Autocrine and Paracrine Transcriptional Regulation of Type IIA Secretory Phospholipase A2 Gene in Vascular Smooth Muscle Cells
Objective— The inflammation that occurs during the development of atherosclerosis is characterized by a massive release of sPLA2-IIA (group IIA secretory phospholipase A2) from vascular smooth muscle cells (VSMCs). We have investigated the autocrine function of sPLA2-IIA in rat aortic and human VSMCs.
Methods and Results— We found that the transcription of the endogenous sPLA2-IIA gene increased by adding a cell supernatant containing human sPLA2-IIA proteins. We show that this effect was independent of the sPLA2 activity using sPLA2-IIA proteins lacking enzyme activity. Transient transfections with various sPLA2-IIA rat promoter-luciferase constructs demonstrated that the C/EBP, NK-κB, and Ets transcription factors are involved in the increase in sPLA2-IIA gene transcription. We also found the M-type sPLA2 receptor mRNA in VSMCs, and we showed that the sPLA2-luciferase reporter gene was induced by the specific agonist of the sPLA2 receptor, aminophenylmannopyranoside (APMP), and that this induction was mediated by the same transcription factor-binding sites. Finally, we used a sPLA2-IIA mutant unable to bind heparan-sulfate proteoglycans to show that the binding of wild-type sPLA2-IIA to proteoglycans is essential for the induction of an autocrine loop.
Conclusions— We have thus identified new autocrine and paracrine pathways activating sPLA2-IIA gene expression in rat and human VSMCs.
Phospholipase A2 (PLA2) enzymes (EC 220.127.116.11) hydrolyze ester bonds at the sn-2 position of glyceroacylphospholipids to produce lysophospholipids and nonesterified fatty acids such as arachidonic acid (C20:4 n-6). The latter is further metabolized, contributing to the proinflammatory actions and later resolution.1 PLA2 enzymes are present in most tissues and 4 groups are distinguished by subcellular location, molecular weight, and calcium dependence. One of these groups is secretory PLA2s (sPLA2s). The sPLA2s are extracellular low-molecular-mass (13 to 18 kDa) enzymes that have 6 to 8 disulfide bridges and require millimolar concentrations of Ca2+.2 The isoform sPLA2-IIA is the most abundant sPLA2 in vascular smooth muscle cells (VSMCs).3 Its synthesis is stimulated by inflammatory cytokines such as interleukin 1β (IL-1β), IL-6, and tumor necrosis factor-1α.4 The plasma of patients with various inflammatory diseases, particularly atherosclerosis, contains high concentrations of sPLA2-IIA.5,6 Transgenic mice that overproduce human sPLA2-IIA have confirmed its involvement in atherosclerosis.7 sPLA2-IIA also functions as a messenger, and this latter function can be independent of its enzymatic activity. For example, sPLA2-IIA triggers a signaling cascade in monocytic cells that has features similar to those found in other cell types and seems to be unrelated to its catalytic activity.8,9 The recent identification of membrane proteins binding sPLA2 has highlighted the biological effects of these enzymes. Several studies have shown that sPLA2-IB and sPLA2-IIA act via specific receptors characterized in several species (M-type receptors) to play roles that are independent of their catalytic activity.9–13 Studies on mice lacking the M-type PLA2 receptor have demonstrated the involvement of the sPLA2-IB/sPLA2 receptor system for the production of proinflammatory cytokines during the endotoxic shock.14 Alternatively, the messenger sPLA2-IIA function may be caused by its binding to proteoglycans. VSMCs produce proteoglycans in vitro and in vivo and this production is increased in proliferating cells.15 sPLA2-IIA is anchored to cell surfaces via its C-terminal heparan sulfate proteoglycans (HSPG)-binding domain.16,17 Some of the functions of sPLA2-IIA9,18–19 depend on its binding to proteoglycans, and the sPLA2 enzymatic activity and/or the M-type sPLA2 receptor binding can also be involved.
This study was performed to identify the molecular events responsible for the amplification of sPLA2-IIA gene expression during atherosclerosis. We analyzed the effect of adding soluble human sPLA2-IIA proteins on sPLA2-IIA gene expression in 2 complementary cell culture models of VSMCs. One was VSMCs isolated from rat aortas, previously used to study the interplay of transcription factors involved in the activation of the rat sPLA2-IIA promoter.20,21 The other one was a line of human VSMCs (AU-1 cells), used as a species homologous model. We demonstrate here that sPLA2-IIA has paracrine/autocrine functions, independent of its catalytic activity and involving the PLA2 receptor and/or proteoglycans.
For an expanded online Methods section, please see http://atvb.ahajournals.org.
Aortic Smooth Muscle Cells
Rat VSMCs were isolated from male Wistar rats by enzymatic digestion of thoracic aortic media (method described previously).21 AU-1 cells are a cell line derived from a human aorta. Their morphology and the staining with monoclonal antibodies to α-actin and desmin confirmed the smooth muscle cell phenotype.
Phospholipase A2 Assay
Phospholipase A2 activity was measured using a selective fluorometric assay as described.22
The constructs containing the V5-His tagged human sPLA2-IIA (hsPLA2-IIA) wild-type or mutated were obtained by polymerase chain reaction (PCR) as described online. [−488;+46] sPLA2-luc and mutant constructs were obtained by site-directed mutagenesis as previously described.23
VSMCs Transfections for Tagged Protein Expression and Protein Purification From the Supernatants
70% confluent P100 dishes (Corning) were transfected by lipofectAMINE plus as recommended by the manufacturer (Life Technologies). The tagged proteins present in the supernatants of transfected cells were purified with Ni-NTA agarose nickel-charged beads as recommended by the manufacturer (Quiagen). The elution product was concentrated with acetone and prepared for SDS-PAGE.
RNA Extraction and Reverse-Transcription PCR
Total RNAs were extracted from P100 dishes with the RNeasyMini Kit (Quiagen) and 1 μg was used as template for retrotranscription. RT-PCR was performed as described previously using GAPDH cDNA as internal control.20
VSMCs transfections and luciferase assays were realized as described previously.23
Ni and V5 Depletions of the Supernatants
8 μg of the V5 antibody (In Vitrogen) were incubated 1 night with 50 μL of proteins A/G agarose beads (Santa Cruz). Supernatants were depleted by a 2-hour incubation with these beads or Ni-NTA agarose nickel-charged beads (Quiagen), filtered, and used to induce VSMCs. We checked the good depletion by measuring sPLA2 activity in the supernatants.
hsPLA2-IIA and b3E supernatants were incubated 1 hour at 4°C with 50 μL of heparin-Sepharose (Pharmacia). NaCl was added to 0.4 mol/L final when necessary. After centrifugation, the sPLA2 activities remaining in the supernatants were assayed.
The data represent the mean ± standard deviation (SD) of at least 3 independent experiments. The Student t test was used to compare 2 means of different experimental conditions. Multiple comparisons were performed by 1-way ANOVA followed by the Student-Newman-Keuls comparison procedure. P<0.05 was considered to be significant.
Influence of a Supernatant Containing hsPLA2-IIA Proteins on the sPLA2-IIA Gene Transcription
Secondary cultures of rat VSMCs were transiently transfected with a human sPLA2-IIA V5-His tagged (hsPLA2-IIA) construct (Figure 1A). The supernatant was collected 24 hours later and added onto untransfected cells (Figure 1B). We checked that the hsPLA2-IIA construct was expressed and secreted by VSMCs by revealing tagged-hsPLA2-IIA proteins in supernatants of cells transfected by hsPLA2-IIA construct (Figure 1C). Prostaglandin E2 (PGE2), a final product of the sPLA2 activity, is a major mediator of inflammation and strongly induces sPLA2 gene expression in VSMCs. To eliminate the hypothesis that the observed upregulation depends on the production of PGE2 in the supernatants of transfected cells, we checked that the supernatants were devoid of PGE2 by enzyme-linked immunosorbent assay (ELISA) (data not shown).
Nontransfected cells were incubated for 24 hours with medium alone (control), medium containing effectors known to induce sPLA2-IIA (IL-1β plus forskolin),21 supernatant of control cells transfected with a Bluescript-KS plasmid (control supernatant), and supernatant of cells transfected with hsPLA2-IIA (hsPLA2-IIA supernatant). Reverse-transcription PCR was then performed (Figure 2). There were little sPLA2-IIA mRNAs in cells incubated under basal conditions; incubation with IL-1 plus forskolin increased transcription of the sPLA2-IIA gene by 6-fold, and incubation with the hsPLA2-IIA supernatant increased the sPLA2-IIA mRNA/GAPDH mRNA ratio >3-fold compared with the control supernatant. This increase reflects the equilibrium between its transcription rate and its degradation rate. We checked that the induction was caused by the tagged hsPLA2-IIA protein by passing the hsPLA2-IIA supernatant through a nickel column, which retained histidine-tagged proteins. The depleted supernatant had no enzymatic activity (result not shown) or effect on endogenous sPLA2-IIA gene transcription. We obtained similar results with a supernatant depleted in hsPLA2-IIA by passing through V5-antibody beads.
The induction of the sPLA2-IIA transcription by the hsPLA2-IIA supernatant was also observed when cells were pretreated for 24 hours with IL-1β (1 to 5 ng/mL). Thus, sPLA2 gene induction was confirmed in this inflammatory context (Figure I, available online at http://atvb.ahajournals.org). Moreover, the enzyme activity measured in hsPLA2 supernatant was in the same range as sPLA2 enzyme activity found in supernatant of cells induced by pathophysiological doses of IL-1β (1 ng/mL), eg, 100 to 150 pmol/min per mL. sPLA2-IIA mRNA induction by the hsPLA2-IIA was also in the same range than sPLA2-IIA induction by IL-1β. Consequently, the autocrine induction of the sPLA2-IIA transcription by the hsPLA2-IIA seems to be relevant in pathophysiological conditions. The major experiments were repeated with human AU-1 cells (Figure II, available online at http://atvb.ahajournals.org), showing that sPLA2-IIA proteins strongly induced the transcription of the endogenous hsPLA2-IIA gene in an autocrine or paracrine manner.
No Need for sPLA2 Enzymatic Activity for the Induction of the sPLA2-IIA Gene Transcription
Because many of the effects of sPLA2s are thought to be unrelated to their enzymatic activity, we determined whether the induction of sPLA2-IIA transcription depended on sPLA2 catalytic activity in VSMCs. We constructed 2 human sPLA2-IIA mutants (Figure 1A). The calcium-binding loop of the sPLA2-IIA protein contains a Glycine (Gly29) essential for Ca2+ binding. In the mutant designated G29S, this Gly29 was replaced by Ser. The second catalytically inactive human sPLA2-IIA mutant, H47E, contained a Glu instead of the His47, which is the catalytic center of the protein.24 We verified that the supernatants of cells transfected with mutant constructs contained no sPLA2 enzymatic activity (Figure 3A), and confirmed the lack of enzymatic activity by a more functional method (Figure III, available online at http://atvb.ahajournals.org). We also checked that these 2 constructs were produced and secreted by rat aortic smooth muscle cells (Figure IV, available online at http://atvb.ahajournals.org).
The supernatants from cells overproducing the G29S or the H47E proteins stimulated sPLA2-IIA gene transcription as the supernatant of cells overproducing the wild-type protein (Figure 3B). The mRNA/GAPDH ratio was equally increased in rat smooth muscle cells and human smooth muscle cells (Figure V, available online at http://atvb.ahajournals.org). The removal of G29S or H47E by passing the supernatants of transfected cells through a nickel column almost completely prevented endogenous sPLA2-IIA gene transcription (results not shown). We conclude that the induction of endogenous sPLA2-IIA gene expression by sPLA2-IIA proteins does not depend on the sPLA2 enzymatic activity.
Involvement of CAAT/Enhancer-binding Protein1, Nuclear Factor-κB, and Winged Helix-Turn-Helix Transcription Factors in the Induction of sPLA2-IIA Transcription by hsPLA2-IIA Proteins
We have previously demonstrated that a 5′flanking region of the rat sPLA2-IIA promoter, up to 500 bp ([−488;+46]), is functionally sufficient to account for the induction by IL-1β and the cAMP transduction signal.23 We first reproduced these inductions by cotransfecting VSMCs with a [−488;+46] sPLA2-luciferase reporter gene and a pCMV–β-galactosidase gene (cytomegalovirus promoter/enhancer) (used to normalize transfection efficiency) (Figure 4). The luciferase activity measured 24 hours after induction reflected the efficiency of the sPLA2-IIA transcription. We then incubated the cotransfected VSMCs with hsPLA2-IIA supernatant or a control supernatant. The supernatant containing human sPLA2 proteins increased the rat sPLA2-IIA promoter activity up to twice that of the control supernatant, which is similar to the induction obtained for the endogenous gene (Figures 2 and 3⇑).
We have previously shown that the best characterized activators of gene expression during inflammation, the nuclear factor-κB (NF-κB), CAAT/enhancer-binding protein (C/EBP), and winged helix-turn-helix transcription factors (Ets-1), are involved in the stimulation of rat sPLA2-IIA gene transcription by IL-1β and cAMP.23 Therefore, we investigated whether these transcription factors were involved in the induction of the sPLA2-IIA transcription by the hsPLA2-IIA supernatant using [-488;+46] luc constructs mutated for the transcription factor binding sites. The luciferase/β-galactosidase activity ratio stimulation obtained with the hsPLA2-IIA supernatant was abolished with the mutant constructs, whereas forskolin still induced the transcription of the mutant Ets and mutant NF-kB constructs (Figure 4). Likewise, mutations of C/EBP, Ets, and NF-kB elements abolished induction by IL-1β as previously described. Thus, the C/EBP, NF-κB, and Ets binding elements all contribute to the induction of sPLA2-IIA transcription by the hsPLA2-IIA supernatant.
Mediation of sPLA2-IIA Gene Transcription by sPLA2 Receptors
The effects of the sPLA2-IIA proteins on VSMCs could be mediated by the M-type sPLA2 receptor located in cellular plasma membranes. M-type sPLA2 receptor mRNAs were detected in our secondary cultures of rat VSMCs and in AU-1 cells by reverse-transcription PCR. A single 488-bp cDNA identified by automatic sequencing as the M-type sPLA2 receptor was found (result not shown). This indirectly suggested that M-type sPLA2 receptors could mediate some of the effects of sPLA2-IIA. We investigated the consequences of inducing M-type receptors on sPLA2 gene expression by transfecting VSMCs with the [−488;+46] luc construct and inducing them 24 hours later with a specific agonist of the receptor, p-amino-phenyl-α D-mannopyranoside (APMP).10,25,26 APMP significantly increased the reporter gene activity in a dose-dependent manner. APMP already enhanced transcription at a concentration of 20 μmol/L (1.7-fold induction) and had a maximal effect at 200 μmol/L (3.1-fold induction). An almost maximal effect at ≈30 μmol/L was observed (Figure 5). The induction by APMP was abolished when the mutated [−488;+46] luc constructs were used (Figure VI, available online at http://atvb.ahajournals.org). Thus, the transcription of sPLA2-IIA is induced after stimulation of the M-type sPLA2 receptor, via the same transcription factors binding sites.
Need for sPLA2 Protein Binding to Proteoglycans for Increased sPLA2-IIA Gene Expression by Cell Supernatant
We constructed a sPLA2-IIA mutant that has normal sPLA2 enzyme activity but impaired proteoglycan binding. This b3E construction contains 3 mutations: K56E, K102E, and R118E (Figure 1A). The mutated amino acids of the resulting protein correspond to the amino acids necessary for the binding of sPLA2 to proteoglycans.17 The b3E protein was correctly synthesized and secreted by VSMCs (Figure IV). The sPLA2 activity in b3E supernatant correlated with the amount of protein visualized by immunoblotting. Incubating transfected cells for 20 minutes with a medium containing 1 mol/L NaCl abolished the interaction of sPLA2 with proteoglycans and released cell surface-associated sPLA2 proteins.15 As can be seen on Figure 6A, the 3 mutations in b3E resulted in the accumulation of more sPLA2 activity in the supernatants, with a concomitant decrease in the sPLA2 activity solubilized from cell surfaces by 1 mol/L NaCl. Enzymatic activity before the NaCl wash is twice as the same as after the wash for the hsPLA2-IIA supernatant, whereas it is 5-times higher for the b3E supernatant. However, the total secreted activity is approximately the same for the wild-type and the b3E supernatant. We used another approach to confirm the decreased affinity of b3E to proteoglycans based on heparin binding capacity of sPLA2 (Figure 6B). At low NaCl concentration, wild-type hsPLA2 activity is retained almost completely on heparin-sepharose beads, resulting in a depleted activity in the supernatant. Incubation with 0.4 mol/L NaCl prevented sPLA2 binding to the beads and led to a higher enzymatic activity in the supernatant. In contrast, the enzymatic activity of the b3E supernatant is not retained on heparin-sepharose beads at low NaCl concentration, which is consistent with a low affinity of this mutant protein for proteoglycans. This confirms that the b3E protein does not bind to proteoglycans at the cell surface and consequently is not associated with the cell surface after secretion. We then used supernatants from cells transfected with b3E to induce nontransfected cells and investigated the endogenous transcription of the sPLA2-IIA gene. The b3E supernatants stimulated the transcription of the endogenous sPLA2-IIA gene less than did wild-type supernatant in rat VSMCs (Figure 6C) as in human VSMCs (Figure VII, available online at http://atvb.ahajournals.org). Thus, sPLA2-IIA must be bound to proteoglycans to allow stimulation of sPLA2-IIA gene transcription.
The phenotypic switch of VSMCs occurring early in atherogenesis is accompanied by an increased production of several extracellular matrix molecules such as vascular cell adhesion molecule-1 and proteoglycans having chemoattractant properties and facilitating the sequestration of secreted proteins at the cell surface.27 The synthesis of sPLA2-IIA is stimulated by pro-inflammatory cytokines and the enzyme is responsible for the production of pro-inflammatory mediators promoting atherogenesis. sPLA2-IIA is thus at the edge of an inflammation amplification mechanism.3 Our previous studies on the pathophysiological functions of sPLA2-IIA in inflammation of the arterial wall have included work on the stimulation of sPLA2-IIA gene expression via the IL-1β and cAMP pathways. We have now examined the autocrine role of sPLA2-IIA together with the mechanisms by which it regulates its own synthesis. We used 2 cell systems: rat vascular VSMCs and a human cell line (AU-1) developed by the group of Bonnet.28 These secondary cultures are good physiological models of the inflammatory process because they express almost all the main markers of inflammation. The use of these 2 types of cells shows that sPLA2-IIA action is not species-specific, because it has been shown that some sPLA2 isoforms may have different functions depending on the species.10
The present study shows that a cell supernatant containing human sPLA2-IIA proteins stimulates the transcription of the sPLA2-IIA gene in rat and human VSMCs, and that this effect is independent of its enzymatic activity. For the first time to our knowledge in VSMCs, we thus demonstrate an autocrine/paracrine role of sPLA2-IIA potentially implicated in the delayed and amplified inflammation observed in atherosclerotic and restenotic vessels. sPLA2-IIA could stimulate activation of VSMCs via 2 mechanisms: interaction with membrane receptors analogous to those binding other sPLA2s, and binding to HSPG accumulated on the cell surface.
Upregulation of the endogenous sPLA2-IIA gene was reproduced with the sPLA2-IIA gene promoter region [−488; +46] fused to an exogene luciferase reporter. The 500-bp promoter seems to contain sufficient sequences to enable the enhancement of sPLA2-IIA transcription by the supernatant containing sPLA2-IIA proteins. Because mutation of the C/EBP, NF-κB, and Ets transcription factor binding sites contributed equally to a dramatic reduction of the promoter activity, we propose that these 3 factors cooperate to stimulate gene transcription. Cooperation and physical interactions between these 3 transcription factors occur in the regulation of several inflammatory genes.29–32 Our results emphasize the interplay of concerted signaling pathways. The present study also demonstrates that the M-type sPLA2 receptor is expressed in VSMCs, and that a specific agonist of the sPLA2 receptor (APMP) can also increase the transcription of the luciferase reporter gene via the same transcription factor binding sites.
Besides, it is known that VSMCs express different subtypes of proteoglycans, and that HSPG secretion by proliferating cells is implicated in atherogenesis.15 Binding of sPLA2-IIA to HSPG may account for the autocrine/paracrine action of sPLA2-IIA proteins. Murakami et al demonstrated that sPLA2-IIA is bound to glypican, and Sartipy et al described the binding of sPLA2-IIA to a cell surface molecule, decorin, in atherosclerotic lesions.17,33 Our results using cell supernatant containing sPLA2-IIA proteins unable to bind proteoglycans (mutant b3E) demonstrated the involvement of proteoglycan interactions in the induction of sPLA2-IIA gene transcription. This finding corroborates recent results obtained in mesangial cells using heparinase-1.19 Unlike this pharmacological compound, the use of sPLA2-IIA mutants eliminates the possibility of a release of truncated heparin sulfate chains from the cell surface. Proteoglycans function as a type of receptor system for fibroblast growth factor and for members of the opiate receptor family.34,35 Proteoglycans are thus able to potentiate transmembrane signaling pathways.
Whereas sPLA2-IIA is the most abundant sPLA2 isoform in atherosclerotic arteries, other sPLA2 isoforms can also bind to proteoglycans (eg, group V) and could have functions similar to those of sPLA2-IIA. However, we did not observe a significant upregulation of group V sPLA2 transcription by IL-1β plus forskolin in rat VSMCs and AU-1 cells (result not shown).
PLA2s could then be implicated in the pathogenesis of atherosclerosis via 2 mechanisms. First, PLA2s may interact with the cell surface of VSMCs and hydrolyze phospholipids of apolipoprotein B100 lipoproteins bound to the extracellular proteoglycans. This could facilitate the binding of aggregated low-density lipoproteins to VSMCs and would lead to the accumulation of pro-atherogenic oxidized lipids in the arterial intima.5 Second, PLA2s absorbed onto the cell surface could increase the susceptibility to atherosclerosis by activating the transcription not only of the sPLA2-IIA gene, but also that of genes involved in proliferation. These autocrine/paracrine mechanisms should thus worsen atherosclerotic disease and prevent any resolution in inflammation.
This work was supported by the Centre National de la Recherche Scientifique (CNRS), the Université Pierre et Marie Curie, the Association pour la Recherche sur le Cancer (ARC) (contract 5969) and Amandine Jaulmes is supported by a doctoral fellowship from the French Ministère de l’Education Nationale de l’Enseignement Supérieur et de la Recherche (MENESR). We are deeply grateful to Jacques Bonnet for the gift of AU-1 cells. We also thank Bruno Feve, Joelle Masliah for critically reading the manuscript, and Owen Parkes for linguistic assistance.
- Received December 22, 2004.
- Accepted March 9, 2005.
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