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T he IL-1 cytokine family consists of three members, IL-1␣, mammary cancer cells fail to grow tumors in the foot pad of IL-1␣
IL-1, and IL-1R antagonist. IL-1 is a crucial regulator of KO mice, whereas progressive tumor growth is observed in wild-
the innate immune system and inflammatory responses (1, type mice. In support of these findings, the expression of IL-1 is
2). A variety of cell types, including lymphocytes, monocytes, fi- significantly increased in a variety of malignant lesions and par-
broblasts, endothelial cells, and epithelial cells, can produce IL-1. ticularly in metastatic human tumor specimens, including non-
The proinflammatory activities of IL-1 result largely from stimu- small cell lung cancer, colorectal carcinoma, and melanoma (7, 8).
lating the expression of genes encoding inflammatory mediators. It Furthermore, antagonism of IL-1R using IL-1R antagonist results
is well-documented that IL-1 increases the expression of cycloox- in significant inhibition of angiogenesis and growth of xenografted
ygenase-2 (COX-2)3 and the production of PGE2 (3, 4). Many human tumors that produce IL-1. These data suggest that the IL-1
biological activities of IL-1 are actually due to increased PGE2 signaling system plays critical roles in neoangiogenesis and tumor
production (5). Inhibition of COX-2 enzyme activity significantly metastasis in a variety of cancers and inhibition of this pathway
reduces IL-1-induced inflammation, suggesting that the COX-2/ may be used for treatment of certain cancers.
PGE2 signaling system plays critical roles in the proinflammatory
A large body of studies indicates that COX-2 exerts pro-onco-
activities of this cytokine.
genic effects on a variety of tumors (9). COX-2 enzyme catalyzes
Accumulative evidence suggests that IL-1 plays critical roles in
the conversion of arachidonic acid to PGG2 and PGH2. PGH2 is
the development of malignant lesions. The most compelling evi-
subsequently converted to a variety of prostaglandins which in-
dence was generated in IL-1 knockout (KO) mouse models.
clude PGE2, PGD2, PGF2␣, PGI2, and thromboxaneA2 by each
Voronov et al. (6) have demonstrated the critical roles of IL-1 in
respective PG synthase. Recent studies provide strong evidence
tumor invasiveness and angiogenesis. Mice solely deficient in
IL-1␣ or IL-1 exhibit dramatically impaired tumor development that PGE2 is a key mediator for proneoplastic actions of COX-2.
and blood vessel growth. B16 melanoma cells do not metastasize PGE2 promotes proliferation of human colorectal carcinoma cells.
to the lung of IL-1 KO mice; however, wild-type mice die from DNA synthesis is increased by PGE2 treatment in several colon
lung metastasis by day 20 after inoculation of B16 cells. DA/3 cancer cell lines (10). PGE2 stimulates the growth of human colo-
rectal cancer cells when grown in extracellular matrix (11–13). In
addition, PGE2 promotes colon cancer cell migration and increases
Department of Surgery and Cancer Center, Indiana University School of Medicine, their metastatic potential (12, 14 –16). Further evidence demon-
Indianapolis, IN 46202 strates that PGE2 promotes intestinal neoplasia through enhancing
Received for publication August 11, 2006. Accepted for publication January 12, 2007. tumor angiogenesis (17–20). Knockout of the EP2 receptor or in-
The costs of publication of this article were defrayed in part by the payment of page hibition of COX-2 enzyme results in a reduction of neoangiogen-
charges. This article must therefore be hereby marked advertisement in accordance esis in APC⌬716 mouse tumors (19, 21). The molecular mecha-
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
nisms by which PGE2 promotes such a wide range of malignant
This work was supported in part by National Institutes of Health Grants DK-065615
(to H.S.) and DK-64593 (to H.S.). phenotypes are poorly understood. A potential mechanism is that
2
Address correspondence and reprint requests to Dr. Hongmiao Sheng, Department PGE2 stimulates the production of a variety of growth factors, so
of Surgery, Indiana University, Indianapolis, IN 46202. E-mail address: hsheng@ that provides a proneoplastic environment. In the present study, we
iupui.edu
attempted to elucidate the role of IL-1␣ in COX-2/PGE2 proneoplas-
3
Abbreviations used in this paper: COX-2, cyclooxygenase-2; KO, knockout; CT, tic actions in colorectal neoplasia. We found that PGE2 induced the
cycle threshold; VEGF, vascular endothelial growth factor; PKA, protein kinase A;
ARE, adenosine uridine-rich element; UTR, untranslated region; siRNA, small-inter- expression of IL-1␣ in colon cancer cells through both transcriptional
fering RNA; DRB, 5,6,-dichlorobenzimidazole riboside; GPCR, G protein-coupled and posttranscriptional mechanisms. IL-1␣ strongly stimulated colon
receptor; CRE, cAMP responsive element; EP, E-prostanoid.
cancer cell migration and induced the expression of VEGF. Our re-
Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00 sults suggest a positive loop between COX-2/PGE2 and IL-1␣ that
www.jimmunol.org
4098 PGE2 INDUCTION OF IL-1␣
may play critical roles in neoangiogenesis and metastasis of colon Table I. Expression of IL-1␣, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7,
cancer. IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18,
IL-19, IL-20, and IL-22 was analyzeda
Real-time RT-PCR All statistical analyses were performed on a personal computer with the
StatView 5.0.1 software (SAS Institute). Analyses between two groups
IL-1␣ expression was quantified using real-time quantitative PCR or were determined using the unpaired Student t test. Differences with a p
TaqMan technique (Applied Biosystems). The sequence of the primer/ value of ⬍0.05 were considered as statistically significant.
probe set was based on IL-1␣ mRNA sequence (GenBank accession no.
NM_000575) and includes the following: forward primer, CCTCTTCT Results
GGGAAACTCACG; reverse primer, AAGTTTGGATGGGCAACTG PGE2 induction of IL-1␣ expression
ATGT. 18S rRNA TaqMan assay reagent was used for internal control.
One-step RT-PCR was performed with 40 ng of RNA for both target gene PGE2 modulates a number of signaling pathways and, therefore,
and endogenous controls. Duplicate cycle threshold (CT) values were an- regulates the expression of an array of genes. To determine
alyzed in Microsoft Excel using the comparative CT (⌬⌬CT) method as
described by the manufacturer (Applied Biosystems). The amount of target whether PGE2 regulates the expression of inflammatory cytokines,
(2⫺⌬⌬CT) was obtained by normalized to 18s and relative to a calibrator. we conducted targeted cDNA arrays, using GEArray Human
Growth Factor/Cytokine Array. LS-174T cells were treated with
ELISA PGE2 at relatively low (0.5 M) or high (10 M) concentrations
Levels of human VEGF and IL-1␣ proteins in cell culture medium and cell for 4, 8, and 24 h; levels of a panel of cytokines were analyzed. A
lysates were quantified using ELISA kits (R&D Systems). Cells were number of cytokines, including IL-1␣, IL-1, IL-7, IL-8, IL-12␣,
seeded in 60-mm plates and serum was deprived for 24 h before PGE2 IL-14, IL-15, IL-18, and IL-19, were expressed in LS-174T cells.
treatment. Culture medium were collected and cell lysates were prepared in
radioimmunoprecipitation assay buffer (1⫻ PBS, 1% Nonidet P-40, 0.5%
IL-1␣ appeared to be the leading cytokine that was strongly in-
sodium deoxycholate, 0.1% SDS, 10 mg/ml PMSF, 10 g/ml aprotinin, duced by PGE2. A 4-h treatment with either low or high concen-
and 1 mM sodium orthovanadate) for ELISA. trations of PGE2 resulted in an ⬃6-fold increase in the expression
The Journal of Immunology 4099
with pCMV-Luc-IL-1␣ 3⬘ UTR (1780 –2054), which did not in- effect of PGE2 and significantly stabilized Luc-IL-1␣ 3⬘ UTR. In-
clude the ARE cluster between nt 2389 and 2445. PGE2-regulated terestingly, addition of EP1 agonist induced luciferase activity as
stabilization of IL-1␣ 3⬘ UTR was fully restored in cells that were well. It was not surprising that an increase in the level of cAMP by
transfected with pCMV-Luc-IL-1␣ 3⬘ UTR (1780 –2503). Next, addition of dibutyryl cAMP reproduced the effects of PGE2 and EP
we inserted a sequence of 106 bp (nt 2355–2460), which includes agonists in LS-174T-Luc-IL-1␣ 3⬘ UTR cells. The stimulatory ac-
the ARE clusters, into the pCMV-Luc reporter vector. This min- tion of PGE2 on Luc-IL-1␣ 3⬘ UTR activity required activation of
imum sequence mimicked the role of the entire IL-1␣ 3⬘ UTR, the PKA pathway because inhibition of PKA by H-89 completely
facilitating the degradation of luciferase mRNA and appeared to be attenuated the PGE2-induced luciferase activity (Fig. 4B). In con-
responsive to PGE2-induced stabilization of IL-1␣ mRNA 3⬘ UTR. trast, an MEK inhibitor, PD-98059, and a PI3K inhibitor, LY-
294002, did not block PGE2-induced Luc-IL-1␣ 3⬘ UTR expres-
Roles of EP/cAMP/PKA in PGE2 stabilization of IL-1␣ 3⬘ UTR sion. In support of these findings, ectopic expression of active
PGE2 acts via specific transmembrane G protein-coupled receptors PKA also reproduced the effect of PGE2 and robustly increased the
(GPCR) (31). Previous studies suggest that PGE2 exerts proneo- luciferase expression of pCMV-Luc-IL-1␣ 3⬘ UTR in LS-174T
plastic actions predominantly through the EP2,4/cAMP/PKA path- cells (Fig. 4C).
way. To elucidate the signaling pathways involved in PGE2 sta-
bilization of IL-1␣ mRNA 3⬘ UTR, pCMV-Luc-IL-1␣ 3⬘ UTR
reporter was stably transfected into LS-174T cells (LS-174T-Luc- Functional roles of IL-1␣ in colon cancer cells
IL-1␣3⬘ UTR). Stimulation with PGE2 increased luciferase activ- Both PGE2 and IL-1 play critical roles in tumor invasiveness.
ity ⬃3-fold (Fig. 4A). Both EP2 and EP4 agonists mimicked the Therefore, we next compared the roles of IL-1␣ and PGE2 in colon
The Journal of Immunology 4101
expression using siRNA. LS-174T and HCA-7 cells were trans- VEGF expression, suggesting that PGE2/EP2 signaling is critical
fected with IL-1␣ siRNA before PGE2 treatment. Efficient down- for increased levels of VEGF in intestinal neoplasm (19). Fukuda
regulation of IL-1␣ protein was achieved 72 h after siRNA trans- et al. (40) have reported that PGE2 induction of VEGF in HCT-116
fection. As a result, PGE2-induced IL-1␣ expression was colon cancer cells is mediated by the transcriptional activator hy-
significantly inhibited by two independent siRNA sequences in poxia-inducible factor 1. We have demonstrated that PGE2 induces
both LS-174T (data not shown) and HCA-7 cells (Fig. 6B). Inter- the transcription of the VEGF1 in LS-174T cells through activation
estingly, transfection of IL-1␣ siRNA also significantly reduced of the -catenin/T cell factor pathway (20). Our results from this
the levels of PGE2-induced VEGF in both LS-174T and HCA-7 study suggest that PGE2 induction of VEGF expression involves a
cell-conditioned medium, suggesting the involvement of IL-1␣ variety of mechanisms. Apparently, activation of the IL-1 pathway
signaling in PGE2 induction of VEGF. To determine the effects of critically contributes to the induction of VEGF by PGE2.
colon cancer cell-released growth factors on tubular organization, PGE2 acts via specific transmembrane GPCR (31). EP1R signals
HUVECs were placed on growth factor-reduced Matrigel. via generation of IP3 and increased intracellular Ca2⫹. EP2 and
HUVEC spontaneously formed tubular structures on extracellular EP4 receptors are coupled to stimulatory G (Gs) proteins and signal
matrix. As expected, conditioned medium collected from PGE2- through increased cAMP, whereas the EP3R is coupled to inhibi-
stimulated HCA-7 cells robustly increased the number and length tory G (Gi) proteins which inhibit the generation of cAMP. PGE2
of HUVEC-formed tubes (Fig. 6C). Transfection with IL-1␣ stimulates the transcription of a number of genes through the
siRNA before PGE2 engagement, however, significantly attenu- cAMP/PKA pathway where the cAMP responsive element (CRE)
ated the stimulatory effects of HCA-7-conditioned medium on tu- within the promoter plays critical roles (13, 41). The CRE consists
bular formation by HUVEC. of an 8-bp palindrome (TGACGTCA) and is typically found
within 100 nt of the TATA box. Although typical CRE/TATA
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