cells in giant cell tumors of bone have many phenotypic and functional characteristics of normal osteoclasts, we have examined the interaction between the ...
Vol. 268, No. 14, Issue of May 15, PP. 10087-10094,1993 Printed in U.S.A.
THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1993 by The American Society for Biochemistry and Molecular Biology, Inc.
5-Lipoxygenase Metabolites of Arachidonic Acid Stimulate Isolated Osteoclasts to Resorb Calcified Matrices* (Received for publication, January 6, 1993)
Wolf E. GallwitzS, GregoryR. Mundy$, ChangH. Lee$, Mei Qiao$, G. David Roodman*, Mark RafteryQ,Simon J. GaskellQ, and LyndaF. BonewaldSlI From the $University of Texas Health Science Center a t Sun Antonio, Sun Antonio, Texas 78284-7877 and the §Baylor College of Medicine, Houston, Texas 77030
for tartrate-resistant acid phosphatase(TRAP),’ possess Bone resorption requires cooperation between osteoclasts and mononuclear accessory cells by mechanisms receptors for calcitonin (Komiya et al., 1990) and lack monowhich have not been elucidated.Since multinucleated cyte-macrophage surface antigens (Goldring et al., 1986). The cells in giantcell tumors of bone have many phenotypic mononuclear cells are comprised of two distinct populations, and functional characteristics ofnormal osteoclasts, one population which does not persist in culture and is posiwe have examined the interaction between the bone- tive for Ia and monocyte-macrophage antigens (Ling et al., resorbing multinucleated cells and the distinct mono- 1988) and another which persists in culture and resembles nuclear stromalcells from these tumors. We have found connective tissuestromal cells, produces TypesI and I11 that these mononuclear cells produce anactivity which collagen, and has receptors for parathyroid hormone (Goldrstimulates both giant cells from giant cell tumors and ing et al., 1986). These latter cells can be readily established rodentosteoclaststoresorbbone in vitro. We have in cell culture. One such cell line (C433) derived from these identifiedthe activity andfoundthatitrepresents several products of the 5-lipoxygenase pathway of ar- cells was shown by us tocause greater increases in osteoclast activity as measured by accumulation of TRAP activity than achidonic acid metabolism, namely B-hydroxyeicosaany of the known osteoblast-like cell lines. The purpose of tetraenoic acid and the leukotrienes. These data indicate that5-lipoxygenasemetabolites stimulate isolatedthis study was to characterize the osteoclast stimulating acosteoclasts to resorb bonein vitro and may represent tivity produced by this cell line. We found that this activity a mechanism by which mononuclear stromal cells in could be ascribed to 5-hydroxyeicosanoids, which are 5-lipoxygenase metabolites of arachidonic acid. human giant cell tumors communicate with the giant cells. In addition, theseresults may explain a possible EXPERIMENTALPROCEDURES mechanism for communication between accessory cells and osteoclasts involved in normal bone resorption. Materials-The inhibitors for various enzymes in the 5-lipoxygen-
The molecular mechanisms by which osteoclasts are activated are unknown. In vitro data indicate that bone-resorbing cytokines and hormones do notact directly on osteoclastsbut rather on osteoblasts or their precursors in the bone marrow environment and that these cells are responsible for osteoclast activation (Rodan and Martin, 1981; McSheehy and Chambers, 1986). This activation may be mediated either by cellcell contact or by locally active soluble factors. In a search for cell sources of such soluble factors, we found that a stromal cell line (C433) derived from agiant cell tumor of bone produced prodigious amounts of osteoclast-stimulating activity greater than anywe found inconditioned media from cells with osteoblast characteristics (Oreffo et al., 1993). Human giant cell tumors of bone are comprised of heterogeneous cell populations, including giant cells with many of the phenotypic and functional characteristics of osteoclasts as well as mononuclear cells. The multinucleated cells are positive for osteoclast surface antigens (Davies et al., 1989),
* This research was supported by National Institutes of Health Grant AR39529. The costsof publication of this articlewere defrayed in part by the payment of page charges. This article must therefore be hereby marked“advertisement” in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact. llTo whom correspondenceshould be addressed University of Texas Health Science Center, Dept. of Medicine, Division of Endocrinology and Metabolism, 7703 Floyd Curl Dr., San Antonio, T X 78284-7877. Tel.: 210-567-6666; Fax: 210-567-6693.
ase pathway were supplied by Dr. Michael Chang (Rhone-Poulenc Rorer, Inc.). The radioimmunoassays for 5-HETE were purchased from Advanced Magnetics, Inc. (Cambridge, MA). The commercially available 5-HETE and leukotrienes, protease enzymes, flurbiprofen, indomethacin, and nordihydroguaiaretic acid were purchased from Sigma. Isolation of Avian Osteoclasts-Avian osteoclasts were isolated from medullary bone of laying White Leghorn hens Gallus domesticus (Pioneer Animal Supply, Kingswheel, OH) asdescribed previously by Zambonin-Zallone and Teti(1981). In brief, bone marrow suspensions from the medullary bone of femora were filtered through Nytex cloth (110 pm, Tetko, Elmsford, NY), centrifuged for 5 rnin at 1200 rpm, and the cell pelletresuspended in 0.2% NaCl for 3 min to lyse erthyrocytes. After layering the cells on 100% fetal bovine serum for 1 h, sedimented cells were further filtered through Nytex filters (55 pm). Cells were harvested, resuspended in a-minimal Eagle’s medium (GIBCO) containing 10% fetal bovine serum, with penicillin (100 units/ml), streptomycin (100 pg/ml), and arabinose-0-D-cytosine furanoside (araC) 3 pg/ml, to inhibit proliferation of nonosteoclastic cells. Cells were plated in 24- or 48-well plates (Costar, Cambridge, MA) at 1 x lo4 cells/well and incubated at 37 “Cin 10% COz humidified air for 48 h, after which they were washed to remove nonadherent cells. When mature osteoclasts were observed experiments were begun and terminated within 48 h. Quantitation of Tartrate-resistant Acid Phosphatase by Fluorescence Spectroscopy-Osteoclast TRAP activity was measured using fluorescence spectroscopy as described by Chambers et al. (1987) with minor modifications. In brief, media from osteoclast cultures were harvested and stored at -70 “C until ready for assay. The cells were
’ The abbreviations used are: TRAP, tartrate-resistant acid phosphatase; HETE, hydroxyeicosatetraenoic acid; HPLC, high pressure measure liquid chromatography; LTC,, LTD,, and LTE,, leukotriene C4, leukotriene D4, and leukotriene El; GC-MS, gas chromatographymass spectrometry; 5-L0,5-lipoxygenase.
10087
5-LO Metabolites ActivateOsteoclasts
10088
A
B
*
c 433 PTH
I
c433
D
. I -
0
c
c433 FIG. 1. Quantitation of pit formation on sperm whale dentine by rat osteoclasts treated with C433-conditioned medium (DMEM, 0.1% BSA, lo-' M 1,25-(OHh vitamin Da), with parathyroid hormone (PTH)(lo-' M) and C = control (DMEM, 0.1%BSA, lo-' M 1,25-(OH)zvitamin Ds)( A )or human giant cells treated with C433-conditioned medium ( B ) .Quantitation of TRAP activity by avian osteoclasts incubated with C433-conditioned medium ( C ) and humangiant cells incubated with C433T / C = treatedlcontrol ratio. conditioned medium (D). 1.0
I
! I
11.0
2000
,
60 Daltonsl I h -0.8 .E
1000
-
i
0.8
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0.6
-0.6
0.4-
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FRACTION NUMBER FIG. 2. Molecular mass determination of C433activity. Fifty ml of C433-conditioned medium was filtered, lyophilized, and reconstituted before applying to a Bio-Gel P-2 column equilibrated in 10 mM ammonium bicarbonate, pH 7.1, and standardized (insulin, >2000 daltons; nonapeptide, 986 daltons; and sodium chloride, 58.5 daltons). Protein was monitored a t 280 nm. Every second fraction was bioassayed utilizing the isolated avian osteoclast TRAP assay. Molecular mass of the activity was determined to be less than 1000 daltons. M U P , methylumbelliferyl phosphate. washed with phosphate-buffered saline and harvested in 0.5 ml of Triton X-100 (0.05%,w/v). Aliquots of media or lysate (30 pl) were incubated with 170 plof 2 mM methylumbelliferyl phosphate, pH 5.0, in 0.48 M acetate buffer (0.48 M sodium acetate, 0.48 M acetic acid, pH 5.0) and 20 mM tartaric acid. Samples were incubated for 30 min
at 37 "C and the reaction terminated with 100 pl of stop solution containing 50 mM glycine, 50 mM EDTA, pH 10.4. Fluorescence was measured a t excitation 360 nm and emission 448 nm using a fluorimeter (Fluoroskan, Flow Instruments). Enzyme activity was expressed as micromoles of methylumbelliferyl phosphate hydrolyzed/ min/pg of protein and theprotein content measured by the technique of Lowry et al. (1951). Disaggregated Neonatal Rat Osteoclast Pit FormationAssayQuantitation of the effects of isolated osteoclasts on calcified matrices was determined using minor modifications of the disaggregated osteoclast resorption assay as described by Boyde et al. (1984). Sperm whale dentine (0.25 X 7 X 7 mm) was prepared using a Buehler low speed diamond saw (Buehler, Lake Bluff, IL) followed by sonication (15 min) in several changes of distilled water. Slices were sterilized using ultraviolet light. Neonatal Sprague-Dawley rats (2-3 days) were sacrificed by decapitation, the femurs and tibias were removed, scraped free of adherent tissue, and trimmed free of the epiphyses. The bones from one litter were combined and quickly minced using a scalpel blade in 2 ml of 199 medium and then vigorously mixed with a pipette in an 8-ml tube, allowed to settle for 10 s, and then100 p1 of the suspension was added to each well for a total of 16 wells in a 48-well microtiter plate containing sperm whale dentine (approximately four neonates are used per 16 wells). The cells were incubated at 37 "C for 30 min at which time the dentine was removed, washed in media, and placed in a fresh 48-well plate containing 250 pl of test medium. Osteoclasts were stained for TRAP and counted and then pit numbers were counted following toluidine blue (0.1% w/v) staining by light microscopy. The plan areaof matrix resorbed was quantitated using acomputer-assisted morphometric program ona Bioquant System IV analysis system (R & M Biometrics, Nashville, TN). Preparation of 2 x 6 Positive Cells from GiantCell Tumors-Human giant cell tumors (normally discarded after surgery) were minced and passed through 1#40SS wire mesh screen, allowed to settle for 5 min, and the cell suspension above the sediment collected. The cells were incubated with 23C6 monoclonal antibody that identifies the osteoclast vitronectin receptor (generously provided by Dr. Michael Horton) (1:lO dilution of hybridoma supernatant/106 cells) at 4 "C in serum-free medium for 30 min, after which immunomagnetic beads coated with anti-mouse IgG (Dynabead, Dynal, Inc., Great Neck, NY) were added to thecell suspension. The cell suspension was mixed for 5 min and then the 23C6 positive cells were separated using a magnet (Dynal, Inc.) on the side of the tube while suction was applied to remove all negative cells.Over 90% of the cells adhering to the magnetic beads were23CG-positive, and these cells were used for induction of TRAP activity and for pit formation on sperm whale dentine. Cells were plated at 60 cells/dentine slice or 4 X lo4 cells/ well in 24-well plates for induction of TRAP activity. Neonatal Mouse Calvarial Assay-The assay was performed as described by Gowen et al. (1983). Timed pregnant mice were injected with 45Ca2 days before parturition. Half-calvaria were removed from the 1-2-day-old pups and preincubated for 24 h in BGJb medium at 37 "C in a humidified atmosphere of 5% COa before transfer tofresh media with or without test substances for 48h. The bones were incubated for a further 72 h and media and bones collected. Boneresorbing activity was expressed as the percentage of the total 45Ca released into themedium. Purification Protocol for the C433 Active Fractions"C433 cells were grownin 10%fetal bovine serum (Whittaker,Walkersville, MD) 50% RPMI, 50% McCoys (Flow, McLean, VA) until a density of lo6 cells/ml was obtained, at which time the cells were harvested, washed, and placed in serum-free DMEM plus lo-' M 1,25-(OH)zD3for 48 h before harvest of conditioned medium. The conditioned medium was brought up to 15% ethanol and acidified to pH of3.5 with concentrated HC1. This conditioned medium was applied to a prewetted CIS Sep-Pak (Millipore, Waters, Milford, MA), and bound material was extracted using ethyl acetate.This material was dried under nitrogen and resuspended inwater, 1%acetic acid and analyzed by high pressure measure liquid chromatography (HPLC) (Waters, Milford, MA) applied to a CSsemipreparative 25 cm X 10 mm reverse phase column (Keystone Scientific, Inc., Bellefonte, PA) at 2 ml/min. The gradient was 0-100% methanol, 1%acetic acid over 60 min with 6ml fractions collected. The fractions were dried under nitrogen, and those fractions to be assayed for osteoclast stimulating activity were stabilized with 20 p1 of 10% bovine serum albumin. Fractions to be used for gas chromatography-mass spectrometric analysis were prepared for derivatization. Gas Chromatography-Mass Spectrometric Analysis for 5-Lipoxygen-
10089
5-LO Metabolites Activate Osteoclasts 3,
1
"
'I
C
UV
C433
1.20
B
C
HEAT
C433
C
A
P
2.70
IC
2.1 6
T
D
. I .
1.62 1.08 0.54
v
-
0.00 C
C433 FLUR
INDO NDGA
C
C433
-
lo6
la"
NDGA-
FIG. 3. Chemical characterizationof activity. A , C433-conditioned medium was treated with ultraviolet light ( U V )for 24 h or placed in a boiling water bath (100 'C) for 15 min and bioassayed. B, amino peptidase M ( A ) , carboxypeptidase ( C ) , or Pronase-CB ( P ) were incubated in the presence of C433-conditioned medium for 12 h at 37 "C. Samples were centrifuged, and each supernatant was bioassayed for activity. C, C433 cells were incubated for 48 h (2 X lo5 cells/ml) in DMEM, 0.1% BSA, lo-@M 1,25-(OH)2D3in the presence of either lo-' M flurbiprofen (FLUR), M indomethacin ( I N D O ) ,or M nordihydroguaiaretic acid (NDGA).The resultant conditioned mediumwas or lo-' M nordihydroguaiaretic acid. Conditioned mediumwas bioassayed for bioassaved for activitv. D.C433were treated with induction of TRAP altivity in avian osteoclasts. TABLEI Effect of lipoxygenase inhibitors on production offactors which stimulate TRAP activity in isolated osteoclasts C433 cells were incubated at 48 h in the presence of various inhibitors at 10 and 100 ng/ml. The resultant conditioned medium was bioassayed for TRAP activity in avian osteoclasts. The properties of the inhibitors are referenced under "Results." Inhibition
activity TRAP Treatment Dose
%
Medium alone
0.89 f 0.08
C433-conditioned medium alone
1.62 f 0.25
genated using a rhodium black catalyst. This procedure yields a common product, 5-hydroxyeicosanoic acid, from 5-lipoxygenase products such as 5-HETE and the peptido-leukotrienes. Each hydrogenated sample was converted to thepentafluorobenzyl ester trimethylsilyl ether derivative. Gas chromatographic-mass spectrometric analyses were performed with selected ion monitoring of ions characteristic of derivative of 5-hydroxyeicosanoic acid and of the ["C4] analogue. Statistical Analysis-Data were analyzed using the Student's t test or the Bonferonni test ( p < 0.05) using a statistical package for the IBM PC, SAS Institutes, Inc. (Cang, NC). RESULTS
RG6797 (5-LO inhibitor)
10 100
1.12 f 0.19" 0.86 f 0.08'
68 104
RG5901 (peptido-leukotriene antagonist and 5-LO inhibitor)
1.50 10 f 0.32 100 1.03 f 0.19"
16 81
RG12525 (LTD, receptor antagonist and 5-LO inhihitor)
1.73 10 t 0.25 100 0.80 -C 0.05'
0 122
RG2871 (mast cell degranulation inhibitor
IO 1.54 t 0.22 100 27 1.42 f 0.30
11
a
Significant difference using Student's t test ( p < 0.05).
* Significant difference using Bonferonni ( p < 0.05).
use Products-Bioactive HPLC fractions were analyzed for the presence of 5-lipoxygenaseproducts using a modification of the procedure described by Balazy and Murphy (1986). Each fraction was supplemented with [8,9,20,11-13C4]LTC4 (Raftery et d . , 1992) and hydro-
The conditioned media harvested from C433 cells stimulated freshly isolated neonatal rat osteoclasts to form resorptionlacunaeonsperm whale dentine. There wasa 3-fold increase in resorbed area/dentine slice compared with controls, and the C433-conditionedmedium contained similar resorbing activity t o maximal concentrations of parathyroid hormone (lo-' M) in this assay (Fig. 1A). The conditioned medium also stimulated giant cells isolated fromhuman giant cell tumors of bone to form resorptionlacunaeonsperm whale dentine. These giantcells were isolated using apanning technique with an antibody, 23C6, which recognizes osteoclasts preferentially (Hortonet al., 1985).The areaof dentine resorbed by thesegiant cells was increased 180-fold over controls (Fig. 1B).When conditionedmedium from C433 cells
Osteoclasts 5-LO Activate Metabolites
10090
be used to monitor purification, because they were obtained from surgical specimens, and tumor availability was not predictable. Initially, attempts were made to estimate the molecular weight of the activity. Using a combination of filtration membranes and gel filtration experiments, we found the activity was present in fractions less than 5 kDa. Fig. 2 represents a Bio-Gel P2 column showing that the activity was eluted from this column between the markers for1000 and 60 daltons. 0 l o 20 30 40 50 60 70 a0 Further chemical characterization of the activity showed that itwas relatively heat-stable but completely destroyed by I 10.125 24 h of ultraviolet light treatment (Fig. 3A). The activity in B C433 was not significantly affected by treatment with proPeptidoteases such as aminopeptidase M, carboxypeptidase, or Proleukotriene naseCB (Fig. 3B).Theactivity was extractableinethyl acetate and appeared tobehave as an organic molecule (data not shown). Thus, the activity appearedbetoa small nonprotein compound. Since prostaglandins and other arachidonic I I I I I I acid metabolites possess these characteristics and areknown 0 20 40 60 80 to have important effects on bone cell function, we determined 1.0 whether arachidonic acid metabolites could be responsible. The initial approach was to determine if production of the activity by C433 cells was blocked by inhibitors of arachidonic acid pathway enzymes. These included flurbiprofen and indomethacin which inhibit prostaglandin synthase, and nordihydroguaiaretic acid, a n inhibitor of the lipoxygenases. FlurM ) and indomethacin M ) had no signifibiprofen cant effects on production of C433 activity (Fig. 3C). The lipoxygenase inhibitor nordihydroguaiaretic acid completely 0 20 40 60 80 blocked production of C433 activityin a dose-dependent rnin manner (Fig. 3, C and D ) . FIG. 4. Partial purification of biological activity from Since nordihydroguaiaretic acid is a nonspecific inhibitor C433-conditioned medium using C8 reverse phase high pres- of lipoxygenases, we obtained more specific inhibitors from sure liquid chromatography ( A ) .The gradient was 0-100% meth- Dr. Michael Chang (Rhone-Poulenc Rorer,Collegeville, PA). anol, 1% acetic acid over 60 min with 6-ml fractions collected. The of C433 factor productionwas RG6797 fractions were dried under nitrogen, and those fractions to be used The strongest inhibitor for bioassay (TRAP induction in avian osteoclasts) were stabilized which is aselective strong 5-lipoxygenase (5-LO) inhibitor and weak 12-LO inhibitor (Huanget al., 1989) (Table I). This with 20 p1 of 10% bovine serum albumin. Fractions to be used for GC-MS analysis were immediately derivatized. Commercially avail- inhibitorprobablyactsas a chelator by preventing5-LO able (Sigma) LTC4/LTD4(1.2 pg) and 5-HETE (5 pg) were applied translocation. RG5901, another 5-LO inhibitor (VanInwegen to a C, reverse phase HPLC under the exact conditions as C433- et al., 1987), also partially inhibited production of the activity. conditionedmedium. B , LTC4/LTD4standard eluted at the same fraction (55% methanol) as activity 2. C, 5-HETE standard eluted at Production of the activity was also blocked by RG12525, an et al., 1990). Although the same fraction (85%methanol) as activity 3. MUP, methylumbel- LTDlreceptorantagonist(Huang RG12525 is ahigh affinity LTDI receptor antagonist, this liferyl phosphate. compoundcan also inhibit5-LO at 100-1000-fold greater concentrations. RG2871, a mast cell degranulation inhibitor was incubated with organ culturesof neonatal mouse calvariae which doesnot inhibitenzymes of the 5-LO pathway (Khandpreviously incorporated which 45Ca,there was a n increase in wala et al., 1987),hadlittle effect onproduction of C433 boneresorption(datanotshown).Thus,theconditioned activity. medium harvested from C433 cells contains an activitywhich Since the inhibitor studies implicated 5-LO metabolites as stimulates isolated osteoclasts to form resorption lacunae mediators and of osteoclast activation, we next devised a purifistimulates bone resorption in organ cultures. cation protocol for lipoxygenase metabolites of the arachiIn order to characterize and identify the bone-resorbing donic acid pathway. Stability of the factors was examined in activity produced by C433 cells, we chose touse TRAP activity various solvents useful for HPLC purification. The activity in isolatedpurified avian osteoclasts aasmeasure of osteoclast was stable in ethanol and methanol but not in triethanolamine stimulation.Measurement of pitson calcified matricesis or pyridine. Acetonitrile was found to be toxic to isolated time-consuming, difficult and imprecise, and is not suitable osteoclasts even after repeated lyophilization, and so methafor a purification assay. TRAP activity has been used previ- nol wasusedfor HPLC.Afterpurification using HPLC, ously as a parameter of osteoclast stimulation (Chambers and bioactivity was observed in unretained material and in three 9 and 10 (activity Fuller, 1984; Zaidi et al., 1988; Oreffo et al., 1990, 1992), so we fractions corresponding to fraction numbers decided to use this measurement to assess the capacity of 1 = 25% MeOH), fraction numbers 17 and 18 (activity 2 = 55% MeOH)andfractionnumber 25 (activity 3 = 85% C433-conditioned medium to activate osteoclasts. Increases inTRAPactivity of up to 5-fold were seenwhen (2433- MeOH) (Fig. 4). The concentrations of 5-LOmetabolitesin each HPLC conditioned medium was added to isolated avian osteoclasts fraction were determined by stable isotope dilution and gas (Fig. 1C). Even greater increases in TRAP activity in response hydrot o C433-conditioned medium were seen in giant cells from chromatography-massspectrometry(GC-MS)after giant cell tumors of bone (Fig. 1D). However, these could not genation and conversion to pentafluorobenzyl ester trimeth-
Is
I
10091
5-LO Metabolites Activate Osteoclasts 1202
Internal Standard (d
Activity 1
FIG. 5. Stable isotope dilution/gas chromatography-mass spectrometric analyses of HPLC fractions exhibiting osteoclast activating activity. [13C4]LTC4(0.5ng)wasadded to each fraction which was then hydrogenated and converted to the pentafluorobenzyl ester trimethylsilyl ether derivative. Ions monitored during GC-MS analysis corresponded to the product of the endogenous 5-LO metabolites ( m / z 399) and the internal standard ( m / z 403). The slightly shorter retention time observed during analysis of the activity 1 fraction reflecteda minor difference in analytical conditions.The relative abundance of the m / z 399 ion was normalized for each analysis to the relative abundance of m / z 403.For quantification, responseratios ( m / z 399 to m/z 403) were based on peakarea calculation.
12:14
I\
Internal Standard
mlz 399
Activity 2
12:15
A
Internal Standard
0 11.40
11.60
11.80
12.00
12.20
12.40
12.60
12.80
13.00
13.20
Retention Time (min)
TABLEI1 Quuntitation of 5-HETE in C433-conditioned media with and without treatment with 1,25-(0H)2 vitaminD,(lo-’ M) A radioimmunoassay (Advanced Magnetics, Inc., Cambridge,MA) was performedto measure 5-HETE in C433 conditioned media.Units are in pg/ml. Assays were performed in duplicate.
appropriate chromatographic retention time, defined by the internal standard. The results (Fig. 5 ) were consistent with the presence of 5-LO metabolites in each of the HPLC fractions exhibiting osteoclast activating activity at amounts corresponding (in “LTC4equivalents”) to 35, 83, and 917 pg/ml for the 25, 55, and 85% fractions, respectively. +1,25-(OHL vitamin D, A commercially available mixture of LTC4/LTD4 was apTime No treatment (lo-’ M) plied to the Cs reverse phase HPLC column under the same h conditions as the extracted C433-conditioned medium. LTC,/ 0 0.62, 0.51 2 (Fig. 4B). LTD, eluted in the same fraction as activity 3.53 6 3.36,0.900.91, Commercially available 5-HETE was also applied to Cs re12 4.98, 5.71 11.6811.07, verse phase HPLC under the same conditions as the extracted C433-conditioned medium. 5-HETE eluted in the same fracylsilyl ether derivatives. Ions characteristic of the product of tion as activity3 (Fig. 4C). The data are therefore consistent endogenous 5-LO metabolites ( m / z 399) and of the internal with the identification of activity 3, the 85%peak, as 5-HETE standard, [13C4]LTC4(m/z 403), were monitored during each and activity 2, the 55% peak, asa peptido-leukotriene, either analysis. Thus, identification of endogenous 5-LO products LTC4, -D4, or -E4. Activity 1, the 25% peak, remains unidenLTB, elutes at 80% MeOH, was based on the observation of the characteristic ion at the tified.Commerciallyavailable
5-LQ Metabolites Activate Osteoclasts
10092
-
N
40
40
A
E
B
3. t
0
x v
30
*
I
*
30
0
0 .-
FIG. 6. UV-treated C433-conditioned medium ( C M ) stabilized the HPLC-purified activity 2 and activity 3 fractions as determined in the isolated rat osteoclast pit formation assay.
a
20
20
10
10
L < U
a,
s0 -
tn
a, C
:433
0
c433 UV-Treated
+
Activity Activity 2 3
A 51
Without UV CM Activity Activity 2 3
B
i
*
T
*
1
*
c433
C433
LTC,/D,
A
5-HETE
*
T
C
C433
LTC,
LTC, I D4 5-HETE
D
*
C
andLTBl was notdetectableinanyfraction by GC/MS analysis. To confirm that C433-conditioned medium contained these 5-LO metabolites and that productionwas hormonally regulated, we performed radioimmunoassays for 5-HETE using commercially availablekits. C433 cells secreted large amounts of 5-HETE (Table 11) and 1,25-(OH)2 vitamin Ds increased the pTOdUCtion of 5-HETE by (3433 cells over control levels. When activity 2 (putative peptido-leukotriene) and activity 3 (putative 5-HETE)were added to the isolated rat osteoclast pit assay in UV-treated C433 conditioned medium, they stimulated bone resorption in a manner similar to that of the untreated C433-conditioned medium (Fig. 6). Commercially available LTC4/LTD4 and 5-HETEwere then tested on both isolated avian osteoclasts and isolated human giantcells. With both avian osteoclasts and human giant cells,LTC4/LTD4 and 5-HETE at10"" M maximally stimulated TRAP activity (Fig. 7, A and B ) . Commercially available LTC4 and LTE4 were tested forcapacity to stimulate pit formation by isolated giant cells (Fig. 7C) and isolated rat osteoclasts (Fig. 7 0 ) . Both compounds stimulated isolated resorbing cells a t 10""
PTH
FIG. 7. Biological effects of commercially available peptido-leukotrienes and 5-HETE. LTCh/LTD4 (10"' M) and 5-HETE (lo-'' M) were incubated for 48 h in the presence of isolated avianosteoclasts ( A ) , isolated 23C6(+) human giant cells of bone ( B ) , andTRAPindiction was measured. LTC4 (10"' M ) was tested on pit formation by isolated 23C6+ human giant cells ( C ) and LTE4(10"" M ) on pit formation by isolated rat osteoclasts ( D ) . *, significant difference using BonferonrIi ( p < 0.05). P T K , parathyroid hormone.
LTE
M. These commercially available metabolites were also tested in the murine neonatal calvaria assay and the fetal rat, long bone assay, both well recognized assays for measuring the effects of bone-resorbing cytokines. The metabolitesinduced resorption in the neonatal murinecalvarial assay but not the fetal long bone assay (data not shown), the same results as that observed with the (2433-conditioned medium (Oreffo et al., 1991). Finally, to determine whether 5-HETE and the peptidoleukotrienes have additive or synergistic biologic effects, commercially available compounds were tested for TRAP induction in isolated avian osteoclasts. The effects are not synergistic but appear additive (Fig. 8). DISCUSSION
In this report, we show that the bone-resorbing activity found in conditionedmedium from a stromal cell line isolated from a giant cell tumor of bone (C433) can be ascribed to metabolites of the 5-lipoxygenase pathway. We purified these metabolites €ram the conditioned medium of C433 cells using an osteoclast stimulationassay. Commercially available5-LO
10093
5-LO Metabolites Activate Osteoclasts
.ea
n
K
Q)
-w
O0.a78 1 0.6
\
.-C
E a
\
0.4 I
3
I I
0.2
a
0.1
3. v
a fy
0.0 FIG. 8. Effect of combining comImercially available 5-HETE and the peptido-leukotriene LTD, on TRAP activity in isolatedavian osteoclasts. Both 5-HETE and LTD, optin mally stimulated TRAP activity a t 10"' c M. Higher molarity (lo-' M) appears to 0 o.8 stimulatesuboptimally. The effects of 0 L combining 5-HETE and LTD, appear a additive. m "
:I
*
c
c433
10
r
I 11
1 0 - l ~ 10 -12 LTD4
1
10 -lolo -12M
1
[ 1 O"oM 5-HETE ] T*
0.5
E
\
a 3
I
c 10c433
-lolo -12
10-12M
5-HETE
[- 10 "OM LTD4-
3
metabolites mimicked the effects of (2433-conditioned me- and will increase adherence, oxygen radical production, and leukocytes dium. LTC,/LTD,, 5-HETE, and C433-conditioned medium lysosomal degranulationinpolymorphonuclear (Goldman et al., 1986). LTCl and LTD, have been shown to caused pit formation by isolated rat osteoclasts and human giant cells and stimulated bone resorption in neonatal mouse promote myeloid colony formation (Ziboh et al., 1986),prolifcalvariae. They also stimulated TRAP activity in avian osteo-eration of glomerular epithelial cells (Band et al., 1985), and secretion of luteinizinghormoneandluteinizinghormone clasts and human giantcells. Eicosanoids are derived from the oxidative metabolism of releasing-hormone (Parker,1987). Anumber of cytokines and will arachidonic acid (Smith, 1989; Parker, 1987). These deriva- growth factors such as interleukin-1 and interleukin-2 of leukotrienes(Parker, tives, which include the prostaglandins, thehydroxyeicosate- induce productionandsecretion traenoicacids,andtheleukotrienesarediverseand have 1987). Significant breakthroughs have been made in identipowerful but short-livedphysiological effects. It hasbeen well fying proteins andenzymes involved in the synthesisof these documented that prostaglandins play a critical role in both compounds. For example, only cells which contain both 5bone resorption and bone formation (Raisz and Martin, 1983), lipoxygenase (5-LO) and a recently cloned protein, 5-lipoxybut little isknown concerning therole of leukotrienes in bone genase-activating protein (FLAP), will produce leukotrienes remodeling. ProstaglandinssuchasprostaglandinEland (Dixon et al., 1990; Reid et al., 1990). Hormones which induce prostaglandin Ez in general are more stable than the leuko- 5-LOmetaboliteproduction mayregulate not only 5-LO trienes and have been shown to inactivate isolated osteoclasts enzymes but FLAP expression. (Chambers and Dunn, 1983). There are previous reports that leukotrienes may be mod5-Lipoxygenase metabolites possess a diverse range of bio- ulators of bone cell function. Meghji et al. (1988) tested logical activities, especially in allergic and inflammatory re- purified leukotrienes in the neonatal mouse calvarial assay sponses. The molecule LTB, is chemotactic for polymorpho- and found significant bone resorption. However, other invesnuclear leukocytes, eosinophils, lymphocytes, andmonocytes tigators have not been able to repeat these results. This may
5-LO Activate Metabolites
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be due to the unstable nature of these compounds. We have found that special precautions are necessary to maintain biological activity. The compounds must be stored under argon in the absence of light. Onceremoved from these conditions the commercial compounds must be diluted quickly and used immediately. Activity of commercially available compounds was detectable at higher concentrations (10"j to M) in the organ cultureassay, whereas activity was detectable at much lower concentrations (10"' to 10"l M ) in the isolated avian osteoclast and isolated human giant cell assays. This may reflect readier access of 5-LO metabolites to target cells in the isolated cell culture system. The C433-conditioned medium maintained activity when stored at 4 "C for 3-4 months (data not shown). However, once purification was initiated, biological activity was quickly lost, especially after HPLC purification. The studies suggest the presence of a stabilizing factor in C433-conditioned medium. Investigations into the natureof the stabilizing activity are currently being pursued. Dziak and co-workers (Mohammed et al., 1989) examined the role of leukotrienes in orthodontic tooth movement, a model used to examine bone remodeling. These investigators found significant inhibition of tooth movement using the leukotriene inhibitor AA 861, even though enhanced levels of prostaglandins were detected in thistreated tissue. They suggested that inhibition of LT synthesis might influence tooth movement and that prostaglandins and leukotrienes might mediate different steps in a cascade of events that results in initiation of bone remodeling. Our data indicate that 5-lipoxygenase metabolites stimulate isolated osteoclasts to resorb bone in vitro and may represent a mechanism by which mononuclear cells in human giantcell tumors communicate with the giant cells. In addition, these results may explain a possible mechanism for communication between accessory cells and osteoclast activation in normal bone resorption. The analytic evidence supports the presence of peptido-leukotriene and 5-HETE, the latter being quantitatively more significant in C433-conditioned medium. The unknown eicosanoid is present in much smaller amounts and mayprove difficult to identify as much larger volumes of conditioned medium will benecessary. Acknowledgments-We thank Dr. Phil Keeting, West Virginia University, Morgantown, VA for helpful advice and insight concerning the role of arachidonic acid metabolites in bone resorption, Dr. Ron Williams, Department of Orthopaedic Surgery, University of Texas HealthScience Center for supplying the giant cell tumor tissue, Dr. Michael Chang, Rhone-Poulenc Rorer for the 5-LO inhibitors, and Dr. Jilly Evans, Merck Frosst, Quebec, Canada for the critical reading of the manuscript. We also thank Gloria Pech6 and Thelma Barrios for excellent secretarial assistance. In memorium, we would like to recognize Dr. June Marshall, Orthopaedic Hospital, LOSAngeles, who established the C433 cell line and without whose contribution this work would not be possible. REFERENCES Athanasou, N. A., Wells, C.A., Quinn, J., Ferguson, D. P., Heryet, A., and McGee, J. 0. (1989) Br. J. Cancer 59,491-498 Balazy, M., and Murphy, R. C. (1986) Aml. Chem. 5 8 , 1098-1101
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