Growth Factors Enhance Interleukin-1β-Induced Persistent Activation of Nuclear Factor-κB in Rat Vascular Smooth Muscle Cells
Objective— Activation of extracellular signal-regulated kinases (ERKs) is required for interleukin-1β to persistently activate nuclear factor (NF)-κB and concomitantly express inducible NO synthase (iNOS) in rat vascular smooth muscle cells (VSMCs). The present study examined whether platelet-derived growth factor (PDGF) or epidermal growth factor (EGF) could influence the VSMC response to interleukin-1β via an ERK-related signaling pathway.
Methods and Results— Treatment of VSMCs with PDGF or EGF alone potently induced ERK phosphorylation and DNA synthesis but did not induce NF-κB activation or iNOS expression. However, either PDGF or EGF markedly enhanced interleukin-1β-induced persistent NF-κB activation and iNOS expression but did not affect the early and transient NF-κB activation. Growth factor-induced DNA synthesis was attenuated in the presence of interleukin-1β. Inhibition of ERK phosphorylation with selective inhibitors (PD98059 or U0126) attenuated interleukin-1β-induced persistent NF-κB activation and iNOS expression in either the absence or presence of the growth factors.
Conclusions— These results indicate that interleukin-1β-induced expression of NF-κB-dependent genes, such as iNOS, is potentiated in the presence of growth factors through a mechanism requiring ERK-dependent enhanced NF-κB activation, and the results also suggest that NF-κB activation is not required for PDGF or EGF to trigger DNA synthesis in VSMCs.
- extracellular signal-regulated kinase
- growth factors
- nuclear factor-κB
- vascular smooth muscle cells
Persistent activation of nuclear factor (NF)-κB has been observed in human atherosclerotic lesions,1–4⇓⇓⇓ in which it may regulate the expression of numerous genes controlling inflammation, cell differentiation, proliferation, and apoptosis. Interleukin (IL)-1β may play a pivotal role in this pathophysiological process. We have recently demonstrated that the extracellular signal-regulated kinases (ERKs) are involved in the IL-1β signaling pathway, leading to prolonged activation of NF-κB and inducible NO synthase (iNOS) gene expression in rat vascular smooth muscle cells (VSMCs).5 The ERK signaling pathway consists of a protein kinase cascade that has been reported to link growth signals with nuclear gene transcription.6 Inhibition of ERK activation by either selective inhibitors or antisense oligodeoxynucleotides directed toward downregulation of ERK synthesis showed little or no effect on IL-1β-induced early activation of NF-κB but effectively reduced the IL-1β-induced prolonged NF-κB DNA binding and iNOS expression,5 suggesting that activation of ERK might be a signal not only for cell proliferation but also for the cellular response to proinflammatory cytokines, such as IL-1β.
Growth factors, such as platelet-derived growth factor (PDGF) and epidermal growth factor (EGF), coexist with IL-1β in atherosclerotic lesions.7–10⇓⇓⇓ However, the relationship of these growth factors with IL-1β in the modulation of VSMC function is not well defined. PDGF and EGF potently activate ERK and cause VSMC proliferation, a response that has been implicated during the progression of vascular changes that occur during atherosclerosis, restenosis, and hypertension.7–10⇓⇓⇓ Several studies have suggested that growth factors, including PDGF and EGF, activate NF-κB in cultured cells.11,12⇓ However, the mechanisms by which growth factors activate NF-κB are vague and controversial. Whether activation of NF-κB is required for cell proliferation is also speculative. The present study demonstrated that either PDGF or EGF alone did not activate NF-κB but dramatically enhanced the IL-1β-induced persistent activation of NF-κB and iNOS expression through an ERK-dependent mechanism and that NF-κB activation is not required for growth factors to initiate DNA synthesis. This finding reveals a potentially important relationship between growth factors and IL-1β in the signal integration that may occur in vascular remodeling or atherogenesis.
DMEM/Ham’s F-12 medium (DMEM/F12) and FCS were purchased from Life Technologies. Recombinant human IL-1β (specific activity ≈1×108 U/mg) was purchased from Genzyme. PDGF (BB isoform) and EGF were from Upstate Biotechnology. PD98059, U0126, SB203580, and wortmannin were from Calbiochem. Monoclonal antibody against iNOS was from Transduction Laboratories. Antibodies against phospho-p44/42 mitogen-activated protein kinase (MAPK, Thr202/Tyr204) and p44/42 MAPK were from Cell Signaling. Antibody against NF-κB p65 was from Santa Cruz. The NF-κB consensus oligonucleotide was from Promega. [γ-32P]ATP and [6-3H]thymidine were from DuPont-New England Nuclear. All other materials used were commercial products of the highest grade available.
Rat VSMCs from the thoracic aorta were isolated and cultured as described previously.13 Cells between passages 5 and 9 were used in the experiments. At confluence, the cells were washed twice with serum-free medium and then maintained in DMEM/F12 with 0.1% FCS for 24 hours. The medium was refreshed just before treatment. The cells were then incubated with or without additions (IL-1β, PDGF, EGF, inhibitors, or vehicle) for designated times, as indicated in Results.
VSMCs were cultured on 4-well Lab-Tek II chamber slides (Nalge Nunc International) under the same conditions described above. After treatment, the cells were washed with cold PBS, fixed for 5 minutes in methanol at −20°C, and air-dried at room temperature. The staining was performed by incubating with 10% normal goat serum in PBS for 20 minutes, followed by incubating with primary antibody (5 μg/mL NF-κB p65 polyclonal antibody [C-20], sc-372, Santa Cruz) for 2 hours in PBS with 1.0% BSA, washing 3 times with PBS, incubating for 1 hour with FITC-conjugated goat anti-rabbit antibody (1:100 dilution, Jackson Laboratory) in PBS with 1.0% BSA, washing 3 times with PBS, and finally mounting with aqueous mounting medium. The images observed under a fluorescence microscope were recorded as TIFF files with BioQuant-TCW95 software, version 2.50.4, on a linked computer.
Determination of Nitrite
The concentration of nitrite, a stable metabolite of NO, in the medium was determined with Griess reagent as described previously.14
Determination of Protein
Protein content of the cell lysates was determined with a BCA protein assay reagent (Pierce), with BSA used as a standard.
Western Blot Analysis
Whole cell lysates were prepared, and Western blot analysis was performed as described previously.14 The images were obtained and analyzed by using the pdi 420oe Scanning Densitometer (pdi, Inc).
Electrophoretic Mobility Shift Assay
Nuclear extracts were prepared, and DNA-binding activity was assessed by electrophoretic mobility shift assay (EMSA) with the use of NF-κB consensus oligonucleotide as described previously.14
Determination of DNA Synthesis
VSMCs cultured in 24-well plates were incubated in DMEM/F12 containing 0.1% FCS, with or without 10 ng/mL PDGF or EGF in the absence or in the presence of 3 ng/mL IL-1β for 18 hours, after which [3H]thymidine (1 μCi per well) was added, and the cells were further incubated for 6 hours. The cells were then washed 3 times with PBS, treated with ice-cold 10% trichloroacetic acid for 15 minutes, and washed with ethanol-ethyl ether (3:1 [vol:vol]). The acid-insoluble material was dissolved in 0.5 mL of 0.3N NaOH. The protein content was determined by BCA method and the radioactivity was determined by liquid scintillation spectrometry.
PDGF and EGF Potentiate IL-1β-Induced iNOS Expression and Nitrite Production Through an ERK-Dependent Pathway
Treatment of VSMCs with IL-1β for 24 hours induced nitrite production, and either PDGF or EGF significantly enhanced the IL-1β-induced nitrite production (Figure 1A). However, treatment of the cells with PDGF or EGF alone for 24 hours did not induce the production of nitrite. The MAPK/ERK kinase (MEK) inhibitor U0126 significantly attenuated the nitrite production induced by either IL-1β alone or IL-1β plus the growth factors.
Western blot analysis showed that the expression of iNOS induced by IL-1β was dramatically enhanced by either PDGF or EGF and was accompanied by the enhanced phosphorylation of ERK (Figure 1B). However, the growth factors alone did not induce iNOS expression (data not shown). ERK phosphorylation and iNOS expression were both dramatically inhibited by U0126. SB203580, a selective inhibitor for p38 MAPK, and wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI3 kinase), showed no effect on the expression of iNOS induced by IL-1β plus PDGF, suggesting that the p38 MAPK pathway and PI3 kinase pathways are not responsible for the enhanced iNOS induction. IL-1β caused a prolonged phosphorylation of ERK in VSMCs (Figure 1C, top). Either PDGF or EGF also induced ERK phosphorylation and sustained the phosphorylation of ERK at a level higher than basal level for several hours (Figure 1C, bottom).
IL-1β Induces ERK-Dependent Persistent Activation of NF-κB
VSMCs were exposed to IL-1β for 1 hour or 16 hours in the absence or presence of PD98059, an inhibitor of ERK phosphorylation, before nuclear extracts were obtained for EMSA. NF-κB activation after exposure to IL-1β for 1 hour (representative of transient activation) and 16 hours (representative of persistent activation) is shown in Figure 2. The activated NF-κB at 1 hour was composed mainly of the p65/p50 heterodimer. However, after 16 hours, the p65/p50 heterodimer and the p50/p50 homodimer were clearly visible. Inhibition of ERK phosphorylation with PD98059 showed little or no influence on the IL-1β-induced NF-κB DNA-binding at 1 hour but resulted in an obviously decreased NF-κB DNA binding at 16 hours, suggesting that ERK activation is required only for the persistent activation of NF-κB involving formation of both the p65/p50 heterodimer and the p50/p50 homodimer complexes.
Immunofluorescence staining (Figure 3) showed the translocation of NF-κB p65 from the cytoplasm to the nucleus after IL-1β treatment for 1 hour and for 16 hours. However, in the presence of either PD98059 or U0126, the nuclear translocation of NF-κB p65 induced by IL-1β at the 16-hour time point was markedly reduced, whereas at the earlier time, the translocation was similar to that observed when cells were treated with IL-1β alone. These immunohistochemical results are consistent with the findings observed in EMSA, suggesting that the inhibition of ERK phosphorylation reduced the activation and translocation of NF-κB in response to IL-1β, which led to a decreased NF-κB binding to DNA.
PDGF and EGF Enhance IL-1β-Induced Persistent Activation of NF-κB Through an ERK-Dependent Pathway
As revealed by EMSA (Figure 4A), VSMCs treated for 1 hour or 16 hours with either PDGF or EGF alone did not show NF-κB activation. However, either PDGF or EGF obviously enhanced the IL-1β-induced persistent (at 16 hours) but not transient (at 1 hour) NF-κB activation. In the presence of U0126, the persistent NF-κB activation was clearly attenuated. To examine whether the growth factors alone could activate NF-κB at other time points, a time course from 15 minutes to 3 hours after PDGF or EGF treatment was examined, with untreated cells and IL-1β-treated cells as the negative and positive controls, respectively. The result from EMSA showed no visible NF-κB activation by either PDGF or EGF at any of the examined time points (Figure 4B).
Activation of NF-κB Is Not Required for PDGF or EGF to Initiate DNA Synthesis
Although NF-κB was not activated, treatment with either PDGF or EGF for 24 hours significantly increased [3H]thymidine incorporation in VSMCs. The basal and growth factor-enhanced [3H]thymidine incorporations were both reduced significantly by IL-1β (Figure 5), despite the obvious NF-κB activation that was dramatically enhanced by either PDGF or EGF, as shown in Figure 4.
NF-κB activation plays a central role in host defense and inflammatory events associated with atherosclerosis, rheumatoid arthritis, asthma, septic shock, lung fibrosis, glomerulonephritis, cancer, and AIDS.15,16⇓ Atherosclerosis has been considered an unusual form of chronic inflammation within the artery wall,7,8⇓ and persistent activation of NF-κB has been found in atherosclerotic lesions or after arterial balloon injury in animal experiments.2,17–19⇓⇓⇓ The mechanisms by which NF-κB is persistently activated have not been completely defined. Little is known about whether or not (or how) local growth factors and cytokines contribute to the regulation and persistent activation of NF-κB in atherosclerotic lesions.
We have recently found that ERK activation is required for IL-1β-induced persistent, but not early and transient, activation of NF-κB in rat VSMCs.5 This suggests that IL-1β may activate NF-κB by 2 different ways: one is rapid but transient and independent of ERK activation; another is slow but persistent and dependent on sustained ERK activation. However, it raised the question of whether or not activation of ERK is sufficient to persistently activate NF-κB. In the present study, we demonstrated that treatment of the cells with either PDGF or EGF alone failed to activate NF-κB, despite potent and sustained ERK activation. However, both growth factors dramatically enhanced IL-1β-induced prolonged activation of NF-κB without influencing the early and transient activation. Inhibition of ERK phosphorylation significantly attenuated IL-1β-induced prolonged activation of NF-κB, either in the absence or in the presence of the growth factors, strongly supporting the hypothesis that the growth factors enhance the prolonged NF-κB activation through an ERK-dependent mechanism. This finding suggests that activation of ERK is not solely responsible but that it is required for the prolonged activation of NF-κB triggered by growth factors and cytokines. A possible mechanism for the involvement of ERK in the persistent activation of NF-κB is shown schematically in Figure 6.
Our data showing that PDGF or EGF alone fail to activate NF-κB is consistent with the report by Rauch et al,20 who demonstrated that PDGF could not activate NF-κB in cultured human VSMCs or fibroblasts. However, an early report by Obata et al11 showed that growth factors, including PDGF, EGF, basic fibroblast growth factor, and insulin-like growth factor-1, activated NF-κB in rat VSMCs. Although it is difficult to explain the discrepancy, it is possible that endotoxin contamination potentiated the effect of growth factors in that study. It has been suggested that NF-κB is a target of the PI3 kinase/Akt pathway in PDGF signaling.12 However, Rauch et al presented evidence that Akt phosphorylation was not involved in NF-κB activation in human VSMCs and fibroblasts. We also observed that either PDGF alone or EGF alone potently induced Akt phosphorylation in rat VSMCs (data not shown) but did not induce NF-κB activation, indicating that ether PDGF or EGF alone cannot activate NF-κB through the PI3 kinase/Akt pathway in rat VSMCs. Our data showing that wortmannin had no effect on iNOS expression induced by IL-1β plus PDGF also indirectly suggest that the PI3 kinase/Akt pathway is not required for NF-κB activation. Habib et al21 recently reported that a high level of EGF receptor expression, a frequent occurrence in human tumors, is optimal for EGF-induced NF-κB activation. In their study, 2 cell lines, including MDA-MB-468 cells with a high level of EGF receptor expression, showed a constitutive activation of NF-κB even without the addition of EGF into the cultures and that the addition of EGF enhanced the constitutive NF-κB activation. Interestingly, various cancer cell lines have been found to be capable of expressing IL-1,22–24⇓⇓ which may account for the upregulation of EGF receptor expression in several cancer cell lines, including MDA-MB-468 cells.25 A well-defined signaling pathway for IL-1β to activate NF-κB has been described as follows: IL-1β→IL-1 receptor-1→TRAF6 (tumor necrosis factor receptor-associated factor 6)→NIK (NF-κB-inducing kinase)→I-κB kinases (α, β, γ)→I-κB (α, β)→NF-κB.26 We have previously shown that ERK may participate in persistent activation of NF-κB, in part, by regulating I-κBβ degradation.5 Removal of IL-1β from cultures could reduce and stop the NF-κB activation.5 These results suggest that a unique signal (most probably from TRAF6 to I-κB kinases) is triggered by IL-1β but cannot be activated by growth factors and might be a prerequisite for the persistent activation of NF-κB (see Figure 6).
Whether or not NF-κB activation is required for growth factors to initiate cell proliferation remains speculative. Our data in the present study indicate that either PDGF or EGF alone did not activate NF-κB but stimulated [3H]thymidine incorporation in rat VSMCs, suggesting that NF-κB activation is not a required proliferative signal. Interestingly, in the presence of IL-1β, the basal level and the growth factor-stimulated [3H]thymidine incorporations were significantly reduced. This occurred despite the sustained activation of ERK and NF-κB and the induction of iNOS. These data suggest that different signal integration in response to growth factors or IL-1β may greatly change the cell phenotype after vascular injury or during atherosclerosis. In the absence of IL-1β, PDGF and EGF stimulate VSMC proliferation, whereas in the presence of IL-1β, PDGF and EGF enhance NF-κB-dependent gene expression (such as iNOS), which in turn may attenuate VSMC proliferation. Therefore, inhibition of ERK activation may inhibit not only cell proliferation but also the persistent activation of NF-κB and the expression of numerous NF-κB-dependent genes. These findings may provide important clues for therapeutic considerations in the diseases thought to involve persistent activation of NF-κB during their pathogenesis.
This work was supported by National Institutes of Health grants HL-55620 and HL-31607 and American Heart Association Research Award 0160251T.
Received August 22, 2002; revision accepted September 3, 2002.
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