Ionotropic purinergic receptor P2X4 is involved in the regulation of chondrogenesis in chicken micromass cell cultures

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 30␮L 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 180␮Mfinalconcen a ion.ADP,UDP,UTP(180␮M)and 137 b adykinin (20␮M) 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 250␮ma a1.5␮L/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), 10␮g/mL Go dox, 10␮g/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 50␮L 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 (20␮L) o e e se ansc ip ase eac- 172 ion (Omnisc ip , Quiagen) con ained 500 ng RNA, 0.25 ␮LRNase 173 inhibi o , 0.25␮Loligo (dT), 1␮L dNTP (200␮M), 1␮l 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 50␮L (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, 1␮L dNTP [200␮M], 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 50␮g 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 (180␮M), and su amin229 (10␮M) 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. 50␮L 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 180␮M 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 (10␮M) 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 180␮M 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 20␮M. 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 180␮M 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 (10␮M) 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) 20␮M 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 180␮M UDP on Ca2+ ansien s measu ed in he absence o ex e nal calcium. Line indica es he applica ion o UDP. (D) 180␮M 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 (50␮g 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 1␮m 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 180␮M 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 (50␮g 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 G Model 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 [1] C. Ma a, J. Fodo , Z. Szijgya o, T. Juhasz, P. Ge gely, L. Cse noch, R. Zakany, 594 Cy osolic eeCa2+ concen a ionexhibi sacha ac e is ic empo alpa e ndu - 595 ing in i o ca ilage di e en ia ion: a possible egula o y ole o calcineu in in 596 Ca-signalling o chond ogenic cells, Cell Calcium 44 (2008) 310–323. 597 [2] M.B. Gold ing, K. Tsuchimochi, K. Iji i, The con ol o chond ogenesis, J. Cell 598 Biochem. 97 (2006) 33–44. 599 [3] W.M. Kulyk, J.L. F anklin, L.M. Ho man, Sox9 exp ession du ing chond ogene- 600 sis in mic omass cul u es o emb yonic limb mesenchyme, Exp. Cell Res. 255 601 (2000) 327–332. 602 [4] V. Le eb e, W. Huang, V.R. Ha ley, P.N. Good ellow, C.B. de, SOX9 is a po en 603 ac i a o o he chond ocy e-specific enhance o he p o alpha1(II) collagen 604 gene, Mol. Cell Biol. 17 (1997) 2336–2346. 605 [5] C.L. Tu, Y. Oda, L. Komu es, D.D. Bikle, The ole o he calcium-sensing ecep o 606 in epide mal di e en ia ion, Cell Calcium 35 (2004) 265–273. 607 [6] M. Zayza oon, Calcium/calmodulin signaling con ols os eoblas g ow h and 608 di e en ia ion, J. Cell Biochem. 97 (2006) 56–70. 609 [7] S. Kawano, K. O su, S. Shoji, K. Yamaga a, M. Hi aoka, Ca(2+) oscilla ions egu- 610 la ed by Na(+)-Ca(2+) exchange and plasma memb ane Ca(2+) pump induce 611 fluc ua ionso memb anecu en sandpo en ialsinhumanmesenchymals em 612 cells, Cell Calcium 34 (2003) 145–156. 613 [8] W.G. Fishe , P.C. Yang, R.K. Medikondu i, M.S. Ja i, NFAT and NFkappaB ac i- 614 a ion in T lymphocy es: a model o di e en ial ac i a ion o gene exp ession, 615 Ann. Biomed. Eng. 34 (2006) 1712–1728. 616 [9] S. Chawla, H. Bading, CREB/CBP and SRE-in e ac ing ansc ip ional egula o s 617 a e as on-o swi ches: du a ion o calcium ansien s specifies he magni ude 618 o ansc ip ional esponses, J. Neu ochem. 79 (2001) 849–858. 619 [10] S. Pie a-Velazquez, D.F. Hawkins, M.K. Whi eca age, D.C. Col e , D.G. S okes, S.A. 620 Jimenez, Regula ion o he human SOX9 p omo e by Sp1 and CREB, Exp. Cell 621 Res. 313 (2007) 1069–1079. 622 [11] M. Tomi a, M.I. Reinhold, J.D. Molken in, M.C. Naski, Calcineu in and NFAT4 623 induce chond ogenesis, J. Biol. Chem. 277 (2002) 42214–42218. 624 [12] R. Zakany, E. Bako, S. Felszeghy, K. Hollo, M. Balazs, H. Ba dos, P. Ge gely, L. 625 Modis, Okadaic acid-induced inhibi ion o p o ein phospha ase 2A enhances 626 chond ogenesis in chicken limb bud mic omass cell cul u es, Ana . Emb yol. 627 (Be l.) 203 (2001) 23–34. 628 [13] P.B. Ah ens, M. Solu sh, R.S. Rei e , S age- ela ed capaci y o limb chond oge- 629 nesis in cell cul u e, De . Biol. 60 (1977) 69–82. 630 [14] F.So o,M. Ga cia-Guzman, W.S uhme ,Cloned ligand-ga edchannelsac i a ed 631 by ex acellula ATP (P2X ecep o s), J. Memb . Biol. 160 (1997) 91–100. 632 [15] R.A. No h, A. Su p enan , Pha macology o cloned P2X ecep o s, Annu. Re . 633 Pha macol. Toxicol. 40 (2000) 563–580. 634 [16] V. Rale ic, G. Bu ns ock, Recep o s o pu ines and py imidines, Pha macol. Re . 635 50 (1998) 413–492. 636 [17] I.R. O iss, G.E. Knigh , S. Ranasinghe, G. Bu ns ock, T.R. A ne , Os eoblas 637 esponses o nucleo ides inc ease du ing di e en ia ion, Bone 39 (2006) 638 300–309. 639 [18] C. Colle , C. S ube, L. Cse noch, N. Mallouk, C. Ojeda, B. Alla d, V. Jacquemond, 640 E ec s o ex acellula ATP on eshly isola ed mouse skele al muscle cells du - 641 ing p e-na al and pos -na al de elopmen , Pfluge s A ch. 443 (2002) 771–778. 642 [19] M. Ry en, A. Hoebe z, G. Bu ns ock, Sequen ial exp ession o h ee ecep o 643 sub ypes o ex acellula ATP in de eloping a skele al muscle, De . Dyn. 221 644 (2001) 331–341. 645