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ABSTRACTS P-111 to P-146 POSTER PRESENTATIONS
Posters will be on display throughout the symposium, but will be attended by their presenting authors as follows: Odd numbers on Friday 11:00 - 12:00, Sunday 11:00 - 12:00
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| Cell Biology: Osteoclasts and Related Cytokines EFFECTS OF REPEATED INGESTION OF CALCIUM-FORTIFIED SPRING WATER ON SERUM C-TERMINAL CROSS-LINKING TELOPEPTIDE OF TYPE I COLLAGEN (CTX) J. A. Guillemant1, C. Accarie2, V. de la Guéronničre3, S. E. Guillemant1,2* 1Faculté de Médecine Pitié-Salpźtričre, Paris, France 2EPHE, Nutrition Hydrominérale, Paris, France 3Pōle Expertise Eau, Bourg-la-Reine, France In a previous study (Nutrition Research, 2002) we showed that repeated ingestion of calcium-rich water could significantly increase the serum concentration of ionized calcium and suppress serum CTX demonstrating that calcium-rich mineral water can effectively suppress bone resorption. Nevertheless, the availability of natural calcium- rich mineral water is limited and the possibility to fortify natural spring water with calcium salt is to be considered. Fifteen young adult males (21-26 y) ingested at three times (08.00, 11.00 and 14.00) 660 ml of either calcium-fortified spring water (300 mg/L of elemental calcium) or calcium-poor (<10 mg/L) mineral water. Blood was collected before 08.00 and at 11.00, 14.00 and 17.00 for measurement of serum ionized calcium and CTX. After every intake of calcium-fortified water, serum ionized calcium increased (from 1.25 to 1.31 mmol/L) while CTX decreased (by 60%) gradually. Since ingestion of calcium-poor mineral water induced a modest but significant decrease in CTX we compared the two sets of trials with repeated- measures two-factor analysis of variance with interaction. Ingestion of calcium- fortified water resulted in a significant decrease in serum CTX (time, P<0.0001; treatment, P<0.0001; time-by-treatment, P<0.0001) demonstrating that usually drinking calcium-fortified water could efficiently suppress bone resorption throughout the day. EFFECT OF RANKL ON THE FORMATION OF OSTEOCLASTS GENERATED FROM PERIPHERAL BLOOD MONONUCLEAR CELLS: CORRELATION BETWEEN AREA OF RESORPTION AND RELEASE OF BETA C-TERMINAL TELOPEPTIDES B. Y. Y. Chan1*, K. A. Buckley2, J. Dutton1, J. A. Gallagher2, W. D. Fraser1 1Department of Clinical Chemistry, Royal Liverpool University Hospital, The University of Liverpool, Liverpool, L69 3GA, UK 2Human Bone Cell Research Group, Department of Human Anatomy and Cell Biology, The University of Liverpool, Liverpool, L69 3GE, UK BACKGROUND: The receptor activator for nuclear factor-kappaB ligand (RANKL) is a critical factor for osteoclast (OC) differentiation, survival and maturation. The development of techniques that generate OCs in vitro from peripheral blood mononuclear cells (PBMCs) have provided methods of producing unlimited supplies of OCs which were previously difficult to obtain. The current method to quantify the resorptive activity of OCs by point counting using reflected light microscopy is time consuming and can lead to inaccuracies. The bone specific resorption marker beta c-terminal telopeptide (beta-CTx) from degradation of type I collagen may provide an alternative measurement for OC resorption. This study evaluated the effect of RANKL in stimulating OC activity, and correlated the release of beta-CTx from dentine wafers with the area of resorption produced by OCs. METHOD: Venous blood from patients with Paget's disease of bone and healthy volunteers was collected and PBMCs were isolated. PBMCs from Paget's subjects were cultured in medium containing either 10% FCS, or 1% or 10% human serum derived from the same individual, 30 nano-grams per millilitre RANKL, and 25 nano- grams per millilitre macrophage-colony-stimulating factor (M-CSF). PBMCs from normal subjects were cultured in the presence of 30, or 60 nano-grams per millilitre RANKL, 25 nano-grams per millilitre M-CSF, and 10% FCS. Cell culture medium was harvested at specific time points (7, 14, and 21 days) during a 3-week incubation for measurement of beta-CTx by electrochemiluminescence immunoassay (ROCHE, Lewes UK). End-point area of resorption was evaluated by point counting using reflected light microscopy. RESULTS: Incubation of Pagetic PBMCs with 10% Pagetic serum or 10% FCS resulted in a significantly higher release of beta-CTx and a greater area of resorption than from cells cultured in 1% Pagetic serum. Beta-CTx was significantly correlated with the area of resorption (r=0.92, n=62). Beta-CTx release from cultures of 'healthy' PBMCs was also significantly correlated with the increasing area of resorption produced from OC formed in the presence of increasing concentrations of RANKL (r=0.97, n=13). CONCLUSION: Use of beta-CTx as a marker for bone resorption activity could replace the laborious method of point counting resorption lacunae produced by recombinant RANKL-generated OCs. SIMULATED RATES OF RESORPTION DURING CANCELLOUS BONE REMODELING USING MICHAELIS-MENTEN (M-M) EQUATIONS AND THE INTERACTIVE EFFECTS OF RANKL, RANK AND OPG ON OSTEOCLAST ACTIVITY M. J. Martin*, J. C. Buckland-Wright GKT School of Biomedical Sciences, King's College, London, UK The development of pharmaceutical treatments for bone disease can be enhanced by predicting the effects of disturbance on the process of bone resorption during cancellous bone remodeling. We present a mathematical model to simulate the rates of cellular activity during the resorption phase of remodeling in healthy bone, controlled by interactions between matrix, marrow and vascular tissue. Rates of cellular activity during resorption within a representative volume of bone are calculated on a daily time step by the M-M kinetic equations that describe enzyme and cellular activity. Osteoclastic activity, AOcl, is simulated by the interaction between RANKL, RANK and the competitive inhibition of OPG, evident as the observed dependency of osteoclast survival and function on the ratio of RANKL to OPG. The ratio of RANKL to OPG is representd as the effective concentration of RANKL, [RANKLEff], and the required presence of stromal cell production of M- CSF is represented as factor FM-CSF, which is equal to 1 in normal, healthy bone. AOcl = [(VOclmax * [RANKLEff]) / ([RANKLEff] + KOclM)] * FM-CSF where VOclmax is the maximum rate of osteoclastic activity, and KOclMis the M-M constant. The release of factors such as TGFbeta1 from the matrix are tracked, and the decrease in osteoclastic resorption with time is simulated by the negative feedback effect of released TGFbeta1 on OPG production by marrow stromal cells. Apoptosis and cessation of osteoclastic resorption is assumed to occur at a threshold level of TGFbeta1 released from the matrix. The rate of collagen fibril removal during the second phase of resorption is simulated by the activity of mono-nucleated lining cells, using the M-M kinetic equation. The model is parameterised using published data (for example, Eriksen et al., 1984; Parfitt et al., 1996; Fuller et al., 1998). This is the first mathematical model that uses the M-M equations adapted for cellular activity to simulate the rate of resorption during the remodeling of cancellous bone. TARTRATE-RESISTANT ACID PHOSPHATASE FUNCTIONS AS A PROTEIN TYROSINE PHOSPHATASE IN MOUSE LUNG TISSUE P. Muhonen*, K. Kaarlonen, H. Ylipahkala, S. L. Alatalo, H. K. Väänänen, J. M. Halleen Institute of Biomedicine, Department of Anatomy, University of Turku, Finland Tartrate-resistant acid phosphatase (TRACP) is an enzyme with unknown biological function. It is expressed primarily in bone-resorbing osteoclasts and activated macrophages. TRACP knock-out mice develop mild osteopetrosis and show reduced clearance of the pathogen S. Aureus, suggesting an important role for the enzyme in bone resorption and immune defense system. We have used TRACP over- expressing (TRACP+) mice to identify potential biological substrates for the phosphatase activity of the enzyme. We performed Western-analysis to lung homogenates from wild-type (WT) and TRACP+ mice using phosphotyrosine and phosphoserine antibodies and quantitated band intensities with image analysis. The specific activity of TRACP was significantly higher in lung homogenates from TRACP+ mice compared with WT mice. Lung homogenates from TRACP+ mice contained significantly lower amount of phosphotyrosine, but the amount of phosphoserine was not different from WT mice. Two bands sized 120 kD and 180 kD were identified that contained 90% less phosphotyrosine in lung homogenates from TRACP+ mice. The band intensities showed a significant negative correlation with the specific activity of TRACP in lung homogenates from TRACP+ mice. These results suggest that TRACP functions as a protein tyrosine phosphatase, but not as a serine phosphatase, and that it may have at least two different natural substrates in lung tissue. CHARACTERIZATION OF CIRCULATING HUMAN OSTEOCLAST PRECURSORS T. A. Hentunen*, H. Michael, H. K. Väänänen Department of Anatomy, Institute of Biomedicine, University of Turku, Turku, Finland Osteoclasts are multinucleated bone-resorbing cells. They are differentiated from hematopoietic precursors, including cells in the peripheral blood. The phenotype of osteoclast precursor is still unknown. First, we developed a method to induce high numbers of resorbing osteoclasts from the total peripheral blood mononuclear cell (PBMC) fraction using a cocktail of growth factors: RANKL, M-CSF, TNF-alfa and dexamethasone. PBMC were enriched using Ficoll isolation method. From one million plated PBMC/bone slice formed between 100-500 tartrate-resistant acid phosphatase (TRACP) -positive multinucleated cells (MNC) with 5 nuclei/cell on an average in a 14-day culture. Osteoclast formation capacity was thus approximately 0.15 %. It appeared that lymphocytes present in the culture interfered osteoclast formation causing variation and inhibition in osteoclast maturation. Next, we purified monocytes up to purity of 99 % using immunomagnetic cell separation system (MACS microbeads, Miltenyi, Germany). CD14+ cells as plated at the cell density of 0.1 million cells/bone slice generated approximately 550 TRACP+ MNC with 5 nuclei/cell. Hence, the osteoclast formation capacity was between 2.75 %. Purified CD14+ cells had 15-20 fold higher osteoclast formation capacity compared to whole PBMC fraction suggesting that osteoclast precursors reside in monocyte population. CD11b+ cells formed approximately 180 TRACP+ MNC. This was 30 % of the number obtained with CD14+ cells. CD61+ cells were also able to differentiate into resorbing TRACP-positive MNC. Their osteoclast formation capacity was slightly lower than the one of CD11b+ cells. CD169+ cells, however, did not form any osteoclastic cells. This was an interesting observation suggesting that in the circulation there may be different monocyte subpopulations, which are already committed to osteoclast, macrophage and dendritic cell lineages. TRACP+ MNC generated in the culture expressed vacuolar proton ATPase and formed typical actin rings on bone slices. Their resorption activity was high, the resorption area being 50- 80 % of the bone slice area. In addition, calcitonin was found to inhibit the resorption activity of osteoclast-like cells formed in the culture. To summarize, circulating human osteoclast precursors are positive for CD14, CD11b and CD61 and negative for CD15 and CD169 and they can be induced to differentiate into osteoclasts with high resorption activity. LOCALIZATION OF TRACP IN RESORBING OSTECLASTS J. Vääräniemi*, J. Halleen, H. K. Väänänen Department of Anatomy, Institute of Biomedicine, University of Turku, Turku, Finland Osteoclasts are multinucleated monocyte-macrophage derivatives responsible for bone resorption. They dissolve bone mineral by secretion of protons and degrade collagen matrix by secretion of proteolytic enzymes that are secreted through the ruffled border (RB) membrane. RB faces the bone matrix and has late endosomal/lysosomal characteristics but it is compositionally different from lysosomes. Resorption products are endocytosed inside the cell through RB and secreted to the extracellular space via a functional secretory domain in the basolateral membrane. This route is defined as transcytotic pathway. Osteoclasts also produce high amounts of reactive oxygen species (ROS) during resorption without known function. In this study we used confocal microscopy to characterize the precence and localization of tartarate resistant acid phosphatase (TRACP) in resorbing and non- resorbing rat osteoclasts cultured on bovine bone. TRACP is a specific marker for osteoclasts in bone and serum TRACP is a potential marker of bone resorption. Biological function and subcellular localization of TRACP remains unclear although TRACP may have functions as a protein tyrosine phosphatase and in iron metabolism. We studied the colocalization of TRACP vesicles and secreted or endocytoted vesicles found in different intravesicular pathways. Our results show that TRACP is present in both resorbing and non-resobing osteoclasts and it is localized perinuclearly. Many TRACP-containing vesicles are big compared to other vesicles. In resorbing osteoclasts some but not all TRACP containing vesicles are acidic as indicated by DAMP that is concentrated in acidic organelles. TRACP colocalizes with bone collagen degrading enzyme cathepsin K in the cytoplasm and near basolateral membrane of resorbing osteoclast. TRACP also colocalizes with fluorescently labelled bone in the cytoplasm but not in the bone surface level. Some colocalization can be seen with transferrin, Rab3a and Rab9 proteins. TRACP does not colocalize or colocalizes very little with late endosomal or lysosomal markers like Rab7 protein or LDL-containing lysosomes. FOCUS ON BONE REMODELING IN CELIAC DISEASE D. Fortunati1*, A. Taranta2, M. Longo1, N. Rucci2, S. Migliaccio2,3, M. L. Bianchi4, M. T. Bardella5, S. Saraifoger4, A. Teti2 1Dept of Histology and Medical Embryology, University of Rome 'La Sapienza', Rome, Italy 2Dept of Experimental Medicine, University of L'Aquila, L'Aquila, Italy 3Dept of Medical Physiopathology, University of Rome 'La Sapienza', Rome, Italy 4Bone Metabolic Unit, Istituto Auxologico Italiano-IRCCS, Milan, Italy 5Cattedra di Gastroenterologia-IRCCS Ospedale Maggiore, Milan, Italy Gluten intolerance causes celiac disease. This inflammatory disorder of the small bowel is often associated with low bone mass density (BMD). To assess whether any factors not related to intestinal malabsorption were involved in altered bone turnover, human peripheral blood monocytes were challenged for three weeks with sera from 21 healthy donors, 17 just-diagnosed, yet untreated celiac patients, and 25 celiac patients on gluten-free diet for at least two years, and assessed for osteoclast formation ability. In cultures containing sera from untreated celiac patients, an early increase in the numbers of TRAP-positive multinucleated cells was observed, relative to treatment with sera from healthy donors, which persists throughout three weeks of culture. A lesser increase was noticed with sera from celiac patients on diet. We therefore investigated whether different sera contained abnormal levels of specific molecules, well-known to affect osteoclastogenesis, or, alternatively, whether they caused altered secretion of these factors by osteoblasts. Unchanged PTH, but increased IL-6 and decreased IL-12, were evidenced in sera from celiac patients. Nevertheless, no significant modulations of IL-6 and IL-12 could be evidenced, by means of RT-PCR analysis, in osteoblasts. Decreased IL-18 and osteoprotegerin (OPG) were observed, in these cells, upon treatment with sera from untreated celiac patient, as well as increased alkaline phosphatase activity, proliferation, and matrix mineralization. Increased proliferation was also obtained with sera from patients on diet. This set of data demonstrates that the osteopenia often found in celiac patients may likely be due to additional factors, beside intestinal malabsorption, causing serum imbalance of molecules affecting bone-turnover. COMPARISON OF OSTEOPROTEGERIN (OPG) AND RECEPTOR ACTIVATOR OF NF-KB LIGAND (RANKL) BIOLOGICAL ACTIVITIES IN OSTEOCLAST CULTURES Y. Wittrant1, S. Theoleyre1, S. Couillaud1, C. Dunstan2, D. Heymann1, F. Rédini1* 1Laboratoire de Physiopathologie de la résorption osseuse et thérapie des tumeurs osseuses primitives, Faculté de Médecine, Nantes, France 2Amgen Inc, Thousand Oaks, USA Introduction : OPG and RANKL are newly discovered molecules that are greatly involved in bone remodelling mechanisms. RANKL, expressed as a membrane- associated protein by osteoblast/stromal cells is essential for osteoclast (OC) differentiation/activation, and functions through its receptor RANK at the surface of osteoclasts. OPG is an osteoblast-secreted decoy receptor that inhibits osteoclast differentiation and activation. Objectives : This study investigated the direct effects of human OPG and RANKL in cultures of purified rabbit osteoclasts. Several parameters were analysed : OC markers expression (TRAP, Cathepsin K), the activities and expression of proteases involved in matrix bone resorption [cathepsin K, MatrixMetalloProteinase (MMP)-9, - 2] and their Tissue Inhibitors of MMP (TIMP)-1 and -2 expressions. Methods : Purified OC were obtained after treatment of total rabbit bone cells with pronase/EDTA. OC were then incubated 24 hours with/without 10, 50 and 100 nanog/ml of OPG or RANKL. The expression of OC parameters was analysed by semi-quantitative RT-PCR. The activities of MMP were analyzed by gelatin zymography. The cathepsin K assay was performed in the presence of the synthetic fluorogenic dipeptide Z-Leu-Arg-AMC as substrate. Results : The expression of two OC markers (TRAP and cathepsin K) were inhibited by 100 nanog/ml OPG, and stimulated by RANKL. Surprisingly, the cathepsin K activity measured in the supernatants of the same cultures was inhibited by both OPG and RANKL. MMP-2 expression and activity could not be quantified in the majority of the assays. MMP-9 expression and activity were both enhanced in the presence of OPG and RANKL. However, the expression of TIMP-1, the natural inhibitor of MMP-9, was enhanced by OPG and decreased by RANKL. Conclusion : Overall results show that RANKL exerts pro-resorptive effects on purified OC cultures with an increase of TRAP, cathepsin K, MMP expressions and a decrease of TIMP-1,2 expressions. In parallel, we also demonstrated that OPG differentially regulates protease expression and activities in the same culture condition, producing a marked inhibitory effect on cathepsin K expression (OC marker), and a stimulation of MMP-9 activity and expression. However, this effect could be balanced by the stimulation of TIMP-1 expression. ACIDOSIS STRONGLY UPREGULATES MRNA FOR CATHEPSIN K, TRAP AND TRAF-6 IN BONE A. Brandao-Burch1*, S. Meghji2, T. R. Arnett1 1Department of Anatomy and Developmental Biology, University College London, London, UK 2Eastman Dental Institute, University College London, London, UK Slight extracellular acidification is the obligatory initial activation step for resorption pit formation by cultured osteoclasts (OC). Resorption in vivo and in bone organ cultures is also highly acidosis-dependent. The great sensitivity of OC to extracellular H+ suggests that this effect is unlikely to be due simply to reduction of the gradient against which OC must pump H+ to dissolve bone mineral. Furthermore, resorption pit formation involves removal of the organic matrix of bone. To determine whether acidosis 'switches on' genes linked to resorption, we compared mRNA expression in mouse calvarial bones maintained for 3d in normal (pH 7.20±0.02) or slightly acidified (pH 7.05±0.02) media. Positive and negative controls were PGE2 and OPG, respectively. Resorption was assessed as Ca 2+release. RT-PCR was performed on DNase-treated total RNA from bones in separate reactions, using GAPDH as control. PCR products separated by gel electrophoresis were stained with ethidium bromide and quantified by densitometric image analysis. Values in table represent means ±SEM; n=3-5. Thus, mRNAs for cathepsin K (a key enzyme for organic matrix degradation by OC and TRAP5 (required for optimal resorption) were elevated strikingly in mild acidosis. Cathepsin K mRNA was undetectable in non-resorbing bones. Surprisingly, mRNA for c-src, thought to be required for OC ruffled border formation was inhibited by acidosis. In parallel experiments, mRNA for TRAF-6, an intracellular factor associated with OC activation, was elevated ~5.8-fold by similar acidosis. Cell function, including that of osteoblasts, is normally impaired by low ambient pH. The unusual stimulatory effect of H+ on OC may represent a primitive 'failsafe' mechanism that evolved to correct systemic acidosis by ensuring release of alkaline bone mineral when the kidneys and lungs are unable to remove sufficient H+ equivalent.
OSTEOPROTEGERIN BIOLOGICAL ACTIVITY AND SIGNALLING IN OSTEOCLAST-LIKE CELLS IS DEPENDENT ON THE PRESENCE OF RECEPTOR ACTIVATOR OF NF-KB LIGAND S. Theoleyre1, Y. Wittrant1, S. Couillaud1, M. Berreur1, P. Vusio2, C. Dunstan3, F. Blanchard1, F. Rédini1, D. Heymann1* 1EE 99-01, Faculté de Médecine, Nantes, France 2INSERM U463, Nantes, France 3Amgen Inc, Thousand Oaks, USA Osteoprotegerin (OPG)/Receptor Activator of Nuclear Factor kB Ligand (RANKL)/RANK, three molecules recently discovered, appear to be the key molecular triad of bone remodelling. Indeed, RANKL, expressed as a membrane- associated protein by osteoblast/stromal cells is essential for osteoclast (OC) differentiation and acts via its receptor RANK located at the surface of osteoclasts. In this system, OPG is an osteoblast-secreted decoy receptor that prevents the RANKL/RANK interactions, thus inhibiting osteoclast differentiation/activation. More recently, we demonstrated that OPG stimulates MMP9 activity of rabbit OC by the ras/MAPK kinase pathway. The purpose of the present study was (i) to better characterize the molecular interactions between OPG/RANKL/RANK, (ii) to investigate the potential direct biological effect of OPG on osteoclast-like and the cell signalling pathways associated. OPG/RANKL/RANK interactions were studied using a BIAcore 2000 system. To examine the signal transduction pathways induced by OPG in osteoclast-like cells, we used RAW264.7 mouse monocytes differentiated in the presence of RANKL (50 nanog/ml). Following the differentiation step, cells were cultured in serum-free medium for 24h and were incubated with RANKL (50 nanog/ml) for 2h in the same culture conditions. The cells were washed and incubated in the presence or absence of OPG (50 nanog/ml), RANKL (50 nanog/ml) and anti-RANKL antibody (4 microg/ml). Cell signalling was then analysed by western blots. BIAcore analysis revealed that RANK and OPG bound to RANKL charged chips with respectively Kd = 6 nanoM and 0.12 nanoM. When OPG was passed over the RANKL-RANK association (steady-state), a response of approximately 1000 RU was observed, demonstrating the formation of an OPG/RANKL/RANK complex. The pre- incubation of RANKL with OPG prevented the subsequent interaction between RANKL and RANK. Concerning cell signalling studies, RANKL induced the phosphorylation of p38 and ERK1, ERK2, in differentiated RAW264.7 cells. Interestingly, OPG activated p38, ERK1, ERK2 pathways whereas the co-incubation of OPG and anti-RANKL antibody abolished the p38 signal transduction. These original data evidence the capability of OPG, RANKL, RANK, to form a molecular complex. Furthermore, this work describes for the first time an OPG- induced signal-transduction pathway in osteoclast-like cells which is dependent on the presence of RANKL via a complex OPG/RANKL/RANK. CAPACITIVE CALCIUM ENTRY AND CALCIUM STORES REFILLING REGULATE OSTEOCLASTIC SURVIVAL AND BONE RESORPTION PROCESS R. Mentaverri*, S. Kamel, M. Brazier UMRO, Faculty of Pharmacy, University of Picardie Jules Verne, Amiens, France Bone resorption is closely dependent of osteoclastic survival and osteoclast apoptotic cell death could represent a key step at the end of this process. The present study was undertaken to determine whether intracellular calcium store replenishment and capacitive calcium entry (CCE) are involved in osteoclastic survival and consequently in bone resorption. We demonstrate that i) thapsigargin (1µM), a Sarco- Endoplasmic Reticulum Calcium ATPase pump (SERCA) blocker, decreases osteoclastic survival from 90±1.5% to 50±1.8% (p<0.001) and bone resorption process from 100±10% to 62±8% (p<0.001). ii) 2APB (100µM) and SKF-96365 (30µM), two store-operated channels (SOCs) blockers, dramatically decrease osteoclastic survival and bone resorption. After 48 h of culture with 2APB or SKF- 96365, a 75% decrease of osteoclastic survival is observed with both compounds (p<0.001). iii) when cells are cultured in calcium-free EGTA containing medium, rendering CCE impossible, a 50% decrease of osteoclastic survival is observed. iiii) thapsigargin and calcium free medium inhibit synergically osteoclastic survival which falls dramatically to a value close of 0% (p<0.001). While, osteoclastic survival increases significantly from 50 ± 1.8% to 66.2 ± 3.7% when thapsigargin-treated cells are cultured in the presence of 20mM of calcium, suggesting that increasing extracellular calcium concentration stimulates osteoclastic survival when the filling of intracellular stores is prevented by a treatment with 1 µM of thapsigargin. Taken together, our data strongly suggest that blockade of calcium stores refilling with SERCA and CCE blockers inhibits osteoclastic survival and consequently bone resorbing process. We suggest that in osteoclasts, calcium movements between different cellular compartments involved in the regulation of calcium signalling such as calcium stores refilling and CCE are closely associated to the regulation of osteoclastic survival and that CCE may at least in part oppose to the thapsigargin- induced osteoclastic apoptosis by a mechanism, which remains to clarify. THE ROLE OF TARTRATE-RESISTANT ACID PHOSPHATASE IN DENDRITIC CELLS E. Esfandiari1*, C. R. Stokes1, T. M. Cox2, M. J. Evans3, A. R. Hayman1 1School of Clinical Veterinary Science, University of Bristol, Bristol, UK 2Department of Medicine, University of Cambridge, Cambridge, UK 3School of Biosciences, University of Cardiff, Cardiff, UK Tartrate-resistant acid phosphatase (TRAP) is a histochemical marker of the osteoclast. It is also characteristic of alveolar macrophages and dendritic cells (DCs). TRAP activity is pathologically increased in Gaucher's disease and hairy cell leukemia, and in conditions where bone resorption is enhanced, including osteoporosis, hyperparathyroidism and Paget's disease. DCs are the most potent antigen-presenting cell of the immune system. They are critically involved in the initiation of primary immune responses, graft rejection, autoimmune diseases and T cell dependent humoral immune responses. Blood-derived human DCs matured by induction with LPS displayed a five-fold increase in TRAP activity compared with immature DCs suggesting a role for TRAP in the immune response. Studies conducted with mice lacking TRAP as a result of targeted gene disruption have an intrinsic defect of bone resorption and display an abnormal immunomodulatory response and cytokine secretion profile in cultured bone marrow and peritoneal macrophages. Our aim in this study was to investigate the significance of TRAP in the dendritic cell using the TRAP knockout mice. DCs were prepared from the bone marrow of TRAP deficient and wild-type mice. Maturation of DCs from wild-type with LPS resulted in a significant increase in the surface expression of MHC class II and the co-stimulatory molecule CD80. In contrast the ability of LPS to upregulate expression of these molecules in TRAP deficient DCs was greatly reduced. Mature DCs from TRAP deficient mice were found to produce significantly more IL-10 and IL-12 (p40) in culture than those from wild-type mice. The addition of anti IL-10 to cultures of LPS stimulated TRAP deficient DCs resulted in an increased expression of MHC-II and CD80 molecules. The role of TRAP was further investigated in vivo by comparing the induction and expression of delayed hypersensitivity responses. TRAP deficient and wild-type mice were sensitised and challenged with the contact sensitising agent picryl chloride. The 24h foot pad response was significantly reduced in the TRAP deficient mice. Together these in vitro and in vivo data would suggest that TRAP may play a role in the maturation of dendritic cells. Acknowledgements: Arthritis Research Campaign DETECTION AND QUANTIFICATION OF RANKL AND OSTEOPROTEGERIN GENE EXPRESSION BY REAL TIME PCR IN ILIAC BONE FROM ELDERLY PATIENTS WITH OSTEOPOROSIS AND OSTEOARTHROSIS B. M. Abdallah1*, L. S. Stilgren3, N. Nissen2,3, M. Kassem1,3, H. R. I. Joergensen2, B. Abrahamsen3 1Clinic for Molecular Endocrine Treatment KMEB, Odense University Hospital, DK- 5000 2Dept of Orthopaedic Surgery O, Middelfart, Odense University Hospital, DK-5500 3Dept of Endocrinology M, Odense University Hospital, DK-5000. Denmark Background: RANK-L is a potent inducer of osteoclastogenesis and this action is blocked by the decoy receptor osteoprotegerin (OPG). Treatment with OPG potently blocks bone resorption in postmenopausal women, but the relationship between serum OPG levels and bone mineral density is unclear, as both positive and negative associations have been reported in the literature. Moreover, serum levels may not directly parallel the local production of these factors within the bone microenvironment. Therefore, the purpose of the present study was to compare the expression of OPG and RANK-L directly in iliac bone from patients with hip fracture ('osteoporosis') and hip replacement ('osteoarthrosis'), to test the hypothesis that the RANK-L/OPG mRNA ratio is raised in osteoporotic bone. Study population: 24 patients with osteoarthrosis of the hip and six patients with hip fracture. Mean age 75.2 ±7.2 vs 77.7 ±9.1 (n.s.). Fem. neck BMD 0.72 g/cm2 ±0.13 vs 0.57 g/cm2 ±0.09 (p<0.02). Biopsies were obtained at the time of surgery after informed consent. Methods: tRNA was extracted from 8 mm iliac bone biopsies, reverse transcribed and real time quantification of RANKL and OPG to beta-actin mRNA was performed with a SYBR Green I real time PCR assay using the comparative CT method for calculating relative gene expression. Results: Beta-actin normalized mRNA amounts were as follows (median and IQ- ranges, Mann-Whitney test) Conclusions: The median RANK-L/OPG ratio was higher in iliac bone biopsis from patients with osteoporosis compared with patients with osteoarthrosis of the hip, though this difference was just short of statistical significance (p=0.09). The present study gives additional support to the involvement of the RANKL/OPG family of cytokines in the pathogenesis of senile osteoporosis.
DISORGANIZATION OF ACTIN-RING IN HUMAN OSTEOCLASTS WITH IMPAIRED CATHEPSIN K EXPRESSION S. Avnet*, A. Lamolinara, D. Granchi, A. Giunti, N. Baldini Laboratory for Pathophysiology, Istituti Ortopedici Rizzoli, and Department of Orthopaedic Surgery, University of Bologna, Bologna, Italy Human cathepsin K is a cysteine protease, that highly and selectively expressed in osteoclasts. Knockout of this enzyme in mice, as well as lack of functional enzyme in human pycnodysostosis, result in osteopetrosis. Cathepsin K targeting may, therefore offer as a promising tool for protection from bone loss whenever elevated resorption activity occurs, such as in osteolytic bone metastasis. Despite the well-proven involvement of cathepsin K in bone resorption, different sites of activity may be suggested for this enzyme in addition to collagen proteolysis. To investigate the contribution of cathepsin K to cytoskeletal organization, osteoclast polarization, and, in turn, osteoclastic bone resorption through continuous cavitation, we studied the effects of cathepsin K completely phosphorothioate antisense oligodeoxynucleotides (AS-ODNs) on human osteoclasts obtained from peripheral blood monocytes. An effective uptake of AS-ODNs by osteoclasts was demonstrated by using fluorescein- labeled AS-ODNs at different times of exposure and dosage. Treatment with antisense significantly decreased the amount of cathepsin K in osteoclasts, as verified by immunofluorescence and Western-blot. Under the same conditions, osteoclast viability was unaffected, as shown by the number of TRAP-positive cells. At a10 mM dose, AS-ODN treatment inihibited pit formation, and, interestingly, also impaired the organization of pseudopods that failed to form regular or complete actin-rings (Figure: a, negative control, b, AS-ODN, c, S-ODN). Sense ODNs that were used as negative controls had no effect on cathepsin K protein level, on actin-ring formation, or on resorption activity of osteoclasts. These data show that inhibition of cathepsin K in human osteoclasts results in a reduction of the resorption activity, associated with a derangement in the cytoskeletal organization of the actin-ring.
ASSOCIATION MEMBRANE RAFTS WITH OSTEOCLAST FUNCTION Z. H. Lee*, H. B. Kwak, H. H. Kim Chosun University Dental College, Gwangju, South Korea Membrane rafts are specialized microdomains enriched in glycosphingolipids, cholesterol, and glycosylphosphatidylinositol (GPI)-anchored proteins. Proteins modified with saturated acyl chain groups, such as GPI-anchored proteins and double acylated proteins, and certain transmembrane proteins have been found to be enriched in rafts. The involvement of rafts has been implicated in many important cellular processes, which include generation and maintenance of cellular polarity, chemotactic migration, and cell surface receptor signaling. Src family tyrosine kinases, modified by myrisylation and palmitation, have been shown to be preferentially associated with rafts. Given the importance of Src in bone resorbing function of osteoclasts, we investigated the potential involvement of membrane rafts in osteoclast activity. Membrane raft microdomains were disrupted by cholesterol depleting or sequestering agents and their effects on mature osteoclasts were examined. We found that disruption of rafts interfere with the actin ring formation by osteoclasts upon stimulation by receptor activator of nuclear factor kappaB ligand (RANKL). Furthermore, dentine resorption activity of mature osteoclasts was greatly reduced by a short exposure to the raft-disrupting agent. In addition, the survival of osteoclasts was hampered even in the presence of RANKL when the raft integrity was destroyed. Our findings indicate that the integrity of membrane raft microdomains is required for proper function of osteoclasts. RANKL SIGNALING TO AKT REQUIRES MEMBRANE RAFT MICRODOMAINS H. H. Kim*, H. Ha, Z. H. Lee Chosun University Dental College, Gwangju, South Korea Lipid rafts are membrane microdomains, rich in sphigolipids and cholesterol. Raft microdomains have been shown to function as a signaling platform for immune cell surface receptors by selectively recruiting and excluding signaling adaptors and protein tyrosine kinases to vicinity of the receptor complexes. A few studies demonstrating raft- association of CD40, a member of the tumor necrosis factor receptor (TNFR) family have been reported. Accordingly, tumor necrosis factor receptor-associated factor (TRAF) 2 and TRAF3 proteins, key signaling adaptor molecules utilized by many TNFR family receptors, were shown to be recruited to raft microdomains during CD40 signaling. In this study we explored the possibility that lipid rafts have a role in the signal transduction of receptor activator of nuclear factor kappaB (RANK), a TNFR member important for osteoclast differentiation and activation. We found that TRAF6 is recruited to rafts by RANKL stimulation in osteoclasts. Furthermore, disruption of rafts with the cholesterol depleting agents resulted in an interference in the RANKL activation of Akt in osteoclasts. This interference by raft disruption was specific as the MAPK and NF-kB signaling pathways were not suppressed under the same experimental conditions. These results suggest that the membrane raft microdomain has a significant role in RANKL signaling during osteoclast differentiation and activation. AN EX-VIVO HUMAN MODEL OF OSTEOCLASTOGENESIS TO INVESTIGATE THE ROLE OF THE PHYTOESTROGEN GENISTEIN S. Lorenzetti*, T. Pennimpede, M. Heim, G. Kampmann, R. Scott, P. Fuchs, P. Weber, W. Hunziker, I. Bendik, F. Branca 1National Institute for Food and Nutrition Research (INRAN), Rome, Italy 2Roche Vitamins Ltd., Research & Development, Human Nutrition and Health, Basel, Switzerland Hormone replacement therapy is known to have a beneficial effect on estrogen deficiency since it stops the high rate of bone turnover typical during postmenopause. Soy phytoestrogens have been recommended as a dietary alternative because the soy isoflavonoid genistein has been shown to modulate osteoclast (OCL) differentiation and resorption. As a model of human osteoclastogenesis we have taken advantage of a recently established ex vivo approach which is based on primary cultures of OCL hematopoietic precursors. OCL precursors are monocytes known to express CD14, CD11b and CD11c receptors, the same markers present in macrophage precursors. By selection of the CD14-positive monocytes is possible to isolate such precursors and, upon RANKL induction, these cells have been shown to leave their default pathway and to be committed as OCLs. Our aim is to investigate the role of genistein during the RANKL-induced human osteoclastogenesis and to compare genistein effects with those of 17beta-estradiol (E2) on the gene expression levels. We have designed TaqMan probes and primers for detecting twenty-five different osteoclast marker genes in order to analyze the expression profiles by quantitative real-time RT-PCR. There was an observed difference between E2 and genistein treatment (at various concentrations) on the transcriptional regulation of some of the investigated genes. In particular, differential regulation was observed for the expression levels of genes encoding the osteoclast differentiation factor receptor (hRANK), the matrix metalloproteinase 2 (MMP-2) and its inhibitor TIMP-2, the cysteine proteinase Cathepsin K, and subunits of the vitronectin receptor (VNR). SERUM TARTRATE-RESISTANT ACID PHOSPHATASE 5B AND OSTEOCALCIN IN NATURALLY OCCURRING OSTEOPETROTIC RATS S. L. Alatalo1*, K. K. Ivaska1, J. M. Halleen1, S. C. Jr Marks2, H. K. Väänänen1 1Institute of Biomedicine, Department of Anatomy, University of Turku, Turku, Finland 2Department of Cell Biology, University of Massachusetts Medical Center, Worcester, MA, USA Osteopetrosis is a rare, genetic disorder due to markedly decreased bone resorption as a result of several different osteoclastic abnormalities. Three different naturally occurring osteopetrotic rat strains have been identified; toothless (tl/tl) containing severely reduced number of osteoclasts with normal ruffled borders, osteopetrotic (op/op) containing slightly reduced number of osteoclasts with poorly developed ruffled borders and incisors absent (ia/ia) containing markedly increased number of osteoclasts without ruffled borders. The defected gene in tl/tl mutation is colony- stimulating factor (CSF-1 or M-CSF), but the mutations in op/op and ia/ia rats are still unknown. We measured the bone resorption marker tartrate-resistant acid phosphatase 5b (TRACP 5b) and the bone formation marker total osteocalcin from the serum of 2- week-old osteopetrotic rats and their normal littermate controls (nlm). The results are shown in table 1. TRACP 5b was significantly decreased and osteocalcin significantly increased in tl/tl rats, while both markers were unchanged in op/op rats. In ia/ia rats, TRACP 5b was significantly increased and osteocalcin unchanged. In vitro osteoclast cultures were made from long bones of 1- to 4-day-old ia/ia rat pups. Despite of their inability to resorb bone, osteoclasts from ia/ia rats secreted more TRACP 5b into the culture medium than osteoclasts from nlm controls. These results suggest that in various osteopetrosis rat strains, secreted TRACP 5b might reflect the number of osteoclasts rather than their activity. Osteocalcin measurements suggest that bone formation is increased in tl/tl rats but not affected in op/op or ia/ia rats.
CXCL5 (ENA-78) IS DIFFERENTIALLY EXPRESSED IN OSTEOCLASTS AND MONOCYTES L. Skelton1*, A. Platt2, M. Murphy1, M. Horton3 1Celltech R&D Ltd., 216 Bath Road, Slough, Berkshire, SL1 4EN, UK 2Celltech R&D Ltd., Granta Park, Great Abington, Cambridge, CB1 6GS, UK 3Royal Free and University College School of Medicine, Bone and Mineral Centre, The Rayne Institute, 5 University Street, London, WC1E 6JJ, UK Osteoclasts are of haemopoietic origin, belonging to the myeloid cell lineage. The importance of chemokines and chemokine receptors in the biology of other myeloid cell types is well documented. With this in mind, a custom made microarray was used to examine mRNA expression of chemokines and chemokine receptors, as well as selected GPCRs, in human monocytes and 'in vitro' monocyte derived osteoclasts. The resulting gene expression data was analysed by hierarchical clustering performed blind to description of cell type. Data are grouped together solely on the similarity between gene expression profiles. The analysis resulted in the formation of three clusters, one containing four monocyte samples, one containing four osteoclast samples and a third containing three osteoclast and two monocyte samples. The overlap of osteoclasts and monocytes in this third cluster is representative of the similarity of expression of the genes studied in this microarray by these two cell types. Several genes show very distinct expression patterns. For example, CXCL5 (ENA-78) showed increased expression in the osteoclast samples. Time course experiments for the production of CXCL5 protein in osteoclast cultures demonstrated a correlation between levels of CXCL5 and the formation of tartrate resistant acid phosphatase (TRAP) positive multinucleated cells. Immunocytochemistry attributed CXCL5 production in these cultures to multinucleated osteoclast cells. Supernatants from osteoclast cultures induced chemotaxis of peripheral blood granulocytes and this chemotaxis could be partially blocked by antibodies to either CXCL5 or CXCL8 (IL- 8). These data demonstrate differential expression of CXCL5 in monocytes and osteoclasts and suggest a role for this chemokine in osteoclast biology. THE INHIBITION OF OSTEOPROTEGERIN PRODUCTION IN HUMAN OSTEOBLASTIC CELLS BY THREE GLUCOCORTICOID ANALOGUES E. L. Humphrey1*, H. L. Smith1, J. H. H. Williams2, M. J. Marshall1 1Charles Salt Centre, RJAH Orthopaedic Hospital, Oswestry, UK 2Chester Centre for Stress Research, Chester College, Chester, UK The long-term treatment of patients with inflammatory disease using glucocorticoid drugs often results in bone loss and fractures. The mechanism of this effect is not understood but is thought to be partly due to inhibition of osteogenesis and partly to increased bone resorption. Bone resorption is largely controlled by the balance between the pro-resorptive cytokine RANKL (receptor activator of NFź-B ligand) and the decoy receptor for RANKL, osteoprotegerin (OPG). OPG production by osteoblastic cells has been shown to be inhibited by glucocorticoids and therefore this may lead to an increase in bone resorption. The glucocorticoid, deflazacort (DEF), is reported to be relatively bone sparing compared to dexamethasone (DEX) and prednisolone (PRED), that is its use results in less loss of bone mineral density. Our aim was to see if DEF had less inhibitory effect on OPG production in osteoblastic cells than DEX or PRED. An enzyme-linked immuno-assay was developed to assay human OPG produced by the osteosarcoma cell line MG63 and by primary human osteoblasts in culture. The production of OPG by MG63 cells over 48 hours was inhibited by the three glucocorticoids compared with vehicle control cultures. DEF showed 52 percent inhibition, DEX showed 67 percent inhibition and PRED showed 69 percent inhibition at 10 nanomolar concentration. When these analogues were used at dose rates that reflected the potency of inhibition of inflammation there were no significant differences in the inhibition of OPG production in these cells. The inhibition of OPG protein production reflected a reduction in messenger RNA for OPG. Thus, any bone sparing effect that DEF may have is unlikely to be mediated through an effect on OPG production. Preliminary results with primary human osteoblastic cells showed a similar relationship. FREQUENCY OF THE P392L MUTATION IN THE SEQUESTOSOME 1 GENE IN BELGIAN PATIENTS WITH PAGET'S DISEASE E. Cleiren1*, G. Beyens1, J. P. Devogelaer2, F. Vanhoenacker3, L. Verbruggen4, J. Van Offel3, W. Van Hul1 1Dept. Medical Genetics, University of Antwerp, Belgium 2Dept. of Rheumatology, UCL, Brussels, Belgium 3Dept. of Radiology, University Hospital of Antwerp, Antwerp, Belgium 4Dept. of Rheumatology, University Hospital of Brussels (VUB), Brussels, Belgium Paget's disease of bone (PDB) is a skeletal disorder characterized by focal areas of increased osteoclastic bone resorption and disorganized formation of new bone. It is the second most common metabolic bone disorder only preceeded by osteoporosis. PDB is genetically heterogeneous with currently already 7 susceptibility loci being reported. Recently, a missense mutation (P392L) was found in PDB cases in the gene encoding sequestosome 1 (SQSTM1) located at the PDB3 locus on chromosome 5q35. The SQSTM1 protein is a component of the NFkappaB signaling pathway, targeting cellular proteins for degradation through the proteasome pathway. Since the mutation affects the ubiquitin-binding domain of the protein, the hypothesis has been formulated that decreased degradation of the SQSTM1 protein leads to increased activation of the NFkappaB signaling pathway. The P392L mutation turned out to be recurrent as being present in 46% of the familial cases from an extended French Canadian population but also in 16% of the sporadic cases from the same population. Analysis of the P392L mutation in an extended set of PDB patients, mainly from British descent, indicated that this mutation accounts for 20% of familial cases and 8.9% of sporadic cases within the British population. We now evaluated the frequency of the P392L mutation in the SQSTM1 gene in a cohorte of PDB patients of Belgian origin. By snapshot analysis (Applied Biosystems), 92 PDB patients without any familial history related to Paget's disease, were screened for the mutation. In only 3 patients (3.3%) the mutation was found. This indicates that the impact of mutations in the SQSTM1 gene as the primary disease cause is highly variable between different population and seems to be correlated with the frequency of familial cases within the population. DIVERGENT EFFECTS OF PASTEURELLA MULTOCIDA TOXIN ON THE DIFFERENTIATION AND ACTIVITY OF MOUSE, RABBIT AND HUMAN OSTEOCLASTS N. W. A. McGowan1*, D. Harmey1, G. Stenbeck2, A. E. Grigoriadis1 1Craniofacial Development, King's College London, London, UK 2Bone and Mineral Centre, University College London, London, UK Pasteurella multocida toxin (PMT) is a bacterially-produced toxin that causes the porcine bone resorbing disease, atrophic rhinitis. PMT is a potent mitogen and stimulates phospholipase C, leading to activation of protein kinase C, and increases in inositol phosphates and intracellular calcium. PMT also signals indirectly through the Ras/MAP kinase pathway in addition to inducing actin rearrangements through the small GTPase Rho. Previous work by our laboratory has established that PMT inhibits osteoblast differentiation, in part via activation of Rho and Rho kinase. Furthermore, PMT inhibits osteoblast-mediated osteoclastogenesis, partly through a reduction in the ratio of RANKL to OPG. Despite the marked effects on osteoblasts, the potential direct effects of PMT on osteoclast precursor differentiation and bone resorption are less clear. In this study, the effects of PMT on osteoclasts were assessed in three well- established systems, sRANKL and MCSF-based cultures of murine bone marrow cells, human peripheral blood mononuclear cells (PBMCs), and primary osteoclasts isolated from neonatal rabbits. In murine cultures, continuous exposure to PMT dose- dependently inhibited the formation of multinucleated TRAP positive cells and resorption pits. Pulse experiments revealed that addition of PMT between days 1-3, 3- 6 or 6-9, inhibited osteoclast formation and resorption. Exposure of primary mature rabbit osteoclasts to PMT for 24h appeared to interfere with F-actin ring formation and induce cellular contraction in some osteoclasts, although surprisingly, with no apparent effect on resorption. In cultures of human PBMCs, PMT inhibited osteoclast differentiation and resorption when present for the duration (0-14 days), or initial stages of culture (0-7 days). Interestingly, however, when PMT was present for the latter stages of culture (10-14 days), there was a ~1.5-2-fold increase in the proportion of actively resorbing osteoclasts compared to control cultures (>40% vs >70%) as assessed by expression of vitronectin receptor and F-actin rings. This correlated with a 3-fold increase in the area resorbed in the presence of PMT. Taken together, these findings indicate that PMT has possible divergent effects on osteoclast differentiation and activity and we are currently investigating the signalling pathways induced by PMT to establish their roles in osteoclast differentiation and activity. INHIBITION OF PROTEIN PRENYLATION BY RISEDRONATE CAUSES ACTIVATION OF RAC, PAK AND P38 J. E. Dunford*, F. P. Coxon, M. J. Rogers Dept. Medicine and Therapeutics, University of Aberdeen, Aberdeen, UK Nitrogen-containing bisphosphonates (N-BPs), e.g. risedronate (RIS), inhibit bone resorption by preventing the synthesis of isoprenoid lipids required for prenylation of small GTPases such as Rho, Rac and Cdc42. Prenylation is essential for the correct function of small GTPases since it enables membrane localisation and interaction with regulatory factors. We sought to confirm that N-BPs affect the activity of small GTPases in J774 macrophages, which undergo apoptosis following treatment with N- BPs. The active, GTP-bound forms of Rac, Cdc42 and Rho were measured using a pull- down assay utilising agarose beads conjugated to the GTPase-binding domain of PAK or Rhotekin. Precipitated Rac, Cdc42 or Rho was then quantitated by western blotting. Treatment of J774 cells for 20 hours with 100microM RIS or other N-BPs caused an increase in the level of GTP-Rac but had no effect on the level of GTP-Cdc42 or GTP- Rho. Inhibition of Rac prenylation by 10microM GGTI-298 also caused an increase in GTP-Rac, but clodronate (a bisphosphonate that does not inhibit protein prenylation) had no effect. Similar results were obtained with PC3 and T47D cells. To confirm that GTP-bound Rac in RIS-treated cells was unprenylated, J774 cells were metabolically labelled with [14C]mevalonate in the absence or presence of RIS. Unlike with control cells, GTP-Rac precipitated from RIS-treated cells was not radiolabelled and hence was in the unprenylated form. We determined next whether active, unprenylated Rac was capable of activating downstream signalling pathways in J774 cells. Treatment for 20 hours with 100microM RIS or 10microM GGTI-298 caused an increase in the kinase activity of PAK immunoprecipitated from cell lysates and a sustained increase in the level of phosphorylated p38 MAP kinase. The timecourse of p38 activation closely resembled the activation of Rac by RIS. Inhibition of p38 with SB203580 caused an increase in apoptosis of RIS-treated J774 cells compared to treatment with RIS alone. These studies demonstrate that inhibition of protein prenylation by RIS causes activation, rather than inhibition, of Rac. In J774 macrophages, the unprenylated, activated form of Rac can activate the downstream effectors PAK and p38. Activation of p38 may confer decreased sensitivity to N-BP-induced apoptosis. NITROGEN-CONTAINING BISPHOSPHONATES INDUCE INTRACELLULAR ATP-ANALOGUE (APPPI) PRODUCTION H. Mönkkönen1*, S. Auriola2, J. Vepsäläinen3, J. Mönkkönen1 1Department of Pharmaceutics, University of Kuopio, Kuopio, Finland 2Department of Pharmaceutical Chemistry, University of Kuopio, Kuopio, Finland 3Department of Chemistry, University of Kuopio, Kuopio, Finland Bisphosphonates (BPs) are a class of drugs developed for the treatment of metabolic bone diseases with high bone turnover, such as Paget's disease, tumor associated osteolysis and osteoporosis. Despite their widespread clinical use, the exact biochemical mechanism of action of bisphosphonates was largely unknown, although very recent research had shed new light on this issue. Bisphosphonates can be divided into two classes with distinct molecular mechanisms of action. Nitrogen-containing BPs (N-BPs), such as alendronate and risedronate, appear to inhibit one or more enzymes of the intracellular mevalonate pathway, thereby preventing the modification of important signalling proteins with isoprenoid lipids. Loss of prenylated proteins causes a loss of osteoclast function and apoptotic cell death. The less potent non-nitrogen-containing BPs (non-N-BPs), such as clodronate and etidronate, do not inhibit isoprenylation but are metabolised intracellularly by osteoclasts to cytotoxic analogues of adenosine triphosphate (ATP), which accumulate in the cell cytoplasm and cause apoptosis at high doses, but has specific anti-inflammatory action at lower doses. It has been previously shown that N-BP:s are not metabolised to ATP-analogue. We found, however, that N-BPs induce intracellular ATP-analogue (Apppi) production via inhibition of mevalonate pathway in vitro by intact mammalian cells, such as J774 macrophages and C6 gliomas. Non-N-BPs do not induce Apppi production. The structure of Apppi (triphosphoric acid 1-adenosin-5'-yl ester 3-(3- methylbut-3-enyl) ester) was determined by using mass spectrometry (MS) and nuclear magnetic resonance (NMR). In contrast to the AppCp-type metabolites of non-N-BPs, Apppi does not contain BP in the molecular structure. It seems that Apppi production correlate the capacity of N-BPs to inhibit FPP-synthase (the enzyme of mevalonate pathway). The potent N-BPs (such as risedronate and zoledronate) strongly inhibit FPP-synthase and also strongly induce Apppi production. Therefore Apppi production may also correlate with bisphosphonate activity. MOLECULAR MECHANISM OF THE ACTION OF TRANSFORMING GROWTH FACTOR-BETA IN OSTEOCLAST DIFFERENTIATION S-J. Park*, E. J. Chang, J. S. Ko, H-M. Kim College of Dentistry, Seoul National University, Seoul, Korea Transforming growth factor-beta (TGF-beta) has been known to be crucial in osteoclastogenesis that is totally inhibited when the autocrine TGF-beta was blocked. Molecular mechanism of the TGF-beta action in osteoclastogenesis, however, has not been studied well. In the present study, receptor activator of NF-kappaB (RANK) signal transduction pathway was examined to understand the action of TGF- beta promoting osteoclastogenesis. Pre-osteoclast cells attached to the surface when mouse bone marrow cells were cultured under influence of macrophage colony stimulating factor (M-CSF) for 3 days, were further treated with M-CSF and receptor activator of NF-kappaB ligand (RANKL) without or with TGF-beta for another 3 days to generate osteoclasts expressing RANK. Much more multinucleated tartrate resistant alkaline phosphatase positive osteoclasts were generated when cells were treated with M-CSF and RANKL with TGF-beta. TGF-beta treatment kept both level of TRAF 6 and activation of NFkappa-B high upon RANKL stimulation, both of which are known necessary conditions in osteoclastogenesis. Action of TGF- beta was transmitted through extracellular signal-regulated kinase (ERK) 1/2 in differentiating osteoclast cells, while smad 2 or c-Jun N-terminal kinase was not activated by TGF-beta. These results indicate that TGF-beta may indirectly promote osteoclastogenesis by potentiating a signal transduction pathway of RANK including TRAF6 as well as NFkappa B which is imperative in osteoclastogenesis after activating ERK 1/2 MAPK. IN VITRO VOLUMETRIC ANALYSIS OF RESORPTION PITS BY IMMUNOFLUORESCENCE CONFOCAL MICROSCOPY B. M. Nicholls*, G. T. Charras, M. A. Horton, S. A. Nesbitt Bone and Mineral Centre, The Rayne Institute, University College London, London, WC1E 6JJ, UK In vitro bone resorption assays are essential for the screening and analysis of bone- sparing drugs prior to their use in vivo. Herein, a novel application of immunofluorescence confocal microscopy provides a new method that allows individual quantification of thousands of resorption pits for number, area, depth and volume. Rabbit osteoclasts were seeded onto dentine slices (pre-labelled with fluoro-X) and cultured for 3 days. A variety of resorption regulators were used at doses to give a range of resorption while maintaining osteoclast numbers; 0.05 mM E-64 (a cathepsin K inhibitor), 0.1 mM alendronate (a bisphosphonate), 25 nM kistrin (an integrin antagonist) and 50 pM TIMP-1 protein (a metaloproteinase inhibitor). After fixation and permeabilisation, the cultures were analysed by immunofluorescent staining and laser scanning confocal microscopy (LSCM) for the non-resorbed matrix with fluoro- X and the resorption pits with Cy5-labelled antibodies to type I collagen. 16 optical xy sections were taken through the resorption sites by LSCM and the image stacks analysed using an in-house computer programme with Pv-wave software. The fluorescence intensity pixels for the Cy5 and fluoro-X images and their collection depths were used to calculate pit number, area, depth and volume. Percentage changes for each of these resorption parameters were assessed by Mann-Whitney statistics. The compounds tested gave a range of resorption and a majority of the resorption parameters were altered significantly (n=18, P < 0.05). Kistrin, alendronate and E-64 inhibited resorption and reduced pit volumes by 33%, 68% and 86%, respectively, with comparable falls in pit areas. In contrast, low concentrations of TIMP-1 proteins, increased pit volumes and areas by 88% and 54%, respectively. Additionally, these quantification methods for bone resorption were assessed further using the E-64 resorption inhibitor in dose response, recovery and spike experiments. Again, significant changes in the resorption parameters were achieved. In summary, these techniques provide an alternative, semi-automated approach to assess in vitro bone resorption and could be further developed to increase the throughput of screening for bone inhibitory drugs. ANALYSIS OF INTRONIC POINT MUTATIONS IN TCIRG1 GENE USING MINIGENE APPROACH L. Susani1*, F. Pagani2, C. Stuani2, A. Frattini1 1Istituto Tecnologie Biomediche, CNR, Milan, Italy 2Molecular Pathology Department, I.C.G.E.B., Trieste, Italy In autosomal recessive osteopetrosis (ARO), a severe bone disease with a fatal outcome generally within the first decade of life, a bone resorption defect leads to increased bone density and defective remodelling. This osteoclast malfunction causes various abnormalities including macrocephaly, progressive deafness, blindness, hepatosplenomegaly and severe anemia. We have analyzed more than 100 ARO patients and we found that about 50% of them bear mutations in TCIRGB, a gene that encodes the osteoclast-specific 116-kD subunit of the vacuolar proton pump. 37.5% of these mutations affect the invariant GT/AG dinucleotide splicing sites. In 7 patients we identify intronic point mutations flanking the splice sites whose effect on splicing cannot be predicted by simple sequence inspection. Since RNAs of many of these patients may not be always available, we evaluate the use of a specific 'minigene splicing-vector'-based approach to study the disease-causing mechanisms. We have analysed two mutations near the canonical GT 5' splice site: a G to T transversion at position +4 of the intron 2 and a G to A transition at position +5 of the intron 14. The genomic regions spanning exon 2 and exons 14, with and without the mutations, were amplified and cloned into pTB vector (Nat Genet 2002,30 426). Wild-type or mutant minigenes were transfected in HeLa cells, the pattern of splicing analysed by RT-PCR on extracted RNA and the resulting PCR products sequenced to check the effect of the mutations. Both mutations caused a splicing defect. The +4 G to T transversion caused the deletion of the last 42 bp in exon 2. Subsequent RT-PCR analysis on patient RNA confirmed the presence of the same deletion. The +5 G to A transition, whose patient RNA was not available, induced skipping of the entire exon 14. Our results demonstrate that this approach is an efficient and reliable method to test the effects of intronic point mutations when only genomic DNA is available. We are currently evaluating the use of the minigene system to provide mechanistic insight and possible new therapeutic strategies to correct these splicing defects. CLCN7 MUTATIONS CAUSE A BROAD SPECTRUM OF BONE DEFECTS A. Frattini, A. Pangrazio*, L. Susani, C. Sobacchi, M. Mirolo, P. Vezzoni, A. Villa Istituto Tecnologie Biomediche, CNR, Milan, Italy Infantile malignant Autosomal Recessive Osteopetrosis (ARO, MIM 259700) is a severe bone disease with a fatal outcome, generally within the first decade of life. Defects in bone resorption due to osteoclast malfunction is associated with a number of severe clinical manifestations, including: macrocephaly, progressive deafness, blindness, hepatosplenomegaly and severe anemia beginning in early infancy or in fetal life. The only available treatment capable of providing sustained improvement of osteoclast function is bone marrow transplantation (BMT). In the present work, we report the clinical and molecular analysis of a large series of 89 patients investigated for the two genes, Atp6i and ClCN7, known to be responsible for this disease. The ATP6i (TCIRG1) codes for a subunit of the proton pump, while the ClCN7 codes for a chloride channel. Both these molecules are involved in the acidification of the extracellular milieu. In our series, 48 cases showed defects in the Atp6i gene, while 13 patients bore mutations in the chloride channel gene, while the remaining cases did not harbour any defect in either gene. The clinical and molecular analysis of this wide number of patients led us to suggest that the spectrum of clinical presentation covered by these two genes is different. Atp6i-dependent abnormalities are restricted to classical severe ARO, and most of them are null mutations, consisting of nonsense, deletions and splicing defects. On the contrary, the clinical spectrum of ClCN7 mutations is wider and many of them are missense substitutions which cause severe as well as benign ADO. In addition, our findings suggest that ClCN7 mutations are also responsible for a particular subset of intermediate forms, showing that ClCN7 defects represent a continuum with both ARO and ADO. Moreover, we found that the same mutation can cause a relatively broad range of phenotypes even in the same family. In conclusion, our findings contribute to further highlight the heterogeneity of human osteopetroses showing that ClCN7 dependent forms cover a wider range of clinical pictures. BUTANEDIOL ESTERS AS NOVEL INHIBITORS OF BONE RESORPTION R. J. van 't Hof*, I. R. Greig, A. I. I. Mohamed, S. H. Ralston Dept. Medicine and Therapeutics, University of Aberdeen, Aberdeen, UK We have recently shown that non-steroidal anti-inflammatory drugs (NSAIDs) that contain a nitric oxide (NO)-donor group are potent inhibitors of osteoclast activity and formation. However, the mechanism of action of these agents is at present unknown and appears to be independent of both COX-inhibition and the NO-donor properties. As the NO-donor group is linked to the NSAID by means of a butanediol link, we investigated the possible anti-resorptive properties of alkane diol esters. Co-cultures of murine osteoblasts (8x103 cells/well) and bone marrow cells (2x105 cells/well) were performed on dentine in 96-well plates in the presence of 10 nM 1,25- (OH)2 -vitamin D3. Drugs and IL-1beta (10U/ml) were added to the cultures on day 7 and the cultures were terminated on day 10. Osteoclasts (OC) were identified by tartrate resistant acid phosphatase staining, and resorption area was measured using a reflected light microscope coupled to an image analysis system. In the co-culture system, the butanediol ester of biphenyl carboxylic acid (ABD56) acted as a potent inhibitor of OC formation and activity (IC50: 23microM). When biphenyl carboxylic acid was linked to alkane diols of different chain length (3, 5 or 6 carbons), only the hexanediol ester had some inhibitory effect on osteoclasts, however with an IC50 >100microM. The terminal hydroxyl moiety of ABD56 is crucial for its activity, as the biphenyl carboxylic acid ester of butanol had no effect on OC formation or activity. To assess the importance of the biphenyl moiety for drug potency, we tested the 1-naphthyl (4N), homoveratryl (4H), 4-biphenylacetyl (4BPA), 1-biphenylcarboxy (4BPX) and 4-phenyltoluyl (4PT) derivatives. 4BPX, 4H, 4N and 4PT were totally inactive at concentrations up to 100 microM, and only 4BPA showed any activity (IC50: 55microM). To assess possible inhibitory effects on bone formation, we studied the effects of ABD56 on cultures of primary murine osteoblasts. At concentrations of up to 100 microM, ABD56 did not affect osteoblast proliferation or alkaline phosphatase expression under both basal and PTH-stimulated conditions. In conclusion, butanediol esters like ABD56 form a new class of potent anti- resorptive agents with possible therapeutic use in diseases characterized by increased bone resorption such as osteoporosis, rheumatoid arthritis and Paget's disease of bone. NO-NSAIDS INDUCE APOPTOSIS IN MACROPHAGES AND MATURE OSTEOCLASTS BUT NOT PRIMARY OSTEOBLASTS A. I. I. Mohamed1*, P. Del Soldato2, S. H. Ralston1, R. J. van 't Hof1 1Dept. Medicine and Therapeutics, University of Aberdeen, Aberdeen, UK 2Nicox SA, Sophia Antipolis, France We have previously shown that non-steroidal anti-inflammatory drugs (NSAIDs) that contain a nitric oxide (NO)-donor group inhibit osteoclast (OC) activity and formation. However, the mechanism of action of these compounds is presently unknown, and is not dependent on either COX- inhibition or NO-donor properties. Here, we investigate whether the NO-NSAID HCT1026 and its non-nitrosylated derivative HCT1027 induce apoptosis in rabbit OC and J774 macrophage-like cells. Rabbit OC were isolated form the long bones of newborn rabbits, and osteoblasts were isolated from the calvaria of newborn mice. Drugs were added at 50 or 100 microM, and at the end of the experiments, cells were fixed and the nuclei stained using DAPI. Apoptotic cells were identified by condensed nuclear morphology, or by TUNEL staining. Activation of caspases was measured using the APO-ONE caspase activity kit (Promega) in cultures of J774 cells. The profile and identity of the metabolites of HCT1026 and HCT1027 were determined using HPLC. Both HCT1026 and HCT1027 were potent inducers of apoptosis in both osteoclasts and J774 cells as evidenced by numbers of apoptotic nuclei. In J774 cultures, exposure to HCT1026 and HCT1027 resulted in DNA fragmentation detected by the TUNEL assay and gel electrophoresis. Furthermore, HCT1026 caused a rapid (within 12h) induction of caspase-3 and -7 activity in J774 cells. Treatment of primary osteoblasts with HCT1026 for up to 48 hours failed to induce apoptosis, suggesting that these compounds act only on cells of the monocyte-macrophage lineage. As cells of the monocyte-macrophage lineage express high levels of esterases, we studied the possible metabolic breakdown/modification of HCT1026 and HCT1027. When incubated with purified esterases (0.1 microg/ml), both compounds were rapidly hydrolysed into Flurbiprofen, the parent compound. However, in lysates from J774 cells treated with either HCT1026 or HCT1027, only the original compounds were observed, suggesting that the activity of these drugs does not depend on the formation of metabolites. Our results indicate that HCT1026 and HCT1027 are powerful inducers of apoptosis in osteoclast. However, their exact molecular mechanism of action is currently unknown and will require further investigation. PARATHYROID-HORMONE RELATED PEPTIDE EXPRESSION AT SITES OF BONE EROSION IN RHEUMATOID ARTHRITIS D. O'Gradaigh*, S. Bord, J. E. Compston Bone Research Group, University of Cambridge School of Clinical Medicine, Addenbrooke's Hospital, Cambridge, UK Parathyroid-hormone related peptide (PTHrP), first identified in patients with humoral hypercalcaemia of malignancy is also found in the synovial fluid of patients with rheumatoid arthritis (RA). Its expression by fibroblast-like synovial cells can be induced by the inflammatory cytokines TNF-alpha and IL-1. Tumour invasion of bone is facilitated by expression of PTHrP by malignant cells which activates local osteoclasts, the resulting bone resorption releasing TGFbeta which then stimulates further expression of PTHrP. As rheumatoid erosion progresses relentlessly until the joint is destroyed or surgically replaced, we hypothesised that a similar positive- feedback loop occurred at sites of osteoclastic bone erosion in RA involving PTHrP and TGFbeta. We used an indirect immunoperoxidase technique to stain serial sections of formalin-fixed paraffin-embedded RA bone and attached synovial tissue (pannus) obtained at joint replacement surgery. Primary antibodies were a monoclonal antibody to N-terminal human PTHrP, and rabbit polyclonal antibodies to TGFbeta isoforms 1- 3. Control sections were stained with murine IgG or pre-immune rabbit serum respectively. Osteoclasts were identified by expression of tartrate-resistant acid phosphatase by substrate reaction. Using image analysis software, fifteen joints were studied, comparing the area of positive staining at the bone-pannus interface at sites of osteoclastic erosion with non-eroded sites in the same section. PTHrP expression was localised to the lining layer of synovial pannus, to vascular structures in the sub-lining layer and to chondrocytes adjacent to areas of erosion. The proportion of synovial tissue positively staining for PTHrP was significantly higher (11.5%, sd 3.6) at sites of active erosion (as identified by TRAP-stained osteoclasts) compared with sites where pannus was attached to bone without erosion (6.55% sd 3.4; p=0.02). TGFbeta was not identified in the majority of sections, with no significant differences in expression at eroded versus non-eroded sites. In conclusion, we have found that PTHrP is specifically associated with bone erosion in RA. As we did not identify increased expression of TGFbeta at these sites, it is likely that PTHrP expression is instead regulated by inflammatory cytokines in this condition. HUMAN OSTEOCLAST ESTROGEN RECEPTOR EXPRESSION DIFFERS FROM THAT OF OSTEOBLASTS T. Pennimpede1*, S. Lorenzetti2, M. Heim1, G. Kampmann1, R. Scott1, P. Fuchs1, F. Branca2, W. Hunziker1, P. Weber1, I. Bendik1 1Roche Vitamins Ltd., Research & Development, Human Nutrition and Health, Basel, Switzerland 2National Institute for Food and Nutrition Research (INRAN), Rome, Italy Estrogens, including the natural hormone 17beta-estradiol (E2), exhibit effects on various tissues by binding to and transactivating the estrogen receptor (ER). There exists extensive evidence surrounding the significance of the preservation of healthly levels of estrogens on bone health. Investigations have shown that estrogens are important regulators in the coupling of osteoblast and osteoclast. This has led to speculation about the use of E2 and other estrogenic compounds to prevent and to treat osteoporosis. Our group has previously profiled the nuclear hormone receptors in human trabecular bone-derived osteoblast-like cells (HOBs). In this study we used quantitative real-time RT-PCR to profile the expression levels of several isoforms of the estrogen receptor and their splice variants, along with other members of the nuclear hormone receptor superfamily, and various osteoclast marker genes in human osteoclast cells (OCLs). This osteoclast cell model results from the differentiation of CD14+ cells in the presence of receptor activator of nuclear-factor-kB ligand (RANKL) and macrophage-colony stimulating factor. The main differences in expression levels between hOBs and hOCLs were seen in ER-beta1, 2 and 5, peroxisome proliferator-activated receptor gamma (PPARgamma), calcitonin receptor (CR), tartrate-resistant acid phosphatase (TRAP) and RANKL. OSTEOCLAST RUFFLED BORDER HAS DISTINCT SUBDOMAINS FOR SECRETION AND DEGRADED MATRIX UPTAKE M. T. K. Mulari*, H. Zhao, P. Lakkakorpi, H. K. Väänänen Institute of Biomedicine, University of Turku, Finland Subosteoclastic bone resorption is a result of HCl and proteinase secretion through the late endosomal ruffled border. As bone matrix is degraded, it enters osteoclasts' transcytotic vesicles for further processing and is then finally exocytosed to the intercellular space. The present study clarifies the spatial relationship between these vesicle fusion and matrix uptake processes at the ruffled border. Our results show the presence of V-H+-ATPase, small GTPase rab7 as well as dense aggregates of F-actin at the peripheral ruffled border, where basolaterally endocytosed transferrin and cathepsin K are delivered. Also multivesicular body -like vesicles are occasionally observed to release their inner membranes to the lacuna thus emphasizing its late endosomal nature. On the contrary, rhodamine-labelled bone matrix enters transcytotic vesicles at the central ruffled border, where the vesicle budding proteins such as clathrin, AP-2 and dynamin II are also localized. This mechanism of membrane turnover appears to differ from that in neuronal synapses, where numerous separated subdomains for membrane fusion and transmitter uptake are present. We present a model for the mechanism of ruffled border turnover and suggest that, due to its late endosomal characteristics, the ruffled border serves as a valuable model for studying the dynamic organization of other endosomal compartments as well. SEVERE HYPOXIA STIMULATES FORMATION BUT NOT ACTIVITY OF RODENT OSTEOCLASTS J. C. Utting*, I. R. Orriss, A. Brandao-Burch, T. R. Arnett Department of Anatomy & Developmental Biology, University College London, London, UK Reduced O2 is well-known to act as a powerful stimulator of the formation of marrow-derived cells. We compared the short-term effects of hypoxia on the survival and activity of mature osteoclasts (OC) with its effects on the formation and activity of marrow-derived OC in longer-term cultures. Mixed cell populations containing mature OC obtained from fragmented neonatal rat bones were sedimented onto 5mm ivory discs. The discs were cultured in sealed flasks purged with 0.2 to 20% O2 (plus 5% CO2, balance N2) in MEM / 10% FCS acidified to pH 7.0 (1 ml / disc). After 27h, discs were fixed and TRAP-stained to enable counting of viable, multinucleate OC; discs were restained with toluidine blue to visualise resorption pits after OC removal. For a representative experiment, in 20, 12, 5, 2, 1 & 0.2% O2, OC numbers were 30.5±4.6, 26.2±4.0, 31.2±6.6, 16.5±2.9, 15.3±1.4 & 6.5±2.0*, respectively; numbers of pits were 87.2±13.5, 93.8±18.6, 113.3±24.4, 53.0±8.6, 68.3±8.9 & 22.3±3.4, respectively; values are means ±SEM (n=6); * p<0.05 vs. 20% O2 value (Bonferroni). Medium pH was not altered by O2 level. Thus, O2 levels below 5% reduce mature OC adhesion or survival and pit formation in roughly equal measure. To study longer-term effects of very low O2 we cultured initially non-adherent mononuclear cells from mouse marrow on ivory discs (2x106 cells seeded / 5 mm disc) with 20 ng/ml M-CSF and 10 ng /ml RANKL in flasks gassed as above (1 ml medium / disc). Cultures were maintained for 10d at pH 7.3, then acidified for the final 3d, and regassed daily. For a representative experiment, in 20, 12, 5, 2, 1 & 0.2% O2, OC numbers were 120±14, 241±48, 338±55*, 425±46**, 307±46 & 282±64, respectively; the proportion of the disc surface covered by resorption pits was 2.8±0.3, 9.1±2.5, 18.7±3.2**, 26.6±2.1***, 21.3±4.4** & 13.1±1.4%, respectively; values are means ± SEM (n=8); *** p<0.001 ** p<0.01, * p<0.05 vs. 20% O2 value (Bonferroni). Medium pH was not altered by O2 level. Thus, OC formation peaks at 2% O2 but is still appreciable at 0.2% O2. OC formed in low O2 were larger, thus hypoxia stimulated resorption more than absolute OC numbers. CALPAIN CONTRIBUTES TO THE REGULATION OF OSTEOCLAST ATTACHMENT AND SPREADING M. Marzia*, L. Neff, R. Chiusaroli, R. Baron, W. C. Horne School of Medicine, Yale University, New Haven, CT, USA The high motility of osteoclasts is thought to require rapid transient podosome formation. Calcitonin (CT) inhibits motility, suggesting that it might regulate at least some aspects of podosome function. CT also induces an increase in intracellular Ca2+, which might in turn activate calpains, Ca2+-dependent proteases that are thought to promote cell spreading and locomotion by modifying adhesion complexes and by facilitating rear-end detachment. We therefore examined whether calpains might be associated with adhesion structures in OCs and if CT affected calpain activity. Immunofluorescence analysis showed high levels of mu-calpain associated with the OC actin ring. CT treatment induced the rapid (1-3 min) dispersion of the actin ring and the associated mu-calpain, followed soon after by cell retraction (5 min). The actin ring, again with associated mu-calpain, reappeared 20-60 min after the addition of CT. OCs treated with three different calpain inhibitors were less spread, and the staining of both F-actin and mu-calpain in the actin ring was more intense than in untreated cells. In the calpeptin-treated OCs, CT still induced the loss of the actin ring but little or no cell retraction, and the recovery of the actin ring occurred more rapidly than in the control OCs (3-5 min). Two known substrates of calpain, filamin A and talin were also associated with the OC actin ring, and treatment with CT induced the dispersion of both filamin and talin in parallel with that of the actin ring. High MW cleavage products of both filamin A and talin and were detected by Western blotting of lysates of untreated OC, but not in lysates of calpeptin-treated OCs, suggesting that filamin and talin may be continuously cleaved by calpain in untreated OCs. CT treatment also transiently reduced the amounts of filamin A and talin fragments. The demonstration that a calpain inhibitor blocks the cleavage of the podosome-associated filamin and talin and affects the dynamic stability of the actin ring suggest that calpain is involved in the regulation of podosome assembly and/or disassembly. Our results also suggest that the mechanism of CT-induced retraction of OCs may involve the transient reduction of calpain activity. BIGLYCAN REDUCES LPS-STIMULATED OSTEOLYSIS BY INHIBITING OSTEOCLAST DIFFERENTIATION Y. Bi*, T. Kilts, X-D. Chen, T. Xu, M. F. Young CSDB, NIDCR, NIH, Bethesda, Maryland, USA Small leucine-rich proteoglycans (SLRPs) are extracellular molecules that bind to collagens and growth factors to regulate cell growth and matrix assembly. We have previously shown that biglycan (bgn), a member of class I SLRPs, plays an important role in the differentiation of osteoblast precursors. Since osteoblasts and their precursors regulate the differentiation of osteoclasts through cell-cell and cell-matrix contact, we hypothesized that bgn is involved in regulating osteoclast differentiation. To test this, we used a quantitative version of the murine calvarial model of osteolysis to compare the effect of LPS on 7-week-old wildtype and bgn knockout mice. Titanium particles, as a LPS-carrier, were implanted onto the parietal bones of each mouse. After the indicated periods of time, parietal bones were harvested and processed for x-ray using a faxitron. The extent of osteolysis in each bone was determined from the x-ray image, in a blinded fashion, by computer-assisted histomorphometry. Time course experiments showed that LPS induced osteolysis occurred more rapidly and extensively in bgn knockout mice compared to wildtype mice. There was 55% less bone resorption in wildtype mouse parietal bones than that in bgn knockout mouse parietal bones when osteolysis reached its peak at day 7. To further understand the mechanism of action, we determined the effects of bgn on 1,25- vitamin D3-induced osteoclast differentiation in vitro. In those experiments, we co- cultured spleen derived osteoclast precursors with primary calvarial cells from wild type or bgn knockout mice. Time course and dose response experiments showed that TRAP+ multinuclear cells (MNCs) appeared earlier and more extensively in the co- cultures containing calvarial cells from bgn knockout mice than wildtype mice, regardless of the source of spleen cells. Thus, the inhibition of bgn on osteoclast differentiation is mediated by calvarial cells. Further studies using PCR analysis of mRNA from those co-cultures indicated that the increased osteoclast differentiation might occur via a RANK/RANKL-independent mechanism. Bgn has been shown to bind and regulate cytokines and growth factors in bone matrix, such as, TGF-beta. We propose that bgn might inhibit osteoclast differentiation and bone resorption indirectly by modulating production, storage and/or activity of cytokines that regulate osteoclasts.
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