical roles in a variety of cellular processes, including cell motility/migration, mechanotranduction, and cancer development. Based on fluorescent resonance energy transfer (FRET), we have developed and characterized a genetically encoded single-molecule Src biosensor, which enables the imaging and quantification of temporal-spatial activation of Src in live cells. In this paper, we summarize the application of this biosensor to study a variety of cellular functions. First, we introduced a local mechanical stimulation by applying laser-tweezer-induced traction on fibronectin-coated beads adhered to the cells. Using a membrane-anchored Src biosensor, we observed a wave propagation of Src activation in a direction opposite to the applied force. This Src reporter was also applied to visualize the interplays between cell-cell and cell-ECM adhesions. The results indicate that integrin-ligation can induce Src activation around cell-cell junctions and cause the disruption of adherens junctions. Lastly, the flow-induced dynamic Src activation at subcellular levels was visualized by the FRET biosensor simultaneously with actin-fused mCherry, a red fluorescence protein. Our results indicate that shear stress induced a moderate up-regulation of Src activation in the whole cell, but a significant translocation of active Src from perinuclear regions toward cell periphery. In summary, our novel Src biosensor has made it possible to monitor key signaling transduction cascades involving Src in live cells with temporal-spatial characterization in mechanobiology.Proc SPIE 02/2008;