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Originally published online as doi:10.1189/jlb.0806517 on October 19, 2006

Published online before print October 19, 2006
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(Journal of Leukocyte Biology. 2007;81:557-566.)
© 2007 by Society for Leukocyte Biology

Mechanisms underlying TGF-ß1-induced expression of VEGF and Flk-1 in mouse macrophages and their implications for angiogenesis

Seong-Hyun Jeon*, Byung-Chul Chae*, Hyun-A Kim*, Goo-Young Seo*, Dong-Wan Seo*, Gie-Taek Chun*, Nam-Soo Kim*, Se-Won Yie*, Woo-Hyeon Byeon*, Seok-Hyun Eom*, Kwon-Soo Ha{dagger}, Young-Myeong Kim{dagger} and Pyeung-Hyeun Kim*,{dagger},1

* Department of Molecular Bioscience, School of Bioscience and Biotechnology, and
{dagger} Vascular System Research Center, Kangwon National University, Chunchon, Korea

1 Correspondence: Department of Molecular Bioscience, School of Bioscience and Biotechnology, Kangwon National University, Chunchon 200-701, Korea. E-mail: phkim{at}kangwon.ac.kr


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
TGF-ß induces vascular endothelial growth factor (VEGF), a potent angiogenic factor, at the transcriptional and protein levels in mouse macrophages. VEGF secretion in response to TGF-ß1 is enhanced by hypoxia and by overexpression of Smad3/4 and hypoxia-inducible factor-1{alpha}/ß (HIF-1{alpha}/ß). To examine the transcriptional regulation of VEGF by TGF-ß1, we constructed mouse reporters driven by the VEGF promoter. Overexpression of HIF-1{alpha}/ß or Smad3/4 caused a slight increase of VEGF promoter activity in the presence of TGF-ß1, whereas cotransfection of HIF-1{alpha}/ß and Smad3/4 had a marked effect. Smad2 was without effect on this promoter activity, whereas Smad7 markedly reduced it. Analysis of mutant promoters revealed that the one putative HIF-1 and two Smad-binding elements were critical for TGF-ß1-induced VEGF promoter activity. The relevance of these elements was confirmed by chromatin immunoprecipitation assay. p300, which has histone acetyltransferase activity, augmented transcriptional activity in response to HIF-1{alpha}/ß and Smad3/4, and E1A, an inhibitor of p300, inhibited it. TGF-ß1 also increased the expression of fetal liver kinase-1 (Flk-1), a major VEGF receptor, and TGF-ß1 and VEGF stimulated pro-matrix metalloproteinase 9 (MMP-9) and active-MMP-9 expression, respectively. The results from the present study indicate that TGF-ß1 can activate mouse macrophages to express angiogenic mediators such as VEGF, MMP-9, and Flk-1.

Key Words: MMP-9 • HUVEC • promoter


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Macrophages play a key role in inflammatory and tumor angiogenesis [1 ]. The angiogenic activity of macrophages is known to be associated with their secretory properties. Macrophages produce a variety of angiogenic factors including vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), TGF-{alpha}, TNF-{alpha}, platelet-derived growth factor (PDGF), IL-8, and extracellular matrix (ECM)-degradative enzymes such as matrix metalloproteinase 9 (MMP-9), an important angiogenic factor [2 3 4 5 6 7 ]. It is believed that macrophages do not participate in angiogenesis unless activated [8 ]. Thus, microenvironmental influences on macrophages are important in the regulation of angiogenesis in wounds and malignant tumors [9 ]. The role of VEGF in angiogenesis is well-documented. VEGF expression is regulated by pathological conditions such as hypoxia and hypoglycemia [10 , 11 ], as well as by cytokines and growth factors such as PDGF, TNF-{alpha}, IL-1, and IL-6 [12 13 14 15 ]. It is not known whether TGF-ß can stimulate macrophages to secrete VEGF.

TGF-ß is a 25-kDa homodimeric protein, which regulates a variety of cellular responses, such as proliferation, differentiation, migration, and apoptosis [16 , 17 ]. TGF-ß1 is produced by a variety of cells including macrophages, T cells, and tumor cells [18 ], and TGF-ß signal transduction is mediated by a heteromeric complex of serine/threonine kinase receptors type I (TßR-I) and type II (TßR-II) and Smads [19 , 20 ]. The first in vivo evidence for a role of TGF-ß in angiogenesis came from the observation of new capillary formation after injection of the factor into mice [21 ]. Since then, several studies have shown that TGF-ß is involved in angiogenesis, leading to tumor progression [22 ]. One of the functions of TGF-ß in angiogenesis seems to be associated with its ability to induce VEGF, for example, in human tumor cells [23 ], mouse fibroblasts [24 ], and epithelial cells [25 ]. Smad3 and hypoxia-inducible factor-1{alpha} (HIF-1{alpha}) cooperate with TGF-ß to induce VEGF transcription in humans [26 ]. It is not known whether TGF-ß can induce mouse macrophages to produce VEGF and if so, what mechanisms are involved. TGF-ß induces MMP-9 expression in dermal fibroblasts and epidermal keratinocytes [27 ], but as in the case of VEGF, it is not clear whether TGF-ß can induce the production of MMP-9 in macrophages.

In the present study, we show that TGF-ß-activated macrophages produce VEGF and that Smad3/4 mediate this response together with HIF-1{alpha}. TGF-ß1 also markedly increased levels of fetal liver kinase-1 [Flk-1; the major VEGF receptor (VEGFR)] and MMP-9. These results imply that TGF-ß-activated mouse macrophages may contribute to angiogenesis via the production of these angiogenic factors.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mice
BALB/c mice were purchased from Orient Co. Ltd. (Gyeonggi-do, Korea) and maintained on an 8- to 16-h light-dark cycle in an animal environmental control chamber (Myung Jin Inst. Co., Seoul, Korea). Animals were fed Purina Laboratory Rodent Chow 5001 ad libitum. Eight- to 12-week old mice were used in this study. Animal care was in accordance with the institutional guidelines set forth by Kangwon National University (Korea).

Preparation of peritoneal macrophages
Mice were injected i.p. with 3 ml 3% thioglycolate broth (Sigma Chemical Co., St. Louis, MO) in PBS. Following 3 days, peritoneal cells were harvested from peritoneal lavages of PBS containing 2% FBS and washed twice with HBSS. Isolated cells were resuspended in 10% FBS-DMEM and dispensed into a 100-mm culture dish. Cells were then incubated in a CO2 incubator for 2 h at 37°C. Adherent cells were used as peritoneal macrophages.

Cell culture
Peritoneal macrophages and WEHI3 mouse myelomonocytes were cultured in DMEM supplemented with 10% FBS or 1% FBS in normoxic conditions (5% CO2, 21% O2, 74% N2) using a humidified CO2 incubator. Hypoxic exposure was carried out under 5% CO2, 1% O2, and 94% N2 at 37°C using a BactronTM anaerobic chamber (Sheldon Manufacturing, Cornelius, OR). HUVECs were isolated from human umbilical cord veins by collagenase treatment as described [28 ] and used in Passages 2–7. The cells were grown in M199 medium (Invitrogen, Carlsbad, CA), supplemented with 20% FBS, 100 units/ml penicillin, 100 µg/ml streptomycin, 3 ng/ml bFGF (Upstate Biotechnology, Lake Placid, NY), and 5 units/ml heparin at 37°C in a humidified CO2 incubator.

ELISA for mouse VEGF
VEGF produced in cultures was determined by an ELISA developed in our lab. Antimouse VEGF antibody (R&D Systems, Minneapolis, MN) was added at 0.4 µg/ml in 0.05 M bicarbonate buffer (pH 9.3) to 96-well, U-bottom, polyvinyl microplates (Becton Dickinson and Co., Oxnard, CA). After incubation overnight at 4°C, the plates were washed and blocked with 1% gelatin for 1 h. Samples (50 µl) or standard protein (mouse recombinant VEGF, R&D Systems) diluted in 0.5% gelatin were added to the wells. After incubation for 1 h at 37°C, the plates were washed again, and 50 ng/ml biotinylated antimouse VEGF antibody (R&D Systems) was added for 1 h at 37°C. The plates were then washed and incubated with streptavidin-HRP for 1 h at 37°C. After washing, 0.2 mM ABTS (Sigma Chemical Co.) was added to the wells, and after 10 min, the colorimetric reaction was measured at 405 nm with an ELISA reader VERSAmax (Molecular Devices, Sunnyvale, CA).

RT-PCR
RNA preparation, RT, and PCR were performed as described previously [29 ]. Primers for PCR were synthesized by Bioneer Corp. (Seoul, Korea). The primers for mouse VEGF were: forward primer 5'-CAG GCT GCT GTA ACG ATG AA-3' and reverse primer 5'-CAG GAA TCC CAG AAA CAA CC-3'. Primers spanning the mouse VEGF gene amplified three mRNA variants, VEGF120, VEGF164, and VEGF188, as the expected 522-, 636-, and 708-bp products, respectively. The primers for Flk-1 were: forward primer 5'-GAT GGG AAC CGG AAC CT-3' and reverse primer 5'-AAT TCC ACA ATC ACC ATG AG-3', and for mouse MMP-9: forward primer 5'-ACT CAC ACG ACA TCT TCC AG-3' and reverse primer 5'-AGA AGG AGC CCT AGT TCA AG-3'. All reagents were from Promega (Madison, WI).

Assay of endothelial tube formation
WEHI3 cells (2x107 cells) were transfected with Smad3/4 (5 µg each) and HIF-1{alpha}/ß (15 µg each) and cultured in the presence of TGF-ß1 for 24 h. The supernatant was harvested, and its ability to promote the formation of tubular structures in vitro was examined using the tube formation assay. Briefly, 250 µl ice-cold Matrigel (BD Biosciences, San Jose, CA) was added to a 24-well plate and allowed to solidify at 37°C for 30 min. HUVECs (500 µl/well; at 2x105 cells/ml) and 500 µl conditioned medium were added. The HUVECs were incubated for 12 h, and tube formation was determined with a light microscope (x100).

Plasmid constructions
Two different mouse VEGF promoter DNA fragments (–1216 to +399 and –451 to +399) were amplified from mouse spleen genomic DNA by PCR. The PCR primers were based on the sequences reported previously [30 ] (Accession No. U41383). The VEGF promoter fragments were subcloned into pGL3 (Promega). The reporters under the control nt –1216 to +399 and –451 to +399 of the VEGF promoter were named pVEGF(W) and pVEGF1, respectively. Mutations were introduced into pVEGF(W) by QuikChangeTM site-directed mutagenesis (Stratagene, La Jolla, CA). Likewise, a mouse MMP-9 promoter-reporter was constructed by cloning mouse MMP-9 genomic DNA fragment –707 to +30 into pGL3-basic plasmid. The PCR primers were based on the promoter sequence reported previously [31 ] (Accession No. D15060). pcDNA3-HIF-1{alpha} and pcDNA3-HIF-1ß were kindly provided by Dr. L. Eric Huang (Brigham and Women’s Hospital, Harvard Medical School, Boston, MA). The mammalian expression vectors for Smad2, Smad3, Smad4, and Smad7 in N-terminal, Flag-tagged pcDNA3 were generously provided by Dr. Masahiro Kawabata (The Cancer Institute, Tokyo, Japan).

Transfection and luciferase assay
Transfection was performed by electroporation with a Gene Pulser II (Bio-Rad Laboratories, Hercules, CA) [29 ]. Reporter plasmids were cotransfected with expression plasmids and plasmid cytomegalovirus-ß-galactosidase (PCMVß-gal; Stratagene) and luciferase and ß-gal assays performed as described [29 ].

Chromatin immunoprecipitation (ChIP) assay
ChIP assays were performed using a ChIP assay kit (Upstate Biotechnology, Lake Placid, NY). WEHI3 cells (1x106 cells) were fixed with 1% formaldehyde, washed, resuspended in lysis buffer, and sonicated. After removing cell debris by centrifugation, the supernatant was diluted tenfold with ChIP dilution buffer and precleared with salmon sperm DNA/Protein A agarose-50% slurry. The supernatant fraction was transferred to a fresh tube with 10 µg/ml anti-Flag antibody (Sigma Chemical Co.) or 5 µg/ml anti-hemagglutinin (HA) antibody (Zymed Laboratories, South San Francisco, CA) and incubated overnight at 4°C. Salmon sperm DNA/Protein A agarose-50% slurry (60 µl) was added to the immune complexes, and after incubation for 1 h at 4°C, the supernatant was discarded. The histone-DNA cross-links were reversed by digestion with 2 µl 10 mg/ml proteinase K, and the DNA was extracted. PCR was performed to amplify the 935- to –916-bp VEGF promoter sequences from the transcription start site with primers: forward 5'-GCC AGA CTA CAC AGT GCA TA-3' and reverse 5'-GCT TAT CTG AGC CCT TGT CTG-3', and the products were resolved by electrophoresis on 2% agarose gels.

Immunoprecipitation and Western blot analysis
For immunoprecipitation experiments, WEHI3 cells were transfected with the appropriate expression vectors. After 24 h incubation, cells were collected, lysed, and subjected to immunoprecipitation with anti-FLAG antibody (Sigma Chemical Co.) using protein A-Sepharose (Amersham Pharmacia Biotech, Uppsala, Sweden). For Western blot analysis, total cell lysates or immunoprecipitates were subjected to SDS-PAGE under reducing conditions, and proteins were transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad Laboratories). Specific immunodetection was carried out by incubation with anti-HIF-1{alpha} (BD PharMingen, San Diego, CA) or anti-FLAG antibodies, followed by peroxidase-conjugated goat antimouse IgG (Pierce, Rockford, IL). The presence of specific proteins was revealed using a chemiluminescence assay (Supersignal detection kit, Pierce).

Flow cytometry
Cultured cells were suspended in DMEM, 5% FBS, and 0.1% NaN3 at a density of 1 x 106 cells/ml. Rabbit antimouse Flk-1 (Santa Cruz Biotechnology, CA) was added to the cell suspension and placed at 4°C for 30 min. After washing three times with HBSS, FITC-conjugated, goat antirabbit IgG (Sigma Chemical Co.) was added and incubated at 4°C for 30 min. The cells were then washed and resuspended in PBS-1% formalin. Flow cytometric analysis was performed with a FACScan (Becton Dickinson, Mountain View, CA).

Gelatin zymography
Samples were subjected to electrophoresis on 10% SDS-polyacrylamide gels containing 0.1% (w/v) gelatin. The gels were washed with 2.5% Triton X-100 and incubated at 37°C for 12 h in buffer containing 10 mM CaCl2, 0.15 M NaCl, 50 mM Tris (pH 7.5), and 0.02% NaN3 (w/v). They were then stained with 0.25% Coomassie brilliant blue R 250 in 45% methanol and 10% acetic acid for 30 min and destained in 20% ethanol and 5% acetic acid until bands were visible.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of TGF-ß1 on expression of VEGF in mouse macrophages
Although TGF-ß can induce VEGF expression in a human tumor cell line [32 ], it is not known whether TGF-ß influences expression of VEGF in murine macrophages. As macrophages are one of the major sources of VEGF during angiogenesis [9 ], we first measured VEGF expression in WEHI3 cells, a mouse macrophage cell line, by ELISA, in the presence of TGF-ß1. As shown in Figure 1A , TGF-ß1 increased VEGF secretion twofold, and secretion was greater in serum-free medium. In addition, basal and TGF-ß1-induced VEGF secretion was augmented by hypoxia. As Smad3 and -4 are well-known components of the TGF-ß-signaling pathway, we explored their involvement in VEGF expression. Overexpression of Smad3/4 hardly affected VEGF secretion under normoxic conditions but increased it substantially under hypoxic conditions (Fig. 1B) .


Figure 1
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  		Figure 1. TGF-ß1 enhances VEGF expression in mouse macrophage cells. (A) Effects of hypoxic and serum-free conditions on VEGF production by macrophages stimulated with TGF-ß1. WEHI3 cells (4x105 cells/ml) were cultured with TGF-ß1 (2 ng/ml) in normoxic or hypoxic conditions for 24 h. VEGF was determined by ELISA. (B) Effect of overexpressed Smad3/4 on VEGF production under the influence of TGF-ß1 and hypoxia. WEHI3 cells (2x107) were transfected with Smad3/4 (5 µg each) and cultured with TGF-ß1 (2 ng/ml) in serum-free, normoxic/hypoxic conditions for 24 h. (C) Effect of overexpressed HIF-1{alpha}/ß on TGF-ß1-induced, Smad3/4-mediated VEGF production. WEHI3 cells (2x107) were transfected with Smad3/4 (5 µg each) and HIF-1{alpha}/ß (15 µg each) and cultured with TGF-ß1 (2 ng/ml) in serum-free, normoxic/hypoxic conditions for 24 h. Data are means of triplicate samples ± SEM. (D) Overexpression of Smad3/4 and HIF-1{alpha}/ß enhances endogenous VEGF transcription by TGF-ß1-stimulated macrophage cells. WEHI3 cells were transfected with Smad3/4 and HIF-1{alpha}/ß as indicated. After transfection, cells were treated with 2 ng/ml TGF-ß1 and cultured in serum-free condition for 12 h. Levels of VEGF mRNA were measured by RT-PCR. (E) Effect of supernatants of TGF-ß1-stimulated macrophages on tube formation by HUVECs. Conditioned media were prepared from WEHI3 cells transfected with Smad3/4 and HIF-1{alpha}/ß as shown in D. HUVECs were seeded in 24-well plates coated with Matrigel and treated with conditioned medium for 12 h. Tube formation was determined with a light microscope (x100). To neutralize VEGF, the conditioned medium was preincubated with 5 µg/ml anti-VEGF antibody (Ab) for 1 h at 37°C. (F) Effect of TGF-ß1 on the transcriptional and the secretion levels of VEGF in normal mouse macrophages. Mouse peritoneal macrophages (1x106 cells/well) were treated with TGF-ß1 (1 ng/ml) in hypoxic and serum-free conditions. Levels of VEGF mRNA were measured by RT-PCR 12 h later, and VEGF secretion was determined by ELISA after 72 h cultured. Data are means of triplicate samples ± SEM.

 
As the HIF-1 complex is the major mediator of the hypoxia signaling pathway [33 34 35 ], we investigated whether HIF-1{alpha}/ß was involved in TGF-ß1-dependent VEGF secretion. In the event, overexpression of HIF-1{alpha}/ß enhanced TGF-ß1-induced, Smad3/4-mediated VEGF secretion (Fig. 1C) . We detected three alternative transcripts of VEGF by RT-PCR using primers derived from exon3 and exon8 of the mouse VEGF gene, and as shown in Figure 1D , TGF-ß1 stimulated transcription of VEGF164, the predominant form of VEGF. Overexpression of Smad3/4 or HIF-1{alpha}/ß increased this further, and the combination of the two molecules had the greatest effect. The responses of VEGF120 and VEGF188 mRNAs were similar to that of VEGF164 (Fig. 1D) . Further, we performed a tube formation assay using HUVECs to see whether the VEGF produced by TGF-ß-activated macrophages had angiogenic activity (Fig. 1E) and found that conditioned medium from WEHI3 cells transfected with Smad3/4 and HIF-1{alpha}/ß markedly enhanced tube formation. The increase was comparable with that obtained with 10 ng/ml VEGF (data not shown), and the effect of the conditioned medium was inhibited completely by anti-VEGF antibody. Finally, to obtain the direct evidence that TGF-ß1 is implicated in VEGF regulation in normal macrophages, we determined the effect of TGF-ß1 on VEGF expression in peritoneal macrophages. As shown in Figure 1F , TGF-ß1 significantly increased the VEGF expression at the transcriptional and at the protein level.

Construction and analysis of mouse VEGF promoters
We searched the putative VEGF promoter sequence for potential transcription factor-binding elements with a GEMS Launcher and identified multiple Smad-binding elements (SBEs; CAGACs) and a putative hypoxia-responsive element (HRE; nnncnnaCGTGsn, capital letters denote the core sequence). We constructed two mouse VEGF promoter reporters, pVEGF(W) (1615 bp, –1217 to +399, retaining the putative SBEs and HRE) and pVEGF1 (850 bp, –451 to +399, lacking the putative SBEs and HRE) [30 ] (Supplemental Fig. 1). TGF-ß1 increased the activity of the pVEGF(W) promoter, particularly in hypoxic and serum-free conditions (Fig. 2A ). Overexpression of HIF-1{alpha}/ß in the presence of TGF-ß1 increased promoter activity threefold, as compared with the basal promoter activity, and overexpressed Smad3/4 had a marginal effect. Overexpression of the two factors together in the presence of TGF-ß1 led to a 14-fold increase in promoter activity, indicating that Smad3/4 and HIF-1{alpha}/ß are required for optimal VEGF transcription (Fig. 2B) . We next asked whether Smad7, a negative regulator of the TGF-ß signaling pathway [36 ], affected the pVEGF(W) promoter activity. Overexpression of Smad7 substantially inhibited the stimulation of promoter activity by overexpression of HIF-1{alpha}/ß and Smad3/4 in the presence of TGF-ß1 but not in the absence of TGF-ß1 (Fig. 2B) . This suggests that Smad7 specifically inhibits TGF-ß1-mediated VEGF transcription. In contrast with pVEGF(W), overexpression of HIF-1{alpha}/ß or Smad3/4 or both did not increase the promoter activity of pVEGF1, even in the presence of TGF-ß1 (Fig. 2B) , indicating that the upstream VEGF DNA segment containing the putative HRE and SBEs is indispensable for promoter activity.


Figure 2
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  		Figure 2. Analysis of mouse VEGF promoter reporters. (A) Effects of TGF-ß1 and hypoxia on pVEGF(W) promoter activity in 10% FBS or serum-free medium. pVEGF(W) (15 µg) was transfected into WEHI3 cells, and they were cultured for 24 h. Transcriptional activity was measured using the luciferase reporter assay. (B) Effect of HIF-1{alpha}/ß, Smad3/4, and Smad7 on VEGF promoter activity. pVEGF(W) or pVEGF1 was cotransfected into WEHI3 cells (2x107) with expression vectors coding for HIF-1{alpha}/ß (15 µg, each), Smad3/4 (5 µg each), and Smad7 (10 µg), and the cells were cultured with TGF-ß1 (2 ng/ml) for 24 h. Data are means of triplicate samples ± SEM.

 
To determine whether the putative HRE and SBEs are important for promoter activity, we tested the effect of mutating them (see Fig. 3A ). Promoter reporters carrying mS1 and mS2 had virtually no activity in response to TGF-ß1, and overexpression of the mutant Smad3/4 and HIF-1{alpha}/ß reporters produced strikingly diminished activity, showing that the SBEs and HRE are indispensable for TGF-ß1-induced VEGF promoter activity (Fig. 3B) . It has been reported that hypoxia induces VEGF promoter activity by HIF-dependent and HIF-independent pathways [37 ]. Therefore, it was interesting to determine how the promoter reporter with the mutated HRE responded to TGF-ß1 and hypoxia. In fact, the mH reporter was inactive under these conditions (Fig. 3C) , indicating that hypoxia contributes to TGF-ß1-induced VEGF transcription, mainly via a HIF/HRE-dependent pathway in mouse macrophages.


Figure 3
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  		Figure 3. Functional assays of the HRE and SBEs in the mouse VEGF promoter. (A) Construction of the three different mutant VEGF reporters. Two to three nucleotides (underlined, bold letters) within HRE and SBE were substituted as indicated. (B) Effects of overexpressed HIF-1{alpha}/ß and Smad3/4 on the mutant VEGF reporters. Reporters (each 15 µg) were cotransfected with expression vectors coding for HIF-1{alpha}/ß (15 µg each) and Smad3/4 (5 µg each) into WEHI3 cells (2x107), and the cells were cultured with TGF-ß1 (2 ng/ml) for 24 h. (C) Effects of hypoxia and overexpressed Smad3/4 on HRE-mutated VEGF reporters. Reporters (each 15 µg) were cotransfected with expression vectors coding for Smad3/4 (5 µg each) into WEHI3 cells (2x107). The cells were cultured with TGF-ß1 (2 ng/ml) for 24 h in hypoxic condition. Data are means of triplicate samples ± SEM. mS1, Reporter with mutated SBE1; mS2, reporter with mutated SBE2; mH, reporter with mutated HRE.

 
Binding of HIF-1{alpha} and Smad proteins to the mouse VEGF promoter
We investigated the binding of Smad3/4 and HIF-1{alpha} proteins to the HRE and SBEs of the mouse VEGF promoter by ChIP assays (Fig. 4 A-1 ). TGF-ß1 markedly increased binding of Smad3/4 to the promoter (Fig. 4 A-2) . Binding of HIF-1{alpha} also increased in response to TGF-ß1 and was enhanced further by cotransfection with Smad3/4 (Fig. 4 A-3) . These results demonstrate that Smad3/4 binds to SBE1 or SBE2 or both and that HIF-1{alpha}, in cooperation with Smad3/4, binds to HRE.


Figure 4
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  		Figure 4. ChIP assay for Smad and HIF-1{alpha} binding (A) and immunoprecipitation for Smad and HIF-1{alpha} interaction (B). (A-1) Diagram of the DNA amplified in this study. Primer pairs to amplify a 210-bp segment encompassing SBE1, HRE, and SBE2 were: forward 5'-GCC AGA CTA CAC AGT GCA TA-3' and reverse 5'-GCT TAT CTG AGC CCT TGT CTG-3'. WEHI3 cells (1x107) were transfected with Flag-tagged Smad3/4 (2 µg each) and/or HA-tagged HIF-1{alpha} and HIF-1ß (5 µg each). After transfection, the cells were treated with 2 ng/ml TGF-ß1 and cultured in serum-free medium for 12 h. (A-2) ChIP assays for binding of Smad3. Anti-flag antibody was used to detect promoter-bound Smad3/4. (A-3) ChIP assays for binding of HIF-1{alpha}. Anti-HA antibody was used to detect promoter-bound HIF-1{alpha}. (B) Immunoprecipitation (IP) and Western blot (WB) for the binding between Smad3 and HIF-1{alpha}. WEHI3 cell lysates were immunoprecipitated with anti-Flag antibodies. Total extracts and immunoprecipitates were separated by SDS-PAGE and transferred to PVDF membranes, and membranes were incubated with anti-HIF-1{alpha} or anti-FLAG antibodies. The presence of HIF-1{alpha} or Smad3 was revealed using a chemiluminescence assay.

 
Physical interaction between Smad3 and HIF-1{alpha}
Smad proteins regulate transcription in collaboration with other transcription factors through direct protein–protein interactions [38 ]. To determine the molecular basis of the cooperation between Smad3 and HIF-1{alpha}, we cotransfected WEHI3 cells with expression vectors encoding both transcription factors (Fig. 4B) . Immunoprecipitation with anti-Flag antibodies followed by the immunodetection with anti-HIF-1{alpha} demonstrated the association between Smad3 and HIF-1{alpha}. This interaction could be detected in the absence of TGF-ß1 stimulation, although TGF-ß1 treatment improved the amount of HIF-1{alpha} coprecipitated with Smad3. These results suggest that HIF-1{alpha} interacts with Smad3.

Effects of Smad2, p300, and E1A on mouse VEGF promoter activity
Like Smad3, Smad2 is a receptor-activated Smad known as a mediator of the TGF-ß1 signaling pathway. Phosphorylated Smad2 and Smad3 form heterodimeric complexes with Smad4, and overexpression of Smad3/4 or Smad2/4 enhances TGF-ß1-induced p3TP-Lux promoter activity [39 ]. Furthermore, Smad2, Smad3, and Smad4 synergize to activate the TGF-ß1-inducible plasminogen activator inhibitor-1 promoter [40 ]. By contrast, others have demonstrated antagonistic interactions between Smad2 and Smad3 [41 , 42 ]. We found that unlike Smad3/4, cotransfection of Smad2/4 together with HIF-1{alpha}/ß did not increase the reporter activity of pVEGF1, and overexpression of Smad2 actually reduced Smad3/4-mediated promoter activity (Fig. 5A ).


Figure 5
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  		Figure 5. Effects of Smad2 and p300 on VEGF promoter activity. (A) Smad2 does not mediate TGF-ß1-induced VEGF promoter activity. WEHI3 cells (2x107) were cotransfected with pVEGF(W) (15 µg) and expression vectors coding for Smad2 (5 µg), Smad3 (5 µg), Smad4 (5 µg), and HIF-1{alpha}/ß (15 µg each). The cells were cultured with TGF-ß1 (2 ng/ml) for 24 h. Data are means of triplicate samples ± SEM. (B) p300 cooperates with Smad3/4 and HIF-1{alpha}/ß to enhance VEGF promoter activity. WEHI3 cells (2x107) were cotransfected with pVEGF(W) (15 µg) and expression vectors coding for HIF-1{alpha}/ß (15 µg each), Smad3/4 (5 µg each), p300 (5 µg), and E1A (5 µg). Data are means of triplicate samples ± SEM.

 
p300 interacts with Smad3 and enhances TGF-ß1-induced transcription [43 ], and interaction of the adenoviral oncoprotein E1A with p300 inhibits this effect [44 ]. Moreover, interaction of p300 with HIF-1{alpha} leads to induction of hypoxia-responsive genes [45 ]. We assessed the effect of p300 on the mouse VEGF promoter. Overexpressed p300 enhanced basal and TGF-ß1-induced promoter activity, and activity was enhanced further by cotransfection with Smad3/4 in the presence of HIF-1{alpha}/ß, and overexpression of E1A abolished the combined effect of Smad3/4 and p300 (Fig. 5B) . Thus, these results show that p300 acts as a coactivator and E1A as a repressor of VEGF transcription mediated by Smad3/4 and HIF-1{alpha}/ß.

Effect of TGF-ß1 and VEGF on expression of the VEGFR
VEGF binds to two tyrosine kinase receptors, VEGFR-1 [fetal liver tyrosine kinase-1 (Flt-1)] and VEGFR-2 (kinase domain region/Flk-1), which are expressed on monocytes/macrophages [46 ]. Flk-1 mediates most of the actions of VEGF [47 ], and TGF-ß1 down-regulates its expression in vascular endothelial cells and lung mesenchyme [48 , 49 ]. By contrast, TGF-ß1 up-regulates Flk-1 expression in mouse mammary epithelial cells [50 ]. Therefore, we assessed the effect of TGF-ß1 and VEGF on expression of Flk-1 in mouse macrophages. As shown in Figure 6A , TGF-ß1 alone had little effect on endogenous Flk-1 transcription, whereas overexpression of Smad3/4 or HIF-1{alpha}/ß together with TGF-ß1 stimulated transcription, and the effect of the two factors together was greater than that of either on its own. Flt-1 expression was virtually unaffected under the same conditions. Thus, TGF-ß1 specifically stimulates mouse macrophages to express Flk-1 and Smad3/4, and HIF-1{alpha}/ß mediates this effect. As cytokines tend to behave in an autocrine manner, and TGF-ß1 induces VEGF secretion (Fig. 1) , we determined the effect of VEGF on Flk-1 expression in WEHI3 cells. Addition of VEGF increased the level of Flk-1 transcripts but not of VEGF transcripts themselves (Fig. 6B) . These results indicate that TGF-ß1 may increase Flk-1 expression by inducing VEGF secretion. Moreover, as TGF-ß1 and overexpressed Smad3/4 and HIF-1{alpha}/ß increased surface expression of Flk-1 (Fig. 6C) , it seems clear that TGF-ß1 is involved in the expression of this VEGFR. However, the increase in expression of Flk-1 was not fully abrogated by anti-VEGF-neutralizing antibody: Surface expression was only reduced by ~30%, suggesting that TGF-ß1 up-regulates Flk-1 expression by additional mechanism(s). Although it has been shown that VEGF attenuates TGF-ß action in human endothelial cells [51 ], we found that in mouse macrophages, VEGF has little effect on TGF-ß1-induced VEGF transcription (Supplemental Fig. 2).


Figure 6
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  		Figure 6. Effect of TGF-ß1 and VEGF on expression of Flk-1, VEGF, and MMP-9. (A) WEHI3 cells (1x107) were transfected with Smad3/4 (2 µg) and HIF-1{alpha}/ß (5 µg) and incubated with 2 ng/ml TGF-ß1 in serum-free medium for 36 h. Flk-1 mRNA was measured by RT-PCR. (B) Cells were starved in serum-free medium for 12 h and then exposed to 5 ng/ml mouse VEGF. RT-PCR was performed following incubation for 12 h. (C) WEHI3 cells (1x107) were transfected with Smad3/4 (2 µg) and HIF-1{alpha}/ß (5 µg), exposed to 2 ng/ml TGF-ß1 and/or 75 ng/ml antineutralizing VEGF antibody, and cultured for 48 h. Cells were stained with FITC-labeled anti-Flk-1 antibody and analyzed by FACS. (D) WEHI3 cells (2x106) were incubated with TGF-ß1 (2 ng/ml) or VEGF (5 ng/ml or 20 ng/ml as indicated) in serum-free medium. After 48 h, the conditioned media were collected, and MMP-9 activity was analyzed by gelatin zymography. The arrows indicate the 92-kDa (pro-) and 86-kDa (active-) MMP-9-specific bands.

 
Why is Flk-1 expressed in macrophages, and what is the meaning of its expression in relation to angiogenesis? We assumed that Flk-1 expression is associated with angiogenesis, as TGF-ß1 and VEGF induced Flk-1 expression in our earlier study, and in addition, macrophages can produce MMP-9, a powerful angiogenic factor [7 ]. Therein, we compared the effects of TGF-ß1 and VEGF on gelatinolytic activation of MMP-9 by zymography. Both factors increased pro-MMP-9 expression (92 kDa). In contrast, high doses of VEGF markedly induced the appearance of active-MMP-9 (86 kDa), whereas TGF-ß1 did not (Fig. 6D) . These data show that TGF-ß1 and VEGF preferentially increase pro-MMP-9 and active-MMP-9 expression, respectively, in mouse macrophages.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Although VEGF is expressed under the influence of TGF-ß1 in several cell types [23 24 25 ], it was not known if TGF-ß1 has such an effect on macrophages. The present studies demonstrate that TGF-ß1 stimulates mouse macrophages to produce VEGF and that a typical TGF-ß signaling pathway is involved. As reported for various TGF-ß target genes [17 ], Smad3 and Smad4 promoted VEGF expression, and Smad7 inhibited it. However, from an analysis of VEGF promoter activity, we observed that TGF-induced VEGF promoter activity was profoundly dependent on hypoxic conditions. VEGF is readily expressed in hypoxic conditions, and hypoxia, hypoglycemia, and nutrient deficiency is a key characteristic of solid tumors. Moreover, activated macrophages accumulate in large numbers in such hypoxic/ischemic tissues and influence each phase of the angiogenic process by releasing growth factors such as VEGF [1 , 52 ]. In fact, we found that TGF-ß1 enhances VEGF expression at the transcriptional and the protein levels under the hypoxic and serum-deprived conditions in normal peritoneal macrophages. In contrast, VEGF secretion was not induced by TGF-ß1 in bone marrow-derived macrophages [53 ]. The discrepancy between two results is likely a result of the adopted culture conditions and the source of macrophages.

It is not surprising that VEGF is readily induced by hypoxic and low serum conditions. What was unexpected was that overexpression of Smad3/4 had little effect on VEGF expression at the transcriptional or protein level in response to TGF-ß1. A positive effect of overexpressed Smad3/4 was only noticeable in hypoxic conditions or when HIF-1{alpha}/ß was overexpressed. This finding was confirmed by further analysis of mutant VEGF promoters: VEGF promoter activity was reduced substantially when the putative HRE was mutated, although Smad3/4 was overexpressed, or conditions were hypoxic. These observations indicate that the putative HRE (–917 to –910) is critical for TGF-ß1-induced VEGF expression. The two putative SBEs are also likely to be essential, as reporters with either of these two sites mutated had no TGF-induced activity. Thus, Smad3/4 and HIF-1{alpha}/ß may act together in transcription of VEGF in response to TGF-ß1. This view is supported by the observation that overexpression of Smad3/4 increased binding of HIF-1{alpha} to the promoter (Fig. 4 A-3) , reflecting the fact that Smad3/4 and HIF-1{alpha} interact to bind to the promoter. Moreover, HIF-1{alpha} physically associates with Smad3 so that HIF-1{alpha} and Smad3 have a synergistic effect on transcription of human VEGF in response to TGF-ß [26 ]. We also observed this interaction in mouse macrophages. Therefore, it appears that HIF-1{alpha} also cooperates with Smad3/4 to mediate TGF-ß1-induced VEGF gene expression in mouse macrophages. It remains to be determined whether HIF-1{alpha} and Smad3/4 bind to their consensus motifs in a coordinated manner.

We recently demonstrated that Smad3/4 and Runx3 synergize to induce Ig germ-line {alpha} transcription, leading to IgA isotype switching in TGF-ß1-activated mouse B cells [54 ]. However, in the present study, Runx3 did not affect the mouse VEGF promoter activity at all (data not shown). In contrast, p300 acted as a coactivator and E1A as a repressor of Smad3/4-mediated VEGF promoter activity, as in our previous studies [54 , 55 ]. It thus appears that the difference in regulation between the Ig germ-line {alpha} and VEGF promoters is a result of the different roles of Runx3 and HIF-1. In fact, because of their relatively low DNA-binding specificity, the individual Smad proteins must cooperate with other DNA-binding proteins to elicit specific transcriptional responses. Smad3/4 appear to be general mediators of TGF-ß signaling, and target specificity is probably conferred by a transcription factor such as Runx in the Ig germ-line {alpha} gene [56 ]. We propose that HIF-1 is the specific transcriptional factor in TGF-ß-induced VEGF gene expression, although this remains to be established definitely.

What could be the physiological relevance of TGF-ß-induced VEGF production by macrophages, which are thought to play a key role in inflammatory and tumor angiogenesis [1 ] because of their ubiquity in normal tissues and especially in inflamed regions? Macrophages are usually in a nonactivated, nonangiogenic state. Once activated, they can influence every phase of angiogenesis, by altering the local ECM, stimulating endothelial cells to migrate or proliferate, and inhibiting vascular growth with the formation of differentiated capillaries [1 , 57 ]. Thus, it is conceivable that TGF-ß1 triggers angiogenesis by inducing VEGF production by macrophages. Moreover, we found that TGF-ß1, although less potent than VEGF (Supplemental Fig. 3), stimulated macrophages to express MMP-9 as well as to express Flk-1 (VEGFR-2). Possible pathways regarding regulation of VEGF, Flk-1, and MMP-9 by TGF-ß1 are presented in Figure 7 . Although it still remains to be determined whether this complex scenario occurs in the angiogenesis model, our results reveal that there are multiple checkpoints to inhibit the gene expression of angiogenic factors by TGF-ß-activated macrophages. In this regard, known inhibitors such as an inhibitor of TGF-ß Type I receptor kinase activity [58 ] or inhibitors of HIF-1{alpha} expression [59 , 60 ] are potentially useful in altering the progression of tumor angiogenesis. We are currently exploring the regulation of Flk-1 promoter activity by TGF-ß1. To obtain further information about tumor angiogenesis, it will be interesting to elucidate the mechanisms by which other factors including TGF-ß1 regulate expression and activation of MMP-9 in macrophages as well as in tumors.


Figure 7
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  		Figure 7. Proposed mechanisms by which TGF-ß1 stimulates mouse macrophages (M{phi}) to express VEGF, Flk-1, and MMP-9. Upon stimulation by TGF-ß1, Smad3 becomes phosphorylated by the activated TGF-ß receptors and forms complexes with Smad4. These Smad complexes translocate into the nucleus, where they bind SBEs, and HIF-1 binds HRE in the VEGF promoter in hypoxic conditions, thereby activating transcription. In this, p300 can act as the coactivator of Smad3 and HIF-1. The secreted VEGF in turn enhances the expression of Flk-1 and MMP-9 via binding to Flk-1. Meanwhile, TGF-ß1 can stimulate the gene expression of Flk-1 and also presumably MMP-9. The VEGF and MMP-9 secreted by TGF-ß1-activated macrophages may function during angiogenesis.

 


   ACKNOWLEDGEMENTS
 
This work was supported by a Vascular System Research Center grant from the Korea Science and Engineering Foundation and by the second stage of the Brain Korea 21 program. We thank Dr. L. Eric Huang for the generous gift of pcDNA3-HIF-1{alpha} and pcDNA3-HIF-1ß and Dr. Masahiro Kawabata for providing us the mammalian expression vectors Smad2, Smad3, Smad4, and Smad7 in N-terminal, Flag-tagged pcDNA3.

Received August 16, 2006; revised September 18, 2006; accepted September 25, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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Mol. Pharmacol., August 1, 2007; 72(2): 440 - 449.
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