Role of an Endoplasmic Reticulum Ca -Independent Phospholipase A2 in Cisplatin-Induced Renal Cell Apoptosis

It has been demonstrated recently that rabbit renal proximal tubule cells (RPTC) express a novel Ca -independent phospholipase A2 (iPLA2) whose activity localizes to the endoplasmic reticulum (ER-iPLA2) and is similar to group VIB PLA2. In this study, the expression of group VIB PLA2 was examined and the role of ER-iPLA2 in cisplatin-induced apoptosis was determined. Cisplatin induced both timeand concentration-dependent RPTC apoptosis as determined by p53 nuclear localization, annexin V staining, caspase 3 activity, and chromatin condensation. Inhibition of ER-iPLA2 with bromoenol lactone (5 M) reduced cisplatin-induced annexin V binding 40%, chromatin condensation 55%, and caspase 3 activity 42%, but had no effect on p53 nuclear localization. Treatment of RPTC with the protein kinase C stimulator phorbol 12-myristate 13-acetate increased the activity of ER-iPLA2 2-fold and increased cisplatin-induced RPTC apoptosis. These studies demonstrate that group VIB PLA2 is expressed in RPTC and suggest that RPTC ER-iPLA2 is the rabbit homolog of group VIB PLA2. These data also demonstrate that ER-iPLA2 acts downstream of p53 and upstream of caspase 3 to mediate cisplatin-induced RPTC apoptosis. Finally, ER-iPLA2 seems to be regulated by protein kinase C. Phospholipase A2 (PLA2) are esterases that hydrolyze glycerophospholipids at the sn-2 position of the glycerol backbone, releasing a fatty acid and a lysophospholipid (Six and Dennis, 2000). PLA2 are classified according to their nucleotide and amino acid sequence into 19 separate groups (Balsinde et al., 2002). Historically, PLA2 have been classified into three groups: secretory PLA2 (sPLA2), cytosolic (cPLA2), and Ca 2 -independent PLA2 (iPLA2). sPLA2 are approximately 15 kDa, use a histidine amino acid to facilitate hydrolysis, and, with one exception (group XII), require Ca for activity (Six and Dennis, 2000; Balsinde et al., 2002). In contrast to sPLA2, cPLA2 (group IVA, B, and C) use a serine at their catalytic site and are larger than sPLA2, ranging in size from 61 to 110 kDa (Six and Dennis, 2000). Groups IVA and B PLA2 require Ca 2 for translocation to membranes upon activation (Balsinde et al., 2002), whereas Group IVC is membrane associated and Ca -independent (Underwood et al., 1998). Group VI members are typically referred to as iPLA2 because they do not require Ca 2 for either their activity or translocation to the membrane upon activation (Balsinde et al., 2002) and use a serine to catalyze hydrolysis (Six and Dennis, 2000). Group VI PLA2 represent some of the newest PLA2 to be characterized and include group VIA-1, VIA-2, and VIB PLA2. Group VIA-1 and VIA-2 PLA2 are splice variants of the same gene, are 85-kDa, and reside in the cytosol. Group VIA-1 and VIA-2 PLA2 also are referred to as iPLA2 . In contrast, group VIB PLA2 (also referred to as iPLA2 ) is associated with the microsomal fraction (Mancuso et al., 2000; Tanaka et al., 2000) and is transcribed from a different gene than group VIA PLA2. Whereas group VIB PLA2 shares little DNA sequence homology to group VIA PLA2, group VIA-1 and VIA-2 PLA2 and group VIB PLA2 are similar in size (85 kDa) and are inhibited by the suicide substrate (E)-6-(bromoethylene)-3-(1-naphthaleny)-2Htetrahydropyran-2-one (bromoenol lactone, BEL) (Balsinde et al., 1995; Balsinde and Dennis, 1996; Mancuso et al., 2000; Tanaka et al., 2000). However, group VIA PLA2 is sensitive to the PLA2 inhibitors arachidonyl trifluoromethylketone This work was supported by a National Research Service Award DK-10079 (to B.S.C.) and a National Institutes of Health Grant DK-62028 (to R.G.S. and J.M.). Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. DOI: 10.1124/jpet.103.060541. ABBREVIATIONS: PLA2, phospholipase A2; ER-iPLA2, endoplasmic reticulum-Ca 2 -independent phospholipase A2; sPLA2, secretory phospholipase A2; cPLA2, cytosolic phospholipase A2; iPLA2, Ca 2 -independent phospholipase A2; BEL, bromoenol lactone; AAOCF2, arachidonyl trifluoromethylketone; MAFP, methyl arachidonyl fluorophosphonate; RPTC, rabbit renal proximal tubule cell(s); ER, endoplasmic reticulum; PMA, phorbol 12-myristate 13-acetate; DAPI, 4 ,6-diamidino-2-phenylindole-dihydrochloride; PI, propidium iodide; DMSO, dimethyl sulfoxide; RT-PCR, reverse transcriptase-polymerase chain reaction; PBS, phosphate-buffered saline; bp, base pair. 0022-3565/04/3083-921–928$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 308, No. 3 Copyright © 2004 by The American Society for Pharmacology and Experimental Therapeutics 60541/1128137 JPET 308:921–928, 2004 Printed in U.S.A. 921 at A PE T Jornals on N ovem er 9, 2017 jpet.asjournals.org D ow nladed from (AAOCF3) and methyl arachidonyl fluorophosphonate (MAFP) (Lio et al., 1996; Balsinde, 2002), whereas group VIB is relatively insensitive to these compounds (Cummings et al., 2002). Rabbit renal proximal tubule cells (RPTC) contain an endoplasmic reticulum (ER) iPLA2 activity that is inhibited by BEL and is relatively insensitive to AAOCF3 and MAFP (Schnellmann et al., 1994; Cummings et al., 2002). Using immunoblot analysis and an antibody directed against cytosolic group VIA PLA2, an immunoreactive 85-kDa protein was identified in RPTC microsomes. In contrast, no protein was identified in the RPTC cytosol. This study identified and characterized an ER-iPLA2 activity and suggested that the ER-iPLA2 is a group VIB PLA2, not group VIA PLA2. The roles of group VI PLA2 in cell physiology or death are not well understood. Group VIA PLA2 has been implicated in the maintenance of membrane integrity (Balsinde and Dennis, 1996), in insulin signaling in pancreatic islet -cells (Ma et al., 2001), and in cAMP response element-binding protein phosphorylation during ischemia in cardiomyocytes (Williams and Ford, 2001). Recent reports also have hypothesized that the activity of group VI PLA2, like group IV PLA2, may be increased in response to oxidative-induced damage (Balboa and Balsinde, 2002) or by protein kinase C (Steer et al., 2002). Compared with group VIA PLA2, little is known concerning the role of microsomal iPLA2, such as ER-iPLA2, in either cell physiology or injury. Inhibition of RPTC ER-iPLA2 activity using BEL potentiated oxidant-induced lipid peroxidation and oncosis in renal cells (Cummings et al., 2002). Furthermore, the role of ER-iPLA2 in oncosis seems to be specific to that produced by oxidants because BEL treatment did not potentiate oncosis induced by the nonoxidant antimycin A. Thus, one role for microsomal group VIB PLA2, such as ER-iPLA2, is the protection of cells from oxidant-induced oncosis. A role for ER-iPLA2 in apoptosis has not been examined. However, several studies have demonstrated that the cytosolic group VIA PLA2 mediates apoptosis (Enari et al., 1996; Sapirstein et al., 1996; Atsumi et al., 1998; De Valck et al., 1998; Atsumi et al., 2000). For example, group VIA PLA2mediated arachidonic acid release correlates to caspase 3 activation and the progression of apoptosis (Atsumi et al., 1997, 1998, 2000). The goals of this study were to determine whether ER-iPLA2 mediates RPTC apoptosis and to determine where in apoptosis signaling ER-iPLA2 acts. Materials and Methods Materials. Female New Zealand White rabbits (1.5–2.0 kg) were purchased from Myrtle’s Rabbitry (Thompson Station, TN). L-Ascorbic acid-2-phosphate (magnesium salt) was obtained from Wako Chemicals USA (Richmond, VA). DEVD-afc (caspase 3 substrate), IETD-afc (caspase 8 substrate), and LEHD-afc (caspase 9 substrate) were purchased from BioVision (Palo Alto, CA). The rat kidney cDNA library was purchased from Stratagene (La Jolla, CA). The caspase 3 inhibitor DEVD-fmk, the general pan caspase inhibitor ZVAD-fmk, and annexin-FITC were obtained from R&D Systems (San Diego, CA). The antibody to p53, AAOCF3, phorbol 12-myristate 13-acetate (PMA), and 4 ,6-diamidino-2-phenylindole-dihydrochloride (DAPI) were purchased from Calbiochem (La Jolla, CA). PMA, cisplatin, propidium iodide (PI), and all other chemicals and materials were obtained from Sigma-Aldrich (St. Louis, MO). Isolation of Proximal Tubules and Culture Conditions. Rabbit renal proximal tubules were isolated using the iron oxide perfusion method and grown in 35-mm tissue culture dishes under improved conditions as described previously (Nowak and Schnellmann, 1995, 1996). The cell culture medium was a 1:1 mixture of Dulbecco’s modified Eagle’s medium/Ham’s F-12 (without D-glucose, phenol red, or sodium pyruvate) supplemented with 15 mM HEPES buffer, 2.5 mM L-glutamine, 15 mM sodium bicarbonate, and 6 mM lactate. Hydrocortisone (50 nM), selenium (5 ng/ml), human transferrin (5 g/ml), bovine insulin (10 nM), and L-ascorbic acid-2-phosphate (50 M) were added to fresh culture medium immediately before daily media change. In general, confluent RPTC were treated with inhibitors or diluent control [DMSO at 0.1% (v/v)] for 30 min before treatment with cisplatin. Reverse-Transcriptase-Polymerase Chain Reaction. RTPCR using total RNA isolated from primary cultures of RPTC or PCR using a rat kidney cDNA library (Stratagene) was performed using primers designed against the sequences of group VIB and VIA1 and A2 PLA2 reported in Mancuso et al. (2000) and Ma et al. (1998), respectively (Table 1). For group VIB PLA2, the RT step was performed at 50°C for 30 min followed by 2 min at 92°C to inactivate the RT. PCR was then performed with 35 cycles of 30 s at 72°C, 90 s at 55°C, and 30 s at 92°C followed by a final extension step of 2 min at 72°C. For group VIA PLA2, the conditions were exactly the same as reported by Ma et al. (1998). RT-PCR products were analyzed by agarose gel electrophoresis. Candidate bands were excised and extracted from the gel using Millipore Ultrafree-DA extraction columns (Bedford, MA), ethanol precipitated, and subjected to automated fluorescence based sequencing at the Biotechnology Resource Laboratory at the Medical University of South Carolina. Candidate sequences were compared with those previously entered into GenBank. Measurement of iPLA2 Activity. PLA2 activity was determined under linear conditions in microsomes and cytosol as described previously (McHowat et al., 1998). Activity was measured using synthetic (16:0, [H]18:1) plasmenylcholine and diacylcholine substrates (100 M) in the absence of Ca (presence of 4 mM EGTA). For PLA2 activity inhibition studies, confluent RPTC were exposed to either a solvent control [DMSO 0.1% (v/v)] or 5 M BEL for 30 min (Cummings et al., 2002). Measurement of Annexin V and PI Staining. Annexin V and PI staining were determined using flow cytometry as described previously (Schutte et al., 1998; Goldberg et al., 1999; Meijerman et al., 1999) with modifications (Cummings and Schnellmann, 2002). Briefly, media were removed, RPTC were washed twice with PBS, and incubated in binding buffer (10 mM HEPES, 140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1.8 mM CaCl2, pH 7.4) containing annexin V-FITC (25 g/ml) and PI (25 g/ml) for 10 min. Cells were washed three times in binding buffer, released from the monolayers using a rubber policeman, and staining quantified using a BD Biosciences FacsCalibur flow cytometer. For each measurement 10,000 events