From the Department of Microbiology and Immunology and § Internal Medicine, University of Texas Medical Branch, Galveston, Texas 77555-1070
Received for publication, July 26, 2000, and in revised form, November 9, 2000
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ABSTRACT |
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Prostaglandins (PG), which are responsible for a
large array of biological functions in eukaryotic cells, are produced
from arachidonic acid by phospholipases and cyclooxygenase enzymes COX-1 and COX-2. We demonstrated that PG levels in cells were partly
controlled by a regulatory protein, phospholipase A2
(PLA2)-activating protein (PLAA). Treatment of murine
macrophages with lipopolysaccharide, interleukin-1 Inflammation is a hallmark of many diseases (e.g.
peritonitis, carditis, and arthritis) and is characterized by edema and tissue injury due to the release of numerous chemotactic and
pro-inflammatory cytokines (e.g.
IL-1 Once AA is liberated from cell membranes, it becomes the substrate for
cyclooxygenases or lipoxygenases to form prostaglandins/thromboxanes or
leukotrienes, respectively. There are two isoforms of cyclooxygenase, constitutive COX-1 and inducible COX-2. COX-2-induced PG production has
been implicated in inflammation, as elevated levels of this protein
have been found in synovial cells treated with inflammatory mediators
or from patients with rheumatoid arthritis (33-37). The expression of
the cox-2 gene appears to be highly regulated by a number of
transcription factors, in particular by NF- Prostaglandin PGE2 is particularly important, since it is a
bronchodilator and vasodilator in the lungs and stimulates adenylate cyclase with the formation of cAMP, which stimulates intestinal chloride secretion during noninflammatory and inflammatory diarrheas (7, 8). PGE2 has been shown to increase cPLA2
and COX-2 levels and thereby amplify its own production, as well as to
increase synthesis of some inflammatory cytokines resulting in a
perpetuation of the immune response (23, 41, 42).
Several drugs have been developed to interrupt PG formation and,
therefore, the severity of the inflammatory response. Two central
targets have been COX-2 and PLA2 (43). Recently, a
phospholipase A2-activating protein (PLAA) cDNA has
been cloned from a human monocyte cell line (U937) by our research
group (44). The cDNA-encoding PLAA was originally cloned from
murine BC3H1 cells using antibodies to melittin, a 26-amino
acid (aa)-long peptide and a major component in bee venom (45). At the
aa level, murine, rat, and human PLAA exhibit 93-97% homology (44).
When murine PLAA was injected into the synovium of rabbits, an
increased amount of inflammation occurred that correlated with an
increase in sPLA2 activity (46). Likewise,
sPLA2 and PLAA have been implicated in a number of other cell functions, including Golgi tubulation and retrograde trafficking, peroxisomal motility in Chinese hamster ovary cells, and calcium oscillations/amylase secretion in rat pancreatic acini (47-49).
Inflammation is a prominent characteristic of inflammatory bowel
diseases (IBD), which include Crohn's disease (CD) and ulcerative colitis (UC) (50); however, the etiology and the pathogenesis of these
diseases are not yet fully understood (6). It is widely accepted that
there are two central components to IBD (8). The first is the
initiation of an aberrant immune response, which may result from
absorption of foreign antigens of microbial origin, possibly from
resident flora in the intestinal lumen. The second is the amplification
and perpetuation of this aberrant immune response (7, 8), which is
responsible for tissue injury due to the involvement of cytokines,
eicosanoids, reactive oxygen species, and complement (6). Examination
of biopsy specimens from patients with UC and CD has revealed a marked
increase in all of these factors and of PLA2 and
PGE2 (4, 51). We initiated studies to assess the
involvement of PLAA in PLA2-associated eicosanoid production leading to inflammation in patients with UC and CD. Our
studies using immunohistochemistry detected PLAA in inflammatory cells
from patients with UC and CD and in mice chemically treated with
dextran sodium sulfate, which induced colitis (52). The tissues from
normal patients and untreated mice were devoid of any staining for
PLAA.
The objectives of this study were as follows: 1) to establish whether
PLAA was induced in inflammatory cells (e.g. macrophages) by
LPS, IL-1 Materials--
Polyclonal antibodies to a synthetic peptide
representing aa residues 185-199 (NSKDFVTTSEDRSLR) of murine PLAA were
generated in rabbits (52). This region was highly conserved in murine, rat, and human PLAA (44). Polyclonal antibodies to cPLA2,
COX-2, and all of the secondary antibodies used in enzyme-linked
immunosorbent assay (ELISA) and Western blot analysis were purchased
from Santa Cruz Biotechnology (Santa Cruz, CA). The polyclonal
antibodies to cPLA2 and COX-2 were to regions conserved
between mouse, rat, and human. Monoclonal anti-human sPLA2
type II antibodies were from Roche Molecular Biochemicals. Polyclonal
antibody to phosphorylated proteins (serine, threonine, and tyrosine)
was purchased from Zymed Laboratories Inc. (South San
Francisco, CA). Bacterial lipopolysaccharide (LPS) from
Escherichia coli, interleukin-1 LPS/IL-1 Immunoprecipitation of Phosphorylated Proteins--
RAW264.7
cells previously treated with LPS (10 ng/ml) and analyzed for PLAA
expression via Western blot analysis were subjected to
immunoprecipitation studies. An aliquot of a protein sample (20 µg)
from untreated and LPS-stimulated RAW cells was incubated with 10 µg
of an antibody against three phosphorylated proteins (serine,
threonine, and tyrosine) for 1 h at 37 °C. Following incubation, 100 µl of protein G-Sepharose was added, and the slurry was mixed at room temperature for 1 h. The slurry was centrifuged at 13,000 × g for 5 min, and the resulting supernatant
was transferred to microcentrifuge tubes. The pellet was washed twice
with PBS. Both the pellet and the supernatant were boiled for 5 min in
200 µl of SDS sample buffer and loaded onto an SDS-12%
polyacrylamide gel as described previously. Western blot was probed
with anti-peptide antibodies to PLAA.
Treatment of Macrophages with Various Inhibitors and Northern
Blot Analysis--
RAW264.7 cells (1 × 106/ml) were
treated with various phospholipase/protease inhibitors concurrently
with LPS (10 ng/ml) or with melittin (1 µg/ml). Cells were treated
with 25 µM LY311727 (sPLA2 inhibitor), 10 µM BTEE, and TPCK (chymotrypsin-like serine protease
inhibitors), 50 µM D609 (PLC inhibitor) or 70 µM propranolol (PLD inhibitor) for periods of 0, 10, 30 min and 2, 4, 8, 24 h. Supernatants were collected and stored at
Gel Shift Assay--
Gel shift assays were performed according
to the manufacturer's recommendations (Promega, Madison, WI). Briefly,
1 × 106 cells/ml were washed twice with 1× PBS,
followed by the addition of 1 ml of PBS, and the cells were scraped
into a cold Eppendorf tube. Cells were spun down at 3,000 rpm for
30 s, and the resulting supernatant was removed. Solution A (50 mM HEPES, pH 7.4, 10 mM KCl, 1 mM
EDTA, 1 mM EGTA, 1 mM dithiothreitol, 0.1 µg/ml phenylmethylsulfonyl fluoride, 1 µg/ml pepstatin A, 1 µg/ml
leupeptin, 10 µg/ml soybean trypsin inhibitor, 10 µg/ml aprotinin,
and 0.5% Nonidet P-40) was added to the pellet in a 2:1 ratio (v/v)
and allowed to incubate on ice for 10 min. The cells were pelleted at
6,000 × g for 30 s, and the supernatant was
removed. Solution B (Solution A + 1.7 M glucose) was added
to the pellet in a 1:1 ratio (v/v) and centrifuged at 12,000 × g for 10 min followed by removal of the supernatant. Solution C (solution A + 10% glycerol and 400 mM KCl) was
added to the pellet in a 2:1 ratio (v/v) and vortexed on ice for 20 min. The cells were centrifuged at 12,000 × g for 20 min, and the resulting nuclear extract supernatant was collected in a
chilled Eppendorf tube. Protein concentrations of nuclear extracts were quantitated using a BCA assay kit (Pierce). Consensus oligonucleotides were end-labeled using T4 polynucleotide kinase and
[ PLA2 Activity and PGE2 Assay from
Stimulated Macrophages--
Cell culture supernatants and whole cell
lysates with the original culture medium were assayed for
PLA2 activity according to protocols described previously
(54). Briefly, 10 µl of supernatant or cell lysate from stimulated
RAW cells was incubated with 10 µl of [3H]AA E. coli cells, 30 µl of high pressure liquid chromatography grade
water, and 10 µl of buffer (500 mM Tris-HCl, pH 8.0, 500 mM NaCl, 1 mM CaCl2, 5 mg/ml bovine
serum albumin, 50% glycerol) for 15 min at 37 °C. After incubation,
150 µl of ice-cold PBS was added, and the solution was centrifuged at
13,000 × g for 10 min. An aliquot (50 µl) of the
supernatant was transferred to scintillation vials and counted for the
release of radioactivity. Prostaglandin levels were determined using an
EIA kit according to the manufacturer's instructions (Amersham
Pharmacia Biotech). Briefly, 50 µl of standard/sample was pipetted
into a precoated goat anti-mouse 96-well plate. Aliquots (50 µl) of a
mouse polyclonal PGE2 antibody and PGE2
conjugated to alkaline phosphatase were added to each well and allowed
to incubate at room temperature for 1 h. After incubation, wells
were washed six times with 400 µl of 1× PBS + 0.5% Tween 20, followed by the addition of 150 µl of 3,3',5,5'-tetramethylbenzidine
substrate (Sigma). Wells were read at 670 nm with an ELISA reader
(Molecular Dynamics, Sunnyvale, CA) 30 min after addition of substrate.
Pretreatment of Macrophages with Antisense plaa
Oligonucleotide/cPLA2 Antisense Oligonucleotide Prior to
Stimulation with LPS--
RAW264.7 cells at a concentration of 1 × 106 cells/ml were incubated with 3H-labeled
AA overnight (1 µCi/ml) and washed three times with Dulbecco's
modified Eagle's medium to remove unincorporated label. Cells were
pretreated for 4 h with 25 µg/ml antisense plaa
oligonucleotide (5'-TGGAGTGGCCGCTCATACAGTGCAT-3') or a random
oligonucleotide of similar base composition and length (44). Following
pretreatment, cells were stimulated with 10 ng/ml LPS for 24 h,
and aliquots of the supernatants were collected and counted to
determine AA release. Similarly a synthetic oligonucleotide to
antisense cPLA2 was purchased from Cayman Chemical Co. (Ann
Arbor, MI) and applied to cells (10 µg/ml) using Clonfectin
(CLONTECH) for 4 h prior to stimulation with
LPS. Cell supernatants were collected and assayed for PGE2
levels. The cell lysates were analyzed for cPLA2 and COX-2
antigen levels by Western blot analysis.
Biopsy Specimens--
Over 200 biopsy specimens were collected
under an Institutional Review Board-approved protocol from
individuals undergoing endoscopic procedures for diagnostic purposes,
cancer surveillance, colon cancer screening, and assessment of diarrhea
and of responses to therapy. In addition to these specimens, which were
categorized as either normal control samples or other types of
intestinal inflammation based on pathology reports, we collected
specimens from patients diagnosed with IBD (UC and CD), and these
samples consisted of both macroscopically inflamed and uninflamed areas.
Statistical Analysis--
Data were analyzed by a multiple group
comparison test (Scheffé), which allowed all groups to be
compared with each other, and hence is the most stringent statistical
method available. In addition, the Student's t test was
performed on a number of samples for statistical significance between
two variables. A p value of LPS Stimulates PLAA Production in Macrophages--
To ascertain
whether PLAA was increased by inflammatory mediators, we stimulated
RAW264.7 cells with LPS (10 ng to 10 µg/ml) followed by Western
analysis for PLAA using anti-PLAA peptide antibodies. As seen in Fig.
1A, 10 ng/ml LPS resulted in
increased levels of a 72-kDa polypeptide, which corresponded to the
size of full-length PLAA. PLAA levels became maximal within 10-30 min (3-fold increase) followed by a reduction at 4 h, which continued to decrease through the last time point tested of 24 h.
Significant increases in PLAA levels were observed at higher
concentrations of LPS (10 µg/ml, data not shown). We selected a more
physiological relevant concentration (10 ng/ml) of LPS in all of our
subsequent experiments. The PGE2 levels in the culture
supernatants of these cells were similarly elevated with significant
increases within 30 min, becoming maximal at 24 h (Fig.
2). The PLA2 activity, as
measured by AA release, also revealed a significant increase within 30 min in culture supernatants and cell lysates. The PLA2 activity decreased at 2 h followed by an increase up to 8 h
(Fig. 2). Other enzymes utilized for prostaglandin formation were also elevated upon stimulation of macrophages with LPS. Cytosolic
PLA2 (cPLA2) showed an increase within 30 min
and continued to increase for 8 h, resulting in a 5-fold increase
in protein levels at 8 h based on Western blot analysis (Fig.
1B). Interestingly, neither COX-2 nor secretory
PLA2 (sPLA2) protein levels increased until 4 h after LPS stimulation of macrophages and continued to increase until the last time point tested of 24 h with 50- and 5-fold
increases in protein levels, respectively, based on Western blot
analysis (Fig. 1, C and D).
IL-1 PLAA Is a Phosphorylated Protein Based on Immunoprecipitation
Analysis--
Immunoprecipitation studies of PLAA were conducted to
ascertain whether the activity of PLAA might be regulated by
phosphorylation due to its early up-regulation similar to other
signaling molecules. Western blot analysis of phosphorylated proteins
(immunoprecipitated with a phosphospecific antibody) using antibodies
to PLAA revealed a similarly sized polypeptide both in 0- and 10-min
samples after LPS stimulation, indicating that PLAA was phosphorylated
before stimulation. We selected the 10-min time point, as maximum PLAA production was noted within the first 30 min of LPS stimulation. The
0-min sample served as a control. No PLAA-specific band was detected in
the supernatant portion of the immunoprecipitation reaction (data not shown).
Release of [3H]AA from LPS-stimulated Macrophages Is
Reduced in Cells Pretreated with plaa-specific Oligonucleotide--
To
establish a more definitive role for PLAA in increasing phospholipase
activity and subsequently PGE2 production, RAW264.7 cells
were pretreated with antisense plaa oligonucleotide for 4 h prior to stimulation with 10 ng/ml LPS. Results showed that LPS elicited a dramatic decrease in AA release compared with cells that
had been pretreated with a scrambled oligonucleotide of similar length
and base composition (Fig. 4). Similar
results were obtained when the concentration of LPS used to stimulate
the cells was increased to 1 ng/ml (data not shown). Our data also
indicated that plaa antisense oligonucleotide-treated cells
exhibited a reduced level of cPLA2 compared with cells
treated with LPS and random oligonucleotide based on Western analysis,
indicating a direct effect of PLAA in regulating cPLA2,
which controls AA levels in cells.
Melittin Induces Expression of COX-2- and TNF PGE2 Is Formed in LPS-stimulated Macrophages Via
Various Phospholipases--
The paucity of literature as to the
identity of the specific phospholipases involved in LPS-induced AA
release from phospholipids prompted us to initiate studies to ascertain
the contribution of various phospholipases in LPS-induced
PGE2 production in macrophages. LPS-stimulated RAW264.7
cells concurrently treated with the sPLA2 inhibitor
LY311727, PLC inhibitor D609, or PLD inhibitor propranolol showed
significant decreases (~90%) in PGE2 production, as
compared with cells treated with LPS alone (Fig.
7). To address the specificity of these
various inhibitors, we examined cox-2 gene expression. It is
evident from Fig. 8A that
sPLA2 inhibitor LY311727 abrogated LPS-induced
cox-2 gene expression in macrophages when compared with
cells treated with LPS alone (Fig. 8C). However, the PLC inhibitor D609 (data not shown) and PLD inhibitor propranolol did not
alter LPS-induced cox-2 gene expression, as indicated by
both Northern and Western blot analyses (Fig. 8, B and
C). In addition to the various inhibitors, we also tested
the effects of pretreatment of LPS-stimulated cells with an antisense
oligonucleotide to cPLA2 upon PGE2 production.
As shown in Fig. 7, pretreatment of cells with a specific antisense
oligonucleotide to cPLA2 reduced LPS-stimulated
PGE2 production by ~60%, as compared with cells treated
with LPS alone. We further demonstrated that cells pretreated with
cPLA2 antisense oligonucleotide prior to LPS stimulation reduced cPLA2 levels based on Western blot analysis;
however, the levels of COX-2 remained unaffected (data not shown),
indicating specificity of the antisense oligonucleotide used. The data
in Fig. 7 pointed to the role of both cPLA2 and
sPLA2 as well as PLC and PLD in PG production in
LPS-stimulated macrophages.
Protease Inhibitors and sPLA2 Inhibitor Abrogated cox-2
Gene Expression by Preventing LPS-induced Translocation of NF- Inflammatory Mediators Are Up-regulated in Biopsy Specimens from
Patients with Inflammation--
We have screened over 200 patient
tissue specimens for antigen levels of PLAA, cPLA2, COX-2,
and sPLA2 as well as for PLA2 activity.
Representative biopsy specimens were from patients with Crohn's
disease (including both quiescent and actively diseased tissue
samples), unclassified IBD samples, inflamed colon samples, and normal
samples. In addition to these biopsy specimens, patients with other
types of intestinal inflammation, including Clostridium difficile colitis, melanosis coli, and ischemic colitis were also analyzed. Western blot analysis of these specimens for PLAA,
cPLA2, COX-2, and sPLA2 revealed that actively
diseased and inflamed tissues contained elevated levels of these
antigens as compared with quiescent and normal tissue sections (Fig.
10, A-D). The increase in
these antigen levels coincided with an increase in PLA2
activity in these tissues, which was 3-5-fold higher (data not shown)
compared with that of patients in which no apparent inflammation was
evident. Likewise, of the 14 analyzed cases of actively diseased IBD,
71% had elevated levels of these antigens as compared with normal controls (data not shown).
For an inflammatory response to occur in the host, there must be
an initial event (e.g. infection and trauma). This event either triggers an explosive cellular infiltration response that may
result in injury to the tissues of the host or a protective innate
immune response. This protective immune response may become uncontrolled due to predisposing factors, such as the genetics of the
individual in cases of UC or CD (7). Patients with UC and CD exhibit
increased permeability of their intestinal mucosa (6), which allows
bacterial products, such as LPS, to penetrate the lamina propria from
the intestinal lumen and to bind to its receptor, CD14, on macrophages
to induce transcriptional activation of a number of genes. Likewise,
antiglycan antibodies to the cell wall mannan of Saccharomyces
cerevisiae and mycobacterial antigens are highly correlated with
Crohn's disease (56-58). LPS is a known stimulator of the immune
response and of the pro-inflammatory cytokines IL-1 Among various genes activated by LPS in host cells, we sought to
delineate the role of PLAA, a G-like regulatory protein (59), in
PGE2 production, as its level was up-regulated within
minutes in LPS-stimulated macrophages (Fig. 1A). To define
the role of PLAA in eicosanoid formation, we used plaa
antisense oligonucleotide, and we demonstrated that it decreased levels
of AA release in LPS-stimulated cells (Fig. 4). In addition, the
plaa antisense oligonucleotide-pretreated cells reduced
LPS-induced cPLA2 levels, indicating the direct role of
PLAA in controlling cPLA2. These data demonstrated the
importance of PLAA in the regulation of eicosanoid formation at the
initial rate-limiting step of substrate (AA) formation. The ability of
PLAA to increase sPLA2 activity in rheumatoid arthritis as
well as in cell extracts of BC3H1 cells has been reported,
thus making PLAA an important regulatory component in inflammation (1,
60, 61). Furthermore, the possibility that PLAA may regulate additional
phospholipases (e.g. PLC and PLD) cannot be ruled out, and
these studies are currently under investigation. The ability of
melittin, which exhibits homology within the sequence of PLAA (62), to
produce PGE2 via activation of PLC and PLD, in addition to
PLA2 (Fig. 6A), points to the possible role of
PLAA in regulating various phospholipases.
In addition to LPS, stimulation of macrophages was examined with the
pro-inflammatory cytokines IL-1 Most reports on PLAA have focused primarily on cell culture, with few
clinical correlations. Likewise, most Western analysis on PLAA has
utilized antibodies to melittin, rather than to PLAA. This has resulted
in some confusion regarding the size of PLAA, as antibodies to melittin
resulted in the identification of PLAA in IL-1 The purported role of melittin as an sPLA2 activator is
widely known (45, 68-71). Melittin is also highly hemolytic to sheep red blood cells; however, downstream effects of melittin on cells have
not been described (72). The homology of PLAA to melittin led us to
ascertain the effects melittin had directly on macrophages, which could
then be correlated back to PLAA. As shown in Fig. 5, melittin caused
increases in abundance of message for both TNF Melittin also blocked many of the toxic effects of Lipid A, such as
pyrogenicity in the rabbit model and lethality of chick embryos by
preventing Lipid A from binding its receptor (73). However, the effects
melittin has on LPS-induced PGE2 production have not been
examined. Interestingly, melittin reduced PGE2 levels in
LPS-stimulated RAW cells by ~75% (Fig. 6B). The exact
mechanism of reduced PGE2 production by melittin in
LPS-stimulated cells is currently unknown, but it has been hypothesized
that melittin could prevent binding of LPS to its receptor (73).
Alternatively, melittin has been reported to be a noncompetitive
inhibitor of PLA2 (62). Based on these reports (62, 73),
melittin would be expected to antagonize the effect of LPS on
PLA2 activity and subsequent PGE2 production.
Synthetic peptides of PLAA that share this region of homology with
melittin could be developed to block LPS-induced PGE2
production to help abrogate the immune response.
Since it is unknown whether PLAA regulates more than one kind of
phospholipase and since the relative contribution of each of the
phospholipases upon PG production has not been thoroughly investigated
in the literature, we sought to establish the involvement of
PLA2, PLC, and PLD in PG synthesis. Through the use of
various inhibitors and antisense oligonucleotides, our results
indicated that several phospholipases are involved in PG production as
inhibitors to sPLA2, cPLA2, PLC, and PLD
significantly reduced PG production in LPS-stimulated macrophages (Fig.
7). Since our studies demonstrated the role of cPLA2 early
in AA metabolism, we used methyl arachidonyl fluorophosphonate (5 µM) to show whether cPLA2 was also involved in late PG production, as cPLA2 antigen levels remained
high at 8-24 h based on Western blot analysis (Fig. 1B).
Our results indicated that this inhibitor could itself induce
PGE2 production (data not shown). These results were
recently substantiated by Lin and Chen (74) who indicated that
cPLA2 inhibitor methyl arachidonyl fluorophosphonate
induced PGE2 production via activation of COX-2. To
circumvent this problem, we used a cPLA2 antisense
oligonucleotide, which reduced LPS-induced PGE2 production
in macrophages by 60% after 24 h (Fig. 7), indicating
cPLA2 to be involved in both early and late phase
production of PGE2 (21). Our data also supported the role
of sPLA2 in late PGE2 production based on
Western blot analysis (Fig. 1D) and the ability of
cPLA2 antisense oligonucleotide to block only 60% of
PGE2 production (Fig. 7). At present, we are investigating
cross-talk between cPLA2 and sPLA2 that leads to sustained PGE2 production over a period of 24 h.
The ability of various phospholipases (e.g.
PLA2, PLC, and PLD) to reduce significantly PG production
also suggests some communication among these phospholipases as well,
with recent studies by Ueno et al. (75) indicating
cross-talk between sPLA2 and PLD.
The sPLA2 inhibitor LY311727 reduced PGE2
production in RAW cells (Fig. 7); however, we noted that its effect was
partly nonspecific as this inhibitor also reduced cox-2 gene
expression (Fig. 8). On the contrary, both the PLC and PLD inhibitors
did not alter cox-2 gene expression but significantly
reduced PGE2 levels in LPS-stimulated macrophages (Figs. 7
and 8). These results indicated that COX-2 production was not regulated
by availability of AA substrate and that the cox-2 gene was
independently expressed in LPS-stimulated macrophages. The presence of
two binding sites for NF- Our results indicated that PLAA potentially could play an important
role in many inflammatory diseases such as IBD. Reports that purified
PLAA can induce synthesis of the proinflammatory cytokines IL-1 To substantiate that PLAA is a major contributor to the inflammation
seen in IBD, we examined biopsy specimens from patients with CD, UC,
and from other patients undergoing endoscopy for increases in PLAA,
cPLA2, COX-2, and sPLA2 antigen levels (Fig. 10, A-D). Most IBD cases examined were from patients with
CD; however, similar results were seen in patients with UC. We have
analyzed over 200 samples consisting of both normal, IBD, and non-IBD
inflamed tissue, with results indicating that only those tissues that
were pathologically inflamed showed increased levels of the above
proteins and elevated PLA2 activity. These results
corroborate our tissue culture studies and support the role of PLAA in
inflammation. In addition, the level of PLC and PLD in biopsy specimens
is currently under investigation to confirm their role in inflammation.
Since many of the responses seen in IBD, such as elevated cytokine and eicosanoid production, are common to many other inflammatory diseases, we would expect that regulation of PLAA would not only help patients with intestinal inflammation but improve inflammation in patients with
other general types of inflammation such as allergies, asthma, and
arthritis. Given that PLAA is an upstream mediator of PLA2 activity, and the ability of PLAA to regulate other phospholipases is
unknown, it is important that we determine in the future the direct
effect of PLAA on other phospholipases. Furthermore, since PLC and PLD
significantly contribute to PG production, we can target both PLAA and
these phospholipases as therapeutic tools to control inflammation.
, and tumor
necrosis factor-
increased PLAA levels at early time points (2-30
min), which correlated with an up-regulation in cytosolic
PLA2 and PGE2 levels. Both COX-2 and secretory
PLA2 were also increased in lipopolysaccharide-stimulated
macrophages, however, at later time points of 4-24 h. The role of PLAA
in eicosanoid formation in macrophages was confirmed by the use of an
antisense plaa oligonucleotide. Within amino acid residues
503-538, PLAA exhibited homology with melittin, and increased
PGE2 production was noted in macrophages stimulated with
melittin. In addition to PLA2, we demonstrated that
activation of phospholipase C and D significantly controlled
PGE2 production. Finally, increased antigen levels of PLAA,
COX-2, and phospholipases were demonstrated in biopsy specimens from
patients with varying amounts of intestinal mucosal inflammation, which
corresponded to increased levels of phospholipase activity. These
results could provide a basis for the development of new therapeutic
tools to control inflammation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
,1 TNF
, and IL-8),
phospholipase A2 (PLA2), and eicosanoids
(e.g. prostaglandins (PGE2) and leukotriene
B4) (1-7). Phospholipase and eicosanoid production are
inextricably linked through a pathway originating from arachidonic acid
(AA), a 20-carbon, unsaturated fatty acid that is hydrolyzed from
membrane phospholipids by secretory PLA2
(sPLA2) and cytosolic PLA2 (cPLA2)
(8). Among these PLA2s, a 14-kDa type IIa
sPLA2, which hydrolyzes phospholipids from the sn-2 position, has been extensively studied. The
sPLA2 requires millimolar amounts of calcium for activity
and has been linked to many other inflammatory diseases, notably adult
respiratory distress syndrome (5), rheumatoid arthritis (1), and asthma (2, 9, 10). Recently, cPLA2 (85-110 kDa) has been
characterized and found to preferentially use glycerophospholipids as a
substrate and requires submicromolar amounts of calcium for activity
(11). A third PLA2 is calcium-independent PLA2
(iPLA2), and, although not as extensively characterized as the
others, iPLA2 is thought to be responsible for the late phase
production of prostaglandins (PGs) when intracellular calcium levels in
cells have been depleted (12-14). Several groups (15-20) have
reported that sPLA2 is the primary phospholipase involved
in PG production in target cells. Conversely, other investigators have
documented that cPLA2 is the predominant phospholipase
involved in PG formation (13, 21-27). The contribution of the other
phospholipases (PLC and PLD) in PG production has not been as
extensively studied (28). It is plausible that various phospholipases
may be involved in PG production, and the induction of a particular
phospholipase is dependent on the cell type involved, the stimulus
given, and the activation state of the cell before induction (11,
29-32).
B (35, 37-40).
, and TNF
; 2) whether an antisense oligonucleotide to
plaa cDNA would inhibit AA release from stimulated
cells, which could lead to the development of potentially important
therapeutic PLAA peptides controlling inflammation; 3) whether other
phospholipases (e.g. PLC and PLD) contribute to
PGE2 production; and 4) whether levels of PLAA,
phospholipases, and COX-2 could be correlated to the extent of
inflammation in patients with IBD.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(IL-1
), and protease inhibitors N-tosyl-L-phenylalanine chloromethyl
ketone (TPCK) and N-benzoyl-L-tyrosine ethyl
ester (BTEE) were from Sigma. Tumor necrosis factor-
(TNF
) was
obtained from PeproTech, Inc. (Rocky Hill, NJ). LY311727 was a gift
from Lilly. Phospholipase C inhibitor D609 and phospholipase D
inhibitor propranolol were purchased from Biomol (Plymouth Meeting,
PA). Prostaglandin E2 enzyme immunoassay kit and
protein G-Sepharose were purchased from Amersham Pharmacia Biotech. Gel
shift assay kits were from Promega (Madison, WI). The probe for
cox-2 cDNA was from Cayman Chemical Co. (Ann Arbor, MI).
The probe for TNF
encoding cDNA was generated by reverse transcription-polymerase chain reaction using specific TNF
reverse transcription-polymerase chain reaction amplimers
(CLONTECH, Palo Alto, CA) and total RNA from
macrophage cell line RAW264.7. A 354-base pair cDNA fragment was
cloned into TA cloning vector pCR2.1 (Invitrogen, Carlsbad CA). The
recombinant plasmid was digested with the EcoRI restriction
enzyme to obtain the fragment. The probe for glyceraldehyde-3-phosphate
dehydrogenase cDNA was purchased from CLONTECH.
Synthetic melittin, a 26-aa-long peptide (53), was synthesized and
purified by high pressure liquid chromatography at the M.D. Anderson
Cancer Center (Houston, TX).
/TNF
/Melittin Stimulation of Macrophages and
Western Blot Analysis--
RAW264.7 cells (ATCC, Manassas, VA), a
murine macrophage-like cell line (1 × 106 cells/ml),
were cultured in Dulbecco's modified Eagle's medium and 10% fetal
bovine serum with 50 µg/ml gentamicin and 100 units/ml penicillin/streptomycin at 37 °C, 5% CO2. The cells
were treated either with 10 ng to 10 µg/ml LPS, 10 ng/ml IL-1
,
100-500 ng/ml TNF
, and 1 µg/ml synthetic melittin for selected
times of 2, 10, 30, and 60 min and 2, 4, 8, and 24 h. After
incubation with LPS, IL-1
, TNF
, or melittin, cell supernatants
were collected and stored at
70 °C until further analysis. The
cell monolayer was rinsed with phosphate-buffered saline (PBS) at room
temperature and then lysed in RIPA buffer (1× PBS, 1% Nonidet P-40,
0.5% sodium deoxycholate, 0.1% SDS) containing protease inhibitors
(100 µg/ml phenylmethylsulfonyl fluoride, aprotinin at 30 µl/ml,
and 100 mM sodium orthovanadate at 10 µl/ml) as described
by the manufacturer (Santa Cruz Biotechnology). The cell lysate was
passed through a 21-gauge needle to shear the DNA and centrifuged at
15,000 × g for 20 min to remove cellular debris. The
protein concentration of the resulting supernatant was determined using
a BCA protein assay kit according to the manufacturer's instructions
(Pierce). Equal amounts of protein were loaded onto an SDS-12%
polyacrylamide gel and electrophoresed at 200 V for 45 min followed by
transfer to a nitrocellulose membrane. The blots were briefly rinsed in 1× Tris-buffered saline (TBS) and then incubated overnight in 3%
gelatin to block nonspecific binding of antibodies to the membrane. The
blots were then washed twice for 10 min with 1× TBS containing 0.5%
Tween 20 (TTBS) and incubated with the appropriate primary antibody (1 µg/ml) for 2 h followed by washing twice for 10 min each with
1× TTBS. The blots were then incubated with the appropriate secondary
antibody (1:25,000) for 1 h. Finally, blots were washed three
times with 1× TTBS and once with 1× TBS for 10 min each followed by
development using an enhanced chemiluminescence (ECL) kit (Pierce)
(54). The blots were scanned using a gel documentation system (Bio-Rad)
to calculate fold increase in the antigen level.
70 °C for further analysis. RNA was isolated using TriZol reagent
according to protocols outlined by Life Technologies, Inc. Briefly, 1 ml of TriZol reagent was added to each 35-mm dish and allowed to shake
for 5 min. Cell lysate was transferred to a microcentrifuge tube with
subsequent addition of 200 µl of chloroform and shaken for 15 s,
followed by centrifugation at 12,000 × g for 15 min.
The aqueous phase was transferred to a new microcentrifuge tube, and
the RNA was precipitated using 0.5 ml of isopropyl alcohol. RNA was
allowed to precipitate at room temperature for 10 min and centrifuged at 12,000 × g for 10 min. The supernatant was removed,
and the RNA pellet was washed with 1 ml of 80% ethanol followed by
centrifugation at 7,500 × g for 5 min. The RNA pellet
was air-dried for 5 min and resuspended in diethyl
pyrocarbonate-treated water. RNA was quantitated using a
spectrophotometer (Amersham Pharmacia Biotech). Subsequently, RNA was
separated on a 3% formaldehyde/agarose gel and visualized by staining
with ethidium bromide (55). RNA was transferred to a nylon membrane and
baked at 80 °C for 2 h. Membranes were prehybridized for 15 min
at 68 °C with Quikhyb (Stratagene, La Jolla CA), followed by the
addition of an appropriately labeled [
-32P]dCTP probe
(1 × 106 cpm/ml) (ICN, Costa Mesa CA) and 10 mg/ml
salmon sperm DNA (Stratagene) in the hybridization solution as
described (54). Hybridization was performed for 2 h at 68 °C
followed by 4 washes for 20 min each with 2× SSC (1× SSC is 0.15 M sodium chloride plus 0.015 sodium citrate, pH 7.4) + 0.1% SDS (twice) and 1× SSC + 0.1% SDS (twice) at 68 °C.
Membranes were allowed to air dry for 5 min before being exposed to
film overnight at
70 °C. Diethyl pyrocarbonate-treated water was
used to prepare all the reagents used for the RNA work.
-32P]dATP (ICN) for 10 min at 37 °C and the
unincorporated 32P removed (54). Gel shift reactions were
assembled as follows where the term sample denotes nuclear extracts
from treated RAW cells: negative control (7 µl of water and 2 µl of
gel shift binding buffer), positive control/sample (5 µl of water, 2 µl of gel shift binding buffer, 2 µl of HeLa nuclear extract/sample
(10 µg/ml), specific competitor of a transcription factor (4 µl of
water, 2 µl of gel shift binding buffer, 2 µl of HeLa nuclear
extract/sample (10 µg/ml), 1 µl of unlabeled competitor
oligonucleotide), supershift reaction (3 µl of water, 2 µl of gel
shift binding buffer, 2 µl of HeLa nuclear extract/sample (10 µg/ml), 2 µl of specific supershift antibody (e.g.
p50/p65)) (Santa Cruz Biotechnology). Reactions were assembled and
allowed to incubate at room temperature for 10 min followed by the
addition of 1 µl (50,000-200,000 cpm) of 32P-labeled
oligonucleotide and another 20 min of incubation at room temperature.
Subsequently 1 µl of gel loading buffer was added to each reaction
and loaded onto a 4% nondenaturing gel and electrophoresed until the
dye was three-fourths of the way down the gel. The gel was dried at
80 °C for 2 h and exposed to film overnight at
70 °C.
0.05 was considered
statistically significant.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Western blot analysis on proteins from LPS
(10 ng/ml)-treated murine macrophage RAW264.7 cell line. Fifty
micrograms of total proteins were loaded into each lane of an SDS-12%
PAGE, and the blots were probed with polyclonal anti-peptide antibodies
to PLAA (A), polyclonal antibodies to cPLA2
(B), polyclonal antibodies to COX-2 (C), or
monoclonal antibodies to sPLA2 (D). Cell lysates
(A-C) were prepared in RIPA buffer with appropriate
protease inhibitors as described under "Experimental Procedures."
Cell supernatants (D) were directly loaded on to the gel.
Appropriate secondary antibodies conjugated with horseradish peroxidase
were used, followed by an enhanced chemiluminescence substrate kit
(Pierce) to develop the blots. The blots were scanned to calculate fold
increase in the antigen level. Three independent experiments were
performed, and a representative blot is shown.
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Fig. 2.
LPS increases PLA2 activity and
PGE2 levels in macrophages. PLA2 and
PGE2 levels were measured in culture supernatants or cell
lysates from LPS (10 ng/ml)-treated murine macrophage RAW264.7 cell
line. The PLA2 activity was measured using oleic
acid-labeled E. coli as a substrate, whereas
PGE2 was measured by a radioimmunoassay kit (for details
see "Experimental Procedures"). Three independent experiments were
performed, and an average mean with standard errors was plotted.
Asterisks denote statistically significant values at
p 0.05
and TNF
Stimulates PLAA Production in
Macrophages--
In addition to LPS, we also examined other
inflammatory mediators such as pro-inflammatory cytokines for their
effects on PLAA and PLA2 production. Stimulation of
RAW264.7 cells with 10 ng/ml IL-1
revealed an increase in PLAA
antigen levels within 2 min as determined by Western blot analyses
(Fig. 3A). PLAA levels continued to increase for up to 30 min with a 3-fold increase in
antigen levels (Fig. 3A). A similar pattern was noted for
cPLA2 levels, and the increases in the levels of PLAA and
cPLA2 coincided with the increase in PLA2
activity (data not shown). RAW264.7 cells stimulated with various
concentrations of TNF
(100 ng-500 ng) followed by Western blot
analysis for PLAA revealed a band of ~72 kDa, which increased in
intensity within 10 min, followed by a decrease at 120 min for
concentrations of 100 and 200 ng/ml (Fig. 3B). However,
TNF
concentration of 500 ng/ml resulted in an increase in PLAA
within 10 min that continued to increase up to the last time point
tested of 2 h after stimulation, with a 50-fold increase in
antigen level (Fig. 3B).
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Fig. 3.
Western blot analysis on proteins from
IL-1 (10 ng/ml)-treated murine macrophages
RAW264.7 cell line (A) and TNF
(100-500 ng/ml)-treated RAW cells (B). 25 µg of total proteins were loaded into each lane of an SDS-12% PAGE,
and the blots were probed with polyclonal anti-peptide antibodies to
PLAA. Cell lysates were prepared in RIPA buffer with appropriate
protease inhibitors as described under "Experimental Procedures."
Appropriate secondary antibodies conjugated with horseradish peroxidase
were used, followed by enhanced chemiluminescence substrate kit
(Pierce) to develop the blots. The blots were scanned to calculate fold
increase in the antigen level. Three independent experiments were
performed, and a representative blot is shown.
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Fig. 4.
Antisense oligonucleotide to plaa
reduced [3H]AA release from
LPS-stimulated RAW cells. RAW264.7 cells were radiolabeled with
[3H]AA overnight, and excess unincorporated label was
removed. Cells were treated with 25 µg/ml of an antisense
oligonucleotide to plaa or a random oligonucleotide of
similar base composition and length for 4 h prior to stimulation
with LPS. Aliquots of cell supernatants were assayed for percent
release of radiolabeled AA using a -counter. Cells treated with LPS
alone served as a positive control. Three independent experiments were
performed, and an arithmetic means ± S.E. are presented.
Asterisks denote statistical significance at
p < 0.05.
-encoding Genes in
Macrophages--
The sequence of melittin, which has a high degree of
homology within a region of PLAA (aa residues 503-538), prompted us to examine the effect of synthetic melittin on COX-2 and TNF
production in macrophages. RAW cells treated with a sub-lethal concentration of
melittin (1 µg/ml) resulted in increased abundance of TNF
and
COX-2 mRNA (Fig. 5). The maximum
increase in the message for TNF
was noted at 2 h but then
declined thereafter. The increase in abundance of COX-2 mRNA
occurred at 4 h and was maximally increased at 8 h. As an
internal control, a cDNA probe for glyceraldehyde-3-phosphate dehydrogenase was used (Fig. 5). After macrophages were stimulated with
melittin, an increase in PGE2 levels occurred in a
time-dependent fashion with maximum PGE2 levels
observed in 24 h (data not shown). PGE2 levels in
melittin-stimulated cells appeared to be primarily produced via PLC, as
an inhibitor to PLC reduced PGE2 levels in melittin-treated
cells by 77% as compared with sPLA2 and PLD inhibitors, which reduced PGE2 levels by 43 and 37%, respectively
(Fig. 6A). Most interestingly,
melittin was a potent inhibitor of LPS-induced PGE2
production when both were applied to the cells at the same time. As
seen in Fig. 6B, melittin (1 µg/ml) reduced
PGE2 production in LPS-stimulated macrophages by
~75%.
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Fig. 5.
Synthetic melittin-stimulated transcription
of genes coding for TNF and COX-2 as
determined by Northern blot analysis. RAW264.7 cells were treated
with 1 µg/ml synthetic melittin for various times. RNA was isolated
using TRIzol® reagent, and equal amounts were separated on a 3%
formaldehyde/agarose gel. RNA was transferred to nylon membranes and
probed with
-32P-labeled cDNA probe for TNF
. The
blots were exposed to film overnight. After exposure, the blots were
stripped (50% formamide + 2× SSC, 2 h 68 °C) to remove probe
and reprobed with
-32P-labeled cDNA probe for COX-2
and glyceraldehyde-3-phosphate dehydrogenase (G3PDH) to
normalize for RNA loading. Three independent experiments were
performed, and the results from a representative experiment are
shown.
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Fig. 6.
Melittin induced PGE2 production
via PLC and reduced LPS-induced PGE2 production in RAW
cells. RAW264.7 cells were treated concurrently with 1 µg/ml
synthetic melittin and a phospholipase inhibitor to either
sPLA2 (LY311727 (25 µM)), PLC (D609 (50 µM)), or PLD (propranolol (70 µM))
(A). RAW264.7 cells were treated with 10 ng/ml LPS alone or
with 1 µg/ml synthetic melittin and LPS at the same time
(B). The supernatants were collected and analyzed for
PGE2 levels using an enzyme immunoassay. Three independent
experiments were performed, and an arithmetic means ± S.E. are
shown. Asterisks denote statistical significance at
p < 0.05.
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Fig. 7.
Various phospholipase inhibitors reduced
PGE2 levels in RAW264.7 cells. RAW cells were treated
with LPS (10 ng/ml) and LY311727 (25 µM), propranolol (70 µM), D609 (50 µM), BTEE (10 µM), TPCK (10 µM), or with antisense
cPLA2 oligonucleotide for 24 h as described under
"Experimental Procedures." Supernatants from variously treated
cells were collected and assayed for PGE2 levels using an
enzyme immunoassay kit (Amersham Pharmacia Biotech) as described under
"Experimental Procedures." Arithmetic means ± S.E. are
plotted, and asterisks indicate statistically significant
differences at p < 0.05, compared with LPS alone from
four different experiments.
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Fig. 8.
Inhibitors to sPLA2 reduced
cox-2 gene expression in RAW cells, whereas inhibitors
to PLD did not, based on Northern and Western blot analysis.
RAW264.7 cells were treated with LPS (10 ng/ml) and indicated
inhibitors at the same time for various time points. RNA was isolated
using TRIzol® reagent, and equal amounts of RNA were separated on a
formaldehyde/agarose gel. RNA was transferred to nylon membranes and
probed with -32P-labeled cDNA probe for COX-2. Equal
amounts of protein (15 µg/ml) isolated from cells were separated on
an SDS-12% PAGE gel and transferred to a nitrocellulose membrane.
Membranes were then probed with COX-2 antibodies, followed by
appropriate secondary antibody, and the blots developed with a
chemiluminescence substrate. A, Northern and Western blots
of LPS- and LY311727 (25 µM)-treated cells. B,
Northern and Western blots of LPS- and propranolol (70 µM)-treated cells. C, Northern and Western
blots of cells treated with LPS alone (10 ng/ml). Four independent
experiments were performed, and the results from a representative
experiment are shown.
B in
the Nucleus--
To define the mechanisms(s) that leads to
down-regulation of cox-2 gene expression in LPS-stimulated
cells by protease inhibitors (e.g. TPCK and BTEE) and
LY311727, we determined their effect on the activation of transcription
factor NF-
B. Both protease inhibitors and LY311727 decreased
PGE2 production in LPS-stimulated cells by 60-97% (Fig.
7). These protease inhibitors also abrogated cox-2 gene
expression in LPS-induced cells (data not shown). Both TPCK and BTEE
significantly reduced LPS-induced NF-
B (p50/p65 heterodimer) that
was translocated into the nucleus as compared with cells stimulated
with LPS alone (data not shown). Likewise, the sPLA2
inhibitor (LY311727) reduced translocation of p50/p65 in LPS-induced
macrophages as observed for protease inhibitors TPCK and BTEE (data not
shown). In contrast, no significant effect on NF-
B p50/p65
heterodimer translocation was observed in macrophages treated with
propranolol and LPS (Fig. 9A)
when compared with cells treated with LPS alone (Fig. 9B).
Similarly, no effect on NF-
B translocation was observed when cells
were treated with a PLC inhibitor, D609 (data not shown).
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Fig. 9.
PLD inhibitor did not alter
NF- B translocation in LPS-stimulated
macrophages. Gel shift analysis of RAW264.7 cells treated with
propranolol (70 µM) and LPS (10 ng/ml) (A) or
LPS alone (B) for indicated times. Gel shift nuclear
extracts from stimulated RAW cells were isolated according to protocols
outlined under "Experimental Procedures" and incubated with
-32P-labeled consensus oligonucleotide of NF-
B for 20 min prior to separation on a nondenaturing 4% PAGE. After
electrophoresis, the gel was dried and exposed to film overnight.
Arrows indicate p50/50 homodimer and p50/p65 heterodimer
bands of NF-
B. Three independent experiments were performed, and the
results from a representative experiment are shown.
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Fig. 10.
Up-regulation of inflammatory mediators in
patients with IBD. Representative specimens from patients
diagnosed with IBD (CD) consisting of both actively inflamed tissue and
quiescent tissue and from patients with normal tissue were analyzed for
cPLA2 (A), COX-2 (B), PLAA
(C), and sPLA2 (D) by Western blot
analysis. A and B were from the same patient,
whereas the CD and uncategorized IBD samples in D were from
different patients. Equal concentrations of protein (50 µg/ml) were
loaded onto an SDS-12% PAGE, and the blots were probed with the
indicated antibodies. Appropriate secondary antibodies conjugated to
horseradish peroxidase were used, followed by enhanced
chemiluminescence substrate kit (Pierce) to develop the blots.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and TNF
,
which can lead to an inflammatory state. LPS also increases the
production of PGs, which are synthesized from AA liberated from cell
membranes by phospholipases. Several phospholipases have been
characterized (e.g. PLA2, PLC, and PLD), but the
contribution of these to PG production has not been thoroughly studied.
Furthermore, it is unknown how various phospholipases are regulated in
a cell. These studies therefore were designed to demonstrate the
contribution of various phospholipases in PG production and to
determine the role of a regulatory protein (PLAA) in controlling
phospholipase activity in a cell.
and TNF
, which also resulted in
increased levels of PLAA at early (within 10 min) time points (Fig. 3,
A and B). However, saturation of cells with the
highest tested concentration of TNF
(500 ng/ml) also resulted in
PLAA expression at a later time point of 120 min (Fig. 3B). These findings, in conjunction with the reported ability of PLAA to
increase cytokine production (63-65), led to the possibility of an
autocrine role of PLAA, which could result in perpetuating the
inflammatory response. The rapid induction of the plaa gene in macrophages by LPS, IL-1
, and TNF
coincided with an early increase in cPLA2 and PGE2 levels, with
concomitant increases in the PLA2 activity (Figs.
1B, 2, and 3A), indicating the role of PLAA and
cPLA2 in initiating AA metabolism. The role of
cPLA2 in the production of early phase PGE2 has
been reported in mast cells and fibroblasts (17, 21). However, some
studies have indicated a role for cPLA2 in
COX-2-dependent, delayed PGE2 production (23,
26, 66).
-stimulated rabbit
distal colon cells at ~45 kDa plausibly representing a degraded
version of PLAA (67), whereas antibodies to PLAA synthetic peptides we
have generated resulted in the identification of a band of ~72 kDa,
which is the correct size of the full-length PLAA (44). The ability of
the plaa gene to be up-regulated rapidly after induction,
and its homology with numerous G protein
-subunits, which regulate
the activities of many cellular enzymes (59), prompted us to determine
whether PLAA requires phosphorylation for its activity.
Immunoprecipitation studies with cells stimulated with LPS revealed
PLAA to be phosphorylated both at 0- and 10-min time points, indicating
that although phosphorylation may be needed for the function of PLAA,
it does not regulate its activity, as is the case with many other
intermediate signaling molecules.
and COX-2 as measured
by Northern blot analysis. PGE2 levels were elevated in
macrophages treated with melittin, and these increased levels were
mediated primarily by PLC and to a lesser extent by PLD and
PLA2 (Fig. 6A).
B upstream of the cox-2 gene
prompted us to ascertain whether the nonspecific effect of LY311727 on
cox-2 gene expression might be due to interference with
NF-
B activation. Indeed, sPLA2 inhibitor LY311727
reduced NF-
B translocation and may in part explain the effects this
inhibitor had upon reduced PGE2 production. Protease
inhibitors that prevent the degradation of I
B, the inhibitor component of the NF-
B complex, should reduce translocation of NF-
B and subsequently the cox-2 gene expression (35, 38). We therefore examined the role of transcription factor NF-
B on cox-2 gene expression using protease inhibitors BTEE and
TPCK. BTEE and TPCK significantly impaired NF-
B translocation and
cox-2 gene expression with a concomitant decrease in
PGE2 production (data not shown). Both PLC and PLD
inhibitors did not alter NF-
B translocation as compared with cells
stimulated with LPS alone, indicating the specificity of these
inhibitors (Fig. 9). Taken together, these data showed that in addition
to PLA2, PLC and PLD are important phospholipases involved
in PGE2 production.
and
TNF
led to the possibility that both an autocrine and paracrine loop
can form, which would help perpetuate the aberrant immune response in
IBD (7, 63-65). However, the purified PLAA used in the above-mentioned
studies was isolated using anti-melittin affinity chromatography and
exhibited a size of 28 kDa, which probably represented a degraded
version of the full-length PLAA (76). We recently reported purification
of full-length human PLAA (44), which will now be used to demonstrate
possible autocrine and paracrine roles of this regulatory molecule.
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ACKNOWLEDGEMENTS |
---|
We thank X-J. Xu for providing technical help and Mardelle Susman for editorial assistance.
![]() |
FOOTNOTES |
---|
* This work was supported in part by a grant from the Crohn's and Colitis Foundation of America and American Heart Association (to A. K. C.).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.
Supported by The James W. McLaughlin Predoctoral Fellowship Fund.
¶ To whom correspondence should be addressed: Dept. of Microbiology and Immunology, 301 University Blvd., University of Texas Medical Branch, Galveston, TX 77555-1070. Tel.: 409-747-0578; Fax: 409-747-6869; E-mail: achopra@utmb.edu.
Published, JBC Papers in Press, November 27, 2000, DOI 10.1074/jbc.M006690200
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ABBREVIATIONS |
---|
The abbreviations used are:
IL, interleukin;
PLA2, phospholipase A2;
sPLA2,
secretory PLA2;
cPLA2, cytosolic
PLA2;
PLAA, phospholipase A2-activating
protein;
PG(s), prostaglandin(s);
LPS, lipopolysaccharide;
TNF, tumor necrosis factor
;
AA, arachidonic acid;
IBD, inflammatory
bowel diseases;
CD, Crohn's disease;
UC, ulcerative colitis;
aa, amino
acids;
ELISA, enzyme-linked immunosorbent assay;
TPCK, N-tosyl-L-phenylalanine chloromethyl ketone;
BTEE, N-benzoyl-L-tyrosine ethyl ester;
PBS, phosphate-buffered saline;
TBS, Tris-buffered saline.
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