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J Biol Chem, Vol. 274, Issue 39, 27726-27733, September 24, 1999

Secretory Group IIA Phospholipase A₂ Generates Anti-apoptotic Survival Signals in Kidney Fibroblasts^*

Yingpei Zhang, John Lemasters^§, and Brian Herman^§¶

From the  Department of Cellular and Structural Biology, The University of Texas Health Science Center at San Antonio, San Antonio, Texas 78284-7762 and the ^§ Department of Cell Biology and Anatomy, University of North Carolina, Chapel Hill, North Carolina 27599-7090

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Mammalian group IIA phospholipase A₂ (PLA₂) is believed to play important roles in inflammation, cell injury, and tumor resistance. However, the cellular site of action has not been clearly defined as it has long been recognized that group IIA PLA₂ is both a secretory and mitochondrial protein. The purpose of this study was to determine the subcellular target of the group IIA PLA₂ and its role in apoptosis stimulated by growth factor withdrawal. Cloning of the rat liver group IIA PLA₂ demonstrated a typical secretory signal and no alternative splicing of the primary transcript. When a sequence including the signal peptide and first 8 residues in the mature enzyme or the entire PLA₂ (including the signal peptide) was fused to enhanced green fluorescent protein, the fusion protein was directed to the secretory pathway rather than mitochondria in baby hamster kidney (BHK) cells. To examine the role of group IIA PLA₂ in cell injury, wild type (wt) rat group IIA PLA₂ and a mutant group IIA PLA₂ containing a His-47  Gln mutation (at the catalytic center) were transfected into BHK cells and cells stably expressing these constructs were isolated. After deprivation of growth factors, both normal BHK cells and BHK cells expressing mutant PLA₂ underwent massive apoptosis, while BHK cells expressing wt PLA₂ showed considerable resistance to growth factor withdrawal-induced apoptosis. The secretory PLA₂ inhibitors 12-epi-scalaradial and aristolochic acid abrogated resistance to apoptosis in the wt PLA₂ expressing cells. These two inhibitors did not induce cell death in the presence of fetal bovine serum, suggesting that they induce cell death by blocking PLA₂ generated survival signals. This study demonstrates that group IIA PLA₂ generates anti-apoptotic survival signals in BHK cells targeting the secretory pathway, and suggests that high levels of group IIA PLA₂ accumulated at inflammatory sites may not only regulate inflammation, but also may protect cells from unnecessary death induced by pro-inflammatory agents.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Mammalian group IIA phospholipase A₂ (PLA₂)¹ catalyzes the release of sn-2 fatty acid from membrane phospholipids. This reaction produces arachidonic acid (AA) and lysophosphatidic acid (LPA) (1-5), both of which are multifunctional lipid mediators. Group IIA PLA₂ also has anti-bacterial activity and functions as a modifier of tumor multiplicity (6-8). In addition to being a secretory/plasma membrane-associated protein (9), group IIA PLA₂ has been isolated from purified mitochondria (10-13), and has been suggested to be localized at inner and outer membrane contact sites (14). Sequence analysis of this mitochondrial group IIA PLA₂ reveals the protein to be nearly identical to the mature secretory enzyme (15), indicating that the pre-protein of mitochondrial group IIA PLA₂ is likely processed at the exactly the same site as the secretory group IIA PLA_2. As discussed in a recent review, mitochondria are thought to be a target of group IIA PLA₂ (2). Using PLA₂ inhibitors and antisense oligonucleotides specific for group IIA PLA₂, we found that group IIA PLA₂ contributed to cell injury during chemical hypoxia in isolated rat hepatocytes (16, 17). Mitochondrial injury is a critical step in cell death during chemical hypoxia (18). Data from our laboratory as well as others have demonstrated that products of PLA₂ catalyzed reactions can induce the mitochondrial permeability transition (MPT), and reagents known to inhibit PLA₂ activity can block induction of the MPT in isolated mitochondria (19, 20). Thus, the mitochondria appear to be a logical target of group IIA PLA₂ where it could induce the MPT and onset of cell death.

The recent discovery of other low molecular PLA₂s (i.e. group V PLA₂) has drawn into question some of the functions previously attributed to group IIA PLA₂ (2). For example, late phase release of AA during mast cell activation was believed to be mediated by group IIA PLA_2, but has recently been found to be normal in a cell line lacking demonstrable group IIA PLA₂ activity (21). Certain reagents used to modulate group IIA PLA₂ activity such as inhibitors, antisense oligonucleotides, and antibodies thought to be specific to group IIA PLA₂, have now been shown to act nonspecifically via group V PLA₂. Given these concerns, the most direct way of elucidating group IIA PLA₂ function is to study cellular phenotype changes after expression of group IIA PLA₂ and its mutants. In this study, we generated cells stably expressing rat group IIA PLA₂ and its His-47 (a residue essential for its catalytic activity) to Gln mutant (22). We found that the group IIA PLA₂ generated anti-apoptotic survival signals in BHK cells. This finding sheds new light on the understanding of the function of group IIA PLA₂ in inflammation and other pathological processes.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Vectors and Reagents-- Enhanced GFP (EGFP) fusion vector pEGFP-N1, expression vector pIRES1neo, Quick Clone rat liver cDNA, and ApoAlert DNA Fragmentation Assay Kit were purchased from CLONTECH (Palo Alto, CA). Restriction enzymes were purchased from New England Biolab (Beverly, MA). T4 DNA ligase and CELLTiter 96 Aqueous One Solution Cell Proliferation Assay were from Promega (Madison, WI). The Expand High Fidelity PCR System and Apoptotic DNA Ladder Kit were from Roche Molecular Biochemicals (Indianapolis, IN). QIAprep Miniprep, QIAEX II Gel Extraction Kit, and SuperFect Transfection Reagent were purchased from QIAGEN (Santa Clarita, CA). BODIPY TR ceramide and MitoTracker Red CM-H₂Xros were purchased from Molecular Probes (Eugene, OR). 12-epi-Scaradial, aristolochic acid, and lysophosphatidic acid were purchased from BIOMOL (Plymouth Meeting, PA).

Vector Construction-- The PCR amplification program used was as follows: 94 °C 5 min, 50 °C 2 min, 72 °C, 3 min (first cycle); 94 °C 1 min, 50 °C 2 min, 72 °C, 3 min (30 cycles), 94 °C 1 min, 50 °C, 2 min, 72 °C 10 min (last cycle). To clone the sequence encoding the mature rat liver group IIA PLA₂ protein, PCR reaction was performed using upstream primer F3B, 5'-AATTCAGCTCGAGATGAGCCTTCTGGAGTTTGGGCAAATG-3' and downstream primer R1B, 5'-CGAAGATGCTGCAGTAAGCAACTGGGCGTCTTCCCTTTGCAAAACTTGTTGGGGTAGAAC-3' and rat liver cDNA as template. The fragment was cut with XhoI/PstI and inserted into pEGFP-N1. The resulting plasmid was named pF3B. To clone the complete cDNA sequence of the rat group IIA PLA₂, PCR reaction was performed using upstream primer F5, 5'-GAGAGCGGCCGCATGAAGGTCCTCCTGTTGCTAGCAGT-3' and downstream primer R1D, 5'-GATGTCGAATTCAGCAACTGGGCGTCTTCCCTTTGC-3' and rat liver cDNA as template. The PCR fragment was digested with NotI/EcoRI and inserted into pIRES1neo, resulting in pPLA₂. To create an in-frame fusion between the full-length PLA₂ and EGFP, PCR reaction was performed using upstream primer F4B 5'-AATTCAGCTCGAGATGAGCCTTCTGGAGTTTGGGCAAATG-3' and downstream primer R1B and rat liver cDNA as template. The PCR fragment was digested with PstI/XhoI, then cloned into PstI/XhoI sites upstream of the N terminus of EGFP in pEGFP-N1. The resulting plasmid was named pF4B. The PLA₂-EGFP coding region was removed by digestion with NdeI (a site in the CMV promoter upstream of the PLA₂-EGFP region) and NotI, and was cloned into pIRES1neo, resulting in pFPG. To construct MHIS that carries a His-47  Gln mutation in PLA₂ (22), we used the upstream primer MHISF 5'-CAGAGGATCCCCCAAGGATGCCACAGATTGGTGCTGTGTGACTCAAGACTGTTGTTAC-3', which overlaps the single BamHI site in the PLA₂ and carried a CAT (His) to CAA (Gln), and the downstream primer MHISR 5'-ATGGTGGCGACCGGTGGATCCCGGGCCCGC-3' and pF4B as template in the PCR reaction. The fragment carrying the Gln mutation was digested with BamHI and recovered from agarose gel. The His-47 in pF4B was then replaced with Gln using the following procedure: pF4B was digested with BamHI to remove the fragment carrying His-47. The remaining vector sequence (including part of the PLA₂ sequence) was then ligated to the fragment carrying the mutation at 16 °C for 1 h. PCR was then performed using F4B and R1B as primers with the ligation reaction as template. The fragment corresponding to the correct size was recovered from the agarose gel, digested with XhoI/PstI, and cloned into pEGFP-N1. The resulting plasmid was named pMHIS. To clone the signal peptide sequence, PCR was performed using primer SIGF 5'-GTGTATCATATGCCAAGTACG-3' and primer SIGR 5'-CAGAATCTGCAGCCCAAACTCCAGAAGGCTCCCCTGGACCTGAATTGAG-3' and pFPG as template. The fragment was recovered and digested with NdeI/PstI, then cloned into the corresponding sites in pF4B, resulting in in-frame fusion with EGFP coding region. The resulting plasmid was named pSIG/G. To generate the group IIA PLA₂ mutants carrying a His-47  Gln mutation, PCR was performed using F5 and R1D primers and pMIHS template. The fragment was cloned as described for pPLA₂. In all the fusion proteins, the stop codon of PLA₂ (TGA) was changed to TTA which encodes a Leu residue. The sequences of all PCR-derived constructs were confirmed.

Transfection-- Baby hamster kidney (BHK) cells were cultured at 37 °C in 5% CO₂ in Dulbecco's modified Eagle's medium (DMEM) (Life Technologies, Inc., Gaitherburg, MD) containing 10% fetal bovine serum (FBS, Sigma). In some experiments, DMEM/F-12 (Life Technologies, Inc.) was used. For transient expression, cells were grown on coverslips in a 35-mm culture dish to about 50% confluence. In a typical transfection experiment, 2 µg of DNA was mixed with 100 µl of medium (FBS- and antibiotic-free) and 25 µl of SuperFect Transfection Reagent (QIAGEN) and incubated at room temperature for 30 min. 1 ml of medium containing 10% FBS was added and the mixture immediately added to the cells. Following incubation at 37 °C for 2 h, the cells were washed with phosphate-buffered saline, then cultured in fresh medium. EGFP fluorescence usually could be observed after overnight incubation. To obtain stable clones, Geneticin (500 µg/ml, Life Technologies, Inc.) was added to medium 2-3 days post-transfection. The cells were selected for 2 weeks.

RNA Isolation and Reverse Transcriptase (RT)-PCR-- RNA was isolated from cells using High Pure RNA Isolation Kit (Roche Molecular Biochemicals) according to the manufacturer's manual. RT-PCR was performed using Access RT-PCR System (Promega). A 50-µl reaction contained 25 µl of nuclease-free water, 10 µl of avian myeloblastosis virus/Tf1 5 × buffer, 1 µl of 10 mM dNTP mixture, 5 µl of F5 primer (4 µM), 5 µl of R1D primer (4 µM), 2 µl of 25 mM MgSO_4, 1 µl of avian myeloblastosis virus reverse transcriptase (5 units/µl), 1 µl of Tf1 DNA polymerase (5 units/µl), and 2 µl of RNA. Reverse transcription was carried out at 48 °C for 1 h. After incubation at 95 °C for 4 min to inactivate reverse transcriptase, PCR amplification was performed using 95 °C 1 min, 60 °C 1 min, 70 °C 1 min for 30 cycles. The DNA was separated on 1% agarose gel.

Fluorescent Microscopy-- Final concentrations of the probes were 10 nM for MitoTracker and 15 µM for BODIPY TR ceramide. To stain organelles, cells were cultured in medium containing the appropriate probes for 10-30 min, then washed three times with phosphate-buffered saline. The coverslips were then mounted on slides and sealed with rubber cement. Fluorescence was examined with Olympus IX-70 microscope and recorded with an ORCA100 CCD camera (Hamamatsu). The filter settings for EGFP fluorescence were a 488/20 nm excitation band-pass filter, a 495-nm dichroic mirror, and a 513/30 nm emission filter. The filter settings for red fluorescence (MitoTracker, BODIPY TR ceramide) were a 530-550 nm excitation band-pass filter, a 570-nm dichroic mirror, and a 590-nm (long pass) emission filter.

Cell Viability and Apoptosis Detection-- Cell viability was determined with CELLTiter 96 AQueous One Solution Cell Proliferation Assay (Promega) according the manufacture's instruction. DNA ladders were detected either with Apoptotic DNA Ladder kit (Roche Molecular Biochemicals) or with partial lysis of plasma membrane with 0.5% Triton X-100 with some modification (23) and preparing low molecular DNA. TUNEL assay was performed using ApoAlert DNA fragmentation Assay Kit (CLONTECH).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cloning and Characterization of Rat Group IIA PLA₂-- The full-length sequence (including signal peptide) of secretory group IIA PLA₂ from rat platelets (secretory/membrane-associated) is available in GenBank (accession number D00523). Rat liver group IIA PLA₂ has been reported to be localized in mitochondria (12), and the sequence of the mature liver/mitochondrial group IIA PLA₂ is also available in GenBank (accession number X74364). The sequence of these two proteins (rat platelet secretory group IIA PLA₂ versus rat liver group IIA PLA₂) differs by only 1 amino acid residue, i.e. Arg-94 in the liver/mitochondrial group IIA PLA₂ versus Ala-94 in the secretory group IIA PLA₂ (15, 24, 25).

Because the presequence of the mitochondrial group IIA PLA₂ has not been identified, it is unclear whether these two proteins contain the same signal peptide, and whether group IIA PLA₂ can localize to both mitochondria and secretory pathways. Based on the N- and C-terminal sequence of the liver/mitochondrial group IIA PLA₂, we cloned the cDNA sequence encoding mature PLA₂ from rat liver by PCR amplification. The sequence we cloned was identical to that of the secretory group IIA PLA₂ including an Ala at position 94. It is likely that the inaccuracy of earlier versions of Taq polymerase caused the previously reported difference in the sequence of mitochindrial versus secretory PLA₂. Using primers corresponding to the signal peptide sequence of the secretory PLA₂ and the COOH terminus of mitochondrial/secretory group IIA PLA₂, we amplified the full-length cDNA sequence of the group IIA PLA₂ from rat liver. The sequence, including signal peptide, was again identical to that of the rat secretory group IIA PLA₂. We then examined whether there were any alternatively spliced forms of group IIA PLA₂ that may bear different presequence/or signal peptide. Using primers corresponding to the 5' terminus of the primary transcript (PSTART in Fig. 1A) (26) and to the C terminus of mitochondrial/secretory group IIA PLA₂ (R1B) to amplify any possible alternatively spliced mRNAs, only one band was detected in the reaction product (Fig. 1B). To examine whether this band contained more than one DNA fragment, the band was recovered and subjected to direct sequencing using PSTART and R1B as primers. No heterogeneity was found (Fig. 1C). Therefore, we conclude that there is only one group IIA PLA₂ molecule in rat liver. The data also indicate that before post-translational processing, mitochondrial group IIA PLA₂ bears the same signal peptide as the secretory group IIA PLA₂ does.

View larger version (31K): [in this window] [in a new window]   Fig. 1.   Examination of alternatively spliced mRNA by PCR. A, genomic structure of rat group IIA PLA2 gene. Primers, transcription initiation site, and intron 1 are indicated by arrows. Exons are shown as boxes. B, agarose gel (1%) electrophoresis of PCR products. C, sequence of the fragment recovered from the gel (PSTART primer). MW, /HindIII DNA.

To examine whether rat liver group IIA PLA₂ targets to mitochondria, we fused the N-terminal 29 residues (including the 21-residue signal peptide and 8 residues of the mature enzyme) to the N terminus of EGFP (SIG/G in Fig. 2). When expressed in BHK cells, the SIG/G fusion protein localized mainly at one pole of the nucleus (Fig. 3B), which is characteristic for the Golgi body. In contrast, when expressed alone EGFP was evenly distributed in the cytosol (Fig. 3A). In some cells, fusion proteins were also found in typical secretory organelles, including Golgi stacks (Fig. 3C), vesicles detaching from Golgi body (Fig. 3D), endoplasmic reticulum (Fig. 3E), and vesicles (Fig. 3F). This reflects the different stages of secretory protein undergoing the maturation-secretion process.

View larger version (38K): [in this window] [in a new window]   Fig. 2.   Gene constructs. SIG, signal peptide. H47Q, His-47  Gln mutation.

View larger version (56K): [in this window] [in a new window]   Fig. 3.   SIG/G fluorescence. A, cells transfected with pEGFP-N1 (control). B-F, cells transfected with pSIG/G. A was taken with × 10 objective lens; B with × 20; C-F with × 100.

We also generated fusion proteins composed of the full-length rat group IIA PLA₂ (including signal peptide) and EGFP (FPG or MHIS in Fig. 2). MHIS is identical to FPG except it contains a His-47  Gln mutation. When expressed in BHK cells, these fusion proteins also displayed a distribution pattern typical for a secretory protein. Fig. 4, A and B (FPG) and C (MHIS), show the fusion protein localized mainly in vesicles. Fig. 4, D-F (FPG), show the localization of FPG fusion protein in the Golgi body. Fig. 4, G-I (FPG), demonstrate that the fusion protein does not localize in mitochondria. From these results, it is concluded that the rat liver group IIA PLA₂ is a secretory protein. Therefore, during cell injury, group IIA PLA₂ cannot directly regulate mitochondrial function and induce the MPT. Thus, all of the physiological effects elicited by rat liver group IIA PLA₂ should be considered as the result of its interaction on the membranes of the secretory pathways, and in particular, the plasma membrane.

View larger version (30K): [in this window] [in a new window]   Fig. 4.   Localization of FPG and MHIS fusion protein. A (FPG), B (FPG), and C (MHIS) show the fusion proteins in vesicles rather that mitochondria. D (FPG), E (BODYPI TR ceramide), and F (overlaid fluorescence of D and E) show the localization of FPG in Golgi body. G (FPG), H (MitoTracker), and I (overlaid fluorescence of G and H) show that the protein does not localize in mitochondria. A was taken with × 10 objective lens, and all others with × 100.

Group IIA PLA₂ Generates Anti-apoptotic Survival Signals in BHK Cells-- To study the biological function of the secretory group IIA PLA₂, we generated cells stably expressing this gene. For this purpose, we used a bicistronic expression system (pIRES1neo, CLONTECH). In this system, a single cytomegalovirus promoter directs the transcription of mRNA consisting of both the group IIA PLA₂ and the selection marker neo^r. The group IIA PLA₂ is located upstream of neo^r in the bicistronic transcript. Because the two genes are simultaneously expressed, after transfection and selection nearly all the G418-resistant cells express the group IIA PLA₂. This allowed us to rapidly obtain a pool of PLA₂ expressing cells without cloning. A total of 35 G418-resistant clones were obtained. After harvesting and pooling of these cells (NP), the expression of rat group IIA PLA₂ was confirmed (Fig. 5A). To our surprise, stable expression of group IIA PLA₂ was not associated with cytotoxicity. In fact, when cultured in serum-free medium, normal BHK cells underwent massive apoptosis, while apoptosis in the NP cells was significantly lower (Fig. 5B).

View larger version (72K): [in this window] [in a new window]   Fig. 5.   Detection of the rat group IIA PLA2 expression and apoptosis. A, detection of rat group IIA PLA2 expression in BHK, HQ, and NP cells by RT-PCR. B, DNA ladder. BHK and NP cells were recovered by trypsin digestion. After washing twice with serum-free medium, 2 × 106 cells were plated into 35-mm dishes with DMEM/F12 medium and 0.5% FBS. After 3 days, DNA was isolated using Apoptotic DNA ladder kit and resolved on 1.2% agarose gel. MW1, /HindIII marker. C, DNA ladder. After washing twice with serum-free medium, 107 cells were plated into 90-mm dishes in DMEM/F12 medium and 1% FBS. 24 h later, the cells were washed once with serum-free medium, and cultured in serum-free medium for 24 h. DNA ladder was prepared by Triton X-100 lysis. MW2, 123-base pair DNA ladder. D, TUNEL assay. HQ cells were washed twice with medium then plated onto glass coverslip in DMEM/F-12 medium and 1% FBS. 24 h later, the cells were washed once with medium, and cultured in serum-free medium. After overnight culture, DNA fragmentation was detected with ApoAlert DNA Fragmentation Assay Kit (CLONTECH). a, fluorescent image (show DNA fragmentation). b, transmitted light image (× 40 objective lens).

To examine whether resistance to apoptosis of the NP cells was due to expression of the rat group IIA PLA₂, we generated a mutant PLA₂, in which His-47, the residue essential for the hydrolytic activity (by binding of an H₂O molecule to the catalytic center) of PLA₂, was mutated to Gln (Fig. 2) (22). A pool of cells (HQ) expressing the His-47 mutant was obtained using the same method as described for the NP cells, and the expression of the mutant PLA₂ was confirmed (Fig. 5A). As shown in Fig. 5, C and D, the HQ cells underwent massive apoptosis after withdrawal of FBS.

We then compared the viability of BHK, HQ, and NP cells in medium lacking growth factors. Fig. 6, A-C, show NP cells survived better than BHK and HQ cells in serum-free medium. Morphological changes observed in serum-free medium is also more substantial for normal BHK and HQ cells compared with NP cells (Fig. 6D).

View larger version (37K): [in this window] [in a new window]   Fig. 6.   Cell viability after deprivation of growth factors. A-C, 24-72 h viability. BHK, HQ, and NP cells were washed twice with serum-free medium, then plated into 96-well plate at 4 × 104 cells/well in DMEM/F-12 and 1% FBS. 24 h later, the cells were washed once with serum-free medium and cultured in either serum-free medium or supplemented with 5% FBS. The cell viability was determined with CELLTiter 96 AQueous One Solution Cell Proliferation Assay. D, cell morphology (× 10 objective lens) 8 h after serum withdraw. The experiments were done in triplicate.

If resistance to apoptosis of NP cells is caused by rat group IIA PLA₂ expression, then secretory PLA₂ inhibitors should be able to abrogate this effect. 12-epi-Scalaradial (SCA) is a specific inhibitor for secretory PLA₂ (27-29). A dose-dependent inhibition of cell survival in serum-free medium was found in NP cells in the presence of SCA (Fig. 7A). SCA also inhibited the survival of BHK and HQ cells. A possible explanation is that it might abrogate the endogenous sPLA₂ function. Another widely used PLA₂ inhibitor, aristolochic acid (ArA) (30-32), also abrogated the survival effect in NP cells (Fig. 7B). In the presence of FBS, these inhibitors did not have significant effects on cell survival (Fig. 7, C and D). Taken together, these results strongly suggest that expression of rat group IIA PLA₂ produces survival signals in this model system.

View larger version (38K): [in this window] [in a new window]   Fig. 7.   PLA2 inhibitors and LPA on cell survival in serum-free medium. A, SCA. B, ArA. C, SCA plus serum. D, ArA plus serum. E, LPA. The cells were plated into 96-well plates and washed 24 h later as described in the legend to Fig. 6. The cells were then treated with the SCA, ArA, SCA + FBS, ArA + FBS for 24 h, or with LPA for 96 h, then the viability was determined. In FBS + SCA/or + ArA treatment, 10% FBS was used. The experiments were done in triplicate.

Group IIA PLA₂ is also known to generate lipid mediators including AA and LPA (5). To study whether they might mediate group IIA PLA₂ survival activity in the NP cells, we treated the NP cells with LPA and AA. At high concentration (30 µM), AA itself induced cell death. At lower concentration, AA did not have apparent protective effects (data not shown). Similarly, LPA did not have any protective effect in either BHK or HQ cells, but it did increase cell survival of NP cells (Fig. 7E). Therefore, neither AA nor LPA can replace the signals elicited by rat group IIA PLA₂.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The major goal of our study was to elucidate the role of rat liver group IIA PLA₂ in apoptosis induced by serum starvation. It is known that increased group IIA PLA₂ expression is associated with hypoxia/reperfusion injury in rat hepatocytes (16, 17), and that the products of PLA₂-catalyzed reactions can induce the MPT in isolated mitochondria. In addition, a number of papers have proposed that mitochondrial PLA₂ might induce the MPT following a variety of insults (33-35). These data, in conjunction with the report detailing the biochemical purification of mitochondrial localized group IIA PLA₂ from rat liver 10 years ago (12), support the hypothesis that group IIA PLA₂ is part of the injury pathway in cells and exerts this injury promoting activity by targeting the mitochondria. However, it has generally been accepted that mammalian group IIA PLA₂ can be both a secretory and mitochondrial protein (2, 3, 12-14), and to date, most work aimed at elucidating the activity of group IIA PLA₂ have focussed on secretory group IIA PLA₂. Because of this, we decided to visualize the location of this PLA₂ in normal and stressed cells using an EGFP-PLA₂ fusion protein to determine whether mitochondria are a target of group IIA PLA₂.

We cloned group IIA PLA₂ from rat liver (reportedly localized in mitochondria (12)), and the sequence we cloned was identical to secretory group IIA PLA₂. Furthermore, we did not find any alternatively spliced variants of the primary transcript of group IIA PLA₂ that might generate a different signal peptide. These results indicate that there is only one type of group IIA PLA₂ molecule in rat liver.

We then studied the subcellular localization of the rat group IIA PLA₂. To accomplish this, we used EGFP as a tag to follow the maturation and intracellular location of the fusion protein in living cells. This method has been demonstrated to work for both secretory and mitochondrial proteins (36-38). To our surprise, EGFP fusion protein consisting of either the signal peptide or full-length sequence of group IIA PLA₂ was found exclusively localized to the secretory pathways, but never in mitochondria. This distribution pattern is not cell-type (BHK) specific, as similar results were obtained in the mouse hepatocyte cell line AML12 (data not shown). Taken together, these data indicate that group IIA PLA₂ is not a mitochondrially localized protein. It is therefore unlikely that group IIA PLA₂ has any direct role in regulating mitochondrial function and onset of the MPT.

Secretory group IIA PLA₂ is believed to regulate a number of physiological as well as pathological processes by generating AA and other lipid mediators. High levels of group IIA PLA₂ have been found at inflammatory sites and increased serum group IIA PLA₂ levels are thought to be related to the severity of sepsis (39-41). In isolated hepatocytes, expression of group IIA PLA₂ was up-regulated during chemical hypoxia and its contribution to hypoxic injury has been demonstrated (17). Despite numerous in vitro data that suggest the important role of the group IIA PLA₂ in inflammation and cell injury, it should be noted that in vivo models of injury have not always supported such a destructive role for group IIA PLA₂. For example, transgenic mice expressing high levels of group IIA PLA₂ do not develop any overt inflammatory conditions. No abnormalities were found in any tissue other than skin, where alopecia, epidermal hyperplasia, adnexal hyerplasia, and hyperkeratosis were observed (42). Several mouse strains have been found to contain a frameshift mutation in group IIA PLA₂, resulting in loss of the enzyme activity. Infection of group IIA PLA₂ (/) mice (C57BL/6) with Helicobacter felis, a gastroduodenal pathogen, lead to increased apoptosis and proliferation compared with group IIA PLA₂ (+/+) BALB/c and C3H/HeJ mouse (43). Much more marked inflammatory and proliferative response to H. felis was also observed in another group IIA PLA₂ (/) mouse SV129 than in PLA₂ (+/+) mice (43). No satisfactory explanation has been proposed for this contradiction. One possible reason is that sPLA₂ contributes to some extent in the destruction of pathogen in PLA₂ (+/+) mice.

During the course of these studies, we noted that cells stably expressing group IIA PLA₂ were more resistant to apoptosis induced by serum starvation. This protective role of group IIA PLA₂ in serum withdrawal-induced apoptosis was further confirmed using a number of other approaches including PLA₂ inhibitors and site-directed mutagenesis of group IIA PLA₂ His-47. Because the lipid mediators AA and LPA could not mimic the effects of group IIA PLA₂, it appears that other mechanisms are responsible for the protective physiological function of group IIA PLA₂. Interestingly, although LPA had no effect in BHK and HQ cells, it did promote survival in NP cells. As LPA can transduce survival signals via phosphatidylinositol 3-kinase (43, 44), it is possible that the group IIA PLA₂ and LPA might synergistically transduce survival signals in fibroblasts.

How group IIA PLA₂ generates survival signals remains to be elucidated. Group I PLA₂ can stimulate cell proliferation by binding to a receptors (45). Whether group IIA PLA₂ has its own receptors or can bind to group I PLA₂ receptors of BHK cells is unknown. Given the similarity between group I and IIA PLA₂, it is possible that group IIA PLA₂ may activate a survival pathway by a similar mechanism. If so, this might also explain why PLA₂ possesses some functions independent of its hydrolytic activity.

Secretory PLA₂ has been identified as enhancing factor that enhances the binding of epidermal growth factor to cells (47). It is possible that rat group IIA PLA₂ may trigger anti-apoptotic signaling via epidermal growth factor. Alternatively, group IIA PLA₂ may affect the receptor signaling via modifying membrane topology.

The anti-apoptotic activity of group IIA PLA₂ also may explain the pathogenesis of certain clinical diseases. It has been reported that the use of certain Chinese herbs containing aristolochic acid for weight loss may cause severe renal failure (48). As aristolochic acid inhibits group IIA PLA₂ activity, it seems likely that aristolochic acid inhibition of PLA₂-mediated signaling may contribute to the renal injury seen in these patients. The immunosuppressive drug cyclosporin A has the well known side effect of nephrotoxicity (49). Cyclosporin A has been shown to down-regulate mRNA level of group IIA PLA₂ in mesangial cells (50). If group IIA PLA₂ generates anti-apoptotic signals in kidney, long-term use of cyclosporin A may impair group IIA PLA₂-medited survival signal transduction.

Group IIA PLA₂ is transcriptionally up-regulated by inflammatory cytokines, partially via NFB (50). NFB is believed to protect against tumor necrosis factor-induced cell death by inducing the expression of anti-apoptotic genes (51). High levels of group IIA PLA₂ is present at inflammatory sites, and the physiological role of the protein in inflammation remains largely unknown. Our results suggest the possibility that group IIA PLA₂ might counteract the effects caused by some pro-inflammatory cytokines, such as tumor necrosis factor that promotes cell death.

	ACKNOWLEDGEMENTS

We thank Dr. Akiyuki Takahashi, Yongming Liu, and other members in Dr. Herman's laboratories for their help.

	FOOTNOTES

^* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: Dept. of Cellular and Structural Biology, The University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Dr., San Antonio, TX 78284-7762. E-mail: hermanb@uthscsa.edu.

	ABBREVIATIONS

The abbreviations used are: PLA₂, phospholipase A₂; SCA, 12-epi-scalaradial; ArA, aristolochic acid; EGFP, enhanced green fluorescent protein, MPT, mitochondrial permeability transition; AA, arachidonic acid; LPA, lysophosphatidic acid; PCR, polymerase chain reaction; BHK, baby hamster kidney; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; RT, reverse transcriptase.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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