UNCORRECTED PROOF
YCECA 972 1—14
Please ci e his a icle in p ess as: C. Ma a, e al., Cy osolic ee Ca2+ concen a ion exhibi s a cha ac e is ic empo al
pa e n du ing in i o ca ilage di e en ia ion: A possible egula o y ole o calcineu in in Ca-signalling o chond ogenic
cells, Cell Calcium (2008), doi:10.1016/j.ceca.2007.12.010
ARTICLE IN PRESS
+Model
YCECA 972 1—14
Cell Calcium (2008) xxx, xxx—xxx
1
jou nal homepage: www.else ie .com/loca e/ceca
Cy osolic ee Ca2+ concen a ion exhibi s a
Q1
cha ac e is ic empo al pa e n du ing in i o
ca ilage di e en ia ion: A possible egula o y ole
o calcineu in in Ca-signalling o chond ogenic cells
2
3
4
5
Csaba Ma aa,1,J
´
anos Fodo b,1, Zsol Sz
´
ıjgy´
a ´
oc,Tam
´
as Juh´
asza,
P´
al Ge gelyc,L
´
aszl´
o Cse nochb,R
´
oza Z´
ak´
anya,∗
6
7
aDepa men o Ana omy, His ology and Emb yology, Medical and Heal h Science Cen e, Uni e si y o Deb ecen,
Q2
Nagye dei k . 98, H-4032 Deb ecen, Hunga y
8
9
bDepa men o Physiology, Medical and Heal h Science Cen e, Uni e si y o Deb ecen, Hunga y10
cCell Biology and Signalling Resea ch G oup o he Hunga ian Academy o Sciences, Depa men o Medical Chemis y,
Resea ch Cen e o Molecula Medicine, Uni e si y o Deb ecen, Hunga y
11
12
Recei ed 24 July 2007; ecei ed in e ised o m 15 No embe 2007; accep ed 29 Decembe 200713
KEYWORDS
Chond ogenesis;
High-densi y cul u e;
In acellula Ca2+
concen a ion;
Fu a-2;
Cyclospo ine A;
Sox9
Summa y We measu ed changes o cy osolic Ca2+ concen a ion du ing chond ogenesis, which
occu s in high-densi y cul u es (HDC) o chond i ying chicken mesenchymal cells. A significan ,
ansien ele a ion was de ec ed in Fu a-2-loaded cells on day 3 o cul u ing, when majo i y o
chond ogenic cells o HDC become di e en ia ed. This 140 nM peak o cy osolic Ca2+ concen-
a ion is a esul o inc eased Ca-influx and is indispensable o p ope chond ogenesis, because
addi ion o 0.8 mM EGTA o cul u e medium on day 2 o 3 significan ly dec eased he in acel-
lula Ca2+ concen a ion abolishing he Ca2+-peak o day 3 and inhibi ed ca ilage o ma ion.
Uncon olled Ca2+ influx e oked by a Ca2+ ionopho e exe ed dual e ec s on chond ogenesis in a
14
15
16
17
18
19
20
21
Abb e ia ions: AAS, a omic abso p ion spec ome y; AM, ace oxy-me hyles e ; BMP, bone mo phogenic p o ein; CMF-PBS, calcium
and magnesium ee phospha e bu e ed saline; CPA, cyclopiazonic acid; CsA, cyclospo ine A; DMMB, dime hylme hylene blue; DMSO,
dime hyl sul oxide; dNTP, deoxy ibonucleo ide iphospha e; DTT, di hio h ei ol; ECM, ex acellula ma ix; EGTA, e hylene glycol
e aace ic acid; ER, endoplasmic e iculum; FACS, fluo escence ac i a ed cell so e ; FCS, oe al cal se um; FGF, fib oblas g ow h
ac o ; GAPDH, glyce aldehide-3-phospha e dehyd ogenase; HDC, high-densi y cell cul u e; IGF, insulin-like g ow h ac o ; IP3,
inosi ol-1,4,5- isphospha e; MAPK, mi ogen-ac i a ed p o ein kinase; MTT, 3-(4,5-dime hyl hiazolyl-2)-2,5-diphenyl e azolium b omide;
N-CAM, neu al cell adhesion molecule; OA, okadaic acid; PBS, phospha e bu e ed saline; PBST, phospha e bu e ed saline wi h 0.1%
Tween 20; PKC, p o ein kinase C; PMCA, plasma memb ane Ca2+ ATPase; PMSF, phenylme hylsulphonyl; RT-PCR, e e se ansc ip ion
and polyme ase chain eac ion; RyR, yanodine ecep o ; SDS-PAGE, sodium dodecyl sulpha e polyac ilamide gel elec opho esis; Se ,
se ine; SERCA, sa co(endo)plasmic e iculum Ca2+ ATPase; SOCE, s o e-ope a ed calcium en y; Th , h eonine; TRPV, ansien ecep o
po en ial ion channel.
∗Co esponding au ho . Tel.: +36 52 416 392; ax: +36 52 432 290.
Q3
E-mail add ess: [email p o ec ed] (R. Z´
ak´
any).
1These wo au ho s con ibu ed equally o his wo k.
0143-4160/$ — see on ma e © 2008 Else ie L d. All igh s ese ed.
doi:10.1016/j.ceca.2007.12.010
UNCORRECTED PROOF
YCECA 972 1—14
Please ci e his a icle in p ess as: C. Ma a, e al., Cy osolic ee Ca2+ concen a ion exhibi s a cha ac e is ic empo al
pa e n du ing in i o ca ilage di e en ia ion: A possible egula o y ole o calcineu in in Ca-signalling o chond ogenic
cells, Cell Calcium (2008), doi:10.1016/j.ceca.2007.12.010
ARTICLE IN PRESS
+Model
YCECA 972 1—14
2 C. Ma a e al.
concen a ion-dependen manne ; 0.1 mg/L A23187 inc eased, whe eas 5 mg/L A23187 almos
o ally blocked ca ilage o ma ion. In acellula Ca-s o es seemed no o ha e any significan
pa icipa ion in he egula ion o changes o cy osolic Ca2+ concen a ion o chond i ying cells.
Ac i i y o Ca—calmodulin-dependen p o ein phospha ase, calcineu in esponded o changes o
in acellula Ca2+ concen a ion induced by EGTA o A23187 in a di e en ia ion s age-dependen
manne . Since inhibi ion o calcineu in wi h cyclospo ine A elimina ed he peak in he cy osolic
Ca2+ concen a ion, an ac i e egula o y ole o calcineu in on Ca2+ influx o chond i ying cells
can be supposed.
22
23
24
25
26
27
28
29
© 2008 Else ie L d. All igh s ese ed.30
In oduc ion31
Hyaline ca ilage is an impo an elemen o he e e-32
b a e skele al sys em. I p o ides p imo dia o bones o med33
by endochond al ossifica ion and emains he majo shock-34
abso bing s uc u e o he a icula su aces o join s.35
Chond ogenic mesenchymal cells can be de i ed om di -36
e en emb yonic s uc u es: he c anial pa o neu al37
c es is he sou ce o ca ilage p imo dia o se e al c an-38
io acial bones; scle o ome o somi es di e en ia es in o39
e eb ae; appendicula bones de i e om mesenchymal40
cells o soma opleu a [1].
41
High-densi y cell cul u e sys em (HDC) es ablished om42
chond ogenic mesenchymal cells isola ed om limb buds
43
o 4-day-old chicken emb yos is a well-known model o in44
i o ca ilage di e en ia ion [2—4]. This simple model can45
p o ide in o ma ion on he molecula s eps leading o di -
46
e en ia ion o chond op ogeni o cells o chond oblas s.47
In HDC, o ma ion o ca ilage s a s wi h he ec ui -
48
men o chond op ogeni o mesenchymal p ogeni o cells49
ha a e condensa ion and nodule o ma ion, di e en i-50
a e in o chond oblas s and chond ocy es. Condensa ion and51
nodule o ma ion ake place on he fi s day o cul u ing
52
and a e pa ly egula ed by ansien appea ance o Ca2+-53
dependen in e cellula junc ions like N-CAM (neu al cell54
adhesion molecule) and N-cadhe in [5]. Chond op ogeni o 55
cells di e en ia e in o chond oblas s on he second and56
hi d day o cul u ing [4,6], con olled by nume ous g ow h57
ac o s and o he signal molecules, e.g. FGF, BMP, Wn , IGF
58
and membe s o Hedgehog and Sox ansc ip ion ac o ami-59
lies [7]. In pa allel o he in acellula changes, ex acellula 60
ma ix (ECM) su ounding he di e en ia ing chond ogenic
61
cells is also subjec o p o ound changes: di e en ia ing62
cells s a o sec e e ca ilage-specific ma ix componen s,63
such as collagen ype II and agg ecan on he hi d day o cul-
64
u ing pe iod [8]. The unique composi ion and o ganiza ion
65
o ECM is c ucial o main enance o he p ope mo phol-66
ogy and unc ion o hese cells [9]. Exp ession o collagen67
ype II and co e p o ein o agg ecan is con olled by Sox9, a68
high-mobili y-g oup domain con aining ansc ip ion ac o
69
[10—12]. De ec ion o he exp ession le el and he phospho-70
yla ion s a us o Sox9, as well as moni o ing he exp ession
71
o he co e p o ein o agg ecan a e a eliable ma ke s o 72
chond ogenesis.73
Calcium ion is a ubiqui ous cellula signal. The concen-74
a ion o in acellula ee Ca2+ (∼10−7M) is 104 imes75
lowe han ha o he ex acellula fluid. This dis ibu-
76
ion p o ides he po en ial o he influx o Ca2+ in o cells,77
whe e i can ac as a second messenge . Va ious s imuli78
p omo e he mo emen o Ca2+ ei he om he ex acel-79
lula space o om in acellula s o es in o he cy osol. 80
The ele a ed le el o cy osolic ee Ca2+ exe s a a ie y 81
o specific changes in cellula unc ion, such as ac i a- 82
ion o p o ein kinases and p o ein phospha ases, which, 83
in u n, egula e o he p ocesses, like p oli e a ion o di - 84
e en ia ion [13]. The molecula s eps leading o ca ilage 85
di e en ia ion, among o he ac o s a e egula ed by Ca2+ 86
sensi i e enzymes like one o he Se /Th specific p o- 87
ein kinases, PKCalpha [14] o he Se /Th -specific p o ein 88
phospha ase calcineu in [15,16], ha is unique among phos- 89
pha ases o i s abili y o sense changes o in acellula 90
Ca2+ concen a ion h ough i s ac i a ion by i s calcium 91
binding subuni and calmodulin. Calcineu in is bes known 92
as a egula o o T-lymphocy e ac i a ion, since i s pha - 93
macological inhibi o s, cyclospo ine A (CsA), ac olimus, 94
pimec olimus and apamycin a e all used in he clinical p ac- 95
ice as immunosupp essan s [17]. Calcineu in is also known 96
o pa icipa e in se e al di e en ia ion p ocesses, such as 97
de elopmen o di e en muscle issues and he ne ous 98
sys em [18].99
In his s udy we measu ed he cy osolic ee Ca2+ concen- 100
a ion du ing ca ilage di e en ia ion in he chond ogenic 101
cells o HDC. A cha ac e is ic empo al pa e n in he 102
changes o cy osolic Ca2+ concen a ion could be obse ed; 103
he e was a significan and ansien ele a ion on he hi d 104
cul u ing day, he c ucial day o chond ocy e di e en ia- 105
ion. Mo eo e , beside he changes o he basal cy osolic 106
Ca2+ le el, cells o chond i ying mic omass cul u es also 107
exhibi spon aneous calcium e en s, a phenomenon cha - 108
ac e is ic o se e al o he p ima y cell cul u es [19,20].We 109
p o ide e idence ha he empo al pa e n o he changes 110
o cy osolic ee Ca2+ concen a ion in chond i ying cells is 111
indispensible o p ope ca ilage o ma ion and depends on 112
ex acellula Ca2+ a he han he a ailabili y o in acellu- 113
la Ca-s o es. We also demons a e ha calcineu in can play 114
a dual ole in Ca-signalling o chond ogenic cells: i s ac i i y 115
is modula ed by cy osolic Ca2+ concen a ion and he inhibi- 116
ion o calcineu in wi h CsA elimina es he Ca2+ peak o HDC 117
esul ing in a p onounced dec ease in ca ilage o ma ion. 118
This second obse a ion aises he possibili y o he ac i e 119
egula o y e ec o his enzyme on he enhancemen o Ca2+ 120
influx o chond i ying cells. 121
Ma e ials and me hods 122
Cell cul u e 123
Dis al pa s o he limb buds o 4-day-old Ross hyb id 124
chicken emb yos (Hambu ge —Hamil on s ages 22—24 [21])125
UNCORRECTED PROOF
YCECA 972 1—14
Please ci e his a icle in p ess as: C. Ma a, e al., Cy osolic ee Ca2+ concen a ion exhibi s a cha ac e is ic empo al
pa e n du ing in i o ca ilage di e en ia ion: A possible egula o y ole o calcineu in in Ca-signalling o chond ogenic
cells, Cell Calcium (2008), doi:10.1016/j.ceca.2007.12.010
ARTICLE IN PRESS
+Model
YCECA 972 1—14
Possible egula o y ole o calcineu in in Ca-signalling o chond ogenic cells 3
we e emo ed and p ima y mic omass cul u es o chon-126
d i ying mesenchymal cells we e es ablished om a cell127
suspension wi h a densi y o 1.5 ×107cells/mL. Fi een128
mic oli e s d ople s o he suspension we e inocula ed129
on ound co e glasses (diame e : 30 mm; Menzel-Gl¨
ase ,130
Menzel GmbH, B aunschweig, Ge many) placed in o plas-131
ic Pe i dishes (Nunc, Nape ille, IL, USA). Cells we e132
allowed o a ach o he su ace o 2 h a 37 ◦C. Day133
o inocula ion is conside ed as day 0. Colonies we e
134
g own in Ham’s F12 medium (Sigma, Budapes , Hun-135
ga y) supplemen ed wi h 10% oe al cal se um (Gibco,136
Gai he sbu g, MD, USA) and we e kep a 37 ◦C in an a mo-137
sphe e o 95% ai and 5% CO2and 80% humidi y. The138
medium was changed on e e y second day o a e ea -139
men s.140
De e mina ion o cy osolic ee Ca2+ concen a ion141
Measu emen s we e pe o med on di e en days o cul u ing142
using he calcium-dependen fluo escen dye Fu a-2. Cul-143
u es we e ans e ed o 2 mL esh Ham’s F12 medium
144
con aining 10 ␮L Fu a-2-AM (10 ␮M) and 4 ␮L neos igmin145
(0.3 nM), in o de o inhibi ex acellula choline es e ases.
146
A e 60 min o incuba ion a 37 ◦CinaCO
2incuba o ,147
cul u es we e washed wice in Ty ode’s solu ion con-
148
aining 137 mM NaCl, 5.4 mM KCl, 0.5 mM MgCl2, 1.8 mM149
CaCl2, 11.8 mM Hepes—NaOH, 1 g/L glucose, pH 7.4, in150
o de o emo e he Fu a-2-AM a ached o he ex a-151
cellula ma ix. Fu a-2-loaded cells we e placed on he152
s age o an in e ed fluo escen mic oscope (Diapho , Nikon,
153
Kowasaki, Japan) and iewed using a 40×oil imme -154
sion objec i e. Measu emen s we e ca ied ou in he
155
same sal solu ion in a pe usion chambe using a dual156
wa eleng h monoch oma o (Del aScan, Pho on Technolo-157
gies In e na ional, Law ence ille, KY, USA) equipmen .158
All measu emen s we e pe o med a oom empe a u e.
159
Fluo escence o Fu a-2-loaded cells was measu ed using
160
exci a ion wa eleng hs o 340 and 380 nm and an emis-161
sion wa eleng h o 510 nm. In acellula Ca2+ concen a ions162
we e calcula ed om he a ios o in ensi ies (R=F340/F380)163
as desc ibed by G ynkiewicz e al. [22]. In acellula Ca2+
164
le els o HDC we e measu ed 2 h a e inocula ion on165
ound co e glasses hen on cul u ing days 1—6 a he same
166
pe iod o each day. In acellula Ca2+ le els o un ea ed167
con ol cul u es and cul u es ea ed wi h EGTA, calcium168
ionopho e A23187, cyclopiazonic acid (CPA) o CsA we e169
assayed in 5 independen expe imen s measu ing 30 cells170
in each case. All measu emen s we e ca ied ou di ec ly
171
a e ea men s wi h EGTA, calcium ionopho e A23187,
172
CPA o CsA. Da a we e s a is ically analyzed by S uden ’s173
- es .174
T ea men s wi h cyclopiazonic acid and ca eine
175
To de e mine he ole o in acellula Ca-s o es in he176
changes o he cy osolic Ca2+ concen a ion, he in acel-177
lula Ca2+ pump inhibi o CPA was adminis e ed o HDC.178
Fo single cell measu emen s, CPA was dissol ed in DMSO179
and dilu ed in a modified, Ca2+- ee T yode’s solu ion180
(con aining 5 mM EGTA, wi hou CaCl2) o a final concen-181
a ion o 10 ␮M. The inhibi o was di ec ly pe used in
182
he close p oximi y (app oxima ely 50 ␮m) o cells. P io 183
o adminis a ion o CPA, cells we e washed wi h Ca2+-184
ee Ty ode’s solu ion o emo e all aces o ee Ca2+ 185
om he medium. To in es iga e p olonged e ec o deple- 186
ion o in acellula Ca2+ sou ce, CPA was adminis e ed 187
di ec ly in o he cul u e medium o 12 h on cul u ing 188
day 2 o 3 a a final concen a ion o 10 ␮M. Ca eine 189
(15 mM, dilu ed in Ty ode’s solu ion), an agonis o yan- 190
odine ecep o (RyR) was adminis e ed a close p oximi y 191
(abou 50 ␮m) o cells o 100 s du ing single cell measu e- 192
men s. 193
Measu emen and analysis o spon aneous calcium 194
ansien s 195
Spon aneous calcium ansien s we e moni o ed using LSM 196
510 META Lase Scanning Con ocal Mic oscope (Zeiss, 197
Obe kochen, Ge many). Cells o high-densi y mic omass 198
cell cul u es we e incuba ed o 1 h a 37 ◦C wi h 10 ␮M199
Fluo-4-AM in Ham’s F12 medium. Calcium imaging was pe - 200
o med in no mal Ty ode’s solu ion (see abo e). x—yanalysis 201
and line scan images we e aken o moni o he fluo es- 202
cence in ensi y du ing spon aneous ac i i ies. Fi y images 203
we e eco ded du ing a 48.4 s in e al o pe o m he x—y204
analysis. Line scan images we e eco ded a 1.54 ms/line, 205
512 pixels/line and 4096 lines using a 63×wa e imme sion 206
objec i e. Fluo-4-loaded cells we e exci ed wi h a 488 nm 207
a gon ion lase and emi ed fluo escence was collec ed 208
a 500—570 nm. Images we e analyzed using an au oma ic 209
e en de ec ion p og am de eloped in he Depa men o 210
Physiology. 211
De e mina ion o Ca concen a ion o he cul u e 212
medium 213
Ca concen a ion o he cul u e medium (F12 supplemen ed 214
wi h 10% FCS) was de e mined wi h a omic abso p ion 215
spec ome y (AAS) on Philips PU9200X equipmen in ai - 216
ace ylene flame. Measu emen s we e pe o med a he 217
wa eleng h o 422.7 nm; each sample was measu ed h ee 218
imes o 4 s. De e mina ion o Ca concen a ion was ca ied 219
ou wi h s anda d addi ion me hod; samples we e dilu ed in 220
0.1 M ni ic acid [23].221
T ea men s wi h EGTA, A23187 calcium ionopho e 222
and cyclospo ine A 223
In o de o examine he e ec s o emo al o Ca2+ om 224
he ex acellula fluid, cell cul u es we e ed a cul u e 225
medium con aining 0.8 mM EGTA (Am esco, Solon, OH, USA; 226
pH adjus ed o 7.4) o 12 h on day 2 o 3 o cul u ing. 227
To assess he e ec o ele a ed in acellula Ca2+, cul- 228
u es we e ed wi h a cul u e medium con aining calcium 229
ionopho e A23187 (Sigma, Budapes , Hunga y) a concen- 230
a ions o 0.1 and 5 mg/L o 1 h on cul u ing days 2 and 231
3. Calcium ionopho e A23187 was dissol ed in DMSO hen 232
dilu ed in cul u e medium. Ac i i y o calcineu in was inhib- 233
i ed wi h he con inuous applica ion o 2 ␮M cyclospo ine A 234
(Sigma, Budapes , Hunga y) s a ed on day 1.
UNCORRECTED PROOF
YCECA 972 1—14
Please ci e his a icle in p ess as: C. Ma a, e al., Cy osolic ee Ca2+ concen a ion exhibi s a cha ac e is ic empo al
pa e n du ing in i o ca ilage di e en ia ion: A possible egula o y ole o calcineu in in Ca-signalling o chond ogenic
cells, Cell Calcium (2008), doi:10.1016/j.ceca.2007.12.010
ARTICLE IN PRESS
+Model
YCECA 972 1—14
4 C. Ma a e al.
Ligh mic oscopical analysis o ca ilage235
di e en ia ion236
Ca ilage ma ix was isualized by s aining wi h dime hyl-
237
me hylene blue (DMMB, Ald ich, Ge many) as desc ibed238
p e iously [2]. The amoun o sulpha ed ma ix compo-239
nen s was de e mined wi h a semi-quan i a i e me hod,240
by measu ing he op ical densi y o ex ac ed oluidine241
blue (Reanal, Budapes , Hunga y) bound o glycosamino-242
glycans in ma u e HDC. Six-day-old cell cul u es we e243
fixed in a solu ion con aining 28% e hanol, 4% o ma-244
lin and 2% ace ic acid, s ained wi h 0.1% oluidine blue
245
dissol ed in glycine—HCl bu e (pH 1.8) o 15 min, he
246
unbound oluidine blue was washed in glycine—HCl bu e 247
o 1 h. The dye bound o highly sulpha ed p o eogly-
248
cans and glycosaminoglycans was ex ac ed in 8% HCl249
dissol ed in absolu e e hanol. Abso bance o samples con-250
aining ex ac ed oluidine blue was measu ed a he251
wa eleng h o 625 nm on a mic opla e eade (Chameleon,252
Hidex, Tu ku, Finland). Samples om 10 cul u es o each253
expe imen al g oup we e de e mined in 5 independen 254
expe imen s. Da a we e s a is ically analyzed wi h S uden ’s255
- es .256
Measu emen o cell p oli e a ion wi h257
3H- hymidine labelling and mi ochond ial ac i i y258
wi h MTT assay
259
Fo measu emen o p oli e a ion a e o cells in HDC260
15 ␮L d ople s o cell suspension we e inocula ed in o261
wells o special, opaque 96-well mic o i e pla es (Wal-262
lac, Pe kinElme Li e and Analy ical Sciences, Shel on, CT,263
USA). Ham’s F12 medium con aining 1 ␮Ci/mL (185 GBq/mM)264
3H- hymidine (dilu ed om me hyl-3H- hymidine solu ion,265
Ame sham Biosciences, Budapes , Hunga y) was added o266
he wells o 16 h on di e en days o cul u ing. A e 267
washing wice wi h PBS (phospha e bu e ed saline), p o-268
eins we e p ecipi a ed wi h ice-cold 5% ichlo oace ic269
acid, washed wi h PBS again, and placed in an exsicca-270
o con aining phospho ous pen oxide in o de o abso b271
mois u e. P io o measu emen s, 50 ␮L scin illa ion solu-272
ion (MaxiLigh ; Hidex, Finland) was added o each well,273
and adioac i i y was coun ed by a liquid scin illa ion274
coun e (Chameleon, Hidex, Tu ku, Finland). Ten sam-275
ples o each expe imen al g oup om fi e independen 276
expe imen s we e s a is ically analyzed wi h S uden ’s -277
es .
278
Cellula me abolic ac i i y was de e mined by MTT assay,279
a means o measu ing he ac i i y o li ing cells ia mi o-280
chond ial dehyd ogenases. Cells cul u ed in wells o 96-well281
mic o i e pla es we e used and 10 ␮L MTT eagen [3-
282
(4,5-dime hyl hiazolyl-2)-2,5-diphenyl e azolium b omide;283
5 mg MTT/1 mL PBS] was added in o each well. Cells we e
284
incuba ed o 2 h a 37 ◦C in MTT-con aining Ham’s F12285
medium. Following addi ion o 100 ␮L MTT solubilizing solu-286
ion (10% T i on X-100 and 0.1 M HCl dissol ed in anhyd ous287
isop opanol) op ical densi y was measu ed a 570 nm on
288
a mic opla e eade (Chameleon, Hidex, Tu ku, Finland).
289
Ten samples o each expe imen al g oup om ou sepa-
290
a e expe imen s we e s a is ically analyzed wi h S uden ’s291
- es .292
De e mina ion o cell su i al by FACS analysis 293
One hund ed mic oli e s d ople s o cell suspension we e 294
inocula ed in o plas ic Pe i dishes. Following ea men s 295
o ei he A23187 o EGTA as gi en in he ‘‘T ea men s 296
wi h EGTA, A23187 calcium ionopho e and cyclospo ine A’’ 297
sec ion, cul u es we e washed wice wi h CMF-PBS (cal- 298
cium and magnesium ee PBS), incuba ed wi h 250 ␮L299
annexin-V DY647 eagen (Cen al Eu opean Biosys ems, 300
Budapes , Hunga y) and/o 10 ␮g/mL p opidium-iodide o 301
10 min a oom empe a u e, washed again wi h CMF-PBS, 302
and ypsinized o 15 min o ob ain cell suspension. Cells 303
we e collec ed by cen i uga ion a 800 ×g o 10 min, esus- 304
pended in 1 mL FACS bu e (PBS supplemen ed wi h 1% 305
BSA and 0.05% NaN3) and a e o cell su i al was de e - 306
mined using a CyFlow®space Flow Cy ome e (Pa ec GmbH, 307
M¨
uns e , Ge many). Annexin-V DY647 was moni o ed a 308
670 nm, p opidium-iodide was measu ed a 620 nm. Mea- 309
su emen lowe h eshold we e se on cell-size pa icles. 310
Analysis was pe o med wi h WinMDI 2.8 So wa e. 311
RT-PCR analysis 312
To al RNA was isola ed om cells using RNA Isola ion Ki 313
acco ding o he manu ac u e ’s ins uc ion (Gen a Sys- 314
ems Inc., Minneapolis, MN, USA). The assay mix u e o 315
e e se ansc ip ase eac ion con ained 2 ␮g RNA, 0.112 ␮M316
oligo(dT), 0.5 mM dNTP, 200 uni s M-MLV RT in 1 ×RT bu e . 317
The sequences o p ime pai s used o PCR eac ions we e 318
as ollows: o chicken agg ecan 5-CAA TGC AGA GTA CAG 319
AGA-3and 5-TCT GTC TCA CGG ACA CCG-3, o chicken 320
Sox9 5-CCC CAA CGC CAT CTT CAA-3and 5-CTG CTG ATG 321
CCG TAG GTA-3, o chicken calcineu in 5-CTG CTC TGA 322
TGA ACC AAC AGT T-3and 5-ACG GCA AGG ACC AGG TAA 323
ACA-3, o chicken GAPDH 5-GAG AAC GGG AAA CTT GTC 324
AT-3and 5-GGC AGG TCA GGT CAA CAA-3, o chicken 325
inosi ol-1,4,5- isphospha e ecep o (IP3R) ype-1 5-CGG 326
CTG TGG TCT GAG ATA C-3and 5-GGT AAT AGG GAA GAT 327
GGT AGT G-3, o chicken IP3R ype-2 5-AAG CCT ACC 328
TTA TGA CCT CC-3and 5-CAT TGT TTC CTC CAT CCT G- 329
3, and o chicken IP3R ype-3 5-TGT GGG TGG ACA AGA 330
AAG G-3and 5-GCA GGA ACT GAT GGG TGA A-3. Ampli- 331
fica ions we e pe o med in a he mocycle (PCR Exp ess 332
Tempe a u e Cycling Sys em, Hybaid, UK) as ollows: 94 ◦C, 333
1 min, ollowed by 30 cycles (94 ◦C, 30 s, 54 ◦C, 30 s, 72 ◦C, 334
30 s) and hen 72 ◦C, 5 min. Thi y-fi e cycles we e used 335
a 57 ◦C o IP3- ecep o s. PCR p oduc s we e analyzed by 336
elec opho esis in 1.2% aga ose gel con aining e hidium b o- 337
mide. 338
Wes e n-blo analysis 339
To al cell lysa es o endoplasmic e iculum (ER) ac ion o 340
HDC we e examined by Wes e n blo . Cell cul u es we e 341
ha es ed immedia ely a e ea men s on espec i e days 342
o cul u ing. Cell pelle s we e suspended in 100 ␮Lo 343
homogeniza ion bu e [con aining 50 mM T is—HCl bu e 344
(pH 7.0), 0.5 mM di hio h ei ol, 10 ␮g/mL Go dox, 10 ␮g/mL 345
leupep in, 1 mM phenylme hylsulphonyl (PMSF), 5 mM ben- 346
zamidine and 10 ␮g/mL ypsin inhibi o ]. A e s o ing hem 347
a −70 ◦C, suspensions we e sonica ed by pulsing bu s o 348
UNCORRECTED PROOF
YCECA 972 1—14
Please ci e his a icle in p ess as: C. Ma a, e al., Cy osolic ee Ca2+ concen a ion exhibi s a cha ac e is ic empo al
pa e n du ing in i o ca ilage di e en ia ion: A possible egula o y ole o calcineu in in Ca-signalling o chond ogenic
cells, Cell Calcium (2008), doi:10.1016/j.ceca.2007.12.010
ARTICLE IN PRESS
+Model
YCECA 972 1—14
Possible egula o y ole o calcineu in in Ca-signalling o chond ogenic cells 5
ou imes 30 s by 50 cycles (B anson Sonifie , Danbu y,349
USA). ER ac ion o HDC was p epa ed om 3-day-old cul-350
u es g own in Pe i dishes. Cells we e homogenized using351
a Dounce homogenise in a bu e con aining 5 mM HEPES,352
320 mM suc ose and p o ease inhibi o s [3.4 ␮g/mL Go dox,353
3.4 ␮g/mL leupep in, 1 mM phenylme hylsulphonyl (PMSF),354
1.6 mM benzamidine and 3.4 ␮g/mL ypsin inhibi o , pH355
7.4]. A e cen i uga ion a 4500 ×g o 20 min, supe -356
na an was collec ed and cen i uged a 10,000 ×g o
357
15 min. Supe na an was cen i uged a 150,000 ×g o 358
120 min. Pelle con aining endoplasmic e iculum esicles359
(mic osome ac ion) was collec ed in lysis bu e (50 mM360
T is—HCl, pH 7.2 con aining p o ease inhibi o s, see abo e),361
snap- ozen in liquid ni ogen and s o ed a −70 ◦C. Sam-362
ples o SDS-PAGE we e p epa ed by he addi ion o 1/5363
olume o fi e old concen a ed elec opho esis sample364
bu e (310 mM T is—HCl, pH 6.8; 10% SDS, 50% glyc-
365
e ol, 100 mM DTT, 0.01% b omophenol blue) o cell lysa es366
and boiled o 10 min. Abou 40 ␮g o p o ein was sepa-367
a ed by 7.5% SDS-PAGE gel o immunological de ec ion368
o Sox9, phospho-Sox9, RyR, IP3- ecep o and calcineu in.369
P o eins we e ans e ed elec opho e ically o ni ocel-370
lulose memb ane. A e blocking in 5% non- a d y milk371
in PBS, memb anes we e incuba ed wi h he ollowing
372
p ima y an ibodies o e nigh a 4 ◦C: polyclonal an i-Sox9373
an ibody (Abcam L d., Camb idge, UK) in 1:200 dilu ion,
374
polyclonal an i-phospho-Sox9 an ibody (Sigma, Budapes ,
375
Hunga y) in 1:200 dilu ion, monoclonal an i-RyR an ibody376
(A fini y BioReagen s, Golden, CO, USA) in 1:1000 dilu ion,377
polyclonal an i-IP3R ype 1 an ibody (Sigma, Budapes , Hun-378
ga y) in 1:250 dilu ion and polyclonal an i-calcineu in (␣379
subuni ) an ibody (Ups a e, Dundee, Sco land, UK) in 1:200380
dilu ion. A e washing h ee imes o 10 min wi h PBST (PBS
381
supplemen ed wi h 0.1% Tween 20), memb anes we e incu-
382
ba ed wi h a seconda y an ibody, an i-mouse IgG (Sigma,383
Budapes , Hunga y) in 1:1000 dilu ion o RyR and an i-384
abbi IgG (Sigma, Budapes , Hunga y) in 1:1000 dilu ion
385
o de ec ion o IP3 ecep o , Sox9, phospho-Sox9 and cal-386
cineu in in PBS con aining 5% non- a d y milk o 1 h. Signals
387
we e de ec ed by enhanced chemiluminescence (Ame sham388
Biosciences, Budapes , Hunga y).389
Enzyme ac i i y assay o calcineu in390
Ac i i y o calcineu in was measu ed by he elease391
o 32Pi om 32P-labelled p o ein phospha ase inhibi o -
392
1 (780 cpm/pmol) as desc ibed by Yang e al. [24]
393
wi h some modifica ions [25]. Thi y mic oli e s o he
394
assay mix u e (50 mM T is—HCl bu e pH 7.0) con ain-395
ing 0.16 mM di hio h ei ol, 3.4 ␮g/mL Go dox, 3.4 ␮g/mL396
leupep in, 1 mM phenylme hylsulphonyl (PMSF), 1.6 mM ben-397
zamidine, 3.4 ␮g/mL ypsin inhibi o , 40 ␮g/mL calmodulin,
398
0.2 mM CaCl2, 100 nM okadaic acid (OA), 2 nM p o ein399
phospha ase inhibi o -2, ex ac con aining abou 80 ␮g400
p o ein and 32P-labelled p o ein phospha ase inhibi o -1401
(20—30,000 cpm/ eac ion mix u e) was incuba ed a 30 ◦C
402
o 20 min. The eac ion was e mina ed by he addi ion o 403
100 ␮L o 20% ichlo oace ic acid and 100 ␮L o 6 mg/mL
404
bo ine se um albumin. A e cen i uga ion, 32Pi-con en o 405
180 ␮L o he supe na an ac ion was de e mined in a liq-406
uid scin illa ion coun e .407
Resul s 408
Cy osolic ee Ca2+ concen a ion o un ea ed cell 409
cul u es shows a cha ac e is ic age-dependen 410
pa e n 411
Cy osolic ee Ca2+ concen a ion was de e mined in Fu a- 412
2-loaded cells on di e en days o cul u ing. Basal le el o 413
in acellula Ca2+ concen a ion o chond oblas s was ound 414
o ha e an age-dependen pa e n (Fig. 1A). Ini ially, Ca2+ 415
le el is low, wi h a s a ing concen a ion o abou 75 nM on 416
day 0, hen i sligh ly inc eases in pa allel wi h he p og es- 417
sion o di e en ia ion. A 140 nM peak o he cy osolic ee 418
Ca2+ concen a ion was obse ed on day 3 o cul u ing in 419
cells o un ea ed con ol cul u es. I should be no ed ha 420
chond ogenic cells o HDC di e en ia e in o chond oblas s 421
on his day o cul u ing [3]. F om day 4, Ca2+ le el d ops, 422
howe e , i e ains a sligh ly ele a ed concen a ion (abou 423
100 nM) as compa ed o days 0—2. 424
Di e en ia ing chicken chond op ogeni o cells exhibi 425
pe iodical inc eases in cy osolic ee Ca2+ (Fig. 1B—D). These 426
oscilla ions we e de ec able mos ly on cul u ing day 3. F e- 427
quencies o oscilla ions we e simila in all he cells obse ed: 428
he pe iod was 4 ±1.2 s (mean ±s anda d e o o he mean; 429
n= 20), maximum ampli udes we e 15—20% highe han he 430
mean basal fluo escence in ensi y. 431
Changes o cy osolic Ca2+ concen a ion o HDC 432
ha dly depend on in acellula sou ces 433
We aimed o de e mine whe he he ex acellula Ca2+ o 434
he in acellula Ca-s o es could be he sou ce o ele a ed 435
cy osolic Ca2+ le els. The Ca concen a ion o F12 medium 436
con aining 10% FCS p o ed o be app oxima ely 0.78 mM 437
acco ding o AAS measu emen s (da a no shown). In o de 438
o educe he concen a ion o ee Ca2+ in he cul u e 439
medium, EGTA was applied in equimola (0.8 mM) concen a- 440
ion. EGTA ea men significan ly dec eased he cy osolic 441
Ca2+ le el o app oxima ely 60% o ha o un ea ed con ol 442
cells (Fig. 2A). Twel e hou s o EGTA ea men p o ed o be 443
e ec i e; when main ained in 0.8 mM EGTA h oughou he 444
cul u ing pe iod, cul u es de ached om he glass o plas- 445
ic su ace and died. To in es iga e he ole o in acellula 446
Ca-s o es, cyclopiazonic acid (CPA), an inhibi o o he Ca2+ 447
pump o smoo h endoplasmic e iculum was adminis e ed a 448
a concen a ion o 10 ␮M o 8 min. To emo e ee Ca2+ om 449
he medium, cells we e washed wi h Ca2+- ee Ty ode’s p io 450
o adminis a ion o CPA. A e addi ion o Ca2+- ee Ty ode’s 451
he basal cy osolic Ca2+ le el dec eased om 140 o 120 nM 452
showing he dependence o his pa ame e on he ex a- 453
cellula Ca2+ concen a ion. App oxima ely 60 s a e he 454
adminis a ion o 10 ␮M CPA, cy osolic Ca2+ le el s a ed o 455
inc ease e y slowly (Fig. 2B). The sligh ele a ion in cy oso- 456
lic Ca2+ clea ly shows ha he in acellula Ca2+-s o es a e 457
no emp y, howe e , he amoun o s o ed Ca2+ is ei he low 458
o he a e o leak is small. When adminis a ion o bo h CPA 459
and Ca- ee Ty ode’s ceased and we e washed ou wi h no - 460
mal Ca2+ con aining solu ion, a well-defined peak in cy osolic 461
Ca2+ le el could be obse ed owing o he en y o ex acel- 462
lula Ca2+ in o he cy osol, eflec ing on a possible ac i a ion 463

UNCORRECTED PROOF
YCECA 972 1—14
Please ci e his a icle in p ess as: C. Ma a, e al., Cy osolic ee Ca2+ concen a ion exhibi s a cha ac e is ic empo al
pa e n du ing in i o ca ilage di e en ia ion: A possible egula o y ole o calcineu in in Ca-signalling o chond ogenic
cells, Cell Calcium (2008), doi:10.1016/j.ceca.2007.12.010
ARTICLE IN PRESS
+Model
YCECA 972 1—14
6 C. Ma a e al.
Figu e 1 Day-by-day a ia ion o basal in acellula Ca2+ le els in chond i ying cells o un ea ed con ol HDC (A). Ca2+ concen-
a ions we e de e mined in Fu a-2-loaded cells as desc ibed in he ‘‘Ma e ials and me hods’’ sec ion. Rep esen a i e da a o fi e
independen expe imen s showing mean alues o basal in acellula Ca2+ le els o 30 cells ±s anda d e o o he mean. S a is ical
analysis by S uden ’s - es compa ing he espec i e da a o he p e ious cul u ing day, *P< 0.01. Spon aneous calcium e en s in
cells o chond i ying high-densi y mic omass cell cul u es on cul u ing day 3 (B—D). Calcium ansien s we e de e mined in Fluo-4-AM
loaded cells as gi en in he ‘‘Ma e ials and me hods’’ sec ion. Changes o fluo escence in ensi y we e eco ded in a 50 s in e al.
x—yanalysis o a ep esen a i e cell is shown in panel B, ime cou se o mean in ensi y is shown in panel C. A ep esen a i e calcium
e en measu emen pe o med by line scan imaging is shown in panel D.
UNCORRECTED PROOF
YCECA 972 1—14
Please ci e his a icle in p ess as: C. Ma a, e al., Cy osolic ee Ca2+ concen a ion exhibi s a cha ac e is ic empo al
pa e n du ing in i o ca ilage di e en ia ion: A possible egula o y ole o calcineu in in Ca-signalling o chond ogenic
cells, Cell Calcium (2008), doi:10.1016/j.ceca.2007.12.010
ARTICLE IN PRESS
+Model
YCECA 972 1—14
Possible egula o y ole o calcineu in in Ca-signalling o chond ogenic cells 7
Figu e 2 Basal in acellula Ca2+ le els o 0.8 mM EGTA- ea ed cul u es on days 2 and 3 (A). Measu emen s we e ca ied ou
ollowing 12 h o EGTA ea men s. Da a ep esen mean ±s anda d e o o he mean o in acellula Ca2+ le els o 30 cells measu ed
in 5 ep esen a i e expe imen s. E ec o cyclopiazonic acid (CPA) on he elease o Ca2+ om in acellula s o es (B). Rep esen a i e
eco d ou o 30 cells in 5 independen expe imen s. As e isks indica e significan (*P< 0.01) dec ease in basal Ca2+ concen a ion
as compa ed o he espec i e con ol.
o s o e-ope a ed Ca2+ en y (SOCE) channels [26]. The exac 464
na u e o his phenomenon, howe e , emains o be u he 465
in es iga ed.466
To in es iga e he possible ole o in e nal Ca-s o es in467
he egula ion o Ca-homeos asis o di e en ia ing chon-468
d op ogeni o cells, u he expe imen s we e pe o med.469
Ten mic omola CPA was adminis e ed o he cul u e medium
470
o HDC on day 2 o 3 o 12 h. This p olonged inhibi ion o 471
he Ca-pump o smoo h endoplasmic e iculum mus ha e472
esul ed in a comple e abolishmen o in acellula s o es,
473
howe e , no de imen al e ec on chond ogenesis could be474
obse ed (Fig. 3A). CPA- ea men sligh ly ele a ed cy oso-475
lic Ca2+ le el (Fig. 3B), which could be ela ed o he unc ion476
o SOCE channels. Combined ea men s wi h EGTA and CPA477
o 12 h (i.e., inhibi ion o Ca2+ en y om bo h ex acellu-478
la and in acellula sou ces) esul ed in a comple e loss o 479
me ach oma ically s ained ca ilage ma ix demons a ing
480
he Ca2+ dependence o in i o chond ocy e di e en ia-481
ion (da a no shown). This phenomenon may implica e he482
insu ficien capaci y o in acellula Ca-s o es o eplenish483
he unc ion o Ca2+ en y pa hways in chond ogenic cells.484
The impo ance o Ca2+-influx om ex acellula space485
is u he suppo ed by in es iga ing he endoplasmic486
e iculum yanodine ecep o (RyR) and he inosi ol-1,4,5-487
isphospha e (IP3) ecep o s. RyR was no de ec able by488
Wes e n blo analyses pe o med on o al cell lysa es o HDC 489
and only a weak band was obse ed in samples con aining 490
sepa a ed endoplasmic e iculum ac ion o HDC (Fig. 4A). 491
Fu he mo e, no esponse was de ec ed when ca eine, an 492
agonis o RyR was adminis e ed du ing single cell measu e- 493
men s (Fig. 4B). These esul s indica e ha hough p esen , 494
he low amoun o RyR loca ed in he endoplasmic e icu- 495
lum o cells o HDC may no be unc ioning and p obably 496
does no significan ly con ibu e o he ele a ion o basal 497
cy osolic Ca2+. Amplifica ion o IP3 ecep o iso o ms by RT- 498
PCR shows ha only he mRNA o IP3R ype 1 is exp essed 499
by cells o HDC (Fig. 4C). The IP3 ecep o p o ein could be 500
ha dly de ec ed in samples p epa ed om he endoplasmic 501
e iculum ac ion o chond ogenic cells by Wes e n blo ing 502
(Fig. 4D). 503
Dec eased ex acellula Ca2+ concen a ion inhibi s 504
ca ilage o ma ion and educes he exp ession o 505
chond ogenic mas e ansc ip ion ac o Sox9 506
Cul u es ea ed wi h EGTA o 12 h on day 2 o 3 o cul u - 507
ing showed a p o ound dec ease in me ach oma ic s aining 508
pe o med on day 6 o cul u ing (9% o 17% o un ea ed 509
con ol cul u es, espec i ely, Fig. 5A), demons a ing a sig- 510
Figu e 3 E ec o 10 ␮M CPA on ca ilage o ma ion in chond i ying mic omass cul u es (A). Me ach oma ic ca ilage a eas in
6-day-old high-densi y colonies isualized wi h DMMB dissol ed in 3% ace ic acid. Op ical densi y (OD625) o samples con aining
oluidine blue ex ac ed wi h 8% HCl dissol ed in absolu e e hanol. Da a a e mean alues ±s anda d e o o he mean (±7%) o
each expe imen al g oup ou o 10 measu emen s. Basal in acellula Ca2+ le els o 10 ␮M CPA- ea ed cul u es on days 2 and 3 (B).
Measu emen s we e ca ied ou di ec ly a e he 12-h ea men wi h CPA. Da a ep esen mean ±s anda d e o o he mean o
in acellula Ca2+ le els o 30 cells measu ed in 5 independen expe imen s.
UNCORRECTED PROOF
YCECA 972 1—14
Please ci e his a icle in p ess as: C. Ma a, e al., Cy osolic ee Ca2+ concen a ion exhibi s a cha ac e is ic empo al
pa e n du ing in i o ca ilage di e en ia ion: A possible egula o y ole o calcineu in in Ca-signalling o chond ogenic
cells, Cell Calcium (2008), doi:10.1016/j.ceca.2007.12.010
ARTICLE IN PRESS
+Model
YCECA 972 1—14
8 C. Ma a e al.
Figu e 4 De ec ion o he exp ession and unc ion o RyR and IP3R in HDC. Wes e n blo analysis o RyR in ER ac ion o cells
o HDC on cul u ing day 3 (A). Rep esen a i e da a o fi e independen expe imen s. E ec o he RyR-agonis ca eine (15 mM) on
basal cy osolic Ca2+ le el o cells o HDC on cul u ing day 3 (B). Rep esen a i e eco d ou o 30 cells in 5 independen expe imen s.
Amplifica ion o chicken IP3R ype 1 ecep o (C). Rep esen a i e esul ou o h ee independen expe imen s. Wes e n blo analysis
o IP3R in ER ac ion o cells in HDC (D). Rep esen a i e analysis ou o h ee independen expe imen s.
nifican educ ion o ca ilage o ma ion. Adminis a ion o 511
EGTA a e day 5 did no ha e any significan e ec on ma ix512
p oduc ion (da a no shown).513
Inhibi ion o chond ogenesis was moni o ed by de ec ion514
o mRNAs o agg ecan co e p o ein and Sox9, he majo
515
ca ilage-specific ansc ip ion ac o . A significan dec ease516
in he mRNA le els o bo h agg ecan co e p o ein and Sox9
517
was obse ed unde he e ec o EGTA on each day o ea -
518
men s, demons a ing ha educed in acellula Ca2+ le el519
dec eases ca ilage o ma ion, a leas pa ly, ia inhibi ion
520
o ca ilage di e en ia ion (Fig. 5B). These findings we e
521
u he suppo ed by Wes e n blo analyses showing ha 522
ea men wi h EGTA sligh ly educed he p o ein le el o 523
Sox9 on day 2, and a significan dec ease was obse ed in524
he phospho yla ion le el o Sox9 on day 3 (Fig. 5C).
525
Ca2+ ionopho e has dual concen a ion-dependen 526
e ec s on ca ilage o ma ion 527
Abou 0.1 mg/L concen a ion o he Ca2+ ionopho e A23187 528
aised he in acellula Ca2+ le els o app oxima ely 125% o 529
un ea ed con ol cells, and 5 mg/L concen a ion o Ca2+ 530
ionopho e esul ed in an e en highe inc ease (abou 150%) 531
in cy osolic Ca2+ le els (Fig. 6A). On he o he hand, he wo 532
concen a ions o ionopho e applied had opposi e e ec s 533
on ca ilage o ma ion. Following ea men wi h he lowe 534
concen a ion (0.1 mg/L) o A23187 on bo h days 2 and 3 o 535
one hou , an ex ensi e ca ilage o ma ion occu ed by day 536
6; when ea men was pe o med on day 3 only, he amoun 537
o me ach oma ically s ained ca ilage ma ix inc eased o 538
153% o con ol cul u es (Fig. 6B). On he con a y, highe 539
Figu e 5 E ec o 0.8 mM EGTA on ca ilage de elopmen o chond i ying mic omass cul u es (A). Me ach oma ic ca ilage a eas
in 6-day-old high-densi y colonies isualized wi h DMMB dissol ed in 3% ace ic acid. Op ical densi y (OD625) o samples con aining
oluidine blue ex ac ed wi h 8% HCl dissol ed in absolu e e hanol. Da a a e mean alues ±s anda d e o o he mean (±5%) o each
expe imen al g oup ou o 10 measu emen s. E ec o EGTA on he exp ession o agg ecan and he exp ession and phospho yla ion
o Sox9 ansc ip ion ac o in HDC o a ious ages (B and C). Fo RT-PCR eac ions GAPDH was used as a con ol. Rep esen a i e
da a o fi e independen expe imen s. As e isks indica e significan (*P< 0.01) dec ease in op ical densi y o ex ac ed oluidine
blue as compa ed o he espec i e con ol.
UNCORRECTED PROOF
YCECA 972 1—14
Please ci e his a icle in p ess as: C. Ma a, e al., Cy osolic ee Ca2+ concen a ion exhibi s a cha ac e is ic empo al
pa e n du ing in i o ca ilage di e en ia ion: A possible egula o y ole o calcineu in in Ca-signalling o chond ogenic
cells, Cell Calcium (2008), doi:10.1016/j.ceca.2007.12.010
ARTICLE IN PRESS
+Model
YCECA 972 1—14
Possible egula o y ole o calcineu in in Ca-signalling o chond ogenic cells 9
Figu e 6 E ec s o he Ca2+ ionopho e A23187 on ca ilage de elopmen o chond i ying mic omass cul u es. Basal in acellula
Ca2+ le els o 0.1 o 5 mg/L A23187- ea ed cul u es on days 2 and 3 (A). Measu emen s we e ca ied ou di ec ly a e he 30-min
ea men wi h A23187. Da a ep esen mean ±s anda d e o o he mean o in acellula Ca2+ le els o 30 cells measu ed in 5
independen expe imen s. Me ach oma ic ca ilage a eas in 6-day-old high-densi y colonies isualized wi h DMMB dissol ed in 3%
ace ic acid (B). Op ical densi y (OD625) o samples con aining oluidine blue ex ac ed wi h 8% HCl dissol ed in absolu e e hanol.
Da a shown a e mean alues ±s anda d e o o he mean (±6%) o each expe imen al g oup ou o 10 measu emen s. E ec o
A23187 on he mRNA exp ession o agg ecan and Sox9 ansc ip ion ac o (C) and p o ein exp ession and phospho yla ion o Sox9
(D) in HDC o a ious ages. Fo RT-PCR eac ions GAPDH was used as a con ol. Rep esen a i e da a o fi e independen expe imen s.
As e isks indica e significan (*P< 0.01) inc ease in basal Ca2+ concen a ions o significan (*P< 0.01) change in op ical densi y o
ex ac ed oluidine blue as compa ed o he espec i e con ol.
concen a ion (5 mg/L) o A23187 caused a ma ked inhibi-
540
ion o ca ilage o ma ion, especially when adminis e ed541
on day 2, educing he amoun o me ach oma ically s ained542
ca ilage ma ix o app oxima ely 36% o un ea ed con ol543
cul u es (Fig. 6B). Since mRNA le els o ca ilage di e en-544
ia ion ma ke s Sox9 and agg ecan co e p o ein, and bo h
545
p o ein le el and phospho yla ion o Sox9 showed a sligh 546
inc ease a he han inhibi ion unde he e ec o 5 mg/L547
ionopho e (Fig. 6C and D), he mechanism o he dec ease o 548
ca ilage o ma ion should be u he in es iga ed. Admin-549
is a ion o A23187 a e day 5 did no ha e any significan 550
e ec on ma ix p oduc ion (da a no shown).
551
E ec s o EGTA, A23187 Ca2+ ionopho e and CPA552
on a e o p oli e a ion, mi ochond ial ac i i y and
553
cellula iabili y o cells o HDC554
Cell p oli e a ion, mi ochond ial ac i i y and cellula iabil-555
i y assays we e pe o med ollowing ea men s wi h EGTA,556
he Ca2+ ionopho e A23187 and CPA, since dec eased ca -557
ilage ma ix p oduc ion could be accoun ed o ei he by
558
he inhibi ion o he di e en ia ion o chond ogenic mes-559
enchymal cells o chond oblas s o by he dec ease in cell560
numbe s. Al hough he p oli e a ion a e o cells in mic o- 561
mass cul u es was significan ly educed unde he e ec o 562
0.8 mM EGTA (Fig. 7A), he mi ochond ial ac i i y o cells 563
was no a ec ed (Fig. 7B) as e ealed by 3H- hymidine 564
inco po a ion and MTT assay, espec i ely. Cell p oli e a- 565
ion was sligh ly s imula ed by low concen a ion o he Ca2+ 566
ionopho e A23187 on day 3 (Fig. 7A). High concen a ion 567
(5 mg/L) o A23187 educed he a e o cell p oli e a ion 568
on bo h days, bu i did no cause any significan dec ease 569
in mi ochond ial ac i i y (Fig. 7B). T ea men wi h CPA did 570
no influence he p oli e a ion a e o cells (Fig. 7A) and 571
i did no p o e o be cy o oxic acco ding o MTT assays 572
(Fig. 7B). 573
Cellula iabili y ollowing ea men s wi h EGTA o he 574
Ca2+ ionopho e A23187 was also analyzed by FACS (Fig. 7C575
and D). The ionopho e did no influence he dis ibu ion o 576
cells compa ed o he con ol. The pe cen age inc ease o 577
dead cells we e as ollows: 3.2% and 0.7% (in he p esence o 578
0.1 mg/L) and 1.8% and 3.5% (in he p esence o 5 mg/L) on 579
days 2 and 3, espec i ely. T ea men wi h 0.8 mM EGTA led 580
o a highe a e o cell dea h, hus he pe cen age inc ease 581
o dead cells p o ed o be 10% and 9.1% on days 2 and 3, 582
espec i ely. Apop osis was no de ec ed in he ionopho e- 583
ea ed HDC (da a no shown).