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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

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
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

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Vascular cell responses are regulated by soluble and structural agonists and modulated by mechanical signals induced by hemodynamic forces with effects on signal transduction and gene activation.^1–3 Development of atherosclerosis has the hallmarks of a response to injury with a significant inflammatory component,^4,5 regulated in part by the transcription factor NF-B, activated in vascular cells during development of the disease.⁶ NF-B signal transduction and gene activation are induced by soluble mediators⁷ and by flow-mediated shear stress,^8,9 and contribute significantly to development of altered transcriptional profiles and distinct cellular phenotypes during progression of atherosclerosis.^1,10–13

The significance of NF-B in inflammation is demonstrated by the findings that its activation is a universal feature of inflammatory responses, that cognate binding sites are present in all inflammatory genes, and that NF-B knockout mice show disrupted inflammatory responses.¹⁴ NF-B transcription factors are hetero- or homodimers of Rel family proteins,¹⁵ with the heterodimer NF-B1/relA (p50/p65 NF-B) constituting the predominant species in many cell types and demonstrated to play a role in mechanical stimulation of the pathway.¹⁶ In the inactive state, NF-B is retained in the cytoplasm, complexed to inhibitors of NF-B (IBs). Activation of the pathway by cytokines or growth factors causes phosphorylation and degradation of the inhibitor and release of the NF-B transcription factor, resulting in its nuclear translocation and binding to cognate binding sites.^17–19 In addition, NF-B activation has been demonstrated to be regulated through intercompartmental trafficking of both free and complexed signaling components, resulting in distinct effects on pathway activation and DNA binding,^20–23 and to be tightly controlled by concentrations of pathway intermediates.^23–26

To determine the specific molecular mechanisms characterizing flow-mediated activation of NF-B, we have developed a system that allows real-time analysis of shear stress–induced signaling events at the single cell level. The experiments show that laminar flow has distinct effects on NF-B control in both endothelial and smooth muscle cells, resulting in alterations in the pathway steady state, which could underlie induction of the phenotypic changes characterizing cell populations at sites of development of atherosclerosis.¹

	Materials and Methods

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Plasmid Constructs
The IL-8 construct, containing the promoter and transcription start site, was subcloned into pEGFP1 (Clonetech) yielding plasmid pIL-8EGFP1.²⁷ The IB construct, a kind gift of Ronald Hay, University of St Andrews, Scotland, UK,²⁸ was cloned into pCMV-controlled pEGFP-N2 (Clontech) and into pEYFP-N1 (Clontech).^25,26 Plasmid pEGFPrelA was constructed by subcloning pBluescript RelA (AIDS Research and Reference Reagent Programme)²⁹ into pEGFP-C1, as described,^23,24 or into pECFP-C1 (Clontech).²⁶ N17Ras, a kind gift of Allan Hall, Imperial College, London, UK,³⁰ was cloned into a pCMV plasmid.³¹ All sequences were confirmed by sequence analysis.

Tissue Culture and Transfection
Monkey smooth muscle cells, a kind gift from Elaine Raines, University of Washington, Seattle, Wash, endothelial cells (HMEC-1, CDC,E-036 to 91/0, Ades, Lawley, and Candal), or HeLa cells were maintained in Dulbecco modified Eagle medium (DMEM, Gibco) [10% fetal calf serum (FCS), penicillin, and streptomycin (100 µg/mL) at 37°C, in 5% CO₂].^23–27,31 Cells were plated on fibronectin (Sigma) coated coverslips in 6-well plates at 50 000 or 100 000 cells/well, 24 or 48 hours before transfection, and transiently transfected with single constructs, described earlier (2.2 to 4.4 µg cDNA/50 000 cells), or cotransfected (6.6 µg cDNA), using calcium phosphate coprecipitation with glycerol shock, as previously.^23–26 Transfection levels, at a range of cDNA concentrations, were correlated with levels of respective endogenous protein, using immunostaining and Western analysis, and comparing with a series of standards, to determine nuclear and cytoplasmic concentrations, and nuclear/cytoplasmic ratios.^24,26 At fluorescent readings of less than 2 units, corresponding to at most 7 and 15 times endogenous levels of IB and relA, respectively, cells were characterized by cytoplasmic localization of the fusion protein. Further, cells transefected at these levels demonstrated the same biological responses as untransfected cells,^24,26,32 and were therefore used in all experiments.

Flow Chamber and Single Cell Analysis
Twenty-four or forty-eight hours after transfection, cells were transferred to a flow chamber, characterized for parameters related to velocity (m/s) and shear stress (N/m²), under steady state conditions using the fluid dynamics program CFX (ANSYS Inc) (Figure 1), which was then inserted into a Molecular Dynamics confocal laser scanning microscope (Model 2010) fitted with a 37°C stage incubator. EGFP, fusion proteins were visualized through a Nikon Diaphot microscope (Model 300) and a Silicon Graphics workstation (laser power 10 mW, band selection 488 nm, PMT voltage 750), using a PlanApo objective (20x, N.A. 0.75), and a 50-µm confocal pinhole aperture generating an optical section of 1.9 µm, as previously.^23,27,31 For FRET (fluorescence resonance energy transfer) analysis, images of ECFP, EYFP, and FRET, obtained using a 60x Plan Apo oil immersion objective (NA 1.4), a digital camera (12-bit Hamamatsu C4742–95), driven by OpenLab software (Improvision), and a series of filter sets (Omega Optical) as previously.²⁶ The flow chamber was fitted with a syringe pump (Alaris, NAC P6000) or a peristaltic pump (Watson/Marlow 101U/R). Infusion rates of 50 to 1500 mL/h (0.3 to 10 x10^–2 Nm^–2) were applied for 1 or 2 hours for analysis of signal transduction, and for up to 8 hours for analysis of gene activity. Fluorescence intensity was determined during continuous flow or, to optimize accuracy of the reading, during a brief interruption, at 5- or 10-minute intervals, or for gene regulation, once every hour. Using an automated stage drive, the same set of cells were visualized and images collected at each time point.^24,26 In some experiments, cells were instead stimulated with saturated levels of IL-1ß (10^–9 mol/L=nmol/L), a kind gift of Steve Poole, NIBSC.

View larger version (31K): [in this window] [in a new window]   Figure 1. Characterization of a system for administration of flow-mediated shear stress and confocal microscopy. A, Cross section (top) and longitudinal section (bottom) through the flow-chamber demonstrating the velocity profile, with a value of 2.5x10–2 ms–1 (for 250 mL/hr, scale 0.0025 to 0.0922 ms–1), in the central area, used for confocal analysis (square), with lower levels at the walls of the chamber (open arrowhead). Arrow indicates central axis and direction of flow. B, Shear stress distribution within the flow chamber demonstrating values of 1.38 to 1.48x10–2 Nm–2 overall (for 250 mL/hr; scale 0.0138 to 0.0148 Nm–2), and of 1.40 to 1.45x10–2 Nm–2 (4% variation) in the central area used for confocal analysis (square). Higher levels at the lateral part of the chamber are indicated by the superimposed crossbar (arrowhead). Arrow indicates central axis and direction of flow.

Quantitation of fluorescent protein concentration was done using scans taken horizontally through each nucleus, calculated from 2 to 3 cytoplasmic or nuclear readings for each cell, and data analyzed using NIH image and Matlab (Mathworks. Inc), as previously.^23–26 Data were expressed relative to levels of fusion protein at time 0 for each cell and averages determined using cell numbers as indicated, all in excess of 25 to 30 random cells for each condition, demonstrated to be representative of the culture as a whole.^23–26 All images were corrected for background, and FRET images in addition for overspill.^24,26 Control experiments for confocal microscopy showed that laser induced bleaching constituted no more than 1% to 3%, and for the FRET analysis that emission at 545 nm was totally eliminated by photobleaching at 500 nm (EYFP), with a linear correlation between the reduction in yellow and increase in cyan fluorescence. Averages of two or three independent experiments were determined for each protocol, and presented as mean±SEM.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Computational analysis of the velocity and shear stress gradients demonstrated an even distribution with a small range of variation within the flow chamber. The fluid velocity was maximal in the center of the chamber (2.5x10^–2 m/s) and approached zero toward the walls (Figure 1A). Conversely, the shear stress gradient was minimal in the center, demonstrating levels of 1.4x10^–2 Nm^–2 (0.14 dyne cm^–2) at 250 mL/hr, assuming a viscosity of 1 mPa·s, with higher levels at the walls (Figure 1B). Calculations performed for series of flow rates demonstrated that the velocity profile did not change appreciably over much of the chamber, and showed that variations in wall shear stress were less that 4% in the central area, used for data collection. Initial experiments using this system together with GFP-based methods demonstrated a successive increase in IL-8 promoter activity over time during application of flow-mediated shear stress (Figure 2A). Quantitation showed a nearly 2-fold enhancement in gene activation over 8 hours at a shear stress of 0.6x10^–2 Nm^–2 (100 mL/hr). In addition, these experiments showed that once activated at this rate for 3 hours, the increase in gene activation was maintained at significantly reduced levels of shear stress (0.14x10^–2 Nm^–2, 25 mL/hr) (data not shown).

View larger version (14K): [in this window] [in a new window]   Figure 2. Flow-mediated shear stress activates NF-B regulated genes. A, Cells transfected with an EGFP-containing construct induced through the IL-8 promoter were examined by confocal microscopy during application of flow-mediated shear stress. Confocal micrographs of the same areas were taken at time intervals during continuous flow of 100 mL/hr over 8 hours. Bar=10 µm. B, Data obtained by continuous single-cell readings from 3 random fields at low magnification such as in A, each containing 20 to 30 cells, at times indicated, were analyzed for fluorescent intensity using the NIH image program. Data are presented relative to levels before induction of flow and shown are the mean±SEM for two experiments.

Analysis of regulation of NF-B activation demonstrated that flow mediate shear stress caused a decrease in cytoplasmic levels of IB over time (Figure 3A). Quantitation of single cell readings over a range of flow rates (50 to 1500 mL/hr), corresponding to shear stresses of 0.3 to 10x10^–2 Nm^–2, demonstrated a positive correlation between the level of shear stress and the reduction in cytoplasmic IB (Figure 3B). Lower flow rates caused a gradual decrease in inhibitor levels of 20% to 30%. At rates of 150 mL/hr (1x10² Nm^–2) and higher, a pronounced reduction in cytoplasmic inhibitor levels was induced, corresponding to 60% (Figure 3B). The effect was transient or biphasic, characterized by an initial rapid decline during the first 10 to 20 minutes, followed by an increase in cytoplasmic IB and a second significant reduction (Figure 3B).

View larger version (33K): [in this window] [in a new window]   Figure 3. Flow-mediated effects on cytoplasmic IB levels demonstrate a biphasic pattern. A, Confocal micrographs of cells transfected with IB EGFP, as described in Materials and Methods, and subjected to flow-mediated shear stress. Micrographs were taken at intervals indicated, during induction of flow at 100 mL/hr. Bar=10 µm. B, Data from a series of experiments as in A using cells transfected with IBEGFP and subjected to flow-mediated shear stress at various rates ranging from 50 to 1500 mL/h, as indicated, corresponding to shear stresses of 0.3 to 10x10–2 Nm–2. Cytoplasmic levels of the IB fusion protein were monitored continuously, and micrographs taken at various times, as indicated, for each condition, and quantitated as described in the Material and Methods. Readings from a total of 172 single cells were expressed relative to initial levels for each cell in the unperturbed state, and data presented as averages for each flow rate and time, to demonstrate IB cytoplasmic levels in relation to both parameters. Lines show profiles for individual flow rates over time. SEM=1% to 7%.

Subsequent experiments designed to assess involvement of intercompartmental trafficking demonstrated that flow-mediated activation, in contrast to cytokine-induced responses, caused a pronounced increase in nuclear IBEGFP, which, in a significant proportion of the cells, reached levels comparable to that recorded for the transcription factor subunit relA (Figure 4A). Quantitation of this type of data demonstrated a pronounced increase in nuclear IB, over a range of shear stress levels (1 to 10x10^–2 Nm^–2) (Figure 4B), which corresponded to up to 70% of the concomitant reduction in cytoplasmic inhibitor levels. The effect, observed in both types of vascular cells, although in general somewhat more pronounced in endothelial cells, was characterized by oscillatory behavior with an overall successive increase in nuclear IB of 60% to 150% during 60 minutes of flow (Figure 4B). Parallel experiments demonstrated that the flow-induced reduction in cytoplasmic inhibitor levels of 60% was comparable to that caused by cytokine activation, as previously.^25,26 Further, they confirmed that, in contrast, the nuclear translocation of IB was observed during the shear stress–induced response only (Figure 4Ci and 4Cii). In comparison, experiments using EGFP tagged relA showed that flow-mediated effects on compartmental distribution of the NF-B subunit were comparable to those induced during cytokine stimulation (Figure 4Biii and 4Biv). However, similarly to effects on IB, shear stress–regulated nuclear translocation of relA demonstrated a biphasic or cyclic pattern, not observed during IL-1 activation.

View larger version (20K): [in this window] [in a new window]   Figure 4. Flow-mediated shear-stress induces nuclear translocation of both IB and relA. A, Cells transfected with IBEGFP (top row) or EGFPrelA (bottom row) were subjected to continuous flow at a rate of 150 mL/hr, demonstrating a pronounced increase in nuclear levels of both signaling components. Bar=10 µm. B, Quantitation of fluorescence intensities from single cell readings of cultures transfected with IBEGFP and subjected to flow-mediated shear stress as in A, demonstrating the same biphasic reductions in cytoplasmic IBEGFP concomitant with increases in nuclear inhibitor levels, characterized by a cyclic pattern, at flow rates of 150 (, ), 500 (, ), and 1500 (, ) mL/hr, corresponding to shear stresses of 1, 3, and 10x10–2 Nm–2. Data represent the averages of a total of 148 nuclear and cytoplasmic readings at each time-point; shown are the mean±SEM from 3 experiments, for each condition. Nuclear levels (filled symbols); cytoplasmic levels (unfilled symbols). C, Quantitation of data as in A, comparing flow-mediated and cytokine induced effects on nuclear and cytoplasmic levels of IBEGFP and EGFPrelA fusion proteins, over time. Nuclear (filled squares) and cytoplasmic (unfilled squares) fluorescence intensities from single cell readings of cultures subjected to flow-mediated shear stress as in A (62 cells), or to IL-1 (1 nmol/L) stimulation (75 cells), were quantitated using NIH image, and expressed relative to initial levels for each cell, and averaged for each time-point as indicated. Each graph represents the mean±SEM of two independent experiments. i, Cells transfected with IBEGFP and NF-B activity induced by flow-mediated shear stress as in A. ii, Cells transfected with IBEGFP and NF-B activity induced by IL-1ß (1 nmol/L). iii, Cells transfected with EGFPrelA and NF-B activity induced by flow-mediated shear stress, as in A. iv, Cells transfected with EGFPrelA and NF-B activity induced by IL-1ß (1 nmol/L).

The similar profiles obtained for nuclear translocation of NF-B and IB prompted analysis of complex formation using FRET (Figure 5A). These demonstrated, as earlier (see Figure 4A), a successive nuclear translocation of ECFPrelA, and showed a pronounced reduction in cytoplasmic IBEYFP, concomitant with an enhancement in nuclear levels. In contrast, the levels of FRET, proportional to the concentration of IBEYFP/ECFPrelA complexes, were successively reduced in both nucleus and cytoplasm. Quantitation of such data demonstrated the same profiles for relA (i) and IB (ii) as observed using EGFP-tagged components (see Figure 4B), and in addition, showed a successive reduction in FRET in both cellular compartments during continuous administration of flow (Figure 5Bii).

View larger version (22K): [in this window] [in a new window]   Figure 5. Shear-stress induced nuclear translocation of IB involves non-relA complexed inhibitor. A, Cells transfected with ECFPrelA (left) and IBEYFP (center), as described in the Material and Methods, were analyzed for nuclear and cytoplasmic levels of signaling intermediates, as above. In addition, the levels of FRET (right) in the nuclear and cytoplasmic compartments were determined during flow-mediated stress induced at 150 mL/hr. Bar=10 µm. B, Graphs demonstrating results of quantitation of experiments as in A. RelA (i) nuclear () and cytoplasmic levels (). IB (ii) nuclear () and cytoplasmic levels (), and FRET levels (ii), reflecting complexed signaling intermediates, in the nucleus () and cytoplasm (). Data from 162 nuclear and cytoplasmic single cell measurements at each time-point were expressed relative to levels at time 0 for each cell and averaged. Shown are the mean±SEM of three experiments.

Further experiments designed to assess the role of upstream structural regulators in flow-mediated NF-B activation demonstrated the involvement of the Ras GTP-ase (Figure 6A). Cotransfection with the dominant-negative N17 Ras significantly reduced the impact of shear stress on both cytoplasmic and nuclear levels of IB (Figure 6A). Quantitation demonstrated a reduction in the effects on cytoplasmic inhibitor levels of between 30% and 60%, during 60 minutes of flow, correlating with the level of the induced change (Figure 6B). In addition, these experiments showed that cells subjected to shear stress in the presence of N17Ras consistently demonstrated low nuclear levels, reflecting total inhibition of the flow-mediated nuclear translocation of IB (Figure 6B).

View larger version (21K): [in this window] [in a new window]   Figure 6. Flow-mediated effects on IB are regulated through the Ras GTP-ase. A, Cells were transfected with IBEGFP only, or cotransfected with N17Ras as indicated, and subjected to continuous flow-mediated shear stress induced at 150 mL/hr, and confocal micrographs taken at the times indicated. Bar=10 µm. B, Quantitation of effects of flow-mediated shear stress on IBEGFP turnover over time, using cells cotransfected with empty vector (control) or with the dominant-negative N17Ras. Fluorescence intensity readings were obtained from 134 single cell nuclear () and cytoplasmic () measurements at each time-point indicated and data quantitated using NIH image, as described in the Materials and Methods and expressed as a percent of flow-induced changes in control cultures. Graph shows mean±SEM for two experiments.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Using a well-controlled in vitro system, allowing continuous, real-time, single cell measurements of signal transduction events during flow-mediated stress, we demonstrate distinct activation of the inflammatory regulator NF-B. Whereas the NF-B transcription factor relA was regulated in a manner similar to that induced during activation by soluble mediators, flow-mediated control of IB was induced, to a significant extent, by a nondegradation-dependent mechanism, involving nuclear translocation of the inhibitor.

The biological significance of the flow-mediated nuclear translocation of IB was supported by its induction within the range of physiological shear stress levels estimated for endothelial and smooth muscle cells^33,34 and demonstrated to impact vascular permeability,³⁵ adhesion,³⁶ and control of regulatory mediators.^37–40 In addition, the relevance of the observed effects was supported by a correlation with flow-mediated induction of NF-B–regulated genes. The significance of nuclear translocation of IB as a general mechanism in vascular shear stress–mediated pathway activation was further demonstrated by its induction in both endothelial and smooth muscle cells. Although endothelial cells are continuously and directly affected by flow-mediated inputs, the impact on smooth muscle cells is induced by transvascular fluid filtration³³ and after injury to the intima.⁴¹ The consistency of the effect on IB regulation, in both cell types, and over a range of shear-stress levels, demonstrating a pattern distinct from that induced through cytokines and growth-factors,^7,23–26 supports the notion that this mechanism of activation, involving a biphasic or oscillatory response, is characteristic and significant in flow-mediated regulation of NF-B in vascular cells.

The distinct regulation of flow-mediated activation, controlled only to a minor extent through the mechanism induced by cytokine signaling, involving degradation and subsequent de novo synthesis of IB,¹⁴ will likely be of significance during coregulation of soluble and structural agonists during development of inflammatory vascular disease. The relative impact of the distinct mechanisms on pathway activation will be determined in part by the soluble ligand concentration and receptor binding, possibly influenced by convective effects.^42–44 Further, shear stress–mediated NF-B activation, involving bidirectional trafficking of IB,^20–23 is likely less sensitive to limiting events related to degradation-dependent mechanisms controlling cytokine-mediated activation. The data, supported by the FRET analysis, indicate that flow-mediated activation causes dissociation of the IB/NF-B complex, and suggest that both components remain intact, possibly as a direct consequence of structural changes involving the cytoskeleton and GTP-ase–related proteins.⁴⁵ Alterations in the balance between degradation and transport of the inhibitor could result from changes in the level of agonist-induced inhibitor phosphorylation, or ubiquitination, required for proteosome-mediated degradation.¹⁷ Regulation through effects on IB phosphorylation is supported by the demonstrated role of IKK in shear-stress induced NF-B activation,⁴⁶ and by ongoing studies showing involvement of the NF-B inducing kinase, NIK, in flow-mediated activation of the pathway (Qwarnstrom, unpublished data, 2004). Changes in the relative impact of various mechanisms of regulation, possibly induced through a select set of upstream signaling intermediates, are further in agreement with the demonstrated involvement of the Ras GTP-ase.^31,32

The pronounced reduction in the flow-mediated alterations observed in the presence of the dominant-negative Ras is consistent with its recently demonstrated significance in fluid shear stress.⁴⁷ Related effects on cell shape and the cytoskeleton through mechanotransduction of signaling events^2,30,48,49 could in part be responsible for the induced IB translocation. Total inhibition of flow-mediated nuclear transport of IB, in the presence of N17Ras, likely reflects regulation of active and specific control,^50,51 because in the absence of biomechanical input, enhanced Ras activity causes cytoplasmic retention of the inhibitor.⁵² The resulting enhancement in nuclear IB levels could impact reestablishing a cytoplasmic pool of inactivated NF-B after pathway induction.^14,21 In addition, the shear stress–induced intercompartmental trafficking of noncomplexed inhibitor is likely to result in altered profiles of gene targeting by selectively blocking DNA/NF-B binding.^20,53 Flow-mediated translocation of free IB, together with induction of stress responsive elements (SSRE) in the presence of a low level of IB degradation–dependent activation, could account for qualitative and selective alterations in gene expression, resulting in unique patterns of transcriptional profiles and translated into distinct functional phenotypes.^54,55

Changes in nuclear concentration of signaling intermediates is expected to ultimately impact cytokine-mediated events, at the level of signal transduction and gene regulation, through effects on nuclear/cytoplasmic shuttling of both NF-B and its inhibitor.^22–23 Specifically, intracompartmental shuttling of IB likely affects NF-B–mediated responses by altering the relative contributions of the various inhibitor isoforms and disturbing the coordinated control of signals required to generate the characteristic NF-B activation profile.⁵⁶ Considering the distinct roles for the various IB family members, enhanced levels of free IB, resulting from decreased proteosome degradation, is in agreement with a reduced relative impact of the dampening effects of the ß and isoforms and with sustained oscillations during activation.⁵⁶

In addition, the tight interdependence between the relative levels of cytoplasmic IB and relA is expected to be significantly affected by flow-induced nuclear translocation of the inhibitor, resulting in fundamental changes in pathway control during activation. Thus, although activation of NF-B can be induced over a range of concentrations of endogenous signaling components,³³ pathway functionality is highly controlled by changes in the NF-B/IB ratio. This has been demonstrated by single cell analysis,^24,26 and confirmed by mathematical modeling, showing IB turnover and nuclear translocation of NF-B to be tightly controlled by relative concentrations of the complex components (Dower and Qwarnstrom, unpublished data, 2004; Pogson et al, unpublished data, 2005). Changes in IB concentration, such as induced through flow-mediated stress are therefore likely to have potent effects on both resting conditions, and on regulation in the context of cytokine and growth-factor mediated responses by altering the balance determined by relative levels of signaling components and thereby disrupting the system steady state.

In summary, using single cell analysis, we have demonstrated distinct regulation of NF-B by flow-mediated activation in endothelial and smooth muscle cells, which is consistent with enhanced and altered inflammatory responses during development of atherosclerosis. We find that flow-induced shear stress affects select pathway-regulatory events, impacting relative levels of signaling components, with consequences for the system steady state. Such changes are thought to affect both specificity and extent of gene induction, and to be amplified in the context of activation through growth-factors and cytokines. Induction of compensatory mechanisms related to these flow-induced alterations, together with ensuing changes in regulation of inflammatory responses, could underlie the challenge in phenotypic stability of vascular cells in atherosclerotic lesions.

	Acknowledgments

 
The confocal microscopy facility was cofunded by the Wellcome Trust and the Medical Research Council. The work was supported by grants from the Medical Research Council, Biotechnology and Biological Sciences Research Council, and Nuffield Foundation (to E.E.Q.) and from the Biotechnology and Biological Sciences Research Council (to R.S.).

	Footnotes

 
Present address for L. Persson is the Department of Physics, Lund Institute of Technology, University of Lund, Sweden; for I. Evans, Department of Medicine, Royal Free & University College Medical School, Rayne Institute, London, UK; for L. Yang, F Brigham Women‘s Hospital, Center for Excellence in Vascular Biology, New Research Building/Harvard Institution of Medicine, Boston, Mass.

*These authors contributed equally to the work.

Original received August 17, 2004; revision received February 10, 2005; accepted February 11, 2005.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Gimbrone MA. Vascular endothelium, hemodynamic forces, and atherogenesis. Am J Pathol. 1999; 155: 1–5.[Free Full Text]
2. Ingber DE. Mechanical signaling and the cellular response to extracellular matrix in angiogenesis and cardiovascular physiology. Circ Res. 2002; 91: 877–887.[Abstract/Free Full Text]
3. Davies PF. Flow-mediated endothelial mechanotransduction. Physiol Rev. 1995; 75: 519–560.[Abstract/Free Full Text]
4. Raines EW, Dower SK, Ross R. Interleukin-1 Mitogenic Activity for Fibroblasts and Smooth Muscle Cells is due to PDGF-AA. Science. 1989; 243: 393–396.[Abstract/Free Full Text]
5. Ross R. The pathogenensis of atherosclerosis: a perspective for the 1990s. Nature. 1993; 362: 801–809.[CrossRef][Medline] [Order article via Infotrieve]
6. Brand K Page S, Rogler G, Bartsch A, Brandl R, Knuechel R, Page M, Kaltschmidt C, Baeuerle PA, Neumeier D. Activated transcription factor nuclear factor kappa B is present in the atherosclerotic lesion. J Clin Invest. 1996; 97: 1715–1722.[Medline] [Order article via Infotrieve]
7. Bauerle PA, Baltimore D. NF-B: ten years after. Cell. 1996; 87: 13–20.[CrossRef][Medline] [Order article via Infotrieve]
8. Khachigian L, Resnick N, Gimbrone MJ, Collins T. Nuclear factor-B interacts functionally with the platelet-derived growth-factor B-chain shear-stress response element in vascular endothelial cells exposed to fluid shear stress. J Clin Invest. 1995; 96: 1169–1175.[Medline] [Order article via Infotrieve]
9. Lan Q, Mercurius K, Davies P. Stimulation of transcription factors NF-B and AP 1 in endothelial cells, subjected to shear stress. Biochem Biophys Res Commun. 1994; 201: 950–956.[CrossRef][Medline] [Order article via Infotrieve]
10. Garcia-Cardena G, Comander JI, Blackman BR, Anderson KR, Gimbrone MA. Mechanosensitive endothelial gene expression profiles: scripts for the role of hemo-dynamics in atherogenesis? Ann NY Acad Sci. 2001; 947: 1–6.[Abstract/Free Full Text]
11. Garcia-Cardena G, Comander JI, Anderson KR, Blackman BR, Gimbrone MA, Biomechanical activation of vascular endothelium as a determinant of its functional phenotype Proc Natl Acad Sci USA. 2001; 98: 4478–4485.[Abstract/Free Full Text]
12. Takayama K, Garcia-Cardena G, Comander J, Gimbrone MA. Unveiling anti-inflammatory circuitry in human macrophages by transcriptional profiling. Circulation. 2001; 104: 1531–1539.
13. Blackman BR, Garcia-Cardena G, Gimbrone MA. A new in vitro model to evaluate differential responses of endothelial cells to simulated arterial shear stress waveforms J Biomech Eng-Trans. 2002; 124: 397–407.
14. Beg AA, Baltimore D. An essential role for NF-kappa B in preventing TNF-alpha-induced cell death. Science. 1996; 274: 782–784.[Abstract/Free Full Text]
15. Verma IM, Stevenson JK, Schwarz EM, van Antwerp D, Miyamotot S. Rel/ NF-kappa-B/I-kappa-B family: intimate tales of association and dissociation. Genes Dev. 1995; 9: 2723–2735.[Free Full Text]
16. Hishikawa K, Oemar BS, Yang Z. Pulsatile stretch stimulates superoxide production and activates nuclear factor-B in human coronary smooth muscle. Circ Res. 1997; 81: 797–803.[Abstract/Free Full Text]
17. Finco TS, Baldwin AS. Mechanistic aspects of NF-B regulation: the emerging role of phosphorylation and proteolysis. Immunity. 1995; 3: 263–272.[CrossRef][Medline] [Order article via Infotrieve]
18. Zandi E, Rothwarf DM, Delhase M, Hayakawa M, Karin M. The I kappa B kinase complex (IKK) contains two kinase subunits, IKK alpha and IKK beta, necessary for IB phosphorylation and NF-B activation. Cell. 1997; 91: 243–252.[CrossRef][Medline] [Order article via Infotrieve]
19. Hu Y, Baud V, Delhase M, Zhang PL, Deerinck T, Ellisman M, Johnson R, Karin M. Abnormal morphogenesis but intact IKK activation in mice lacking the IKK alpha subunit of IB kinase. Science. 1999; 284: 316–320.[Abstract/Free Full Text]
20. Arenzana-Seisdedos F, Thompson J, Rodrigues MS, Bachelerie F, Thomas D, Hay, RT. Inducible nuclear expression of newly synthesised IB alpha negatively regulates DNA-binding and transcriptional activities of NF-B. Mol Cell Biol. 1995; 15: 2689–2696.[Abstract]
21. Arenzana-Seisdedos F, Turpin P, Rodrigues MS, Thomas D, Hay RT, Virelizier JL, Dargemont C. Nuclear localisation of IB alpha promotes active transport of NF-B from the nucleus to the cytoplasm. J Cell Sci. 1997; 110: 369–378.[Abstract]
22. Johnson C, van Antwerp D, Hope TJ. AnN-terminal nuclear export signal is required for the nucleocytoplamsic shuttling of IkappaB alpha. EMBO J. 1999; 18: 6682–6693.[CrossRef][Medline] [Order article via Infotrieve]
23. Carlotti F, Dower SK, Qwarnstrom EE. Dynamic shuttling of NF-B between the nucleus and the cytoplasm as a consequence of inhibitor dissociation. J Biol Chem. 2000; 275: 41028–41034.[Abstract/Free Full Text]
24. Carlotti F, Chapman R, Dower SK, Qwarnstrom EE. Activation of nuclear factor B in single living cells: dependence of nuclear translocation and anti-apoptotic function on EGFPRELA concentration. J Biol Chem. 1999; 274: 37941–37949.[Abstract/Free Full Text]
25. Yang L, Chen H, Qwarnstrom EE. Degradation of IB during NF-B activation is limited by a post-phosphorylation/ubiquitination event. BBRC. 2001; 285: 603–608.[Medline] [Order article via Infotrieve]
26. Yang L, Ross K, Qwarnstrom E E. RelA Regulation of IB phosphorylation: A positive feedback-loop for high affinity NF-B complexes. J Biol Chem. 2003; 278: 30881–30888.[Abstract/Free Full Text]
27. Kiss-Toth E, Guesdon FMJ, Wyllie D, Qwarnstrom EE, Dower SK. A novel mammalian expression screen exploiting green fluorescent protein–based transcription detection in single cells. J Immunol Methods,. 2000; 239: 125–135.[CrossRef][Medline] [Order article via Infotrieve]
28. Rodrigues MS, Michalopoulos I, Arenzana-Seisdedos F, Hay RT. Inducible degradation of IB alpha in vitro and in vivo requires the acidic C-terminal domain of the protein. Mol Cell Biol. 1995; 15: 2413–2419.[Abstract]
29. Duckett CP, Perkins ND, Kowalik TF, Schmid RM, Huang ES, Baldwin AS, Nabel GJ. Dimerization of NF-B2 with relA (p65) regulates DNA binding, transcriptional activation an inhibition by an IB-alpha (MAD-3). Mol Cell Biol. 1993; 13: 1315–1322.[Abstract/Free Full Text]
30. Hall A. Rho GTP-ases and the actin cytoskeleton. Science. 1998; 279: 509–514.[Abstract/Free Full Text]
31. Caunt J, Kiss-Toth E, Carlotti F, Chapman R, Qwarnstrom EE. Ras controls Tumor Necrosis Receptor-associated Factor (TRAF) 6-dependent induction of nuclear factor-B: selective regulation through receptor signalling components. Biol Chem. 2001; 276: 6280–6288.
32. Schooley K, Zhu P, Carlotti F, Dower SK, Qwarnstrom EE. Activation of relA containing NF-B dimers by Interleukin-1: evidence for complex dynamics at the single cell level. Biochem J. 2003; 369: 331–339.[CrossRef][Medline] [Order article via Infotrieve]
33. Kim MH, Harris NR, Korzick DH, Tarbell JM. Control of the arteriolar myogenic response by transvascular fluid filtration. Microvasc Res. 2004; 68: 30–37.[CrossRef][Medline] [Order article via Infotrieve]
34. Wang DM, Tarbell JM. Modelling interstitial flow in an artery wall allows estimation of wall shear stress on smooth muscle cells. J Biomech Eng. 1995; 117: 358–363.[Medline] [Order article via Infotrieve]
35. Jo H, Dull RO, Hollis TM, Tarbell JM. Endothelial albumine permeability is shear dependent, time dependent, and reversible. Am J Physiol. 1991; 260: H1992–H1996.[Medline] [Order article via Infotrieve]
36. Boyce JA, Mellor EA, Perkins B, Lim YC, Luscinskas FW. Human mast cell progenitors use alpha 4-integrin, VCAM-1, and PSGL-1 E-selectin for adhesive interactions with human vascular endothelium under flow conditions. Blood. 2002; 99: 2890–2896.[Abstract/Free Full Text]
37. Alshihabi S, Chang Y, Frangos JA, Tarbell JM. Shear stress-induced release of PGI₂ and RGE₂ by vascular smooth muscle cells. Biochem Biophys Res Commun. 1996; 224: 808–814.[CrossRef][Medline] [Order article via Infotrieve]
38. Papadaki M, Tilton RG, Eskin SJ, McIntire LV, Nitric oxide production by cultured human aortic smooth muscle cells: stimulation by fluid flow. Am J Physiol Heart Circ Physiol. 1998; 274: H616–H626.[Abstract/Free Full Text]
39. Wang S, Tarbell JM. Effect of fluid flow on smooth muscle cells in a 3-dimensional collagen gel model. Arterioscler Thromb Vasc Biol. 2000; 20: 2220–2225.[Abstract/Free Full Text]
40. Ziegler T, Bouzourene K, Harrison VJ, Brunner HR, Hayoz D. Influence of oscillatory and unidirectional flow environments on the expression of endothelin and nitric oxide synthase in cultured endothelial cells. Artherioscler Thromb Vasc Biol. 1998; 18: 686–692.[Abstract/Free Full Text]
41. Lipowsky H. Shear stress in the circulation. In: Koller C, Karley G. eds. Flow Dependent Regulation of Vascular Function. New York, NY: Oxford University Press, 1995; 28–45.
42. Mo M, Eskin SG, Schilling WP. Flow-induced changes in Ca²⁺ signalling of vascular endothelial cells: effect of shear stress and ATP. Am J Physiol. 1991; 29: H1698–H1707.
43. Dull RO, Davies PF. Flow modulation of agonist (ATP)-response (Ca²⁺) coupling in vascular endothelial cells. Am J Physiol. 1991; 262: H149–H154.
44. Dull RO, Tarbell JM, Davies PF. Mechanisms of flow-mediated signal transduction in endothelial cells: kinetics of ATP surface concentrations. J Vasc Res. 1992; 29: 410–419.[Medline] [Order article via Infotrieve]
45. Fenwick C, Na SY, Voll RE, Zhong H, Im SY, Lee JW, Ghosh S. A subclass of Ras proteins that regulate the degradation of IkappaB. Science. 2000; 287: 869–873.[Abstract/Free Full Text]
46. Bhullar IS, Li Y-S, Miao H, Zandi E, Lim M, Shyy JY-J, Chien S. Fluid shear stress activation of IB kinase is integrin dependent. J Biol Chem. 1998; 273: 30544–30549.[Abstract/Free Full Text]
47. Gudi S, Huvar I, White CR, McKnight NL, Dusserre N, Boss GR, Frangos JA. Rapid activation of Ras by fluid flow is mediated by G alpha(q) and G beta gamma subunits of heterotrimeric G proteins in human endothelial cells. Arterioscler Thromb Vasc Biol. 2003; 23: 994–1000.[Abstract/Free Full Text]
48. Downward J. Ras signalling and apoptosis. Curr Opin Genet Develop. 1998; 8: 49–54.[CrossRef][Medline] [Order article via Infotrieve]
49. Wang N, Butler JP, Ingber DE. Mechanotransduction across the cell-surface and through the cytoskeleton. Science. 1993; 260: 1124–1127.[Abstract/Free Full Text]
50. Sachdev S, Hoffman A, Hannink M. Nuclear localisation of IB is mediated by the second ankyrin repeat: the IB ankyrin repeats define a novel class of cis-acting nuclear import sequences. Mol Cell Biol. 1998; 18: 2524–2534.[Abstract/Free Full Text]
51. Turpin P, Hay RT, Dargemenont C. Characterisation of IB nuclear import pathway. J Biol Chem. 1999; 274: 6804–6812.[Abstract/Free Full Text]
52. Prigent M, Barlat I, Langen H, Dargemont C. IkappaBalpha and IkappaBalpha /NF-kappa B complexes are retained in the cytoplasm through interaction with a novel partner, RasGAP SH3-binding protein 2. J Biol Chem. 2000; 275: 36441–36449.[Abstract/Free Full Text]
53. Zabel U, Baeurle PA. Purified human IB can rapidly dissociate the complex of the NF-B transcription factor with its cognate DNA Cell. 1990; 61: 255–265.[CrossRef][Medline] [Order article via Infotrieve]
54. Nagel T, Resnick N, Dewey CF, Gimbrone MA. Vascular endothelial cells respond to spatial gradients in fluid shear stress by enhanced activation of transcription factors. Artheriosc Thromb and Vasc Biol. 1999; 19: 1825–1834.
55. Gimbrone MA, Topper JN, Nagel T, Anderson KR, Garcia-Cardena G. Endothelial dysfunction, hemodynamic forces, and atherogenesis. Ann NY Acad Sci. 2000; 902: 230–240.[Abstract/Free Full Text]
56. Hoffman A, Levchenko A, Scott ML, Baltimore D. The NF-B signalling module: temporal control and selective gene activation. Science. 2002; 298: 1241–1245.[Abstract/Free Full Text]

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