ToxSci Advance Access originally published online on April 11, 2006
Toxicological Sciences 2006 92(1):311-320; doi:10.1093/toxsci/kfj194
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c-Src Is the Primary Signaling Mediator of Polychlorinated Biphenyl–Induced Interleukin-8 Expression in a Human Microvascular Endothelial Cell Line
* Molecular Neuroscience and Vascular Biology Laboratory, Department of Surgery and College of Agriculture, University of Kentucky, Lexington, Kentucky 40536
1 To whom correspondence should be addressed at Molecular Neuroscience and Vascular Biology Laboratory, Department of Surgery, Division of Neurosurgery, University of Kentucky Medical Center, 593 Wethington Building, 900 South Limestone, Lexington, KY 40536. Fax: (859) 323-2705. E-mail: michal.toborek{at}uky.edu.
Received January 6, 2006; accepted March 29, 2006
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ABSTRACT |
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Interleukin-8/CXCL8 (IL-8) is a prominent factor that modulates endothelial cell proliferation, migration, and angiogenesis. Therefore, the present study focused on the regulatory mechanisms of IL-8 expression induced by environmental pollutants such as polychlorinated biphenyls (PCBs). Treatment of human microvascular endothelial cells (HMECs) with specific PCB congener, 2,2',4,6,6'-pentachlorobiphenyl (PCB 104), dose dependently increased levels of IL-8 mRNA and secreted protein. IL-8–neutralizing antibody inhibited migration of endothelial cells stimulated by conditioned media derived from PCB 104–treated HMECs. Site-directed mutagenesis of the IL-8 promoter– and DNA-binding assays revealed that activator protein 1 (AP-1) and nuclear factor
B (NF-B) sites are required for PCB 104–induced IL-8 transcription. Most importantly, pharmacological inhibition of Src kinase activity or overexpression of dominant-negative c-src in HMECs resulted in a significant decrease in IL-8 expression and promoter activity. In contrast, ectopic expression of activated c-Src markedly increased promoter activity of IL-8. These stimulatory effects of dominant-positive c-src were abrogated by mutagenesis of AP-1– and NF-B–binding sites in the IL-8 promoter.
Key Words: IL-8; endothelial cells; c-Src; AP-1; NF-B; angiogenesis; PCB.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Migration of endothelial cells is a crucial process involved in various pathophysiological conditions, including neovascularization, tumor growth, and metastasis formation (Heidemann et al., 2003
). Interleukin-8/CXCL8 (IL-8), a member of the chemokine family, is a potent chemotactic, proangiogenic, and prometastatic factor. It is secreted as a 72– or 77–amino acid protein by a wide variety of cell types including endothelial and epithelial cells (Koch et al., 1992; Nakamura et al., 1991; Xie, 2001). Substantial evidence exists that IL-8, via its paracrine effects, can regulate angiogenesis and contribute to metastatic potential and tumor progression, leading to enhanced cell proliferation, migration, and matrix metalloprotease (MMP) expression in endothelial cells (Li et al., 2003; Matsuo et al., 2004; Murdoch et al., 1999). To further demonstrate its importance in metastasis formation, IL-8 was identified as a major endothelium-secreted chemokine in metastatic melanoma cells that posses CXCR1 receptor (Ramjeesingh et al., 2003).
IL-8 expression is regulated primarily at the level of gene transcription, and its promoter region contains functional binding sites for a variety of transcription factors, including nuclear factor B (NF-B) and activator protein 1 (AP-1) (Mukaida et al., 1994; Sica et al., 1990; Yasumoto et al., 1992). The signaling mechanisms leading to IL-8 overexpression in endothelial cells are not fully understood. One of the candidate pathways for IL-8 production may involve the Src family. Indeed, activation of the Src family kinases has been known to play an important role in cell migration, angiogenesis, and chemokine induction (Frame et al., 2002). It was also shown that Src signaling is involved in expression of another critical proangiogenic factor, vascular endothelial growth factor (VEGF) (Ellis et al., 1998; Mukhopadhyay et al., 1995; Summy and Gallick, 2003). Finally, c-Src may contribute to the activation of NF-B and AP-1 and thus stimulate expression of inflammatory mediators (Liu et al., 2001; Trevino et al., 2005; Yeh et al., 2004).
Vascular endothelial cells form a continuous monolayer that functions as a selective barrier between blood and the underlying layers of vessel walls. Therefore, endothelial cells may be targeted by a variety of environmental toxicants, including polychlorinated biphenyls (PCBs) (Annas et al., 1998). PCBs are a class of polychlorinated aromatic hydrocarbons composed of 209 discrete congeners. Due to their high lipophilicity and structural stability, PCBs are persistent environmental pollutants (Kimbrough, 1995). Evidence indicates that chronic exposures to PCBs can induce several harmful effects, including neurotoxicity, inflammatory responses, and tumor promotion (Andersson et al., 1999; Hennig et al., 2002; Kodavanti et al., 1995). Our research demonstrated that exposure to PCBs can induce potent endothelial cell activation, enhance cellular oxidative stress, and stimulate a variety of potential prometastatic processes, such as increased permeability across the vascular endothelium and induction of adhesion molecules (Choi et al., 2003; Eum et al., 2004; Hennig et al., 2002). We demonstrated that both coplanar and highly ortho-substituted noncoplanar PCB congeners, such as 2,2',4,6,6'-pentachlorobiphenyl (PCB 104), are effective in the activation of endothelial cells and induction of prometastatic properties of the microvasculature. For example, treatment with PCB 104 induced endothelial hyperpermeability and markedly increased transendothelial migration of the MDA-MB-231 cells (a highly metastatic breast cancer cell line) across human microvascular endothelial cells (HMECs) (Eum et al., 2004). PCB 104 exposures also resulted in enhanced adhesiveness of the THP-1 cells (a human acute monocytic leukemia cell line) to human endothelial cells (Choi et al., 2003). PCB-mediated hyperpermeability was associated with overexpression of VEGF, whereas transmigration of tumor cells was regulated by increased expression of MMP-3 (Eum et al., 2003, 2004).
Due to the importance of environmental pollutants in vascular responses leading to formation of tumor metastases, the aim of the present study was to determine the role of IL-8 in PCB 104–mediated migration of endothelial cells. In addition, we evaluated the signaling mechanisms of PCB 104–induced IL-8 overexpression in endothelial cells. We provided evidence that exposure of microvascular endothelial cells to PCB 104 results in an increase in IL-8 mRNA and protein expression. Most importantly, we indicated that the c-Src kinase–mediated activation of NF-B and AP-1 is the major signaling pathway that regulates PCB 104–induced transcriptional increase in IL-8 mRNA.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Reagent and plasmids.
PCB 104 (> 99% pure) was purchased from AccuStandard (New Haven, CT). PP2, PP3, LY294002, and PD98059 were purchased from Calbiochem (La Jolla, CA). PP1 was obtained from Biomol (Plymouth Meeting, PA). Anti-Akt antibody and antibodies used for supershift assays were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies against phospho-ERK1/2, phospho-Akt, phospho-Src, and Src were obtained from Cell Signaling (Beverly, MA). Neutralizing anti–IL-8 antibodies were purchased from R&D Systems (Minneapolis, MN). All other chemicals and reagents were purchased from Sigma (St Louis, MO).
Luciferase reporter constructs containing human IL-8 promoter (– 133 to + 44 bp; pIL-8Luc), NF-B–mutated IL-8 promoter (pIL-8Luc-mNF-B), and AP-1–mutated IL-8 promoter (pIL-8Luc-mAP-1) were generous gifts from Dr Mukaida at Kanazawa University, Japan. Dominant-negative c-src (c-src-DN) and dominant-positive c-src (c-src-DP) vectors and empty pUSEamp(–) vector were purchased from Upstate (Waltham, MA). pNF-B-Luc and pAP-1-Luc reporter constructs were obtained from Stratagene (La Jolla, CA).
Cell cultures and PCB treatment.
HMECs were a generous gift from Dr Eric Smart (University of Kentucky Medical Center, Lexington, KY). HMEC is an immortalized cell line obtained by the transformation of HMECs with the SV40 large T antigen. These cells retain the endothelial cell phenotype and functional characteristics (True et al., 2000). HMECs were cultured in MCDB 131 medium enriched with 10% fetal bovine serum (FBS), 2mM L-glutamine, 50 units/ml penicillin, 50 µg/ml streptomycin, 1 µg/ml hydrocortisone, and 0.01 µg/ml epidermal growth factor (EGF) in 5% CO2 atmosphere at 37°C. Prior to each experiment, the cells were serum starved in experimental medium containing 1% FBS without EGF for at least 12 h. All assays were performed at least in triplicate.
Serum concentrations of PCBs can reach approximately 3µM in people exposed to these toxicants (Jensen, 1989; Wassermann et al., 1979). However, local microenvironmental levels of PCBs in the extracellular space are not known. Therefore, in the present study, cells were treated with PCB 104 at the concentration range of 2–15µM.
In selected experiments, HMECs were pretreated with inhibitors of specific signaling pathways for 1 h prior to adding PCB 104. The inhibitors were then maintained in the media throughout the PCB 104 exposure. A stock solution of PCB 104 was prepared in DMSO, and the same amounts of DMSO as in PCB-treated cells were added to control cultures. Levels of DMSO in experimental media were less than 0.1% and did not affect endothelial cells. Treatment with 10µM PCB 104 with or without pharmacological inhibitors for 24 h did not affect cell viability as determined by the (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT) conversion assay (data not shown). In addition, there were no apparent cellular morphological changes under these experimental conditions.
Transient transfection.
HMECs were seeded on 24-well culture plates and cultured in MCDB 131 medium containing 10% FBS. When cultures reached 90% confluency, (usually 24 h after seeding), transfections were performed in serum-free MCDB 131 containing 4 µl of Lipofectin transfection reagent (Invitrogen Carlsbad, CA), 0.95 µg plasmid DNA (e.g., 0.4 µg firefly luciferase reporter construct, 0.05 µg renilla luciferase control vector [pRL-TK], and 0.5 µg c-src vector or empty vector) per well. Following a 5-h incubation with the transfection mixture, cells were allowed to recover for 24 h in normal medium (MCDB 131 with 10% FBS). Cells were then stimulated with PCB 104 for 16 h and analyzed using Dual-Glo Luciferase Assay System (Promega, Madison, WI). The results were normalized according to Renilla luciferase activity.
Endothelial cell migration assay.
Transwell chambers (6.5-mm ID) (Corning Costar, Cambridge, MA) with polycarbonate membranes containing 8.0-µm pores were coated with Matrigel (BD Biosciences, Franklin Lakes, NJ). Calcein (5µM)-labeled endothelial cells (5 x 104) were plated onto polycarbonate membranes of the upper chamber of the Transwell system containing normal MCDB 131 medium. The conditioned medium generated by the exposure of HMECs to PCB 104 at the concentrations of 2, 5, or 10µM was added to the lower chamber. In specific experiments, neutralizing anti–IL-8 antibody was added to the conditioned medium 30 min prior the cell migration assay. Following an 18 h incubation with the conditioned media, cells were fixed with 4% formaldehyde and washed extensively with PBS. To remove the nonmigrating cells, cells on the upper face of the membranes were gently scraped using a cotton swab. The cells that migrated through the polycarbonate membranes were counted under a fluorescent microscope using five random microscope fields under x200 magnification.
Enzyme-linked immunosorbent assay.
Medium protein levels of IL-8 were quantified using the Quantikine human IL-8 immunoassay ELISA kit (R&D Systems) according to the manufacturer's instructions. Briefly, HMECs were cultured in fibronectin-coated wells of the 24-well plates to confluency and then treated with PCB 104 (2, 5, or 10µM) for 9 h. The media were collected, centrifuged, and used for ELISA to determine IL-8 protein levels. In addition, HMECs were lysed with 1 N NaOH, and the protein content was determined for each well using the Bradford assay (Sigma). The amount of IL-8 released into the media was normalized according to the cellular protein levels.
Real-time RT-PCR.
Total RNA was isolated from PCB 104–treated HMECs using the RNeasy mini kit (Qiagen, Valencia, CA) according to the manufacturer's instructions. First-strand cDNA was generated from 1 µg of the total RNA using the Reverse Transcription System kit (Promega) with random hexamer primers. IL-8 mRNA expression was determined by real-time RT-PCR using the ABI Prism 7000 Sequence Detection System (Applied Biosystems, Foster City, CA). PCR amplification was performed using TaqMan Universal PCR Master Mix and predeveloped human IL-8 primer pair and probe (Applied Biosystems). PCR cycles consisted of an initial denaturation step at 95°C for 10 min, followed by 95°C for 15 s and 60°C for 60 s (for up to 45 cycles). PCR amplification of actin (a housekeeping gene) was performed for each sample to normalize IL-8 mRNA levels.
Electrophoretic mobility shift assay.
Nuclear extracts were prepared as described previously (Lee et al., 2003). Double-stranded oligonucleotides were end-labeled with 32P-ATP using bacteriophage T4 polynucleotide kinase (Promega). The following double-stranded oligonucleotide probes were used: NF-B (5'-ATC GTG GAA TTT CCT CTG A-3'), mutated NF-B (mNF-B) (5'-ATC GTt aAc TTT CCT CTG A-3'), AP-1 (5'-GTG ATG ACT CAG GTT TG-3'), and mutated AP-1 (mAP-1) (5'-GTG Ata tCT gtG GTT TG-3'). The sequences of mutated probes (indicated by the lower case letters) correspond to the mutations of the NF-B– and AP-1–binding sites introduced in the IL-8 promoter by Mukaida et al. (1994). All oligonucleotides were synthesized by Integrated DNA Technologies (Coralville, IA). Binding reactions for AP-1 were performed with 2 µg of nuclear protein extracts in a 20-µl volume of reaction mixture (10mM Tris-Cl, pH 7.5, 50mM NaCl, 1mM EDTA, 0.1mM dithiothreitol, 10% glycerol, and 2 µg of poly[dI-dC]). To determine NF-B DNA binding, 4 µg of nuclear protein extracts was incubated with a 20-µl volume of reaction mixture (20mM HEPES, pH 8.0, 100mM KCl, 0.2mM EDTA, 20% glycerol, 0.5mM dithiothreitol, 1 µg salmon sperm DNA, 2 µg of poly[dI-dC]). For supershift analysis, antibodies (2 µg) against AP-1 subunits (c-Fos, c-Jun, or Jun family) or NF-B subunits (c-Rel, p65, or p50) were added to the reaction mixture. Protein-DNA complexes were analyzed on a nondenaturing 7% polyacrylamide gel using 0.25xTBE buffer (50mM Tris-HCl, 45mM boric acid, 0.5mM EDTA, pH 8.4).
Immunoblotting.
Treated HMECs were washed with cold PBS and lysed with lysis buffer (1.0% Nonidet P-40, 20mM Tris-HCl [pH 7.6], 1mM EDTA, 0.5mM EGTA, 10mM MgCl2, 1mM Na3VO4, 2mM dithiothreitol, 1 µg/ml of aprotinin, 1 µg/ml of leupeptin, and 1mM phenylmethylsulfonyl fluoride) for 20 min at 4°C, followed by centrifugation at 12,000 x g at 4°C. Then, 4x SDS sample buffer (250nM Tris-HCl [pH 6.8], 40% glycerol, 8% SDS, and 4% ß-mercaptoethanol) was added in the ratio of 3:1 to the aliquots of supernatants containing 20 µg of protein. The samples were boiled for 3 min, briefly centrifuged, electrophoresed in 10 or 12.5% SDS-PAGE, and transferred onto a Hybond-enhanced chemiluminescence membrane (Amersham Biosciences, Piscataway, NJ). The membrane was blocked for 1 h with 3% (wt/vol) bovine serum albumin in Tris-buffered saline (TBS) containing 0.1% (vol/vol) Tween-20 (TBS-T) and incubated overnight at 4°C with the primary antibody (1 µg antibody/ml blocking solution). The membrane was then washed three times with TBS-T and incubated for 2 h with horseradish peroxidase–conjugated secondary antibody. After washing three times with TBS, immunoreactive protein bands were visualized with the enhanced chemiluminescence system (Amersham Biosciences) using Kodak image station 2000R (Eastman Kodak Co., Rochester, NY).
Cell viability assay (MTT conversion assay).
HMECs were cultured on 96-well plates. Confluent cultures were exposed for 24 h to 10µM PCB 104 and/or a series of pharmacological inhibitors employed in the present study. At the end of treatment time, cells were rinsed with Hank's balanced salt solution and incubated with MTT solution (1 mg/ml medium) for 3 h at 37°C. The insoluble colored formazan salts were dissolved in DMSO, and the absorbance was assessed at 570 nm.
Statistical analysis.
Results are expressed as means ± SDs. Data were statistically analyzed using one-way ANOVA, and the pairwise comparison was performed using Student-Newman-Keuls post hoc test. Statistical probability of p < 0.05 was considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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PCB 104 Exposure Stimulates IL-8 Expression in HMECs
IL-8 has recently been shown to contribute to cancer progression by acting as a mitogenic and angiogenic factor (Xie, 2001
). Therefore, we first determined the effects of PCB 104 on IL-8 mRNA and protein expression. As indicated in Figure 1A, exposure to PCB 104 for 3.5 h significantly increased IL-8 mRNA levels in HMECs. To address the question whether PCB 104 induces IL-8 upregulation at the transcriptional level, confluent HMECs were pretreated for 1 h with actinomycin D, the inhibitor of RNA transcription. Actinomycin D significantly reduced the stimulatory effects of PCB 104, while basal level of IL-8 mRNA was slightly reduced by treating actinomycin D alone without statistical significance. These results indicated that IL-8 overexpression in PCB 104–treated HMECs requires new synthesis of mRNA (Fig. 1A).
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To establish whether PCB 104–mediated IL-8 mRNA overexpression is transcriptionally active, we used a promoter activity assay with the pIL-8Luc-133 reporter construct. Figure 1B shows that PCB 104 treatment in a dose-dependent manner increased IL-8 promoter activity. The maximum increase in luciferase activity was observed in HMECs treated with 10µM PCB 104. In addition, treatment with PCB 104 significantly and in a dose-dependent manner increased the IL-8 protein production in HMECs as measured by ELISA (Fig. 1C).
IL-8 Is Involved in PCB-Mediated Endothelial Cell Migration
Migration of microvascular endothelial cells is an important component of the angiogenic response and a prerequisite for cancer cell metastasis (Koch et al., 1992). Therefore, we next determined the effects of PCB 104 on the HMEC migration using the Matrigel-coated Transwell system. In these experiments, cells were treated with the conditioned media generated by exposing separate HMEC cultures to different concentrations of PCB 104 for 9 h. As indicated in Figure 2A, the HMEC migration was markedly increased by the PCB 104–conditioned media as compared to controls. The conditioned medium from cultures exposed to PCB 104 at a concentration as low as 2µM was sufficient to effectively stimulate endothelial cell migration. Direct (i.e., not in the conditioned medium) exposure to 10µM PCB 104 did not affect cell migration (data not shown). In selected experiments, cell migration assays were performed in the presence of neutralizing IL-8 antibodies. As shown in Figure 2B, the anti–IL-8 antibody significantly and dose dependently abrogated the HMEC migration induced by conditioned media from the PCB 104–exposed cultures.
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Transcription Factors NF-
B and AP-1 Are Required for PCB 104–Induced IL-8 ExpressionThe promoter region of IL-8 contains the cis elements for NF-
B and AP-1 binding between – 133 and + 44 bp (Mukaida et al., 1990; Yasumoto et al., 1992). Therefore, we performed a series of DNA-binding assays using oligonucleotide probes that directly correspond to these binding sites. As indicated in Figures 3A and 3B, a 1-h treatment with 10µM PCB 104 markedly activated NF-B and AP-1. The specificity of these assays was confirmed by using mutated oligonucleotide probes and nonradiolabeled oligonucleotides for the binding reactions.
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We also performed supershift analyses with antibodies against NF-
B subunits (c-Rel, p65, or p50) or AP-1 subunits (c-Fos, c-Jun, or Jun family). The assays indicated that c-Rel and p50 (but not p65) were included in the PCB 104–induced NF-B–DNA binding complex (Fig. 3A). In addition, c-Fos was identified as a major component of the AP-1 complex (Fig. 3B). Although addition of anti–c-Jun antibody did not affect the formation of the AP-1–DNA complex, a general antibody against the Jun family markedly attenuated AP-1–DNA binding (Fig. 3B). Thus, it appears that the PCB 104–induced AP-1 complex in HMECs is composed of the c-Fos/Jun family protein heterodimer.
To further indicate the importance of NF-B and AP-1 in PCB 104–mediated IL-8 overexpression, we transfected HMECs with the IL-8 promoter construct containing mNF-B– and mAP-1–binding sites. As indicated in Figure 3C, mutation in these binding elements significantly decreased stimulatory effects of PCB 104 on IL-8 promoter activity.
c-Src Kinase Regulates PCB 104–Induced IL-8 mRNA Expression
Our next series of experiments focused on a possible involvement of c-Src activation in PCB 104–induced IL-8 expression. Regulation of c-Src activity is mediated through phosphorylation/dephosphorylation of specific tyrosine residues. Phosphorylation of tyrosine 418 (Y418) in the catalytic domain is an activating signal of c-Src.
We assessed the phosphorylation pattern of c-Src kinase in PCB 104–exposed HMECs using antibodies against phospho-c-Src kinase (Fig. 4A). Phosphorylation of tyrosine 418 was transiently and rapidly induced 5 min after exposure to PCB 104 (Fig. 4A).
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In order to determine the importance of the c-Src kinase family in IL-8 expression, HMECs were pretreated with PP1 and PP2 (two distinctive pharmacological inhibitors of the Src kinases) prior to treatment with PCB 104. In addition, PP3 was used as a negative control of PP2. As shown in Figure 4B, both PP1 and PP2, but not PP3, significantly and in a dose-dependent manner inhibited PCB 104–induced IL-8 mRNA expression. Moreover, PP2 markedly abolished the increase in IL-8 promoter activity induced by cotransfection with the IL-8Luc-133 construct and the c-src-DP vector (Fig. 4C).
PP1 and PP2 are general inhibitors of the Src kinase family. To determine the specific involvement of c-Src kinase in PCB 104–induced IL-8 expression, we cotransfected HMECs with the IL-8Luc-133 construct and the c-src-DN vector. Figure 4D illustrates that the effect of PCB 104 on IL-8 promoter activation in this cell model was significantly less pronounced as compared to control cultures transfected with the IL-8Luc-133 and an empty vector instead of the c-src-DN vector. These results indicate that c-Src kinase may be a key signaling molecule for IL-8 mRNA production in PCB 104–exposed HMECs.
NF-B and AP-1 Are Involved in c-Src–Mediated IL-8 Transcription
To determine whether c-Src kinase can regulate NF-B activation and AP-1 activation, we cotransfected HMECs with the c-src-DP vector and the cis reporter luciferase plasmids containing five repeats of the NF-B–binding element (pNF-B-Luc) or seven consensus repeats of the AP-1–binding element (pAP-1-Luc). Overexpression of c-Src resulted in a significant increase in luciferase activity of both pNF-B-Luc and AP-1 as compared to cells transfected with an empty vector instead of the c-src-DP vector (Figs. 5A and 5B). In addition, transactivation of both NF-B and AP-1 induced by c-src-DP was significantly inhibited by pretreatment with PP2 but not PP3.
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We next evaluated whether activation of c-Src kinase can regulate IL-8 transcription activity through activation of NF-
B and AP-1. HMECs were cotransfected with the c-src-DP vector and the IL-8 promoter construct with mutated binding sites for NF-B and AP-1 (pIL-8Luc-mNF-B and pIL-8Luc-mAP-1, respectively). As illustrated in Figure 5C, overexpression of c-Src markedly enhanced IL-8 promoter activity. However, this stimulatory effect was significantly less pronounced in cells cotransfected with the pIL-8Luc-mNF-B or the pIL-8Luc-mAP-1.
ERK1/2 and PI3K/Akt Are Involved in c-Src–Mediated IL-8 mRNA Expression
The ERK1/2– and PI3K/Akt–signaling pathways can regulate the activation of AP-1 and NF-B, respectively. Therefore, we examined an interaction between c-Src, ERK1/2, and PI3K/Akt signaling in PCB 104–treated HMECs. As shown in Figure 6A, inhibition of Src kinase activity by PP1 markedly attenuated the PCB 104–induced phosphorylation of both ERK1/2 and Akt. In addition, a blockage of PI3K did not affect ERK1/2 phosphorylation, and the inhibition of ERK1/2 had no influence on PI3K activation. These Western blot results indicate that Src kinase is an upstream regulator of the ERK1/2 and PI3K/Akt pathways; however, there is no cross talk at the level of ERK1/2 and Akt in PCB 104–exposed HMECs.
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In order to evaluate whether the PI3K/Akt and ERK1/2 pathways are involved in PCB-induced IL-8 expression, HMECs were treated with PCB 104 in the presence of LY294002 (an inhibitor of PI3K) or with PD98059 (an inhibitor of MEK1, a kinase upstream from ERK1/2). As indicated in Figure 6B, treatments with both LY294002 and PD98059 dose dependently inhibited the PCB 104–induced IL-8 mRNA expression.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We hypothesize that exposure to environmental pollutants such as PCBs can induce the development of vascular alterations and thus contribute to the formation of blood-borne metastases. In the present study, we demonstrate that treatment of HMECs with PCB 104 induced IL-8 production and subsequently augmented endothelial cell migration. PCB 104–mediated IL-8 overexpression is regulated by an increased transcription rate via c-Src kinase–mediated activation of NF-
B and AP-1. These results suggest that exposure to selected noncoplanar PCB congeners, like PCB 104, may induce the proinflammatory and chemotactic effects and stimulate endothelial cell migration. Such events are critical elements of neovascularization, leading to tumor progression and subsequent metastasis formation. Indeed, it was recently demonstrated that another noncoplanar PCB congener, 2,2',4,4',5,5'-hexachlorobiphenyl, can positively modulate angiogenic processes in endothelial cells (Tavolari et al., 2006). There is considerable evidence that angiogenesis and inflammation occur in an interactive and overlapping manner (Gillitzer and Goebeler, 2001). For example, IL-8 was initially identified as a major proinflammatory cytokine in the pathogenesis of vascular disease (Uguccioni et al., 1999). Subsequent evidence indicated that IL-8 can contribute directly to the modulation of endothelial cell proliferation, migration, and regulation of angiogenesis via its paracrine effects on endothelial cells (Matsuo et al., 2004; Murdoch et al., 1999; Shi et al., 2001; Xie, 2001).
The results of the present study indicate that endothelial cells stimulated by PCB 104 can be a major source of IL-8. This effect appears to be regulated primarily at the transcriptional level. Specifically, a blockage of RNA transcription by actinomycin D markedly (although not completely) reduced IL-8 mRNA expression (Fig. 1A). In addition to transcriptional regulation, it has been reported that IL-8 expression can be regulated by the alterations of mRNA stability. Indeed, the 3'-flanking region of the IL-8 gene contains the repetitive ATTTA motif, which is responsible for mRNA stability of various cytokines (Chen and Shyu, 1995). Therefore, we cannot exclude the possibility that increased IL-8 mRNA levels in PCB 104–exposed HMECs results also from posttranscriptional changes.
The IL-8 promoter region contains multiple cis elements for transcription factors, NF-B, AP-1, CCAAT enhancer-binding protein (C/EBP), and Signal transducers and activators of transcription (STAT), all of which have been implicated in IL-8 transcription by various stimuli (Mukaida et al., 1990, 1994; Roebuck et al., 1995; Yatsunami et al., 1997). However, the specific involvement of individual promoter elements in the regulation of IL-8 mRNA expression is highly dependent on a cell type and type of stimuli (Lakshminarayanan et al., 1998; Yatsunami et al., 1997). For example, H2O2 and tumor necrosis factor (TNF)-
induce differential binding of AP-1 and NF-B to the IL-8 promoter region in endothelial and epithelial cells (Lakshminarayanan et al., 1998). In epithelial cells, treatment with H2O2 activated AP-1 but not NF-B, whereas exposure to TNF- stimulated both transcription factors. In contrast, TNF- activated NF-B but not AP-1, whereas H2O2 did not activate either of the transcription factors in endothelial cells. In the present study, we observed that PCB 104 can stimulate DNA binding of both NF-B and AP-1 (Figs. 3A and 3B). In addition, using mutated IL-8 promoter constructs, we indicated that NF-B and AP-1 binding is equally critical for PCB 104–induced IL-8 transcription in HMECs. Thus, it appears that NF-B and AP-1 work in concert for a maximal activation of IL-8 promoter in PCB 104–stimulated endothelial cells.
To study the upstream regulatory mechanisms of IL-8 overexpression in PCB 104–stimulated endothelial cells, we focused on the Src pathway. Endothelial cells can express at least three members of the Src kinase family (namely, c-Src, c-Fyn, and c-Yes), which have partially overlapping functions (Trevino et al., 2005). Treatment with PCB 104 induced phosphorylation of tyrosine 418, a pattern of phosphorylation changes consistent with c-Src activation. In addition, our results with pharmacological inhibitors of Src kinases indicate that this signaling pathway can induce IL-8 expression in PCB 104–treated HMECs. Most importantly, transfection experiments with c-src-DN and c-src-DP vectors specifically identified c-Src as the critical kinase responsible for activation of transcription factors, leading to PCB 104–induced IL-8 overexpression.
Although the involvement of c-Src in AP-1 activation is relatively well established (Chandrasekar et al., 2005; Funakoshi-Tago et al., 2003), the role of NF-B in c-Src–mediated IL-8 transcription in the human endothelial cell has not been consistently demonstrated. For instance, it was reported that overexpression of v-src can activate the IL-8 in an NF-B–dependent manner (Eicher et al., 1994). In contrast, IL-1–mediated stimulation of c-Src kinase was involved in AP-1 but not in NF-B activation. In the same report, a kinase-dead mutant of c-Src induced NF-B activation but was unable to stimulate AP-1 or Akt (Funakoshi-Tago et al., 2003). In the present study, we provide two independent pieces of evidence that c-Src kinase is involved in PCB 104–mediated activation of not only AP-1 but also NF-B. Firstly, the transfection of HMECs with the c-src-DP vector markedly activated both NF-B and AP-1 transactivation (Figs. 5A and 5B). Secondly, mutation of NF-B– or AP-1–binding sites in the IL-8 promoter significantly abolished IL-8 promoter activation induced by the c-src-DP vector (Fig. 5C).
The mechanisms of c-Src–mediated NF-B activation may involve several mechanisms. It was reported that expression of a kinase-dead c-Src augmented IL-1–mediated NF-B activation through physical interaction with IKK-
in glioblastoma cells (Funakoshi-Tago et al., 2003, 2005). However, IB was also suggested to be a critical mediator for NF-B activation induced by c-Src (Davis et al., 2004; Fan et al., 2003; Huang et al., 2003). Indeed, inhibition of Src by PP1 blocked the shear stress–induced IB phosphorylation and NF-B activation (Davis et al., 2004). Similar effects were also induced by overexpression of c-src-DN in epithelial cells exposed to TNF- (Huang et al., 2003). Finally, PP2 or c-Src (–/–) knockout cell lines prevented the c-Src–mediated IB- phosphorylation and NF-B activation induced by hypoxia and reoxygenation (Fan et al., 2003).
The regulatory role of c-Src–mediated activation of NF-B and AP-1 in PCB 104–mediated induction of IL-8 expression does not preclude a possible involvement of additional factors in transcription upregulation of the IL-8 gene. Recently, a putative STAT3-binding site containing the interferon--activated site (GAS) consensus sequence was identified in the upstream region of the human IL-8 promoter between – 430 to – 438 bp (Yeh et al., 2004). The IL-8 reporter plasmid used in this study contained a region from – 133 to + 43 bp and did not include the STAT3-binding element. Thus, our results of promoter activation with pIL-8Luc-133 were unable to reflect a possible contribution of STAT3 to PCB 104–induced IL-8 expression. It should be pointed out that PP1, PP2 (Fig. 4B), and the c-src-DN vector (Fig. 4D) did not completely abolish PCB 104–induced activation of the IL-8. These results may suggest that not only NF-B and AP-1 but also other transcription factors, e.g., STAT3, can contribute to the c-Src–mediated IL-8 mRNA expression in PCB 104–treated HMECs.
The present study also demonstrates that PI3K/Akt and ERK1/2 are downstream signaling mediators of c-Src kinase in the pathway leading to IL-8 induction in PCB 104–treated HMECs. Indeed, inhibition of Src activity by PP1 resulted in decreased phosphorylation of both Akt and ERK1/2. However, inhibition of PI3K by LY294402 did not affect PCB 104–induced phosphorylation of ERK1/2. Similarly, blocking ERK1/2 activity by PD98059 had no influence on Akt phosphorylation in PCB 104–treated HMECs (Fig. 6B). Thus, it appears that PI3K/Akt and ERK1/2 are independent downstream signaling pathways that are regulated by c-Src activation. The importance of the PI3K/Akt and ERK1/2 signaling is related to activation of NF-B and AP-1 (Chandrasekar et al., 2005; De Martin et al., 2000; Eum et al., 2004; Karin, 1995). In addition, PI3K has been recognized as a critical mediator of endothelial angiogenic responses, such as actin reorganization and chemotaxis (Hooshmand-Rad et al., 1997). Indeed, overexpression of the catalytic subunit of PI3K as well as the constitutively active Akt increased in vivo angiogenesis, as measured by the chicken chorioallantoic membrane assay (Jiang et al., 2000). These data are in agreement with the findings of the present study that the PI3K/Akt and ERK1/2 pathways can participate in c-Src–mediated increased production of IL-8 and migration of endothelial cells.
In conclusion, the present study indicates that exposure of HMECs to noncoplanar ortho-chlorinated PCBs, such as PCB 104, can induce endothelial cell migration via production of proinflammatory and proangiogenic IL-8. In addition, our study provides strong evidence that PCB 104–induced IL-8 overexpression is regulated via c-Src–mediated activation of NF-B and AP-1. However, further studies are advocated to determine the structure-activity relationship between specific PCB congeners and their proinflammatory and proangiogenic effects.
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NOTES |
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Disclaimer: The authors certify that all research involving human subjects was done under full compliance with all government policies and the Helsinki Declaration.
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ACKNOWLEDGMENTS |
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We thank Dr Naofumi Mukaida for the kind gift of IL-8 promoter reporter plasmids. This study was supported by National Institutes of Health/National Institute of Environmental Health Sciences (NIH/NIEHS). (P42 ES 07380) and Kentucky Lung Cancer Research Program.
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