Degradation of Group V Secretory Phospholipase A₂ in Lung Endothelium is Mediated by Autophagy

Abstract

Group V secretory phospholipase A₂ (gVPLA₂) is a potent inflammatory mediator in mammalian tissues that hydrolyzes phospholipids and initiates eicosanoid biosynthesis. Previous work has demonstrated that multiple inflammatory stimuli induce its expression and secretion in several cell types, including the lung endothelium. However, little is known about the mechanism(s) by which gVPLA₂ inflammatory signaling is subsequently downregulated. Therefore, in this study we characterized potential clearance mechanisms for gVPLA₂ in lung endothelial cells (EC). We observed that exogenous gVPLA₂ is taken up rapidly by nutrient-starved human pulmonary artery EC (HPAEC) in vitro, and its cellular expression subsequently is reduced over several hours. In parallel experiments performed in pulmonary vascular EC isolated from mice genetically deficient in gVPLA₂, the degradation of exogenously applied gVPLA₂ occurs in a qualitatively similar fashion. This degradation is significantly attenuated in EC treated with ammonium chloride or chloroquine, which are lysosomal inhibitors that block autophagic flux. In contrast, the proteasomal inhibitor MG132 fails to prevent the clearance of gVPLA₂. Both immunofluorescence microscopy and proximity ligation assay demonstrate the co-localization of LC3 and gVPLA₂ during this process, indicating the association of gVPLA₂ with autophagosomes. Nutrient starvation, a known inducer of autophagy, is sufficient to stimulate gVPLA₂ degradation. These results suggest that a lysosome-mediated autophagy pathway contributes to gVPLA₂ clearance from lung EC. These novel observations advance our understanding of the mechanism by which this key inflammatory enzyme is downregulated in the lung vasculature.

Introduction

Secretory phospholipase A₂ (sPLA₂) is a family of lipolytic enzymes that catalyze the cleavage of fatty acids from the sn-2 position of phospholipids, leading to the generation of free fatty acids and lysophospholipids (1, 2). The gVPLA₂ member of this family is a 14-kDa enzyme with proinflammatory activity that produces several biological effects including airway inflammation, airway hyperresponsiveness, cell adhesion, transcellular communication, and generation of lipid mediators (3–5). In the activated state, intracellular gVPLA₂ translocates from the submembrane compartment to the outer cell membrane or nuclear membrane and cleaves membrane phosphatidylcholine resulting in the generation of fatty acids and arachidonic acid metabolites (4–7). An expanding body of work implicates the activation of this enzyme in the pathophysiology of acute lung injury syndromes such as ARDS (acute respiratory distress syndrome) (8). Its activity increases pulmonary endothelial cell (EC) permeability through direct hydrolysis of the cell membrane, and it has a functional role in the inflammatory responses associated with LPS- or ventilator-induced lung injury (4, 9, 10). Thus, a detailed understanding of how the activation and expression of gVPLA₂ are regulated in lung tissue may provide important insights into the pathophysiology of inflammatory lung injury. Previously, Kim et al reported that exogenous gVPLA₂ added to neutrophils was internalized within 20 minutes and then degraded 45 minutes after addition (3). However, the mechanism that regulates this critical process of gVPLA₂ inactivation and clearance has not yet been defined; nor has gVPLA₂ degradation been explored in other target cell types such as lung EC.

To maintain cellular homeostasis, protein expression is regulated by the tightly balanced processes of synthesis and degradation. Eukaryotic cells have several fundamental pathways that are involved in protein degradation: the ubiquitin-proteasome, the endo-/lysosomal (endocytosis), and the phago-/lysosomal (autophagy) proteolysis pathways (11). For example, recent work has identified the ubiquitin-proteasome pathway as a key mediator of acute lung injury syndromes (12). Autophagy is an alternative process by which cells autodigest specific portions of their cytoplasmic contents (13, 14). This process is essential for optimal cell function and can play important roles in the development of disease (15, 16). In this study, we sought to characterize the mechanism by which gVPLA₂ is degraded in lung EC and identified an important role for autophagy in this process.

Materials and Methods

Reagents

Recombinant human gVPLA₂ protein and monoclonal antibody were purchased from Cayman Chemical (Ann Arbor, Mich.) Chloroquine and ammonium chloride were purchased from Sigma (St. Louis, MO). LC3B and SQSTM1/p62 antibodies were acquired from Cell Signaling (Danvers, MA). Immunofluorescent reagents Alexa Fluor 546 and 488 were obtained from Molecular Probes (Grand Island, NY). Duolink Detection Kit was purchased from Olink Bioscience (Uppsala, Sweden).

Human Lung Endothelial Cell Culture

Human pulmonary artery endothelial cells (HPAEC) were obtained from Lonza (Walkersville, MD) and cultured according to the manufacturer’s instructions as previously described (9). EC (Passages 6–9) were grown in Endothelial Growth Medium-2 (EGM-2, Lonza), supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich), at 37°C in a 5% CO₂ incubator.

Isolation and Culture of Mouse Pulmonary Vascular Endothelial Cells (mPVECs)

All experiments involving mice were approved by the Office of Animal Care and Institutional Biosafety for the University of Illinois at Chicago. Pulmonary vascular EC were isolated in a modified approach (17) from previously described mice that are genetically deficient in the PLA2G5 gene that encodes for gVPLA₂ (18) and wild-type mice (C57BL/6). For each isolation, 2–3 mice (6–8 weeks old) were used. The mice were anesthetized with ketamine/xylazine prior to exposure of the lungs by thoracotomy. The lungs were perfused with 30 ml PBS containing 10 U/ml heparin via the right cardiac ventricle and then excised and placed in a 60-mm dish containing DMEM, Collagenase (Worthington Biochemical, New Jersey), Dispase and DNase (Sigma-Aldrich). The tissues were minced under sterile conditions with scissors then strained through a 70 μm cell strainer (BD Bioscience, Germany). Cells were washed and then incubated with the following antibodies: anti-mouse CD16/32, anti-mouse CD31-PECy7, anti-mouse CD45-Alexa 700 (Biolegend, San Diego, California) for 15 minutes at 4°C. Cells were washed and FACS was performed using MoFlo Astrios Cell Sorter (Beckman-Coulter Diagnostics). Endothelial cells were isolated by CD31+/CD45− sorting and then seeded 1.0 × 10⁶ cells/well on gelatin coated 6-well plates and grown in EGM-2 at 37°C in a 5% CO₂ incubator.

Cell treatment

Starvation

Cells (human and mouse EC) were grown to confluence, and then the medium was replaced with basal medium (without FBS) 2 hours prior to the addition of recombinant gVPLA_2. Cells were incubated for various times as specified in each experimental set. For one comparison set of experiments, HPAEC were treated with recombinant gVPLA₂ in complete media (media containing 10% FBS).

Treatment with Proteasomal and Lysosomal Inhibitors

Cells were grown to confluence, and then the culture growth medium was replaced with basal medium without FBS and containing 10 μM MG132, 10–100 μM chloroquine, or 10 mM ammonium chloride. After 30 minutes, 100 nM recombinant gVPLA₂ was added to the medium and incubated for various times as specified in individual experiments.

Immunoblotting analysis

Treated EC were subsequently washed with cold Ca²⁺/Mg²⁺-free PBS and lysed with 0.3% SDS buffer containing protease and phosphatase inhibitors (1 mM EDTA, 1 mM PMSF, 1 mM sodium orthovanadate, 1mM sodium fluoride, 0.2 TIU/ml aprotinin, 10 μM leupeptin, 5 μM pepstatin A). Sample proteins were separated with 15% SDS-PAGE gels and transferred onto nitrocellulose membranes (Bio-Rad). Membranes were then immunoblotted with primary antibodies (1:500–1000, 4°C, overnight) followed by secondary antibodies conjugated to HRP (1:5000, room temperature, 1 hour). Protein expression was detected with enhanced chemiluminescence (Pierce ECL or SuperSignal West Dura, Pierce Biotechnology, Rockford, Ill.) on Biomax MR film (Kodak, Rochester, NY). Multiple blots were scanned and quantitatively analyzed using ImageJ software (National Institutes of Health, Bethesda, Maryland, USA).

Immunofluorescence

EC were grown on gelatinized cover slips before exposure to various conditions as described for individual experiments. EC were then fixed in 3.7% formaldehyde for 10 minutes, permeabilized with 0.25% Triton-X100 for five minutes, washed in PBS, blocked with 2% BSA in TBS-T for one hour, and then incubated for one hour at room temperature with the primary antibody of interest. After washing, EC were incubated with the appropriate secondary antibody conjugated to immunofluorescent dyes for 1 hour at room temperature. Final washing was performed with TBS-T, and coverslips were mounted using Prolong Anti-Fade Reagent (Invitrogen) and analyzed using a Nikon Eclipse TE2000-s inverted microscope and Adobe Photoshop 7.0.

Proximity Ligation Assay for Quantification of Co-localization

Proximity ligation was performed according to the manufacturer’s protocol using the Duolink Detection Kit with PLA PLUS and MINUS Probes for mouse and rabbit (Olink Bioscience, Uppsala, Sweden). In brief, an oligonucleotide-conjugated probe (used as secondary antibody) is directed against primary antibodies raised against gVPLA₂ or LC3. HPAEC grown on glass slides were incubated with chloroquine and/or gVPLA₂ human recombinant protein and then serum starved for 18 hours. Cells were washed with chilled PBS and fixed with 4% paraformaldehyde for 15 min, blocked, and permeabilized with 0.2% Triton-X100 containing 3% BSA, and incubated overnight with appropriate antibodies. DAPI stain was included in the Duolink Detection Kit, while anti-Phalloidin Alexa488 at 1:5000 (Invitrogen) was added during the detection reaction. Specimens were mounted with Vectashield mounting media (Vector Laboratories, Burlingame, CA) and examined with an epi-fluorescence microscope under a ×60 oil objective. Texas-Red signal was analyzed via BlobFinder Imaging Software, developed and optimized for the analysis of images generated by the in situ PLA (Uppsala Science Park, Sweden). Five fields were randomly chosen for analysis and averaged, and four separate samples were examined per condition (approximately 60 cells).

Statistical Analysis

Data are expressed as means (±SEM). Student’s t-test was used for the comparison of two groups. One-way ANOVA was used for multiple-group comparisons, followed by post-hoc analysis with Dunnett’s or Tukey’s test. Statistical significance was defined as P ≤ 0.05.

RESULTS

Time-dependent gVPLA₂ degradation in lung EC

When gVPLA₂ is secreted into the extracellular space, it acts directly on lung EC to induce permeability (9) and is rapidly internalized by cells (3). In the current study, we first determined the time course of gVPLA₂ degradation after being internalized by cultured lung EC. Human recombinant gVPLA₂ (100 nM) was added to serum-starved HPAECs and incubated from 30 minutes up to 6 hours. This timeframe was selected to model the exposure to extracellular gVPLA₂ that occurs in lung EC during the critical early phase of acute lung injury syndromes (4, 7, 9). Whole cell lysates were then analyzed by immunoblotting to demonstrate gVPLA₂ levels in EC over time. A time-dependent decline in gVPLA₂ levels was observed, with gVPLA₂ expression at 6 hours decreased by almost 70% as compared to 30 minutes (0.68±0.04 vs 2.20±0.04, p=.001) (Fig. 1A). Because human lung EC can synthesize gVPLA₂ in response to some inflammatory signals (9), we next isolated pulmonary vascular EC (mPVECs) from mice genetically deficient in gVPLA₂ (18) to observe the degradation process of exogenous gVPLA₂ without the potential confounder of endogenous EC production. After the addition of human recombinant gVPLA₂ (100 nM), mPVECs from gVPLA₂ knockout mice demonstrated a slower rate of decline in gVPLA₂ expression compared to HPAECs (Fig. 1B), with a 50% decline at 48 hours. Similarly, for wild-type murine EC, gVPLA₂ protein degradation occurs after 18 hours with a maximum effect at 48 hours (Fig. 1C). No degradation was observed at time points earlier than 4 hours for mouse EC (data not shown).

Figure 1. Time-dependent gVPLA₂ degradation in human and mouse lung EC.

(A) Serum-starved HPAECs were stimulated with recombinant gVPLA₂ protein (100 nM). Cell lysates were then collected at various time points post incubation, and western blot analysis of gVPLA₂ expression was performed. N=3 independent experiments. **P<0.01 vs. the 30 min time point. Mouse PVECs isolated from (B) gVPLA₂ knockout mice or (C) wild-type (WT) mice were serum starved and stimulated with recombinant gVPLA₂ protein (100 nM). Cell lysates were then collected at various time points post incubation, and western blot analysis of gVPLA₂ expression was performed. N=3–5 independent experiments. *p<0.05, **p<0.01 vs. control. Levels of gVPLA₂ were normalized to β-actin protein. Representative blots and densitometric quantification are shown.

Degradation of gVPLA₂ is not dependent on the ubiquitin-proteasome pathway

To ascertain whether gVPLA₂ degradation in lung EC is mediated by the ubiquitin-proteasomal pathway, which is known to play a role in acute lung injury syndromes (12), serum-starved HPAECs were pretreated with the proteasomal inhibitor MG132 (19) 30 minutes before the addition of gVPLA₂. Interestingly, proteasomal inhibition with MG132 not only failed to inhibit the degradation of gVPLA₂ over time, but instead it was associated with a trend toward accelerated clearance of gVPLA₂ that was significant at the later time point (Fig. 2). In gVPLA₂-deficient mPVECs, the effects of proteasome inhibition could not be adequately assessed because MG132 caused extensive apoptosis and cell loss at the relevant 48 hour time point. MG132 is known to induce apoptosis in some EC through a caspase-dependent pathway under some conditions (19). Overall, these MG132 data argue against an important role for the proteasomal pathway in gVPLA₂ degradation in lung EC.

Figure 2. Proteasomal inhibition does not prevent the degradation of gVPLA_2.

Serum-starved HPAECs were treated with MG132 (10 μM) for 30 minutes before the addition of 100 nM gVPLA₂ recombinant protein. Cell lysates were then collected at 4, 6 and 18-hour time points for western blot analysis of gVPLA₂ expression. N=3 independent experiments. *p<0.05 vs. gVPLA₂ alone for the indicated time point. Levels of gVPLA₂ were normalized to β-actin protein. Representative blots and densitometric quantification are shown.

Degradation of gVPLA₂ in lung EC is dependent on a lysosomal pathway

To determine if the lysosomal/autophagic pathway participates in gVPLA₂ clearance in lung EC, serum-starved HPAECs were pretreated with the lysosomal inhibitors ammonium chloride (NH₄Cl) or chloroquine (CQ) 30 minutes prior to adding gVPLA₂ recombinant protein. CQ is a classical anti-malarial drug that inhibits lysosomal acidification and blocks autophagosome-lysosome fusion (20–23). Both NH₄Cl and CQ neutralize the lysosomal pH to decrease lysozyme activity, cause autophagosome accumulation, and block lysosomal protein degradation (24, 25). In the presence of NH₄Cl, gVPLA₂ levels were significantly increased compared to controls at 6 and 18 hours (Fig. 3A–B). Similarly, in HPAEC pretreated with CQ, gVPLA₂ protein degradation is blocked (Fig. 3C). A similar trend was observed in mPVECs isolated from gVPLA₂ knockout and WT mice (Fig. 4A–B).

Figure 3. Lysosomal inhibition attenuates the degradation of gVPLA₂ in HPAEC.

Serum-starved HPAECs were treated with lysosomal inhibitors, (A-B) 10 mM ammonium chloride (NH₄Cl), or (C) 100 μM chloroquine, 30 minutes prior to adding 100 nM gVPLA₂ recombinant protein. Cell lysates were collected at indicated time points for western blot analysis of gVPLA₂ expression. N=3–5 independent experiments. Levels of gVPLA₂ were normalized to β-actin protein. *p≤0.05 vs. gVPLA₂ alone. Representative blots and densitometric quantification are shown.

Figure 4. Lysosomal inhibition attenuates the degradation of gVPLA₂ in murine lung EC.

Lung ECs were isolated from (A) gVPLA₂ KO or (B) wild-type (WT) mice. Serum-starved EC were treated with ammonium chloride (NH₄Cl, 10 mM), or chloroquine (100 μM) 30 minutes prior to addition of 100 nM gVPLA₂ recombinant protein. Cell lysates were collected after 48 hours for western blot analysis of gVPLA₂ expression. Depicted are representative blots of three independent experiments.

Both NH₄Cl and CQ block autophagic flux and cause autophagosome accumulation. Because of their effect on inhibiting autophagy, proteins associated with the autophagosomes cannot be degraded. To confirm that NH₄Cl or CQ inhibit autophagic flux in gVPLA₂-treated EC, we assessed autophagosome accumulation in HPAEC and murine EC by determining the levels of LC3-II and p62 expression (26, 27). We observed a significant increase in the LC3-II/I ratio, a hallmark of autophagy (28, 29), in HPAEC receiving NH₄Cl or CQ and gVPLA₂ compared to control cells (Fig. 5A–B), while gVPLA₂ alone did not increase the LC3-II/I ratio in these cells under these conditions. A similar pattern was observed in mPVECs isolated from gVPLA₂ knockout and WT mice that were incubated with NH₄Cl or CQ and gVPLA₂ (Fig. 5C–D). For the mouse cells, LC3-I signal was too weak, and therefore only LC3-II is depicted. Furthermore, the levels of the essential autophagy protein p62/SQSTM1 were increased after treatment with CQ and NH₄Cl in HPAECs (Fig. 6A) and gVPLA₂-deficient and WT mPVECs (Fig. 6B–C). Overall, these results support the hypothesis that lysosomal proteolysis/autophagy participates in the degradation of gVPLA₂.

Figure 5. Effects of chloroquine and ammonium chloride on LC3-II expression in gVPLA₂-treated lung EC.

Serum-starved HPAECs were pretreated either with (A) 10 mM NH₄Cl or (B) 10–100 μM chloroquine (CQ), for 30 minutes before the addition of 100 nM gVPLA₂ recombinant protein. Cell lysates were collected after 6 hours for western blot analysis of LC3-II/LC3-I expression. N=3 independent experiments. * p<0.05; ** p<0.01 vs. control untreated EC. Mouse PVECs isolated from (C) gVPLA₂ knockout or (D) wild-type (WT) mice were pre-treated with 10 mM NH₄Cl or 100 μM CQ for 30 minutes, prior to the addition of 100 nM gVPLA₂. Cell lysates were collected after 48 hours for western blot analysis of LC3-II expression. N=3 independent experiments. *p<0.05, **p<0.01, ***p<0.001 vs. gVPLA₂ alone and vs. control untreated EC. Representative blots and densitometric quantification are shown.

Figure 6. Effects of chloroquine and ammonium chloride on p62/SQSTM1 expression in gVPLA₂-treated lung EC.

Serum-starved HPAECs were pretreated either with (A) 10 mM NH₄Cl or (B) 100 μM chloroquine (CQ), for 30 minutes before the addition of 100 nM gVPLA₂ recombinant protein. Cell lysates were collected after 6 hours for western blot analysis of p62/SQSTM1 expression. N=5 independent experiments. * p<0.05 vs. control untreated EC. Mouse PVECs isolated from (B) gVPLA₂ knockout and (C) wild-type (WT) mice were pre-treated with 10 mM NH₄Cl or 100 μM CQ for 30 minutes, prior to the addition of 100 nM gVPLA₂. Cell lysates were collected after 48 hours for western blot analysis of p62/SQSTM1 expression. N=3 independent experiments. *p<0.05, **p<0.01, ***p<0.001 vs. control untreated EC. Representative blots and densitometric quantification are shown.

These data indicate that gVPLA₂ degradation is blocked when autophagy is inhibited, suggesting that autophagy needs to be triggered for gVPLA₂ clearance. Moreover, our data demonstrate that gVPLA₂ alone does not have a significant effect on the autophagy markers (LC3-II and p62), suggesting that exogenous gVPLA₂ does not activate the autophagy process. To further examine whether the experimental conditions under which gVPLA₂ is degraded are associated with autophagy, we investigated whether starvation is sufficient to activate autophagy and initiate gVPLA₂ degradation. For these experiments, gVPLA₂ was added to HPAEC incubated in complete or basal media (starvation). As depicted in Figure 7A, LC3-II expression is increased in nutrient starvation conditions, while p62 levels are decreased, indicating that autophagy is activated. Moreover, gVPLA₂ is degraded only in starved HPAEC, while the levels of gVPLA₂ remained stable in the unstarved cells (Fig. 7B). Taken together, our data suggest that starvation induces autophagy and gVPLA₂ degradation.

Figure 7. Effect of starvation on gVPLA₂ expression.

Recombinant gVPLA₂ was added to HPAEC grown in complete media (no starvation) vs basal media (starvation). (A) Cell lysates were collected after 6 hours for western blot analysis of LC3 and p62 expression to assess the autophagy status. (B) gVPLA₂ levels were assessed under these serum conditions by western blotting. Representative blots of three independent experiments are shown.

During autophagy, the target protein colocalizes with LC3 prior to lysosomal degradation (14). To determine if gVPLA₂ colocalizes with LC3, HPAEC were starved and treated with either CQ alone, gVPLA₂ alone, or both CQ and gVPLA₂ for 18 hours. Immunofluorescence with anti-gVPLA₂ and anti-LC3 antibodies revealed cytoplasmic vesicular structures or “puncta” in cells stained with anti-LC3 that overlay with gVPLA₂ in the presence of CQ (Fig. 8). To further confirm that gVPLA₂ and LC3 colocalize in lung EC, analysis by proximity ligation assay was then performed. The average signal intensity for each condition was quantified and analyzed via BlobFinder Imaging Software. Five fields per condition were randomly chosen for analysis and averaged (approximately 60 cells) (Fig. 9). This PLA analysis yielded positive signals as discrete fluorescent spots constituting evidence of gVPLA₂-LC3 interactions occurring at a maximum distance of 40 nm. These interactions were significantly increased in the presence of CQ.

Figure 8. LC3 co-localizes with gVPLA₂ in lung EC treated with chloroquine.

Serum-starved HPAECs were incubated for 18 hours with either vehicle control, 100 μM chloroquine alone, 100 nM human recombinant gVPLA₂ alone, or both CQ and gVPLA₂. The cells were then subjected to immunofluorescence using anti-LC3 (RED) and anti-gVPLA₂ (GREEN) antibodies. Nuclei were stained with DAPI (BLUE). Cytoplasmic vesicular structures or “puncta” were observed. Overlay of images demonstrate co-localization (YELLOW) of LC3 and gVPLA₂ in the presence of CQ. Images are representative of 4 independent experiments.

Figure 9. Chloroquine significantly increases LC3-gVPLA₂ co-localization in lung ECs.

(A) Serum-starved HPAECs were incubated for 18 hours with either vehicle control, 100 μM chloroquine alone, 100 nM human recombinant gVPLA₂ alone, or both CQ and gVPLA₂. The close association of gVPLA₂ with LC3B was detected by in situ proximity ligation assay (red dots). Representative images are shown. (B) Average signal intensity for each condition was quantified and analyzed via BlobFinder Imaging Software, developed and optimized for the analysis of images generated by the in situ PLA (Uppsala Science Park, Sweden). Five fields per condition were randomly chosen for analysis and averaged (~60 cells). N = 5/condition, * p < 0.05 vs. control.

DISCUSSION

To our knowledge, this is the first study to explore the mechanism by which the potent inflammatory mediator, gVPLA₂, is degraded and eliminated from lung endothelial cells. Prior work by our group and others has demonstrated that gVPLA₂ expression is increased in lung EC in several models of ARDS in vitro and in vivo, while inhibition of gVPLA₂ expression or activity is protective in these models (4, 7, 9, 10). Therefore, it is important to characterize how gVPLA₂ expression is downregulated in order to better understand the regulation of this inflammatory enzyme. Previous studies have demonstrated that extracellular gVPLA₂ is rapidly internalized and degraded in neutrophils (3), but the pathway involved in gVPLA₂ degradation has not been determined. In addition, gVPLA₂ degradation has not been characterized in other target cell types such as lung EC. For our current study, we focused on clearance of gVPLA₂ from human pulmonary artery EC (HPAEC). However, because human lung EC can synthesize gVPLA₂ in response to inflammatory signals (9), we also characterized gVPLA₂ degradation in mPVECs isolated from mice genetically deficient in gVPLA₂ (18) to observe the degradation process of exogenous gVPLA₂ without the potential confounder of endogenous EC production. In general, the observed results were qualitatively similar between HPAECs and mPVECs in our experiments.

Eukaryotic cells have several major pathways available for protein degradation: the ubiquitin-proteasome, the endo-/lysosomal (endocytosis), and the phago-/lysosomal (autophagy) proteolysis pathways (11). Of these, our results implicate the autophagy/lysosomal pathway as a route of gVPLA₂ clearance in lung EC. Autophagy involves the formation of a double membrane vesicle, called the autophagosome, from discrete portions of the cytosol. Once completely formed, the cytoplasmic material trapped inside the autophagosome is delivered to a lysosome and fused for degradation. In mammalian cells, multiple proteins are involved in autophagy and its regulation. Among these, the microtubule-associated protein 1 light chain 3 (LC3), a mammalian homologue of the Aut7/Apg8 component of the yeast membrane, is a specific autophagosomal marker (11, 30). LC3 attaches to the autophagosomal membrane, and this attachment persists for most of the lifespan of the autophagosome, including the early stages after fusion with the lysosome. Sequestosome-1(p62) binds LC3 and ubiquitinylated proteins and likely serves as a cargo receptor for autophagic degradation (31).

Our studies support the conclusion that gVPLA₂ is degraded through autophagy induction and lysosomal degradation in vascular EC. First, inhibitors of lysosomal, but not proteasomal degradation, significantly increased cellular content of gVPLA₂ in HPAEC after the addition of exogenous gVPLA₂ (Fig. 2–3). In contrast, proteasomal inhibition did not increase levels of gVPLA₂ but instead appeared to increase its degradation. This interesting result is consistent with a previous study which demonstrated that MG132 can induce autophagy (32). Secondly, gVPLA₂ is degraded when autophagy is induced by nutrient starvation (Fig. 7), a well-established stimulus for autophagy activation (33). Indeed, starved HPAEC had increased levels of LC3-II and decreased levels of p62 compared to non-starved cells, indicating the successful activation of autophagy. Moreover, both immunofluorescence microscopy and proximity ligation assays (Fig. 8–9) demonstrate the colocalization of LC3 and gVPLA₂ in the presence of CQ, which in this context serves to arrest the autophagy process so that these interactions can be better observed. This interpretation is further supported by Rhee et al who reported that internalized gVPLA₂ colocalized in HEK293 cells with LAMP-2, a lysosome marker (34).

Previous in vitro studies have demonstrated that other members of the sPLA₂ family, sPLA₂-IB and sPLA₂-X, are internalized and degraded via the lysosomal pathway after binding to the M-type phospholipase A₂ receptor (PLA₂R) (35, 36). These studies implicate a possible role for this receptor in the clearance of extracellular sPLA₂. Moreover, PLA₂R^−/− mice exhibit higher levels of sPLA₂-IB and sPLA₂-X in bronchoalveolar lavage fluid after ovalbumin treatment than wild type mice, further supporting an important role for PLA₂R in the clearance of sPLA₂-IB and X (37, 38). PLA₂R has a high affinity for sPLA₂-IB, -IIA, -IIE, -IIF, and –X (39, 40), but there is no receptor for gVPLA₂ (3, 39). It has been reported that gVPLA₂ binds to cell membrane via heparan sulfate proteoglycans (3), is associated with lipid rafts, and internalized in a flotillin-dependent pathway (34). In the current study, we observed a substantial colocalization of gVPLA₂ with marker proteins specific for autophagosomal degradation (LC3, Fig. 8–9), supporting the conclusion that gVPLA₂ degradation requires autophagy induction. In addition, LC3-II levels, a hallmark of autophagy (28, 29), and p62/SQSTM1 expression, another marker for autophagic flux (31, 41), were increased in HPAEC receiving NH₄Cl or CQ and gVPLA₂ compared to control cells (Fig. 5–6). Thus, conditions associated with inhibition of gVPLA₂ degradation (NH₄Cl or CQ) are also associated with autophagy inhibition and subsequent autophagosome accumulation.

An important question raised by these observations is whether the endosomal pathway is involved in gVPLA₂ degradation. In general, autophagy degrades cytoplasmic components within lysosomes, while endocytosis mediates lysosomal degradation of extracellular and plasma membrane proteins. However, recent studies have found that several membrane proteins are degraded by autophagy rather than endocytosis, including transient receptor potential vanilloid type 1 (42) and connexin 43 (30, 43). As activated and cell membrane-bound gVPLA₂ is internalized in a flotillin-dependent pathway (34), it is possible that gVPLA₂ is associated with the endosomal trafficking pathway and is transferred to autophagosomes through the endosomes. However, its degradation would not be completed unless autophagy is activated by starvation or other stimuli. Whether gVPLA₂ can also be degraded through an endosomal-lysosomal pathway without the involvement of autophagy remains unclear, and further studies are required to answer these questions.

In conclusion, our data indicate that degradation of extracellular gVPLA₂ in human lung EC is mediated by intracellular autophagy and lysosomal degradation. These novel observations improve our understanding of the regulation of gVPLA₂ expression and provide important insights into how this key inflammatory enzyme is downregulated in the lung vasculature. This work has potential relevance to the pathophysiology of ARDS and other inflammatory lung syndromes.