Please
ci e
his
a icle
in
p ess
as:
J.
Fodo ,
e
al.,
S o e-ope a ed
calcium
en y
and
calcium
in lux
ia
ol age-ope a ed
calcium
channels
egula e
in acellula
calcium
oscilla ions
in
chond ogenic
cells,
Cell
Calcium
(2013),
h p://dx.doi.o g/10.1016/j.ceca.2013.03.003
ARTICLE IN PRESS
G
Model
YCECA
1472
1–16
Cell
Calcium
xxx (2013) xxx–
xxx
Con en s
lis s
a ailable
a
SciVe se
ScienceDi ec
Cell
Calcium
j
ou na
l
ho
me
page:
www.else ie .com/loca e/ceca
S o e-ope a ed
calcium
en y
and
calcium
in lux
ia
ol age-ope a ed
calcium
channels
egula e
in acellula
calcium
oscilla ions
in
chond ogenic
cells
1
2
János Fodo a,1,
Csaba
Ma ab,1,
Tamás
Oláha,
Tamás
Juhászb,
Roland
Takácsb,
Ad ienn
Tó ha,
Q1
Bea ix
Dienesa,
László
Cse nocha,
Róza
Zákányb,∗
3
4
aDepa men
o
Physiology,
Medical
and
Heal h
Science
Cen e,
Uni e si y
o
Deb ecen,
Nagye dei
k .
98,
H-4032
Deb ecen,
Hunga y5
bDepa men
o
Ana omy,
His ology
and
Emb yology,
Medical
and
Heal h
Science
Cen e,
Uni e si y
o
Deb ecen,
Nagye dei
k .
98,
H-4032
Deb ecen,
Hunga y6
7
a
i
c
l
e
i
n
o8
9
A icle
his o y:10
Recei ed
10
Janua y
201311
Recei ed
in
e ised
o m
11
Ma ch
201312
Accep ed
21
Ma ch
201313
A ailable online xxx
14
Keywo ds:15
SOCE16
STIM17
O ai118
Sox9
19
P oli e a ion20
a
b
s
a
c
Chond ogenesis
is
known
o
be
egula ed
by
calcium-dependen
signalling
pa hways
in
which
empo al
aspec s
o
calcium
homeos asis
a e
o
key
impo ance.
We
aimed
o
be e
cha ac e ise
calcium
in lux
and
elease
unc ions
wi h
espec
o
apid
calcium
oscilla ions
in
cells
o
chond i ying
chicken
high
densi y
cul u es.
We
ound
ha
di e en ia ing
chond ocy es
exp ess
he
!1subuni
o
ol age-ope a ed
calcium
channels
(VOCCs)
a
bo h
mRNA
and
p o ein
le els,
and
ha
hese
ion
channels
play
impo an
oles
in
gene a ing
Ca2+ in lux
o
oscilla ions
as
ni edipine
in e e ed
wi h
epe i i e
calcium
ansien s.
Fu -
he mo e,
VOCC
blockade
ab oga ed
chond ogenesis
and
almos
comple ely
blocked
cell
p oli e a ion.
The
con ibu ion
o
in e nal
Ca2+ s o es
ia
s o e-ope a ed
Ca2+ en y
(SOCE)
seems
o
be
indispensable
o
bo h
Ca2+ oscilla ions
and
chond ogenesis.
Mo eo e ,
his
is
he
i s
s udy
o
show
he
unc ional
exp ession
o
STIM1/STIM2
and
O ai1,
molecules
ha
o ches a e
SOCE,
in
chond ogenic
cells.
Inhibi ion
o
SOCE
combined
wi h
ER
calcium
s o e
deple ion
abolished
di e en ia ion
and
se e ely
diminished
p oli e a ion,
sugges ing
he
impo an
ole
o
in e nal
pools
in
calcium
homeos asis
o
di e en ia ing
chond ocy es.
Finally,
we
p esen
an
in eg a ed
model
o
he
egula ion
o
calcium
oscilla ions
o
di e -
en ia ing
chond ocy es
ha
may
ha e
impo an
implica ions
o
s udies
o
chond ogenesis
induced
in
a ious
s em
cell
popula ions.
© 2013 Else ie L d. All igh s ese ed.
Abb e ia ions:
AM,
ace oxy-me hyles e ;
ARC,
a achidona e- egula ed
Ca2+-
sensi i e
channel;
ATP,
adenosine
iphospha e;
CaMKII,
Ca2+–calmodulin
depend-
en
p o ein
kinase
II;
CPA,
cyclopiazonic
acid;
CRAC,
Ca2+ elease-ac i a ed
Ca2+
channel;
CREB,
cAMP- esponse
elemen
binding
p o ein;
DMMB,
dime hylme hy-
lene
blue;
ECM,
ex acellula
ma ix;
ER,
endoplasmic
e iculum;
FBS,
oe al
bo ine
se um;
FTHM,
ull
ime
a
hal
maximum;
HDC,
high
densi y
cell
cul u e;
HRP,
ho se
adish
pe oxidase;
IP3,
inosi ol-1,4,5- isphospha e;
IP3R,
inosi ol-1,4,5-
isphospha e
ecep o ;
MSC,
mesenchymal
s em
cell;
NCX,
Na+–Ca2+ exchange ;
NFAT,
nuclea
ac o
o
ac i a ed
T
lymphocy es;
NMDA,
N-me hyl-d-aspa a e
ecep o ;
PKC,
p o ein
kinase
C;
PLC,
phospholipase
C;
PMCA,
plasma
memb ane
Ca2+-ATPase;
RMP,
es ing
memb ane
po en ial;
RT-PCR,
e e se
ansc ip ion
ollowed
by
polyme ase
chain
eac ion;
RyR,
yanodine
ecep o ;
SDS–PAGE,
sodium
dodecyl
sulpha e–polyac ylamide
gel
elec opho esis;
SERCA,
sa coplas-
mic/endoplasmic
e iculum
Ca2+ ATPase;
SOC,
s o e-ope a ed
calcium
channel;
SOCE,
s o e-ope a ed
Ca2+ en y;
STIM,
s omal
in e ac ion
molecule;
TB,
oluidine
blue;
TRP,
ansien
ecep o
po en ial
ecep o ;
TRPC,
canonical
ansien
ecep-
o
po en ial
ecep o ;
TRPV,
ansien
ecep o
po en ial
ecep o
anilloid;
VOCC,
ol age-ope a ed
calcium
channel.
∗Co esponding
au ho .
Tel.:
+36
52
255
567;
ax:
+36
52
255
115.
E-mail
add esses:
[email p o ec ed]
(T.
Juhász),
oza@ana .med.unideb.hu
(R.
Zákány).
1These
au ho s
con ibu ed
equally
o
he
wo k.
1.
In oduc ion
21
One
o
he
ini ial
s eps
du ing
o ma ion
o
he
emb yonic
22
skele on
is
he
di e en ia ion
o
chond op ogeni o
cells
in o
23
ex acellula
ma ix-sec e ing
chond oblas s.
La e
on,
ma u e
24
chond ocy es
a e
o med
ha
unde go
e minal
di e en ia ion
p e-
25
ceding
endochond al
ossi ica ion
[1].
Ca ilage
o ma ion,
including
26
di e en ia ion
and
p oli e a ion
o
chond ogenic
cells,
is
igh ly
27
egula ed
by
complex
in e play
be ween
nume ous
amilies
o
28
signalling
p o eins,
e en ually
leading
o
ac i a ion
o
Sox9,
as
29
well
as
Sox6
and
L-Sox5— ansc ip ion
ac o s
ha
a e
essen ial
30
o
chond ogenesis;
hey
a e
in ol ed
in
he
speci ica ion
o
he
31
chond ogenic
lineage
and
ac i a e
he
exp ession
o
chond ogenic
32
ma ke
genes
(e.g.
COL2A1,
AGR1)
[2,3].
33
Du ing
he
ea ly
s eps
o
chond ogenesis,
in acellula
signalling
34
unde goes
p o ound
changes
o
ini ia e
speci ic
gene
ac i a ion
35
ha
equi es
ansloca ion
o
lineage-speci ic
ansc ip ion
ac o s
36
in o
he
cell
nucleus.
In
pa icula ,
he
nuclea
localisa ion
signal
37
a
he
N- e minus
o
he
chond ocy e-speci ic
ansc ip ion
ac o
38
Sox9
was
shown
o
con ain
a
calmodulin-binding
egion
and
39
Ca2+–calmodulin
has
been
epo ed
o
be
in ol ed
in
he
nuclea
40
en y
o
Sox9
[4].
Calcium
ions
(Ca2+)
a e
sugges ed
o
be
key
41
0143-4160/$
–
see
on
ma e ©
2013 Else ie L d. All igh s ese ed.
h p://dx.doi.o g/10.1016/j.ceca.2013.03.003
Please
ci e
his
a icle
in
p ess
as:
J.
Fodo ,
e
al.,
S o e-ope a ed
calcium
en y
and
calcium
in lux
ia
ol age-ope a ed
calcium
channels
egula e
in acellula
calcium
oscilla ions
in
chond ogenic
cells,
Cell
Calcium
(2013),
h p://dx.doi.o g/10.1016/j.ceca.2013.03.003
ARTICLE IN PRESS
G
Model
YCECA
1472
1–16
2J.
Fodo
e
al.
/
Cell
Calcium
xxx (2013) xxx–
xxx
ac o s
in ol ed
in
con olling
di e en ia ion
o
cells,
including42
human
mesenchymal
s em
cells
(MSCs)
[5]
and
chond ocy es
[6].43
In
gene al,
Ca2+- egula ed
cellula
e en s
equi e
ansien ly44
ele a ed
cy osolic
Ca2+ concen a ion
o
ac i a e
Ca2+-sensi i e45
signalling
componen s
[7].
Cells
u ilise
wo
main
sou ces
o
Ca2+ o 46
ini ia ing
and
gene a ing
signals.
On
he
one
hand,
Ca2+ en y
ac oss47
he
plasma
memb ane
ia
ei he
ol age-ope a ed
Ca2+ channels48
(VOCCs)
o
agonis -dependen
and
ol age-independen
Ca2+ en y49
pa hways,
amongs
which
a e
he
ecep o
ion
channels
(e.g.
P2X50
[8],
NMDA- ecep o s
[9],
TRPV
channels
[10];
he
la e
g oup51
also
ac
as
senso s
o
osmo ic
p essu e,
olume,
s e ch,
and
a i-52
ous
chemical
and
mechanical
s imuli
[11]),
he
s o e-independen 53
(e.g.
a achidona e-dependen
non-capaci i e
Ca2+ en y
[12])
and54
‘s o e-ope a ed’
Ca2+ (SOC)
channels
(e.g.
TRPs
[5]);
and
on
he55
o he
hand,
Ca2+ elease
om
in e nal
s o es
such
as
he
smoo h56
endoplasmic
e iculum
(ER)
ia
he
inosi ol-1,4,5- isphospha e57
ecep o
(IP3R)
o
he
yanodine
ecep o
(RyR),
as
well
as
Ca2+
58
induced
Ca2+ elease
(CICR)
o
ia
he
exchange
p o ein
di ec ly59
ac i a ed
by
cyclic
AMP
(Epac)
[13,14].
Since
changes
o
in a-60
cellula
Ca2+ concen a ion
a e
ela ed
o
cell
p oli e a ion
and61
di e en ia ion
ha
a e
impo an
unc ions
o
undi e en ia ed
62
cells
such
as
MSCs,
he
Ca2+ homeos asis
o
s em
cells
has
been63
ho oughly
in es iga ed
[15].64
I
is
well
documen ed
ha
di e en
spa ial
and
empo al
pa -65
e ns
o
in acellula
ee
Ca2+ concen a ion
play
dis inc
oles
in66
he
egula ion
o
a ious
cellula
p ocesses.
No
only
a
s able
ise,67
bu
also
pe iodic
oscilla o y
changes
o
cy osolic
Ca2+ concen a-68
ion
ep esen
a
nea ly
uni e sal
signalling
mechanism
e en
in69
non-exci able
cells
[16].
Signal
ansduc ion
pa hways
igge ed
by70
Ca2+ oscilla ions
a e
gene ally
accep ed
o
con ol
cellula
di e -71
en ia ion
ia
educing
he
h eshold
o
he
ac i a ion
o
di e en
72
Ca2+-dependen
ansc ip ion
ac o s,
including
nuclea
ac o
o 73
ac i a ed
T
lymphocy es
(NFAT),
nuclea
ac o -"B
(NF-"B),
Jun
N-74
e minal
kinase-1
(JNK1),
myocy e
enhance
ac o -2
(MEF2)
and75
he
cAMP- esponse
elemen
binding
p o ein
(CREB),
depending
on76
he
equency
and
ampli ude
o
he
Ca2+ ansien s
[17,18].
Spon a-77
neous
Ca2+ oscilla ions
we e
epo ed
in
se e al
non-exci able
cells78
unde going
di e en ia ion
o
p oli e a ion
[19].
In
human
MSCs,79
he
majo
sou ce
o
Ca2+ o
he
obse ed
oscilla ions
is
Ca2+ elease80
om
ER
ia
IP3Rs,
bu
Ca2+ in lux
ia
non-capaci i e
SOCs
is
also81
equi ed
o
sus ain
hese
oscilla ions,
wi hou
a
signi ican
con i-82
bu ion
om
VOCCs
[5].
Unlike
MSCs,
spon aneous
Ca2+ oscilla ions83
in
mos
o
he
cells
examined,
including
di e en ia ing
limb
bud-84
de i ed
mesenchymal
cells,
seemed
o
depend
on
he
a ailabili y
o 85
ex acellula
Ca2+.
These
obse a ions
a e
in
a
good
co ela ion
wi h86
ou
p e ious
esul s
implying
ha
mainly
Ca2+ in lux,
a
leas
in
pa 87
ia
he
ligand-ga ed
pu ine gic
ca ion
channel
P2X4,
is
equi ed
o 88
main aining
sus ained
aised
cy osolic
Ca2+ le els
in
di e en ia -89
ing
chond ogenic
cells
[20,21].
Ne e heless,
in
hese
s udies,
he90
con ibu ion
om
in e nal
Ca2+ s o es,
in
spi e
o
he
unc ional91
exp ession
o
IP3Rs,
seemed
o
be
less
impo an
in
di e en ia ing92
chicken
chond ogenic
cells.
Many
Ca2+ en y
and
elease
p ocesses93
ha e
been
documen ed
in
ma u e
chond ocy es
[22];
howe e ,
94
knowledge
ega ding
he
p ecise
egula ion
and
unc ion
o
Ca2+
95
homeos asis,
including
high- equency
spon aneous
oscilla ions,96
du ing
in
i o
chond ogenesis
is
s ill
spa se.97
In
his
s udy,
we
aimed
a
u he
cha ac e ising
he
Ca2+
98
homeos asis
o
di e en ia ing
chicken
chond ocy es
wi h
spe-99
cial
emphasis
on
he
con ibu ion
o
in e nal
Ca2+ s o es
and100
he
in ol emen
o
VOCCs
in
gene a ing
and
main aining
high-
101
equency
epe i i e
Ca2+ ansien s
o
modula e
cellula
unc ions102
such
as
di e en ia ion
and
p oli e a ion.
We
unde ook
o
ana-
103
lyse
in acellula
Ca2+ dynamics
in
indi idual
chond i ying
cells
a 104
high
spa ial
and
empo al
esolu ion
using
LIVE
con ocal
Ca2+ imag-105
ing
mic oscopy.
Ha ing
pe o med
a
de ailed
in es iga ion
o
he
106
con ibu ion
o
in e nal
Ca2+ s o es
and
s o e-ope a ed
Ca2+ en y107
(SOCE)
o
he
Ca2+ homeos asis
o
di e en ia ing
chond ocy es
and
108
he
in ol emen
o
Ca2+ in lux
ia
VOCCs
in
gene a ing
o
main-
109
aining
spon aneous
Ca2+ oscilla ions,
we
p o ide
a
e ined
model
110
o
Ca2+ signalling
e en s
including
Ca2+ in lux
and
elease
unc ions
111
in
di e en ia ing
cells
o
chond i ying
mic omass
cul u es
du ing
112
in
i o
chond ogenesis.
113
2.
Ma e ials
and
me hods
114
2.1.
P ima y
high
densi y
chond i ying
cell
cul u es
115
A
well-known
and
easily
ep oducible
in
i o
expe imen al
116
model
o
s udy
hyaline
ca ilage
o ma ion
was
i s
desc ibed
by 117
Ah ens
e
al.
[23].
In
hese
high
densi y
cell
cul u es
(HDC),
chicken
118
limb
bud-de i ed
chond op ogeni o
mesenchymal
cells
spon a-
119
neously
di e en ia e
o
chond oblas s
and
chond ocy es
on
days
2120
and
3
o
cul u ing,
and
a
well-de ec able
amoun
o
hyaline
ca i-
121
lage
ex acellula
ma ix
(ECM)
is
p oduced
by
day
6.
122
To
es ablish
p ima y
mic omass
cell
cul u es
o
chond i-
123
ying
mesenchymal
cells,
Ross
hyb id
chicken
emb yos
o
124
Hambu ge –Hamil on
de elopmen al
s ages
22–24
(4.5-day-old)
125
we e
used.
Wo k
on
ea ly
chick
emb yos
in
i o
does
no
equi e
126
a
license
om
he
E hics
Commi ee
o
he
Uni e si y
o
Deb e-
127
cen.
Dis al
pa s
o
o elimbs
and
hindlimbs
o
emb yos
we e
128
isola ed
and
dissocia ed
in
0.25%
ypsin–EDTA
(Sigma,
S .
Louis,
129
MO,
USA;
pH
7.4)
a
37◦C
o
1
h.
The
enzyma ic
diges ion
was
e -
130
mina ed
by
he
addi ion
o
equal
olume
o
oe al
bo ine
se um
131
(FBS;
Gibco,
Gai he sbu g,
MD,
USA)
and
diges ed
limb
buds
we e
132
il e ed
h ough
a
20-#m
po e
size
plas ic
il e
uni
(Millipo e,
133
Bille ica,
MA,
USA)
o
yield
a
single
cell
suspension
o
chond o-
134
genic
mesenchymal
cells.
A e
a
b ie
cen i uga ion
(a
800
×
g
o
135
10
min),
cells
we e
esuspended
in
Ham’s
F12
medium
(Sigma)
sup-
136
plemen ed
wi h
10%
FBS
a
a
concen a ion
o
1.5
×
107cells
mL−1137
and
100–100
#L
d ople s
we e
inocula ed
in o
plas ic
cell
cul-
138
u e
pla es
(O ange
Scien i ique,
B aine-l’Alleud,
Belgium).
A e 139
allowing
he
cells
o
a ach
o
he
su ace
o
120
min
a
37◦C
140
in
a
CO2incuba o
(5%
CO2and
80%
humidi y),
2
mL
o
Ham’s
141
F12
supplemen ed
wi h
10%
FBS,
0.5
mM
s abile
l-glu amine
142
and
an ibio ics/an imico ics
(penicillin
50
U
mL−1,
s ep omycin
143
50
#g
mL−1,
ungizone
1.25
#g
mL−1;
TEVA,
Deb ecen,
Hunga y)
144
was
added.
Day
o
inocula ion
was
conside ed
as
day
0
o
cul u -
145
ing.
Cul u es
we e
kep
a
37◦C
in
a
CO2incuba o
o
6
days.
The
146
medium
was
changed
on
e e y
second
day.
147
2.2.
Con ocal
mic oscopy
148
2.2.1.
Line-scan
analysis
149
Spon aneous
Ca2+ ansien s
and
he
e ec s
o
modi ied
ex a-
150
cellula
ionic
milieu
o
a ious
d ugs
on
Ca2+ oscilla ions
we e
151
moni o ed
using
an
LSM
510
META
Lase
Scanning
Con ocal
152
Mic oscope
(Zeiss,
Obe kochen,
Ge many).
All
measu emen s
we e
153
pe o med
a
oom
empe a u e.
Cells
o
1-
and
2-day-old
high
den-
154
si y
mic omass
cell
cul u es
we e
incuba ed
o
30
min
a
37◦C
155
wi h
10
#M
Fluo-4-AM
in
Ham’s
F12
medium.
Calcium
imag-
156
ing
was
ca ied
ou
in
no mal
(in
mM:
137
NaCl,
5.4
KCl,
0.5
157
MgCl2,
1.8
CaCl2,
11.8
HEPES;
1
g
L−1glucose;
pH
7.4)
o
Ca2+- ee
158
(con aining
5
mM
EGTA,
wi hou
CaCl2)
Ty ode’s
solu ion.
The
sa -
159
coplasmic/endoplasmic
e iculum
Ca2+-ATPase
(SERCA)
inhibi o
160
cyclopiazonic
acid
(CPA,
Sigma)
was
used
a
a
inal
concen a ion
161
o
10
#M
in
no mal
Ty ode’s
solu ion
(s ock:
10
mM,
in
DMSO).
162
SOCE
blocke s,
i.e.
he
non-speci ic
TRPC
an agonis
YM-58483 163
(a
py azole
de i a i e,
also
known
as
BTP-2;
Sigma)
[24]
and
164
LaCl3(Sigma)
[5]
we e
used
a
1
#M
and
500
#M
inal
concen-
165
a ions,
espec i ely,
dilu ed
in
no mal
Ty ode’s
solu ion
(s ocks: 166
300
mM
and
1
mM
in
dis illed
wa e
and
DMSO,
espec i ely).
167
Please
ci e
his
a icle
in
p ess
as:
J.
Fodo ,
e
al.,
S o e-ope a ed
calcium
en y
and
calcium
in lux
ia
ol age-ope a ed
calcium
channels
egula e
in acellula
calcium
oscilla ions
in
chond ogenic
cells,
Cell
Calcium
(2013),
h p://dx.doi.o g/10.1016/j.ceca.2013.03.003
ARTICLE IN PRESS
G
Model
YCECA
1472
1–16
J.
Fodo
e
al.
/
Cell
Calcium
xxx (2013) xxx–
xxx 3
(These
compounds
could
no
be
applied
o
oscilla ing
cells
in
Ca2+-168
ee
Ty ode’s
since
emo al
o
ee
ex acellula
Ca2+ ions
alone169
abolished
Ca2+ oscilla ions,
which
would
ha e
ende ed
he
e al-170
ua ion
o
he
e ec s
o
hese
blocke s
impossible.)
Acquisi ion
o 171
line-scan
images
s a ed
immedia ely
a e
changing
he
solu ion172
on
he
cul u es.
Du ing
measu emen s,
only
cells
exhibi ing
Ca2+
173
oscilla ions
we e
in es iga ed,
and
o he
cells
we e
dis ega ded.174
Line-scan
images
we e
acqui ed
a
0.8
ms/line,
512
pixels/line
wi h175
7
ms
in e als,
eco ding
8192
lines
using
a
63×wa e
imme sion176
objec i e.
Measu emen s
we e
ca ied
ou
in
cells
om
3
indepen-177
den
expe imen s.
Images
we e
analysed
using
an
au oma ic
e en 178
de ec ion
so wa e
de eloped
in
he
Depa men
o
Physiology
o 179
he
Uni e si y
o
Deb ecen,
Medical
and
Heal h
Science
Cen e.
180
2.2.2.
X–Y
moni o ing181
Se ies
o
X–Y
images
we e
eco ded
om
andom
isual
ields
o 182
1-
and
2-day-old
Fluo-4-loaded
chond i ying
cul u es
wi h
LIVE
5183
Lase
Scanning
Con ocal
Mic oscope
(Zeiss,
Obe kochen,
Ge many)
184
using
EC
Plan-Neo lua
20×/0.50
M27
objec i e
wi h
2×
digi al185
zoom.
Calcium
imaging
was
pe o med
in
no mal
and
Ca2+- ee186
Ty ode’s
solu ions
(see
abo e).
All
measu emen s
we e
pe o med187
a
oom
empe a u e.
LaCl3(500
#M),
YM-58483
(1
#M),
and188
ni edipine
(10
#M)
we e
dilu ed
in
no mal
Ty ode’s
solu ion
(con-189
aining
1.8
mM
Ca2+;
see
abo e).
F ame
acquisi ion
a e
was
10
s−1.190
A
o al
numbe
o
1000
images
we e
eco ded
du ing
con ol
191
condi ions
on
days
1
and
2.
When
he
e ec s
o
pha macons
on192
spon aneous
Ca2+ ansien s
we e
examined
du ing
ime
se ies193
eco dings,
he
same
isual
ields
on
each
cul u e
we e
obse ed
1,
3194
and
5
min
a e
eplacing
he
ba h
solu ion
om
no mal
Ty ode’s
o195
he
es
solu ion
(LaCl3–YM-58483;
o
ni edipine).
Du ing
hese
la -196
e
expe imen s,
a
o al
numbe
o
500
X–Y
scans
we e
eco ded
a 197
each
ime
poin .
Da a
analysis
was
ca ied
ou
using
Zeiss
Enhanced
198
Na iga ion
(ZEN
2009)
so wa e.
The
ound-shaped
chond ocy es
199
we e
ma ked
as
egion
o
in e es
(ROI)
on
each
isual
ield.
Time-200
dependen
luo escen
in ensi ies
o
ROIs
we e
analysed
using
an201
au oma ic
e en
de ec ion
so wa e
de eloped
in
he
Depa men
202
o
Physiology.203
2.3.
Single
cell
luo escen
Ca2+ measu emen s204
Measu emen s
we e
pe o med
on
day
2
o
cul u ing
using205
he
calcium
dependen
luo escen
dye
Fu a-2
as
desc ibed
p e-206
iously
[6].
B ie ly,
cul u es
we e
ans e ed
o
2
mL
esh
Ham’s207
F12
medium
con aining
10
#L
Fu a-2-ace oxy-me hyles e
(AM;208
10
#M)
and
4
#L
neos igmin
(0.3
nM;
o
inhibi
ex acellula 209
choline-es e ase
ac i i y).
Fu a-2-loaded
cells
we e
hen
placed
on210
he
s age
o
an
in e ed
luo escen
mic oscope
(Diapho ;
Nikon,211
Kowasaki,
Japan)
and
iewed
using
a
40×
oil
imme sion
objec i e.212
Measu emen s
we e
pe o med
in
no mal
and
Ca2+- ee
Ty ode’s213
solu ions
(see
abo e).
LaCl3(500
#M),
YM-58483
(1
#M),
and
CPA214
(10
#M)
we e
dilu ed
in
Ca2+- ee
Ty ode’s
solu ion.
Exci a ion215
wa eleng h
was
al e ed
be ween
340
and
380
nm
(F340 and
F380)216
by
a
mic ocompu e -con olled
dual-wa eleng h
monoch oma o 217
(Del aScan;
Pho on
Technologies
In e na ional,
New
B unswick,
NJ,218
USA).
Emission
was
moni o ed
a
510
nm
a
10
Hz
acquisi ion
a e219
using
a
pho omul iplie .
Backg ound
luo escence
was
sub ac ed
220
on-line
om
F340 and
F380 signals
by
he
da a
acquisi ion
so wa e.221
In acellula
[Ca2+]
was
calcula ed
om
he
a io
o
measu ed222
luo escence
in ensi ies
(R
=
F340/F380)
as
desc ibed
by
G ynkiewicz223
e
al.
[25].
The
measu ing
ba h
was
cons an ly
pe used
wi h
no -224
mal
Ty ode’s
solu ion
a
a
a e
o
2
mL
min−1(EconoPump;
Bio-Rad225
Labo a o ies,
CA,
USA).
Tes
solu ions
we e
di ec ly
applied
o
he226
cells
h ough
a
pe usion
capilla y
ube
(Pe usion
PencilTM;
Au o-227
Ma e
Scien i ic,
San
F ancisco,
CA,
USA)
wi h
an
in e nal
diame e
o
228
250
#m
a
a
a e
o
1.5
#L
s−1,
using
a
local
pe usion
sys em
(Val e229
BankTM 8
e sion
2.0,
Au oMa e
Scien i ic).
All
measu emen s
we e
230
pe o med
a
oom
empe a u e.
231
2.4.
Modula ion
o
Ca2+ in lux
ia
ol age-ope a ed
Ca2+ 232
channels
wi h
ni edipine
and
inhibi ion
o
SOCE
233
In
o de
o
assess
longe - e m
e ec s
o
in e e ence
wi h
Ca2+ 234
homeos asis
o
di e en ia ing
chond ocy es,
he
abo e
compounds
235
we e
also
added
o
he
cul u e
medium
o
HDC.
The
L- ype
VOCC-
236
blocke
ni edipine
was
adminis e ed
o
he
cul u e
medium
om
237
he
beginning
o
he
i s
cul u ing
day
a
a
inal
concen a ion
o
238
10
#M.
Fo
con ol
expe imen s,
he
ehicle
(DMSO,
Sigma)
was
239
added
o
cul u es
a
equal
olumes.
To
assess
he
ole
o
in e -
240
nal
Ca2+ s o es,
ollowing
s o e
deple ion
wi h
10
#M
CPA,
SOCE
241
was
blocked
by
co-applica ion
o
1
#M
YM-58483
and
500
#M
242
LaCl3.
CPA,
YM-58483
and
LaCl3we e
added
o
he
cul u e
medium
243
on
cul u ing
day
2
o
24
h.
Fo
con ol
expe imen s,
he
ehicles
244
(DMSO
and
s e ile
wa e )
we e
added
o
cul u es
a
equal
olumes. 245
2.5.
Quali a i e
and
semi-quan i a i e
de e mina ion
o
ca ilage
246
ma ix
p oduc ion 247
Fo
isualisa ion
o
ca ilage
ma ix
in
HDC,
low
pH
me ach o-
248
ma ic
s aining
was
pe o med
wi h
dime hyl
me hylene
blue
249
(DMMB;
Sigma)
dissol ed
in
3%
ace ic
acid
on
day
6
o
cul u -
250
ing.
The
amoun
o
sulpha ed
ma ix
componen s
was
de e mined
251
wi h
a
semi-quan i a i e
me hod,
by
measu ing
he
op ical
den-
252
si y
o
ex ac ed
oluidine
blue
(TB;
Reanal,
Budapes ,
Hunga y)
253
bound
o
glycosaminoglycans
in
6-day-old
HDC.
Bo h
quali a i e
254
and
semi-quan i a i e
s aining
p ocedu es
we e
desc ibed
p e i-
255
ously
in
mo e
de ail
[6].
256
2.6.
Measu emen
o
cell
p oli e a ion
and
mi ochond ial
ac i i y
257
Ra e
o
cellula
p oli e a ion
in
HDC
was
de e mined
by
mea-
258
su ing
he
adioac i i y
o
inco po a ed 3H- hymidine
du ing
a
259
16-h-long
pe iod
on
day
3
(as
desc ibed
ea lie
[6])
s a ed
260
p omp ly
a e
combined
ea men
wi h
CPA,
YM-58483
and
LaCl3,
261
o
ni edipine.
Fo
he
in es iga ion
o
mi ochond ial
ac i i y,
cells
262
cul u ed
in
wells
o
96-well
pla es
we e
used
and
MTT-assay
263
was
pe o med
immedia ely
a e
ea men s
on
day
3
as
i
was
264
desc ibed
p e iously
[6].
Un ea ed
3-day-old
HDC
we e
used
as
265
con ols
o
bo h
assays.
Measu emen s
we e
ca ied
ou
in
6
sam-
266
ples
o
each
expe imen al
g oup
in
3
independen
expe imen s.
267
2.7.
Re e se
ansc ip ion
ollowed
by
PCR
analysis
268
To al
RNA
om
HDC
was
isola ed
as
desc ibed
p e iously
[6].
269
The
assay
mix u e
(20
#L)
o
e e se
ansc ip ase
(RT)
eac ions
270
con ained
500
ng
o al
RNA,
0.25
#L
RNase
inhibi o ,
2
#L
andom
271
p ime s,
0.8
#L
dNTP
Mix
(4
mM),
50
uni s
(1
#L)
Mul iSc ibeTM 272
RT
in
1×
RT
bu e
(High
Capaci y
RT
ki ;
Applied
Biosys ems,
273
Fos e
Ci y,
CA,
USA)
and
complemen a y
cDNA
was
ansc ibed
274
a
37◦C
o
2
h.
Ampli ica ions
o
speci ic
cDNA
sequences
we e
275
achie ed
wi h
speci ic
p ime
pai s
ha
we e
designed
based
on
276
chicken
nucleo ide
sequences
published
in
GenBank
and
pu chased
277
om
In eg a ed
DNA
Technologies,
Inc.
(IDT;
Co al ille,
IA,
USA).
278
Nucleo ide
sequences
o
o wa d
and
e e se
p ime s
and
eac-
279
ion
condi ions
a e
shown
in
Table
1.
PCR
eac ions
we e
ca ied
280
ou
in
a
inal
olume
o
25
#L
con aining
1–1
#L
o wa d
and
281
e e se
p ime s
(10
#M),
0.5
#L
cDNA,
0.5
#L
dNTP
Mix
(200
#M),
282
and
1
uni
(0.2
#L)
P omega
GoTaq®DNA
polyme ase
in
1×
G een
283
GoTaq®Reac ion
Bu e
in
a
p og ammable
he mal
cycle
(Labne
284
Mul iGeneTM 96-well
G adien
The mal
Cycle ;
Labne
In e na-
285
ional,
Edison,
NJ,
USA)
wi h
he
ollowing
se ings:
2
min
a
95◦C
286
Please
ci e
his
a icle
in
p ess
as:
J.
Fodo ,
e
al.,
S o e-ope a ed
calcium
en y
and
calcium
in lux
ia
ol age-ope a ed
calcium
channels
egula e
in acellula
calcium
oscilla ions
in
chond ogenic
cells,
Cell
Calcium
(2013),
h p://dx.doi.o g/10.1016/j.ceca.2013.03.003
ARTICLE IN PRESS
G
Model
YCECA
1472
1–16
4J.
Fodo
e
al.
/
Cell
Calcium
xxx (2013) xxx–
xxx
Table
1
Nucleo ide
sequences,
ampli ica ion
si es,
GenBank
accession
numbe s,
amplicon
sizes
and
PCR
eac ion
condi ions
o
each
p ime
pai
a e
shown.
Gene
P ime
Nucleo ide
sequence
(5′→
3′)
GenBank
ID
Annealing
empe a u e
Amplicon
size
(bp)
CaV1.2
(CACNA1C) Sense
CAA
CAG
AGC
CAA
AGG
ACT
AAA
(3054–3074) XM
416388
54◦C
477
An isense
GTG
ACG
ATG
ACG
AAA
CCA
A
(3512–3530)
CaV1.3
(CACNA1D) Sense
AGG
CTC
ATC
AAT
CAC
CAC
A
(2704–2722) NM
205034
54◦C
387
An isense
AAA
GAC
GCA
CTG
AAC
AAC
G
(3072–3090)
CaV2.2
(CACNA1B) Sense
CTA
CGC
CAC
GAC
CCT
ACA
C
(2442–2460) NM 204293 61◦C 408
An isense
TTC
TCA
ACG
CCT
TCT
TCC
A
(2831–2849)
CaV2.3
(CACNA1E) Sense
TCA
CCA
ACT
CCG
ACC
GTA
AC
(3347–3366) XM
422255 60◦C
500
An isense
CAC
CTC
CAT
CTT
GTT
CTT
CTC
AT
(3824–3846)
CaV3.1
(CACNA1G) Sense
CAC
TGA
ATC
CGT
CCA
TAG
CAT
C
(1989–2010) XM 001232653 61◦C 423
An isense
CTG
TCT
GAG
TCC
GTC
TCG
TTG
T
(2390–2411)
CaV3.2
(CACNA1H) Sense
CCC
TGG
AAG
GAT
GGG
TTG
A
(1256–1274) XM 414830 61◦C 371
An isense
CTG
CCC
GTT
TGT
GGT
GTT
G
(1608–1626)
CaV3.3
(CACNA1I) Sense
CTG
AGG
ACG
GAT
ACA
GGA
GAT
(2281–2301) XM
425474
59◦C
437
An isense
TTG
CGT
GAA
GAG
TTG
GAG
AC
(2698–2717)
O ai1 Sense
TAG
CAA
CGT
GCA
TAA
TCT
CAA
(264–284) NM
001030658
57◦C
257
An isense
TCA
GTC
CAA
AGG
GAA
CCA
T
(502–520)
STIM1 Sense
GGT
GGT
GTC
CAT
CGT
CAT
CG
(426–445) NM 001030838 62◦C 356
An isense
GCT
CCT
TCT
CGG
CGT
TCT
TC
(762–781)
STIM2 Sense
CAA
TTA
GCA
ATC
GCC
AAA
G
(1177–1195) XM
420749
57◦C
495
An isense
CAC
AGA
AAG
GAT
GTC
AGG
GT
(1652–1671)
Agg ecan
co e
p o ein
(AGR1)
Sense
CAA
TGC
AGA
GTA
CAG
AGA
(276–294) XM 001232949 54◦C 430
An isense
TCT
GTC
TCA
CGG
ACA
CCG
(688–704)
Collagen
II
(COL2A1) Sense
GGA
CCC
AAA
GGA
CAG
ACG
G
(1191–1210) NM 204426 59◦C 401
An isense
TCG
CCA
GGA
GCA
CCA
GTT
(1573–1591)
Sox9 Sense
CCC
CAA
CGC
CAT
CTT
CAA
(713–731) NM
204281
54◦C
381
An isense
CTG
CTG
ATG
CCG
TAG
GTA
(1075–1093)
GAPDH Sense
GAG
AAC
GGG
AAA
CTT
GTC
AT
(238–258) NM
204305
54◦C
556
An isense
GGC
AGG
TCA
GGT
CAA
CAA
(775–793)
o
ini ial
dena u a ion
ollowed
by
35
epea ed
cycles
o
dena u -287
a ion
a
94◦C
o
30
s,
p ime
annealing
o
45
s
a
an
op imised288
empe a u e
o
each
p ime
pai
(see
Table
1),
and
ex ension
a 289
72◦C
o
90
s.
A e
he
inal
cycle,
u he
ex ension
was
allowed290
o
p oceed
o
ano he
7
min
a
72◦C.
PCR
p oduc s
we e
analysed291
using
a
1.2%
e hidium
b omide-con aining
aga ose
gel.
Op ical292
densi y
o
PCR
p oduc
signals
was
de e mined
by
using
ImageJ
293
(Image
P ocessing
and
Analysis
in
Ja a)
e sion
1.46
eewa e294
(h p:// sbweb.nih.go /ij/).295
2.8.
SDS–PAGE
and
Wes e n
blo
analysis296
To al
cell
lysa es
o
HDC
o
sodium
dodecyl
sul-297
pha e–polyac ylamide
gel
elec opho esis
(SDS–PAGE)
we e298
p epa ed
as
desc ibed
p e iously
[26].
50
#g
o
p o ein
was
sep-299
a a ed
by
7.5%
SDS–PAGE
gel
o
immunological
de ec ion
o
key300
p o eins
o
Ca2+ in lux
o
elease
unc ions
(i.e.
pan
!1subuni 301
o
ol age-ga ed
Ca2+ channels
and
STIM1),
as
well
as
p o ein302
exp ession
and
phospho yla ion
s a us
o
he
chond ogenic
mas e 303
ansc ip ion
ac o
Sox9.
P o eins
we e
ans e ed
elec opho e -304
ically
o
ni ocellulose
memb anes.
A e
blocking
in
5%
non- a
d y305
milk
in
PBS,
memb anes
we e
incuba ed
wi h
p ima y
an ibodies
306
o e nigh
a
4◦C
as
ollows:
abbi
polyclonal
an i-CaVpan
!1
307
subuni
in
1:200,
epi ope:
in acellula
C- e minus
(Alomone308
Labs,
Je usalem,
Is ael);
and
mouse
monoclonal
an i-STIM1
in
309
1:500,
epi ope:
25–139
in
human
ha
has
a
high
simila i y
o
he310
chicken
sequence
(BD
Biosciences,
F anklin
Lakes,
NJ,
USA);
abbi
311
polyclonal
an i-Sox9
an ibody
(Abcam,
Camb idge,
UK)
in
1:600;312
abbi
polyclonal
an i-P-Sox9
an ibody
(Sigma)
in
1:800;
abbi 313
polyclonal
an i-ac in
an ibody
(San a
C uz
Bio echnology,
Inc.,
314
San a
C uz,
CA,
USA)
and
abbi
polyclonal
an i-GAPDH
an ibody315
(Abcam).
A e
washing
o
30
min
in PBST,
memb anes
we e
incu- 316
ba ed
wi h
he
HRP-conjuga ed
seconda y
an ibody,
an i- abbi
317
IgG
(Bio-Rad)
in
1:1500
dilu ion.
Memb anes
we e
de eloped
by 318
enhanced
chemiluminescence
eac ion
(Millipo e,
Bille ica,
MA,
319
USA)
acco ding
o
he
ins uc ions
o
he
manu ac u e .
Op ical 320
densi y
o
signals
was
measu ed
by
using
ImageJ
1.46.
321
2.9.
S a is ical
analysis
322
All
da a
a e
ep esen a i e
o
a
leas
h ee
independen
expe -
323
imen s.
A e ages
a e
exp essed
as
mean
±
SEM
(s anda d
e o
o
324
he
mean;
n,
numbe
o
cells
measu ed).
S a is ical
analysis
was
325
pe o med
by
using
S uden ’s
- es .
Th eshold
o
s a is ically
sig-
326
ni ican
di e ences
as
compa ed
o
espec i e
con ol
cul u es
was
327
se
a
*P
<
0.05.
328
3.
Resul s
329
3.1.
Rapid
spon aneous
Ca2+ oscilla ions
a e
de ec able
in
330
di e en ia ing
chond ocy es
331
To
in es iga e
spon aneous
epe i i e
ansien
inc eases
in
332
cy osolic
Ca2+ concen a ion
in
indi idual
cells
o
p ima y
chon-
333
d i ying
mic omass
cul u es
de i ed
om
emb yonic
limb
buds,
334
Fluo-4
luo escen
Ca2+ imaging
echnique
was
applied
on
days
1
335
and
2
using
LIVE
con ocal
mic oscopy.
As
hese
cul u es
a e
he e o-
336
geneous
by
na u e
in
e ms
o
cellula
composi ion
(epi helial
cells 337
and
muscle
p ogeni o s
wi h
dis inc
mo phology
can
also
be
ound
338
in
ela i ely
small
numbe s
along
wi h
os eochond op ogeni o
339
cells;
see
[21]),
only
cells
wi h
ound
mo phology
we e
included 340
in
his
s udy,
while
o he s
we e
dis ega ded.
Se ies
o
X–Y
images
341
Please
ci e
his
a icle
in
p ess
as:
J.
Fodo ,
e
al.,
S o e-ope a ed
calcium
en y
and
calcium
in lux
ia
ol age-ope a ed
calcium
channels
egula e
in acellula
calcium
oscilla ions
in
chond ogenic
cells,
Cell
Calcium
(2013),
h p://dx.doi.o g/10.1016/j.ceca.2013.03.003
ARTICLE IN PRESS
G
Model
YCECA
1472
1–16
J.
Fodo
e
al.
/
Cell
Calcium
xxx (2013) xxx–
xxx 5
Fig.
1.
Spon aneous
Ca2+ oscilla ions
in
cells
o
HDC
on
day
2
o
cul u ing.
P io
o
measu emen s,
cells
we e
loaded
wi h
Fluo-4-AM
o
30
min
Ca2+ oscilla ions
we e
obse ed
wi hou
agonis
s imula ion
in
Ty ode’s
solu ion
con aining
1.8
mM
Ca2+
a
oom
empe a u e.
(A)
Se ies
o
X–Y
images
we e
eco ded
om
andom
isual
ields
o
chond i ying
cul u es
wi h
Zeiss
LIVE
5
Lase
Scanning
Con ocal
Mic o-
scope.
These
ou
ep esen a i e
ames
we e
acqui ed
a
6.5,
18.3,
29.6
and
54.4
s
du ing
measu emen s.
A ows
indica e
di e en ia ing
chond ocy es
wi h
epe i i e
in acellula
Ca2+ oscilla ions.
(B)
Time
cou se
o
luo escence
in ensi ies
o
he
cells
ma ked
wi h
a ows
in
panel
(A).
Fluo-4
luo escence
in ensi y
alues
no malised
o
baseline
luo escence
(F/F0)
a e
plo ed
s.
ime.
Wide
anges
o
equency
and
ampli ude
o
oscilla ing
cells
we e
obse ed.
we e
eco ded
om
andom
isual
ields
o
Fluo-4-loaded
chond i-
342
ying
cul u es
on
days
1
and
2
o
cul u ing.
Ca2+ oscilla ions
we e343
eco ded
om
cells
ba hed
in
Ty ode’s
solu ion
con aining
1.8
mM344
Ca2+ wi hou
agonis
s imula ion
a
oom
empe a u e.
Fou
ep e-345
sen a i e
X–Y
images
eco ding
Fluo-4-loaded
cells
in
a
2-day-old346
cul u e
aken
a
6.5,
18.3,
29.6
and
54.4
s
du ing
measu emen s347
a e
shown
in
Fig.
1A.
Wide
anges
o
equency
and
ampli ude
o 348
oscilla ing
cells
we e
obse ed
(Fig.
1B;
see
Supplemen a y
Video).349
On
cul u ing
day
1,
45
o
240
cells
in es iga ed
(19%)
exhibi ed350
spon aneous
Ca2+ oscilla ions;
whe eas
on
day
2,
he
p opo ion351
Fig.
2.
Pooled
da a
o
Ca2+ oscilla ions
ga he ed
om
se ies
o
X–Y
images
acqui ed
om
andom
isual
ields
o
Fluo-4
loaded
HDC
on
cul u ing
days
1
and
2
wi h
Zeiss
LIVE
5
Lase
Scanning
Con ocal
Mic oscope.
(A)
Ra io
o
oscilla ing
cells
and
e-
quency
o
epe i i e
Ca2+ ansien s
on
days
1
and
2
o
cul u ing.
Numbe s
abo e
ba s
indica e
he
numbe
o
oscilla ing
cells
compa ed
o
all
cells
eco ded.
(B)
Ampli ude
and
ull
ime
a
hal
maximum
(FTHM)
o
Ca2+ oscilla ions
in
di e en ia ing
cells
o
HDC
on
cul u ing
days
1
and
2.
Fo
bo h
panels
(A)
and
(B),
while
calcula ing
he
pa ame e s
o
Ca2+ oscilla ions,
only
oscilla ing
cells
wi h
ound,
chond oblas -like
mo phology
we e
conside ed.
Measu emen s
we e
ca ied
ou
on
cul u es
om
4
independen
expe imen s.
Da a
ep esen
mean
±
s anda d
e o
o
he
mean
(SEM).
Numbe s
in
pa en heses
abo e
ba s
indica e
he
numbe
o
cells
measu ed.
As e isks
(*)
ma k
signi ican
di e ences
(*P
<
0.05)
be ween
pa ame e s
o
oscilla ing
cells
in
1-
and
2-day-old
HDC.
o
cells
o
show
ansien
inc eases
in
cy osolic
Ca2+ concen a-
352
ion
was
subs an ially
highe
(175
o
317
cells;
55%)
(Fig.
2A).
No 353
only
he
p opo ion
o
oscilla ing
cells,
bu
also
hei
pa ame e s
354
exhibi ed
subs an ial
changes
du ing
he
cou se
o
di e en ia ion
355
(Fig.
2A
and
B).
While
he
equency
o
oscilla ions
we e
ound
o
356
be
signi ican ly
smalle
on
day
2
(0.06
±
0.003
Hz;
n
=
175)
s.
day
1
357
(0.08
±
0.01
Hz;
n
=
45;
P
=
0.01)
(Fig.
2A),
he
a e age
ampli ude
o
358
ansien
inc eases
in
cy osolic
Ca2+ concen a ion- ela ed
luo es-
359
cence
a io
signi ican ly
inc eased
om
day
1
(exp essed
as
!F/F0:
360
0.97
±
0.11;
n
=
45)
o
cul u ing
day
2
(1.27
±
0.06;
n
=
175;
P
=
0.03)
361
(Fig.
2B).
The
hi d
a iable
ha
was
used
o
desc ibe
he
du a ion
362
o
indi idual
spon aneous
ansien s
( ull
ime
a
hal
maximum;
363
FTHM)
did
no
p o e
o
be
s a is ically
di e en
on
he
wo
cul u -
364
ing
days
in es iga ed
(2.37
±
0.29
s;
n
=
45
on
day
1
s.
2.57
±
0.15
s;
365
n
=
175
on
day
2;
P
=
0.55)
(Fig.
2B).
366
3.2.
Al e ed
ex acellula
ionic
milieu
and
Ca2+ en y
blocke s
367
modi y
he
appea ance
and
quan i a i e
pa ame e s
o
apid
368
spon aneous
Ca2+ oscilla ions
in
a
ime-dependen
manne
369
Pa ame e s
o
spon aneous
Ca2+ oscilla ions
we e
examined
on
370
day
2
o
cul u ing
by
eco ding
se ies
o
X–Y
images.
In
each
cul-
371
u e,
a
andom
isual
ield
wi h
oscilla ing
cells
was
se ,
and
ames
372

Please
ci e
his
a icle
in
p ess
as:
J.
Fodo ,
e
al.,
S o e-ope a ed
calcium
en y
and
calcium
in lux
ia
ol age-ope a ed
calcium
channels
egula e
in acellula
calcium
oscilla ions
in
chond ogenic
cells,
Cell
Calcium
(2013),
h p://dx.doi.o g/10.1016/j.ceca.2013.03.003
ARTICLE IN PRESS
G
Model
YCECA
1472
1–16
6J.
Fodo
e
al.
/
Cell
Calcium
xxx (2013) xxx–
xxx
we e
acqui ed
in
con ol
condi ions
(no mal
Ty ode’s
solu ion;
con-373
aining
1.8
mM
Ca2+)
a
he
0
ime
poin ,
and
hen
in
he
p esence374
o
es
solu ions
(ei he
10
#M
ni edipine;
o
500
#M
LaCl3and375
1
#M
YM-58483);
1,
3
and
5
min
a e
changing
he
ba h
o
he376
es
solu ions.
Con ol
measu emen s
we e
pe o med
on
andom377
isual
ields
in
cul u es
a
0-,
1-,
3-
and
5-min
ime
poin s
in
no -378
mal
Ty ode’s
solu ion.
I
should
be
no ed
ha
all
h ee
pa ame e s379
o
Ca2+ oscilla ions
in es iga ed
in
his
s udy
(i.e.
a io
o
oscilla ing380
cells;
ampli ude
and
equency
o
oscilla ions)
ollowed
a
s eady381
decline
in
he
5-min
ime
ame
e en
in
con ol
condi ions;
he e-382
o e,
all
pa ame e s
ollowing
an
in e en ion
we e
no malised
o383
hei
espec i e
con ol
alues
(da a
no
shown).384
Applica ion
o
he
dihyd opy idine
L- ype
Ca2+ channel
blocke 385
ni edipine
did
no
cause
signi ican
changes
in
he
a io
o
oscil-386
la ing
cells
as
compa ed
o
un ea ed
con ol
cells
eco ded387
a
3-
and
5-min
ime
poin s
(80.93
±
25.78%
[P
=
0.56];
and388
91.73
±
28.95
[P
=
0.84],
espec i ely;
n
=
43
o
all
ime
poin s;389
Fig.
3A),
and
no
signi ican
changes
we e
obse ed
in
he
ampli-390
ude
ei he
(87.58
±
11.89%
[P
=
0.5];
69.44
±
10.26%
[P
=
0.16];391
and
75.83
±
6.26%
[P
=
0.13]
a
1-, 3-
and
5-min
ime
poin s;392
n
=
25,
16
and
11,
espec i ely;
Fig.
3B).
A
he
same
ime,
393
ni edipine
dec eased
he
equency
(53.97
±
8.52%
[P
=
0.01];
and394
64.46
±
12.88%
[P
=
0.15]
a
3-
and
5-min
ime
poin s;
n
=
16
and
11,395
espec i ely;
Fig.
3C)
o
Ca2+ oscilla ions;
his
pa ame e
was
ound396
o
be
signi ican ly
di e en
om
he
con ol
a
he
3-min
ime
poin .397
These
esul s
indica e
ha
ni edipine
in e e ed
wi h
he
equency398
o
oscilla ions,
a he
han
he
ac ual
numbe
o
oscilla ing
cells399
and
he
ampli ude
o
he
Ca2+ ansien s.
In
o he
wo ds,
ea men 400
wi h
ni edipine
did
no
abolish
epe i i e
Ca2+ ansien s,
sugges -401
ing
ha
VOCCs
a e
no
he
p ima y,
al hough
impo an
ac o s
o402
media e
his
phenomenon.
403
By
con as ,
adminis a ion
o
he
SOCE
blocke s
signi ican ly404
dec eased
he
a io
o
oscilla ing
cells
a
all
h ee
ime
poin s405
(11.59
±
6.95%
[P
=
0.0006];
23.05
±
13.58%
[P
=
0.008];
and
0.0%406
[P
=
0.0]
a
1-,
3-
and
5-min
ime
poin s,
espec i ely;
n
=
29
o 407
all
ime
poin s)
compa ed
o
he
con ol
(Fig.
3A);
in
pa icula ,408
no
oscilla ing
cells
could
be
obse ed
in
andom
isual
ields
a e 409
5
min.
A
he
same
ime,
ampli udes
o
epe i i e
Ca2+ ansien s410
we e
also
educed
(45.35
±
7.53%
[P
=
0.24];
and
58.33
±
12.85%
411
[P
=
0.34]
a
1-
and
3-min
ime
poin s;
n
=
2
and
3,
espec i ely;412
Fig.
3B),
and
hese
blocke s
also
dec eased
he
equency
o
oscil-413
la ions
a
he
3-min
ime
poin
(45.45
±
22.72%
[P
=
0.13];
n
=
3;414
Fig.
3C).415
Since
hese
esul s
sugges
ha
ER
Ca2+ s o es
play
a
de e min-416
ing
ole
in
egula ing
epe i i e
Ca2+ ansien s
in
di e en ia ing417
cells
o
HDC,
we
aimed
o
u he
analyse
he
e ec s
o
blockade
418
o
s o e-ope a ed
Ca2+ channels,
as
well
as
emo al
o
ee
ex a-419
cellula
Ca2+ ions
om
he
ba h
solu ion
wi h
a
highe
empo al420
esolu ion
on
line-scan
diag ams.
In
hese
measu emen s,
only421
ound-shaped
di e en ia ing
cells
ha
exhibi ed
p ominen
Ca2+
422
oscilla ions
we e
included
and
o he
cells
we e
dis ega ded.
Line-423
scan
images
o
oscilla ing
cells
we e
eco ded
on
day
2
o
cul u ing424
in
no mal
Ty ode’s
solu ion
(Fig.
4A).
In
he
46
cells
examined,
he
425
equency
and
he
ampli ude
o
Ca2+ oscilla ions
we e
ound
o
be426
0.08
±
0.007
Hz
and
1.44
±
0.15,
espec i ely.
Then,
measu emen s427
we e
con inued
by
changing
he
en i e
olume
o
he
ba h
solu ion428
on
he
cul u es
o
he
es
solu ions.
When
LaCl3and
YM-58483429
we e
applied
o
oscilla ing
cells
(n
=
10),
Ca2+ oscilla ions
we e430
blocked,
and
a
se e e
dis u bance
in
cy osolic
Ca2+ was
obse ed:431
small-ampli ude
a hy hmic
luc ua ions
in
basal
cy osolic
Ca2+
432
concen a ion,
a he
han
pe iodic
Ca2+ ansien s,
we e
eco ded;433
he e o e,
he
analysis
o
Ca2+ concen a ion- ela ed
changes
in434
ela i e
luo escence
in ensi ies
could
no
be
pe o med
(Fig.
4B).435
Wi h
he
ER
Ca2+ s o es
deple ed—i.e.
when
he
SERCA-blocke 436
CPA
was
co-applied
wi h
LaCl3and
YM-58483—Ca2+ oscilla ions437
we e
immedia ely
elimina ed
(n
=
10)
and
no
changes
in
cy osolic438
Fig.
3.
Pooled
da a
o
Ca2+ oscilla ions
ob ained
om
se ies
o
X–Y
images
acqui ed
om
Fluo-4
loaded
HDC
in
esponse
o
a ious
ea men s.
Measu emen s
we e
ca ied
ou
wi h
Zeiss
LIVE
5
Lase
Scanning
Con ocal
Mic oscope.
A
o al
numbe
o
500
images
we e
eco ded
a
each
ime
poin
o
each
isual
ield;
ame
acquisi ion
a e
was
10
s−1.
(A)
Pe cen age
o
oscilla ing
cells
be o e
ea men
(con ol),
and
1,
3
o
5
min
a e
he
applica ion
o
ba h
solu ion
con aining
10
#M
ni edipine,
o
500
#M
LaCl3and
1
#M
YM-58483.
Values
we e
no malised
o
he
un ea ed
cells
measu ed
a
0
min
(con ol),
and
hen
a
1,
3
and
5
min.
Numbe s
in
pa en heses
abo e
ba s
show
he
numbe
o
cells
measu ed.
(B)
Ampli ude
o
Ca2+ oscilla ions,
no malised
o
alues
o
un ea ed
con ol
cells.
Numbe s
in
pa en heses
abo e
ba s
ep esen
he
numbe
o
oscilla ing
cells
measu ed.
(C)
F equency
o
Ca2+ oscilla ions
no malised
o
he
con ol.
Numbe s
in
pa en heses
abo e
ba s
show
he
numbe
o
cells
measu ed.
Fo
panels
(A)–(C),
oscilla ing
cells
wi h
ound
mo phology
in
he
same
andom
isual
ield
we e
eco ded
a
all
ou
ime
poin s.
Di e en ia ing
ca ilage
colonies
we e
only
used
o
a
single
measu emen
se ies
and
hen
we e
disca ded.
G aphs
ep esen
pooled
da a
o
3
independen
expe imen s,
measu ing
andom
isual
ields
o
5
colonies
o
each
ea men .
As e isks
(*)
ma k
signi ican
di e ences
(*P
<
0.05)
be ween
pa ame e s
o
ea ed
s.
con ol
cells
a
espec i e
ime
poin s.
Please
ci e
his
a icle
in
p ess
as:
J.
Fodo ,
e
al.,
S o e-ope a ed
calcium
en y
and
calcium
in lux
ia
ol age-ope a ed
calcium
channels
egula e
in acellula
calcium
oscilla ions
in
chond ogenic
cells,
Cell
Calcium
(2013),
h p://dx.doi.o g/10.1016/j.ceca.2013.03.003
ARTICLE IN PRESS
G
Model
YCECA
1472
1–16
J.
Fodo
e
al.
/
Cell
Calcium
xxx (2013) xxx–
xxx 7
Fig.
4.
Pha macological
modula ion
o
spon aneous
Ca2+ oscilla ions
and
e ec s
o
al e ed
ex acellula
ionic
milieu
in
Fluo-4
loaded
di e en ia ing
chond ocy es
o
2-day-old
HDC.
Rep esen a i e
con ocal
line-scan
images
and
ime
cou ses
o
Fluo-4
luo escence
in ensi ies
a e
shown;
ho izon al
and
e ical
calib a ions
a e
he
same
o
all
aces
in
panels
(A)–(D).
Ho izon al
lines
unde
aces
show
he
du a ion
o
ea men s
wi h
pha macons
o
al e ed
ex acellula
ionic
milieu.
Acqui-
si ion
o
line-scan
images
s a ed
immedia ely
a e
changing
he
ba h
solu ion
on
he
cul u es.
P io
o
ha ,
no mal
unc ions
we e
de ec ed
on
each
cul u e.
(A)
Spon aneous
Ca2+ oscilla ions
in
no mal
([Ca2+]e=
1.8
mM)
Ty ode’s
solu ion.
(B)
A e
he
non-selec i e
ca ion
channel-media ed
Ca2+ en y
blocke
LaCl3(500
#M)
and
he
s o e-ope a ed
Ca2+ en y
and
Ca2+ elease-ac i a ed
Ca2+ (CRAC)
chan-
nel
blocke
YM-58483
(1
#M)
we e
applied
in
no mal
([Ca2+]e=
1.8
mM)
Ty ode’s,
Ca2+ oscilla ions
ceased,
al hough
i egula
luc ua ions
in
basal
cy osolic
Ca2+ con-
cen a ion
emained
de ec able.
(C)
When
he
SERCA-blocke
CPA
(10
#M)
was
co-adminis e ed
wi h
500
#M
LaCl3and
1
#M
YM-58483
in
1.8
mM
[Ca2+]e,
Ca2+
oscilla ions
we e
o ally
elimina ed.
(D)
3
min
a e
changing
he
ba h
solu ion
o
Ca2+- ee
Ty ode’s,
pe iodic
oscilla ions
could
no
be
de ec ed.
Line-scan
diag ams
on
panels
(A)–(D)
a e
ep esen a i e
da a
ou
o
4
independen
expe imen s.
Ca2+ le els
could
be
de ec ed,
e en
in
he
p esence
o
ex e nal439
Ca2+ in
he
ba h
solu ion
(Fig.
4C).
When
oscilla ing
cells
(n
=
10)440
we e
ba hed
wi h
Ca2+- ee
Ty ode’s
solu ion,
he
ampli udes441
o
oscilla ions
g adually
dec eased,
and
only
disappea ed
a e 442
se e al
minu es.
The
ep esen a i e
line-scan
diag am
(Fig.
4D)443
shows
lack
o
epe i i e
Ca2+ ansien s
3
min
a e
changing
he
444
ba h
solu ion
o
Ca2+ ee
Ty ode’s.445
3.3.
Di e en ia ing
chond ocy es
exp ess
he
˛1subuni
o
VOCCs446
a
bo h
mRNA
and
p o ein
le els447
Since
ni edipine
was
ound
o
educe
he
equency
o
spon-448
aneous
Ca2+ oscilla ions;
u he mo e,
applica ion
o
120
mM449
KCl
e oked
la ge
Ca2+ ansien s
in
he
same
expe imen al450
model
in
ou
p e ious
expe imen s
[21],
we
can
hypo hesise
ha
451
ol age-ope a ed
Ca2+ channels
may
be
exp essed
and
unc ion452
on
di e en ia ing
chond ocy es.
O
he
se e al
di e en
subuni s453
ha
comp ise
unc ional
VOCCs,
he
Ca2+ selec i e
po e- o ming454
!1subuni
is
he
one
ha
p ima ily
de e mines
he
channel455
p ope ies.
The e o e,
we
i s
downloaded
a ailable
sequence
456
da a
o
chicken
!1subuni
mRNAs,
and
ca ied
ou
RT-PCR
457
eac ions
wi h
p ime
pai s
speci ic
o
each
ype
o
VOCC
designed
458
by
P ime
P emie
5.0
so wa e
(P emie
Bioso ,
Palo
Al o,
CA,
459
USA).
Two
ion
channel
subuni
mRNA
ansc ip s
(CaV1.2
and
460
CaV1.3)
o
L- ype
(o
dihyd opy idine-sensi i e)
Ca2+ channels
461
we e
ound
o
be
exp essed
by
chond ocy es,
wi h
CaV1.2
showing
462
a
cons an
exp ession
le el,
while
CaV1.3
exhibi ed
a
peak-like
463
pa e n
wi h
almos
5- old
s onge
signals
on
days
2–4
o
cul-
464
u ing
(Fig.
5A).
By
con as ,
he
R- ype
CaV2.3
!1subuni
mRNA
465
showed
a
ma ked
exp ession
in
chond op ogeni o
mesenchymal
466
cells,
and
g adually
disappea ed
om
di e en ia ed
chond oblas s
467
and
chond ocy es.
The
h ee
T- ype
(CaV3.1,
CaV3.2
and
CaV3.3)
468
ion
channel
subuni s,
in e es ingly,
ollowed
a
e y
simila
mRNA
469
exp ession
p o ile
o
wha
has
been
obse ed
in
case
o
L- ype
470
channels;
CaV3.1
and
CaV3.2
ollowed
a
cons an
exp ession
(wi h
471
he
la e
exhibi ing
signs
o
down egula ion
in
ma u e
chon- 472
d ocy es),
whe eas
CaV3.3
also
showed
s onge
signals
du ing
473
di e en ia ion
o
chond op ogeni o
cells
on
days
2–4
(Fig.
5A).
474
Ha ing
con i med
mRNA
exp essions
o
a ious
VOCCs,
we
also 475
wan ed
o
check
he
p esence
o
!1subuni s
a
he
p o ein
le el.
476
By
using
a
polyclonal
an ibody
aised
agains
CaV!1subuni s,
477
immuno eac i e
bands
we e
de ec ed
a
he
expec ed
molecula
478
weigh
(app ox.
130
kDa).
The
p o ein
showed
s ong
exp essions
479
in
o al
cell
lysa es
o
HDC
h oughou
he
en i e
cul u ing
pe iod
480
(Fig.
5B).
These
indings,
oge he
wi h
da a
ob ained
om
Ca2+ 481
imaging
expe imen s,
demons a e
he
unc ional
exp ession
o
482
a ious
VOCCs
in
di e en ia ing
chond ogenic
cells.
483
Nex ,
we
also
unde ook
o
cha ac e ise
molecules
ha
enable
484
Ca2+ en y
ollowing
s o e
deple ion
(also
known
as
Ca2+ elease-
485
ac i a ed
Ca2+ o
CRAC
channels).
Al hough
O ai1
and
STIM1,
486
essen ial
media o s
o
CRAC
channel
unc ion,
ha e
been
desc ibed
487
in
2005
and
2006,
espec i ely
[27],
hei
exp ession
and
unc-
488
ion
ha e
no
been
in es iga ed
in
chond ocy es.
To
his
end,
by
489
designing
speci ic
p ime s,
we
we e
able
o
demons a e
he
con-
490
s an
mRNA
exp ession
o
STIM1,
STIM2
and
O ai1
h oughou
he
491
en i e
cul u ing
pe iod
(Fig.
5B),
p obably
e lec ing
on
hei
essen-
492
ial
ole
in
Ca2+ homeos asis.
Fu he mo e,
we
also
demons a ed
493
he
p esence
o
STIM1
p o ein
in
o al
cell
lysa es
o
HDC
on
all
494
cul u ing
days,
wi h
a
cons an
exp ession
pa e n
(Fig.
5B).
The
495
iden i y
o
he
uppe
immunogenic
band
a
∼90
kDa
is
unknown;
i
496
can
well
be
a
glycosyla ed
o m
o
a
splice
a ian
o
STIM1.
No e-
497
wo hy
ha
wo
STIM1
p o eins
o
di e en
size
we e
ound
o
be
498
exp essed
in
mu ine
issues:
besides
he
well-known
STIM1
iso-
499
o m,
a
new
115
kDa
STIM1
e e ed
o
as
STIM1L
has
also
been
500
ecen ly
epo ed
[28].
Owing
o
lack
o
comme cially
a ailable
501
chicken-speci ic
an ibodies
aised
agains
O ai1,
Wes e n
blo
anal-
502
ysis
o
his
p o ein
could
no
be
pe o med.
503
3.4.
Modula ion
o
ei he
VOCC
unc ion
o
SOCE
in
di e en ia ing
504
chond ocy es
de imen ally
a ec s
in
i o
chond ogenesis
505
To
assess
he
long- e m
e ec s
o
he
dihyd opy idine
L- ype
506
Ca2+ channel
blocke
ni edipine
(applied
a
10
#M
con inuously
507
om
day
1,
due
o
cons an
exp ession
o
!1subuni s)
and
SOCE
508
inhibi ion
combined
wi h
ER
Ca2+ s o e
deple ion
(by
500
#M
509
LaCl3,
1
#M
YM-58483
and
10
#M
CPA
adminis e ed
on
day
2
o
510
24
h)
on
ca ilage
ma ix
p oduc ion
in
i o,
u he
expe imen s
511
we e
pe o med.
Con inuous
ea men
wi h
ni edipine
signi i-
512
can ly
a enua ed
ca ilage
ma ix
p oduc ion
by
cul u ing
day
6
513
as
e ealed
by
me ach oma ic
s aining
p ocedu es
(Fig.
6A).
Wi h
514
ER
Ca2+ s o es
deple ed,
blockade
o
SOCE
o
only
24
h
on
day
2
515
esul ed
in
an
equally
p ominen
inhibi o y
e ec
(Fig.
6A),
e lec -
516
ing
on
he
impo an
oles
o
bo h
pa hways
(i.e.
Ca2+ en y
ac oss
517
he
plasma
memb ane
ia
VOCCs
and
Ca2+ elease
om
in e nal
518
Ca2+ s o es)
in
Ca2+ homeos asis
o
di e en ia ing
chond ocy es.
519
Please
ci e
his
a icle
in
p ess
as:
J.
Fodo ,
e
al.,
S o e-ope a ed
calcium
en y
and
calcium
in lux
ia
ol age-ope a ed
calcium
channels
egula e
in acellula
calcium
oscilla ions
in
chond ogenic
cells,
Cell
Calcium
(2013),
h p://dx.doi.o g/10.1016/j.ceca.2013.03.003
ARTICLE IN PRESS
G
Model
YCECA
1472
1–16
8J.
Fodo
e
al.
/
Cell
Calcium
xxx (2013) xxx–
xxx
Fig.
5.
Exp ession
p o iles
o
key
molecules
o
Ca2+ in lux
and
elease
on
a ious
days
o
cul u ing.
(A)
mRNA
exp ession
pa e ns
o
!1subuni s
o
ol age-ga ed
(L-,
R-,
and
T- ype)
Ca2+ channels
we e
de ec ed
by
RT-PCR.
(B)
mRNA
and
p o ein-le el
exp ession
p o iles
o
molecules
(STIM1/STIM2
and
O ai1)
ha
o ches a e
he
SOCE
mechanism,
and
p o ein-le el
exp ession
o
he
Ca2+ selec i e
po e- o ming
!1subuni
o
ol age-ga ed
Ca2+ channels
on
a ious
days
o
cul u ing,
de ec ed
wi h
RT-PCR
and
Wes e n
blo
analyses,
espec i ely.
Fo
he
STIM1
immunoblo ,
he
lowe
band
co esponds
o
he
expec ed
(70
kDa)
molecula
weigh
(ma ked
by
a ows).
Fo
PCR
eac ions
shown
in
panels
(A)
and
(B),
GAPDH
was
used
as
a
con ol
(only
shown
in
panel
B).
Fo
Wes e n
blo s,
ac in
was
used
as
a
con ol.
Da a
shown
in
panels
(A)
and
(B)
a e
ep esen a i e
ou
o
3
independen
expe imen s.
Numbe s
below
bands
ep esen
in eg a ed
densi ies
o
signals
de e mined
by
ImageJ
eewa e
ha
we e
no malised
o
he
alue
o
day
0.
No ewo hy
ha
combined
ea men s
(wi h
CPA,
YM-58483
and520
LaCl3)
longe
han
24
h
comple ely
ab oga ed
chond ogenesis
(da a521
no
shown).
Day
2
was
chosen
o
assess
he
e ec s
o
SOCE
inhibi-
522
ion
on
he
di e en ia ion
s ep
o
chond op ogeni o
cells.523
Nex ,
we
looked
a
whe he
hese
ea men s
in e e ed
wi h524
he
mRNA
and
p o ein
exp ession
o
key
genes
o
chond ogene-525
sis;
as
mRNA
exp ession
o
ype
2
collagen
(COL2A1)
and
agg ecan
526
co e
p o ein
(AGR1),
as
well
as
mRNA
and
p o ein
exp ession
and527
phospho yla ion
s a us
o
he
key
chond ogenic
ma ke
Sox9
we e528
moni o ed
on
cul u ing
day
3
by
RT-PCR
and
Wes e n
blo
analy-
529
ses,
espec i ely.
Al hough
con inuous
ea men
wi h
ni edipine
530
did
no
in e e e
wi h
he
mRNA
exp ession
o
hese
genes,
Sox9531
p o ein
exp ession
was
ound
o
be
ma kedly
educed,
wi hou
a532
de ec able
change
in
i s
phospho yla ion
le el
(Fig.
6B
and
C).
By533
con as ,
in e e ence
o
ER
Ca2+ s o e
unc ions
caused
a
s ong534
down egula ion
o
mRNAs
o
ECM
componen s,
al hough
mRNA535
ansc ip
le els
o
Sox9
we e
no
al e ed
(Fig.
6B).
A
he
p o ein536
le el,
Sox9
p o ein
exp ession
and
phospho yla ion
s a us
ollowed537
he
same
changes
as
obse ed
o
ea men s
wi h
ni edipine;
Sox9538
p o ein
le el
was
also
ound
o
be
dec eased,
and
he
phospho y-539
la ion
s a us
was
also
only
sligh ly
modi ied
ollowing
SOCE
block540
(Fig.
6C).541
Since
in
addi ion
o
modula ing
in
i o
chond ogenesis
a
he
542
molecula
le el,
hese
ea men s
could
ha e
al e ed
me abolic543
ac i i y
and/o
he
cell
cycle
o
di e en ia ing
chond ocy es;
he e-
544
o e,
mi ochond ial
ac i i y
and
a e
o
p oli e a ion
we e
also
545
de e mined
on
day
3
by
MTT
es
and 3H- hymidine
inco po a-
546
ion
assays,
espec i ely.
While
nei he
ni edipine
no
combined
547
ea men
o
p e en
Ca2+ e-up ake
in o
deple ed
in e nal
s o es
548
modula ed
cellula
me abolic
ac i i y,
bo h
ea men s
almos 549
comple ely
ab oga ed
cell
p oli e a ion
(Fig.
6D).
Consequen ly,
550
he
obse ed
dec ease
in
me ach oma ic
ma ix
p oduc ion
can
551
pa ially
be
a ibu ed
o
he
de ec ed
d ama ic
inhibi ion
o
cell
552
p oli e a ion.
553
3.5.
SOCE
blocke s
al e
he
pa ame e s
o
s o e-ope a ed
Ca2+ 554
en y
induced
by
s o e
deple ion
du ing
luo escen
single
cell
555
Ca2+ measu emen s
556
In acellula
Ca2+ concen a ion
measu emen s
we e
pe o med
557
in
2-day-old
cul u es
loaded
wi h
Fu a-2
o
assess
he
pa ame e s
558
o
Ca2+ ansien s
igge ed
ia
SOCE
in
he
absence
(Fig.
7A)
and
in
559
he
p esence
o
SOCE
blocke s
(500
#M
LaCl3and
1
#M
YM-58483;
560
Fig.
7B).
A
he
beginning
o
eco dings,
in e nal
Ca2+ s o es
o
561
cells
we e
emp ied
by
p e- ea men
wi h
he
SERCA-inhibi o
CPA
562
(10
#M)
dissol ed
in
Ca2+- ee
Ty ode’s
(no
shown).
The
changes
563
in
cy osolic
Ca2+ concen a ion
e oked
by
e-es ablishing
he
no -
564
mal
(1.8
mM)
ex acellula
Ca2+ concen a ion
we e
hen
eco ded.
565
Please
ci e
his
a icle
in
p ess
as:
J.
Fodo ,
e
al.,
S o e-ope a ed
calcium
en y
and
calcium
in lux
ia
ol age-ope a ed
calcium
channels
egula e
in acellula
calcium
oscilla ions
in
chond ogenic
cells,
Cell
Calcium
(2013),
h p://dx.doi.o g/10.1016/j.ceca.2013.03.003
ARTICLE IN PRESS
G
Model
YCECA
1472
1–16
J.
Fodo
e
al.
/
Cell
Calcium
xxx (2013) xxx–
xxx 9
Fig.
6.
E ec s
o
he
dihyd opy idine
L- ype
Ca2+ channel
blocke
ni edipine
(applied
con inuously
om
day
1
a
10
#M)
and
SOCE
inhibi ion
combined
wi h
ER
Ca2+ s o e
Q2
deple ion
(by
500
#M
LaCl3,
1
#M
YM-58483
and
10
#M
CPA
adminis e ed
on
day
2
o
24
h)
on
ca ilage
ma ix
p oduc ion
in
i o.
(A)
Me ach oma ic
ca ilage
a eas
in
6-day-old
high
densi y
colonies
we e
isualised
wi h
DMMB
dissol ed
in
3%
ace ic
acid
(pH
1.8).
Me ach oma ic
(pu ple)
s uc u es
ep esen
ca ilaginous
nodules
o med
by
many
cells
and
a
ca ilage
ma ix
ich
in
polyanionic
GAGs.
O iginal
magni ica ion
was
2×.
Scale
ba ,
1
mm
op ical
densi y
(OD625)
was
de e mined
in
supe na an s
o
6-day-old
cul u es
con aining
oluidine
blue
ex ac ed
wi h
8%
HCl
dissol ed
in
absolu e
e hanol.
(B)
mRNA
ansc ip s
o
key
chond ogenic
and
ECM
ma ke
genes
on
day
3
analysed
by
RT-PCR.
GAPDH
was
used
as
a
con ol.
(C)
P o ein
exp ession
and
phospho yla ion
s a us
o
he
mas e
chond ogenic
ansc ip ion
ac o
Sox9
in
3-day-old
cul u es.
GAPDH
was
used
as
a
con ol.
(D)
Mi ochond ial
ac i i y
and
a e
o
p oli e a ion
on
day
3
de e mined
by
MTT
es
and 3H-Thymidine
inco po a ion
assays,
espec i ely.
S a is ically
signi ican
(*P
<
0.05)
di e ences
in
ex inc ion
(OD625)
o
samples
o
TB
and
in
a e
o
p oli e a ion
a e
ma ked
by
as e isks
(*).
Abb e ia ions
used
o
panels
(B)–(D):
C,
con ol;
Ni e,
ni edipine;
SB,
SOCE
block
by
LaCl3,
YM-58483
and
CPA.
Rep esen a i e
da a
ou
o
3
independen
expe imen s
a e
shown.
(Fo
in e p e a ion
o
he
e e ences
o
colou
in
his
igu e
legend,
he
eade
is
e e ed
o
he
web
e sion
o
he
a icle.)
Compa ed o
un ea ed
con ol
cells,
in
he
p esence
o
he
SOCE
566
blocke s
bo h
he
ampli ude
(114.4
±
15.6;
n
=
16
s.
58.4
±
5.6;567
n
=
10;
P
=
0.01;
Fig.
7C)
and
he
maximal
a e
o
ise
(3.9
±
0.5;
n
=
16
568
s.
1.5
±
0.2;
n
=
10;
P
=
0.001;
Fig.
7D)
o
SOCE
we e
signi ican ly
569
dec eased.570
4.
Discussion571
4.1.
Ca2+ homeos asis
in
di e en ia ing
chond ocy es572
I
is
gene ally
accep ed
ha
Ca2+ is
he
mos
e sa ile
second573
messenge .
The e
is
accumula ing
e idence
ha
Ca2+ signalling574
pa hways
a e
key
media o s
o
cellula
e en s
in ol ed
in
di -575
e en ia ion
p ocesses
also
in
non-exci able
cells
including
MSCs576
and
chond ocy es.
A
sus ained
ise
in
cy osolic
Ca2+ concen a ion
577
induces
di e en ia ion
o
MSCs:
inc eased
in acellula
Ca2+ was
578
epo ed
o
exe
a
biphasic
egula o y
ole
in
adipocy e
di e en-
579
ia ion,
inhibi ing
he
ea ly
s ages
while
p omo ing
he
la e
s age
580
o
di e en ia ion
[29].
Di e en ia ion
o
mesenchymal
cells
in o
581
chond ocy es
is
also
con olled
by
Ca2+ dependen
pa hways:
high
582
concen a ion
o
ex acellula
Ca2+ was
ound
o
p omo e
chond o-
583
genic
di e en ia ion
in
chicken
HDC
[30].
By
con as ,
ex acellula
584
Ca2+ was
epo ed
o
modula e
di e en ia ion
du ing
skele oge-
585
nesis
in
chicken
emb yonic
cal a ia,
whe e
low
concen a ions
586
enabled
chond ogenesis
[31].
The
impo ance
o
Ca2+ in lux
ia
587
plasma
memb ane
ion
channels
du ing
chond ogenesis
in
mouse
588
limb
bud-de i ed
HDC
was
con i med
by
he
ac
ha
ea men
589
wi h
he
L- ype
channel-speci ic
blocke s
ni edipine
and
e apamil 590
Please
ci e
his
a icle
in
p ess
as:
J.
Fodo ,
e
al.,
S o e-ope a ed
calcium
en y
and
calcium
in lux
ia
ol age-ope a ed
calcium
channels
egula e
in acellula
calcium
oscilla ions
in
chond ogenic
cells,
Cell
Calcium
(2013),
h p://dx.doi.o g/10.1016/j.ceca.2013.03.003
ARTICLE IN PRESS
G
Model
YCECA
1472
1–16
16 J.
Fodo
e
al.
/
Cell
Calcium
xxx (2013) xxx–
xxx
[55]
H.J.
Kwon,
Y.
Ohmiya,
K.I.
Honma,
S.
Honma,
T.
Nagai,
K.
Sai o,
K.
Yasuda,
Syn-1265
ch onized
ATP
oscilla ions
ha e
a
c i ical
ole
in
p echond ogenic
condensa ion1266
du ing
chond ogenesis,
Cell
Dea h
&
Disease
3
(2012)
e278.1267
[56]
H.J.
Kwon,
Ex acellula
ATP
signaling
ia
P2X(4)
ecep o
and
cAMP/PKA1268
signaling
media e
ATP
oscilla ions
essen ial
o
p echond ogenic
condensa ion,1269
Jou nal
o
Endoc inology
214
(2012)
337–348.1270
[57]
J.A.
Tsai,
O.
La sson,
H.
Kindma k,
Spon aneous
and
s imula ed
ansien s
in1271
cy oplasmic
ee
Ca(2+)
in
no mal
human
os eoblas -like
cells:
aspec s
o
hei 1272
egula ion,
Biochemical
and
Biophysical
Resea ch
Communica ions
263
(1999)1273
206–212.1274
[58]
C.
Nguyen,
M.
Liebe he ,
C.
Bo da ,
F.
Vela d,
D.
Come,
F.
Lio e,
H.K.
Ea,
In a-1275
cellula
calcium
oscilla ions
in
a icula
chond ocy es
induced
by
basic
calcium1276
phospha e
c ys als
lead
o
ca ilage
deg ada ion,
Os eoa h i is
and
Ca ilage1277
20
(2012)
1399–1408.
1278
[59]
M.
Tomi a,
M.I.
Reinhold,
J.D.
Molken in,
M.C.
Naski,
Calcineu in
and1279
NFAT4
induce
chond ogenesis, Jou nal o Biological Chemis y
277
(2002)
1280
42214–42218.1281
[60] R.
Zakany,
Z.
Szijgya o,
C.
Ma a,
T.
Juhasz,
C.
Cso os,
K.
Szucs,
G.
Czi a,
T.1282
Bi o,
L.
Modis,
P.
Ge gely,
Hyd ogen
pe oxide
inhibi s
o ma ion
o
ca ilage
in1283
chicken
mic omass
cul u es
and
dec eases
he
ac i i y
o
calcineu in:
impli-1284
ca ion
o
ERK1/2
and
Sox9
pa hways, Expe imen al
Cell
Resea ch
305
(2005)1285
190–199.1286
[61] P.
De
Koninck,
H.
Schulman,
Sensi i i y
o
CaM
kinase
II
o
he
equency
o 1287
Ca2+ oscilla ions,
Science
279
(1998)
227–230.1288
[62]
A.
Shimazaki,
M.O.
W igh ,
K.
Ellio ,
D.M.
Sal e ,
S.J.
Millwa d-Sadle ,1289
Calcium/calmodulin-dependen
p o ein
kinase
II
in
human
a icula
chond o-1290
cy es,
Bio heology
43
(2006)
223–233.
1291
[63]
G.
Dupon ,
A.
Goldbe e ,
CaM
kinase
II
as
equency
decode
o
Ca2+ oscilla ions,1292
Bioessays
20
(1998)
607–610.
[64]
M.M.
Knigh ,
S.R.
McGlashan,
M.
Ga cia,
C.G.
Jensen,
C.A.
Poole,
A icula
chon-
1293
d ocy es
exp ess
connexin
43
hemichannels
and
P2
ecep o s—a
pu a i e
1294
mechano ecep o
complex
in ol ing
he
p ima y
cilium?
Jou nal
o
Ana omy
1295
214
(2009)
275–283.
1296
[65]
L.
E b,
Z.
Liao,
C.I.
Seye,
G.A.
Weisman,
P2
ecep o s:
in acellula
signaling,
1297
P luge s
A chi
452
(2006)
552–562.
1298
[66]
R.B.
Cla k,
C.
Kondo,
D.D.
Belke,
W.R.
Giles,
Two-po e
domain
K(+)
channels
1299
egula e
memb ane
po en ial
o
isola ed
human
a icula
chond ocy es, The
1300
Jou nal
o
Physiology
589
(2011)
5071–5089.
1301
[67] A.
Mobashe i,
R.
Lewis,
J.E.
Maxwell,
C.
Hill,
M.
Womack,
R.
Ba e -Jolley,
Cha -
1302
ac e iza ion
o
a
s e ch-ac i a ed
po assium
channel
in
chond ocy es,
Jou nal
1303
o Cellula Physiology
223
(2010)
511–518. 1304
[68]
R.B.
Cla k,
N.
Ha ano,
C.
Kondo,
D.D.
Belke,
B.S.
B own,
S.
Kuma ,
B.J.
Vo a,
1305
W.R.
Giles,
Vol age-ga ed
K+cu en s
in
mouse
a icula
chond ocy es
egula e
1306
memb ane
po en ial,
Channels
(Aus in)
4
(2010)
179–191.
1307
[69] A.
A gen a o,
H.
Sim,
S.
Kelly,
S.
P eiss,
A.
Clay on,
D.A.
Jans,
V.R.
Ha ley,
A
1308
SOX9
de ec
o
calmodulin-dependen
nuclea
impo
in
campomelic
dys-
1309
plasia/au osomal
sex
e e sal, Jou nal o Biological Chemis y
278
(2003) 1310
33839–33847.
1311
[70] R.
Williams,
I.M.
Khan,
K.
Richa dson,
L.
Nelson,
H.E.
McCa hy,
T.
Analbelsi,
1312
S.K.
Singh ao,
G.P.
Dow hwai e,
R.E.
Jones,
D.M.
Bai d,
H.
Lewis,
S.
Robe s,
H.M. 1313
Shaw,
J.
Dudhia,
J.
Fai clough,
T.
B iggs,
C.W.
A che ,
Iden i ica ion
and
clonal
1314
cha ac e isa ion
o
a
p ogeni o
cell
sub-popula ion
in
no mal
human
a icula 1315
ca ilage,
PLoS
ONE
5
(2010)
e13246.
1316
[71]
S.
Koelling,
J.
K uegel,
M.
I me ,
J.R.
Pa h,
B.
Sadowski,
X.
Mi o,
N.
Miosge,
Mig a-
1317
o y
chond ogenic
p ogeni o
cells
om
epai
issue
du ing
he
la e
s ages
o
1318
human
os eoa h i is,
Cell
S em
Cell
4(2009) 324–335. 1319