Store-operated calcium entry and calcium influx via voltage-operated calcium channels regulate intracellular calcium oscillations in chondrogenic cells

Please ci e his a icle in p ess as: J. Fodo , e al., S o e-ope a ed calcium en y and calcium in lux ia ol age-ope a ed calcium channels egula e in acellula calcium oscilla ions in chond ogenic cells, Cell Calcium (2013), h p://dx.doi.o g/10.1016/j.ceca.2013.03.003 ARTICLE IN PRESS G Model YCECA 1472 1–16 Cell Calcium xxx (2013) xxx– xxx Con en s lis s a ailable a SciVe se ScienceDi ec Cell Calcium j ou na l ho me page: www.else ie .com/loca e/ceca S o e-ope a ed calcium en y and calcium in lux ia ol age-ope a ed calcium channels egula e in acellula calcium oscilla ions in chond ogenic cells 1 2 János Fodo a,1, Csaba Ma ab,1, Tamás Oláha, Tamás Juhászb, Roland Takácsb, Ad ienn Tó ha, Q1 Bea ix Dienesa, László Cse nocha, Róza Zákányb,∗ 3 4 aDepa men o Physiology, Medical and Heal h Science Cen e, Uni e si y o Deb ecen, Nagye dei k . 98, H-4032 Deb ecen, Hunga y5 bDepa men o Ana omy, His ology and Emb yology, Medical and Heal h Science Cen e, Uni e si y o Deb ecen, Nagye dei k . 98, H-4032 Deb ecen, Hunga y6 7 a i c l e i n o8 9 A icle his o y:10 Recei ed 10 Janua y 201311 Recei ed in e ised o m 11 Ma ch 201312 Accep ed 21 Ma ch 201313 A ailable online xxx 14 Keywo ds:15 SOCE16 STIM17 O ai118 Sox9 19 P oli e a ion20 a b s a c Chond ogenesis is known o be egula ed by calcium-dependen signalling pa hways in which empo al aspec s o calcium homeos asis a e o key impo ance. We aimed o be e cha ac e ise calcium in lux and elease unc ions wi h espec o apid calcium oscilla ions in cells o chond i ying chicken high densi y cul u es. We ound ha di e en ia ing chond ocy es exp ess he !1subuni o ol age-ope a ed calcium channels (VOCCs) a bo h mRNA and p o ein le els, and ha hese ion channels play impo an oles in gene a ing Ca2+ in lux o oscilla ions as ni edipine in e e ed wi h epe i i e calcium ansien s. Fu - he mo e, VOCC blockade ab oga ed chond ogenesis and almos comple ely blocked cell p oli e a ion. The con ibu ion o in e nal Ca2+ s o es ia s o e-ope a ed Ca2+ en y (SOCE) seems o be indispensable o bo h Ca2+ oscilla ions and chond ogenesis. Mo eo e , his is he i s s udy o show he unc ional exp ession o STIM1/STIM2 and O ai1, molecules ha o ches a e SOCE, in chond ogenic cells. Inhibi ion o SOCE combined wi h ER calcium s o e deple ion abolished di e en ia ion and se e ely diminished p oli e a ion, sugges ing he impo an ole o in e nal pools in calcium homeos asis o di e en ia ing chond ocy es. Finally, we p esen an in eg a ed model o he egula ion o calcium oscilla ions o di e - en ia ing chond ocy es ha may ha e impo an implica ions o s udies o chond ogenesis induced in a ious s em cell popula ions. © 2013 Else ie L d. All igh s ese ed. Abb e ia ions: AM, ace oxy-me hyles e ; ARC, a achidona e- egula ed Ca2+- sensi i e channel; ATP, adenosine iphospha e; CaMKII, Ca2+–calmodulin depend- en p o ein kinase II; CPA, cyclopiazonic acid; CRAC, Ca2+ elease-ac i a ed Ca2+ channel; CREB, cAMP- esponse elemen binding p o ein; DMMB, dime hylme hy- lene blue; ECM, ex acellula ma ix; ER, endoplasmic e iculum; FBS, oe al bo ine se um; FTHM, ull ime a hal maximum; HDC, high densi y cell cul u e; HRP, ho se adish pe oxidase; IP3, inosi ol-1,4,5- isphospha e; IP3R, inosi ol-1,4,5- isphospha e ecep o ; MSC, mesenchymal s em cell; NCX, Na+–Ca2+ exchange ; NFAT, nuclea ac o o ac i a ed T lymphocy es; NMDA, N-me hyl-d-aspa a e ecep o ; PKC, p o ein kinase C; PLC, phospholipase C; PMCA, plasma memb ane Ca2+-ATPase; RMP, es ing memb ane po en ial; RT-PCR, e e se ansc ip ion ollowed by polyme ase chain eac ion; RyR, yanodine ecep o ; SDS–PAGE, sodium dodecyl sulpha e–polyac ylamide gel elec opho esis; SERCA, sa coplas- mic/endoplasmic e iculum Ca2+ ATPase; SOC, s o e-ope a ed calcium channel; SOCE, s o e-ope a ed Ca2+ en y; STIM, s omal in e ac ion molecule; TB, oluidine blue; TRP, ansien ecep o po en ial ecep o ; TRPC, canonical ansien ecep- o po en ial ecep o ; TRPV, ansien ecep o po en ial ecep o anilloid; VOCC, ol age-ope a ed calcium channel. ∗Co esponding au ho . Tel.: +36 52 255 567; ax: +36 52 255 115. E-mail add esses: [email p o ec ed] (T. Juhász), oza@ana .med.unideb.hu (R. Zákány). 1These au ho s con ibu ed equally o he wo k. 1. In oduc ion 21 One o he ini ial s eps du ing o ma ion o he emb yonic 22 skele on is he di e en ia ion o chond op ogeni o cells in o 23 ex acellula ma ix-sec e ing chond oblas s. La e on, ma u e 24 chond ocy es a e o med ha unde go e minal di e en ia ion p e- 25 ceding endochond al ossi ica ion [1]. Ca ilage o ma ion, including 26 di e en ia ion and p oli e a ion o chond ogenic cells, is igh ly 27 egula ed by complex in e play be ween nume ous amilies o 28 signalling p o eins, e en ually leading o ac i a ion o Sox9, as 29 well as Sox6 and L-Sox5— ansc ip ion ac o s ha a e essen ial 30 o chond ogenesis; hey a e in ol ed in he speci ica ion o he 31 chond ogenic lineage and ac i a e he exp ession o chond ogenic 32 ma ke genes (e.g. COL2A1, AGR1) [2,3]. 33 Du ing he ea ly s eps o chond ogenesis, in acellula signalling 34 unde goes p o ound changes o ini ia e speci ic gene ac i a ion 35 ha equi es ansloca ion o lineage-speci ic ansc ip ion ac o s 36 in o he cell nucleus. In pa icula , he nuclea localisa ion signal 37 a he N- e minus o he chond ocy e-speci ic ansc ip ion ac o 38 Sox9 was shown o con ain a calmodulin-binding egion and 39 Ca2+–calmodulin has been epo ed o be in ol ed in he nuclea 40 en y o Sox9 [4]. Calcium ions (Ca2+) a e sugges ed o be key 41 0143-4160/$ – see on ma e © 2013 Else ie L d. All igh s ese ed. h p://dx.doi.o g/10.1016/j.ceca.2013.03.003 Please ci e his a icle in p ess as: J. Fodo , e al., S o e-ope a ed calcium en y and calcium in lux ia ol age-ope a ed calcium channels egula e in acellula calcium oscilla ions in chond ogenic cells, Cell Calcium (2013), h p://dx.doi.o g/10.1016/j.ceca.2013.03.003 ARTICLE IN PRESS G Model YCECA 1472 1–16 2J. Fodo e al. / Cell Calcium xxx (2013) xxx– xxx ac o s in ol ed in con olling di e en ia ion o cells, including42 human mesenchymal s em cells (MSCs) [5] and chond ocy es [6].43 In gene al, Ca2+- egula ed cellula e en s equi e ansien ly44 ele a ed cy osolic Ca2+ concen a ion o ac i a e Ca2+-sensi i e45 signalling componen s [7]. Cells u ilise wo main sou ces o Ca2+ o 46 ini ia ing and gene a ing signals. On he one hand, Ca2+ en y ac oss47 he plasma memb ane ia ei he ol age-ope a ed Ca2+ channels48 (VOCCs) o agonis -dependen and ol age-independen Ca2+ en y49 pa hways, amongs which a e he ecep o ion channels (e.g. P2X50 [8], NMDA- ecep o s [9], TRPV channels [10]; he la e g oup51 also ac as senso s o osmo ic p essu e, olume, s e ch, and a i-52 ous chemical and mechanical s imuli [11]), he s o e-independen 53 (e.g. a achidona e-dependen non-capaci i e Ca2+ en y [12]) and54 ‘s o e-ope a ed’ Ca2+ (SOC) channels (e.g. TRPs [5]); and on he55 o he hand, Ca2+ elease om in e nal s o es such as he smoo h56 endoplasmic e iculum (ER) ia he inosi ol-1,4,5- isphospha e57 ecep o (IP3R) o he yanodine ecep o (RyR), as well as Ca2+ 58 induced Ca2+ elease (CICR) o ia he exchange p o ein di ec ly59 ac i a ed by cyclic AMP (Epac) [13,14]. Since changes o in a-60 cellula Ca2+ concen a ion a e ela ed o cell p oli e a ion and61 di e en ia ion ha a e impo an unc ions o undi e en ia ed 62 cells such as MSCs, he Ca2+ homeos asis o s em cells has been63 ho oughly in es iga ed [15].64 I is well documen ed ha di e en spa ial and empo al pa -65 e ns o in acellula ee Ca2+ concen a ion play dis inc oles in66 he egula ion o a ious cellula p ocesses. No only a s able ise,67 bu also pe iodic oscilla o y changes o cy osolic Ca2+ concen a-68 ion ep esen a nea ly uni e sal signalling mechanism e en in69 non-exci able cells [16]. Signal ansduc ion pa hways igge ed by70 Ca2+ oscilla ions a e gene ally accep ed o con ol cellula di e -71 en ia ion ia educing he h eshold o he ac i a ion o di e en 72 Ca2+-dependen ansc ip ion ac o s, including nuclea ac o o 73 ac i a ed T lymphocy es (NFAT), nuclea ac o -"B (NF-"B), Jun N-74 e minal kinase-1 (JNK1), myocy e enhance ac o -2 (MEF2) and75 he cAMP- esponse elemen binding p o ein (CREB), depending on76 he equency and ampli ude o he Ca2+ ansien s [17,18]. Spon a-77 neous Ca2+ oscilla ions we e epo ed in se e al non-exci able cells78 unde going di e en ia ion o p oli e a ion [19]. In human MSCs,79 he majo sou ce o Ca2+ o he obse ed oscilla ions is Ca2+ elease80 om ER ia IP3Rs, bu Ca2+ in lux ia non-capaci i e SOCs is also81 equi ed o sus ain hese oscilla ions, wi hou a signi ican con i-82 bu ion om VOCCs [5]. Unlike MSCs, spon aneous Ca2+ oscilla ions83 in mos o he cells examined, including di e en ia ing limb bud-84 de i ed mesenchymal cells, seemed o depend on he a ailabili y o 85 ex acellula Ca2+. These obse a ions a e in a good co ela ion wi h86 ou p e ious esul s implying ha mainly Ca2+ in lux, a leas in pa 87 ia he ligand-ga ed pu ine gic ca ion channel P2X4, is equi ed o 88 main aining sus ained aised cy osolic Ca2+ le els in di e en ia -89 ing chond ogenic cells [20,21]. Ne e heless, in hese s udies, he90 con ibu ion om in e nal Ca2+ s o es, in spi e o he unc ional91 exp ession o IP3Rs, seemed o be less impo an in di e en ia ing92 chicken chond ogenic cells. Many Ca2+ en y and elease p ocesses93 ha e been documen ed in ma u e chond ocy es [22]; howe e , 94 knowledge ega ding he p ecise egula ion and unc ion o Ca2+ 95 homeos asis, including high- equency spon aneous oscilla ions,96 du ing in i o chond ogenesis is s ill spa se.97 In his s udy, we aimed a u he cha ac e ising he Ca2+ 98 homeos asis o di e en ia ing chicken chond ocy es wi h spe-99 cial emphasis on he con ibu ion o in e nal Ca2+ s o es and100 he in ol emen o VOCCs in gene a ing and main aining high- 101 equency epe i i e Ca2+ ansien s o modula e cellula unc ions102 such as di e en ia ion and p oli e a ion. We unde ook o ana- 103 lyse in acellula Ca2+ dynamics in indi idual chond i ying cells a 104 high spa ial and empo al esolu ion using LIVE con ocal Ca2+ imag-105 ing mic oscopy. Ha ing pe o med a de ailed in es iga ion o he 106 con ibu ion o in e nal Ca2+ s o es and s o e-ope a ed Ca2+ en y107 (SOCE) o he Ca2+ homeos asis o di e en ia ing chond ocy es and 108 he in ol emen o Ca2+ in lux ia VOCCs in gene a ing o main- 109 aining spon aneous Ca2+ oscilla ions, we p o ide a e ined model 110 o Ca2+ signalling e en s including Ca2+ in lux and elease unc ions 111 in di e en ia ing cells o chond i ying mic omass cul u es du ing 112 in i o chond ogenesis. 113 2. Ma e ials and me hods 114 2.1. P ima y high densi y chond i ying cell cul u es 115 A well-known and easily ep oducible in i o expe imen al 116 model o s udy hyaline ca ilage o ma ion was i s desc ibed by 117 Ah ens e al. [23]. In hese high densi y cell cul u es (HDC), chicken 118 limb bud-de i ed chond op ogeni o mesenchymal cells spon a- 119 neously di e en ia e o chond oblas s and chond ocy es on days 2120 and 3 o cul u ing, and a well-de ec able amoun o hyaline ca i- 121 lage ex acellula ma ix (ECM) is p oduced by day 6. 122 To es ablish p ima y mic omass cell cul u es o chond i- 123 ying mesenchymal cells, Ross hyb id chicken emb yos o 124 Hambu ge –Hamil on de elopmen al s ages 22–24 (4.5-day-old) 125 we e used. Wo k on ea ly chick emb yos in i o does no equi e 126 a license om he E hics Commi ee o he Uni e si y o Deb e- 127 cen. Dis al pa s o o elimbs and hindlimbs o emb yos we e 128 isola ed and dissocia ed in 0.25% ypsin–EDTA (Sigma, S . Louis, 129 MO, USA; pH 7.4) a 37◦C o 1 h. The enzyma ic diges ion was e - 130 mina ed by he addi ion o equal olume o oe al bo ine se um 131 (FBS; Gibco, Gai he sbu g, MD, USA) and diges ed limb buds we e 132 il e ed h ough a 20-#m po e size plas ic il e uni (Millipo e, 133 Bille ica, MA, USA) o yield a single cell suspension o chond o- 134 genic mesenchymal cells. A e a b ie cen i uga ion (a 800 × g o 135 10 min), cells we e esuspended in Ham’s F12 medium (Sigma) sup- 136 plemen ed wi h 10% FBS a a concen a ion o 1.5 × 107cells mL−1137 and 100–100 #L d ople s we e inocula ed in o plas ic cell cul- 138 u e pla es (O ange Scien i ique, B aine-l’Alleud, Belgium). A e 139 allowing he cells o a ach o he su ace o 120 min a 37◦C 140 in a CO2incuba o (5% CO2and 80% humidi y), 2 mL o Ham’s 141 F12 supplemen ed wi h 10% FBS, 0.5 mM s abile l-glu amine 142 and an ibio ics/an imico ics (penicillin 50 U mL−1, s ep omycin 143 50 #g mL−1, ungizone 1.25 #g mL−1; TEVA, Deb ecen, Hunga y) 144 was added. Day o inocula ion was conside ed as day 0 o cul u - 145 ing. Cul u es we e kep a 37◦C in a CO2incuba o o 6 days. The 146 medium was changed on e e y second day. 147 2.2. Con ocal mic oscopy 148 2.2.1. Line-scan analysis 149 Spon aneous Ca2+ ansien s and he e ec s o modi ied ex a- 150 cellula ionic milieu o a ious d ugs on Ca2+ oscilla ions we e 151 moni o ed using an LSM 510 META Lase Scanning Con ocal 152 Mic oscope (Zeiss, Obe kochen, Ge many). All measu emen s we e 153 pe o med a oom empe a u e. Cells o 1- and 2-day-old high den- 154 si y mic omass cell cul u es we e incuba ed o 30 min a 37◦C 155 wi h 10 #M Fluo-4-AM in Ham’s F12 medium. Calcium imag- 156 ing was ca ied ou in no mal (in mM: 137 NaCl, 5.4 KCl, 0.5 157 MgCl2, 1.8 CaCl2, 11.8 HEPES; 1 g L−1glucose; pH 7.4) o Ca2+- ee 158 (con aining 5 mM EGTA, wi hou CaCl2) Ty ode’s solu ion. The sa - 159 coplasmic/endoplasmic e iculum Ca2+-ATPase (SERCA) inhibi o 160 cyclopiazonic acid (CPA, Sigma) was used a a inal concen a ion 161 o 10 #M in no mal Ty ode’s solu ion (s ock: 10 mM, in DMSO). 162 SOCE blocke s, i.e. he non-speci ic TRPC an agonis YM-58483 163 (a py azole de i a i e, also known as BTP-2; Sigma) [24] and 164 LaCl3(Sigma) [5] we e used a 1 #M and 500 #M inal concen- 165 a ions, espec i ely, dilu ed in no mal Ty ode’s solu ion (s ocks: 166 300 mM and 1 mM in dis illed wa e and DMSO, espec i ely). 167 Please ci e his a icle in p ess as: J. Fodo , e al., S o e-ope a ed calcium en y and calcium in lux ia ol age-ope a ed calcium channels egula e in acellula calcium oscilla ions in chond ogenic cells, Cell Calcium (2013), h p://dx.doi.o g/10.1016/j.ceca.2013.03.003 ARTICLE IN PRESS G Model YCECA 1472 1–16 J. Fodo e al. / Cell Calcium xxx (2013) xxx– xxx 3 (These compounds could no be applied o oscilla ing cells in Ca2+-168 ee Ty ode’s since emo al o ee ex acellula Ca2+ ions alone169 abolished Ca2+ oscilla ions, which would ha e ende ed he e al-170 ua ion o he e ec s o hese blocke s impossible.) Acquisi ion o 171 line-scan images s a ed immedia ely a e changing he solu ion172 on he cul u es. Du ing measu emen s, only cells exhibi ing Ca2+ 173 oscilla ions we e in es iga ed, and o he cells we e dis ega ded.174 Line-scan images we e acqui ed a 0.8 ms/line, 512 pixels/line wi h175 7 ms in e als, eco ding 8192 lines using a 63×wa e imme sion176 objec i e. Measu emen s we e ca ied ou in cells om 3 indepen-177 den expe imen s. Images we e analysed using an au oma ic e en 178 de ec ion so wa e de eloped in he Depa men o Physiology o 179 he Uni e si y o Deb ecen, Medical and Heal h Science Cen e. 180 2.2.2. X–Y moni o ing181 Se ies o X–Y images we e eco ded om andom isual ields o 182 1- and 2-day-old Fluo-4-loaded chond i ying cul u es wi h LIVE 5183 Lase Scanning Con ocal Mic oscope (Zeiss, Obe kochen, Ge many) 184 using EC Plan-Neo lua 20×/0.50 M27 objec i e wi h 2× digi al185 zoom. Calcium imaging was pe o med in no mal and Ca2+- ee186 Ty ode’s solu ions (see abo e). All measu emen s we e pe o med187 a oom empe a u e. LaCl3(500 #M), YM-58483 (1 #M), and188 ni edipine (10 #M) we e dilu ed in no mal Ty ode’s solu ion (con-189 aining 1.8 mM Ca2+; see abo e). F ame acquisi ion a e was 10 s−1.190 A o al numbe o 1000 images we e eco ded du ing con ol 191 condi ions on days 1 and 2. When he e ec s o pha macons on192 spon aneous Ca2+ ansien s we e examined du ing ime se ies193 eco dings, he same isual ields on each cul u e we e obse ed 1, 3194 and 5 min a e eplacing he ba h solu ion om no mal Ty ode’s o195 he es solu ion (LaCl3–YM-58483; o ni edipine). Du ing hese la -196 e expe imen s, a o al numbe o 500 X–Y scans we e eco ded a 197 each ime poin . Da a analysis was ca ied ou using Zeiss Enhanced 198 Na iga ion (ZEN 2009) so wa e. The ound-shaped chond ocy es 199 we e ma ked as egion o in e es (ROI) on each isual ield. Time-200 dependen luo escen in ensi ies o ROIs we e analysed using an201 au oma ic e en de ec ion so wa e de eloped in he Depa men 202 o Physiology.203 2.3. Single cell luo escen Ca2+ measu emen s204 Measu emen s we e pe o med on day 2 o cul u ing using205 he calcium dependen luo escen dye Fu a-2 as desc ibed p e-206 iously [6]. B ie ly, cul u es we e ans e ed o 2 mL esh Ham’s207 F12 medium con aining 10 #L Fu a-2-ace oxy-me hyles e (AM;208 10 #M) and 4 #L neos igmin (0.3 nM; o inhibi ex acellula 209 choline-es e ase ac i i y). Fu a-2-loaded cells we e hen placed on210 he s age o an in e ed luo escen mic oscope (Diapho ; Nikon,211 Kowasaki, Japan) and iewed using a 40× oil imme sion objec i e.212 Measu emen s we e pe o med in no mal and Ca2+- ee Ty ode’s213 solu ions (see abo e). LaCl3(500 #M), YM-58483 (1 #M), and CPA214 (10 #M) we e dilu ed in Ca2+- ee Ty ode’s solu ion. Exci a ion215 wa eleng h was al e ed be ween 340 and 380 nm (F340 and F380)216 by a mic ocompu e -con olled dual-wa eleng h monoch oma o 217 (Del aScan; Pho on Technologies In e na ional, New B unswick, NJ,218 USA). Emission was moni o ed a 510 nm a 10 Hz acquisi ion a e219 using a pho omul iplie . Backg ound luo escence was sub ac ed 220 on-line om F340 and F380 signals by he da a acquisi ion so wa e.221 In acellula [Ca2+] was calcula ed om he a io o measu ed222 luo escence in ensi ies (R = F340/F380) as desc ibed by G ynkiewicz223 e al. [25]. The measu ing ba h was cons an ly pe used wi h no -224 mal Ty ode’s solu ion a a a e o 2 mL min−1(EconoPump; Bio-Rad225 Labo a o ies, CA, USA). Tes solu ions we e di ec ly applied o he226 cells h ough a pe usion capilla y ube (Pe usion PencilTM; Au o-227 Ma e Scien i ic, San F ancisco, CA, USA) wi h an in e nal diame e o 228 250 #m a a a e o 1.5 #L s−1, using a local pe usion sys em (Val e229 BankTM 8 e sion 2.0, Au oMa e Scien i ic). All measu emen s we e 230 pe o med a oom empe a u e. 231 2.4. Modula ion o Ca2+ in lux ia ol age-ope a ed Ca2+ 232 channels wi h ni edipine and inhibi ion o SOCE 233 In o de o assess longe - e m e ec s o in e e ence wi h Ca2+ 234 homeos asis o di e en ia ing chond ocy es, he abo e compounds 235 we e also added o he cul u e medium o HDC. The L- ype VOCC- 236 blocke ni edipine was adminis e ed o he cul u e medium om 237 he beginning o he i s cul u ing day a a inal concen a ion o 238 10 #M. Fo con ol expe imen s, he ehicle (DMSO, Sigma) was 239 added o cul u es a equal olumes. To assess he ole o in e - 240 nal Ca2+ s o es, ollowing s o e deple ion wi h 10 #M CPA, SOCE 241 was blocked by co-applica ion o 1 #M YM-58483 and 500 #M 242 LaCl3. CPA, YM-58483 and LaCl3we e added o he cul u e medium 243 on cul u ing day 2 o 24 h. Fo con ol expe imen s, he ehicles 244 (DMSO and s e ile wa e ) we e added o cul u es a equal olumes. 245 2.5. Quali a i e and semi-quan i a i e de e mina ion o ca ilage 246 ma ix p oduc ion 247 Fo isualisa ion o ca ilage ma ix in HDC, low pH me ach o- 248 ma ic s aining was pe o med wi h dime hyl me hylene blue 249 (DMMB; Sigma) dissol ed in 3% ace ic acid on day 6 o cul u - 250 ing. The amoun o sulpha ed ma ix componen s was de e mined 251 wi h a semi-quan i a i e me hod, by measu ing he op ical den- 252 si y o ex ac ed oluidine blue (TB; Reanal, Budapes , Hunga y) 253 bound o glycosaminoglycans in 6-day-old HDC. Bo h quali a i e 254 and semi-quan i a i e s aining p ocedu es we e desc ibed p e i- 255 ously in mo e de ail [6]. 256 2.6. Measu emen o cell p oli e a ion and mi ochond ial ac i i y 257 Ra e o cellula p oli e a ion in HDC was de e mined by mea- 258 su ing he adioac i i y o inco po a ed 3H- hymidine du ing a 259 16-h-long pe iod on day 3 (as desc ibed ea lie [6]) s a ed 260 p omp ly a e combined ea men wi h CPA, YM-58483 and LaCl3, 261 o ni edipine. Fo he in es iga ion o mi ochond ial ac i i y, cells 262 cul u ed in wells o 96-well pla es we e used and MTT-assay 263 was pe o med immedia ely a e ea men s on day 3 as i was 264 desc ibed p e iously [6]. Un ea ed 3-day-old HDC we e used as 265 con ols o bo h assays. Measu emen s we e ca ied ou in 6 sam- 266 ples o each expe imen al g oup in 3 independen expe imen s. 267 2.7. Re e se ansc ip ion ollowed by PCR analysis 268 To al RNA om HDC was isola ed as desc ibed p e iously [6]. 269 The assay mix u e (20 #L) o e e se ansc ip ase (RT) eac ions 270 con ained 500 ng o al RNA, 0.25 #L RNase inhibi o , 2 #L andom 271 p ime s, 0.8 #L dNTP Mix (4 mM), 50 uni s (1 #L) Mul iSc ibeTM 272 RT in 1× RT bu e (High Capaci y RT ki ; Applied Biosys ems, 273 Fos e Ci y, CA, USA) and complemen a y cDNA was ansc ibed 274 a 37◦C o 2 h. Ampli ica ions o speci ic cDNA sequences we e 275 achie ed wi h speci ic p ime pai s ha we e designed based on 276 chicken nucleo ide sequences published in GenBank and pu chased 277 om In eg a ed DNA Technologies, Inc. (IDT; Co al ille, IA, USA). 278 Nucleo ide sequences o o wa d and e e se p ime s and eac- 279 ion condi ions a e shown in Table 1. PCR eac ions we e ca ied 280 ou in a inal olume o 25 #L con aining 1–1 #L o wa d and 281 e e se p ime s (10 #M), 0.5 #L cDNA, 0.5 #L dNTP Mix (200 #M), 282 and 1 uni (0.2 #L) P omega GoTaq®DNA polyme ase in 1× G een 283 GoTaq®Reac ion Bu e in a p og ammable he mal cycle (Labne 284 Mul iGeneTM 96-well G adien The mal Cycle ; Labne In e na- 285 ional, Edison, NJ, USA) wi h he ollowing se ings: 2 min a 95◦C 286 Please ci e his a icle in p ess as: J. Fodo , e al., S o e-ope a ed calcium en y and calcium in lux ia ol age-ope a ed calcium channels egula e in acellula calcium oscilla ions in chond ogenic cells, Cell Calcium (2013), h p://dx.doi.o g/10.1016/j.ceca.2013.03.003 ARTICLE IN PRESS G Model YCECA 1472 1–16 4J. Fodo e al. / Cell Calcium xxx (2013) xxx– xxx Table 1 Nucleo ide sequences, ampli ica ion si es, GenBank accession numbe s, amplicon sizes and PCR eac ion condi ions o each p ime pai a e shown. Gene P ime Nucleo ide sequence (5′→ 3′) GenBank ID Annealing empe a u e Amplicon size (bp) CaV1.2 (CACNA1C) Sense CAA CAG AGC CAA AGG ACT AAA (3054–3074) XM 416388 54◦C 477 An isense GTG ACG ATG ACG AAA CCA A (3512–3530) CaV1.3 (CACNA1D) Sense AGG CTC ATC AAT CAC CAC A (2704–2722) NM 205034 54◦C 387 An isense AAA GAC GCA CTG AAC AAC G (3072–3090) CaV2.2 (CACNA1B) Sense CTA CGC CAC GAC CCT ACA C (2442–2460) NM 204293 61◦C 408 An isense TTC TCA ACG CCT TCT TCC A (2831–2849) CaV2.3 (CACNA1E) Sense TCA CCA ACT CCG ACC GTA AC (3347–3366) XM 422255 60◦C 500 An isense CAC CTC CAT CTT GTT CTT CTC AT (3824–3846) CaV3.1 (CACNA1G) Sense CAC TGA ATC CGT CCA TAG CAT C (1989–2010) XM 001232653 61◦C 423 An isense CTG TCT GAG TCC GTC TCG TTG T (2390–2411) CaV3.2 (CACNA1H) Sense CCC TGG AAG GAT GGG TTG A (1256–1274) XM 414830 61◦C 371 An isense CTG CCC GTT TGT GGT GTT G (1608–1626) CaV3.3 (CACNA1I) Sense CTG AGG ACG GAT ACA GGA GAT (2281–2301) XM 425474 59◦C 437 An isense TTG CGT GAA GAG TTG GAG AC (2698–2717) O ai1 Sense TAG CAA CGT GCA TAA TCT CAA (264–284) NM 001030658 57◦C 257 An isense TCA GTC CAA AGG GAA CCA T (502–520) STIM1 Sense GGT GGT GTC CAT CGT CAT CG (426–445) NM 001030838 62◦C 356 An isense GCT CCT TCT CGG CGT TCT TC (762–781) STIM2 Sense CAA TTA GCA ATC GCC AAA G (1177–1195) XM 420749 57◦C 495 An isense CAC AGA AAG GAT GTC AGG GT (1652–1671) Agg ecan co e p o ein (AGR1) Sense CAA TGC AGA GTA CAG AGA (276–294) XM 001232949 54◦C 430 An isense TCT GTC TCA CGG ACA CCG (688–704) Collagen II (COL2A1) Sense GGA CCC AAA GGA CAG ACG G (1191–1210) NM 204426 59◦C 401 An isense TCG CCA GGA GCA CCA GTT (1573–1591) Sox9 Sense CCC CAA CGC CAT CTT CAA (713–731) NM 204281 54◦C 381 An isense CTG CTG ATG CCG TAG GTA (1075–1093) GAPDH Sense GAG AAC GGG AAA CTT GTC AT (238–258) NM 204305 54◦C 556 An isense GGC AGG TCA GGT CAA CAA (775–793) o ini ial dena u a ion ollowed by 35 epea ed cycles o dena u -287 a ion a 94◦C o 30 s, p ime annealing o 45 s a an op imised288 empe a u e o each p ime pai (see Table 1), and ex ension a 289 72◦C o 90 s. A e he inal cycle, u he ex ension was allowed290 o p oceed o ano he 7 min a 72◦C. PCR p oduc s we e analysed291 using a 1.2% e hidium b omide-con aining aga ose gel. Op ical292 densi y o PCR p oduc signals was de e mined by using ImageJ 293 (Image P ocessing and Analysis in Ja a) e sion 1.46 eewa e294 (h p:// sbweb.nih.go /ij/).295 2.8. SDS–PAGE and Wes e n blo analysis296 To al cell lysa es o HDC o sodium dodecyl sul-297 pha e–polyac ylamide gel elec opho esis (SDS–PAGE) we e298 p epa ed as desc ibed p e iously [26]. 50 #g o p o ein was sep-299 a a ed by 7.5% SDS–PAGE gel o immunological de ec ion o key300 p o eins o Ca2+ in lux o elease unc ions (i.e. pan !1subuni 301 o ol age-ga ed Ca2+ channels and STIM1), as well as p o ein302 exp ession and phospho yla ion s a us o he chond ogenic mas e 303 ansc ip ion ac o Sox9. P o eins we e ans e ed elec opho e -304 ically o ni ocellulose memb anes. A e blocking in 5% non- a d y305 milk in PBS, memb anes we e incuba ed wi h p ima y an ibodies 306 o e nigh a 4◦C as ollows: abbi polyclonal an i-CaVpan !1 307 subuni in 1:200, epi ope: in acellula C- e minus (Alomone308 Labs, Je usalem, Is ael); and mouse monoclonal an i-STIM1 in 309 1:500, epi ope: 25–139 in human ha has a high simila i y o he310 chicken sequence (BD Biosciences, F anklin Lakes, NJ, USA); abbi 311 polyclonal an i-Sox9 an ibody (Abcam, Camb idge, UK) in 1:600;312 abbi polyclonal an i-P-Sox9 an ibody (Sigma) in 1:800; abbi 313 polyclonal an i-ac in an ibody (San a C uz Bio echnology, Inc., 314 San a C uz, CA, USA) and abbi polyclonal an i-GAPDH an ibody315 (Abcam). A e washing o 30 min in PBST, memb anes we e incu- 316 ba ed wi h he HRP-conjuga ed seconda y an ibody, an i- abbi 317 IgG (Bio-Rad) in 1:1500 dilu ion. Memb anes we e de eloped by 318 enhanced chemiluminescence eac ion (Millipo e, Bille ica, MA, 319 USA) acco ding o he ins uc ions o he manu ac u e . Op ical 320 densi y o signals was measu ed by using ImageJ 1.46. 321 2.9. S a is ical analysis 322 All da a a e ep esen a i e o a leas h ee independen expe - 323 imen s. A e ages a e exp essed as mean ± SEM (s anda d e o o 324 he mean; n, numbe o cells measu ed). S a is ical analysis was 325 pe o med by using S uden ’s - es . Th eshold o s a is ically sig- 326 ni ican di e ences as compa ed o espec i e con ol cul u es was 327 se a *P < 0.05. 328 3. Resul s 329 3.1. Rapid spon aneous Ca2+ oscilla ions a e de ec able in 330 di e en ia ing chond ocy es 331 To in es iga e spon aneous epe i i e ansien inc eases in 332 cy osolic Ca2+ concen a ion in indi idual cells o p ima y chon- 333 d i ying mic omass cul u es de i ed om emb yonic limb buds, 334 Fluo-4 luo escen Ca2+ imaging echnique was applied on days 1 335 and 2 using LIVE con ocal mic oscopy. As hese cul u es a e he e o- 336 geneous by na u e in e ms o cellula composi ion (epi helial cells 337 and muscle p ogeni o s wi h dis inc mo phology can also be ound 338 in ela i ely small numbe s along wi h os eochond op ogeni o 339 cells; see [21]), only cells wi h ound mo phology we e included 340 in his s udy, while o he s we e dis ega ded. Se ies o X–Y images 341 Please ci e his a icle in p ess as: J. Fodo , e al., S o e-ope a ed calcium en y and calcium in lux ia ol age-ope a ed calcium channels egula e in acellula calcium oscilla ions in chond ogenic cells, Cell Calcium (2013), h p://dx.doi.o g/10.1016/j.ceca.2013.03.003 ARTICLE IN PRESS G Model YCECA 1472 1–16 J. Fodo e al. / Cell Calcium xxx (2013) xxx– xxx 5 Fig. 1. Spon aneous Ca2+ oscilla ions in cells o HDC on day 2 o cul u ing. P io o measu emen s, cells we e loaded wi h Fluo-4-AM o 30 min Ca2+ oscilla ions we e obse ed wi hou agonis s imula ion in Ty ode’s solu ion con aining 1.8 mM Ca2+ a oom empe a u e. (A) Se ies o X–Y images we e eco ded om andom isual ields o chond i ying cul u es wi h Zeiss LIVE 5 Lase Scanning Con ocal Mic o- scope. These ou ep esen a i e ames we e acqui ed a 6.5, 18.3, 29.6 and 54.4 s du ing measu emen s. A ows indica e di e en ia ing chond ocy es wi h epe i i e in acellula Ca2+ oscilla ions. (B) Time cou se o luo escence in ensi ies o he cells ma ked wi h a ows in panel (A). Fluo-4 luo escence in ensi y alues no malised o baseline luo escence (F/F0) a e plo ed s. ime. Wide anges o equency and ampli ude o oscilla ing cells we e obse ed. we e eco ded om andom isual ields o Fluo-4-loaded chond i- 342 ying cul u es on days 1 and 2 o cul u ing. Ca2+ oscilla ions we e343 eco ded om cells ba hed in Ty ode’s solu ion con aining 1.8 mM344 Ca2+ wi hou agonis s imula ion a oom empe a u e. Fou ep e-345 sen a i e X–Y images eco ding Fluo-4-loaded cells in a 2-day-old346 cul u e aken a 6.5, 18.3, 29.6 and 54.4 s du ing measu emen s347 a e shown in Fig. 1A. Wide anges o equency and ampli ude o 348 oscilla ing cells we e obse ed (Fig. 1B; see Supplemen a y Video).349 On cul u ing day 1, 45 o 240 cells in es iga ed (19%) exhibi ed350 spon aneous Ca2+ oscilla ions; whe eas on day 2, he p opo ion351 Fig. 2. Pooled da a o Ca2+ oscilla ions ga he ed om se ies o X–Y images acqui ed om andom isual ields o Fluo-4 loaded HDC on cul u ing days 1 and 2 wi h Zeiss LIVE 5 Lase Scanning Con ocal Mic oscope. (A) Ra io o oscilla ing cells and e- quency o epe i i e Ca2+ ansien s on days 1 and 2 o cul u ing. Numbe s abo e ba s indica e he numbe o oscilla ing cells compa ed o all cells eco ded. (B) Ampli ude and ull ime a hal maximum (FTHM) o Ca2+ oscilla ions in di e en ia ing cells o HDC on cul u ing days 1 and 2. Fo bo h panels (A) and (B), while calcula ing he pa ame e s o Ca2+ oscilla ions, only oscilla ing cells wi h ound, chond oblas -like mo phology we e conside ed. Measu emen s we e ca ied ou on cul u es om 4 independen expe imen s. Da a ep esen mean ± s anda d e o o he mean (SEM). Numbe s in pa en heses abo e ba s indica e he numbe o cells measu ed. As e isks (*) ma k signi ican di e ences (*P < 0.05) be ween pa ame e s o oscilla ing cells in 1- and 2-day-old HDC. o cells o show ansien inc eases in cy osolic Ca2+ concen a- 352 ion was subs an ially highe (175 o 317 cells; 55%) (Fig. 2A). No 353 only he p opo ion o oscilla ing cells, bu also hei pa ame e s 354 exhibi ed subs an ial changes du ing he cou se o di e en ia ion 355 (Fig. 2A and B). While he equency o oscilla ions we e ound o 356 be signi ican ly smalle on day 2 (0.06 ± 0.003 Hz; n = 175) s. day 1 357 (0.08 ± 0.01 Hz; n = 45; P = 0.01) (Fig. 2A), he a e age ampli ude o 358 ansien inc eases in cy osolic Ca2+ concen a ion- ela ed luo es- 359 cence a io signi ican ly inc eased om day 1 (exp essed as !F/F0: 360 0.97 ± 0.11; n = 45) o cul u ing day 2 (1.27 ± 0.06; n = 175; P = 0.03) 361 (Fig. 2B). The hi d a iable ha was used o desc ibe he du a ion 362 o indi idual spon aneous ansien s ( ull ime a hal maximum; 363 FTHM) did no p o e o be s a is ically di e en on he wo cul u - 364 ing days in es iga ed (2.37 ± 0.29 s; n = 45 on day 1 s. 2.57 ± 0.15 s; 365 n = 175 on day 2; P = 0.55) (Fig. 2B). 366 3.2. Al e ed ex acellula ionic milieu and Ca2+ en y blocke s 367 modi y he appea ance and quan i a i e pa ame e s o apid 368 spon aneous Ca2+ oscilla ions in a ime-dependen manne 369 Pa ame e s o spon aneous Ca2+ oscilla ions we e examined on 370 day 2 o cul u ing by eco ding se ies o X–Y images. In each cul- 371 u e, a andom isual ield wi h oscilla ing cells was se , and ames 372 Please ci e his a icle in p ess as: J. Fodo , e al., S o e-ope a ed calcium en y and calcium in lux ia ol age-ope a ed calcium channels egula e in acellula calcium oscilla ions in chond ogenic cells, Cell Calcium (2013), h p://dx.doi.o g/10.1016/j.ceca.2013.03.003 ARTICLE IN PRESS G Model YCECA 1472 1–16 6J. Fodo e al. / Cell Calcium xxx (2013) xxx– xxx we e acqui ed in con ol condi ions (no mal Ty ode’s solu ion; con-373 aining 1.8 mM Ca2+) a he 0 ime poin , and hen in he p esence374 o es solu ions (ei he 10 #M ni edipine; o 500 #M LaCl3and375 1 #M YM-58483); 1, 3 and 5 min a e changing he ba h o he376 es solu ions. Con ol measu emen s we e pe o med on andom377 isual ields in cul u es a 0-, 1-, 3- and 5-min ime poin s in no -378 mal Ty ode’s solu ion. I should be no ed ha all h ee pa ame e s379 o Ca2+ oscilla ions in es iga ed in his s udy (i.e. a io o oscilla ing380 cells; ampli ude and equency o oscilla ions) ollowed a s eady381 decline in he 5-min ime ame e en in con ol condi ions; he e-382 o e, all pa ame e s ollowing an in e en ion we e no malised o383 hei espec i e con ol alues (da a no shown).384 Applica ion o he dihyd opy idine L- ype Ca2+ channel blocke 385 ni edipine did no cause signi ican changes in he a io o oscil-386 la ing cells as compa ed o un ea ed con ol cells eco ded387 a 3- and 5-min ime poin s (80.93 ± 25.78% [P = 0.56]; and388 91.73 ± 28.95 [P = 0.84], espec i ely; n = 43 o all ime poin s;389 Fig. 3A), and no signi ican changes we e obse ed in he ampli-390 ude ei he (87.58 ± 11.89% [P = 0.5]; 69.44 ± 10.26% [P = 0.16];391 and 75.83 ± 6.26% [P = 0.13] a 1-, 3- and 5-min ime poin s;392 n = 25, 16 and 11, espec i ely; Fig. 3B). A he same ime, 393 ni edipine dec eased he equency (53.97 ± 8.52% [P = 0.01]; and394 64.46 ± 12.88% [P = 0.15] a 3- and 5-min ime poin s; n = 16 and 11,395 espec i ely; Fig. 3C) o Ca2+ oscilla ions; his pa ame e was ound396 o be signi ican ly di e en om he con ol a he 3-min ime poin .397 These esul s indica e ha ni edipine in e e ed wi h he equency398 o oscilla ions, a he han he ac ual numbe o oscilla ing cells399 and he ampli ude o he Ca2+ ansien s. In o he wo ds, ea men 400 wi h ni edipine did no abolish epe i i e Ca2+ ansien s, sugges -401 ing ha VOCCs a e no he p ima y, al hough impo an ac o s o402 media e his phenomenon. 403 By con as , adminis a ion o he SOCE blocke s signi ican ly404 dec eased he a io o oscilla ing cells a all h ee ime poin s405 (11.59 ± 6.95% [P = 0.0006]; 23.05 ± 13.58% [P = 0.008]; and 0.0%406 [P = 0.0] a 1-, 3- and 5-min ime poin s, espec i ely; n = 29 o 407 all ime poin s) compa ed o he con ol (Fig. 3A); in pa icula ,408 no oscilla ing cells could be obse ed in andom isual ields a e 409 5 min. A he same ime, ampli udes o epe i i e Ca2+ ansien s410 we e also educed (45.35 ± 7.53% [P = 0.24]; and 58.33 ± 12.85% 411 [P = 0.34] a 1- and 3-min ime poin s; n = 2 and 3, espec i ely;412 Fig. 3B), and hese blocke s also dec eased he equency o oscil-413 la ions a he 3-min ime poin (45.45 ± 22.72% [P = 0.13]; n = 3;414 Fig. 3C).415 Since hese esul s sugges ha ER Ca2+ s o es play a de e min-416 ing ole in egula ing epe i i e Ca2+ ansien s in di e en ia ing417 cells o HDC, we aimed o u he analyse he e ec s o blockade 418 o s o e-ope a ed Ca2+ channels, as well as emo al o ee ex a-419 cellula Ca2+ ions om he ba h solu ion wi h a highe empo al420 esolu ion on line-scan diag ams. In hese measu emen s, only421 ound-shaped di e en ia ing cells ha exhibi ed p ominen Ca2+ 422 oscilla ions we e included and o he cells we e dis ega ded. Line-423 scan images o oscilla ing cells we e eco ded on day 2 o cul u ing424 in no mal Ty ode’s solu ion (Fig. 4A). In he 46 cells examined, he 425 equency and he ampli ude o Ca2+ oscilla ions we e ound o be426 0.08 ± 0.007 Hz and 1.44 ± 0.15, espec i ely. Then, measu emen s427 we e con inued by changing he en i e olume o he ba h solu ion428 on he cul u es o he es solu ions. When LaCl3and YM-58483429 we e applied o oscilla ing cells (n = 10), Ca2+ oscilla ions we e430 blocked, and a se e e dis u bance in cy osolic Ca2+ was obse ed:431 small-ampli ude a hy hmic luc ua ions in basal cy osolic Ca2+ 432 concen a ion, a he han pe iodic Ca2+ ansien s, we e eco ded;433 he e o e, he analysis o Ca2+ concen a ion- ela ed changes in434 ela i e luo escence in ensi ies could no be pe o med (Fig. 4B).435 Wi h he ER Ca2+ s o es deple ed—i.e. when he SERCA-blocke 436 CPA was co-applied wi h LaCl3and YM-58483—Ca2+ oscilla ions437 we e immedia ely elimina ed (n = 10) and no changes in cy osolic438 Fig. 3. Pooled da a o Ca2+ oscilla ions ob ained om se ies o X–Y images acqui ed om Fluo-4 loaded HDC in esponse o a ious ea men s. Measu emen s we e ca ied ou wi h Zeiss LIVE 5 Lase Scanning Con ocal Mic oscope. A o al numbe o 500 images we e eco ded a each ime poin o each isual ield; ame acquisi ion a e was 10 s−1. (A) Pe cen age o oscilla ing cells be o e ea men (con ol), and 1, 3 o 5 min a e he applica ion o ba h solu ion con aining 10 #M ni edipine, o 500 #M LaCl3and 1 #M YM-58483. Values we e no malised o he un ea ed cells measu ed a 0 min (con ol), and hen a 1, 3 and 5 min. Numbe s in pa en heses abo e ba s show he numbe o cells measu ed. (B) Ampli ude o Ca2+ oscilla ions, no malised o alues o un ea ed con ol cells. Numbe s in pa en heses abo e ba s ep esen he numbe o oscilla ing cells measu ed. (C) F equency o Ca2+ oscilla ions no malised o he con ol. Numbe s in pa en heses abo e ba s show he numbe o cells measu ed. Fo panels (A)–(C), oscilla ing cells wi h ound mo phology in he same andom isual ield we e eco ded a all ou ime poin s. Di e en ia ing ca ilage colonies we e only used o a single measu emen se ies and hen we e disca ded. G aphs ep esen pooled da a o 3 independen expe imen s, measu ing andom isual ields o 5 colonies o each ea men . As e isks (*) ma k signi ican di e ences (*P < 0.05) be ween pa ame e s o ea ed s. con ol cells a espec i e ime poin s. Please ci e his a icle in p ess as: J. Fodo , e al., S o e-ope a ed calcium en y and calcium in lux ia ol age-ope a ed calcium channels egula e in acellula calcium oscilla ions in chond ogenic cells, Cell Calcium (2013), h p://dx.doi.o g/10.1016/j.ceca.2013.03.003 ARTICLE IN PRESS G Model YCECA 1472 1–16 J. Fodo e al. / Cell Calcium xxx (2013) xxx– xxx 7 Fig. 4. Pha macological modula ion o spon aneous Ca2+ oscilla ions and e ec s o al e ed ex acellula ionic milieu in Fluo-4 loaded di e en ia ing chond ocy es o 2-day-old HDC. Rep esen a i e con ocal line-scan images and ime cou ses o Fluo-4 luo escence in ensi ies a e shown; ho izon al and e ical calib a ions a e he same o all aces in panels (A)–(D). Ho izon al lines unde aces show he du a ion o ea men s wi h pha macons o al e ed ex acellula ionic milieu. Acqui- si ion o line-scan images s a ed immedia ely a e changing he ba h solu ion on he cul u es. P io o ha , no mal unc ions we e de ec ed on each cul u e. (A) Spon aneous Ca2+ oscilla ions in no mal ([Ca2+]e= 1.8 mM) Ty ode’s solu ion. (B) A e he non-selec i e ca ion channel-media ed Ca2+ en y blocke LaCl3(500 #M) and he s o e-ope a ed Ca2+ en y and Ca2+ elease-ac i a ed Ca2+ (CRAC) chan- nel blocke YM-58483 (1 #M) we e applied in no mal ([Ca2+]e= 1.8 mM) Ty ode’s, Ca2+ oscilla ions ceased, al hough i egula luc ua ions in basal cy osolic Ca2+ con- cen a ion emained de ec able. (C) When he SERCA-blocke CPA (10 #M) was co-adminis e ed wi h 500 #M LaCl3and 1 #M YM-58483 in 1.8 mM [Ca2+]e, Ca2+ oscilla ions we e o ally elimina ed. (D) 3 min a e changing he ba h solu ion o Ca2+- ee Ty ode’s, pe iodic oscilla ions could no be de ec ed. Line-scan diag ams on panels (A)–(D) a e ep esen a i e da a ou o 4 independen expe imen s. Ca2+ le els could be de ec ed, e en in he p esence o ex e nal439 Ca2+ in he ba h solu ion (Fig. 4C). When oscilla ing cells (n = 10)440 we e ba hed wi h Ca2+- ee Ty ode’s solu ion, he ampli udes441 o oscilla ions g adually dec eased, and only disappea ed a e 442 se e al minu es. The ep esen a i e line-scan diag am (Fig. 4D)443 shows lack o epe i i e Ca2+ ansien s 3 min a e changing he 444 ba h solu ion o Ca2+ ee Ty ode’s.445 3.3. Di e en ia ing chond ocy es exp ess he ˛1subuni o VOCCs446 a bo h mRNA and p o ein le els447 Since ni edipine was ound o educe he equency o spon-448 aneous Ca2+ oscilla ions; u he mo e, applica ion o 120 mM449 KCl e oked la ge Ca2+ ansien s in he same expe imen al450 model in ou p e ious expe imen s [21], we can hypo hesise ha 451 ol age-ope a ed Ca2+ channels may be exp essed and unc ion452 on di e en ia ing chond ocy es. O he se e al di e en subuni s453 ha comp ise unc ional VOCCs, he Ca2+ selec i e po e- o ming454 !1subuni is he one ha p ima ily de e mines he channel455 p ope ies. The e o e, we i s downloaded a ailable sequence 456 da a o chicken !1subuni mRNAs, and ca ied ou RT-PCR 457 eac ions wi h p ime pai s speci ic o each ype o VOCC designed 458 by P ime P emie 5.0 so wa e (P emie Bioso , Palo Al o, CA, 459 USA). Two ion channel subuni mRNA ansc ip s (CaV1.2 and 460 CaV1.3) o L- ype (o dihyd opy idine-sensi i e) Ca2+ channels 461 we e ound o be exp essed by chond ocy es, wi h CaV1.2 showing 462 a cons an exp ession le el, while CaV1.3 exhibi ed a peak-like 463 pa e n wi h almos 5- old s onge signals on days 2–4 o cul- 464 u ing (Fig. 5A). By con as , he R- ype CaV2.3 !1subuni mRNA 465 showed a ma ked exp ession in chond op ogeni o mesenchymal 466 cells, and g adually disappea ed om di e en ia ed chond oblas s 467 and chond ocy es. The h ee T- ype (CaV3.1, CaV3.2 and CaV3.3) 468 ion channel subuni s, in e es ingly, ollowed a e y simila mRNA 469 exp ession p o ile o wha has been obse ed in case o L- ype 470 channels; CaV3.1 and CaV3.2 ollowed a cons an exp ession (wi h 471 he la e exhibi ing signs o down egula ion in ma u e chon- 472 d ocy es), whe eas CaV3.3 also showed s onge signals du ing 473 di e en ia ion o chond op ogeni o cells on days 2–4 (Fig. 5A). 474 Ha ing con i med mRNA exp essions o a ious VOCCs, we also 475 wan ed o check he p esence o !1subuni s a he p o ein le el. 476 By using a polyclonal an ibody aised agains CaV!1subuni s, 477 immuno eac i e bands we e de ec ed a he expec ed molecula 478 weigh (app ox. 130 kDa). The p o ein showed s ong exp essions 479 in o al cell lysa es o HDC h oughou he en i e cul u ing pe iod 480 (Fig. 5B). These indings, oge he wi h da a ob ained om Ca2+ 481 imaging expe imen s, demons a e he unc ional exp ession o 482 a ious VOCCs in di e en ia ing chond ogenic cells. 483 Nex , we also unde ook o cha ac e ise molecules ha enable 484 Ca2+ en y ollowing s o e deple ion (also known as Ca2+ elease- 485 ac i a ed Ca2+ o CRAC channels). Al hough O ai1 and STIM1, 486 essen ial media o s o CRAC channel unc ion, ha e been desc ibed 487 in 2005 and 2006, espec i ely [27], hei exp ession and unc- 488 ion ha e no been in es iga ed in chond ocy es. To his end, by 489 designing speci ic p ime s, we we e able o demons a e he con- 490 s an mRNA exp ession o STIM1, STIM2 and O ai1 h oughou he 491 en i e cul u ing pe iod (Fig. 5B), p obably e lec ing on hei essen- 492 ial ole in Ca2+ homeos asis. Fu he mo e, we also demons a ed 493 he p esence o STIM1 p o ein in o al cell lysa es o HDC on all 494 cul u ing days, wi h a cons an exp ession pa e n (Fig. 5B). The 495 iden i y o he uppe immunogenic band a ∼90 kDa is unknown; i 496 can well be a glycosyla ed o m o a splice a ian o STIM1. No e- 497 wo hy ha wo STIM1 p o eins o di e en size we e ound o be 498 exp essed in mu ine issues: besides he well-known STIM1 iso- 499 o m, a new 115 kDa STIM1 e e ed o as STIM1L has also been 500 ecen ly epo ed [28]. Owing o lack o comme cially a ailable 501 chicken-speci ic an ibodies aised agains O ai1, Wes e n blo anal- 502 ysis o his p o ein could no be pe o med. 503 3.4. Modula ion o ei he VOCC unc ion o SOCE in di e en ia ing 504 chond ocy es de imen ally a ec s in i o chond ogenesis 505 To assess he long- e m e ec s o he dihyd opy idine L- ype 506 Ca2+ channel blocke ni edipine (applied a 10 #M con inuously 507 om day 1, due o cons an exp ession o !1subuni s) and SOCE 508 inhibi ion combined wi h ER Ca2+ s o e deple ion (by 500 #M 509 LaCl3, 1 #M YM-58483 and 10 #M CPA adminis e ed on day 2 o 510 24 h) on ca ilage ma ix p oduc ion in i o, u he expe imen s 511 we e pe o med. Con inuous ea men wi h ni edipine signi i- 512 can ly a enua ed ca ilage ma ix p oduc ion by cul u ing day 6 513 as e ealed by me ach oma ic s aining p ocedu es (Fig. 6A). Wi h 514 ER Ca2+ s o es deple ed, blockade o SOCE o only 24 h on day 2 515 esul ed in an equally p ominen inhibi o y e ec (Fig. 6A), e lec - 516 ing on he impo an oles o bo h pa hways (i.e. Ca2+ en y ac oss 517 he plasma memb ane ia VOCCs and Ca2+ elease om in e nal 518 Ca2+ s o es) in Ca2+ homeos asis o di e en ia ing chond ocy es. 519 Please ci e his a icle in p ess as: J. Fodo , e al., S o e-ope a ed calcium en y and calcium in lux ia ol age-ope a ed calcium channels egula e in acellula calcium oscilla ions in chond ogenic cells, Cell Calcium (2013), h p://dx.doi.o g/10.1016/j.ceca.2013.03.003 ARTICLE IN PRESS G Model YCECA 1472 1–16 8J. Fodo e al. / Cell Calcium xxx (2013) xxx– xxx Fig. 5. Exp ession p o iles o key molecules o Ca2+ in lux and elease on a ious days o cul u ing. (A) mRNA exp ession pa e ns o !1subuni s o ol age-ga ed (L-, R-, and T- ype) Ca2+ channels we e de ec ed by RT-PCR. (B) mRNA and p o ein-le el exp ession p o iles o molecules (STIM1/STIM2 and O ai1) ha o ches a e he SOCE mechanism, and p o ein-le el exp ession o he Ca2+ selec i e po e- o ming !1subuni o ol age-ga ed Ca2+ channels on a ious days o cul u ing, de ec ed wi h RT-PCR and Wes e n blo analyses, espec i ely. Fo he STIM1 immunoblo , he lowe band co esponds o he expec ed (70 kDa) molecula weigh (ma ked by a ows). Fo PCR eac ions shown in panels (A) and (B), GAPDH was used as a con ol (only shown in panel B). Fo Wes e n blo s, ac in was used as a con ol. Da a shown in panels (A) and (B) a e ep esen a i e ou o 3 independen expe imen s. Numbe s below bands ep esen in eg a ed densi ies o signals de e mined by ImageJ eewa e ha we e no malised o he alue o day 0. No ewo hy ha combined ea men s (wi h CPA, YM-58483 and520 LaCl3) longe han 24 h comple ely ab oga ed chond ogenesis (da a521 no shown). Day 2 was chosen o assess he e ec s o SOCE inhibi- 522 ion on he di e en ia ion s ep o chond op ogeni o cells.523 Nex , we looked a whe he hese ea men s in e e ed wi h524 he mRNA and p o ein exp ession o key genes o chond ogene-525 sis; as mRNA exp ession o ype 2 collagen (COL2A1) and agg ecan 526 co e p o ein (AGR1), as well as mRNA and p o ein exp ession and527 phospho yla ion s a us o he key chond ogenic ma ke Sox9 we e528 moni o ed on cul u ing day 3 by RT-PCR and Wes e n blo analy- 529 ses, espec i ely. Al hough con inuous ea men wi h ni edipine 530 did no in e e e wi h he mRNA exp ession o hese genes, Sox9531 p o ein exp ession was ound o be ma kedly educed, wi hou a532 de ec able change in i s phospho yla ion le el (Fig. 6B and C). By533 con as , in e e ence o ER Ca2+ s o e unc ions caused a s ong534 down egula ion o mRNAs o ECM componen s, al hough mRNA535 ansc ip le els o Sox9 we e no al e ed (Fig. 6B). A he p o ein536 le el, Sox9 p o ein exp ession and phospho yla ion s a us ollowed537 he same changes as obse ed o ea men s wi h ni edipine; Sox9538 p o ein le el was also ound o be dec eased, and he phospho y-539 la ion s a us was also only sligh ly modi ied ollowing SOCE block540 (Fig. 6C).541 Since in addi ion o modula ing in i o chond ogenesis a he 542 molecula le el, hese ea men s could ha e al e ed me abolic543 ac i i y and/o he cell cycle o di e en ia ing chond ocy es; he e- 544 o e, mi ochond ial ac i i y and a e o p oli e a ion we e also 545 de e mined on day 3 by MTT es and 3H- hymidine inco po a- 546 ion assays, espec i ely. While nei he ni edipine no combined 547 ea men o p e en Ca2+ e-up ake in o deple ed in e nal s o es 548 modula ed cellula me abolic ac i i y, bo h ea men s almos 549 comple ely ab oga ed cell p oli e a ion (Fig. 6D). Consequen ly, 550 he obse ed dec ease in me ach oma ic ma ix p oduc ion can 551 pa ially be a ibu ed o he de ec ed d ama ic inhibi ion o cell 552 p oli e a ion. 553 3.5. SOCE blocke s al e he pa ame e s o s o e-ope a ed Ca2+ 554 en y induced by s o e deple ion du ing luo escen single cell 555 Ca2+ measu emen s 556 In acellula Ca2+ concen a ion measu emen s we e pe o med 557 in 2-day-old cul u es loaded wi h Fu a-2 o assess he pa ame e s 558 o Ca2+ ansien s igge ed ia SOCE in he absence (Fig. 7A) and in 559 he p esence o SOCE blocke s (500 #M LaCl3and 1 #M YM-58483; 560 Fig. 7B). A he beginning o eco dings, in e nal Ca2+ s o es o 561 cells we e emp ied by p e- ea men wi h he SERCA-inhibi o CPA 562 (10 #M) dissol ed in Ca2+- ee Ty ode’s (no shown). The changes 563 in cy osolic Ca2+ concen a ion e oked by e-es ablishing he no - 564 mal (1.8 mM) ex acellula Ca2+ concen a ion we e hen eco ded. 565 Please ci e his a icle in p ess as: J. Fodo , e al., S o e-ope a ed calcium en y and calcium in lux ia ol age-ope a ed calcium channels egula e in acellula calcium oscilla ions in chond ogenic cells, Cell Calcium (2013), h p://dx.doi.o g/10.1016/j.ceca.2013.03.003 ARTICLE IN PRESS G Model YCECA 1472 1–16 J. Fodo e al. / Cell Calcium xxx (2013) xxx– xxx 9 Fig. 6. E ec s o he dihyd opy idine L- ype Ca2+ channel blocke ni edipine (applied con inuously om day 1 a 10 #M) and SOCE inhibi ion combined wi h ER Ca2+ s o e Q2 deple ion (by 500 #M LaCl3, 1 #M YM-58483 and 10 #M CPA adminis e ed on day 2 o 24 h) on ca ilage ma ix p oduc ion in i o. (A) Me ach oma ic ca ilage a eas in 6-day-old high densi y colonies we e isualised wi h DMMB dissol ed in 3% ace ic acid (pH 1.8). Me ach oma ic (pu ple) s uc u es ep esen ca ilaginous nodules o med by many cells and a ca ilage ma ix ich in polyanionic GAGs. O iginal magni ica ion was 2×. Scale ba , 1 mm op ical densi y (OD625) was de e mined in supe na an s o 6-day-old cul u es con aining oluidine blue ex ac ed wi h 8% HCl dissol ed in absolu e e hanol. (B) mRNA ansc ip s o key chond ogenic and ECM ma ke genes on day 3 analysed by RT-PCR. GAPDH was used as a con ol. (C) P o ein exp ession and phospho yla ion s a us o he mas e chond ogenic ansc ip ion ac o Sox9 in 3-day-old cul u es. GAPDH was used as a con ol. (D) Mi ochond ial ac i i y and a e o p oli e a ion on day 3 de e mined by MTT es and 3H-Thymidine inco po a ion assays, espec i ely. S a is ically signi ican (*P < 0.05) di e ences in ex inc ion (OD625) o samples o TB and in a e o p oli e a ion a e ma ked by as e isks (*). Abb e ia ions used o panels (B)–(D): C, con ol; Ni e, ni edipine; SB, SOCE block by LaCl3, YM-58483 and CPA. Rep esen a i e da a ou o 3 independen expe imen s a e shown. (Fo in e p e a ion o he e e ences o colou in his igu e legend, he eade is e e ed o he web e sion o he a icle.) Compa ed o un ea ed con ol cells, in he p esence o he SOCE 566 blocke s bo h he ampli ude (114.4 ± 15.6; n = 16 s. 58.4 ± 5.6;567 n = 10; P = 0.01; Fig. 7C) and he maximal a e o ise (3.9 ± 0.5; n = 16 568 s. 1.5 ± 0.2; n = 10; P = 0.001; Fig. 7D) o SOCE we e signi ican ly 569 dec eased.570 4. Discussion571 4.1. Ca2+ homeos asis in di e en ia ing chond ocy es572 I is gene ally accep ed ha Ca2+ is he mos e sa ile second573 messenge . The e is accumula ing e idence ha Ca2+ signalling574 pa hways a e key media o s o cellula e en s in ol ed in di -575 e en ia ion p ocesses also in non-exci able cells including MSCs576 and chond ocy es. A sus ained ise in cy osolic Ca2+ concen a ion 577 induces di e en ia ion o MSCs: inc eased in acellula Ca2+ was 578 epo ed o exe a biphasic egula o y ole in adipocy e di e en- 579 ia ion, inhibi ing he ea ly s ages while p omo ing he la e s age 580 o di e en ia ion [29]. Di e en ia ion o mesenchymal cells in o 581 chond ocy es is also con olled by Ca2+ dependen pa hways: high 582 concen a ion o ex acellula Ca2+ was ound o p omo e chond o- 583 genic di e en ia ion in chicken HDC [30]. By con as , ex acellula 584 Ca2+ was epo ed o modula e di e en ia ion du ing skele oge- 585 nesis in chicken emb yonic cal a ia, whe e low concen a ions 586 enabled chond ogenesis [31]. The impo ance o Ca2+ in lux ia 587 plasma memb ane ion channels du ing chond ogenesis in mouse 588 limb bud-de i ed HDC was con i med by he ac ha ea men 589 wi h he L- ype channel-speci ic blocke s ni edipine and e apamil 590 Please ci e his a icle in p ess as: J. Fodo , e al., S o e-ope a ed calcium en y and calcium in lux ia ol age-ope a ed calcium channels egula e in acellula calcium oscilla ions in chond ogenic cells, Cell Calcium (2013), h p://dx.doi.o g/10.1016/j.ceca.2013.03.003 ARTICLE IN PRESS G Model YCECA 1472 1–16 16 J. Fodo e al. / Cell Calcium xxx (2013) xxx– xxx [55] H.J. 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