Download Roles of Myofibroblasts in Prostaglandin E2–Stimulated Intestinal

Research Article
Roles of Myofibroblasts in Prostaglandin E2–Stimulated
Intestinal Epithelial Proliferation and Angiogenesis
1
2
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Jinyi Shao, George G. Sheng, Randy C. Mifflin, Don W. Powell, and Hongmiao Sheng
1
1
Department of Surgery and Cancer Center, Indiana University School of Medicine, Indianapolis, Indiana; 2Department of Surgery,
University of Cincinnati, Cincinnati, Ohio; and 3Department of Medicine, University of Texas Medical Branch, Galveston, Texas
advantage to intestinal neoplasms (6, 7). In contrast, genetic disruption of the cyclooxygenase-2 (COX-2) gene or the E-prostanoid
receptor 2 (EP2) results in a substantial reduction of polyps in
APC knockout mice (8, 9). Further evidence shows that PGE2
promotes intestinal neoplasia through enhancing tumor angiogenesis (9–11). Knockout of the EP2 receptor or inhibition of COX-2
enzyme results in a reduction of neoangiogenesis in APC D716
mouse tumors (9, 12). Understanding the precise mechanisms by
which PGE2 promotes intestinal epithelial growth and angiogenesis
remains a significant challenge. It has been shown that PGE2
directly stimulates the proliferation of transformed intestinal
epithelial cells (6, 13, 14) and increases the expression of
proangiogenic growth factors in colon cancer cells (15, 16).
However, the effects of PGE2 on cell growth and angiogenesis
in vivo are considerably complex. Interactions between intestinal
epithelial cells and stromal cells, which include fibroblasts,
myofibroblasts, endothelial cells, and other cell types, may
dramatically influence the homeostasis and transformation of the
intestinal epithelium (17).
A large body of studies has shown that intestinal subepithelial
myofibroblasts (ISEMF) play crucial roles in intestinal organogenesis (18–21), proliferation, and differentiation of intestinal
epithelial cells (19), mucosal protection, and wound healing (22).
ISEMFs are located in the lamina propria throughout the
gastrointestinal tract (23, 24) and act through the secretion of
growth factors, cytokines, and chemokines. ISEMFs express and
produce a large number of growth factors, including hepatocyte
growth factor (HGF; ref. 25), insulin-like growth factor (26),
basic fibroblast growth factor (bFGF; ref. 27), platelet-derived
growth factor (PDGF; ref. 28), transforming growth factor-h
(TGF-h; ref. 29), colony stimulating factor (30), nerve growth
factor (30), and stem cell factor (31). Furthermore, immunohistochemical studies reveal that fibroblasts are the predominant
cell type in the lamina propria of normal colon; however, in
both hyperplastic and neoplastic polyps, interstitial fibroblasts are
replaced by myofibroblasts, suggesting that myofibroblasts play
critical roles in colorectal neoplasia (32).
18Co cells were derived from human colonic mucosa and exhibit
many properties of intestinal subepithelial myofibroblasts (33).
Expression of COX-1 is constitutive in 18Co cells, whereas COX-2
can be induced by a variety of stimuli (34). Interleukin-1-activated
18Co cells produce a significant amount of PGE2 (35). Given the
critical functions of both ISEMF and PGE2 in the intestine, we
hypothesized that PGE2 may induce the production of certain
growth factors by ISEMF, which, in turn, stimulate the growth and
transformation of intestinal epithelium. In the present study, we
show that PGE2 exposure increased the expression and secretion
of amphiregulin (AR), HGF, and vascular endothelial growth factor
(VEGF) in 18Co cells and as well as in primary human colonic
myofibroblasts. PGE2-activated 18Co cells stimulated the proliferation and migration of intestinal epithelial cells. Conditioned
Abstract
Prostaglandins (PG) are produced throughout the gastrointestinal tract and are critical mediators for a complex array
of physiologic and pathophysiologic processes in the intestine.
Intestinal myofibroblasts, which express cyclooxygenase
(COX) and generate PGE2, play important roles in intestinal
epithelial proliferation, differentiation, inflammation, and
neoplasia through secreting growth factors and cytokines.
Here, we show that PGE2 activated human intestinal subepithelial myofibroblasts (18Co) through Gs protein–coupled
E-prostanoid receptors and the cyclic AMP/protein kinase A
pathway. 18Co cells and primary colonic myofibroblast isolates expressed a number of growth factors; several of them
were dramatically regulated by PGE2. An epidermal growth
factor–like growth factor, amphiregulin (AR), which was not
expressed by untreated cells, was strongly induced by PGE2.
Expression of vascular endothelial growth factor A (VEGFA)
was rapidly increased by PGE2 exposure. Hepatocyte growth
factor (HGF) was elevated in PGE2-treated myofibroblasts at
both mRNA and protein levels. Thus, PGE2-activated myofibroblasts promoted the proliferation and migration of intestinal epithelial cells, which were attenuated by neutralizing
antibodies to AR and HGF, respectively. Moreover, in the
presence of PGE2, myofibroblasts strongly stimulated the migration and tubular formation of vascular endothelial cells.
Neutralizing antibody to VEGFA inhibited the observed
stimulation of migration. These results suggest that myofibroblast-generated growth factors are important mediators for
PGE2-induced intestinal epithelial proliferation and angiogenesis, which play critical roles in intestinal homeostasis,
inflammation, and neoplasia. (Cancer Res 2006; 66(2): 846-55)
Introduction
Prostaglandins (PG) are generated throughout the gastrointestinal tract and play critical roles in an array of physiologic and
pathophysiologic processes (1, 2). PGs exert a trophic effect on
small intestinal mucosa and stimulate intestinal epithelial cell
proliferation (3). Short-term administration of PGE2 causes
significant stimulation of DNA synthesis; prolonged PGE2 treatment markedly increases the weight and DNA content of the
intestinal mucosa (4). PGE2 and prostacyclin stimulate intestinal
epithelial cell migration and therefore promote intestinal restitution (5). Moreover, PGE2 exerts growth-stimulatory effects on
intestinal tumors, and administration of PGE2 provides a growth
Requests for reprints: Hongmiao Sheng, Department of Surgery, Indiana
University, Indianapolis, IN 46202. Phone: 317-274-2630; E-mail: [email protected].
I2006 American Association for Cancer Research.
doi:10.1158/0008-5472.CAN-05-2606
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PGE2 Activation of Myofibroblasts
RNA extraction and Northern blot analysis. Extraction of total cellular
RNA was carried out as previously described (39). RNA samples were
separated on formaldehyde-agarose gels and blotted onto nitrocellulose
membranes. Blots were hybridized with cDNA probes labeled with
[a-32P]dCTP by random primer extension (Stratagene, La Jolla, CA). After
hybridization and washes, the blots were subjected to autoradiography.
Reverse transcription-PCR. Expression of EP receptors in 18Co cells
was determined using reverse transcription-PCR (RT-PCR) as described
previously (14). Human HGF and VEGF primer pairs were purchased from
R&D Systems. RT-PCR was carried out using ProStar RT-PCR system
(Stratagene) according to the manufacturer’s instructions.
ELISA. Levels of human HGF, AR, and VEGF proteins in cell culture
media were quantified using ELISA kits (R&D Systems). Cells were seeded in
24-well plate, and serum was deprived for 24 hours before PGE2 treatment.
Culture media were collected and stored at 80jC until assays.
Transient transfection and luciferase assay. Assays to determine
transcriptional activity were described previously (39). Briefly, 18Co cells
were transfected with 0.5 Ag of AR reporter plasmid ( 850 to 87) or VEGF
reporter plasmid ( 2279 to +54) along with 0.1 Ag of the pRL-SV plasmid,
containing the Renilla luciferase gene (Promega, Madison WI), using the
FuGENE 6 procedure (Roche, Indianapolis, IN) as described in the
manufacturer’s protocol. Transfected cells were lysed at indicated times
for luciferase assay. Firefly and Renilla luciferase activities were measured
using a Dual-Luciferase Reporter assay system (Promega) and a luminometer. Firefly luciferase values were standardized to Renilla values.
Immunoblot analysis. Immunoblot analysis was done as previously
described (14). Anti–phosphorylated extracellular signal-regulated kinase
(pERK) and anti-phosphorylated Akt (pAkt) antibodies were purchased
from Cell Signaling Technology (Beverly, MA).
Data analysis. All statistical analyses were done on a personal computer
with the StatView 5.0.1 software (SAS Institute, Inc., Cary, NC). Analyses
between two groups were determined using the unpaired Student’s t test.
Differences with P < 0.05 were considered as statistically significant.
media from PGE2-activated 18Co cells promoted the migration and
tubular formation of vascular endothelial cells. Thus, our study
suggests that myofibroblasts may play critical roles in PGE2induced intestinal growth and transformation.
Materials and Methods
Cell culture and reagents. 18Co cells were purchased from American
Type Culture Collection (Manassas, VA) and grown in MEM supplemented
with 10% fetal bovine serum (FBS) and nonessential amino acids. 18Co cells
used for this study were passages 12 to 14. Primary colonic myofibroblast
(CMF, passages 4-10) cultures were established from histologically normal
margins of surgically resected colonic tissue using the outgrowth method
described by Mahida et al. (36, 37). The myofibroblast phenotype was
verified by immunohistochemistry and flow cytometry. All are positive for
a-SMA, myosin heavy chain, and vimentin but are negative for cytokeratin
(epithelial cell marker), desmin (smooth muscle cell marker), factor VIII
(endothelial cell marker), CD45 (bone marrow–derived hematopoetic cell
marker), CD83 and ILT3 (dendritic cells), lysozyme and MAC 387 (both for
macrophages), and other markers of dendritic cells, B cells, or endothelia.
Rat intestinal epithelial (RIE) cells were a generous gift from Dr. Susan
Kirkland (University of London) and grown in DMEM with 10% FBS. Human
umbilical vein endothelial cells (HUVEC) were purchased from Cascade
Biologics (Portland, OR) and grown in Medium 200 supplemented with
low serum growth supplement. PGE2, 17-phenyl-trinor-PGE2, Butaprost,
Sulprostone, and PGE1 alcohol were purchased from Cayman Chemical
(Ann Arbor, MI). H-89, LY-294002, and PD-98059 were purchased from
Calbiochem (San Diego, CA). AR, HGF, and neutralizing antibodies were
purchased from R&D Systems (Minneapolis, MN).
Growth factor array. To determine the relative expression levels of
growth factors, GEArray Q Series Human Growth Factor Gene Array
(SuperArray Bioscience Corporation, Frederick, MD) was carried out
according to the manufacturer’s instructions. Biotin-labeled probe was
synthesized from total RNA and hybridized with a nylon membrane printed
with cDNAs of 96 growth factors and cytokines. The array image was
captured with chemiluminescence detection and analyzed using the
software of GEArray Expression Analysis Suite.
RIE cell-18Co cell coculture system and DNA synthesis. RIE cells
(5 103) suspended in 400 AL complete medium were placed in Transwell
chambers (0.4 Am, Corning Costar Co., Cambridge, MA) and then grown in
serum-free medium for 24 hours. Separately, confluent 18Co cells were
grown in a 24-well plate and treated with 0.5 Amol/L PGE2 for 24 hours.
Subsequently, Transwell chambers containing RIE cells were inserted into
the 24-well plate and grown for 24 hours. [3H]thymidine (1 ACi) was added
to the lower chambers 5 hours before harvest. The Transwell chambers
were washed thrice with 10% trichloroacetic acid times, and the filters
were collected from the chambers. Incorporation of [3H]thymidine was
determined using a scintillation counter.
Cell migration assay. 18Co cells were grown in 24-well plates, serum
starved for 24 hours, and treated with vehicle or PGE2. RIE or HUVEC cells
suspended in 400 AL serum-free McCoy’s 5A medium were placed in
uncoated Transwell chamber (8 Am, Corning Costar). The Transwell
chambers were then inserted into the 24-well plate containing 18Co cells.
After an incubation period of 5 hours at 37jC, cells on the upper surface of
the filter of Transwell chambers were removed with a cotton swab. The
filters were fixed and stained with 0.5% crystal violet solution. Three
microscope fields (200) from each Transwell chamber were randomly
selected, and cells adhering to the undersurface of the filter were counted.
HUVEC tube formation. HUVECs were suspended in 0.1 mL of
indicated conditioned media and placed on growth factor reduced Matrigel
(Collaborative Biomedical Products, Bedford, MA) in 96-well plates.
Morphology of the cells was documented using a digital camera attached
to an inverted microscope. Three photographs from random fields of each
microtiter well (quadruplicate wells for each group) were analyzed. Tubes
were defined as straight cellular extensions joining two cell masses (38).
Tube formation was assessed by the numbers of tubular structures and the
length of tubes.
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Results
PGE2 induced stellate transformation of 18Co myofibroblasts. In response to increased levels of intracellular cyclic AMP
(cAMP), myofibroblasts undergo stellate transformation (40).
Agents that increase cAMP levels including forskolin, cholera
toxin, and PGE2 induce stellate morphology in 18Co cells (33).
Confluent 18Co cells were grown in serum-deprived medium for
24 hours and then treated with 0.5 Amol/L PGE2. Cells acquired a
stellate shape with dendritic-like processes by 2 hours following the
addition of PGE2 (Fig. 1A). By 24 hours, most stellate-transformed
cells returned to their regular fibroblastoid morphology. A selective
protein kinase A (PKA) inhibitor, H-89 (5 Amol/L), completely
attenuated the PGE2-induced stellate transformation of 18Co cells.
PGE2 acts via specific transmembrane G protein-coupled receptors;
four E type prostaglandin (EP) receptor subtypes have been
identified (41). EP2 and EP4 are known to increase intracellular
cAMP levels and activate the PKA pathway. Expression of all of
four EP receptors was detected in 18Co cells by RT-PCR (Fig. 1B).
To determine which EP receptor mediated the PGE2-induced
stellate transformation, selective agonists were employed. Butaprost, a selective EP2 agonist, strongly transformed 18Co cells;
almost all cells acquired a stellate appearance by 2 hours after
the treatment. Activation of EP4 receptor modestly induced
stellation; a small portion of 18Co cells was transformed by PGE1
alcohol (Fig. 1B). As expected, EP1 and EP3 agonists (17-phenyltrinor-PGE2 and Sulprostone) did not induce any morphologic
transformation of 18Co cells (data not shown).
The functional role of PGE2-induced stellation of 18Co cells is
complex (42). We found that PGE2 did not alter the proliferation of
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Figure 1. PGE2 induction of stellate transformation of 18Co cells. A, confluent 18Co cell cultures were serum deprived for 24 hours before addition of vehicle (V ) or
0.5 Amol/L PGE2 (E2 ). H-89 (5 Amol/L) was added 15 minutes before PGE2 treatment. Cells were photographed at the indicated times. B, expression of EP receptors
were analyzed by RT-PCR (top ). Butaprost or PGE1 alcohol at 0.5 Amol/L was added to serum-deprived 18Co cells. Morphologic alterations were documented
using a digital camera attached to an inverted microscope (bottom ). C, 2 105 18Co cells were suspended in serum-free medium and seeded in 8-Am Transwell
chambers. Vehicle or 0.5 Amol/L PGE2 were added into the lower chambers. After a 24-hour incubation, filters were fixed and stained with 0.5% crystal violet solution.
Cells adhering to the undersurface of the filter were photographed (left and middle ) and counted (right ). Columns, mean of cell numbers done in triplicate; bars, SD.
*, P < 0.05. Cell migration assays were done at least three times independently.
(Fig. 2C). The stimulatory action of PGE2 on AR production required
activation of the cAMP/PKA pathway; inhibition of PKA by H-89
completely attenuated the PGE2-induced AR (Fig. 2D). In contrast,
a mitogen-activated protein (MAP)/ERK kinase (MEK) inhibitor
(PD-98059) and a phosphatidylinositol 3-kinase (PI3K) inhibitor
(LY-294002) did not block PGE2-induced AR expression. Activation
of either EP2 receptor or EP4 receptor increased the production of
AR; however, the EP2 signaling seemed to be the predominant
pathway mediating AR induction (Fig. 2E). To determine the
regulatory mechanism mediating PGE2 induction of AR, an AR
promoter-driven reporter plasmid was introduced into 18Co cells.
PGE2 induced the activity of the AR promoter f7-fold, which was
completely blocked by H-89 (Fig. 2F). Although Butaprost exerted
a similar stimulatory effect on the AR promoter, PGE1 alcohol did
not have any effect on AR transcription in 18Co cells.
PGE2 induced the expression of HGF. The Growth Factor
cDNA Array showed that HGF was constitutively expressed by
18Co cells and significantly increased by 4 hours after PGE2
exposure. RT-PCR analysis revealed that PGE2 did not significantly
change the expression of HGF at 1 hour; however, levels of HGF
mRNA were robustly increased at 2 and 4 hours after PGE2
treatment (Fig. 3A). HGF protein was detected in 18Co culture
media at a concentration of f0.4 ng/mL. Addition of PGE2
increased the levels of HGF f5-fold by 24 hours (Fig. 3B).
Inhibition of the PKA pathway by H-89 attenuated the
PGE2-induced production and secretion of HGF. Addition of either
Butaprost or PGE1 alcohol increased HGF production, suggesting
the involvement of both EP2 and EP4 signals in PGE2 induction
of HGF.
PGE2-activated 18Co cells stimulated intestinal epithelial
proliferation and migration. To determine whether PGE2activated 18Co cells were able to stimulate the proliferation of
18Co cells in the absence of serum (data not shown); however,
PGE2 significantly stimulated the migration of 18Co cells,
determined by a modified Boyden chamber assay (Fig. 1C).
Numbers of migrating 18Co cells increased f1-fold in the presence
of 0.5 Amol/L PGE2 compared with the cells treated with vehicle.
18Co cells expressed an array of growth factors. To
determine which growth factors were expressed by 18Co cells, we
carried out targeted cDNA arrays using GEArray Human Growth
Factor Array. 18Co cells were treated with either vehicle or PGE2
for 1, 2, or 4 hours. The expression profile of 96 growth factors,
cytokines, and chemokines was analyzed. Changes in gene
expression z2-fold were shown. As summarized in Table 1, 18Co
cells expressed a number of growth factors, which belong to the
epidermal growth factor (EGF), FGF, PDGF, HGF, TGF-h, and
neuronal growth factor families. Expression of several growth
factors was significantly regulated by PGE2, including amphiregulin
(AR), HGF, neuregulin 1, and VEGFA. Although AR was not
expressed under regular circumstances, AR expression was strongly
induced by PGE2. A 27-fold increase was detected after 18Co cells
were exposed to PGE2 for 1 hour. HGF was constitutively expressed
by 18Co cells, which was increased 5.8-fold at 4 hours after PGE2
exposure. VEGFA was expressed at low levels in vehicle-treated
18Co cells, which was increased 5-fold at the earliest time point.
PGE2 increased the expression of AR. To validate the findings
made from the Growth Factor cDNA Array, we first investigated the
regulation of AR by PGE2. AR mRNA was not detected in vehicletreated 18Co cells; however, PGE2 rapidly increased the levels of AR
mRNA, noted by Northern blot (Fig. 2A) and real-time RT-PCR
analysis (Fig. 2B). The PGE2-induced expression of AR remained at
least 24 hours. Secretion of AR was not detected in untreated 18Co
cell culture medium. AR secretion, however, was robustly increased
in PGE2-stimulated 18Co cells, reaching f100 pg/mL by 24 hours
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PGE2 Activation of Myofibroblasts
a cuboidal appearance. Placing 18Co cells in the bottom chamber
significantly increased the motility of RIE cells. However, when
activated by PGE2, 18Co cells strongly stimulated the migration
of RIE cells; the number of migrating cells increased by f100%
(Fig. 5A, right). Additionally, it was noted that the migrating RIE
cells acquired a widely stretched morphology when cocultured
with PGE2-activated 18Co cells (Fig. 5A, left, d, arrows). To
determine which growth factor mediated the promigratory action
Table 1. PGE2 regulation of 18Co-expressed growth
factors
Gene
symbol
AR
BDNF
CSF1
CTGF
FGF2
FGF13
FGF7
FIGF
GDNF
HGF
IGF2
NRG1
NRP1
NRP2
PDGFA
PDGFC
PDGFD
PTN
TGF-b1
TGF-b2
VEGFA
VEGFB
Gene name
Amphiregulin
Brain-derived neurotrophic factor
Colony stimulating factor 1
Connective tissue growth factor
Fibroblast growth factor 2
Fibroblast growth factor 13
Fibroblast growth factor 7
c-fos-induced growth factor
Glial cell derived neurotrophic factor
Hepatocyte growth factor
Insulin-like growth factor
Neuregulin 1
Neurophilin 2
Neurophilin 2
Platelet-derived growth factor A
Platelet-derived growth factor C
Platelet-derived growth factor D
Pleiotrophin
Transforming growth factor-h1
Transforming growth factor-h2
Vascular endothelial growth factor-A
Vascular endothelial growth factor-B
Fold change
1h
2h
4h
27.0
2.8
2.3
2.8
2.3
3.2
2.0
3.0
2.7
2.0
2.7
2.4
5.8
2.1
2.3
15.6
5.0
2.5
NOTE: Listed are the growth factors expressed by 18Co cells. Results
of growth hormone, cytokines, and chemokines are not shown. Fold
changes of differential expression are expressed as PGE2/vehicle–
treated 18 Co cells.
intestinal epithelial cells, we carried out experiments using
nontransformed RIE cells. Previous studies have shown that the
EGF receptor (EGFR) is restricted to the basolateral compartment
of intestinal epithelial cells (43, 44). Therefore, when EGFR ligands
are added to the apical compartment, no mitogenic response is
observed. In contrast, basolateral administration of EGFR ligands
to intestinal epithelial cells grown on Transwell filters results
in proliferation (44). RIE cells were insensitive to the growthstimulatory effect of AR and HGF when grown on plastic dishes. AR
slightly increased RIE cell proliferation only at relatively high
concentrations (10-100 ng/mL; Fig. 4A). However, when RIE cells
were plated on Transwell filters (0.4 Am); addition of AR to the
lower chamber strongly stimulated the proliferation of RIE cells
(Fig. 4B). Based on these results, RIE cells and 18Co cells were
cocultured in a similar system, in which RIE cells were grown in the
upper chamber and 18Co cells were grown in the bottom chamber.
Neither PGE2 nor 18Co cells stimulated the proliferation of RIE
cells; however, PGE2-activated 18Co cells increased DNA synthesis
of RIE cells by f100% (Fig. 4C). Addition of anti-AR–neutralizing
antibody significantly attenuated the growth advantage of RIE cells
that were stimulated by PGE2-treated 18Co conditioned media,
indicating the mitogenic effect of 18Co cell–generated AR (Fig. 4D).
We next investigated if PGE2-stimulated 18Co cells modulate the
motility of intestinal epithelial cells. In a modified Boyden chamber
assay, PGE2 did not stimulate the migration of RIE cells and the
cells that migrated through the polycarbonate membrane retained
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Figure 2. PGE2 induction of AR expression. A, 18Co cells were serum deprived
for 24 hours before PGE2 treatment. Levels of AR mRNA were analyzed by
Northern blot. B, 18Co cells were serum deprived for 24 hours before PGE2
treatment. Levels of AR mRNA were analyzed by real-time RT-PCR. C, 18Co
cells were serum deprived for 24 hours and treated with vehicle (V ) or PGE2
(E2 ) for the indicated times. Levels of AR protein in cell culture media were
determined by ELISA assay. Columns, mean of AR content done in triplicate;
bars, SD. *, P < 0.05. ELISA assays were done at least three times
independently. D, 18Co cells were treated with vehicle, 5 Amol/L H-89 (H ),
25 Amol/L PD-98059 (P), or 10 Amol/L LY-294002 (L) for 15 minutes before the
addition of 0.5 Amol/L PGE2. After a 24-hour incubation, levels of AR in cell
culture media were determined by ELISA assay. Columns, mean of AR done in
triplicate; bars, SD. *, P < 0.05. E, 18Co cells were serum deprived for 24 hours
before treatments (V = ethanol, E2 = 0.5 Amol/L PGE2, EP1/3 = 0.5 Amol/L
17-phenyl-trinor-PGE2, EP2 = 0.5 Amol/L Butaprost, EP3 = 0.5 Amol/L
Sulprostone, and EP4 = 0.5 Amol/L PGE1 alcohol). After 24 hours, levels of AR
protein were determined by ELISA assay. Columns, mean of AR content done in
triplicate; bars, SD. *, P < 0.05. F, 18Co cells were transiently transfected
with an AR promoter reporter vector. Cells were then treated with ethanol (V ),
0.5 Amol/L PGE2 (E2 ), 0.5 Amol/L Butaprost (But ), or 0.5 Amol/L PGE1 alcohol (E1 )
(E1 ) along with 5 Amol/L H-89 or DMSO (V ) for 6 hours. Firefly and Renilla
luciferase activities were measured and standardized. Columns, mean of Renilla
adjusted luciferase activity done in quadruplicate; bars, SD. *, P < 0.05.
Representative of three separate experiments.
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However, PGE2-activated 18Co conditioned media exerted significantly stronger effects on activation of both MEK/ERK and PI3K/
Akt pathways (Fig. 5D).
PGE2 increased the production of VEGF in 18Co cells. COX2/PGE2 mediates hypoxic induction of VEGF in hepatic stellate
cells (46). Our results of Growth Factor Array showed that PGE2
exposure induced the expression of VEGFA, suggesting that VEGFA
is a PGE2 target gene in 18Co cells. Exposure to PGE2 rapidly
increased the levels of VEGFA mRNA in 18Co cells, noted by
Northern analysis (Fig. 6A). Similar results were observed using
real-time RT-PCR; levels of VEGFA mRNA increased f3.5-fold
after the 18Co cells were treated with PGE2 for 1 hour (Fig. 6B).
Moreover, PGE2 treatment increased the production and secretion
of VEGFA protein. Levels of VEGFA protein were elevated f3.5fold in PGE2-stimulated 18Co culture media (Fig. 6C). Induction of
VEGFA production was mediated by both EP2 and EP4 receptors,
because both Butaprost and PGE1 alcohol increased the levels of
VEGFA mRNA (Fig. 6D) and protein (Fig. 6E). Addition of H-89
completely attenuated the PGE2-induced expression of VEGFA at
both mRNA and protein levels. Furthermore, PGE2 stimulated
VEGF transcription through both EP2 and EP4 signaling. Addition
of PGE2, Butaprost, and PGE1 alcohol increased the activity of
VEGF promoter f1-fold (Fig. 6F).
PGE2-activated 18Co cells enhanced angiogenesis. Because
PGE2 induced the expression of VEGF in 18Co cells, it was of
interest to determine whether PGE2-activated 18Co cells enhanced
angiogenesis. Proangiogenic factors, including VEGF, stimulate
neoangiogenesis by inducing endothelial cell proliferation, migration, and tubular organization. The effects of 18Co cells on the
migration of endothelial cells were evaluated in a coculture system
similar to the system described in Fig. 4D. HUVECs were seeded in
the Transwell; 18Co cells were placed in the bottom chamber.
Addition of PGE2 into the bottom chamber without 18Co cells
slightly stimulated the migration of HUVECs. The presence of 18Co
cells in the bottom chamber significantly increased the motility of
HUVECs. When cocultured with PGE2-activated 18Co cells,
HUVECs acquired a widely stretched morphology (Fig. 7A), and
Figure 3. Expression of HGF in 18Co cells. A, 18Co cells were serum deprived
for 24 hours before addition of ethanol (V ) or 0.5 Amol/L PGE2 (E2). Total
RNA was extracted at the indicated time points, and the expression of HGF
mRNA was analyzed by RT-PCR. B, 18Co cells were serum deprived for
24 hours before treatments (V = ethanol, E2 = 0.5 Amol/L PGE2, EP1/3 =
0.5 Amol/L 17-phenyl-trinor-PGE2, EP2 = 0.5 Amol/L Butaprost, EP3 = 0.5 Amol/L
Sulprostone, and EP4 = 0.5 Amol/L PGE1 alcohol). Supernatants of the cell
cultures were collected after 24 hours, and levels of HGF protein were
determined by ELISA assay. Columns, mean of HGF content done in triplicate;
bars, SD. *, P < 0.05. ELISA assay was repeated at least three times.
of PGE2-activated 18Co, RIE cells were stimulated with AR or HGF.
We found that AR did not increase RIE cell migration, but HGF
significantly stimulated the migration of RIE cells (Fig. 5B). In
agreement with this observation, anti-HGF–neutralizing antibody
completely blocked the PGE2/18Co–induced RIE cell migration
(Fig. 5C).
Proliferation and migration of intestinal epithelial cells require
activation of the MAP kinase and the PI3K pathways (45). To
determine whether PGE2-stimulated 18Co cells activated these
signaling pathways in intestinal epithelial cells, levels of pERK and
pAkt in RIE cells were analyzed. Addition of PGE2 did not change
the levels of pERK and pAkt in RIE cells (data not shown). 18Co
conditioned media rapidly increased the levels of pERK and pAkt.
Figure 4. RIE cell proliferation in coculture with 18Co cells. A, RIE cells (2.5 104) were seeded in 24-well plates and subjected to serum deprivation for 24 hours. AR
or HGF at indicated concentrations were added. After a 24-hour incubation, DNA synthesis was analyzed by [3H]thymidine incorporation. Columns, mean of CPM
done in quadruplicate; bars, SD. Representative of three separate experiments. B, 4 103 RIE cells were seeded in a Transwell (0.4 Am) and serum deprived for
24 hours. AR or HGF at the indicated concentrations were added to the lower chambers. After 24 hours, [3H]thymidine (1 A Ci) was added. The filters containing RIE
cells were collected, and [3H]thymidine incorporation was measured using a scintillation counter. C, in a coculture system, 4 103 RIE cells were seeded in the
Transwell (0.4 Am) and serum deprived for 24 hours before being inserted into a 24-well plate, where serum-starved 18Co cells were stimulated with vehicle (V) or
0.5 Amol/L PGE2 for 24 hours. [3H]thymidine incorporation in RIE cells was determined. D, 18Co cells were treated with vehicle (V ) or PGE2 (E2) for 24 hours, and
then 18Co conditioned media were collected. Normal goat IgG (IgG , 20 Ag/mL), anti-AR–neutralizing antibody (aAR , 20 Ag/mL), or anti-HGF–neutralizing antibody
(aHGF , 20 Ag/mL) was added to the 18Co conditioned media and incubated at 4jC for 30 minutes. RIE cells (4 103) were seeded in the Transwell (0.4 Am) and serum
deprived for 24 hours before being inserted into a 24-well plate, which contained pretreated 18Co conditioned media as indicated. [3H]thymidine incorporation in
RIE cells was determined after a 24-hour incubation and a 5-hour pulse.
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PGE2 Activation of Myofibroblasts
Figure 5. RIE cell migration in coculture with 18Co cells.
A, 2 105 RIE cells suspended in serum-free medium were
seeded in Transwell chambers (8 Am). The chambers were then
inserted into 24-well plates, where 18Co cells were grown and
stimulated with vehicle (V ) or 0.5 Amol/L PGE2 for 24 hours. After a
5-hour incubation, filters were fixed and stained with 0.5% crystal
violet solution. Cells adhering to the undersurface of the filter
were photographed (left ). Cell numbers in three microscope fields
(200) from each Transwell were counted. Columns, mean of
migrating cells done in triplicate; bars, SD. B, 2 105 RIE cells
suspended in serum-free medium were seeded in Transwell
chambers (8 Am). Vehicle (V ), 100 ng/mL AR or 10 ng/mL HGF
were added to the lower chambers. RIE cell migration was
determined after a 5-hour incubation as above. C, 2 105 RIE
cells suspended in serum-free medium were seeded in Transwell
chambers (8 Am). Vehicle (V ) or PGE2 (E2 )–stimulated 18Co
conditioned media that were pretreated with normal IgG (IgG ) or
20 Ag/mL anti-HGF–neutralizing antibody (aHGF ) were added to
the lower chambers. RIE cell migration was determined after a
5-hour incubation. D, 18Co cells were serum deprived for 24 hours
and then stimulated with vehicle (V ) or 0.5 Amol/L PGE2 (E2) for
24 hours before collecting the conditioned media. To eliminate
the direct effect of PGE2, 0.5 Amol/L PGE2 was added to
vehicle-treated conditioned media. 18Co conditioned media were
then added to serum-deprived RIE cells, and cellular protein was
extracted at the indicated time points. Levels of pERKs and
pAkt were determined by Western analysis.
Figure 6. Expression of VEGF in 18Co cells. A and B, 18Co cells were serum deprived for 24 hours before addition of ethanol (V ) or 0.5 Amol/L PGE2 (E2). Total RNA
was extracted at the indicated time points, and levels of VEGF mRNA were analyzed by Northern blot (A) and real-time RT-PCR (B ). C, 18Co cells were serum
deprived for 24 hours before PGE2 exposure. After 24 hours, conditioned media were collected, and levels of VEGF protein were determined by ELISA assay.
Columns, mean of VEGF content done in triplicate; bars, SD. *, P < 0.05. D and E, 18Co cells were serum deprived for 24 hours before treatments (V = ethanol,
E2 = 0.5 Amol/L PGE2) in the presence of a PKA inhibitor (5 Amol/L H-89) or DMSO (V ). Cells were also treated with EP agonists (EP1/3 = 0.5 Amol/L
17-phenyl-trinor-PGE2, EP2 = 0.5 Amol/L Butaprost, EP3 = 0.5 Amol/L Sulprostone, and EP4 = 0.5 Amol/L PGE1 alcohol). D, levels of VEGF mRNA were
determined by RT-PCR after the cells were treated for 2 hours. E, after a 24-hour incubation, levels of VEGF protein in culture media were determined by ELISA assay.
Columns, mean of VEGF content done in triplicate; bars, SD. *, P < 0.05. F, 18Co cells were transiently transfected with a VEGF promoter reporter vector. Cells
were subjected to the indicated treatments for 6 hours (V = ethanol, E2 = 0.5 Amol/L PGE2, But = 0.5 Amol/L Butaprost, and E1 = 0.5 Amol/L PGE1 alcohol). Firefly and
Renilla luciferase activities were measured and standardized. Columns, mean of Renilla -adjusted luciferase activity done in quadruplicate; bars, SD.
*, P < 0.05. Representative of three separate experiments.
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may actually occur in the human intestine, the induction of growth
factors by PGE2 was evaluated in human primary subepithelial
CMF. Three CMF primary isolates (4, 5, and 7) were treated with
PGE2; levels of AR, HGF, and VEGFA were determined by RT-PCR.
PGE2 exposure robustly increased the expression of AR mRNA in all
CMF cultures, which normally did not express AR (Fig. 8A). HGF
and VEGFA mRNAs were induced by PGE2 in a majority of CMF
isolates. Moreover, ELISA assay revealed that whereas HGF and
VEGF proteins were increased f1-fold in PGE2-stimulated CMF
culture media, the presence of PGE2-induced protein levels of AR
>10-fold in all CMF isolates (Fig. 8B).
the number of migrating cells was robustly increased (Fig. 7B, left),
which, however, was significantly attenuated by the addition of
anti-VEGFA–neutralizing antibody (Fig. 7B, right).
To determine the effects of 18Co cell–released growth factors on
tubular organization, HUVECs were placed on growth factor–
reduced Matrigel. HUVECs spontaneously form tubular structures
on extracellular matrix. Addition of PGE2 stimulated HUVEC tube
formation by 8 hours, as quantitated by the numbers and the
length of tubes (Fig. 7C). Conditioned media that collected from
PGE2-stimulated 18Co cells robustly increased the number and
length of tubes of HUVECs. Moreover, HUVEC-formed tubes were
dissociated by 24 hours (Fig. 7D). Addition of PGE2-treated 18Co
conditioned media prevented HUVEC tube dissociation. Interestingly, the presence of anti-VEGFA–neutralizing antibody did not
significantly reduce the PGE2/18Co–induced HUVEC tube formation (data not shown), suggesting the involvement of a complex
mechanism.
PGE2 induction of growth factors in primary myofibroblasts. To determine whether the observations from 18Co cells
Discussion
COX-2 is not expressed in normal intestinal mucosa; its activity
increases dramatically in inflammation, injury, and neoplasia of the
intestine (47). In studies of human colorectal cancer, COX-2 levels
are increased in about 90% of cancers and f50% of premalignant
colorectal adenomas, but the enzyme is not usually detected in
Figure 7. HUVEC migration and tubular
formation in coculture with 18Co cells.
A and B, 1 105 HUVECs suspended in
serum-free medium were seeded in
Transwell chambers (8 Am). The chambers
were then inserted into 24-well plates,
where 18Co cells were grown and
stimulated with vehicle (V ) or 0.5 Amol/L
PGE2 for 24 hours. After a 5-hour
incubation, filters were fixed and stained
with 0.5% crystal violet solution. A, cells
adhering to the undersurface of the
filter were photographed. B, numbers of
migrating cells in three microscope fields
(200) from each Transwell were counted.
Columns, mean of migrating cells done in
triplicate (left ); bars, SD. HUVEC migration
was assessed when vehicle (V ) or PGE2
(E2)–stimulated 18Co conditioned media
that were pretreated with normal IgG
(IgG ) or anti-VEGF–neutralizing antibody
(aVEGF , 20 Ag/mL) were added to the
lower chambers (right ). C, 1 104 HUVEC
suspended in serum-free medium
containing vehicle (V ), 0.5 Amol/L PGE2
(E2), vehicle-treated 18Co conditioned
media, or PGE2-activated 18Co
conditioned media were placed onto growth
factor–reduced Matrigel. After an 8-hour
incubation, cells were photographed
(left , 100). Numbers of tubes were
counted (middle ), and the relative length
of the tubular structure was measured
(right ). D, 5 103 HUVEC cells
suspended in serum-free medium
containing vehicle (V ), 0.5 Amol/L PGE2
(E2), vehicle-treated 18Co conditioned
media, or PGE2-activated 18Co
conditioned media were placed onto growth
factor–reduced Matrigel. After a 24-hour
incubation, cells were photographed
(left , 100).
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PGE2 Activation of Myofibroblasts
Figure 8. PGE2 induction of growth factors in human colonic
myofibroblasts. A, human primary colonic myofibroblast cell
isolates CMF4, CMF5, and CMF7 were serum deprived for
24 hours before PGE2 treatment. Total RNA was extracted at the
indicated time points. Levels of AR, HGF, VEGF, and h-actin
were analyzed by RT-PCR. B, CMF4, CMF5, and CMF7 cells were
serum deprived for 24 hours before vehicle (V ) or PGE2 (E2)
treatment. After a 24-hour incubation, protein levels of AR, HGF,
and VEGFA in conditioned media were measured by ELISA assay.
with a dramatic down-regulation of AR. In contrast, an EP2-specific
agonist strongly increases the expression of AR in mammary cancer
cell lines, suggesting that AR is a mediator for COX-2/PGE2–induced
mammary gland hyperplasia. In another study, Moraitis et al. (58)
reported that tobacco smoke stimulates the expression of COX-2
through activating the EGFR signaling system in human oral
mucosa. Overexpression of AR and TGF-a was determined to be the
mechanism for the tobacco smoke–induced EGFR activity and
COX-2 expression, suggesting a positive loop between the COX-2/
PGE2 pathway and AR/EGFR signaling.
The growth of solid tumors requires a blood supply that is
achieved through neoangiogenesis. VEGF is one of the major
regulators for neoangiogenesis, which induces endothelial cell
proliferation, migration, and tubular organization (19). PGE2
induces the expression of VEGF in colon cancer cells and in
APC min/+ polyps (16). EP2-mediated PGE2 signaling plays critical
roles in neoangiogenesis. Homozygous deletion of the EP2 receptor
significantly reduces the number and size of intestinal polyps in
APC D716 mice that is associated with a reduction of VEGF
expression, suggesting that PGE2/EP2 signaling is critical for
increased levels of VEGF in intestinal neoplasm (9). Our data show
that PGE2 increased the expression, production, and secretion of
VEGF in 18Co cells and that PGE2-activated 18Co cells promoted
the migration and tubular formation of HUVEC. This suggests that
myofibroblasts may provide proangiogenic factors for intestinal
remodeling and transformation. In addition to VEGF, a number of
members of the FGF family and the TGF-h family were expressed
by 18Co cells and regulated by PGE2; their functional roles in PGE2
proangiogenic actions are still under investigation.
HGF is a known myofibroblast-derived growth factor that
regulates epithelial cell proliferation, differentiation, motility, and
morphology (59). HGF is expressed in the stomach, small intestine,
and colon (60). The biological functions of HGF in the small
and large intestines are not clear, but it is likely that HGF exerts
growth-stimulatory effects on intestinal epithelial cells. When
colon cancer T84 cells are grown in type I collagen gel, HGF does
not induce their differentiation but stimulates their growth (61).
Our results showed that PGE2-activated myofibroblasts increased
the production of HGF, which predominantly stimulated the
migration of intestinal epithelial cells.
adult intestinal tissues (48, 49). Although there are conflicting data
regarding which cell types express COX-2 in intestinal tumors,
COX-2 expression increases in both the epithelial and the stromal
compartments (50). Indeed, PGs can be produced by a variety of
cell types, including normal and transformed intestinal epithelial
cells, myofibroblasts, and macrophages (34, 51, 52). PGs serve as
autocrine or paracrine lipid mediators to signal changes within
their immediate environment, suggesting that PGE2 may mediate
interactions between intestinal epithelial cells and stromal cells
through both autocrine and paracrine mechanisms. PGE2 derived
from both stromal and epithelial compartments may stimulate
stromal cells to release growth factors, which, in turn, provide a
pro-neoplastic environment for the intestinal epithelium. In the
present study, we found that exogenous PGE2 induced the
expression and secretion of several pro-proliferative and proangiogenic growth factors in intestinal subepithelial myofibroblasts,
providing the evidence that myofibroblasts may be a critical
mediator for COX-2/PGE2–mediated intestinal epithelial growth,
transformation, and neoangiogenesis.
Our results show that exogenous PGE2 induced the expression of
AR at both mRNA and protein levels in 18Co cells. AR is a member
of the EGF growth factor family and a ligand of the EGFR. It has
been shown that AR is a primary mitogen for hepatocytes and
critical in the early steps of liver regeneration; AR-null mice display
an impaired proliferative response after partial liver resection (53).
Moreover, AR exerts tumor-promoting effects on colorectal
carcinomas. AR mRNA is expressed in 60% to 70% of primary and
metastatic human colorectal carcinomas but in only 2% to 7% of
normal colonic mucosa samples studied (54). AR plays critical roles
in colon cancer cell proliferation and transformation that are
required for the growth of human colon carcinoma xenografts (55).
We have reported that in response to PGE2 exposure, the expression
of AR is significantly increased in transformed intestinal epithelial
cells, which stimulates the growth of colon cancer cells via an
autocrine mechanism (39, 56). Recent studies have further stressed
the critical role of AR in the transformation of a variety of epithelial
cell types. Chang et al. (57) showed that COX-2 overexpression in the
mammary gland of transgenic mice induced mammary cancer.
Interestingly, genetic deletion of the EP2 receptor significantly
reduced the COX-2-induced mammary cancer, which is associated
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In summary, our studies suggest that myofibroblasts are a
potential mediator of the growth-stimulatory effect and proneoplastic action of PGE2 in the intestine. Myofibroblasts may
receive PGE2 stimulation via autocrine and paracrine pathways.
Upon activation by PGE2, myofibroblasts increasingly produce and
secrete growth factors, which stimulate intestinal epithelial cell
growth and promote angiogenesis. Given the special localization of
myofibroblasts in normal intestine and intestinal neoplasia, the
interaction between myofibroblasts and intestinal epithelial cells
may play important roles in intestinal epithelial growth and
transformation.
PGE2 signals through specific receptors, including EP1, EP2, EP3,
and EP4. Our results showed that PGE2-induced expression and
production of growth factors was mediated by the EP2,4/cAMP/
PKA pathway, because both Butaprost and PGE1 alcohol reproduced PGE2 actions on induction of HGF, AR, and VEGF. In
support of these findings, a selective PKA inhibitor attenuated the
stimulatory effects of PGE2. Although both EP2 and EP4 pathways
acted quite similarly, differences were observed. For example, AR
was regulated by PGE2 at the transcriptional level, which was
mediated by the EP2/cAMP/PKA pathway only. PGE1 alcohol did
not stimulate the transcription of AR but modestly increased the
levels of AR protein in 18Co culture media, suggesting the
involvement of a post-transcriptional regulation. Interestingly,
activation of both EP2 and EP4 increased the transcription of
VEGF. Because PGE1 alcohol also binds to other EP receptors (62),
its specificity to EP4 is relative. Further experiments are required
to determine the precise function of EP4 in PGE2 activation of
18Co cells.
Acknowledgments
Received 7/25/2005; revised 10/10/2005; accepted 11/8/2005.
Grant support: NIH grants DK-065615, DK-064593 (H. Sheng), and DK-55783
(D.W. Powell).
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
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Roles of Myofibroblasts in Prostaglandin E2−Stimulated
Intestinal Epithelial Proliferation and Angiogenesis
Jinyi Shao, George G. Sheng, Randy C. Mifflin, et al.
Cancer Res 2006;66:846-855.
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