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JBC/2015/668277-R1 Bile acid and S1PR2-mediated signaling in CCA
1
Taurocholate Induces Cyclooxygenase-2 Expression via the Sphingosine 1-phosphate
Receptor 2 in a Human Cholangiocarcinoma Cell Line
Runping Liu1, 2, Xiaojiaoyang Li1, Lan Luo3, Xiaoyan Qiang1,2, Phillip B. Hylemon2,4,
Zhenzhou Jiang1, 3*, Luyong Zhang1, 3 * and Huiping Zhou2,4*,
1Jiangsu Key Laboratory of Drug Screening, China Pharmaceutical University, Nanjing,
Jiangsu, China
2Department of Microbiology and Immunology, Virginia Commonwealth University,
Richmond, Virginia, USA
3Jiangsu Center for Pharmacodynamics Research and Evaluation, Nanjing, Jiangsu, China
2McGuire Veterans Affairs Medical Center, Richmond, Virginia, USA
*Running title: Bile acid and S1PR2-mediated signaling in CCA
To whom correspondence should be addressed: Huiping Zhou, Department of Microbiology
and Immunology and McGuire Veterans Affairs Medical Center, Richmond, VA, USA 23298.
Tel:804-828-6817; Fax:804-828-0676; Email:hzhou@vcu.edu. Or Luyong Zhang, Jiangsu
Center for Drug Screening, China Pharmaceutical University, Nanjing, Jiangsu, China
210009. Tel: 86-25-83271500; Fax: 86-25-83271500; Email: lyzhang@cpu.edu.cn. Or
Zhenzhou Jiang, Jiangsu Center for Pharmacodynamics Research and Evaluation, China
Pharmaceutical University, Nanjing, Jiangsu, China 210009. Tel: 86-25-83271142; Fax: 86-
25-83271142; Email: beaglejiang@cpu.edu.cn.
Keywords: Bile acids, Sphingosine 1-phosphate, Biliary cancer, NF-B, EGFR
Background: Cyclooxygenase-2 (COX-2)
and sphingosine 1-phosphate receptor 2
(S1PR2) are highly expressed in human
cholangiocarcinoma (CCA) and taurocholate
(TCA) promotes CCA cell growth via S1PR2.
Results: TCA-mediated activation of S1PR2
contributed to COX-2 expression and CCA
cell growth.
Conclusion: S1PR2 plays a critical role in
TCA-induced COX-2 expression and CCA
growth.
Significance: S1PR2 represents a novel
therapeutic target for CCA.
ABSTRACT
Cholangiocarcinoma (CCA) is a rare, but
highly malignant primary hepatobiliary
cancer with a very poor prognosis and
limited treatment options. Our recent studies
reported that conjugated bile acids (CBAs)
promote the invasive growth of CCA via
activation of sphingosine 1-phosphate
receptor 2 (S1PR2). Cyclooxygenase-2
(COX-2)-derived prostaglandin E2 (PGE2) is
the most abundant prostaglandin in various
human malignancies including CCA.
Previous studies have indicated that COX-2
was highly expressed in CCA tissues, and
the survival rate of CCA patients was
negatively associated with high COX-2
expression levels. It has also been reported
that CBAs induce COX-2 expression, while
free bile acids inhibit COX-2 expression in
CCA mouse models. However, the
underlying cellular mechanisms and
http://www.jbc.org/cgi/doi/10.1074/jbc.M115.668277The latest version is at
JBC Papers in Press. Published on October 30, 2015 as Manuscript M115.668277
Copyright 2015 by The American Society for Biochemistry and Molecular Biology, Inc.
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connection between S1PR2 and COX-2
expression in CCA cells have still not been
fully elucidated. In the current study, we
examined the role of S1PR2 in conjugated
bile acid (taurocholate, TCA)-induced COX-
2 expression in a human HuCCT1 CCA cell
line and further identified the potential
underlying cellular mechanisms. The results
indicated that TCA-induced invasive growth
of human CCA cells was correlated with
S1PR2-medated upregulation of COX-2
expression and PGE2 production. Inhibition
of S1PR2 activation with chemical
antagonist (JTE-013) or downregulation of
S1PR2 expression with gene-specific
shRNA not only reduced COX-2 expression,
but also inhibited TCA-induced activation of
EGFR and ERK1/2/Akt-NF-B signaling
cascade. Conclusion: S1PR2 plays a critical
role in TCA-induced COX-2 expression and
CCA growth and may represent a novel
therapeutic target for CCA.
Cholangiocarcinoma (CCA) is the most
common fatal biliary malignancy and
accounts to 10% to 15% of primary hepatic
cancer originating from the epithelial lining of
bile ducts (1-4). Recent epidemiological
studies indicate that the incidence of CCA is
increasing worldwide, although the risk
factors differ geographically (3). Unlike other
gastrointestinal carcinomas, CCA is
relatively rare and has not been extensively
studied. Certain environment-linked risk
factors, such as liver fluke infection and toxic
chemicals, may play a critical role in the
disease development and progression of
CCA in some patients. Recent studies have
identified several interacting molecular
pathways as vital promoting factors in the
development of CCA , such as cholestasis,
chronic inflammation, oxidative stress, and
dysregulation of the cell injury repair
processes (1-3,5,6). However, the role of
conjugated bile acids (CBA) in CCA
pathogenesis remain largely unknown.
The current available therapeutic options are
mainly limited to surgical resection or liver
transplantation (7).
Cyclooxygenase-2 (COX-2)-derived
prostaglandins (PG) have been implicated in
various carcinogenesis including
cholangiocarcinogenesis (8,9).
Overexpression of COX-2 promoted
cholangiocarcinoma cell growth and survival,
while depletion or pharmacological inhibition
of COX-2 reduced CCA cell proliferation (9-
11). COX-2-derived PGE2 is the most
abundant prostanoid and mediates many
physiological and pathological effects via
activating different G protein coupled
receptors (GPCRs) EP1-4. Both enhanced
expression of COX-2 and increased
production of PGE2 have been associated
with various cancers including CCA (12-14).
Occurrence of CCA is often associated with
chronic cholestasis, and the accumulation of
bile acids, especially conjugated bile acids,
in the liver and bile duct is linked to the
disease progression of CCA. It also has
been reported that CBAs induce COX-2
expression via the epidermal growth factor
receptor (EGFR) and NF-B pathways in
CCA cells (15,16). In CCA patients, the
composition of taurine- and glycine-
conjugated cholic acids is significantly
increased when compared to patients with
benign biliary diseases (17). Our recent
studies showed that CBAs promoted the
proliferation and invasion of both rodent and
human CCA cells through the activation of
sphingosine 1-phosphate receptor 2
(S1PR2)(18). S1PR2 was the predominant
S1PRs expressed in CCA cell and tissues.
Although several recent studies reported
that S1PR2-mediated signaling pathways
were involved in promoting tumor growth and
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progression, the underlying
cellular/molecular mechanisms remain to be
elucidated (19). Whether conjugated bile
acid-mediated activation of S1PR2
contributes to the upregulation of COX-2
expression and PGE2 production in CCA
cells has not been determined and is the
major focus of this study.
In the current study, we examined the
effect of TCA on COX-2 expression and
elucidated the role of S1PR2 in TCA-induced
COX-2 expression and further identified
potential signaling pathways in human CCA
cells.
EXPERIMENTAL PROCEDURES
Materials-S1P and JTE-013 were
purchased from Cayman Chemical (Boston,
MA, USA). pcDNA3.1-3xHA-tagged human
S1PR2 (N-terminus) was obtained from
UMR cDNA Resource Center (Rolla, MO);
Celecoxib, U0126 and MK-2206 were
purchased from Selleck Chemicals (Boston,
MA, USA). Taurocholate (TCA) and dimethyl
sulfoxide (DMSO), monoclonal anti-HA
antibody were obtained from Sigma-Aldrich
(St. Louis, MO, USA). BD Biocoat Matrigel
Invasion Chamber was purchased from BD
biosciences (USA). A QIAzol lysis reagent
was from QIAGEN Sciences (Valencia, CA,
USA). Thermo Hygreen supermix reagent
was purchased from Thermo (Waltham, MA,
USA). RIPA lysis buffer and nuclear protein
isolation kit were obtained from Beyotime
(Nanjing, Jiangsu, China). Bio-Rad protein
assay reagent and 4x sample buffer were
from BIO-RAD (Hercules, CA, USA). Primary
antibodies against COX-2, p-ERK1/2, ERK1,
ERK2, p-Akt, Akt, p-IKKα/β, IKKα/β, p-NF-κB
p65, NF-κB p65, p-EGFR, EGFR, β-actin
and lamin B, as well as HRP-conjugated
secondary antibodies for Western blot were
from Santa Cruz Biotechnology (Santa Cruz,
CA, USA). Alexa Fluor® 488 conjugated
secondary antibodies for
immunofluorescence, High-capacity cDNA
Reverse Transcription Kit, Lipofectamine
2000, cell cultures medium and supplement
components were all from Life Technologies
(Grand Island, NY, USA).
Cell culture and treatment-The human
HuCCT1 CCA cell line was obtained from
Guangzhou Jenniobio Biotechnology Co.,
Ltd (Guangzhou, China) and cultured in
RPMI1640 medium, supplemented with 10%
FBS, 2 mM of L-glutamine, and 50 μg/mL of
gentamicin. TCA was dissolved in serum-
free culture medium. S1P, JTE-013,
Celecoxib, U0126, MK-2206 were prepared
in DMSO to desired concentrations. Drugs
were delivered by directly adding into cell
culture medium. For the cell invasion assay,
drugs were added into medium in the upper
chamber of the matrigel pre-coated transwell.
Transfection of shRNA for down-
regulating S1PR2-The stem loop sequences
of short hairpin RNA (shRNA) specifically
targeting human S1PR2 and the scrambled
control shRNA plasmid were gifts from Dr.
Murthy Karnam (Department of Physiology
and Biophysics, Virginia Commonwealth
University, Richmond, VA). HuCCT1 cells
were transiently transfected with the control
shRNA or S1PR2 shRNA plasmids using
Lipofectamine 2000, as described previously
(20).
Western blot analysis-Total cell proteins
were lysed with RIPA lysis buffer (Beyotime,
China). Nuclear and cytosol proteins were
isolated using a nuclear protein isolation Kit
(Beyotime, China). 50 µg of the proteins
were resolved on a 10% SDS-PAGE gel and
transferred to Nitrocellulose membranes.
Membranes were blocked for 1 h at room
temperature with 5% BSA in TBS buffer and
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then incubated with specific primary
antibodies for overnight at 4C.
Immunoreactive bands were detected using
horseradish peroxidase–conjugated
secondary antibodies and ECL reagents
(Thermo, USA). Images were recorded
using Bio-Rad ChemiDoc XRS imaging
system (Bio-Rad, USA) and further analyze
using Quantity One (Bio-Rad, USA).
RNA isolation and real-time RT-PCR-
Total cellular RNA was isolated using TRIzol
Reagent (QIAGEN, Valencia, CA, USA) and
reverse transcribed into first-strand cDNA
using the High-capacity cDNA Reverse
Transcription Kit (Life Technologies) as
described previously(18). IL-6, TNF-α,
matrix metalloproteinase-2 (MMP-2) and
MMP-9 mRNA levels were determined by
real-time PCR using Thermo Hygreen
supermix reagents and normalized using
GAPDH as an internal control as described
previously(18,20). Primer sequences will be
provided upon request.
Enzyme-linked Immunosorbent Assays
(ELISA) of PGE2-Following different
treatment, cell culture medium was collected
and centrifuged to remove cell debris. PGE2
levels in the supernatant were determined
using a high sensitivity ELISA kit (Enzo Life
Sciences, USA) according to the
manufacturer’s instruction. Safire2
microplate reader (Tecan, Switzerland) was
used to detect the absorbance at 405 nM.
Cell invasion assay-HuCCT1 cells and
HuCCT1 cells transfected with control
shRNA or S1PR2 shRNA were seeded in the
upper chamber of the BD Biocoat Matrigel
Invasion Chamber (BD Bioscience, USA)
and cultured in complete medium with
1%FBS. Cells were pretreated with JTE-013
(10 μM) or Celecoxib (40 μM) or DMSO
(vehicle control) for 1 h, then treated with
TCA (100 μM), or S1P (100 nM), or DMSO
and incubated at 37°C for 24 h. At the end of
treatment, noninvasive cells were removed
from the top surface of upper chamber
membrane. Invasive cells that migrated to
lower surface were fixed with 3.7%
paraformaldehyde and stained with 0.1%
crystal violet solution. Invasive cells were
counted and the invasion index was
analyzed as previously described (18).
Immunofluorescence staining of NF-
B p65-
HuCCT1 cells were seeded into 96-well
plates with glass bottom (Matrical bioscience,
USA) and cultured overnight in serum-free
medium. After treatment with TCA or S1P
with or without JTE-013, cells were fixed with
3.7% formaldehyde in PBS for 45 min,
permeabilized with 0.1% Triton-X-100 for 3
min, and blocked with 3% BSA for 45 min.
Cells were then incubated with rabbit anti-
NF-B p65 antibody (1:100 dilution)
overnight at 4◦C, followed by incubation with
Alexa Fluor-488 labeled goat anti-rabbit
secondary antibody for 30 min at RT. After
washing with PBS, staining of the
intracellular and nuclear NF-B p65 was
visualized under an Olympus IX81 motorized
inverted fluorescence microscope with a 60
x oil objective (Olympus, Japan).
Detection of S1PR2 internalization-HEK293
cells were plated into 6-well plates with
coverslips. Cells were cultured overnight to
60% confluence and transfected with 1 µg of
plasmid DNA (pcDNA3.1-3xHA-tagged
S1PR2) using PolyJetTMDNA In Vitro
Transfection Reagent according to
manufactural instruction. After 24 h of
transfection, cells were cultured in serum-
free medium for overnight, then stimulated
with TCA (100 µM), or S1P (100 nM) or
vehicle control for 10 mins. Cells were fixed
with 3.7% formaldehyde in PBS for 15 mins.
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The expression and localization of S1PR2
were monitored using immunofluorescence
staining with anti-HA antibody and Alexa
Fluor-488 labeled goat anti-mouse
secondary antibody. The images were
recorded using Zeiss Axio Imager A1
fluorescence microscope with a 100 x oil
objective (Jenamed, Carl Zeiss, Germany).
Statistical analysis-All data were expressed
as the mean ± standard error of the mean.
One-way ANOVA and student’s t-test were
employed to analyze the differences
between sets of data. Statistical analysis
was performed using Prism 5.0 (GraphPad,
San Diego, CA) as described previously
(18,20). A value of p<0.05 was considered
statistically significant.
RESULTS
TCA induces COX-2 expression and
chronic inflammation via activation of
S1PR2- COX-2 is a key enzyme involved in
production of prostaglandins and has been
implicated in various cell transformations
including cholangiocytes (21-25). Previous
studies reported that CBAs induced COX-2
expression and promoted growth in human
CCA cells in culture (15,16). Our recent
studies showed that conjugated bile acid
(TCA) promoted invasive cell growth via
activation of S1PR2 in both rat and human
CCA cell lines (18). However, whether
activation of S1PR2 also contributes to
conjugated bile acid-mediated expression of
COX-2 and prostaglandin (PG) synthesis
remained unknown. Therefore, we first
examined the effect of TCA on COX-2
expression in human HuCCT1 cells. As
shown in Fig.1A-B, TCA significantly
increased COX-2 protein levels in a time-
dependent manner, peaking at 8 h. TCA-
induced COX-2 expression was also dose-
dependent (Fig.1C-D). In order to define the
role of S1PR2 in TCA-induced COX-2
expression, a chemical antagonist of S1PR2,
JTE-013, was used. As shown in Fig.2A-B,
both S1P- and TCA-induced COX-2
expression was markedly inhibited by JTE-
013. Similarly, as shown in Fig. 2C-E, down-
regulation of S1PR2 using a S1PR2 shRNA
significantly blocked TCA-induced COX-2
expression.
PGE2, the final enzymatic product of
COX-2, has been implicated in various
chronic inflammation-related cell
transformation including CCA (14). Previous
study from Dr. Wu’s lab reported that COX-
2-derived PGE2 regulates human CCA cell
growth (8). Our study also showed that TCA
significantly increased PGE2 production in
HuCCT1 cells (Fig. 2F). Furthermore,
TCA-induced PGE2 production was
significantly inhibited by JTE-013. However,
both S1P and TCA had no effect on COX-1
protein expression in HuCCT1 cells at
protein level (Fig.3A-B). In addition, we
examined the effect of S1P and TCA on the
mRNA expression of two inflammatory
cytokines, IL-6 and TNF-α. As shown in Fig.
3C-D, both S1P and TCA significantly
induced mRNA expression of IL-6 and TNF-
α, which was attenuated by JTE-013. These
results suggest that S1PR2 may be a key
regulator in TCA-mediated COX-2
expression, PGE2 production and chronic
inflammation.
TCA enhances invasion of HuCCT1
cells through S1PR2-dependent COX-2
activation-Although several studies have
reported that CBAs promote CCA cell growth
and invasion, the contribution of TCA-
induced S1PR2 activation and COX-2
expression on CCA cell invasion has not
been examined (15,26,27). Our recent
studies showed that S1P- and TCA-induced
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invasive growth was blocked by inhibition of
S1PR2 activation in rat CCA cells (18). To
further verify the effect of TCA on the
invasion of human CCA cells, HuCCT1 cells
were plated in the upper chamber of Matrigel
pre-coated transwell inserts and treated with
TCA (100 μM), S1P (100 nM, as a positive
control), or vehicle control, in the presence
or absence of JTE-013 (10 μM). Similar to
our findings in rat CCA cells, the invasion
indexes of TCA and S1P groups were
significantly increased in human HuCCT1
cells, which were significantly inhibited by
JTE-013 (Fig. 4A-B). Down-regulation of
S1PR2 expression using a shRNA also
blocked TCA-induced increase of invasion
index (Fig. 4C-D). In order to further define
the role of TCA-induced S1PR2 activation
and COX-2 expression in CCA cell invasion,
a specific chemical inhibitor of COX-2,
Celecoxib, was used. As shown in Fig. 4E-F,
in the presence of Celecoxib (40 µM), TCA-
induced increase of the invasion index was
also markedly inhibited in HuCCT1 cells.
These results suggest that TCA promotes
CCA cell invasion via activation of S1PR2,
increased COX-2 expression, and synthesis
of PGE2.
TCA activates NF-
B via activation of
S1PR2- The transcription factor NF-B is a
well-known evolutionarily conserved
signaling molecule with many biological
activities. NF-B can be activated by cell
signaling pathways which activate IB
kinase (IKKα/β). Activated IKKα/β further
phosphorylates IB and leads to degradation
of IB and nuclear translocation of NF-B
p65 (28). In addition, IKKα/β are also
involved in the direct phosphorylation of NF-
B p65. Phosphorylation of NF-B p65 not
only enhances the efficiency of DNA binding,
but also provides an additional interaction
site for transcriptional co-activator CBP/p300
(29). Previous studies have shown that bile
acids activate NF-B signaling pathways in
cancer cells (15,30). In order to determine
whether TCA also can activate NF-B
signaling pathways, we examined the
protein levels of phosphorylated IKKα/β (p-
IKKα/β) and phosphorylated NF-B p65 (p-
NF-B p65). As shown in Fig. 5, TCA
significantly increased protein levels of p-
IKKα/β and p-NF-B p65 in both a time-
dependent and dose-dependent manner. In
addition, TCA significantly increased nuclear
translocation of NF-B p65 (Fig. 6). To
further determine whether the NF-B
activation depends on TCA-mediated
activation of S1PR2, we examined the effect
of JTE-013 on TCA-induced activation of NF-
B in HuCCT1 cells. As shown in Fig. 7 and
Fig.8, JTE-013 not only inhibited the TCA-
induced increase of p-IKKα/β and p-NF-B
p65, but also blocked TCA-induced nuclear
translocation of NF-B p65.
TCA activates EGF receptor via S1PR2-
Previous studies reported that bile acids
transactivate the EGF receptor (EGFR) in
cholangiocytes via a TGF-alpha-dependent
mechanism (31). Bile acid-induced
activation of EGFR/ERK1/2 also induced
COX-2 expression in CCA cells (16). In
human CCA, EGFR expression level is
significantly associated with tumor
progression and poor prognosis (32). To
further understand the relationship between
S1PR2 and EGFR/ERK1/2 activation, we
first examined the effect of TCA on the level
of EGFR phosphorylation in HuCCT1 cells.
As shown in Fig. 9, TCA rapidly and dose-
dependently induced phosphorylation of
EGFR. The presence of JTE-013 or
downregulation of S1PR2 using gene-
specific shRNA completely blocked TCA-
induced activation of EGFR, but had no
effect on total protein expression (Fig.10).
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One of the most common mechanisms of
GPCR-mediated activation of EGFR is the
release of the mature EGFR ligands from
transmembrane precursors by matrix
metalloproteinases (MMPs). MMP2 and
MMP9 are members of the MMP family
reported to mediate EGFR activation
through GPCRs (33). We further examined
whether activation of S1PR2 had any effect
on the expression of MMP2 and MMP9 in
HuCCT1 cells. As shown in Fig.11, both
TCA and S1P significantly increased the
mRNA levels as well as the protein levels of
MMP2 and MMP9, but these increases were
blocked by JTE-013.
Role of S1PR2 in TCA-mediated ERK1/2
and Akt activation in HuCCT1 cells-Bile acid-
induced EGFR activation is linked to the
activation of ERK1/2 and Akt (34). Our
previous studies also showed that TCA
induced ERK1/2 and AKt activation via
S1PR2 (18,20). To confirm the role of
S1PR2 activation in TCA-induced ERK1/2
and Akt activation in HuCCT1 cells, we
examined the protein levels of p-ERK1/2 and
p-Akt in TCA-treated HuCCT1 cells. As
shown in Fig.12A-B, TCA rapidly induced
ERK1/2 and Akt activation in a time-
dependent manner, peaking at 4 h. Similar
to TCA-induced EGFR activation, TCA also
dose-dependently induced ERK1/2 and Akt
activation (Fig.12C-D). Furthermore, both
TCA- and S1P-induced ERK1/2 and Akt
activation was markedly inhibited by JTE-
013 (Fig.12E-F).
Effect of TCA-induced ERK1/2 and Akt
activation on COX-2 expression-It has been
well documented that COX-2 expression is
regulated by ERK1/2 and Akt in various cell
types (8,35-38). To further delineate the role
of S1PR2-mediated ERK1/2 and Akt
activation in TCA-induced COX-2
expression in HuCCT1 cells, selective
chemical inhibitors of MEK1/2 and Akt were
used. As shown in Fig.13A-B, in the
presence of U0126 (10 µM), both TCA- and
S1P-induced ERK1/2 activation was
completely blocked. TCA- and S1P-induced
COX-2 expression was also significantly
inhibited by U0126 (Fig. 14A-B). Similarly,
both TCA- and S1P-induced Akt activation
and COX-2 expression were markedly
inhibited by MK2206, a highly selective
inhibitor of Akt1/2/3 (Figs.13C-D and 14C-D).
S1P and TCA induce S1PR2 internalization-
Our previous studies and current study
strongly indicated that TCA activates S1PR2.
In order to delineate whether TCA is able to
directly interact with S1PR2, we examined
the effect of TCA on S1PR2 internalization in
HEK293 cells. Cells were transfected with
HA-tagged human S1PR2 and then treated
with TCA (100 μM) or S1P (100 nM) or
vehicle control for 10 mins at 37°C. To stop
the ligand-mediated internalization process,
cells were fixed with 3.7% formaldehyde at
4°C. The expression and localization of
HA-S1PR2 were monitored by
immunofluorescence staining using specific
anti-HA antibody. As shown in Fig.15, both
TCA and S1P rapidly induced S1PR2
internalization. These results suggest that
TCA directly interact with S1PR2 as
previously suggest from molecular modeling
experiments (20).
DISCUSSION
CCA is very difficult to diagnose at
early stages due to its anatomic location.
Delayed diagnosis precludes many CCA
patients from surgical intervention, the
treatment option with the best prognosis (1).
Concurrently, the outcomes of current
conventional chemotherapies and radiation
are also very poor (3). Therefore,
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identification of novel therapeutic targets for
CCA is extremely urgent. Although
significant amount of effort has been put in
understanding CCA development and
progression during last two decades, the
etiopathogenesis of this cancer are still
largely unknown.
Accumulation of bile acids in bile
ducts and liver is associated with an
increased incidence of CCA (39). However,
the exact role of bile acids in
cholangiocarcinogenesis is still unclear.
Several studies suggested that bile acid-
mediated activation of the nuclear receptor
FXR (farnesoid X receptor) and the GPCR,
TGR5, might be involved in regulating the
proliferation and survival of cholangiocytes
(40). However, our recent studies also
identified that conjugated bile acid-mediated
activation of S1PR2 plays a critical role in
invasive growth of CCA cells in culture (18).
Impaired bile flow is often accompanied with
chronic inflammation and high basal level of
COX-2 expression in the biliary tracts (1),
while the underlying cellular mechanisms
are still not fully identified. Inflammation is
a key stimulator of carcinogenesis,
promoting tumor cell invasion and migration
in various cancers including CCA (41-43).
Recently, there has been significant
progress in identifying the critical role of
COX-2 plays in tumorgenesis of various
cancers including CCA (4,9,44), and PGE2,
the enzymatic product of COX-2, has been
identified as a strong promoter for
mitogenesis, cell proliferation, and invasion
of CCA (9,45,46).
The principal findings of the current
study directly link conjugated bile acid-
mediated activation of S1PR2 to both COX-
2 expression and PGE2 production in human
CCA cells. The results indicated that TCA-
induced activation of S1PR2 is responsible
for activation of EGFR/ ERK1/2/Akt signaling
pathways, which further activates NF-B and
increases COX-2 expression in CCA cells. A
schematic diagram of potential pathways
involved in TCA-induced COX-2 expression
in CCA cells is illustrated in Fig.16. The
current results provide novel mechanisms
underlying conjugated bile acid-mediated
chronic inflammation in CCA.
S1PR2 is one of the five S1PRs
identified so far and has been implicated in
various physiological and pathological
conditions (19). A recent study reported
that extracellular S1P induced COX-2
expression and PGE2 production via
activation of S1PR2, as well as subsequent
activation of ERK1/2 in rat renal mesangial
cells (47). It also has been reported that
S1PR2 signaling is involved in macrophage
retention and inflammatory secretions in the
atherosclerotic lesions (48). However, the
role of S1PR2 in tumor growth and
progression is controversial in different types
of cancers, probably due to its coupling to
different G proteins in different types of cells
(19). In this study, we demonstrated that
TCA dose-dependently induced expression
of COX-2 via activation of S1PR2 in human
HuCCT1 cells. The protein level of COX-2
was significantly increased even at a
concentration of 25 µM (Fig.1). Inhibition of
S1PR2 activation by a chemical antagonist
or down-regulation of S1PR2 with gene-
specific shRNA completely blocked TCA-
induced COX-2 expression and CCA cell
invasion (Figs.2 and 4). Previous studies
reported that bile acids induced COX-2
expression via activation of EGFR/MAPK
signaling pathways in CCA cells (16). We
also have reported that bile acids activated
MAPK via EGFR in hepatocytes (34). In this
study, we found that TCA also significantly
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9
activated EGFR/MAPK signaling pathways
in HuCCT1 cells (Fig.10). A recent study
reported that taurolithocholic acid (TLCA)
promotes CCA cell growth through the
activation of the muscarinic acetylcholine
receptor and the EGFR/ERK1/2 signaling
pathway(49). However, the concentration of
TLCA is relative low compared to conjugated
primary bile acids in CCA as cholestasis
decreases the formation of secondary bile
acids. Clinical studies have shown that the
concentrations of conjugated primary bile
acids (cholic and chenodeoxycholic acid) are
significantly higher in the bile of CCA
patients than in those with the benign
disease (17). In addition, we have reported
that S1PR2 can be activated by TCA, and
S1PR2 is the predominant S1PR expressed
in CCA cells and human tumor tissues
(18,20). Our current study further confirmed
that TCA rapidly induced S1PR2
internalization, indicating that TCA may
directly activates S1PR2 (Fig.15). Therefore,
S1PR2 appears to play a key role in
conjugated bile acid-mediated COX-2
expression and PGE2 synthesis in CCA cells.
It has been shown that activation of
Src kinase or GPCR-mediated activation of
Gi protein can increase the expression of
MMPs, which play an important role in
forming active EGFR ligands (16,31,33,46).
Similarly, in this study, we also found that
both S1P and TCA significantly increased
the expression of MMP2 and MMP9 in
HuCCT1 cells, which was markedly inhibited
by JTE-013 (Fig. 11). These results suggest
that TCA-induced activation of S1PR2
contributes to the increase of MMPs and
subsequent activation of EGFR/ERK/Akt
signaling pathways. In addition, S1PR2 can
also directly activate ERK and Akt directly
through Gαi (18). It has been well
established that activation of ERK/Akt
signaling pathways is essential for activation
of NF-B. NF-B is a key transcription factor
involved in the regulation of various
inflammatory factors including COX-2.
Consistent with previous reports, we also
found that TCA-induced
S1PR2/EGFR/ERK/Akt activation was linked
to NF-B activation in CCA cells (Figs 5-10),
and JTE-013 inhibited not only TCA-/S1P-
induced NF-B activation, but also the
nuclear translocation of NF-B. By using
specific chemical inhibitors of MEK1 and Akt,
we further demonstrated that TCA-induced
activation of S1PR2/ERK1/2/Akt is
responsible for COX-2 expression in CCA
cells (Fig.14). Inhibition of ERK1/2/Akt also
blocked TCA-induced nuclear translocation
of NF-kB (data not shown). Furthermore,
S1P- and TCA-induced expression of
inflammatory cytokines (IL-6 and TNF-α)
was also blocked by JTE-013 (Fig.3).
In summary, as illustrated in Fig.16,
we determined that S1PR2 is a key player in
TCA-induced COX-2 expression in HuCCT1
cells. Activation of S1PR2 by TCA activates
ERK1/2 and Akt signaling pathways via G
proteins and EGFR. Activated ERK1/2 and
Akt further leads to the activation of NF-B
and subsequent COX-2 expression and
PGE2 synthesis. Our study provides further
evidence indicating that S1PR2 represents a
novel therapeutic target for CCA.
Acknowledgement: This study was supported by National Natural Science Fondation of
China Grants (81320108029 to LZ; 81070245, 81270489 to HZ); China National 12th Five-
year Plan “Major Project of Science and Technologies for Signifiant Drug Discovery”
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JBC/2015/668277-R1 Bile acid and S1PR2-mediated signaling in CCA
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(2012ZX09504001-001 to LZ), National Institutes of Heath Grant R01 DK-057543-11 (to PBH
and HZ), VA Merit Awards (to HZ); VCU Massey Cancer Pilot grant (A35362 to HZ).
Conflict of interest: The authors declare no competing financial interest with the contents of
this article.
Author contributions: RL, PBH, ZJ, LZ and HZ conceived the original ideas, designed the
study, analyzed the data and wrote the manuscript; RL, XL, XQ and LL carried out the
experiments and data analysis. ZJ, LZ and HZ contributed equally.
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FOOTNOTES
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To whom correspondence may be addressed: Huiping Zhou, Department of Microbiology and
Immunology and McGuire Veterans Affairs Medical Center, Richmond, VA, USA 23298.
Tel:804-828-6817; Fax:804-828-0676; Email:hzhou@vcu.edu. Or Luyong Zhang, Jiangsu
Center for Drug Screening, China Pharmaceutical University, Nanjing, Jiangsu, China 210009.
Tel: 86-25-83271500; Fax: 86-25-83271500; Email: lyzhang@cpu.edu.cn. Or Zhenzhou Jiang,
Jiangsu Center for Pharmacodynamics Research and Evaluation, China Pharmaceutical
University, Nanjing, Jiangsu, China 210009. Tel: 86-25-83271142; Fax: 86-25-83271142;
Email: beaglejiang@cpu.edu.cn.
The abbreviations used are: Akt, protein kinase B; CBA: conjugated bile acid; CCA,
Cholangiocarcinoma; COX-2, cyclooxygenase-2; EGF, epidermal growth factor; EGFR,
epidermal growth factor receptor; EP, Prostaglandin E receptor, ERK, extracellular regulated
protein kinases; IκB, Inhibitor of κB; IKK α/β, IκB kinase α/β; IL-6, interleukin-6, MMP-2/9,
matrix metalloproteinases 2/9; NF-B, Nuclear factor kappa-light-chain-enhancer of activated
B cells; PI3K, phosphatidylinositide 3-kinases; PGE2, prostaglandin E2; S1P, sphingosine 1-
phosphate; S1PR2, sphingosine 1-phosphate receptor 2; TCA, taurocholate; TLCA;
taurolithocholic acid; TNF-α, tumor necrosis factor-α
FIGURE 1. The effect of TCA on COX-2 expression in HuCCT1 cells. Cells were cultured
in serum-free medium overnight and then treated with either 100 μM TCA for different time
periods (0-24 h) or different concentrations of TCA (0, 25, 50, 100 or 200 μM) for 8h. At the
end of each treatment, cells were harvested and protein levels of COX-2 were detected by
Western blot analysis. (A and C) Representative images of the immunoblots for COX-2 and
actin are shown. (B and D) Relative densities of COX-2 were analyzed using Quantity One
software using actin as a loading control. Values represent the mean ± S.E. of three
independent experiments. Statistical significance relative to the vehicle control: **p<0.01, ***p
<0.001.
FIGURE 2. The role of S1PR2 in TCA-induced COX-2 expression in HuCCT1 cells. Cells
were cultured in serum-free medium overnight and then treated with S1P (100 nM) or TCA
(100 μM) with or without JTE-013 (10 μM) for 8h. At the end of treatment, total protein was
isolated to determine COX-2 protein levels using Western blot analysis. (A) Representative
images of the immunoblots for COX-2 and actin are shown. (B) Relative densities of COX-2
were determined using actin as a loading control. Values represent the mean ± S.E. of three
independent experiments. Statistical significance relative to the vehicle control: *p<0.05,
***p<0.001; Statistical significance relative to corresponding treatment group without JTE-013:
#p<0.05, ###p<0.001. (C) Cells were transfected with either a control scrambled shRNA or
S1PR2-specifc shRNA expression vector for 48h. Relative mRNA levels of S1PR2 were
detected by real-time RT-PCR and normalized using GAPDH as an internal control as
described in “Methods”. (D) Cells were treated with vehicle control or TCA (100 μM) for 8 h
after transfection with control or S1PR2 shRNA for 48 h. Protein levels of COX-2 was
determined by Western blot analysis. Representative images of the immunoblots for COX-2
and actin are shown. (E) Relative densities of COX-2 were determined using actin as a loading
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control. Values represent the mean ± S.E. of three independent experiments. Statistical
significance relative to the vehicle control: *p<0.05; Statistical significance relative to the TCA-
treated group with control shRNA: #p<0.05. (F) Cells were pretreated with JTE-013 (10 μM)
for 1 h before being treated with TCA (100 μM) for 8 hours. At the end of the treatment, cell
culture medium was collected for detection of PGE2 secretion as described in “Methods”.
Values represent the mean ± S.E. of three independent experiments. Statistical significance
relative to the vehicle control: **p<0.01; relative to the TCA treatment group, #p<0.05.
Figure 3. (A-B) Effect of TCA on COX-1 expression. HuCCT1 cells were cultured in serum-
free medium overnight and then treated with S1P (100 nM) or TCA (100 μM) with or without
JTE-013 (10 μM) for 8h. At the end of treatment, total proteins were isolated. The protein level
of COX-1 was detected by Western blot analysis. (A) Representative images of immunoblots
for COX-1 and Actin are shown. (B) Relative protein levels of COX-1 were analyzed using
Actin as a loading control. Values represent the mean ± S.E. of three independent experiments.
(C-D) Effect of TCA on inflammatory cytokines expression. HuCCT1 cells were cultured
in serum-free medium overnight and then treated with S1P (100 nM) or TCA (100 μM) with or
without JTE-013 (10 μM) for 8h. At the end of treatment, total cellular RNA was isolated.
Relative mRNA levels of (A) IL-6 and (B) TNF-α were determined by real-time RT-PCR and
normalized using GAPDH as an internal control. Values represent the mean ± S.E. of three
independent experiments. Statistical significance relative to the vehicle control, *p<0.05,
**p<0.01, ***p<0.001; relative to S1P or TCA treatment group: #p<0.05, ##p<0.01, ###p<0.001.
FIGURE 4. The effect of S1PR2 activation and COX-2 expression on TCA-induced
invasiveness of HuCCT1 cells. (A and B): Cells were cultured inside of the matrigel pre-
coated transwell inserts and pretreated with JTE-013 (10 μM) for 1 hour and then treated with
either S1P (100 nM) or TCA (100 μM) for 24 h. (C and D): Cells were transfected with either
control shRNA or S1PR2 shRNA for 48h. Transfected cells were cultured inside of the matrigel
pre-coated transwell inserts and then treated with TCA (100 μM) for 24 h. (E and F): Cells
were cultured inside of the matrigel pre-coated transwell inserts and treated with TCA (100
μM) for 24 h with or without Celecoxib (40 µM). At the end of treatment, the number of invasive
cells and invasive index were analyzed as described in “Methods”. Representative Images for
each treatment from three independent experiments are shown. Statistical significance
relative to the vehicle control: *p<0.05, **p<0.01, ***p<0.001; relative to the corresponding
S1P or TCA treatment group: #p<0.05, ##p<0.01, ###p<0.001.
FIGURE 5. The effect of TCA on activation of IKKα/β-NF-B pathways in HuCCT-1 cells.
(A-C) Time course of TCA-induced activation of IKKα/β-NF-B pathways. Cells were
cultured in serum-free medium overnight and then treated with TCA (100 μM) for different
treatment periods (0-24 h). The protein levels of phosphorylated IKKα/β (p-IKKα/β), total
IKKα/β, phosphorylated NF-B p65 (p-NF-B p65) and total NF-B p65 were determined by
Western blot analysis. (D-F) TCA-induced dose-dependent activation of IKKα/β-NF-B
pathways. Cells were cultured in serum-free medium overnight and then treated with
different concentrations of TCA (0, 25, 50, 100 or 200 μM) for 4 h. At the end of each treatment,
cells were harvested, and total protein was isolated. The protein levels of p-IKKα/β, total
IKKα/β, p-NF-B p65 and total NF-B p65 were determined by Western blot analysis. (A
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and D) Representative images of p-IKKα/β, total IKKα/β, p-NF-B p65 and total NF-B p65
are shown. (B-C and E-F) Relative protein levels of p-IKKα/β/ IKKα/β and p-NF-B p65/NF-
B p65 were determined. Values represent the mean ± S.E. of three independent experiments.
Statistical significance relative to vehicle control: *p<0.05, **p<0.01, ***p<0.001.
Figure 6. The effect of TCA on nuclear translocation of NF-B. HuCCT1 cells were
cultured in serum-free medium for overnight and then treated with TCA (100 μM) for different
treatment periods (0, 2, 4, 8 or 24 h). At the end of each treatment, cytosol and nuclear proteins
were isolated as described in “Methods”. Protein levels of NF-B p65 were determined by
Western Blot analysis. Lamin B and actin were used as loading controls for nuclear and cytosol
protein, respectively. (A) Representative images of the immunoblots for NF-B p65, lamin B
and actin are shown. (B) Relative protein levels of NF-B p65 in the nucleus and cytosol.
Values represent the mean ± S.E. of three independent experiments. Statistical significance
relative to the vehicle control: ***p<0.001. (C-D) Immunofluorescence staining of NF-B p65.
HuCCT1 cells were cultured in serum-free medium overnight and treated with either 100 μM
TCA for different treatment periods (0, 2, 4, or 8 h) or different concentrations of TCA (0, 25,
50, or 100 μM) for 4 h. At the end of treatment, immunofluorescence staining was performed
to detect subcellular location of NF-B p65. Representative images for each group are shown.
FIGURE 7. The effect of chemical antagonist of S1PR2 on S1P- and TCA-induced
activation of IKKα/β- NF-κB pathway. HuCCT1 cells were cultured in serum-free medium
overnight and pre-treated with JTE-013 (10 µM) for 1 h, and then treated with S1P (100 nM)
or TCA (100 μM) for 4h. (A and B) Total protein was isolated, and protein levels of p-IKKα/β,
total IKKα/β, p-NF-B p65 and total NF-B p65 were determined by Western blot analysis.
The relative densities of p-IKKα/β/total IKKα/β and p-NF-B p65/total NF-Bp65 were
determined. (C and D) Cytosol and nuclear proteins were isolated as described in “Methods”.
Protein levels of NF-κB p65, lamin B and actin were determined by Western blot analysis.
Lamin B and actin were used as a loading control for nuclear and cytosol protein, respectively.
(A and C) Representative images of the immunoblots for p-IKKα/β, total IKKα/β, p-NF-B p65
and total NF-B p65 are shown. (B) Relative protein levels of p-IKKα/β/total IKKα/β and p-NF-
B p65/total NF-B p65. (D) Relative protein levels of nuclear NF-B p65/lamin B and cytosol
NF-B p65/actin. Values represent the mean ± S.E. of three independent experiments.
Statistical significance relative to the vehicle control: *p<0.05, **p<0.01, ***p<0.001; relative
to S1P or TCA treatment group: #p<0.05, ##p<0.01, ###p<0.001.
Figure 8. The effect of JTE-013 on S1P- and TCA-induced nuclear translocation of NF-
B. HuCCT1 cells were cultured in serum-free medium overnight and then treated with either
100 μM TCA for different treatment periods (0, 2, 4, 8 or 24 h) or different concentrations of
TCA (0, 25, 50, or 100 μM) for 4 h. At the end of treatment, immunofluorescence staining was
performed to detect subcellular location of NF-B p65. Representative images for each group
are shown.
Figure 9. The effect of TCA on EGFR transactivation in HuCCT1 cells. HuCCT1 cells
were cultured in serum-free medium overnight and then treated with either 100 μM TCA for
different time periods (0, 0.17, 0.5, 1, 2, 4 or 8 h), or with different concentrations of TCA (0,
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JBC/2015/668277-R1 Bile acid and S1PR2-mediated signaling in CCA
17
25, 50, 100 or 200 μM) for 15 min. At the end of treatment, total protein was isolated. Protein
levels of phosphorylated EGFR (p-EGFR), total EGFR (t-EGFR) were detected by Western
Blot analysis. (A and C) Representative images of the immunoblots for p-EGFR and t-EGFR
are shown. (B and D) Relative protein levels of p-EGFR/t-EGFR were calculated from three
independent experiments. Values represent the mean ± S.E. of three independent
experiments. Statistical significance relative to the vehicle control: *p<0.05, **p<0.01,
***p<0.001; relative to S1P or TCA treatment group: ##p<0.01, ###p<0.001.
FIGURE 10. The role of S1PR2 in TCA-induced EGFR transactivation in HuCCT1 cells.
(A-B) Cells were cultured in serum-free medium and pretreated with JTE-013 (10μM) for 1h,
and then treated with S1P (100 nM) or TCA (100 μM) for 15 min. (C-D) HuCCT1 cells were
transfected with control shRNA or S1PR2 shRNA for 48 h and then treated with TCA (100 μM)
for 15 min. At the end of treatment, total protein was isolated. Protein levels of p-EGFR and
t-EGFR were detected by Western Blot analysis. (A and C) Representative images of the
immunoblots for p-EGFR and t-EGFR are shown. (B and D) Relative protein levels of p-
EGFR/t-EGFR were calculated from three independent experiments. Values represent the
mean ± S.E. of three independent experiments. Statistical significance relative to the vehicle
control: **p<0.01, ***p<0.001; relative to the S1P or TCA treatment group: ##p<0.01,
###p<0.001.
Figure 11. The effect of TCA on MMP-2 and MMP9 expression. HuCCT1 cells were
cultured in serum-free medium overnight and then treated with S1P (100 nM) or TCA (100 μM)
with or without JTE-013 (10 μM) for 8h. At the end of treatment, total cellular RNA and protein
lysates were isolated. Relative mRNA levels of (A) MMP-2 and (B) MMP-9 were determined
by real-time RT-PCR and normalized using GAPDH as an internal control. Protein levels of
MMP2 and MMP9 were detected by Western Blot analysis. Representative images of the
immunoblots for MMP2 and MMP9 are shown (C and E). Relative protein levels of MMP2/Actin
and MMP9/Actin were calculated from three independent experiments. Values represent the
mean ± S.E. of three independent experiments. Statistical significance relative to the vehicle
control: **p<0.01, ***p<0.001; relative to S1P or TCA treatment group: #p<0.05, ##p<0.01.
Figure 12. The effect of TCA on ERK and Akt activation in HuCCT1 cells. (A-B) Time
course of TCA-induced ERK and Akt activation. HuCCT1 cells were cultured in serum-free
medium overnight and then treated with TCA (100 μM) for different treatment periods (0, 0.5,
1, 2, 4, 8 or 24 h) (C-D) TCA-induced dose-dependent activation of ERK and Akt.
HuCCT1 cells were cultured in serum-free medium overnight and then treated with different
concentrations of TCA (0, 25, 50, or 100 μM) for 1 h. (E-F) The effect of JTE-013 on TCA-
/S1P-induced ERK and Akt activation. HuCCT1 cells were cultured in serum-free medium
overnight and pre-treated with JTE-013 (10 μM) for 1 hour and then treated with S1P (100 nM)
or TCA (100 μM) for 1 h. At the end of each treatment, cells were harvested and total protein
was isolated. The protein levels of phosphorylated ERK1/2 (p-ERK), total ERK1/2 (t-ERK),
phosphorylated Akt1/2/3 (p-Akt) and total Akt1/2/3 (t-Akt) were detected by Western blot
analysis. (A, C and E) Representative images of the immunoblots for p-ERK, t-ERK, p-Akt,
and t-Akt are shown. (B, D and F) Relative levels of p-ERK/t-ERK and p-Akt/t-Akt. Values
represent the mean ± S.E. of three independent experiments. Statistical significance relative
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JBC/2015/668277-R1 Bile acid and S1PR2-mediated signaling in CCA
18
to the vehicle control: *p<0.05, **p<0.01, ***p<0.001; relative to S1P or TCA treatment group:
##p<0.01, ###p<0.001.
Figure 13. Inhibition of TCA-induced ERK1/2 and Akt activation by chemical inhibitors
in HuCCT1 cells. HuCCT1 cells were cultured in serum-free medium overnight and
pretreated with a selective inhibitor of MEK1/2, U0126 (10 μM) or a selective inhibitor of Akt,
MK2206 (3 μM) for 1 h and then treated with S1P (100 nM) or TCA (100 μM) for 1 h. At the
end of treatment, total protein was isolated. Protein levels of p-ERK1/2, total ERK1/2, p-Akt,
and t-Akt were detected by Western blot analysis. (A and C) Representative images of the
immunoblots for p-ERK1/2, t-ERK1/2, p-Akt, and t-Akt are shown. (B and D) Relative protein
levels of p-ERK1/2/t-ERK1/2 and p-Akt/t-Akt were calculated from three independent
experiments. Values represent the mean ± S.E. of three independent experiments. Statistical
significance relative to vehicle control: **p<0.01, ***p<0.001; relative to S1P or TCA treatment
group: ##p<0.05, ###p<0.001.
FIGURE 14 . Effect of TCA-induced ERK1/2 and Akt activation on COX-2 expression in
HuCCT1 cells. Cells were cultured in serum-free medium overnight and pretreated with a
selective inhibitor of MEK1/2, U0126 (10 μM) for 1 h and then treated with S1P (100 nM) or
TCA (100 μM) for 1 h. At the end of treatment, total protein was isolated and protein levels of
COX-2 and actin were detected by Western blot analysis. (A and C) Representative images
of the immunoblots for COX-2 and actin are shown. (B and D) Relative protein levels of COX-
2/Actin were calculated from three independent experiments. Values represent the
mean ± S.E. of three independent experiments. Statistical significance relative to the vehicle
control: *p<0.05, **p<0.01; relative to the S1P or TCA treatment group: #p<0.05, ###p<0.01.
Figure 15. Effect of TCA and S1P on S1PR2 internalization. HEK293 cells were transfected
with 3xHA-tagged human S1PR2 as described in “Methods”. Cells were stimulated with
vehicle control or TCA (100 µM) or S1P (100 nM) for 10 mins, the internalization process was
stopped by fixing with 3.7% formaldehyde at 4C. The localization of S1PR2 was monitored
by immunofluorescence staining. Representative images of each treatment group are shown.
Figure 16. Proposed signaling pathways involved in TCA-induced COX-2 expression in
human CCA cells. Activation of S1PR2 by TCA directly activates ERK1/2/AKt via activated
G proteins or indirectly activates ERK1/2/Akt via EGFR. Activated ERK1/2/Akt further activate
NF-B and COX-2 expression by inducing phosphorylation of IKKα/β and IB, ubiquitination
and degradation of p-IB, and nuclear translocation of NF-B.
at VIVA, VA Commonwealth Univ on December 15, 2015http://www.jbc.org/Downloaded from
Figure 1
A
COX-2
Actin
0 0.5 1 2 4 8 24
TCA (100 μM)
Time (h)
0 25 50 100 200
TCA (μM)
COX-2
Actin
C
Time ( 8 h)
00.5 1 2 4824
0.0
2.0
4.0
6.0
** **
***
Time (h)
Relative protein levels
of COX-2
B
025 50 100 200
0.0
1.0
2.0
3.0
4.0
** **
***
TCA (M)
Relative protein levels
of COX-2
D
at VIVA, VA Commonwealth Univ on December 15, 2015http://www.jbc.org/Downloaded from
COX-2
Actin
S1P - + - - + -
TCA - - + - - +
JTE-013
A
COX-2
Actin
Con TCA Con TCA
Control
shRNA
S1PR2
shRNA
D
Control shRNA S1PR2 shRNA
0.0
0.5
1.0
1.5
**
Reltive mRNA levels
of S1PR2
C
B
0.0
1.0
2.0
3.0
4.0
5.0
*
***
####
- + - - + -
- - + - - +
- - - + + +
JTE-013
TCA
S1P
Relative protein levels
of COX-2
F
Control JTE-013 TCA TCA+JTE-013
0
50
100
150
200
**
#
PGE2
(pg/ml)
E
0.0
1.0
2.0
3.0
4.0
5.0
*
#
ControlControl TCA TCA
Control shRNA S1PR2 shRNA
Relative protein leve ls
of COX-2
Figure 2
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0.0
2.0
4.0
6.0
8.0
10.0
*
***
#
###
- + - - + -
- - + - - +
- - - + + +JTE-013
TCA
S1P
Relative mRNA levels
of IL-6
0.0
1.0
2.0
3.0
4.0
** **
## #
- + - - + -
- - + - - +
- - - + + +JTE-013
TCA
S1P
Relative mRNA levels
of TNF-
Figure 3
C D
0
1
2
JTE-013
TCA
S1P - + - - + -
- - + - - +
- - - + + +
Relative protein levels
of COX-1
COX-1
Actin
S1P - + - - + -
TCA - - + - - +
JTE-013
A B
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JTE-013
S1P+JTE-013
TCA+JTE-013
Control
TCA
S1P
A
0.0
1.0
2.0
3.0
***
##
#
JTE-013
TCA
S1P - - + + - -
- - - - + +
- + - + - +
Relative inv as ion inde x
B
Control TCA
Control TCA
Control
shRNA
S1PR2
shRNA
C
Control
TCA
Control
TCA
0.0
1.0
2.0
3.0
**
Control shRNA S1PR2 shRNA
###
Relative inv as ion inde x
D
Control TCA
Celecoxib Celecoxib+TCA
E
0.0
1.0
2.0
3.0
TCA
Celecoxib
- - + +
- + - +
***
##
Relative inv as ion inde x
F
Figure 4
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Figure 5
TCA (100μM)
p-NF-κB p65
NF-κB p65
p-IKKα/β
IKKα/β
0 0.5 1 2 4 8 24
Time (h)
A
0
0.5
1
2
4
8
24
0.0
0.5
1.0
1.5
2.0
2.5
Time (h)
** *** ***
Relative protein levels of
p-IKK//IKK/
0
0.5
1
2
4
8
24
0.0
0.5
1.0
1.5
2.0
** **
Time (h)
Relative protein levels of
p-NF-Bp65 /NF-Bp65
B
Time (4h)
p-IKKα/β
IKKα/β
0 25 50 100 200
TCA (μM)
p-NF-κB p65
NF-κB p65
D
0
25
50
100
200
0.0
1.0
2.0
3.0
**
***
***
TCA (M)
***
Relative protein levels
of p-IKK //IKK/
0
25
50
100
200
0.0
1.0
2.0
3.0
4.0
TCA (M)
*
**
Relative protein levels of
p-NF-Bp65/NF-Bp65
E
CF
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Figure 6
0
2
4
8
12
24
0
1
2
3
4
Time (h)
*** ***
Relative protein levels
of NF-B in nuclear
0
2
4
8
12
24
0.0
0.5
1.0
1.5
Time (h)
Relative protein levels
of NF-B in cytosol
B
D
025 50 100
TCA (μM)
Time (4h)
Cytosol
NF-B p65
Actin
TCA (100μM)
A
NF-B p65
Lamine B
Nuclear
0 2 4 8 12 24
Time (h)
024 8
TCA (100μM)
C
Time (h)
Hochest Rabbit IgG
Negative control
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p-NF-κB p65
NF-κB p65
p-IKKα/β
IKKα/β
S1P - + - - + -
TCA - - + - - +
JTE-013
Figure 7
A
0.0
0.5
1.0
1.5
2.0
2.5
** **
## ##
- + - - + -
- - + - - +
- - - + + +JTE-013
TCA
S1P
Relative protein levels of
p-IKK//IKK/
0.0
1.0
2.0
3.0
**
##
- + - - + -
- - + - - +
- - - + + +JTE-013
TCA
S1P
Relative protein levels of
p-NF-B p65/NF-B p65
B
Nuclear
Lamin B
NF-κB p65
NF-κB p65
Actin
Cytosol
S1P - + - - + -
TCA - - + - - +
JTE-013
C
0.0
1.0
2.0
3.0
**
***
## ###
- + - - + -
- - + - - +
- - - + + +JTE-013
TCA
S1P
Relative protein levels of
NF-B p65 in nucleus
0.0
0.5
1.0
1.5
- + - - + -
- - + - - +
- - - + + +JTE-013
TCA
S1P
Relative protein levels of
NF-B p65 in c ytosol
D
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Figure 9
A
00.17 0.5 1248
0.0
1.0
2.0
3.0
4.0
Time (h)
*
*** *** **
Relative protein levels of
p-EGFR/t-EGFR
B
C
t-EGFR
p-EGFR
0 25 50 100 200
TCA (μM)
Time (15 min)
0
25
50
100
200
0.0
2.0
4.0
6.0
TCA (M)
**
***
Relative protein levels of
p-EGFR/t-EGFR
D
t-EGFR
p-EGFR
0 0.17 0.5 1 2 4 8
TCA (100 μM)
Time (h)
at VIVA, VA Commonwealth Univ on December 15, 2015http://www.jbc.org/Downloaded from
Figure 10
t-EGFR
p-EGFR
S1P - + - - + -
TCA - - + - - +
JTE-013
A
0.0
1.0
2.0 ***
**
### ##
- + - - + -
- - + - - +
- - - + + +
JTE-013
S1P
TCA
Relative protein levels of
p-EGFR/t-EGFR
B
C
0.0
1.0
2.0
3.0
ControlControl TCA TCA
Control shRNA S1PR2 shRNA
**
##
Relative protein levels of
p-EGFR/t-EGFR
D
t-EGFR
p-EGFR
Control TCA Control TCA
Control
shRNA
S1PR2
shRNA
at VIVA, VA Commonwealth Univ on December 15, 2015http://www.jbc.org/Downloaded from
A
0
1
2
3
**
***
#
##
JTE-013
TCA
S1P - + - - + -
- - + - - +
- - - + + +
Relative mRNA levels
of MMP-2
0
1
2
3
4
**
***
##
##
JTE-013
TCA
S1P - + - - + -
- - + - - +
- - - + + +
Relative mRNA levels
of MMP-9
C
B
D
Figure 11
MMP-2
S1P - + - - + -
TCA - - + - - +
JTE-013
Actin
0
1
2
3
4
JTE-013
TCA
S1P - + - - + -
- - + - - +
- - - + + +
**
#
Relative protein
levels of MMP-2
E F
MMP-9
Actin
0
1
2
3
JTE-013
TCA
S1P - + - - + -
- - + - - +
- - - + + +
**
*
#
Relative protein
levels of MMP-9
S1P - + - - + -
TCA - - + - - +
JTE-013
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Figure 12
A
p-ERK1/2
t-ERK1/2
0 0.5 1 2 4 8 24
TCA (100 μM)
p-Akt
t-Akt
Time (h)
C
p-ERK1/2
t-ERK1/2
Time (1 h)
p-Akt
t-Akt
0 25 50 100 200
TCA (μM)
p-
ERK1/2
t-ERK1/2
S1P - + - - + -
TCA - - + - - +
JTE-013
p-Akt
t-Akt
E
0
0.5
1
2
4
8
24
0.0
0.5
1.0
1.5
2.0
*** ** ***
Time (h)
Relative protein levels of
p-ERK1/2/t-ERK1/2
0
0.5
1
2
4
8
24
0
1
2
3
Time (h)
**
***
*
Relative protein levels of
p-Akt/t-Akt
B
0
25
50
100
200
0.0
0.5
1.0
1.5
2.0
2.5
**
TCA (M)
Relative protein levels of
p-ERK1/2/t-ERK1/2
0
25
50
100
200
0.0
1.0
2.0
3.0
TCA (M)
**
Relative protein levels of
p-Akt/t-Akt
D
0.0
1.0
2.0
3.0
**
***
## ###
- + - - + -
- - + - - +
- - - + + +
JTE-013
TCA
S1P
Relative protein levels of
p-Akt/t-Akt
0.0
1.0
2.0
3.0
*
**
##
##
Relative protein levels of
p-ERK1/2/t-ERK1/2
F
at VIVA, VA Commonwealth Univ on December 15, 2015http://www.jbc.org/Downloaded from
p-ERK1/2
t-ERK1/2
S1P - + - - + -
TCA - - + - - +
U0126
A
0.0
1.0
2.0
3.0
** ***
*** ### ###
- + - - + -
- - + - - +
- - - + + +U0126
TCA
S1P
Relative protein levels of
p-ERK1/2/t-ERK1/2
B
C
Figure 13
p-Akt
t-Akt
S1P - + - - + -
TCA - - + - - +
MK2206
0.0
1.0
2.0
3.0
*** **
###
##
- + - - + -
- - + - - +
- - - + + +
MK-2206
TCA
S1P
Relative protein levels of
p-Akt/t-Akt
D
at VIVA, VA Commonwealth Univ on December 15, 2015http://www.jbc.org/Downloaded from
0.0
1.0
2.0
3.0
4.0
**
#
- + - - + -
- - + - - +
- - - + + +
U0126
TCA
S1P
Relative protein levels
of COX-2/Actin
B
Figure 14
A
COX-2
Actin
S1P - + - - + -
TCA - - + - - +
U0126
C
0.0
1.0
2.0
3.0
4.0
*
**
#
##
- + - - + -
- - + - - +
- - - + + +
MK-2206
TCA
S1P
Relative protein levels
of COX-2/Actin
D
COX-2
Actin
S1P - + - - + -
TCA - - + - - +
MK2206
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MMPs
Pro-EGF EGF
EGFR
PLCβ
S1PR2
ERK1/2 and Akt
TCA JTE-013
Transcription
Coactivator
Activated
IKKK
Iκ-B kinase
(IKK) Activated IKK-
I-B NF-BMigration of NF-B
into nucleus
Ubiquitylation and
degradation of p-I-B
In proteasome
COX-2
Figure 16
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Zhou
Zhenzhou Jiang, Luyong Zhang and Huiping
Xiaoyan Qiang, Phillip B. Hylemon,
Runping Liu, Xiaojiaoyang Li, Lan Luo,
Cholangiocarcinoma Cell Line
Receptor 2 in a Human 1-phosphateExpression via the Sphingosine
Taurocholate Induces Cyclooxygenase-2
Gene Regulation:
published online October 30, 2015J. Biol. Chem.
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