Inhibition of receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast formation by pyrroloquinoline quinine (PQQ).

Abstract

The effect of pyrroloquinoline quinine (PQQ) on receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast formation was examined using RAW 264.7 macrophage-like cells. RANKL led to the formation of osteoclasts identified as tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells in the culture of RAW 264.7 cells. However, PQQ inhibited the appearance of osteoclasts and prevented the decrease of F4/80 macrophage maturation marker on RANKL-stimulated cells, suggesting a preventive action of PQQ on RANKL-induced osteoclast differentiation. PQQ inhibited the activation of nuclear factor of activated T cells (NFATc1), a key transcription factor of osteoclastogenesis, in RANKL-stimulated cells. On the other hand, PQQ did not inhibit the signaling pathway from RANK/RANKL binding to NFATc1 activation, including NF-κB and mitogen-activated protein kinases (MAPKs). PQQ augmented the expression of type I interferon receptor (IFNAR) and enhanced the IFN-β-mediated janus kinase (JAK1) and signal transducer and activator of transcription (STAT1) expression. Moreover, PQQ reduced the expression level of c-Fos leading to the activation of NFATc1. Taken together, PQQ was suggested to prevent RANKL-induced osteoclast formation via the inactivation of NFATc1 by reduced c-Fos expression. The reduced c-Fos expression might be mediated by the enhanced IFN-β signaling due to augmented IFNAR expression.

Full text

Page 1

Immunology
Letters
142 (2012) 34–
40
Contents
lists
available
at SciVerse
ScienceDirect
Immunology
Letters
j ourna
l h o me
page:
www.elsevier.com/locate/immlet
Inhibition
osteoclast
of
receptor
activator
of
nuclear
factor-?B
ligand
(RANKL)-induced
formation
by
pyrroloquinoline
quinine
(PQQ)
Erdenezaya
Shoji
Odkhuu,
Naoki
Koide,
Abedul
Haque,
Bilegtsaikhan
Tsolmongyn,
Yoshikazu
Naiki,
Hashimoto,
Takayuki
Komatsu,
Tomoaki
Yoshida,
Takashi
Yokochi∗
Department
of Microbiology
and
Immunology,
Aichi
Medical
University
School
of
Medicine,
Nagakute,
Aichi
480-1195,
Japan
a
r
t
i
c
l
e

i
n
f
o
Article
Received
Received
Accepted
Available online 13 December 2011
history:
16 September
2011
in revised
form
9 November
2011
5 December
2011
Keywords:
Pyrroloquinoline
Receptor
ligand
Osteoclast
Nuclear
c-Fos
Type
quinine
activator
of
nuclear
factor-?B
factor
of activated
T cells
I interferon
receptor
a
b
s
t
r
a
c
t
The
induced
formation
cells
vented
preventive
nuclear
stimulated
to
the
(JAK1)
the
to
sion.
IFNAR
effect
of
pyrroloquinoline
quinine
(PQQ)
on
receptor
activator
of
nuclear
factor-?B
ligand
(RANKL)-
osteoclast
formation
was
examined
using
RAW
264.7
macrophage-like
cells.
RANKL
led
to
the
of osteoclasts
identified
as
tartrate-resistant
acid
phosphatase
(TRAP)-positive
multinucleated
in
the
culture
of
RAW
264.7
cells.
However,
PQQ
inhibited
the
appearance
of
osteoclasts
and
pre-
the
decrease
of
F4/80
macrophage
maturation
marker
on
RANKL-stimulated
cells,
suggesting
a
action
of
PQQ
on
RANKL-induced
osteoclast
differentiation.
PQQ
inhibited
the
activation
of
factor
of activated
T
cells
(NFATc1),
a
key
transcription
factor
of
osteoclastogenesis,
in
RANKL-
cells.
On
the
other
hand,
PQQ
did
not
inhibit
the
signaling
pathway
from
RANK/RANKL
binding
NFATc1
activation,
including
NF-?B
and
mitogen-activated
protein
kinases
(MAPKs).
PQQ
augmented
expression
of
type
I
interferon
receptor
(IFNAR)
and
enhanced
the
IFN-?-mediated
janus
kinase
and
signal
transducer
and
activator
of
transcription
(STAT1)
expression.
Moreover,
PQQ
reduced
expression
level
of
c-Fos
leading
to
the
activation
of
NFATc1.
Taken
together,
PQQ
was
suggested
prevent
RANKL-induced
osteoclast
formation
via
the
inactivation
of
NFATc1
by
reduced
c-Fos
expres-
The
reduced
c-Fos
expression
might
be
mediated
by
the
enhanced
IFN-?
signaling
due
to
augmented
expression.
© 2011 Elsevier B.V. All rights reserved.
1. Introduction
Osteoclasts
hematopoietic
tor
(RANKL)
[2,3].
triggers
factor
mitogen-activated
lar
are
bone-resorbing
multinuclear
cells
derived
from
stem
cells
[1].
The
interaction
between
recep-
activator
of
nuclear
factor
(NF)-?B
(RANK)
and
RANK
ligand
is
essential
for
osteoclast
differentiation
and
activation
The
binding
of
RANKL
and
RANK
on
osteoclast
progenitor
cells
the
activation
of
tumor
necrosis
factor
receptor-associated
6 (TRAF6)
[4]
and
subsequently
the
activation
of
NF-?B and
protein
kinases
(MAPKs),
such
as
extracellu-
signal-regulated
kinase
1/2
(ERK1/2),
p38
and
stress-activated
Abbreviations:
AP1,
activated
protein
1; CREB,
cAMP
responsive
element
bind-
ing
receptor;
kinase
vated
receptor
kinase/c-Jun
and
factor
∗Corresponding
E-mail
protein;
ERK,
extracellular
signal-regulated
kinase;
IFNAR,
type
I interferon
IFN,
interferon;
iNOS,
inducible
type
of
nitric
oxide
synthase;
JAK1,
janus
1; MAPK,
mitogen-activated
protein
kinase;
NFATc1,
nuclear
factor
of
acti-
T cells
1;
NF-?B,
nuclear
factor-?B; PQQ,
pyrroloquinoline
quinine;
RANKL,
activator
of
nuclear
factor-?B ligand;
SAPK/JNK,
stress-activated
protein
N-terminal
kinase;
SD,
standard
deviation;
STAT1,
signal
transducer
activator
of
transcription
1; TRAF6,
tumor
necrosis
factor
receptor-associated
6; TRAP,
tartrate-resistant
acid
phosphatase.
author.
Tel.:
+81
561
62 3311x2269;
fax:
+81
561
63 9187.
address:
yokochi@aichi-med-u.ac.jp
(T.
Yokochi).
protein
factor
tion
role
of osteoclastogenesis
including
calcitonin
is
and
On
inhibitor
togenesis
Pyrroloquinoline
ranging
PQQ
iments
compromised
performance
via
the
PQQ
pathways,
dependent
kinase/c-Jun
N-terminal
kinase
(SAPK/JNK)
[5,6]. Nuclear
of
activated
T
cells
(NFATc1)
is a downstream
transcrip-
factor
in the
RANKL/RANK
signal
pathway
and
plays
a crucial
on
the
osteoclastogenesis
[3,7]. NFATc1
as
a key
molecule
induces
a series
of
osteoclast-specific
genes,
cathepsin
K,
tartrate-resistant
acid
phosphatase
(TRAP),
receptor
and
osteoclast-associated
receptor
[7,8].
c-Fos
also
an essential
transcription
factor
for
osteoclastogenesis
[9]
positively
regulates
osteoclastogenesis
via
NFATc1
activation.
the
other
hand,
c-Fos
induces
interferon
(IFN)-?
[10]
as
a strong
of
osteoclastogenesis
and
negatively
regulates
osteoclas-
through
type
I interferon
receptor
(IFNAR)
[11].
quinine
(PQQ)
possesses
a
variety
of
functions
from
classical
vitamin
to anti-
and
pro-oxidant
[12,13].
is widely
dispersed
in animals
[13–15].
Dietary
in vivo
exper-
reveal
that
PQQ
deficiency
exhibits
growth
impairment,
immune
responsiveness
and
abnormal
reproductive
[16,17].
PQQ
might
be
involved
in bone
metabolism
nitric
oxide
biosynthesis
[12]. However,
there
is
no report
on
role
of PQQ
on
RANKL-induced
osteoclast
formation.
Recently,
has
been
reported
to regulate
several
intracellular
signaling
including
Ras-related
ERK1/2
activation
[18], CREB-
mitochondriogenesis
[19], and
JAK/STAT
activation
[13].
0165-2478/$
doi:10.1016/j.imlet.2011.12.001
– see
front
matter ©

2011 Elsevier B.V. All rights reserved.

Page 2

E. Odkhuu
et al.
/ Immunology
Letters
142 (2012) 34–
40
35
It is of
osteoclast
nal
affected
macrophage-like
report
clast
interest
to clarify
the
effect
of
PQQ
on
RANKL-induced
formation,
especially
RANKL-mediated
intracellular
sig-
pathway.
In this
study
we
aimed
to clarify
if and
how
PQQ
RANKL-induced
osteoclast
formation
in RAW
264.7
murine
cells
or
mouse
peritoneal
macrophages.
Here,
we
a putative
inhibitory
mechanism
of RANKL-induced
osteo-
formation
by PQQ.
2.
Materials
and
methods
2.1.
Materials
Recombinant
was
(Osaka,
p38,
and
ing
were
USA)
(Franklin
from
murine
soluble
RANKL
and
PQQ
disodium
salt
purchased
from
PeproTech
EC
(Princeton,
NJ,
USA)
and
Wako
Japan),
respectively.
A
series
of
antibodies
to p65
NF-?B,
SAPK/JNK,
ERK1/2,
STAT1
and
their
phosphorylated
forms,
c-Jun,
c-Fos
and
rabbit
IgG
were
purchased
from
Cell
Signal-
Technology
(Beverly,
MA,
USA).
Antibodies
to TRAF6
and
IFNAR
purchased
from
Santa
Cruz
Biotechnology
(Santa
Cruz,
CA,
and
an antibody
to NFATc1
and
JAK1
were
from
BD Biosciences
Lakes,
NJ,
USA).
Anti-mouse
IgG
antibody
was
obtained
Pierce
(Rockford,
IL,
USA).
2.2.
Cell
culture
The
murine
macrophage
cell
line,
RAW
264.7,
was
obtained
from
medium
Invitrogen,
essential
5%
SLC,
of
Thioglycollate-elicited
ing
Gaithersburg,
containing
of
were
The
Care
Riken
Cell
Bank
(Tsukuba,
Japan)
and
maintained
in ?-MEM
containing
5% heat
inactivated
fetal
calf
serum
(Gibco-
Carlsbad,
CA,
USA),
antibiotics,
antimycotics
and
non
amino
acid
(Invitrogen,
Carlsbad,
CA,
USA)
at 37◦C under
CO2. Peritoneal
cells
were
collected
from
BALB/c
mice
(Japan
Hamamatsu,
Japan)
3 days
after
an
intraperitoneal
injection
1 ml
sterile
10%
thioglycollate
(Remel,
Kansas
City,
MO,
USA).
peritoneal
cells
were
obtained
by
wash-
out
the
peritoneal
cavity
with
DMEM
medium
(Gibco-BRL,
MD).
The
cells
were
suspended
in DMEM
medium
10%
fetal
calf
serum
and
incubated
for
3 h.
After
removal
nonadherent
cells,
adherent
cells
as
peritoneal
macrophages
further
cultured
for
24 h and
used
for
osteoclast
formation.
animal
experiments
were
carried
out
following
the
Guide
for
and
Use
of Laboratory
Animals,
Aichi
Medical
University.
2.3.
Cell
viability
Cell
viability
was
assessed
by a MTT
assay
and
trypan
blue
dye
with
24
(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium
(Dojindo,
cal
Trypan
previously
interference
exclusion
test,
respectively.
RAW
264.7
cells
were
treated
various
concentrations
of
PQQ
in a 96-well
plate
for
h.
Cell
viability
was
determined
by MTT
activity
using
3-
bromide
Kumamoto,
Japan)
according
to the
instruction.
The
opti-
density
at 570
nm was
determined
with
a microplate
reader.
blue
dye
exclusion
tested
6 day
after
osteoclastogenesis
as
described
[20]. All
measurements
were
corrected
for
the
of
PQQ
at this
wavelength.
2.4.
Osteoclast
formation
RAW
264.7
cells
were
cultured
in 96-well
dishes
at a density
of
cells
at
100
50
culture
the
5000
cells
per
well
and
were
allowed
to adhere
overnight.
The
were
cultured
with
0.25
ml
fresh
media
containing
RANKL
100
ng/ml.
Peritoneal
macrophage
were
treated
with
RANKL
at
ng/ml
and
macrophage-colony
stimulating
factor
(M-CSF)
at
ng/ml
(R and
D systems,
Minneapolis,
MN,
USA)
for
8 days.
The
medium
including
reagents
was
replaced
every
3 days
after
cultivation.
RAW
264.7
cells
and
peritoneal
macrophages
were
stained
cells
for
TRAP
activity
at day
6 or 8,
respectively.
Osteoclast
like
were
counted
as
previously
described
[21,22].
2.5.
TRAP
staining
A
TRAP
staining
kit
was
obtained
from
Primary
Cell
(Sapporo,
Japan).
turer’s
taken
TRAP
staining
was
carried
out
according
to the
manufac-
instruction
as
described
elsewhere
[23]. The
images
were
with
a
digital
camera
attached
to the
microscope.
2.6.
Immunoblotting
Immunoblotting
Briefly,
of
buffer.
by
tein
protein
were
amounts
gel
membranes
phosphate-buffered
priately
reacted
conjugated
Finally,
cence
AE6955
For
ern
15
tein
used
was
performed
as
described
previously
[24].
cells
were
cultured
with
or
without
PQQ
in the
presence
RANKL
(100
ng/ml)
for
indicated
time
and
then
lysed
in a lysis
Cytoplasmic
and
nuclear
fractions
of
proteins
were
isolated
a nuclear
extract
kit
(Active
Motif,
Carlsbad,
CA,
USA).
Pro-
concentrations
were
measured
using
bicinchoninic
acid
(BCA)
assay
reagent
(Pierce,
Rockford,
IL,
USA)
and
cell
lysates
diluted
using
2× sample
buffer
and
boiled
for
5 min.
Equal
of protein
(0.02
mg)
were
analyzed
by
polyacrylamide
electrophoresis
under
reducing
conditions
and
transferred
to
by electroblotting.
After
blocking
with
5% skim
milk
in
saline,
membranes
were
treated
with
appro-
diluted
antibodies.
Resulting
immune
complexes
were
with
1:2000
or
1:500
dilutions
of horseradish
peroxidase-
goat
anti-rabbit
and
anti-mouse
antibody,
respectively.
labeled
antigen
bands
were
detected
with
a chemilumines-
reagent,
supersignal
west
dura
(Pierce)
and
analyzed
using
an
light
capture
system
with
a CS
analyser
(Atto,
Tokyo,
Japan).
reprobing,
the
membranes
were
stripped
with
the
restore
west-
blot
striping
buffer
(Thermo
Scientific,
Rockford,
IL,
USA)
for
min
and
treated
with
corresponding
antibodies.
Prestained
pro-
markers
from
BioDynamics
Laboratory
(Tokyo,
Japan)
were
to estimate
molecular
mass.
2.7.
Laser
flow
cytometry
RAW
264.7
cells
were
seeded
at
1 × 106cells/ml
in a
35-mm
cul-
ture
in
bated
Diego,
30
rescence
and
dish
for
12
h and
then
treated
with
or without
PQQ
(10
?M)
the
presence
of
RANKL
(100
ng/ml)
for
24
h.
The
cells
were
incu-
with
a FITC
conjugated
antibody
to F4/80
(Biolegend,
San
CA,
USA)
or an isotype-matched
irrelevant
antibody
for
min.
The
cell
surface
expression
of F4/80
was
analyzed
by
a
fluo-
activated
cells
sorter
(BD
FACS
Calibur,
San
Jose,
CA,
USA)
the
fluorescence
intensity
is
shown
in a log
scale.
2.8.
Statistical
analysis
Statistical
ues
performed
mean
analysis
was
performed
using
Student’s
t-test
and
val-
of
P < 0.05
were
considered
significant.
All
experiments
were
independently
at
least
three
times.
Data
represent
the
value
of triplicates
± SD.
3.
Results
3.1.
The
effect
of
PQQ
on RANKL-induced
osteoclast
formation
Since
RAW
264.7
macrophage-like
cells
differentiate
into
osteoclast-like
on
cells.
tions
A
osteoclasts
cells
as
described
elsewhere
[25], the
effect
of
PQQ
RANKL-induced
osteoclast
formation
was
examined
using
the
RAW
264.7
cells
were
incubated
with
various
concentra-
of
PQQ
in the
presence
of RANKL
(100
ng/ml)
for 6 days.
number
of
TRAP-positive
and
multinuclear
cells
identified
as
appeared
in the
culture
with
the
addition
of
RANKL.

Page 3

36
E.
Odkhuu
et al.
/ Immunology
Letters
142 (2012) 34–
40
On
induced
prevent
ited
RANKL-induced
peritoneal
incubated
RANKL
clast
cells.
peritoneal
No
exclusion
the
other
hand,
PQQ
at 10
?M significantly
inhibited
RANKL-
osteoclast
formation
(Fig.
1A).
PQQ
at 0.1
?M
did
not
the
appearance
of
TRAP-positive
cells
but
markedly
inhib-
that
of
multinuclear
cells.
The
inhibitory
action
of
PQQ
against
osteoclast
formation
was
also
examined
by
mouse
macrophages
(Fig.
1B).
Peritoneal
macrophages
were
with
various
concentrations
of
PQQ
in the
presence
(100
ng/ml)
and
M-CSF
(50
ng/ml).
PQQ
inhibited
the
osteo-
formation
in peritoneal
macrophages
as
well
as
RAW
264.7
The
cytotoxicity
of
PQQ
against
RAW
264.7
cells
and
mouse
macrophages
were
shown
with
a MTT
assay
in Fig.
1C.
cytotoxicity
of
PQQ
was
also
confirmed
by a trypan
blue
dye
test
(data
not
shown).
3.2.
maturation
The
effect
of
PQQ
on
the
expression
of
the
F4/80
macrophage
marker
on RANKL-treated
RAW
264.7
cells
The
expression
of
a macrophage
maturation
marker
F4/80
is
reported
tic
surface
264.7
ence
FITC-conjugated
indicating
sis.
RANKL
to be
downregulated
during
RANKL-induced
osteoclas-
differentiation
[26]. Therefore,
the
effect
of
PQQ
on
the
F4/80
expression
on
RANKL-treated
cells
was
examined.
RAW
cells
were
cultured
with
or without
PQQ
(10
?M)
in the
pres-
of
RANKL
(100
ng/ml)
for
24
h.
The
cells
were
stained
with
anti-F4/80
antibody
and
the
fluorescence
intensity
the
F4/80
expression
was
determined
by
FACS
analy-
PQQ
prevented
the
reduction
in the
F4/80
expression
although
significantly
reduced
the
fluorescence
intensity
(Fig.
2).
3.3.
RANKL-induced
Characterization
of
an inhibitory
action
of PQQ
on
osteoclast
formation
The
dose-dependent
appearing
bated
(100
was
ber
markedly
PQQ
cells
PQQ
period
osteoclast
stages
[27].
osteoclast
effect
of
PQQ
on
the
number
of
osteoclasts
was
examined
(Fig.
3A).
RAW
264.7
cells
were
incu-
with
various
concentrations
of
PQQ
in the
presence
of
RANKL
ng/ml)
for
6 days
and
the
number
of
osteoclasts
appearing
determined.
PQQ
at
0.1
?M significantly
reduced
the
num-
of
osteoclasts
appearing
at day
6.
The
osteoclast
number
was
inhibited
in
the
presence
of
PQQ
at 10
?M.
In addition,
was
confirmed
to exhibit
no cytotoxicity
against
RAW
264.7
with
a
MTT
assay
and
trypan
blue
dye
exclusion
test.
Next,
was
added
into
the
cultures
at
various
stages
of
the
culture
to clarify
the
crucial
stage
for
inhibition
of
RANKL-induced
formation.
The
culture
period
was
subdivided
into
three
(days
0–2,
days
2–4,
and
days
4–6)
as
described
elsewhere
The
addition
of
PQQ
significantly
inhibited
RANKL-induced
formation
at any
stage
of the
culture
period
(Fig.
3B).
3.4.
expression
The
effect
of
PQQ
on
RANKL-induced
NFATc1
and
c-Fos
NFATc1
induced
RANKL-induced
264.7
presence
NFATc1
bition
RANKL
consisting
RANKL-induced
inhibited
time
followed
RANKL-induced
the
is
known
as
a
key
transcription
factor
triggering
RANKL-
osteoclast
formation
[8,28].
Therefore,
the
effect
of
PQQ
on
NFATc1
activation
was
examined
(Fig.
4A).
RAW
cells
were
cultured
with
various
concentrations
of
PQQ
in the
of
RANKL
(100
ng/ml)
for
24
h.
RANKL
definitely
induced
expression
whereas
PQQ
significantly
inhibited
it.
The
inhi-
was
roughly
dependent
on
the
concentrations
of
PQQ.
Since
leads
to the
NFATc1
expression
via
the
activation
of
AP1
of
c-Fos
and
c-Jun
[6,29,30],
the
effect
of
PQQ
on
the
c-Fos
expression
was
also
examined
(Fig.
4A).
PQQ
the
expression
of
c-Fos
as
well
as
NFATc1.
Further,
the
course
of
RANKL-induced
NFATc1
and
c-Fos
expression
was
in the
presence
or absence
of
PQQ
(Fig.
4B).
PQQ
inhibited
NFATc1
and
c-Fos
expression
24,
48
and
72
h after
RANKL
treatment.
Fig.
were
for
in
was
as
264.7
cells
1. Effect
of
PQQ
on
RANKL-induced
osteoclast
formation.
(A)
RAW
264.7
cells
cultured
with
or
without
PQQ
(10
?M)
in the
presence
of
RANKL
(100
ng/ml)
6 days.
(B)
Peritoneal
macrophages
were
cultured
with
or
without
PQQ
(10
?M)
the
presence
of
RANKL
(100
ng/ml)
and
M-CSF
(50
ng/ml)
for
8 days.
TRAP
staining
carried
out
to identify
osteoclasts.
Scale
bar
indicates
100
?M.
Values
are
shown
Mean
± SD.
*P < 0.05
vs RANKL
+ M-CSF.
(C)
The
cytotoxicity
of PQQ
against
RAW
cells
and
peritoneal
macrophages
was
determined
by
a
MTT
assay.
RAW
264.7
and
peritoneal
cells
were
treated
with
RANKL
and
RANKL
+ M-CSF,
respectively.

Page 4

E. Odkhuu
et al.
/ Immunology
Letters
142 (2012) 34–
40
37
Fig.
RAW
RANKL
conjugated
result
2.
Effect
of
PQQ
on the
expression
of
F4/80
macrophage
maturation
marker.
264.7
cells
were
cultured
with
or without
PQQ
(10
?M)
in the
presence
of
(100
ng/ml)
for
24 h.
The
cells
were
stained
with
an anti-F4/80
antibody
with
FITC.
The
fluorescent
intensity
is expressed
on
a log
scale.
A typical
of
three
independent
experiments
is shown.
3.5.
activation
The
effect
of PQQ
on RANKL-induced
NF-?B and
MAPKs
RANKL
the
PQQ
ined
in
(Fig.
phorylation
treatment
lation.
of those
ther,
in the
activates
TRAF6
via
RANK
[4]
and
subsequently
triggers
activation
of
NF-?B and
a series
of
MAPKs
[31]. The
effect
of
on
RANKL-induced
NF-?B and
MAPKs
activation
was
exam-
(Fig.
5).
RAW
264.7
cells
were
cultured
with
or without
PQQ
the
presence
of
RANKL
(100
ng/ml)
for
30 min
as
an early
stage
5A).
PQQ
(10
?M)
alone
as
well
as
RANKL
induced
the
phos-
of
p38,
ERK1/2,
JNK,
IKK?
and
p65
NF-?B.
Combined
with
PQQ
and
RANKL
also
resulted
in their
phosphory-
There
was
no significant
difference
in the
phosphorylation
signaling
molecules
among
the
experimental
groups.
Fur-
RAW
264.7
cells
were
cultured
with
or
without
PQQ
(10
?M)
presence
of
RANKL
(100
ng/ml)
for
6,
12
or
18
h as
a late
Fig.
264.7
PQQ
are
3.
Inhibitory
actions
of
PQQ
on
RANKL-induced
osteoclast
formation.
RAW
cells
were
cultured
with
various
concentrations
of
PQQ
for
24
h (A)
or
with
(10
?M)
for
various
exposure
time
periods
of
RANKL
(100
ng/ml)
(B).
Values
shown
as
Mean
±
SD.
**P
<
0.01,
*P < 0.05
vs
RANKL
alone.
Fig.
264.7
RANKL
the
quantified
independent
4. Effect
of
PQQ
on
RANKL-induced
NFATc1
and
c-Fos
expression.
(A)
RAW
cells
were
cultured
with
various
concentrations
of PQQ
in the
presence
of
(100
ng/ml)
for
24
h.
(B)
RAW
264.7
cells
were
cultured
with
PQQ
(10
?M)
in
presence
of
RANKL
(100
ng/ml)
for
24,
48 or 72
h.
Immunoblotting
bands
were
by densitometry
and
normalized
against
?-actin.
A
typical
result
of
three
experiments
is shown.
stage
phosphorylation
RANKL-induced
?B and
TRAF6
the
excluding
(Fig.
5B).
PQQ
did
not
affect
RANKL-induced
NF-?B
and
MAPKs
at 6,
12
and
18 h,
suggesting
that
PQQ
did
not
affect
NF-?B and
MAPKs
activation.
RANKL
activates
NF-
MAPKs
via
TRAF6
[4].
Therefore,
the
effect
of PQQ
on
the
expression
was
examined
(Fig.
5A and
B).
PQQ
did
not
affect
TRAF6
expression
30
min,
6 h, 12
h or 18 h after
the
treatment,
an impaired
TRAF6
expression.
3.6.
cells
The
effect
of PQQ
on the
IFN-ˇ signaling
in RANKL-stimulated
IFN-? is
clastogenesis
fact,
Therefore,
osteoclast
duction.
production
tured
(100
an
?
examined
PQQ
and
PQQ
effect
of
significantly
produced
in response
to RANKL
and
inhibits
osteo-
via
down-regulation
of
c-Fos
protein
level
[10,11]. In
this
study
demonstrated
reduced
expression
of
c-Fos
(Fig.
4).
a possibility
was
raised
that
PQQ
might
inhibit
the
formation
through
affecting
RANKL-induced
IFN-? pro-
Therefore,
the
effect
of
PQQ
on
RANKL-induced
IFN-?
was
examined
(Fig.
6A).
RAW
264.7
cells
were
cul-
with
various
concentrations
of
PQQ
in the
presence
of RANKL
ng/ml)
for
8 h and
the
IFN-? production
was
determined
with
enzyme-linked
immunosorbent
assay.
RANKL
induced
the
IFN-
production
whereas
PQQ
did
not
significantly
affect
it.
Next,
we
the
effect
of
PQQ
on
the
expression
of
IFNAR
(Fig.
6B).
significantly
augmented
the
expression
of
IFNAR
at 6, 12,
24
48
h when
RAW
264.7
cells
were
cultured
with
or
without
(10
?M)
in the
presence
of
RANKL
(100
ng/ml).
Moreover,
the
of
PQQ
on
the
expression
of
downstream
signaling
molecules
IFNAR,
such
as
JAK1
and
STAT1,
was
examined
(Fig.
6C).
PQQ
enhanced
phosphorylation
of
STAT1
(pSTAT)
as
well

Page 5

38
E.
Odkhuu
et al.
/ Immunology
Letters
142 (2012) 34–
40
Fig.
(100
experiments
5.
Effect
of
PQQ
on
RANKL-induced
NF-?B and
MAPKs
activation.
(A)
RAW
264.7
cells
were
cultured
with
various
concentrations
of
PQQ
in the
presence
of
RANKL
ng/ml)
for
30
min.
(B)
RAW
264.7
cells
were
cultured
with
PQQ
(10
?M)
in the
presence
of
RANKL
(100
ng/ml)
for
6,
12
or 18
h.
A
typical
result
of
three
independent
is
shown.
as
RANKL
nitric
markedly
tion
of
translocation
augmented
expression
of
JAK1
and
STAT1
in the
presence
of
at 24 h.
Furthermore,
the
expression
of
an
inducible
type
of
oxide
synthase
(iNOS),
which
is
IFN-? inducible
protein,
was
enhanced
by PQQ.
The
effect
of
PQQ
on
nuclear
transloca-
of pSTAT1
was
also
examined
in order
to confirm
the
activation
JAK/STAT
signaling.
PQQ
significantly
augmented
the
nuclear
of pSTAT1
at 24 h (Fig.
6D).
4. Discussion
In
the
present
study
we
demonstrate
that
PQQ
inhibits
RANKL-
induced
which
osteoclast
expression
osteoclast
formation
via
impaired
activation
of NFATc1,
is a
key
transcription
factor
regulating
RANKL-induced
differentiation
[8,28].
The
RANKL-induced
NFATc1
is mediated
by the
activation
of
AP1
consisting
of
c-Fos
Fig.
(100
264.7
fraction
immunoblotting.
6.
Effect
of
PQQ
on
the
IFN-? signaling
in RANKL-stimulated
cells.
(A)
RAW
264.7
cells
were
cultured
with
various
concentrations
of
PQQ
in the
presence
of
RANKL
ng/ml)
for
8 h and
the
level
of
IFN-? in the
culture
supernatant
was
determined
with
an enzyme-linked
immunosorbent
assay.
Values
are
shown
as Mean
±
SD.
(B)
RAW
cells
were
cultured
with
or
without
PQQ
(10
?M)
in the
presence
of
RANKL
(100
ng/ml)
for
6,
12,
24
or
48 h.
(C and
D)
The
total
protein
(C)
or
cytoplasmic
and
nuclear
(D)
were
isolated
24 h after
stimulation
of
RANKL
(100
ng/ml)
with
or
without
PQQ
(10
?M)
and
the
expression
of
a series
of
signaling
molecules
were
analyzed
with
Bands
were
quantified
by
densitometry
and
normalized
against
?-actin
or CREB.
A typical
result
of three
independent
experiments
is shown.

Page 6

E. Odkhuu
et al.
/ Immunology
Letters
142 (2012) 34–
40
39
and
c-Fos
expression
followed
The
back
and
IFN-inducible
c-Fos
causes
activation.
augments
suggesting
augmented
ing
expression.
JAK/STAT
increases
compared
enhance
IFNAR
the
formation
Osteoprotegerin
the
activation
RANKL-induced
TRAF6-NF-?B
RANKL
[3], which
[31].
NF-?B
ulation.
the
p38? MAPK
respectively.
experimental
in
A macrophage
osteoclast
uration
RANKL-induced
ulation
PQQ
macrophage
PQQ
264.7
surface
clast
For
PQQ
biological
neonatal
are
immune,
diac
currently
[13].
of
In
through
cells.
IFN-?
NFATc1
uitously
through
c-Jun
[6,29,30].
The
present
study
demonstrates
the
reduced
expression
in PQQ-treated
cells.
Therefore,
the
reduced
c-Fos
is suggested
to cause
the
impaired
activation
of
NFATc1,
by
the
inhibition
of
RANKL-induced
osteoclast
formation.
c-Fos
expression
is regulated
by IFN-? as
a
negative
feed-
mechanism
[10,11]. IFN-? is
produced
in response
to RANKL
triggers
the
JAK/STAT
signaling
via
IFNAR
[32]. Subsequently,
genes
activated
by
JAK/STAT
signaling
inhibit
the
expression
[10]. Therefore,
the
augmented
IFN-? signaling
reduced
c-Fos
expression
and
further
impaired
NFATc1
Surprisingly,
the
present
study
demonstrates
that
PQQ
the
expression
of
IFNAR
but
not
the
production
of IFN-?,
that
PQQ
enhances
the
activation
of
JAK1
and
STAT1
via
IFNAR
expression.
The
augmented
JAK1/STAT1
signal-
may
reduce
the
c-Fos
expression,
leading
to impaired
NFATc1
In fact,
PQQ
repletion
is reported
to upregulate
the
pathways
[33]. Further,
the
number
of
osteoclasts
notably
in the
bone
of
the
IFN-?−/−[10]
or
IFNAR−/−[32]
mice,
to the
wild-type
mice.
Therefore,
PQQ
is possible
to
the
IFN-?-dependent
JAK/STAT
pathway
via
augmented
expression.
However,
it is
still
unknown
how
PQQ
augments
expression
of
IFNAR.
In addition,
RANKL-induced
osteoclast
is negatively
regulated
via
several
other
mechanisms.
as
the
decoy
receptor
of
RANKL
is known
to inhibit
osteoclastogenesis
[34]. IFN-? inhibits
it via
impaired
TRAF6
[35].
NFATc1
activation
is dependent
on
both
the
and
the
MAPK-AP1
(c-Fos)
[3,11].
The
binding
of
to RANK
induces
the
trimerization
of
RANK
and
TRAF6
leads
to the
activation
of
NF-?B and
a
series
of
MAPKs
In this
study,
PQQ
does
not
affect
the
activation
of
TRAF6,
and
MAPKs
at the
early
and
late
stage
after
RANKL
stim-
However,
PQQ
has
been
recently
reported
to influence
MAPK
pathway.
MAPKKK12,
an
activator
of
the
JNK/SAPK,
and
are
upregulated
by
PQQ
repletion
and
deprivation
[33],
The
discrepancy
might
be
due
to the
difference
in the
system
between
the
in vitro
culture
system
and
the
vivo
system.
maturation
marker,
F4/80,
is not
expressed
on
[36]. Moreover,
the
expression
of
a
macrophage
mat-
marker
F4/80
is reported
to be
downregulated
during
osteoclast
differentiation
[26]. In fact,
RANKL
stim-
reduces
the
F4/80
expression
on
RAW
264.7
cells
whereas
prevents
the
reduction
in the
surface
expression
of
F4/80
maturation
marker.
It is
consistent
with
the
idea
that
inhibits
the
RANKL-induced
osteoclast
differentiation
in RAW
cells
and
keeps
the
cells
as
macrophages.
No
alteration
in the
expression
of
F4/80
also
supports
the
inhibition
of
osteo-
differentiation
by
PQQ.
animals
and
humans,
there
has
been
constant
exposure
to
[14]. In animals,
PQQ
is reported
to participate
in a range
of
functions
with
apparent
survival
benefits
(e.g.,
improved
growth
[16]
and
reproductive
performance
[17]).
There
also
benefits
from
PQQ
supplementation
related
to cognitive,
and
antioxidant
functions,
as
well
as
protection
from
car-
and
neurological
ischemic
events
[13,37]. Although
PQQ
is
not
viewed
as
a vitamin,
PQQ
could
be defined
as
vital
to life
We
have
for
the
first
time
demonstrated
the
regulatory
action
PQQ
on
RANKL-induced
osteoclastogenesis.
conclusion,
PQQ
inhibits
RANKL-induced
osteoclastogenesis
the
inactivation
of
NFATc1
in RAW
264.7
macrophage-like
PQQ
augments
the
IFNAR
expression
and
subsequently
the
signaling.
The
enhanced
IFN-? signaling
causes
the
impaired
activation
via
reduced
c-Fos
expression.
PQQ
present
ubiq-
in mammal
tissues
may
control
osteoclastic
resorption
regulating
osteoclastogenesis.
Conflict
of
interest
statement
The
authors
report
no conflict
of
interest.
The
authors
alone
are
responsible
for
the
content
and
writing
of
the
paper.
Acknowledgements
This
work
was
supported
in part
by
a
Grant-in-Aid
for Scien-
tific
Culture
aided
Culture,
(S1101027).
the
Research
from
the
Ministry
of
Education,
Science,
Sports
and
of
Japan
and
a
grant
of
Strategic
Research
Foundation
Grant-
Project
for
Private
Universities
from
Ministry
of
Education,
Sports,
Science,
and
Technology,
Japan
(MEXT),
2011–2015
We
are
grateful
to K.
Takahashi
and
A.
Morikawa
for
technical
assistance.
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