International Journal of

Molecular Sciences

Article
TNF Increases RANKL Expression via
PGE2-Induced Activation of NFATc1
Hyun-Jung Park 1 , Kyunghwa Baek 2 , Jeong-Hwa Baek 1, * and Hyung-Ryong Kim 3, *
1 Department of Molecular Genetics, School of Dentistry and Dental Research Institute,
Seoul National University, Seoul 08826, Korea; in2010@snu.ac.kr
2 Department of Pharmacology, College of Dentistry and Research Institute of Oral Science,
Gangneung-Wonju National University, Gangwon-do 25457, Korea; daedanhae@gmail.com
3 Graduate School, DGIST, Daegu 42988, Korea
* Correspondence: baekjh@snu.ac.kr (J.-H.B.); hrkim@dgist.ac.kr (H.-R.K.);
Tel.: +82-2-880-2322 (J.-H.B.); +82-53-785-2601 (H.-R.K.); Fax: +82-53-785-2602 (H.-R.K.)

Academic Editor: Masatoshi Maki

Received: 23 December 2016; Accepted: 20 February 2017; Published: 24 February 2017

Abstract: Tumor necrosis factor (TNF) is known to upregulate the expression of receptor
activator of NF-B ligand (RANKL). We investigated the role of the calcineurin/nuclear factor
of activated T-cells (NFAT) signaling pathway in TNF-induced RANKL expression in C2C12
and primary cultured mouse calvarial cells. TNF-induced RANKL expression was blocked
by the calcineurin/NFAT pathway inhibitors. TNF increased NFAT transcriptional activity
and subsequent RANKL promoter binding. Mutations in the NFAT-binding element (MT(N))
suppressed TNF-induced RANKL promoter activity. TNF increased prostaglandin E2 (PGE2 )
production, which in turn enhanced NFAT transcriptional activity and binding to the RANKL
promoter. MT(N) suppressed PGE2 -induced RANKL promoter activity. TNF and PGE2 increased
the expression of RANKL, NFAT cytoplasmic-1 (NFATc1), cAMP response element-binding protein
(CREB), and cyclooxygenase 2 (COX2); which increment was suppressed by indomethacin, a COX
inhibitor. Mutations in the CRE-like element blocked PGE2 -induced RANKL promoter activity.
PGE2 induced the binding of CREB to the RANKL promoter, whereas TNF increased the
binding of both CREB and NFATc1 to this promoter through a process blocked by indomethacin.
The PGE2 receptor antagonists AH6809 and AH23848 blocked TNF-induced expression of RANKL,
NFATc1, and CREB; transcriptional activity of NFAT; and binding of NFATc1 or CREB to the
RANKL promoter. These results suggest that TNF-induced RANKL expression depends on PGE2
production and subsequent transcriptional activation/enhanced binding of NFATc1 and CREB to the
RANKL promoter.

Keywords: TNF; RANKL; PGE2 ; NFATc1; CREB

1. Introduction
Receptor activator of nuclear factor-B ligand (RANKL), a critical regulator of osteoclastogenesis,
is primarily produced by stromal cells or osteoblasts in bone tissue. RANKL, together with colony
stimulating factor 1, induces the differentiation of osteoclasts from hematopoietic precursors and
stimulates bone resorption of mature osteoclasts [1,2]. Various hormones and cytokines regulate
RANKL expression in osteoblast or stromal cells through the activation of intracellular signaling
pathways, including the cAMP/protein kinase A (PKA), calcineurin/nuclear factor of activated T-cells
(NFAT), hedgehog, Wnt/-catenin, and gp130/STAT pathways [38].
Tumor necrosis factor (TNF) is a multifunctional cytokine that regulates various
cellular and biological processes such as cell proliferation, differentiation, apoptosis, immunity,

Int. J. Mol. Sci. 2017, 18, 495; doi:10.3390/ijms18030495 www.mdpi.com/journal/ijms

Int. J. Mol. Sci. 2017, 18, 495 2 of 15

and inflammation [9]. TNF is known to directly induce bone resorption by activating mature
osteoclasts and stimulating the proliferation and differentiation of osteoclast precursors or indirectly
by inducing the expression of osteoclastogenic factors in stromal cells or osteoblasts [1013].
Several mechanistic pathways have been proposed to determine how TNF induces the expression
of RANKL [14,15]. For example, p38 mitogen-activated protein kinase (MAPK) pathway activation
mediates TNF-induced RANKL expression and osteoclast differentiation in precursor bone marrow
cells [16,17]. Cyclooxygenase (COX)/prostaglandin E (PGE) signaling is also considered a mechanistic
pathway by which TNF induces RANKL expression. Prostaglandin E2 (PGE2 ) belongs to the
family of prostanoid, autocrine, and paracrine lipid mediators produced by cells following injury
or cytokine or growth factor stimulation [18]. PGE2 has been described as a potent stimulator of
osteoclastic bone resorption in the context of inflammatory diseases such as rheumatoid arthritis and
osteomyelitis [1922]. PGE2 elevates RANKL expression in cultured mouse primary osteoblasts [20,23]
and human periodontal fibroblasts [24] and is known to bind to any of four G protein-coupled receptors
(EP1, EP2, EP3, or EP4) in various target cells [24,25]. COX is a prostaglandin endoperoxide synthase
that catalyzes prostaglandin synthesis. Expression of the COX isoform cyclooxygenase 2 (COX2), which
is thought to mediate inflammatory events, is rapidly induced by proinflammatory mediators [26,27].
TNF is known to induce COX2 expression and PGE2 production in human gingival fibroblasts
via activation of the NFB pathway [28]. Studies have also reported that TNF increases RANKL
expression through the COX2/PGE2 /EP4/protein kinase A (PKA) signaling pathway [12,19,29].
We previously reported that the cAMP/PKA and calcineurin/NFAT signaling pathways must
cooperate to induce parathyroid hormone-related protein (PTHrP)-induced RANKL expression in
mouse osteoblastic cells [6]. The NFAT family comprises five members: NFAT cytoplasmic-1 (NFATc1)
through NFAT5. Calcium signaling pathways dephosphorylate NFATc1 through NFATc4 via the
activated calcineurin serine or threonine phosphatase. Dephosphorylated NFATs translocate to
the nucleus and regulate the expression of target genes. The calcineurin/NFAT pathway plays
an important role in bone resorption, and NFATc1 is a particularly critical transcription factor for
osteoclast differentiation [30]. In mice, NFATc1 overexpression in osteoblasts led to increased osteoclast
generation and bone resorption [31]. However, the role of the calcineurin/NFAT signaling pathway in
TNF-induced RANKL expression remains unexplored.
In the present study, we demonstrated that TNF induces the transcriptional activation of NFAT
via PGE2 production and that activation of the calcineurin/NFAT signaling pathway is involved in the
TNF/COX2/PGE2 -mediated induction of RANKL expression.

2. Results

2.1. Calcineurin/Nuclear Factor of Activated T-Cells (NFAT) Signaling Is Involved in Tumor Necrosis Factor
(TNF)-Induced Receptor Activator of Nuclear Factor-B Ligand (RANKL) Expression
To confirm the effect of TNF on RANKL expression in C2C12 cells, cells were incubated for
0, 1, 4, 6, 12, and 24 h in the presence of TNF (10 ng/mL); subsequently, the RANKL expression
patterns were examined. TNF clearly upregulated the expression of both RANKL mRNA and protein,
and TNF-induced RANKL expression reached a peak at 24 h (Figure 1A). Therefore, we chose
an incubation period of 24 h for the following experiments.
To determine whether the calcineurin/NFAT pathway is activated by TNF, we used an NFAT
reporter assay with reporter plasmid containing an NFAT response element [32]. TNF induced
an approximately 4.5-fold increase in NFAT reporter activity (Figure 1B). Next, we explored whether the
calcineurin/NFAT pathway is involved in TNF-induced RANKL expression. C2C12 cells were treated
with TNF for 24 h in the presence or absence of the calcineurin phosphatase inhibitors FK506 and
cyclosporin A (CsA). Although FK506 did not affect the basal RANKL expression level, TNF-induced
expression of both RANKL mRNA and protein was blocked (Figure 1C). CsA also downregulated both
the basal and TNF-induced RANKL protein levels and significantly suppressed TNF-induced
RANKL mRNA expression despite increasing the basal RANKL mRNA expression (Figure 1C).
Int. J. Mol. Sci. 2017, 18, 495 3 of 14

Int. J. Mol. Sci. 2017,both

downregulated 18, 495the basal and TNF-induced RANKL protein levels and significantly suppressed
3 of 15
TNF-induced RANKL mRNA expression despite increasing the basal RANKL mRNA expression
(Figure 1C). These results indicate that calcineurin/NFAT pathway activation plays a role in TNF-
These results indicate that calcineurin/NFAT pathway activation plays a role in TNF-mediated
mediated RANKL induction in C2C12 cells.
RANKL induction in C2C12 cells.

Figure 1.1.Calcineurin/nuclear
Calcineurin/nuclearfactor factorof of
activated T-cells
activated (NFAT)
T-cells activation
(NFAT) is involved
activation in tumorinnecrosis
is involved tumor
factor
necrosis (TNF)-induced receptor activator
factor (TNF)-induced of nuclearoffactor-B
receptor activator ligand (RANKL)
nuclear factor-B expressionexpression
ligand (RANKL) in C2C12
cells. (A) TNF
in C2C12 increased
cells. (A) RANKL expression
TNF increased in a time-dependent
RANKL expression manner. C2C12
in a time-dependent manner. cellsC2C12
were incubated
cells were
in the presence of 10 ng/mL TNF for the indicated time periods and
incubated in the presence of 10 ng/mL TNF for the indicated time periods and subjected subjected to quantitative reverseto
transcription-polymerase chain reaction (RT-PCR)
quantitative reverse transcription-polymerase and
chain western(RT-PCR)
reaction blot analyses. Quantitative
and western data are
blot analyses.
presented
Quantitative as means
data are presented
standard deviations
as means (SD); (B) TNF
standard induces(SD);
deviations NFAT (B)transcriptional
TNF inducesactivity.
NFAT
C2C12 cells wereactivity.
transcriptional transfected
C2C12withcells
a reporter plasmid containing
were transfected an NFATplasmid
with a reporter responsecontaining
element, exposed
an NFAT to
TNF for 24 h, and subjected to a luciferase assay. Data are presented as firefly
response element, exposed to TNF for 24 h, and subjected to a luciferase assay. Data are presented luciferase activity levels
relative Renilla activity;
as fireflytoluciferase activity(C) Inhibition
levels relativeof to
theRenilla
calcineurin/NFAT pathway blocked
activity; (C) Inhibition TNF-mediated
of the calcineurin/NFAT
RANKL
pathwayexpression. C2C12 cells pretreated
blocked TNF-mediated with FK506
RANKL expression. (10 g/mL)
C2C12 or cyclosporin
cells pretreated with FK506 A (10
(10g/mL)
g/mL)
were treated with
or cyclosporin A (10TNF
g/mL) for were
24 h treated
and subjected
with TNF to RT-PCR
for 24 hand
andwestern
subjectedblot analyses;and
to RT-PCR (D)western
TNF
increases NFAT binding to the mouse RANKL promoter. C2C12 cells
blot analyses; (D) TNF increases NFAT binding to the mouse RANKL promoter. C2C12 cells were were incubated for 24 h with
TNF, after which a chromatin immunoprecipitation (ChIP) assay was
incubated for 24 h with TNF, after which a chromatin immunoprecipitation (ChIP) assay was performed using an antibody
against
performed NFATc1,
usingwith IgG serving
an antibody as a NFATc1,
against negative control.
with IgGThe RANKL
serving as promoter
a negativeregion control.containing
The RANKL the
NFAT
promoter binding
regionelement was amplified
containing the NFATvia PCR. Quantitative
binding element wasChIP data were
amplified normalized
via PCR. to the input
Quantitative ChIP
and
dataarewerepresented
normalizedas values
to therelative
input andto vehicle-treated
are presented control
as values samples
relative (CON) (E) TNF increased
to vehicle-treated control
RANKL promoterreporter activity in an NFAT binding element-dependent
samples (CON) (E) TNF increased RANKL promoterreporter activity in an NFAT binding manner. C2C12 cells
element-
were transfected with a wild-type (RANKL-WT-luc) or NFAT-binding
dependent manner. C2C12 cells were transfected with a wild-type (RANKL-WT-luc) or NFAT- site mutant (RANKL-MT(N)-luc)
RANKL
bindingpromoter
site mutantreporter, incubated for 24 hRANKL
(RANKL-MT(N)-luc) in the presence
promoter of TNF or NFAT
reporter, overexpression
incubated for 24 h vector,
in the
and to a luciferase assay (* p
presence of TNF or NFAT overexpression vector, and subjected to a luciferase assay (* p <to0.05,
subjected < 0.05, compared to control; # p < 0.05, compared the
indicated pair).
compared to control; # p < 0.05, compared to the indicated pair).

chromatin immunoprecipitation
Next, a chromatin immunoprecipitation (ChIP) assay was performedperformed to to investigate
investigate whether
whether
TNF-activated NFAT
TNF-activated NFAT could
coulddirectly
directlytransactivate
transactivatethe RANKL
the RANKL gene by by
gene binding to itstopromoter.
binding The
its promoter.
PCRPCR
The amplification resultresult
amplification revealed that TNF
revealed that increased the NFATc1
TNF increased binding binding
the NFATc1 to the RANKLto thepromoter
RANKL
(Figure 1D).
promoter To further
(Figure confirm
1D). To that
further the binding
confirm of NFAT
that the binding toof
theNFAT
RANKL promoter
to the RANKL was functionally
promoter was
important inimportant
functionally TNF-induced RANKL expression,
in TNF-induced RANKL luciferase
expression,(luc) reporter
luciferase assays
(luc) wereassays
reporter conducted.
were
RANKL promoterreporter
conducted. contains 2174
RANKL promoterreporter to +1
contains bp2174
of the mouse
to +1 bp ofRANKL geneRANKL
the mouse [4]. gene [4].
Int. J. Mol. Sci. 2017, 18, 495 4 of 15

Int. J. Mol. Sci. 2017, 18, 495 4 of 14

When the RANKL-WT-luc reporter was used, TNF induced an approximately three-fold increase
in luciferase
When activity; however, thereporter
the RANKL-WT-luc insertion of used,
was mutations
TNFinto the NFAT
induced binding element
an approximately (941 to
three-fold
936 bp) partially
increase inhibited
in luciferase thehowever,
activity; TNF-mediated induction
the insertion of RANKL
of mutations intopromoter
the NFATactivity
binding(Figure
element1E).
NFATc1 overexpression (positive control) resulted in an approximately two-fold
(941 to 936 bp) partially inhibited the TNF-mediated induction of RANKL promoter activityinduction that was
completely
(Figure 1E).blocked by mutations
NFATc1 in the NFAT
overexpression binding
(positive element
control) (Figure
resulted in 1E).
an These results indicate
approximately that
two-fold
TNF-activated
induction that wasNFATc1 directlyblocked
completely binds tobyand transactivates
mutations the RANKL
in the NFAT bindingpromoter.
element (Figure 1E). These
results indicate that TNF-activated NFATc1 directly binds to and transactivates the RANKL promoter.
2.2. Inhibition of Calcineurin/NFAT Signaling Partially Reduces TNF-Induced Prostaglandin E2
(PGE ) Production
2.2.2Inhibition of Calcineurin/NFAT Signaling Partially Reduces TNF-Induced Prostaglandin E2
(PGE 2) Production
Previous studies have demonstrated that TNF increases RANKL expression through the
COX2/PGE 2 /EP4/PKA
Previous signaling
studies have pathway [12,19,29]
demonstrated that TNF andincreases
that NFAT activation
RANKL enhances
expression both COX2
through the
expression and PGE
COX2/PGE2/EP4/PKA 2 production [33,34]. Therefore, we examined whether calcineurin/NFAT
signaling pathway [12,19,29] and that NFAT activation enhances both COX2 signaling
pathway activation
expression and PGE would mediate TNF-induced
2 production COX2we
[33,34]. Therefore, expression
examined andwhether
PGE2 production.
calcineurin/NFAT
In C2C12
signaling cells,activation
pathway TNF treatment increased
would mediate the expression
TNF-induced COX2levels of both
expression andCOX2 mRNA and
PGE2 production.
protein;
In these
C2C12 levels
cells,peaked at 24 h (Figure
TNF treatment 2A).the
increased Consistent
expression with thisofresult,
levels both TNF
COX2 treatment
mRNA andfor
24protein; theseinduced
h strongly levels peaked
PGE2 at 24 h (Figurewhich
production, 2A). Consistent
was blockedwithby
thisthe
result,
COXTNF treatment
inhibitor for 24 h
indomethacin
strongly
(Figure induced
2C). FK506PGE production,
did 2not which
significantly was blocked
decrease basalby the COX
COX2 mRNAinhibitor indomethacin
expression or PGE(Figure 2C).
2 production
FK5062B,C).
(Figure did notHowever,
significantly
thedecrease
additionbasal COX2resulted
of FK506 mRNA expression or PGE2 40%
in approximately production
and 20% (Figure 2B,C).in
decreases
However, theCOX2
TNF-induced addition of FK506
mRNA resulted
expression in PGE
and approximately 40% and 20% decreases in TNF-induced
2 production, respectively (Figure 2B,C). These results
COX2 mRNA expression and PGE production, respectively (Figure
suggest that the calcineurin/NFAT signaling pathway plays a non-critical
2 2B,C). These results
role insuggest that the
TNF-induced
calcineurin/NFAT
COX2/PGE signaling pathway plays a non-critical role in TNF-induced COX2/PGE2 expression.
expression.
2

Figure
Figure 2. The calcineurin/NFAT
2. The calcineurin/NFAT signaling pathway
signaling mediates
pathway TNF-induced
mediates TNF-inducedcyclooxygenase 2 (COX2)
cyclooxygenase 2
expression and prostaglandin
(COX2) expression E2 (PGE2 ) production
and prostaglandin in C2C12 cells.
E2 (PGE2) production (A) TNF
in C2C12 cells.stimulated
(A) TNFCOX2 mRNA
stimulated
and protein
COX2 mRNAexpression in a time-dependent
and protein expression in manner; (B) TNF-mediated
a time-dependent manner; (B) COX2 expression decreased
TNF-mediated COX2
following
expression inhibition
decreasedof following
the calcineurin/NFAT signaling
inhibition of the pathway. signaling
calcineurin/NFAT C2C12 cells pretreated
pathway. C2C12 with the
cells
pretreatedphosphatase
calcineurin with the calcineurin
inhibitorphosphatase inhibitor
FK506 (10 g/mL) FK506
were (10 g/mL)
incubated in thewere incubated
presence in theof
or absence
presence
TNF for 24orhabsence of TNF
and subjected tofor 24 h and
RT-PCR subjected
analyses; (C) to RT-PCR analyses;
TNF-induced PGE2(C) TNF-induced
production PGE2
was blocked
production was blocked by indomethacin, a COX inhibitor. Treatment
by indomethacin, a COX inhibitor. Treatment with FK506 slightly suppressed TNF-induced PGE2 with FK506 slightly
suppressedC2C12
production. TNF-induced PGE2 production.
cells were incubated with FK506C2C12 cells were(20incubated
or indomethacin M) in the with FK506
presence or
of TNF
forindomethacin (20 M)
24 h and subjected to in the 2presence
a PGE parameterof TNF forp 24
assay (* h and
< 0.05, subjectedtotocontrol;
compared a PGE2#parameter
p < 0.05). assay
(* p < 0.05, compared to control; # p < 0.05).
Int. J. Mol. Sci. 2017, 18, 495 5 of 15
Int. J. Mol. Sci. 2017, 18, 495 5 of 14

2.3.
2.3. PGE
PGE22 Enhances
Enhances RANKL
RANKL Promoter
Promoter Activity
Activity in
in an
an NFAT Binding Element-Dependent
NFAT Binding Element-Dependent Manner
Manner
We next investigated
investigated whether
whether calcineurin/NFAT
calcineurin/NFAT signaling
signaling plays
plays aa role
role in
in PGE
PGE2-induced RANKL
We next 2 -induced RANKL
expression. When C2C12 cells were incubated in the presence of PGE 2 (50 nM) for 1, 4, 6, 12, and 24 h,
expression. When C2C12 cells were incubated in the presence of PGE2 (50 nM) for 1, 4, 6, 12,
the
and levels of levels
24 h, the RANKL of mRNA
RANKL and mRNA protein were slightly
and protein decreased
were slightly at an at
decreased early time time
an early point,point,
but
remarkably
but remarkably increased in a intime-dependent
increased a time-dependent manner afterafter
manner 6 h 6(Figure 3A).3A).
h (Figure PGE 2 also induced an
PGE 2 also induced
approximately three-fold increase in NFAT-luc reporter activity (Figure 3B).
an approximately three-fold increase in NFAT-luc reporter activity (Figure 3B). In addition,In addition, a ChIPa assay
ChIP
with an NFATc1 antibody revealed that PGE 2 enhanced NFATc1 binding to the RANKL promoter
assay with an NFATc1 antibody revealed that PGE2 enhanced NFATc1 binding to the RANKL promoter
(Figure 3C). Furthermore,
Furthermore, PGEPGE2 significantly increased luciferase activity when the RANKL-WT-luc
(Figure 3C). 2 significantly increased luciferase activity when the RANKL-WT-luc
reporter, but not when the RANKL-MT(N)-luc,
reporter, but not when the RANKL-MT(N)-luc, was was used
used (Figure
(Figure 3D).
3D). These
These results
results indicate
indicate that
that PGE
PGE2
2
activates
activates the calcineurin/NFAT signaling
the calcineurin/NFAT signaling pathway
pathway and and that
that PGE
PGE22-induced
-induced NFATNFAT directly activates
directly activates
RANKL transcription by
RANKL transcription by binding
binding to to the
the RANKL
RANKL promoter.
promoter.

Figure 3.
Figure PGE22 stimulates
3. PGE RANKL expression
stimulates RANKL expression by by activating
activating the
the calcineurin/NFAT pathway in
calcineurin/NFAT pathway in C2C12
C2C12
cells. (A) PGE increased RANKL expression in a time-dependent manner. C2C12
cells. (A) PGE22increased RANKL expression in a time-dependent manner. C2C12 cells were incubated cells were incubated
in the
in the presence
presenceof ofPGE
PGE (50
(50
22 nM)
nM) forfor
thethe indicated
indicated time
time periods,
periods, followed
followed by quantitative
by quantitative RT-PCRRT-PCRand
and western
western blot analyses;
blot analyses; (B) PGE (B) PGE2 increased
2 increased NFAT transcriptional
NFAT transcriptional activity.
activity. C2C12 cellsC2C12 cells were
were transfected
transfected
with withplasmid
a reporter a reporter plasmid an
containing containing an NFATelement,
NFAT response responsetreated
element,
withtreated
PGE2 with
for 24PGE for
h, 2and
24 h, and subjected to a luciferase assay; (C) PGE induced NFAT binding
subjected to a luciferase assay; (C) PGE2 induced NFAT binding to the mouse RANKL promoter.
2 to the mouse RANKL
promoter.
C2C12 cellsC2C12
were cells were
treated treated
with PGE2with for 24PGE 2 fora24
h and h and
ChIP a ChIP
assay wasassay was performed;
performed; (D) PGE2
(D) PGE2 increased
RANKL
RANKL promoter-reporter activity in an NFAT binding element-dependent manner. C2C12 cellsC2C12
increased promoter-reporter activity in an NFAT binding element-dependent manner. were
cells were transfected
transfected with RANKL-WT-luc
with RANKL-WT-luc or RANKL-MT(N)-luc,
or RANKL-MT(N)-luc, incubated incubated
for 24 hfor 24 hpresence
in the in the presence
of PGEof 2,
PGE 2 , and subjected to a luciferase assay (* p < 0.05, compared to
and subjected to a luciferase assay (* p < 0.05, compared to control; # p < 0.05).control; # p < 0.05).

2.4. Cyclooxygenase
2.4. Cyclooxygenase (COX)
(COX) Inhibitor
Inhibitor Blocks
Blocks TNF-Induced
TNF-Induced Binding
Binding of
of NFATc1
NFATc1and
andcAMP
cAMPResponse
Response
Element-Binding Protein
Element-Binding Protein (CREB)
(CREB) to
to the
the RANKL
RANKL Promoter
Promoter
Significant increases
Significant increases inin the
the mRNA
mRNA and and protein
protein expression
expression of RANKL, NFATc1,
of RANKL, NFATc1, cAMP response
cAMP response
element-binding protein
element-binding protein (CREB),
(CREB), andand COX2
COX2 were
were observed
observed in in C2C12
C2C12 cells
cells incubated
incubated with
with PGE for
PGE22 for
24 h (Figure 4A). TNF induced similar levels of gene expression, and this process
24 h (Figure 4A). TNF induced similar levels of gene expression, and this process was inhibited by was inhibited by
indomethacin (Figure 4A). Further treatment with PGE in the presence of
indomethacin (Figure 4A). Further treatment with PGE22 in the presence of indomethacin and TNFindomethacin and TNF
partially rescued
partially rescued the the expression
expression of RANKL, NFATc1,
of RANKL, NFATc1, CREB,
CREB, and and COX2.
COX2. These
These results
results suggest that
suggest that
COX/PGE mediates the TNF-induced expression of RANKL,
COX/PGE22mediates the TNF-induced expression of RANKL, NFATc1, and CREB. NFATc1, and CREB.
We previously
We previously demonstrated
demonstrated that that PTHrP-induced
PTHrP-induced cAMP/PKA signaling promoted
cAMP/PKA signaling promoted NFATNFAT
transcriptional activity
transcriptional activityand
andthat NFAT
that NFAT andandCREB cooperate
CREB to transactivate
cooperate the gene
to transactivate theencoding RANKL
gene encoding
in mice [6]. Because our above data demonstrate that PGE enhances the
RANKL in mice [6]. Because our above data demonstrate2 that PGE2 enhances the transcriptional transcriptional activity of
NFAT and
activity its binding
of NFAT and to
its the RANKL
binding to promoter,
the RANKL wepromoter,
next investigated
we next whether PGEwhether
investigated 2 , produced
PGEin2,
response to TNF, would enhance the binding of CREB to the RANKL
produced in response to TNF, would enhance the binding of CREB to the RANKL promoter and promoter and subsequent
transactivation.
subsequent The RANKL The
transactivation. promoterreporter assay demonstrated
RANKL promoterreporter that mutationsthat
assay demonstrated inserted in the
mutations
CRE-likeinelement
inserted (1093
the CRE-like to 1086
element bp) prevented
(1093 to 1086 bp)PGE 2 from enhancing
prevented PGE2 fromreporter
enhancingactivity (Figure
reporter 4B).
activity
In addition, a ChIP assay with a CREB antibody revealed that PGE enhanced the
(Figure 4B). In addition, a ChIP assay with a CREB antibody revealed2 that PGE2 enhanced the binding binding of CREB
to the
of CREB RANKL
to thepromoter (Figure 4C).
RANKL promoter Consistent
(Figure with thesewith
4C). Consistent results, TNF
these significantly
results, increased
TNF significantly
the binding
increased theof binding
CREB and NFATc1and
of CREB to the RANKL
NFATc1 to promoter
the RANKL in apromoter
process blocked by indomethacin
in a process blocked by
Int. J.J. Mol.
Int. Mol. Sci.
Sci.2017, 18,495
2017,18, 495 66 of
of 14
15

indomethacin (Figure 4D). These results suggest that TNF-induced COX/PGE2 increases the binding
(Figure 4D). These results suggest that TNF-induced COX/PGE2 increases the binding of both CREB
of both CREB and NFAT to the RANKL promoter and transcriptional activation of the RANKL gene.
and NFAT to the RANKL promoter and transcriptional activation of the RANKL gene.

Figure
Figure 4. The
The COX
COX inhibitor
inhibitor indomethacin
indomethacin blocks blocks TNF-induced
TNF-inducedbinding bindingof ofNFATc1
NFATc1 and and cAMP
cAMP
response
response element-binding
element-bindingprotein protein (CREB)
(CREB) to the
to RANKL
the RANKL promoter in C2C12
promoter cells. (A)
in C2C12 cells. increased
PGE2 (A) PGE2
the expression of RANKL, NFATc1, CREB, and COX2, whereas indomethacin
increased the expression of RANKL, NFATc1, CREB, and COX2, whereas indomethacin suppressed suppressed TNF-
mediated RANKL,
TNF-mediated NFATc1,
RANKL, CREB,CREB,
NFATc1, and COX2and COX2 expression. Treatment
expression. Treatmentwithwith
PGEPGE2 in 2the presence
in the presenceof
indomethacin
of indomethacin andandTNFTNF partially
partially rescued
rescued thethe expression
expression ofofRANKL,
RANKL,NFATc1,NFATc1,CREB,CREB,and and COX2.
COX2.
C2C12
C2C12 cells
cellswere
wereincubated
incubated with
withthetheindicated
indicatedreagents for 24for
reagents h and
24 h subjected to RT-PCR
and subjected and western
to RT-PCR and
blot analyses;
western (B) PGE2 (B)
blot analyses; increased RANKL promoterreporter
PGE2 increased RANKL promoterreporter activity inactivity
a CREBinbinding
a CREBelement-
binding
dependent manner. manner.
element-dependent C2C12 cellsC2C12were transfected
cells with RANKL-WT-luc
were transfected with RANKL-WT-luc or a CREB-binding site mutant
or a CREB-binding site
mutant (RANKL-MT(C)-luc)
(RANKL-MT(C)-luc) RANKLRANKL promoter, promoter,
incubated incubated
for 24 for
h in24the
h in the presence
presence of PGEof 2PGE
, and2 ,subjected
and subjected
to a
to a luciferase
luciferase assay;assay;
(C) PGE (C)2 induced
PGE2 induced CREB binding
CREB binding to theRANKL
to the mouse RANKL promoter.
mousepromoter. C2C12 cellsC2C12were
cells were for
incubated incubated
24 h with forPGE
24 h2 with PGE2 andtosubjected
and subjected to a ChIP
a ChIP assay assay with
with CREB CREB and
and control IgG control IgG
antibodies.
antibodies. The RANKL promoter region containing the CREB-binding
The RANKL promoter region containing the CREB-binding element was then amplified; (D) element was then amplified;
(D) Indomethacin
Indomethacin prevented
prevented TNF-induced
TNF-induced NFAT NFAT andand CREB
CREB binding
binding toto themouse
the mouseRANKLRANKLpromoter.
promoter.
C2C12 cells were
C2C12 wereincubated
incubatedfor for2424h hwith
with thethe
indicated
indicated reagents
reagentsandandsubjected
subjectedto a to
ChIP (* p <(*0.05,
assayassay
a ChIP p<
compared
0.05, to control;
compared # p < #0.05).
to control; p < 0.05).

2.5. The
2.5. The PGE
PGE22Receptor
ReceptorAntagonists
AntagonistsAH6809
AH6809and
and AH23848
AH23848 Blocked
Blocked TNF-Induced
TNF-Induced Activation
Activation of NFAT and
Binding of NFATc1 and CREB to the RANKL Promoter
and Binding of NFATc1 and CREB to the RANKL Promoter
We next
We next investigated
investigated whether
whether PGE receptor antagonists
PGE22 receptor antagonists wouldwould inhibit
inhibit TNF-induced
TNF-induced RANKL RANKL
expression and NFAT activation. AH6809 has the highest affinity for the EP2
expression and NFAT activation. AH6809 has the highest affinity for the EP2 receptor, but also actsreceptor, but also acts
as aa weak
as weak antagonist
antagonist against
against EP1
EP1 and
and prostaglandin
prostaglandin D2 D2 receptor
receptor 11(DP1) (DP1)receptors
receptorsininthe
themouse
mouse[35].
[35].
AH23848 is a dual antagonist of the EP4 and thromboxane (TP1) receptors [36,37].
AH23848 is a dual antagonist of the EP4 and thromboxane (TP1) receptors [36,37]. C2C12 cells were C2C12 cells were
incubated with
incubated with AH6809
AH6809 (5 M) in
(5 M) inthe
thepresence
presenceofofTNFTNFfor for1,1,4,4,6,6,12,
12,and
and2424
h,h,
and
andthen
thenthethe
levels of
levels
RANKL mRNA and protein were examined. AH6809 did not significantly
of RANKL mRNA and protein were examined. AH6809 did not significantly suppress TNF-induced suppress TNF-induced
RANKL expression
RANKL expression atat an
anearly
earlytime
timepoint
point(6(6hhororearlier),
earlier),but
butinhibited
inhibitedthe theTNF-mediated
TNF-mediated induction
induction of
RANKL
of RANKL thereafter (Figure
thereafter 5A).
(Figure Incubation
5A). Incubationfor for
24 h
24inh the presence
in the presence of AH6809
of AH6809 or AH23848
or AH23848 blocked the
blocked
TNF-mediated induction of RANKL, NFATc1, and CREB mRNA expression
the TNF-mediated induction of RANKL, NFATc1, and CREB mRNA expression in C2C12 cells in C2C12 cells (Figure 5B).
Compared
(Figure with
5B). NFATc1 mRNA
Compared expression,
with NFATc1 mRNATNF-induced
expression, NFAT protein levelsNFAT
TNF-induced decreased moderately
protein levels
in the presence of AH6809 or AH23848 (Figure 5B). However, TNF-induced
decreased moderately in the presence of AH6809 or AH23848 (Figure 5B). However, TNF-induced NFAT transcriptional
NFAT transcriptional activity was abolished by the addition of AH6809 or AH23848 (Figure 5C).
Int. J. Mol. Sci. 2017, 18, 495 7 of 15

Int. J. Mol. Sci. 2017, 18, 495 7 of 14

activity was abolished by the addition of AH6809 or AH23848 (Figure 5C). Furthermore, ChIP
assays with antibodies
Furthermore, to NFATc1
ChIP assays or CREB demonstrated
with antibodies to NFATc1 orthat PGEdemonstrated
CREB 2 receptor antagonists
that PGEsuppressed
2 receptor

TNF-induced binding of
antagonists suppressed NFATc1 or CREB
TNF-induced to theofRANKL
binding NFATc1 promoter
or CREB (Figure
to the 5D). These
RANKL results indicate
promoter (Figure
that
5D). TNF-induced RANKL expression
These results indicate depends onRANKL
that TNF-induced the production of PGE
expression ,
depends
2 which
onsubsequently
the productionbinds
of
to EP2 or EP4 receptor and enhances the binding and transcriptional activity of NFATc1 and
PGE2, which subsequently binds to EP2 or EP4 receptor and enhances the binding and transcriptional CREB in
the mouse
activity RANKL promoter.
of NFATc1 and CREB in the mouse RANKL promoter.

Figure 5.
Figure 5. The
The PGE
PGE22receptor
receptorantagonists
antagonists AH6809
AH6809 and
and AH23848
AH23848 suppress
suppress the
the TNF-induced
TNF-induced activation
activation
of NFAT and binding of NFATc1 and CREB to the RANKL promoter in C2C12 cells. (A) PGE22 receptor
of NFAT and binding of NFATc1 and CREB to the RANKL promoter in C2C12 cells. (A) PGE receptor
antagonists significantly
antagonists significantly inhibited
inhibited TNF
TNF induction
induction of
of RANKL
RANKL onlyonly after
after incubation
incubation forfor 12
12 and
and 24
24 h;
h;
(B) AH6809 and AH23848 blocked TNF-mediated RANKL, NFATc1, and
(B) AH6809 and AH23848 blocked TNF-mediated RANKL, NFATc1, and CREB expression. AH6809 CREB expression. AH6809
and AH23848
and AH23848 partially
partially suppressed
suppressed TNF-induced
TNF-induced NFATc1
NFATc1 protein
protein expression.
expression. C2C12 cells were
C2C12 cells were
incubated with
incubated with AH6809
AH6809(5(5M) M)ororAH23848
AH23848(5(5M)M)inin
thethe
presence
presenceor or
absence
absenceof TNF
of TNFfor 24
forh;24(C)
h;
PGE receptor antagonists abolished TNF-induced NFAT transcriptional activity;
(C) PGE2 receptor antagonists abolished TNF-induced NFAT transcriptional activity; (D) PGE2
2 (D) PGE 2 receptor
antagonists
receptor prevented
antagonists TNF-induced
prevented TNF-induced NFATNFATand CREB
and CREB binding to to
binding thethemouse
mouseRANKLRANKL promoter
promoter
(* pp <
(* < 0.05,
0.05, compared
compared to to control;
control; ## pp <
< 0.05).
0.05).

2.6. TNF
2.6. Enhances RANKL
TNF Enhances RANKL Expression
Expression in
in aa PGE
PGE2 Production-
Production- and
and NFAT
NFATActivation-Dependent
Activation-DependentManner
Mannerin
2
in Primary Cultured Mouse Calvarial Cells
Primary Cultured Mouse Calvarial Cells
We next
next confirmed
confirmedthe
theroles of of
roles PGE 2 and and
PGE 2 calcineurin/NFAT signaling
calcineurin/NFAT with regard
signaling to TNF-
with regard to
regulated RANKLRANKL
TNF-regulated expression in primary
expression culturedcultured
in primary mouse calvarial (MC) cells.
mouse calvarial (MC)Consistent with our
cells. Consistent
observations
with in C2C12 in
our observations cells, TNF
C2C12 induced
cells, TNFboth RANKL
induced and
both COX2 and
RANKL mRNACOX2andmRNA
proteinand
expression
protein
(Figure 6A). PCR amplification of the DNA region containing the NFAT binding element or CRE-like
region revealed that TNF enhanced the binding of NFATc1 or CREB to the RANKL promoter
(Figure 6B). FK506-mediated inhibition of NFAT activation significantly reduced TNF-induced
Int. J. Mol. Sci. 2017, 18, 495 8 of 15

expression (Figure 6A). PCR amplification of the DNA region containing the NFAT binding
element or 2017,
Int. J. Mol. Sci. CRE-like
18, 495region revealed that TNF enhanced the binding of NFATc1 or CREB to the
8 of 14
RANKL promoter (Figure 6B). FK506-mediated inhibition of NFAT activation significantly reduced
RANKL expression
TNF-induced RANKL (Figure 6C). Indomethacin,
expression (Figure 6C). AH6809, and AH23848
Indomethacin, AH6809,also
andinhibited
AH23848TNF-induced
also inhibited
RANKL mRNA RANKL
TNF-induced expressionmRNA(Figure 6D). These
expression results
(Figure further
6D). Thesesuggest
results that PGEsuggest
further 2 mediates
thatTNF-
PGE2
induced RANKL
mediates expression
TNF-induced through
RANKL a processthrough
expression that involves enhanced
a process bindingenhanced
that involves of NFAT binding
and CREB of
to the RANKL
NFAT and CREB promoter.
to the RANKL promoter.

Figure 6.
Figure 6. PGE
PGE22 production
production andand NFAT activation are
NFAT activation are necessary
necessary forfor TNF-induced
TNF-induced RANKL
RANKL expression
expression
in primary cultured mouse calvarial cells. (A) TNF enhanced
enhanced the the expression
expression of
of RANKL
RANKL and and COX2.
COX2.
indicated time periods and subjected
Calvarial cells were incubated in the presence of TNF for the indicated
to RT-PCR and western blot analyses; (B) ChIP assays revealedrevealed thatthat TNF
TNF increased
increased NFATc1
NFATc1 and
CREB binding to their cognate binding sites in the mouse RANKL promoter; promoter; (C)
(C) A calcineurin/NFAT
signaling inhibitor
inhibitor suppressed
suppressedTNF-induced
TNF-inducedRANKLRANKLexpression.
expression.Calvarial cells
Calvarial were
cells incubated
were incubated for
24 h24inh the
for presence
in the presenceof of
FK506
FK506and
andTNF;
TNF;(D)(D)Treatment
Treatmentwith
withaa COX2
COX2 inhibitor
inhibitor or PGE22 receptor
antagonists suppressed
antagonists suppressedTNF-induced
TNF-inducedRANKL
RANKLexpression.
expression.(*(*pp<< 0.05,
0.05, compared
comparedto to control;
control;## pp << 0.05).

3. Discussion
study, we
In the present study, we demonstrated
demonstrated that that TNF-induced
TNF-induced RANKLRANKL expression involves both
PGE22-induced
-induced activation
activation of of the
the calcineurin/NFAT
calcineurin/NFATpathway pathwayand and subsequent
subsequentbinding
bindingof of NFATc1
NFATc1 to
the RANKL promoter. The following results of our our study
study support
support the
the involvement
involvement of of PGE
PGE22 and the
signalingpathway
calcineurin/NFAT signaling pathway in in TNF-induced
TNF-induced RANKL
RANKL expression:
expression: (i) TNF
(i) TNF stimulated
stimulated COX2
COX2 expression
expression and PGE and PGE2 production;
2 production; (ii) a COX(ii) inhibitor
a COX inhibitor or EP antagonists
or EP antagonists blockedblocked TNF-induced
TNF-induced RANKL
RANKL expression;
expression; (iii) TNF-induced
(iii) TNF-induced RANKL expression
RANKL expression was significantly
was significantly downregulated downregulated by
by inhibition
inhibition of the calcineurin/NFAT pathway; (iv) TNF enhanced the transcriptional
of the calcineurin/NFAT pathway; (iv) TNF enhanced the transcriptional activity of NFATc1 and activity of
NFATc1 of
binding and binding
NFATc1 of NFATc1
to the to the RANKL
RANKL promoter; promoter; (v) RANKL
(v) TNF-induced TNF-induced RANKL promoter
promoterreporter activity
reporter
was activityby
attenuated was attenuated
mutations by NFAT
in the mutations in the
binding NFAT and
element; binding
(vi) aelement; and (vi)oraEP
COX inhibitor COX inhibitor
antagonists
or EP antagonists
blocked TNF-enhancedblocked TNF-enhanced
transcriptional transcriptional
activity and binding of activity
NFAT and binding
to the RANKLofpromoter.
NFAT to the
RANKL promoter.
It has been well demonstrated that RANKL expression increases with TNF treatment in
It has been wellmanner
a dose-dependent demonstrated
and 10 thatng/mL RANKL expression
of TNF increases
was used as a with TNF treatment
representative dose inin several
a dose-
dependent
studies manner
[3843]. and 10 ng/mL
Therefore, of TNF
we adopted was used as aof
a concentration representative
10 ng/mL indose in several
the present studies [3843].
study.
Therefore, we adopted a concentration of 10 ng/mL in the present study.
The ability of TNF to stimulate PGE2 production via COX2 has been well demonstrated [28,4446],
and PGE2 is known to activate the cAMP/PKA signaling pathway in target cells [19]. Of the PGE2
receptor subtypes, EP4 has the highest expression in fibroblasts. Compared to control mice, EP4-
deficient mice exhibited reduced inflammatory responses in a collagen-induced arthritis model,
Int. J. Mol. Sci. 2017, 18, 495 9 of 15

The ability of TNF to stimulate PGE2 production via COX2 has been well
demonstrated [28,4446], and PGE2 is known to activate the cAMP/PKA signaling pathway
in target cells [19]. Of the PGE2 receptor subtypes, EP4 has the highest expression in fibroblasts.
Compared to control mice, EP4-deficient mice exhibited reduced inflammatory responses in
a collagen-induced arthritis model, fewer osteoclasts, and reduced RANKL expression in osteoblast
cells [20,4749]. These reports suggest that EP4 is critical to the stimulation of bone resorption.
Another report demonstrated that in MC osteoblastic cells, PGE2 -induced cAMP production and
RANKL expression are mainly mediated by EP4, although EP4 and EP2 must cooperate to ensure
a full response to PGE2 [23]. In the present study, either an EP2 or EP4 antagonist completely blocked
TNF-induced RANKL expression. In addition, both antagonists abolished TNF-induced NFAT
reporter activity and the binding of NFATc1 and CREB to the RANKL promoter in C2C12 cells.
In primary cultured MC cells, both antagonists had similar partial inhibitory effects on TNF-induced
RANKL expression. These results suggest that both EP2 and EP4 are involved in PGE2 -induced
RANKL expression. However, it is not clear why the responses of primary MC cells and C2C12 cells to
EP antagonists differ.
In the present study, we did not examine the receptor subtypes involved in TNF-induction of
COX2 and PGE2 production. TNF regulates cellular activities via the activation of either of two
receptors: TNF receptor 1 (TNFR1) and TNFR2. TNFR1 is expressed ubiquitously, whereas TNFR2
expression is restricted to specific cell types, including immune cells and neurons [50]. Previous reports
have demonstrated that TNF induces COX2 expression and PGE2 production via TNFR1 in synovial
fibroblasts [51,52]. Furthermore, induction of RANKL by TNF was abolished in TNFR1-deficient
mouse gingival epithelial cells [29]. These studies suggest that the activation of TNFR1 contributes to
TNF-induced activation of COX2 and production of PGE2 in C2C12 and mouse calvarial cells.
Although the PGE2 -induced cAMP/PKA signaling is known to induce RANKL expression,
the effector molecules downstream of PKA have not yet been clearly elucidated. Previously, PKA was
shown to target CREB and thus increase the extent of CREB binding to distal enhancers of RANKL [53].
Assuming that PGE2 /PKA signaling mediates TNF-induced RANKL expression, we propose NFAT
to be another transcription factor that acts downstream of PGE2 to induce RANKL transcription.
Similar to a previous report in which CREB and NFATc1 cooperation was necessary to induce
RANKL transcription via the PTHrP/cAMP/PKA pathway [6], TNF-induced PGE2 enhanced the
binding of CREB and NFATc1 to the mouse RANKL promoter, and mutations in either NFAT-binding
element or the CRE-like element blocked PGE2 -induced RANKL promoter activity. In addition,
indomethacin and EP antagonists prevented TNF-induced binding of NFATc1 and CREB to the
RANKL promoter. These data support a role for NFATc1 as another mediator linking PGE2 /PKA
signaling to RANKL transcription.
A rich body of literature demonstrates that NFAT activation directly regulates COX expression
and PGE2 production. Notably, NFAT activation regulates COX2-encoding genes in human colon
carcinoma [33]. In this study, COX2 expression and PGE2 production were induced in response to
NFAT stimuli and blocked when CsA or FK506 was used to inhibit calcineurin phosphatase activity.
Lipopolysaccharide-induced NFAT activation was shown to regulate PGE2 synthesis in dendritic
cells [34], and NFAT was found to induce COX2 transcription in human glioblastoma cells [54] and
to regulate constitutive COX2 expression in the renal medulla [55]. Furthermore, PTH was shown
to induce COX2 transcription through cross-talk between the cAMP/PKA and calcineurin/NFAT
signaling pathways in murine osteoblastic cells [56]. Therefore, we determined whether inhibition
of the calcineurin/NFAT pathway would suppress COX2-induced PGE2 production. FK506 partially
suppressed TNF-induced COX2 expression and PGE2 production. However, functional PGE2
inhibition by EP antagonists completely abolished the TNF-induced transcriptional activity of NFAT,
indicating that TNF-induced NFAT activation is PGE2 -dependent and subsequently contributes to
the further induction of COX2 expression.
Int. J. Mol. Sci. 2017, 18, 495 10 of 15

In the present study, TNF-induced COX2 expression was obvious only after incubation for 4 h,
although TNF-induced RANKL expression was significant even after 1 h-incubation in C2C12 cells.
In addition, PGE2 antagonist AH6809 did not significantly suppress TNF-induced RANKL expression
at an early time point (6 h or earlier). Given the data demonstrating the difference between the
early vs. late response, it is suggested that there is a PGE2 -independent pathway in RANKL induction
of TNF in the immediate response. Considering the continuing effect of TNF and PGE2 on RANKL
induction after 12 h, PGE2 seems to mediate the slow, delayed, long-term RANKL expression by TNF,
which is more likely to be associated with pathologic conditions such as inflammatory bone resorption.
In conclusion, the present study demonstrates that TNF-induced RANKL expression depends
on PGE2 production and subsequent transcriptional activation, as well as on the enhanced binding of
NFATc1 and CREB to the RANKL promoter in mouse osteoblastic cells.

4. Materials and Methods

4.1. Reagents and Antibodies

Recombinant human TNF was purchased from R&D Systems (Minneapolis, MN, USA). FK506,
cyclosporin A, SB203580, H89, PGE2 , and indomethacin were purchased from Sigma (St. Louis,
MO, USA). AH6809 and AH23848 were purchased from Cayman Chemical (Ann Arbor, MI, USA).
RANKL antibody was purchased from R&D Systems. Antibodies to NFATc1, COX2, and -actin
and horseradish peroxidase (HRP)-conjugated secondary antibodies were obtained from Santa Cruz
Biotechnology (Dallas, TX, USA). CREB antibody was purchased from Cell Signaling Technology
(Danvers, MA, USA).

4.2. Cell Culture

C2C12 cells, a murine mesenchymal cell line that can be differentiated into osteoblasts [57],
were maintained in Dulbeccos modified Eagles medium (DMEM) supplemented with 10% fetal
bovine serum (FBS), 100 U/mL of penicillin, and 100 g/mL of streptomycin.
MC cells were isolated from the frontal and parietal bones of neonatal ICR mice, as previously
described [58]. Before preparation of MC cells, animals were weaned in mouse gang cages following
Institutional Animal Care and Use Committee policies. The animal study was reviewed and approved
by the Special Committee on Animal Welfare, Seoul National University, Seoul, Republic of Korea
(approval no. SNU-20140228-1-5). MC cells were cultured in -minimum essential medium (-MEM)
supplemented with 10% FBS, 100 U/mL of penicillin, and 100 g/mL of streptomycin. DMEM,
-MEM, and FBS were obtained from Hyclone (Walkersville, MD, USA).

4.3. Plasmid Construction

Construction of the NFATc1 expression vector and a NFAT reporter plasmid has been described
in previous publications [32,59]. Construction of a RANKL promoter-luciferase reporter plasmid
(RANKL-WT: 2174 to +1 bp of the mouse RANKL promoter) and a function-defective mutant reporter
containing mutations in the NFAT-binding site (RANKL-MT-N; GGAAAAGCttAA) [4] or in the
CRE-like element (RANKL-MT-C; TGAGGTCATGAGGagg) have been described in previous
publications [6,60,61].

4.4. Reverse Transcription-Polymerase Chain Reaction

Quantitative reverse transcription-polymerase chain reaction (RT-PCR) was performed as
described previously [6]. PCR primers with the following sequences were synthesized by
Cosmogenetech (Seoul, Korea): RANKL-forward (F) 50 -CAG AAG ATG GCA CTC ACT GCA-30 ,
RANKL-reverse (R) 50 -CAC CAT CGC TTT CTC TGC TCT-30 ; NFATc1-F 50 -AAT AAC ATG CGA
GCC ATC ATC-30 , NFATc1-R 50 -TCA CCC TGG TGT TCT TCC TC-30 ; CREB-F 50 -AGC TGC CAC
TCA GCC GGG TA-30 , CREB-R 50 -TGG TGC TCG TGG GTG CTG TG-30 ; COX2-F 50 -CCA GCA
Int. J. Mol. Sci. 2017, 18, 495 11 of 15

CTT CAC CCA TCA GTT-30 ; COX2-R 50 -ACC CAG GTC CTC GCT TAT GA-30 ; glyceraldehyde
3-phosphate dehydrogenase (GAPDH)-F 50 -TCA ATG ACA ACT TTG TCA AGC-30 ; and GAPDH-R
50 -CCA GGG TTT CTT ACT CCT TGG-30 . GAPDH was used as a reference to normalize each sample
for quantification.

4.5. Western Blot Analysis

The western blot analysis was performed as described previously [6]. Immune complexes were
visualized using EZ-Western Lumi Pico (Daeillab Service Co., Seoul, Korea), and chemiluminescence
was detected using a MicroChemi device (DNR, Jerusalem, Israel).

4.6. Chromatin Immunoprecipitation Assay

The chromatin immunoprecipitation (ChIP) assay was performed as described previously [4].
Cross-linked DNA fragments were subjected to pre-clearing with blocked protein G agarose,
and immunoprecipitation was performed with an NFATc1 or CREB antibody or species-matched
control IgG. After purification, DNA sequences were analyzed by PCR amplification of the mouse
RANKL promoter region encompassing either the NFAT-binding element (amplified region: 1070 to
858 bp) or CRE-like element (amplified region: 1147 to 967 bp). PCR primers with the following
sequences were used: NFAT-binding element forward, 50 -GCA AGC TCC AGG CCA GCC TAG-30
and reverse, 50 -CCA ATA AGA CGG CTC AGC TG-30 ; CRE-like element forward, 50 -AGG AGG CAG
AGA TGG CAG AG-30 and reverse, 50 -ACA CGC GCG CGC GCA AAT A-30 .

4.7. Luciferase Reporter Assay

The luciferase reporter assay was performed as described previously [6]. In order to normalize
transfection efficiency, Renilla luciferase plasmid was co-transfected with RANKL promoter reporters.

4.8. PGE2 Assay

Cells were incubated for 24 h in medium supplemented with the indicated reagents, after which
supernatants were collected. The concentration of PGE2 in the supernatant was determined using
Parameter PGE2 (R&D Systems) according to the manufacturers instructions.

4.9. Statistical Analysis

Statistical significance was determined using Students t-test. For the multiple comparisons,
one way ANOVA was performed. When significant main effects were detected, post hoc analyses were
conducted with the least squares means error test. Differences were considered significant at p < 0.05.
Data were analyzed by using the SAS program (version 9.1; SAS Institute, Cary, NC, USA).

Acknowledgments: This research was supported by the Basic Science Research Program through the
National Research Foundation of Korea (NRF), funded by the Ministry of Education, Science, and Technology
(NRF-2012R1A1A2002638).
Author Contributions: Jeong-Hwa Baek and Hyung-Ryong Kim conceived and designed the experiments;
Hyun-Jung Park performed the experiments; Hyun-Jung Park, Kyunghwa Baek, and Jeong-Hwa Baek
analyzed the data; Hyun-Jung Park contributed reagents, materials, and analysis tools; Hyun-Jung Park and
Kyunghwa Baek wrote the paper; Kyunghwa Baek and Jeong-Hwa Baek revised the manuscript contents;
and Jeong-Hwa Baek and Hyung-Ryong Kim approved the final version.
Conflicts of Interest: The authors declare no conflict of interest.

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