1 Department of Pharmaceutical Sciences, Medical University of South Carolina, Charleston, South Carolina 29425; and 2 Department of Pathology, Saint Louis University, St. Louis, Missouri 63104
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ABSTRACT |
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Phospholipase A2 (PLA2) hydrolyzes the sn-2 ester bond in phospholipids, releasing a fatty acid and a lysophospholipid. Recently, a novel 85-kDa membrane-bound-Ca2+-independent PLA2 (iPLA2) was identified in insect and bacterial cells transfected with candidate PLA2 sequences. However, few data exist demonstrating a membrane-bound-iPLA2 in mammalian cells, its subcellular localization, or its physiological role. Herein, we demonstrate the expression of an 85-kDa endoplasmic reticulum (ER)-Ca2+-iPLA2 (ER-iPLA2) in rabbit renal proximal tubule cells (RPTC) that is plasmalogen selective and is inhibited by the specific Ca2+-iPLA2 inhibitor bromoenol lactone (BEL). RPTC exposed to tert-butylhydroperoxide for 24 h exhibited 20% oncosis compared with 2% in controls. Inhibition of ER-iPLA2 with BEL before tert-butylhydroperoxide exposure resulted in 50% oncosis. To determine whether this effect was common to oxidants, we tested the ability of BEL to potentiate oncosis induced by cumene hydroperoxide, menadione, duraquinone, cisplatin, and the nonoxidant antimycin A. All oxidants tested produced oncosis after 24 h, and prior inhibition of ER-iPLA2 potentiated oncosis at least twofold. In contrast, inhibition of ER-iPLA2 did not alter antimycin A-induced oncosis. Lipid peroxidation increased from 1.4- to 5.2-fold in RPTC treated with BEL before oxidant exposure, whereas no change was seen in antimycin A-treated RPTC. These results are the first to demonstrate the expression and subcellular localization of an ER-iPLA2. These results also suggest that ER-iPLA2 functions to protect against oxidant-induced lipid peroxidation and oncosis.
kidney; oxidant-induced cell death; lipid peroxidation
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INTRODUCTION |
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PHOSPHOLIPASE A2 (pla2) IS a superfamily of enzymes that hydrolyzes the sn-2 ester bond in phospholipids, resulting in the release of a free fatty acid and a lysophospholipid (6). It is ubiquitous in nature and plays critical roles in the generation of arachidonic acid, prostaglandins, and leukotrienes (8). PLA2 is classified according to its nucleotide sequence, and to date there are at least 12 groups (I-XII) comprising 16 individual PLA2 (29). Historically, a PLA2 is classified on the basis of whether it is secreted from the cell (secretory PLA2), Ca2+ dependent and located in the cytosol (cPLA2), or Ca2+ independent (iPLA2). cPLA2 (groups IVA-B) is one of the most widely studied and is found in the cytosol of many types of cells, including the pancreas, heart, and kidney (6, 7). cPLA2 requires micromolar amounts of Ca2+ to facilitate translocation to cellular membranes on activation but does not require Ca2+ for its catalytic activity. cPLA2 range in size from 85 to 100 kDa (7). iPLA2 (groups IVC, VI, VIIB, and VIII) does not require Ca2+, ranges in molecular size from 29 to 85 kDa, and typically is found in the cytosol. A key difference between cPLA2 and iPLA2 is that iPLA2 is inhibited by the suicide substrate (E)-6-(bromoethylene)-3-(1-naphthaleny)-2H-tetrahydropyran-2-one [or bromoenol lactone (BEL)], whereas cPLA2 is not. This fact has been used in a number of studies to distinguish between cPLA2 and iPLA2 activities (1, 10). In addition, iPLA2 can be distinguished from cPLA2 activity on the basis of its selectivity for phospholipid substrates and Ca2+ requirement.
A number of iPLA2 activities have been described in a
variety of tissues, including those of hamsters (32), mice
(4), rats (4), and humans (14,
15). To date, the best characterized iPLA2 from all
species is an 85-kDa protein that is inhibited by BEL and is present in
multiple isoforms, a result of alternative splicing. This
iPLA2 is commonly called iPLA2 and is
cytosolic. Recently, Mancuso et al. (16) and Tanaka et al.
(31) have characterized novel membrane-associated
iPLA2 that share 91% DNA sequence homology to each other,
but only limited homology to iPLA2
, confined to the
ATP-binding motif and the lipase site. This iPLA2 has been
termed iPLA2
. Mancuso et al. (16)
demonstrated that translation of the DNA sequence for
iPLA2
in insect Sf-9 cells resulted in the expression of
two proteins with molecular masses of 67-77 kDa. Activity and
expression of these proteins localized to membrane fractions and were
inhibited by BEL. In contrast, Tanaka et al. (31) reported
that translation of a candidate iPLA2
sequence in
bacterial COS-7 cells resulted in one 85-kDa protein whose expression
and activity also localized to membrane fractions. The activity of this
85-kDa iPLA2 was inhibited by BEL. The only other reported
iPLA2 expressed in the membrane is an 80-kDa
iPLA2 expressed in the plasma membrane of cardiomyocytes (17).
The above studies did not examine membrane-bound iPLA2 protein or activity in mammalian cells, determine the selectivity of iPLA2 for specific phospholipids, or determine the membrane location in which it was expressed. In addition, a physiological role for this iPLA2 was not addressed. In this study, we investigated an iPLA2 in renal proximal tubular cells (RPTC) with respect to expression, subcellular localization, inhibition profile, and selectivity for specific phospholipid substrates. In addition, we tested the hypothesis that iPLA2 plays a role in mediating oxidant-induced renal cell oncosis and lipid peroxidation.
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MATERIALS AND METHODS |
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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
(Richmond, VA). The antibody to iPLA2 was purchased from
Cayman Chemical (Ann Arbor, MI), and annexin V-FITC was obtained from
R&D Systems (San Diego, CA). The antibody to the heat shock protein
GRP78 was purchased from Calbiochem (La Jolla, CA), and the antibody
against the peroxisomal protein PMP70 was obtained from Affinity
BioReagents (Golden, CO). The endoplasmic reticulum (ER) and DNA stains
(DiOC6)3 and 4,6-diamidino-2-phenylindole were obtained
from Molecular Probes (Eugene, OR). Cisplatin, propidium idodide,
duraquinone, menadione, antimycin A, tert-butylhydroperoxide
(TBHP), cumene hydroperoxide, and all other chemicals were purchased
from Sigma (St. Louis, MO).
Isolation of proximal tubules and culture conditions. Rabbit RPTC were isolated using the iron oxide perfusion method and grown in 35-mm tissue culture dishes under improved condition, as previously described (21, 22). The cell culture medium was a 1:1 mixture of DMEM/Ham's F-12 (without D-glucose, phenol red, or sodium pyruvate) supplemented with 15 mM HEPES buffer, 2.5 mM L-glutamine, 1 µM pyridoxine HCl, 15 mM sodium bicarbonate, and 6 mM lactate. Hydrocortisone (50 nM), selenium (5 ng/ml), human transferrin (5 µg/ml), bovine insulin (10 nM), and L-ascorbic acid-2-phosphate (50 µM) were added to fresh culture medium immediately before daily media change.
Immunoblot analysis.
Microsomal and cytosolic fractions were isolated from RPTC as
previously described (17). Immunoblot analysis of both
microsomal and cytosolic protein (20 µg) from RPTC was performed as
described previously using a polyclonal rabbit anti-iPLA2
antibody that recognizes 85-kDa iPLA2 (17).
Immunocytochemical analysis.
Immunocytochemical analysis of iPLA2, ER, peroxisomes, and
nuclei staining were performed using confocal laser scanning
microscopy. Confluent RPTC monolayers were washed with PBS, fixed in
3.7% formaldehyde, and permeabilized with 0.1% (vol/vol) Triton
X-100. Nonspecific binding was blocked using 8% BSA, and RPTC were
incubated overnight with a goat anti-rabbit polyclonal antibody to
85-kDa iPLA2 (100-fold dilution) followed by exposure to
a secondary antibody (1,000-fold dilution) conjugated to either a
480/510- or 535/610-nm excitation/emission fluorochrome. For
colocalization studies, RPTC were exposed to markers of the ER
[(DiOC6)3 or GRP78 antibody] or the peroxisomes (PMP70
antibody). Nuclei were stained using 4,6-diamidino-2-phenylindole
(350/390-nm excitation/emission). Secondary antibodies for both GRP78
and PMP70 were conjugated to a 480/510-nm excitation/emission
fluorochrome. After the final wash, RPTC were covered with mounting
media and coverslips. Staining was visualized with a 410 Zeiss confocal
laser scanning microscope.
Measurement of PLA2 activity. PLA2 activity was determined under linear conditions in microsomes and cytosol as described previously (18). Activity was measured by using synthetic (16:0, [3H]18:1) plasmenylcholine and phosphatidylcholine substrates (100 µM) in the absence of Ca2+ (presence of 4 mM EGTA). For PLA2 activity inhibition studies, confluent RPTC were exposed to either a solvent control [DMSO <0.1% (vol/vol)] or 1-10 µM BEL for 30 min.
Measurement of oncosis. Oncosis was monitored by assessment of both annexin V (a marker for apoptosis) and propidium iodide (PI; a marker for oncosis) staining using flow cytometry. Briefly, RPTC were exposed to the oxidants TBHP (200 µM), cumene hydroperoxide (200 µM), duraquinone (400 µM), and cisplatin (400 µM) for 24 h and to menadione (10 µM) for 12 h. In addition RPTC were exposed to the nonoxidant antimycin A (0.3 µM) for 4 h. Annexin and PI staining were monitored using confocal laser scanning microscopy and flow cytometry as described previously, with modifications (9, 19, 28). After treatment, the medium was removed, and RPTC were washed twice and incubated in binding buffer [(in mM) 10 HEPES, 140 NaCl, 5 KCl, 1 MgCl2, 1.8 CaCl2, pH = 7.4] containing annexin V-FITC (2.5 µg/ml) and PI (25 µg/ml). Cells were washed three times in binding buffer and prepared for either flow cytometry or microscopy. For flow cytometry, RPTC were released from the monolayer by using a rubber policeman, and staining was quantified by using a Becton Dickinson FacsCalibur flow cytometer. Minimal binding of annexin V in the absence of PI staining was detected in these samples (<10%), demonstrating that the major mode of cell death was oncosis.
Measurement of lipid peroxidation. Lipid peroxidation in RPTC was monitored as the production of malondialdehyde (MDA) as described previously (26). RPTC were treated with the indicated compound for 2 h and isolated by scraping the dish with a rubber policeman. RPTC suspensions were treated with 10% trichloroacetic acid and centrifuged, the supernatant was removed, and it was combined with 2-thiobarbituric acid (0.76%), heated, cooled, and absorbance measured at 532 nm with 1,1,3,3-tetraethoxypropane as a standard.
Statistical analysis.
RPTC isolated from one rabbit represent one experiment
(n = 1). The appropriate ANOVA was performed for each
data set by using SigmaStat statistical software. Individual means were
compared with Fisher's protected least significant difference test
with P 0.05 being considered a statistically
significant difference between mean values. Linear regression was also
performed by using Sigma Stat statistical software.
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RESULTS |
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Expression and activity of iPLA2 in RPTC.
Microsomal and cytosolic fractions from confluent cultures of RPTC were
subjected to immunoblot analysis using a primary antibody that
recognizes cytosolic 85-kDa iPLA2. This antibody
recognized a single band in immunoblots of microsomal protein from RPTC
and comigrated with purified iPLA
(a gift from Genetics
Institute, Cambridge, MA). No band was detected in immunoblots
of cytosolic RPTC protein (Fig. 1). No
staining was detected in the IgG control. These data show that a
protein with sequence homology to iPLA2
, a cytosolic
protein, is expressed in RPTC microsomal fractions.
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Role of ER-iPLA2 in RPTC cell death.
Little is known concerning the role of iPLA2, particularly
ER-iPLA2, in cell injury and death. To test the hypothesis
that ER-iPLA2 plays a role in RPTC oxidant-induced cell
death, RPTC were treated with either solvent control [DMSO at < 0.1% (vol/vol)] or BEL (5 µM) before 2-, 4-, 12-, and 24-h
exposures to 200 µM TBHP. TBHP caused time-dependent increases in
cell death, resulting in ~20% oncosis at 24 h (Fig.
4). TBHP also induced
concentration-dependent cell death, with a concentration of 500 µM
resulting in 69 ± 3% oncosis after 24 h. The level of
oncosis in controls was ~2-3% and remained constant over time.
Confocal microscopy confirmed the time-dependent increase in oncosis
(data not shown). Treatment of cells with BEL before exposure to TBHP
potentiated oncosis at all time points. Linear regression analysis
revealed a strong correlation (r2 = 0.98)
between BEL inhibition of ER-iPLA2 activity and BEL
potentiation of TBHP-induced oncosis (Fig.
5). These data suggest a direct link
between ER-iPLA2 inhibition and potentiation of
oxidant-induced oncosis.
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Role of ER-iPLA2 in oxidant-induced lipid peroxidation.
We hypothesized that the potentiation of oncosis caused by
ER-iPLA2 inhibition was the result of increased retention
of peroxidized phospholipids during oxidative stress. To test this
hypothesis, RPTC were pretreated with BEL (5 µM) or solvent control
[DMSO at <0.1% (vol/vol)] and exposed to TBHP, cisplatin,
cumene hydroperoxide, menadione, duraquinone, and antimycin A for
2 h. No cell death was evident in RPTC treated with oxidants at
this time. Basal levels of MDA formation in control RPTC were 0.3 nmol/mg, and treatment of RPTC with BEL did not increase this value
(Fig. 7). Treatment of RPTC with oxidants
or antimycin A for 2 h did not increase MDA formation above
control. Longer exposures of cells to oxidants, but not antimycin A,
did result in increased MDA formation (data not shown). In contrast to
oxidants alone, treatment of RPTC with BEL before oxidant exposure
produced MDA formation. Treatment of RPTC with BEL before exposure to
antimycin A did not increase MDA formation.
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DISCUSSION |
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In the past few years, a number of PLA2 have been
identified and progress has been made in elucidating the functions of
many types of PLA2, especially cPLA2. However,
iPLA2 are incompletely explored, and their physiological
and pathological functions remain under investigation. For example,
iPLA2 is a cytosolic 85-kDa PLA2 that is
expressed in the pancreas, heart, brain, and macrophages (5, 6,
14, 15, 24, 34). It also has been demonstrated to exist as
multiple proteins as a result of alternative splicing (15). In normally functioning cells, iPLA2
has been suggested to mediate phospholipid remodeling by means of
mechanisms that are not fully determined (34).
iPLA2
also may mediate cell death induced by Fas ligand
and hypoxia (2, 3, 35).
Recently, Mancuso et al. (16) and Tanaka et al.
(31), using sequences derived from the human genome
project, identified, sequenced, cloned, and expressed a novel
iPLA2 (iPLA2) in insect and bacterial cells.
iPLA2
is similiar in size to iPLA2
and an
iPLA2 found in the plasma membrane of myocytes
(17) but is expressed in intracellular membranes. Neither
Mancuso et al. (16) nor Tanaka et al. (31)
determined the specific intracellular membrane in which
iPLA2
is expressed, its preference for specific phospholipids, or its physiological function within the cell. We report
for the first time the expression of an 85-kDa iPLA2 in
primary cultures of kidney cells whose activity and expression were in
microsomes, demonstrated selectivity for plasmalogen phospholipids, and
were inhibited by BEL. This ER-iPLA2 appears to be
constitutively expressed in microsomal membranes, because freshly
isolated cells had the same iPLA2 expression and activity
as primary cultures (data not shown). The ER location of RPTC
iPLA2 is not the result of translocation of
iPLA2
to the membrane during isolation or culture.
First, no immunoreactive band was detected in the cytosolic fractions
isolated from these cells at any time point of isolation or treatment.
Immunocytochemistry of untreated cells, using the same antibody used
for immunoblot analysis, demonstrated that iPLA2 was
expressed in a punctate manner similar to proteins localized to the ER
membrane as opposed to those expressed in the cytosol. On the basis of
the similarities in membrane expression, size, activity, and inhibition
profile, we propose that ER-iPLA2 is the rabbit
iPLA2
.
The majority of iPLA2 activity in RPTC was present in the
microsomal fraction and displayed substrate selectivity for
plasmenylcholine over phosphatidylcholine. A small amount of
iPLA2 activity was detected in the cytosolic fraction
(~1,000 pmol/mg), which may represent the 28-kDa cytosolic
iPLA2 or a small amount of ER contamination (23). BEL resulted in 90% inhibition of plasmenylcholine
hydrolysis and totally inhibited phosphatidylcholine hydrolysis by RPTC
ER-iPLA2. Neither AAOCF3 nor MAFP inhibited
RPTC ER-iPLA2 activity at concentrations under 10 µM and
only minimally inhibited activity at higher concentrations. Thus RPTC
ER-iPLA2 activity is specifically inhibited by BEL as opposed to AAOCF3 or MAFP. RPTC ER-iPLA2
decreased sensitivity to inhibition by AAOCF3 and MAFP
compared with BEL is different from the inhibition profile seen for
iPLA2 (6). These differences may be a
result of the membrane location of ER-iPLA2 or may reflect variations in key amino acids in the active sight of each enzyme.
The role of cPLA2 and secretory PLA2 in
cellular physiology and injury is well established (3, 6, 20,
25). In comparison, the role of iPLA2 in normal cell
physiology and injury is not as well characterized. In contrast to
iPLA2, nothing is known concerning the role of
ER-iPLA2 in normal cell physiology and cell injury and
death. Data presented in this study suggest that ER-iPLA2
protects against oxidative-induced cell death. Inhibition of
ER-iPLA2 using BEL potentiated oncosis induced by each
oxidant used in this study but not that of a nonoxidant. The increase in oxidant-induced RPTC oncosis after inhibition of
ER-iPLA2 correlated with increases in lipid peroxidation
early in the genesis of oncosis. Finally, a strong correlation exists
between the percent inhibition of ER-iPLA2 with BEL and
potentiation of cell death by BEL (r2 = 0.98), suggesting that these events are linked. Thus we conclude that
the potentiation of oxidant-induced oncosis in RPTC is a result of
inhibition of ER-iPLA2.
Studies on the role of ER-iPLA2 in cell death were conducted with the previously characterized iPLA2-inhibitor BEL. In the absence of an alternative method to inhibit ER-iPLA2, we verified that the potentiation of oxidant-induced cell death in the presence of BEL was not the result of decreases in cellular GSH, ATP, GTP, and oxygen consumption levels (data not shown).
MDA is an end product of lipid peroxidation, which itself is a result of a free radical attack on an unsaturated double bond of a phospholipid (i.e., initiation) (33). The free radical propagates from one phospholipid to another and eventually leads to the formation of smaller lipid fragments, one of which is MDA. The reaction is stopped or terminated when the free radical is quenched or when it is removed from the site of injury. We propose that oxidant-induced lipid peroxidation results in enhanced metabolism of phospholipids by ER-iPLA2. Metabolism of peroxidized phospholipids by ER-iPLA2 would cleave and remove these damaging species before their conversion to MDA and prohibit propagation of the free radical. If this model is correct, then inhibition of ER-iPLA2 with BEL would result in an increase in lipid peroxides before increases in cell death.
In conclusion, we have identified and characterized a novel
ER-iPLA2 that is similar to iPLA2 but
distinct from iPLA2
in that it is expressed at an
intracellular membrane and inhibited specifically by BEL. Inhibition of
RPTC ER-iPLA2 during oxidant-induced renal cell oncosis
resulted in an increase in the level of lipid peroxides, suggesting
that this enzyme may play a key role in the metabolism and removal of
these damaging species during injury induced by diverse stimuli.
Collectively, these data suggest that ER-iPLA2 plays a
protective role during oxidative stress by removing oxidized phospholipids.
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ACKNOWLEDGEMENTS |
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Purified iPLA2 was a gift from Genetics Institute,
Cambridge, MA.
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FOOTNOTES |
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This work was supported by National Research Service Award DK-10079 (B. S. Cummings) and National Institutes of Health Grants DK-62028 (R. G. Schnellmann) and HL-54907 (J. McHowat).
Address for reprint requests and other correspondence: R. G. Schnellmann, Dept. of Pharmaceutical Sciences, Medical Univ. of South Carolina, 280 Calhoun St., PO Box 250140, Charleston, SC 29425 (E-Mail: schnell{at}musc.edu).
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.
April 16, 2002;10.1152/ajprenal.00022.2002
Received 16 January 2002; accepted in final form 14 April 2002.
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