Distinct NF-B Regulation by Shear Stress Through Ras-Dependent IB Oscillations
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循环研究杂志 2005年第4期
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
the Department of Computer Science (R.S.), University of Sheffield, UK.
Abstract
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-BeCregulated 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
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.1eC3 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.1,10eC13
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.17eC19 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,20eC23 and to be tightly controlled by concentrations of pathway intermediates.23eC26
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 stresseCinduced 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
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 e/mL) at 37°C, in 5% CO2].23eC27,31 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 e cDNA/50 000 cells), or cotransfected (6.6 e cDNA), using calcium phosphate coprecipitation with glycerol shock, as previously.23eC26 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-e confocal pinhole aperture generating an optical section of 1.9 e, 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 C4742eC95), 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 x10eC2 NmeC2) 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-1 (10eC9 mol/L=nmol/L), a kind gift of Steve Poole, NIBSC.
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.23eC26 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.23eC26 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
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 (2.5x10eC2 m/s) and approached zero toward the walls (Figure 1A). Conversely, the shear stress gradient was minimal in the center, demonstrating levels of 1.4x10eC2 NmeC2 (0.14 dyne cmeC2) 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 0.6x10eC2 NmeC2 (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 (0.14x10eC2 NmeC2, 25 mL/hr) (data not shown).
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 10x10eC2 NmeC2, 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 NmeC2) 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).
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 10x10eC2 NmeC2) (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 stresseCinduced 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 stresseCregulated nuclear translocation of relA demonstrated a biphasic or cyclic pattern, not observed during IL-1 activation.
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).
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).
Discussion
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 cells33,34 and demonstrated to impact vascular permeability,35 adhesion,36 and control of regulatory mediators.37eC40 In addition, the relevance of the observed effects was supported by a correlation with flow-mediated induction of NF-BeCregulated genes. The significance of nuclear translocation of IB as a general mechanism in vascular shear stresseCmediated 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,7,23eC26 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.42eC44 Further, shear stresseCmediated NF-B activation, involving bidirectional trafficking of IB,20eC23 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-aseeCrelated 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 events2,30,48,49 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 stresseCinduced 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 degradationeCdependent 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.22eC23 Specifically, intracompartmental shuttling of IB likely affects NF-BeCmediated 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 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.).
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.
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the Division of Clinical Engineering (R.B.), School of Clinical Sciences, Faculty of Medicine, University of Liverpool
the Department of Computer Science (R.S.), University of Sheffield, UK.
Abstract
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-BeCregulated 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
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.1eC3 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.1,10eC13
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.17eC19 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,20eC23 and to be tightly controlled by concentrations of pathway intermediates.23eC26
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 stresseCinduced 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
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 e/mL) at 37°C, in 5% CO2].23eC27,31 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 e cDNA/50 000 cells), or cotransfected (6.6 e cDNA), using calcium phosphate coprecipitation with glycerol shock, as previously.23eC26 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-e confocal pinhole aperture generating an optical section of 1.9 e, 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 C4742eC95), 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 x10eC2 NmeC2) 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-1 (10eC9 mol/L=nmol/L), a kind gift of Steve Poole, NIBSC.
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.23eC26 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.23eC26 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
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 (2.5x10eC2 m/s) and approached zero toward the walls (Figure 1A). Conversely, the shear stress gradient was minimal in the center, demonstrating levels of 1.4x10eC2 NmeC2 (0.14 dyne cmeC2) 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 0.6x10eC2 NmeC2 (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 (0.14x10eC2 NmeC2, 25 mL/hr) (data not shown).
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 10x10eC2 NmeC2, 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 NmeC2) 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).
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 10x10eC2 NmeC2) (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 stresseCinduced 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 stresseCregulated nuclear translocation of relA demonstrated a biphasic or cyclic pattern, not observed during IL-1 activation.
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).
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).
Discussion
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 cells33,34 and demonstrated to impact vascular permeability,35 adhesion,36 and control of regulatory mediators.37eC40 In addition, the relevance of the observed effects was supported by a correlation with flow-mediated induction of NF-BeCregulated genes. The significance of nuclear translocation of IB as a general mechanism in vascular shear stresseCmediated 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,7,23eC26 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.42eC44 Further, shear stresseCmediated NF-B activation, involving bidirectional trafficking of IB,20eC23 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-aseeCrelated 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 events2,30,48,49 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 stresseCinduced 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 degradationeCdependent 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.22eC23 Specifically, intracompartmental shuttling of IB likely affects NF-BeCmediated 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 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.).
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.
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