Cytosolic free Ca2+ concentration exhibits a characteristic temporal pattern during in vitro cartilage differentiation: a possible regulatory role of calcineurin in Ca-signalling of chondrogenic cells

UNCORRECTED PROOF YCECA 972 1—14 Please cite this article in press as: C. Matta, et al., Cytosolic free Ca2+ concentration exhibits a characteristic temporal pattern during in vitro cartilage differentiation: A possible regulatory role of calcineurin in Ca-signalling of chondrogenic cells, Cell Calcium (2008), doi:10.1016/j.ceca.2007.12.010 ARTICLE IN PRESS +Model YCECA 972 1—14 Cell Calcium (2008) xxx, xxx—xxx 1 journal homepage: www.elsevier.com/locate/ceca Cytosolic free Ca2+ concentration exhibits a Q1 characteristic temporal pattern during in vitro cartilage differentiation: A possible regulatory role of calcineurin in Ca-signalling of chondrogenic cells 2 3 4 5 Csaba Mattaa,1,J ´ anos Fodorb,1, Zsolt Sz ´ ıjgy´ art´ oc,Tam ´ as Juh´ asza, P´ al Gergelyc,L ´ aszl´ o Csernochb,R ´ oza Z´ ak´ anya,∗ 6 7 aDepartment of Anatomy, Histology and Embryology, Medical and Health Science Centre, University of Debrecen, Q2 Nagyerdei krt. 98, H-4032 Debrecen, Hungary 8 9 bDepartment of Physiology, Medical and Health Science Centre, University of Debrecen, Hungary10 cCell Biology and Signalling Research Group of the Hungarian Academy of Sciences, Department of Medical Chemistry, Research Centre for Molecular Medicine, University of Debrecen, Hungary 11 12 Received 24 July 2007; received in revised form 15 November 2007; accepted 29 December 200713 KEYWORDS Chondrogenesis; High-density culture; Intracellular Ca2+ concentration; Fura-2; Cyclosporine A; Sox9 Summary We measured changes of cytosolic Ca2+ concentration during chondrogenesis, which occurs in high-density cultures (HDC) of chondrifying chicken mesenchymal cells. A significant, transient elevation was detected in Fura-2-loaded cells on day 3 of culturing, when majority of chondrogenic cells of HDC become differentiated. This 140 nM peak of cytosolic Ca2+ concentration is a result of increased Ca-influx and is indispensable to proper chondrogenesis, because addition of 0.8 mM EGTA to culture medium on day 2 or 3 significantly decreased the intracellular Ca2+ concentration abolishing the Ca2+-peak of day 3 and inhibited cartilage formation. Uncontrolled Ca2+ influx evoked by a Ca2+ ionophore exerted dual effects on chondrogenesis in a 14 15 16 17 18 19 20 21 Abbreviations: AAS, atomic absorption spectrometry; AM, acetoxy-methylester; BMP, bone morphogenic protein; CMF-PBS, calcium and magnesium free phosphate buffered saline; CPA, cyclopiazonic acid; CsA, cyclosporine A; DMMB, dimethylmethylene blue; DMSO, dimethyl sulfoxide; dNTP, deoxyribonucleotide triphosphate; DTT, dithiothreitol; ECM, extracellular matrix; EGTA, ethylene glycol tetraacetic acid; ER, endoplasmic reticulum; FACS, fluorescence activated cell sorter; FCS, foetal calf serum; FGF, fibroblast growth factor; GAPDH, glyceraldehide-3-phosphate dehydrogenase; HDC, high-density cell culture; IGF, insulin-like growth factor; IP3, inositol-1,4,5-trisphosphate; MAPK, mitogen-activated protein kinase; MTT, 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide; N-CAM, neural cell adhesion molecule; OA, okadaic acid; PBS, phosphate buffered saline; PBST, phosphate buffered saline with 0.1% Tween 20; PKC, protein kinase C; PMCA, plasma membrane Ca2+ ATPase; PMSF, phenylmethylsulphonyl; RT-PCR, reverse transcription and polymerase chain reaction; RyR, ryanodine receptor; SDS-PAGE, sodium dodecyl sulphate polyacrilamide gel electrophoresis; Ser, serine; SERCA, sarco(endo)plasmic reticulum Ca2+ ATPase; SOCE, store-operated calcium entry; Thr, threonine; TRPV, transient receptor potential ion channel. ∗Corresponding author. Tel.: +36 52 416 392; fax: +36 52 432 290. Q3 E-mail address: [email protected] (R. Z´ ak´ any). 1These two authors contributed equally to this work. 0143-4160/$ — see front matter © 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.ceca.2007.12.010 UNCORRECTED PROOF YCECA 972 1—14 Please cite this article in press as: C. Matta, et al., Cytosolic free Ca2+ concentration exhibits a characteristic temporal pattern during in vitro cartilage differentiation: A possible regulatory role of calcineurin in Ca-signalling of chondrogenic cells, Cell Calcium (2008), doi:10.1016/j.ceca.2007.12.010 ARTICLE IN PRESS +Model YCECA 972 1—14 2 C. Matta et al. concentration-dependent manner; 0.1 mg/L A23187 increased, whereas 5 mg/L A23187 almost totally blocked cartilage formation. Intracellular Ca-stores seemed not to have any significant participation in the regulation of changes of cytosolic Ca2+ concentration of chondrifying cells. Activity of Ca—calmodulin-dependent protein phosphatase, calcineurin responded to changes of intracellular Ca2+ concentration induced by EGTA or A23187 in a differentiation stage-dependent manner. Since inhibition of calcineurin with cyclosporine A eliminated the peak in the cytosolic Ca2+ concentration, an active regulatory role of calcineurin on Ca2+ influx of chondrifying cells can be supposed. 22 23 24 25 26 27 28 29 © 2008 Elsevier Ltd. All rights reserved.30 Introduction31 Hyaline cartilage is an important element of the verte-32 brate skeletal system. It provides primordia of bones formed33 by endochondral ossification and remains the major shock-34 absorbing structure of the articular surfaces of joints.35 Chondrogenic mesenchymal cells can be derived from dif-36 ferent embryonic structures: the cranial part of neural37 crest is the source of cartilage primordia of several cran-38 iofacial bones; sclerotome of somites differentiates into39 vertebrae; appendicular bones derive from mesenchymal40 cells of somatopleura [1]. 41 High-density cell culture system (HDC) established from42 chondrogenic mesenchymal cells isolated from limb buds 43 of 4-day-old chicken embryos is a well-known model of in44 vitro cartilage differentiation [2—4]. This simple model can45 provide information on the molecular steps leading to dif46 ferentiation of chondroprogenitor cells to chondroblasts.47 In HDC, formation of cartilage starts with the recruit48 ment of chondroprogenitor mesenchymal progenitor cells49 that after condensation and nodule formation, differenti-50 ate into chondroblasts and chondrocytes. Condensation and51 nodule formation take place on the first day of culturing 52 and are partly regulated by transient appearance of Ca2+-53 dependent intercellular junctions like N-CAM (neural cell54 adhesion molecule) and N-cadherin [5]. Chondroprogenitor55 cells differentiate into chondroblasts on the second and56 third day of culturing [4,6], controlled by numerous growth57 factors and other signal molecules, e.g. FGF, BMP, Wnt, IGF 58 and members of Hedgehog and Sox transcription factor fami-59 lies [7]. In parallel to the intracellular changes, extracellular60 matrix (ECM) surrounding the differentiating chondrogenic 61 cells is also subject to profound changes: differentiating62 cells start to secrete cartilage-specific matrix components,63 such as collagen type II and aggrecan on the third day of cul64 turing period [8]. The unique composition and organization 65 of ECM is crucial for maintenance of the proper morphol-66 ogy and function of these cells [9]. Expression of collagen67 type II and core protein of aggrecan is controlled by Sox9, a68 high-mobility-group domain containing transcription factor 69 [10—12]. Detection of the expression level and the phospho-70 rylation status of Sox9, as well as monitoring the expression 71 of the core protein of aggrecan are a reliable markers of72 chondrogenesis.73 Calcium ion is a ubiquitous cellular signal. The concen-74 tration of intracellular free Ca2+ (∼10−7M) is 104times75 lower than that of the extracellular fluid. This distribu76 tion provides the potential for the influx of Ca2+ into cells,77 where it can act as a second messenger. Various stimuli78 promote the movement of Ca2+ either from the extracel-79 lular space or from intracellular stores into the cytosol. 80 The elevated level of cytosolic free Ca2+ exerts a variety 81 of specific changes in cellular function, such as activa82 tion of protein kinases and protein phosphatases, which, 83 in turn, regulate other processes, like proliferation or dif84 ferentiation [13]. The molecular steps leading to cartilage 85 differentiation, among other factors are regulated by Ca2+ 86 sensitive enzymes like one of the Ser/Thr specific pro87 tein kinases, PKCalpha [14] or the Ser/Thr-specific protein 88 phosphatase calcineurin [15,16], that is unique among phos89 phatases for its ability to sense changes of intracellular 90 Ca2+ concentration through its activation by its calcium 91 binding subunit and calmodulin. Calcineurin is best known 92 as a regulator of T-lymphocyte activation, since its phar93 macological inhibitors, cyclosporine A (CsA), tacrolimus, 94 pimecrolimus and rapamycin are all used in the clinical prac95 tice as immunosuppressants [17]. Calcineurin is also known 96 to participate in several differentiation processes, such as 97 development of different muscle tissues and the nervous 98 system [18].99 In this study we measured the cytosolic free Ca2+ concen100 tration during cartilage differentiation in the chondrogenic 101 cells of HDC. A characteristic temporal pattern in the 102 changes of cytosolic Ca2+ concentration could be observed; 103 there was a significant and transient elevation on the third 104 culturing day, the crucial day of chondrocyte differentia105 tion. Moreover, beside the changes of the basal cytosolic 106 Ca2+ level, cells of chondrifying micromass cultures also 107 exhibit spontaneous calcium events, a phenomenon char108 acteristic to several other primary cell cultures [19,20].We 109 provide evidence that the temporal pattern of the changes 110 of cytosolic free Ca2+ concentration in chondrifying cells is 111 indispensible to proper cartilage formation and depends on 112 extracellular Ca2+ rather than the availability of intracellu113 lar Ca-stores. We also demonstrate that calcineurin can play 114 a dual role in Ca-signalling of chondrogenic cells: its activity 115 is modulated by cytosolic Ca2+ concentration and the inhibi116 tion of calcineurin with CsA eliminates the Ca2+ peak of HDC 117 resulting in a pronounced decrease in cartilage formation. 118 This second observation raises the possibility of the active 119 regulatory effect of this enzyme on the enhancement of Ca2+ 120 influx to chondrifying cells. 121 Materials and methods 122 Cell culture 123 Distal parts of the limb buds of 4-day-old Ross hybrid 124 chicken embryos (Hamburger—Hamilton stages 22—24 [21])125 UNCORRECTED PROOF YCECA 972 1—14 Please cite this article in press as: C. Matta, et al., Cytosolic free Ca2+ concentration exhibits a characteristic temporal pattern during in vitro cartilage differentiation: A possible regulatory role of calcineurin in Ca-signalling of chondrogenic cells, Cell Calcium (2008), doi:10.1016/j.ceca.2007.12.010 ARTICLE IN PRESS +Model YCECA 972 1—14 Possible regulatory role of calcineurin in Ca-signalling of chondrogenic cells 3 were removed and primary micromass cultures of chon-126 drifying mesenchymal cells were established from a cell127 suspension with a density of 1.5 ×107cells/mL. Fifteen128 microliters droplets of the suspension were inoculated129 on round coverglasses (diameter: 30 mm; Menzel-Gl¨ aser,130 Menzel GmbH, Braunschweig, Germany) placed into plas-131 tic Petri dishes (Nunc, Naperville, IL, USA). Cells were132 allowed to attach to the surface for 2 h at 37 ◦C. Day133 of inoculation is considered as day 0. Colonies were 134 grown in Ham’s F12 medium (Sigma, Budapest, Hun-135 gary) supplemented with 10% foetal calf serum (Gibco,136 Gaithersburg, MD, USA) and were kept at 37 ◦C in an atmo-137 sphere of 95% air and 5% CO2and 80% humidity. The138 medium was changed on every second day or after treat-139 ments.140 Determination of cytosolic free Ca2+ concentration141 Measurements were performed on different days of culturing142 using the calcium-dependent fluorescent dye Fura-2. Cul-143 tures were transferred to 2 mL fresh Ham’s F12 medium 144 containing 10 ␮L Fura-2-AM (10 ␮M) and 4 ␮L neostigmin145 (0.3 nM), in order to inhibit extracellular choline esterases. 146 After 60 min of incubation at 37 ◦CinaCO 2incubator,147 cultures were washed twice in Tyrode’s solution con148 taining 137 mM NaCl, 5.4 mM KCl, 0.5 mM MgCl2, 1.8 mM149 CaCl2, 11.8 mM Hepes—NaOH, 1 g/L glucose, pH 7.4, in150 order to remove the Fura-2-AM attached to the extra-151 cellular matrix. Fura-2-loaded cells were placed on the152 stage of an inverted fluorescent microscope (Diaphot, Nikon, 153 Kowasaki, Japan) and viewed using a 40×oil immer-154 sion objective. Measurements were carried out in the 155 same salt solution in a perfusion chamber using a dual156 wavelength monochromator (DeltaScan, Photon Technolo-157 gies International, Lawrenceville, KY, USA) equipment.158 All measurements were performed at room temperature. 159 Fluorescence of Fura-2-loaded cells was measured using 160 excitation wavelengths of 340 and 380 nm and an emis-161 sion wavelength of 510 nm. Intracellular Ca2+ concentrations162 were calculated from the ratios of intensities (R=F340/F380)163 as described by Grynkiewicz et al. [22]. Intracellular Ca2+ 164 levels of HDC were measured 2 h after inoculation on165 round coverglasses then on culturing days 1—6 at the same 166 period of each day. Intracellular Ca2+ levels of untreated167 control cultures and cultures treated with EGTA, calcium168 ionophore A23187, cyclopiazonic acid (CPA) or CsA were169 assayed in 5 independent experiments measuring 30 cells170 in each case. All measurements were carried out directly 171 after treatments with EGTA, calcium ionophore A23187, 172 CPA or CsA. Data were statistically analyzed by Student’s173 t-test.174 Treatments with cyclopiazonic acid and caffeine 175 To determine the role of intracellular Ca-stores in the176 changes of the cytosolic Ca2+ concentration, the intracel-177 lular Ca2+ pump inhibitor CPA was administered to HDC.178 For single cell measurements, CPA was dissolved in DMSO179 and diluted in a modified, Ca2+-free Tryode’s solution180 (containing 5 mM EGTA, without CaCl2) to a final concen-181 tration of 10 ␮M. The inhibitor was directly perfused in 182 the close proximity (approximately 50 ␮m) of cells. Prior 183 to administration of CPA, cells were washed with Ca2+-184 free Tyrode’s solution to remove all traces of free Ca2+ 185 from the medium. To investigate prolonged effect of deple186 tion of intracellular Ca2+ source, CPA was administered 187 directly into the culture medium for 12 h on culturing 188 day 2 or 3 at a final concentration of 10 ␮M. Caffeine 189 (15 mM, diluted in Tyrode’s solution), an agonist of ryan190 odine receptor (RyR) was administered at close proximity 191 (about 50 ␮m) of cells for 100 s during single cell measure192 ments. 193 Measurement and analysis of spontaneous calcium 194 transients 195 Spontaneous calcium transients were monitored using LSM 196 510 META Laser Scanning Confocal Microscope (Zeiss, 197 Oberkochen, Germany). Cells of high-density micromass 198 cell cultures were incubated for 1 h at 37 ◦C with 10 ␮M199 Fluo-4-AM in Ham’s F12 medium. Calcium imaging was per200 formed in normal Tyrode’s solution (see above). x—yanalysis 201 and line scan images were taken to monitor the fluores202 cence intensity during spontaneous activities. Fifty images 203 were recorded during a 48.4 s interval to perform the x—y204 analysis. Line scan images were recorded at 1.54 ms/line, 205 512 pixels/line and 4096 lines using a 63×water immersion 206 objective. Fluo-4-loaded cells were excited with a 488 nm 207 argon ion laser and emitted fluorescence was collected 208 at 500—570 nm. Images were analyzed using an automatic 209 event detection program developed in the Department of 210 Physiology. 211 Determination of Ca concentration of the culture 212 medium 213 Ca concentration of the culture medium (F12 supplemented 214 with 10% FCS) was determined with atomic absorption 215 spectrometry (AAS) on Philips PU9200X equipment in air216 acetylene flame. Measurements were performed at the 217 wavelength of 422.7 nm; each sample was measured three 218 times for 4 s. Determination of Ca concentration was carried 219 out with standard addition method; samples were diluted in 220 0.1 M nitric acid [23].221 Treatments with EGTA, A23187 calcium ionophore 222 and cyclosporine A 223 In order to examine the effects of removal of Ca2+ from 224 the extracellular fluid, cell cultures were fed a culture 225 medium containing 0.8 mM EGTA (Amresco, Solon, OH, USA; 226 pH adjusted to 7.4) for 12 h on day 2 or 3 of culturing. 227 To assess the effect of elevated intracellular Ca2+, cul228 tures were fed with a culture medium containing calcium 229 ionophore A23187 (Sigma, Budapest, Hungary) at concen230 trations of 0.1 and 5 mg/L for 1 h on culturing days 2 and 231 3. Calcium ionophore A23187 was dissolved in DMSO then 232 diluted in culture medium. Activity of calcineurin was inhib233 ited with the continuous application of 2 ␮M cyclosporine A 234 (Sigma, Budapest, Hungary) started on day 1. UNCORRECTED PROOF YCECA 972 1—14 Please cite this article in press as: C. Matta, et al., Cytosolic free Ca2+ concentration exhibits a characteristic temporal pattern during in vitro cartilage differentiation: A possible regulatory role of calcineurin in Ca-signalling of chondrogenic cells, Cell Calcium (2008), doi:10.1016/j.ceca.2007.12.010 ARTICLE IN PRESS +Model YCECA 972 1—14 4 C. Matta et al. Light microscopical analysis of cartilage235 differentiation236 Cartilage matrix was visualized by staining with dimethyl237 methylene blue (DMMB, Aldrich, Germany) as described238 previously [2]. The amount of sulphated matrix compo-239 nents was determined with a semi-quantitative method,240 by measuring the optical density of extracted toluidine241 blue (Reanal, Budapest, Hungary) bound to glycosamino-242 glycans in mature HDC. Six-day-old cell cultures were243 fixed in a solution containing 28% ethanol, 4% forma-244 lin and 2% acetic acid, stained with 0.1% toluidine blue 245 dissolved in glycine—HCl buffer (pH 1.8) for 15 min, the 246 unbound toluidine blue was washed in glycine—HCl buffer247 for 1 h. The dye bound to highly sulphated proteogly248 cans and glycosaminoglycans was extracted in 8% HCl249 dissolved in absolute ethanol. Absorbance of samples con-250 taining extracted toluidine blue was measured at the251 wavelength of 625 nm on a microplate reader (Chameleon,252 Hidex, Turku, Finland). Samples from 10 cultures of each253 experimental group were determined in 5 independent254 experiments. Data were statistically analyzed with Student’s255 t-test.256 Measurement of cell proliferation with257 3H-thymidine labelling and mitochondrial activity258 with MTT assay 259 For measurement of proliferation rate of cells in HDC260 15 ␮L droplets of cell suspension were inoculated into261 wells of special, opaque 96-well microtiter plates (Wal-262 lac, PerkinElmer Life and Analytical Sciences, Shelton, CT,263 USA). Ham’s F12 medium containing 1 ␮Ci/mL (185 GBq/mM)264 3H-thymidine (diluted from methyl-3H-thymidine solution,265 Amersham Biosciences, Budapest, Hungary) was added to266 the wells for 16 h on different days of culturing. After267 washing twice with PBS (phosphate buffered saline), pro-268 teins were precipitated with ice-cold 5% trichloroacetic269 acid, washed with PBS again, and placed in an exsicca-270 tor containing phosphorous pentoxide in order to absorb271 moisture. Prior to measurements, 50 ␮L scintillation solu-272 tion (MaxiLight; Hidex, Finland) was added to each well,273 and radioactivity was counted by a liquid scintillation274 counter (Chameleon, Hidex, Turku, Finland). Ten sam-275 ples of each experimental group from five independent276 experiments were statistically analyzed with Student’s t-277 test. 278 Cellular metabolic activity was determined by MTT assay,279 a means of measuring the activity of living cells via mito-280 chondrial dehydrogenases. Cells cultured in wells of 96-well281 microtiter plates were used and 10 ␮L MTT reagent [3282 (4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide;283 5 mg MTT/1 mL PBS] was added into each well. Cells were 284 incubated for 2 h at 37 ◦C in MTT-containing Ham’s F12285 medium. Following addition of 100 ␮L MTT solubilizing solu-286 tion (10% Triton X-100 and 0.1 M HCl dissolved in anhydrous287 isopropanol) optical density was measured at 570 nm on 288 a microplate reader (Chameleon, Hidex, Turku, Finland). 289 Ten samples of each experimental group from four sepa290 rate experiments were statistically analyzed with Student’s291 t-test.292 Determination of cell survival by FACS analysis 293 One hundred microliters droplets of cell suspension were 294 inoculated into plastic Petri dishes. Following treatments 295 of either A23187 or EGTA as given in the ‘‘Treatments 296 with EGTA, A23187 calcium ionophore and cyclosporine A’’ 297 section, cultures were washed twice with CMF-PBS (cal298 cium and magnesium free PBS), incubated with 250 ␮L299 annexin-V DY647 reagent (Central European Biosystems, 300 Budapest, Hungary) and/or 10 ␮g/mL propidium-iodide for 301 10 min at room temperature, washed again with CMF-PBS, 302 and trypsinized for 15 min to obtain cell suspension. Cells 303 were collected by centrifugation at 800 ×gfor 10 min, resus304 pended in 1 mL FACS buffer (PBS supplemented with 1% 305 BSA and 0.05% NaN3) and rate of cell survival was deter306 mined using a CyFlow®space Flow Cytometer (Partec GmbH, 307 M¨ unster, Germany). Annexin-V DY647 was monitored at 308 670 nm, propidium-iodide was measured at 620 nm. Mea309 surement lower threshold were set on cell-size particles. 310 Analysis was performed with WinMDI 2.8 Software. 311 RT-PCR analysis 312 Total RNA was isolated from cells using RNA Isolation Kit 313 according to the manufacturer’s instruction (Gentra Sys314 tems Inc., Minneapolis, MN, USA). The assay mixture for 315 reverse transcriptase reaction contained 2 ␮g RNA, 0.112 ␮M316 oligo(dT), 0.5 mM dNTP, 200 units M-MLV RT in 1 ×RT buffer. 317 The sequences of primer pairs used for PCR reactions were 318 as follows: for chicken aggrecan 5-CAA TGC AGA GTA CAG 319 AGA-3and 5-TCT GTC TCA CGG ACA CCG-3, for chicken 320 Sox9 5-CCC CAA CGC CAT CTT CAA-3and 5-CTG CTG ATG 321 CCG TAG GTA-3, for chicken calcineurin 5-CTG CTC TGA 322 TGA ACC AAC AGT T-3and 5-ACG GCA AGG ACC AGG TAA 323 ACA-3, for chicken GAPDH 5-GAG AAC GGG AAA CTT GTC 324 AT-3and 5-GGC AGG TCA GGT CAA CAA-3, for chicken 325 inositol-1,4,5-trisphosphate receptor (IP3R) type-1 5-CGG 326 CTG TGG TCT GAG ATA C-3and 5-GGT AAT AGG GAA GAT 327 GGT AGT G-3, for chicken IP3R type-2 5-AAG CCT ACC 328 TTA TGA CCT CC-3and 5-CAT TGT TTC CTC CAT CCT G329 3, and for chicken IP3R type-3 5-TGT GGG TGG ACA AGA 330 AAG G-3and 5-GCA GGA ACT GAT GGG TGA A-3. Ampli331 fications were performed in a thermocycler (PCR Express 332 Temperature Cycling System, Hybaid, UK) as follows: 94 ◦C, 333 1 min, followed by 30 cycles (94 ◦C, 30 s, 54 ◦C, 30 s, 72 ◦C, 334 30 s) and then 72 ◦C, 5 min. Thirty-five cycles were used 335 at 57 ◦C for IP3-receptors. PCR products were analyzed by 336 electrophoresis in 1.2% agarose gel containing ethidium bro337 mide. 338 Western-blot analysis 339 Total cell lysates or endoplasmic reticulum (ER) fraction of 340 HDC were examined by Western blot. Cell cultures were 341 harvested immediately after treatments on respective days 342 of culturing. Cell pellets were suspended in 100 ␮Lof 343 homogenization buffer [containing 50 mM Tris—HCl buffer 344 (pH 7.0), 0.5 mM dithiothreitol, 10 ␮g/mL Gordox, 10 ␮g/mL 345 leupeptin, 1 mM phenylmethylsulphonyl (PMSF), 5 mM ben346 zamidine and 10 ␮g/mL trypsin inhibitor]. After storing them 347 at −70 ◦C, suspensions were sonicated by pulsing burst for 348 UNCORRECTED PROOF YCECA 972 1—14 Please cite this article in press as: C. Matta, et al., Cytosolic free Ca2+ concentration exhibits a characteristic temporal pattern during in vitro cartilage differentiation: A possible regulatory role of calcineurin in Ca-signalling of chondrogenic cells, Cell Calcium (2008), doi:10.1016/j.ceca.2007.12.010 ARTICLE IN PRESS +Model YCECA 972 1—14 Possible regulatory role of calcineurin in Ca-signalling of chondrogenic cells 5 four times 30 s by 50 cycles (Branson Sonifier, Danbury,349 USA). ER fraction of HDC was prepared from 3-day-old cul-350 tures grown in Petri dishes. Cells were homogenized using351 a Dounce homogeniser in a buffer containing 5 mM HEPES,352 320 mM sucrose and protease inhibitors [3.4 ␮g/mL Gordox,353 3.4 ␮g/mL leupeptin, 1 mM phenylmethylsulphonyl (PMSF),354 1.6 mM benzamidine and 3.4 ␮g/mL trypsin inhibitor, pH355 7.4]. After centrifugation at 4500 ×gfor 20 min, super-356 natant was collected and centrifuged at 10,000 ×gfor 357 15 min. Supernatant was centrifuged at 150,000 ×gfor358 120 min. Pellet containing endoplasmic reticulum vesicles359 (microsome fraction) was collected in lysis buffer (50 mM360 Tris—HCl, pH 7.2 containing protease inhibitors, see above),361 snap-frozen in liquid nitrogen and stored at −70 ◦C. Sam-362 ples for SDS-PAGE were prepared by the addition of 1/5363 volume of fivefold concentrated electrophoresis sample364 buffer (310 mM Tris—HCl, pH 6.8; 10% SDS, 50% glyc365 erol, 100 mM DTT, 0.01% bromophenol blue) to cell lysates366 and boiled for 10 min. About 40 ␮g of protein was sepa-367 rated by 7.5% SDS-PAGE gel for immunological detection368 of Sox9, phospho-Sox9, RyR, IP3-receptor and calcineurin.369 Proteins were transferred electrophoretically to nitrocel-370 lulose membrane. After blocking in 5% non-fat dry milk371 in PBS, membranes were incubated with the following 372 primary antibodies overnight at 4 ◦C: polyclonal anti-Sox9373 antibody (Abcam Ltd., Cambridge, UK) in 1:200 dilution, 374 polyclonal anti-phospho-Sox9 antibody (Sigma, Budapest, 375 Hungary) in 1:200 dilution, monoclonal anti-RyR antibody376 (Affinity BioReagents, Golden, CO, USA) in 1:1000 dilution,377 polyclonal anti-IP3R type 1 antibody (Sigma, Budapest, Hun-378 gary) in 1:250 dilution and polyclonal anti-calcineurin (␣379 subunit) antibody (Upstate, Dundee, Scotland, UK) in 1:200380 dilution. After washing three times for 10 min with PBST (PBS 381 supplemented with 0.1% Tween 20), membranes were incu382 bated with a secondary antibody, anti-mouse IgG (Sigma,383 Budapest, Hungary) in 1:1000 dilution for RyR and anti-384 rabbit IgG (Sigma, Budapest, Hungary) in 1:1000 dilution 385 for detection of IP3receptor, Sox9, phospho-Sox9 and cal-386 cineurin in PBS containing 5% non-fat dry milk for 1 h. Signals 387 were detected by enhanced chemiluminescence (Amersham388 Biosciences, Budapest, Hungary).389 Enzyme activity assay of calcineurin390 Activity of calcineurin was measured by the release391 of 32Pifrom 32P-labelled protein phosphatase inhibitor392 1 (780 cpm/pmol) as described by Yang et al. [24] 393 with some modifications [25]. Thirty microliters of the 394 assay mixture (50 mM Tris—HCl buffer pH 7.0) contain-395 ing 0.16 mM dithiothreitol, 3.4 ␮g/mL Gordox, 3.4 ␮g/mL396 leupeptin, 1 mM phenylmethylsulphonyl (PMSF), 1.6 mM ben-397 zamidine, 3.4 ␮g/mL trypsin inhibitor, 40 ␮g/mL calmodulin, 398 0.2 mM CaCl2, 100 nM okadaic acid (OA), 2 nM protein399 phosphatase inhibitor-2, extract containing about 80 ␮g400 protein and 32P-labelled protein phosphatase inhibitor-1401 (20—30,000 cpm/reaction mixture) was incubated at 30 ◦C 402 for 20 min. The reaction was terminated by the addition of403 100 ␮L of 20% trichloroacetic acid and 100 ␮L of 6 mg/mL 404 bovine serum albumin. After centrifugation, 32Pi-content of405 180 ␮L of the supernatant fraction was determined in a liq-406 uid scintillation counter.407 Results 408 Cytosolic free Ca2+ concentration of untreated cell 409 cultures shows a characteristic age-dependent 410 pattern 411 Cytosolic free Ca2+ concentration was determined in Fura412 2-loaded cells on different days of culturing. Basal level of 413 intracellular Ca2+ concentration of chondroblasts was found 414 to have an age-dependent pattern (Fig. 1A). Initially, Ca2+ 415 level is low, with a starting concentration of about 75 nM on 416 day 0, then it slightly increases in parallel with the progres417 sion of differentiation. A 140 nM peak of the cytosolic free 418 Ca2+ concentration was observed on day 3 of culturing in 419 cells of untreated control cultures. It should be noted that 420 chondrogenic cells of HDC differentiate into chondroblasts 421 on this day of culturing [3]. From day 4, Ca2+ level drops, 422 however, it retains a slightly elevated concentration (about 423 100 nM) as compared to days 0—2. 424 Differentiating chicken chondroprogenitor cells exhibit 425 periodical increases in cytosolic free Ca2+ (Fig. 1B—D). These 426 oscillations were detectable mostly on culturing day 3. Fre427 quencies of oscillations were similar in all the cells observed: 428 the period was 4 ±1.2 s (mean ±standard error of the mean; 429 n= 20), maximum amplitudes were 15—20% higher than the 430 mean basal fluorescence intensity. 431 Changes of cytosolic Ca2+ concentration of HDC 432 hardly depend on intracellular sources 433 We aimed to determine whether the extracellular Ca2+ or 434 the intracellular Ca-stores could be the source of elevated 435 cytosolic Ca2+ levels. The Ca concentration of F12 medium 436 containing 10% FCS proved to be approximately 0.78 mM 437 according to AAS measurements (data not shown). In order 438 to reduce the concentration of free Ca2+ in the culture 439 medium, EGTA was applied in equimolar (0.8 mM) concentra440 tion. EGTA treatment significantly decreased the cytosolic 441 Ca2+ level to approximately 60% of that of untreated control 442 cells (Fig. 2A). Twelve hours of EGTA treatment proved to be 443 effective; when maintained in 0.8 mM EGTA throughout the 444 culturing period, cultures detached from the glass or plas445 tic surface and died. To investigate the role of intracellular 446 Ca-stores, cyclopiazonic acid (CPA), an inhibitor of the Ca2+ 447 pump of smooth endoplasmic reticulum was administered at 448 a concentration of 10 ␮M for 8 min. To remove free Ca2+ from 449 the medium, cells were washed with Ca2+-free Tyrode’s prior 450 to administration of CPA. After addition of Ca2+-free Tyrode’s 451 the basal cytosolic Ca2+ level decreased from 140 to 120 nM 452 showing the dependence of this parameter on the extra453 cellular Ca2+ concentration. Approximately 60 s after the 454 administration of 10 ␮M CPA, cytosolic Ca2+ level started to 455 increase very slowly (Fig. 2B). The slight elevation in cytoso456 lic Ca2+ clearly shows that the intracellular Ca2+-stores are 457 not empty, however, the amount of stored Ca2+ is either low 458 or the rate of leak is small. When administration of both CPA 459 and Ca-free Tyrode’s ceased and were washed out with nor460 mal Ca2+ containing solution, a well-defined peak in cytosolic 461 Ca2+ level could be observed owing to the entry of extracel462 lular Ca2+ into the cytosol, reflecting on a possible activation 463 UNCORRECTED PROOF YCECA 972 1—14 Please cite this article in press as: C. Matta, et al., Cytosolic free Ca2+ concentration exhibits a characteristic temporal pattern during in vitro cartilage differentiation: A possible regulatory role of calcineurin in Ca-signalling of chondrogenic cells, Cell Calcium (2008), doi:10.1016/j.ceca.2007.12.010 ARTICLE IN PRESS +Model YCECA 972 1—14 6 C. Matta et al. Figure 1 Day-by-day variation of basal intracellular Ca2+ levels in chondrifying cells of untreated control HDC (A). Ca2+ concentrations were determined in Fura-2-loaded cells as described in the ‘‘Materials and methods’’ section. Representative data of five independent experiments showing mean values of basal intracellular Ca2+ levels of 30 cells ±standard error of the mean. Statistical analysis by Student’s t-test comparing the respective data to the previous culturing day, *P< 0.01. Spontaneous calcium events in cells of chondrifying high-density micromass cell cultures on culturing day 3 (B—D). Calcium transients were determined in Fluo-4-AM loaded cells as given in the ‘‘Materials and methods’’ section. Changes of fluorescence intensity were recorded in a 50 s interval. x—yanalysis of a representative cell is shown in panel B, time course of mean intensity is shown in panel C. A representative calcium event measurement performed by line scan imaging is shown in panel D. UNCORRECTED PROOF YCECA 972 1—14 Please cite this article in press as: C. Matta, et al., Cytosolic free Ca2+ concentration exhibits a characteristic temporal pattern during in vitro cartilage differentiation: A possible regulatory role of calcineurin in Ca-signalling of chondrogenic cells, Cell Calcium (2008), doi:10.1016/j.ceca.2007.12.010 ARTICLE IN PRESS +Model YCECA 972 1—14 Possible regulatory role of calcineurin in Ca-signalling of chondrogenic cells 7 Figure 2 Basal intracellular Ca2+ levels of 0.8 mM EGTA-treated cultures on days 2 and 3 (A). Measurements were carried out following 12 h of EGTA treatments. Data represent mean ±standard error of the mean of intracellular Ca2+ levels of 30 cells measured in 5 representative experiments. Effect of cyclopiazonic acid (CPA) on the release of Ca2+ from intracellular stores (B). Representative record out of 30 cells in 5 independent experiments. Asterisks indicate significant (*P< 0.01) decrease in basal Ca2+ concentration as compared to the respective control. of store-operated Ca2+ entry (SOCE) channels [26]. The exact464 nature of this phenomenon, however, remains to be further465 investigated.466 To investigate the possible role of internal Ca-stores in467 the regulation of Ca-homeostasis of differentiating chon-468 droprogenitor cells, further experiments were performed.469 Ten micromolar CPA was administered to the culture medium 470 of HDC on day 2 or 3 for 12 h. This prolonged inhibition of471 the Ca-pump of smooth endoplasmic reticulum must have472 resulted in a complete abolishment of intracellular stores, 473 however, no detrimental effect on chondrogenesis could be474 observed (Fig. 3A). CPA-treatment slightly elevated cytoso-475 lic Ca2+ level (Fig. 3B), which could be related to the function476 of SOCE channels. Combined treatments with EGTA and CPA477 for 12 h (i.e., inhibition of Ca2+ entry from both extracellu-478 lar and intracellular sources) resulted in a complete loss of479 metachromatically stained cartilage matrix demonstrating 480 the Ca2+ dependence of in vitro chondrocyte differentia-481 tion (data not shown). This phenomenon may implicate the482 insufficient capacity of intracellular Ca-stores to replenish483 the function of Ca2+ entry pathways in chondrogenic cells.484 The importance of Ca2+-influx from extracellular space485 is further supported by investigating the endoplasmic486 reticulum ryanodine receptor (RyR) and the inositol-1,4,5-487 trisphosphate (IP3) receptors. RyR was not detectable by488 Western blot analyses performed on total cell lysates of HDC 489 and only a weak band was observed in samples containing 490 separated endoplasmic reticulum fraction of HDC (Fig. 4A). 491 Furthermore, no response was detected when caffeine, an 492 agonist of RyR was administered during single cell measure493 ments (Fig. 4B). These results indicate that though present, 494 the low amount of RyR located in the endoplasmic reticu495 lum of cells of HDC may not be functioning and probably 496 does not significantly contribute to the elevation of basal 497 cytosolic Ca2+. Amplification of IP3receptor isoforms by RT498 PCR shows that only the mRNA of IP3R type 1 is expressed 499 by cells of HDC (Fig. 4C). The IP3receptor protein could be 500 hardly detected in samples prepared from the endoplasmic 501 reticulum fraction of chondrogenic cells by Western blotting 502 (Fig. 4D). 503 Decreased extracellular Ca2+ concentration inhibits 504 cartilage formation and reduces the expression of 505 chondrogenic master transcription factor Sox9 506 Cultures treated with EGTA for 12 h on day 2 or 3 of cultur507 ing showed a profound decrease in metachromatic staining 508 performed on day 6 of culturing (9% or 17% of untreated 509 control cultures, respectively, Fig. 5A), demonstrating a sig510 Figure 3 Effect of 10 ␮M CPA on cartilage formation in chondrifying micromass cultures (A). Metachromatic cartilage areas in 6-day-old high-density colonies visualized with DMMB dissolved in 3% acetic acid. Optical density (OD625) of samples containing toluidine blue extracted with 8% HCl dissolved in absolute ethanol. Data are mean values ±standard error of the mean (±7%) of each experimental group out of 10 measurements. Basal intracellular Ca2+ levels of 10 ␮M CPA-treated cultures on days 2 and 3 (B). Measurements were carried out directly after the 12-h treatment with CPA. Data represent mean ±standard error of the mean of intracellular Ca2+ levels of 30 cells measured in 5 independent experiments. UNCORRECTED PROOF YCECA 972 1—14 Please cite this article in press as: C. Matta, et al., Cytosolic free Ca2+ concentration exhibits a characteristic temporal pattern during in vitro cartilage differentiation: A possible regulatory role of calcineurin in Ca-signalling of chondrogenic cells, Cell Calcium (2008), doi:10.1016/j.ceca.2007.12.010 ARTICLE IN PRESS +Model YCECA 972 1—14 8 C. Matta et al. Figure 4 Detection of the expression and function of RyR and IP3R in HDC. Western blot analysis of RyR in ER fraction of cells of HDC on culturing day 3 (A). Representative data of five independent experiments. Effect of the RyR-agonist caffeine (15 mM) on basal cytosolic Ca2+ level of cells of HDC on culturing day 3 (B). Representative record out of 30 cells in 5 independent experiments. Amplification of chicken IP3R type 1 receptor (C). Representative result out of three independent experiments. Western blot analysis of IP3R in ER fraction of cells in HDC (D). Representative analysis out of three independent experiments. nificant reduction of cartilage formation. Administration of511 EGTA after day 5 did not have any significant effect on matrix512 production (data not shown).513 Inhibition of chondrogenesis was monitored by detection514 of mRNAs of aggrecan core protein and Sox9, the major 515 cartilage-specific transcription factor. A significant decrease516 in the mRNA levels of both aggrecan core protein and Sox9 517 was observed under the effect of EGTA on each day of treat518 ments, demonstrating that reduced intracellular Ca2+ level519 decreases cartilage formation, at least partly, via inhibition 520 of cartilage differentiation (Fig. 5B). These findings were 521 further supported by Western blot analyses showing that522 treatment with EGTA slightly reduced the protein level of523 Sox9 on day 2, and a significant decrease was observed in524 the phosphorylation level of Sox9 on day 3 (Fig. 5C). 525 Ca2+ ionophore has dual concentration-dependent 526 effects on cartilage formation 527 About 0.1 mg/L concentration of the Ca2+ ionophore A23187 528 raised the intracellular Ca2+ levels to approximately 125% of 529 untreated control cells, and 5 mg/L concentration of Ca2+ 530 ionophore resulted in an even higher increase (about 150%) 531 in cytosolic Ca2+ levels (Fig. 6A). On the other hand, the two 532 concentrations of ionophore applied had opposite effects 533 on cartilage formation. Following treatment with the lower 534 concentration (0.1 mg/L) of A23187 on both days 2 and 3 for 535 one hour, an extensive cartilage formation occurred by day 536 6; when treatment was performed on day 3 only, the amount 537 of metachromatically stained cartilage matrix increased to 538 153% of control cultures (Fig. 6B). On the contrary, higher 539 Figure 5 Effect of 0.8 mM EGTA on cartilage development of chondrifying micromass cultures (A). Metachromatic cartilage areas in 6-day-old high-density colonies visualized with DMMB dissolved in 3% acetic acid. Optical density (OD625) of samples containing toluidine blue extracted with 8% HCl dissolved in absolute ethanol. Data are mean values ±standard error of the mean (±5%) of each experimental group out of 10 measurements. Effect of EGTA on the expression of aggrecan and the expression and phosphorylation of Sox9 transcription factor in HDC of various ages (B and C). For RT-PCR reactions GAPDH was used as a control. Representative data of five independent experiments. Asterisks indicate significant (*P< 0.01) decrease in optical density of extracted toluidine blue as compared to the respective control. UNCORRECTED PROOF YCECA 972 1—14 Please cite this article in press as: C. Matta, et al., Cytosolic free Ca2+ concentration exhibits a characteristic temporal pattern during in vitro cartilage differentiation: A possible regulatory role of calcineurin in Ca-signalling of chondrogenic cells, Cell Calcium (2008), doi:10.1016/j.ceca.2007.12.010 ARTICLE IN PRESS +Model YCECA 972 1—14 Possible regulatory role of calcineurin in Ca-signalling of chondrogenic cells 9 Figure 6 Effects of the Ca2+ ionophore A23187 on cartilage development of chondrifying micromass cultures. Basal intracellular Ca2+ levels of 0.1 or 5 mg/L A23187-treated cultures on days 2 and 3 (A). Measurements were carried out directly after the 30-min treatment with A23187. Data represent mean ±standard error of the mean of intracellular Ca2+ levels of 30 cells measured in 5 independent experiments. Metachromatic cartilage areas in 6-day-old high-density colonies visualized with DMMB dissolved in 3% acetic acid (B). Optical density (OD625) of samples containing toluidine blue extracted with 8% HCl dissolved in absolute ethanol. Data shown are mean values ±standard error of the mean (±6%) of each experimental group out of 10 measurements. Effect of A23187 on the mRNA expression of aggrecan and Sox9 transcription factor (C) and protein expression and phosphorylation of Sox9 (D) in HDC of various ages. For RT-PCR reactions GAPDH was used as a control. Representative data of five independent experiments. Asterisks indicate significant (*P< 0.01) increase in basal Ca2+ concentrations or significant (*P< 0.01) change in optical density of extracted toluidine blue as compared to the respective control. concentration (5 mg/L) of A23187 caused a marked inhibi540 tion of cartilage formation, especially when administered541 on day 2, reducing the amount of metachromatically stained542 cartilage matrix to approximately 36% of untreated control543 cultures (Fig. 6B). Since mRNA levels of cartilage differen-544 tiation markers Sox9 and aggrecan core protein, and both 545 protein level and phosphorylation of Sox9 showed a slight546 increase rather than inhibition under the effect of 5 mg/L547 ionophore (Fig. 6C and D), the mechanism of the decrease of548 cartilage formation should be further investigated. Admin-549 istration of A23187 after day 5 did not have any significant550 effect on matrix production (data not shown). 551 Effects of EGTA, A23187 Ca2+ ionophore and CPA552 on rate of proliferation, mitochondrial activity and 553 cellular viability of cells of HDC554 Cell proliferation, mitochondrial activity and cellular viabil-555 ity assays were performed following treatments with EGTA,556 the Ca2+ ionophore A23187 and CPA, since decreased car-557 tilage matrix production could be accounted for either by 558 the inhibition of the differentiation of chondrogenic mes-559 enchymal cells to chondroblasts or by the decrease in cell560 numbers. Although the proliferation rate of cells in micro561 mass cultures was significantly reduced under the effect of 562 0.8 mM EGTA (Fig. 7A), the mitochondrial activity of cells 563 was not affected (Fig. 7B) as revealed by 3H-thymidine 564 incorporation and MTT assay, respectively. Cell prolifera565 tion was slightly stimulated by low concentration of the Ca2+ 566 ionophore A23187 on day 3 (Fig. 7A). High concentration 567 (5 mg/L) of A23187 reduced the rate of cell proliferation 568 on both days, but it did not cause any significant decrease 569 in mitochondrial activity (Fig. 7B). Treatment with CPA did 570 not influence the proliferation rate of cells (Fig. 7A) and 571 it did not prove to be cytotoxic according to MTT assays 572 (Fig. 7B). 573 Cellular viability following treatments with EGTA or the 574 Ca2+ ionophore A23187 was also analyzed by FACS (Fig. 7C575 and D). The ionophore did not influence the distribution of 576 cells compared to the control. The percentage increase of 577 dead cells were as follows: 3.2% and 0.7% (in the presence of 578 0.1 mg/L) and 1.8% and 3.5% (in the presence of 5 mg/L) on 579 days 2 and 3, respectively. Treatment with 0.8 mM EGTA led 580 to a higher rate of cell death, thus the percentage increase 581 of dead cells proved to be 10% and 9.1% on days 2 and 3, 582 respectively. Apoptosis was not detected in the ionophore583 treated HDC (data not shown).