Translocation of protein kinase C isoenzymes by elevated extracellular Ca2+ concentration in cells...

Bone Vol. 17, No. 2 August 1995:175-183

ELSEVIER

Translocation of Protein Kinase C Isoenzymes by Elevated Extracellular Ca 2+ Concentration in Cells From a Human Giant Cell Tumor of Bone

A N N A TETI , I A N D R E A H U W I L E R , 2 R O S S E L L A P A N I C C I A , 3 G I A N C A R L O S C I O R T I N O , I and

J O S E F P F E I L S C H I F T E R 2

1 Department of Experimental Medicine, School of Medicine, Universi~ of L'Aquila, Italy 2 Department of Pharmacology, University of Basel, Switzerland 3 Institute of Human Anatomy, School of Medicine, Universi~ of Bari and Istituto Dermopatico dell'lmmacolata. Rome and Institute of Histology and General Embryology, University "La Sapienza," Rome, Italy

In this study we investigated the protein kinase C isoenzymes expressed by human osteoclast-like cells harvested from a giant cell tumor of bone (GCT23 cells), and by freshly isolated rat osteoclasts, lmmunoblotting analysis revealed that the -ct, -fi, and -¢, PKC isoforms, but not the - [3 isoenzyme, are expressed by GCT23 cells. Immunofluorescence studies demonstrated that PKC.ct, -fi, and -e are homogeneously expressed by both mononuclear and muitinucleated GCT23 cells, as well as by rat osteoclasts. Similar to authentic osteoclasts, GCT23 cells responded to an increase of extracel- hilar Ca 2+ concentration ([Ca2+]o) with a dose-dependent elevation of the cytosolic free Ca 2+ concentration ([Ca2+]i). An increase of [Ca2+]o stimulated the translocation of PKC-ct from the cytosolic to the particulate fraction, sug- gesting the involvement of this isoenzyme in the signal transduction mechanism prompted by stimulation of the [Ca2+]o sensing. By contrast, PKC-8 was not altered by exposure to elevated [Ca2÷] o, whereas PKC-e underwent reciprocal translocation, disappearing from the insoluble fraction and increasing in the cytosol. The effects of PKC on GCT23 cell functions were investigated by treatment with phorboi 12- myristate, 13-acetate (PMA). We observed that activation of PKC by PMA failed to affect adhesion onto the substrate, but down-regulated the [Ca2÷]o-induced [Ca2+] i increases. The latter effect was specific, since it was reversed by treatment with the PKC inhibitors staurosporine and chelerythrine. (Bone 17:175-183; 1995)

Key Words: Protein kinase C; Cell signals, Ca2+; Ca 2+ sensing; Giant cell tumor of bone; Osteoclast-like cells.

Introduction

Protein kinase C (PKC) is a Ca 2 +- and phospholipid-dependent kinase, activated by diacylglycerol (DAG), which phosphory- lates proteins on serine and threonine residues. 32"34"36

Molecular cloning has demonstrated that PKC consists of a large family of at least ten different isoenzymes 2~'22'35'39'43 classified in 2 groups: the PKC-ot, -13 I, -1311, and -~t (cPKC), and

Address for correspondence and reprints: Anna Teti, Ph. D., Department of Experimental Medicine, Via Vetoio, Coppito 2, 67100 L'Aquila, Italy.

PKC-6, -¢, -¢', -'q, -0 and -4 (nPKC), with -131 and -1~11' and -¢ and -~' derived by alternate splicing of the same gene tran- scripts. 23'43 Although the isoforms have small differences in their dependency on lipid cofactors and overlapping substrate specificity, a major difference is their distinct tissue and cellular distribution. Isoforms -a, -6, and -4 are widely distributed, whereas the others are expressed in selected organs. 37"4°'56 The cPKC group displays an N-terminal regulatory region with two highly conserved C 1 and C2 regions. C 1 contains two "zinc finger" cystein-rich regions, and the phospholipid and DAG- binding sites. C 2 contains a calcium-binding sequence, respon- sible for the Ca 2 ÷ -dependent activity of this group. 16.21.37.38,49 The nPKC group is Ca 2 ÷ -independent, reflecting the absence of a Ca 2+ -binding C 2 domain. 26''.9

DAG-dependent activation of PKC induces translocation of the enzyme from the cytosolic to the membrane fraction of the cell. 36'47 This occurs when a cell binds a ligand for a phospholipase C (PLC)- or D-associated receptor. 2"36 The C~ region of the PKC N terminal not only binds DAG, but also binds tumor promoting phorbol esters such as phorbol-12-myristate and 13- acetate (PMA), which mimic DAG-activating PKC even in the absence of a receptor agonist. 33

Over recent years, several reports described the physiologic properties of a receptor-like mechanism that detects an increase of extracellular calcium concentration ([Ca 2 ÷ ]o) expressed by selected cell types, including parathyroid cells, 3t thyroid C cells, s osteoclasts, 27"s8 and cytothrophoblastic cells.19 The effects of Ca 2 ÷ on the function of these cells are analogous to those produced by receptor-regulated mechanisms in other cell types. 4 A high [Ca 2÷ ]o evokes transient, followed by sustained, concentration-dependent elevations of the second messenger cytosolic free Ca 2÷ concentrations ([Ca2+]i).27"31'58 This sequence of changes in [Ca 2÷ ]~ closely resembles that produced by signals activating conventional Ca 2÷ mobilizing receptors. 2 Several physiologic properties of the [CaZ+]o sensing are known. Thus, increases of [Ca 2÷]i seem to be accompanied by rapid increases in inositol trisphosphate (IP3), 5"15'28 and to be dependent upon both mobilization of Ca 2÷ from intracellular deposits and Ca 2 + influx across the plasma membrane. 4'27'32'5s Several divalent cations such as Cd 2 ÷, Mg 2+ , Ba 2 +, and Ni 2 ÷ can mimic the effect of Ca 2 +, producing [Ca 2+]i elevations as well. 27"32"51 The response to elevated [CaZ+]o appears to be down-regulated by acidic pH.~l Finally, activation of the [Ca 2 + ]o sensing causes intracellular pH changes ~1 and other eel-

© 1995 by Elsevier Science Inc. 175 8756-3282/95/$9.50 SSDI 8756-3282(95)00172-A

176 A. Teti et al. Bone Vol. 17, No. 2 Protein kinase C in osteoclast-like cells August 1995:175-183

lular modifications 29'32 that lead to changes of cellular activity. Taken together, these characteristics indicate that the [Ca 2+ ]o sensing has several properties of a putative, PLC-associated receptor. 4 These properties have recently been confirmed by molecular cloning of the [Ca 2+ ]o-sensing receptor of parathyroid cells. 6 Molecular cloning is, however, still lacking for the [ Ca2 + ]o sensing of other cell types, including osteoclasts.

Despite the number of reports recently appearing in the lit- erature on this issue, to our knowledge little is known on the activation of PKC by the lEa 2+ ]o sensing expressed in cells resident in the bone tissue, either normal or malignant. In this article, we studied the expression of PKC isoforms in osteoclast- like cells from a human giant cell tumor of bone (GCT23 cells) and in disaggregated rat osteoclasts, and investigated whether stimulation of the [Ca 2+ ]o sensing activates PKC isoenzymes in GCT23 cells. Since both elevation of [Ca 2+ ]o and activation of PKC affect adhesion in bone-resorbing cells, we also investigated whether these two factors modify anchorage of GCT23 cells onto a fetal bovine serum (FBS)-coated substrate. Finally, due to the fact that PKC modifies the response to the agonist in several receptor-mediated signal transduction mechanisms, we addressed the question of whether PKC plays a role in the feedback regulation of the [Ca 2 + ]o sensing of GCT23 cells.

Materials and Methods

Materials

Dulbecco's minimum essential medium (DMEM), Iscove- modified MEM (IDMEM), penicillin, streptomycin, and fetal bovine serum (FBS) were from Eurobio (Paris, France). Phorbol 12-myristate, 13-acetate (PMA), staurosporine, chelerythrine chloride, and fura-2/AM were from Sigma Chemical Co. (St. Louis, MO). Ionomycin was from Calbiochem (La Jolla, CA). Sterile plasticware was from Falcon (Lincoln Park, N J) or Costar (Cambridge, MA). All other reagents were from Carlo Erba (Milan, Italy) or from Merck (Darmstadt, Germany).

Cell Culture

Cells, kindly donated by Dr. Alberta Zambonin Zallone, Istituto di Anatomia Umana Normale, University of Bad, and by Dr. Massimo Serra, Laboratorio di Ricerca Oncologica, Istituto Or- topedico Rizzoli, Bologna, were harvested from a human giant cell tumor (GCT) of bone. m.45 Briefly, cells were disaggregated by pipetting surgically removed fragments of the tumor. Re- leased cells were plated at a density of 10,000/cm 2 in 75 cm 2 flasks, and incubated in IDMEM supplemented with 10% FBS, 100 IU/mL penicillin, 100 Ixg/mL streptomycin, 50 IU/mL mycostatin, and 2.5 Ixg/mL streptomycin, 50 IU/mL mycostatin, and 2.5 ttg/mL amphotericin B, at 37°C in a water-saturated atmosphere containing 5% CO 2. At confluence, cells were re- leased by trypsinization and subcultured. The population was formed by an average of 30% multinucleated cells, containing 3 to 13 nuclei. Histochemical analysis of tartrate-resistant acid phosphatase activity revealed expression of the enzyme mostly by multinucleated cells, whereas 91% of the cells (both multinucleated and mononuclear) showed specific binding to 1251- human calcitonin. Cells resorbed bone and responded to calcitonin with an increase of intracellular cAMP, which led to inhibition of bone resorption. These cells have been classified as GCT23. m'45 For this study, cells were used in passages 9-15, without apparent changes of the responses between early and late passages.

Rat osteoclasts were isolated from 4-day-old Wistar rats. Briefly, long bones were dissected, cut in two longitudinal halves, reduced in small fragments, and pipetted into 1.8 mL 10 mmol/L HEPES-buffered DMEM (pH 7.25) to release osteoclasts from the endosteal surface. Cells were allowed to settle onto 12 mm diameter round coverslips for 30 min at 37°C, then the medium was replaced with fresh DMEM and cells were incubated for 1 h at 37°C in a humidified atmosphere of 95% air and 5% CO 2. Osteoclasts were identified by phase contrast mi- croscopy as multinucleated cells, with nuclei and organelles lo- cated in the central granular area, surrounded by organelle-free pseudopodia. These cells retracted in response to calcitonin, were positively stained for tartrate-resistant acid phosphatase, and formed resorption pits on bone slices.

Analysis of PKC Isoenzymes

Semiconfluent GCT23 cells in 150 cm 2 flasks were incubated in 20 mL of serum-free but otherwise complete IDMEM, containing 0.2% of fatty acid-free BSA, with or without 10 mmol/L Ca 2 + or 10-7 mol/L PMA. After the indicated time periods, the medium was removed and cells were washed twice with ice-cold Dulbecco's phosphate buffer saline (PBS). Cells were then scraped into 1.0 mL of ice-cold homogenization buffer [20 mmol/L Tris-HC1 pH 7.5, 1 mmol/L EDTA, 1 mmol/L EGTA, 2 mmol/L dithiothreitol, l mmol/L phenylmethanesulphonyl flu- oride (PMSF), 10 mmol/L benzamidine, 25 ixg/mL leupeptin, and 6 ~,g/mL aprotinin] with a rubber policeman. All the subsequent steps were carried out at 4°C. The cells were lysed with three 10-sec bursts with a Branson B15 sonifier (setting 4.0) and centrifuged for 1 h at 100,000g. Supernatants were used as a source of cytosolic protein. Pellets were resonicated in 1.0 mL of the same buffer containing 1% (v/v) Triton X-100 and centrifuged for 1 h at 100,000g, yielding the solubilized particulate fractions. Protein content was determined according to Brad- ford. 3 The cell protein fractions were subjected to SDS-PAGE (8% acrylamide gel), and proteins were transferred onto nitrocellulose paper for 1 h at 250 rnA using a Bio-Rad Transblot apparatus. The blotting buffer used was 25 mmol/L Tris-HC1 (pH 7.4), 190 mmol/L glycine in 20% (v/v) methanol. After the transfer, nitrocellulose filters were washed extensively in distilled water and blocked in blocking buffer [50 mmol/L Tris- HC1, pH 7.4, 200 mmol/L NaCI, 0.2% (v/v) Triton X-100, 3% (w/v) BSA, and 10% (v/v) horse serum] for 1 h at 25°C. Filters were then incubated for 4 h at 25°C with monoclonal antibodies raised against the PKC-a and -13 isoenzymes or antiserum reac- tive with PKC-~ and -e isoenzymes (diluted in blocking buffer as indicated in the legend of Figure 1). The detailed characteriza- tion of the antibodies is described elsewhere. 14.17.1s After wash- ing in buffer A (50 mmol/L Tris-HCl, pH 7.4, 200 mmol/L NaCI, 0.2% v/v Triton X-100; 4 × 5 min), the filters were incubated for 1 h with horseradish-peroxidase-conjugated anti- mouse IgG antibodies (PKC-ot and -13) or antirabbit IgG antibodies (PKC-~ and -~), in blocking buffer. Thereafter, filters were washed again (4 x 5 min) in buffer A and finally, for color reaction the filters were incubated in PBS containing 0.5 mg/mL of 3,3'-diaminobenzidine and 0.03% H20 2 for 10 min and then washed extensively in distilled water. Immunoblots were repeated at least 3 times.

The cellular distribution of PKC isoforms within the GCT23 cell population and in disaggregated rat osteoclasts was studied by immunofluorescence. Cells were plated in 3 cm culture dish containing 12 mm diameter round glass coverslips, fixed in 4% paraformaldehyde in PBS (10 min, 4°C), permeabilized with

Bone Vol. 17, No. 2 A. Teti et al. 177 August 1995:175-183 Protein kinase C in osteoclast-like cells

Figure 1. Immunocharacterization of PKC isoenzymes in GCT23 cells. GCT cells were homogenized and fractionated into cytosol (c) and particulate (p) fractions as described in the Materials and Methods section. Samples containing 70 p,g of protein were subjected to SDS-PAGE and transferred to nitrocellulose. Western-blot analysis was performed using either monoclonal antibodies against PKC-ct and -13 at a dilution of l:100, or antisera against PKC-8 and -e at a dilution of 1:1000. Bands were detected with horseradish peroxidase.

0.05 Triton X-100 in PBS (10 rain, 4°C), washed in PBS, and incubated in PBS with 1% fatty acid-free BSA (PBS+BSA) added for 10 min at 22°C. Cells were then incubated with anti- PKC antibodies (dilution l:100, 1 h, 22°C), washed in PBS + BSA (15 min), and incubated with the appropriate (anti- mouse or -rabbit) fluorescein-conjugated secondary antibody (1 h, 22°C). Coverslips were then washed in PBS + BSA, mounted with glycerol/Tris 0.1 mmol/L (pH 9.0, 6:4 v/v), and observed with a Zeiss Axiophot fluorescence microscope equipped with a 40× objective. Cells were photographed with Kodak T-MAX 400 film.

Adhesion

Adhesion was evaluated by a colorimetric method. 96 well multiplates were coated overnight at 4°C with 100 ~L FBS as a source of adhesion proteins. At the end of the incubation, FBS was removed by aspiration and replaced with 60% methanol for 1 h at 0°C. After methanol removal, wells were filled with 100 ILL of 50 mmol/L Tris-HCl buffer, pH 7.8, containing 0.1 mmol PMSF. After 30 min at 22°C, the buffer was removed and wells were rinsed three times with IDMEM supplemented with 0.5% fatty acid-free BSA. Cells were plated in FBS-coated wells at a density of 125,000/cm 2, and incubated in serum-free, 0.5% BSA-containing IDMEM at 37°C, 5% CO 2, for I h with or without increasing concentrations of Ca 2 +, PMA, or staurospo- fine. At the end of incubation, wells were rinsed three times with PBS (equilibrated at 37°C) to remove nonadhering cells, then cells attached to the substrate were fixed with 20% methanol for 30 min at 22°C. Evaluation of adhesion was performed upon staining of the cells with 0.5% crystal violet in 20% methanol for 5 min, extensive rinsing with distilled water, air drying for 15 min, and solubilization of the dye incorporated into the cells with 100 v.L 0.1 N Na citrate in 50% ethanol. Multiplates were then inserted in a Bio-Rad 450 microplate spectrophotometer and optical density, which was linearly proportional to the rate of adhesion, was measured at 550 nm wavelength.

Measurement of [Ca 2 + ]i

[ Ca2÷ ]i was measured by dual wavelength microfluorometry in single cells loaded with Ca 2 ÷-sensitive intracellular probe fura-

2. Cells seeded onto glass coverslips at a density of 2000/cm 2 were loaded with 3 v.mol/L fura-2/AM in serum-free, but otherwise complete, IDMEM at 37°C and 5% CO 2, for 60 min. Coverslips were then washed twice with Krebs-Henseleit- HEPES buffer (KHH: 140.7 mmol/L Na ÷, 5.3 mmol/L K ÷ 132.4 mmol/L CI- , 0.98 mmol/L PO42- , 1.25 mmol/L Ca 2+ 0.81 mmol/L Mg 2+, 20.3 mmol/L HEPES, and 5.5 mmol/L glucose). [CaZ+]i-dependent fluorescence was monitored with an AR-CM microfluorometer (Spex Industries, Inc., Edison, N J) connected with a Diaphot TMD inverted microscope equipped with a CF x40 fluor objective (Nikon Corporation, Tokyo, Ja- pan). Real-time recordings of fluorescence emission at 510 nmol/L from 340 and 380 nm excitation wavelengths, and 340 to 380 nm ratio were recorded by an ASEM Desk 2010 com- puter (ASEM S.p.A., Buia, Italy). [Ca 2+ ]i was calibrated at the end of each experiment by maximally increasing intracellular Ca2+-dependent fura-2 fluorescence with 5 v, mol/L of the Ca2+-ionophore ionomycin, followed by recording minimal fluorescence on addition of 7.5 mmol/L EGTA and 60 mmol/L Tris-HCl, pH 10.5. [Ca 2+ ]i was calculated according to Grynck- iewicz et al. t2

Statistics

Statistic evaluation was performed by the one-way analysis of variance (ANOVA) test. Data are expressed as average --- SEM.

Results

Expression and Distribution of PKC lsoenzymes

Figure 1 illustrates the immunoblotting analysis of PKC isoenzymes -ct, -13, -5, and -¢ in GCT23 cells. Analysis of the cytosolic fraction demonstrated that isoforms -or, -8, and -¢ were expressed by GCT23 cells, whereas immunoreactivity for the isoform -13 was not detectable. Specificity of the PKC-13 antibody was tested towards its respective antigen using insect cell extracts expressing recombinant PKC-131 and PKC-13- 2 isoenzymes. ~7.t8 10 ixg of protein were resolved on SDS-PAGE and immunoblotted using the anti-PKC-13 antibody providing the specific signal. This antibody recognized PKC-I3t and PKC-132 equally well. Apparent molecular weights for PKC-ct, -~, and -e were similar to those established for these isoenzymes in other cell types (approximately 80, 78, and 90 kD, respectively). lmmunofluorescence analysis demonstrated that both mononuclear and multinucleated GCT23 cells expressed PKC-ct, -8, and -e isoforms (Figure 2). Interestingly, PKC-ot, -8, and -e isoenzymes were also expressed by authentic osteoclasts isolated from neonatal rat long bones (Figure 3).

Regulation of PKC lsoenzymes by Extracellular C a 2 +

and PMA

Figure 4 shows immunoblotting of PKC isoenzymes in the cytosolic and particulate fractions of cultured GCT23 cells treated with 10 mmol/L C a 2 + for 30 min. Exposure of cells to 10 mmol/L Ca 2 + resulted in activation and subsequent translocation of PKC-ot from the cytosolic to the membrane compartment. PKC-¢ was partially associated with the particulate fraction even in control conditions. Upon stimulation with [Ca2+] o, PKC-¢ displayed a reciprocal translocation disappearing from the insoluble fraction and increasing in the cytosolic fraction. Whether such a reciprocal shift of PKC-¢ was accompanied by decreased enzyme activity is not known. These observations indicate that


Figure 3. Immunofluorescence analysis of PKC-ct, -13, -8, and -, isoenzymes in freshly isolated rat osteoclasts. PKC-ct (A), -8 (B), and -t (C) isoenzymes were expressed in rat osteoclasts. (D) Immanoreaction for PKC-13 isoenzyme was negative in a rat osteoclast (large arrow). The osteoclast was surrounded by mononuclear PKC-13 positive (example, medium arrow) and PKC-13 negative (example, small arrow) bone mar- row mononuclear cells. (E) Phase contrast microphotograph of the same cells illustrated in (D) [large arrow, osteoclast; medium arrow, same PKC-13 positive mononuclear cell as in (D); small arrow, same PKC-13 negative as in (D)]. (A)-(C) Original magnification × 250; (D)-(E) Orig- inal magnification ×200.

Figure 2. Immunofluorescence analysis of PKC-ct, -13, -8, and -e isoenzymes in GCT23 cells. PKC-et (A), -8 (B), and -e (C) isoenzymes were expressed by both mononuclear (small arrows) and multinucleated (large arrows) cells. (D) Immunoreaction for PKC-13 isoenzyme was negative both in multinucleated (large arrow) and in mononuclear (small arrow) cells. Original magnification × 160.

min. Upon stimulation with PMA, both PKC-ot and -e translocated to the particulate fraction.

It has been demonstrated in several cell types that the effect of PMA on activation of PKC is time-dependent and that up- regulation of the enzyme in short-term exposure is followed by a dramatic down-regulation in long-term treatment. ~'9"13 To in- vestigate whether GCT23 cell PKC-ot and -e isoenzymes were also down-regulated by long exposure to PMA, we treated the cells with 10 - 7 mol/L PMA for 24 h. Results are illustrated in the immunoblotting analysis of Figure 5B. Detection of both PKC isoenzymes -et, and -~ was significantly reduced upon long- term treatment with PMA.

PKC-et and -e may be involved in the signal transduction mechanisms triggered by stimulation of the [Ca 2 + ]o sensing. In contrast to the PKC-ot and -¢ isoenzymes, PKC-~ did not respond to an increase in [Ca 2+ ]o with translocation (Figure 4).

Figure 5A illustrates immunoblots of PKC-et and -E isoenzymes in GCT23 cells treated with PMA (100 nmol/L) for 20

Adhesion

Osteoclast-substrate recognition and anchorage are important mechanisms leading to cell polarization and bone resorption. In order to determine whether activation of the C a 2 + sensing and/or of PKC isoenzymes altered the ability of GCT23 cells to adhere

Bone Vol. 17, No. 2 A. Teti et al. 179 August 1995:175-- 183 Protein kinase C in osteoclast-like cells

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C c 0 P Figure 4. Immunoblot detection of C a 2 + -induced translocation of PKC isoenzymes in GCT23 cells. GCT23 cells were treated with vehicle (v; lanes 1 and 4) or 10 mmol/L CaCI z (t; lanes 2 and 3) for 30 min and the cytosolic (c; lanes 1 and 2) and particulate (p; lanes 3 and 4) fractions were prepared as described in the Materials and Methods section. Sam- ples (70 I~g of protein, measured according to Bradford) were subjected to SDS-PAGE and transferred to nitrocellulose. Western-blot analysis was performed using either a monoclonal antibody against PKC-a at a dilution of 1:100 or antisera against PKC-B and -e at a dilution of I : 1000. Bands were detected with horseradish peroxidase.

to a culture substrate, we measured the rate of anchorage by a colorimetric analysis. Wells were previously coated overnight with FBS to provide a source of extracellular adhesion mole- cules. This treatment induced nearly 80% adhesion of GCT23 cells within I h. To determine whether elevated [Ca 2+ ]o and activation of PKC altered the rate of anchorage to a FBS-coated substrate, GCT23 cells were exposed to mcreasing concentrations of Ca 2÷, PMA, and of the PKC inhibitor staurosporine. The results (Figure 6) demonstrated no significant difference in the adhesion rate of cells treated with Ca 2+ (i to 10 mmol/L Ca 2 ÷ added to the 1.5 mmol/L Ca 2 + -containing IDMEM, final concentrations 2.5 to 11.5 mmol/L, PMA (10 - 9 tO 10-7 mol/L, and staurosporine ( 1 0 - 9 tO 10 - 7 mol/L). This indicates that neither stimulation of the Ca 2 ÷ sensing nor alteration of PKC activity resulted in modification of the anchorage rate of GCT23 cells.

Regulation of the [Ca 2 +]o Sensing

Several receptor-mediated mechanisms are known to be regulated by PKC. Herein, we studied the effect of exposure of GCT23 cells to 10 - 7 mol/L PMA, 10 - 9 mol/L staurosporine, and 10 -~ mol/L chelerythrine on [Ca 2÷ ]o-sensing activity. We evaluated the response of the cells to elevated [Ca 2 ÷ ]o by measurement of the second messenger [Ca 2 ÷ L, which is known to be

increased, in a concentration-dependent manner, by treatment with high [Ca 2÷ ]o. Jr.27 Results from multinucleated cells are shown. No differences were observed in mononuclear compared to multinucleated cells.

Basal [Ca 2 ÷ ]i in GCT23 cells was 117 --- 7 nmol/L (n = 12), a concentration similar to that of a number of other cell types, including bone cells from other species. Basal [Ca 2÷ ]i was stable for at least 30 min. No fluctuations were observed during the observation time, either in untreated or treated cells. Real-time recordings of fura-2 fluorescence, calibrated for [Ca2÷]i (Fig- ures 7 and 8) showed that single GCT23 cells responded to 10 mmol/L Ca 2÷ with prompt [Ca2÷]~ increments (n = 30). In 33% of the cells treatment triggered a rapid [Ca2+]i transient consisting of a peak reached within 89 --- 9 sec, followed by a sustained phase that stabilized at lower levels and lasted several min. The rest of the cells responded to administration of elevated [ Ca2 + ]o by a monophasic [Ca 2 ÷ ]i increase, consisting of the rapid peak only (46% of the cells) or a sustained phase without the initial peak (21%). The average peak increase achieved 333

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Figure 5. lmmunodetection of PMA-induced translocation and down- regulation of PKC isoenzymes in GCT23 cells. GCT23 cells were treated with vehicle (v; lanes 2 and 4) or l0 7 mmol/L PMA (t: lanes l and 3) for 20 min (A) and 24 h (B), and the cytosolic (lanes c; l and 2) and particulate (p; lanes 3 and 4) fractions were prepared as described in the Materials and Methods section. Samples (70 ~tg of protein) were subjected to SDS-PAGE and transferred to nitrocellulose. Western-blot analysis was performed using either a monoclonal antibody against PKC-ct at a dilution of 1:100, or an antiserum against PKC-¢ at a dilution of 1:1000. Bands were detected with horseradish peroxidase.


--- 35 nmol/L, the sustained increase was 135 --+ 10 nmol/L. Responses were not desensitized. Cells treated with repeated administrations of high [Ca2÷]o showed [Ca2+] i elevations of similar amplitude and temporal pattern (not shown).

Figure 7A illustrates events triggered by an increase of [Ca2+]i in a single GCT23 cell treated with or without 10 -7 mol/L PMA. The cell, initially exposed to 10 mmol/L Ca 2÷ in the absence of PMA, showed a "c lass ic" [Ca2+]i transient, considered as an internal control. Subsequently, the cell was washed and treated with 10 - 7 mol/L PMA, which failed to significantly modify [Ca2+] i. However, addition of 10 mmol/L Ca 2 + in cells treated with PMA for 3 min resulted in a significant down-regulation of the [Ca 2÷ ]i transient compared to inter-

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Figure 6. Spectrophotometric analysis of adhesion of GCT23 cells. GCT23 cells were allowed to adhere for l h onto FBS-coated wells and stained with crystal violet. The dye was solubilized by Na citrate and the optical density (OD) measured at 550 nm. Before plating, cells were resuspended in media containing increasing concentrations of Ca 2+ , PMA, or staurosporine as indicated on the abscissa. Results are from three independent experiments performed in quadruplicate, and expressed as average percentage of OD +- SEM vs. internal untreated control.

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Figure 7. PKC-dependent regulation of the [Ca 2÷ ]o-induced signal in GCT23 cells. Real time recordings of fura-2 fluorescence, calibrated for [Ca2+]i in single GCT23 cells treated with agents regulating PKC activity prior to addition of 10 mmol/L Ca 2 +. Due to the variability of the responses to the agonist, which are typically observed in measurements made on single cells, GCT23 cells were first challenged with l0 mmol/L Ca 2+ (Ca), then washed (w) to return [Ca2+]o to near basal levels, and obtain the internal control for each treatment. 10 - 7 mol/L PMA (A), 10 - 9 mol/L staurosporine (B, ST), and l0 -~ mol/L chelerythrine (C, CH) were then added 3 min prior to a subsequent addition of 10 mmol/L C a 2 + ( C a ) .

nal control (n = 9, Figure 8A). In Figure 7B, a single cell was treated with 10-9 mol/L staurosporine prior to the addition of 10 mmol/L Ca 2 ÷. Staurosporine itself failed to induce changes of [ Ca2 + ]i, while a significant potentiation of the [Ca 2+ ]o-induced [ Ca2+ ]i elevation was observed in comparison with the internal control (n = 7, Figure 8B). Similar results were observed treat- ing GCT23 cells with the more specific PKC inhibitor, chelerythrine (n = 6, Figures 7C, 8C), indicating that up-regulation of the Ca 2 ÷ signals was indeed due to inhibition of PKC.

Discussion The present results demonstrate that PKC is a part of the signal transduction mechanism activated by stimulation of the [Ca z ÷ ]o sensing of cells from a human giant cell tumor of bone. Among the four isoenzymes tested (-a, -13, -~, and -~), the isoforms -a , -5, and -e, but not -13, have been detected in resting cells. In- terestingly, freshly isolated rat osteoclasts showed a similar pattern of PKC distribution, with the -or, -5, and -e isoforms expressed and detectable by immunofluorescence. Specificity of the PKC-13 antibody was tested towards its respective recombinant antigen expressed in insect cells. Obviously, we cannot exclude that GCT23 cells express very low amounts of PKC-13 that are below the detection sensitivity of the PKC-13 antibody. PKC-et and -5 are ubiquitously expressed in tissues and cells,

Bone Vol. 17, No. 2 A. Teti et al. 181 August 1995:175-183 Protein kinase C in osteoclast-like cells

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Figure 8. Effect of PKC-regulating agents on [Ca 2 ÷ ]o-induced [Ca 2 + ]i transients in GCT23 cells, expressed as average±SEM of several experiments. Ceils were pretreated with 10 - 7 mol/L PMA, 10 - 9 mol/L staurosporine, or 10 -s mol/L chelerythrine as shown in Figure 7, then 10 mmol/L Ca 2+ were added. Data represent peak [Ca2+]i of at least six independent experiments.

whereas the -e isoform has been detected only in selected organs including brain and kidney, s6 Two -e isoforms are known to date, the -e, and -¢', the latter being a truncate splice variant (approximately 55 kD molecular weight) of the former, s6 Since we found an unique band of apparent molecular weight of approximately 90 kD, we assume that the -e, and not the -e' isoenzyme, is expressed by GCT23 cells in culture.

Challenging the GCT23 cells with elevated [Ca 2÷ ]o for 30 min induced a reciprocal modification of the -or and -e PKC isoenzymes, but not of the PKC-5 isoform. Since we have not performed a detailed kinetic analysis, we cannot exclude the possibility that the PKC-5 isoenzyme may be activated at a dif-

ferent time. Interestingly, the PKC-¢ isoform was partially associated with the membrane even in control conditions, and was reciprocally translocated to the cytosol upon treatment with elevated [Ca 2+ ]o- These results indicate that the -a and the -e isoenzymes are part of the [Ca2+]o sensing-associated signal transduction mechanism. Nevertheless, the role played by the reciprocal activation of these two isoenzymes remains to be elucidated. PKC-et contains a N-terminal regulatory region including the C 2 Ca 2 ÷-binding domain, which is lacking in the PKC-e isoform. This suggests that elevated [Ca 2 + ]o may control PKC-ct activity by both generation of DAG and mobilization of Ca 2 +. On the other hand, Ca 2 ÷ may trigger changes in phospholipid- derived second messengers that subsequently contribute to the translocation of PKC-¢ from the membrane to the cytosol. To our knowledge, the latter observation is novel and has not been previously reported. Both isoforms were translocated to the membrane by short exposure to PMA, a phorbol ester that mimics the effect of DAG without affecting the receptor activity.

Phorbol esters are known to down-regulate PKC activity over several hours of exposure.7" 17.47 This seems to occur in GCT23 cells as well, since our study demonstrated that a 24 h exposure to PMA apparently reduced the expression of PKC-a and -¢ in both the cytosolic and the membrane fraction of the cells. In other cell types, such as a fetal rat skin keratinocyte cell line, it has been demonstrated that down-regulation of PKC depends upon nonlysosomal proteolysis. 7 The decrease of PKC membrane activity is associated in these cells with an increase in cytosolic activity of a protein kinase of smaller molecular weight, independent on Ca2÷/phosphatidylserine/DAG, which does not bind phorbol esters. This kinase may be formed by cleavage of PKC and may represent an active catalytic site of the enzyme. We have not prezently examined the PMA-dependent proteolytic cleavage of PKC over a period of 24 h, so that further experimentation will be necessary to address this question.

As far as the role of PKC in the cell function is concerned, a number of specific cellular activities, including anchorage to the substrate, are regulated by this enzyme. The effects of PKC regulating agents on adhesion of different cell types are, however, unpredictable, since either reduction or enhancement of adhesiveness have been described. 2°'5° Since anchorage to the substrate is most relevant for bone cell functions, we analyzed whether PKC alters the adhesion rate of GCT23 cells over a period of 1 h. The results indicate that activation of PKC by treatment with PMA, as well as exposure to elevated [Ca 2÷ ]o, failed to alter anchorage of cells onto a FBS-coated substrate. Similarly, inhibition of PKC with staurosporine did not significantly modify the adhesion. These results suggest that the cellular targets for such Ca2÷-act ivated pathway and PKC- dependent phosphorylation are not related to the capability of GCT23 cells to adhere to the substrate. Nevertheless, alteration of PKC activity affects GCT23 cell function. In fact, similar to what is observed in authentic osteoclasts by Murrills et al. 3° and by Sue t al. 53, phorbol esters inhibited GCT23 cell bone resorption (unpublished observation).

Phorbol ester-generated stimulation of PKC is known to regulate signal pathways associated with membrane-bound receptors, affecting multiple different sites in receptor-mediated signal transduction systems. It has been established that PKC may in- hibit membrane Ca 2 ÷ influx through Ca 2 + channels, or stimulate Ca 2 + efflux through the Ca 2 ÷ ATPase or Na ÷/Ca 2 + ex- change, thus reducing agonist-mediated [Ca2÷] i increase. Fur- thermore, activators of PKC may decrease IP 3 generation, enhance IP 3 hydrolysis, or directly desensitize PLC-coupled receptors. 9,24'2s'44'46'48"Ss's6 This sequence of effects results in a down-regulation of the agonist/receptor pathway, with suppres-

182 A. Teti et at. Bone Vol. 17, No. 2 Protein kinase C in osteoclast-like cells August 1995:175--183

s ion o f the related cell s ignal ing. W e prev ious ly observed that the [Ca 2÷ ]o sens ing o f ch icken and rabbit os teoclas ts did not undergo this typical regula t ion, but , by contrast , P M A - i n d u c e d act ivat ion o f P K C up-regula ted the [Ca 2 ÷ ]o sens ing , leading to a 2- to 3-fold potent ia t ion o f the [Ca2÷]o- induced [Ca2+]i increase. ~'5'* Herein , we present ev idence that P K C - d e p e n d e n t feedback regula t ion o f the [Ca 2 ÷ ]o sens ing o f cells f rom a giant cell t umor o f bone is at var iance with those o f ch icken and rabbit os teoclas ts , in that it is down- regu la ted (by approximate ly 58%) by P M A . Th i s effect is, by contrast , s imi lar to that observed in parathyroid cells . 4"5z The effect o f P M A was specif ic, s ince t r ea tmen t wi th 10 - 9 m o l / L s t au rospo r ine , concen t r a t i on at which P K C , but not P K A , is inhibited, or with 10 - 5 mol /L chelerythr ine, a novel specif ic P K C inhibitor reversed this effect , inducing a 1.5- and a 2-fold potent ia t ion o f the response to h igh [Ca2÷]o, respect ively. The analys is pe r fo rmed in this s tudy, however , does not a l low one to draw conc lus ions concern ing the site o f PKC-dependen t regula t ion o f the h igh [Ca 2 ÷ ]o-aSsociated s ignal ing pa thway . T hus , the molecu la r m e c h a n i s m s involved remain to be e lucidated.

In conc lus ion , this s tudy provides ev idence that giant cells f rom a h u m a n giant cell t umor o f bone, as well as authent ic rat os teoclas ts , express P K C -or, -~, and -e i soenzymes , and that the GCT23 cell PKC-ct and - t are act ivated in a reciprocal fash ion by s t imula t ion o f the [Ca 2 ÷ ]o sens ing . Act ivat ion o f these i soforms , ei ther by an increase o f [Ca 2÷ ]o or by t rea tment with phorbol esters , does not affect the rate o f adhes ion o f GC T 23 cells to a FBS-coa ted substrate . By contrast , the s ignal t ransduct ion mech- an i sm induced by s t imula t ion o f the [Ca 2 ÷ ]o sens ing is inhibited by act ivat ion o f P K C , indicat ing a negat ive feedback control o f a still u n k n o w n site o f the pa thway leading to [Ca 2 ÷ ]o-induced [ Ca2 ÷]i e levat ions . W h e t h e r or not such resul ts migh t be extended to " n o r m a l " os teoclas ts , wh ich G C T 2 3 cells funct ional ly resemble , to.45 remains to be elucidated.

Acknowledgments: This work was supported by grants from the Italian Ministry of University (quota 60% 93/1925 and 94/1502; quota 40% 92/7069), from the National Research Council (93/2081CTI4 and 93/ 4693CT04) to A.T. and by a grant from the Italian Ministry of University to the Institute of Histology and General Embryology, University "La Sapienza" of Rome. We are indebted to Dr. Elio Ziparo, Dr. Mado Molinaro, and Dr. Mado Stefanini, who kindly made available to us facilities and instrumentation of the Institute of Histology and General Embriology, University "La Sapienza" of Rome, to Dr. Franco Mangia, Istituto di Anatomia Comparata, University "La Sapienza" of Rome, who allowed us to employ the Zeiss Axiophot fluorescent microscope and to Dr. Paolo Men6, Cattedra di Nefrologia, Istituto di Clinica Medica 2, University "La Sapienza" of Rome, for helpful discussion in the preparation of this manuscript. We also thank Dr. Alberta Zambonin Zallone, lstituto di Anatomia Umana Normale, University of Bad, and Dr. Massimo Serra, Laboratorio di Ricerca Oncologica, Istituto Ortope- dico Rizzoli, Bologna, Italy, who kindly donated the CGT23 cells.

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Date Received: October 10, 1994 Date Revised: April 3, 1995 Date Accepted: May 9, 1995

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