Tumor Necrosis Factor α Induces the Expression of the Endothelial Cell-Specific Receptor Roundabout4 through the Nuclear Factor-κB Pathway

Abstract

Roundabout4 (Robo4) is an endothelial cell-specific receptor that regulates vascular stability. Recently, Robo4 has been shown to regulate vascular permeability in inflammation. However, the mechanisms regulating the Robo4 gene in the context of inflammation are poorly understood. In this study, we found that intravenous injection of tumor necrosis factor (TNF) α increased Robo4 expression in mouse organs. In vitro analyses showed that TNFα increased Robo4 expression in human primary endothelial cells, but not in cells pretreated with a nuclear factor (NF)-κB inhibitor. Reporter assays using wild-type and mutant Robo4 promoters indicated that TNFα activated the Robo4 promoter and that both the −2753 and −2220 NF-κB motifs were essential for this activation. Electrophoretic mobility shift assays demonstrated that the NF-κB p65–p50 heterodimer bound to these motifs. These findings were further supported by chromatin immunoprecipitation assays in endothelial cells. Taken together, these results indicated that TNFα induced Robo4 expression by facilitating NF-κB p65–p50 heterodimer binding to the −2753 and −2220 motifs in the Robo4 promoter in endothelial cells in the context of inflammation.

Roundabout (Robo) 4 is a transmembrane receptor that is specifically expressed in endothelial cells (ECs).¹⁾ Although Robo4 expression is observed in the microvasculature of normal tissues such as the lung and heart,^2–4) its expression is particularly high in angiogenic blood vessels in embryos,¹⁾ placentas and tumors.^2,4,5) Robo4 has been shown to be associated with EC migration, proliferation, and angiogenesis as well as blood vessel stabilization.^6–8) Recent studies have indicated that Robo4 is involved in the regulation of cytokine production and vascular permeability in the context of inflammation, such as sepsis.^9,10) Thus, although Robo4 contributes to the inflammatory response via variations in its expression level, it is unclear whether the expression of Robo4 is altered in response to inflammatory stimulation.

The expression of human Robo4 is regulated by its 3-kb promoter region.³⁾ The proximal promoter, to which the transcription factors GA-binding protein and specificity protein 1 bind, is essential for basal promoter activity. The proximal promoter also regulates EC-specific promoter activation through DNA methylation.^3,11,12) Additionally, the upstream promoter contributes to promoter activation in vivo through the binding of activator protein (AP)-1 complexes (c-Jun–cJun and c-Jun–Fra-1) to the −2.8 kb motif.^12,13) In addition to the AP-1 motif, several nuclear factor (NF)-κB consensus motifs are found in the upstream promoter, implying that Robo4 may be regulated by inflammatory signaling, through the binding of NF-κB to those motifs.

NF-κB proteins comprise a group of transcription factors that form homo- and heterodimers. NF-κB subunits including RelA (p65), RelB, c-Rel, NF-κB1 (p50/p105), and NF-κB2 (p52/p100) form dimers in various combinations.^14,15) The most common NF-κB complex is the p65–p50 heterodimer, which binds to the sequence 5′-GGGRNNYYCC-3′.¹⁶⁾ NF-κB is activated through the degradation of inhibitor of NF-κB (IκB) by inflammatory mediators such as tumor necrosis factor (TNF) α, lipopolysaccharide, and interleukin (IL)-1β.¹⁷⁾ NF-κB activation in ECs induces the expression of various inflammatory-response genes including intercellular cell adhesion molecule (ICAM)-1, E-selectin, IL-6, and IL-8,^18–21) and regulates angiogenesis, proliferation, and survival.²²⁾ Thus, NF-κB is an essential transcription factor that mediates the functions of ECs in the inflammatory state by regulating gene expression.

In this study, we analyzed whether inflammatory mediators alter Robo4 expression. Our results showed that TNFα increased Robo4 expression in mice and in human primary ECs. We also demonstrated that this activation was induced through the binding of the NF-κB heterodimer p65–p50 to the NF-κB motif in the Robo4 upstream promoter. These findings provided evidence for a novel mechanism, through which EC function is regulated by Robo4 in the context of inflammation.

RESULTS

Inflammatory Stimulation by TNFα Increased Robo4 Expression in Vivo

To investigate whether Robo4 expression was regulated by inflammatory mediators in mice, we intravenously injected TNFα into mice and measured Robo4 mRNA levels in organs that express Robo4 under normal conditions by real-time PCR. Robo4 mRNA levels were significantly increased in the lungs, kidneys, heart, and liver following injection of TNFα compared to that in control mice (Fig. 1). Thus, these data indicated that TNFα increased Robo4 expression in vivo.

Fig. 1. Effects of TNFα on Robo4 Expression in Mice

Male C57BL/6 mice (8–9 weeks old) were injected intravenously with TNFα (10 µg/mouse) or PBS as a control. The mice were sacrificed 3 h after injection, and the expression of Robo4 and GAPDH (for normalization) mRNA was analyzed in the lungs, kidneys, heart, and liver by real-time PCR. Data are means±standard deviation (S.D.) (n=3).

TNFα Induced Robo4 Expression in ECs via NF-κB Activation

To investigate whether TNFα regulated Robo4 expression in ECs, we treated two types of human primary ECs, human umbilical vein endothelial cells (HUVECs) and human coronary artery endothelial cells (HCAECs), with TNFα and measured Robo4 mRNA levels by real-time PCR. TNFα treatment significantly increased Robo4 mRNA expression in HUVECs and HCAECs (Fig. 2A). This result indicated that TNFα induced Robo4 expression in ECs.

Because TNFα is known to regulate inflammatory–response genes including E-selectin, ICAM-1, and IL-6 through NF-κB activation, we next investigated whether NF-κB activation was involved in the induction of Robo4 mRNA by TNFα. We analyzed the induction of Robo4 mRNA in response to TNFα in HUVECs pretreated with the NF-κB inhibitor ammonium pyrrolidinedithiocarbamate (PDTC). In untreated cells, PDTC did not affect the basal expression of Robo4 mRNA. However, pretreatment with PDTC abolished the TNFα-induced upregulation of Robo4 mRNA (Fig. 2B). These results indicated that Robo4 induction by TNFα is regulated by NF-κB activation.

Fig. 2. Regulation of TNFα-Dependent Robo4 Induction by NF-κB in Endothelial Cells (ECs)

(A) Primary human ECs, human umbilical vein endothelial cells (HUVECs) and human coronary artery endothelial cells (HCAECs), were treated with TNFα or PBS for 8 h. Expression of Robo4 and GAPDH mRNA was analyzed by real-time PCR. Data are means±S.D. (n=4). * p<0.05 versus PBS. (B) HUVECs pretreated with or without 50–100 µM ammonium pyrrolidinedithiocarbamate (PDTC) for 1 h were treated with TNFα or PBS. Expression of Robo4 mRNA was analyzed by real-time PCR. Data are means±S.D. (n=4). * p<0.05 versus PBS. ^# p<0.05 versus treatment with TNFα alone.

TNFα Induced Robo4 Expression through Two NF-κB Motifs in the Robo4 Promoter in ECs

To determine whether an NF-κB–response element was active in the Robo4 promoter, we searched promoter sequences in the JASPAR database and identified potential NF-κB-binding motifs located at −2905, −2753, −2556, −2220, and −1820. We mutated each of the motifs in the promoter and analyzed their functions using reporter assays in HCAECs (Fig. 3A). TNFα activated the wild-type Robo4 promoter. However, this activation was decreased after mutation of either the −2753 or −2220 motif, but not after mutation of the other motifs. Furthermore, double mutation of both the −2753 and −2220 motifs completely abolished TNFα-mediated promoter activation (Fig. 3B). These results indicated that induction of Robo4 expression by TNFα was regulated by promoter activation via the −2753 and −2220 NF-κB motifs.

Fig. 3. Identification of the Response Element for TNFα-Induced Robo4 Promoter Activation in Endothelial Cells

(A, B) Reporter assays were performed using wild-type and mutant Robo4 promoters, including a mutation in each NF-κB motif (A) or mutations in both the −2753 and −2220 motifs (B). Data are means±S.D. (n=6). * p<0.05 versus the wild-type promoter with PBS treatment. ^# p<0.05 versus the wild-type with TNFα treatment.

NF-κB Was Found to Bind to the Robo4 Promoter via the NF-κB Motifs

To identify the transcription factors that bind to the NF-κB motifs, electrophoretic mobility shift assays were performed. Incubation of a radiolabeled probe spanning the −2753 NF-κB motif with nuclear extracts from HUVECs treated with TNFα yielded a shifted band. This band was abolished by the addition of a wild-type competitor, but not a mutant competitor. Addition of antibodies against NF-κB p65 and p50 but not a control antibody also abolished the production of shifted bands (Fig. 4A). Similar results were obtained from assays employing a probe spanning the −2220 NF-κB motif. Thus, these data suggested that the p65–p50 dimer binds to both the −2753 and −2220 NF-κB motifs.

To confirm the induced binding of p65 and p50 to the NF-κB motifs in the endogenous Robo4 promoter in ECs, chromatin immunoprecipitation (ChIP) assays were performed (Fig. 4B). Binding of either p65 or p50 to the −2.7 and −2.2 kb upstream regions of the endogenous Robo4 promoter was barely detectable in unstimulated HUVECs. In contrast, significantly increased binding of both p65 and p50 was detected in HUVECs stimulated with TNFα. These data indicated that TNFα induces binding of both p65 and p50 to the −2.7 and −2.2 kb regions of the Robo4 promoter in ECs.

Fig. 4. Binding of NF-κB p65 and p50 to the −2735 and −2220 Motifs in Endothelial Cells

(A) Electrophoretic mobility shift assays were performed using a radiolabeled probe spanning the −2735 and −2220 NF-κB motifs and nuclear extracts from human umbilical vein endothelial cells (HUVECs) treated with or without TNFα. Competition assays and super shift assays were performed using wild-type or mutant competitors and antibodies against p65, p50, or control IgG, respectively. The arrowheads indicate the shifted bands derived from the p65–p50 heterodimer. (B) ChIP assays were performed using HUVECs treated with or without TNFα using antibodies against p65, p50, or control IgG. Immunoprecipitated DNA fragments were analyzed by real-time PCR with specific primers to amplify each region containing the −2753 or −2220 NF-κB motif. Data are means±S.D. (n=5). * p<0.05 versus PBS.

DISCUSSION

In this study, we demonstrated that the inflammatory mediator TNFα increases Robo4 expression in mice and in human primary ECs. To the best of our knowledge, this is the first study to show the transcriptional regulation of Robo4 by inflammatory stimuli. Since Robo4 and its ligand Slit comprise a signaling pathway that inhibits inflammation-induced endothelial permeability in mouse models of sepsis and influenza,^4,10) Robo4 regulation by TNFα might represent a negative feedback loop involved in preventing excessive increases in vascular permeability during inflammation.

Robo4 is highly expressed in tumor blood vessels compared to normal blood vessels.^1,2) Our current findings demonstrating that Robo4 is regulated by TNFα might explain this vessel type-dependent Robo4 expression. In other words, high concentrations of inflammatory cytokines including TNFα in tumors^23,24) could induce Robo4 expression in ECs of tumor blood vessels. Another mechanism is regulation by shear stress, which has been shown to inhibit Robo4 expression.^12,25) Low shear stress in tumor blood vessels increases Robo4 expression in ECs of tumors. It is possible that Robo4 regulation by shear stress also causes variations in Robo4 induction in vivo and in vitro. Although Robo4 was significantly induced both in vivo and in vitro in our study, the observed induction of Robo4 in vivo was stronger than that in vitro. We speculate that basal Robo4 expression might be upregulated in primary ECs in culture conditions lacking shear stress, and this upregulation might attenuate Robo4 induction in vitro.

Our previous studies suggested that the proximal region of the promoter regulates EC-specific promoter activation, whereas the upstream region regulates promoter activity. Indeed, the AP-1 complex binds to the −2875 motif in the upstream region and activates Robo4 promoter activity.¹³⁾ In this study, we demonstrated that NF-κB also binds to the −2753 and −2220 motifs of the upstream region and activates the promoter in response to TNFα stimulation. These results suggest that the upstream region between −2.2 and −2.9 kb function as an enhancer region to regulate promoter activity. Additionally, we previously demonstrated that the chromatin structure at the −2.5 kb region, which is located in the middle of the enhancer, is highly condensed.^12,13) We speculate that this specific-chromatin structure might enable the upstream enhancer to access the transcription start site. Further studies are needed to elucidate the detailed mechanisms through which NF-κB activates the Robo4 promoter.

EXPERIMENTAL

Animals

Male C57BL/6 mice (8–9 weeks of age) were injected intravenously with TNFα (10 µg/mouse; Wako Pure Chemical Industries, Ltd., Osaka, Japan). The mice were sacrificed 3 h after injection, and tissues were collected and stored in RNAlater RNA Stabilization Reagent (Qiagen, Hilden, Germany). Total RNA was isolated from the homogenized tissues using RNeasy Plus Mini Kit (Qiagen). All animal experiments were approved by the Experimental Animal Care and Use Committee at Osaka University.

Cell Culture

HUVECs and HCAECs were purchased from Lonza (Basel, Switzerland) and cultured at 37°C in an atmosphere containing 5% CO₂ in EGM-2 MV medium (Lonza, Basel, Switzerland).

Real-Time PCR

HUVECs were cultured in EBM-2 medium (Lonza) containing 0.5% fetal bovine serum for 20 h and then treated with 80 ng/mL TNFα or phosphate-buffered saline (PBS; as a control). Total RNA was extracted from these cells using an RNeasy Mini Kit (Qiagen), and 500 ng RNA was used for cDNA synthesis with Superscript III Reverse Transcriptase (Invitrogen, Carlsbad, CA, U.S.A.). Expression levels of Robo4 and glyceraldehyde-3 phosphate dehydrogenase (GAPDH) mRNA were analyzed by real-time PCR using synthesized cDNA with specific primers (Supplementary Table S1) and the QuantiTect SYBR Green PCR Kit (Qiagen). Copy numbers were calculated from standard curves constructed using known amounts of plasmids that contain target sequences. Expression was normalized to that of GAPDH mRNA, and data were collected from at least three independent experiments.

Plasmid Preparation

The luciferase plasmid containing the Robo4 promoter (pGL3-Robo4) was reported previously.³⁾ Robo4 promoter mutants were prepared by PCR-based site-directed mutagenesis using pGL3-Robo4 as a template. The mutated promoter fragments were isolated by digestion with KpnI and EcoRI (for −2905, −2753, and −2556 motifs) or EcoRI and SmaI (for −2220 and −1820 motifs) and inserted into the KpnI and EcoRI sites or the EcoRI and SmaI sites of pGL3-Robo4. To prepare a plasmid with double mutations, the promoter fragment with the −2753 motif mutation was isolated by digestion with KpnI and EcoRI and inserted into the KpnI and EcoRI sites of pGL3-Robo4 with the −2220 motif mutation.

Luciferase Assay

HCAECs were seeded in 6-well plates (1.0×10⁵ cells/well) and cultured for 24 h. The cells were then transfected with 1 µg of a reporter plasmid and 50 ng of a Renilla luciferase plasmid using Fugene6 (Promega, Madison, WI, U.S.A.), and cultured for 24 h. The cells were then treated with TNFα (80 ng/mL) or PBS for 24 h and assayed for luciferase activity using a luminometer. Firefly luciferase activity was normalized to Renilla luciferase activity. Data were collected from at least three independent experiments.

Electrophoretic Mobility Shift Assay

Nuclear extracts were harvested from HUVECs treated with TNFα or PBS for 8 h using a Nuclear Extract Kit (Active motif, Carlsbad, CA, U.S.A.). ³²P-labeled oligonucleotide probes spanning each NF-κB motif (Supplementary Table S1) and nuclear extracts were mixed in binding buffer (10 mM N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonic acid (HEPES)-KOH [pH 7.8], 12.5% glycerol, 0.5 mM ethylenediaminetetraacetic acid (EDTA), 1 mM dithiothreitol, 50 mM KCl, 5 mM MgCl₂, and 2 µg/µL poly dI-dC) for 30 min at 4°C with or without antibodies against p65 (C-20) and p50 (H-119) (Santa Cruz Biotechnology, Santa Cruz, CA, U.S.A.). The resulting samples were analyzed by electrophoresis at 120 V for 2 h on a 4% native polyacrylamide gel in 0.5×TBE buffer.

Chromatin Immunoprecipitation (ChIP) Assay

HUVECs were seeded in 15-cm plates (1.0×10⁷ cells/well) and cultured for 24 h. Cells were treated with TNFα (80 ng/mL) or PBS for 8 h and then crosslinked with a solution containing 50 mM HEPES–KOH (pH 7.5), 100 mM NaCl, 1 mM EDTA, 0.5 mM ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid, and 11% formaldehyde, and sonicated for 20 cycles at 15 s/cycle using a Sonifer Model 250 (Branson, Danbury, CT, U.S.A.) with an output control of 3 and a duty cycle of 30%. The resulting extract was incubated at 4°C for 24 h with Dynabeads pre-coated with 3–10 µg antibodies against p65, p50, and control immunoglobulin G (IgG). DNA–protein complexes were collected using a magnet, and de-crosslinked in a solution containing 50 mM Tris–HCl (pH 8.0), 10 mM EDTA, and 1% sodium dodecyl sulfate. The resulting DNA was analyzed by real-time PCR with specific primers (Supplementary Table S1).

Statistical Analysis

The statistical significance of differences between means was determined by Student’s t-test or Dunnett’s test.

Acknowledgments

This work was supported by JSPS KAKENHI (Grant number JP 26293014) and the Project for Cancer Research and Therapeutic Evolution (P-CREATE) from Japan Agency for Medical Research and Development, AMED.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

The online version of this article contains supplementary materials

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