Association of cPLA2-{alpha} and COX-1 with the Golgi apparatus of A549 human lung epithelial cells

Seema Grewal, Sreenivasan Ponnambalam and John H. Walker*

School of Biochemistry and Molecular Biology, University of Leeds, Leeds, LS2 9JT, UK

* Author for correspondence (e-mail: j.walker{at}leeds.ac.uk)

Accepted 27 February 2003


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Cytosolic phospholipase A2-{alpha} (cPLA2-{alpha}) is an 85 kDa, Ca2+-sensitive enzyme involved in receptor-mediated prostaglandin synthesis. In airway epithelial cells, the release of prostaglandins is crucial in regulating the inflammatory response. Although prostaglandin release has been studied in various epithelial cell models, the subcellular location of cPLA2-{alpha} in these cells is unknown. Using high-resolution confocal microscopy of the human A549 lung epithelial cell line, we show that cPLA2-{alpha} relocates from the cytosol and nuclei to a juxtanuclear region following stimulation with the Ca2+ ionophore A23187. Double staining with rhodamine-conjugated wheat germ agglutinin confirmed this region to be the Golgi apparatus. Markers specific for Golgi subcompartments revealed that cPLA2-{alpha} is predominantly located at the trans-Golgi stack and the trans-Golgi network following elevation of cytosolic Ca2+. Furthermore, treatment of cells with the Golgi-disrupting agent brefeldin A caused a redistribution of cPLA2-{alpha}, confirming that cPLA2-{alpha} associates with Golgi-derived membranes. Finally, a specific co-localization of cPLA2-{alpha} with cyclooxygenase-1 but not cyclooxygenase-2 was evident at the Golgi apparatus. These results, combined with recent data on the role of PLA2 activity in maintaining Golgi structure and function, suggest that Golgi localization of cPLA2-{alpha} may be involved in membrane trafficking in epithelial cells.

Key words: Golgi, Phospholipase A2, Cyclooxygenase, Arachidonic acid, Calcium, Ca2+


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Prostaglandins (PGs) are a group of potent lipid mediators that act as local hormones in regulating a variety of physiological responses. They are also implicated in the pathogenesis of numerous disease states, including asthma, atherosclerosis and cancer (Earnest et al., 1992Go; Vane and Botting, 1995Go). The rate-limiting step in the production of PGs is the liberation of free arachidonic acid from membrane phospholipids by the enzyme cPLA2-a. The released arachidonic acid is then metabolized by cyclooxygenase (COX) enzymes to form prostaglandin H2 (PGH2), and the subsequent cell-specific expression of downstream synthases is responsible for the production of various other PGs.

cPLA2-a belongs to a growing family of phospholipase A2 enzymes that catalyse the hydrolysis of the sn-2 fatty-acyl bond of phospholipids to liberate free fatty acids (Dennis, 1997Go). Since cPLA2-a preferentially liberates arachidonic acid, it is considered the key PLA2 enzyme involved in receptor-mediated PG production. This 85 kDa, Ca2+-sensitive protein is subject to complex regulation at the transcriptional and post-translational level (reviewed by Clark et al., 1991Go). Previous studies have shown that cPLA2-a is present in the cytosol of resting cells and relocates to intracellular membranes following stimulation with a variety of agonists (Evans et al., 2001Go; Glover et al., 1995Go; Hirabayashi et al., 1999Go; Peters-Golden et al., 1996Go; Schievella et al., 1995Go; Sierra-Honigmann et al., 1996Go). This translocation process is mediated by its Ca2+-dependent lipid-binding (CaLB) or C2 domain, which promotes binding to phospholipids upon elevation of intracellular Ca2+ concentrations (Gijon et al., 1999Go).

Downstream of cPLA2-a, COX converts arachidonic acid into PGH2. COX exists as two isoforms encoded by distinct genes. COX-1 is a constitutively expressed housekeeping gene, whereas COX-2 is an inducible gene expressed at higher levels in response to inflammatory and mitogenic stimuli (Smith and Langenbach, 2001Go). Despite similarity in structure and catalytic properties, COX-1 and COX-2 have been shown to use different intracellular pools of arachidonic acid and are believed to have distinct cellular functions (Reddy and Herschman, 1997Go). In addition, accumulating evidence suggests that both the COX isoenzymes are functionally coupled to cPLA2-a to control the immediate and delayed phases of PG synthesis (Croxtall et al., 1996Go).

Airway epithelial cells play a crucial role in the modulation of the inflammatory response by controlling the level of PGs that they produce. Human lung epithelial cells release increased levels of PGs in response to various stimuli, including cytokines such as interleukin-1ß (IL-1ß), interferon-g (IFN-{gamma}) and tumour necrosis factor-{alpha} (TNF-{alpha}), or Ca2+-mobilizing agents such as bradykinin and Ca2+ ionophores (Choudhury et al., 2000Go; Mitchell et al., 1999Go; Saunders et al., 1999Go). Such release has been shown to be dependent on elevation of cytosolic Ca2+ levels and activation of cPLA2-{alpha} (Tokumoto et al., 1994Go).

Surprisingly, the intracellular membrane to which cPLA2-{alpha} relocates following elevation of cytosolic Ca2+ in airway epithelial cells has not been investigated previously. In addition, although the expression levels and activities of the COX-1 and COX-2 enzymes have been studied extensively in epithelial cells, their subcellular locations have not been determined. In this study, we show that cPLA2-{alpha} relocates to the Golgi apparatus of human A549 airway epithelial cells in a Ca2+-dependent manner, where its distribution overlaps that of the COX-1 isoform.


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Materials
Goat polyclonal antibodies to cPLA2-{alpha} were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-COX-1 and COX-2 antibodies were from Cayman Chemical Company (Ann Arbor, MI). Mouse monoclonal antibody against ß-COP was obtained from Sigma (Poole, UK). Mouse monoclonal antibodies against ERGIC53 and ß-1,4-galactosyltransferase (GalT) were kindly provided by H. P. Hauri (Basel, Switzerland) and T. Suganuma (Miyazaki, Japan), respectively. Mouse polyclonal antibody against TGN46 was prepared as described previously (Towler et al., 2000Go). Rhodamine-conjugated wheat germ agglutinin (WGA) and concanavalin A (ConA) were purchased from Sigma. Secondary anti-goat and anti-mouse AlexaFluor488- and 594-conjugated antibodies were from Molecular Probes (Eugene, OR) and anti-goat HRP antibodies were from Pierce (Tattenhall, UK). All other standard reagents and chemicals were from Sigma or BDH (Poole, UK).

Cell culture
A549 human lung carcinoma epithelial cells (ATCC, Rockville, MD) were cultured at 37°C in a humid atmosphere containing 5% CO2 in air. Cells were grown in DMEM supplemented with 10% foetal bovine serum, penicillin (100 U/ml), streptomycin (100 µg/ml) and 2 mM L-glutamine.

Immunofluorescence microscopy
The method for immunofluorescence microscopy was adapted from earlier protocols (Barwise and Walker, 1996Go; Heggeness et al., 1977Go). Cells were grown on glass coverslips in 6-well dishes overnight. Media was removed and the cells were washed three times with pre-warmed (37°C) PBS and fixed in pre-warmed 10% formalin in neutral buffered saline (approximately 4% formaldehyde; Sigma) for 5 minutes. All subsequent steps were performed at room temperature. After fixation, the cells were permeabilized with 0.1% Triton X-100 in PBS for 5 minutes and fixed once again for 5 minutes. The cells were then washed three times with PBS and incubated in sodium borohydride solution (1 mg/ml in PBS) for 5 minutes. Following three further PBS wash steps, the cells were blocked in 5% rabbit serum in PBS for 3 hours. The cells were then incubated with primary antibodies (diluted 1:100 into PBS-5% serum) overnight. Cells were washed eight times with PBS then incubated with AlexaFluor488- and 594-conjugated secondary antibodies, or rhodamine-conjugated WGA or ConA (10 µg/ml) for 3 hours. The cells were then washed eight times with PBS and mounted onto slides in Citifluor mounting medium (Agar Scientific, Stansted, UK).

A23187 and brefeldin A (BFA) treatment
Cells were grown on coverslips in 6-well dishes overnight. Prior to stimulation, media was removed and the cells were washed three times with pre-warmed (37°C) PBS. For A23187 stimulations, PBS was removed and 5 µM A23187 in HEPES/Tyrode's buffer containing 1 mM Ca2+ was added. The cells were then incubated at 37°C for 1 minute, after which time the buffer was removed and fixative was added immediately. For BFA treatment, cells were stimulated as above and then BFA was added to the cells at a final concentration of 10 µg/ml. Cells were incubated at 37°C for 30 minutes then fixed immediately.

Confocal imaging
Confocal fluorescence microscopy was performed using a Leica TCS NT spectral confocal imaging system coupled to a Leica DM IRBE inverted microscope. Each confocal section was the average of four scans to obtain optimal resolution. The system was used to generate individual sections that were 0.485 µm thick. All figures shown in this study represent 0.485 µm sections taken through the centre of the nucleus. To capture double-labelled samples, sequential scanning of each fluorescence channel was performed (according to the manufacturers guidelines) to avoid cross-contamination of fluorescence signals. To quantify the degree of cPLA2-{alpha} overlap with Golgi markers and the COX isoforms, the number of cPLA2-{alpha}+ structures was calculated by visual inspection of 0.485 µm thick sections from four individual cells. For each marker, the number of cPLA2-{alpha}-positive structures that were labelled by the specific marker were counted and expressed as a percentage of the total number of cPLA2-{alpha}-positive structures.

SDS-PAGE and western blotting
Proteins (20 µg per well) were separated on SDS-polyacrylamide gels using a discontinuous buffer system (Laemmli, 1970Go). For western blot analysis, proteins were transferred to nitrocellulose (Towbin et al., 1979Go). Subsequently, the nitrocellulose blots were blocked in 5% non-fat milk in PBS-0.1% Triton X-100 for 1 hour. Primary antibody incubations (1:1000) were carried out overnight at room temperature, followed by 1 hour incubations with the appropriate horseradish peroxidase-conjugated secondary antibody. For antigenic adsorption, the antibody was incubated with its corresponding blocking peptide (1:5 ratio of mg antibody:mg antigen) for 30 minutes at room temperature prior to being incubated with the nitrocellulose blot. Immunoreactive bands were visualized using an enhanced chemiluminescence (ECL) detection kit (Pierce) according to the manufacturer's instructions. Developed films were photographed and captured using the FujiFilm Intelligent dark Box II with the Image Reader Las-1000 package.


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Ca2+-induced relocation of cPLA2-{alpha}
To determine the subcellular location of cPLA2-{alpha} in A549 epithelial cells, immunofluorescence microscopy was carried out using an antibody that is specific for the {alpha}-isoform of cPLA2 (Fig. 1A, lane 1). Antigenic adsorption of this antibody with its corresponding blocking peptide abolished detection of cPLA2-{alpha} by both western blotting (Fig. 1A, lane 2) and immunofluorescence microscopy (data not shown).



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Fig. 1. Distribution of cPLA2-{alpha} in resting and A23817-stimulated A549 cells. (A) cPLA2-{alpha} was detected in A549 cell lysates (20 µg protein) using a goat polyclonal antibody (lane 1). Also shown is the control corresponding to goat polyclonal antibody pre-incubated with blocking peptide (lane 2). (B) Cells were grown on coverslips and incubated with buffer alone (control) or stimulated with 5 µM A23187 (in HEPES/Tyrode's buffer) in the presence of 1 mM extracellular Ca2+ for 1 minute. Cells were then fixed and permeabilized, and cPLA2-{alpha} was detected using immunofluorescence microscopy. Bar, 10 µm.

 

Using this specific antibody, a comparison of the location of cPLA2-{alpha} in resting and Ca2+ ionophore-treated A549 cells was carried out (Fig. 1B). In nonstimulated cells, cPLA2-{alpha} was present in the cytosol and in the nucleus. Following elevation of the cytosolic Ca2+ concentration using the Ca2+ ionophore A23187, a specific relocation of cPLA2-{alpha} to a compact juxtanuclear reticulum was evident (Fig. 1B). Secondary antibody and peptide-adsorbed antibody controls gave no staining (data not shown), confirming that the staining observed corresponded to specific staining of cPLA2-{alpha}. Identical staining patterns, corresponding to juxtanuclear relocation, were also seen following stimulation with the physiological stimulus histamine (data not shown).

A strong possibility was that the juxtanuclear staining pattern observed in ionophore-treated cells corresponded to the Golgi apparatus. To test this possibility, ionophore-treated cells were counterstained with rhodamine-conjugated WGA, a lectin that selectively labels N-acetyl-ß-D-glucosaminyl residues, which are found on proteins in the Golgi apparatus as well in the plasma membrane and the nuclear envelope (Virtanen et al., 1980Go). The results (Fig. 2A) revealed co-localization of cPLA2-{alpha} with WGA-positive perinuclear structures corresponding to the Golgi complex. No overlap was seen at the plasma membrane or nuclear membrane. Counterstaining cells with ConA, a lectin that can be used as a marker for the endoplasmic reticulum (ER) (Virtanen et al., 1980Go), revealed very little overlap (Fig. 2B), indicating that cPLA2-{alpha} was not relocating to the ER.



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Fig. 2. Distribution of cPLA2-{alpha} compared with Golgi and ER markers. Cells were stimulated with 5 µM A23187 (in HEPES/Tyrode's buffer) in the presence of 1 mM extracellular Ca2+ for 1 minute. Cells were then incubated with goat polyclonal anti-cPLA2-{alpha} followed by AlexaFluor488-conjugated anti-goat antibody with either rhodamine-conjugated WGA (A) or rhodamine-conjugated ConA (B). Cells were visualized using immunofluorescence microscopy. Bar, 10 µm.

 

cPLA2-{alpha} co-localizes with trans-Golgi stack and trans-Golgi network (TGN) markers
In order to determine the precise subcompartment of the Golgi apparatus to which cPLA2-{alpha} was relocating, cells were labelled with antibodies specific for various Golgi subcompartments. First, in order to determine whether cPLA2-{alpha} was localized to the ER-Golgi intermediate compartment (ERGIC), ionophore-treated cells were selectively labelled with antibodies against the 53 kDa ERGIC-resident protein ERGIC53 (Schweizer et al., 1988Go; Schweizer et al., 1990Go). Double labelling of cells with this antibody and an anti-cPLA2-{alpha} antibody (Fig. 3A) demonstrated that, although the juxtanuclear staining pattern given by each of these antibodies was similar, only a small amount of overlap (indicated by yellow in the merged image) was evident. Thus, cPLA2-{alpha} does not appear to relocalize to the ERGIC upon ionophore stimulation.



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Fig. 3. Distribution of cPLA2-{alpha} compared with markers for the ER-Golgi intermediate compartment and the cis-Golgi cisternae. Cells were stimulated with 5 µM A23187 (in HEPES/Tyrode's buffer) in the presence of 1 mM extracellular Ca2+ for 1 minute. Cells were then incubated with goat polyclonal anti-cPLA2-{alpha}, with either mouse monoclonal anti-ERGIC53 (A) or mouse monoclonal anti-ß-COP (B) followed by anti-goat AlexaFluor488 and anti-mouse AlexaFluor594 secondary antibodies. Cells were visualized using immunofluorescence microscopy. Bar, 10 µm.

 

In order to label specifically the cis-Golgi network and the cis-cisternae of the Golgi apparatus, cells were labelled with an antibody against ß-COP, a subunit of the Golgi-associated coatamer protein complex COPI (Wieland and Harter, 1999Go). Again, this antibody gave a staining pattern similar to that observed for cPLA2-{alpha} (Fig. 3B). However, only partial overlap of the two staining patterns was observed and close inspection revealed that cPLA2-{alpha} and ß-COP were associated with neighbouring structures that were not superimposed (Fig. 3B, merge).

The distribution of cPLA2-{alpha} was also compared with the distribution of proteins of the trans-Golgi cisternae and the TGN. First, cells were specifically labelled with antibodies against GalT, an enzyme found in both the trans-Golgi cisternae and the TGN (Nilsson et al., 1993Go; Roth et al., 1985Go). The results (Fig. 4A) demonstrated that a high degree of overlap was evident, indicating that cPLA2-{alpha} is localized predominantly to the trans side of the Golgi complex. To investigate this further, cells were counterstained with an antibody against TGN46, a TGN-resident protein (Prescott et al., 1997Go). Again, a high degree of overlap between the two antibodies was observed (Fig. 4B), indicating that cPLA2-{alpha} locates to both the trans-Golgi stack and the TGN subcompartments following ionophore stimulation.



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Fig. 4. Distribution of cPLA2-{alpha} compared with markers for the trans-Golgi stack and the TGN. Cells were stimulated with 5 µM A23187 (in HEPES/Tyrode's buffer) in the presence of 1 mM extracellular Ca2+ for 1 minute. Cells were then incubated with goat polyclonal anti-cPLA2-{alpha}, with either mouse monoclonal anti-GalT (A) or mouse monoclonal anti-TGN46 (B) followed by anti-goat AlexaFluor488 and anti-mouse AlexaFluor594 secondary antibodies. Bar, 10 µm.

 

Quantification of the degree of overlap between cPLA2-{alpha} and the various Golgi and ER markers was performed as described in the Materials and Methods. The results (Fig. 5) confirm that cPLA2-{alpha} overlaps predominantly with markers of the Golgi apparatus, whereas little overlap is seen with the ER marker (approximately 72% overlap with WGA compared with only 23% with ConA). In particular, cPLA2-{alpha} showed approximate 70% and 60% overlap with the trans-Golgi markers, GalT and TGN46, respectively. By contrast, markers of the cis-Golgi (ERGIC53, ß-COP) showed only 20-25% overlap.



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Fig. 5. Analysis of the degree of cPLA2-{alpha} overlap with markers specific for Golgi subcompartments. The percentage overlap was calculated as described in the Materials and Methods. The graph represents results obtained from four independent experiments (±s.d.).

 

Ca2+-induced Golgi localization of cPLA2-{alpha} can be disrupted with BFA
To test whether cPLA2-{alpha} was relocating to the lipid bilayer membranes of the trans-Golgi stack and the TGN, and was not just in close proximity to these membranes, cells were treated with BFA, a potent fungal metabolite that is known to inhibit trafficking through the secretory pathway (reviewed by Klausner et al., 1992Go). Previous studies using BFA have shown that it blocks protein transport into the Golgi complex, resulting in the redistribution of Golgi-stack proteins and TGN-resident proteins to the ER and endosomal systems, respectively (Lippincott-Schwartz et al., 1990Go; Lippincott-Schwartz et al., 1991Go; Wood et al., 1991Go).

Cells were treated with BFA (10 µg/ml) for 30 minutes following stimulation, and the location of cPLA2-{alpha} was analysed by immunofluorescence microscopy. The results indicate that BFA treatment caused a redistribution of cPLA2-{alpha} from the Golgi to dispersed cytosolic structures (Fig. 6).



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Fig. 6. Effects of BFA treatment on the localization of cPLA2-{alpha}. Cells were stimulated with 5 µM A23187 (in HEPES/Tyrode's buffer) in the presence of 1 mM extracellular Ca2+ for 1 minute. BFA (10 µg/ml) was then added to the cells for 30 minutes and the cells were fixed and permeabilized. cPLA2-{alpha} was then detected by immunofluorescence microscopy. Bar, 10 µm.

 

cPLA2-{alpha} co-localizes with COX-1 but not with COX-2
The data above demonstrated that, in A549 lung epithelial cells, cPLA2-{alpha} relocates primarily to the Golgi complex and the TGN. In order to determine whether COX enzymes showed a similar localization, their distribution was analysed by confocal microscopy. Cells were double labelled with anti-cPLA2-{alpha} antibodies and specific monoclonal antibodies raised against either COX-1 or COX-2. The results (Fig. 7) demonstrated that the two COX isoforms were present at distinct subcellular locations in A549 lung epithelial cells. COX-1 was constitutively localized at the Golgi complex and in cytoplasmic structures in both control and ionophore-stimulated cells, whereas COX-2 was always present at the nuclear membrane and in cytosolic speckles. Prior to stimulation, no co-localization of cPLA2-{alpha} with either of the COX isoenzymes was observed (data not shown). Interestingly, however, following stimulation with A23187, a specific co-localization of cPLA2-{alpha} with COX-1 but not with COX-2 was evident in regions of the Golgi apparatus (Fig. 7A and 7B, merged images). Quantification of the degree of overlap revealed that 63±14% of the cPLA2-{alpha}+ structures overlapped COX-1 immunoreactivity, whereas only a 3±5% overlap was evident for COX-2 (Fig. 7C).



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Fig. 7. (A) Distribution of cPLA2-{alpha} compared with COX-1 and COX-2. Cells were grown on coverslips and stimulated with 5 µM A23187 (in HEPES/Tyrode's buffer) in the presence of 1 mM extracellular Ca2+ for 1 minute. Cells were then incubated with goat polyclonal anti-cPLA2-{alpha}, with either mouse monoclonal COX-1 (A) or mouse monoclonal COX-2 (B) followed by anti-goat AlexaFluor488 and anti-mouse AlexaFluor594 secondary antibodies. Bar, 10 µm. (C) Quantification of the degree of cPLA2-{alpha} overlap with COX-1 and COX-2. Percentage overlap was calculated as described in the Materials and Methods. The graph represents results obtained from four independent experiments (±s.d.).

 


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The relocation of cPLA2-{alpha} to the Golgi complex
The A549 human lung epithelial cell line is a popular model for the study of PG release by airway epithelial cells. Although the activation of cPLA2-{alpha} and the subsequent release of arachidonic acid have been studied extensively in these cells, to date the subcellular location of cPLA2-{alpha} in A549 lung epithelial cells has not been determined. Using high-resolution confocal immunofluorescence microscopy, we have shown here that cPLA2-{alpha} is present in the cytosol and in the nuclei of resting A549 cells. Following stimulation with the Ca2+ ionophore A23187, a specific relocation of cPLA2-{alpha} to the region of the Golgi apparatus was observed. Double labelling with the Golgi-binding lectin WGA confirmed that cPLA2-{alpha} was localized at the Golgi apparatus (Fig. 2). This finding is consistent with previous studies on Madin-Darby canine kidney epithelial cells (Evans et al., 2001Go) and kidney LLC-PK1 epithelial cells (Choukroun et al., 2000Go), both of which also revealed Golgi localization of cPLA2-{alpha}.

Further investigation of the Golgi localization using markers for specific Golgi subcompartments (Figs 3, 4) revealed that cPLA2-{alpha} was localized primarily at the trans-Golgi cisternae of the Golgi complex and at the TGN. Only a small amount of cPLA2-{alpha} was found associated with the ERGIC or the cis face of the Golgi stack. In addition, disruption of the Golgi apparatus using BFA caused redistribution of cPLA2-{alpha} to cytosolic structures, as expected for a Golgi-associated protein (Fig. 6). This finding is consistent with previous work on Madin-Darby canine kidney epithelial cells, which demonstrated that the Golgi localization of over-expressed GFP-cPLA2-{alpha} could be disrupted with BFA (Evans et al., 2001Go).

Previous immunofluorescence studies on non-epithelial cells have shown that cPLA2-{alpha} relocates predominantly to the ER, the nuclear envelope or the perinuclear region (Gijon and Leslie, 1999Go; Glover et al., 1995Go; Hirabayashi et al., 1999Go; Peters-Golden et al., 1996Go; Schievella et al., 1995Go). However, here we found very little relocation of cPLA2-{alpha} to the ER of A549 cells. Thus, it appears that Golgi localization of cPLA2-{alpha} may be specific for epithelial cells, suggesting that the precise relocation of cPLA2-{alpha} to intracellular membranes is dependent on cell type.

Co-localization of cPLA2-{alpha} with COX-1
Following our observations on the cell-specific relocation of cPLA2-{alpha} to the Golgi apparatus, we investigated the subcellular location of the COX enzymes that lie downstream of cPLA2-{alpha} in PG synthesis. Our results revealed that, in A549 cells, the COX-1 and COX-2 isoforms have distinct subcellular locations. COX-1 was constitutively localized to the Golgi apparatus and small cytoplasmic structures, whereas COX-2 was detected mainly on ER-like punctate structures and the nuclear membrane. Previous studies on bovine aortic endothelial cells have also shown distinct intracellular locations for these two proteins, with COX-1 localized at the nuclear membrane and ER, and COX-2 present in cytosolic vesicular structures (Liou et al., 2000Go). It is feasible that such distinct spatial organization of the COX isoforms may, in part, provide an explanation for their observed differences in function. Although the two COX enzymes show similar enzymatic properties in vitro, recent evidence has shown that their role in the regulation of PG synthesis is clearly segregated (reviewed in Smith and Langenbach, 2001Go). COX-1 has been shown to be pivotal in the immediate phase of PG synthesis, which is elicited by Ca2+-mobilizing agonists, whereas COX-2 is involved in the delayed response, which lasts for several hours following activation by proinflammatory stimuli. Thus, it is possible that the distinct subcellular location of the two isoenzymes is crucial for mediating specific responses that depend on the length and magnitude of the stimulus.

Most interestingly, our results here revealed a specific co-localization of a pool of COX-1 with cPLA2-{alpha} at the Golgi complex. By contrast, no co-localization with COX-2 was evident. Whether this is due to direct interaction of the COX-1 isoform with cPLA2-{alpha} remains to be investigated. If this is the case, the constitutive localization of COX-1 at the Golgi complex could be involved in recruiting cPLA2-{alpha} specifically to this site following stimulation. Alternatively, both proteins could favour localization in phospholipid membranes of a certain composition, which are present only in specific subdomains of the Golgi apparatus.

The co-localization of cPLA2-{alpha} with the COX-1 isoform presented here is a novel finding. Previous studies have shown that COX-1 but not COX-2 co-localizes with prostacyclin synthase in phorbol ester-induced endothelial cells (Liou et al., 2000Go), further suggesting that enzymes involved in eicosanoid production might be complexed or in very close proximity to one another. Such an association could ensure the immediate synthesis of PGs following an increase in intracellular Ca2+. It must be noted, however, that the studies carried out here were performed on cells that displayed basal, uninduced levels of COX-2 expression (Croxtall et al., 1996Go). Following exposure to mitogenic or inflammatory stimuli, cells exhibit increased levels of COX-2 expression (Smith and Langenbach, 2001Go) and it is possible that this induced COX-2 shows an alternative intracellular localization. Furthermore, whether or not any of this induced COX-2 co-localizes with cPLA2-{alpha} under these conditions remains to be investigated.

Potential roles for cPLA2-{alpha} in the Golgi complex
Previous reports have implicated PLA2 activity in the maintenance of correct Golgi structure and function. These include studies on Golgi formation (Choukroun et al., 2000Go), tubulation (de Figueiredo et al., 1999Go) and membrane remodelling (Schmidt et al., 1999Go). In addition, several studies using PLA2 inhibitors have highlighted the importance of PLA2 activity in trafficking between the ER and the Golgi (de Figueiredo et al., 2000Go; Drecktrah and Brown, 1999Go; Slomiany et al., 1992Go), endocytosis (Mayorga et al., 1993Go), exocytosis (Slomiany et al., 1998Go) and the intracellular trafficking of secretory proteins (Choukroun et al., 2000Go; Tagaya et al., 1993Go). Since most of the inhibitors used in such studies were broad-range inhibitors, the precise species of PLA2 involved in these processes is unclear. Nevertheless, studies related directly to cytosolic PLA2-{alpha} do confirm that this member of the PLA2 family is involved in trafficking events and maintenance of Golgi structure and function (Choukroun et al., 2000Go; Slomiany et al., 1992Go).

The exact role of cPLA2 in Golgi structure and function is unclear. It is evident that PLA2s have several features that could be potentially very useful in controlling the membrane fusion processes that mediate intracellular trafficking. PLA2 activity generates two bioactive compounds, lysophospholipid and arachidonic acid. It has been shown previously that lysophospholipid is able to stimulate secretion (Murakami et al., 1991Go) and arachidonic acid is known to promote membrane bilayers to form a HII phase (Burger and Verkleij, 1990Go), a lipid arrangement that may be imperative during membrane fusion events. Alternatively, the PLA2-dependent changes in trafficking observed might be due to a direct effect of the enzyme on the lipid composition of the Golgi membrane. It is known that the asymmetry of the lipid bilayer is necessary to maintain the fundamental properties of the various intracellular compartments within the cell (Verkade and Simons, 1997Go). Following this, it is possible that hydrolysis of membrane phospholipids by PLA2 could modify the composition of the two leaflets, thereby playing a crucial role in controlling the structure and trafficking of Golgi membranes. Furthermore, whether the cell-specific Golgi localization of cPLA2-{alpha} in A549 cells is of particular importance in maintaining the polarity of these epithelial cells remains to be investigated. Epithelial cell polarity is derived from the selective distribution of their proteins and lipids into distinct plasma membrane domains. Previous studies have implied that cPLA2 activity has effects on the distribution of only a subset of proteins (Choukroun et al., 2000Go); thus, it is possible that cPLA2-{alpha} may be playing a role in selective membrane trafficking in these cells.

In conclusion, the studies presented here demonstrate that cPLA2-{alpha} relocates in a Ca2+-dependent manner to the Golgi apparatus of human A549 lung epithelial cells where it co-localizes with COX-1. The functional relevance of this co-localization and the mechanisms that underlie the specific targeting to this region remain to be investigated. Nevertheless, these data, together with recent studies on the role of PLA2 activity in maintaining Golgi structure and function, suggest that the Golgi location of cPLA2-{alpha} might play an important role in membrane trafficking events.


    References
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 Summary
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 Materials and Methods
 Results
 Discussion
 References
 

Barwise, J. L. and Walker, J. H. (1996). Annexins II, IV, V and VI relocate in response to rises in intracellular calcium in human foreskin fibroblasts. J. Cell Sci. 109,247 -255.[Abstract/Free Full Text]

Burger, K. N. and Verkleij, A. J. (1990). Membrane fusion. Experientia 46,631 -644.[Medline]

Choudhury, Q. G., McKay, D. T., Flower, R. J. and Croxtall, J. D. (2000). Investigation into the involvement of phospholipases A(2) and MAP kinases in modulation of AA release and cell growth in A549 cells. Br. J. Pharmacol. 131,255 -265.[Abstract/Free Full Text]

Choukroun, G. J., Marshansky, V., Gustafson, C. E., McKee, M., Hajjar, R. J., Rosenzweig, A., Brown, D. and Bonventre, J. V. (2000). Cytosolic phospholipase A(2) regulates golgi structure and modulates intracellular trafficking of membrane proteins. J. Clin. Invest. 106,983 -993.[Abstract/Free Full Text]

Clark, J. D., Lin, L. L., Kriz, R. W., Ramesha, C. S., Sultzman, L. A., Lin, A. Y., Milona, N. and Knopf, J. L. (1991). A novel arachidonic acid-selective cytosolic PLA2 contains a Ca(2+)-dependent translocation domain with homology to PKC and GAP. Cell 65,1043 -1051.[Medline]

Croxtall, J. D., Newman, S. P., Choudhury, Q. and Flower, R. J. (1996). The concerted regulation of cPLA2, COX2, and lipocortin 1 expression by IL-1ß in A549 cells. Biochem. Biophys. Res. Commun. 220,491 -495.[CrossRef][Medline]

de Figueiredo, P., Polizotto, R. S., Drecktrah, D. and Brown, W. J. (1999). Membrane tubule-mediated reassembly and maintenance of the Golgi complex is disrupted by phospholipase A2 antagonists. Mol. Biol. Cell 10,1763 -1782.[Abstract/Free Full Text]

de Figueiredo, P., Drecktrah, D., Polizotto, R. S., Cole, N. B., Lippincott- Schwartz, J. and Brown, W. J. (2000). Phospholipase A2 antagonists inhibit constitutive retrograde membrane traffic to the endoplasmic reticulum. Traffic 1, 504-511.[CrossRef][Medline]

Dennis, E. A. (1997). The growing phospholipase A2 superfamily of signal transduction enzymes. Trends Biochem. Sci. 22,1 -2.[CrossRef][Medline]

Drecktrah, D. and Brown, W. J. (1999). Phospholipase A(2) antagonists inhibit nocodazole-induced Golgi ministack formation: evidence of an ER intermediate and constitutive cycling. Mol. Biol. Cell 10,4021 -4032.[Abstract/Free Full Text]

Earnest, D. L., Hixson, L. J. and Alberts, D. S. (1992). Piroxicam and other cyclooxygenase inhibitors: potential for cancer chemoprevention. J. Cell. Biochem. Suppl. 16I,156 -166.

Evans, J. H., Spencer, D. M., Zweifach, A. and Leslie, C. C. (2001). Intracellular calcium signals regulating cytosolic phospholipase A2 translocation to internal membranes. J. Biol. Chem. 276,30150 -30160.[Abstract/Free Full Text]

Gijon, M. A. and Leslie, C. C. (1999). Regulation of arachidonic acid release and cytosolic phospholipase A2 activation. J. Leukocyte Biol. 65,330 -336.[Abstract]

Gijon, M. A., Spencer, D. M., Kaiser, A. L. and Leslie, C. C. (1999). Role of phosphorylation sites and the C2 domain in regulation of cytosolic phospholipase A2. J. Cell Biol. 145,1219 -1232.[Abstract/Free Full Text]

Glover, S., de Carvalho, M. S., Bayburt, T., Jonas, M., Chi, E., Leslie, C. C. and Gelb, M. H. (1995). Translocation of the 85-kDa phospholipase A2 from cytosol to the nuclear envelope in rat basophilic leukemia cells stimulated with calcium ionophore or IgE/antigen [published erratum appears in J. Biol. Chem. (1995). 270, 20870]. J. Biol. Chem. 270,15359 -15367.[Abstract/Free Full Text]

Heggeness, M. H., Wang, K. and Singer, S. J. (1977). Intracellular distributions of mechanochemical proteins in cultured fibroblasts. Proc. Natl. Acad. Sci. USA 74,3883 -3887.[Abstract]

Hirabayashi, T., Kume, K., Hirose, K., Yokomizo, T., Iino, M., Itoh, H. and Shimizu, T. (1999). Critical duration of intracellular Ca2+ response required for continuous translocation and activation of cytosolic phospholipase A2. J. Biol. Chem. 274,5163 -5169.[Abstract/Free Full Text]

Klausner, R. D., Donaldson, J. G. and Lippincott-Schwartz, J. (1992). Brefeldin A: insights into the control of membrane traffic and organelle structure. J. Cell Biol. 116,1071 -1080.[Medline]

Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227,680 -685.[Medline]

Liou, J. Y., Shyue, S. K., Tsai, M. J., Chung, C. L., Chu, K. Y. and Wu, K. K. (2000). Colocalization of prostacyclin synthase with prostaglandin H synthase-1 (PGHS-1) but not phorbol ester-induced PGHS-2 in cultured endothelial cells. J. Biol. Chem. 275,15314 -15320.[Abstract/Free Full Text]

Lippincott-Schwartz, J., Donaldson, J. G., Schweizer, A., Berger, E. G., Hauri, H. P., Yuan, L. C. and Klausner, R. D. (1990). Microtubule-dependent retrograde transport of proteins into the ER in the presence of brefeldin A suggests an ER recycling pathway. Cell 60,821 -836.[Medline]

Lippincott-Schwartz, J., Yuan, L., Tipper, C., Amherdt, M., Orci, L. and Klausner, R. D. (1991). Brefeldin A's effects on endosomes, lysosomes, and the TGN suggest a general mechanism for regulating organelle structure and membrane traffic. Cell 67,601 -616.[Medline]

Mayorga, L. S., Colombo, M. I., Lennartz, M., Brown, E. J., Rahman, K. H., Weiss, R., Lennon, P. J. and Stahl, P. D. (1993). Inhibition of endosome fusion by phospholipase A2 (PLA2) inhibitors points to a role for PLA2 in endocytosis. Proc. Natl. Acad. Sci. USA 90,10255 -10259.[Abstract]

Mitchell, R. A., Metz, C. N., Peng, T. and Bucala, R. (1999). Sustained mitogen-activated protein kinase (MAPK) and cytoplasmic phospholipase A2 activation by macrophage migration inhibitory factor (MIF). Regulatory role in cell proliferation and glucocorticoid action. J. Biol. Chem. 274,18100 -18106.[Abstract/Free Full Text]

Murakami, M., Kudo, I., Fujimori, Y., Suga, H. and Inoue, K. (1991). Group II phospholipase A2 inhibitors suppressed lysophosphatidylserine-dependent degranulation of rat peritoneal mast cells. Biochem. Biophys. Res. Commun. 181,714 -721.[Medline]

Nilsson, T., Pypaert, M., Hoe, M. H., Slusarewicz, P., Berger, E. G. and Warren, G. (1993). Overlapping distribution of two glycosyltransferases in the Golgi apparatus of HeLa cells. J. Cell Biol. 120,5 -13.[Abstract]

Peters-Golden, M., Song, K., Marshall, T. and Brock, T. (1996). Translocation of cytosolic phospholipase A2 to the nuclear envelope elicits topographically localized phospholipid hydrolysis. Biochem. J. 318,797 -803.[Medline]

Prescott, A. R., Lucocq, J. M., James, J., Lister, J. M. and Ponnambalam, S. (1997). Distinct compartmentalization of TGN46 and ß 1,4-galactosyltransferase in HeLa cells. Eur. J. Cell Biol. 72,238 -246.[Medline]

Reddy, S. T. and Herschman, H. R. (1997). Prostaglandin synthase-1 and prostaglandin synthase-2 are coupled to distinct phospholipases for the generation of prostaglandin D2 in activated mast cells. J. Biol. Chem. 272,3231 -3237.[Abstract/Free Full Text]

Roth, J., Taatjes, D. J., Lucocq, J. M., Weinstein, J. and Paulson, J. C. (1985). Demonstration of an extensive trans-tubular network continuous with the Golgi apparatus stack that may function in glycosylation. Cell 43,287 -295.[Medline]

Saunders, M. A., Belvisi, M. G., Cirino, G., Barnes, P. J., Warner, T. D. and Mitchell, J. A. (1999). Mechanisms of prostaglandin E2 release by intact cells expressing cyclooxygenase-2: evidence for a `two-component' model. J. Pharmacol. Exp. Ther. 288,1101 -1106.[Abstract/Free Full Text]

Schievella, A. R., Regier, M. K., Smith, W. L. and Lin, L. L. (1995). Calcium-mediated translocation of cytosolic phospholipase A2 to the nuclear envelope and endoplasmic reticulum. J. Biol. Chem. 270,30749 -30754.[Abstract/Free Full Text]

Schmidt, A., Wolde, M., Thiele, C., Fest, W., Kratzin, H., Podtelejnikov, A. V., Witke, W., Huttner, W. B. and Soling, H. D. (1999). Endophilin I mediates synaptic vesicle formation by transfer of arachidonate to lysophosphatidic acid. Nature 401,133 -141.[CrossRef][Medline]

Schweizer, A., Fransen, J. A., Bachi, T., Ginsel, L. and Hauri, H. P. (1988). Identification, by a monoclonal antibody, of a 53-kD protein associated with a tubulo-vesicular compartment at the cis-side of the Golgi apparatus. J. Cell Biol. 107,1643 -1653.[Abstract]

Schweizer, A., Fransen, J. A., Matter, K., Kreis, T. E., Ginsel, L. and Hauri, H. P. (1990). Identification of an intermediate compartment involved in protein transport from endoplasmic reticulum to Golgi apparatus. Eur. J. Cell Biol. 53,185 -196.[Medline]

Sierra-Honigmann, M. R., Bradley, J. R. and Pober, J. S. (1996). `Cytosolic' phospholipase A2 is in the nucleus of subconfluent endothelial cells but confined to the cytoplasm of confluent endothelial cells and redistributes to the nuclear envelope and cell junctions upon histamine stimulation. Lab. Invest. 74,684 -695.[Medline]

Slomiany, A., Grzelinska, E., Kasinathan, C., Yamaki, K., Palecz, D. and Slomiany, B. L. (1992). Function of intracellular phospholipase A2 in vectorial transport of apoproteins from ER to Golgi. Int. J. Biochem. 24,1397 -1406.[Medline]

Slomiany, A., Nowak, P., Piotrowski, E. and Slomiany, B. L. (1998). Effect of ethanol on intracellular vesicular transport from Golgi to the apical cell membrane: role of phosphatidylinositol 3-kinase and phospholipase A2 in Golgi transport vesicles association and fusion with the apical membrane. Alcohol Clin. Exp. Res. 22,167 -175.[Medline]

Smith, W. L. and Langenbach, R. (2001). Why there are two cyclooxygenase isozymes. J. Clin. Invest. 107,1491 -1495.[Free Full Text]

Tagaya, M., Henomatsu, N., Yoshimori, T., Yamamoto, A., Tashiro, Y. and Fukui, T. (1993). Correlation between phospholipase A2 activity and intra-Golgi protein transport reconstituted in a cell-free system. FEBS Lett. 324,201 -204.[CrossRef][Medline]

Tokumoto, H., Croxtall, J. D. and Flower, R. J. (1994). Differential role of extra- and intracellular calcium in bradykinin and interleukin 1 {alpha} stimulation of arachidonic acid release from A549 cells. Biochim. Biophys. Acta 1211,301 -309.[Medline]

Towbin, H., Staehelin, T. and Gordon, J. (1979). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76,4350 -4354.[Abstract]

Towler, M. C., Prescott, A. R., James, J., Lucocq, J. M. and Ponnambalam, S. (2000). The manganese cation disrupts membrane dynamics along the secretory pathway. Exp. Cell Res. 259,167 -179.[CrossRef][Medline]

Vane, J. R. and Botting, R. M. (1995). New insights into the mode of action of anti-inflammatory drugs. Inflamm. Res. 44,1 -10.[Medline]

Verkade, P. and Simons, K. (1997). Robert Feulgen Lecture 1997. Lipid microdomains and membrane trafficking in mammalian cells. Histochem. Cell Biol. 108,211 -220.[CrossRef][Medline]

Virtanen, I., Ekblom, P. and Laurila, P. (1980). Subcellular compartmentalization of saccharide moieties in cultured normal and malignant cells. J. Cell Biol. 85,429 -434.[Abstract]

Wieland, F. and Harter, C. (1999). Mechanisms of vesicle formation: insights from the COP system. Curr. Opin. Cell Biol. 11,440 -446.[CrossRef][Medline]

Wood, S. A., Park, J. E. and Brown, W. J. (1991). Brefeldin A causes a microtubule-mediated fusion of the trans-Golgi network and early endosomes. Cell 67,591 -600.[Medline]