Group IID heparin-binding secretory phospholipase A2is expressed in human colon carcinoma cells and human mast cells and up-regulated in mouse inflammatory tissues Makoto Murakami1, Kumi
Trang 1Group IID heparin-binding secretory phospholipase A2
is expressed in human colon carcinoma cells and human mast cells and up-regulated in mouse inflammatory tissues
Makoto Murakami1, Kumiko Yoshihara1, Satoko Shimbara1, Masatsugu Sawada2, Naoki Inagaki2,
Hiroichi Nagai2, Mikihiko Naito3, Takashi Tsuruo3, Tae Churl Moon4, Hyeun Wook Chang4and Ichiro Kudo1
1
Department of Health Chemistry, School of Pharmaceutical Sciences, Showa University, Tokyo;2Pharmacological Department, Gifu College of Pharmacy, Gifu, Japan;3Institute of Molecular and Cellular Biosciences, University of Tokyo, Tokyo, Japan;
4 College of Pharmacy, Yeungnam University, Gyonsan, Korea
Group IID secretory phospholipase A2 (sPLA2-IID), a
heparin-binding sPLA2that is closely related to sPLA2-IIA,
augments stimulus-induced cellular arachidonate release in a
manner similar to sPLA2-IIA Here we identified the
resi-dues of sPLA2-IID that are responsible for heparanoid
binding, are and therefore essential for cellular function
Mutating four cationic residues in the C-terminal portion of
sPLA2-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
sPLA2s, which were equally active on A23187- and
IL-1-primed cellular membranes, sPLA2-IID showed apparent
preference for A23187-primed membranes Several human colon carcinoma cell lines expressed sPLA2-IID and sPLA2
-X constitutively, the former of which was negatively regu-lated by IL-1 sPLA2-IID, but not other sPLA2 isozymes, was expressed in human cord blood-derived mast cells The expression of sPLA2-IID was significantly altered in several tissues of mice with experimental inflammation These results indicate that sPLA2-IID may be involved in inflam-mation in cell- and tissue-specific manners under particular conditions
Keywords: phospholipase A2; colon carcinoma; mast cell; inflammation
Phospholipase A2(PLA2), 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 PLA2 (sPLA2), cytosolic PLA2 (cPLA2), Ca2+
-inde-pendent PLA2 (iPLA2) and platelet-activating factor
acetylhydrolase [1] The sPLA2 family comprises Ca2+
-dependent, disulfide-rich and low molecular mass
(14–18 kDa) enzymes with histidine residue in the catalytic
center To date, 10 sPLA2isozymes (IB, IIA, IIC, IID, IIE,
IIF, III, V, X, and XII) have been identified in mammals
[1,2] A subset of sPLA2s contributes to the release of
arachidonic acid for eicosanoid generation and can also
participate in a variety of physiological events
The regulatory functions of sPLA2-IIA, a prototypic proinflammatory sPLA2, 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 sPLA2s (IIC, IID, IIE, IIF, and V), the genes for which are clustered in the same chromosome locus, have been identified [19–24] Among them, sPLA2-V has the ability
to augment cellular arachidonic acid release often more efficiently than does sPLA2-IIA [8–12,25,26], whereas the functions of the other novel group II sPLA2s are obscure sPLA2-IB (pancreatic PLA2) 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] sPLA2
-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 sPLA2receptor [30,31] sPLA2-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 sPLA2s (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: mako@pharm.showa-u.ac.jp
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, stemcell 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)
Trang 2(HSPG) on cell surfaces In the former situation, the
glycosylphosphatidylinositol-anchored HSPG glypican
supports the arachidonic acid-releasing function of the
HSPG-binding sPLA2s by sorting theminto 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 sPLA2s
[34,35] Thus, in addition to their enzymatic characteristics,
the HSPG-binding properties of sPLA2s also dictate their
cellular behaviors and functions Cationic amino acid
clusters in the N- and/or C-terminal domains of sPLA2
-IIA [7,36] and sPLA2-V [8,34] are responsible for their
functional association with HSPGs
sPLA2-IID, an isozyme most related to sPLA2-IIA, is
reportedly expressed in immune and digestive organs and is
proposed to replace sPLA2-IIA under certain conditions
[21,22] We have recently shown that sPLA2-IID, like
sPLA2-IIA, binds to the HSPG glypican and augments the
arachidonic acid-releasing response in HEK293 cells [12]
To better understand the regulatory functions of sPLA2
-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 sPLA2-IID is regulated both
positively and negatively by proinflammatory stimuli
M A T E R I A L S A N D M E T H O D S
Materials
HEK293 cells (Human Science Research Resources Bank,
Osaka, Japan) and colon carcinoma cell lines (American
Type Culture Collection) were cultured in RPMI 1640
m edium (Nissui Pharm aceutical Co., Tokyo, Japan)
con-taining 10% fetal bovine serum[8–12] cDNAs for human
and mouse sPLA2s, human cyclooxygenase (COX)-2 and
human microsomal prostaglandin E2 (PGE2)
(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/Piand 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/Piand
grown in tissue culture flasks in AIM-V medium (Life
Technologies) in the presence of 100 ngÆmL)1recombinant
human stem cell factor (SCF) for 8 weeks Non-adherent
cells were then cultured for an additional 2 weeks with
100 ngÆmL)1SCF and 50 ngÆmL)1human 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 Fcereceptor 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)
The enzyme immunoassay kit for PGE2 was from Cayman Chemicals (Ann Arbor, MI, USA) Rabbit antihuman COX-1 and antihuman cPLA2a antibodies were fromSanta Cruz Anti-human cytosolic PGE2 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 fromZymed A23187 was from CalBiochem Human IL-1b was obtained fromGenzyme Construction of sPLA2-IID mutants
Mouse sPLA2-IID mutants were produced by PCR with the Advantage cDNA polymerase mix (Clontech) The condi-tion of PCR was 25 cycles at 94C, 55 C and 72 C for
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 sPLA2-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 lg total RNA from 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 cPLA2a 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 sPLA2-IB sense, 5¢-ATGAAACTCCTTGTGCTAGCTG-3¢ and anti-sense, 5¢-TCAACTCTGACAATACTTCTTGG-3¢; human sPLA2-hIIA sense, 5¢-CAGAATGATCAAGTTGACGAC AG-3¢ and antisense, 5¢-TCAGCAACGAGGGGTGCTC CTC-3¢; human sPLA2-hIID sense, 5¢-ATGGAACTTGCA CTGCTGTGTG-3¢ and antisense, 5¢-CAGTCGCTTCTG GTAGGTGTCC-3¢; human sPLA2-IIE sense, 5¢-ATGAA ATCTCCCCACGTGCTGG-3¢ and antisense, 5¢-TGTAG GTGCCCAGGTTGCGGCG-3¢; human sPLA2-IIF sense, 5¢-ATGAAGAAGTTCTTCACCGTG-3¢ and antisense, 5¢-CTAGCAGGTGACCTCCTCAGG-3¢; human sPLA2
-V sense, 5¢-ATGAAAGGCCTCCTCCCACTGG-3¢ and antisense, 5¢-GGCCTAGGAGCAGAGGATGTTG-3¢; and human sPLA2-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 94C for 5 s and 68 C for
4 min, using the Advantage cDNA polymerase mix RT-PCR of glyceraldehyde-3-phosphate dehydrogenase
Trang 3(GAPDH) was performed using specific primers (Clontech).
The PCR products were analyzed by 1% agarose gel
electrophoresis with ethidiumbromide 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) to the ears of m ice result in contact
hypersensitivity of the ears as well as significant elevation
of serumIgE levels, accompanied by the increased TH1
response for the onset of skin dermatitis and the TH2
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
sPLA2activity were performed as described in our previous
reports [8–12]
R E S U L T S
Determination of the heparin-binding site of mouse
sPLA2-IID
The amino-acid sequences of mouse and human sPLA2-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 sPLA2-IIAs and rat and human sPLA2-Vs serve
as functional heparin-binding sites [7,8,34,36], we replaced
some of these cationic residues in mouse sPLA2-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 sPLA2s 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 1M 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 sPLA2-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 formof sPLA 2 s PLA 2 activities in the secreted (S) and cell membrane-bound (i.e NaCl-solubilized) (C) fractions were measured.
Trang 4This observation is in line with previous studies on the
HSPG-binding of sPLA2-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 [3H]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 [3H]arachidonic acid release, which was
accompanied by PGE2generation (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 PGE2 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 sPLA2-IID are correlated with its membrane-binding property, and lend further support for the notion that this enzyme, as does sPLA2-IIA [7–12], acts on cells through an HSPG-dependent mechanism in this setting
sPLA2-IID prefers Ca2+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 sPLA2 -IID released arachidonic acid after A23187 stimulation more efficiently than it did after IL-1 stimulation under the condition where sPLA2-IIA and -V released equivalent levels of arachidonic acid in both responses (Fig 3A) Thus, A23187-induced arachidonic acid release by these three sPLA2s reached comparable levels (net 4–6%), whereas IL-1-stimulated arachidonic acid release by sPLA2-IID (net 0.7%) was apparently lower than that by sPLA2-IIA and -V (net 4–5%) (Fig 3A)
When cells expressing sPLA2-IID were cocultured with those coexpressing COX-2 and mPGES and then stimulated (transcellular prostaglandin biosynthesis [9]), the increased production of PGE2in response to A23187 was higher than that in response to IL-1 (Fig 3B, left) In comparison, coculture of cells expressing sPLA2-V with those coexpress-ing COX-2 and mPGES increased both the immediate and delayed PGE2-biosynthetic responses almost equally (Fig 3B, right) These results indicate that sPLA2-IID secreted fromthe transfectants acts preferentially on the A23187-elicited membranes of neighboring cells, where the arachidonic acid released by the paracrine or juxtacrine action of sPLA2-IID is supplied to downstreamCOXs and mPGES
sPLA2-IID expression in human colon carcinoma cell lines Although sPLA2-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 sPLA2 isozyme remains obscure We therefore surveyed the expression of sPLA2-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 [3H]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 [3H]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) [3H]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 generation AA, arachidonic acid.
Trang 5WiDr and HCT2998 cells (Fig 4A) Unexpectedly,
treat-ment of these cells with IL-1 consistently decreased the
expression of sPLA2-IID in a time-dependent manner
sPLA2-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) sPLA2-V was detected only in IL-1-stimulated
HT29 cells, and sPLA2-IIA was weakly and constitutively
expressed in HT29, KM12 and KM20L2 cells (Fig 4B)
The expression of other sPLA2s (IB, IIE and IIF) was
undetectable
The expression of other enzymes involved in the PGE2
-biosynthetic pathway in these colon carcinoma cell lines was
also investigated (Fig 4C) cPLA2a 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 PGE2-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 cPLA2a, COX-1, cPGES
and mPGES in each cell line were unaltered Among these
cell lines, only WiDr cells produced a substantial amount of
PGE in response to IL-1 (Fig 4D), most likely because
COX-2 is a rate-limiting step for IL-1-dependent PGE2 biosynthesis [6–12]
sPLA2-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 sPLA2s [41] RT-PCR analyses revealed that, unlike mouse mast cells, human mast cells developed in the presence of SCF and IL-6 fromcord blood cells [38] expressed only sPLA2-IID, but not the other sPLA2s including -IB, -IIA, -IIE, -IIF, -V and -X (Fig 5) The expression of cPLA2a was readily detected under the same experimental conditions (Fig 5) The expression of sPLA2-IID and cPLA2a 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)
sPLA2-IID expression in mouse tissues during inflammation
The expression of sPLA2-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)1IL-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.
Trang 6RT-PCR (Fig 6A) After administration of LPS, sPLA2
-IID expression was upregulated in the lung, thymus and
heart in a dose-dependent manner Conversely, sPLA2-IID
expression was decreased in the kidney of LPS-treated mice
In the spleen, intestine and colon, in which the basal sPLA2
-IID expression was high, as well as in the brain and liver,
sPLA2-IID expression was largely unchanged after LPS
challenge In the ears of mice with DNFB-induced atopic
dermatitis, there was a marked increase in sPLA2-IID
expression (Fig 6B)
D I S C U S S I O N
sPLA2-IID, which was originally identified by searching
nucleic acid data bases for expressed sequence tags
repre-senting parts of genes for sPLA2homologs, displays all of
the specific features of sPLA2-IIA: the homology between
these two enzymes is 50% [21,22] sPLA2-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 sPLA2-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 sPLA2-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 sPLA2-IID also
crucially influences its binding to cellular HSPG (Fig 1)
Most importantly, as in the cases of sPLA2-IIA and -V,
enzymes that act on rearranged cellular membranes
through the HSPG-dependent pathway [7,34,36], mutation
of these basic residues of sPLA2-IID led to a m arked
reduction of its ability to release arachidonic acid, produce
PGE2and 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 sPLA2-IID augments arachidonic
acid release fromactivated cells through the pathway
dependent upon the HSPG glypican or other HSPG
molecules [12] The three-dimensional structure of sPLA
-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 sPLA2-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 sPLA2-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 fromvarious 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 fromhuman 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 ethidiumbromide, samples were taken for
Southern blotting using cDNA probes for the mixture of these sPLA 2 s.
Trang 7Our transfection studies have revealed a subtle but
substantial difference between sPLA2-IID and other group
II subfamily enzymes (sPLA2-IIA and -V) These enzymes
are in common active on rearranged cellular membranes
that have been primed by various cell activators [6–12], yet
sPLA2-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 sPLA2 isozyme exerts its
arachidonic acid-releasing function more effectively in the
Ca2+ evoked immediate response than in the
cytokine-induced delayed response The membrane rearrangement
that renders cells more susceptible to sPLA2s 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, sPLA2
-IID may be better suited to the particular perturbed
membrane structures that are formed during prompt
Ca2+ signaling than to those formed during sustained
cytokine signaling
In search of human cell lines that endogenously express
sPLA2-IID, we found that several colon carcinoma cell
lines constitutively express this particular sPLA2 isozyme
(Fig 4) Most of these cell lines also express sPLA2-X, an
observation reminiscent of the recent report by Morioka
et al [32] demonstrating the elevated expression of sPLA2
-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
PGE2, a major COX-2 product, is involved in this process
[46–48] Furthermore, targeted disruption of the cPLA2a
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
sPLA2-X [32,49], sPLA2-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 PGD2, LTC4, and
cytokines) mediators following engagement of the Fce
receptor I on their surfaces by IgE and cognate antigen
Previous studies have established that mast cells represent a
potent source of sPLA2s; mouse IL-3-dependent bone
marrow-derived mast cells express all or some of the group
II subfamily sPLA2s according to culture conditions [41,50],
mouse mast cell line MMC-34 cells express sPLA2-V [51],
and rat peritoneal mast cells express sPLA2-IIA [52] These
sPLA2s 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-IID but not the other isozymes (Fig 5) Given the
experimental evidence that sPLA2-IID, as do the other group II subfamily sPLA2s, 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 sPLA2-IID may display similar functions in human mast cells In this regard, sPLA2-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 sPLA2 -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 sPLA2-IIA [55] We also recently found that sPLA2-V is located in mast cells in tissues from patients with allergic symptoms (
& I Kudo, unpublished data)
Increased expression of sPLA2-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 sPLA2s are inducible enzymes Consistent with our results, Ishizaki
et al [22] have shown that sPLA2-IID expression is increased in the thymus and lung of LPS-treated rats, and Shakhov et al [56] have shown that sPLA2-IID expression
is markedly reduced in lymphoid tissues of lymphotoxin a-deficient mice However, this rather tissue-restricted induction of sPLA2-IID differs fromthe induction of sPLA2-IIA and -V [57,58], which is more widespread among tissues Moreover, LPS treatment resulted in reduced expression of sPLA2-IID in the kidney (Fig 6A),
in which the expressions of sPLA2-IIA and -V [57,58] exhibit a reciprocal pattern Decreased expression of sPLA2-IID, relative to increased expression of sPLA2-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 sPLA2-IID and those of sPLA2-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 sPLA2-IID gene, consistent with its proinflammatory signal-associated inducible nature In comparison, the putative promoter region of the human sPLA2-Vgene 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 sPLA2-IIA gene, albeit with a different alignment [59,60] Such differences among the promoter regulatory regions of these sPLA2s may account for their distinct expression and induction
The present study implies that the structurally related group II subfamily sPLA2 isozymes are not always functionally compensatory, even if they utilize common regulatory machinery under particular conditions The expression and induction profiles of each sPLA2 isozyme during inflammatory responses are tissue- and cell-specific
It is therefore likely that functional redundancy and segregation of sPLA2 isozymes must occur in different physiological and pathological states and in different cells and tissues
Trang 8A C K N O W L E D G E M E N T S
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
fromthe Ministry of Education, Science, Culture, Sports and
Technology of Japan.
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