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Originally published In Press as doi:10.1074/jbc.M706927200 on January 3, 2008

J. Biol. Chem., Vol. 283, Issue 12, 7754-7762, March 21, 2008
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TRAIL Stimulates Proliferation of Vascular Smooth Muscle Cells via Activation of NF-{kappa}B and Induction of Insulin-like Growth Factor-1 Receptor*

Mary M. Kavurma, Supported by a C. J. Martin Fellowship from the National Health and Medical Research Council of Australia (ID 300587){ddagger}§1, Michael Schoppet{ddagger}2, Yuri V. Bobryshev, Levon M. Khachigian§, and Martin R. Bennett{ddagger}

From the {ddagger}Division of Cardiovascular Medicine, University of Cambridge, Box 110, Addenbrooke's Hospital, Cambridge CB2 2QQ, United Kingdom and the §Centre for Vascular Research and Faculty of Medicine, University of New South Wales, Kensington 2052, Australia

Received for publication, August 20, 2007 , and in revised form, November 29, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
TRAIL/Apo2L (tumor necrosis factor-related apoptosis-inducing ligand) is a multifunctional protein regulating homeostasis of the immune system, infection, autoimmune diseases, and apoptosis. However, its function in normal, nontransformed tissues is not clear. Here we show that TRAIL increases vascular smooth muscle cell (VSMC) proliferation in vitro, effects that can be blocked with neutralizing antibodies to TRAIL receptors DR4 and DcR1. In aortocoronary saphenous vein bypass grafts in vivo, TRAIL co-localizes with VSMC, proliferating cell nuclear antigen, and insulin-like growth factor type 1 receptor (IGF1R) expression but not active caspase-3. TRAIL is required for serum-inducible IGF1R expression, and antisense IGF1R inhibits TRAIL-induced VSMC proliferation. At 1 ng/ml, TRAIL stimulates IGF1R mRNA expression greater than insulin-like growth factor-1 and also activates the IGF1R promoter 7-fold. TRAIL-inducible IGF1R expression requires NF-{kappa}B activation. Consistent with this, ammonium pyrrolidine dithiocarbamate, a pharmacological inhibitor of NF-{kappa}B, blocks TRAIL-induced IGF1R expression, and p65 overexpression increases IGF1R protein levels. In addition, NF-{kappa}B binds a novel TRAIL-responsive element on the IGF1R promoter. Our findings suggest that the biological functions of TRAIL in VSMC extend beyond its role in promoting apoptosis. Thus, TRAIL may play an important role in atherosclerosis by regulating IGF1R expression in VSMC in an NF-{kappa}B-dependent manner.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Tumor necrosis factor (TNF)3-related apoptosis-inducing ligand (TRAIL/Apo2L) is a type II transmembrane protein, which can be proteolytically cleaved at its C terminus to form a soluble ligand. TRAIL is a member of the TNF family of ligands with close homology to Fas ligand. Both the membrane-bound and soluble forms of TRAIL rapidly induce apoptosis in multiple cell lines (1, 2). TRAIL induces an apoptosis signal following engagement with its specific death domain-containing receptors, DR4 and DR5 (3, 4). Studies have identified additional membrane-bound decoy receptors of TRAIL, DcR1 and DcR2, which lack death domains or functional death domains and cannot induce apoptosis (3–7). Osteoprotegerin (OPG) is the only known soluble decoy receptor for TRAIL (8).

Although TRAIL induces apoptosis in transformed cells, soluble TRAIL has little proapoptotic activity in nontransformed cells (9). Furthermore, although TRAIL, DR4, and DR5 are expressed in normal and in diseased atherosclerotic tissue (10–12), the role of TRAIL in atherosclerosis is poorly understood, particularly its effect on vascular smooth muscle cell (VSMC) biology. TRAIL can induce apoptosis of VSMC (12–14). For example, a recent study by Sato et al. (14) demonstrated that plaque-derived CD4+ T cells induce VSMC apoptosis in a TRAIL-dependent manner (14). TRAIL-induced apoptosis of vascular cells may therefore regulate cell turnover in the vessel wall. In contrast to these findings, recombinant human TRAIL administration attenuated development of plaques in diabetic ApoE-null animals and affected cellular composition of plaque lesions by inducing apoptosis of macrophages and increasing VSMC content (15). Although this study suggests that TRAIL may protect VSMC, it is not clear if the increase in VSMC content was a direct effect of TRAIL or due to changes in other cell types.

One of the most potent antiapoptotic growth factors for VSMC is insulin-like growth factor-1 (IGF1), which binds the type 1 receptor (IGF1R). The IGF1R possesses intrinsic tyrosine kinase activity, activating a number of downstream mediators, including phosphatidylinositol 3-kinase and mitogen-activated protein kinase (16, 17). IGF1R activation initiates signaling pathways involved in cell proliferation, survival, differentiation, and transformation (18). Interestingly, dominant negative IGF1R inhibits neointimal formation through suppression of VSMC proliferation, migration, and induction of apoptosis by Akt inhibition (19). Reduction of IGF1R expression in vascular pathologies, such as intimal thickening in atherosclerosis, instent neointimal proliferation after percutaneous coronary interventions, or pulmonary hypertension, might be beneficial given that down-regulation of IGF1R expression correlates with increased sensitivity to apoptosis in VSMC (20, 21). In contrast, human plaque-derived VSMC have reduced IGF1R expression and a subsequent defect in IGF1R-dependent survival (21). This may be potentially detrimental in advanced atherosclerosis, given that apoptosis of VSMC can lead to plaque vulnerability and rupture (22), processes that can precipitate myocardial infarction and sudden death.

We therefore examined the direct effects of soluble TRAIL on VSMC and its role in IGF1R signaling. We show that TRAIL promotes VSMC proliferation at low concentrations. The prosurvival and proproliferative effects of TRAIL on VSMC are IGF1R-dependent, and TRAIL up-regulates IGF1R via NF-{kappa}B-signaling. We have also identified a novel NF-{kappa}B site on the IGF1R promoter responsible for TRAIL-induced IGF1R expression and subsequent proliferation of VSMC.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture—Primary human VSMC were isolated from the aortas of patients undergoing cardiac transplantation under informed consent with ethical approval from the Huntingdon Local Ethics Committees. Human cells were cultured in Dulbecco's modified Eagle's medium (Sigma) supplemented with 20% fetal calf serum, and WKY12-22 rat VSMC were cultured in Waymouth's medium (Invitrogen) with 10% fetal calf serum. Additional supplements included penicillin (10 units/ml), streptomycin (10 µg/ml), and L-glutamine (1 mM). Cells were maintained at 37 °C in a humidified atmosphere of 5% CO2. Human VSMC were not used beyond passage 10. Unless specified, WKY12-22 VSMC were used in all transfection experiments and luciferase assays. WKY12-22 cells were seeded into 6-well titer plates and at 40% confluence were starved for 24 h. The following day, the cells were transfected in serum-free medium with the indicated constructs and 10 ng/well pRL-SV40 using FuGENE6 transfection reagent (Roche Applied Science). TRAIL was added at the same time as transfection. For transfections involving the NF-{kappa}B reporter (pNF-{kappa}B-Luc), TNF{alpha} was added 24 h after the transfection. The cells were stimulated for a further 24 h prior to harvest. Luciferase activity was quantified using the Dual Luciferase assay system (Promega). Luciferase activity was normalized to the internal control pRL-SV40.

Immunohistochemistry—Antibodies to TRAIL (1:100 dilution; R&D Systems MAB375), IGF1Rβ (1:100 dilution; Santa Cruz Biotechnology sc-713), proliferating cell nuclear antigen (1:50 dilution; Dako M879), active caspase-3 (1:50 dilution; BD Pharmingen 557035), and smooth muscle-{alpha}-actin (1:25 dilution, Novocastra ASM-1) were employed on consecutive paraffin sections of formalin-fixed aortocoronary saphenous vein bypass grafts with angiographic luminal stenosis of >75% explanted at redo operation at St. Vincent's Hospital (Sydney, Australia). Informed consent was obtained from each patient. The study protocol conforms to the ethical guidelines of the 1975 Declaration of Helsinki and was approved by the Human Research Committee of St Vincent's Hospital Sydney. Prior to staining, deparaffinized sections were treated with 3% hydrogen peroxide (peroxidase blocking) and boiled in citrate buffer, pH 6.0, to retrieve antigenicity. The standard avidin-biotin complex immunoperoxidase technique was used (23). After washing in Tris-buffered saline, pH 7.6, sections were incubated in the primary antibody for 60 min, followed by incubation with the appropriate secondary antibody (horse anti-mouse, vector BA-2000 or goat anti-rabbit, vector BA-1000) for 20 min, and finally with avidin-biotin complex (Elite Vector PK-6100) for 30 min. Immunogenicity was visualized by treatment in 3,3'-diaminobenzidine solution for 2 min, which produced brown coloration. Sections were counterstained with Mayer's hematoxylin. As a negative control, the primary antibody was omitted, or the sections were treated with the immunoglobulin fraction of suitable nonimmune serum as a substitute for the primary antibody. No positive staining was observed in any of the negative control sections (data not shown). Human plaque tissue was obtained after informed consent with ethical approval from the Huntingdon or Cambridge Local Ethics Committees, respectively. Double labeling was performed as described by R&D Systems using the R&D cell and tissue kit, where the substrate was 5-bromo-4-chloro-3-indolyl phosphate, and for smooth muscle {alpha}-actin (SMA) the substrate vector was 3,3'-diaminobenzidine. TRAIL antibodies (1:25 dilution; R&D Systems) and SMA (1:500 dilution; DAKO) were used in this assay.

Plasmid Constructs—The rat IGF1R promoter construct -476/+640 and -188/+640 was kindly provided by Haim Werner (Tel-Aviv University, Israel). pRL-SV40 was purchased from Promega. pebgNLS and pebgSp1 were obtained from Gerald Thiel (Institute for Genetics, University of Cologne). mNF-{kappa}B-476/+640 IGF1R was constructed using the QuikChange mutagenesis kit (Stratagene) and oligonucleotides (mutation in boldface type): F-mNF-{kappa}B, 5'-GACTCCAAAAATGAATGCCTTGGGCGCCG-3'; R-mNF-{kappa}B, 5'-CGGCGCCCAAGGCATTCATTTTTGGAGTC-3'. pNF-{kappa}B-Luc was purchased from Stratagene.

Proliferation and BrdUrd Incorporation Assays—Human VSMC were seeded into a 96-well titer plate (4000 cells/well). The following day, cells were starved for 24 h followed by the addition of human recombinant TRAIL (R&D Systems) for a further 24 h in serum-free medium. Where indicated, recombinant human IGF1 (Pepro Tech) was used as a positive control. Cells were harvested with trypsin/EDTA, and trypan blue exclusion or Coulter counting was utilized to analyze viable cells. For BrdUrd incorporation studies, the Cell Proliferation enzyme-linked immunosorbent assay system (Roche Applied Science) was used. Briefly, BrdUrd (10 µM) was added to the cells 24 h prior to harvest. Cells were fixed, incubated with anti-BrdUrd for 90 min, and washed in PBS, and immune complexes were detected by substrate reaction at A450. Absorbance correlates directly with DNA synthesis. For antisense (AS) and sense (S) assays targeting the IGF1R (24), human VSMC were starved in serum-free medium for 6 h prior to transfection with AS or S using FuGENE6. The cells were arrested for a further 18 h. The following day, a second transfection with AS and S was performed in either TRAIL-containing medium (1 ng/ml, for proliferation assays) or DMEM containing 20% fetal calf serum (for protein expression). The cells were harvested 24 h later for cell counting or Western blotting. Sequences used were as follows: AS, 5'-TCCGGAGCCAGACTTCATTC-3';S, 5'-GAATGAAGTCTGGCTCCGGA-3' (24). In experiments involving TRAIL receptors, neutralizing antibodies to DR4 (Abcam), DcR1 (R&D Systems), DcR2 (R&D Systems), and OPG (R&D Systems) at the indicated concentrations were added at the same time as recombinant TRAIL stimulation at 1 ng/ml. For DR5, human recombinant DR5-Fc (Alexis Chemicals) was used in proliferation assays. An IgG control antibody or recombinant human IgG Fc were used as controls. Cells were harvested for counting 24 h later.


Figure 1
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  		FIGURE 1.
TRAIL at low concentrations stimulates proliferation of human VSMC without inducing apoptosis. A, TRAIL increases total cell numbers and proliferation of VSMC at 24 h, as assayed by Coulter counting total cell counts (left) and BrdUrd incorporation (right). *, p < 0.05 compared with serum-free medium (SFM) control. Recombinant human IGF1 was used as a positive control. B, TRAIL at 0.1–100 ng/ml does not induce apoptosis of human VSMC. Flow cytometry of annexin V-positive/propidium iodide-negative VSMC treated with the indicated concentrations of TRAIL for 24 h. *, p < 0.05 compared with 0 ng/ml TRAIL. C, TRAIL at 400 ng/ml inhibits human VSMC proliferation in 24 h as assayed by Coulter counting total cell counts (left) and BrdUrd incorporation (right). *, p < 0.05 compared with serum-free medium control. Results are means ± S.D. (n = 3).

 
Flow Cytometry for Annexin V/Propidium Iodide (PI) Staining—At 60–70% confluence, human VSMC were starved for 24 h followed by the addition of TRAIL at the indicated concentrations for a further 24 h. Cells were harvested as previously described (25). Annexin V staining was analyzed by flow cytometry within 1 h. Results are expressed as annexin V staining as a percentage of the total cell population.

Western Blot Analysis—Human VSMC were treated with TRAIL for 24 h after serum starvation for 24 h. For experiments involving ammonium pyrrolidine dithiocarbamate (APDC; Calbiobiochem), APDC was added to cells at the same time as TRAIL treatment. The cells were harvested using radioimmunoprecipitation buffer (150 mM NaCl, 50 mM Tris-HCl, pH 7.5, 1% deoxycholate, 0.1% SDS, 100% Triton X-100) or SDS-sample buffer, together with a mixture of protease inhibitors (Sigma). Proteins were resolved by 8% SDS-PAGE and transferred onto Immobilon-P membranes (Millipore). Membranes were blocked overnight containing 5% skimmed milk and 0.05% Tween 20. IGF1Rβ-chain was detected using a rabbit polyclonal antibody (1:2500; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), Sp1 was detected using a rabbit polyclonal antibody (1:1000; Santa Cruz Biotechnology), and β-actin was detected using a mouse monoclonal antibody (1:30,000; Sigma). Primary antibodies were detected with horseradish peroxidase-conjugated secondary antibodies and visualized by chemiluminescence (GE Healthcare).

Real Time Polymerase Chain Reaction—Total RNA was isolated using STAT-60 (Tel-Test, Inc., Friendswood, TX) as described by the manufacturer. cDNA was generated from 1–5 µg of total RNA and analyzed using the Rotor-Gene 3000 (Corbett Research) by absolute quantification. Taqman expression assay systems (Applied Biosystems) for human IGF1R, DR4, DR5, DcR1, DcR2, OPG, and β-actin were used. Values were normalized to β-actin.

Electrophoretic Mobility Shift Assays (EMSA)—Human recombinant NF-{kappa}B p50 and p65 were purchased from Promega Corp. and Active Motif, respectively. Nuclear extracts were prepared as previously described (25). In binding reactions, recombinant protein or nuclear extract was incubated at room temperature for 20 min in binding buffer (10 mM Tris, pH 7.5, 50 mM NaCl, 1 mM DTT, 1 mM EDTA, and 5% glycerol), together with 1 µg of poly(dI-dC) and the indicated 32P-double-stranded oligonucleotides in a total volume of 20 µl. In supershift assays, extracts were incubated with antibody for 15 min, prior to the addition of the probe. Samples were resolved by 6% nondenaturing polyacrylamide gel electrophoresis and visualized by autoradiography. Oligonucleotide sequences were as follows: (i) NF-{kappa}B consensus annealed oligonucleotide purchased from Santa Cruz Biotechnology (forward oligonucleotide, 5'-ACTTGAGGGGACTTTCCCAGGC-3'; reverse oligonucleotide, 5'-CGGACCCTTTCAGGGGAGTTGA-3'); (ii) NF-{kappa}B IGF1R oligonucleotide F-NF-{kappa}B IGF1R (5'-GACTCCAAAAAACAATGCCCCGGGCGCCG-3') and R-NF-{kappa}B IGF1R (5'-CGGCGCCCGGGGCATTGTTTTTTGGAGTG 3'); (iii) mutant NF-{kappa}B IGF1R (mutation in boldface type), F-mNF-{kappa}B IGF1R (5'-GACTCCAAAAATGAATGCCCCTTGCGCCG-3') and R-mNF-{kappa}B IGF1R (5'-CGGCGCAAGGGGCATTCATTTTTGGAGTG-3').


Figure 2
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  		FIGURE 2.
TRAIL stimulates human VSMC proliferation via DR4 and DcR1. A, expression of TRAIL, DR4, DR5, DcR1, DcR2, and OPG mRNA in normal human VSMC as determined by quantitative PCR. Expression of TRAIL and its receptors were normalized to β-actin. Results are means ± S.E. (n = 3). B, DR4- and DcR1-neutralizing antibodies inhibit TRAIL-induced proliferation of VSMC. Cells were starved for 24 h, followed by stimulation with TRAIL at 1 ng/ml. Neutralizing antibodies or Fc proteins to TRAIL receptors (0.1 ng/ml) were added at the same time as TRAIL treatment. Cells were harvested for counting by Coulter counter 24 h later. Results are means ± S.D. (n = 3). #, p < 0.05 compared with serum-free medium (SFM) control. *, p < 0.05 compared with irrelevant control.

 


Figure 3
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  		FIGURE 3.
TRAIL is expressed in atherosclerotic tissue. A, TRAIL is expressed in saphenous vein bypass sections in regions of VSMC proliferation. Sections were stained for smooth muscle-{alpha}-actin (SMA), TRAIL, proliferating cell nuclear antigen (PCNA), active caspase-3, and IGF1R. Top, x200 magnification; bottom, x400 magnification. B, TRAIL is expressed in VSMC of human atherosclerotic tissue. Blue, TRAIL expression; brown, SMA expression. The arrows represent double-labeled cells. Results are representative of at least two independent determinations. Top, x100 magnification; bottom, x600 magnification.

 
Statistics—Data were analyzed using Microsoft Excel. Data were assessed for significance by Student's t test. Comparisons between three or more unpaired groups were made using analysis of variance. p < 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
TRAIL at Low Concentrations Stimulates Proliferation via DR4 and DcR1 without Inducing Human VSMC Apoptosis—We first examined the direct effects of soluble TRAIL on human VSMC. TRAIL stimulated human VSMC cell counts within 24 h with a peak concentration at 1 ng/ml compared with cells in serum-free medium (Fig. 1A, left). Consistent with these findings, increased BrdUrd incorporation was also observed, again with a peak effect at 1 ng/ml (Fig. 1A, right), suggesting that TRAIL stimulates proliferation of human VSMC. Interestingly, TRAIL-stimulated proliferation appears biphasic with only modest effects at higher concentrations. In addition, TRAIL treatment between 0.1 and 100 ng/ml did not induce apoptosis of human VSMC compared with untreated VSMC as determined by annexin V and PI staining (Fig. 1B). A significant increase in apoptosis was only observed at TRAIL concentrations of 400 ng/ml (Fig. 1B). These findings were supported by reduced proliferation of VSMC exposed to 400 ng/ml (Fig. 1C).


Figure 4
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  		FIGURE 4.
TRAIL regulates serum-inducible IGF1R expression and IGF1R-dependent proliferation of VSMC. A, Western blotting demonstrating that a neutralizing antibody to TRAIL blocks serum-inducible IGF1R protein expression in human VSMC. The isotype control antibody at the same concentration has minimal effect. β-actin demonstrates unbiased loading. The intensity of IGF1R expression relative to β-actin was determined by densitometry (bottom). Results are means ± S.D. (n = 3). *, p < 0.05 compared with isotype control. B, TRAIL-induced proliferation of human VSMC is blocked by AS (antisense) oligonucleotides targeting the IGF1R. Cells were starved for 6 h, followed by initial transfection with AS and S (sense IGF1R) oligonucleotides. The following day, a second transfection was performed, together with TRAIL stimulation for a further 24 h. Cells were harvested for counting by Coulter counter or trypan blue exclusion 24 h later. Results are means ± S.E. (n = 3). *, p < 0.05 compared with serum-free medium (SFM) control. #, p < 0.05 compared with S oligonucleotide control. C, Western blot demonstrating inhibition of IGF1R expression by AS in human VSMC compared with the S oligonucleotide control (0.25 µM). β-Actin (bottom) demonstrates unbiased loading. The results are representative of at least two independent determinations.

 
Consistent with a direct effect of TRAIL on VSMC, we found that TRAIL, DR4, DR5, DcR1, DcR2, and OPG mRNA are all expressed in human VSMC, although at varying levels (Fig. 2A). To investigate which TRAIL receptor is responsible for the proproliferative effect of soluble TRAIL, VSMC were incubated with neutralizing antibodies or recombinant Fc protein to DR4, DR5, DcR1, DcR2, and OPG or an irrelevant control. Neutralizing DR4 and DcR1 antibody inhibited TRAIL-mediated proliferation at 0.1 µg/ml concentration, an effect not observed by the control, or by DR5, DcR2, and OPG (Fig. 2B). These findings illustrate that TRAIL at low concentrations stimulates proliferation via DR4 and DcR1, without inducing apoptosis of VSMC.

TRAIL Is Expressed in Saphenous Vein Bypass Sections in Regions of VSMC Proliferation and IGF1R Expression—To examine whether TRAIL is present in human vessels and to analyze any association with VSMC proliferation, we examined human aortocoronary saphenous vein bypass grafts showing graft failure for TRAIL by immunohistochemical analysis (Fig. 3A). Strong SMA staining was observed in the intima at sites of graft stenosis, implying high VSMC content (Fig. 3A). TRAIL was expressed in regions that were SMA-positive (Fig. 3A). Consistent with this, TRAIL and SMA double-labeling was also evident in human atherosclerotic tissue (Fig. 3B). TRAIL also co-localized with proliferating cell nuclear antigen (Fig. 3A), a marker of proliferation, and consistent with our in vitro findings, TRAIL staining did not correlate with increased apoptosis, as demonstrated by immunohistochemistry for active caspase-3 (Fig. 3A). The IGF1R is important for proliferation of many cell types, including VSMC (27). TRAIL expression also co-localized in part with that of the IGF1R (Fig. 3A). Thus, TRAIL is expressed in regions of VSMC proliferation in human disease and co-localizes with increased IGF1R expression.

TRAIL Is Important for Inducible IGF1R Expression and IGF1R-dependent Proliferation of VSMC—To determine if TRAIL is important for IGF1R expression, serum-inducible IGF1R expression in VSMC was evaluated following treatment with a neutralizing antibody directed to TRAIL. TRAIL neutralizing antibody inhibited serum-inducible IGF1R expression of human VSMC compared with the isotype control (Fig. 4A). In addition, TRAIL-inducible VSMC proliferation was blocked by AS oligonucleotides targeting the IGF1R (Fig. 4B). In contrast, the S control had no effect (Fig. 4B). Western blotting for IGF1R expression in human VSMC confirmed the inhibitory effect of AS on IGF1R expression (Fig. 4C). These findings suggest that TRAIL is produced by VSMC in an autocrine or paracrine manner and that TRAIL-induced proliferation of VSMC is dependent on the IGF1R.

TRAIL Induces IGF1R Gene Expression—To examine whether TRAIL directly regulates IGF1R expression in VSMC, we treated human VSMC with TRAIL for 24 h and performed quantitative PCR for IGF1R mRNA. TRAIL induced IGF1R mRNA expression over a range of concentrations (Fig. 5A), with a peak at 1 ng/ml, the same peak concentration that induced VSMC proliferation. IGF1-stimulated cells were used as a positive control (Fig. 5A). TRAIL, at the lower concentrations (0.1–1 ng/ml), also stimulated IGF1R protein production as assessed by Western blot, which was comparable with levels achieved by serum (Fig. 5B). TRAIL also increased IGF1R promoter activity (Fig. 5C), again with a peak at 1 ng/ml, suggesting that TRAIL regulates IGF1R transcription. These findings taken together demonstrate a positive effect of TRAIL on IGF1R expression in VSMC.

TRAIL-inducible IGF1R Does Not Involve Sp1—To examine the molecular and transcriptional mechanisms of TRAIL-induced IGF1R expression, we used deletion constructs of the IGF1R promoter previously described (28). Whereas -476/+640 of the IGF1R promoter was activated upon treatment with TRAIL at 1 ng/ml, -188/+640 failed to respond to TRAIL treatment (Fig. 6A). Within this region (-476/-188) there are six putative Sp1 elements (29). We therefore examined the effect of dominant negative Sp1 (30) on TRAIL-induced IGF1R expression. Basal expression of IGF1R protein was reduced in cells transiently expressing dominant-negative Sp1, consistent with previous reports (29) (Fig. 6C). Overexpression of dominant-negative Sp1 increased Sp1 protein levels compared with the backbone control (Fig. 6B). Interestingly, TRAIL-induced IGF1R protein expression in VSMC was not blocked by this mutant construct (Fig. 6C). Furthermore, dominant negative Sp1 overexpression did not affect TRAIL-induced IGF1R promoter activity (data not shown). These findings taken together suggest that TRAIL-inducible IGF1R expression does not require Sp1.


Figure 5
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  		FIGURE 5.
TRAIL induces IGF1R gene expression and promoter activity. A, IGF1R mRNA expression of human VSMC treated with the indicated concentrations of TRAIL for 24 h as determined by quantitative PCR. IGF1 was used as a positive control, and results were normalized to β-actin. Results are mean ± S.D. (n = 3). *, p < 0.05 compared with untreated control. B, Western blot for IGF1R protein expression in human VSMC treated with TRAIL for 24 h. β-Actin demonstrates unbiased loading. C, IGF1R promoter activity in WKY12-22 rat VSMC transfected with -476/+640 IGF1R promoter-luciferase reporter (1 µg/well of a 6-well titer plate) and simultaneously treated with TRAIL for 16 h as determined by the Dual Luciferase assay system. Results are mean ± S.D. (n = 2). *, p < 0.05 compared with untreated control.

 


Figure 6
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  		FIGURE 6.
TRAIL-inducible IGF1R does not involve Sp1. A, IGF1R promoter activity in WKY12-22 rat VSMC treated with TRAIL. Cells were starved for 24 h prior to transient transfection with -476/+640 and -188/+640 IGF1R promoter-luciferase reporter (1 µg/well of a 6-well titer plate), simultaneously treated with TRAIL (1 ng/ml) for a further 16 h as determined by the Dual Luciferase assay system. Results are mean ± S.D. (n = 2). *, p < 0.05 compared with untreated control. B, Western blotting to demonstrate overexpression of pebgSp1 (dominant negative Sp1) or its backbone control (pebgNLS). 3 µg of indicated constructs were transfected into WKY12-22 rat VSMC for 24 h using FuGENE6. β-Actin demonstrates unbiased loading. C, Western blotting for IGF1R expression in WKY12-22 rat VSMC transfected with dominant-negative Sp1. Cells were starved for 24 h prior to transient transfection with 3 µg of pebgSp1 or its backbone control for 24 h in a 6-well titer plate. Cells were treated with TRAIL (1 ng/ml) at the same time of transfection. These findings are representative of at least two independent determinations.

 
TRAIL Regulates IGF1R Expression via NF-{kappa}B—TRAIL activates NF-{kappa}B in endothelial cells (31). Consistent with these findings, TRAIL activated NF-{kappa}B in VSMC (Fig. 7A). Using an NF-{kappa}B consensus oligonucleotide for EMSA analysis, TNF{alpha} induced multiple bands, which could be supershifted with antibodies to p65 (RelA) and p50, components of NF-{kappa}B (Fig. 7A). Three nucleoprotein complexes were induced after TRAIL treatment at 1 ng/ml, also common to TNF{alpha}-treated cells (Fig. 7A, double-headed arrows). Furthermore, TNF{alpha} activated an NF-{kappa}B reporter vector (p-NF-{kappa}B-Luc), which was blocked by APDC, a pharmacological inhibitor of NF-{kappa}B (Fig. 7B). EMSA for NF-{kappa}B binding was performed with nuclear extracts of VSMC exposed to TRAIL at multiple concentrations. TRAIL increased NF-{kappa}B DNA binding at low concentrations (0.1–10 ng/ml) but not at the subapoptotic 100 ng/ml concentration (Fig. 7C). TRAIL at 1 ng/ml also activated p-NF-{kappa}B-Luc. In contrast, 400 ng/ml TRAIL did not (Fig. 7D). Furthermore, APDC blocked TRAIL-inducible IGF1R protein expression (Fig. 7E), suggesting that NF-{kappa}B plays a role in TRAIL-induced up-regulation of IGF1R. The role of NF-{kappa}B was further confirmed by transient transfection of the p65 component of NF-{kappa}B, which increased IGF1R expression in VSMC, whereas the backbone control, pcDNA3, had no effect (Fig. 7F). Taken together, these findings suggest that TRAIL activates NF-{kappa}B and that NF-{kappa}B regulates IGF1R expression in VSMC.


Figure 7
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  		FIGURE 7.
TRAIL regulates IGF1R expression via NF-{kappa}B. A, TRAIL and TNF{alpha} increase NF-{kappa}B DNA binding using an NF-{kappa}B consensus probe as demonstrated by EMSA. Human VSMC were starved for 24 h followed by TRAIL (1 ng/ml) and TNF{alpha} (500 units/ml) addition for 30 min. Nuclear extracts were prepared as described under "Experimental Procedures." S, NF-{kappa}B supershifts, {alpha}, denotes antibody. B, TNF{alpha} activates pNF-{kappa}B-Luc reporter activity, which is inhibited by APDC. WKY12-22 rat VSMC were starved for 24 h. The cells were then transfected with 1 µg of pNF-{kappa}BLuc and 10 ng of pRL-SV40 per well (6-well titer plate). The following day, 10 µM APDC was added 1 h prior to 10 ng/ml TNF{alpha}. Cells were stimulated with TNF{alpha} for 24 h and then assayed as determined by the Dual Luciferase assay system. Results are mean ± S.D. (n = 3). *, p < 0.05 compared with untreated control. C, TRAIL induces NF-{kappa}B DNA binding using an NF-{kappa}B consensus probe as demonstrated by EMSA. Human VSMC were starved in serum-free medium (SFM) for 24 h followed by the addition of TRAIL at multiple concentrations (ng/ml) for 30 min. Nuclear extracts were prepared as described under "Experimental Procedures." The arrows represent increased NF-{kappa}B binding. D, TRAIL at 1 ng/ml activates pNF-{kappa}B-Luc reporter activity. This increase in luciferase activity is no longer observed with the addition of 400 ng/ml TRAIL. WKY12-22 rat VSMC were starved for 24 h. The cells were then transfected with 1 µg of pNF-{kappa}B-Luc and 10 ng of pRL-SV40 per 6-well titer plate. TRAIL was added at the same time as transfection. 24 h later, the cells were harvested and assayed as determined by the Dual Luciferase assay system. Results are mean ± S.D. (n = 3). *, p < 0.05 compared with untreated control. E, APDC at 10 µM blocks TRAIL-induced IGF1R protein expression as determined by Western blotting for the IGF1R. WKY12-22 rat VSMC were starved for 24 h, followed by TRAIL stimulation at 1 ng/ml for 24 h. APDC at various concentrations was used. β-Actin demonstrates unbiased loading. F, Western blotting for the IGF1R with cells transiently overexpressing p65 (RelA) NF-{kappa}B. WKY12-22 rat VSMC were starved for 24 h followed by transient transfection with pcDNA3 and p65 (3 µg/well in a 6-well titer plate) for a further 24 h. β-Actin demonstrates unbiased loading. These findings are representative of at least two independent determinations.

 
Identification of a Functional NF-{kappa}B Site on the IGF1R Promoter—By sequence analysis, we identified a 100% putative consensus (RRRCWN0–13WGYY) NF-{kappa}B site between -476 and -188 of the IGF1R promoter at positions -325/-315 (5'-GGGCATTGTTT-3') relative to the Inr element. To confirm that NF-{kappa}B can bind the IGF1R promoter in VSMC, we performed EMSA using an oligonucleotide that contains this site and recombinant NF-{kappa}B p50. NF-{kappa}B p50 was able to bind this region of the promoter as demonstrated by EMSA (Fig. 8A, left). Recombinant p65 also bound the IGF1R promoter although with much lesser affinity (data not shown). Nuclear extracts of human VSMC treated with TRAIL formed an inducible complex that was supershifted with antibodies directed to NF-{kappa}B p50 and p65 (Fig. 8B), suggesting that this complex contains both p50 and p65 NF-{kappa}B. The specificity of the inducible complex was also demonstrated by cold competition assays using cold excess NF-{kappa}B consensus oligonucleotide (data not shown). To assess the functionality of the NF-{kappa}B site, a mutation was introduced into the NF-{kappa}B-IGF1R oligonucleotide and -476/+640 (mNF-{kappa}B-476/+640). NF-{kappa}B binding was no longer supported with this mutant oligonucleotide (Fig. 8A, right). Furthermore, TRAIL-induced activation of the IGF1R promoter was no longer observed with construct mNF-{kappa}B-476/+640 compared with wild type (Fig. 8C). These findings demonstrate that TRAIL-inducible NF-{kappa}B binds the IGF1R promoter and regulates IGF1R expression in VSMC.


Figure 8
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  		FIGURE 8.
Identification of a functional NF-{kappa}B site on the IGF1R promoter. A, recombinant p50 NF-{kappa}B binds the putative NF-{kappa}B site on the IGF1R promoter as determined by EMSA (NF-{kappa}B IGF1R oligonucleotide). The mutant NF-{kappa}B (mNF-{kappa}B) IGF1R oligonucleotide does not bind p50 NF-{kappa}B. 1 and 3 µl of human recombinant p50 NF-{kappa}B were used. B, human VSMC treated with TRAIL (1 ng/ml) induce NF-{kappa}B binding to the IGF1R promoter as determined by EMSA. S, denotes supershift, {alpha}, denotes antibody. C, IGF1R promoter activity in WKY12-22 rat VSMC co-transfected with -476/+640 IGF1R promoter-luciferase reporter and mNF-{kappa}B -476/+640 IGF1R promoter-luciferase reporter (1 µg/well of a 6-well titer plate) as determined with the Dual Luciferase assay system. Cells were starved for 24 h prior to transfection and stimulation with TRAIL (1 ng/ml) for a further 16 h. Results are mean ± S.E. (n = 3). *, p < 0.05 compared with the untreated control.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
TRAIL is a promising cytokine in cancer therapy and has been implicated in the innate immunosurveillance against malignancies. Although it does not demonstrate unwanted systemic toxicity so far, little is known about its physiological role in vivo or its potential side effects, especially in the vascular system. We have found that TRAIL has proliferative effects on VSMC induced by NF-{kappa}B-dependent up-regulation of the IGF1R, beyond its tumoricidal activity. This is the first demonstration of the positive regulation of the IGF1R by TRAIL in any cell type. The effects of TRAIL are significant, since TRAIL-induced proliferation at 1 ng/ml is equivalent to serum and IGF1, a known mitogen. The significance is also demonstrated by the reduction of IGF1R protein expression by ~70% following the addition of a neutralizing antibody to TRAIL. Furthermore, TRAIL at 1 ng/ml induces IGF1R mRNA stronger than IGF1 itself (10-fold) and increases IGF1R promoter activity by 7-fold. Indeed, the proproliferative effects that occur at 1 ng/ml are 400 times less than the proapoptotic effects observed at 400 ng/ml. Consistent with this, TRAIL expression in vivo in sections of saphenous vein bypass grafts co-localizes with proliferating VSMC and the IGF1R but not cell death (active caspase-3). These findings suggest that TRAIL promotes VSMC proliferation in vivo.

TRAIL-induced apoptosis of VSMC has been described (12, 14). In our hands, soluble TRAIL did not induce apoptosis of VSMC even at 100 ng/ml, consistent with our in vivo observations of little or no active caspase-3 staining in saphenous vein bypass sections expressing TRAIL. In contrast, a significant increase in apoptosis following TRAIL treatment in VSMC was only observed at 400 ng/ml, where NF-{kappa}B activation is no longer observed. Interestingly, others have reported 50% killing of VSMC by recombinant human TRAIL at the same concentration (14). We only observed an apoptosis rate of 13%. This may be due to the different techniques employed, since we used flow cytometry compared with nuclear fragmentation/condensation as assayed by 4',6-diamidino-2-phenylindole (14).

Proliferation induced by TRAIL has been reported in multiple cell types, including leukemia cells, endothelial cells, VSMC, eosinophils, and fibroblasts (32–36). We found that at 1 ng/ml, TRAIL induces NF-{kappa}B activation, IGF1R expression, and VSMC proliferation. In contrast, higher TRAIL concentrations did not activate NF-{kappa}B; nor did they increase IGF1R expression and VSMC proliferation. To our knowledge, this is the first illustration of a biphasic effect of TRAIL. Although TRAIL-induced apoptosis requires lipid rafts as plasma membrane platforms for death receptor-initiated signals (37), it appears that TRAIL-DISC assembly in the nonraft phase of the plasma membrane leads to inactivation of caspase-8 and activation of NF-{kappa}B and ERK (38). Perhaps at low concentrations of TRAIL (0.1–1 ng/ml), nonrafts mediate the nonapoptotic functions of TRAIL in VSMC (including proliferation), and at high concentrations (400 ng/ml) lipid rafts mediate TRAIL-induced apoptotic signals. However, the mechanism(s) of action for this observed effect by TRAIL in VSMC has not been established.

The proliferative effects of TRAIL in fibroblasts involve DR5 and activation of ERK, p38, and phosphatidylinositol 3-kinase signaling (36). Although all receptors for TRAIL were expressed in VSMC at varying levels, we found that a neutralizing antibody to DR4 and DcR1 was able to block TRAIL-induced VSMC proliferation. Our studies are in contrast to those in fibroblasts (36) and implicate the TRAIL-DR4- and TRAIL-DcR1-mediated pathway in VSMC proliferation. Furthermore, the proliferative effect of TRAIL on VSMC was dependent on IGF1R signaling, since antisense oligonucleotides targeting the IGF1R were able to block TRAIL-induced proliferation of VSMC.

IGFIR expression plays an important role in the survival, proliferation, and migration of VSMC, processes involved in vascular remodeling. Indeed, TRAIL induced IGF1R mRNA, protein, and promoter activity, suggesting that TRAIL regulates IGF1R transcription in VSMC. Although the nonapoptotic functions of TRAIL described in vascular cells are NF-{kappa}B-independent and involve activation of ERK1/2 or Akt pathways (39), in our studies, TRAIL-induced IGF1R expression is NF-{kappa}B-dependent. TRAIL activates NF-{kappa}B in endothelial cells, although with lesser affinity than TNF{alpha} (31). We too saw similar effects of NF-{kappa}B activity in VSMC treated with 1 ng/ml TRAIL and TNF{alpha}. Furthermore, we have defined a novel NF-{kappa}B binding site on the IGF1R promoter, responsive to TRAIL, different from the NF-{kappa}B-occupying AngII-responsive element on the IGF1R promoter previously described (40). In fact, TRAIL-inducible NF-{kappa}B did not bind this latter site (data not shown). In contrast, TRAIL treatment supported NF-{kappa}B binding on the IGF1R promoter at positions -325/-315, which was inhibited by mutation of this NF-{kappa}B consensus site. Subsequent overexpression studies with p65 increased IGF1R expression in VSMC. Furthermore, a pharmacological inhibitor to NF-{kappa}B blocked TRAIL-induced IGF1R expression, confirming a role for NF-{kappa}B in TRAIL-induced signaling of VSMC.

In summary, this study defines a novel role for TRAIL in vascular biology, such that TRAIL has proliferative effects on VSMC that are IGF1R-dependent. By inducing VSMC proliferation, TRAIL may have negative effects in early atherosclerosis or in instent restenosis after percutaneous coronary interventions. On the contrary, TRAIL-induced VSMC proliferation may stabilize the atheromatous plaque and prevent it from rupture in the later stages of atherosclerosis. Given that IGF1R expression is reduced in human atherosclerotic plaques (41) and serum levels of soluble TRAIL are reduced in patients with coronary artery disease (11, 42), a trend also observed in patients with type II diabetes (26), our data suggest that TRAIL-mediated IGF1R expression is an important process in the development of cardiovascular disease.


   FOOTNOTES
 
* This work was supported in part by British Heart Foundation Grant PG/04/005/16497 and RG/04/001 (to M. R. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

2 Supported by Deutsche Forschungsgemeinschaft Grant Ho 1875/5-2 and a grant from the Prof. Dr. A. Schmidtmann Foundation. Back

1 To whom correspondence should be addressed: The Centre for Vascular Research, The University of New South Wales, Sydney, New South Wales 2052, Australia. Tel.: 61-2-9385-8109; Fax: 61-2-9385-1389; E-mail: m.kavurma{at}unsw.edu.au.

3 The abbreviations used are: TNF, tumor necrosis factor; OPG, osteoprotegerin; VSMC, vascular smooth muscle cell; IGF1, insulin-like growth factor-1; IGF1R, insulin-like growth factor type 1 receptor; SMA, smooth muscle {alpha}-actin; BrdUrd, bromodeoxyuridine; AS, antisense; S, sense; APDC, ammonium pyrrolidine dithiocarbamate; EMSA, electrophoretic mobility shift assay; PI, propidium iodide. Back


   ACKNOWLEDGMENTS
 
We thank Nicola Figg for technical expertise in immunohistochemistry.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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