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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2000;20:385.)
© 2000 American Heart Association, Inc.

Vascular Biology

Convergence of Redox-Sensitive and Mitogen-Activated Protein Kinase Signaling Pathways in Tumor Necrosis Factor-–Mediated Monocyte Chemoattractant Protein-1 Induction in Vascular Smooth Muscle Cells

Gilles W. De Keulenaer; Masuko Ushio-Fukai; QiQin Yin; Andrew B. Chung; P. Reid Lyons; Nobukazu Ishizaka; Kalpana Rengarajan; W. Robert Taylor; R. Wayne Alexander; Kathy K. Griendling

From the Division of Cardiology, Emory University School of Medicine, Atlanta, Ga.

Correspondence to Kathy K. Griendling, PhD, Emory University, Division of Cardiology, 1639 Pierce Dr, 319 WMB, Atlanta, GA 30322. E-mail kgriend{at}emory.edu

	Abstract

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Abstract—Monocyte chemoattractant protein-1 (MCP-1) is an important component of the inflammatory response of the vessel wall and has been shown to be regulated by cytokines, such as tumor necrosis factor- (TNF-). However, the precise signaling pathways leading to MCP-1 induction have not been fully elucidated in vascular smooth muscle cells (VSMCs). Cytokine signal transduction involves protein kinases as well as reactive oxygen species (ROS). The relation between these 2 factors is not clear. In this study, we show that TNF- induces a parallel phosphorylation of extracellular signal–regulated kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (p38MAPK) and increases MCP-1 mRNA expression in cultured VSMCs. Inhibition of ERK1/2 but not p38MAPK caused a partial attenuation of MCP-1 induction (43±10% inhibition). Incubation of VSMCs with multiple antioxidants (diphenylene iodonium, liposomal superoxide dismutase, catalase, N-acetylcysteine, dimethylthiourea, and pyrrolidine dithiocarbamate) had no effect on TNF-–mediated MCP-1 upregulation. However, simultaneous blockade of the ERK1/2 and ROS pathways by using PD098059 combined with diphenylene iodonium or N-acetylcysteine potently enhanced the ability of MAPK kinase inhibitors to abrogate MCP-1 mRNA expression (100±2% inhibition). Thus, parallel ROS-dependent and ERK1/2-dependent pathways converge to regulate TNF-–induced MCP-1 gene expression in VSMCs. These data unmask a complex but organized integration of ROS and protein kinases that mediates cytokine-induced vascular inflammatory gene expression.

Key Words: monocyte chemoattractant protein-1 • smooth muscle cells • reactive oxygen species • cytokines • tyrosine kinase

	Introduction

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Monocyte attachment to the endothelium and accumulation in the subendothelial space of the arterial wall are critical initial steps in atherosclerotic lesion formation. This process is the result of complex activation reactions in the vessel wall, including an increase in chemotactic activity and induction of proinflammatory genes, such as adhesion molecules and chemoattractant factors. Expression of one of the main chemoattractant proteins for monocytes, appropriately named monocyte chemoattractant protein-1 (MCP-1), is increased during atherogenesis^{1 2} and is sensitive to regulation by cytokines, including tumor necrosis factor- (TNF-), in several cell types.³

The precise signaling pathways leading to MCP-1 induction have not been fully elucidated. Conflicting reports in the literature implicate reactive oxygen species (ROS)-mediated nuclear factor B (NF-B)–dependent mechanisms as well as tyrosine kinase pathways in this process.^{4 5 6 7 8} To date, there has been no unifying integrated hypothesis to explain these apparently disparate results. TNF- activates multiple signaling pathways, most notably, mitogen-activated protein kinases (MAPKs).⁹ MAPKs are serine/threonine kinases that transduce signals from the cell membrane to the nucleus.^{10 11 12} Four groups of MAPKs have been identified in mammalian cells: the extracellular signal–regulated kinases 1 and 2 (ERK1/2, also termed p42/44 MAPK), the c-Jun NH₂-terminal kinases (JNK, also named stress-activated protein kinase [SAPK]), p38MAPK (also called CSBP), and big MAPK 1 (BMK1, also termed ERK5).^{10 12 13} ERK1/2 is stimulated by growth factors and mitogenic stimuli,^{10 12} whereas p38MAPK, JNK, and BMK-1 are primarily activated by cellular stresses, including proinflammatory cytokines.^{13 14} Stimulation of these MAPKs leads to activation of distinct transcription factors, including C-Fos, c-Jun, and possibly NF-B.^{10 15} Binding sites for each of these transcription factors are present in the mouse MCP-1 promoter.¹⁶

Although TNF- stimulation of MAPKs has not been demonstrated in vascular smooth muscle cells (VSMCs), we have previously shown that TNF- activates a membrane-associated nonmitochondrial oxidase that preferentially hydrolyzes NADH to produce superoxide.¹⁷ These findings raise the possibility that redox-sensitive pathways contribute to TNF-–induced MCP-1 expression in VSMCs. This has been shown to be true in other cell types,¹⁸ and NF-B and activator protein-1 (AP-1) binding sites¹⁶ represent possible targets for ROS. In addition, ROS can selectively stimulate different MAPKs, making the relation between NADH/NADPH oxidase–mediated pathways and MAPK-mediated protein phosphorylation events in the induction of MCP-1 expression by TNF- unclear. In the present study, therefore, we have analyzed the redox sensitivity and the involvement of MAPKs in TNF-–induced MCP-1 gene expression in VSMCs in an attempt to provide an integrated understanding of their respective roles in this process. We found that ROS-sensitive and redox-insensitive MAPK pathways mediate the induction of MCP-1 by TNF- in a convergent, nonserial, parallel signaling cascade circuit, emphasizing the highly organized interactive nature of intracellular signals leading to gene induction.

	Methods

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Materials
All chemicals were of analytical grade or better. BSA and phenylmethylsulfonyl fluoride were from Boehringer-Mannheim. Soybean trypsin inhibitor, glutamine, penicillin, streptomycin, calf serum, and trypsin-EDTA were purchased from GIBCO. TRI reagent was from Molecular Research Center. [³²P]dCTP was purchased from NEN Life Science Products. Magna NT nylon membranes and Prime-It II probe labeling kits were purchased from Stratagene, and Biospin columns were from Bio-Rad. The enhanced chemiluminescence (ECL) Western blotting detection system was purchased from Amersham Life Sciences. Diphenylene iodonium (DPI) was purchased from Toronto Research Chemicals. SB203580, PD098059, and PD169316 were purchased from Calbiochem. Liposomal superoxide dismutase was a kind gift from Dr Bruce Freeman (University of Alabama, Birmingham). The Anti-ACTIVE MAPK polyclonal antibody that was used for the detection of phosphorylated forms of ERK1/2 (Thr183/Tyr185) and U0126 were from Promega. Phospho-specific p38MAPK (Thr180/Tyr182) and ATF-2 antibodies were obtained from New England Biolabs, Inc. Common buffer salts were purchased from Fisher. All other chemicals and reagents, including DMEM with 25 mmol/L HEPES and 4.5 g/L glucose, were from Sigma Chemical Co.

Cell Culture
VSMCs were isolated from rat thoracic aorta by enzymatic digestion as described previously.¹⁹ Cells were grown in DMEM supplemented with 10% calf serum, 2 mmol/L glutamine, 100 U/mL penicillin, and 100 µmol/L streptomycin, passaged twice a week by harvesting with trypsin-EDTA, and seeded into 80-cm² flasks. For experiments, cells between passages 6 and 20 were seeded into 100-mm dishes, fed every other day, and used at confluence.

In some experiments, we used VSMCs that had been stably transfected with antisense p22phox.²⁰ In these cells, p22phox mRNA expression is completely inhibited, and protein expression is markedly attenuated.

Northern Blot Analysis
Total RNA was isolated by using TRI reagent. RNA (10 µg) was separated on 1% denaturing formaldehyde agarose gels, transferred to Nytran membranes (Schleicher & Schuell) by overnight upward capillary blotting with 10x SSC, and immobilized by UV cross-linking as described previously.²¹ Consistency of total RNA loading between samples was controlled by densitometric analysis of 28S RNA ultraviolet fluorescence in the presence of ethidium bromide. Mouse MCP-1 cDNA was labeled by use of a random primer labeling kit (Prime-It II) and [³²P]dCTP. Blots were prehybridized at least 2 hours and hybridized overnight at 42°C in the following solution: 1 mol/L NaCl, 50 mmol/L Tris HCl (pH 7.4), 5x Denhardt’s solution, 50% formamide, 0.5% SDS, and 100 µg/mL sheared and denatured salmon sperm DNA. Denhardt’s solution was omitted during hybridization. After hybridization, the blots were washed 3 times in 1x SSC and 0.1% SDS at 56°C. The blots were autoradiographed by using Hyperfilm-MP at -80°C, and the relative density of each band was determined by laser densitometry. Staining of the 28S band by ethidium bromide after transfer to the membrane was used for normalization.

Detection of p38MAPK and ERK1/2 by Immunoblotting
VSMCs at 80% to 90% confluence in 100-mm dishes were made quiescent by incubation with DMEM containing 0.1% calf serum for 48 hours. Cells were stimulated with agonist at 37°C for specified durations. After treatment, cells were washed 3 times with ice-cold PBS and put on ice. Cells were lysed with 500 µL ice-cold lysis buffer at pH 7.4 (mmol/L: HEPES 50, EDTA 5, and NaCl 50) containing 1% Triton X-100, protease inhibitors (10 µg/mL aprotinin, 1 mmol/L phenylmethylsulfonyl fluoride, and 10 µg/mL leupeptin), and phosphatase inhibitors (mmol/L: sodium fluoride 50, sodium orthovanadate 1, and sodium pyrophosphate 10). Solubilized proteins were centrifuged at 12 000g in a microfuge (4°C) for 30 minutes, and supernatants were stored at -80°C. Extracted protein was quantified by the micro-Bradford assay. Proteins (25 µg) were separated by using 10% SDS-PAGE and transferred to Hybond-ECL nitrocellulose membranes at 100 V for 1 hour. Membranes were blocked overnight at room temperature with PBS containing 6% nonfat dry milk and 0.1% Tween 20. The blots were incubated for 1 hour with primary antibodies (rabbit polyclonal phospho-specific p38, ERK1/2 antibodies that detect MAPK only when activated by phosphorylation on TXY) at 1:2000 and 1:40 000 in PBS containing 1% nonfat dry milk and 0.1% Tween 20, respectively. After incubation with secondary antibodies (horseradish peroxidase–conjugated goat anti-rabbit antibody, 1:1000) for 1 hour in PBS containing 1% nonfat dry milk and 0.1% Tween 20, phosphorylated forms of proteins were detected by ECL chemiluminescence. It has been shown that phosphorylation of MAPKs is associated with the activation of MAPKs^{22 23 24} ; therefore, phosphorylation was taken as a measure of MAPK enzymatic activity.

Western Analysis of MCP-1 Protein
VSMCs at 80% to 90% confluence were made quiescent by incubation with DMEM containing 0.1% calf serum for 48 hours and stimulated with TNF- at 37°C for the specified durations. After treatment, conditioned medium (10 mL) was collected and concentrated to 1 mL with Centricon filters (10 000g for 30 to 40 minutes). Proteins were separated on 4% to 20% Tris-glycine gels and transferred to nitrocellulose membranes. Membranes were blocked overnight at room temperature with PBS containing 5% nonfat dry milk and 0.1% Tween 20 and were incubated for 1 hour with primary rabbit MCP-1 antibodies at 1:500. After incubation with secondary antibodies (horseradish peroxidase–conjugated goat anti-rabbit antibody, 1:1000), MCP-1 proteins were detected by ECL chemiluminescence.

Statistical Analysis
Comparisons between groups were made by the unpaired Student t test. Results are expressed as mean±SE.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Induction of MCP-1 by TNF- and ROS
To compare the ability of TNF- and ROS to induce MCP-1 mRNA expression in VSMCs, we exposed cells to TNF-, H₂O₂, or xanthine/xanthine oxidase (to generate H₂O₂ and superoxide) and measured MCP-1 levels by Northern analysis. As shown in Figure 1A, TNF- (50 to 800 U/mL) dose-dependently increased MCP-1 expression as early as 2 hours, and levels remained elevated for at least 24 hours (Figure 1B). MCP-1 protein accumulation increased by 24 hours after TNF- (400 U/mL) by 13.1±1.6-fold (n=2). ROS were also able to induce MCP-1 mRNA: a 2-hour incubation of H₂O₂ upregulated MCP-1 mRNA in a dose-dependent manner (Figure 1B), and xanthine/xanthine oxidase (400 µmol/L, 20 mU/mL, 5 minutes), which generates superoxide and H₂O₂, caused a 2-fold increase in MCP-1 mRNA levels (data not shown).

View larger version (35K): [in this window] [in a new window]   Figure 1. Effect of TNF- and extracellular ROS on MCP-1 mRNA expression. RNA was extracted and prepared for Northern analysis as described in Methods. A, Dose-response relation of TNF-–induced MCP-1 mRNA expression is shown. At the top, VSMCs were exposed to medium alone (0) or medium with the indicated dose of TNF- for 2 hours. On the bottom, densitometric data (mean±SE) are shown for 3 independent experiments, in which MCP-1 mRNA expression in unstimulated cells was defined as 100%. B, On the left, VSMCs were exposed to medium alone or to medium with TNF- (400 U/mL) and harvested at the indicated time points. On the right, VSMCs were exposed to H2O2 (100 to 400 µmol/L) and harvested after 2 hours of incubation. These blots are representative of 3 identical experiments.

Role of ROS in TNF-–Induced MCP-1 Upregulation
We have previously shown that TNF- activates a membrane-bound p22phox-based NADH/NADPH oxidase and induces superoxide production,¹⁷ but the potential role of this oxidase in MCP-1 induction has not been investigated. To determine whether ROS are involved in TNF-–induced MCP-1 upregulation, we examined the effects of various antioxidants on this response. Figure 2A shows the effect of NADH/NADPH oxidase inhibition by preincubation with DPI, an inhibitor of flavin-containing oxidases,²⁵ and stable transfection with antisense p22phox.²⁰ Previous experiments have shown that both interventions inhibited TNF-–induced NADH oxidase activity, without affecting activity in control cells.¹⁷ However, neither DPI nor p22phox antisense transfection influenced the level of MCP-1 induced by TNF-, although antisense p22phox decreased the baseline. Similarly, inhibitors of xanthine oxidase (allopurinol, 10 mmol/L) and lipoxygenase/cytochrome P-450 monooxygenase (5,8,11,14-eicosatetraynoic acid, 50 µmol/L) did not attenuate TNF-–induced MCP-1 induction (data not shown).

View larger version (60K): [in this window] [in a new window]   Figure 2. A, Representative Northern blot showing the effect of DPI and p22phox antisense transfection on TNF-–induced MCP-1 mRNA expression. VSMCs were exposed to TNF- (400 U/mL) and harvested after 4 hours of incubation. Cells were preincubated with DPI (10 µmol/L) for 30 minutes before addition of TNF-. RNA was extracted and prepared for Northern analysis as described in Methods. B, Representative Northern analysis showing the effect of antioxidants on TNF-–induced MCP-1 mRNA expression. VSMCs were exposed to TNF- (400 U/mL) and harvested after 4 hours of incubation. Antioxidants NAC (10 mmol/L), dimethylthiourea (DMTU, 10 mmol/L), pyrrolidine dithiocarbamate (PDTC, 100 µmol/L), and liposomal superoxide dismutase (lip SOD, 200 U/mL) were preincubated 30 to 60 minutes before addition of TNF-. Catalase (CAT, 3000 U/mL) was added 72 hours before administration of TNF- and refreshed every 24 hours. RNA was extracted and prepared for Northern analysis as described in Methods. All examples are representative of 2 or 3 experiments.

To confirm further the lack of involvement of ROS in TNF-–induced MCP-1 upregulation, we used a series of antioxidant interventions that inhibit ROS-mediated reactions by different mechanisms. Neither N-acetylcysteine (NAC, 10 mmol/L), dimethylthiourea (10 mmol/L), pyrrolidine dithiocarbamate (100 µmol/L), liposomal superoxide dismutase (200 U/mL), nor catalase (3000 U/mL) had any effect on MCP-1 mRNA induction by TNF- (Figure 2B). Each of these interventions has been shown to successfully inhibit ROS-mediated events in VSMCs and other cell types when used under the circumstances and concentrations of the present study.^{18 26 27 28 29} These data suggest that blocking redox-mediated reactions alone in VSMCs is not sufficient to inhibit TNF-–mediated MCP-1 gene induction.

Role of MAPK Pathways in TNF-–Induced MCP-1 Upregulation
Activation of MAPKs depends on tyrosine phosphorylation. As a first approach to assess a possible involvement of MAPK pathways in TNF-–induced MCP-1 expression, we examined the effect of the nonselective tyrosine kinase inhibitor genistein. As shown in Figure 3, genistein dose-dependently reduced MCP-1 mRNA in both unstimulated and TNF-–stimulated cells. The extent of inhibition by genistein was greater in cells exposed to TNF-, indicating that MCP-1 induction by TNF- is at least partially dependent on a tyrosine kinase pathway. These data raise the possibility that MAPKs may be involved in this response.

View larger version (41K): [in this window] [in a new window]   Figure 3. Top, Representative Northern analysis showing the effect of genistein (25 to 150 µmol/L) on TNF-–induced MCP-1 mRNA expression. VSMCs were preincubated with genistein 30 to 60 minutes before addition of TNF- (400 U/mL) for 4 hours. RNA was extracted and prepared for Northern analysis as described in Methods. Bottom, Relative density of each band determined by laser densitometry and normalized to the 28S band by ethidium bromide.

To assess the ability of TNF- to activate MAPKs in these cells, we measured phosphorylation of ERK1/2 and p38MAPK, which correlates with activation. As shown in Figure 4A, TNF- induced a rapid biphasic increase in ERK1/2 phosphorylation, which was detected as early as 2 minutes after addition of TNF- and reached a maximum after 15 minutes. In contrast, TNF- activation of p38MAPK (Figure 4B) was monophasic and peaked at 10 minutes

View larger version (22K): [in this window] [in a new window]   Figure 4. Time course of TNF- stimulation of ERK1/2 and p38MAPK phosphorylation in VSMCs. Preparation of cell lysates, Western blotting, and detection of phosphorylation were performed as described in Methods. Growth-arrested VSMCs were stimulated with 400 U/mL TNF- for the indicated times, and ERK1/2 (A) and p38MAPK (B) phosphorylation were measured. At the top of panels A and B are representative immunoblots of TNF-–induced phosphorylation of ERK1/2 and p38MAPK. At the bottom of panels A and B are averaged data quantified by densitometry of immunoblots using NIH image 1.61, expressed as fold increase in phosphorylation, in which the phosphorylation observed in cells at time 0 was defined as 1.0 (control). Values are the mean±SE for 3 independent experiments. *P<0.05 vs control.

Because TNF- strongly activated both ERK1/2 and p38MAPKs, we examined the involvement of these pathways in TNF-–stimulated MCP-1 mRNA expression in VSMCs. PD098059 (100 µmol/L) and U0126 (50 µmol/L) are specific inhibitors of MAPK kinase, the upstream kinase responsible for activation of ERK1/2. These compounds completely inhibit ERK1/2 phosphorylation induced by the agonist.^{30 31} Similarly, the downstream effects of p38MAPK, such as ATF-2 phosphorylation,³¹ can be prevented by incubation of the cells with 10 µmol/L SB203580 or PD169316.^{31 32} As shown in Figure 5, inhibition of ERK1/2 alone partially attenuated TNF-–induced upregulation of MCP-1 mRNA (43±10%, n=16). Inhibition of p38MAPK alone had no effect on this response, nor did it enhance the ability of ERK1/2 inhibitors to block MCP-1 induction (P<0.05). This observation suggests that the ERK1/2 pathway is partly responsible for TNF-–stimulated MCP-1 upregulation.

View larger version (43K): [in this window] [in a new window]   Figure 5. Effect of ERK1/2 and p38MAPK inhibition on TNF-–induced MCP-1 expression. Growth-arrested VSMCs were pretreated in the absence or presence of the p38MAPK inhibitors SB203580 (SB, 10 µmol/L) or PD169316 (PD16, 10 µmol/L), the MAPK kinase inhibitors PD098059 (PD98, 100 µmol/L) or U0126 (U01, 50 µmol/L), or the indicated combination for 30 minutes. The cells were then stimulated with 400 U/mL TNF- for 2 hours in the continuous presence of inhibitors. Open bars indicate absence of TNF-; solid, hatched, crosshatched, or stippled bars indicate presence of TNF-. Data are mean±SE densitometric data (number of replicates is given above bars), with MCP-1 mRNA expression in unstimulated cells defined as 100%. *P<0.05 vs response in the absence of inhibitors.

Interaction of ROS and ERK1/2 in MCP-1 Regulation
The inhibition of TNF-–induced MCP-1 mRNA expression by ERK1/2 inhibitors is only partial. In VSMCs, ERK1/2 activation is largely redox insensitive, and others have shown that ROS can transactivate MCP-1, raising the possibility that both ERK1/2 and a redox-sensitive pathway combine to regulate MCP-1 expression. Figure 6 shows that this was indeed the case. DPI (10 µmol/L) alone did not affect TNF-–stimulated MCP-1 expression, but in the presence of PD098059, it completely abolished the effect of TNF- (100±2% inhibition, Figure 6). Similarly, reduction of oxidant stress by incubation of the cells with NAC (10 mmol/L) in the presence of PD098059 substantially reduced TNF-–induced MCP-1 mRNA upregulation (data not shown). In contrast, coincubation of DPI and SB203580 had no effect on TNF-–induced MCP-1 mRNA expression (data not shown). These data suggest that ROS and ERK1/2 converge to regulate MCP-1.

View larger version (55K): [in this window] [in a new window]   Figure 6. Effect of ERK1/2 and ROS inhibition on TNF-–induced MCP-1 expression. Growth-arrested VSMCs were pretreated in the presence or absence of 10 µmol/L DPI and/or 100 µmol/L PD098059 (PD) for 30 minutes. The cells were then stimulated with 400 U/mL TNF- for 2 hours in the continuous presence of inhibitors. Top, Representative Northern blot for MCP-1 mRNA. Ethidium bromide staining of 28S rRNA is also shown as an indication of loading. Bottom, Densitometric data (mean±SE) for 3 independent experiments, in which MCP-1 mRNA expression in unstimulated cells was defined as 100%. *P<0.01 vs response in the absence of inhibitors.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The present study provides evidence for a novel role of ERK1/2 and ROS in TNF-–induced MCP-1 mRNA expression in VSMCs. ERK1/2 is necessary, but not sufficient, for MCP-1 induction. An additional redox-sensitive pathway also appears to be necessary, because inhibition of ROS generation by DPI or NAC enhanced the effect of ERK1/2 inhibition. These observations support a potential interaction of ROS with MAPKs in the regulation of inflammatory gene expression in VSMCs.

The present literature suggests a critical role for ROS in MCP-1 gene expression. Numerous studies have shown that exogenous ROS can induce MCP-1 mRNA,^{6 18 33} and in general, agonist-induced or mechanical force–induced MCP-1 expression can be inhibited by antioxidants.^{6 34 35} We have previously shown that TNF- activates a p22phox-based NADH/NADPH oxidase in VSMCs¹⁷ ; thus, our present finding that multiple classes of antioxidants had no effect on TNF-–induced MCP-1 mRNA expression in VSMCs was somewhat unexpected. It has been shown, however, that TNF- stimulates the binding of several transactivating factors to the MCP-1 promoter,¹⁶ only some of which are redox sensitive, indicating that MCP-1 expression may be the result of integrated activation of multiple components of a complex regulatory system.

The present observation that genistein partially attenuates TNF-–stimulated MCP-1 induction suggests that MAPKs or other tyrosine kinase–dependent mechanisms are involved. In VSMCs, TNF- stimulates ERK1/2 and p38MAPK (Figure 4 and References 13, 36, and 37^{13 36 37} ), as well as JNK.³⁸ Furthermore, we have previously shown that p38MAPK but not ERK1/2 is downstream from agonist-induced ROS production in these cells.^{17 31} The role of MAPK in TNF-–mediated MCP-1 induction, however, has not been examined. In the present study, we found that 2 chemically distinct MAPK kinase inhibitors achieved significant partial attenuation of TNF-–stimulated MCP-1 mRNA expression. This suggests that ERK1/2 is necessary, but not sufficient, for induction of this gene. Similar results have been found for angiotensin II induction of MCP-1 in these cells.³⁹ Interestingly, blockade of ROS-dependent mechanisms by DPI or NAC enhanced the ability of inhibitors of the ERK1/2 pathway to abrogate MCP-1 expression. In other systems, p38MAPK has been shown to participate in TNF-–induced MCP-1 expression,^{40 41} but this does not appear to be the case in VSMCs. The TNF-–stimulated ROS-sensitive pathway that participates in induction of MCP-1 is apparently independent of p38MAPK, because SB203580 and PD169316 alone had no effect on MCP-1 expression, nor were they able to substitute for antioxidants in augmenting the effects of MAPK kinase inhibitors. Taken together, these data suggest that ROS-dependent and ERK1/2 pathways converge to regulate MCP-1 mRNA expression.

The identity of the ROS-sensitive pathway remains to be defined. As noted above, TNF- activates JNK in some cell types,³⁸ and JNK has been shown to be redox sensitive.⁴² However, to date, no role for JNK in MCP-1 mRNA upregulation has been demonstrated.⁴⁰ It is likely that other, non-MAPK signaling mechanisms also contribute to agonist-mediated MCP-1 gene expression. In other systems, phosphatidylinositol 3-kinase has been shown to be involved in platelet-derived growth factor–stimulated MCP-1 upregulation⁴³ and has been postulated to be redox sensitive.⁴⁴ Evidence also exists for a role of NF-B in MCP-1 expression,^{45 46} but the redox sensitivity of NF-B activation is controversial and may depend on the agonist.⁴³

As discussed above, MCP-1 expression depends on the interaction of multiple regulatory factors whose activity is temporally controlled. Thus, although ERK1/2 phosphorylation is transient, activation of this signaling pathway may initiate subsequent downstream events whose effects are more sustained. Alternatively, prolonged induction of MCP-1 mRNA by TNF- may at least in part be due to MAPK-independent pathways (see above). Although inhibition of ERK1/2 does partially attenuate TNF- induction of MCP-1, the lack of effect of antioxidants on the MCP-1 response to TNF- suggests that ROS alone do not fully activate transcription. However, when MAPK kinase inhibitors are combined with antioxidants, TNF- induction of MCP-1 mRNA levels is completely blocked, suggesting an interaction between the 2 pathways. Interestingly, Ahmad et al⁴⁷ have recently shown that TNF- activates AP-1–associated signaling components that regulate the nuclear activity of NF-B in endothelial cells. This raises the possibility that ERK1/2, via activation of AP-1, may modify NF-B or other unidentified ROS-sensitive transcription factors.

Recent work has revealed that many biological processes are sensitive to the biochemical action of oxidizing metabolites in the cell. In contrast to previous speculations that ROS have only toxic biological properties, these studies have demonstrated that cell growth and hypertrophy,^{20 25 28} inflammation,²⁹ paracrine communication,^{48 49} and apoptosis⁵⁰ can be initiated and/or propagated by fluctuations in the redox state. Imbalances in production or scavenging of ROS can lead to pathological processes, including atherogenesis. These processes have been shown to be mediated by cytokines, critical determinants of the inflammatory response. Our recent discovery that TNF- activates NADH oxidase,¹⁷ together with the present data demonstrating its involvement in TNF-–mediated proinflammatory gene induction, underscores the potential importance of NADH/NADPH oxidase and ROS in vascular disease.

In summary, we showed in the present study that TNF- activates both ERK1/2 and p38MAPK but that only ERK1/2 is involved in the upregulation of MCP-1. However, concomitant and parallel production of ROS is necessary for full induction of MCP-1. Thus, ROS-dependent and ERK1/2-dependent pathways converge to modulate TNF-–induced MCP-1 expression. These data provide insight into the complex, but highly organized, interactions of ROS and protein kinases that mediate cytokine-induced vascular inflammatory gene expression.

	Acknowledgments

 
This study was supported by National Institutes of Health grant HL-58863 and a fellowship from the Belgian American Educational Foundation (to Dr De Keulenaer). We thank Carolyn Morris for excellent secretarial assistance.

Received July 12, 1999; accepted August 16, 1999.

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