UNCORRECTED PROOF
Please ci e his a icle in p ess as: J. Fodo , e al., Iono opic pu ine gic ecep o P2X4is in ol ed in he egula ion o chond ogenesis in
chicken mic omass cell cul u es, Cell Calcium (2009), doi:10.1016/j.ceca.2009.02.004
ARTICLE IN PRESS
G Model
YCECA10501–10
Cell Calcium xxx (2009) xxx–xxx
Con en s lis s a ailable a ScienceDi ec
Cell Calcium
jou nal homepage: www.else ie .com/loca e/ceca
Iono opic pu ine gic ecep o P2X4is in ol ed in he egula ion
o chond ogenesis in chicken mic omass cell cul u es
1
2
János Fodo a,1, Csaba Ma ab,1, Tamás Juhászb, Tamás Oláha, Mónika Gönczia,
Zsol Szíjgyá óc, Pál Ge gelyc, 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, 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
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,
Medical and Heal h Science Cen e, Uni e si y o Deb ecen, Hunga y
7
8
9
a icle in o10
11
A icle his o y:12
Recei ed 5 Augus 200813
Recei ed in e ised o m 2 Feb ua y 200914
Accep ed 9 Feb ua y 2009
15
A ailable online xxx16
17
Keywo ds:18
In i o ca ilage o ma ion19
High densi y cell cul u e20
Fu a-221
Single cell Ca measu emen 22
P2X ecep o s
23
P2Y ecep o s24
ATP sec e ion25
Immunocy ochemis y26
abs ac
We ha e p e iously demons a ed ha ele a ion o ee cy osolic Ca2+ concen a ion a he ime o di e -
en ia ion o chond oblas s was mainly due o a Ca2+ influx and i was indispensable o ca ilage o ma ion
in chicken high densi y mesenchymal cell cul u es (HDC) [C. Ma a, J. Fodo , Z. Szijgya o, T. Juhasz, P. Q1
Ge gely, L. Cse noch, R. Zakany, 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 44 (2008) 310–323]. He e, we epo ha chond ogenic cells sec e ed
ATP and adminis a ion o ATP o he cul u e medium e oked Ca2+ ansien s exclusi ely in he p es-
ence o ex acellula Ca2+ and only on day 3 o cul u ing, when he final commi men o chond oblas s
occu s. Mo eo e , ATP caused ele a ed p o ein exp ession o he chond ogenic ansc ip ion ac o Sox9
and s imula ed ca ilage ma ix p oduc ion. Exp ession pa e n o di e en ypes o bo h iono opic and
me abo opic pu ine gic ecep o s was de ec ed. Agonis s o me abo opic ecep o s, ADP and UDP did
no e oke any Ca2+ ansien s andhad noinfluence on ca ilage o ma ion, while UTP caused ansien ele-
a ion o cy osolic Ca2+ concen a ion in 3-day-old HDC wi hou s imula ing ma ix p oduc ion. Su amin,
whichblocks all P2X ecep o sbu no P2X4didno impede he e ec s o ATP, u he mo e,P2X4appea ed
in he plasma memb ane ac ion and ga e signals wi h immunocy ochemis y only om day 3. In sum-
ma y, we sugges a ole o iono opic pu ine gic signalling o P2X4in he gene a ion o ATP-dependen
Ca2+ ansien s o di e en ia ing chond oblas s.
© 2009 Published by Else ie L d.
1. In oduc ion27
Complex egula o y and signalling ne wo ks in ol ing28
cell–ma ix and cell–cell in e ac ions, including igh ly egula ed
29
gene exp ession, media e he successi e s ages o p oli e a ion,30
nodule o ma ion and di e en ia ion ha p oduce hyaline ca i-
31
lage [2]. One o he key egula o s o hese signalling p ocesses32
in chond ogenic cells is Sox9. Since he exp ession o collagen33
ype II and he co e p o ein o agg ecan a e con olled by his
34
ansc ip ion ac o , Sox9 is o en e e ed o as he mas e gene o
35
chond ogenesis [3,4]. Ano he impo an ac o in he egula ion36
o molecula s eps leading o chond ogenic di e en ia ion is he37
ansien ele a ion o he in acellula Ca2+ concen a ion [1]. The38
igh ly egula ed le el o cy osolic Ca2+ is in ol ed in a numbe o 39
∗Co esponding au ho . Fax: +36 52 432 290.
E-mail add ess: [email p o ec ed] e.hu (R. Zákány).
1These wo au ho s con ibu ed equally o he wo k.
signalling p ocesses in a a ie y o cell ypes. Among non-exci able 40
cells, he ole o in acellula Ca2+ in he di e en ia ion p ocess 41
o ke a inocy es [5] and os eoblas s [6] has been es ablished. 42
Cy osolic- ee Ca2+ concen a ion changes a e cha ac e ized by 43
long- e m, high ampli ude changes, and by sho - e m, spon a- 44
neous, pe iodic Ca2+ concen a ion changes, so called oscilla ions 45
in di e en ia ing mesenchymal s em cells [7]. Mo eo e , he wo 46
di e en ypes o Ca2+ concen a ion changes influence he ac i i y 47
o di e en ansc ip ion ac o s: oscilla ions ac i a e CREB, while 48
long- ime sus ained Ca2+ concen a ion ele a ions ac i a e NFAT 49
[8,9]. Bo h ansc ip ion ac o s ha e impo an unc ions du ing 50
chond ogenesis [10,11].51
High densi y cell cul u e (HDC) es ablished om chond ogenic 52
mesenchymal cells isola ed om dis al limb buds o 4-day-old 53
chicken emb yos is a widely accep ed model o in i o ca ilage 54
di e en ia ion [12,13], p o iding da a on he molecula egula- 55
ion o he di e en ia ion o chond op ogeni o mesenchymal cells 56
o chond oblas s. In his model a spon aneous ca ilage o ma- 57
ion occu s; he ini ial appea ance o chond oblas s and ca ilage 58
0143-4160/$ – see on ma e © 2009 Published by Else ie L d.
doi:10.1016/j.ceca.2009.02.004
UNCORRECTED PROOF
Please ci e his a icle in p ess as: J. Fodo , e al., Iono opic pu ine gic ecep o P2X4is in ol ed in he egula ion o chond ogenesis in
chicken mic omass cell cul u es, Cell Calcium (2009), doi:10.1016/j.ceca.2009.02.004
ARTICLE IN PRESS
G Model
YCECA10501–10
2J. Fodo e al. / Cell Calcium xxx (2009) xxx–xxx
specific ex acellula ma ix molecules akes place on day 3 o cul-59
u ing, and he majo i y o cells di e en ia e in o chond ocy es by60
day 6, when a high amoun o ca ilage ma ix can be de ec ed.61
In ou p e ious wo k [1] we epo ed ha a igh egula ion o 62
ee cy osolic Ca2+ le els (be ween 80 and 140 nM) is needed o 63
p ope chond ogenesis in cells o HDC. We also desc ibed a cha -64
ac e is ic empo al pa e n o he changes o cy osolic Ca2+ le els65
du ing chond ogenic di e en ia ion wi h a defini i e peak on day66
3 o cul u ing, he day on which chond op ogeni o cells di e -67
en ia e o chond oblas s. Al hough in acellula elemen s o Ca2+
68
homeos asis (e.g. RyR, IP3 ecep o and SERCA) we e de ec ed in69
chond ogeniccells, we ailed oshowanye idenceconce ning hei 70
con ibu ionine okingCa2+ ansien sin di e en ia ingchond ob-71
las s. The e o e, he ex acellula space has been p o ed o be he72
sou ce o he ele a ed cy osolic Ca2+ concen a ion.73
In he p esen wo k, we aimed o de e mine ansmemb ane74
p o eincandida es esponsible o heCa2+ influxin ochond ogenic75
cells. The amily o pu ine gic ecep o s is ubiqui ously p esen 76
in a numbe o cell ypes and p o ides ecep o s o ex acellula 77
nucleo ides ac ing as pa ac ine o au oc ine media o s. Pu ine gic78
ecep o s ha e wo majo ypes: P1 ecep o amilies a e sen-
79
si i e o adenosine, while P2 ecep o amilies a e sensi i e o80
ATP, ADP, and UTP. The la e is u he di ided in o wo majo 81
ecep o sub ypes: P2Y and P2X. Membe s o he me abo opic82
P2Y sub ype a e 7 ansmemb ane domain-con aining ecep o s83
coupled o G p o eins and linked o PLC signalling ansduc ion84
pa hways ha lead o he elease o in acellula Ca2+ om inosi ol-85
1,4,5- isphospha e (IP3)-sensi i e Ca2+ s o es. The iono opic P2X86
ecep o s a e ATP-ga ed ion channels allowing Ca-influx. Se en87
P2X subuni s (P2X1–P2X7) ha e been desc ibed and cloned so a 88
[14,15]. On he o he hand, eigh P2Y iso o ms ha e been desc ibed
89
in human issues: P2Y1, P2Y2, P2Y4, P2Y6, P2Y11 , P2Y12, P2Y13 and90
P2Y14 ecep o s. Al hough, based on sequence analysis P2Y3and91
P2Y5we e supposed o be membe s o he P2Y sub amily, u -92
he s udies e ealed ha hey ha e di e en phamacology and a e93
in ol ed in non-pu ine gic signalling pa hways as e iewed in [16].94
The ac ha P2X ecep o sa esequen iallyexp essedin emb yonic95
a and mouse skele al muscle cells and os eoblas s [17-19] aised96
he possibili y o he in ol emen o hese channels in he Ca2+
97
homeos asis o di e en ia ing chond ogenic mesenchymal cells.
98
In his s udy, we epo ha cells o HDC esponded o ex a-99
cellula ATP by ele a ing hei in acellula Ca2+ le els mainly a 100
he ime o chond oblas o ma ion and adminis a ion o ATP o101
he cul u e medium s imula ed chond ogenesis. Recep o s espon-102
sible o he ele a ion o Ca2+ concen a ion seemed o be membe s103
o he P2X amily, and based on ou da a, we p opose ha P2X4
104
ecep o scon ibu e o heele a iono cy osolicCa2+ le elso chon-105
d ogenic cells on day 3 o cul u ing. Mo eo e , cells o HDC sec e ed106
ATP in o he cul u ing medium, which suppo s ou heo y ha a107
pu ine gic au oc ine egula ion is in ol ed in he p ope con ol o 108
chond ogenesis.109
2. Ma e ials and me hods110
2.1. Cell cul u e111
High densi y cell cul u es we e p epa ed as desc ibed in [1].112
B iefly,dis alpa so helimbbudso 4-day-oldRosshyb idchicken113
emb yos (Hambu ge –Hamil on s ages 22–24 [20])we e emo ed
114
and chond i ying mic omass cul u es o mesenchymal cells we e
115
es ablished. 15 o 30L d ople s o he suspension con aining
116
1.5 ×107cells/mL we e inocula ed on ound co e glasses (diam-117
e e : 30 o 10mm; Menzel–Gläse , Menzel GmbH, B aunschweig,118
Ge many)placedin oplas icPe idishes(Nunc,Nape ille,IL,USA).
119
Cells we e allowed o a ach o he su ace o 2ha 37◦C. Day o 120
inocula ion is conside ed as day 0. Colonies we e g own in Ham’s 121
F12 medium (Sigma, Budapes , Hunga y) supplemen ed wi h 10% 122
e al cal se um (Gibco, Gai he sbu g, MD, USA), an ibio ics and 123
an imyco ics, and we e kep a 37◦C in an a mosphe e o 95% ai 124
and 5% CO2and 80% humidi y. The medium was changed on e e y 125
second day. 126
2.2. Single cell Ca2+ measu emen s 127
Measu emen s we e pe o med on di e en days o cul u ing 128
using he calcium dependen fluo escen dye Fu a-2 as desc ibed 129
p e iously [1]. Fu a-2-loaded cells we e placed on he s age o 130
an in e ed fluo escen mic oscope (Diapho , Nikon, Kowasaki, 131
Japan) and iewed using a 40×oil imme sion objec i e. Mea- 132
su emen s we e pe o med in no mal (137mM NaCl, 5.4mM KCl, 133
0.5mM MgCl2, 1.8mM CaCl2, 11.8mM HEPES, 1 g/L glucose, pH 7.4) 134
o calcium- ee (con aining 5mM EGTA, wi hou CaCl2) Ty ode’s 135
solu ion. ATP solu ion was p epa ed om no mal and Ca2+- ee 136
Ty ode’sa 180Mfinalconcen a ion.ADP,UDP,UTP(180M)and 137
b adykinin (20M) we e p epa ed om Ca2+- ee Ty ode’s. Be o e 138
applica ion o Ca2+- ee ATP, ADP, UDP and UTP, cells we e ea ed 139
in Ca2+- ee Ty ode’s o 150 s. Su amin solu ion was p epa ed in 140
Ty ode’s and used a a final concen a ion o 10 M. Exci a ion 141
wa eleng h was al e ed be ween 340 and 380nm and fluo escence 142
in ensi ies (F340 and F380) we e measu ed as desc ibed p e iously 143
[1]. Tes solu ions we e di ec ly applied o he cells h ough a pe - 144
usion capilla y ube (Pe usion PencilTM; Au oMa e Scien ific, San 145
F ancisco,CA,USA)wi hanin e naldiame e o 250ma a1.5L/s 146
a e, using a local pe usion sys em (Val e BankTM 8 e sion 2.0, 147
Au oMa e Scien ific). All measu emen s we e pe o med a oom 148
empe a u e. Da a we e s a is ically analyzed by S uden ’s - es . 149
2.3. P epa a ion o cell ex ac s 150
Cell cul u es we e ha es ed on each day o cul u ing. Cell pel- 151
le s we e suspended in 100 L o homogeniza ionbu e con aining 152
50mM T is–HCl bu e (pH 7.0), 10g/mL Go dox, 10g/mL leu- 153
pep in, 1mM phenylme hylsulphonyl (PMSF), 5mM benzamidine, 154
10 g/mL ypsin inhibi o and 0.5% T i on X-100. Samples we e 155
snap- ozen in liquid ni ogen, and we e s o ed a −70 ◦C. Samples 156
we e sonica ed o ou imes 30s by 50 cycles (B anson Soni- 157
fie , Danbu y, USA). Fo Wes e n blo analyses, o al cell lysa es 158
and plasma memb ane ac ions we e used. Fo isola ion o plasma 159
memb ane ac ion o HDC, sonica ed samples we e cen i uged a 160
50,000×g o 90min a 4◦C. Pelle was i u a ed con inuously in 161
50L homogeniza ion bu e supplemen ed wi h 1% T i on X-100 162
a 4◦C. A e 1h o i u a ion samples we e cen i uged again a 163
50,000×g o 55min a 4◦C, and supe na an con aining plasma 164
memb ane ac ion was used o Wes e n blo analyses. 165
2.4. RT-PCR analysis 166
Fo RT-PCR analysis, ca ilage colonies we e washed h ee imes 167
wi h RNase- ee physiological sodium chlo ide, snap- ozen in liq- 168
uid ni ogen and s o ed a −70 ◦C. To al RNA was isola ed om cells 169
o HDC o a ious ages using Quiagen RNeasy®Mic o Ki acco d- 170
ing o he ins uc ions o he manu ac u e (Quiagen, Budapes , 171
Hunga y). The assay mix u e (20L) o e e se ansc ip ase eac- 172
ion (Omnisc ip , Quiagen) con ained 500 ng RNA, 0.25 LRNase 173
inhibi o , 0.25Loligo (dT), 1L dNTP (200M), 1l M-MLV 174
RT in 1×RT bu e . Amplifica ions o specific cDNA sequences 175
we e pe o med wi h specific p ime s (In eg a ed DNA Technolo- 176
gies, Co al ille, IA, USA) ha we e designed based on published 177
chicken nucleo ide sequences ( o sequences o p ime pai s, see 178
Supplemen a y Ma e ial, Table 1). PCR eac ions we e allowed o 179
p oceed in a final olume o 50L (con aining 2 L o wa d and 180
UNCORRECTED PROOF
Please ci e his a icle in p ess as: J. Fodo , e al., Iono opic pu ine gic ecep o P2X4is in ol ed in he egula ion o chond ogenesis in
chicken mic omass cell cul u es, Cell Calcium (2009), doi:10.1016/j.ceca.2009.02.004
ARTICLE IN PRESS
G Model
YCECA10501–10
J. Fodo e al. / Cell Calcium xxx (2009) xxx–xxx 3
e e se p ime s, 1L dNTP [200M], and 5 uni s P omega GoTaq®
181
DNA polyme ase in 1× eac ion bu e ) in a p og ammable he -182
mocycle (Eppendo Mas e cycle, Ne hele , Hinz GmbH, Hambu g,183
Ge many) wi h he ollowing se ings: 2min a 95◦C o ini ial184
dena u a ion ollowed by epea ed cycles o dena u a ion a 94◦C185
o 1min, p ime annealing o 60s a an op imized empe a u e,186
and ex ension a 72◦C o 1min30s.A e he final cycle, u he 187
ex ension was allowed o p oceed o ano he 10 min a 72◦C. PCR188
p oduc s we e analyzed using a 1.5% e hidium b omide-s ained189
aga ose gel.190
2.5. Wes e n blo analysis191
To al cell lysa es and plasma memb ane ac ions we e exam-192
ined by Wes e n blo . Samples o SDS-PAGE we e p epa ed by193
he addi ion o 1/5 olume o 5- old concen a ed elec opho e-194
sis sample bu e (310 mM T is–HCl, pH 6.8; 10% SDS, 50% glyce ol,195
100 mM DTT, 0.01% b omophenol blue) o cell lysa es and boiled196
o 5min. Abou 50g o p o ein was sepa a ed by 7.5% SDS-PAGE197
gel o immunological de ec ion o P2X- ecep o s. P o eins we e
198
ans e ed elec opho e ically o ni ocellulose memb anes. A e 199
blocking in 5% non- a d y milk in PBS, memb anes we e incu-200
ba ed wi h p ima y an ibodies aised agains he ca boxy e mini201
o P2X- ecep o s (Alomone Labs, Je usalem, Is ael) and P2Y4 ecep-202
o (Sigma, Budapes , Hunga y), amino e minus o P2Y1 ecep o 203
(Sigma, Budapes , Hunga y) and 3 d in acellula loop o P2Y2
204
ecep o (AlomoneLabs,Je usalem,Is ael)o e nigh a 4◦Cin1:200205
dilu ion. A e washing h ee imes o 10min wi h PBST (PBS sup-206
plemen ed wi h 0.1% Tween 20), memb anes we e incuba ed wi h207
a seconda y an ibody, an i- abbi IgG (Sigma, Budapes , Hunga y)
208
in 1:1000 dilu ion in PBS con aining 5% non- a d y milk o 1h.209
Signals we e de ec ed by enhanced chemiluminescence eac ion210
(Ame sham Biosciences, Budapes , Hunga y).211
2.6. Immunocy ochemical s aining o P2X ecep o s212
3-day-old cul u es we e washed wice wi h PBS and fixed in213
4% pa a o maldehyde o 15min a 4◦C. A e washing in PBS, cells214
we e pe meabilized wi h 0.1% T i on X-100 in PBS o 30min. Non-215
specific binding si es we e blocked by 30min p eincuba ion in 1%
216
bo ine se um albumin (BSA) in PBS, ollowed by incuba ion wi h217
he p ima y an ibodies dilu ed in 1:100 a 4 ◦C o e nigh . Subse-218
quen ly, he cul u es we e washed h ee imes wi h PBS o 10min,
219
and we e incuba ed wi h a FITC-conjuga ed an i- abbi IgG (Vec-220
o Labo a o ies, Bu lingame, CA, USA) dilu ed 1:500 in PBS o 1h.221
Cul u es we e washed h ee imes wi h PBS and moun ed wi h222
Vec ashield®moun ing medium con aining DAPI (Vec o Labo a-223
o ies, Bu lingame, CA, USA). Con ol expe imen s we e ca ied224
ou wi h p ima y an ibodies incuba ed wi h hei con ol pep ides225
acco ding o he ins uc ions o he manu ac u e (da a no shown).
226
2.7. Adminis a ion o ex acellula ATP, ADP, UDP, UTP and227
su amin228
ATP (100 M), ADP, UDP and UTP (180M), and su amin229
(10M) we e adminis e ed o cells o HDC on a ious days o cul-
230
u ing. The nucleo ides and su amin we e dilu ed in he cul u e231
medium.E ec sonme ach oma icca ilagema ix o ma ionwe e232
examinedbyme ach oma ics ainingwi hdime hylme hyleneblue233
and oluidine blue as desc ibed p e iously [1].234
2.8. De e mina ion o ex acellula ATP in he cul u e medium235
Concen a ion o ex acellula ATP sec e ed by cells o high den-
236
si y cell cul u es was de e mined using Adenosine 5- iphospha e237
(ATP) Bioluminescen Assay Ki (Sigma, Budapes , Hunga y). Mea- 238
su emen s we e ca ied ou acco ding o he ins uc ions o he 239
manu ac u e , wi h mino modifica ions. B iefly, 20 d ople s o he 240
cell suspension (100 L each) we e inocula ed in o Pe i dishes 241
(diame e : 200mm, O ange Scien ifique, B aine-l’Alleud, Belgium) 242
and we e ed wi h 20mL cul u e medium. Concen a ion o ATP 243
sec e edbycells o HDC in o hecul u emedium wasde e mineda 244
app oxima ely he same pe iod o each cul u ing day. The medium 245
was changed e e y day ollowing measu emen s. 50L o he cul- 246
u e medium (pH adjus ed o 7.8) was used o de e mine he 247
amoun o ATP in he cul u e medium in 2 pa allel expe imen s. 248
Backg ound ligh emission was de e mined using blanks (bo h s e - 249
ile wa e and Ham’s F12 cul u e medium). Since he ATP Assay Mix 250
is no s able o a long pe iod, a g adual dec ease in he sensi i i y 251
may occu . The e o e, a new s anda d cu e was p epa ed each day 252
p io o measu emen s (concen a ions o ATP s anda d solu ions 253
we e as ollows: 10−6,10
−7,10
−8,10
−9and 10−10 M). Lumines- 254
cence o samples was de e mined using a mic owell pla e eade 255
(Chameleon, Hidex, Tu ku, Finland). 256
3. Resul s 257
3.1. Cells o HDC espond o ex acellula ATP by ele a ing 258
in acellula Ca2+ concen a ion 259
ATP a a cons an concen a ion o 180M was adminis e ed o 260
he close p oximi y o cells o HDC on a ious days o cul u ing. 261
Fig. 1 (A–E) shows ha adminis a ion o ATP could induce a an- 262
sien inc easein in acellula Ca2+ le elsincells o aCa2+ con aining 263
ba hing solu ion. No e ha bo h he ampli ude o he a e age 264
esponse (maximal inc ease in in acellula Ca2+ concen a ion) 265
and he ime o exposu e o ATP needed o induce he ansien 266
exhibi ed a di e en ia ion-dependen pa e n (see also Fig. 1F–G). 267
The sho es exposu e o ATP needed o e oke a Ca2+ ansien was 268
cha ac e is ic o cul u ing days 3 and 4, and a he same ime Ca2+ 269
ansien s wi h he highes ampli ude (179 and 165nM, espec- 270
i ely) we e also eco ded on hese days. On he fi s cul u ing day 271
ATP, e en hough adminis e ed o a long pe iod (120 s), could no 272
e oke any changes in he in acellula Ca2+ concen a ion (Fig. 1A), 273
u he mo e, we could ha dly de ec cha ac e is ic peaks in 2-day- 274
old cells ei he (Fig. 1B). By day 6, he ampli ude o he ansien s 275
dec easedandonlya p olongedapplica iono ATPcoulde okesuch 276
esponses (Fig. 1E). 277
S iking di e ences we e also obse ed ega ding he numbe o 278
cells esponding o ATP (Fig. 1F and G). While mos o he cells (90%) 279
esponded oATPin3-day-oldcul u es,essen iallynonedida day1 280
o cul u ing. Adminis a ion o ATP on o he days could also induce 281
calcium ansien s, bu he p opo ion o cells ha esponded was 282
ha dly compa able wi h ha on day 3 (Fig. 1F). 283
To es ablish whe he me abo opic o iono opic pu ine gic 284
ecep o s we e esponsible o hese e ec s, ATP was adminis e ed 285
o cells in a Ty ode’s solu ion lacking ee Ca2+ (Fig. 2A and B). In he 286
30 cells examined, no esponse was de ec ed on ei he days o cul- 287
u ing in he absence o ex acellula Ca2+. This obse a ion fi mly 288
suppo edou heo y ha influx o ex acellula Ca2+ wasneeded o 289
e oke he e ec o ex acellula ATP and he ecep o o ATP could 290
be a membe o he iono opic pu ine gic ecep o amily (P2X), bu 291
did no exclude he ole o me abo opic pu ine gic ecep o s and 292
in acellula Ca2+ s o es. 293
To find candida es among P2X ecep o s, hei non-specific 294
an agonis su amin was es ed on cells o HDC. I is known ha 295
su amin inhibi s all P2X ecep o s excep P2X4and P2X6[16].296
Cells ea ed wi h su amin (10M) showed no significan al e - 297
a ion in he in acellula Ca2+ le el ollowing he adminis a ion 298
o ATP (Fig. 2C). Repe i i e adminis a ion o ATP could induce 299
UNCORRECTED PROOF
Please ci e his a icle in p ess as: J. Fodo , e al., Iono opic pu ine gic ecep o P2X4is in ol ed in he egula ion o chond ogenesis in
chicken mic omass cell cul u es, Cell Calcium (2009), doi:10.1016/j.ceca.2009.02.004
ARTICLE IN PRESS
G Model
YCECA10501–10
4J. Fodo e al. / Cell Calcium xxx (2009) xxx–xxx
Fig. 1. E ec o 180M ATP on he cy osolic Ca2+ le els o Fu a-2-loaded cells o HDC. (A–E) Ca2+ ansien s e oked by adminis a ion o ATP in cells on di e en days o
cul u ing. Rep esen a i e eco ds o 5 independen expe imen s. Lines indica e he applica ion o ATP. (F) Ca2+ ansien s we e measu ed in he p esence o 1.8mM ex e nal
calcium. Ra io o cells esponding o ATP on each day o cul u ing. Rep esen a i e da a o 5 independen expe imen s. (G) Changes in he peak ampli ude o calcium ansien s
de ec ed on di e en days o cul u ing. Numbe s indica e he p opo ion o cells esponding o ATP. Rep esen a i e da a o 5 independen measu emen s.
epe i i e and ansien ele a ions in in acellula Ca2+ concen a-300
ion in 3-day-old HDC (Fig. 2D). Rela i ely sho pe iods o washing
301
(app oxima ely 100s) we e equi ed o allow he cells o eco e 302
om he 30-sec-long exposu es o ATP. This obse a ion, oge he 303
wi h he lack o significan desensi iza ion (Fig. 2D) aised he pos-304
sibili y o he p esence and unc ion o P2X4and/o P2X6 ecep o 305
sub ypes.306
Al hough he abo e esul s clea ly sugges ed ha P2X ecep-
307
o s play he decisi e ole, we also ca ied ou expe imen s o
308
ob ain da a on he unc ion o me abo opic P2Y ecep o s. Fi s 309
we in ended o examine whe he in acellula Ca2+ s o es a e310
p esen andcon ain eleasableCa2+ by heac i a iono IP3pa hway.311
Since b adykinin ecep o s a e known o ac i a e his pa hway and312
a e desc ibed as being exp essed by chond ocy es [21], he e o e313
b adykinin was adminis e ed o 3-day-old cells a a concen a ion314
o 20M. A sligh ele a ion (30nM) o ee cy osolic Ca2+ concen-315
a ion was obse ed in 60% o cells p o ing he p esence and ac i e316
unc ioning o IP3signalling (Fig. 3A).317
ADP, UDP and UTP a e non-specific agonis s o me abo opic318
pu ine gic ecep o s (P2Y). These compounds we e adminis e ed319
o cells o HDC a a concen a ion o 180M on day 3 o cul u ing320
(Fig. 3B–D). Sligh ele a ion o cy osolic Ca2+ was de ec ed only in321
50% o cells measu ed du ing he adminis a ion o UTP. The a e -
322
age ampli ude o UTP-e oked Ca2+ ansien s was 57nM. On he323
o he hand, adminis a ion o ADP and UDP did no esul in any
324
significan Ca2+ ansien s.325
3.2. Chond ogenic mesenchymal cells exp ess a ious P2X and 326
P2Y ecep o sub ypes du ing di e en ia ion 327
To iden i y he p esence and exp ession pa e n o a ious 328
pu ine gic ecep o sdu ingchond ogenicdi e en ia iono chicken 329
mesenchymal cells, RT-PCR eac ions we e pe o med. mRNA 330
sequences o chicken P2X ecep o s, bu no o P2X6(no ye pub- 331
lished) as well as P2Y1, P2Y3, and P2Y5 ecep o s we e downloaded 332
om GenBank and specific p ime pai s o each mRNA sequence 333
we e designed o amplifica ion (see Supplemen a y Ma e ial). 334
Amplime s o expec ed sizes we e iden ified o all he a ail- 335
able mRNAs, excep o P2X2, whe e only e y weak signals we e 336
de ec ed(Fig.4A). mRNAexp essiono P2X1and P2X7 ecep o sub- 337
ypes ollowed a peak-like pa e n du ing di e en ia ion wi h he 338
highes exp ession le els on days 3 o 4, espec i ely. P2X3 ecep o 339
sub ype mRNA exhibi ed a a he a iable exp ession p ofile: he 340
s onges bands we e de ec ed be ween days 1 and 3 o cul u ing. 341
P2X4and P2X5 ecep o sub ypes showed he s onges exp es- 342
sion le els. Bo h ecep o s exp essed ma kedly on day 1 hen he 343
signal became g adually weake . mRNAs o P2Y1, P2Y3and P2Y5344
showed cons an exp ession le els h oughou he cul u ing pe iod 345
(Fig. 6B). 346
Wes e n blo analysis showed a di e en exp ession p ofile o 347
he di e en P2X ecep o sub ypes (Fig. 4B). We could no de ec 348
he P2X2 ecep o sub ype, and no signals we e isible o P2X3349
and P2X6, ei he in o al lysa es o in isola ed plasma memb ane 350
UNCORRECTED PROOF
Please ci e his a icle in p ess as: J. Fodo , e al., Iono opic pu ine gic ecep o P2X4is in ol ed in he egula ion o chond ogenesis in
chicken mic omass cell cul u es, Cell Calcium (2009), doi:10.1016/j.ceca.2009.02.004
ARTICLE IN PRESS
G Model
YCECA10501–10
J. Fodo e al. / Cell Calcium xxx (2009) xxx–xxx 5
Fig. 2. Responses o cells o HDC o adminis a ion o ATP on day 3 o cul u ing. (A) Reco d showing he lack o ATP-e oked Ca2+ ansien s in he absence o ex e nal calcium.
(B) Basal in acellula Ca2+ le els and he peak ampli ude o ATP-e oked Ca2+ ansien s in he p esence and absence o ex e nal calcium. Numbe s in pa en heses show he
numbe o cells measu ed. Da a ep esen mean±s anda d e o o he mean o in acellula Ca2+ le els o cells assayed in 5 independen expe imen s. As e isk indica es
significan (*P<0.01) inc ease in peak ampli ude o ATP-e oked Ca2+ ansien s as compa ed o he espec i e con ol. (C) E ec o he P2X an agonis su amin (10M) on
ATP-e oked calcium ansien s in he p esence o ex e nal calcium. (D) Calcium ansien s e oked by epea ed adminis a ion o ATP in he p esence o ex e nal calcium
showing he lack o desensi iza ion o P2X ecep o s in di e en ia ing chond ocy es. Rep esen a i e eco d o 5 independen expe imen s is p esen ed in panels A, C o D.
Lines in panels A, C and D indica e he applica ion o ATP. P eceding he applica ion o Ca- ee ATP cells we e ea ed in Ca- ee Ty ode’s o 150 s.
ac ions (da a no shown). P o ein exp ession o P2X1sub ype in351
o al lysa es ollowed a simila p ofile o he mRNA exp ession, bu 352
in he plasma memb ane ac ions s ong bands we e de ec ed on353
days 1–3, he ea e he p o ein le els ma kedly dec eased. In con-354
as wi h he esul s o RT-PCR eac ions, p o ein le els o P2X5
355
ecep o sub ype we e ha dly de ec able in o al cell lysa es, bu 356
in plasma memb ane ac ions o day 4 was cha ac e ized by a
357
s onge signal.358
Fo P2X7, a p ofile showing a a iable exp ession pa e n was359
obse ed in o al lysa es wi h he s onges bands on days 2 and360
4, espec i ely, howe e , in he plasma memb ane ac ions a361
peak-like pa e n wi h s onges bands on days 2, 3 and 4 was 362
obse ed. 363
Ne e heless, p o ein exp ession o P2X4 ecep o sub ype 364
p o ed o be he mos in e es ing (Fig. 4B). While in o al cell lysa es 365
i showed a a he a iable p ofile, in isola ed plasma memb ane 366
ac ions i fi s appea ed on day 3 wi h a s ong band, and by 367
days 4 and 6 i s exp ession apidly diminished. I is impo an o 368
no e ha he as majo i y o chond ogenic mesenchymal cells 369
esponded o ATP on his day o cul u ing, which also coincides 370
wi h he day o di e en ia ion cha ac e ized by ele a ed cy oso- 371
lic Ca2+ le els epo ed ea lie [1]. P esence o P2X1, P2X4and 372
Fig. 3. Func ional cha ac e iza ion o P2Y ecep o s in cells o HDC on day 3 o cul u ing. (A) 20M b adykinin-e oked Ca2+ ansien s measu ed in he absence o ex e nal
calcium. Line indica es he applica ion o b adykinin. (B) E ec o 180 M ADP on Ca2+ ansien s measu ed in he absence o ex e nal calcium. Line indica es he applica ion
o ADP. (C) E ec o 180M UDP on Ca2+ ansien s measu ed in he absence o ex e nal calcium. Line indica es he applica ion o UDP. (D) 180M UTP-e oked Ca2+ ansien s
in he absence o ex e nal calcium. Line indica es he applica ion o UTP. Rep esen a i e eco ds o 3 independen expe imen s.
UNCORRECTED PROOF
Please ci e his a icle in p ess as: J. Fodo , e al., Iono opic pu ine gic ecep o P2X4is in ol ed in he egula ion o chond ogenesis in
chicken mic omass cell cul u es, Cell Calcium (2009), doi:10.1016/j.ceca.2009.02.004
ARTICLE IN PRESS
G Model
YCECA10501–10
6J. Fodo e al. / Cell Calcium xxx (2009) xxx–xxx
Fig. 4. Exp ession pa e n o P2X ecep o sub ypes in cells o HDC on di e en days o cul u ing. (A) mRNA exp ession pa e n o a ious P2X ecep o s was de ec ed by
RT-PCR eac ions. P2X ecep o sub ypes (bu no P2X6) we e amplified using specific p ime s and de ec ed a expec ed sizes. GAPDH was used as a con ol. (B) Wes e n
blo analysis o P2X ecep o p o eins in cells o HDC. To al p o ein and memb ane ac ion samples we e used (50g in each lane) o examine he p o ein exp ession le el.
Rep esen a i e da a each o 3 independen expe imen s, pe o med in iplica es. (C) Immunocy ochemical s aining o 3-day-old HDC demons a ing p esence o P2X1, P2X4
and P2X7 ecep o s. A ows indica e he accumula ion o immunoposi i e signal in he plasma memb ane o chond ogenic cells. Images we e eco ded om 1m hick
op ical slices. O iginal magnifica ion was 40×. Rep esen a i e images o 3 independen expe imen s, each pe o med in iplica es.
P2X7 ecep o swasalsop o edbyimmunocy ochemicals ainingo 373
HDC. A memb ane-bound localiza ion was clea ly isible o P2X4
374
ecep o s (Fig. 4C). Fu he mo e, p esence o P2X4in ca ilagineous375
p imo diain de elopinglimbso chicken emb yoswas also demon-376
s a eda ade elopmen als age(8-day-oldemb yo)co esponding377
o app oxima ely 4-day-old HDC (da a no shown).
378
We also de ec ed he exp ession o me abo opic pu ine gic379
ecep o s in HDC. As P2Y3and P2Y5 ecep o s a e no ega ded as380
unc ional membe s o his amily o ecep o s [16], we in es iga ed
381
P2Y1, P2Y2, and P2Y4p o ein in o al cell lysa es and plasma mem-382
b ane ac ions. On day 1, he p o ein o P2Y1was no exp essed383
by cells o HDC, hen i was p esen a a cons an le el bo h in384
o al cell lysa es and in plasma memb ane ac ions un il day 6,
385
when i showed a small decline. The P2Y2 ecep o p o ein was386
ound o be exp essed in a peak like pa e n in o al lysa es wi h387
s onges signals ondays2-4.Howe e ,wede ec edacons an le el
388
o exp ession in he plasma memb ane ac ion wi h he excep ion
389
o day 6, when he signal became weake . We could only de ec 390
specific signals o P2Y4 ecep o in he plasma memb ane ac-391
ion wi h a s onge band on day 2, bu no immunoposi i i y was392
obse ed in o al lysa es (Fig. 6C).393
3.3. Adminis a ion o ex acellula ATP on day o di e en ia ion 394
inc eases ma ix p oduc ion 395
In o de o suppo ou hypo hesis ha he en ance o ex a- 396
cellula Ca2+ in o chond ogenic mesenchymal cells is ia P2X 397
ecep o s, especially ia P2X4sub ype, u he expe imen s we e 398
pe o med. ATP was adminis e ed a a ious concen a ions ocells 399
o high densi y cul u es on day 3 o cul u ing. A he concen a ion 400
o 100 M, ex ensi e ma ix p oduc ion occu ed by day 6 (Fig. 5A) 401
demons a ed by bo h DMMB and TB s ainings. mRNA le els o col- 402
lagen II and he co e p o ein o agg ecan also eflec ed he sligh ly 403
highe a e o ma ix p oduc ion unde he e ec o ATP (Fig. 5B). 404
Al hough mRNA exp ession le el o Sox9 did no change (Fig. 5B), 405
p o ein exp ession o his ansc ip ion ac o became highe as 406
a esul o he adminis a ion o ATP (Fig. 5C). T ea men o HDC 407
wi h ATP on days 2 and 4 o cul u ing did no al e he ca ilage 408
ma ix p oduc ion (da a no shown). These esul s indica e ha 409
ATP has a posi i e e ec on bo h ca ilage ma ix p oduc ion and 410
chond oblas di e en ia ion when i is applied a he ime o final 411
commi men o chond op ogeni o cells and ATP does no exe any 412
e ec on p ema u e o ma u e chond oblas s. 413
UNCORRECTED PROOF
Please ci e his a icle in p ess as: J. Fodo , e al., Iono opic pu ine gic ecep o P2X4is in ol ed in he egula ion o chond ogenesis in
chicken mic omass cell cul u es, Cell Calcium (2009), doi:10.1016/j.ceca.2009.02.004
ARTICLE IN PRESS
G Model
YCECA10501–10
J. Fodo e al. / Cell Calcium xxx (2009) xxx–xxx 7
Fig. 5. E ec s o ATP (100 M) and su amin (10 M) on ca ilage de elopmen o chond i ying mic omass cul u es. Bo h chemicals we e adminis e ed on day 3 o cul u ing. (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 o each expe imen al g oup ou o 5 measu emen s. S anda d e o s o he means we e
wi hin±8%. As e isks indica e significan (*P<0.01) inc ease in op ical densi y o ex ac ed oluidine blue as compa ed o he espec i e con ol. (B) mRNA exp ession o
collagen ype II, co e p o ein o agg ecan and sox9 a e ea men wi h ATP and/o su amin. GAPDH was used as a con ol. Rep esen a i e da a o 3 independen expe imen s,
each pe o med in iplica es. (C) E ec o ATP and/o su amin on he p o ein exp ession o Sox9. Rep esen a i e da a o 3 independen expe imen s, each pe o med in
iplica es.
Adminis a ion o he non-specific P2X ecep o an agonis
414
su amin alone o combina ion wi h ATP did no cause any signifi-415
can al e a ion in he amoun o ca ilage ma ix p oduced by he416
end o he 6-day-old cul u ing pe iod (Fig. 5A). Mo eo e , he ATP-
417
s imula ed Sox9 exp ession was no a ec ed by su amin ea men ,
418
u he suppo ing ou heo y ha P2X4 ecep o could be in ol ed419
in he ansmissiono hechond ogenesisp omo inge ec o ex a-420
cellula ATP (Fig. 5B and C).
421
3.4. Adminis a ion o ADP, UDP and UTP o he cul u e medium422
has no e ec on ca ilage o ma ion423
We examined he adminis a ion o he nucleo ides on ca -424
ilage ma ix p oduc ion o HDC o elucida e a pu a i e ole o 425
me abo opic pu ine gic ecep o s. The nucleo ides applied a a426
concen a ion o 180M in o he cul u ing medium on day 3 did427
no exe any e ec on he amoun o ca ilage ma ix p oduced by428
heend o he 6-day-longcul u ing pe iod as e ealedbyme ach o-429
ma ic s aining (Fig. 6A).430
3.5. Cells o high densi y cul u es sec e e ATP in o he cul u e
431
medium
432
The demons a ion o he e ec i eness o adminis a ion o ATP433
on ma ix p oduc ion aises he ques ion whe he he chond o-
434
genic mesenchymal cells sec e e ATP in o he cul u e medium as
435
an au oc ine media o o p omo e and acili a e hei own di e en-
436
ia ion. To in es iga e his, he cul u e medium was emo ed om437
he cells o high densi y cul u es on each day o cul u ing and ATP438
assays we e pe o med. We ound ha on each day o cul u ing a
439
small amoun o ATP was de ec able in he cul u e medium in he440
ange o 2–10nM, which is compa able o da a measu ed in cul u e 441
medium o o he non-exci able cells [22].442
4. Discussion 443
In i o chond ogenesis is a dynamic, mul is ep p ocess egu- 444
la ed by a a ie y o molecula p ocesses, many o which in ol e 445
ac i a ion and deac i a ion o p o ein kinases and phospha ases 446
sensi i e ochangeso in acellula Ca2+ le els.Inchickenhigh den- 447
si y mesenchymal cell cul u es, chond ogenic mesenchymal cells 448
di e en ia e in o chond oblas s and hen o chond ocy es du ing a 449
6-day-long cul u ing pe iod. The majo i y o chond oblas s, cha - 450
ac e ized by he abili y o p oduc ion o a ca ilage specific ECM, 451
appea om cul u ing day 3. 452
We ha e p e iously demons a ed ha cy oplasmic- ee Ca2+ 453
concen a ion o chond ogenic cells exhibi ed a cha ac e is ic an- 454
sien ele a ion on day 3 o cul u ing. This has been ound o be 455
indispensable o p ope di e en ia ion and he essen ial ole o he 456
influx o ex acellula Ca2+ has been documen ed [1]. In acellula 457
Ca2+ s o es ha e been shown o con ain eleasable Ca2+, bu he a e 458
o leak was low and ee cy oplasmic Ca2+ concen a ion became 459
only sligh ly highe in he absence o ex acellula Ca2+. Mo eo e , 460
RyR and IP3 ecep o s ha e been ound o be exp essed weakly and 461
s imula ion o RyR did no esul in he ele a ion o cy oplasmic 462
Ca2+. Ou da a ha e unde lined he ole o Ca2+ influx om ex a- 463
cellula space in he gene a ion o he cy oplasmic Ca2+ peak. The 464
in acellula s o es seemed o be con ibu ing o he main enance 465
o cy osolic basal Ca2+ concen a ion [1].466
In he p esen s udy, we epo he possible in ol emen o 467
P2X and P2Y, ligand-ga ed pu ine gic ecep o s, in he egula ion 468
o he Ca2+ homeos asis o chond ogenic cells pa icula ly du ing 469
UNCORRECTED PROOF
Please ci e his a icle in p ess as: J. Fodo , e al., Iono opic pu ine gic ecep o P2X4is in ol ed in he egula ion o chond ogenesis in
chicken mic omass cell cul u es, Cell Calcium (2009), doi:10.1016/j.ceca.2009.02.004
ARTICLE IN PRESS
G Model
YCECA10501–10
8J. Fodo e al. / Cell Calcium xxx (2009) xxx–xxx
Fig. 6. (A) Me ach oma ic ca ilage a eas in 6-day-old HDC 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 om HDC wi h 8% HCl dissol ed in absolu e e hanol. Da a a e mean alues o each expe imen al g oup ou o 4–4 pa allel samples o 3 independen measu emen s.
S anda d e o s o he means we e wi hin ±8%. (B) mRNA exp ession pa e n o a ious P2Y ecep o s de ec ed by RT-PCR eac ions. P2Y ecep o sub ypes we e amplified
using specific p ime s and de ec ed a expec ed sizes. GAPDH was used as a con ol. Rep esen a i e da a o 3 independen expe imen s, each pe o med in iplica es. (C)
Wes e n blo analysis o P2Y1P2Y2and P2Y4 ecep o p o eins in cells o HDC. To al p o ein and memb ane ac ion samples we e used (50g in each lane) o examine he
p o ein exp ession le el. Rep esen a i e da a o 3 independen expe imen s, each pe o med in iplica es.
hei di e en ia ion. Pu ino ecep o s a e known o be exp essed in470
emb yonic issues [23] and a e p obably in ol ed in he di e en ia-
471
ion p ocess o exci able [24] and non-exci able [25] cells. Howe e ,
472
no da a a e a ailable conce ning he possible in ol emen o such473
p ocesses in he di e en ia ion o chond oblas s om mesenchy-474
mal cells. Du ing single cell measu emen s using Fu a-2-loaded475
cells, ATP, an agonis o pu ine gic ecep o s, was adminis e ed on476
a ious days o cul u ing. We ound ha he cells o HDC esponded477
o ATP by cha ac e is ic Ca2+ ansien s. We also ound ha mos o 478
he cells only esponded o ATP on day 3 o cul u ing, a he ime o
479
di e en ia ion o chond oblas s. The phenomenon ha some cells480
showed esponse on o he days han day 3 can be explained by481
conside ing he ac ha he cells o HDC exhibi some he e ogene-482
i y in hei s age o di e en ia ion, and hough he as majo i y483
di e en ia es on day 3 o cul u ing, he e a e some cells, which
484
could each his s age o de elopmen somewha ea lie o la e .485
We also showed ha p obably he membe s o he iono opic P2X
486
ecep o sub amily can be accoun ed o he influx o ex acellula 487
Ca2+.488
We also es ed he e ec o P2Y ecep o agonis s on Ca2+ an- 489
sien s in cells o 3-day-old HDC. When ATP was adminis e ed o he 490
cellsinaTy ode’ssolu ionlacking eeCa2+,ATP ailed oele a e he 491
in acellula Ca2+ concen a ion. ADP, he agonis o P2Y1, and UDP, 492
heligand o P2Y6 ecep o , nei he e okedanysignifican ele a ion 493
in he ee cy oplasmic Ca2+ concen a ion, no did hey influence 494
ca ilage o ma ion o HDC. Howe e , he agonis o P2Y2and P2Y4495
ecep o s, UTP caused a ansien ele a ion o cy osolic- ee Ca2+ in 496
50% o cells in es iga ed. RT-PCR and Wes e n blo analyses p o ed 497
he p esence o P2Y1, P2Y2, P2Y3, P2Y4and P2Y5 ecep o s in cells 498
o HDC. 499
All he P2X ecep o mRNAs in es iga ed, excep ha o P2X2,500
we e exp essed by cells o HDC showing a iable exp ession p o- 501
files. The phenomenon o age-dependen exp ession sugges s he 502
in ol emen o pu ine gic signalling in he media ion o chond o- 503
genic di e en ia ion. Ou findings ha P2X ecep o sub ypes a e 504
exp essed by di e en ia ing chond ogenic cells in a di e en ia- 505
ion s age-dependen manne a e compa able o s udies conduc ed 506
on hema opoie ic cell lines [26]. A he p o ein le el, cells o HDC 507
UNCORRECTED PROOF
Please ci e his a icle in p ess as: J. Fodo , e al., Iono opic pu ine gic ecep o P2X4is in ol ed in he egula ion o chond ogenesis in
chicken mic omass cell cul u es, Cell Calcium (2009), doi:10.1016/j.ceca.2009.02.004
ARTICLE IN PRESS
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YCECA10501–10
J. Fodo e al. / Cell Calcium xxx (2009) xxx–xxx 9
exp essed only ecep o sub ypes P2X1, P2X4, P2X5and P2X7and508
he exp ession p ofiles in o al cell lysa es and plasma memb ane509
ac ions we e di e en . This di e ence was excep ionally in e -510
es ing in he case o P2X4: his ecep o s a ed o appea on511
day 3 in he plasma memb ane ac ion wi h a e y s ong sig-512
nal, whe eas weake signals we e de ec ed on days 4 and 6. The513
cha ac e is ic exp ession p ofile o P2X4 aised he possibili y ha 514
his ligand-ga ed ecep o could be an impo an channel h ough515
which ex acellula Ca2+ en e he cy osol and con ibu e o he516
ele a edCa2+ le elneeded o hechond ogenicdi e en ia ionp o-517
cess. Al hough o he P2X ecep o s we e also p esen in plasma518
memb ane ac ions, ATP-e oked Ca2+ ansien s we e no elimi-519
na ed when su amin and ATP we e applied simul aneously. Since520
su amin is no a P2X4an agonis bu inhibi s o he P2X ype521
pu ine gic ecep o s exp essed by cells o HDC, ou da a sugges 522
ha hese ecep o s may con ibu e o he main enance o he basal523
cy osolic Ca2+ concen a ion.524
The ATP-media ed unc ion o P2X4 ecep o du ing he di e -525
en ia ion p ocess was p o ed by he addi ion o ex acellula ATP526
o he cul u e medium. I esul ed in an inc ease o he exp es-527
sion o Sox9, he mas e ansc ip ion ac o o chond ogenesis. We
528
de ec ed highe amoun o me ach oma ic ca ilage ma ix p o-529
duced in ATP- ea ed HDC by he end o he 6-day-long cul u ing530
pe iod. This e ec was achie ed exclusi ely when ATP was admin-531
is e ed on day 3 o cul u ing. When ATP was added p io o (day 3)532
o a e he di e en ia ion pe iod o chond ogenic cells (day 4 o 533
cul u ing), i did no esul in any significan e ec on he ma ix534
o ma ion o HDC. This obse a ion unde lines he impo ance o 535
ATP in he acili a ion o ca ilage di e en ia ion and may ule ou 536
i s ole in he s imula ion o ma ix p oduc ion o ma u e ca ilage.537
Al hough applica ion o me abo opic P2Y ecep o agonis s o he
538
cul u e medium on day 3 caused Ca2+ ansien s simila o hose539
gene a ed by ATP, bu did no al e he amoun o ca ilage ma ix540
p oduced by he end o he 6-day-long cul u ing pe iod. The e o e541
we sugges ha me abo opic P2Y ecep o s a he con ibu e o542
he main enance o basal cy osolic Ca2+ concen a ion in cells o 543
HDC.544
Al hough su amin did no elimina e he ATP-e oked Ca2+ an-545
sien s o chond ogenic cells, he inc eased me ach oma ic ca ilage
546
ma ix p oduc ion caused by ATP was comple ely diminished. This
547
e ec does no seem o be exe ed ia he inhibi ion o chond ogen-548
esis, since he exp ession o Sox9, was no educed by su amin, and549
he mRNA exp ession le els o nei he collagen ype II no agg e-550
can we e a ec ed. Su amin has been epo ed o inhibi hyalu onic551
acid syn hesis o fib oblas s [27], and hyalu onic acid is esponsi-552
ble o holding agg ega es o agg ecan oge he in ca ilage ma ix.
553
The educed amoun o hyalu onic acid may cause inc eased loss o 554
agg ecan du ing me ach oma ic s aining p ocedu es o HDC, caus-555
ing i ual educ ion o he de ec ed amoun o ca ilage ma ix.556
We also showed ha cells o HDC sec e ed ATP in o he cul u e557
medium. This ATP may ac as an au oc ine media o o acili a e and558
p omo e hei own di e en ia ion. Chond ogenic cells sec e ed ATP559
h oughou he cul u ing pe iod, and hey esponded o he ex a-
560
cellula ly adminis e ed ATP wi h a peak-like ele a ion o ic. Ca2+
561
concen a ion only a he ime o di e en ia ion. This ac u he 562
suppo s he pu ine gic concep in he con ol o chond ogenesis.563
Ou da a on ATP concen a ions sec e ed in o he cul u e medium
564
seem significan ly less han he concen a ions applied o ma ix565
p oduc ion assays. I is impo an o emphasize ha ATP assays566
we e pe o med in 20mL o cul u e medium. Fu he mo e, he ol-
567
ume o he cell cul u e i sel and he olume o he cul u e medium
568
di e bya ac o o app oxima ely1000. The e o e, he ATPsec e ed
569
by he cells could ha e eached much highe concen a ions a he570
close p oximi y o cells. Thus he de ec ed concen a ions o ATP571
sec e ed by chond ogenic cells should be in he ange in which P2X
572
ecep o s espond o his ligand [28,29].573
In summa y, ou obse a ions p o ide he fi s e idence on 574
he possibili y o a pu ine gic au o egula ion o chond ogenesis. 575
Pu ine gic ecep o s, membe s o he Ca2+ ool ki used by cells o 576
HDCcan beone o he keyelemen s in he egula ion o he ele a ed 577
cy osolic Ca2+ le els du ing ca ilage di e en ia ion in i o and in 578
i o.579
Acknowledgemen s 580
The au ho s hank M s. K isz ina Bí ó and M s. Ibolya Va ga o 581
hei excellen assis ance. We also hank Ádám Jóna o his wo k 582
in pe o ming immunohis ochemical s ainings o P2X4 ecep o on 583
pa a fin-embedded chicken emb yos. This wo k was suppo ed by 584
g an s om he Hunga ian Science Resea ch Fund (OTKA K60620 585
and OTKA T49151) and om he Hunga ian Minis y o Heal h (ETT 586
083/2006). R.Z. is suppo ed by a Mecena u a g an (29/ME-02) 587
om he Medical and Heal h Science Cen e, Uni e si y o Deb e- 588
cen, Hunga y. 589
Appendix A. Supplemen a y da a 590
Supplemen a y da a associa ed wi h his a icle can be ound, in 591
he online e sion, a doi:10.1016/j.ceca.2009.02.004.592
Re e ences 593
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