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Cardiovascular Research 2006 72(3):447-455; doi:10.1016/j.cardiores.2006.09.012
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Copyright © 2006, European Society of Cardiology

Role of NADPH oxidase 4 in lipopolysaccharide-induced proinflammatory responses by human aortic endothelial cells

Hye Sun Parka,1, Jung Nyeo Chuna,1, Hye Young Junga, Chulhee Choib,* and Yun Soo Baea,*

aDivision of Molecular Life Sciences, Center for Cell Signaling Research, Ewha Womans University, 11-1 Daehyun-Dong, Seodaemoon-Gu, Seoul 120-750, Republic of Korea
bDepartment of Biosystems, Korea Advanced Institute of Science and Technology, 373-1 Guseong-Dong, Yuseong-Gu, Daejeon, Republic of Korea

* Corresponding authors. Bae is to be contacted at Tel.: +82 2 3277 2729; fax: +82 2 3277 3760. Choi, Tel.: +82 42 869 4321; fax: +82 42 869 4310. Email address: cchoi{at}kaist.ac.kr baeys{at}ewha.ac.kr

Received 3 May 2006; revised 21 July 2006; accepted 18 September 2006


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: We investigated the role of NADPH oxidase 4 (Nox4) on lipopolysaccharide (LPS)-induced proinflammatory responses by human aortic endothelial cells (HAECs).

Methods and results: Yeast two-hybrid and glutathione-S-transferase pull-down assays indicated that the cytosolic Toll/IL-1R region of Toll-like receptor 4 (TLR4) (amino acids 739–769) is the responsible domain for interaction with the COOH terminal of Nox4 (amino acids 451–530). Consistently, overexpression of the COOH-terminal region of Nox4 inhibited nuclear factor-{kappa}B activation in response to LPS. Downregulation of Nox4 by transfection of siRNA specific to Nox4 in HAECs resulted in a failure to induce reactive oxygen species (ROS) generation and subsequent expression of intercellular adhesion molecule-1 (ICAM-1) and chemokines such as IL-8 and monocyte chemoattractant protein-1 (MCP-1) in response to LPS. Furthermore, transient transfection of endothelial cells with Nox4 siRNA led to a decrease in migration and adhesion of monocytes in response to LPS by 36% and 52%, respectively.

Conclusions: Nox4 plays a central role in LPS-induced proinflammatory responses by endothelial cells in an ROS-dependent manner.

KEYWORDS NADPH oxidase 4; Toll-like receptor 4; Lipopolysaccharide; Reactive oxygen species; Endothelial cells; Atherosclerosis


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Leukocyte–endothelial interaction is a critical step for development of inflammatory vascular lesions. Chemokines and adhesion molecules are upregulated by proinflammatory cytokines and contribute to initiation and progression of the interactions between endothelial cells and leukocytes. Key steps for the induction of inflammation include adhesion of inflammatory cells to the endothelium followed by transendothelial migration of adherent cells into underlying tissues. These events involve sequential monocytic–endothelial interactions such as rolling, firm adhesion and transendothelial migration, which are mediated by specific interactions between endothelial cells and monocytes [1].

Endothelial cells respond to LPS in a TLR4-dependent manner leading to the subsequent release of inflammatory cytokines such as IL-6, IL-8, and MCP-1 and enhanced expression of adhesion molecules including ICAM and VCAM [2]. Chemokines are the members of a superfamily of small polypeptides that mediate migration, proliferation and activation of a variety of inflammatory cells including monocytes. MCP-1 and IL-8 are known to play a crucial role in the recruitment of monocytes, neutrophils, and T lymphocytes to inflammatory lesions [3,4]. ICAM-1, a 76–115 kDa cell surface glycoprotein with five extracellular immunoglobulin-like domains, is a ligand for LFA-1, Mac-1 and CD43 [5]. ICAM-1 is constitutively expressed at low levels on endothelial cells. However, in response to various pathophysiological stimuli, ICAM-1 is robustly induced and mediates the interaction between endothelial cells and blood cells central to the development of inflammation [6]. It has been reported that NF-{kappa}B, a key transcription factor in inflammation, controls the expression of MCP-1 and ICAM-1 protein in site of endothelial dysfunction [7]. Moreover, the activation of NF-{kappa}B can be controlled by reactive oxygen species (ROS) such as superoxide anion and H2O2 [8,9].

Several lines of evidence indicate that LPS enhances the generation of reactive oxygen species (ROS) such as superoxide anion and hydrogen peroxide as second messengers in cell signaling [10]. The primary form of the free radical is superoxide anion (O2–) that is mainly generated by NADPH oxidase (Nox). The radical anion is spontaneously or enzymatically converted to H2O2 in cells. ROS are produced in mammalian cells in response to the activation of various cell surface receptors and contribute to intracellular signaling processes which in turn regulate various biological activities including host defense and metabolic conversions [11–13]. To date, seven homologs (Nox1, Nox3, Nox4, Nox5, Duox1, and Duox2) of gp91phox (Nox2) have been identified in various nonphagocytic cells [14]. Among them, Nox4 is highly expressed in the endothelial cells. Expression of Nox1 and Nox2 was also detected in the endothelial cells, but the expression level was significantly lower [15]. It has been demonstrated that LPS-induced proinflammatory responses such as NF-{kappa}B activation and subsequent expression of chemokines and adhesion molecules can be controlled by ROS [8,9,16]. We have recently shown that Nox4 is involved in LPS-mediated ROS generation and NF-{kappa}B activation in human HEK293T cells and U937 monocytic cells [11]. Here, we propose the role of Nox4 as a key player in LPS-induced proinflammatory responses by human endothelial cells.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1. Cell culture
Human aortic endothelial cells (HAECs) were purchased from Cambrex Corp. HAECs were grown to confluence in endothelial growth medium (EGM2) supplemented with 2% FBS, 50 µg/ml gentamicin sulfate, ascorbic acid, heparin, VEGF, hEGF, hFGF-B, R3-IGF-1 and hydrocortisone at 37 °C under 5% CO2 in 95% air. All experiments were carried out with HAEC in sixth or seventh passage. Human monocytic U937 cells were cultured in RPMI 1640 (JBI) supplemented with 10% FBS, penicillin G (100 U/ml), streptomycin (100 µg/ml) and L-glutamine (2 mmol/l).

2.2. Construction of siRNA for Nox4 (Nox4 siRNA) and transfection
A sequence of 21-nucleotide residues in length (GTCAACATCCAGCTGTACCdTdT) specific to the human Nox4 cDNA (nucleotide residues, 1474 to 1492) was selected for synthesis of a small interfering RNA (siRNA). Control siRNA (Dharmacon Cat# D-001210-02-20) was purchased from Dharmacon. HAECs were transiently transfected with Nox4 siRNA and control RNA (200 nmol/l) using OligofectamineTM reagent following the manufacturer's instruction (Invitrogen) [17]. After 48 h of transfection with siRNA, HAECs were used for the subsequent experiments. Depletion of endogenous Nox4 by the siRNA was confirmed by RT-PCR or quantitative fluorescent real time RT-PCR.

2.3. Measurement of intracellular H2O2 production
HAECs (2–3x105) cultured in 35 mm culture dish were transfected with 200 nmol/l control siRNA or Nox4 siRNA for 36–48 h with Oligofectamine. Intracellular levels of H2O2 were assayed after stimulation of cells with 100 ng/ml LPS (Escherichia coli 055:B5) (Sigma) for 30 min. Dishes of confluent cells were washed with HBSS and incubated for 5 min in the dark at 37 °C with HBSS containing 5 µmol/l 2,7-dichlorofluorescin-diacetate (DCF-DA) (Molecular Probes). Analysis of intracellular H2O2 production with scanning confocal microscope (LSM 510, Carl Zeiss) was performed as described previously [11,12]. All experiments were repeated at least three times.

2.4. Yeast two-hybrid assay
Direct interaction of TLR4-C with Nox4-C terminal was examined by yeast two-hybrid assay (BD Clontech, Palo Alto, CA). The yeast cell line EGY48 pre-transformed with p80p-LacZ construct were cotransformed with one of the pB42AD-TLR4-C mutants (C1, amino acid 678–706; C2, amino acid 704–737; C3, amino acid 739–769; C4, amino acid 767–801; C5, amino acid 800–833) and one of the pLexA-Nox4-C mutants (C1, amino acid 354–450; C2, amino acid 451–530; C3, amino acid 531–578) as shown in Fig. 2. Following the selection for Trp+ and His+ phenotype, the Leu-dependent growth and βgal activity were tested in induction medium (SD/galactose/Raffinose). As the positive and negative controls, yeast cell lines EGY48/p80p-LacZ cotransfected with pLexA53 and pB42ADT and EGY48/p80p-LacZ cotransfected with pLexA/pB42AD were used, respectively.


Figure 2
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  		Fig. 2 Interaction of TLR4 with Nox4. A and B, Schematic representation of TLR4 and Nox4 and various constructs of TLR4 and Nox4. C, The yeast EGY48 transformed with p80p-LacZ cells were cotransformed with a pair of pB42AD-TLR4 mutant and pLexA-Nox4-C mutant as shown in A and B. Following the selection for Trp+ and His+ phenotype, its Leu-dependent growth and β-galactosidase activity were tested in induction medium (SD/galactose/Raffinose). D, Glutathione-Sepharose 4B bead-conjugated GST or GST-TLR4 mutant proteins were incubated with lysates (500 µg) of HEK293T cells transfected with pcDNA3.0-HA-Nox4-C for 4 h at 4°C in 20 mM phosphate buffer (pH 7.0) containing 150 mM NaCl, 1% Triton X-100 and 0.5% NP40. After washing with cold lysis buffer for 3 times, proteins retained specifically by the glutathione-Sepharose 4B beads were subjected to immunoblot analysis with antibodies to HA.

 
2.5. Immunoblot analysis
HAECs (2x105) were plated in 6-well plates and incubated with LPS (100 ng/ml) for the indicated time. Cells were deprived of serum for 3–4 h and subsequently incubated for the indicated time at 37 °C in the absence or presence of LPS (100 ng/ml). Incubation with the agonist was terminated by addition of the lysis buffer (50 mM Tris–HCl (pH 7.4), 1 mM EDTA, 1% NP40, 150 mM NaCl, Na-deoxycholate 0.25%, 1 ìg/ml aprotinin, 1 ìg/ml leupeptin, 1 mM PMSF, 1 mM Na3Vo4, 1 mM NaF, phosphatase inhibitor cocktail 1 (Sigma; PP1, PP2A, microcystin LR, cantharidin, (-)-p-bromotetramisole). Cell lysates (20 µg of protein) were electrophoresed in 12% SDS gels, and proteins were transferred to nitrocellulose membrane. Human I{kappa}B{alpha} and β-actin were detected with anti-human I{kappa}B{alpha} monoclonal antibody (Santa Cruz) and anti-human β-actin monoclonal antibody (Sigma) followed by incubation with an HRP-conjugated goat anti-mouse IgG antibodies (Santa Cruz). The membrane was developed using Enhanced Chemiluminescence Detection reagents (Pierce).

2.6. Measurement of NF-B{kappa} (p65) binding activity in the nuclear extracts
HAECs (5x105) were transfected with 200 nmol/l control siRNA or Nox4 siRNA for 36–48 h with Oligofectamine. After transfection, HAECs were stimulated with LPS for 1 h, rinsed twice with cold PBS, and scraped and centrifuged for 5 min at 300 xg. The pellet was the resuspended in a hypotonic buffer (20 mM Hepes, 5 mM NaF, 10 mM Na2MoO4, 0.1 mM EDTA). After 15 min incubation on ice, Nonidet P-40 was added to 0.5% and the lysate was further incubated on ice for 30 min with gentle rocking. The lysate was then centrifuged for 10 min at 14,000 xg. The resulting supernatant constitutes the total protein extract. The protein concentration of the lysate was determines by Bradford assay (Bio-Rad, Hercules, CA). The presence of nuclear proteins was assayed for the DNA binding activity of p65. Activation of the subunit in 4 ìg of protein extract was determined using an NF-{kappa}B p65 enzyme-linked immunosorbent assay (ELISA)-based transcription factor assay kit, TransAM NF-{kappa}B assay kit (ActiveMotif, Carlsbad, CA), according to the manufacturer's protocol. The NF-{kappa}B detecting antibody recognizes an epitope on p65 that is accessible only when NF-{kappa}B is activated.

2.7. Reverse transcription-PCR (RT-PCR)
Total RNA was prepared from each cell culture using Trisol (Invitrogen). Reverse transcription was performed using the total RNA as the template with the RT-for-PCR kit (Promega). PCR amplification of Nox4 mRNA were carried out with the primers 5'-CTCAGCGGAATCAATCAGCTGTG-3' and 5'-AGAGGAACACGACAATCAGCCTTAG-3'. GAPDH (glyceraldehydes 3-phosphate dehydrogenase) served as the loading control.

2.8. Determination of MCP-1 or IL-8 protein levels
HAECs (2–3x105) cultured in 6-well plates were transfected as described above. HAECs were left untreated or treated with LPS (100 ng/ml), and the medium was collected. MCP-1 or IL-8 protein levels were determined by ELISA assay kit according to the manufacturer's instruction (R&D systems).

2.9. Monocyte migration assay
Migration assays were performed in a Trans system (Transwell, Costar; Cambridge, MA). Transwells (polycarbonate membranes with 8 µm pore sizes) in 24-well plates with polycarbonate membranes pre-coated on their underside with gelatin B (1 µg/ml) were used. HAEC monolayers were activated with 100 ng/ml LPS for 8 h, and U937 monocytes (105 cells in 100 µl media) were added to the upper compartment. After 2 h of incubation, the membrane was recovered and fixed in methanol and stained by H&E. The number of migrated cells was determined using a light microscope. Blocking experiments were performed by incubating the cells with 10 µg/ml of the neutralizing {alpha}-MCP-1 antibody (R&D systems) for 30 min at room temperature or with the corresponding isotype control antibody.

2.10. Monocyte adhesion assay
U937 monocytes were incubated in the absence or presence of IFN-{gamma} (100 U/ml) and LPS (100 ng/ml) for 24 h. HAECs were treated in the absence or presence of LPS (100 ng/ml) for 6 h, U937 cells were stained with 5 µmol/l TMRE (Molecular Probes) for 30 min and then unstimulated or activated U937 cells (1x106 cells) were added and allowed to adhere to HAECs. After 30 min, cells were washed twice to remove any nonadherent U937 cells, and adherent U937 cells were analyzed under an inverted microscope. In the blocking experiments, anti-CD54 antibody (BD PharMingen; San Diego, CA) was added to HAECs for 1 h prior to cell adhesion. Mouse IgG1 was used as isotype control antibody (BD PharMingen).

2.11. Measurement of ICAM-1 protein expression level
The expression of ICAM-1 was determined by flow cytometric analysis as previously described [18]. HAECs were stained with an Phycoerythrin (PE) anti-mouse CD54 (ICAM-1) (1:500) (BD PharMingen). Control was stained using a mouse IgG1 isotype antibody conjugated with PE (1:100). HACEs were stimulated with LPS (100 ng/ml) for 6 h. Cells were washed twice with PBS and removed from the culture flask by trypsinization. Subsequently, 5x105 cells were incubated with saturating amounts of the respective monoclonal antibody for 1 h at 4 °C. Cells were washed twice with PBS, resuspended in PBS and subjected to flow cytometry in a fluorescence-activated cell sorter (FACScan, Becton and Dickinson, Mountain View, CA). After excitation at 488 nm, the emission of PE was recorded through specific band pass filters (515 to 545 nm).

2.12. Statistical analysis
Data are means±standard deviation (SD) of values from over three independent experiments. Levels of significance for comparisons between samples were determined by one-way analysis of variance using InStat software (GraphPad Software Inc., San Diego, CA).


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1. Nox4 is essential for LPS-induced ROS generation and NF-{kappa}B activation by human aortic endothelial cells
To verify the role of Nox4 isozyme in LPS-mediated ROS generation in HAECs, we subjected the cells to transient transfection with small interference RNA specific for the Nox4 (Nox4 siRNA). Transfection of HAECs with Nox4 siRNA specifically reduced Nox4 expression (Fig. 1C). Quantitative fluorescent real time-PCR indicated that the level of Nox4 mRNA was reduced over 80% in the HAECs transfected with Nox4 siRNA compared to the control cell transfected with scramble RNA (data not shown). Importantly, we confirmed that Nox4 siRNA does not interfere the expression of Nox1 or Nox2 isozymes (data not shown). HAECs transfected with Nox4 siRNA failed LPS-induced ROS generation, whereas the cells transfected with scramble siRNA exhibited an increase of ROS in response to LPS, suggesting that Nox4 is also essential for LPS-induced ROS production in HAECs (Fig. 1A and B).


Figure 1
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  		Fig. 1 Effect of Nox4 isozyme knockdown on LPS-mediated H2O2 generation. A and B, After transfection of Nox4 siRNA to HAECs for 48 h, the generation of H2O2 was monitored by confocal microscopic analysis of DCF fluorescence in the absence or presence of LPS (100 ng/ml for 30 min). Representative microscopic fields are shown in (A) and the fluorescence intensity relative to nonstimulated cells is shown in (B). Data in B are mean±SD of three independent experiments. *Comparison with control groups, P<0.001; **significantly different from the groups stimulated with LPS alone, P<0.01. C, Total RNA was prepared and assayed by RT-PCR. GAPDH serves as a loading control.

 
Although we have previously shown that COOH-terminal region of Nox4 interacts with TIR of TLR4, the detailed molecular nature of this interaction has yet to be determined. To study the interaction between Nox4 and TLR4, we utilized yeast two-hybrid experiment and GST pull-down assay. We transformed each of the Nox4 derivatives, pLexA-Nox4-C1 (amino acid 354–450), pLexA-Nox4-C2 (amino acid 451–530), and pLexA-Nox4-C3 (amino acid 531–578), into yeast harboring one of the five TLR4 derivatives, pB42AD-TLR4-C1 (amino acid 678–706), pB42AD-TLR4-C2 (amino acid 704–737), pB42AD-TLR4-C3 (amino acid 739–769), pB42AD-TLR4-C4 (amino acid 767–801), and pB42AD-TLR4-C5 (amino acid 800–833) (Fig. 2A and B). Yeast cells expressing pB42AD-TLR4-C2 or pB42AD-TLR4-C3 together with pLexA-Nox4-C2 showed normal growth and produced blue colony without leucine in the presence of X-gal (Fig. 2C). Interaction of pLexA-Nox4-C2 with pB42AD-TLR4-C3 was stronger than that with pB42AD-TLR4-C2 as judged by the growth of blue colony (Fig. 2C). We prepared GST fusion proteins containing various TIR domain of TLR4 (GST-TLR4-C1, amino acids 678–706; GST-TLR4-C2, amino acids 704–737; GST-TLR4-C3, amino acids 739–769; GST-TLR4-C4, amino acid 767–801; GST-TLR4-C5, amino acids 800–833) and conjugated then to glutathione-Sepharose 4B beads. Various GST-conjugated TIR domains were incubated with lysates of HEK293T cells expressing HA-tagged Nox4-C (amino acid 250–573) and then subjected to immunoblot analysis with antibody against HA. The results indicated that GST-TLR4-C3 or GST-TLR4-C4 domains specifically interact with Nox4-C, whereas control GST, GST-TLR4-C1, or GST-TLR4-C5 does not (Fig. 2D). Moreover, Nox4-C has stronger affinity with GST-TLR4-C3 than that with GST-TLR4-C4. Both experiments thus clearly demonstrated that TLR4-C3 (amino acid 739–769) is the responsible domain for interaction with COOH-terminal region of Nox4 (Fig. 2).

We have shown that Nox4 is involved in LPS-mediated NF-{kappa}B activation in HEK293T cells [11]. Therefore, we measured the LPS-mediated degradation of the cytoplasmic inhibitor protein, I{kappa}B{alpha} as an indication of NF-{kappa}B activation. Stimulation of HAECs with LPS resulted in markedly reduced I{kappa}B{alpha} levels, whereas the degradation of I{kappa}B{alpha} was blocked by knockdown of Nox4 isozyme suggesting that Nox4 is involved in NF-{kappa}B activation in response to LPS stimulation (Fig. 3A). To examine LPS-dependent NF-{kappa}B activation in further detail, we measured the NF-{kappa}B DNA binding activity in lysates of HAEC. LPS-stimulated NF-{kappa}B (p65) binding activity was enhanced by 20% compared to the control cells (Fig. 3B). However, in the protein extracts of HAECs transfected with Nox4 siRNA, such NF-{kappa}B (p65) binding activity was decreased by 58% compared to the cells transfected with scrambled siRNA (Fig. 3B). We next evaluated the effect of direct interaction of COOH-terminal region of Nox4 with TIR of TLR4 on the activation of NF-{kappa}B in response to LPS. The stimulation of HEK293T cells expressing TLR4/CD14/MD2 with LPS resulted in a 2.5-fold increase NF-{kappa}B activity (Fig. 4A). In contrast, contransfection of COOH-terminal region of Nox4 (Nox4-C, amino acid 250-573, Fig. 4B) to HEK293T cells expressing TLR4/CD14/MD2 showed a markedly decrease NF-{kappa}B activity in response to LPS (Fig. 4A and B). The most likely explanation is that Nox4-C blocks interaction of Nox4 with TLR4 by competing for TIR of TLR4 and thereby acting in a dominant-negative manner. These results demonstrate that COOH-terminal region of Nox4 is involved in LPS-mediated NF-{kappa}B activation.


Figure 3
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  		Fig. 3 Effect of Nox4 on NF-{kappa}B activation. A, HAECs were transfected with control siRNA or Nox4 siRNA and further cultured in a serum-free medium for 4 h. The cells were treated with LPS for varying time periods (0–60 min). Then, total proteins (20 µg) were subjected to immunoblot analysis with antibodies against I{kappa}B{alpha} and actin, respectively. Degradation of I{kappa}B{alpha} was quantitatively analyzed by gel scanning program (Un-Scan it, Silk Scientific Co.). B, HAECs (5x105) were transfected with 200 nM control siRNA or Nox4 siRNA for 48 h with Oligofectamine (Invitrogen). After transfection, HAECs were treated with LPS (100 ng/ml) for an additional 1 h. Nuclear extracts (4 µg total protein) were assayed for the binding DNA activity of p65 using the TransAM NF-{kappa}B assay kit (ActiveMotif, Carlsbad, CA) according to the manufacturer's manual. *Comparison with control groups, P<0.001; **significantly different from the groups stimulated with LPS alone, P<0.01.

 

Figure 4
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  		Fig. 4 Effect of COOH-terminal region of Nox4 on LPS-induced NF-{kappa}B activation. A, HEK293T cells were grown until 50% confluent and were transfected transiently with an NF-{kappa}B-dependent luciferase reporter construct (({kappa}B)3-IFN-luciferase) and constructs of pcDNA3.0-HA-Nox4-C (amino acid 250–573), pFLAG-CMV-TLR4, pFLAG-CMV-CD14, pFLAG-CMV-MD2, and pCMV-β galactosidase (βgal) plasmid using Effectine (Qiagen, Valencia, CA). Empty vectors were used as controls. After 48 h, cells were stimulated with 1 µg/ml LPS. After 6 h, luciferase activity was measured using the Luciferase Reporter Assay System (Promega, Madison, WI) according to manufacturer's instructions and normalized relative to βgal activity. B, Cells transfected as in (A), were lysed and subjected to immunoblot analysis with antibodies to HA; filters were reprobed with antibodies to actin.

 
3.2. Effect of Nox4 isozyme on the LPS-induced expression of chemokines and migration
Several lines of evidence demonstrate that LPS-mediated NF-{kappa}B activation leads the production of chemokines [16]. To test whether Nox4 could affect the production of cytokines in HAECs, we measured IL-8 and MCP-1 secretion levels. Incubation of Nox4 siRNA-transfected HAECs with LPS (100 ng/ml) resulted in a significant decrease of the secretion of IL-8 and MCP-1 by 55% and 30%, respectively (Fig. 5A and B). These results indicate that Nox4 isozyme is involved in LPS-induced IL-8 and MCP-1 secretion.


Figure 5
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  		Fig. 5 Effect of Nox4 knockdown on LPS-induced cell migration. A and B, HAECs transfected with control siRNA or Nox4 siRNA were stimulated in the absence or presence of LPS (100 ng/ml). For MCP-1 and IL-8 measurement, the medium was collected at 8 h and at 6 h after LPS treatment, respectively. Cytokine levels were determined by ELISA assay kit according to the manufacturer's instruction. *Comparison with control groups, P<0.001; **significantly different from the groups stimulated with LPS alone, P<0.01. C, HAECs transfected with control siRNA or Nox4 siRNA were stimulated in the absence or presence of LPS (100 ng/ml). U937 cells were allowed to migrate on gelatin-coated Transwell plates for 2 h. After fixing and H&E staining, the number of migrated cells was determined. *Comparison to the value of the control groups, P<0.001; #significantly different from the value of LPS alone stimulated, P<0.001.

 
MCP-1 is a key mediator in activation and initiation of migration of monocytic cells. Therefore, we explored the role of Nox4 isozyme in migration of monocytic cells to LPS-induced dysfunction area of endothelial cells. Endothelial cells were stimulated with LPS for 8 h, and then the migration of monocytic U937 cells to endothelial cells through Transwell pores was measured. LPS-induced migration of monocytic U937 cells could be blocked by pretreatment with antibodies against MCP-1, whereas control antibody (IgG1k) had no inhibitory effect on U937 cell migration (Fig. 5C). The result suggests that MCP-1 is essential for the migration of monocyte cells. As shown in Fig. 5C, LPS-induced migration of monocytic U937 cells through the pore of transwells were inhibited by downregulation of Nox4 compared to the control cells transfected with control siRNA (2.7-fold increase with Nox4 siRNA versus 4.2-fold increase with control siRNA). The result demonstrated that sequential activation of Nox4 and NF-{kappa}B by LPS is essential for the MCP-1 production and migration of monocytic cells to endothelial cells.

3.3. Inhibition of Nox4 isozyme suppresses LPS-induced ICAM-1 expression and adhesion between endothelial cells and monocytic cells
Expression of adhesion molecules on the surface of endothelial cells is critical for the association of monocytic cells in circulation. LPS enhances the expression of adhesion molecules on the surface of HAECs in NF-{kappa}B activation-dependent manner, ultimately leading to the association of ECs with monocytes [19]. We investigated the role of Nox4 in the expression of ICAM-1, an important adhesion molecule, in HAECs in response to LPS. Cells were stimulated with LPS for 6 h, and the expression of ICAM-1 was measured by flow cytometry. Upon stimulation with LPS, ICAM-1 expression was increased in a time- and dose-dependent manner in HAECs. The optimal induction was achieved by 100 ng/ml LPS (data not shown). HAECs transfected with the Nox4 siRNA showed a similar basal expression level of ICAM-1 compared to the control HAECs. PE-fluorescence intensity as an indication of ICAM-1 expression in control cells increased about 6-fold in response to LPS. However, HAECs-transfected with Nox4 siRNA showed a reduction of the expression of ICAM-1 by 30% (Fig. 6A). Pretreatment of HAECs with 5 µmol/l MG-132 as an inhibitor of NF-{kappa}B activation resulted in the suppression of LPS-induced ICAM-1 expression by 45% (Fig. 6A).


Figure 6
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  		Fig. 6 Effect of Nox4 knockdown on LPS-induced adhesion of U937 monocytic cells to HAECs through ICAM-1. A, For ICAM-1 protein expression, HAECs were pretreated with MG-132 (5 µmol/l) for 1 h, then stimulated with LPS (100 ng/ml) for 6 h. ICAM-1 protein expression was determined by flow cytometric analysis. 10,000 cells were routinely analyzed using WinMDI 3.2 program in FACScalibur (Becton Dickinson). *Comparison to the value of the control groups, P<0.001; MFI, Mean Fluorescence Intensity. B, After transfection of Nox4 siRNA to HAECs, HAECs were stimulated with LPS (100 ng/ml) for 24 h. After co-cultured with U937 cells for 30 min, adherent U937 cells were counted under an inverted epifluorescence microscope. #Significantly different from the value of stimulation with LPS stimulated alone, P<0.001.

 
We next examined monocytic U937 cells adhesion to endothelial cells. Endothelial cells treated with LPS for 6 h, and then monocyte adhesion to endothelial cells was determined. Monocytic adhesion to LPS-stimulated endothelial cell was increased compared to the untreated control cells by 71-fold (Fig. 6B). Addition of antibodies against CD54 (ICAM-1) and CD18 (LFA-1/Mac-1) to LPS-stimulated endothelial cells resulted in inhibited adhesion of monocytic cells to endothelial cells compared to the addition of control antibodies (Fig. 6B). Moreover, CD18 (LFA-1/Mac-1) expression induced by LPS/IFN-{gamma} on monocytic cell surface was increased in time-dependent manners (data not shown). Downregulation of Nox4 by transfection of Nox4 siRNA in HAECs significantly inhibited LPS-induced adhesion of monocytic cells with endothelial cells (34-fold increase with Nox4 siRNA versus 71-fold increase with control siRNA) (Fig. 6B). These results demonstrate that TLR4–Nox4–ROS–NF-{kappa}B pathway is critical for the expression of ICAM-1 and adhesion of monocytic cells with endothelial cells.


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
It is now widely accepted that inflammation contributes to various stages of atherogenesis [1,20,21]. The process of inflammation is defined as an infiltration of blood leukocytes to tissues with aided by chemokines and adhesion molecules. Several lines of evidence indicate that reactive oxygen species (ROS) play an important role in this process [22]. ROS generation in response to a variety of stimuli has been shown to be required for intracellular signaling in many cell types [10–12]. ROS can regulate intracellular kinase activities through reversible inactivation of phosphatase and can activate transcription factors including NF-{kappa}B which in turn leads to expression of downstream numerous genes [8,10]. It is well established that redox-sensitive transcriptional factor, NF-{kappa}B plays an important role in immune responses and inflammation processes [8,9]. For example, during the course of serious bacterial infections, bacterial LPS interacts with TLR4 resulting in an activation of transcription factor NF-{kappa}B. This process is crucial in upregulating the transcription of genes encoding proinflammatory cytokines.

Several reports indicated that ROS are likely involved in the expression of inflammatory cytokines such as IL-6, IL-8 and MCP-1 [23]. Recent studies have shown that Rac1 and NADPH oxidase are involved in the regulation of MCP-1 gene expression in endothelial cells and that antioxidants repress LPS-induced IL-8 secretion in THP-1 cells [24]. Additionally, ROS production has an important role in the surface expression of ICAM-1 and monocyte adhesion [17]. However, the manner in which ROS regulates monocyte adhesion is unclear.

Although we previously showed that COOH-terminal region of Nox4 becomes directly coupled with the TIR region of TLR4 in response to LPS and that the complex of Nox4 with TLR4 in turn induces ROS generation and NF-{kappa}B activation, molecular details of the interaction have not been defined. Several lines of evidence suggest that COOH-terminal region of NADPH oxidase isozymes is responsible for the interaction with cytosolic regulatory proteins [25,26]. In this report, we showed that Nox4-C2 (amino acid 451–530) strongly interacts with TIR of TLR4 (Fig. 2). Amino acid sequence in the region of Nox4 is highly similar to the interacting sequences in Nox1 and Nox2. Moreover, the overexpression of COOH-terminal region of Nox4 inhibited LPS-mediated NF-{kappa}B activation indicating that the region by itself can act as dominant negative form in the cells (Fig. 4). In the present study, we characterized the functional role of Nox4 in inflammatory signaling in HAECs in response to LPS. We showed that depletion of Nox4 represses the IL-8 and MCP-1 secretion by 55% and 30%, respectively, and completely blocks LPS-induced ROS generation (Figs. 1 and 5Go). These results suggest that Nox4 directly affects the production of inflammatory cytokines. As seen in Fig. 6, the expression of ICAM-1 in response to LPS in HAECs transfected with Nox4 siRNA was reduced by 30% as compare to the level in the control cells. Even though ICAM-1 expression was reduced by 30%, the monocyte adhesion to HAECs was markedly blocked (Fig. 6). Taken together, Nox4 generated ROS signal appears to play a critical role in mediating LPS-induced monocyte adhesion.

Bacterial infection can enhance the development of early atherosclerotic lesions by stimulating the interaction of endothelial cells with monocytes [2,27]. This interaction is enhanced by ICAM-1 expression on endothelial cells. LPS induces adhesion molecule expression on endothelial cells which is tightly linked with vascular disease development [27]. Several reports have documented that TLR-mediated cell signaling contributes to vascular pathogenesis including atherosclerotic processes. Specifically, the expression of TLR1, TLR2, and TLR4 has been demonstrated in vascular tissues with atherosclerotic lesion [28]. Minimally modified LDL (mm-LDL) is recognized by TLR4 and CD14 on macrophage surface resulting in intracellular actin polymerization and spreading of macrophages [29]. Bacterial and human heat shock protein 60 (HSP60) stimulates TLR4, leading to the expression of proinflammatory cytokines [30]. These results suggest that bacterial and viral infections promote the progress of proinflammatory stage in atherosclerosis. Moreover, two recent reports studying animal model with TLR4 signaling defects indicated that innate immunity plays a critical role in proinflammatory responses leading to atherosclerosis [31,32]. Blockage of TLR4 signaling in mice with MyD88 deficiency resulted in a decrease in lipid contents and expression of proinflammatory cytokines for atherogenesis. Based on experimental results in animal model and in vitro experiment in this report, we propose a more detailed molecular mechanism regarding the linkage between TLR4 signaling and atherosclerosis.

In conclusion, we propose that Nox4-derived reactive oxygen species play a critical role in LPS-induced signaling cascades leading to migration and adhesion of monocyte to HAECs. Our conclusion is based on the observations that LPS-induced NF-{kappa}B activation is mediated by Nox4-induced ROS generation, that NF-{kappa}B regulates in turn the secretion of chemokines such as IL-8 and MCP-1, and that expression of adhesion molecule ICAM-1 leads to a proinflammatory condition in endothelial cells.


   Acknowledgements
 
This work is supported by the NCRC program of MOST/KOSEF (Grant # R15-2006-020-00000-0) through the Center for Cell Signaling & Drug Discovery Research at Ewha Womans University, by 21st century Frontier Functional Proteomics Project (FPR05C2-510) from the Ministry of Science and Technology, by a grant of the Korea Health 21 R&D Project (grant number A06-00043579), Ministry of Health and Welfare and by Seoul R&BD program (grant number 10527). We thank Dr. Jaesang Kim for critical reading.


   Notes
 
1 Both authors contributed equally to this work. Back

Time for primary review 38 days


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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