Published online before print April 9, 2009, doi: 10.1161/CIRCRESAHA.108.190751
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© 2009 American Heart Association, Inc.
Molecular Medicine |
Antiinflammatory Effects of the ETS Factor ERG in Endothelial Cells Are Mediated Through Transcriptional Repression of the Interleukin-8 Gene
From the Divisions of Cardiology (L.Y., V.N.-K., Y.Z., M.K., P.O.), Molecular and Vascular Medicine (L.Y., V.N.-K., Y.Z., M.K., L.V., K.Y., C.V.C., W.C.A., P.O.), and Interdisciplinary Medicine and Biotechnology (M.B.); Department of Medicine; and Center for Vascular Biology Research (L.Y., V.N.-K., Y.Z., M.K., L.V., K.Y., C.V.C., W.C.A., P.O.), Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Mass.
Correspondence to Dr. Peter Oettgen, Division of Cardiology, Department of Medicine, and the Center for Vascular Biology Research, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215. E-mail joettgen{at}bidmc.harvard.edu
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Abstract |
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ERG (Ets-related gene) is an ETS transcription factor that has recently been shown to regulate a number of endothelial cell (EC)-restricted genes including VE-cadherin, von Willebrand factor, endoglin, and intercellular adhesion molecule-2. Our preliminary data demonstrate that unlike other ETS factors, ERG exhibits a highly EC-restricted pattern of expression in cultured primary cells and several adult mouse tissues including the heart, lung, and brain. In response to inflammatory stimuli, such as tumor necrosis factor-
, we observed a marked reduction of ERG expression in ECs. To further define the role of ERG in the regulation of normal EC function, we used RNA interference to knock down ERG. Microarray analysis of RNA derived from ERG small interfering RNA– or tumor necrosis factor-–treated human umbilical vein (HUV)ECs revealed significant overlap (P<0.01) in the genes that are up- or downregulated. Of particular interest to us was a significant change in expression of interleukin (IL)-8 at both protein and RNA levels. Exposure of ECs to tumor necrosis factor- is known to be associated with increased neutrophil attachment. We observed that knockdown of ERG in HUVECs is similarly associated with increased neutrophil attachment compared to control small interfering RNA–treated cells. This enhanced adhesion could be blocked with IL-8 neutralizing or IL-8 receptor blocking antibodies. ERG can inhibit the activity of the IL-8 promoter in a dose dependent manner. Direct binding of ERG to the IL-8 promoter in ECs was confirmed by chromatin immunoprecipitation. In summary, our findings support a role for ERG in promoting antiinflammatory effects in ECs through repression of inflammatory genes such as IL-8.
Key Words: ETS factor • interleukin-8 • inflammation • Tumor necrosis factor-
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Introduction |
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The ETS transcription factors consist of approximately 30 family members. All ETS factors share a highly conserved 80- to 90-aa-long DNA-binding domain, the ETS domain.1 Through this conserved DNA-binding domain, these proteins recognize DNA with an internal conserved DNA-binding sequence core motif "GGAA/T." In addition, there are conserved protein–protein interacting domains such as the PNT (pointed) domain.2–8 ETS factors were originally shown to play a central role in regulating genes involved in hematopoiesis. Many monocytic-, B-, and T-cell–specific genes are regulated by ETS factors. ETS factors also function as protooncogenes and have been causally linked to a number of human cancers.1 For example, ERG was originally recognized for its role as a protooncogene in the development of several types of human cancers, especially in prostate cancer.9
We and others have demonstrated a critical role for selected ETS factors in the regulation of several vascular-specific genes, including Tie1, Tie2, vascular endothelial growth factor (VEGF) receptor 1, VEGF receptor 2, von Willebrand factor (vWF), Robo4, platelet/endothelial cell adhesion molecule-1, VE-cadherin, and endothelial nitric oxide.10–17 The ETS factor Tel is required for the development of extraembryonic blood vessels. Targeted disruption of this gene leads to abnormalities in vitelline vein development.18 Homozygous deletion of the ETS factor Fli-1 also leads to abnormalities in vascular development that are associated with reductions in the level of Tie2, loss of vascular integrity particularly in the brain, that lead to bleeding into the meninges and embryonic lethality at day 11.5.19
Although a number of ETS factors participate in the regulation of endothelial cell (EC)-restricted genes as described above, most ETS factors do not exhibit an EC-restricted pattern. Several recent studies support that ERG exhibits an EC-restricted pattern of expression.20–23 Furthermore, a few studies have pointed to the potential role of the ETS factor ERG in the regulation of a selected number of endothelial-specific genes including angiopoietin-2, endoglin, vWF, and VE-cadherin.24–28 Evidence also supports the involvement of ERG in EC differentiation and angiogenesis.29 In this study, we confirm that ERG is more EC-restricted than other ETS factors, raising the possibility that ERG may play a unique role in regulating EC function.
Others have shown that ERG protein expression levels are markedly downregulated in ECs in the presence of tumor necrosis factor (TNF)-.30 TNF- is known to be a potent mediator of inflammation in vivo and in vitro. Exposure of ECs to TNF- is associated with the induction of multiple inflammatory genes including interleukin (IL)-1β and IL-8, which in turn promote neutrophil attachment. Exposure of ECs to TNF- also leads to downregulation of selected genes, such as intercellular adhesion molecule-2.
The purpose of this study was to begin to characterize the role of ERG in regulating EC function in the setting of inflammation. Our results show that under noninflammatory conditions, ERG exhibits a highly EC-restricted pattern of expression in vitro and in vivo. In response to inflammatory stimuli, such as TNF-, lipopolysaccharide (LPS), or IL-1β, ERG expression is dramatically reduced. Our data indicate that ERG regulates IL-8 expression. ERG can bind to specific ETS sites within the IL-8 promoter. Downregulation of ERG leads to a significant induction of IL-8 mRNA and protein expression levels. Furthermore, downregulation of ERG is also associated with an increase in neutrophil attachment, which is abolished by the presence of blocking antibodies directed against IL-8 or IL-8 receptors. In summary, our results support a role for ERG in promoting antiinflammatory effects in ECs through suppression of selected proinflammatory genes.
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Materials and Methods |
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Mouse Models of Endotoxemia
The model used is as described previously.31 Briefly, 8 male 8-week-old C57BL/6 mice were injected IP with normal saline (control) or LPS (18 mg/kg) from Escherichia coli serotype 0111:B4 (Sigma-Aldrich, St Louis, Mo), or treated with cecal ligation and puncture (CLP). Mice were anesthetized by intraperitoneal injection of xylazine (5 mg/kg) and ketamine (80 mg/kg) after 6 or 24 hours of LPS or CLP treatment to collect tissues.
Small Interfering RNA Transfection
Human umbilical vein (HUV)ECs were plated at a density to achieve 80% to 90% confluence on the day of transfection. Lipofectamine 2000 (Invitrogen, Carlsbad, Calif) and small interfering (si)RNAs were first incubated in Opti-MEM I Reduced-Serum Medium (Invitrogen) and added to media from HUVECs. The siRNA used in this study are as follows: ERG siRNA, 5'-GGACAGACUUCCAAGAUGAUU-3'; control siRNA, 5'-UAGCGACUAAACACAUCAA-3', produced by Dharmacon (Lafayette, Colo).
Neutrophil Preparation and Adhesion
Neutrophils were isolated following the method described previously.32 Briefly, neutrophils were prepared from human whole blood (collected in the presence of citrate and dextran) by standard Ficoll-Hypaque (Sigma) buoyant density centrifugation, followed by brief osmotic lysis of red blood cells. HUVECs expressing either ERG siRNA or control siRNA were plated at 90% confluence and cultured for 12 hours with medium alone, medium containing TNF-, or selected blocking antibody before the experiment. Freshly isolated neutrophils were washed and resuspended and then added to HUVEC monolayers and incubated at 37°C for 4 hours. Samples were washed and imaged live using a 10 x phase-contrast objective, and the number of adherent neutrophils was counted.
Luciferase Reporter Gene Constructs
Human IL-8 promoter (–1396- to +27-bp) fragments were cloned from human BAC clone RP11–126P1 by PCR. The primer sequences were: sense, 5'-AGCTAGCCAGACAAACCTTTTTGGAAAG-3'; antisense, 5'-TCTCGAGGTCTCTGAAAGTTTGTGCCTTAT-3', each of which contains NheI and XhoI cutting sites respectively. The –1396- to +27-bp fragment was inserted into the NheI-XhoI site and subcloned into the pGL3 Basic luciferase reporter vector (Promega, Madison, Wis).
IL-8–Blocking Studies
Culture media were mixed with antibodies against IL-8, CXCR1, or CXCR2 (R&D Systems, Minneapolis, Minn) or with control normal IgG at a final concentration of 10 µg/mL. Neutrophil attachment assays were then carried out.
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Results |
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Previous studies on ERG have indicated a selective expression of ERG in ECs by in situ hybridization or traditional RT-PCR. In this study, we evaluated ERG expression more extensively in a variety of human and mouse cells using quantitative real-time PCR (Q-PCR). Among these, ERG expression was only detected in ECs and not in other cell types including smooth muscle cell (SMC) and cells of hematopoietic origin (Figure 1A and 1B). Moreover, ERG was expressed in all the ECs we studied, despite their different tissue origins. Evaluation of ERG protein expression by immunofluorescence demonstrated that ERG protein is predominantly localized to the nuclei of ECs such as HUVECs, whereas no staining was observed in HASMC (Figure 1C). ERG expression was also evaluated in a variety of mouse tissues using Q-PCR. ERG expression levels were highest in the heart and lung, with lower levels in the brain and minimal or no detectable expression in the liver (Figure 2A). ERG expression was next evaluated by immunofluorescence in tissue sections from mouse heart and brain (Figure 2B). These studies demonstrated a close association of ERG in the nuclei of cells expressing the EC-specific marker VE-cadherin. DAPI staining showed many cells that do not express either ERG or VE-cadherin.
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Others have shown that ERG expression is downregulated in ECs after stimulation with TNF-.30 In this study, we observed a 50% reduction of ERG protein expression at 6 hours and nearly undetectable levels 24 hours after TNF- stimulation of HUVECs (Figure 3A). This was also observed for LPS (Figure 3B) and IL-1β (data not shown). A similar reduction in ERG expression levels was observed in mouse hearts 24 hours after systemic administration of LPS by immunofluorescence staining (Figure 3C). A significant reduction in ERG expression was also confirmed at both 6- and 24-hour time points in this model by Q-PCR (Figure I in the online data supplement, available at http://circres.ahajournals.org). Although injection of endotoxin has been widely used as a model of sepsis, limitations of this model include the transient nature of the response based on a single intraperitoneal injection of endotoxin and that it is not a model of bacterial sepsis.11 To further validate our initial findings, we used as second mouse model of sepsis, the CLP model, that may more closely mimic bacterial sepsis that occurs in patients.12,13 A similar reduction of ERG expression was observed in the mouse hearts using the CLP model, as detected by immunofluorescence staining (Figure 3D).
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The fact that ERG is downregulated in the setting of inflammation suggested to us that ERG might play an important role in the regulation of inflammatory responses in ECs. To test this hypothesis, we used ERG-specific siRNA to downregulate ERG expression. A total of 5 siRNAs directed against ERG were screened, 4 of which led to significant (>85%) reductions in ERG expression at the level of protein (Online Figure II) and mRNA (data not shown). The ERG siRNA that yields the greatest reduction is shown in Figure 4A and 4B. An early event that occurs in the setting of inflammation is activation of the endothelium. This leads to increased attachment and transmigration of neutrophils and other inflammatory cells. To evaluate the effect of ERG on neutrophil attachment, freshly isolated human neutrophils were incubated with HUVECs treated with either ERG siRNA or control siRNA as a control. Downregulation of ERG was associated with a significant increase in neutrophil attachment (100% increase in comparison with control siRNA–treated cells), however, less than what was observed with maximal TNF- stimulation (Figure 4C and 4D).
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Based on the fact that ERG expression is suppressed in response to inflammatory mediators such as TNF-, we postulated that ERG might be involved in the transcriptional regulation of a subset of genes downstream of TNF-. To identify potential ERG gene targets, we performed microarray analysis of ERG siRNA– and control siRNA–treated HUVECs. Comparisons were made with publicly available microarray data for TNF-–treated HUVECs. The comparison of the ERG siRNA differentially expressed genes and TNF- differentially expressed genes demonstrates significant overlap (P<0.01). The detailed expression of the overlapping genes in the ERG siRNA and TNF- datasets is shown in Online Figure III, A and B, respectively. Interestingly, approximately 20% of the genes significantly up- or downregulated by suppression of ERG with siRNA (>2.0 fold change) overlap with changes in gene expression that are observed after TNF- stimulation, although the total number of genes differentially expressed after TNF- stimulation was much greater than after ERG siRNA treatment (Figure 5 and Online Tables I and II). Among the list of genes identified, several are involved in modulating inflammatory responses in ECs including IL-8, CD44, urokinase, and plasminogen activator inhibitor-1. The complete list of the differentially regulated genes as a result of ERG knockdown in HUVECs has been submitted to the Geo database (accession no. GSE14801; http://www.ncbi.nlm.nih.gov/geo). Changes in the expression of selected genes were validated by Q-PCR (Figure 6A). Furthermore, evaluation of changes in the secreted expression of proteins in the supernatant of HUVECs by ELISA after treatment with control or ERG siRNA demonstrated that IL-8 is dramatically upregulated (above 25 ng/mL; Figure 6B and 6C) as a result of ERG downregulation. To ensure the specificity of the ERG siRNA, the expression levels of a panel of other ETS factors including Ets-1, Ets-2, Fli-1, Ese-1, ELF-1, and Nerf1 was examined. No change in the expression of any of the other ETS factors except ERG was observed after ERG siRNA treatment (Online Figure IV). We also used a second ERG-directed siRNA and observed a similar induction of IL-8 and CD44 expression (Online Figure V).
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To further characterize whether the increase in neutrophil attachment to HUVECs observed as a result of ERG suppression is IL-8–dependent, IL-8–blocking antibodies were used. In the presence of blocking antibodies to IL-8 or the IL-8 receptors (CXCR1 and CXCR2), the stimulatory effect of decreased ERG expression on neutrophil attachment was abolished (Figure 6D). The presence of equivalent concentrations of isotype-matched control antibodies IgG had no effect (Figure 6D). These results support the overall concept that ERG is a critical regulator of neutrophil attachment in ECs that appears to be IL-8–dependent.
To evaluate whether IL-8 is a direct target of ERG, we analyzed the nucleotide sequence of the proximal 1.4 kb of the IL-8 promoter for putative ERG-binding sites. Several downstream targets of ERG have been identified in ECs.24–26 Based on a comparison of the nucleotide sequences of the known ERG-binding sites in the promoters of these genes, we constructed the following ERG consensus binding site: (A/G)(G/C)AGGAA(A/G). Using this sequence, we identified at least 2 putative ERG-binding sites within the proximal IL-8 promoter (chromatin immunoprecipitation [ChIP]1 and ChIP2; Figure 7A). ERG binding was observed by ChIP to DNA fragments containing ChIP2 but not ChIP1 (Figure 7B). ERG binding was markedly diminished in response to TNF- treatment (Online Figure VI). The proximal 1.4 kb of the human IL-8 promoter was subcloned into the PGL3 luciferase reporter vector. Transactivation assays demonstrated that cotransfection with increasing amounts of ERG cDNA is associated with decreased IL-8 promoter activity, suggesting that ERG represses IL-8 expression (Figure 7D). In contrast, cotransfection of Fli-1, another ETS family member that is highly homologous to ERG, did not significantly affect IL-8 promoter activity (Online Figure VII). To examine whether the ERG-binding site within the ChIP2 region is required for the suppression of the IL-8 promoter by ERG, site-directed mutagenesis was used to create mutations in the critical nucleotides required for ERG binding to the IL-8 promoter. The transactivation assay demonstrated that suppression of the promoter by ERG was largely abolished by the mutation of the ERG-binding site (Figure 7C).
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Discussion |
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IL-8 is widely recognized as a potent chemotactic factor for leukocyte recruitment to the endothelium in the setting of inflammation.33 The release of IL-8 is triggered by inflammatory signals from a variety of cells including neutrophils, smooth muscle cells and ECs. It plays a key role in host defense mechanisms through its effect on neutrophil activation. However, sustained elevation of IL-8 levels may result in tissue damage. IL-8 administration alone can induce the conversion of rolling to stationary adhesion of neutrophils in as little as 80 ms after the initial attachment.34 Electron microscopic studies have demonstrated that radiolabeled interstitial IL-8 is internalized by venular ECs abluminally and is transcytosed to the luminal surface, where it is presented to the adherent leukocytes predominantly through EC projections.35 Furthermore, TNF-
–mediated transendothelial neutrophil migration has been shown to be IL-8–dependent.35
IL-8 expression is induced in ECs by a number of inflammatory mediators, including TNF-, oxidized low-density lipoprotein, and endotoxin.36,37 In cultured ECs, exposure to TNF-, for example, is associated with sustained increases in IL-8 protein in the supernatant of the cells for greater than 24 hours. The induction of IL-8 by these inflammatory mediators has been shown to occur predominantly at the level of gene transcription.37 Furthermore, the transcriptional elements required for induction were shown to be contained within the proximal 1.4 kb of the IL-8 gene promoter, and induction of a luciferase reporter construct containing this region by TNF- was at least partially dependent on activation by nuclear factor (NF)-
B. The increased expression of IL-8, and other cytokines including IL-6 and granulocyte-macrophage colony-stimulating factor, in ECs by TNF- can be inhibited by the 2 inhibitors of NF-B, pyrrolidine dithiocarbamate and N-acetylcysteine.38 In our study, we observed that downregulation of ERG in HUVECs was associated with a significant increase in neutrophil attachment, which is less than what was observed with maximal TNF- stimulation, suggesting that the combined effects of NF-B activation and ERG suppression may be necessary for maximal IL-8 induction and neutrophil attachment. Another mechanism by which expression of the IL-8 gene can be induced in ECs is in response to hypoxia. IL-8 expression is rapidly induced in human pulmonary and dermal microvascular ECs exposed to hypoxia or cobalt.39 This occurs at the transcriptional level and is associated with activation and increased binding of hypoxia-inducible factor-1, NF-B, and activator protein-1, to the IL-8 promoter.
We and others have shown a role for selected members of the ETS transcription factor family in the regulation of inflammatory responses in ECs. For example, the Ets factor ESE-1 is rapidly induced in cultured ECs in response to proinflammatory cytokines including IL-1β and TNF- and in vivo in response to endotoxin administration.40 Target genes of ESE-1 include nitric oxide synthase 2 and cyclooxygenase-2. Similarly Ets-1 induction occurs in ECs in response to several inflammatory stimuli including proinflammatory cytokines and angiotensin II. Furthermore, Ets-1 is a critical mediator of the generation of reactive oxygen species and inflammatory gene expression in ECs in vivo in response to systemic infusion of angiotensin II.41,42 Target genes of Ets-1 include monocyte chemotactic protein-1, p47phox, and plasminogen activator inhibitor-1.
Although most ETS factors function as transcriptional activators, selected members of the ETS family members have also been shown to function as transcriptional repressors of gene transcription. For example, the ETS factor Elk-3 represses the expression of hemoxygenase-1 in primary macrophages.43 When macrophages are exposed to endotoxin, Elk-3 levels rapidly diminish, with an associated increase in hemoxygenase-1 expression. Elk-3 has also been shown to be a repressor of the nitric oxide synthase 2 gene under noninflammatory conditions.44 The results of our study suggest a similar role for ERG as a transcriptional repressor of IL-8 and several other genes in ECs. Interestingly, in contrast to our findings for ERG in ECs, the ETS factor MEF can induce the expression of IL-8 in hematopoietic cells.45
In addition to its role as a transcriptional repressor, ERG has also been shown to function as a positive regulator of a number of EC-restricted genes, including vWF, VE-cadherin, angiopoietin-2, and endoglin.24–28 Of particular interest to us was the fact that, in contrast to other ETS factors, ERG appears to exhibit a much more EC-restricted expression pattern both in cultured cells and in vivo in several different organs. Taken together, the results of our studies demonstrate that ERG functions as a transcriptional repressor of a selected number of genes. In the setting of inflammation, ERG expression in ECs is markedly diminished, leading to the increased expression of IL-8 and other proinflammatory genes. Future studies directed at understanding how ERG levels can be maintained or upregulated in ECs could lead to novel approaches toward inhibiting vascular inflammatory responses.
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Acknowledgments |
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Sources of Funding
This work was supported by NIH grant P01 HL76540 (to P.O. and W.C.A.), American Heart Association Established Investigator Award EIA0740012 (to P.O.), T32 training grant HL07374-26 (to L.Y.), and an Arthritis Foundation Arthritis Investigator Award (to C.V.C.).
Disclosures
None.
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Footnotes |
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Original received November 5, 2008; revision received March 27, 2009; accepted April 1, 2009.
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References |
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