Published online before print July 31, 2008, doi: 10.1161/CIRCRESAHA.108.182097
© 2008 American Heart Association, Inc.
Integrative Physiology |
p21-Activated Kinase Signaling Regulates Oxidant-Dependent NF-
B Activation by Flow
From the Robert M. Berne Cardiovascular Research Center (A.W.O., C.H., B.R.B., M.A.S.), Departments of Microbiology (A.W.O., M.A.S.) and Biomedical Engineering (B.R.B.), Mellon Prostate Cancer Research Center (M.A.S.), University of Virginia, Charlottesville; and Department of Pathology (A.W.O.), Louisiana State University Health Sciences Center, Shreveport, La.
Correspondence to Martin Alexander Schwartz, University of Virginia, Cardiovascular Research Center, 415 Lane Rd, Charlottesville, VA 22908. E-mail maschwartz{at}virginia.edu
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Abstract |
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Disturbed blood flow induces inflammatory gene expression in endothelial cells, which promotes atherosclerosis. Flow stimulates the proinflammatory transcription factor nuclear factor (NF)-
B through integrin- and Rac-dependent production of reactive oxygen species (ROS). Previous work demonstrated that NF-B activation by flow is matrix-specific, occurring in cells on fibronectin but not collagen. Activation of p21-activated kinase (PAK) followed the same matrix-dependent pattern. We now show that inhibiting PAK in cells on fibronectin blocked NF-B activation by both laminar and oscillatory flow in vitro and at sites of disturbed flow in vivo. Constitutively active PAK rescued flow-induced NF-B activation in cells on collagen. Surprisingly, PAK was not required for flow-induced ROS production. Instead, PAK modulated the ability of ROS to activate the NF-B pathway. These data demonstrate that PAK controls NF-B activation by modulating the sensitivity of cells to ROS.
Key Words: endothelial cell dysfunction • extracellular matrix • fluid shear stress • NF-B • reactive oxygen species
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Introduction |
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Atherosclerosis, a chronic inflammatory disease of the artery wall, is highly affected by risk factors such as hyperlipidemia, smoking, and diabetes. These factors, however, are relatively uniform throughout the vasculature, whereas atherosclerosis occurs mainly at vessel curvatures, branch points, and bifurcations that show disturbances in blood flow.1,2 Endothelial cells (ECs) in these regions show decreased flow-induced nitric oxide release and enhanced inflammatory gene expression, so called EC dysfunction.3 Systemic risk factors stimulate these sites to develop into fatty streaks, regions of lipid-laden tissue macrophages, and subsequently into atherosclerotic plaques.
Flow patterns critically regulate EC function in vitro. Applying laminar flow to EC monolayers triggers transient activation of signaling events, including increased integrin affinity and activation of Rho family GTPases, nuclear factor (NF)-B and c-Jun N-terminal kinase (JNK).4 However, these events are downregulated at later times as cells adapt. Prolonged laminar flow decreases oxidative stress, EC turnover, and inflammatory gene expression.5 By contrast, disturbed flow stimulates sustained activation of inflammatory events and endothelial turnover.6–8
The NF-B family of transcription factors is an important component of the endothelial inflammatory response. NF-B consists of heterodimeric protein complexes, the most studied involving the p65 and p50 subunits (hereafter referred to as NF-B), that stimulate antiapoptotic and proinflammatory gene expression.9 Inactive p65 is held in the cytoplasm by inhibitory IB proteins.9 When activated, the upstream IKK kinases phosphorylate IB, leading to its ubiquitination and degradation, thereby allowing p65 to translocate to the nucleus. IKKs also phosphorylate p65 on a critical serine (S536) that modulates transcriptional activity.10 Multiple atherogenic stimuli, including disturbed flow, inflammatory cytokines, and reactive oxygen species (ROS) activate NF-B.11 Atherosclerosis-prone arterial regions show chronic NF-B activation and NF-B–dependent gene expression, including adhesion molecules and inflammatory cytokines.12,13 Endothelial NF-B is thereby thought to contribute to atherogenesis by modulating inflammatory gene expression.
The pathway by which flow stimulates NF-B has been studied extensively. Flow appears to act directly on a complex of proteins at cell–cell junctions, resulting in stimulation of phosphatidylinositol 3-kinase and conversion of integrins to a high affinity state.14,15 Newly activated integrins bind extracellular matrix (ECM) proteins, which initiates intracellular signals that include activation of the small GTPase Rac.14 Rac activates the NADPH oxidase complex to produce ROS,16 which stimulates NF-B–inducing kinase (NIK) and IKKβ,17,18 critical kinases in the classic NF-B activation pathway. All of these components are required for NF-B activation by flow.14,17,19,20
The composition of the subendothelial ECM dictates which of the many EC integrins bind ligand following flow-induced activation.21 In accordance with the above model, the subendothelial ECM also strongly influences signaling in response to flow through the distinct signaling properties of different integrins. For example, flow activates NF-B in ECs on fibronectin (FN) and fibrinogen, which are found mainly at sites of injury and inflammation, but not on collagen (Coll) or laminin, components of the normal basement membrane.22 Importantly, there is little FN or fibrinogen beneath the endothelium in most of the vasculature, but these proteins are found are at sites of disturbed flow in vivo.22 This matrix remodeling correlates closely with endothelial inflammatory markers such as intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1. Interestingly, deletion of an alternatively spliced domain of FN that reduces its assembly into matrix decreases atherosclerosis in hypercholesterolemic ApoE–/– mice.23 Taken together, these data support a role for matrix remodeling in EC dysfunction and atherosclerosis.
PAK1 to -3 are a group of highly homologous Ser/Thr protein kinases that serve as effectors for Rac and Cdc42.24 PAK1 and -2 are found in ECs, whereas PAK3 is found largely in the brain. PAK is maintained in an inactive state by its N-terminal autoinhibitory domain (AID), which binds and blocks the kinase domain. Activation results in dissociation of the AID–kinase domain complex and phosphorylation of residues that further block autoinhibition. More than 25 substrates for PAKs have been identified, including many cytoskeletal proteins, mitogen-activated protein (MAP) kinase pathway components, and regulators of cell survival. PAK also regulates NF-B activation in a few systems.25,26 However, this control is by no means universal,27 and constitutively active PAK does not activate NF-B.27,28 Flow activates PAK in ECs, and active PAK regulates junctional integrity and monolayer or vessel permeability.29 Interestingly, PAK shows the same matrix-dependence as NF-B, occurring in ECs on FN or fibrinogen but not on Coll or basement membrane protein. Furthermore, PAK activation in mouse arteries correlates with areas of FN deposition and inflammatory gene expression similar to NF-B.29
These findings prompted us to investigate the relationship between matrix-specific activation of PAK and NF-B in this system. These studies identified a novel role for PAK in matrix-specific NF-B activation by modulating the ability of ROS to activate NF-B.
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Materials and Methods |
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Briefly, our studies used bovine aortic endothelial cells and human umbilical vein ECs treated with various PAK inhibitors and exposed to either laminar flow or oscillatory flow in a parallel plate flow chamber. NF-
B activation was measured by immunoblotting and immunocytochemistry. NF-B–dependent gene expression was determined by real-time quantitative PCR. Quantification of ROS production was performed using the dye 2,7-dichlorodihydrofluorescein diacetate (H2-DCFDA). In vivo studies were carried out using C57Bl/6J mice, in which effects of PAK inhibition on NF-B were analyzed by immunohistochemistry. An expanded Materials and Methods section is available in the online data supplement at http://circres.ahajournals.org.
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Results |
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PAK Is Required for NF-
B Activation by Onset of FlowTo determine whether PAK is required for flow-induced NF-
B activation in cells on FN, we first used a previously described cell-permeant peptide corresponding to the N-terminal proline-rich sequence of PAK that binds the SH3 adapter protein Nck.30 This peptide prevents the interaction between PAK and Nck and blocks PAK-dependent changes in endothelial monolayer permeability, migration, and angiogenesis.29,30 Activation of the classic NF-B pathway involves IKK-dependent phosphorylation and degradation of the inhibitor IB, as well as Ser536 phosphorylation and nuclear translocation of p65.9 Pretreatment of ECs with this peptide completely blocked p65 nuclear translocation in response to flow compared to inactive control peptide (Figure 1A and 1B). Results with control peptide were indistinguishable from untreated cells (not shown). Flow-induced p65 phosphorylation was also substantially reduced by the PAK-Nck peptide (Figure 1C).
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To confirm these results, we examined 2 other PAK inhibitors. Expression of a PAK AID construct that blocks kinase activity did not affect basal NF-B activity but flow-induced p65 nuclear translocation was significantly inhibited (Figure 1D). Transfecting cells with the PAK p21-binding domain, which binds and inhibits the upstream GTPases Rac and Cdc42, also blocked flow-induced NF-B nuclear translocation (Figure 1E). Finally, the induction of the NF-B target gene ICAM-1 in response to acute onset of flow was significantly inhibited by pretreatment with the PAK-Nck peptide (Figure 1F).
Disturbed Flow In Vitro and In Vivo
Both laminar and oscillatory flow initially activate proinflammatory pathways; however, ECs adapt to laminar flow and proinflammatory signals return to baseline or below baseline, although these signals are maintained in disturbed flow.31 In vitro, both PAK and NF-B are activated transiently by acute onset of flow29,32 but in a sustained manner under disturbed flow.8,29 Both PAK and NF-B are also activated at sites of disturbed flow in vivo.12,29 To test whether PAK is required for sustained NF-B activation by disturbed flow, human umbilical vein ECs transfected with the PAK AID construct (
80% transfection efficiency) were plated on FN and exposed to oscillatory flow for 18 hours. The PAK AID completely inhibited both basal (49% reduction, P<0.01) and flow-induced PAK activation (67% reduction, P<0.01) (Figure 2A). AID expression also blocked the increase in NF-B p65 phosphorylation (Figure 2A) and nuclear translocation (Figure 2B). Oscillatory flow-induced expression of the proinflammatory genes ICAM-1 and interleukin-8 were inhibited as well (Figure 2C and 2D). Thus, PAK is required for both transient activation of NF-B in laminar shear and sustained activation in oscillatory shear.
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We next asked whether PAK is required for NF-B activation at regions of disturbed flow in arteries in vivo. NF-B activation in regions of disturbed flow colocalizes with FN in the subendothelial ECM,22 and we previously found that injecting mice with the PAK-Nck peptide reduced vascular permeability at these sites.29 C57Bl/6 mice have modest PAK and NF-B activation at locations of disturbed flow in the absence of other markers of atherosclerosis.12,29 Mice therefore received injections of control or PAK-Nck peptide for 3 days, at which time arteries were examined by immunohistochemistry. Similar to untreated mice,12 mice treated with control peptide showed nuclear NF-B in ECs at the expected sites, which was decreased in PAK-Nck peptide treated mice (Figure 3A and 3B). Taken together, these results show that PAK is critical for flow-induced NF-B activation in vitro and in vivo.
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Rescue by Active PAK
ECs plated on basement membrane proteins, such as Coll and laminin, do not activate either PAK or NF-B.22,29 To test whether low PAK activity is rate-limiting for NF-B activation under these conditions, cells were transfected with WT or active T423E PAK. Active PAK did not directly activate NF-B in cells on Coll but rescued both p65 nuclear translocation (Figure 4A) and p65 phosphorylation on Ser 536 (Figure 4B) in response to flow, compared to cells transfected with wild-type PAK. These data provide strong evidence that differential PAK activation mediates matrix-specific NF-B activation by flow.
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Relationship to p38 MAP Kinase
Previous results demonstrated that p38 MAP kinase was preferentially activated in cells on Coll and that blocking p38 partially restored NF-B activation by flow.22 We therefore investigated the relationship between p38 and PAK signaling in this system. Flow does not activate PAK in cells on Coll, suggesting that Coll-specific p38 activation could prevent NF-B activation by inhibiting PAK. However, inhibiting p38 in cells on Coll did not increase flow-induced PAK activation (Figure 4C). To test the converse hypothesis, that PAK stimulates NF-B activation in cells on FN by suppressing p38, we transfected cells with active PAK and plated them on Coll. Whereas active PAK is sufficient to rescue NF-B activation in cells on Coll, active PAK increased rather than decreased p38 activation both with and without flow (Figure 4D). Furthermore, inhibiting PAK signaling in cells on FN did not enhance p38 activation (Figure I in the online data supplement). Thus, PAK does not promote NF-B by inhibiting p38; rather, the data suggest that the inhibitory effect of p38 cannot overcome the effect of active PAK. Therefore, these results show that the matrix-specific regulation of PAK and p38 are independent events, with PAK being the major determinant of matrix-specific NF-B activation.
Role of NF-B–Inducing Kinase
We next turned our attention to the mechanism by which PAK regulates flow-induced NF-B activation. We previously found that activation of IKKβ by flow is matrix-specific, occurring in cells on FN but not Coll.22 NIK phosphorylates and activates IKK
and IKKβ,33 is activated by ROS,18 and is required for NF-B activation by both flow17,19 and constitutively active Rac.26,28 To test the involvement of NIK, cells plated on Coll or FN were stimulated with flow and NIK activation assayed using a phosphorylation-specific antibody to Thr559, a key phosphorylation site in the activation loop of NIK that regulates NIK kinase activity.34 In cells on FN, flow induced a 2.5- to 3-fold increase in NIK phosphorylation, whereas cells on Coll showed only a slight and transient response (Figure 5A). Inhibiting PAK in cells on FN with the PAK-Nck peptide reduced flow-mediated NIK phosphorylation (Figure 5B) and expression of constitutively active T423E PAK in cells on Coll rescued flow-induced NIK phosphorylation (Figure 5C). Thus, NIK activation is matrix-specific and PAK-dependent. These data indicate that PAK regulates NF-B activation either at or upstream of NIK.
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Role of ROS
Flow-induced NF-B activation depends on the production of ROS, because both antioxidants and genetic deletion of p47phox prevent flow-induced NF-B activation.20,35 In neutrophils, PAK regulates the NADPH oxidase complex through phosphorylation of both the p67phox36 and p47phox37 subunits. We therefore considered whether PAK might regulate flow-induced ROS production in ECs. To test this idea, we measured ROS production in ECs on either Coll or FN using the cell-permeant redox sensitive compound H2-DCFDA. H2-DCFDA is oxidized primarily by H2O2, a metabolite of superoxide.38 Surprisingly, the ability of shear stress to increase H2-DCFDA fluorescence was matrix-independent (Figure 6A). Additionally, the inhibitory PAK peptide had no effect on flow-induced ROS production in cells on FN (Figure 6B). Static controls were not lysed until the end of the 30 minute time point to ensure that increases in ROS production were not attributable to accumulation of mitochondrial-derived ROS over the course of the assay. Thus, PAK does not act by controlling ROS production.
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The lack of flow-induced NF-B activation in cells on Coll, despite the production of ROS, suggests that matrix regulates cellular sensitivity to ROS. Addition of exogenous H2O2 is sufficient to activate NF-B,39 and the H2O2 scavenger catalase blocks both flow-induced inflammatory gene expression40 and atherosclerosis in vivo.41 Consistent with these results, polyethylene glycol-catalase inhibited NF-B activation by oscillatory flow (supplemental Figure II). We therefore tested the sensitivity of cells to ROS by addition of exogenous H2O2. Cells on FN showed much higher H2O2-induced p65 phosphorylation compared to cells on Coll (Figure 7A). Similar to flow-induced NF-B activation, blocking PAK in cells on FN with the inhibitory peptide abolished both H2O2-induced p65 nuclear translocation (Figure 7B) and phosphorylation (Figure 7C). This effect was not attributable to enhanced antioxidant activity on Coll or in response to the peptide inhibitors because neither treatment affected the oxidation of H2-DCFDA by flow. Similar to the peptide inhibitors, the PAK AID construct also diminished both H2O2-induced p65 nuclear translocation (Figure 7D) and phosphorylation (Figure 7E) in cells on FN. Finally, expression of the constitutively active T423E PAK construct in cells on Coll rescued H2O2-induced p65 nuclear translocation (Figure 7F). Although NF-B activation by exogenous H2O2 required concentrations above that physiologically produced (500 to 750 µmol/L compared to 300 to 400 µmol/L), endogenously produced oxidants can act locally and may therefore be effective at lower doses.
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To confirm these results, we also examined NIK activation. ECs on FN showed much higher H2O2-induced NIK activation compared to cells on Coll (Figure 8A). The PAK-Nck peptide abolished H2O2-induced NIK phosphorylation in cells on FN (Figure 8B), as did expression of the PAK AID (Figure 8C). Taken together, these data provide strong evidence that matrix-specific PAK activation regulates flow-induced NF-B activation by modulating the ability of ROS to activate NIK and NF-B.
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Discussion |
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The present work defines PAK as a critical upstream mediator of matrix-specific NF-
B activation by flow. This conclusion is based on results showing that PAK inhibitors blocked NF-B activation by both acute onset of flow and oscillatory flow in cells on FN; conversely, active PAK restored activation of NF-B by flow in cells on Coll. Blocking PAK also decreased NF-B activation in atherosclerosis-prone regions of the mouse carotid sinus in vivo. Active PAK did not, however, affect basal NF-B activity in the absence of flow. These data suggest that PAK-dependent regulation of NF-B activation is highly specific and demonstrate that NF-B activation in this system requires multiple inputs.
Previous work showed that activation of NF-B by flow20,35 or Rac42 requires ROS. PAK can regulate the NADPH oxidase complex in neutrophils, where NOX2 is a critical NADPH oxidase subunit.36,37 However, neither matrix composition nor PAK inhibition affected flow-induced ROS production in ECs. These cells use mainly NOX1 to generate ROS in response to flow.40 Thus, a distinct requirement for PAK is not surprising. Instead, we found that activation of both NF-B and NIK by exogenous H2O2 was higher in cells on FN compared to Coll. Furthermore, the response to H2O2 was decreased by PAK inhibitors in cells on FN and increased by activating PAK in cells on Coll. Whereas H2O2 did not stimulate PAK activation on any matrix, PAK activity was moderately but significantly higher in cells on FN compared to Coll (supplemental Figure III), suggesting that a baseline threshold of PAK activity may be required for this pathway to proceed. Taken together, these data provide strong evidence that PAK modulates the pathway by which H2O2 triggers NF-B activation.
Relatively little is presently known concerning the role of PAK in the inflammatory response. Migration of leukocytes to CXCL143 and CXCL1244 requires PAK1, and the PAK-Nck inhibitory peptide reduces neutrophil activation and infiltration in lipopolysaccharide-induced lung injury in mice.45 Active PAK can stimulate the activation of the JNK and p38 MAP kinase pathways, both of which are implicated in proinflammatory gene expression.46 However, reports of PAK involvement in NF-B activation have been inconsistent. Constitutively active Rac activates NF-B through production of ROS,42 which is blocked by dominant negative NIK and IKKβ.26,28 Active Rac mutants incapable of activating PAK still activate NF-B,27 and the active T423E PAK construct is insufficient to activate NF-B.27,28 Thus, PAK is not a central component of the pathway linking Rac to NF-B. However, dominant negative PAK inhibits NF-B activation by some stimuli, including expression of activated Rac.25,26 These data can be reconciled by a model in which PAK sensitizes the NIK/IKKβ pathway to activation by ROS. As in other signaling networks, the relative importance of PAK would then depend on both the strength and the nature of the upstream signal.47
In addition to flow and atherosclerosis, oxidant-induced activation of NF-B has been implicated in responses to cigarette smoke, proinflammatory cytokines such as interleukin-1β, aging, ischemia/reperfusion injury, myocardial infarction, cancer, and diabetic renal failure.48,49 The ability of PAK to regulate oxidant-dependent NF-B activation may therefore be important in multiple pathologies and suggests that PAK is a potential therapeutic target. However, long-term global PAK inhibition is likely to be deleterious, because strong immunosuppression increases the risk of infection and cancer.50 Furthermore, PAK3 is important in brain function and PAK inhibition using a different cell-permeable peptide inhibitor results in symptoms resembling Alzheimer’s disease in mice.51 However, multiple endogenous proteins can inhibit PAK signaling, including nischarin, hPIP, POPX1/2, and protein kinase A.24 These endogenous negative feedback mechanisms, especially those that primarily affect the vasculature, could be useful therapeutic targets in limiting endothelial activation and atherosclerosis.
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Acknowledgments |
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We acknowledge Andy Pryor for assistance with the immunohistochemistry, Lynn Hedrick and Brian Wamhoff (University of Virginia) for RT-PCR primers, and David Scott (University of Virginia) for assistance with plasmid production and useful discussions.
Sources of Funding
This work was supported by United States Public Health Service grants RO1 HL75092 (to M.A.S.), HL80956 (to M.A.S. and B.R.B.), and HL082836 (to B.R.B.) and by American Heart Association Scientist Development Grant 0735308N (to A.W.O.).
Disclosures
None.
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Footnotes |
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Original received March 17, 2008; revision received July 22, 2008; accepted July 23, 2008.
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References |
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