S. Fukumoto et al.

Fig. 3. Effect of MAPK inhibitors on LPS-induced cox-2 mRNA expression. RAW 264.7 cells were treated withPD98059 (50 µM), SB203580 (10 µM) or a combination of both for 1 h and were stimulated with LPS (100 ng/mL)
for 3 h. Total RNA and total protein were obtained from the cells and cox-2 and β -actin mRNA expressions were assessed by semi-quantitative RT-PCR (A). The cox-2 mRNA level was quantiﬁed and was normalized to β -actin. The
relative cox-2 mRNA levels are presented as percentages of the LPS-induced control in the absence of MAPK inhibitor (B). The cox-2 protein expression was assessed by Western blot analysis (C). One representative of 2 independent
experiments is shown.

jected to semi-quantitative RT-PCR or Western
blot analysis, respectively. SB203580 (Fig. 3A,Lane 2) and PD98059 (Lane 3) suppressed cox-2mRNA expression by 75% or 52%, respectively,
and cox-2 protein production (Fig. 3C, Lanes 2,3, respectively). The combination of both inhibitors reduced the cox-2 mRNA (Fig. 3A, Lane 4)and cox-2 protein (Fig. 3C, Lane 4) to nearly null.
Figure 3B shows the quantitative relations.
Next we examined the effect of ES products
in combination with MAPK inhibitor(s) on cox-2
mRNA expression by Northern blot analysis. ESproducts or MAPK inhibitors reduced LPS-induced cox-2 mRNA expression, and the combination with ES products and each MAPK inhibitor
additively suppressed cox-2 mRNA expression(Fig. 4).

Fig. 4. Additive suppression of LPS-induced cox-2
mRNA expression by a combination of MAPK inhibitorand ES products. RAW 264.7 cells were left untreatedor preincubated with ES products (5 µg/mL) for 6 h prior
to medium change. The cells were then left untreated ortreated either with PD98059, SB203580 or a combinationof both for 1 h and were then stimulated with LPS (100ng/mL) for 3 h. Total RNA was obtained from the cells
and was subjected to Northern blot analysis (A). Thecox-2 mRNA level was quantiﬁed and was normalized togapdh. The relative cox-2 mRNA levels are presented aspercentages of the LPS-induced control in the absence ofES products and MAPK inhibitor (100%) (B). The ﬁgure
is representative of the 3 independent experiments.

Destabilization of LPS-induced cox-2 mRNAby ES products
The level of cox-2 mRNA is regulated both transcriptionally and post-transcriptionally (Wadleighet al., 2000; Alcaide and Fresno, 2004). Therefore, we examined the effect of ES products
on cox-2 mRNA stability in LPS-stimulated
macrophages using a transcriptional inhibitor,
44

Parasite inhibits cox-2 gene expression�����Fig. 5. Destabilization of LPS-induced cox-2 mRNA by ES products. RAW 264.7 cells were left untreated or preincubted with ES products (5 µg/mL) prior to medium change. The cells were then stimulated with LPS (100 ng/mL)
for 3 h and treated with actinomycin D (10 µg/mL). Total RNA was obtained at different time points (0, 15, 30, 60,90 and 120 min) after the addition of actinomycin D. Total RNA was obtained from the cells and was subjected to
Northern blot analysis. The cox-2 mRNA expression was quantiﬁed and was normalized to gapdh. One representative experiment of three is shown. Act D, actinomycin D.

cruzi, a protozoan parasite, is reported to destabilize cox-2 mRNA (Alcaide and Fresno, 2004),
this is the ﬁrst report that shows that the parasitic
helminth suppresses cox-2 gene expression.
It is well known that PGE2 potentiates LPSinduced cox-2 mRNA expression via an adenyryl
cyclase/cAMP-dependent pathway (Hinz et al.,2000). As the plerocercoids released PGE2 in thesupernatants of the in vitro culture (Fukushimaet al., 1993; Gao et al., 1998), we also examinedthe effect of ES products on cox-2 mRNA expression stimulated by the cAMP-dependent pathway.
Stimulation by dbcAMP, a cell-permeable cAMPanalogue, could not induce cox-2 gene expression, but enhanced the induction of cox-2 mRNAby LPS, and the ES products suppressed the enhanced cox-2 mRNA expression. These results
suggest that ES products may inhibit the autocrine
feed-forward loop of PGE 2 in LPS-stimulated
macrophages around the plerocercoids of S. erinaceieuropaei.
In agreement with other studies (Chanmugamet al., 1995; Hwang et al., 1997), we showed that
ERK1/2 and p38 MAPK pathways are crucial forcox-2 gene expression and protein production inLPS-activated RAW 264.7 macrophages. Of note,
PD98059 (an inhibitor for the ERK1/2 pathway)
and SB203580 (an inhibitor of the p38 MAPKpathway) additively suppressed gene expression(Figs. 3 and 4). The ES products and both ofMAPKs inhibitors also additively affected cox-2

actinomycin-D. As shown previously, the macrophages were treated with ES products for 6 h,washed, and stimulated with LPS for 3 h. Thenthe cells were further treated with actinomycin D(10 µg/mL) at varying intervals. Total RNA was
subjected to Northern blot hybridization to examine the remaining cox-2 mRNA. As seen in Fig.
5, cox-2 mRNA was degraded rapidly in macrophages treated with ES products, in comparison tocells without treatment of ES products. The cox-2
mRNA levels were normalized to gapdh mRNAlevels. The remaining cox-2 mRNA level at 90
min was 43% in the absence of ES products or17% in the presence of ES products. The level at
120 min was 25% in the absence of ES products
or 3% in the presence of ES products. ES products reduced the stability of cox-2 mRNA.

Discussion
Some parasites such as ﬁlarial nematodes secrete
immunomodulatory molecules that protect the organism (Maizels et al., 2001; Harnett e al., 2004).
The cox-2 isozyme has been known as one of thepotent inflammatory factors of host inflammation through PGs (Vane and Booting, 1998; Kimet al., 2004). We found that ES products fromplerocercoids of S. erinaceieuropaei inhibit cox-2
gene expression in LPS-activated macrophages.
Although AgC10, a mucin from Trypanosoma
45

S. Fukumoto et al.

gene expression (Fig. 4). Recently, we reported
that ES products suppress TNF-α mRNA expression by inhibiting ERK1/2 and p38 MAPK pathways in RAW 264.7 macrophages stimulated withLPS (Dirgahayu et al., 2002). We also showedthat ES products do not affect LPS-induced nuclear translocation of NF-κB by electrophoretic
mobility shift assay (Dirgahayu et al., 2002),
while the activation of NF-κB is important for theinduction of cox-2 mRNA in the LPS-activatedmonocyte-macrophage lineage (D’Acquisto et al.,1998). Although the mechanism of suppression
by ES products needs to be investigated further,
these lines of data suggest the possibility that ES
products suppress LPS-induced cox-2 gene expression by inhibiting ERK1/2 and p38 MAPK
pathways.
Recently, p38 MAP kinase has been reported to play a major role in mRNA stabilization
through AU-rich elements in the 3'-untranslatedregions (Winzen et al., 1999; Lasa et al., 2000 and2001). The 3'-untranslated region of cox-2 contains 22 AUUUA motifs that are recognized to
be important determinants of mRNA instability(Chen and Shyu, 1995; Zubiaga et al., 1995). We
observed that ES products reduce cox-2 mRNAstability (Fig. 5), and suppose that ES products
destabilize cox-2 mRNA by inhibiting the phosphorylation of p38 MAPK.
The present investigation showed that ESproducts suppress cox-2 mRNA expression andcox-2 protein production, which may attenuate inﬂammation around the plerocercoids.

References
1 Ajizian SJ, English BK, Meals EA. Speciﬁc inhibitors of p38 and extracellular signal-regulated kinase
mitogen-activated protein kinase pathways block inducible nitric oxide synthase and tumor necrosis factor accumulation in murine macrophages stimulatedwith lipopolysaccharide and interferon-γ. J Infect
Dis 1999;179:939–944.
2 Alcaide P, Fresno M. AgC10, a mucin from Trypanosoma cruzi, destabilizes TNF and cyclooxygenase-2 mRNA by inhibiting mitogen-activated protein
kinase p38. Eur J Immunol 2004;34:1695–1704.
3 Alessi DR, Cuenda A, Cohen P, Dudley DT, Saltiel
AR. PD 098059 is a speciﬁc inhibitor of the activation of mitogen-activated protein kinase kinase in vitro and in vivo. J Biol Chem 1995;270:27489–27494.
4 Chanmugam P, Feng L, Liou S, Jang BC, Boudreau
M, Yu G, et al. Radicicol, a protein tyrosine kinase
inhibitor, suppresses the expression of mitogeninducible cyclooxygenase in macrophages stimulatedwith lipopolysaccharide and in experimental glomerulonephritis. J Biol Chem 1995;270:5418–5426.
5 Chen CY, Shyu AB. AU-rich elements: characterization and importance in mRNA degradation.
Trends Biochem Sci 1995;20:465–470.
6 D’Acquisto F, Sautebin L, Iuvone T, Di Rosa M,
Carnuccio R. Prostaglandins prevent inducible nitric oxide synthase protein expression by inhibiting
nuclear factor-κB activation in J774 macrophages.
FEBS Lett 1998;440:76–80.
7 Dirgahayu P, Fukumoto S, Miura K, Hirai K. Excretory/secretory products from plerocercoids of Spirometra erinaceieuropaei suppress the TNF-α gene
expression by reducing phosphorylation of ERK1/2
and p38 MAPK in macrophages. Int J Parasitol
2002;32:1155–1162.
8 Dirgahayu P, Fukumoto S, Tademoto S, Kina Y,Hirai K. Excretory/secretory products from plerocercoids of Spirometra erinaceieuropaei suppress
interleukin-1β gene expression in murine macrophages. Int J Parasitol 2004;34:577–584.
9 Fukumoto S, Hirai K, Tanihata T, Ohmori Y, Stuehr
DJ, Hamilton TA. Excretory/secretory products
from plerocercoids of Spirometra erinacei reduce
iNOS and chemokine mRNA levels in peritoneal
macrophages stimulated with cytokines and/or LPS.
Parasite Immunol 1997;19:325–332.
10 Fukushima T, Isobe A, Hojo N, Shiwaku K, Yamane
Y, Torii M. The metabolism of arachidonic acid toprostaglandin E2 in plerocercoids of Spirometra erinacei. Parasitol Res 1993;79:634–638.
11 Gao T, Fukushima T, Isobe A, Hojo N, Shiwaku
K, Yamane Y. Arachidonic acid metabolism toprostaglandin E2 , D2 , F 2α , and I 2 in the plerocer-Acknowledgments: The authors are grateful to Dr.
Kazutoyo Miura, Division of Molecular Medical Zoology, Department of Microbiology and Pathology, Schoolof Medicine, Tottori University Faculty of Medicine, for
his useful comments on the manuscript.
This work was supported by Grants-in-Aid for Scientiﬁc Research (numbers 12670231 and 16590339) from
the Ministry of Education, Culture, Sports, Science andTechnology of Japan.

46

Parasite inhibits cox-2 gene expression

Clark AR. Regulation of cyclooxygenase 2 mRNAstability by the mitogen-activated protein kinase p38
signaling cascade. Mol Cell Biol 2000;20:4265–4274.
21 Lasa, M, Brook M, Saklatvala J, Clark AR. Dexamethasone destabilizes cyclooxygenase 2 mRNAby inhibiting mitogen-activated protein kinase p38.
Mol Cell Biol 2001;21:771–780.
22 Maizels RM, Gomez-Escobar N, Gregory WF, Murray J,Zang X. Immune evasion genes from ﬁlarial nematodes. Int J Parasitol 2001;31:889–898.
23 Miura K, Fukumoto S, Digahayu P, Hirai K, Excretory/secretory products from plerocercoids of Spirometra erinaceieuropaei suppress gene expressions
and production of tumor necrosis factor-α in murinemacrophages stimulated with lipopolysaccharide orlipoteichoic acid. Int J Parasitol 2001;31:39–47.
24 O’Neill GP, Ford-Hutchinson AW. Expression ofmRNA for cyclooxygenase-1 and cyclooxygenase-2
in human tissues. FEBS Lett 1993;330:156–160.
25 Vane JR, Botting RM. Anti-inﬂammatory drugs andtheir mechanism of action. Inflamm Res 1998;47
Suppl 2:S78–87.
26 Wadleigh DJ, Reddy ST, Kopp E, Ghosh S, Herschman HR. Transcriptional activation of the cyclooxygenase-2 gene in endotoxin-treated RAW 264.7
macrophages. J Biol Chem 2000;275:6259-6266.
27 Winzen R, Kracht M, Ritter B, Wilhelm A, ChenCY, Shyu AB, et al. The p38 MAPK pathway
signals for cytokine-induced mRNA stabilization
via MAP kinase-activated protein kinase 2 andAU-rich region-targeted mechanism. EMBO J
1999;18:4969-4980.
28 Zubiaga AM, Belasco JG, Greenberg ME. The nonamer UUAUUUAUU is the key AU-rich sequencemotif that mediates mRNA degradation. Mol Cell
Biol 1995;15:2219–2230.

coids of Spirometra erinaceieuropaei. J Parasitol
1998;84:1107–1111.
12 Giroux M, Descoteaux A. Cyclooxygenase-2 expression in macrophages: modulation by protein kinase C-α. J Immunol 2000;165:3985–3991.
13 Harnett W, McInnes IB, Harnett MM. ES-62, afilarial nematode-derived immunomodulator withanti-inﬂammatory potential. Immunol Lett 2004;94:27–33.
14 Hinz B, Brune K, Pahl A. Prostaglandin E2 upregulates cyclooxygenase-2 expression in lipopolysaccharide-stimulated RAW 264.7 macrophages. Biochem
Biophys Res Commun 2000;272:744–748.
15 Ho FM, Lai CC, Huang LJ, Kuo TC, Chao CM, LinWW. The anti-inflammatory carbazole, LCY-2CHO, inhibits lipopolysaccharide-induced inflammatory mediator expression through inhibition ofthe p38 mitogen-activated protein kinase signaling
pathway in macrophages. Br J Pharmacol 2004;141:1037–1047.
16 Hwang D, Jang BC, Yu G, Boudreau M. Expression of mitogen-inducible cyclooxygenase inducedby lipopolysaccharide: mediation through both
mitogen-activated protein kinase and NF-κB signaling pathways in macrophages. Biochem Pharmacol
1997;54:87–96.
17 Kim BH, Kang KS, Lee YS. Effect of retinoids on
LPS-induced COX-2 expression and COX-2 associated PGE2 release from mouse peritoneal macrophages and TNF-α release from rat peripheral bloodmononuclear cells. Toxicol Lett 2004;150:191–201.
18 Kudesia S, Indira DB, Sarala D, Vani S, Yasha TC,Jayakumar PN, et al. Sparganosis of brain and spinal cord: unusual tapeworm infestation (report oftwo cases). Clin Neurol Neurosurg 1998;100:148–152.
19 Kujubu DA, Fletcher BS, Varnum BC, Lim RW, Herschman HR. TIS10, a phorbol ester tumor promoterinducible mRNA from Swiss 3T3 cells, encodes anovel prostaglandin synthase/cyclooxygenase homologue. J Biol Chem 1991;266:12866–12872.
20 Lasa M, Mahtani KR, Finch A, Brewer G, Saklatvala J,

Received November 17, 2005;
accepted November 25, 2005
Corresonding author: Soji Fukumoto, MD, PhD

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