Báo cáo Y học: Group IID heparin-binding secretory phospholipase A2 is expressed in human colon carcinoma cells and human mast cells and up-regulated in mouse inflammatory tissues doc

Group IID heparin-binding secretory phospholipase A
2
is expressed in human colon carcinoma cells and human mast cells
and up-regulated in mouse inflammatory tissues
Makoto Murakami
1
, Kumiko Yoshihara
1
, Satoko Shimbara
1
, Masatsugu Sawada
2
, Naoki Inagaki
2
,
Hiroichi Nagai
2
, Mikihiko Naito
3
, Takashi Tsuruo
3
, Tae Churl Moon
4
, Hyeun Wook Chang
4
and Ichiro Kudo
1
1
Department of Health Chemistry, School of Pharmaceutical Sciences, Showa University, Tokyo;
2
Pharmacological Department,

Gifu College of Pharmacy, Gifu, Japan;
3
Institute of Molecular and Cellular Biosciences, University of Tokyo, Tokyo, Japan;
4
College of Pharmacy, Yeungnam University, Gyonsan, Korea
Group IID secretory phospholipase A
2
(sPLA
2
-IID), a
heparin-binding sPLA
2
that is closely related to sPLA
2
-IIA,
augments stimulus-induced cellular arachidonate release in a
manner similar to sPLA
2
-IIA. Here we identified the resi-
dues of sPLA
2
-IID that are responsible for heparanoid
binding, are and therefore essential for cellular function.
Mutating four cationic residues in the C-terminal portion of
sPLA
2
-IID resulted in abolition of its ability to associate
with cell surface heparan sulfate and to enhance stimulus-
induced delayed arachidonate release, cyclooxygenase-2
induction, and prostaglandin generation in 293 cell trans-

fectants. As compared with several other group II subfamily
sPLA
2
s, which were equally active on A23187- and IL-1-
primed cellular membranes, sPLA
2
-IID showed apparent
preference for A23187-primed membranes. Several human
colon carcinoma cell lines expressed sPLA
2
-IID and sPLA
2
-
X constitutively, the former of which was negatively regu-
lated by IL-1. sPLA
2
-IID, but not other sPLA
2
isozymes,
was expressed in human cord blood-derived mast cells. The
expression of sPLA
2
-IID was significantly altered in several
tissues of mice with experimental inflammation. These
results indicate that sPLA
2
-IID may be involved in inflam-
mation in cell- and tissue-specific manners under particular
conditions.
Keywords: phospholipase A

2
; colon carcinoma; mast cell;
inflammation.
Phospholipase A
2
(PLA
2
), which catalyzes the hydrolysis of
the ester bond of the sn-2 position of glycerophospholipid to
liberate free fatty acid and lysophospholipid, is structurally
and functionally subdivided into four major classes: secre-
tory PLA
2
(sPLA
2
), cytosolic PLA
2
(cPLA
2
), Ca
2+
-inde-
pendent PLA
2
(iPLA
2
) and platelet-activating factor
acetylhydrolase [1]. The sPLA
2
family comprises Ca

2+
-
dependent, disulfide-rich and low molecular mass
(14–18 kDa) enzymes with histidine residue in the catalytic
center. To date, 10 sPLA
2
isozymes (IB, IIA, IIC, IID, IIE,
IIF, III, V, X, and XII) have been identified in mammals
[1,2]. A subset of sPLA
2
s contributes to the release of
arachidonic acid for eicosanoid generation and can also
participate in a variety of physiological events.
The regulatory functions of sPLA
2
-IIA, a prototypic
proinflammatory sPLA
2
, have been investigated in a
number of studies [3–18]. In general, this enzyme is
exocytosed or newly synthesized and secreted by the cells
after stimulation with proinflammatory agents [3–6] and
plays an augmentative role in arachidonic acid release and
prostaglandin generation [4–12], elimination of infectious
bacteria [13–15], and other pathophysiological events
[16–18]. Subsequently, several new group II subfamily
sPLA
2
s (IIC, IID, IIE, IIF, and V), the genes for which are
clustered in the same chromosome locus, have been

identified [19–24]. Among them, sPLA
2
-V has the ability
to augment cellular arachidonic acid release often more
efficiently than does sPLA
2
-IIA [8–12,25,26], whereas the
functions of the other novel group II sPLA
2
s are obscure.
sPLA
2
-IB (pancreatic PLA
2
) and -X, the genes for which
each map to distinct chromosomes, have a unique
N-terminal prepropeptide and proteolytic removal of this
prepropeptide produces an active enzyme [27–29]. sPLA
2
-
IB plays a role in the digestion of dietary phospholipids in
the gastrointestinal tract and stimulates cellular responses
by acting as a ligand for the sPLA
2
receptor [30,31]. sPLA
2
-X
potently promotes arachidonic acid release through acting
on the external plasma membrane of target cells, an event
depending on its interfacial binding to zwitterionic phos-

phatidylcholine [11,12,29,32,33].
Accumulating evidence has suggested that the cellular
functions of the heparin-binding group II subfamily of
sPLA
2
s (IIA and V) are influenced both positively [7–12]
and negatively [34,35] by heparan sulfate proteoglycan
Correspondence to M. Murakami, the Department of Health
Chemistry, School of Pharmaceutical Sciences, Showa University,
1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan.
Fax: + 81 3 3784 8245, Tel.: + 81 3 3784 8197,
E-mail:
Abbreviations:sPLA
2
, secretory phospholipase A
2
; cPLA
2
, cytosolic
phospholipase A
2
; COX, cyclooxygemase; mPGES, microsomal
PGE
2
synthase; cPGES, cytosolic PGE
2
synthase; HSPG, heparan
sulfate proteoglycan; IL, interleukin; SCF, stem cell factor; LPS,
lipopolysaccharide; DNFB, 2,4-dinitroflurobenzene; GAPDH,
glyceraldehyde-3-phosphate dehydrogenase.

Enzyme: phospholipase A
2
(EC 3.1.1.4).
(Received 28 January 2002, revised 15 April 2002,
accepted 17 April 2002)
Eur. J. Biochem. 269, 2698–2707 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02938.x
(HSPG) on cell surfaces. In the former situation, the
glycosylphosphatidylinositol-anchored HSPG glypican
supports the arachidonic acid-releasing function of the
HSPG-binding sPLA
2
s by sorting them into particular
caveolin-rich punctate and perinuclear compartments
[10,12]. Conversely, certain HSPG moieties facilitate inter-
nalization and subsequent proteolytic degradation, thereby
leading to inactivation of the HSPG-binding sPLA
2
s
[34,35]. Thus, in addition to their enzymatic characteristics,
the HSPG-binding properties of sPLA
2
s also dictate their
cellular behaviors and functions. Cationic amino acid
clusters in the N- and/or C-terminal domains of sPLA
2
-
IIA [7,36] and sPLA
2
-V [8,34] are responsible for their
functional association with HSPGs.

sPLA
2
-IID, an isozyme most related to sPLA
2
-IIA, is
reportedly expressed in immune and digestive organs and is
proposed to replace sPLA
2
-IIA under certain conditions
[21,22]. We have recently shown that sPLA
2
-IID, like
sPLA
2
-IIA, binds to the HSPG glypican and augments the
arachidonic acid-releasing response in HEK293 cells [12].
To better understand the regulatory functions of sPLA
2
-
IID, we have determined its functional HSPG-binding site
by site-directed mutagenesis. Furthermore, we show that
this isozyme is expressed in human colon carcinoma cell
lines and human mast cells as well as various mouse tissues.
Importantly, the expression of sPLA
2
-IID is regulated both
positively and negatively by proinflammatory stimuli.
MATERIALS AND METHODS
Materials
HEK293 cells (Human Science Research Resources Bank,

Osaka, Japan) and colon carcinoma cell lines (American
Type Culture Collection) were cultured in RPMI 1640
medium(NissuiPharmaceuticalCo.,Tokyo,Japan)con-
taining 10% fetal bovine serum [8–12]. cDNAs for human
and mouse sPLA
2
s, human cyclooxygenase (COX)-2 and
human microsomal prostaglandin E
2
(PGE
2
)
1
synthase
(mPGES) and their HEK293 cell transfectants were
described previously [8–12,37].
To obtain human cord blood-derived mast cells [38],
heparin-treated umbilical cord blood was obtained from
normal full-term vaginal deliveries under auspices of the
Kyungpook National University Hospital. Cord blood was
diluted with the same volume of NaCl/P
i
and layered over
Histopaque-1077 (Sigma) at room temperature within 4 h
of delivery. The cord blood monoculcear cell fraction was
obtained after centrifugation at 1000 g for 20 min at room
temperature. The cells were washed twice with NaCl/P
i
and
grown in tissue culture flasks in AIM-V medium (Life

Technologies) in the presence of 100 ngÆmL
)1
recombinant
human stem cell factor (SCF) for 8 weeks. Non-adherent
cells were then cultured for an additional 2 weeks with
100 ngÆmL
)1
SCF and 50 ngÆmL
)1
human interleukin (IL)-
6 in AIM-V medium. The mast cells thus obtained were
> 97% tryptase- and  70% chymase-positive as demon-
strated by immunocytostaining using specific antibodies,
expressed functional c-kit and Fc
e
receptor I as assessed by
flow cytometry, and responded to immunological and
nonimmunological stimuli to secrete granule contents (T. C.
Moon, M. Murakami, I. Kudo & H. W. Chang, unpub-
lished data)
2
.
The enzyme immunoassay kit for PGE
2
was from
Cayman Chemicals (Ann Arbor, MI, USA). Rabbit
antihuman COX-1 and antihuman cPLA
2
a antibodies
were from Santa Cruz. Anti-human cytosolic PGE

2
synthase (cPGES) antibody was prepared as described
previously [39]. Lipofectamine PLUS reagent, Opti-MEM
medium, geneticin and TRIzol reagent were from Life
Technologies. Horseradish peroxidase-conjugated antigoat
and antirabbit IgGs were from Zymed. A23187 was from
CalBiochem. Human IL-1b was obtained from Genzyme.
Construction of sPLA
2
-IID mutants
Mouse sPLA
2
-IID mutants were produced by PCR with the
Advantage cDNA polymerase mix (Clontech). The condi-
tion of PCR was 25 cycles at 94 °C, 55 °Cand72°Cfor
30 s each. The primers used were as follows: mIID-5¢,5¢-AT
GAGACTCGCCCTGCTGTGTG-3¢;KE2,5¢-TTAGCA
TGCTGGAGTCTCGCCTTCGCAAC-3¢; and KE2RS2,
5¢-GCATGCTGGAGTCTCGCCTTCGCAACAGGGCC
ACCAGTA-3¢. PCR was preformed with mIID-5¢ and
KE2 or KE2RS2 using mouse sPLA
2
-IID cDNA as a
template. Each PCR product was ligated into pCR3.1
(Invitrogen) and was transfected into Top10F¢ supercom-
petent cells (Invitrogen). The plasmids were isolated and
sequenced using a Taq cycle sequencing kit (Takara, Ohtsu,
Japan) and an autofluorometric DNA sequencer DSQ-1000
L (Shimadzu, Tokyo, Japan) to confirm the sequences.
RT-PCR and Southern blotting

Synthesis of cDNAs was performed using avian myeloblas-
tosis virus reverse transcriptase and 0.5 lgtotalRNAfrom
mouse tissues and human cell lines, according to the
manufacturer’s instructions supplied with the RNA PCR
kit (Takara). Subsequent amplification of the cDNA
fragments was performed using 1 lL of the reverse-
transcribed mixture as a template with specific oligonucle-
otide primers (Greiner Japan) as follows: mIID-5¢ and
mIID-3¢ (see above); human cPLA
2
a sense, 5¢-ATGTCATT
TATAGATCCTTACC-3¢ and antisense, 5¢-TCAAAGTT
CAAGAGACATTTCAG-3¢; human mPGES sense, 5¢-AT
GCACTTCCTGGTCTTCCTCG-3¢ and antisense, 5¢-GC
TTCCCCAGGAAGGCCACGG-3¢; human sPLA
2
-IB
sense, 5¢-ATGAAACTCCTTGTGCTAGCTG-3¢ and anti-
sense, 5¢-TCAACTCTGACAATACTTCTTGG-3¢; human
sPLA
2
-hIIA sense, 5¢-CAGAATGATCAAGTTGACGAC
AG-3¢ and antisense, 5¢-TCAGCAACGAGGGGTGCTC
CTC-3¢; human sPLA
2
-hIID sense, 5¢-ATGGAACTTGCA
CTGCTGTGTG-3¢ and antisense, 5¢-CAGTCGCTTCTG
GTAGGTGTCC-3¢; human sPLA
2
-IIE sense, 5¢-ATGAA

ATCTCCCCACGTGCTGG-3¢ and antisense, 5¢-TGTAG
GTGCCCAGGTTGCGGCG-3¢; human sPLA
2
-IIF sense,
5¢-ATGAAGAAGTTCTTCACCGTG-3¢ and antisense,
5¢-CTAGCAGGTGACCTCCTCAGG-3¢; human sPLA
2
-
Vsense,5¢-ATGAAAGGCCTCCTCCCACTGG-3¢ and
antisense, 5¢-GGCCTAGGAGCAGAGGATGTTG-3¢;
and human sPLA
2
-X sense, 5¢-ATGCTGCTCCTGCTAC
TGCCG-3¢ and antisense, 5¢-TCAGTCACACTTGGGC
GAGTC-3¢. PCR conditions were 94 °C for 30 s and then
30 cycles of amplification at 94 °Cfor5sand68°Cfor
4 min, using the Advantage cDNA polymerase mix.
RT-PCR of glyceraldehyde-3-phosphate dehydrogenase
Ó FEBS 2002 Analyses of group IID phospholipase A2 (Eur. J. Biochem. 269) 2699
(GAPDH) was performed using specific primers (Clontech).
The PCR products were analyzed by 1% agarose gel
electrophoresis with ethidium bromide staining. The gels
were further subjected to Southern blot hybridization using
appropriate cDNAs as probes.
Lipopolysaccharide treatment of mice
Lipopolysaccharide (LPS) (5 mgÆkg
)1
) was administered
intraperitoneally to 4-week-old male C57BL/6 mice (Nip-
pon Bio-Supply Center, Tokyo, Japan). After 24 h, mice

were sacrificed by bleeding, their organs were removed, and
RNA was extracted by homogenization in TRIzol reagent
using 10 strokes of a Potter homogenizer at 1000 r.p.m.
3
Mouse ear atopic dermatitis
Five repeated topical applications of 2,4-dinitrofluoroben-
zene(DNFB)totheearsofmiceresultincontact
hypersensitivity of the ears as well as significant elevation
of serum IgE levels, accompanied by the increased T
H1
response for the onset of skin dermatitis and the T
H2
response in the lymph node [40]. The ears of C57BL/6 mice
(Nippon Bio-Supply Center) were painted with 25 lL
0.15% (w/v) DNFB or vehicle (acetone/olive oil 3 : 1) once
a week. The ears were removed 24 h after the fifth painting
and subjected to RNA extraction. Replicate ear sections
were fixed by formalin, embedded in paraffin and stained
with hematoxylin and eosin to verify the progress of
inflammation. All procedures and analyses of other param-
eters are detailed elsewhere [40].
Other procedures
Northern and Western blottings, establishment and activa-
tion of HEK293 transfectants, and measurement of in vitro
sPLA
2
activity were performed as described in our previous
reports [8–12].
RESULTS
Determination of the heparin-binding site of mouse

sPLA
2
-IID
The amino-acid sequences of mouse and human sPLA
2
-IIDs
reveal the presence of multiple cationic amino acid residues
in their C-terminal regions [21,22]. Since the multiple cationic
residues in the corresponding C-terminal portions of mouse
and human sPLA
2
-IIAs and rat and human sPLA
2
-Vs serve
as functional heparin-binding sites [7,8,34,36], we replaced
some of these cationic residues in mouse sPLA
2
-IID with
neutral and/or anionic amino acids by site-directed muta-
genesis. The KE2 mutant, in which two lysine residues near
the C-terminal end (Lys138 and Lys140) were replaced by
glutamic acid, and the KE2RS2 mutant, in which two
conserved arginine residues (Arg136 and Arg138) were
additionally mutated to serine, were constructed (Fig. 1A).
cDNAs for the native and mutant enzymes were transfected
into HEK293 cells to establish drug-resistant stable clones.
Comparable expression of the mutant and native enzymes
was confirmed by Northern blotting (Fig. 1B).
As the membrane distribution of sPLA
2

s expressed in
HEK293 cells largely reflects their association with cell
surface HSPG [7–12], we measured the enzyme activity in
the supernatant and membrane-bound (i.e. 1
M
NaCl-
solubilized) fractions of the established transfectants
(Fig. 1C). Consistent with our recent reports [7–12], the
membrane-bound fraction contained more than 50% of the
native enzyme (Fig. 1C). The distribution of the KE2
mutant between the two fractions was similar to that of the
native enzyme (Fig. 1C). In contrast, the activity of the
KE2RS2 mutant was detected mainly in the supernatants,
with only a minor portion being recovered from the
membrane-bound fraction (Fig. 1C). Thus, simultaneous
mutation of the four cationic residues in the C-terminal
domain of sPLA
2
-IID led to a marked reduction of its
membrane-binding (and therefore HSPG-binding) capacity.
Fig. 1. Mutation of basic amino acid residues near the C-terminus of
sPLA
2
-IID affects its association with the cell surface. (A) Amino acid
sequences of the C-terminal part of mouse sPLA
2
-IID (mIID) and its
mutants, KE2 and KE2RS2. Two and four basic amino acids are
replaced by glutamic acid or serine in KE2 and KE2RS2, respectively.
(B) Expression of the wild-type (WT) and two mutants of mIID in

HEK293 cells, as assessed by RNA blotting. (C) Membrane binding of
the WT and two mutants of mIID. After collecting the culture sup-
ernatants, the cells were incubated for 30 min with medium containing
1
M
NaCl, which solubilizes the cell surface HSPG-bound form of
sPLA
2
s. PLA
2
activities in the secreted (S) and cell membrane-bound
(i.e. NaCl-solubilized) (C) fractions were measured.
2700 M. Murakami et al. (Eur. J. Biochem. 269) Ó FEBS 2002
This observation is in line with previous studies on the
HSPG-binding of sPLA
2
-IIA, in which multiple cationic
residues in the C-terminal domain are required for its proper
association with heparanoids [7,8,34,36].
When the cells were prelabeled with [
3
H]arachidonic acid
and were then stimulated with A23187 for 30 min (Fig. 2A)
or with IL-1 for 4 h (Figs 2,B,C) as models for the
immediate and delayed responses, respectively [8–12], a
marked elevation of [
3
H]arachidonic acid release, which was
accompanied by PGE
2

generation (Fig. 2C), was observed
in cells transfected with the native enzyme or KE2 mutant,
but not appreciably in those transfected with the KE2RS2
mutant. In the absence of stimulus, there were no increases
in arachidonic acid release and PGE
2
generation even in
cells transfected with the native enzyme (data not shown).
Furthermore, IL-1-stimulated COX-2 expression was faci-
litated in cells transfected with the native enzyme or KE2
mutant, whereas it occurred only minimally in cells trans-
fected with KE2RS2 (Fig. 2D). These observations suggest
that the cellular functions of sPLA
2
-IID are correlated with
its membrane-binding property, and lend further support
for the notion that this enzyme, as does sPLA
2
-IIA [7–12],
acts on cells through an HSPG-dependent mechanism in
this setting.
sPLA
2
-IID prefers Ca
2+
ionophore-induced perturbed
membrane
While studying the arachidonic acid-releasing functions of
the three heparin-binding group II subfamily enzymes (IIA,
IID and V) in HEK293 transfectants, we noted that sPLA

2
-
IID released arachidonic acid after A23187 stimulation
more efficiently than it did after IL-1 stimulation under the
condition where sPLA
2
-IIA and -V released equivalent
levels of arachidonic acid in both responses (Fig. 3A). Thus,
A23187-induced arachidonic acid release by these three
sPLA
2
s reached comparable levels (net 4–6%), whereas
IL-1-stimulated arachidonic acid release by sPLA
2
-IID (net
0.7%) was apparently lower than that by sPLA
2
-IIA and -V
(net 4–5%) (Fig. 3A).
When cells expressing sPLA
2
-IID were cocultured with
those coexpressing COX-2 and mPGES and then stimulated
(transcellular prostaglandin biosynthesis [9]), the increased
production of PGE
2
in response to A23187 was higher than
that in response to IL-1 (Fig. 3B, left). In comparison,
coculture of cells expressing sPLA
2

-V with those coexpress-
ing COX-2 and mPGES increased both the immediate and
delayed PGE
2
-biosynthetic responses almost equally
(Fig. 3B, right). These results indicate that sPLA
2
-IID
secreted from the transfectants acts preferentially on the
A23187-elicited membranes of neighboring cells, where the
arachidonic acid released by the paracrine or juxtacrine
action of sPLA
2
-IID is supplied to downstream COXs and
mPGES.
sPLA
2
-IID expression in human colon carcinoma cell lines
Although sPLA
2
-IID has been reported to be expressed
in tissues related to the immune response (spleen and
thymus) and digestion (small and large intestines) of both
human and mouse [21,22], which types of cell express this
sPLA
2
isozyme remains obscure. We therefore surveyed
the expression of sPLA
2
-IID in various human cell lines,

and found that its transcript, as assessed by RT-PCR,
was constitutively expressed in several human colon
carcinoma cell lines, including HT29, KM12, KM20L2,
Fig. 2. Mutation of basic amino acid residues near the C-terminus of
sPLA
2
-IID affects its cellular arachidonic acid-releasing function. (A)
Immediate arachidonic acid release. Control HEK293 cells and cells
transfected with the WT or mutant mIID were prelabeled with
[
3
H]arachidonic acid and then stimulated for 30 min with 10 l
M
A23187 to assess [
3
H]arachidonic acid release. (B–D) Delayed
arachidonic acid release and PGE
2
generation. Control cells and cells
transfected with the WT or mutant mIID were stimulated for 4 h with
IL-1b to assess [
3
H]arachidonic acid release (B), PGE
2
production (C)
and COX-2 induction (D). In (D), COX-2 expression was assessed by
RNA blotting. Equal loading of RNA on each lane was verified by
ribosomal RNA staining with ethidium bromide (not shown). AA,
arachidonic acid.
Fig. 3. sPLA

2
-IID elicits the immediate response in preference to the delayed response. (A) [
3
H]arachidonic acid release by control HEK293 cells and
cells transfected with sPLA
2
-IIA, -IID or -V in response to A23187 (30 min) or IL-1b (4 h). (B) Transcellular PGE
2
production by sPLA
2
-IID (left)
and sPLA
2
-V (right). Control, and COX-2/mPGES-coexpressing cells were cocultured for 4 days with control cells (–) or sPLA
2
-expressing cells
(+), and were then stimulated for 4 h with IL-1b to assess PGE
2
generation. AA, arachidonic acid.
Ó FEBS 2002 Analyses of group IID phospholipase A2 (Eur. J. Biochem. 269) 2701
WiDr and HCT2998 cells (Fig. 4A). Unexpectedly, treat-
ment of these cells with IL-1 consistently decreased the
expression of sPLA
2
-IID in a time-dependent manner.
sPLA
2
-X was also detected in these cell lines, in which its
expression was unaffected by IL-1 except for HCT2998
cells, in which there was a slight increase in its expression

(Fig. 4B). sPLA
2
-V was detected only in IL-1-stimulated
HT29 cells, and sPLA
2
-IIA was weakly and constitutively
expressed in HT29, KM12 and KM20L2 cells (Fig. 4B).
The expression of other sPLA
2
s (IB, IIE and IIF) was
undetectable.
The expression of other enzymes involved in the PGE
2
-
biosynthetic pathway in these colon carcinoma cell lines was
also investigated (Fig. 4C). cPLA
2
a was detected in KM12,
KM20L2 and WiDr cells. COX-1 was highly expressed in
HT29 and WiDr cells and weakly expressed in KM20L2
cells. COX-2 was detected only in WiDr cells. The two
terminal PGE
2
-biosynthetic enzymes, cPGES and mPGES,
were expressed in all cell lines. Following IL-1 treatment,
COX-2 expression was markedly induced in WiDr cells,
whereas the expression levels of cPLA
2
a,COX-1,cPGES
and mPGES in each cell line were unaltered. Among these

cell lines, only WiDr cells produced a substantial amount of
PGE
2
in response to IL-1 (Fig. 4D), most likely because
COX-2 is a rate-limiting step for IL-1-dependent PGE
2
biosynthesis [6–12].
sPLA
2
-IID expression in human cultured mast cells
We have previously reported that mouse bone marrow-
derived cultured mast cells developed in the presence of IL-3
express all the group II subfamily sPLA
2
s[41].RT-PCR
analyses revealed that, unlike mouse mast cells, human mast
cells developed in the presence of SCF and IL-6 from cord
blood cells [38] expressed only sPLA
2
-IID, but not the other
sPLA
2
s including -IB, -IIA, -IIE, -IIF, -V and -X (Fig. 5).
The expression of cPLA
2
a was readily detected under the
same experimental conditions (Fig. 5). The expression of
sPLA
2
-IID and cPLA

2
a in human mast cells was
unchanged after treatment with various mast cell-poietic
cytokines and immunological stimuli (T. C. Moon,
M. Murakami, I. Kudo & H. W. Chang, unpublished data)
4
.
sPLA
2
-IID expression in mouse tissues
during inflammation
The expression of sPLA
2
-IID in several tissues of mice
before and 24 h after injection of LPS was examined by
Fig. 4. Expression of sPLA
2
-IID and other PGE
2
-biosynthetic enzymes in human colon carcinoma cell lines. (A) Cells were stimulated for the
indicated periods with 1 ngÆmL
)1
IL-1b, and the expression of sPLA
2
-IID was assessed by 30 cycles of RT-PCR. After staining of the gel with
ethidium bromide (top), samples were subjected to Southern blotting using
32
P-labeled human sPLA
2
-IID cDNA as a probe (middle). Equal

loading of samples on each lane was verified by the expression of GAPDH, as assessed by RT-PCR (bottom). (B) The same samples [with (+) or
without (–) 12-h stimulation with IL-1b] were subjected to RT-PCR (30 cycles) followed by Southern blotting to assess the expression of sPLA
2
-X, -V
and -IIA. (C) Expression of cPLA
2
a, COX-1, COX-2, cPGES and mPGES with or without 12-h stimulation with IL-1b. The expression of cPLA
2
a,
COX-1 and cPGES was assessed by immunoblotting, COX-2 by RNA blotting, and mPGES by RT-PCR (30 cycles) followed by Southern
blotting. (D) Cells were stimulated for 12 h with IL-1b and PGE
2
released into the supernatants was quantified.
2702 M. Murakami et al. (Eur. J. Biochem. 269) Ó FEBS 2002
RT-PCR (Fig. 6A). After administration of LPS, sPLA
2
-
IID expression was upregulated in the lung, thymus and
heart in a dose-dependent manner. Conversely, sPLA
2
-IID
expression was decreased in the kidney of LPS-treated mice.
In the spleen, intestine and colon, in which the basal sPLA
2
-
IID expression was high, as well as in the brain and liver,
sPLA
2
-IID expression was largely unchanged after LPS
challenge. In the ears of mice with DNFB-induced atopic

dermatitis, there was a marked increase in sPLA
2
-IID
expression (Fig. 6B).
DISCUSSION
sPLA
2
-IID, which was originally identified by searching
nucleic acid data bases for expressed sequence tags repre-
senting parts of genes for sPLA
2
homologs, displays all of
the specific features of sPLA
2
-IIA: the homology between
these two enzymes is  50% [21,22]. sPLA
2
-IID and -IIA
also possess several common properties, one of which is
their high affinity for heparanoids [7–12]. The major
heparin-binding site of sPLA
2
-IIA is located near the
C-terminus, where a highly localized site of basic residues
affects its heparanoid affinity with diffuse basic residues
throughout the molecule having a modifying role [7,36].
Similarly, the C-terminal basic amino acid cluster contri-
butes to the binding of sPLA
2
-V to heparanoids [8,34]. In

the present study, we have shown that a similar cluster of
basic amino acids near the C-terminus of sPLA
2
-IID also
crucially influences its binding to cellular HSPG (Fig. 1).
Most importantly, as in the cases of sPLA
2
-IIA and -V,
enzymes that act on ÔrearrangedÕ cellular membranes
through the HSPG-dependent pathway [7,34,36], mutation
of these basic residues of sPLA
2
-IIDledtoamarked
reduction of its ability to release arachidonic acid, produce
PGE
2
and induce COX-2 in HEK293 cells (Fig. 2), despite
the fact that the mutation does not have a profound effect
on enzyme activity (Fig. 1C). These results agree with our
recent observation that sPLA
2
-IID augments arachidonic
acid release from activated cells through the pathway
dependent upon the HSPG glypican or other HSPG
molecules [12]. The three-dimensional structure of sPLA
2
-
IIA demonstrates that the C-terminal heparin-binding
domain is located on the opposite side of a globular
molecule to the interfacial binding surface [34], implying

that this enzyme can interact simultaneously with substrates
and heparanoids. Given the assumption that sPLA
2
-IID
has a similar ternary structure, it is conceivable that its
anchoring on the heparan sulfate chains of glypican (or
other HSPG) through the C-terminal cationic surface allows
sPLA
2
-IID to be locally concentrated and interact efficiently
with phospholipids in adjacent cellular membranes.
Fig. 6. Expression of sPLA
2
-IID in mouse during inflammation. RNAs
obtained from various tissues of mice 24 h after administration of the
indicated doses of LPS (A) and the ears of mice with or without five
repeated treatments with DNFB (B) were subjected to RT-PCR (30
cycles) followed by Southern blotting to assess the expression of
sPLA
2
-IID. To verify equal loading of RNA on each lane, RT-PCR
(25 cycles) for GAPDH was also performed. R and L in (B) indicate
right and left ears, respectively.
Fig. 5. Expression of sPLA
2
-IID in human cord blood-derived mast
cells. RNA obtained from human cord blood-derived mast cells was
subjected to RT-PCR (30 cycles) using specific primers for human
sPLA
2

-IB, IIA, IID, IIE, IIF, V and X (left) and for cPLA
2
a (right).
After staining of the gel with ethidium bromide, samples were taken for
Southern blotting using cDNA probes for the mixture of these sPLA
2
s.
Ó FEBS 2002 Analyses of group IID phospholipase A2 (Eur. J. Biochem. 269) 2703
Our transfection studies have revealed a subtle but
substantial difference between sPLA
2
-IID and other group
II subfamily enzymes (sPLA
2
-IIA and -V). These enzymes
are in common active on ÔrearrangedÕ cellular membranes
that have been primed by various cell activators [6–12], yet
sPLA
2
-IID, relative to -IIA and -V, shows apparent
preference for A23187-primed rather than IL-1-primed
cellular membranes (Fig. 3). This is, in our hands, the first
demonstration that a particular sPLA
2
isozyme exerts its
arachidonic acid-releasing function more effectively in the
Ca
2+
evoked immediate response than in the cytokine-
induced delayed response. The membrane rearrangement

that renders cells more susceptible to sPLA
2
s involves
several processes, such as altered membrane phospholipid
asymmetry (i.e. exposure of anionic phospholipids in the
outer leaflet of the membrane), accelerated membrane
oxidation and possibly sphingomyelin breakdown [1].
Although the precise mechanisms are still unclear, sPLA
2
-
IID may be better suited to the particular perturbed
membrane structures that are formed during prompt
Ca
2+
signaling than to those formed during sustained
cytokine signaling.
In search of human cell lines that endogenously express
sPLA
2
-IID, we found that several colon carcinoma cell
lines constitutively express this particular sPLA
2
isozyme
(Fig. 4). Most of these cell lines also express sPLA
2
-X, an
observation reminiscent of the recent report by Morioka
et al. [32] demonstrating the elevated expression of sPLA
2
-

X in human colon adenocarcinoma neoplastic cells and
tissues. A growing body of evidence has shown that
nonsteroidal anti-inflammatory drugs that inhibit COX-2
can suppress colorectal tumorigenesis [42–45] and that
PGE
2
, a major COX-2 product, is involved in this process
[46–48]. Furthermore, targeted disruption of the cPLA
2
a
gene has provided unequivocal evidence that this enzyme
contributes significantly, if not solely, to the expansion of
colorectal cancer, most probably by acting as a major
supplier of arachidonic acid to COX-2 [49]. Our present
results raise the intriguing possibility that, in addition to
sPLA
2
-X [32,49], sPLA
2
-IID may also be able to promote
certain phases of colorectal cancer development. Unfor-
tunately, none of the cell lines used in this study (even
WiDr cells, which express COX-2) turned out to depend
on the COX products for their growth (data not shown),
and the confirmation of this hypothesis awaits future
study.
Mast cells are highly specialized effector cells in the
immune system, where they release a number of granule-
associated preformed (e.g. histamine, serotonin, and pro-
teases) and newly synthesized (e.g. PGD

2
, LTC
4
,and
cytokines) mediators following engagement of the Fc
e
receptor I on their surfaces by IgE and cognate antigen.
Previous studies have established that mast cells represent a
potent source of sPLA
2
s; mouse IL-3-dependent bone
marrow-derived mast cells express all or some of the group
II subfamily sPLA
2
s according to culture conditions [41,50],
mouse mast cell line MMC-34 cells express sPLA
2
-V [51],
and rat peritoneal mast cells express sPLA
2
-IIA [52]. These
sPLA
2
s play augmentative roles in stimulus-coupled
degranulation and lipid mediator generation in rodent mast
cells [41,50–52]. Here we show that human cord blood-
derived mast cells developed in SCF and IL-6 [38] express
sPLA
2
-IID but not the other isozymes (Fig. 5). Given the

experimental evidence that sPLA
2
-IID, as do the other
group II subfamily sPLA
2
s, has the ability to augment IgE/
antigen-dependent exocytosis of granule-associated media-
tors and generation of eicosanoids in rodent mast cells
[12,41], it is tempting to speculate that sPLA
2
-IID may
display similar functions in human mast cells. In this regard,
sPLA
2
-IID may represent a novel therapeutic and prophy-
lactic target for allergic diseases. It should be noted,
however, that this finding does not necessarily mean that
all mast cells distributed in human tissues express sPLA
2
-
IID only, since mast cell phenotypes is crucially influenced
by tissue microenvironments [53,54]. Indeed, a recent
immunohistochemical analysis has demonstrated that
human intestinal mast cells contain sPLA
2
-IIA [55]. We
also recently found that sPLA
2
-V is located in mast cells in
tissues from patients with allergic symptoms (

5
M. Murakami
& I. Kudo, unpublished data).
Increased expression of sPLA
2
-IID was observed in
some tissues (lung, thymus and heart) of mice with LPS-
induced systemic inflammation and in the ears of mice
with atopic dermatitis (Fig. 6), providing further support
for the notion that the group II subfamily of sPLA
2
sare
inducible enzymes. Consistent with our results, Ishizaki
et al. [22] have shown that sPLA
2
-IID expression is
increased in the thymus and lung of LPS-treated rats, and
Shakhov et al.[56]haveshownthatsPLA
2
-IID expression
is markedly reduced in lymphoid tissues of lymphotoxin
a-deficient mice. However, this rather tissue-restricted
induction of sPLA
2
-IID differs from the induction of
sPLA
2
-IIA and -V [57,58], which is more widespread
among tissues. Moreover, LPS treatment resulted in
reduced expression of sPLA

2
-IID in the kidney (Fig. 6A),
in which the expressions of sPLA
2
-IIA and -V [57,58]
exhibit a reciprocal pattern. Decreased expression of
sPLA
2
-IID, relative to increased expression of sPLA
2
-V,
by proinflammatory stimulus was also observed in human
colon carcinoma cell lines (Fig. 4A,B). These results argue
that the regulatory mechanisms for gene expression, and
perhaps functions, of sPLA
2
-IID and those of sPLA
2
-IIA
and -V are not entirely identical and are even cell- and
tissue-specific. Searching the nucleic acid database reveals
the presence of the TATA box and the binding motifs for
AP-1 and NFjB in the putative 5¢-flanking promoter
region of the human sPLA
2
-IID gene, consistent with its
proinflammatory signal-associated inducible nature. In
comparison, the putative promoter region of the human
sPLA
2

-V gene contains the C/EBP and CREB motifs as
well as distal AP-1, NFjB and glucocorticoid-responsive
elements. These motifs are also present in the promoter
region of the sPLA
2
-IIA gene, albeit with a different
alignment [59,60]. Such differences among the promoter
regulatory regions of these sPLA
2
s may account for their
distinct expression and induction.
The present study implies that the structurally related
group II subfamily sPLA
2
isozymes are not always
functionally compensatory, even if they utilize common
regulatory machinery under particular conditions. The
expression and induction profiles of each sPLA
2
isozyme
during inflammatory responses are tissue- and cell-specific.
It is therefore likely that functional redundancy and
segregation of sPLA
2
isozymes must occur in different
physiological and pathological states and in different cells
and tissues.
2704 M. Murakami et al. (Eur. J. Biochem. 269) Ó FEBS 2002
ACKNOWLEDGEMENTS
We thank G. Lambeau (CNRS-UPR) and M.H. Gelb (University of

Washington) for providing us cDNAs for human and mouse sPLA
2
-
IIDs. This work was supported by Grant-in-Aid for Scientific Research
from the Ministry of Education, Science, Culture, Sports and
Technology of Japan.
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