Secreted phospholipase A2-IIA-induced a phenotype of activated microglia in BV-2 cells requires epidermal growth factor receptor transactivation and proHB-EGF shedding
© Martín et al.; licensee BioMed Central Ltd. 2012
Received: 7 December 2011
Accepted: 4 June 2012
Published: 2 July 2012
Activation of microglia, the primary component of the innate immune response in the brain, is a hallmark of neuroinflammation in neurodegenerative disorders, including Alzheimer’s disease (AD) and other pathological conditions such as stroke or CNS infection. In response to a variety of insults, microglial cells produce high levels of inflammatory cytokines that are often involved in neuronal injury, and play an important role in the recognition, engulfment, and clearance of apoptotic cells and/or invading microbes. Secreted phospholipase A2-IIA (sPLA2-IIA), an enzyme that interacts with cells involved in the systemic immune/inflammatory response, has been found up-regulated in the cerebrospinal fluid and brain of AD patients. However, despite several approaches, its functions in mediating CNS inflammation remain unknown. In the present study, the role of sPLA2-IIA was examined by investigating its direct effects on microglial cells.
Primary and immortalized microglial cells were stimulated by sPLA2-IIA in order to characterize the cytokine-like actions of the phospholipase. The hallmarks of activated microglia analyzed include: mitogenic response, phagocytic capabilities and induction of inflammatory mediators. In addition, we studied several of the potential molecular mechanisms involved in those events.
The direct exposure of microglial cells to sPLA2-IIA stimulated, in a time- and dose-dependent manner, their phagocytic and proliferative capabilities. sPLA2-IIA also triggered the synthesis of the inflammatory proteins COX-2 and TNFα. In addition, EGFR phosphorylation and shedding of the membrane-anchored heparin-binding EGF-like growth factor (pro-HB-EGF) ectodomain, as well as a rapid activation/phosphorylation of the classical survival proteins ERK, P70S6K and rS6 were induced upon sPLA2-IIA treatment. We further demonstrated that the presence of an EGFR inhibitor (AG1478), a matrix metalloproteinase inhibitor (GM6001), an ADAM inhibitor (TAPI-1), and a HB-EGF neutralizing antibody abrogated the phenotype of activated microglia induced by the sPLA2-IIA.
These results support the hypothesis that sPLA2-IIA may act as a potent modulator of microglial functions through its ability to induce EGFR transactivation and HB-EGF release. Accordingly, pharmacological modulation of EGFR might be a useful tool for treating neuroinflammatory diseases characterized by sPLA2-IIA accumulation.
KeywordsMicroglia Secreted phospholipase A2-IIA Proliferation Phagocytosis Epidermal growth factor receptor
Microglial cells are considered as central nervous system (CNS)-resident professional macrophages. They constantly survey the brain parenchyma and react immediately to changes in the microenvironment, becoming readily activated in response to infection or injury . They may play a dual role, participating in host defense of the brain and tissue repair, as well as acting as phagocytes to engulf tissue debris and dead cells. However, microglia can also contribute to the establishment or exacerbation of tissue damage depending on the type or intensity of the harmful stimulus.
Cerebral ischemia and other neurodegenerative disorders such as Alzheimer's disease (AD), Parkinson’s disease, and multiple sclerosis, among others, are associated with proliferation and activation of microglia [2–6]. The activated microglia undergo dramatic morphological changes, from a resting ramified form to an activated amoeboid shape, and secrete a host of immunomodulatory and neurotoxic factors. Whilst significant advances have been made to identify the contribution of the cytotoxic agents released from microglia to the neurodegenerative process, it is less clear and remains to be determined which factors trigger microglial activation in these various disorders.
In neurodegenerative diseases such as Alzheimer’s, for example, players involved in the inflammatory process include S100a9, β-amyloid peptides (Aβ), macrophage colony-stimulating factor and acute-phase proteins such as C-reactive protein, amyloid P and secreted phospholipase A2-IIA (sPLA2-IIA), among others [2, 7–12]. Recent studies have revealed that S100a9, Aβ and macrophage colony-stimulating factor themselves can promote the reactivity of microglia to enhance their neurotoxicity. However, any role that sPLA2-IIA might play in microglia activation is still unknown.
Secreted phospholipases A2 represent a family of eleven low molecular mass, calcium- dependent lipolytic enzymes. They catalyze the hydrolysis of the sn-2 ester bond of glycerophospholipids present in cell membranes to form essential cell-signaling molecules. They are widely distributed in human tissues including brain, where their specific function is still largely unclear [13, 14], although current evidence suggests that sPLA2s may affect some neuronal functions, such as neuritogenesis, neurotoxicity, neurotransmitter release and survival [15–20]. Different levels of sPLA2 activity have been found in various regions of the central nervous system in both humans and rodents, and the subtypes identified include sPLA2-IIA, IIC, IIE, II, V, X and XII.
Secreted PLA2-IIA (sPLA2-IIA) was first identified in synovial fluid, and then characterized as an acute-phase protein under the transcriptional control of pro-inflammatory cytokine signaling . Later on, its presence in tears was reported and it came to be considered a powerful innate ocular surface barrier against Gram-positive bacteria . Its serum levels dramatically increase under pathological conditions that involve systemic inflammatory processes such as sepsis, rheumatoid arthritis, and cardiovascular disease (up to 1000-fold and >1 μg/ml). Additionally, enhanced expression of sPLA2-IIA has also been found in certain neurological disorders and as a result of brain insult, it being associated with CNS injuries such as cerebral ischemia or mechanical damage to spinal cord tissue [18, 23–26]. Recent reports have shown it to be up-regulated in both cerebrospinal fluid and brain of patients with Alzheimer’s disease. In fact, increased immunoreactivity for sPLA2-IIA has been reported in reactive astrocytes of the cortex and hippocampus (restricted mainly to the dentate gyrus and CA3 field) around Aβ-containing plaques [11, 12].
sPLA2-IIA, as well as other sPLA2 subtypes, can also exert various biological functions and transduce signals independently of their catalytic activity through receptors or binding proteins such as M-type receptor, factor Xa, integrin αvβ3 and α4β1, heparan sulfate and proteoglycans, etcetera [27–31]. Indeed, it has been reported that sPLA2-IIA influences survival of some cellular types within the CNS including oligodendrocytes and neurons, independently of its catalytic activity [19, 24].
In this study, we provide data demonstrating the functional consequences of microglial cell exposure to the activating agent sPLA2-IIA. We have measured proliferative responses, phagocytic capabilities and synthesis and release of several molecules with pro-inflammatory activities, for example, tumor necrosis factor-α (TNFα) and cycloxigenase-2 (COX-2), as indices of microglial activation. In addition, we have characterized several of the potential molecular mechanisms involved in these events.
A C127 mouse fibroblast cell line, stably transfected with the coding sequence of sPLA2-IIA from human placenta, was kindly provided by Dr Olivier (Nancy University Hospital, Nancy Cedex, France) and used as a source of human recombinant enzyme (hr-sPLA2-IIA) in some experiments to ascertain specificity . sPLA2-IIA was obtained and purified as described previously . The absence of lipopolysaccharide (LPS) in the preparation was confirmed by the limulus amebocyte lysate assay test in the batches used for the experiments. Moreover, experiments are conducted in the absence of fetal calf serum (FCS), which ensures that the effect is observed in the absence of LPS binding protein, necessary for the action of low concentrations of LPS.
Bee venom sPLA2-III (structurally related to human group III) and human recombinant sPLA2-V were from Cayman (Tallinn, Estonia). Rapamycin, pyrazole pyrimidine-type 2 (PP2), porcine sPLA2-IB, LPS, both anti-rabbit and anti-mouse fluorescein isothiocyanate (FITC) secondary antibodies, FITC-dextran and other chemicals were from Sigma Chemical Co. (St. Louis, MO, USA.). PD98059 and AG1478 inhibitors were from Tocris Biosciece (Bristol, UK). Policlonal anti-heparin-binding epidermal growth factor (HB-EGF) neutralizing antibody and the inhibitors GM6001, chloromethylketone (CMK) and TNFα proteinase inhibitor-1 (TAPI-1) were from Calbiochem (San Diego, CA, USA). Rabbit anti-mitogen-activated protein kinase (MAPK) was from Zymed Laboratories (San Francisco, CA, USA). Rabbit antibody phosphorylated (phospho)-ERK1/2 (Thr202/Tyr204), phospho-S6 ribosomal protein (Ser235/236) and phospho-P70S6 kinase (Thr389) were from Cell Signaling Technology, Inc. (Danvers, MA, USA). The Rabbit phosphor-Src (Ser473), phospho-EGF (Tyr1173), phospho-EGF (Tyr845), anti-actin, and COX-2 antibodies were from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Hybond-P membrane was from Amersham Biosciences (GE Healthcare Europe GmbH, Barcelona, Spain). DMEM and the cell culture supplements, including FCS, were purchased from Gibco BRL (Burlington, Canada).
BV-2 murine microglia cells, a generous gift from Dr JR Bethea (University of Miami School of Medicine, Miami, Florida, USA), were cultured at 37°C in a humidified atmosphere of 5% CO2 in high sucrose DMEM, supplemented with 100U/ml penicillin, 100 μg/ml streptomycin, 50 μg/ml gentamicin, 2 mM glutamine, and 10% heat-inactivated fetal calf serum (FCS).
Primary microglia-enriched cultures were obtained from primary mixed glial cultures from 2- to 4-day-old neonatal C57BL/6 mice. To obtain mixed glial cultures, cerebral cortices were dissected, carefully stripped of their meninges, and digested with 0.25% trypsin-EDTA solution (Invitrogen, Life Technologies S.A., Madrid, Spain) for 25 minutes at 37°C. Trypsinization was stopped by adding an equal volume of culture medium, to which 0.02% deoxyribonuclease I (Sigma) was added. The culture medium consisted of DMEM-F-12 nutrient mixture supplemented with 10% FCS, 0.1% penicillin-streptomycin, and 0.5 μg/mL amphotericin B (Fungizone, Invitrogen). Cells were pelleted (5 minutes, 200 g), re-suspended in culture medium, and brought to a single cell suspension by repeated pipetting followed by passing through a 105 μm-pore mesh. Cells were seeded at a density of 3.5 × 105 cells/ml (1.2 × 105 cells/cm2) and cultured at 37°C in a 5% CO2 humidified atmosphere. Medium was replaced every 5 to 7 days. Microglial cultures were prepared by the mild trypsinization method previously described by Saura et al. . Briefly, after 19 to 21 days in vitro, mixed glial cultures were treated for 30 minutes with 0.06% trypsin in the presence of 0.25 mM EDTA and 0.5 mM Ca2+. This resulted in the detachment of an intact layer of cells containing virtually all the astrocytes, leaving a population of firmly attached cells identified as > 98% microglia. The microglial cultures were treated 24 h after isolation by this procedure. Experiments were carried out in accordance with the Guidelines of the European Union Council (86/609/EU), following the Spanish regulations (BOE 67/8509-12, 1988) for the use of laboratory animals, and approved by the Animal Ethics Committee of the Universidad de Valladolid. Cultures were found to be 99% microglia by staining with FITC-conjugated Griffonia (Bandeiraea) simplicifolia lectin I-B4 isolectin (1:100, Sigma St. Louis, MO, USA), a lectin that recognizes microglia, and an antibody against glial fibrillary acidic protein (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), to identify astrocytes.
Primary and immortalized microglial cells were serum-starved 24 h before the experiments, and then were stimulated for different times, as indicated, in the presence or absence of inhibitors.
Cell proliferation was quantified using the Promega kit, Cell Titer 96RAqueous One Solution Cell Proliferation Assay (Promega Corporation, Madison, Wisconsin, USA, according to the manufacturer's recommendations. Briefly, primary and immortalized BV-2 microglial cells were seeded in 96-well tissue culture plates and serum-starved for 24 h. Then, cells were treated in quadruplicate with the stimuli, in the presence or absence of the indicated inhibitors. After 24 h of incubation, formazan product formation was assayed by recording the absorbance at 490 nm in a 96-well plate reader. The results were expressed as optical density (OD) values, as an assessment of the number of metabolically active cells. Microglia cell viability was also assessed by trypan blue exclusion.
Western blot analysis
After treatment, cells were washed twice with PBS and harvested in Laemmli SDS sample buffer. Protein extracts were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes, which were incubated for 18 h at 4°C with the indicated antibodies, including ERK 1/2, p-ERK1/2, p-P70S6K, p-rS6, COX-2 and actin. After washing with Tris-Tween buffered saline (TTBS), a 1:2.000 (v/v) dilution of horseradish peroxidase-labeled immunoglobulin (IgG) was added at room temperature for 30 h. The blots were developed using enhanced chemiluminescence.
Flow cytometric analysis
BV-2 cells, 5 × 106/flask, were treated with 1 μg/ml of sPLA2-IIA for different periods of time at 37°C. Cells to be analyzed for expression of epidermal growth factor receptor (EGFR) were fixed in a mixture of 4% paraformaldehyde and 0.2% Triton X-100 in PBS for 15 minutes at room temperature, before incubation with FITC-conjugated anti-mouse EGFR antibody for 1 h at 4°C, as previously described . For EGFR phosphorylation analysis, cells were fixed in 4% paraformaldehyde for 15 minutes, washed with PBS, permeabilizaed with 0.3% Triton X-100 for 5 minutes, washed, incubated with anti-phospho EGFR (Tyr1173) or EGFR (Tyr845) antibody for 1 h at 4°C, and then with an FITC-labelled secondary antibody for 45 min at 4°C. After washing, the cells were analyzed with a Flow Cytometer (GalliosTM; Beckman Coulter, Brea, CA, USA). Data analysis was performed using WinMDI 2.7 software (Scripps Institute, La Jolla, CA, USA).
Induction of apoptosis
Jurkat T cells were cultured in RPMI 1640 with 10% FBS at 37°C in 5% CO2. Apoptosis was induced in Jurkat T cells (106 cells/ml) by overnight exposure to 400 μM H2O2 in serum-free RPMI medium. To distinguish between cells in the early or late stages of apoptosis, staining with Annexin V-FITC was combined with propidium iodide (PrI) staining. Afterwards, cells were immediately analyzed by flow cytometry (GalliosTM; Beckman Coulter, USA). Cells in the early stage of apoptosis were negative for PrI but stained with Annexin V-FITC, whereas in the late stage apoptotic cells stained for both PrI and Annexin V-FITC. Jurkat T cells treated in this way were about 90% late-stage apoptotic cells.
Phagocytosis of particles
Microglial cells seeded in 96-well plates or in 25-mm2 flasks were incubated with medium, 1 μg/ml of sPLA2-IIA, 100 UI/ml of interferon-γ (IFNγ) at 37°C for 24 h, in the presence or absence of the indicated inhibitors. After 24 h, the phagocytic ability of the cells was measured using FITC-dextran as a tracer . Briefly, cells were exposed to 0.1 mg/ml of FITC-labelled dextran (MW 40,000) for 2 h. Non-internalized particles were removed by vigorously washing three times with cold PBS (pH 7.4) prior to measuring fluorescence at 480 nm excitation and 520 nm emission on either a Flow Cytometer (GalliosTM; Beckman Coulter, USA) or a Fluoroskan multiwell plate reader (TECAN Genios Pro; Tecan Group Ltd, Männedorf, Switzerland). As a background, the cultures without FITC-dextran were used (blank wells). Each culture condition was performed in quadruplicate, and three independent experiments were performed. To visualize the internalized dextran, cells were also analyzed on a Leica TCS SP5X confocal microscope with a ×60 oil objective.
Phagocytosis of apoptotic cells (efferocytosis)
Phagocytic assays were performed on BV-2 cells (effector cells) after 24 h incubation in the presence of the inflammatory stimuli. Apoptotic Jurkat T cells were used as target cells. Briefly, PrI-labeled apoptotic Jurkat T cells were added to the BV-2 cells at a 8 to 10:1 ratio (apoptotic cell to BV-2) and incubated at 37°C in 5% CO2 for 2 h in DMEM medium. Then, BV-2 cells were washed gently with cold PBS and trypsinized by incubating them with a solution 0.25% trypsin/EDTA for 5 minutes to remove uningested cells. Afterwards, cells were fixed, stained with a PE-conjugated-CD68 antibody and analyzed by flow cytometry. PE fluorescence was analyzed in FL2 (555 to 600 nm), while red fluorescence from PrI was analyzed in FL3 (555 to 600 nm). To quantify phagocytosis, PrI fluorescence was analyzed only in the cell populations exhibiting PE-CD68 positive staining (BV-2 microglia cells). The BV-2 microglia cells were positive for PrI fluorescence only if they had ingested PrI-labeled Jurkat T cells. To confirm efferocytosis, a Leica TCS SP5X confocal microscope was used with the Leica LAS AF acquisition software and a ×60 oil objective. For confocal microscopy, BV-2 cells were plated onto 12-mm round cover slips and stained with an Alexa-fluor-CD11b antibody. We used 4',6'-diamidino-2-phenylindole hydrochloride (DAPI) to identify nuclei in BV-2 cells.
All data were expressed as the mean ± SD and analyzed by one-way ANOVA followed by post-hoc comparisons (Bonferroni test) using the GraphPad Prism Version 4 software (San Diego, CA, USA). P < 0.05 was considered statistically significant.
sPLA2-IIA triggers microglial proliferation
This effect on growth was paralleled by the activation/phosphorylation of key proteins involved in cell survival and proliferation such as ERK, P70S6K and rS6. Activated forms of these proteins from whole cell lysates were monitored using specific anti-phospho antibodies that recognize only their activated/phosphorylated form. To determine whether the mTORC1 pathway was activated following sPLA2-IIA stimulation, we used an antibody that detects phosphorylation of P70S6K on threonine 389, a site well known to be selectively phosphorylated by mTORC1 and widely used to monitor mTORC1 activation.
As shown in Figure 1D, sPLA2-IIA treatment induced a rapid and sustained increase in ERK, P70S6K and rS6 phosphorylation in BV-2 cells. This effect was blocked in the presence of specific pharmacological inhibitors, including PD98059 (MEK inhibitor), rapamicin (mTOR inhibitor) and PP2 (Src kinase inhibitor), which also affected the proliferative response (Figure 1E, F). Thus, ERK and mTORC1 are key components of the intracellular signals regulating cell growth.
Involvement of epidermal growth factor receptor (EGFR) transactivation in sPLA2-IIA-enhanced microglial cell proliferation
sPLA2-IIA induces a proliferative response in microglial cells via an epidermal growth factor receptor (EGFR)-ligand-dependent mechanism
sPLA2-IIA treatment enhances phagocytosis and efferocytosis in BV-2 microglia cells
In subsequent experiments, we examined whether transactivation of EGFR is also a key step for controlling sPLA2-IIA-mediated efferocytosis. Consistent with the signaling mechanism recruited by the secreted phospholipase to promote proliferation of BV-2, we found that the presence of the selective inhibitors GM6001, CMK and TAPI-1 also abolished the phagocytic response triggered by the sPLA2-IIA on microglial cells (Figure 6D), as it previously did on sPLA2-IIA-enhanced cell growth.
sPLA2-IIA promotes synthesis and secretion of inflammatory mediators in BV-2 cells
Lastly, we further examined whether blockage of EGFR signaling at different levels, as demonstrated in previous sections, affects the expression of these inflammatory proteins induced by sPLA2-IIA. Figure 8C and D show that sPLA2-IIA-induced up-regulation of COX-2 and secretion of TNFα was significantly inhibited by the presence of the inhibitors AG1478, GM6001, TAPI-1 and CMK, as well as by the polyclonal anti-HB-EGF antibody. Similarly, IFNγ-induced COX-2 expression was also abrogated by the presence of the neutralizing anti-HB-EGF antibody.
All these studies clearly pointed to a crucial role of EGFR transactivation, through MMP-mediated cleavage of mature forms of EGFR ligands, in the signaling and functional activity of the sPLA2-IIA.
The importance of sPLA2-IIA in neurodegenerative diseases, especially in those associated with inflammatory processes has started to emerge in recent years. Several studies have shown an increase in the expression of sPLA2-IIA in reactive astrocytes both in experimental models of cerebral ischemia and in specific regions of human brains in AD associated with amyloid plaques [11, 12, 18, 23, 26]. It has been suggested that the interaction of astrocytes with Aβ and other inflammatory stimuli, such as IL-1β or TNFα, are responsible for this sPLA2-IIA induction which could be associated in the early inflammatory events. Although the ability of sPLA2-IIA to affect the functional activities and the survival or death of astrocytes, neurons and oligodendrocytes has been explored, this is the first study in which the effect of sPLA2-IIA on microglial cells has been addressed. Our interest in microglia owes to the fact that these cells, in conjunction with astrocytes, are responsible for coordinating inflammatory responses in the brain and elicit immune responses against pathological stimuli.
Several pro-inflammatory and immunoregulatory responses associated with certain secreted PLA2 types have been reported in previous studies. Thus, sPLA2-IIA induces differentiation of monocytes into monocyte-derived dendritic cells or alternatively activated macrophages ; both human and bee venom type III trigger maturity of dendritic cells, which is accompanied by up-regulation of surface markers and by an increase in their migratory and immunostimulatory capacity [43, 44]. Furthermore, type V regulates phagocytosis on macrophages by modulating phagosome maturation . sPLA2-IIA also enhances the expression of COX-2 in mast cells and promotes degranulation and cytokine release in human eosinophils, as well as up-regulation of certain surface activation markers . In addition, sPLA2-IIA, IB, X and III elicit proliferative signals, in vitro, in several cell types [30, 33, 47–49], and type IIA has proven to be protective even against oxysterol-induced apoptosis in oligodendrocytes .
In this study we showed that sPLA2-IIA, as well as type III, IB and V, enhance the proliferative and phagocytic capacity of BV-2 microglia cells to a similar extent as IFNγ, one of the cytokines up-regulated in the brain in different disorders and a well-known inducer of an activated state in microglial cells. Focusing on type IIA actions, two kind of phagocytosis have been evaluated: phagocytosis of inert particles (as a measure of non-specific phagocytosis) and of apoptotic cells (as a measure of disease-relevant processes of phagocytosis). The ability of microglia to phagocytose inert material and apoptotic cells is critical for the clearance of pathogen/cell debris and dead cells under pathological conditions. We demonstrated that sPLA2-IIA increases the uptake of apoptotic Jurkat T cells as well as dextran beads, thus indicating that sPLA2-IIA from the microenvironment might contribute to the innate immune response on the CNS by modulating the phagocytic efficiency of microglial cells. These findings are in concordance with the responses reported for other CNS soluble factors, including IFNγ, as well as for various secreted sPLA2s on other myeloid-lineage cells [36, 43, 44].
To our knowledge, there are no studies, either in vivo or in vitro, describing production and secretion of sPLA2-IIA by microglial cells, while astrocytes have been identified as a key cellular source of sPLA2-IIA in the CNS under different pathological conditions [11, 12, 18, 23, 51]. Therefore, we propose that the sPLA2-IIA, once released by astrocytes, might act on the microglia, in a paracrine manner, to promote microglial activation and to further stimulate phagocytosis and production of inflammatory mediators such TNFα or COX-2 , thereby affecting the inflammatory environment of the brain and contributing to additional neuronal cell damage.
These results have led us to question the possible mechanisms - signaling molecules and receptors - underlying the functional effects of sPLA2-IIA. It has previously been reported that the biological activities induced by sPLA2s can be dependent on both enzymatic and nonenzymatic mechanisms. Whereas the ability of types X and III to stimulate cell growth has been found to be mostly dependent on their intrinsic catalytic activity, the mitogenic response induced by type IB and IIA seems to be unrelated to its enzymatic activity. Both an integrin-dependent  and an EGFR-dependent pathway  have been characterized as new sPLA2-IIA putative signaling mechanisms. In this study, we found that sPLA2-IIA induced a phenotype of activated microglia in BV-2 cells which is linked to the activation of the classical MAPK/ERK and mTOR/P70S6K pathways through MMP-dependent ectodomain shedding of the transmembrane precursor pro-HB-EGF and subsequent transactivation of the EGFR.
The EGFR is expressed ubiquitously in the mammalian brain, being detected in neurons and glia cells [40, 52]. It has been hypothesized that EGFR activation is a master signal transduction pathway of the cellular activation process in response to different brain injuries and causes the characteristics of the reactive astrocyte/microglia phenotype [53–55]. Thus, activation of the EGFR pathway is responsible for the hypertrophy, proliferation and migration of reactive astrocytes, and perhaps of activated microglia, at the site of neural injury [40, 56, 57]. We have herein showed that sPLA2-IIA induces a sustained EGFR phosphorylation at Tyr 1176 and Tyr 845 residues that is abolished or diminished in the presence of the selective EGFR inhibitor, AG1478. To understand the mechanisms by which phospholipase causes EGFR phosphorylation, we used a general matrix metalloprotease inhibitor (GM6001) and an ADAMs inhibitor (TAPI-1), which are known to block the proteolytic cleavage of various membrane-anchored EGFR pro-ligands such as pro-EGF, pro-TGFα, pro-HB-EGF, and pro-amphiregulin. We have found that the presence of these inhibitors blocked the effect of sPLA2-IIA on EGFR phosphorylation as well as on ectodomain shedding of HB-EGF, suggesting a possible role of ADAMs and HB-EGF in sPLA2-IIA-induced EGFR transactivation. Although it is possible that other EGFR ligands could be also involved in sPLA2-IIA-induced EGFR transactivation, the fact that the presence of a HB-EGF-neutralizing Ab prevented the molecular and biological effects of the phospholipase suggests that HB-EGF plays a major role in the response induced by the sPLA2-IIA. We focused mainly on HB-EGF because of the extensive literature showing its role in cell survival and proliferation, both in vivo and in vitro[58–61]. Whether the remnant C-terminal fragment generated, HB-EGF-CTF, translocates to the nucleus and plays any role in sPLA2-IIA signaling should be investigated in greater detail in the future. Interestingly, transactivation of EGFR upon microglial stimulation with IFNγ also involves HB-EGF shedding, and is critical for the mitogenic and pro-inflammatory activity of this cytokine. This cross-talk mechanism between different signaling systems allows the integration of the great diversity of stimuli and supports the key role of the EGFR in diverse pathophysiological disorders.
Additionally, we showed that sPLA2-IIA induces rapid phosphorylation on Src at Tyr 416, and by using the selective inhibitor PP2 we demonstrated that Src participates in both HB-EGF shedding and EGFR phosphorylation at Tyr 845 and at Tyr 1173. Likewise, as already mentioned, EGFR phosphorylation at Tyr 845 (a Src phosphorylation site) is also diminished by MMP inhibitors, which indicates that products of MMPs are necessary for Src-mediated phosphorylation of EGFR at Tyr-845. Thus, it raises the possibility that EGFR ligands generated by MMP-mediated cleavage of membrane precursors collaborate with Src kinases in promoting sPLA2-IIA-induced EGFR transactivation. Therefore, our results suggest that Src contributes to sPLA2-IIA-induced EGFR transactivation at various steps: Src may serve as an upstream component of EGFR transactivation by phosphorylating Tyr 845 directly and indirectly by a MMPs/ADAMs/HB-EGF-dependent mechanism. These findings are consistent with abundant evidence indicating that external stimuli can transactivate EGFR in complex Src-dependent signaling [62–64]. Further studies are required to clarify the precise role of Src in this system, as well as to determine which member(s) of the family (Src, Fyn, and Yes) is involved in sPLA2-IIA-induced EGFR transactivation and BV-2 cells activation. It is possible that a particular member is involved in HB-EGF shedding and another one in EGFR phosphorylation at Tyr 845.
In contrast to Src signaling, sPLA2-IIA-activated MEK/ERK/MAPK and mTOR/P70S6K signaling pathways effectively seem to be downstream of EGFR transactivation. Thus, whereas the experimental conditions that affect HB-EGF release and EGFR phosphorylation abrogate phosphorylation of ERK, P70S6K and rS6, the presence of the specific inhibitors PD98059 (for MEK), or rapamicin (for mTOR) scarcely affects sPLA2-IIA-stimulated HB-EGF shedding and EGFR phosphorylation. In addition, our data suggest a complex, not linear, signaling network involving these two cascades, as the inhibition of any of those pathways prevents sPLA2-IIA-promoted activation of BV-2 microglia cells. It has been described that both pathways cross-talk extensively and may regulate each other both positively and negatively . mTOR can be considered a key node of these complex signaling cascades, and exists as two different entities: the raptor-mTOR complex and the rictor-mTOR complex. Thus, it has been reported that phosporylation of P70S6K and its substrate, rS6, can take place in a rapamycin-dependent manner [66, 67], or independently of mTOR, being Akt, ERK and even phosphatidic acid, direct upstream effector molecules [68, 69]. Moreover, inhibition of the raptor-mTOR complex can trigger activation of the ERK/MAPK cascade, while inhibition of the rictor-mTOR complex inhibits Akt and ERK phosphorylation . We have found that rapamycin, as well as PD98059, at concentrations that diminish or even suppress the proliferative and fagocytic capabilities of sPLA2-IIA-activated BV-2 cells, also suppress phosphorylation of ERK, P70S6K and rS6. In this study there was no attempt to investigate more deeply the effect of sPLA2-IIA on the sequential activation of these signaling proteins or the cross-talk between the raptor-mTOR/rictor-mTOR complexes. However, the relationship between these signaling pathways certainly deserves further, independent study due to the complex link existing between their components.
In conclusion, our results reveal that sPLA2-IIA activates primary and immortalized BV-2 microglia cells; EGFR plays a key role as a critical regulator of this sPLA2-IIA-mediated effect, and also indicates that shedding of pro-HB-EGF is a crucial step in this response. Accordingly, the possibility that sPLA2-IIA may affect immune system function in the CNS in certain pathologies should be carefully considered.
A disintegrin and metalloproteinase
Central nervous system
Dulbecco’s modified Eagle’s medium
Enzyme-linked immunosorbent assay
Epidermal growth factor receptor
Fetal calf serum
Heparin-binding epidermal growth factor
Secreted phospholipase A2-IIA human recombinant enzyme
Heparin-binding EGF-like growth factor
Secreted phospholipase A2-IIA
Tumor necrosis factor-α
TNFα proteinase inhibitor-1
Tris-Tween buffered saline
Transforming growth factor.
We thank C Sánchez for assistance with confocal microscoy and MI Cabero for her valuable technical support. We also thank Dr J Saura, from the University of Barcelona, for his assistance in primary cultured microglia, and A DeMarco for his editorial assistance. This work was supported by grants SAF2009-08407 from the Spanish Ministry of Science and Innovation, and CSI11A08 from the Government of Castilla y León. Claudia Cordova was funded by the FPI Program from the Government of Castilla y León (co-funded by FSE).
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