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(The FASEB Journal. 2006;20:1727-1729.)
© 2006 FASEB

Activation of sPLA2-IIA and PGE2 production by high mobility group protein B1 in vascular smooth muscle cells sensitized by IL-1ß

Amandine Jaulmes*, Sylvain Thierry{dagger}, Brigitte Janvier*, Michel Raymondjean* and Vincent Maréchal{dagger},1

UMR Physiologie et Physiopathologie, Université Pierre et Marie Curie, CNRS,
* Bâtiment A, Paris, France; and

{dagger} Centre de recherches biomédicales des Cordeliers, Paris, France

1Correspondence: Centre de recherches biomédicales des Cordeliers, 15 rue de l’Ecole de Médecine, Paris 75270, CEDEX 06, France. E-mail: vincent.marechal{at}snv.jussieu.fr

ABSTRACT

Lipid mediators such as prostaglandin E2 (PGE2) play a central role during atherogenesis as a consequence of inflammation. PGE2 is produced from phospholipids by a cascade of enzymatic reactions involving phospholipase A2 (PLA2), cyclooxygenase (COX), and prostaglandin E synthase (PGES). It is released by several cell types, including vascular smooth muscle cells (VSMCs). Recent work has shown that the secretory PLA2-IIA (sPLA2-IIA), the most abundant isoform of secreted PLA2 in VSMCs, acts as a potent cytokine and activates VSMCs through a positive feedback loop. High mobility group protein 1 (HMGB1), also known as amphoterin, is a ubiquitous protein that plays various roles in the nucleus. HMGB1 is released by necrotic cells and by immune cells in response to various inflammatory mediators and acts as a potent proinflammatory cytokine. The present study investigates the role of HMGB1 in the activation of sPLA2-IIA expression and PGE2 production in VSMCs. Recombinant HMGB1 slightly activated the sPLA2-IIA, COX-2, and mPGES-1 genes but dramatically stimulated these genes in VSMCs that had been incubated with the proinflammatory cytokine IL-1ß for 24 h. This effect was accompanied by significantly increased PGE2 release. Induction of the three known receptors of HMGB1, namely RAGE, TLR-2, and TLR-4, by IL-1ß suggests that proinflammatory cytokines sensitize VSMCs to HMGB1. This provides new insights into the role of HMGB1 in VSMCs, suggesting it may be essential for the progression of atherosclerosis.—Jaulmes, A., Thierry, S., Janvier, B., Raymondjean, M., Maréchal, V. Activation of sPLA2-IIA and PGE2 production by high mobility group protein B1 in vascular smooth muscle cells sensitized by interleukin (IL)-1ß.


Key Words: inflammation • atherosclerosis • VSMCs • sPLA2-IIA

THERE IS NOW considerable evidence indicating that inflammatory processes are involved in the pathogenesis of atherosclerosis. Many macrophages and activated T cells are present in atherosclerotic plaques, and these cells release proinflammatory cytokines and other mediators that act by autocrine and paracrine mechanisms (1) . Vascular smooth muscle cells (VSMCs) also play a central role in atherosclerosis since they proliferate, migrate, and express genes encoding adhesion molecules and proteins involved in proinflammatory reactions and in extracellular matrix (ECM) remodeling (2) .

VSMCs also synthesize lipid mediators that have physiological or pathophysiological actions. These include prostaglandins that contribute to the chronic inflammatory response associated with atherosclerosis. One of them, prostaglandin E2 (PGE2), mediates various functions in vascular biology, including the regulation of vascular tone, thrombocyte function, and inflammatory responses (3 , 4) . PGE2 is produced from arachidonic acid by a cascade of enzymatic reactions that are performed by cyclooxygenase (COX) and PGE synthase (PGES). Arachidonic acid results from the hydrolysis of phospholipids by phospholipase A2 (PLA2). Secretory PLA2-IIA (sPLA2-IIA) is the most abundant isoform of secreted PLA2 in VSMCs (5) . sPLA2-IIA synthesis is stimulated by inflammatory cytokines such as IL-1ß (IL-1ß), IL-6, and tumor necrosis factor-1 (TNF-{alpha}) (6) . Recent work in our laboratory provided evidence that sPLA2-IIA could activate its own production in VSMCs through a positive activation loop (7) . Whereas the genes encoding COX-1, COX-3, cytosolic PGES, and type 2 microsomal PGES (mPGES-2) are constitutively expressed, those for COX-2 and mPGES-1 are regulated by growth factors and cytokines (8 9 10) . PGE2 synthesis is enhanced in atherosclerotic lesions by the induction of the functionally coupled COX-2/mPGES-1 genes (11) . The precise regulation of sPLA2-IIA, COX-2, and mPGES-1 strongly suggests that the synthesis of prostanoids is tightly regulated during atherogenesis.

HMGB1 has been recognized recently as an important proinflammatory cytokine. It is produced by necrotic cells and by specific cells of the immune system exposed to proinflammatory signals (LPS, TNF-{alpha}, IL-1ß) (12) . It interacts with specific receptors including RAGE (receptor for advanced glycation end products), toll-like receptor (TLR)-2, and TLR-4 (13 , 14) . The binding of HMGB1 to its receptors results in the activation of several kinases such as ERK-1/2, p38 MAPK, and c-Jun NH2-terminal kinase, which ultimately leads to the activation of NF-{kappa}B-dependent genes (15 , 16) .

HMGB1 is abundant in the macrophages of atherosclerotic plaques and less so in endothelial cells and intimal VSMCs (17) . Several cell types, including VSMCs, bear HMGB1 receptors (18 19 20 21) . Degryse et al. have shown that HMGB1 induces chemotaxis and cytoskeleton reorganization in VSMCs in vitro and have provided evidence that this effect is mediated at least in part by RAGE (22) . These data suggest that HMGB1 is recognized as a signal by VSMCs. The present report describes the activation by HMGB1 of the genes involved in the production of eicosanoids in VSMCs. This activation was observed after treatment with IL-1ß. Moreover, IL-1ß was proved to induce the expression of RAGE, TLR-2, and TLR-4 genes and resulted in the accumulation of RAGE at the plasma membrane. We conclude that IL-1ß acts in concert with HMGB1 to trigger a regulatory pathway that may amplify inflammation during atherogenesis.

MATERIALS AND METHODS

Cell culture
Rat VSMCs were isolated by enzymatic digestion of thoracic aortic media from male Wistar rats and cultured as described previously (23) . Peripheral blood monocytic cells (PBMC) were isolated from freshly drawn blood of healthy donors by Ficoll-Paque gradient centrifugation (Pharmacia Biotech, Piscataway, NJ, USA). PBMC were maintained in RPMI medium (Life Technologies, Rockville, MD, USA) supplemented with 10% fetal calf serum, 2 mM glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin in a humidified 5% CO2/95% air incubator.

Purification of recombinant human HMGB1 protein (rHMGB1)
rHMGB1 was purified from E. coli BL21 (DE3)pLysS transformed with pET15b-6His-HMGB1. The expression vector encoded the full-length human HMGB1 protein, which is nearly identical to the rat HMGB1, fused to a polyhistidine tag at its NH2 terminus. Protein expression and purification were conducted under native conditions using Ni-NTA resin (Qiagen, Chatsworth, CA, USA) following the manufacturer’s recommendations. Residual RNA and DNA were removed from the column by incubating the resin with a buffer containing 50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, 10 mM ß-mercaptoethanol, pH 8, DNase (Promega, Madison, WI, USA; 1.5 µg /ml), and RNase A (15 µg/ml) for 2 h at 25°C with continuous shaking. rHMGB1 was then eluted with a buffer containing 50 mM NaH2PO4, 300 mM NaCl, 50 mM imidazole, and 10 mM ß-mercaptoethanol, pH 8. Residual LPS was extracted with Triton X-114. Briefly, 1/20 (v/v) Triton X114 was added to the solution containing the recombinant protein. The mixture was incubated for 1 h at 4°C with constant rotation and for 20 min at 37°C. Then the sample was centrifuged at 4500 g for 15 min at room temperature. The supernatant was collected and extensively dialyzed against PBS. Residual LPS was measured with the E-toxate reagent (Sigma, St. Louis, MO, USA) according to the manufacturer’s recommendations.

Electrophorectic mobility shift assays (EMSA)
Hemicatenanes (hcDNA) were prepared and labeled as described in ref 24 . EMSA were carried out as described by Stros et al. (25) .

Cellular fractionation
VSMCs (10x106) were collected by scraping and homogenized (Dounce glass homogenizer, pestle B) in HB buffer (20 mM Tris-HCl, 1 mM EDTA, and 255 mM sucrose, pH 7.5) containing protease inhibitors (Roche Molecular Biochemicals, Nutley, NJ, USA). The homogenates was centrifuged at 16,000 g for 20 min. The resulting pellet was suspended in HB buffer, loaded onto a sucrose cushion (1.12 M sucrose, 20 mM Tris-HCl, 1 mM EDTA), and centrifuged at 100,000 g for 60 min at 4°C in a SW28 rotor. Cytoplasmic membranes were collected, washed in HB buffer, and pelleted by centrifugation at 48,000 g for 45 min at 4°C. The membrane proteins were quantified by Bradford colorimetric assay.

Western blot
Western blot was carried out as described previously (23) with membrane protein (10 µg), rabbit anti-RAGE antibody (Ab) (TEBU, diluted 1:400), rabbit Ab directed against the G{alpha}i3/{alpha} protein (diluted 1:400; antiserum desalted and purified as in ref 26 ) or a rabbit Ab against human HMGB1 (diluted 1:3000) (27) .

RNA extraction and reverse transcription-polymerase chain reaction (RT-PCR)
Total RNA was extracted using the RNAeasy kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer’s instructions. Total RNA (1 µg) was used as a template for reverse transcription. First-strand cDNA synthesis was performed with murine mammary lentivirus RT (Invitrogen, Carlsbad, CA, USA) plus random hexamers according to the manufacturer’s recommendations (Gibco BRL, Gaithersburg, MD, USA). Quantitative RT-PCR was done with the ABsoluteTM QPCR SYBR Green fluorescein mixture (Abgene, Epsom, UK). The total reaction vol was 25 µl containing SYBR Green PCR core reagents, 2.5 ng cDNA, and 300 nM of each specific primer. Amplification was performed on an iCycler (Bio-Rad Laboratories, Hercules, CA, USA). Samples were denatured for 15 min at 95°C and amplified for 40 cycles as follows: denaturation for 15 s at 95°C, annealing for 30 s at the appropriate hybridization temperature (Thyb), and elongation at 72°C for 30 s. Each real-time PCR run included cDNAs in triplicate in parallel with serial dilutions of a cDNA mix tested for each primer pair to generate a standard curve. The values were normalized to an internal control, hypoxanthine-guanine phosphoribosyl transferase (HPRT). This curve was then used to estimate the relative quantity of the relevant mRNA in each sample. The generation of specific PCR products was confirmed by melting curve analysis. The primers used to amplify rat sPLA2-IIA were 5'-GTGACTCATGACTGTTGTTAC-3' (forward) and 5'-CAAAACATTCAGCGGCAGC-3' (reverse) (Thyb=61°C). Those for rat COX-2 were 5'-TGTATGCTACCATCTGGCTTCGG-3' (forward) and 5'-GTTTGGAACAGTCGCTCGTCATC-3' (reverse) (Thyb=62°C); for rat mPGES-1: 5'-CTGCTGGTCATCAAGATGTACG-3' (forward) and 5'-CCCAGGTAGGCCACGGTGTGT-3' (reverse) (Thyb=58°C); for rat TLR-2: 5'GAGGCCAGCCCTGGTCCATG-3' (forward) and 5'-CTCCAGAGACCAGTGCGGCC-3' (reverse) (Thyb=65°C); for rat TLR-4: 5'-TTGCAGTGGGTCAAGGACCAG-3' (forward) and 5'-GCTACAGTGGCTACCACAAGC-3' (reverse) (Thyb=61°C). Finally, primers used to amplify rat HPRT were 5'-CTCATGGACTGATTATGGACAGGAC-3' (forward) and 5'GCAGGTCAGCAAAGAACTTATAGCC-3' (reverse).

Phospholipase A2 assay
Phospholipase A2 activity was measured by a selective fluorometric assay (28) .

TNF-{alpha} and PGE2 quantification
The PGE2 concentration in the cell culture supernatant was measured using a PGE2 enzyme immunoassay kit (Cayman Chemical Co., Ann Arbor, MI, USA) according to the manufacturer’s recommendation. The TNF-{alpha} concentration was measured using the human TNF-{alpha} ELISA development kit (TEBU).

Statistical analysis
Student’s t test was used to compare the means of two treatments performed under the same experimental condition. Two mean values were considered significantly different when P < 0.05.

RESULTS

Purification and characterization of the recombinant HMGB1 protein (rHMGB1)
HMGB1 has been shown to exhibit various proinflammatory activities through interaction with RAGE, TLR2, and/or TLR4 (14) . HMGB1 purified from E. coli has cytokine activity similar to that of its mammalian counterpart (29) . Native as well as recombinant yeast and bacterial HMGB1 has been shown to activate rat VSMCs (22) . Based on these results, recombinant E. coli HMGB1 (rHMGB1) was used in this study. Polyhistidine-tagged rHMGB1 was synthesized in E. coli and purified under native conditions by Ni-NTA metal chromatography. Preliminary experiments indicated that rHMGB1 was associated with bacterial RNA and DNA. The original protocol was modified to remove all bacterial nucleic acid. LPS was also removed by Triton X-114 extraction, and the residual LPS content was < 0.3 pg/µg rHMGB1. The resulting recombinant protein was > 99% pure by SDS-PAGE (Fig. 1 A). Two major polypeptides were isolated, and mass spectrometry analysis indicated that the faster migrating protein resulted from removal of the C-terminal acidic region of HMGB1 (F. Strauss, personal communication). Since the two forms of HMGB1 have similar proinflammatory activities (30) , experiments were conducted with purified fractions that contained both polypeptides.


Figure 1
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  		Figure 1. Biochemical and cytokine activity of rHMGB1. A) HMGB1 was produced in E. coli. The purified protein (rHMGB1) was analyzed by 10% (w/v) SDS-PAGE. Gels were stained with Coomassie blue (lane 1) and Western-blotted (lane 2). B) rHMGB1 DNA binding activity was analyzed by EMSA. rHMGB1 (100 pg) was incubated with labeled hemicatenanes (hcDNA). Complexes were resolved by PAGE and visualized by autoradiography. C) Purified human monocytes were treated with PBS, 100 ng/ml LPS, or 1 µg/ml rHMGB1. Release of TNF-{alpha} into the supernatant was measured after various incubation times.

Before investigating the effect of rHMGB1 on VSMCs, we designed a series of assays with the aim of confirming that rHMGB1 was biochemically and biologically active. The DNA binding ability of rHMGB1 was analyzed by EMSA using hemicatenanes as a probe. Hemicatenated DNA loops (hcDNA) are synthetic DNA structures that bind HMGB1 with extremely high affinity (Kd<1 pM) (24) . Incubation of labeled hcDNA with rHMGB1 resulted in a specific gel retardation (Fig. 1B ). Additional experiments indicated that the rHMGB1-hcDNA complexes could be supershifted with a polyclonal antibody directed against human HMGB1 (data not shown). Native and recombinant HMGB1 have been shown to activate TNF-{alpha} secretion from human monocytes (31) . The proinflammatory activity of rHMGB1 was evaluated by measuring its ability to promote the release of TNF-{alpha} from primary human monocytes. There was a characteristic time-dependent secretion of TNF-{alpha} when primary human monocytes were incubated with rHMGB1 (1 µg/ml) (Fig. 1C ). Monocytes released TNF-{alpha} only when they were incubated with a high concentration (100 ng/ml) of LPS, but not when incubated with low doses (0.3 pg/ml). Complementary experiments indicated that these fractions were as active as human HMGB1 derived from necrotic extracts in promoting TNF-{alpha} release from human monocytes (data not shown).

HMGB1 induces sPLA2-IIA gene expression in VSMCs
Our experiments were conducted on primary cultures of VSMCs isolated from rat aortas. These cells are especially suitable for studies of inflammatory processes since they undergo a phenotypic change in response to proinflammatory cytokines with an increase in the expression of adhesion proteins (vascular cell adhesion molecule-1 and MCP-1), ECM molecules (metalloproteases), and acute-phase enzymes such as sPLA2-IIA and COX-2 (32) .

As sPLA2-IIA plays both direct and indirect roles in the activation of VSMCs, a series of experiments were conducted to investigate the expression of the sPLA2-IIA gene in the presence of increasing doses of rHMGB1. These experiments were performed with VSMCs incubated for 8 or 24 h with rHMGB1. sPLA2-IIA expression was analyzed by RT-PCR. The amounts of sPLA2-IIA RNA were normalized to that of the housekeeping HPRT gene product, an RNA whose concentration is relatively stable under these conditions. As shown in Fig. 2 , there was a modest but significant dose-dependent increase in sPLA2-IIA mRNA in VSMCs incubated for 24 h with 100 ng to 1 µg/ml rHMGB1. Higher concentrations did not lead to greater sPLA2-IIA gene expression (data not shown). Therefore, all subsequent experiments were performed with 1 µg/ml rHMGB1. This concentration is compatible with HMGB1 concentrations that have been measured in vivo in various situations (33 , 34) (M. Bianchi and V. Marechal, unpublished results). Induction of the sPLA2-IIA gene could not be attributed to residual LPS since the amount of sPLA2-IIA mRNA in cells incubated with 2.5 to 250 pg/ml LPS was unchanged (data not shown). These results were reproduced with >10 different VSMCs pools and 5 different preparations of rHMGB1. sPLA2 activity, used as an indicator of sPLA2-IIA release, was also measured in the cell culture supernatant in the presence or absence of rHMGB1. There was a barely detectable increase in sPLA2 activity in VSMCs incubated with 1 µg/ml rHMGB1, even after 24 h (Fig. 3 A).


Figure 2
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  		Figure 2. HMGB1-induced sPLA2-IIA transcription. sPLA2-IIA transcription was measured by reverse transcription/real-time PCR using the expression of the HPRT gene as an internal standard. The results are expressed as fold induction compared with the expression in cells treated with PBS alone. VSMCs were incubated for 24 h with 1, 10, 100, or 1000 ng/ml rHMGB1. The data are means ± SD of one experiment representative of 5 experiments performed in duplicate. *P < 0.05 significantly different from the control. **P < 0.01 significantly different from the control.


Figure 3
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  		Figure 3. Potentiation of sPLA2-IIA gene expression induction in response to HMGB1 in VSMCs treated with IL-1ß. VSMCs were incubated with 10 ng/ml IL-1ß for 24 h, then with 1 µg/ml rHMGB1 for 8 or 24 h. A) sPLA2 activity was measured in the supernatant and normalized to the cell number. The data are means ± SD of one experiment representative of at least 3 experiments. **P < 0.01 significantly different from the control. §§P < 0.01 significantly different from IL-1ß alone. B) sPLA2-IIA transcription was measured by reverse transcription/real-time PCR using HPRT gene expression as the internal standard. The results are expressed as fold induction compared with the expression in cells treated with PBS alone. The data are means ± SD of one experiment representative of at least 3 experiments performed in duplicate. *P < 0.05 significantly different from the control. **P < 0.01 significantly different from the control. §§P < 0.01 significantly different from IL-1ß alone.

Therefore, rHMGB1 activated sPLA2-IIA gene transcription in a dose-dependent manner, but this activation was associated with a moderate increase in sPLA2 activity (Fig. 3A ).

HMGB1 enhances sPLA2-IIA gene expression in IL-1ß-sensitized VSMCs
In the case of vascular inflammation, particularly during atherogenesis, VSMCs are activated by several proinflammatory cytokines, including IL-1ß (35) . As IL-1ß has been shown to increase sPLA2-IIA gene expression in VSMCs in a dose-dependent manner (36) , we wondered whether low doses of IL-1ß might act in concert with HMGB1 to activate sPLA2-IIA expression. To test this hypothesis, VSMCs were cultivated for 24 h with 10 ng/ml IL-1ß, then incubated for another 8 or 24 h with fresh medium containing 10 ng/ml IL-1ß and 1 µg/ml rHMGB1. As expected, IL-1ß alone induced a 4-fold increase in sPLA2 enzymatic activity (Fig. 3A ). More surprisingly, rHMGB1 increased sPLA2 enzymatic activity by up to 10-fold in cells that had first been incubated with IL-1ß. As expected, the induction of sPLA2-IIA enzymatic activity was associated with an increase in sPLA2-IIA gene expression (Fig. 3B ). Whereas IL-1ß alone induced a 7-fold increase in sPLA2-IIA gene expression compared with untreated cells, rHMGB1 induced a 14-fold increase in IL-1ß-treated cells (Fig. 3B ). We conclude from these experiments that the induction of sPLA2-IIA by rHMGB1 was potentiated by IL-1ß and occurred mainly at the transcriptional level.

HMGB1 induces PGE2 release by IL-1ß-treated VSMCs
sPLA2 activity is essential for the production of PGE2 (37) . As rHMGB1 was shown to promote sPLA2-IIA enzymatic activity in IL-1ß-treated VSMCs, we assumed that PGE2 release could also be induced by HMGB1 under similar experimental conditions. We tested this by incubating IL-1ß-treated VSMCs with 1 µg/ml rHMGB1 for 8 or 24 h, then measuring PGE2 in the cell culture supernatant. Incubation in the presence of rHMGB1 alone did not significantly increase PGE2 release, in agreement with the modest effect of rHMGB1 on sPLA2 activity (Fig. 4 A). However, rHMGB1 increased PGE2 release up to 12-fold compared with untreated cells when VSMCs had been incubated with 10 ng/ml IL-1ß for 24 h. Conversely, IL-1ß alone induced only a 3.5-fold increase in PGE2 release. Thus, rHMGB1 dramatically increased the PGE2 released from VSMCs that had been sensitized with IL-1ß.


Figure 4
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  		Figure 4. PGE2 release, COX-2 and mPGES-1 transcription induced by HMGB1 in IL-1ß-treated VSMCs. VSMCs were treated with 10 ng/ml IL-1ß for 24 h, then incubated with 1 µg/ml rHMGB1for 8 or 24 h. A) PGE2 release into the supernatant was quantified by ELISA. The data are means ± SD of one experiment representative of 3 experiments. **P < 0.01 significantly different from the control. §P < 0.05 significantly different from IL-1ß alone. B) Transcriptions of COX-2 and C) mPGES-1 genes were measured by reverse transcription/real-time PCR using the expression of the HPRT gene as an internal standard. The results are expressed as fold induction compared with the expression in cells treated with PBS alone. The data are means ± SD of one experiment representative of 3 experiments performed in duplicate. *P < 0.05 significantly different from the control. **P < 0.01 significantly different from the control. §P < 0.05 significantly different from IL-1ß alone. §§P < 0.01 significantly different from IL-1ß alone.

HMGB1 induces the expression of the COX-2 and mPGES-1 genes in IL-1ß-treated cells
PGE2 accumulation was shown to be associated with the induction of sPLA2-IIA by rHMGB1. However, other enzymes are responsible for the synthesis of PGE2 from arachidonic acid, including cyclooxygenases (COX) and PGE synthases (PGES). Whereas the genes encoding for COX-1, COX-3, cytosolic PGES, and type 2 microsomal PGES (mPGES-2) are constitutively expressed, those for COX-2 and mPGES-1 are regulated by several growth factors and cytokines (38) . Therefore, we asked whether the expression of COX-2 and mPGES-1 genes was affected in response to rHMGB1 in VSMCs treated or not treated with IL-1ß.

Incubating cells with 1 µg/ml rHMGB1 for 24 h did not alter the expression of COX-2 or mPGES-1 genes, whereas IL-1ß alone increased COX-2 transcription 3-fold and mPGES-1 gene transcription 2.5-fold (Fig. 4B, C ). More strikingly, incubation of IL-1ß-treated cells with rHMGB1 dramatically increased COX-2 (11-fold) and mPGES-1 (7-fold) gene expression. These experiments demonstrated that the induction of PGE2 by rHMGB1 in IL-1ß-sensitized VSMCs is associated with the concomitant activation of several genes involved in the synthesis of PGE-2 from arachidonic acid, namely sPLA2-IIA, COX-2, and mPGES-1.

IL-1ß induces the expression of RAGE, TLR-2, and TLR-4
RAGE, one of the main receptors of HMGB1, is both present and functional in VSMCs (22) . Notably, RAGE was shown to be required for HMGB1 to stimulate the migration of VSMCs. Therefore, we hypothesized that IL-1ß treatment might increase RAGE expression and thus explain the greater sensitivity of IL-1ß-treated VSMCs to the action of rHMGB1. Plasma membrane proteins were extracted and analyzed by Western blot to measure the presence of RAGE at the cell membrane. As illustrated in Fig. 5A, IL-1ß stimulated plasma membrane RAGE whereas G{alpha}i3/{alpha} membrane protein concentration was not altered. VSMCs incubated with 10 ng/ml IL-1ß for 24 h had 4- to 5-fold more RAGE at the plasma membrane than did controls.

Recent studies have indicated that the two others receptors for HMGB1—TLR-2 and TLR-4—are present in vascular and airway smooth muscle cells (39 , 40) . We therefore examined the possible influence of IL-1ß treatment on TLR-2 and TLR-4 gene expression in rat VSMCs. As shown on Fig. 5B , IL-1ß enhanced TLR-2 gene expression >2-fold and increased TLR-4 gene expression as well, albeit to a lesser extent.


Figure 5
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  		Figure 5. Induction of HMGB1 receptors by inflammatory cytokines. A) VSMCs were incubated with 10 ng/ml IL-1ß for 24 h. The plasma membrane proteins were extracted and a 10 µg aliquot was subjected to SDS-PAGE and Western blot using a specific anti-RAGE Ab or an anti-G{alpha}i3/{alpha}0 Ab. B) VSMCs were incubated with 10 ng/ml IL-1ß for 24 h. Transcription of the TLR-2 and TLR-4 genes was measured by reverse transcription/real-time PCR using the expression of the HPRT gene as an internal standard. The results are expressed as fold induction compared with the expression in untreated cells. The data are the means ± SD of 3 experiments performed in duplicate. *P < 0.05 significantly different from the control.

Consequently, the greater sensitivity of IL-1ß-treated VSMCs to HMGB1 could be explained at least in part by the induction of three genes encoding for known receptors for HMGB1, namely RAGE, TLR-2, and TLR-4.

DISCUSSION

In many proinflammatory contexts, HMGB1 is secreted by activated cells or passively released by necrotic cells. In atheroma plaques, HMGB1 is especially abundant (17) . Smooth muscle cells are recognized as key actors in atherogenesis (1) . Recent work by Marco Bianchi’s group demonstrated that large amounts of extracellular HMGB1 (~40 pg/mg tissue/24 h) are produced by atherosclerotic plaques but not by normal arteries (M. Bianchi, personal communication). We have demonstrated that HMGB1 activates the expression of sPLA2-IIA, COX-2, and mPGES-1 in VSMCs sensitized with IL-1ß, a cytokine known to play an essential role in the progression of atherosclerotic plaques (Fig. 3) . As shown here, this results in a dramatic increase in the release of PGE2 by VSMCs. We recently demonstrated that sPLA2-IIA is involved in an autocrine activation loop in VSMCs (7) . Taken together these results suggest that HMGB1 activity might be important in atherosclerotic plaques since it could initiate an amplification loop that eventually leads to the gradual accumulation of two potent soluble factors, i.e., sPLA2-IIA and PGE2.

HMGB1 was shown to stimulate the expression of three inducible genes encoding coupled enzymes: sPLA2-IIA, COX-2, and mPGES-1. These three genes are induced by IL-1ß, at least in part through a NF-{kappa}B-dependent pathway (36 , 41 , 42) . As most if not all known ligands of RAGE, TLR-2, and TLR-4 can also activate NF-{kappa}B, it is likely that NF-{kappa}B-dependent pathways are involved in the activation sPLA2-IIA, COX-2, and mPGES-1 by HMGB1.

Our results indicate that IL-1ß acts by increasing relative levels of the three known receptors for HMGB1, namely RAGE, TLR-2, and TLR-4. RAGE was first recognized as a receptor for advanced glycation end products (AGE), the ultimate result of nonenzymatic glycation and the oxidation of protein and lipids. AGE accumulate in the blood plasma of diabetic patients and are thought to be important in diabetic vascular complications (43) .

So far, the effects of AGE and HMGB1 on RAGE function are indistinguishable. Both AGE and HMGB1 stimulate VSMCs chemotaxis and activate NF-{kappa}B (22 , 44 , 45) . Therefore, it is likely that the induction of RAGE by IL-1ß also sensitizes the VSMCs to the action of AGE and thus contributes to vascular diseases in diabetic patients. This hypothesis is reinforced by recent observations indicating that RAGE is overproduced in atherosclerotic plaques from diabetic patients (46) .

Recent work by Degryse et al. has demonstrated that the migration of VSMCs induced by HMGB1 can be attenuated by anti-RAGE antibodies, suggesting that other receptors for HMGB1 are expressed on VSMCs (22) . Our results confirm that TLR-2 and TLR-4 are also expressed in primary rat VSMCs. We found that the expression of TLR-2, and to a lesser extent TLR-4, was stimulated by IL-1ß. These results agree with previous observations. Indeed, Morris et al. provided evidence that expression of the TLR-2 and TLR-4 genes in airway smooth muscle cells is increased by proinflammatory stimuli, including LPS, IL-1ß, and TNF-{alpha} (39) . The role of TLR-2 and TLR-4 might be more important than initially suspected since clinical investigations have demonstrated that TLR-2 and TLR-4 transcripts are 3-fold more abundant in atheroma plaques than in normal arteries (21) .

In conclusion, this work sheds new light on the contribution of HMGB1 to the activation of VSMCs, notably during the inflammation process associated with atherogenesis.

ACKNOWLEDGMENTS

This work was supported by the Centre National de la Recherche Scientifique (CNRS) and the Université Pierre et Marie Curie. A.J. and S.T. were supported by doctoral fellowships from the French Ministère de l’Education Nationale, de la Recherche et de la Technologie. We also thank François Strauss and Claire Gaillard for providing the pET15–6His-HMGB1-pLysS plasmid and Rachel Boniface for technical assistance. The English text was edited by Owen Parkes and Paul Lazarow.

Received for publication December 6, 2005. Accepted for publication March 31, 2006.

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B. Samuelsson, R. Morgenstern, and P.-J. Jakobsson
Membrane Prostaglandin E Synthase-1: A Novel Therapeutic Target
Pharmacol. Rev., September 1, 2007; 59(3): 207 - 224.
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