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,

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+
-
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

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: [email protected]
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.

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,

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

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

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

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

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

-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,

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

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

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

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

-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

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

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 (

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

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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