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Am J Physiol Cell Physiol 292: C362-C371, 2007. First published August 16, 2006; doi:10.1152/ajpcell.00535.2005
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VASCULAR BIOLOGY

Low shear stress preferentially enhances IKK activity through selective sources of ROS for persistent activation of NF-{kappa}B in endothelial cells

Sumathy Mohan,1 Koichi Koyoma,1 Amalraj Thangasamy,1 Hiroyasu Nakano,4 Randolph D. Glickman,2 and Natarajan Mohan3

Departments of 1Pathology, 2Ophthalmology, and 3Radiation Oncology, University of Texas Health Science Center at San Antonio, San Antonio, Texas; and 4Department of Immunology, Juntendo University School of Medicine, Tokyo, Japan

Submitted 21 October 2005 ; accepted in final form 6 June 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
NF-{kappa}B signaling pathway has been known to play a major role in the pathological process of atherogenesis. Unlike high shear stress, in which the NF-{kappa}B activity is transient, our earlier studies have demonstrated a persistent activation of NF-{kappa}B in response to low shear stress in human aortic endothelial cells. These findings partially explained why low shear regions that exist at bifurcations of arteries are prone to atherosclerosis, unlike the relatively atheroprotective high shear regions. In the present study, we further investigated 1) the role of NF-{kappa}B signaling kinases (IKK{alpha} and beta) that may be responsible for the sustained activation of NF-{kappa}B in low shear stress and 2) the regulation of these kinases by reactive oxygen species (ROS). Our results demonstrate that not only is a significant proportion of low shear-induced-kinase activity is contributed by IKKbeta, but it is also persistently induced for a prolonged time frame. The IKK activity (both {alpha} and beta) is blocked by apocynin (400 µM), a specific NADPH oxidase inhibitor, and diphenyleneiodonium chloride (DPI; 10 µM), an inhibitor of flavin-containing oxidases like NADPH oxidases. Determination of ROS also demonstrated an increased generation in low shear stress that could be blocked by DPI. These results suggest that the source of ROS generation in endothelial cells in response to low shear stress is NADPH oxidase. The DPI-inhibitable component of ROS is the primary regulator of specific upstream kinases that determine the persistent NF-{kappa}B activation selectively in low shear-induced endothelial cells.

upstream {kappa}B kinases; laminar shear stress; oxidative stress; atherogenesis; reactive oxygen species

ENDOTHELIAL CELLS lining the vascular wall respond differentially to different flow shear regimen. Studies from the past have clearly demonstrated that regions of artery that experience low shear stress and reversing flow patterns such as arterial bifurcations and curved segments of the large elastic artery are highly predisposed to the development of atherosclerotic lesions, unlike high shear regions that are relatively resistant to the disease (2, 15). Our earlier in vitro studies (17–19) and in vivo reports from other laboratories (9) have indicated the possible involvement of NF-{kappa}B signaling mechanisms in low shear lesion-prone areas. The NF-{kappa}B family of transcription factors (p65 or RelA, p50, p52, Bcl-3, c-Rel and RelB) regulate many proinflammatory endothelial signals that are activated in the early stages of atheroma formation (29). NF-{kappa}B family members form homo- or heterodimeric complexes that are retained in the cytoplasm in quiescent (unstimulated) cells by associating with a family of inhibitor proteins, called I{kappa}Bs, which include I{kappa}B{alpha} and I{kappa}Bbeta. Activation of NF-{kappa}B by a variety of stimuli is dependent on the phosphorylation of site-specific serine residues such as Ser32 and Ser36 of I{kappa}B{alpha} and Ser19 and Ser23 of I{kappa}Bbeta, followed by the subsequent degradation of the I{kappa}B proteins, which allows the translocation of the active form of NF-{kappa}B into the nucleus to activate downstream target genes. Phosphorylation of I{kappa}B is mediated by a macromolecular complex called the signalsome with a molecular mass of ~900 kDa that is composed of three major proteins, IKK{alpha}, IKKbeta, and IKK{gamma}/NEMO (NF-{kappa}B essential modulator). Both IKK{alpha} and IKKbeta are serine/threonine kinases, which exert kinase activity upon stimulation, whereas IKK{gamma} lacks kinase activity but is required for the activation of IKK{alpha} and IKKbeta in vivo (14). IKK{alpha} and IKKbeta are highly homologous with the characteristic structure of an NH2-terminal protein kinase domain, which contains a canonical mitogen-activated protein kinase kinase activation loop motif of the sequence Ser-X-X-X-Ser. Phosphorylation of the site-specific serine residues in the activation loop, i.e., Ser176 and Ser180 in IKK{alpha} and Ser 177 and Ser181 in IKKbeta, is thought to be important for the regulation of their kinase activity. Replacement of these serine residues to alanine or mutation of these residues inactivates their kinase activity.

The possible involvement of certain components of the NF-{kappa}B signaling pathway that could be influenced by flow shear stress has been reported (4, 10). However, these reports have focused on the effect of physiological high shear stress of 12 and 15 dyn/cm2. Studying the nature of these upstream signaling elements involved in NF-{kappa}B regulation under conditions of low shear stress is important, because this environment is more relevant to the atherogenic process observed at sites of branching arteries. Therefore, the present study has investigated the IKK regulation in low shear flow-adapted human aortic endothelial cells. Our results indicate that even though both IKK{alpha} and IKKbeta activity are turned on by low shear stress, IKKbeta activity contributes to a major proportion of total IKK activity. NF-{kappa}B-inducing kinase (NIK), the upstream member of this signaling pathway, also contributes to in vitro I{kappa}B{alpha} phosphorylation. The IKK activity by low shear stress is significantly downregulated by inhibitors of NADPH oxidase, indicating that the reactive oxygen species (ROS) generated by low shear stress through this enzyme could be responsible for activating the NF-{kappa}B signaling mechanism. The contribution of ROS from xanthine oxidase, however, may not influence the low shear-induced NF-{kappa}B signaling mechanism. These results identify new specific targets that may help to control the atherogenic process that is augmented by low shear stress.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell culture. Human aortic endothelial cells (HAEC; Clonetics, San Diego, CA) were cultured in MCDB-131 medium (Sigma, St. Louis, MO) containing 10% bovine calf serum (BCS; Hyclone, Kansas City, KS) and enriched with 250 ng/ml fibroblast growth factor (PeproTech, Rocky Hill, NJ), 1 mg/ml epidermal growth factor (PeproTech), 1 mg/ml hydrocortisone (Sigma), 100 U/ml penicillin, and 100 mg/ml streptomycin (Media-tech, Herndon, VA). Cells from passages 4 to 7 were used in all the experiments.

Treatment conditions. HAEC maintained in MCDB-131 medium supplemented with 10% BCS were incubated in reduced serum (2%)-containing medium for 20 h before treatments. Cells induced by TNF-{alpha} (Pharmingen, San Diego, CA) at a concentration of 10 ng/ml were used as positive controls. Cells incubated with 400 µM apocynin, 10 µM diphenyleneiodonium chloride (DPI; Sigma), 100 µM pyrrolidine dithiocarbamate (PDTC), 200 µM allopurinol, and 0.5 µM carbonyl cyanide m-chlorophenylhydrazone (CCCP; Sigma) were used for blocking studies. For spectrofluorometric studies, cells were incubated with 10 µg of SOD mimetic [Mn(III) tetrakis(4-benzoic acid) porphyrin chloride; Sigma] for 45 min before shear stress. Concentrations of these inhibitors were chosen on the basis of studies reported previously (5, 11, 24). Cell viability determined using the standard Trypan blue dye exclusion method at these concentrations after 24 or 48h was >98%.

Shear stress experiments. Polyester film (10 x 19-cm Mylar sheets; Regal Plastics, San Antonio, TX) precoated with 2% gelatin was seeded with HAEC that were grown to near confluence. The cells were then incubated in MCDB-131 medium supplemented with 2% serum without growth factors and hydrocortisone for 20 h before the initiation of the flow shear to avoid any influence of growth factors on the induced responses. Flow experiments were performed using the closed-loop flow system as described previously (17–19). Cells were subjected to low (2 dyn/cm2) or high shear stress (16 dyn/cm2) by adjusting the height of separation between the parallel plates. Cells seeded on slips and grown as static cultures at 37°C were used as negative controls.

Expression vectors and transient transfections. Mammalian expression vectors encoding untagged NIK (wild or mutant), Flag-tagged IKK{alpha} and IKKbeta (wild type), the mutant constructs of Flag-tagged IKK{alpha}-KM (K44A) and IKKbeta-KM (K44A), and the substrate glutathione S-transferase fusion protein of I{kappa}B{alpha} with 1–100 amino acids (GST-I{kappa}B{alpha}) were used in this study as described previously (21). Transfection of these constructs was performed as reported earlier (17). In brief, HAEC seeded on plastic slips for shear experiments were incubated in MCDB-131 medium containing 2% serum devoid of growth factors for 24 h. The expression vectors (full-length cDNA, 2 µg) were transfected using the Effectene transfection reagent (Qiagen, Valencia, CA) following the manufacturer's protocol. The DNA-lipid complex was added to the cells slowly and incubated in a limited volume (8–10 ml) of medium for 24 h and then subjected to shear stress. For confirmation studies, parallel experiments were carried out using bovine aortic endothelial cells (BAEC) to test limitations due to reduced transfection efficiencies of human endothelial cells.

NF-{kappa}B-dependent reporter assays. Endothelial cells were transfected with 100 ng of the NF-{kappa}B luciferase reporter system (p4xNF-kB Luc; Mercury pathway profiling luciferase system, Clontech Laboratories) along with each wild-type (IKK{alpha} and IKKbeta) expression construct. The total DNA was kept constant at 2 µg/plate. Transfected cells were subjected to shear stress for 6 h, and the luciferase activity was measured as per the manufacturer's instructions (Promega, Madison, WI) using a TD-20/20 luminometer (Turner Design, Sunnyvale, CA).

In vitro phosphorylation assays. Cells induced by shear stress were washed with ice-cold PBS and lysed for 30 min on ice in 1 ml of lysis buffer containing 1% Nonidet P-40, 50 mM HEPES (pH 7.3), 150 mM NaCl, 2 mM EDTA, 1 µg /ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 1 mM phenylmethylsulfonyl fluoride, 0.1 mM sodium orthovanadate, and 1 mM NaF. The cell lysates were precleared by incubating with protein A-agarose 4B (BD Pharmingen, San Diego, CA) for 1 h at 4°C in the cold room. After centrifugation, the cleared lysates were incubated with anti-Flag monoclonal antibody (1 µg/ml)-tagged protein A-agarose for 2 h at 4°C. The immunoprecipitates were washed three times with the lysis buffer and twice in kinase buffer containing 20 mM HEPES (pH 7.3), 20 mM MgCl2, 20 mM MnCl2, 1 mM EDTA, 1 mM NaF, 0.1 mM sodium orthovanadate, and 1 mM DTT. The immunoprecipitates were then incubated with 1 µg of GST-I{kappa}B{alpha}(1–100) and [{gamma}-32P]ATP (10 µCi) in the kinase buffer for 20 min at 30°C. The reaction was stopped by the addition of the Laemmli's sample buffer. The eluted proteins were subjected to SDS-PAGE, and the autoradiograms were visualized using a PhosphorImager (Bio-Rad). In all cases, expression of the transfected proteins was verified by immunoblotting of aliquots of cell lysates. GST-I{kappa}Bs in the reaction mixtures were also verified by Coomassie blue staining to confirm equal loading.

Inhibition of IKK{alpha} and IKKbeta activity by siRNA transfection. siGenome smartpool small interference (si)RNA reagents specific for IKK{alpha} and IKKbeta were purchased from Dharmacon (Lafayette, CO). The nonspecific control siRNA-transfected cells and untransfected cells were used as controls and to determine the efficiency of transfection. Transfections were performed using Hiperfect transfection reagent (Qiagen). To determine the efficiency of siRNA in blocking IKK activity, we harvested transfected cells after 72 h to measure protein levels of IKK{alpha} and IKKbeta expression by immunoblotting. Furthermore, cells transfected with siRNA for IKK{alpha} and IKKbeta either individually or together were subjected to low shear stress for 6 h or kept at static culture conditions. Upon completion of shear stress, cells were washed with HBSS, fixed with 3.7% paraformaldehyde for 10 min, and permeabilized using 0.2% Triton X-100 for 5 min. Cells were blocked with 1% goat serum for 30 min at 37°C and further incubated with rabbit anti-p65 antibody to determine the translocation of p65 subunit into the nucleus. Alexa Fluor 350-tagged goat anti-rabbit was used as secondary antibody. Nuclear translocation of p65 subunit of NF-{kappa}B induced by low shear stress was examined under a Nikon ES-T-2000u epifluorescence microscope.

Measurement of intracellular ROS induced by shear stress. ROS generation was measured in endothelial cells using dihydrorhodamine 123 (DHR 123; Molecular Probes, Eugene, OR) as described previously (23, 30, 31). Cells were preincubated with 10 µM DHR 123 in PBS for 30 min. DHR 123-loaded cells were incubated with or without ROS inhibitors for specific enzyme sources that were shown to inhibit kinase activity. To ensure the specificity of ROS measurements, we used the general ROS inhibitors PDTC and SOD mimetic. Treated cells were exposed to 1 or 3 h of low or high shear stress. Intracellular levels of generated ROS in the cell lysates were measured using a spectrofluorometer (490-nm excitation, 530-nm emission; Jobin Yvon-SPEX Fluorolog FL-3; Edison, NJ) to quantitate the fluorescence level of control versus shear samples.

Statistical analysis. Each experiment was repeated three times independently. All results were expressed as means ± SD, and P <0.05 was used for significance. Student's t-test was used to determine statistical significance. In some experiments (independent IKK{alpha}, IKKbeta, and NIK activity and kinase activity with wild-type and mutant constructs in combination), data were analyzed using the randomized block analysis of variance (ANOVA), treating experiments as blocks and treatments as repeated measures. Comparisons of the treatments with each other and with the static control condition were done with means from the ANOVA and t-tests.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Both IKK{alpha} and beta activity are independently induced by low shear stress. Our earlier reports (18) have demonstrated the low shear (2 dyn/cm2) stress-induced persistent increase of NF-{kappa}B DNA binding activity with the associated phosphorylation and degradation of inhibitor subunit I{kappa}B{alpha} in HAEC. To further understand whether or not, and to what extent, the upstream kinases in the {kappa}B signaling cascade are responsible for this persistent activation of NF-{kappa}B, we investigated the independent contribution of IKK{alpha} and IKKbeta in response to low shear stress. Since the components of NF-{kappa}B upstream kinases, namely, IKK{alpha}, IKKbeta, and NIK, are very tightly associated complexes of a 900-kDa signalsome, the contribution of kinase activities by independent components of this signalsome is difficult to determine. Therefore, to better understand their independent regulation, we used the approach of forced expression of Flag-tagged IKK{alpha} and IKKbeta components to determine the ectopic kinase activity after shear stress exposure. The transfected cells subjected to high shear (16 dyn/cm2) did not alter the independent activity of either IKK{alpha} or IKKbeta. In contrast, cells subjected to low shear (2 dyn/cm2) showed a 55 ± 7.1% (P < 0.01) and 101 ± 19% (P < 0.001) increase in the IKK{alpha} and IKKbeta kinase activity, respectively, compared with static control (Figs. 1 and 2A). The minimum difference in GST-I{kappa}B{alpha} staining and immunoblotting of Flag-tagged IKK{alpha} and IKKbeta expression clearly indicated the increase in the kinase activity induced by low shear stress was not due to differences in the transfection efficiency and the quantity of substrate added in each lane. In addition, in both the cells transfected with kinase mutant constructs for IKK{alpha} or IKKbeta (plasmids encoding IKK{alpha}-KM and IKKbeta-KM), the IKK{alpha} and IKKbeta activity remained at the basal level similar to that in the static controls, thus conferring the specificity of these assays. These results, therefore, showed that both IKK{alpha} and IKKbeta are independently activated by low shear stress, which is not the case in high shear stress (Figs. 1 and 2A). As demonstrated in Fig. 2B, the independent overexpression of wild-type IKK{alpha} and IKKbeta resulted in enhanced downstream target gene expression when the IKK wild-type constructs were cotransfected with 100 ng of p4xNF-{kappa}B Luc reporter system. Cells transfected with p4xNF-{kappa}B Luc alone exhibited a 1.5-fold increase over control (P < 0.05). Both wild-type IKK{alpha} and IKKbeta cotransfection with NF-{kappa}B Luc reporter system significantly increased luciferase activity independently (8.1 ± 0.2-fold increase with IKK{alpha} and 30.01 ± 1.7-fold increase with IKKbeta; P < 0.01 compared with NF-{kappa}B Luc-transfected cells). Luciferase activity was downregulated when cells were cotransfected with IKK{alpha} mutant (1.2 ± 0.45-fold; P < 0.01). Cotransfection with IKKbeta mutant resulted in an unaltered luciferase activity (P = 0.68), thus indicating the specificity of IKK{alpha}- or IKKbeta-dependent downstream luciferase activity.


Figure 1
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  		Fig. 1. Low shear induces I{kappa}B{alpha} kinase (IKK{alpha}) activity. Human aortic endothelial cell (HAEC) monolayers were transiently transfected with plasmids encoding the Flag epitope-tagged IKK{alpha} wild (wt) or mutant ({Delta}) constructs. The transfected cells were kept as static controls (Cont.) or subjected to a low shear stress (LS) of 2 dyn/cm2 or a high shear stress (HS) of 16 dyn/cm2 for 30 min. The IKK wild or mutant protein was immunoprecipitated with anti-Flag monoclonal antibody for kinase activity assay using glutathione S-transferase (GST)-I{kappa}B{alpha} (1–100 amino acids) and [{gamma}-32P]ATP as substrates. KA denotes the results of kinase activity. WB indicates Western blotting performed with anti-Flag monoclonal antibody showing equal protein expression. Coomassie blue staining of GST-I{kappa}B{alpha} demonstrates the use of an equal amount of substrate in all the samples. Bar graph presents kinase activity as percent increase over control. Data are means ± SD from 3 independent experiments. *P < 0.01 indicates statistical significance.

 

Figure 2
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  		Fig. 2. A: low shear induces IKKbeta activity. HAEC monolayers were transiently transfected with plasmids encoding the Flag epitope-tagged IKKbeta wild or mutant constructs. The transfected cells were kept as static controls or subjected to a low shear stress of 2 dyn/cm2 or a high shear stress of 16 dyn/cm2 for 30 min. The IKK wild or mutant protein was immunoprecipitated with anti-Flag monoclonal antibody for kinase activity assay using GST-I{kappa}B{alpha} (1–100 amino acids) and [{gamma}-32P]ATP as substrates. Bar graphs present kinase activity in terms of percent increases over control. Data are means ± SD from 3 independent experiments. *P < 0.001 indicates statistical significance. B: HAEC monolayers were transiently cotransfected with plasmids encoding the NF-{kappa}B-luciferase reporter (p4xNF-{kappa}B-Luc; 100 ng) along with Flag epitope-tagged IKK{alpha} or IKKbeta wild or mutant constructs. The transfected cells were kept as static controls or subjected to a low shear stress of 2 dyn/cm2 for 6 h. Harvested cells were lysed, and luciferase activity was measured as per the manufacturer's instructions. Bar graphs present luciferase activity in terms of fold increases over control. Data are means ± SD from 3 independent experiments. *P < 0.01 indicates statistical significance compared with pNF-{kappa}B Luc-transfected samples.

 
IKKbeta activity contributes to majority of the total IKK activity in HAEC exposed to low shear stress. To determine whether both IKK{alpha} and IKKbeta overexpression together may yield an additive increase in the total IKK activity under conditions of low shear stress, we transiently transfected endothelial cells with both Flag-tagged IKK{alpha} and IKKbeta constructs and exposed them to shear stress for 30 min or left them as static controls. As demonstrated in Fig. 3, both IKK{alpha} and IKKbeta wild-type constructs together resulted in 53.66 ± 4.5% (P < 0.01) in total IKK activity over (transfected/no-shear control) basal activity (100%). Both the mutant constructs exhibited 5% IKK activity over no-shear control (P > 0.1). Next, to validate the contribution of each IKK isoform on low shear stress-induced total kinase activity, we transfected cells with a combination of either wild-type IKKbeta with {Delta}IKK{alpha} or wild-type IKK{alpha} with {Delta}IKKbeta. Cells that were overexpressed with IKK beta wild type in the presence of IKK{alpha} mutant showed a 30 ± 4.9% increase in activity over static control (P < 0.05), whereas in the presence of IKKbeta mutant, the total IKK activity was only 3.6 ± 2.5% over control.


Figure 3
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  		Fig. 3. Kinase-deficient IKKbeta blocks total IKK activity in HAEC exposed to low shear stress. HAEC monolayers were transiently transfected either with plasmid encoding the Flag epitope-tagged IKKbeta wild (1 µg) and IKK{alpha} mutant constructs (1 µg) or plasmid encoding the Flag epitope-tagged IKKbeta mutant (1 µg) and IKK{alpha} wild constructs (1 µg). As a positive and negative control, cells were transfected with both wild-type constructs (1 µg each) and both mutant constructs (1 µg each), respectively. The transfected cells were kept as static controls or subjected to a low shear stress of 2 dyn/cm2 for 30 min. Cells were harvested, and the lysates were immunoprecipitated with anti-Flag monoclonal antibody for kinase activity assay as described in Fig. 1. *P < 0.01 indicates statistical significance.

 
Forced expression of NIK upregulates in vitro I{kappa}B{alpha} phosphorylation. To examine the contribution of NIK in low shear-induced IKK, cells were forcefully coexpressed with untagged NIK wild or mutant expression vector with Flag-tagged IKK{alpha} and IKKbeta expression plasmid. Since the IKK activation results in the phosphorylation of I{kappa}B{alpha}, we directly measured I{kappa}B{alpha} phosphorylation that is responsible for the translocation of active NF-{kappa}B complex into the nucleus. Cells overexpressed with untagged wild-type NIK demonstrated an increase in I{kappa}B{alpha} phosphorylation (55 ± 10% over control; P < 0.01) under conditions of low shear stress (Fig. 4). This increase was downregulated to 8 ± 15% when the cells were overexpressed with mutant NIK construct, confirming the activity observed was totally mediated by NIK. In contrast, overexpression of NIK in cells exposed to high shear stress slightly increased (10 ± 14% over control) I{kappa}B {alpha} phosphorylation, and the level of activity remained the same in {Delta}NIK overexpressed cells, indicating that high shear stress fails to induce this pathway.


Figure 4
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  		Fig. 4. NF-{kappa}B-inducing kinase (NIK) mediates low shear-induced I{kappa}B{alpha} phosphorylation. HAEC monolayers were transiently cotransfected with plasmids encoding the untagged NIK wild or mutant constructs with Flag-tagged wild-type IKK{alpha} and IKKbeta. The transfected cells were kept as static controls or subjected to a low shear stress of 2 dyn/cm2 or a high shear stress of 16 dyn/cm2 for 30 min. The cell lysates were immunoprecipitated with anti-Flag monoclonal antibody for kinase activity assay using GST-I{kappa}B{alpha} (1–100 amino acids) and [{gamma}-32P]ATP as substrates. Bar graphs present kinase activity in terms of percent increase over control. Data are means ± SD from 3 independent experiments. *P < 0.01.

 
Kinetics of IKKbeta activity under low versus high shear stress. Since IKKbeta activity was enhanced and found to be dominant in low shear-exposed cells, we next investigated the time-dependent activation of IKKbeta under conditions of low shear stress. In vitro kinase assays were performed in cells overexpressed with Flag-tagged IKKbeta and subjected to low shear stress for various time intervals starting from 30 min to 2 h. Cells induced with TNF-{alpha} for 2 and 5 min were used as positive controls (data not shown). As presented in Fig. 5A, IKKbeta activity reached a maximum activity at 30 min (101 ± 19% increase over control; P < 0.05). The levels started decreasing (40 ± 28% over control) at 60 min and again increased at 120 min (66 ± 24% over control), the longest time point examined in this study. In the case of high shear stress, after a slight rise (10.7 ± 8.9% over control) at 30 min, the IKKbeta activity significantly decreased to the basal level (less than no-shear control) at both 60 and 120 min. To determine the responses of other upstream kinases at earlier time periods, we determined IKK{alpha}, IKKbeta, and NIK activities after 15-min onset of shear. IKKbeta activity was not significantly induced in both low and high shear stress compared with static control. IKK{alpha} was significantly reduced in high shear-exposed cells (93 ± 1.4%; P < 0.05), whereas low shear-induced activity did not achieve statistical significance compared with control (138 ± 18%; P = 0.19). NIK activity was significantly increased under conditions of low shear stress (36 ± 0.6%; P < 0.05), whereas the level of activity in high shear stress exhibited insignificant changes (95 ± 14%; P = 0.7) compared with static control.


Figure 5
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  		Fig. 5. A: time kinetics of low shear-induced IKKbeta activity. HAEC transfected with 2 µg of IKKbeta wild-type construct were subjected to low (2 dyn/cm2) or high shear stress (16 dyn/cm2) for 30, 60, and 120 min. Cells were harvested, and lysates were immunoprecipitated with anti-Flag monoclonal antibody for kinase activity assay as described in Fig. 1. *P < 0.05 indicates statistical significance. B: low shear-induced IKK{alpha}, IKKbeta, and NIK activity. HAEC transfected with 2 µg of Flag-tagged IKK{alpha} or IKKbeta with or without untagged NIK (wild) construct were subjected to low (2 dyn/cm2) or high shear stress (16 dyn/cm2) for 15 min to determine early responses after the onset of shear stress. Cells were harvested, and lysates were immunoprecipitated with anti-Flag monoclonal antibody for kinase activity assay. *P < 0.05 indicates statistical significance compared with static control.

 
Low shear-induced nuclear translocation of NF-{kappa}B/p65 subunit is blocked by the inhibition of endogenous IKK activity with IKK{alpha}- and beta-specific siRNA. To test whether inhibition of endogenous IKK{alpha} and IKKbeta activity would abolish the nuclear translocation of NF-{kappa}B p65 subunit, we transfected endothelial cells with siRNA specific for IKK{alpha} and IKKbeta, either independently or together. As shown in Fig. 6A, after 72 h of transfection, siRNA significantly downregulated (>95%) the endogenous IKK{alpha} and IKKbeta (protein) expression in endothelial cells. In parallel experiments, siRNA-transfected cells were kept as static controls (Fig. 6A) or subjected to low shear stress (2 dyn/cm2) for 6 h (Fig. 6B). Low shear stress induced nuclear translocation of NF-{kappa}B/p65 subunit in siRNA control transfected (Fig. 6B) and untransfected cells (data not shown). The IKK{alpha} and IKKbeta siRNA transfection significantly blocked the nuclear translocation of low shear-induced NF-{kappa}B/p65 subunit (Fig. 6, C and D).


Figure 6
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  		Fig. 6. Inhibition of endogenous IKK activity by small interference (si)RNA blocks the low shear-induced translocation of p65 subunit of NF-{kappa}B into the nucleus. Endothelial cells were subjected to 6 h of low shear stress after transfection with siRNA for IKK{alpha} and IKKbeta. At the end of shear run, cells were fixed, permeabilized, and further incubated with NF-{kappa}B antibody followed by Alexa Fluor 350-tagged secondary antibody. Stained cells were observed under an epifluorescent microscope. A: cells transfected with siRNA (IKK{alpha} and IKKbeta) kept as static controls. B: control siRNA-transfected cells exposed to low shear stress for 6 h. C and D: IKK{alpha} and IKKbeta siRNA-transfected cells, respectively, exposed to low shear for 6 h. Cells were also subjected to immunoblotting to confirm inhibition of IKK{alpha} and IKKbeta protein expression (shown at top panel).

 
Specific inhibitors of ROS downregulate IKK activity induced by low shear stress. To identify the upstream mechanism and the mediator that regulates the persistent activity of NF-{kappa}B signaling kinases in low shear stress-induced HAEC, we examined the contribution of ROS that are known to induce NF-{kappa}B-mediated signaling mechanisms. HAEC transiently transfected with Flag-tagged IKK{alpha} and IKKbeta were incubated with both specific and general (known) ROS inhibitors for 45 min before the cells were exposed to low shear stress or kept as no-shear controls. As shown in Fig. 7, increased kinase activity observed at 30 min after low shear stress was not downregulated by CCCP (0.5 µM), allopurinol (200 µM), or PDTC (100 µM). However, apocynin (400 µM), a specific inhibitor of NADPH oxidases, and DPI (10 µM), an inhibitor of flavin-containing oxidases like NADPH oxidase, downregulated the low shear-induced IKK activity by 88.4 ± 17.5 and 76 ± 10.5% (P < 0.05 compared with kinase activity in low shear stress in the absence of the inhibitors), respectively. The basal kinase activity in the presence of inhibitors under no-shear control was not influenced.


Figure 7
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  		Fig. 7. Inhibition of low shear-induced IKK activity by ROS inhibitors. HAEC forcefully expressed with IKK wild-type expression constructs were incubated with ROS inhibitors (as indicated) for 45 min before low shear stress. Cell lysates were prepared immediately after 30 min of shear for kinase activity measurements. Bar graphs represent kinase activity in terms of percent increase over control. *P < 0.05 indicates statistical significance compared with low shear condition in the absence of inhibitors. CCCP, carbonyl cyanide m-chlorophenylhydrazone; PDTC, pyrrolidine dithiocarbamate; DPI, diphenyleneiodonium chloride.

 
Direct measurement of ROS in HAEC exposed to low versus high shear stress. The generation of ROS in low versus high shear stress-exposed endothelial cells was determined using DHR 123. As demonstrated in Fig. 8, low shear exhibited significant increase (P < 0.05) in ROS generation at both 1 h (154.6 ± 4.1%) and 3 h (152 ± 7.55%) after shear stress exposure compared with no-shear control, wherein fluorescence intensity (indicating ROS generation) was considered 100%. ROS generation induced by 3 h of low shear stress was downregulated significantly by preincubating the cells with 10 µg of SOD mimetic agent (105.7 ± 3.05%; P < 0.05 compared with ROS generation in cells exposed to 3 h of low shear), indicating specificity of ROS measurements. When cells were preincubated with inhibitors of specific ROS-generating enzyme sources, intracellular ROS levels were significantly reduced. Cells incubated with DPI (10 µM), an inhibitor of flavin-containing oxidases like NADPH oxidase, exhibited significantly reduced ROS generation by low shear stress (119.7 ± 0.6%; P < 0.05 compared with low shear at 3 h). HAEC exposed to high shear stress for both 1 and 3 h exhibited a significant decrease (P < 0.05 compared with ROS generation in conditions of low shear) in fluorescence intensity. The levels at 1 and 3 h were 81.3 ± 7.8 and 87 ± 25.2%, respectively.


Figure 8
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  		Fig. 8. Synthesis of ROS in endothelial cells exposed to low vs. high shear stress. Endothelial cells were incubated with 10 µM dihydrorhodamine 123 for 30 min at 37°C with or without ROS inhibitors. After incubation, cells were washed and subjected to shear stress for either 1 or 3 h. Cell lysates prepared were subjected to spectroflurometric measurements. Data are means ± SD from 3 independent experiments and are expressed as percentages of control values. *P < 0.05 indicates statistical significance (using Student's t-test) compared with no-shear control. **P < 0.05 indicates statistical significance compared with ROS generation at 3 h of low shear stress.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In this report we have demonstrated the persistent increase in IKK activity, and in particular, IKKbeta activity, after laminar low shear stress in HAEC. Our results indicated the possibility that the low shear-induced increase in IKK activity is regulated by ROS generated by NADPH oxidases. This ROS-mediated persistent IKK activity may be responsible for the prolonged NF-{kappa}B DNA binding activity and the associated endothelium-monocyte adhesion that we observed after exposure to low shear stress in our earlier studies (18, 19). The IKK-dependent translocation of the p65 subunit of NF-{kappa}B into the nucleus has been shown in endothelial cells that are adapted to uniform (both spatially and temporally) laminar flow (4, 10). These studies were carried out after unidirectional high shear stress of 15 or 12 dyn/cm2 with human umbilical vein endothelial cells (HUVEC) in which the endogenous activity of total IKK{alpha} plus IKKbeta was measured or in BAEC in which forcefully expressed total IKK activity was measured using IKK{alpha} and IKKbeta expression vectors. In our studies, we used HAEC, expressing IKK{alpha} and IKKbeta independently or together, exposed to uniform laminar flow generating a unidirectional low shear stress of 2 dyn/cm2. HAEC exposed to unidirectional high shear stress of 16 dyn/cm2 were used as a standard to evaluate the low shear response. Static cultures were used as negative control. Although results reported by Bhullar et al. (4) showed total kinase activity, we sought to measure the activity of individual kinases (i.e., IKK{alpha} or IKKbeta activity) by using GST-I{kappa}B{alpha}(1–100) as a substrate. Interestingly, in contrast to the findings reported previously (4, 10), in our studies high shear stress failed to induce independent IKK{alpha} or IKKbeta activity. Time-course experiments showed a significant downregulation of IKK activity at increasing time points compared with no-shear control. However, total kinase activity after high shear stress has been reported to be transiently induced at 5 (10) and 30 min (4). Consistent with these results, we demonstrated previously that 30 min of high shear stress induced a transient NF-{kappa}B DNA binding activity that decreased after 2 h and showed a small second phase of activity after 16 h (18). Together, these results clearly revealed a distinct pattern of kinase activity in the same cell type from different sites of the vascular bed or from different sources under similar flow shear regimen. Alternatively, measuring the low levels of endogenous total kinase activity in HAEC versus independent kinase activity (IKK{alpha} or IKKbeta activity) that are forcefully expressed with the individual expression vectors may account for those differences described above.

The higher levels of IKK activities noticed in low shear stress confirm that insignificant activities observed in high shear stress are not due to decreased transfection efficiencies of endothelial cells from human origin. Our results (Figs. 1 and 2) demonstrate that IKK{alpha} and IKKbeta are required together to enhance the transient total IKK activity in high shear stress, whereas low shear stress can independently activate significant levels of both IKK{alpha} and IKKbeta activity. The results of triplicate experiments were consistent in showing that IKKbeta activity was enhanced 101 ± 19%, whereas IKK{alpha} activity increased only 55 ± 7.1%, compared with control, indicating that IKKbeta activity may be preferentially activated in low shear stress. This observation is supported by results of Meiler et al. (22) showing that dominant negative IKKbeta overexpression significantly abolished the downstream events of both monocyte rolling and adherence in HUVEC exposed to laminar low shear stress of 2 dyn/cm2.

Induction of IKKs by cytokines, particularly TNF-{alpha}, has been shown to be directional. First, TNF-{alpha} triggers the activation of IKK{alpha}, which then stimulates IKKbeta (33). Similar mechanisms could occur under conditions of low shear stress where unlike TNF-{alpha}, the preferential activation of IKKbeta may regulate IKK{alpha} activity. To investigate whether such directional activation is possible under conditions of low shear stress, we performed cotransfection experiments to determine whether the activity of the heterodimeric IKK{alpha} and IKKbeta complex is influenced by each other, as reported earlier in TNF-{alpha}-mediated activation of NF-{kappa}B (33), or remains independent. Whereas cells coexpressing both IKK{alpha} mutant and IKKbeta wild type under conditions of low shear stress induced a 30 ± 4.9% increase in kinase activity, cells coexpressing IKK{alpha} wild type and IKKbeta mutant showed no enhancement in the amount of total kinase activity. The activity in these cells was very similar to that in cells cotransfected with both IKK{alpha} and IKKbeta mutant constructs. These cotransfection experiments may indicate 1) the increased functionality of IKKbeta subunit, since it is known that IKKbeta exhibits a 20- to 50-fold higher level of activity than IKK{alpha} (21, 33), and 2) IKKbeta activity may be essential for IKK{alpha} activity. However, it is apparent from the results presented in Figs. 1 and 2B that low shear stress could induce the activity of both ectopically overexpressed IKKs in an independent manner, unlike TNF-{alpha} activity (33). Therefore, the possibility that IKKbeta activity may be essential for IKK{alpha} kinase activity is implausible. A plausible model is that low shear stress could activate the upstream kinases like NIK or MEKK3 and then stimulate both IKKs. In our attempts to determine the contribution of MEKK3, low shear stress did not induce IKK activity when endothelial cells were overexpressed with Flag-tagged MEKK construct (unpublished results).

However, overexpression of NIK did have a positive influence over I{kappa}B{alpha} phosphorylation even at an earlier time point (15 min), indicating that NIK could be the only upstream regulator. These cotransfection experiments were also performed in BAEC to confirm the results and to determine whether transfection efficiency of wild and mutant constructs would influence the observed results. In our experience, the basal kinase activity (no-shear controls) in human endothelial cells seems to be higher than in the bovine cells. In addition, because of the limitations of transfection efficiencies, a maximum of 0.5- to 2-fold increase could only be achieved in kinase activity assays using human cells. However, this increase was sufficient to induce a significant downstream gene (luciferase expression as shown in Fig. 2B), thus confirming the functional consequence of the measured kinase activities.

Our time-course experiments clearly indicated that low shear stress exhibited IKK activity up to 120 min, unlike HAEC exposed to high shear stress (used as control) where IKK activity gradually declined to a level even less than that of no-shear controls with increasing hours of exposure to shear stress. Also, it is surprising that IKKbeta and IKK{alpha} independently induced 101 ± 19 and 55 ± 7.1% increases in activity, respectively, but did not yield a cumulative increase when they were cotransfected together. The increase was only 54 ± 4.5% over control. This could be due to the amount of plasmid DNA that was used for transient transfection (with a limit of 2 µg of total plasmid DNA/30-cm2 area of slip, 1 µg of IKK{alpha} and 1 µg of IKKbeta were used in cotransfection experiments, whereas 2 µg of IKK{alpha} or IKKbeta plasmid DNA was used in independent transfections). The transient transfection of IKK{alpha} and IKKbeta with p4xNF-{kappa}B Luc reporter system independently induced 8.1 ± 0.2-fold and 30.01 ± 1.7-fold luciferase activity, respectively, over luciferase activity in the absence of IKK{alpha} or IKKbeta overexpression, indicating the increase in IKK activity is responsible for NF-{kappa}B-dependent downstream target gene expression.

We examined the NIK activity in HAEC overexpressed with untagged NIK expression construct with Flag-tagged IKK{alpha} and IKKbeta plasmid. As presented in Fig. 4, NIK expression increased (55 ± 10% over control) I{kappa}B{alpha} phosphorylation in low shear stress. However, under conditions of high shear stress, the increase in I{kappa}B{alpha} phosphorylation (10 ± 14% over control) was insignificant compared with control. This observation in high shear stress is opposite to the report of Hay et al. (10), who demonstrated NIK involvement in high shear stress in NIK-overexpressed HUVEC, using nuclear translocation of p65 by immunofluorescence as the end point. This discrepancy again could reflect differences in methodology, controls, and cell type used. We performed in vitro kinase assays in untagged NIK-overexpressed HAEC that were cotransfected with Flag-tagged wild-type IKK{alpha} or IKKbeta plasmids. Since the cell lysates were immunoprecipitated with anti-Flag antibody, the resulting kinase activity is due to IKK that is regulated by overexpressed NIK activity. The controls in our studies were HAEC overexpressed with NIK construct but not exposed to shear stress. Although NIK activity was slightly increased in high shear stress, this increase was statistically insignificant compared with the activity that resulted in cells expressing mutant NIK construct and the no-shear control. In the report by Hay et al. (10), it is ambiguous whether the results were compared with no-shear controls. In addition, endothelial cells derived from different vascular beds could have different signaling responses (in particular, differences between venous and arterial cells). Overall, these results strongly support the hypothesis presented in our earlier reports (17–19) stating that the activity of NF-{kappa}B signaling kinases is highly upregulated in endothelial cells under conditions of low shear stress typical of branching arteries, which are predisposed to atherosclerosis. In addition, results from these overexpression studies are further supported by the IKK{alpha} and IKKbeta siRNA blocking experiments, as demonstrated in Fig. 6. IKK{alpha} or IKKbeta siRNA transfected individually or combined together blocked low shear-induced nuclear translocation of p65 subunit in most cells. However, in IKK{alpha} siRNA-transfected cells, we could observe p65 translocation with limited fluorescence in some cells. This could probably be due to the endogenous IKKbeta activity, which remains active even after the suppression of IKK{alpha} activity. Together, both overexpression and blocking studies support our hypothesis that persistent activity of upstream kinases regulates the prolonged NF-{kappa}B activity under conditions of low shear stress.

One of the earliest transcription factors recognized in eukaryotic cells as a redox-sensitive regulator is NF-{kappa}B. Accumulating evidence shows that activation of NF-{kappa}B and the subsequent upregulation of {kappa}B-dependent gene expression are significantly induced by the generation of ROS (1, 5, 27, 28). Stimulation of IKK activity by H2O2 (14) has provided direct evidence for the involvement of ROS in regulating upstream NF-{kappa}B signaling kinases. Use of reducing agents like N-acetylcysteine has been shown to inhibit IKK{alpha} and IKKbeta activity, thus suggesting a pivotal role of IKK{alpha} and IKKbeta in the redox regulation of NF-{kappa}B (26). Using aortic endothelial cells obtained from C57Bl/6 (MAE-C57) and p47phox–/– (MAE-p47–/–) mice, which lack a component of NAD(P)H oxidase, Hwang et al. (13) demonstrated the generation of superoxide produced from p47phox-dependent NADPH oxidase by oscillatory shear stress (±5 dyn/cm2), which in turn leads to monocyte adhesion. Acute exposure (1 h) of mouse aortic endothelial cells to laminar high shear stress (15 dyn/cm2) increased superoxide production, whereas chronic exposure (18 h) reduced it significantly. In both human and bovine aortic endothelial cells, we observed a significant decrease in dihydrorhodamine oxidation after acute (1 h) exposure to laminar high shear stress, indicating reduced generation of ROS. It is not clear at this time whether these discrepancies are due to differences in the genetic make up of the cell types used (mouse vs. human), the method of shear stress application (cone and plate model vs. parallel-plate method), and/or the type of fluorescein probes used [dihydroethidium (DHE) vs. DHR 123].

In general, three fluorescein probes, namely, DHE, DHR 123, and dichlorofluorescein diacetate (H2DCFDA), have been described as useful tools for the measurement of ROS. The redox-sensitive fluoroprobe DHR 123 has been used widely in endothelial cells to measure the generation of ROS (23, 30, 31). In the present study, we preferred the use of DHR 123 to measure ROS generation over H2DCFDA because of the susceptibility of the latter agent to undergo autooxidation during shear stress (7). In addition, we experienced leakage of the dye from cells in a short span of time under shear in parallel-plate flow chambers. DHE has been suggested to be used as a qualitative indicator of superoxide anion but is not recommended to be used in quantitative measure, because it can catalyze the dismutation of superoxide and can be oxidized by cytochrome c within the cell (3). DHR 123 is reported to be more sensitive in detecting ROS than other dyes tested (32, 34). DHR 123 is membrane permeable. It is oxidized by ROS intracellularly to become a stable fluorescein (Rh123) derivative, which is membrane-impermeable and is pumped into mitochondria (6). Therefore, this dye does not leak out of cells after being oxidized. Although H2DCFDA and DHE are reported to specifically target H2O2 and superoxide anions, respectively, DHR 123 has been widely used for testing global ROS. Multiple ROS in particular peroxynitrites convert DHR 123 into a highly fluorescent form (16, 32, 34). As reported by Lopez-Ongil et al. (16), if DHR 123 is more sensitive to peroxynitrite, which is formed after the reaction of superoxide with nitric oxide, it is likely that high shear stress generates minimal peroxynitrite at the initiation of shear stress (there is more nitric oxide and less superoxide release) and thus could have exhibited insignificant DHR 123 fluorescence in our studies. This difference in the sensitivity of the fluorescent dye used in our study could have resulted in differences that we observed in the level of generation of ROS under conditions of high shear stress.

The generation of superoxide (O2–), an important ROS in the vasculature, is mediated by several enzyme systems, including NADPH oxidases, lipoxygenases, xanthine oxidases, and myeloperoxidases (8). Our attempts to identify whether one of these enzyme sources could be involved in the persistent IKK activity in laminar low shear stress-exposed endothelial cells clearly indicated that apocynin- and DPI-inhibitable ROS mediate the enhanced and persistent upstream IKK activity. PDTC, a metal chelator, which blocked the downstream NF-{kappa}B DNA binding activity as shown in our earlier report (19), did not alter the upstream IKK activity (data not shown), indicating that PDTC-inhibitable ROS does not influence upstream IKK activity. Our PDTC data support the earlier report of Hayakawa et al. (11), who reported that TNF-{alpha}-induced IKK activation was not significantly inhibited by PDTC. However, PDTC inhibits NF-{kappa}B DNA binding activity by preventing the downregulation of I{kappa}B{alpha} phosphorylation or directly blocks the ubiquitin ligase toward phosphorylated I{kappa}B{alpha}, therefore blocking nuclear translocation of NF-{kappa}B at the downstream end. These results raise the possibility that different ROS activate specific stages of NF-{kappa}B signaling mechanisms. Alternatively, oxidative radical stress may stimulate IKK through the suppression of protein phosphatase, such as PP2A, because these enzymes are known to be inactivated by oxidative modification (14). Therefore, thorough understanding of the contributing factors and their underlying mechanisms is required to identify potential drug targets to attenuate the enhanced NF-{kappa}B signaling that contributes to atherogenesis.

In addition, the involvement of ROS-generating enzyme sources could be cell type specific. Interleukin-1beta has been shown to activate NF-{kappa}B through the production of ROS by 5-lipoxygenase in lymphoid cells and by NADPH oxidase in monocytic cells (5). Our results suggest that NADPH oxidases are involved in activating the NF-{kappa}B signaling pathway in endothelial cells exposed to laminar low shear stress. Furthermore, endothelial cells incubated with 2-deoxy glucose to block pentose shunt pathway for NADPH production significantly downregulated overexpressed IKK activity (data not shown). This result confirms the contribution of NADPH oxidase in activating IKK under conditions of low shear stress. Our study also suggests that specific species of reactive oxygen intermediates could only induce the upstream NF-{kappa}B signaling kinases. In summary, our study has identified potential targets that are influenced by hemodynamics of the blood flow, which contribute to the development of atherosclerotic lesions at localized regions (bifurcations) of large and muscular arteries that are characterized as low shear stress regions of the vasculature.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by National Heart, Lung, and Blood Institute Grant 1R01 HL-63032-01A1 (to S. Mohan) and partial support as salary for a technician funded by the Office of Science (Biological and Environmental Research), U.S. Department of Energy, Grant DE-FG02-03ER63449. Spectrofluorometer facilities for ROS measurements were supported by an unrestricted grant to the Dept. of Ophthalmology from Research to Prevent Blindness, Inc. (to R. D. Glickman).


   ACKNOWLEDGMENTS
 
We thank Dr. Douglas R. Spitz, Free Radical and Radiation Biology Program, University of Iowa, and Dr. Anthony J. Valente, Department of Medicine, University of Texas Health Science Center at San Antonio, for support, discussion, and valuable suggestions.


   FOOTNOTES
 

Address for reprint requests and other correspondence: S. Mohan, Dept. of Pathology, Univ. of Texas Health Science Center, San Antonio, TX 78229 (e-mail: mohan{at}uthscsa.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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