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Osteoclasts
HUMAN GROWTH HORMONE STIMULATES PROTEINASE ACTIVITIES OF RABBIT BONE CELLS VIA hIGF-I
A. V. Rousselle*, C. Damiens, E. Grimaud, M. Padrines, D. Heymann, F. Redini
Medicine Faculty, EE-99/01, Nantes, France
Human growth hormone (hGH) and human insulin-like growth factor-I (hIGF-I) are known to have a marked influence on osteoclastic formation and bone resorption in an unfractionated rabbit bone cell model. Recently, we reported that the stimulatory effects of hGH on the resorption activity of osteoclasts are partly mediated by hIGF-I produced by stromal cells. Some in vitro studies have provided evidence for the determining role of proteases during bone resorption via the organic bone matrix digestion. The present study investigated the effects of both hGH and hIGF-I on cysteine-proteinase (cathepsins) and matrix metalloproteinase (MMP-2 and MMP-9) activities in total rabbit bone cells and in purified osteoclasts. Cathepsin activities were detected in supernatants by the action of a fluorogenic substrate and metalloproteinase activities by zymography. After 24 hours of incubation with total bone cells, hGH and hIGF-I (50 ng/ml) significantly upmodulated cathepsins, MMP-9 (latent form) and MMP-2 (active form) activities. Similar studies were performed in the presence of human parathyroid hormone (hPTH, 50 ng/ml). hPTH increased MMP-2 and MMP-9 activities whereas it had no effect on the production of cathepsins by bone cells. When neutralizing anti-hIGF-I antiserum was added to the culture, the stimulatory effects of hGH were totally abolished, indicating that hGH-modulated protease activities were partly mediated by local hIGF-I secretion. Cysteine-proteinase activities released by purified osteoclasts were hardly detectable and were not modulated by hGH and hIGF-I. However, hIGF-I but not hGH increased MMP-2 and MMP-9 activities released by purified osteoclasts. In short, hGH markedly stimulates the expression of proteinases in total rabbit bone cells via local hIGF-I production by stromal cells. These results also suggest that hGH- and hIGF-I-induced modulation of cysteine-proteinase activities are related to non-osteoclastic bone cells whereas stimulation of MMP-2 and MMP-9 release by hIGF-I are mainly due to osteoclastic cells in our culture system.
BONE RESORPTION RELEASES IMMUNOREACTIVE OSTEOCALCIN FROM BONE MATRIX IN VITRO
K. Ivaska*, T. Heino, T. Hentunen, H. K. Väänänen
Institute of Biomedicine, Department of Anatomy, University of Turku, Turku, Finland
Osteocalcin is a bone matrix protein produced by osteoblasts. Circulating osteocalcin is considered as an index of osteoblastic activity, but the circulating pool might also contain osteocalcin molecules released from the resorption. Osteocalcin is, however, assumed to be degraded in osteoclasts into small fragments which cannot be detected by immunoassays. In this study we demonstrate that osteoclastic bone resorption in vitro releases osteocalcin which is large enough for immunodetection.
Primary osteoclasts from rat long bones were cultured on devitalized slices of bovine cortical bone. The culture medium was supplemented with 10% bovine serum which was depleted of osteocalcin with immunoaffinitychromatography in order to remove the interfering bovine serum osteocalcin. Osteocalcin concentrations in conditioned medium were measured daily with three sensitive non-commercial immunoassays.
Immunoreactive osteocalcin was observed in culture medium after 48 - 72 hours. The concentration of osteocalcin increased for 4 days after which it remained constant. The concentration of total osteocalcin was up to 6 ng/ml. Interestingly, osteocalcin was also detected with an assay specific for only intact molecules. In control cultures bovine bone slices or rat bone cells alone did not produce significant amounts of immunoreactivity. Inhibitors of bone resorption, e.g. bafilomycinA1, inhibited the release of osteocalcin into medium. Parathyroid hormone and 1,25(OH)2D3, which both stimulate bone resorption, significantly increased osteocalcin concentrations. The increase continued throughout the observation period of 7 days. It was most pronounced in the PTH-treated cultures in which the concentration of total osteocalcin was as high as 40 ng/ml.
Our results indicate that osteoclastic bone resorption is able to release immunodetectable fragments of osteocalcin from bovine bone matrix. This preliminary data also suggests that some of the resorbed osteocalcin might be able to escape the degradation machinery in osteoclasts as intact molecule. It remains to be evaluated if this in vitro result applies also in vivo and arouses a question if intact serum osteocalcin can be regarded only as a marker of bone formation.
THE MECHANISM OF RGD-INSULIN ANTI-OSTEOCLASTIC BONE RESORPTION
Y. Xue*, D. Li
Department of Biochemistry, Beijing Ji Shui Tan Hospital, Beijing, P.R. China
Objective: This study is to explore the mechanism of anti-bone resorption of RGD-insulin using multinuclear giant cells (MGC) as a model of osteoclast-like cells (OLCs) in vitro. RGD-insulin is a new polypeptide containing RGD(Arg-Gly-Asp) sequence with insulin-like structure, but it has no physiologic activity of insulin.
Methods: OLCs were incubated with 10-5, 10-6, 10-7 mol/L RGD-insulin for 24 or 48 hours. The OLCs were stained by tartrate-resistant acid phosphatase (TRAP) and acid phsophatase (AP). OLCs were detected by in situ hydridization for the gene expression of carbonic anhydrase II (CAII) and stained by in situ apoptosis using TUNEL. Also attachment tests were performed after RGD-insulin treatment.
Results: (1) OLCs and osteoclasts had similar characteristics with multinuclei, TRAP positive and bone resorptive function. (2) RGD-insulin was able to inhibit the attachment function of OLCs and induced OLCs apoptosis in dose dependent manners. (3) 10-7mol/L RGD-insulin significantly inhibited the gene expression of CAII, which is relative with bone resorption.
Conclusion: RGD-insulin induced OLCs apoptosis in dose-dependent manners and the apoptosis of OLCs might be related with the expression of CAII mRNA.
OSTEOCLAST-MEDIATED RESORPTION OF HYDROXYAPATITE CERAMIC: SPECIFIC PATTERNS AND ADJUVANT ROLE OF THE SURROUNDING BONE
A. Boyde1*, M. Arora1, A. Corsi2,3, R. Quarto4, R. Cancedda4,5, P. Bianco2,3
1University College London, London, UK
2Universitą dell’Aquila, l'Aquila, Italy
3Universitą "La Sapienza" di Roma, Rome, Italy
4Centro di Biotecnologie Avanzate, Genova, Italy
5Universitą di Genova, Genova, Italy
We previously reported the existence of complementary integration and disintegration mechanisms within hydroxyapatite ceramic (HAC) implants used to replace critical-sized segmental defects in ovine tibiae. However, the relative roles of (1) direct osteoclastic resorption of HA as against (2) mechanical fragmentation with macrophage (polykaryon) engulfment of fine particles and (3) non-specific dissolution remained unclear. We therefore instituted experiments to test for in vitro pit formation. However, it is difficult to recognise resorption pits in HAC because of its inbuilt defects. We therefore also used test samples in which bone had formed on and in HAC in vivo. Thick slabs of HAC or bone+HAC were embedded in PMMA, so that, after cutting and polishing the test substrates, there were no macropores within which to lose the isolated pre-hatch chick OCs seeded on them. Cultures were run for 48 hours. Presence of live OCs was documented by neutral red before and toluidine blue staining after termination. Resorption patches were located by staining with toluidine blue after removal of cells with bacterial enzyme detergent. Samples were carbon coated and studied by conventional reflection LM and reflection confocal microscopic mapping and BSE SEM. Findings: Initial experiments showed little change in medium pH in HAC-only, but a reduction in pH in HAC+bone cultures: medium pH was reduced in later experiments to maximise resorptive activity. BSE showed that dissolution of HA-only does occur, but that OC-etched areas were darker, suggesting preferential attachment of an organic residue, proven by its removal by oxygen plasma ashing. ‘Pits’ were very shallow in HAC compared with bone. Compared with HAC-only, resorption of HA in HAC+bone samples was more frequent and showed sub-HAC-surface attack comparable to that found in vivo. Conclusions: HA materials may be conditioned in vivo to modify the nature of the OC resorptive process.
MACROPHAGE-DEACTIVATING MOLECULES, TRANSFORMING GROWTH FACTOR BETA1 (TGF-BETA), INTERLEUKIN (IL) -4 AND IL-13 STIMULATE THE OSTEOCLAST-FORMING POTENTIAL OF PERIPHERAL BLOOD HAEMOPOIETIC PRECURSORS IN A LYMPHOCYTE-RICH MICROENVIRONMENT
H. M. Massey1*, M. A. Horton2, A. M. Flanagan1
1Bone and Mineral Unit, University College, London, UK
2Royal Free and University College Medical School, London, UK
Osteoclasts are cells which resorb bone; they derive from M-CSF-dependent hemopoietic precursors in the presence of RANKL. TGF-beta, a macrophage deactivator, has been recently found to enhance murine osteoclast formation. We tested the hypothesis that not only TGF-beta but also other macrophage deactivators, including IL-4 and IL-13, enhance human osteoclast formation. We employed a peripheral blood mononuclear cell (PBMC) osteoclast formation assay, plating 2x105 PBMCs per well, and maintained the cultures in M-CSF (25ng/ml) for the duration of the experiment. RANKL (30ng/ml) was added on day 1 or day 4 of the 14 day experiments. We found that all three molecules stimulated human osteoclast formation and bone resorption in a dose-responsive manner; maximal stimulation occurred at 10ng/ml with TGF-beta, at 1ng/ml with IL-4 and at 0.1ng/ ml with IL-13. This stimulatory effect (up to a nine fold increase compared to cultures without these cytokines) was only observed in cultures containing non-adherent PBMCs. In addition, TGF-beta only exerted its effect if present for 4 days prior to the addition of RANKL. The osteoclast-enhancing effect of these cytokines was not mediated through induction of RANKL, or PGE2 production. IL-4/IL-13 (1ng/ml) also strongly suppressed osteoclast formation in lymphocyte-depleted cultures and IL-4 (1ng/ml) stimulated fusion of osteoclast precursors to form large, resorbing syncytia. We also found that removal of the non-adherent PBMCs (lymphocytes) in the osteoclast-forming cultures enhanced osteoclast formation compared to lymphocyte-rich cultures.
Our data support the postulate that the macrophage deactivating molecules enhance the osteoclast-forming potential of the cultures by antagonising the effect of molecules generated in the presence of lymphocytes. This suggests that TGF-beta, IL-4 and IL-13 increase the number of M-CSF-dependent precursors responsive to RANKL, thereby enlarging the population capable of differentiating into osteoclasts. Finally, since others have shown previously that these cytokines suppress human osteoclastogenesis, our data demonstrate that the cytokine milieu and/or state of cell activation determines the response of osteoclast precursors to TGF-beta, IL-4 and IL-13.
OSTEOCLAST DIFFERENTIATION FACTOR IS DOMINANT IN CONTROLLING THE EXTENT OF BONE RESORPTION
N. Fazzalari1,2*, J. Kuliwaba1,2, G. Atkins1, M. Forwood2, D. Findlay3
1Institute of Medical and Veterinary Science
2Adelaide University
3University of Queensland
The cellular and molecular mechanisms that lead to particular trabecular structures in healthy bone are not well understood. Bone remodeling is initiated by osteoclastic resorption followed by osteoblastic formation of new bone. Osteoclast differentiation factor (ODF) is a newly described regulator of osteoclast formation and function, whose activity appears to be a balance between interaction with its receptor, receptor activator of nuclear factor kappa-B (RANK), and with an antagonist binding protein, osteoprotegerin (OPG). We have examined the relationship between the expression of ODF, RANK and OPG, and indices of bone structure and turnover, in human cancellous bone from the proximal femur.
Bone samples were obtained from individuals with osteoarthritis (OA) (n=13; median age 66 years) at joint replacement surgery and from autopsy controls (n=12; median age 68.5 years). These bone samples were processed for histomorphometric analysis and RNA was isolated for semi-quantitative RT-PCR analysis of ODF, RANK and OPG mRNA gene expression.
ODF, OPG and RANK mRNA were abundant in human cancellous bone, with significant differences between control and OA individuals. The ratios of ODF/ OPG and ODF/RANK mRNA were significantly lower in OA (1.78±0.98; 0.59±0.31) compared to the controls (3.41±1.94, p<0.02; 2.53±1.5, p<0.001). Furthermore, eroded bone surface (ES/BS[%]) was significantly lower (p<0.05) in the OA group (6.37±3.17) compared to controls (9.74±4.53). In the controls, trabecular bone volume (BV/TV[%]) was inversely related to a measure of the effective level of ODF, the ratio of ODF/OPG mRNA (r=-0.67; p<0.05). This is consistent with a pro-osteoclastic action of ODF. In addition, strong associations were found between the ratio of ODF/OPG mRNA and the bone resorption index, ES/BS[%] (r=0.93; p<0.001).
These relationships strongly support the concept that our surrogate measures, ODF and OPG mRNA levels, relate directly to levels of expression of the corresponding proteins in the bone tissue. Also, that the effective concentration of ODF is dominant in controlling the extent of bone resorption in human trabecular bone in vivo. In contrast to the controls, the molecular histomorphometric relationships reported above were not evident in trabecular bone from severe primary OA, suggesting that bone turnover may be regulated differently in this disease.
EFFECT OF PROLONGED ADMINISTRATION OF GRADED DOSES OF RECOMBINANT INTERLEUKIN 1-BETA ON SKELETAL MINERAL CONTENT AND ON BIOLOGICAL MARKERS IN RATS
E. del Pozo1*, K. Lippuner1, R. Perrelet1, A. MacKenzie2
1Osteology Unit, University Hospital of Berne, Switzerland
2Novartis Pharma, Basle, Switzerland
The effect of recombinant interleukin-beta (rIL-1beta) on bone remodelling was investigated in 8-week old female rats. Five groups of 10 animals each had miniosmotic pumps (Alzet 2002, 200 ul) implanted subcutaneously and primed to release 100, 500, 1000 or 2000 ng/kg/24h daily for 14 days. On days 1 and 14 measurements of total bone mineral content (BMC) were performed by means of dual energy X-ray absorptiometry (DXA). Blood was drawn on days 1 and 14 and analyzed for ionized Ca, osteocalcin (OC), creatinine and PTH. On the same days 24 h urine samples were collected and assayed for Ca, deoxypyridinoline (Dpd) and creatinine. On completion of the study the animals were sacrificed, both tibiae were dissected and the trabecular volume assessed by histomorphometry on Van Kosa stains.
Results: Biological activity of administered IL-1beta was supported by increasing rectal temperature and elevations of circulating corticosterone. Somatic growth as estimated by weight progression was normal at all doses. Total skeletal mineral content, however, decreased in a dose dependent manner reaching significances at the 1000 and 2000 ng/day doses. Also the tibial trabecular volumes estimated by histomorphometry showed a stepwise reduction in close correlation with the DXA measurements. Urinary Ca and Dpd increased in a parallel manner reflecting enhanced bone mineral and matrix resorption. Blood Ca remained normal throughout the study and OC showed the expected physiological decay for this age group, whereas PTH exhibited a dose dependent fall. It is therefore proposed that graded infusions of recombinant rIL-1beta induced a dose-related skeletal mineral and matrix loss in the absence of repair phenomena as shown by the lack of OC reaction and by decreasing circulating PTH.
Overproduction of IL-1beta in chronic inflammatory diseases such as rheumatoid arthritis may at least in part explain the generalized osteopenia that occurs in these conditions.
HIGH CONCENTRATIONS OF MACROPHAGE COLONY STIMULATING FACTOR AND HIGH MONOCYTE DENSITIES INHIBIT THE FORMATION OF TARTRATE-RESISTANT ACID PHOSPHATASE-POSITIVE OSTEOCLAST-LIKE CELLS
M. N. Rowlands*, C. A. Sharp, M. J. Marshall
Charles Salt Centre, Oswestry, UK
As a preliminary to studying the peptides released during osteoclastic bone resorption on bone slices, we optimised a method for generating osteoclasts from mouse bone marrow-derived cells. The dependence of osteoclast differentiation on macrophage colony stimulating factor (mCSF) and osteoclast differentiation factor (ODF, also known as TRANCE, OPGL and RANKL) is now well established. However, different authors have reported various conditions, in particular, the optimal mCSF concentration for the differentiation of cells of the monocyte lineage into tartrate-resistant acid phosphatase-positive osteoclast-like cells (TRAP+OCL). Our preliminary work suggested that the presence of bone or dentine slices affected the production of TRAP+OCL. The purpose of this work was to establish the requirements for maximum conversion of monocytes into TRAP+OCL.
Mouse bone marrow was placed in a flask with recombinant human mCSF for one day and the non-adherent cells passaged on to a 96-well microtitre plate for 6 days in the presence of recombinant human soluble ODF and mCSF. The effect of concentration of mCSF and of the seeding density on the number of mono- and multinucleate-TRAP+OCL was investigated. There was a dramatic inhibition of both mononuclear and multinuclear TRAP+OCL formation at seeding densities above 25,000 cells per well and at mCSF concentrations above 10ng/ml. Total cell numbers were not significantly affected by mCSF concentrations above 5ng/ml. Further observations on the mechanism of this effect and the influence of these parameters on resorption of bone slices by TRAP+OCL will be reported. In conclusion, high levels of mCSF and high cell densities inhibit the formation of TRAP+OCL.
EFFECTS OF THE NATURAL AND ARTIFICIAL MENSTRUAL CYCLE ON SECRETION OF BONE RESORPTIVE CYTOKINES IN WHOLE-BLOOD CULTURES
B. Abrahamsen1, L. Nickelsen1*, V. Bonnevie2, H. Beck-Nielsen1
1Dept of Endocrinology, Odense Univ. Hospital, Odense, Denmark
2Dept. of Medical Biology, Univ. of South Denmark, Odense, Denmark
Background: Bone remodelling is influenced by resorptive cytokines such as Interleukin 1 (IL-1) and -6 (IL-6), and TNF-alpha. While estradiol may exert some of its effects on bone through reducing the secretion of IL-1 and IL-6, progestagens seem to increase cytokine secretion. Thus, some studies have found increased IL-1 and IL-6 in the luteal phase of the cycle, though others have found lower IL-6 in this phase. The aim of the present study was to compare cytokine production in whole-blood cultures established in the luteal and the follicular phase of the menstrual cycle.
Study population: Healthy female volunteers: Regular menstruating premenopausal women (n=11, mean age 39.4y±6.1). Postmenopausal women (n=11, mean age 56.8y±3.6) receiving cyclic HRT (TrisekvensR, Novo, estradiol and noretisterone acetate).
Methods: Blood samples were diluted 1:4 in RPMI-1640 and cultured for 24h with and w/o lipopolysaccharide (LPS). The supernatant was stored for IL-1 beta and IL-6 ELISA. In addition, serum FSH and estradiol was measured. Serum osteoprotegerin (OPG) measurements are currently in progress.
Results: The LPS-stimulated production of IL-1 and IL-6 was significantly higher (median +48% and +77%, p<0.05) in the luteal phase compared with the follicular phase, whereas unstimulated cultures showed no cyclic changes. The LPS-stimulated IL-1 and IL-6 production was negatively correlated with age (r=0.74, p<0.001, r=0.52, p<0.05) and significantly higher in premenopausal women (p<0.01). In contrast, basal IL-1 production was higher in the postmenopausal group, both in the "follicular" (p<0.01) and "luteal" phase (p<0.01).
Conclusion: The secretion of IL-1 and IL-6 assessed in whole-blood culture increases significantly in the luteal phase of the cycle. Compared with premenopausal women, postmenopausal women receiving HRT showed higher basal IL-1 secretion with a lower capacity for LPS-stimulated IL-1 and IL-6 production. This demonstrates the impact of age, time in the menstrual cycle, and stimulation conditions on cytokine production and explains some of the discrepancies between clinical studies of cytokine production and bone loss.
| IL-1 beta (pg/ml) | IL-6 (pg/ml) | |||
| Follicular | Luteal | Follicular | Luteal | |
| Pre-menop. (n=11) |
3877 (1617-6729) |
5474 * (1791-7408) |
8191 (5100-12525) |
10706 (4799-20581) |
| Post-menop. HRT (n=11) | 1485 (272-4086) |
1301 (667-3517) |
4271 (1943-16498) |
5736 (2054-9344) |
| Total population (n=22) | 2261 (272-6729) |
2698 * (667-7408) |
5847 (1943-16498) |
7371 * (2054-20581) |
| Median and range, LPS-stimulated whole-blood cultures (* p<0.05 Wilcoxon) | ||||
PHAGOCYTOSIS DETERMINES LINEAGE DESTINY OF MACROPHAGE-OSTEOCLAST PRECURSORS INCUBATED IN TNF-ALPHA/RANKL
K. Fuller*, K. E. Bayley, T. J. Chambers
St George's Hospital Medical School, London, UK
It has recently been found that TNF-alpha, like RANKL (also known as TRANCE, OPGL and ODF), can induce osteoclast formation in-vitro from cells of the mononuclear phagocyte lineage, and that TGF-beta augments this. Since TNF-alpha with TGF-beta is considered a cytokine combination that induces the differentiation of wound-healing macrophages in vivo, yet induces osteoclasts in-vitro, additional factors presumably exist that regulate macrophage-osteoclast lineage choice in-vivo. We found that while ingestion of apoptotic thymocytes had no effect on osteoclast differentiation from immature mononuclear phagocytes, phagocytosis of latex beads, glutaraldehyde-fixed red cells, or necrotic thymocytes strongly suppressed osteoclast formation, even under otherwise-optimal osteoclast-inductive conditions. The inhibition of osteoclast formation could not be attributed to particulate-induced nitric oxide production, or to release of osteoclast-inhibitory materials by phagocytosing cells. Nor could it be explained by phagocytosis-induced modulation of TGF-beta and/or TNF-alpha secretion, since inhibition of osteoclast formation occurred despite exogenous cytokines. This suggests that phagocytosis profoundly influences osteoclast-macrophage lineage choice. It appears to do so independently of autocrine-paracrine mechanisms, and the lineage outcome depends on the nature of the phagocytic stimulus.
FLT3 LIGAND CAN SUBSTITUTE FOR M-CSF IN SUPPORT OF OSTEOCLAST DIFFERENTIATION AND FUNCTION
J. M. Lean*, K. Fuller, S. W. Fox, T. J. Chambers
St George's Hospital Medical School, London, UK
Although bone resorption and osteoclast numbers are reduced in osteopetrotic (op/op) mice, they are nevertheless present and functional, despite absence of M-CSF. This suggests alternative factors can partly compensate for the crucial actions of M-CSF in osteoclast-induction. We found that when bone marrow cells were incubated in receptor activator of NFkappaB ligand (RANKL) with Flt3 ligand, TRAP-positive cells were formed, and bone resorption occurred. Osteoclast-induction by Flt3 ligand could not be attributed to synergy by Flt3 ligand with the low levels of endogenous M-CSF produced by the bone marrow cells themselves, since Flt3 ligand also induced osteoclast formation from the hemopoietic cells of op/op mice. Flt3 ligand appears to substitute for M-CSF through induction of RANK and enhanced survival in precursors with little proliferation, so that fewer osteoclasts form than in wild-type. However, the ratio of osteoclasts to macrophages in our cultures was similar in Flt3 ligand vs M-CSF, suggesting that the ability of Flt3 ligand to support osteoclastic differentiation was equivalent to that of M-CSF. The ability of Flt3 ligand to substitute for M-CSF in the induction of RANK but not in proliferation is consistent with the presence of osteoclasts only in small numbers in op/op mice. These observations of cells in-vitro were supported by data from experiments in-vivo. We found that injection of recombinant soluble Flt3 ligand into op/op mice induced a substantial decrease in osteoclast number, suggesting that Flt3 ligand partly compensates in-vivo for absence of M-CSF.
LIMITED RANKL EXPOSURE IN VITRO INDUCES OSTEOCLASTOGENESIS IN HUMAN PBMC
G. C. Nicholson1, C. J. Aitken1, J. M. Hodge1, W. R. Holloway1, T. Gough1, F. Collier1, M. A. Kirkland2, D. E. Myers1*
1Department of Medicine, University of Melbourne, The Geelong Hospital
2The Douglas Hocking Institute, The Geelong Hospital, Barwon Health
M-CSF and RANKL are essential for the proliferation, differentiation and activation of cells during osteoclastogenesis. Whilst RANKL does not induce colony formation in haemopoietic cells (Lacey et al., 1998), it acts as a differentiation factor and RANKL may prevent monocyte/macrophage commitment induced by rhM-CSF. PBMC and CD14+ PBMC incubated on bone with rhM-CSF (Genetics Institute, USA) and rhsRANKL (Peprotech, USA) exhibit strong osteoclastogenesis over a 3 week period (Nicholson et al., 2000). We have investigated the temporal requirement of rhsRANKL in limited addition experiments in which the cytokine was added to PBMC for specified periods on defined days in the presence of rhM-CSF. A limited addition, as short as 1 hour on day 12 of a 21 day culture, still induced multinucleation of monocyte/macrophages with resorption of the bone surface measured at 21 days. PBMC exposed to RANKL between days 8 and 14 only, generated significantly more multinuclear TRAP+ve cells (MuOC) than with uninterrupted RANKL exposure (day 8-14, 275±56 MuOC; day 0-21, 143±40 MuOC; p<0.05). However, the percentage of bone surface resorbed in these two groups was not significantly different (day 8-14, 21.3±4.0%; day 0-21, 25.6±6%; p<0.05) suggesting that RANKL is promoting bone resorption activity in these cells. Treatment with RANKL during the first week alone (RANKL +/-/-) resulted in negligible osteoclastogenesis with 11.43±4 MuOC per bone slice that showed sporadic resorption lacunae over the bone surface (0.45±0.26%). In contrast, treatment during the third week alone (RANKL -/-/+) resulted in a mean generation of 93.8±23.7 MuOC per bone slice with slightly more resorption (10±2.4% of the bone surface). This shows that RANKL is not required in the first week and has limited effect in the third week of these cultures. These findings have both practical and physiological relevance to the study of osteoclastogenesis. For practical purposes, under a limited RANKL protocol, RANKL needs to be added to cultures for the duration of only one media change period (3-4 days) or shorter and simplification of this model will enable more elaborate studies of differentiation, fusion and activation in the absence of RANKL.
Lacey et al. (1998) Cell 93, 165-76
Nicholson et al. (2000) Clin Sci (Colch) 99, 133-140
GLUCOCORTICOIDS MODULATE OSTEOPROTEGERIN/ OSTEOPROTEGERIN LIGAND AND OSTEOCLAST ACTIVITY
L. Mancini*, I. MacIntyre, M. Perretti
The William Harvey Research Institute, St. Bartholomew's and the Royal London SMD, ECIM-6BQ, London, UK
Glucocorticoids (GCs) are potent immunosuppressive and anti-inflammatory agents. They are effective in virtually all forms of chronic inflammation including rheumatoid arthritis. However, while GCs reduce clinical symptoms, long term therapy is associated with severe side-effect, osteoporosis being one of the most important. The discovery of two novel cytokines with bone modulating activity, osteoprotegerin (OPG) and OPG ligand (OPGL) has opened new avenues for therapeutic intervention on this disabilitating disease.
The effects of different GC were tested in a bone resorption assay in order to compare their ability in promoting bone loss and their modulation of OPG and OPGL.
Primary osteoclasts were prepared from neonatal Sprague-Dawley rat femora; cells were plated on a calcium phosphate apatite substrate (OAASTM, OCT Inc). After 24h, cells were removed and the resorbed area was measured.
Prednisolone induced a concentration-dependent increase in bone resorptive activity, maximal at 1nM; the same action was exerted by OPGL. OPG 30ng/ml was able to completely prevent the increase in bone resorption induced by prednisolone and OPGL, suggesting that GCs modulate this new cytokines.
Nitric oxide (NO) has long been known to be able to inhibit osteoclastic resorptive activity. For this reason a novel class of GCs, able to release NO, has been synthesised. NCX-1015 (prednisolone 21-[(4'-nitrooxymethyl)benzoate]) a NO-releasing derivative of prednisolone was also tested for bone resorption (174±31%) The rate of bone resorption by cells treated with 1nM NCX-1015 was comparable with that of control. In order to evaluate if NO was the agent responsible for the protective effect we tested the bone resorptive activity of a compound with the same spacer, but without the nitrooxymethyl group (termed NCX-1016), therefore unable to release nitric oxide. As expected NXC-1016 shares the same ability of prednisolone in inducing bone resorption, suggesting that NO released by NCX-1015 but not by NCX-1016 inhibits bone resorption induced by GC moiety of the compound. It remains to be determined if NCX-1015 inability to activate osteoclasts is linked to a lack of effect on OPG/OPGL synthesis/release, or on the actions of this cytokines (i.e. receptor modulation).
INTERLEUKIN-6, LEUKEMIA INHIBITORY FACTOR AND ONCOSTATIN M STIMULATE BONE RESORPTION AND REGULATE THE EXPRESSION OF RANKL, OPG AND RANK IN MOUSE CALVARIAE
E. Persson1*, P. Palmquist1, H. H. Conaway3, P. Lundberg1,2, U. H. Lerner1,2
1Dept. Oral Cell Biology, Umeå University, Umeå, Sweden
2Centre for Musculoskeletal Research, National Institute for Working Life, Umeå, Sweden
3Dept. Physiology and Biophysics, University of Arkansas for Medical Sciences, Little Rock, Arkansas, USA
Interleukin-6 (IL-6) is abundantly expressed in bone and the expression is regulated by several calciotropic hormones and cytokines. In the present study, the effects of IL-6, in the absence and presence of soluble interleukin-6 receptor (sIL-6R), on bone resorption and on the mRNA expressions of RANKL, RANK and OPG in neonatal mouse calvarial bones have been investigated. The effects of IL-6 were compared to those of leukemia inhibitory factor (LIF) and oncostatin M (OSM), two other peptides in the IL-6 family of cytokines. IL-6 or sIL-6R, by their own, did not affect 45Ca release. However, in the presence of sIL-6R, IL-6 stimulated bone mineral and matrix release in a manner dependent on the concentrations of both IL-6 and sIL-6R. The stimulatory effect on mineral mobilization by IL-6 + sIL-6R was abolished by three different osteoclast inhibitors. 45Ca release induced by IL-6 + sIL-6R was partially (40%) inhibited by indomethacin and flurbiprofen. Parathyroid hormone (PTH) and interleukin- 1 beta (IL-1 beta) stimulate IL-6 production in calvariae; however, sIL-6 R did not potentiate the stimulatory effect of PTH and IL-1 beta on 45Ca release. Semi-quantitative RT-PCR showed that IL-6 + sIL-6R enhanced the expressions of RANKL and OPG, and decreased that of RANK. LIF and OSM stimulated 45Ca release and enhanced the mRNA expression of RANKL and OPG, but did not affect that of RANK. Vitamin D3 stimulated 45Ca release, enhanced RANKL and RANK mRNA expression and decreased that of OPG. OPG inhibited 45Ca release stimulated by RANKL, IL-6 + sIL-6R, LIF, OSM and vitamin D3. An antibody neutralizing mouse gp 130 inhibited 45Ca release induced by IL-6 + sIL-6R. These data show that mouse calvarial bones respond to IL-6 and sIL-6R with enhanced osteoclastic bone resorption and that regulation of RANKL and OPG are downstream effects of cytokine receptors in the IL-6 family of cytokines. The data further suggest that RANK expression in preosteoclasts/osteoclasts can be regulated by both vitamin D3 receptors and by gp 130.
P2 RECEPTOR STIMULATION UPREGULATES EXPRESSION OF OSTEOCLAST DIFFERENTIATION FACTOR (ODF) BY HUMAN OSTEOBLASTS
K. A. Buckley*, J. A. Gallagher, W. B. Bowler
Human Bone Cell Research Group, Department of Human Anatomy and Cell Biology, New Medical School, Ashton Street, University of Liverpool, L69 3GE, UK
Nucleotides, such as ATP, exist extracellularly due to lytic or non-lytic release, where they are agonists at a group of membrane bound receptors termed P2 receptors, numerous members of which have been found to be expressed by both osteoblasts and osteoclasts.
We have previously shown that ATP induces immediate early gene expression in osteoblasts and stimulates resorption in co-cultures containing both osteoclasts and osteoblasts. In contrast ATP was ineffective at stimulating resorption by an osteoclast population alone, as assessed in human Rankligand cultures. Osteoclast differentiation factor (ODF) is expressed by osteoblasts and is known to be fundamental for the fusion of monocytic pre-osteoclasts to form multinucleated osteoclast cells and also stimulates resorption by mature osteoclasts.
In this study, the human osteosarcoma cell line SaOS-2 was stimulated with either ATP (100microM) or PTH (100ng/ml) over a 12-hour period and ODF mRNA expression assessed by semi-quantitative RT-PCR. ATP stimulation resulted in elevated ODF mRNA expression 4 to 12 hours post-stimulation, and as has been previously reported in other osteoblast cells, PTH also induced an increase in ODF mRNA expression. Osteoprotegerin (OPG) mRNA levels were not affected following ATP or PTH treatment.
Therefore in co-cultures containing both osteoblasts and osteoclasts the observed increase in osteoclast resorption seen after ATP stimulation may be due to an increase in expression of ODF mRNA by osteoblasts. These data provide additional evidence for the importance of extracellular nucleotides in the bone micro-environment, and further implicate the role of ATP in localised remodelling events.
P275 T
Withdrawn
IMMUNOLOCALIZATION OF OSTEOCLAST DIFFERENTIATION FACTOR IS ASSOCIATED WITH THE OSTEOCLAST DEVELOPMENT IN RAT PERIODONTIUM
S. Kawamoto1*, S. Ejiri2, K. Hoshi2, E. Nagaoka1, H. Ozawa2
1Kagoshima University, Kagoshima, Japan
2Niigata University, Niigata, Japan
Integrity of the alveolar bone is required for it to function as an osseous support of the tooth, and in the biology of the alveolar bone, the cellular dynamics of the osteoclast in the periodontium is an important subject. Recently, we investigated on the development of osteoclast in ovariectomized rat periodontium, and found that the manifestation of osteoclast differentiation factor (ODF) was associated with the induction of osteoclasts. The aim of the present study was to observe further the immunohistochemical localization of ODF in rat periodontium.
Mandibles of rats were decalcified and embedded in paraffin, and horizontal and frontal sections were prepared for immunohistochemical analysis using antibodies against human RANKL/ODF and human protein gene product 9.5 (PGP9.5). To identify osteoclastic cells, tartrate resistant acid phosphatase activity was detected using the Azo dye method.
In the horizontal sections, the immunolocalization of ODF was markedly observed at the distal area of periodontium of molars in which osteoclasts appeared due to the physiological tooth drift. In the frontal sections, the ODF-immunoreactivity in the periodontium was localized on spindle-shaped mesenchymal cells having a perivascular location near the bone surface. Around blood vessels near the bone surface, reactions were more intense on the cells at the root side than on the alveolar bone side. In addition, immunoreation of ODF was detected on structures, which appeared to be elongated cell processes near the blood vessels in the frontal sections. Immunohistochemical examination for the general antigen of nervous-specific protein (PGP9.5) suggested a morphological similarity between these structures and nerve fibers.
In conclusion, the present results demonstrated the immunolocalization of ODF is variable in rat periodontium, and associated with the location of osteoclasts, although further investigations are needed for the identification of the ODF-immunopositive structures. It is suggested that ODF plays an important role in the development of osteoclasts in the periodontium, and in activation of bone resorption corresponding to physiological tooth migration and/or pathological conditions such as estrogen deficiency.
EXPRESSION OF OSTEOCLASTOGENIC MARKERS IN INFLAMED SYNOVIAL TISSUES IN PROSTHESIS LOOSENING AND ARTHRITIS
A. Sabokbar*, A. Pocock, O. Kudo, S. D. Neale, E. Schulze, L. Danks, N. A. Athanasou
University of Oxford, Oxford, UK
Osteolysis associated with periprosthetic loosening and rheumatoid arthritis (RA) is thought to be due to a number of factors including: (i) activation of local resident cells (e.g. macrophages and T-cells); (ii) release of inflammatory cytokines; (iii) increased macrophage-osteoclast differentiation. Recently, newly identified molecules, RANKL (expressed on stromal cells) and osteoprotegerin (OPG), have been shown to play a central role in osteoclast development. The interaction between RANKL and its receptor RANK, which is expressed on osteoclast precursor cells, is essential for osteoclast formation. This process is blocked by OPG which acts as a soluble decoy receptor. The aim of the present study was to determine the extent of mRNA expression of the major factors controlling osteoclast formation (i.e. RANK, RANKL and OPG) in periprosthetic tissues and RA synovium.
Tissue specimens were taken at surgery from patients undergoing joint replacements due to aseptic loosening (n=4), RA (n=4) or osteoarthritis (OA) (n=6). Giant cell tumour of bone was used as a positive control. Total RNA was extracted from the isolated cells and mRNA expression of RANK, RANKL, OPG and b-actin was determined using RT-PCR. Expression of all these osteoclastogenic markers was noted in all the pathological specimens examined and the level of expression of these factors (expressed as a ratio to the b-actin PCR product) correlated with the severity of osteolysis. RANK mRNA expression was substantially higher in periprosthetic tissues (1.5-2 fold) than in OA and RA synovium. An increase in the RANKL:OPG mRNA ratio in RA synovium compared to periprosthetic tissues and OA was also noted.
This study has shown that the major factors regulating the formation of osteoclasts (i.e. RANK, RANKL and OPG) are present in the inflamed (macrophage-enriched) tissues which are associated with pathological bone resorption in arthritis and aseptic loosening. An increase in RANKL expression or decreased expression of OPG (i.e. the ratio of RANKL to OPG) is likely to be associated with the differences in the extent of osteoclast formation and consequently the osteolysis associated with these inflammatory conditions.
FORMATION AND ISOLATION OF HUMAN OSTEOCLASTS
O. Kudo*, A. Sabokbar, M. Jevon, S. D. Neale, N. A. Athanasou
University of Oxford, Oxford, UK
Analysis of the cytochemical and functional characteristics of osteoclasts has only really been carried out in any detail using avian/mammalian osteoclasts isolated directly from bone. In adult humans, osteoclasts are tightly adherent to bone, few in number and therefore not readily available for extraction by physical, enzymatic or other means. Although osteoclastic cells can be isolated from giant cell tumours of bone, these tumours are rare and the physiological nature of the osteoclastic cells isolated from these tumours is questionable. As it is very difficult to isolate osteoclasts from the bone of adult human subjects (in whom bone biopsy is in any case not usually indicated), the functional and cytochemical activity of osteoclasts in bone diseases such as osteoporosis is unknown. We have recently developed a technique for the isolation of human osteoclasts formed from peripheral blood mononuclear precursors that are present in the monocyte fraction. Following isolation of monocytes from peripheral blood, these cells are cultured with RANKL and human M-CSF and dexamethasone on a Type I collagen gel for 14 days. During this culture period, numerous multinucleated cells form on the gel which is then treated by collagenase, releasing numerous osteoclasts. These cells are then seeded onto coverslips or dentine slices for further study of their cytochemical characteristics and lacunar resorption activity. Numerous TRAP-positive multinucleated cells, capable of lacunar resorption, are derived from these cultures. This technique should make it possible for human osteoclasts from patients with osteolytic disorders such as osteoporosis and Paget's disease to be formed in culture and made available for study of their cytochemical, functional and other characteristics.
EXPRESSION OF MSP RECEPTOR RON IN OSTEOCLAST-LIKE GCT CELL LINES
C. Camerino1*, M. Santoro2, G. Mori1, M. Grano1, P. C. Marchisio3, G. Gaudino2, A. Zambonin Zallone1
1Dept. of Human Anatomy, Univ. of Bari, Italy
2University A. Avogadro, Novara, Italy
3DIBIT, San Raffaele Milano, Italy
Macrophage Stimulating Protein (MSP) is a growth factor with a chemotactic activity on murine peritoneal macrophages. MSP is synthesized in an inactive form and can be cleaved by members of the coagulation cascade. His receptor is the RON gene product, a transmembrane tyrosine kinase, member of the hepatocyte growth factor receptor family. Ron is expressed in epithelial cells, keratinocytes, granulocytes, monocytes and osteoclast. It has previously been shown that MSP and his receptor RON stimulate human osteoclast activity enhancing bone resorption and differentiation.
In this study we examined the signal transduction pathway of MSP-RON in an osteoclast-like cell line, GCT 23, obtained from Giant Cell Tumors of Bone. Using specific anti Ron C-terminal antibody, we detected by western blot RON expression in GCT 23. Binding of MSP to RON induced phosphorilation of RON 150 Kda beta-chain within 10 minutes. GCT 23 treated with MSP showed a time dependent inhibition of proliferation together with marked morphological changes. Indeed, after addition of MSP, the characteristic spread morphology of untreated GCT 23 switched to a spindle motile phenotype. Furthermore by western blot we detected that the activation of RON in GCT 23 produced an increase in MAPK and AKT activity. MAPK and AKT are 2 different but sinergic pathway, both triggering differentiative and antiapoptotic effects due to the phosphorilation of BAD. This study establishes Giant Cell Tumors of bones as a new target for MSP. The action of MSP on GCT 23 may have implication on cell growth and protection from apoptosis.
OSTEOCLAST SIZE CORRELATES WITH DIFFERENCES IN RESORPTIVE ACTIVITY
J. N. M. Heersche*, R. Lees, M. F. Manolson, V. Sabharwal
Faculty of Dentistry, University of Toronto, Toronto, Canada
A local increase in osteoclastic bone resorption is frequently associated with a local increase in both the size and the number of osteoclasts. Whether there is a functional advantage to increased size in terms of resorptive activity or efficiency, and whether differences exist in this regard between large and small osteoclasts, is not clear. To investigate this issue, we evaluated resorptive activity and activity of pH regulatory pathways in large (>10 nuclei) and small (<5 nuclei) resorbing and non-resorbing osteoclasts. For these studies, we used dispersed rabbit osteoclasts cultured on devitalized bone slices or collagen/hydroxyapatite coated coverslips and analyzed their activity using time lapse recordings, quantitation of resorption lacunae and measurement of intracellular pH in the presence or absence of inhibitors of the vacuolar H+-ATPase (V-ATPase) and of the Na+/H+ exchanger. We first established that actively resorbing large and small osteoclasts were equally effective bone resorbers when resorption was calculated as resorption per nucleus, thereby confirming previous observations of others. Interestingly, however, we found that as a population, large osteoclasts were much more effective resorbers. This results from the fact that the proportion of large osteoclasts that are actively resorbing in this culture system is much greater than the proportion of small osteoclasts actively resorbing (40% versus 5%). Increased resorptive activity of both large and small osteoclasts was associated with increased intracellular pH (non-resorbing 7.3-7.4, resorbing 7.6). Selective inhibition of either V-ATPase activity or Na+/H+ exchange activity indicated that in resorbing osteoclasts intracellular pH is primarily regulated by V-ATPase activity, and by Na+/H+ exchange activity in non-resorbing osteoclasts. In agreement with this, in situ hybridization of osteoclasts cultured on plastic substrata revealed that mRNA levels for the putative osteoclast-specific 116 kDa V-ATPase subunit (a3) were on average 3 fold higher in large osteoclasts than in small osteoclasts. We hypothesize, on the basis of our results, that the differences between large and small osteoclasts in terms of the proportion of cells that are actively resorbing are the result of different activation pathways (i.e. differential responses to specific agonists) in large versus small osteoclasts.
EXPRESSION OF FUNCTIONAL HUMAN CALCITONIN RECEPTOR WITH GREEN FLUORESCENT PROTEIN TAG IN HEK293 AND BHK21 CELLS
D. E. Myers1*, C. M. Dark1, J. M. Hodge1, C. J. Aitken1, P. M. Sexton2, M. T. Gillespie3, C. M. Lopez1, M. H. C. Lam3, G. C. Nicholson1
1Department of Medicine, University of Melbourne, The Geelong Hospital, Australia
2The Howard Florey Institute, University of Melbourne, Australia
3St. Vincent's Institute of Medical Research, Australia
Calcitonin receptor (CTR) is a G-protein coupled seven-transmembrane receptor that is expressed on osteoclasts in bone and in other tissues such as neural tissue, kidney tubules and pituitary. Under the influence of regulatory proteins called receptor-activating modifying proteins (RAMPs), CTR can recognise other ligands such as CGRP, amylin and adrenomedullin. CT is an important regulator of bone turnover that causes retraction of osteoclasts thus decreasing their bone resorptive capacity. The aim of this project was to investigate internalization and processing of the calcitonin receptor in mammalian cell lines and to determine functional characteristics of green fluorescent protein (GFP)-tagged hCTR (both 16 aa insert positive, hCTRI1+; and, 16 aa insert negative, hCTRI1-).
Constructs of the human calcitonin receptor were prepared by site-directed cloning of the CTR plasmid into the multicloning site of ClontechR EGFP-C1 and ClontechR EGFP-N1 vectors using the two hCTR isoforms (GFP attached to either the amino (N) terminal or the carboxyl (C) terminal of CTR). The four forms of the constructs were then transiently transfected into Hek293 or BHK21 cells which were analysed for 125I-sCT binding (saturation and competition binding) receptor localisation and internalisation using confocal microscopy. Control (vector only) transfections were also performed.
The C-terminal-tagged form of the CTR exhibited the most efficient binding of 125I-sCT for each of the CTR isoforms and results indicated that N-terminal labelling of the receptor may interfere with the ligand attachment. The extracellular tag was localised at the membrane indicating that these constructs are not internalised. The intracellular tagged form localised in the ER and golgi apparatus indicating that the receptor was processed by conventional pathways. CTR internalisation was demonstrated for both the C-terminal GFP-tagged isoforms (CTRI1- and CTRI1+).
We conclude that the GFP-hCTR constructs can be transfected into mammalian cells and retain ligand-receptor interaction as well as internalisation. This should prove to be a useful model to study receptor trafficking and retroendocytosis of the CTR as well as specificity of ligand-receptor interactions and involvement of the receptor-modifying RAMPs.
APICAL EXOCYTOSIS OF LYSOSOMAL PROTEINS INVOLVES MATRIX TRANSCYTOSIS IN BONE-RESORBING OSTEOCLASTS
S. A. Nesbitt1*, P. P. Lehenkari2, S. E. Gschmeisser3, M. A. Horton1
1University College London, London, UK
2Oulu University, Oulu, Finland
3ICRF, London, UK
During bone resorption, osteoclasts liberate bone matrix and transport it through the cell before its release into the extracellular space from apical exocytotic sites. Herein, characterisation of these exocytotic sites shows that they release degraded bone matrix in large complexes of lysosomal proteins.
Human osteoclasts, derived from osteoclastoma tissue, were cultured for up to 14 days on biotinylated dentine slices. The biotin label was used to ‘track’ the resorbed matrix released by the osteoclasts. The culture media were purified on avidin columns to isolate the resorbed bone matrix before its serial fractionation through membrane filters of cut-off sizes of 0.2 micrometers and 300, 100, 30, and 3 kDa. Dot and Western blotting analysis of the fractions identified large complexes (>300 kDa) of degraded bone matrix comprised of CTx collagen fragments, cathepsin K, TRAP, MMP-9, LAMPs, V-ATPase and annexin II. The morphology of the apical exocytotic sites in the bone-resorbing osteoclasts was assessed by confocal microscopy with 3D reconstructions and by field emission scanning electron microscopy; immunofluorescent and immunogold antibody staining was used to identify several of the proteins in the bone matrix complexes. The exocytotic sites formed large vesicular structures (1-3 micrometers in diameter) and were located in a dense area of pseudopodia at the cell apex. Aggregates of bone matrix, associated with the exocytotic sites, co-expressed CTx, cathepsin K, TRAP, LAMPs and annexin II proteins.
In summary, the transcytosis of resorbed bone matrix through osteoclasts appears to use specialised exocytotic sites which release resorbed bone matrix in complexes with lysosomal proteins. Thus, these data provide evidence that osteoclasts may use transcytotic pathways to clear spent proteins derived from the lysosomal compartments associated with the degradation of the bone matrix.
FOLLOWING OF INTRACELLULAR VESICLE TRAFFIC IN LIVING OSTEOCLASTS USING CONFOCAL MICROSCOPY
J. Vaaraniemi*, H. K. Vaananen
Institute of Biomedicine, Department of Anatomy, University of Turku, Finland
Our aim was to develop a method to measure intravesicular pH and to locate intracellular compartments in actively resorbing rodent osteoclasts and follow the resorption process in time lapse mode. The living osteoclasts grown on bone slices or glass cover slips were transferred under the confocal microscope and the cells were viewed using water immersion objective in heated cell chamber at 37 deg C. Cells were viewed in time-lapse mode using laser scanning confocal microscopy.
The ratio of pH-sensitive fluorescein to pH insensitive rhodamine fluorescence emission intensity was used as an indicator of pH in fluorescein and rhodamine double-labeled transferrin containing vesicles. Results indicated the acidic nature of many of these vesicles and decrease of pH during time and slight increase of pH when osteoclasts were treated with bafilomycin A1. Colocalization of DAMP, which binds to acidic vesicles, and transferrin, was studied in fixed osteoclasts. These studies indicated that at least part of the transferrin containing vesicles were acidic. To study osteoclast metabolism the cells were stained with MitoTracker probe, which passively diffuses across the plasma membrane and accumulates in active mitochondria. The results show that mitochondria are accumulated in the specific cell areas. To measure the changes of mitochondrial membrane potential during assays we followed the retention of the potential-sensitive dye, rhodamine 123. This study shows that confocal laser scanning microscopy is useful tool when observing living cells stained with vital fluorescent probes.
MAMMALIAN OSTEOCLASTS LOSE CENTROSOMAL MICROTUBULE ORGANIZING CENTRES DURING MATURATION
L. Patrikainen*, M. T. K. Mulari, H. K. Väänänen
Institute of Biomedicine, Department of Anatomy, University of Turku, Finland
It has been reported earlier that multinucleated non-resorbing avian osteoclasts have an equal number of nuclei and centrosomal microtubule organizing centers (MTOCs). Our aim was to characterize microtubule organization patterns in both non-resorbing and resorbing mammalian and avian osteoclasts in vitro.
Rat pups and chickens were killed by decapitation. Osteoclasts were mechanically harvested from the endosteal surfaces of long bones after which the cells were cultured on glass coverslips or bovine bone slices for 72 or 48 hours, respectively. After the fixation in minus 20 deg C methanol or paraformaldehyde, the microtubules were visualized with anti-alpha-tubulin antibody and pericentriolar matrix proteins with antibodies against gamma-tubulin and pericentrin. The cells were viewed with confocal laser scanning microscope.
Our results show that - in contrast to chicken osteoclasts - the number of centrosomal MTOCs does not correlate to the number of nuclei in either non-resorbing or resorbing rat osteoclasts. Instead, the microtubules mainly radiate from the vicinity of nuclei where pericentrin is also dispersed. This indicates that rat osteoclasts lose nearly all of their centrosomal MTOCs when mononuclear precursor cells mature whereas in non-resorbing chicken osteoclasts the one nucleus-one centrosomal MTOC -pattern is maintained. Interestingly, we also found out that chicken osteoclasts undergo very profound changes in their microtubule organization pattern when they start to resorb bone: the vast majority of centrosomal MTOCs is lost like in rat osteoclasts.
Our data indicates that there may be an evolutionary difference between avian and mammalian osteoclasts. Rabbit, mouse and human osteoclasts share a similar microtubule organization pattern to rat osteoclasts which differs from that observed in chicken and quail osteoclasts.
INDUCTION OF HUMAN PERIPHERAL BLOOD MONONUCLEAR CELLS TO DIFFERENTIATE INTO RESORBING OSTEOCLASTS
T. A. Hentunen*, H. K. Vaananen
University of Turku, Turku, Finland
Peripheral blood mononuclear cells (PBMC) isolated from different species have been shown to differentiate into active osteoclasts under appropriate conditions. The combination of soluble forms of growth factors, receptor activator of NF-kappaB ligand (RANKL, also called TRANCE/ODF) and macrophage colony-stimulating factor (M-CSF) is powerful for inducing osteoclast formation and resorption activity. By culturing isolated human PBMC for 8-21 days on bovine bone slices in the presence of RANKL (20 ng/ml) and M-CSF (10 ng/ml), we found that tartrate-resistant (TRAP)-positive multinucleated cells (MNC) started to be formed at day 14. Their resorption activity was rapidly increased after that. To find out if osteoclast precursors are adherent or non-adherent, isolated human PBMC were let to attach to a tissue culture dish overnight in the alpha-MEM- 10% foetal calf serum. Non-adherent cells were collected and plated on bone slices (106/slice). Adherent cells were trypsinized from the dish and plated on bone slices (50000/slice). Cells were cultured for 21 days in the presence of RANKL and M-CSF. At the end of the culture, cells were stained for TRAP and TRAP-positive MNC were counted. Cells were then removed from bone slices by brushing and resorption pits were stained using peroxidase-conjugated WGA-lectin. In adherent and non-adherent cell cultures, there were 172.8±62.9 and 240.5±115.0 resorption pits/bone slice, respectively. Adherent cells had 14-fold higher pit formation capacity than non-adherent cells when pit numbers were compared to the number of cells plated. When we used the whole PBMC fraction, there were approximately 490.8±126.8 pits and 88.5±23.7 TRAP-positive MNC on bone slices after a 21-day culture. Dexamethasone at 10-7-10-8 M enhanced the RANKL/M-CSF-simulated resorption so markedly that the number of pits was impossible to count. On the other hand, 17beta-estradiol at 10-9 and 10-7 M inhibited resorption 72% and 93%, respectively. This data suggest that the majority of osteoclast precursors reside in adherent population of human PBMC and that human PBMC culture can be used to study the effect of estrogen on both human osteoclast differentiation and resorption activity.
ACTIVATED ENDOTHELIUM RECRUITS OSTEOCLAST PRECURSORS FROM PERIPHERAL BLOOD
N. W. A. McGowan*, E. J. Walker, S. H. Ralston, M. H. Helfrich
Bone Research Group, Dept. of Medicine and Therapeutics, University of Aberdeen, Aberdeen AB25 2ZD, Scotland, UK
Introduction: Bone remodelling is a focal process of renewal and repair essential for the maintenance of skeletal integrity. Multinucleated osteoclasts (OCs) are formed from haemopoietic progenitors in the bone marrow and from OC progenitors (OCL-Ps) circulating in peripheral blood. OCL-Ps will encounter vascular endothelium when they migrate to sites of bone resorption, and it has been suggested that endothelium may play a role in the regulation of OC formation and in the targeting of the remodelling process. We therefore examined the ability of OCL-Ps to adhere to endothelium in an in vitro culture model.
Methods: PBMCs were isolated from 3 healthy volunteers and allowed to adhere to control or activated BMEC-1 cells (a human bone marrow-derived endothelial cell line) for 90 minutes at 37°C. Endothelial activation involved stimulation of BMEC-1 cells with IL-1beta and TNFalpha for 18 hours before addition of PBMCs. The endothelium-adherent and non-adherent PBMC fractions were then placed in osteoclast formation cultures. Optimal conditions for osteoclast formation from PBMCs had been previously established. PBMCs were cultured for 21 days (14 days at pH 7.4 followed by 7 days at pH 7.0) on dentine slices in the presence of RANKL, M-CSF, PGE2, Dexamethasone, and 1,25-VitD3. Osteoclast formation was assessed by counting cells expressing VNR and F-actin rings and also by quantifying the area of dentine resorbed.
Results: Significantly more active osteoclasts (as demonstrated by cells displaying actin rings) were generated from PBMCs panned onto cytokine-stimulated endothelium than from PBMCs panned onto non-stimulated endothelium (p=0.016). This increase in active osteoclast number was accompanied by a significant increase (p=0.028) in total VNR positive cells and by an increase in bone resorption. In addition, high numbers of osteoclasts were generated from PBMCs that did not adhere to control endothelium and all osteoclast precursors were depleted from non-adherent PBMCs by cytokine-activated endothelium.
Conclusions: Our results show that OCL-Ps rarely adhere to control endothelium, but that all OCL-Ps were actively recruited to endothelium exposed to resorption stimulating cytokines. These findings suggest that endothelium is intimately involved in the regulation of osteoclast formation and bone resorption.
OSTEOCLAST FORMATION IS ENHANCED IN CAMURATI-ENGELMANN DISEASE
N. W. A. McGowan1*, H. MacPherson1, M. H. Helfrich1, K. Janssens2, W. van Hul2, S. H. Ralston1
1Bone Research Group, Dept. of Medicine and Therapeutics, University of Aberdeen, Aberdeen AB25 2ZD, Scotland, UK
2Department of Medical Genetics, University of Antwerp, Belgium
Introduction: Camurati-Engelmann disease (CED) is a rare autosomal dominant bone disorder characterised by increased bone turnover, bone pain and progressive osteosclerosis affecting the diaphysis of long bones. Recent work has shown that the disease is due to mutations affecting residues in the latency-associated peptide (LAP) region of the TGF-beta1 molecule. It has been hypothesised that these mutations might act functionally as "activating" mutations of TGF-beta1 by reducing the ability of the LAP to bind mature TGF-beta1 and increase TGF-beta1 bioavailability, but the functional effects of the mutations are unknown. Since TGF-beta1 is a potent stimulator of osteoclast formation in vitro, we studied OCL formation from PBMCs from CED patients and controls to determine if the mutation was associated with osteoclast activation.
Methods: PBMCs were isolated from the whole blood of 3 patients with CED with a R218C mutation and 3 controls (1 unaffected family control and 2 age matched volunteers). PBMCs were cultured under conditions previously established to be optimal for osteoclast formation in our laboratory. Briefly, PBMCs were incubated at 37°C for 21 days on dentine slices at optimised pH in the presence of RANKL (0, 10, 50 & 100ng/ml), MCSF (20ng/ml), PGE2, Dexamethasone, and 1,25-VitD3 (all 10-8M). Osteoclast activity was assessed by removing all cells and quantifying the area resorbed.
Results: No osteoclast formation was observed in the absence of RANKL in either CED or controls, but osteoclastic bone resorption was significantly enhanced in PBMCs isolated from patients with CED compared to controls at all RANKL concentrations (p<0.001, GLM ANOVA) with pairwise significant differences at 50ng/ml (p=0.028) and 100ng/ml RANKL (p=0.013).
Conclusions: These findings suggest that PBMCs from patients with CED have an increased ability to differentiate into osteoclasts capable of bone resorption when compared to controls, consistent with the hypothesis that the R218C mutation increases TGF-beta1 bioactivity. Further studies are in progress to determine if individuals with the R218C mutation exhibit increased TGF-beta1 bioactivity and to see if the observed increase in osteoclast activity can be blocked by TGF-beta1 antibodies.
RECOMBINANT G-PROTEIN OF VS-VIRUS IS TARGETED TO THE RUFFLED BORDER IN OSTEOCLASTS
M. T. K. Mulari1*, L. Patrikainen1, K. Metsikkö2, H. K. Väänänen1
1Inst. of Biomedicine, Dept. of Anatomy, Univ. of Turku, Finland
2Dept. of Anatomy, Univ. of Oulu, Finland
We have followed the synthesis and delivery of a recombinant vesicular stomatitis virus (recVSV) in osteoclasts. These viruses express a chimeric glycoprotein (G) with the cytoplasmic domains derived from the human CD-4 molecule (Schnell et al., 1998).
Bone marrow cells were harvested from rat long limb bones and cultured on bovine bone slices. After 48 hours of culture, cells were infected with recVSV-recombinant viruses for 60 minutes, followed by G-protein recognition by immunofluorescent and immunoelectron microscopic methods at several time points. During the first hour of infection, an increasing staining of the glycoprotein was observed at the peripheral cytoplasm but not in the vicinity of the ruffled border. This indicated the synthesis of the chimeric G-protein in the rough endoplasmic reticulum. After two hours, labelling was also concentrated perinuclearly corresponding to the pattern of the Golgi distribution. Finally, after three hours, the recVS virus particles were observed at the peripheral ruffled border leaving the central domain free from signal and at the functional secretory domain (FSD).
Our findings suggest that the cytoplasmic CD-4 of the recVSV G-protein targets the protein to the ruffled border. We propose that this pathway may reflect the main protein synthesis pathway in osteoclasts.
Schnell, M. J., Buonocore, L., Boritz, E., Ghosh, H. P., Chernish, R., and Rose, J. K. Requirement for a non-spesific glycoprotein cytoplasmic domain sequence to drive efficient budding of vesicular stomatitis virus. EMBO J 17:1289-1296; 1998.
CONNEXIN-MIMETIC PEPTIDE GAP 27 DECREASES OSTEOCLASTIC ACTIVITY
J. Ilvesaro*, P. Tavi, J. Tuukkanen
University of Oulu, Finland
Introduction: Bone remodeling is dependent on the balance between bone resorbing osteoclasts and bone forming osteoblasts. Gap junctions are specialized regions of cell membrane, which connect cells and provide aqueous intercellular channels for bi-directional movement of small molecules between the cytoplasms of neighboring cells, thus linking the cytosols of the two cells directly. We have shown previously that osteoclasts contain gap-junctional protein connexin-43, and a commonly used gap-junctional inhibitor, heptanol, can inhibit osteoclastic bone resorption. Since heptanol may also have some unspecific effect unrelated to gap-junctional inhibition we wanted to test the importance of gap-junctional communication to osteoclasts using a more specific inhibitor. A synthetic connexin-mimetic peptide, Gap 27, was used to evaluate the contribution of gap-junctional communication to osteoclastic bone resorption.
Methods and Results: Gap 27 was used in the well-characterized pit-formation assay, where rat osteoclasts are cultured on bovine bone slices. The cells were allowed to grow for 48 hours in 500 µM Gap 27 after which number of actin rings and numbers TRAP+ mononuclear and multinucleated cells were counted. Gap 27 caused a remarked decrease in the number of both mononuclear and multinucleated rat osteoclasts. The decrease seemed to be due to osteoclastic apoptosis, whereas the other cells of the culture model seemed unaffected. As seen earlier with heptanol, the ratio of mononuclear and multinucleated osteoclasts was altered using the inhibitors so that the proportion of mononuclear TRAP+ cells was greater than that of multinucleated osteoclasts. This indicates a defect in the maturation and fusion of osteoclast precursors into multinucleated osteoclasts. In addition, we could see significantly reduced number of osteoclasts with actin rings indicating that the activity of osteoclasts was decreased by the peptide.
Conclusions: On the basis of these results we conclude that gap-junctional communication is essential for proper remodeling for bone and to the action of bone-resorbing osteoclasts. The results with the Gap 27 peptide are very similar to those received from our earlier experiments with heptanol. Thus, we believe that functional gap junctions truly are needed in osteoclasts, since the specific blocking of gap junctions in osteoclast cultures drastically decreases osteoclastic activity.
BISPHOSPHONATE-INDUCED OSTEOCLAST APOPTOSIS INVOLVES LOSS OF MITOCHONDRIAL MEMBRANE POTENTIAL AND ACTIVATION OF CASPASE-3
H. L. Benford1*, M. E. Nuttall2, M. J. Rogers1
1University of Aberdeen, Aberdeen, UK
2SmithKline Beecham Pharmaceuticals, Pennsylvania, USA
We have recently demonstrated that both nitrogen-containing bisphosphonates and non-nitrogen-containing bisphosphonates cause apoptosis of rabbit osteoclasts, human osteoclastoma-derived osteoclasts and human osteoclast-like cells generated in cultures of bone marrow in vitro. Osteoclast apoptosis involves characteristic morphological changes and the activation of caspase-3-like proteases with proteolytic DEVDase activity which can be visualised in unfixed, dying osteoclasts and osteoclast-like cells using a cell-permeable, fluorogenic substrate (PhiPhiLux).
To characterise the effector caspase(s) activated during osteoclast apoptosis, we first examined the ability of lysates from purified, bisphosphonate-treated rabbit osteoclasts to cleave a fluorogenic substrate of caspase-3-like proteases (Ac-DEVD-AMC). Treatment of osteoclast cultures for 48 hours with 100microM zoledronic acid, risedronate, alendronate and pamidronate caused an 11-, 7-, 3- and 4-fold increase in DEVDase activity respectively. Zoledronic acid was still effective at lower concentrations (25microM or above). Treatment with 100microM ETI or CLO caused a 4-fold and 5-fold increase in DEVDase activity. Lysates from osteoclasts that had been treated with alendronate or clodronate were also examined for the ability to cleave fluorogenic substrates of caspase-6 (Ac-VEID-AMC) and caspase-7 (AMC-VDQVDGWK-(DNP)-NH2). No increase in the cleavage of caspase-6 or -7 substrates was detected compared with lysates from untreated osteoclasts.
Apoptosis of rabbit osteoclasts was dependent on caspase activation, since the morphological features of apoptosis (nuclear condensation and fragmentation) resulting from alendronate, clodronate or zoledronic acid treatment were suppressed by 50microM or 100microM zVAD-fmk (a broad-range caspase inhibitor) or by 10microM SB-281277 (a specific isatin sulfonamide inhibitor of caspase-3/-7). Furthermore, the p17 fragment of active caspase-3 could be detected in apoptotic human osteoclast-like cells by immunofluorescence staining. Osteoclasts with hyperpolarised mitochondrial membranes were also visualised using rhodamine123, a cationic fluorochrome that accumulates in mitochondria with polarised membrane potential. Following treatment with 100microM alendronate or clodronate, apoptotic osteoclasts failed to sequester rhodamine 123 in mitochondria, indicating loss of membrane potential.
Together, these observations suggest that caspase-3 is the major effector caspase activated in osteoclasts by bisphosphonate treatment. A likely route of caspase-3 activation is via the loss of mitochondrial membrane potential, release of cytochrome C from mitochondria and activation of initiator caspases such as procaspase-9.
A METABOLITE OF CLODRONATE, APPCCL2P, CAUSES OSTEOCLAST APOPTOSIS AND INHIBITS BONE RESORPTION IN VITRO
J. C. Frith1*, J. Monkkonen2, M. J. Rogers1
1Dept of Medicine and Therapeutics, University of Aberdeen, Polwarth Building, Foresterhill, Aberdeen, UK
2Dept of Pharmaceutics, University of Kuopio, Kuopio, Finland.
Although the molecular mechanism of action of nitrogen-containing bisphosphonates has recently been identified, the mechanism of action of clodronate (CLO) remains less clear. We have previously demonstrated that clodronate is metabolised to a non-hydrolysable ATP analogue (AppCCl2p) by osteoclasts both in vitro and in vivo. However, the effect of this metabolite on osteoclasts has not been fully determined. In this study, we used liposome-encapsulated AppCCl2p (lipo-AppCC2p) to investigate whether intracellular accumulation of AppCCl2p within osteoclasts in vitro could account for the inhibitory effects of CLO on bone resorption.
Bone marrow cells were isolated from 4-day old New Zealand White rabbits and seeded onto tissue culture plates or dentine discs. After 2 hours, the cells on dentine were treated with 50microM lipo-CLO, 50microM lipo-AppCCl2p or an equivalent amount of phospholipid. 24 hours after isolation, the osteoclasts in culture plates were purified by pronase-EDTA digestion and then treated with 20microM lipo-CLO, 20microM lipo-AppCCl2p or an equivalent amount of phospholipid. After 48 hours of treatment, lipo-CLO and lipo-AppCCl2p significantly reduced the number of TRAP-positive, multinucleated cells in culture plates and on dentine, compared to control cultures. The reduction in osteoclast number on culture plates was accompanied by a significant increase in the proportion of apoptotic osteoclasts in both lipo-CLO and lipo-AppCCl2p treated cultures. On dentine, treatment with lipo-CLO and lipo-AppCCl2p also caused a significant reduction in resorption. In all cases, the effect of lipo-AppCCl2p was indistinguishable from the effect of lipo-CLO.
These results demonstrate that the formation and accumulation of AppCCl2p within osteoclasts, following internalisation of CLO, accounts for the anti-resorptive effects of CLO. The exact molecular pathways by which AppCCl2p causes osteoclast apoptosis are currently under investigation.
THE EFFECT OF CLODRONATE METABOLISM ON THE INTRACELLULAR ATP CONCENTRATION IN RAW 264 MACROPHAGES
H. Mönkkönen1*, S. Niva1, M. J. Rogers2, J. Mönkkönen1
1Department of Pharmaceutics, University of Kuopio, Finland
2Department of Medicine and Therapeutics, University of Aberdeen, UK
Although bisphosphonates have long been considered to be metabolically inert, we have recently shown that clodronate and other bisphosphonates closely resembling pyrophosphate are metabolized into adenine-containing analogues of ATP in various cell types, including macrophages. The AppCp-type metabolites of bisphosphonates are formed by a back reaction catalyzed by class II aminoacyl-tRNA-synthetase enzymes. These enzymes can probably bind bisphosphonate instead of pyrophosphate in the ATP binding site. Our studies on the metabolism of clodronate indicated that a considerable proportion of clodronate internalized by macrophages in vitro is metabolized into AppCCl2p, which accumulates to high intracellular concentrations during the first 12 hours of exposure and inhibits cytokine release. These data strongly suggest that clodronate acts as a prodrug, affecting macrophages as a result of intracellular accumulation of AppCCl2p. In the present study, we examined the influence of clodronate metabolism on the intracellular ATP concentration in RAW 264 macrophages. Intracellular ATP concentration was measured with a luminometer using a luciferin-luciferase assay.
The influence of clodronate metabolism on the intracellular ATP concentration was studied by collecting samples for 1-24 hours after treatment of RAW 264 cells with 30 µM liposome-encapsulated or 1000 µM free clodronate. The intracellular ATP concentration in untreated RAW 264 cells was 3-5 mM. Free or liposomal clodronate reduced intracellular ATP levels significantly only after 24 hours of exposure.
The effects of AppCCl2p on cell function, such as cytokine secretion, do not appear to be a consequence of decreased ATP levels in the cells, because the intracellular ATP concentration did not drastically decrease in clodronate-treated cells during the first 12 hours of exposure, although the intracellular concentration of AppCCl2p reaches its peak and cytokine secretion is already strongly affected by this time. This suggests that clodronate affects macrophages due to the accumulation of the metabolite AppCCl2p, rather than due to a decrease in intracellular ATP. The intracellular accumulation of AppCCl2p appears eventually to cause a decrease in ATP levels after 24 hours, perhaps as a consequence (rather than a cause) of cell death.
EFFECT OF BISPHOSPHONATES ON PROTEIN KINASE C ACTIVITY IN VITRO
J. Mönkkönen1, E. Ekokoski2, H. Mönkkönen1*, R. Tuominen2
1Department of Pharmaceutics, University of Kuopio, Finland
2Department of Pharmacy, Division of Pharmacology and Toxicology, University of Helsinki, Finland
Bisphosphonates are synthetic pyrophosphate analogues that are used in the treatment of metabolic bone diseases to inhibit excessive bone resorption and hypercalcaemia. In addition to their anti-resorptive properties, some of them have been shown to have anti-inflammatory effects in animal models of rheumatoid arthritis as well as in arthritis in humans. Furthermore, clodronate, a non-nitrogen containing bisphosphonate, can inhibit the release of cytokines from RAW 264 macrophages, while alendronate, as nitrogen-containing bisphosphonate has pro-inflammatory properties. Studies have shown that clodronate is metabolized by various cell types, including osteoclasts and macrophages, to an AppCCl2p analogue of ATP, which may be responsible for the anti-inflammatory properties of clodronate. Protein kinase C (PKC) comprises a family of ubiquitous signal-transducing enzymes, which play a key role in the regulation of various cellular activities. A role for PKC in diverse inflammatory processes has been revealed. In the present study we have investigated whether bisphosphonates have an effect on PKC activity.
In vitro PKC assay experiments were carried out using partially purified rat brain extract as enzyme preparate. Clodronate (50 µM and 500 µM) and alendronate (50 µM and 500 µM) slightly decreased PKC activity, which was initially evoked by addition of diacylglycerol, phosphatidyl serine and Ca2+ to the reaction mixture. The clodronate metabolite, AppCCl2p (15 µM and 50 µM), markedly decreased the PKC activity. The bisphosphonates have high affinity for Ca2+. Therefore the PKC activity experiments were carried out also in conditions were no Ca2+ ions were added to the reaction mixture in order to exclude this chelation effect. In these experiments, both clodronate and alendronate slightly decreased the activity, whereas AppCCl2p effectively inhibited PKC activity. The effect of AppCCl2p on PKC activity was counteracted by increasing concentrations of ATP. Taken together, these results suggest that the metabolite of clodronate, AppCCl2p, possesses anti-inflammatory activity which may be based on PKC inhibition.
BLOCKADE OF P2X7 RECEPTORS INHIBITS OSTEOCLAST FUSION IN VITRO
A. Gartland*, J. A. Gallagher, W. B. Bowler
Human Bone Cell Research Group, Dept of Human Anatomy & Cell Biology, The University of Liverpool, Liverpool, UK
The P2X7 receptor is an ionic channel gated by extracellular ATP, which is capable of forming large pores in the plasma membrane. It is known to be expressed by cells of the lymphohematopoietic system, including multipotent hematopoietic stem cells and macrophages, however the exact physiological role of this receptor is only partially understood. Recent evidence suggests that the P2X7 receptor plays a crucial role in the last step, i.e. fusion, of the complex chain of events leading to generation of macrophage-derived giant cells.
Osteoclasts are multinucleated cells found within the skeleton that have the defining characteristic of the ability to resorb bone. Recently, RANK ligand has been identified as the factor responsible for the commitment of mononuclear cells to the osteoclast lineage. However, the exact mechanism via which these mononuclear cells fuse to form multinucleated osteoclasts is not known.
We have previously reported the expression of P2X7 receptors in human osteoclastoma tissue and RANK ligand-generated human osteoclasts. In this study, we demonstrate that the P2X7 receptors expressed by RANK ligand-generated human osteoclasts are capable of forming transmembrane pores as assessed by agonist-induced ethidium bromide uptake. Blockade of pore formation using the P2X7 antagonist oATP or a P2X7 blocking monoclonal antibody inhibited osteoclast formation by up to 70% in human peripheral blood mononuclear cell cultures stimulated with M-CSF and RANK ligand. These antagonists caused a concomitant reduction in resorption when cultures were grown on dentine substrates. These data provide compelling evidence for the involvement of the P2X7 receptor in the complex chain of events leading to osteoclast formation.
BONE RESORPTION IS DECREASED IN VITRO BY 17B ESTRADIOL AND RALOXIFENE FROM HUMAN PERIPHERAL BLOOD MONONUCLEAR CELLS
M. A. Burde*, Ph Couttet, D. Saintier, M. C. de Vernejoul, M. E. Cohen-Solal
INSERM U349, Paris, France
Hormone replacement therapy decreases in vivo bone resorption in postmenopausal women, but the direct in vitro effect of estradiol on osteoclast differentiation from human monocytes remains uncertain. We therefore evaluated the effect of estradiol and Raloxifene in the osteoclast differentiation model induced by RANK-L and MCS-F from peripheral blood mononuclear cells (PBMC) obtained from 8 healthy volunteers. Adherent PBMC were cultured in phenol red-free medium and 10% FCS and in the presence of RANK-L (30 ng/ml) and MCS-F (25 ng/ml) (Controls:C). Cells were treated with vehicle (C), 10-8M estradiol (E2) or 10-8M Raloxifene (Rlx). Cultures were performed in duplicate to evaluate the number of TRAP+ multinucleated cells and onto bovine bone slices for pit formation. At day 18, in all samples, RANK-L and MCS-F induce a significant increase in number of TRAP+ multinucleated cells (MNC) compared with cultures with complete medium only (9.41±2.53 vs 2.43±0.91, p<0.01). Expression of estrogen receptors (ER) a and b were assessed in RANK-L and MCS-F cultures at day 1 and 18 using nested RT-PCR. We found no expression of ERb at any time, whereas ERa expression was increased at day 18 in resorbing cells. In RANK-L and MCS-F treated-cultures, MNC number was not decreased in the presence of E2 (9.15±2.37), but was reduced with Rlx (5.90±1.75). In contrast, the pit area was significantly decreased in E2-treated cultures (2.22±1.58%) and in Rlx-treated cultures (1.65±1.21%) compared to controls (5.10±2.4%), (all p<0.05). Moreover, both E2 and Rlx did not change the expression of ERa, ERb and neither of RANK. We then assessed the effect of the 2 compounds on osteoclast precursor proliferation. PBMC were cultured with 10-9 and 10-8M of estradiol and Raloxifene. At day 3, there was no difference in proliferation rate between control cells and both estradiol and raloxifene-treated cells, at any dose, indicating that estradiol and Raloxifene has no effect on cell proliferation. In conclusion, our results suggest that 17b estradiol and Raloxifene can directly inhibit osteoclast bone resorption from human PBMC cultured with RANK-L and MCS-F. This inhibitory effect is not mediated through the regulation of RANK.
NITRIC OXIDE INHIBITS TARTRATE RESISTANT ACID PHOSPHATASE ACTIVITY
L. Mancini*, N. Moradi-Bidhendi, I. MacIntyre
The William Harvey Research Institute, St. Bartholomew's and the Royal London SMD, ECIM 6BQ, London, UK
Tartrate resistant acid phosphatase (TRAP) is an iron-containing glycoprotein that can be distinguished from other acid phosphatases due to its resistance to inhibition by tartrate.
It was initially discovered in leukocytes but it was later found that osteoclasts secrete TRAP into the circulation during bone resorption, it is therefore considered a marker of bone resorption in conditions involving increased bone turnover.
TRAP is mainly localised in resorption vacuoles suggesting that it is secreted by the osteoclasts in the site of active resorption although the mechanism by which TRAP promotes bone resorption is still unclear. However, it is clearly established that inhibition of acid phosphatase by chemical and immunological means can abolish osteoclastic bone resorption.
Further, acid phosphatase knock-out mice have mild osteoporosis while genetically engineered mice overexpressing acid phosphatase have an increased bone turnover. Thus acid phosphatase seems to be essential for bone resorption and osteoclast activity.
In this study we analysed the effects of nitric oxide, a free radical gas whose osteoclast inhibitory proprieties are now well established, on TRAP activity.
A human recombinant form of TRAP (kindly provided by T. Cox, Cambridge, UK) was incubated with two NO-donors (sodium nitroprusside, SNP, and DETA NONOate, NOC-18) in a time-course/dose-response experiment and the activity of the enzyme was then measured biochemically with a kit commercially available (Sigma, Poole, UK).
Both SNP and NOC-18 were able to reduce TRAP activity by 35% and 20% respectively after as little as 30 min; and a maximum of inhibition was observed after 18h when the enzyme activity is reduced by 50% by both NO-donors.
NO is known to bind to iron in various enzymes (this is the mechanism by which it activates guanylate cyclase, its recognised second messenger) therefore the ability of NO to reduce TRAP activity can be attributed to its binding to the iron moiety of the protein.
This finding is important since TRAP is directly associated with the ability of osteoclasts to resorb bone and could account for the inhibitory properties exerted by NO.
DIETARY LYCOPENE IS INHIBITORY TO REACTIVE OXYGEN SPECIES-MEDIATED RESORPTION BY OSTEOCLASTS
L. G. Rao1*, N. Krishnadev2, A. Cheng2, L. J-F. Liu1, T. M. Murray1, A. V. Rao2
1Calcium Research and Bone Biology Laboratory, Division of Endocrinology, St Michael's Hospital & Dept of Medicine, University of Toronto, Canada
2Dept of Nutritional Sciences2, Faculty of Medicine, University of Toronto, Canada
The reactive oxygen species (ROS) produced by osteoclasts have been shown to stimulate bone resorption. In this report, we tested whether dietary lycopene, a potent antioxidant carotenoid naturally present in tomatoes, can inhibit the ROS-mediated effects on osteoclasts. We tested its effects on the differentiation of mononucleated cells to tartrate-resistant acid phosphatase positive (TRAP+) multinucleated osteoclasts and on the activity of these cells to resorb bone. Bone marrow cells were isolated from rat femur, plated onto 16-well calcium phosphate coated steologic discs and cultured in alpha-MEM medium supplemented with beta-glycerophosphate and ascorbic acid. The cells were treated with vehicle or PTH and varying doses of water-dispersible lycopene. At day 8, the cells were removed from the discs with detergent and the resorption pits quantified in a Microst Reader. In a parallel experiment, the cells were cultured in 12-well plastic dishes and at day 8, the cells were stained for TRAP. Results showed that PTH stimulated resorption pits from 2% to 4.5%. Both the basal (1-way ANOVA, p<0.05) and PTH-stimulated resorption (p<0.005) were significantly inhibited in a dose-dependent manner by 10-5M to10-7M lycopene. Microscopic examination of the stained cells revealed a nearly complete inhibition of TRAP+ multinucleated cell formation in cultures treated with 10-5M lycopene compare to vehicle or PTH-treated cells. Osteoclasts reduced the nitroblue tetrazolium (NBT) to purple colored formazan, indicating the presence of ROS in these cells. Lycopene at 10-5M concentration decreased the formazan-staining cells, indicating that lycopene inhibited the formation of ROS-secreting osteoclasts. In conclusion, we have shown that lycopene inhibited basal and PTH-stimulated osteoclast resorption, as well as the formation of TRAP+ multinucleaneated osteoclasts. These effects were exerted by inhibition of the activity of ROS produced by osteoclasts. These novel findings may be important in the pathogenesis, treatment and prevention of osteoporosis.
FUNCTIONAL ROLE OF IONOTROPIC NON-NMDA RECEPTORS IN OSTEOCLAST RESORPTIVE ACTIVITY
M. Mukhida*, R. Gilbert, Y-F. Chen, G. I. Anderson
Dalhousie University, Halifax, Nova Scotia, Canada
Bone mass maintenance involves a balance between bone formation and resorption, mediated by osteoblasts and osteoclasts, respectively. When this balance favours bone resorption, pathology such as osteoporosis may occur. Recent investigations have focused on the role of the neurotransmitter, glutamate (Glu), as a potential cellular mediator of bone resorption. Nerve fibers containing Glu have been identified adjacent to bone cells expressing Glu receptors (GluR). Glu produces its effects via interactions with GluRs of both the ionotropic (iGluR) and metabotropic types. iGluRs are multi-subunit, ligand-gated ion channels and the primary mediators of excitatory neurotransmission in the brain. iGluRs are divided into three groups: NMDA, AMPA and kainate (Non-NMDA).
Previously, we identified the expression of various NMDAR and non-NMDAR subunits on osteoblasts and osteoclasts in vivo. A functional role for NMDARs in regulation of osteoclast-mediated bone resorption was demonstrated when NMDAR antagonists were shown to inhibit bone resorption in vitro. The role of non-NMDARs in bone is unclear thus we investigated their role in osteoclast resorption. Osteoclasts were isolated from the longbones of neonatal NZW rabbits and seeded onto bovine cortical bone slices. Cells were grown in osteoclast-supporting media (10-8M Vit D3 +VitC). To investigate the role of various non-NMDARs in osteoclast resorption, selective NMDAR (MK801) and non-NMDAR (NBQX) antagonists were employed (concentrations ranging from 0, 0.01, 0.1, 1, 25, 50 and 100 uM). After 4 days, osteoclasts were removed and the bone slices stained using antitype 1 collagen antibody labelled with DAB. This allowed quantitation of pit resorption area per bone slice using a Leco 2001 image analysis system. MK801 at 100 uM decreased pit area by 85.3% (p<0.05) relative to controls without drug. NBQX, which acts as a selective competitive AMPA antagonist at concentrations lower than 1 uM and an AMPA and kainate receptor blocker at higher concentrations, decreased pit area by 48.3% (p<0.05) at concentrations of 100 uM, relative to controls. Lower NBQX concentrations did not significantly decrease bone resorption. These data suggest that there is a functional role for both kainate and NMDA receptors in osteoclast resorption.
BONE-RESORBING ACTIVITIES OF HIGHLY ENRICHED OSTEOCLASTS IN RESPONSE TO MECHANICAL STRAIN
K. Kurata1*, T. Uemura2,3, A. Nemoto2,3, T. Tateishi2,3,4, T. Murakami1, H. Higaki5, H. Miura1, Y. Iwamoto1
1Kyushu University, Fukuoka, Japan
2NAIR, Tsukuba, Japan
3CREST, JST (Japan Science and Technology)
4Tokyo University, Tokyo, Japan
5Kyushu Sangyo University, Fukuoka, Japan
Adaptive remodeling has been recognized to be responsible for the activities of osteoblasts and osteoclasts, which would sense their mechanical environments and regulate deposition or resorption of bone matrix. The effects of mechanical stimuli on isolated osteoclasts, however, have been scarcely examined because it has proven quite difficult to prepare a number of pure osteoclasts and to cultivate them on mineralized substratum during mechanical stimulation. In current study, we examined whether the mechanical stretching had any influences on the bone-resorbing activities of isolated mature osteoclasts.
We newly developed an experimental system for applying mechanical stretching to the ivory slice/plastic plate component, on which cells could be cultured. The loading apparatus was designed to stretch the ivory surface by applying three-point bending to the plate. The loading frequency, strain rate, and generated strain over an ivory surface could be controlled by a personal computer. Mature and highly enriched osteoclasts were cultured for 2, 12, and 24 h on the ivory/plate component, while being subjected to intermittent tensile strain of 1730 micro-strain at frequency of 1 Hz. Alterations of the activity of the osteoclastic resorption were investigated by comparing the mRNA expressions of two kinds of osteoclast marker enzymes, tartrate resistant acid phosphatase (TRAP) and cathepsin K. Additionally, enhancement of osteoclastic activity was assessed by quantitative measurements of resorption pits on the ivory substrata.
A time-dependent increase was observed in the mRNA expressions, whose increasing rates were accelerated in the stretched groups. TRAP and cathepsin K mRNA levels of the 24 h stretched group respectively induced a 1.71- and 1.36-fold upregulation compared with the control group. Stretching for 24 h significantly increased pit formation, resulting in 1.24- and 2.38-fold increases in the total pit area and the number of pits per ivory, respectively. A stretch-activated cation (SA-cat) channel blocker significantly inhibited the increases of the mRNA level and pit formation after 24 h of stretching.
This study suggested the possibility that the mature osteoclasts responded to mechanical stretching through a mechanism involving SA-cat channel in the absence of mesenchymal cells and, as a result, upregulated their bone-resorbing activity.
OSTEOCLAST SPREADING IS CORRELATED WITH SPONTANEOUS AND OSCILLATORY ACTIVATION OF A CALCIUM- DEPENDENT POTASSIUM CURRENT (IKCA)
L. Espinosa1*, L. Paret1, C. Ojeda2, C. Itzstein1, P. D. Delmas1, C. Chenu1
1Unité INSERM 403, Lyon, France
2Unité INSERM 121, Lyon, France
Cell movement is often linked to [Ca]i oscillations. We studied osteoclast movement during bone resorption together with their electrical activity. Rabbit osteoclasts were isolated from long bones and were recorded by time-lapse videomicroscopy. Electrical recordings were performed simultaneously in the same cell using patch-clamp techniques in whole-cell (perforated patch) and single channel configurations. Osteoclast membrane spreading occurred by waves of lamellipodia and image analysis of the spreading rate showed a discontinuous progression along the axis of movement. Concurrently, the whole-cell membrane current showed oscillatory activation, consisting in peaks (5-20 seconds duration) of variable intensity and frequency that were coincident with peaks of the spreading rate. 95% of spreading cells demonstrated these spontaneous transient variations of the current, while 80% of quiescent cells did not exhibit any variation of the current. Using cell attached configuration, we confirmed that osteoclast spreading was linked to a transient activity of single channels, with cycles of frequent openings ("bursts") followed by periods of rest. In patch-clamp studies, we identified the current underlying such oscillations as a calcium-dependent potassium current (IKCa), and single channels recordings showed a unitary conductance of about 30 pS (145 mM K+). The small conductance of this channel and its lack of sensitivity to low concentrations (<5mM) of tetraethylammonium (TEA) indicate that the observed IKCa is not a classical big conductance calcium-dependent potassium channel (BK). Preliminary studies using RT-PCR analysis revealed the presence in rabbit osteoclasts of the functional BK channel subunit "slo", suggesting the possible involvement of an intermediate channel composed by "slo" and a regulatory subunit such as "slack". The small conductance potassium channels (SK) could also be involved in this current.
Our results demonstrate for the first time the presence of a spontaneous "rhythmic" activity of a calcium-dependent potassium current linked to the rate of osteoclast membrane spreading. Further studies of interactions between integrin membrane receptors, internal calcium handling and cytoskeleton will improve our understanding of osteoclast morphological changes required for bone resorption.
