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Oral Presentations
Abstracts O-1 to O-48
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IS GEOMETRICAL INSTABILITY OF THE PROXIMAL FEMUR A CRITICAL FACTOR IN THE PATHOPHYSIOLOGY OF MINIMALLY TRAUMATIC HIP FRACTURES? M. J. Walters1,2*, J. Meyer2 1Division Of Veterinary and Biomedical Sciences, Murdoch University, Perth, Western Australia 2Department of Anatomy and Human Biology, University of Western Australia, Perth, Western Australia Low bone mineral density (BMD) is a known factor in the pathophysiology of minimally traumatic hip fracture (MTHF). Another variable that appears to contribute to fracture risk is the geometry of the proximal femur. Hip axis length (HAL) has been shown to predict hip fracture in prospective studies, but in retrospective studies femoral neck and shaft widths, and not HAL, vary with fracture incidence. This study sought an explanation of this conundrum through a detailed comparison of the variations in the components of HAL and other aspects of the geometry of the proximal femur in normal aging and in cases of hip fracture. The projected geometry of the proximal femurs of 1132 women aged 18 to 90 years without hip fractures and of the unfractured limbs of 140 women was assessed from dual energy x-ray absorptiometry printouts within 9 days of a MTHF. Decade-by-decade differences in the widths of the femoral neck (FNW) and proximal shaft (PFSW), as well as HAL and its components (femoral head + acetabular breadth (AcH), femoral neck length (FNL) and greater trochanteric width) were evaluated by Analysis of Variance (ANOVA) and those between women with and without fractures <65 years (N=222) by Student's T-test. Amongst women without hip fractures most geometric measures varied significantly and directly with age and inversely with BMD. The exceptions were HAL and FNW. The proportion of HAL made up by the femoral head and acetabulum (AcH/HAL) did demonstrate some age-dependency. In contrast, in women with fractures, geometric dimensions tended to decline directly with BMD and no systematic variation in AcH/HAL was seen with age. There were no statistically significant differences in HAL between women of similar age with and without fractures, but the AcH/HAL ratio was significantly lower in women with fractures. The dimensions of geometric features on the contralateral limbs adjacent to common fracture sites, as well as BMD, differentiated significantly between women with different types of fracture and those without fractures. The results of this study are discussed in the light of possible instabilities of structure and variations in the projected geometry of the proximal femur arising from age, stress and osteoporosis-related remodelling. PEAK BONE MASS IN THE CALCANEUS IN MEN AND THE INFLUENCE OF LIFESTYLE FACTORS U. Pettersson1*, C. Nyman2, R. Lorentzon1, K. Landin-Wilhelmson2, L. Hulthén2, O. Johnell3, R. Kullenberg4, E. Norjavaara5, L. Samuelsson5, D. Mellstrom2 1Sports Medicine Unit, Umeå University, Umeå, Sweden 2Department of Geriatrics and Internal Medicine, Goteborg University, Goteborg, Sweden 3Department of Orthopaedics, Malmo University, Malmo, Sweden 4Department of Radiophysics, Goteborg University, Goteborg, Sweden 5The National Service Administration, Goteborg, Sweden Aims: The aim of this study was to evaluate the time of attaining peak bone mass in the calcaneus in men. The present study is part of an ongoing study investigating relationships between anthropometric parameters, muscle strength, lifestyle factors including physical activity and calcium intake and genetic analysis and BMD in 12000 male military recruits. Methods: Since 1998, 2805 men (age 17.3-19.9 years) have been recruited from the compulsory military service in Gothenburg, Sweden. Bone mineral density (BMD, g/cm2) of the calcaneus was measured with DEXA, Calscan. Isometric muscle strength of the total body was measured in Newton meters (Nm) using Isokai and physical capacity was estimated from a maximal stress test. Lifestyle factors including smoking, use of corticosteroids, training habits and calcium intake were evaluated by a questionnaire. Results: ANCOVA analysis revealed that peak BMD of the calcaneus (0.65±0.10 g/cm2) occurred at the age of 18.3 years. BMD was significantly associated with body weight (r=0.14), BMI (r=0.14), physical capacity (r=0.15), muscle strength (r=0.24), calcium intake (r=0.08), and years of regular physical activity (r=0.30). Smoking was a significant negative predictor (r=-0.06). Stepwise regression analysis revealed that years of regular physical activity were the strongest predictor, explaining 8% of the variation in BMD. Other independent predictors were muscle strength (beta=0.13) and BMI (beta=0.08). All these factors totally explained 11% of the variation of BMD. Conclusion: This study suggests that peak bone mass in the calcaneus in men is attained around 18 years. Years of regular physical activity were the strongest predictor of BMD, indicating the sensitivity of the calcaneus bone for mechanical loading. PEAK BONE MASS: A STUDY OF THE GENETIC AND ENVIRONMENTAL DETERMINANTS IN YOUNG MEN AND WOMEN F. E. McGuigan1*, L. Murray2, A. Gallagher3, G. Davey-Smith4, C. E. Neville3, R. van't Hof1, C. Boreham3, S. H. Ralston1 1Aberdeen University, Aberdeen, Scotland 2Queens University Belfast, N. Ireland 3University of Ulster, N. Ireland 4Department of Social Medicine, University of Bristol, Bristol, UK Attainment of peak bone mass is an important risk factor for the development of osteoporosis in later life. Although it is believed that genetic, intrauterine and environmental factors all contribute to the regulation of bone mass, how they interact to determine peak bone mass is not yet fully understood. In this study we investigated the role of polymorphisms of the vitamin D receptor (VDR), estrogen receptor alpha (ESR) and collagen type I alpha 1 (COLIA1) genes and the contribution of birth weight, diet, exercise and other environmental variables to the regulation of peak bone mass in a population based cohort of 460 men and women whose average age was 22 years. Stepwise multiple regression analysis showed that body weight was the strongest predictor of BMD in women accounting for 16.4% of the variance in spine BMD and 8.4% of the variance in hip BMD. Other predictors at the spine were VDR genotype (3.8%) and carbohydrate intake (1.6%) whereas other predictors at the hip were vitamin D intake (3.4%) and ER genotype (3.4%). In men, physical activity was the strongest predictor of BMD, accounting for 6.7% of the variance at the spine and 5.1% at the hip. Other significant predictors at the spine were body weight (5%), and ER PvuII genotype (2.8%) whereas other predictors at the hip were weight (3.4%), age (2.9%) alcohol intake (2%) and ER PvuII allele (4.1%). Birthweight was not found to be a significant predictor of BMD at either the spine or hip. Interestingly, of all the polymorphisms studied, only COLIA1 genotype was found to be a significant predictor of birthweight in women accounting for 4% of the variance. We conclude that peak bone mass is regulated by an overlapping, but distinct set of environmental and genetic influences which differ in men and women. Much of the variance in peak bone mass was unexplained by the variables studied here however, suggesting either that most of the genes which regulate BMD remain to be discovered, or that major environmental influences on BMD exist that have not yet been identified. BONE REMODELLING IN CELIAC DISEASE: AN IN VITRO STUDY M. Longo1*, A. Taranta1, M. L. Bianchi2, S. Saraifoger2, M. T. Bardella3, A. Teti4 1Department of Histology and Medical Embryology, University of Rome 'La Sapienza', Rome, Italy 2Bone Metabolic Unit, Istituto Auxologico Italiano - IRCCS 3Cattedra di Gastroenterologia - IRCCS Ospedale Maggiore, Milan, Italy 4Department of Experimental Medicine, University of L'Aquila, Italy Celiac disease (CD), an inflammatory disorder of the small bowel due to gluten intolerance, is often associated with low bone mass. The mechanisms altering bone remodelling in celiac patients are still poorly understood. To explore whether CD affects bone formation, sera from patients were tested for their ability to influence human osteoblasts. Three groups of subjects were selected: (1) CD patients on gluten-free diet (GFD) for at least 2 years, in stable conditions, with no sign of disease activity and negative to anti-gliadin and anti-endomysium antibodies; (2) untreated, newly diagnosed CD patients; and (3) age-matched healthy donors. Human osteoblasts were obtained from surgical specimens of healthy subjects and exposed to 5% sera. 3H-thymidine incorporation revealed that sera from both groups of CD patients stimulated osteoblast proliferation (p<0.01 vs. controls). Histochemical and biochemical analyses showed significantly higher alkaline phosphatase activity upon exposition to sera from untreated patients (p<0.005). Consistently, matrix mineralization was also increased, in a semiquantitative analysis, by the sera of CD patients, with the highest levels in the untreated patient group. These data hint to osteoblast stimulation rather than inhibition in CD subjects, possibly due to released factors enhancing their function. On the basis of these observations, we analysed whether CD-sera - treated osteoblasts stimulated bone resorption through the release of cytokines or other growth factors influencing osteoclast function. Transcriptional expression of the osteoclast stimulating cytokines IL-1 and IL-6, and of the specific osteoclast inhibitory factor, osteoprotegerin (OPG), was evaluated, and showed similar mRNA levels for IL-1 and IL-6 in all groups, while OPG mRNA was significantly reduced in the osteoblast exposed to sera of untreated patients (p<0.01). This suggests a partial suppression of the OPG-mediated block of osteoclast function, which may increase bone resorption. We therefore tested the sera for altered osteoclast formation in cultures of human peripheral blood monocytes and observed, consistently, that sera from CD patients increased osteoclastogenesis relative to healthy donors. We conclude that systemic factors may contribute to bone loss in CD patients, partly due to osteoblast-mediated enhancement of osteoclast bone resorption. WOMEN WITH ANTIBODIES AGAINST HELICOBACTER PYLORI HAVE INCREASED RISK OF OSTEOPOROTIC FRACTURES B. L. Langdahl1*, J. M. Hansen2, M. K. Møller1, H. Nielsen2, L. Østergaard3 1Dept. of Endocrinology, Aarhus University Hospital, Aarhus, Denmark 2Dept. of Infectious Diseases, Aalborg Hospital, Aalborg, Denmark 3Dept. of Infectious Diseases, Aarhus University Hospital, Aarhus, Denmark Osteoporosis is a multifactorial disease. From epidemiological studies it is known that thin women have an increased risk of developing osteoporotic fractures. Chronic infection with Helicobacter pylori (H. Pylori) has been associated with various extra-gastrointestinal disorders, such as growth retardation in children and cardiovascular disease. We therefore wanted to investigate if prevalence of antibodies against H. Pylori is associated with osteoporotic fractures. We examined 79 female patients (mean age: 65.4±6.3 years) with osteoporosis defined as having at least one low-energy vertebral fracture and low bone mineral density (BMD) and 79 healthy female controls (mean age: 64.2±7.0 years). Serum samples from each individual were examined for antibodies against H. Pylori by a latex agglutination test. BMD was examined by DXA at the lumbar spine and at the femoral neck. A total of 38.0 % (30/79) osteoporotic women had antibodies against H. Pylori compared with 22.5 % (17/79) of agematched normal women (chi-sq=5.12, p=0.02). The oddsratio for having an osteoporotic fracture if seropositive for H. Pylori is 2.23 (1.11-4.51). BMD corrected for age was 0.821±0.158 g/cm2 (mean±SD) at the lumbar spine in individuals with antibodies against H. Pylori (n=47) compared with 0.861±0.162 g/cm2 in individuals without (n=111), p=0.16. At the femoral neck the corresponding values were 0.683±0.098 g/cm2 and 0.709±0.107 g/cm2, respectively (p=0.16). In conclusion, seropositivity for H. Pylori infection is associated with increased risk of osteoporotic fractures and a tendency towards reduced bone mass in the spine and the hip. PREGNANCY AND LACTATION HAVE NO LONG TERM EFFECTS UPON BONE DENSITY IN A HEALTHY POPULATION: A TWIN STUDY J. D. Wark1*, L. Paton1, J. Alexander1, C. Margerison1, M. Frame1, B. Kaymakci1, C. Nowson2 1Department of Medicine, Royal Melbourne Hospital, University of Melbourne, Australia 2School of Health Sciences, Deakin University, Australia Pregnancy and lactation impose a high maternal calcium demand. To determine whether these stresses have long-term effects upon bone, we sought differences in bone mineral density (BMD) in healthy female twin pairs, (1) who differed in their absolute number of pregnancies beyond 20 weeks, and (2) in a cross- sectional analysis of 1354 individuals twins and their relatives. Study 1 comprised 498 female twin pairs, mean age 42(15.0) years of whom 83 pairs were discordant for ever being pregnant. Subjects in study 2 were grouped according to the number of pregnancies: group A, no pregnancy (n=426), group B, 1 or 2 pregnancies (n=455) and group C, 3 or more pregnancies (n=473). BMD at the lumbar spine (LS) and total hip (HP) and total body (TB) bone mineral content (BMC) were measured. Where appropriate, BMD was adjusted for age, height and fat mass. Study 1: No significant within-pair differences were observed in BMD at the LS, HP or in TB BMC in twins discordant for ever being pregnant (paired t-tests). Pairs were grouped according to the difference in number of pregnancies (range:0-6). Subjects were also divided into three groups: equal number of pregnancies, different by 1 or 2 pregnancies, or different by 3 or more. No within-pair difference in unadjusted/adjusted HP BMD, LS BMD or TBBMC was observed with the difference in number of pregnancies. Study 2: Cross-sectionally, group B and group C subjects had higher adjusted LS BMD (3.0% and 3.2% respectively, both p=0.001) and adjusted TB BMC (2.7% and 3.1%, respectively, both p<0.001) compared with non-parous subjects (group A). Adjusted HP BMD was greater in group C compared with group A (2%, p=0.018). Parous women who breastfed had higher adjusted TB BMC (3.1%, p=0.005) and a marginally higher adjusted HP BMD (2.5%, p=0.04) than parous non-breastfeeders. There was some evidence from the cross-sectional analysis for a positive association between parity and BMD, but there was no observed within-pair difference in bone mineral measures in twins discordant for ever being pregnant or for the number of pregnancies. We detected no long-term detrimental effect of pregnancy or breastfeeding on BMD. A PHOSPHONOCARBOXYLATE ANALOGUE OF RISEDRONATE (NE10790) INHIBITS RAB PRENYLATION IN OSTEOCLASTS IN VIVO AND DISRUPTS THE MEMBRANE LOCALIZATION OF RAB PROTEINS IN OSTEOCLASTS IN VITRO F. P. Coxon1*, E. Mules2, M. C. Seabra2, F. H. Ebetino3, M. J. Rogers1 1Department of Medicine & Therapeutics, University of Aberdeen, Aberdeen, UK 2Department of Cell & Molecular Biology, Imperial College, London, UK 3Procter & Gamble Pharmaceuticals, Mason, Ohio, USA Nitrogen-containing bisphosphonates such as risedronate act by inhibiting farnesyl diphosphate (FPP) synthase, thereby preventing the synthesis of isoprenoid lipids required for the prenylation of small GTPases in osteoclasts. We recently found that a weakly anti-resorptive analogue of risedronate, NE10790, selectively prevents prenylation of Rab GTPases in cells in vitro by inhibiting Rab geranylgeranyl transferase (Rab GGTase). NE10790 has no effect on FPP synthase, farnesyl transferase (FTase) or GGTase I and therefore does not affect the prenylation of other proteins. We have now investigated the effects of NE10790 on rabbit osteoclasts in vivo. Rabbits were subcutaneously injected with 2.1 mg P/kg NE10790. After 24 hours, osteoclasts were purified from a crude preparation of bone cells by immunomagnetic bead separation with an anti-vitronectin receptor (VNR) antibody. Using an in vitro prenylation assay, we found that NE10790 caused the accumulation of unprenylated Rabs in VNR-positive osteoclasts, but not in VNR-negative cells. We have also investigated the effect of NE10790 on subcellular localization of Rab6 by immunostaining and western blotting. Rab6 localized to the Golgi, seen as distinct perinuclear staining in osteoclasts and punctate staining in J774 cells. Treatment of osteoclasts or J774 cells with either NE10790 or risedronate disrupted this localization, resulting in diffuse staining for Rab6 throughout the cell. By contrast, FTI-277 and GGTI-298 (inhibitors of FTase and GGTase I, respectively) had no effect. For western blotting, lysates were fractionated by ultracentrifugation or Triton X-114 separation. Rab6 was almost completely localized to the membrane fraction in untreated J774 cells and osteoclasts, but accumulated in the cytoplasmic fraction in cells treated with NE10790 and risedronate. By contrast, only risedronate caused the accumulation of Rac (which is prenylated by GGTase I) in the cytosol. These data suggest that the anti-resorptive effects of NE10790 are likely due to inhibition of Rab prenylation in osteoclasts, resulting in the cytoplasmic accumulation of unprenylated Rab proteins. TGF-BETA INDUCES HUMAN OSTEOCLAST FORMATION AND BONE RESORPTION IN THE ABSENCE OF SOLUBLE RANKL I. Itonaga, A. Sabokbar, O. Kudo, N. A. Athanasou* Nuffield Department of Orthopaedic Surgery, Nuffield Orthopaedic Centre, University of Oxford, Oxford, UK Osteoclast progenitors can differentiate into mature bone resorbing osteoclasts in the presence of macrophage colony stimulating factor (M-CSF) and RANK ligand (RANKL), which is expressed on bone stromal/osteoblastic cells. Osteoprotegerin (OPG) inhibits RANKL-induced osteoclast formation and bone resorption. It has been reported that tumour necrosis factor-alpha (TNF-alpha), a potent cytokine involved in regulation of osteoclast activity via a primary effect on osteoblasts, can directly (in the presence of M-CSF) induce the differentiation of osteoclast progenitors into mature osteoclasts. These studies revealed that TNF- alpha-induced osteoclast formation is independent of RANK/RANKL interaction. In the present study we sought to determine whether transforming growth factor beta (TGF-beta), another powerful modifier of bone resorption, can similarly induce osteoclast formation and bone resorption in vitro. To address this, mononuclear cells were isolated from peripheral blood of healthy volunteers and cultured for up to 24 days on glass coverslips and dentine slices in the presence of: (i) RANKL and M-CSF; (ii) TGF-beta, M-CSF±OPG; (iii) TGF-beta, M-CSF±anti TNF-alpha antibody. In some experiments, CD14+ mononuclear cells isolated using Mini MACS CD14 Micro Beads cell sorter were cultured. The extent of osteoclast formation and bone resorption was determined by generation of TRAP- positive multinucleated cells, actin ring formation and lacunar resorption on dentine slices. We noted that addition of TGF-beta, in the presence of M-CSF, but in the absence of RANKL, was sufficient to induce the formation of TRAP-positive multinucleated osteoclast-like cells which were capable of forming actin ring and lacunar resorption on dentine slice in vitro. This osteoclast formation was also induced in CD14-sorted mononuclear cell cultures. The addition of OPG or anti TNF-alpha antibody to the cultures containing TGF-beta did not inhibit osteoclast formation and bone resorption, thus suggesting that TGF-beta induces osteoclast formation in manner independent of the RANK/RANKL or TNF-alpha mechanism. Our results indicate that TGF-beta, which is abundant in bone and thought to have potent effects on bone metabolism, can directly induce osteoclast precursors to differentiate into active bone resorbing osteoclasts. PATTERNS OF EXPRESSION OF CHEMOKINES AND THEIR RECEPTORS IN OSTEOCLASTS J. M. Lean*, C. Murphy, K. Fuller, T. J. Chambers St George's Hospital Medical School, London, UK Although much has been learned recently of the mechanisms by which osteoclast differentiation and function are regulated, much less is known of the factors that regulate their migration and localisation. In related cell types, chemokines play a major role in these processes. We therefore systematically tested the expression of RNA for chemokine receptors and their ligands by osteoclasts. Because bone is the natural substrate for osteoclasts and may influence osteoclast behaviour, we also tested expression on bone slices. Quantitative RT-PCR using real time analysis with sybr green was therefore performed on RNA isolated from bone marrow cells after incubation with M-CSF with/without RANKL, on bone or plastic, and assayed for known murine chemokines and their receptors. As a measure of osteoclast differentiation TRAP expression was also measured. We found that RANKL induced a 100-fold increase in TRAP in cells grown on plastic. This was increased a further 10-fold when grown on bone. RANKL reduced the expression of RNA for the Fc receptor (CD16) and c-fms. The chemokines most highly expressed by bone marrow cells incubated on tissue culture plastic with M-CSF and RANKL were: CX3CL (neurotactin/fractalkine), CCL2(MCP-1), CCL3(MIP1-alpha), CCL6(MRP-1), CCL7(MCP3), CCL9(MIP1-gamma), CCL12(MCP-5), CXCL4(PF4) and CXCL6. The most abundant chemokine receptor was CCR1, followed by CX3CR, CCR3, CCR5 and CXCR4. However, we found that the major effect of RANKL was to reduce RNA for chemokines most strongly associated with inflammation. Thus, CCL2(MCP-1), CCL3(MIP1-alpha) and CCL6(MRP-1) were strongly inhibited by RANKL. Cells incubated on bone showed even greater inhibition, with expression of CCL7(MCP3) and CXCL9(MIG) becoming undetectable. RANKL increased expression of CCL9(MIP1-gamma) (up to 50-fold), CCL22(MDC) (up to 10-fold) and CXCL13(BCA-1) (from undetectable). RANKL also upregulated the expression of receptors CCR1 and CCR3. There was a highly significant correlation between the number of copies of mRNA for TRAP and CCL9(MIP1-gamma). The expression of CCL9(MIP1-gamma) and CCL22(MDC) and their corresponding receptors suggest an autocrine or paracrine role for these chemokines in osteoclasts. Initial studies have shown no effect of these agents on osteoclast differentiation or resorption, but both agents stimulate cytoplasmic motility, suggesting that they may play a role in osteoclastic migration/localisation. STIMULATION OF RANKL-DEPENDENT OSTEOCLAST FORMATION BY INFLAMMATORY (RHEUMATOID/CRYSTAL) ARTHRITIS SYNOVIAL FLUID L. Danks, A. Sabokbar, I. Itonaga, N. A. Athanasou* Nuffield Department of Orthopaedic Surgery, Nuffield Orthopaedic Centre, University of Oxford, Oxford, UK Periarticular bone resorption is a feature of arthritis due to crystal deposition and rheumatoid disease. The inflammatory synovial fluid in these conditions contains numerous inflammatory cells and humoral factors which promote osteoclast formation. In order to determine the effect of inflammatory synovial fluid (SF) on human osteoclast formation in this study, SF from rheumatoid arthritis (RA), pyrophosphate crystal arthritis (PPA) and osteoarthritis (OA) patients was added to cultures of peripheral blood mononuclear cells (PBMCs) in the presence and absence of RANKL (30ng/ml) and M-CSF (25ng/ml). The extent of osteoclast formation and lacunar resorption was assessed by tartrate resistant acid phosphatase (TRAP), vitronectin receptor (VNR) and a lacunar resorption assay. The number of TRAP and VNR positive cells was dose dependently increased following the addition of 1, 5 and 10% SF to PBMC cultures. Mononuclear TRAP and VNR positive cells also were noted in PBMC cultures incubated with 10% SF. The mean percentage area of lacunar resorption was significantly increased in cultures incubated with 10 % RA and PPA SF compared to OA SF (p=0.03 and p=0.02, respectively). SF alone was incapable of supporting osteoclast formation from PBMCs. These results indicate that inflammatory synovial fluid stimulates RANKL-induced osteoclast formation and lacunar resorption. Some of these cultures consisted entirely of mononuclear TRAP and VNR positive cells, indicating that multinuclearity is not a sufficient marker for osteoclast formation. The extent of osteoclast formation and lacunar resorption was consistently extensive in these cultures (i.e. 90-100% area resorption). Our findings suggest that factors present in inflammatory synovial fluid of RA and PPA patients is likely to be of pathogetic significance in periarticular bone resorption. MET, A NOVEL MODULATOR OF ESTROGEN INDUCED TRANSCRIPTION S. M. Colley1*, A. Flynn2, M. Norman2, D. Wynick2, J. H. Tobias1 1Rheumatology Unit, Division of Medicine, University of Bristol, Bristol, UK 2University Research Centre for Neuroendocrinology, University of Bristol, Bristol, UK High-dose estrogen is known to stimulate osteoblast activity in postmenopausal women as well as rodent models. To investigate the molecular mechanisms underpinning this effect, we compared the gene expression profiles of mRNA isolated from the tibiae of adult mice treated with either estrogen or vehicle for 4 days by subtractive hybridisation analysis. During the course of this investigation, a gene fragment showing no significant homology with previously characterised sequences was isolated and found to detect a single 3.8Kb transcript by northern analysis. A full length cDNA homologous to this gene was generated by EST directed Rt-PCR using bone marrow cDNA as the source of template. Sequence analysis of the cloned products revealed it coded for a 1031 amino acid protein. This peptide contains a SAF Box DNA binding motif, an RNA binding domain and shares an overall identity of 34% with the estrogen suppresser SAF-B/HET/ HAP. SET has previously been reported bind the estrogen receptor directly, act as a repressor both in breast and bone cell lines and augment the anti-estrogen effects of the SERM Tamoxifen. When the cDNA we have isolated was expressed as a fusion product with enhanced yellow fluorescent protein, it was found to localise exclusively to the nucleus. It was further observed that its expression in MCF-7 cells resulted in a dose dependent reduction by up to 50% in estrogen induced ERE luciferase reporter gene expression. From these data we conclude that this novel sequence represents a previously undescribed Modulator of Estrogen induced Transcription, which we refer to as MET, that is likely to play a significant role in modulating estrogen responses in bone and other tissues. MODULATION OF HUMAN ER ALPHA F PROMOTER ACTIVITY BY A PROTEIN KINASE C - DEPENDENT MECHANISM IN HUMAN DIFFERENTIATED OSTEOBLAST-LIKE CELLS M. Longo1,3*, V. De Luca1,3, S. Denger2, G. F. Caselli3, F. Gannon2, A. Teti3, S. Migliaccio3,4 1Department of Histology & Medical Embryology, University 'La Sapienza', Rome, Italy 2EMBL, Heidelberg, Germany 3Department of Experimental Medicine, University of L'Aquila, Italy 4Department of Medical Physiopathology, University 'La Sapienza', Rome, Italy The gene for human estrogen receptor alpha (ERalpha) gives rise to six different mRNAs driven in a tissue-specific manner by six different promoters (A-F) and translated into a single protein. The F-promoter has been described as specific for the bone tissue, but its modulation has not been clarified yet. To elucidate the role of ERalpha promoters in bone physiology and its regulation, we generated human osteoblast-like cells (SAOS-2) stably transfected with expression vectors carrying human ERalpha A, B, and F promoter sequences upstream of the luciferase reporter gene. No luciferase activity was detected in cells containing promoters A and B relative to cells transfected with empty vectors. In contrast, high basal luciferase activity was found in two clones carrying the F promoter. Our previous results have evidenced significant modulations in ERalpha levels and activity upon confluence-induced differentiation in osteoblasts, involving close interaction with PKC isoforms. This prompted us to analyse F promoter activity during osteoblast differentiation and its possible dependence on PKC isoform expression and/or activity. F promoter activity was evaluated in sub-confluent (proliferating) and post-confluent (differentiated) cells, and found to be constantly up-regulated in differentiated versus proliferating osteoblasts, in identical serum conditions. A two/three-fold higher luciferase activity was detectable 24 hr after confluence, with no significant increase up to 96 hr thereafter. Overnight treatment of post-confluent cells with 10-8M 12-O-tetradecanoylphorbol-13-acetate (TPA), known to abolish PKC isoform expression, resulted in a marked decrease of F promoter activity up to the level detected in proliferating counterparts, which strongly suggests a PKC-dependent up-regulation of the promoter upon differentiation. We also observed that in proliferating cells identical TPA treatments induced a slight increase of promoter activity. In contrast, during proliferation the promoter was unaffected by treatment with calphostin C, a compound inhibiting the activity, but not the expression, of most PKCs, while decreased luciferase activity relative to untreated counterparts was again observed in differentiated cells, but to a lesser extent. These data strongly suggest tight dependence on intact PKC pathways for F promoter regulation, possibly involving both kinase activity -independent and - dependent PKC functions. ACTIVATION OF THE BMP-6 PROMOTER BY THE ESTROGEN RECEPTOR S. M. Colley1*, D. B. Ong1, S. Kitazawa2, M. Norman3, D. Wynick3, J. H. Tobias1 1Rheumatology Unit, Division of Medicine, University of Bristol, Bristol, UK 2Division of Molecular Pathology, Kobe University Graduate School of Medicine, Kobe, Japan 3University Research Centre for Neuroendocrinology, University of Bristol, England High dose estrogen is known to induce osteogenesis in the long bones of adult mice. In searching for factors that play a role in this process, we observed a significant increase in Bone Morphogenetic Protein 6 (BMP-6) transcripts in mRNA isolated from femora of animals treated with 17 beta-estradiol (E2) compared to vehicle (Plant et al. 2001). Based on evidence that E2 increases BMP-6 expression in osteoblasts in a cell autonomous manner in vitro (Rickard et al 1998), we explored the mechanisms by which estrogen induces osteogenesis by studying the regulation of BMP-6 expression in osteoblasts. Initially, the activity of a 1.2Kb BMP-6 promoter sequence coupled to a luciferase reporter (Tamada et al 1998) was compared between ROS and SMER osteosarcoma cells, of which the latter represent ROS cells stably transfected with estrogen receptor (ER) alpha. Basal BMP-6 reporter activity was found to be considerably higher in SMER versus ROS cells, with no further increase after the addition of E2. This suggestion that BMP-6 promoter activity is influenced by ER in a non-ligand dependent manner was confirmed by comparing reporter activity in ROS cells transiently transfected with the BMP-6 reporter and an ER alpha expression vector; co-transfection with ER alpha stimulated BMP-6 reporter activity by four-fold irrespective of the presence of E2. Equivalent findings were observed in liver and renal cell lines (HepG2 and COS cells respectively). Taken together, these results indicate that ER acts to increase the level of BMP-6 promoter activity in a non ligand dependent manner irrespective of cell background, as assessed using a construct based on the 1.2Kb proximal sequence. This suggests that ligand-dependent activation of BMP-6, as observed in previous in vitro and in vivo studies of the effects of estrogen on BMP-6 mRNA, involves an interaction between the proximal portion of the BMP-6 promoter and more distal sites yet to be identified. Plant A. et al (2001) In press: J. Bone Miner. Res Rickard D. (1998) J. Clin. Invest. 101, 413-22. Tamada H. (1998) Biochim. Biophys. Acta 1395, 247-51. MICE DEFICIENT IN 11BETA-HYDROXYSTEROID DEHYDROGENASE TYPE 1 LACK BONE MARROW ADIPOCYTES AND MAINTAIN NORMAL BONE FORMATION J. Justesen1*, L. I. Mosekilde3, K. Stenderup1, M. Holmes4, J. J. Mullins4, J. R. Seckl4, E. F. Eriksen1, M. Kassem1,2 1University Department of Endocrinology and Metabolism, University Hospital of Aarhus, Denmark 2University Department of Endocrinology and Metabolism, University Hospital of Odense, Denmark 3Department of Cell Biology, Institute of Anatomy, University of Aarhus, Denmark 4Molecular Medicine Centre, Western General Hospital, Edinburgh, UK The physiological effects of glucocorticoids (GCs) on the skeleton are complex and poorly understood. The cellular activity of GCs is regulated at a pre- receptor level by 11beta-hydroxysteroid dehydrogenases (HSDs). The type 1 isozyme catalyses the formation of active cortisol (corticosterone) within cells from circulating inert 11-keto forms. This isoform is expressed in bone. Thus, we studied the bone phenotype of HSD1 deficient (HSD1-/-) mice and wild type (Wt) animals using bone histomorphometry applied to proximal tibial metaphysis and vertebrae. In addition, we studied the cellular characteristics of cultured osteoblastic cells obtained from femurs, calvariae and bone marrow from both groups in vitro. See table for histomorphometric results. Osteoblastic cells from both
HSD1-/- and Wt mice exhibited similar growth characteristics, alkaline
phosphatase activity and in vitro mineralization. They were able to form adipocytes in
presence of the dexamethasone, indomethacin, and IBMX. We are currently investigating the
in vivo bone formation potential of the cells.
ROLE OF BETA-ARRESTINS IN THE REGULATION OF PARATHYROID HORMONE (PTH) ACTIVITY IN VITRO AND IN VIVO S. L. Ferrari1*, D. Sternlight2, D. Pierroz1, R. Lefkowitz3, M. Bouxsein2 1Div. of Bone Diseases, University Hospital, Geneva, Switzerland 2Orthopedic Biomechanics Laboratory, Beth Israel Deaconess Medical Center, Boston, MA, USA 3Howard Hughes Medical Institute Laboratories, Duke University Medical Center, Durham, NC, USA Beta-arrestin 1 and 2 (b-arr1 and b-arr2) are cytoplasmic adaptor molecules involved in the regulation of signaling and intracellular trafficking of many G protein-coupled receptors. We have previously reported that recruitment of b-arr2 to the cell membrane upon activation of human PTH/PTH-related protein (PTHrP) receptor (PTH1Rc) is responsible for desensitization of PTH (and PTHrP)-stimulated cAMP signaling and for internalization of ligand-receptor complexes. Using a fully automated fluorescence microscope coupled to an ultrasensitive digital CCD camera, we have now generated a movie showing PTH- stimulated internalization and fusion of membrane vesicles (endosomes) containing the PTH1Rc, b-arr2 and b-arr1 in an isolated living cell. The importance of this phenomenon in the regulation of cAMP signaling is further demonstrated by the development of PTHrP-derived agonists that don't mobilize arrestins nor internalize PTH1Rc, thereby triggering prolonged activation of adenylyl cyclase in vitro. In order to evaluate the influence of beta-arrestins on PTH biological activity in vivo, we have started to investigate mice null for beta-arrestin 2. Adult b-arr2 KO mice and their wild-type (wt) littermates show no gross differences in their apparent phenotype nor in skeletal morphology on conventional radiographs. However, evaluation of bone mineral density (BMD) by pDXA (PIXImus) and of skeletal microarchitecture by high resolution micro-computed tomography and histomorphometry suggests that, compared to wild type littermates, six-months-old b-arr2 KO mice have lower BMD (-9%) and altered trabecular microarchitecture (BV/TV, -17%, connectivity, -55%). These osteoporotic features are compatible with a sustained (continuous) and catabolic effect of endogenous PTH in b-arr2 KO mice. In order to further examine this possibility, the development of the bone phenotype in b-arr2 KO mice is currently being surveyed by longitudinal assessment of BMD at 4, 8, 12 16 and 20 weeks. Moreover, to directly evaluate whether PTH has a prolonged and sustained biological activity in these mice, the calcemic response (at 2, 6, 12 and 24 hours) to acute PTH administration (500microg/kg) is currently being measured. Altogether, these studies will provide further evidence for the role of beta-arrestins in the regulation of PTH biological activity in vitro and in vivo. BONE MORPHOGENETIC PROTEIN-7 PROMOTES TENDON GRAFT INTEGRATION IN THE RECONSTRUCTION OF THE ANTERIOR CRUCIATE LIGAMENT R. Mihelic1*, V. Kusec2, D. Bobinac3, S. Zoricic3, M. Jelic4,5, M. Pecina5, S. Vukicevic4 1Orthopaedic Hospital, Lovran, Croatia 2Clinical Institute of Laboratory Diagnosis, Clinical Hospital Centre, Zagreb, Croatia 3Department of Anatomy, School of Medicine, University of Rijeka, Croatia 4Department of Anatomy, School of Medicine, University of Zagreb, Croatia 5Department of Orthopaedic Surgery, School of Medicine, University of Zagreb, Croatia Reconstruction of the anterior cruciate ligament requires an early integration of the tendon graft in the bone tunnel. However, an undesired outcome, as widening of the bone tunnel (whipe-screen effect) has been described. It has been well documented that bone morphogenetic proteins (BMP) induce bone formation and regeneration of other skeletal-related tissues when applied locally. In this study BMP-7 was injected with the tendon graft in the bone tunnel of the ovine femur and tibia in order to investigate its effect upon graft integration. Ten sheep (1 year old, male, 37 kg) were operated under general anaesthesia, tunnels of 4.5 mm in diameter were drilled in the right femur and tibia, and approximately 5 cm of the m. peroneus tertius tendon inserted and fixed. In the experimental group 25 microg of BMP-7 (OP-1) in 0.2 mL buffer was applied in the bone tunnel of 5 sheep. Helistat was used as carrier in three animals. In the control group (n=5) the entire procedure was carried out identically but without the addition of BMP. Animals were sacrified after three weeks, operated knees were excised and processed for histologic analysis. The outcome was satisfactory in all experimental animals, which was also assessed by X-rays. In all specimens the tendon fibres of the epitendineum invaded the surrounding bone marrow space. Bone formation and remodelling was enhanced with thicker trabeculae on the bone-tendon interface. In the experimental group areas of woven bone with large lacunae and dense trabecular structure were present. This was less pronounced in the specimen without Helistat. These areas of newly formed bone differed in size and were located at different sites along the bone tunnel. This was either not observed or present in a much less extent in control specimen. Invasion of the tendon fibrous tissue into the bone marrow space was significantly (p<0.01) greater for the experimental group (approx. 600- 1800 mm) than in the control animals (approx. 400-800 mm). These results suggest that BMP-7 contributes to better integration of the tendon possibly by stimulating outgrowth of tendon fibers into the marrow space and by inducing rapid new bone formation. THE MULTIPLE FUNCTIONS OF OSTEOBLAST-STIMULATING FACTOR-1 IN BONE DEVELOPMENT H. I. Roach*, R. S. Tare, R. O. C. Oreffo, N. M. P. Clarke University Orthopaedics, University of Southampton, General Hospital, Southampton, UK In search for anabolic agents that increase osteoblast activity, we examined the functions of osteoblast-stimulating factor-1 (OSF-1), an extracellular matrix- associated growth/ differentiation factor. We studied bone development up to 30 weeks of age in transgenic mice, which carried six extra copies of the human OSF- 1 gene, and also examined the effects of OSF-1 on osteogenic cells and cartilage explants in vitro. OSF-1 was synthesized by osteoblasts at an early stage of differentiation, prior to late markers of the osteoblast phenotype, i.e. bone sialoprotein and matrix mineralization. In vivo, OSF-1 was localized (using immunocytochemistry) to sites of new bone formation, i.e. the periosteum, at the groove of Ranvier, and in the primary spongiosa. The factor was secreted and stored in the bone matrix, presumably for future use during the remodeling process. In vitro, OSF-1 increased the osteogenic differentiation of mesenchymal stem cells in mouse bone marrow cultures by ~50% at a concentration of only 10 pg/ml, which was considerably lower than the concentrations of BMP-2 required to achieve a similar effect. However, OSF-1 did not have BMP-like osteoinductive capacity, since it was unable to convert the differentiation pathway of C2C12 pre- myoblastic cells into the osteoblast lineage, whereas BMP-2 was able to do so. Overexpression of OSF-1 in transgenic mice did not result in larger animals. However, the calcium content/ mg bone was ~10% higher in the male transgenic mice. In the transgenic (but not control) mice, OSF-1 was localized in the growth plates and in articular chondrocytes, some of which also synthesized type I collagen. To confirm that excess OSF-1 had provided the stimulus for synthesis of this bone-type collagen, cartilage explants were cultured with exogenous OSF-1 (50ng/ml). This also induced chondrocytes to synthesize type I collagen. Thus the functions of OSF-1 in bone development range from enhancing osteogenic differentiation and increasing bone mineral content to, perhaps, inducing osteogenic differentiation of chondrocytes. The results suggest that OSF-1 is a factor important for bone formation and remodelling. PROTEIN KINASE D MEDIATES ACTIVATION OF MAP KINASES P38 AND JNK INDUCED BY Gq PROTEIN-COUPLED RECEPTORS IN OSTEOBLAST-LIKE CELLS J. Caverzasio*, J. Lemonnier, Ch. Gayor Dept of Medicine, University Hospital, Geneva, Switzerland G protein-coupled receptors (GPCRs) are transducers of the anabolic effect of potent osteotropic factors such as parathyroid hormone and fluoride. Recent studies suggest the implication of several mitogen-activated protein kinases (MAPKs) in the regulation of osteoblastic cell activity by GPCRs. In this study, we investigated the effect of PGF2alpha (PGF2), a GqPCR agonist, on activation of MAPKs in MC3T3-E1 (E1) osteoblast-like cells and the molecular mechanism involved in activation p38 and JNK by PGF2. PGF2 (1 microM) activated the three MAPKs Erk, JNK and p38 with different kinetics. Erk activation was rapid, maximal at 5 min and lasted about 10 min whereas JNK and p38 (JNK/p38) activation was apparent after 15 min, maximal at one hour and lasted about 3 h. Two PKC inhibitors, Go6983 and Go6976 differentially blunted activation of MAPKs induced by PGF2. G06983, an inhibitor of typical PKCs, completely blocked Erk activation without influencing activation of JNK and p38 whereas Go6976 had the opposite effect. Go6976 is a potent inhibitor of protein kinase D (PKD) (also called PKCmu) and the role of this kinase in mediating activation of JNK/p38 by PGF2 was therefore investigated. Kinetic analysis indicated that PGF2 induced a rapid translocation of PKD from the cytosol to a membrane fraction wherein it became phosphorylated on several serine residues (p-PKD) before returning in the cytosol. Interestingly, the return of p-PKD in the cytosol correlated with activation of JNK/p38 by PGF2 and this return was blocked by Go6976. Moreover, in E1 cells stably transfected with a kinase dead PKD mutant (K612W) and having a normal growth, activation of JNK/p38 by PGF2 was impaired (80-90% inhibition). In conclusion, GqPCRs in osteoblast-like cells can stimulate the three MAPKs Erk, JNK and p38 by different mechanisms. Erk activation is mediated by a typical PKC isoform whereas the newly described PKD mediates activation of JNK and p38. The molecular mechanism by which PKD activates JNK and p38 is complex. Indeed, it involves the translocation of PKD from the cytosol to a membrane fraction for its phosphorylation and activation before returning in the cytosol for the stimulation of the JNK and p38 pathways. FIBROBLAST GROWTH FACTOR-2 INHIBITS APOPTOSIS IN HUMAN CALVARIA OSTEOBLASTS THROUGH A PKC- DEPENDENT PATHWAY F. Debiais, E. Hay, P. J. Marie* INSERM U349 Affiliated CNRS, Paris, France We previously showed that Fibroblast Growth Factor-2 (FGF-2) controls osteoblast human calvaria cells through protein kinase C (PKC) and src signalling pathways. In this study, we determined the effects of FGF-2 on apoptosis in human calvaria osteoblasts and we analysed the underlying signaling pathways. Apoptosis was determined in normal human calvaria osteoblasts and in immortalized human neonatal calvaria (IHNC) cells by terminal deoxynucleotidyl transferase-mediated nick-end labeling (TUNEL) or trypan blue staining. Treatment with rhFGF-2 (50 ng/ml) for 96 h increased the number of TUNEL-positive cells in primary human calvaria osteoblasts cultured in serum-deprived medium (P<0.02). In contrast, rhFGF-2 decreased the number of apoptotic cells at early time-points, from 6 to 24 h of treatment (P<0.05). A similar dual effect of FGF-2 on apoptosis was found in IHNC cells. The analysis of mechanisms involved in this dual effect showed that rhFGF-2 (50 ng/ml) decreased caspase-2 activity at 15-48 h and increased this activity at 96 h in serum-deprived IHNC cells (P<0.05). Consequently, rhFGF-2 significantly reduced effector caspase (-3,-6,-7) activity at 15-48 h and increased this activity at 96 h (P<0.05). In contrast, rhFGF-2 did not affect caspase-8 and caspase-9 activities in IHNC cells at any time point. This shows that FGF-2 induced a biphasic effect on apoptosis in human osteoblasts by modulating caspase-2 activity. We then analysed the signaling pathway involved in the anti-apoptotic effect of FGF-2. Treatment of IHNC cells with the PKC inhibitor calphostin C (1 µM) abolished the inhibitory effect of rhFGF-2 on both caspase-2 and caspase-3 activities at 24 h (P<0.05). In contrast, the MAP kinases inhibitors PD 98059 and SB 203580, that inhibit Erk1,2, and p38 respectively, did not suppress the inhibitory effect of FGF-2 on caspase-2 and caspase-3 activities. These results show that 1) FGF-2 induces a dual effect on apoptosis in human osteoblasts through a selective modulation of caspase-2 activity which regulates effector caspases and DNA fragmentation, and 2) the early anti-apoptotic effect of FGF-2 on human calvaria osteoblasts is mediated by PKC signaling. This identifies a novel PKC-dependent anti-apoptotic effect of FGF-2 in human osteoblasts. INHIBITION OF OSTEOBLAST APOPTOSIS BY THROMBIN C. N. Pagel, C. Chinni, L. A. Abraham, E. J. Mackie* School of Veterinary Science, University of Melbourne, Parkville, Australia Maintenance of the skeleton is critically dependent on the availability of osteoblasts, which itself is determined by rates of osteoblast proliferation and apoptosis. The recent observation that steroid-induced osteoporosis is associated with osteoblast apoptosis has highlighted the importance of understanding the factors regulating this process. We demonstrated recently that thrombin is a survival factor for skeletal myoblasts, and instigated the current study to determine whether thrombin also inhibits osteoblast apoptosis. Primary cultures of neonatal rat calvarial osteoblasts and human osteoblast-like (Saos-2) cells were deprived of serum for 24 h, then incubated in the presence or absence of thrombin for 36 h. The percentage of cells undergoing apoptosis was quantitated on the basis of nuclear morphology and the presence of DNA strand breaks (detected by the TUNEL method). In both cell types, thrombin caused a significant reduction in the proportion of cells undergoing apoptosis. The effect was concentration-dependent with a maximal effect (>75% reduction below control levels) at 100 nM. The effect was dependent on thrombin's proteolytic activity, but could not be mimicked by urokinase or factor Xa. Three members of the protease-activated receptor (PAR) family, PARs-1, -3 and -4, have been identified as thrombin receptors. Although PAR-1 is known to be expressed by osteoblasts, thrombin's effect was not mimicked by a peptide activator of PAR-1. In osteoblasts from PAR-1-null mice, thrombin inhibited apoptosis as effectively as in osteoblasts from wildtype mice. In RT-PCR studies, Saos-2 cells were shown to express PAR-3 but not PAR-4, whereas primary osteoblast-like cells were shown to express PAR-4 but not PAR-3. Serum-deprived Saos-2 cells were treated with thrombin for 1 h, then the medium was replaced with fresh medium (without thrombin) which was collected 20 h later. This conditioned medium when applied to serum-deprived osteoblasts caused a significant inhibition of apoptosis. Medium from cells treated concurrently with thrombin and cycloheximide contained no thrombin-induced apoptosis inhibitory activity. These results indicate that thrombin inhibits osteoblast apoptosis through induction of a secreted inhibitor of apoptosis. The receptor mediating thrombin's effect has not yet been identified, but is clearly not PAR-1. COEXPRESSION OF BSP AND CBFA1/RUNX2 IN HUMAN MELANOMA: AN INCOMPLETE EPITHELIAL TO OSTEOGENIC CONVERSION AS THE LINK BETWEEN BSP EXPRESSION AND TUMOR INVASIVENESS? M. Riminucci1,2*, A. Corsi1,2, K. Peris3, L. W. Fisher4, S. Chimenti5, S. Licci1, P. Bianco1 1Department of Experimental Medicine and Pathology, La Sapienza University, Rome, Italy 2Department of Experimental Medicine, Laboratory of Pathology, Univeristy of L'Aquila, Italy 3Department of Biomedical Sciences, Division of Dermatology, University of L'Aquila, Italy 4Craniofacial and Skeletal Diseases Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD, USA 5Department of Dermatology, Tor Vergata University, Rome, Italy Ectopic expression of the osteoblast-specific protein Bone Sialoprotein (BSP) occurs in some malignant epithelial tumors capable of metastatic growth in the skeleton. In breast and prostate cancer,production of BSP has also been positively correlated with tumor progression. For this reason, the expression of BSP as a potential predictive marker is under intensive investigation in early, localized malignant tumors. However, the biological significance of BSP expression in cancer and its underlying mechanisms have remained elusive. We analyzed the expression of BSP in 17 human melanocytic tumors in vivo by immunolocalization. The expression of BSP was detected in 5/5 Clark's level IV melanomas and related lymph node metastasis and in one level III lesion with histological evidence of vascular invasion. By contrast, neither 2 level I nor 1 level II and 1 level III (without vascular invasion) melanomas expressed BSP. In cultures of two human melanoma cell lines (MeWo, SK) the pattern of BSP localization was determined by confocal immunofluorescence and compared to that observed in a rat osteosarcoma cell line (UMR 106-01-BSP) and in normal primary rat osteoblats. In all systems, BSP was immunolocalized to the Golgi apparatus and to secretory vesicles. By contrast, BSP-labelled focal adhesions were obvious in primary osteoblasts and absent both in osteosarcoma and melanoma cells. BSP mRNA was demonstrated in both melanoma cell lines by RT-PCR. These data demonstrated that human melanoma cells express BSP mRNA and protein as a reflection of an invasive behavior both in vivo and in vitro. We then showed that in both melanoma cell lines BSP expression was associated with the expression of CBFA1/Runx2, a pivotal transcriptional regulator of osteoblast - specific genes. This suggests that the expression of BSP in melanoma (and perhaps in other epithelial cancers) may represent an incomplete 'epithelial - to - osteogenic conversion' dictated by the ectopic expression of the CBFA1/Runx2 gene. Since CBFA1/Runx2 is involved in the transcriptional regulation of some matrix degrading enzymes involved in tumor invasion, the production of BSP (and other bone matrix proteins) by some tumors, and its unexplained link to tumor invasiveness would then be traced to a common regulatory mechanism. PROTEIN KINASE C PLAYS AN ESSENTIAL ROLE IN THE REGULATION OF HUMAN CALVARIA OSTEOBLAST PHENOTYPE BY FGF-2, FGFR-2 AND BMP-2 P. J. Marie*, F. Debiais, J. Lemonnier, Ph Delannoy, E. Hay INSERM U349 affiliated CNRS, Paris, France The formation of cranial bone requires the differentiation of osteoblasts from indifferentiated mesenchymal cells. The balance between osteoblast recruitment, proliferation, differentiation and apoptosis is essential for calvarial bone formation. However, the mechanisms controlling the human osteoblast phenotype during normal calvarial bone formation are poorly understood. Using human calvarial osteoblasts, we determined the mechanisms that regulate the differentiated osteoblast phenotype in human calvaria. We first showed that Fibroblast Growth Factor-2 (FGF-2) promotes osteoblast differentiation in human calvaria osteoblasts. Consistently, we found that constitutive activation of FGF receptor-2 (FGFR-2) activates the expression of alkaline phosphatase (ALP), type I collagen and osteocalcin gene expression in human calvaria cells. Using specific PKC inhibitors, we showed that this effect is mediated mainly by activation of protein kinase C (PKC) by FGFR-2 signaling. Bone Morphogenetic Protein-2 (BMP-2) is another factor involved in cranial bone development. We found that BMP-2 activates PKC activity and osteoblast gene expression in human calvaria cells. In addition, we showed that direct activation of PKC by phorbol ester promotes ALP activity, a marker of the osteoblast phenotype, in human calvaria cells. Finally, we investigated the implication of PKC in osteoblast apoptosis, a phenomenon which plays an important role in bone formation. We found that both FGFR-2 activation and BMP-2 increased apoptosis in human calvaria osteoblasts. Moreover, functional studies in human calvaria osteoblasts showed that apoptosis induced by both effectors is mediated by PKC activity, leading to the downstream activation of specific caspases. These studies indicate that PKC is an essential signaling molecule involved in the regulation of the human calvaria osteoblast phenotype by FGF-2, FGFR-2 and BMP-2. MUTATIONS IN TRANSFORMING GROWTH FACTOR-BETA1 CAUSE CAMURATI-ENGELMANN DISEASE BY AFFECTING EITHER SECRETION OR ACTIVATION OF THE PROTEIN K. Janssens1*, S. H. Ralston2, W. Van Hul1 1Center of Medical Genetics, University of Antwerp, Antwerp, Belgium 2Department of Medicine and Therapeutics, University of Aberdeen Medical School, Aberdeen, UK Camurati-Engelmann disease (CED) or Progressive Diaphyseal Dysplasia is a rare bone disorder, inherited in an autosomal dominant mode. Its main clinical symptoms are a waddling gait, pain in the lower and upper limbs, fatigue and reduced muscle mass. Radiologically, CED is characterised by hyperostosis of the diaphyses and metaphyses of the long bones. Sclerosis can also be seen at the base of the skull. In 2000, the gene responsible for CED was located on the chromosomal region 19q13.2 and shortly afterwards, the gene was identified as being TGFB1. So far, 7 mutations have been reported in 17 CED families. With only one exception - a small duplication in the signal peptide - all mutations are missense mutations, located in the latency associated peptide (LAP), which confers latency to the mature TGF-beta1. In order to unravel the underlying mechanism, we performed several functional studies using conditioned medium and/or lysates of cells transfected with overexpression constructs of control and mutant TGFB1. ELISA of conditioned media showed a marked difference between controls and CED-patients. Patients with missense mutation R218C or C225R secrete a normal amount of TGF-beta1, but have a two- to threefold higher amount of active TGF-beta1; the mutations in the LAP apparently influence the ease with which the mature TGF-beta1 is being activated. The duplication in the signal peptide on the contrary decreases TGF-beta1 secretion. ELISA of lysates shows that this diminished secretion is reflected by a retention of TGF-beta1 intracellularly. The results of the ELISA were confirmed with a luciferase reporter assay. The diminished secretion and increased retention of TGF-beta1 by the duplication in the signal peptide could also be confirmed by Western blotting. In conclusion, we were able to illustrate that the mutations found in CED-patients have a profound effect on the function of TGF-beta1, by affecting either the secretion or the activation of the protein. Further experiments are however necessary to reveal how mutations with an apparently opposing effect can give rise to the same disorder. MUTATED SUBUNIT A3 OF VACUOLAR PROTON PUMP IN PATIENTS WITH AUTOSOMAL RECESSIVE OSTEOPETROSIS: GENOTYPE-PHENOTYPE CORRELATION A. Taranta1*, A. Ricci1, S. Migliaccio1, I. Recchia1, G. De Rossi2, E. Lanino3, S. H. Ralston4, A. Villa5, P. Vezzoni5, A. Teti1 1Department of Experimental Medicine, University of L'Aquila, Italy 2Bambino Gesù Hospital, Rome, Italy 3Gaslini Hospital, Genoa, Italy 4Dept. of Medicine and Therapeutics, University of Aberdeen, Scotland 5Istituto di Tecnologìe Biomediche Avanzate, Consiglio Nazionale delle Ricerche, Segrate, Italy Autosomal recessive osteopetrosis (arOP) is a severe genetic disease characterised by dense and fragile bones due to impaired osteoclast bone resorption. Genotyping of arOP patients analysed so far has allowed the identification of ATP6i gene mutation in 50% of the cases. This gene encodes the osteoclast specific a3 V-ATPase subunit which contributes to proton transport and acidification of the osteoclast resorbing compartment. The aim of our study was to identify genotype-phenotype relationship in our ATP6i-dependent arOP patients. Clinical follow-up showed diffuse osteosclerosis and endobone appearance, macro- and hydro-cephaly, pale optic nerve papillae, anaemia, low neutrophil counts, elevated alkaline phosphatase and, remarkably high PTH (parathyroid hormone) levels. The iliac crest biopsies were characterized by osteosclerotic pattern, massive and irregular bone trabeculae and unresorbed mineralized cartilage. Excess of normally polarised osteoclasts and scarce haematopoietic tissue, against a background of diffuse fibrosis, were observed. Osteoclasts obtained from peripheral blood monocytes showed no morphological differences relative to normal osteoclasts and their TRAP (tartrate-resistent acid phosphatase) activity was several fold higher than average. No immunostain was observed with an antibody raised against peptide 811-829 localized in the C-terminus of the a3 subunit, downstream of the mutation. These osteoclasts resorbed bone, but their lacunae were very shallow. In contrast to other forms of OP, cytoskeletal arrangement, podosome pattern and alphaVbeta3 integrin distribution were unremarkable. Pyk2 and c-src appeared normally distributed at the paramarginal area where these tyrosonine kinases contributed to integrin-mediated osteoclast adhesion and outside-in signalling. In conclusion, this is the first genotype-phenotype correlation in ATP6i-dependent osteopetrosis, and shows that mutations of the ATP6i gene increases TRAP activity but do not affect osteoclast morphology and adhesion structures, and that bone resorption is severely impaired but not completely abolished. We hypothesize that the elevated serum PTH levels found in these patients may likely account, at least in part, for the high numbers of osteoclasts observed in bone biopsies. ALBERS-SCHÖNBERG DISEASE (AUTOSOMAL DOMINANT OSTEOPETROSIS, TYPE II) IS CAUSED BY MUTATIONS IN THE CLCN7 CHLORIDE CHANNEL GENE E. Cleiren1, O. Bénichou2, E. Van Hul1, J. Gram3, J. Bollerslev4, F. R. Singer5, K. Beaverson6, M. P. Whyte7, M. C. de Vernejoul2, W. Van Hul1* 1Dept Medical Genetics, University of Antwerp, Belgium 2Laboratoire INSERM U 349, Hôpital Lariboisière, Paris, France 3Dept. Medicine, Ribe County Hospital, Esbjerg, Denmark 4Dept. Endocrinology, Rikshospitalet, Oslo, Norway 5John Wayne Cancer Institute, Santa Monica, CA, USA 6Weill Medical College of Cornell University, New York, NY, USA 7Center for Metabolic Bone Disease and Molecular Research, Shriners Hospitals for Children, St. Louis, MO, USA Albers-Schönberg disease, or autosomal dominant osteopetrosis, type II (ADO II), is the most common form of osteopetrosis, a group of conditions characterised by an increased skeletal mass due to impaired bone and cartilage resorption. However, due to its relatively benign clinical picture, with many patients being asymptomatic and only detected by coincidental radiographic examination, the prevalence of ADO is underestimated. Among families with ADO, two subtypes are generally reported based primarily on radiographic features. Type I (ADOI) features a generalised, diffuse osteosclerosis affecting especially the cranial vault. Clinical manifestations of ADO II include non-traumatic fractures, especially of long bones, cranial nerve palsies, osteoarthritis of the hip and mandibular osteomyelitis. ADO II manifests radiographically with a segmentary osteosclerosis, predominately at the vertebral endplates ('rugger jersey spine'), iliac wings ('bone within bone' sign), and skull base. Recently, a genome wide search led us to assign a gene underlying ADO II to chromosome 16p13.3. Following this gene assignment, we now report seven different mutations in the gene encoding the ClCN7 chloride channel in all 12 ADO II families analysed. This chloride channel is essential for the acidification of the extracellular resorption lacuna necessary for the osteoclast-mediated degradation of bone tissue. Additionally, a patient with the severe, autosomal recessive, infantile form of osteopetrosis (ARO) was identified as being homozygous for a ClCN7 mutation. From genotype-phenotype correlations, it seems that ADO II reflects a dominant negative effect, whereas loss-of-function mutations in ClCN7 do not cause abnormalities in heterozygous individuals. Because some ARO patients have mutations in both copies of the ClCN7 gene, ADO II is allelic with a subset of ARO cases. HARDNESS AND COMPOSITION OF CANCELLOUS BONE IN OSTEOPOROSIS AND OSTEOARTHRITIS A. M. Coats1*, P. Zioupos2, R. M. Aspden3 1Department of Chemistry, University of Aberdeen, Meston Walk, Aberdeen AB24 3UE, UK 2Materials and Medical Sciences, Cranfield University, Shrivenham SN6 8LA, UK 3Orthopaedic Surgery, University of Aberdeen, Polworth Building, Foresterhill, Aberdeen AB25 2ZD, UK Cancellous bone from patients with osteoarthritis (OA) has a reduced material density and appears to be undermineralized. It is hypothesized that this will result in a reduction in the mechanical stiffness and strength of the bone matrix. In this study, bone was obtained from superior and inferior sites, subjected to relatively high and low loads respectively, from human femoral heads retrieved after surgery for osteoporotic hip fracture (OP) or for hip arthroplasty due to osteoarthritis (OA). Microindentation testing and electron probe microanalysis (EPMA) were used to measure the hardness and the elemental composition of cancellous bone from immediately adjacent microscopic sites at various depths from the subchondral bone plate. Overall, OA bone was found to have hardness values that were 7% lower than those from OP bone. Bone from the inferior site was harder than that from the superior in both diseases. There was no variation with depth below the subchondral plate and no difference between sexes. Using EPMA, only the Ca/P ratio was significantly different between disease groups (OA: 1.715, OP: 1.694, P=0.015) and no correlation was found between hardness and any of the composition measurements. Though only an indirect measurement of stiffness, the reduction in hardness values support the hypothesis that OA bone would have reduced mechanical properties. COLIA1 SP1 BINDING SITE POLYMORPHISM PREDISPOSES TO OSTEOPOROTIC FRACTURES BY ALTERING COLLAGEN PRODUCTION AND IMPAIRING BONE MINERALISATION T. L. Stewart1*, P. Roschger2, B. Grabner2, P. Fratzl3, V. Mann1, R. M. Aspden1, K. Klaushofer2, S. H. Ralston1 1Bone Research Group, University of Aberdeen, UK 2Ludwig Boltzman Institute of Osteology, Vienna, Austria 3Erich Schmid Institute of Material Science, Leoben, Austria A polymorphic Sp1 binding site has been identified in the COL1A1 gene which predicts the risk of osteoporotic fractures, independent of differences in bone mineral density. In this study, we examined the functional mechanisms responsible for this observation. Ex-vivo mechanical testing of bone cores from patients of different genotype showed reduced yield strength (adjusted for bone density) in G/T heterozygotes (n=7) when compared with G/G homozygotes (n=10) (4.60±SD 0.3 MPa vs 3.55±SD 0.2 MPa; p=0.034). Further analysis, using quantitative backscattered electron imaging (qBEI), showed reduced mineralisation of bone in G/T heterozygotes (n=7) compared with G/G homozygotes (n=6) (20.2±SD 1.2 % vs 21.5±SD 0.5 %; p=0.049) and increased heterogeneity of mineralisation, as reflected by broadening of the bone mineralisation density distribution peak (4.5±SD 0.5 % vs 3.5±SD 0.5 % p=0.038). Further studies have used an in-vitro model of bone mineralisation, in which calcified bone nodules were generated from long term cultures of primary human osteoblasts from patients of different genotype, and calcium deposition was quantified by Alizarin Red S staining. Cultures from G/T heterozygotes produced an abnormally increased ratio of the collagen type I alpha (1) chain to the alpha I (2) chain (G/T=2.3:1.0) in vitro, compared with G/G homozygotes (1.99:1.0; p=0.007), and formed significantly less mineralised bone nodules than G/G homozygotes (mean±SD 1650 + 140 vs 690±SD 130 microM Alizarin Red S /105 cells; p<0.0001), even though total cell number, collagen production and alkaline phosphatase activity were similar in both genotype groups. Our data indicate that the COLIA1 'T' allele influences the ratio, but not the total amount of collagen type I alpha chains produced by bone cells, leading to abnormal mineralisation of bone both in vivo and in vitro and reduced bone strength. The studies define a novel genetic mechanism of osteoporosis which predisposes to fracture by affecting bone quality rather than bone quantity. INTERACTION BETWEEN POLYMORPHISMS IN THE METHYLENETETRAHYDROFOLATE REDUCTASE (MTHFR) GENE AND THE COLLAGEN TYPE I ALPHA 1 (COLIA1) GENE IN BONE MINERAL DENSITY AND FRACTURE RISK J. B. J. van Meurs1*, P. Arp1, M. van der Klift2, A. Hofman2, H. A. P. Pols1,2, A. G. Uitterlinden1,2 1Department of Internal Medicine, Erasmus University Centre, Rotterdam, The Netherlands 2Department of Epidemiology and Biostatistics, Erasmus University Centre, Rotterdam, The Netherlands Homocystinuria, characterised by high levels of homocysteine, is associated with osteoporosis, possibly due to interference of homocysteine with collagen metabolism. We found earlier that mildly elevated levels of serum homocysteine are associated with increased risk for osteoporotic fractures. We here examined possible genetic influences on this relation. We studied a functional variant of MTHFR (C677T), an essential enzyme in homocysteine metabolism, and interaction between this variant and the Sp1 G/T polymorphism in the COLIA1 gene in a population-based sample of 1532 postmenopausal women in relation to BMD and incident non-vertebral fractures. Homozygote MTHFR 677T-carriers had a 2.5% lower BMD (p=0.01) than homozygote 677C-carriers, but no association was found of this polymorphism with fracture risk. To study interaction between MTHFR and COLIA1 polymorphisms, subjects
were divided our population into four groups based on presence of risk allele(s) (see
Table).
We conclude that the combination of COLIA1 and MTHFR polymorphisms predisposes women to osteoporotic fractures. This risk is only partly explained by differences in bone mineral density, suggesting that factors, other then those reflected by BMD, determine the increased fracture risk. ASSOCIATION STUDY OF THE CDX-2 POLYMORPHISM IN THE 1A PROMOTER REGION OF THE HUMAN VITAMIN D RECEPTOR GENE AND FRACTURE Y. Fang1*, J. B. J. van Meurs1, A. Hofman2, H. P. T. M. van Leeuwen1, C. M. van Duijn2, H. A. P. Pols1,2, A. G. Uitterlinden1,2 1Department of Internal Medicine, Erasmus University Medical Center, Rotterdam, The Netherlands 2Department of epidemiology, Erasmus University Medical Center, Rotterdam, The Netherlands Polymorphisms of the human Vitamin D Receptor (hVDR) gene are reported to be associated with several clinical endpoints including osteoporosis. Recently, a single nucleotide polymorphism (SNP) was discovered in the 1a promoter region of the VDR gene. This G to A substitution in a binding site of the intestinal- specific transcription factor Cdx-2, was found to modulate the transcription of hVDR gene and to be associated with decreased BMD in a small group of postmenopausal Japanese women. We here present a comparison of the frequency of the A allele in the three major human races, and observed a negative correlation between prevalence of the A allele and hip fracture incidence in these ethnic groups, suggesting a protective effect of this allele on fracture risk. To study this putative relation in more detail, we performed an association study of this SNP in a large cohort of Dutch Caucasian elderly. First, we developed an allele-specific multiplex PCR test and then determined the Cdx-2 genotype in 2447 men and women aged 55 year or older. We found the frequency of the G- and the A-allele to be 81% and 19%, respectively. The A allele had a protective effect on occurrence of osteoporotic fractures, especially for non-vertebral fracture in women (OR of AA vs. GG genotype is 0.2, 95%CI is 0.05-0.8). This effect did not vary by adjustment for age, weight and bone minal density (BMD). We conclude that the A allele of the VDR Cdx-2 polymorphism is present in Caucasians, albeit at low frequency. We demonstrate a protective effect of this allele on non-vertebral fracture, which is not explained by differences in BMD in this population. POLYMORPHISMS OF THE TNFR2 GENE ARE ASSOCIATED WITH BONE MINERAL DENSITY IN WOMEN FROM THE UK O. M. E. Albagha*, P. N. Tasker, F. E. A. Mcguigan, D. M. Reid, S. H. Ralston Dept of Medicine and Therapeutics, University of Aberdeen, Aberdeen, UK Genetic factors are important in the pathogenesis of osteoporosis and linkage analysis has identified a candidate locus for hip bone mass on chromosome 1p36 (Devoto et al. Eur J Hum Genet 1998). One of the positional candidate genes within this region is tumour necrosis factor receptor 2 (TNFR2). We investigated the relationship between TNFR2 gene polymorphisms and bone mineral density in 1246 perimenopausal women randomly selected from the local population in Scotland. A single nucleotide polymorphism (SNP) located in exon 6 along with 3 SNPs located in the 3'UTR were studied. The exon 6 genotypes were determined by standard PCR based methods. The 3'UTR polymorphisms, which are located at nucleotides (nt.) 593, 598, and 620 of the TNFR2 gene (Genbank Accession U52165) were typed by DNA sequencing of PCR products spanning the region of interest. Genotype frequencies were similar to those previously reported and we found that women with the genotype A-T-C (corresponding to nt593-A/A, nt598-T/T, and nt620-C/C) had significantly lower femoral neck (FN) BMD (mean Z-score±sem=-0.441±0.084; n=86) than those without this genotype (0.050±0.031; n=1160; p-value<0.00001). A similar trend was observed for LS-BMD but it did not reach statistical significance (p-value=0.39). The A-T-C haplotype also significantly predicted FN BMD (p-value<0.0001) in a multiple linear regression model which included other possible predictors of BMD (such as age, height, weight, years since menopause, and duration of HRT use) with evidence for a gene dose effect. No association was found between exon 6 polymorphism and BMD in the population studied. In summary, we have confirmed that allelic variation in the 3'UTR of the TNFR2 gene is associated with BMD but in contrast with previous work we found an association between hip rather than LS- BMD. These data suggest that the 3'UTR polymorphisms are strongly related to FN-BMD, however further studies are required to investigate whether the 3'UTR polymorphisms directly influence the TNFR2 gene function or whether they are in linkage disequilibrium with causal polymorphisms elsewhere in the TNFR2 gene or in other genes nearby. CONGENITAL PSEUDARTHROSIS OF THE TIBIA: TREATMENT WITH A BONE MORPHOGENETIC PROTEIN (OP-1 DEVICE) D. Anticevic1*, Z. Stanec2, M. Jelic1, S. Vukicevic3 1Department of Orthopedic Surgery, Medical School, Zagreb, Croatia 2Department of Plastic Surgery, Medical School, Zagreb, Croatia 3Department of Anatomy, Medical School, Zagreb, Croatia PURPOSE: To evaluate locally administered osteogenic protein-1 device (OP-1, bone morphogenetic protein-7, BMP-7) on bone regeneration in congenital pseudarthrosis of the tibia (CPT). METHODS (Case report): A six-year old girl with Type III (Crawford) cystic CPT anteriorly angulated for 80 degrees and two inch limb shortening was initially treated with resection of pseudarthrotic and cystic bony lesion. A free vascularized fibular 1.5 inch long graft was then transferred into the defect area. The proximal and distal tibial ends were cut in a step-like fashion to increase contact area with the graft. OP-1 device was carefully placed on the contact area between the tibia and the fibula graft. The bones were fixed with an intramedullary rod and the Ilizarov device that were removed after three and six months, respectively. Gradual weight bearing started three months after surgery and the leg protection with bivalved splint started six months after surgery. RESULTS: Three weeks following surgery a significant amount of new bone was observed along the graft and at both proximal and distal contact surfaces. At four months the proximal part of the graft was fully incorporated with the tibia while the contact area between the tibia and fibula at the distal end was significantly smaller. At seven months following surgery both proximal cortices and medullary canal between the tibial and fibular graft were fused and already remodeled, while distally a much wider contact area between the graft and the tibia was formed. One year following surgery lower limb was one and a half-inch shorter. No valgus ankle deformity was detected. CONCLUSION: The time to bone union was four months on the proximal and seven months on the distal graft contact surface. In patients with CPT adjuvant local application of OP-1 could thus significantly stimulate the tibial bone union and restore a fully weight-bearing lower limb. SIGNIFICANCE: The first child with highly angulated CPT was treated successfully with a recombinant bone morphogenetic protein to enhance bone regeneration and repair. OSTEOPETROSIS WITH SPARSE HAIR AND DENTAL DYSPLASIA - AN UNUSUAL PRESENTATION CASE REPORT I. Baric1*, V. Kusec2, V. Sarnavka3, M. Cuk1, D. Krpan4, S. Mutar-Susic5, I. Skrinjaric5, D. Begovic1 1Department of Pediatrics, Clinical Hospital Centre, Zagreb, Croatia 2Clinical Institute of Laboratory Diagnosis, Clinical Hospital Centre, Zagreb, Croatia 3Department of Dermatology, Clinical Hospital Centre, Zagreb, Croatia 4General Hospital 'Sveti Duh', Zagreb, Croatia 5School of Dentistry, Zagreb, Croatia Increased bone density was diagnosed in the age of 2 years after observing radiodense appearance of skull X-ray. Other clinical features of the 6 year old girl include: radiodense appearance of almost the entire skeleton, dental dysplasia, compression of cranial nerves, occasional seizures, brittle and sparse hair, absence of eyebrows and eyelashes. No fracture has occurred so far despite her lively behaviour. No abnormalities of the internal organs or nail dysplasia are present. No haematological abnormalities or impairment of haematopoiesis have developed. Measurements of markers of bone turnover indicate increased bone formation and resorption, most probably appropriate for age and growing skeleton. Densitometry performed at 3 and 5 years indicated a dramatic increase in bone density Z-score being plus 2.3 and 14.4, respectively. Bone biopsy at the iliac crest revealed increased bone volume (93 percent), almost resembling cortical bone but without cartilage residues. Bone marrow spaces were grossly reduced and contained no haematopoiesis. Bone formation was increased. ATP6i gene mutation was not confirmed in this girl. No treatment has been attempted so far and her general condition has not changed considerably. Despite all efforts a definite diagnosis has not been established. OSTEOPETROSIS - PLAIN FILM RADIOGRAPHS VS MRI - CASE REPORT OF ADOP II TYPE P. Marusic1*, L. J. Vojnic1, H. Landeka2, B. Brkljacic1 1Department of Radiology, University Hospital 'Dubrava', Zagreb, Croatia 2Department of Neurology,University Hospital 'Dubrava', Zagreb, Croatia Osteopetrosis (marble bones, Albers-Schenborg disease) is a group of inherited bone disorders characterized radiographically by variable generalized symetrical skeletal sclerosis due to defective osteoclast function. Autosomal dominant (benign) osteopetrosis - ADOP may be asymptomatic and discovered incidentally on plain radiographs. On the basis of radiographic criteria ADOP has been differentited in two types. Type I is characterized by sclerotic thickening of the calvarium, most prominent in the vault, and litle sclerosis of the spine. Type II is characterized by sclerotic thickening of the base of the skull while the calvarium is nearly normal. The other radiographic findings include hyperostotic vertebral endplates (sandwich sign, 'rugger-jersey spine') and endobones in other bones. We would like to present a case of a 75-year-old woman who was admitted to the hospital, for the first time, because of difficult walking, and pain in her left leg and lumbar region. Plain film of the cranium revealed sclerotic thickening of the basis of the skull and obliteration of the mastoid air cells. The calvarium was normal. Computed tomography of the brain confirmed sclerotic areas on the basis of the skull. Radiographs of the lumbar spine revealed narrowing of the L4/L5 and L5/S1 intervertebral space and impressive endplates sclerotic thickening of the vertebraes' bodies (sandwich sign). MRI of this region revealed disc protrusion at L4/L5 and L5/S1 with decreased bone marrow signal intensity. Plain film of the foot revealed sclerotic changes of the all tarsal bones with forming endobones (bone within bone sign) and scattared increase in bone density of the metetarsal bones. MRI of this region also revealed 'bone within bone sign' especially in calcaneus, talus and cuboid bone. The aim is to compare plain films of the spine and foot with MRI of these regions. The conclusion is that plain film is better in showing the changes of the vertebraes' bodies and MRI of this region should be used only when patient has spinal cord symptoms. MRI gives better results in examination of the foot compared to the plain films because of multiplanar projections used in MRI analysis. NEUROENDOCRINE TUMOR CAUSING FGF23-INDEPENDENT ONCOGENIC HYPOPHOSPHATEMIC OSTEOMALACIA F. Paglia1*, A. Corsi2,3, S. Minisola1, S. Dionisi1, S. De Geronimo1, S. Licci2, A. Marzullo2, F. Malchiodi4, M. Riminucci2,3, P. Bianco2 1Department of Clinical Science, La Sapienza University, Rome, Italy 2Department of Eperimental Medicine and Pathology, La Sapienza University, Rome, Italy 3Department of Experimental Medicine, Laboratory of Pathology, Univeristy of L'Aquila, Italy 4Laboratory of Ultrastructure, ISS, Rome, Italy Oncogenic Hypophosphatemic Osteomalacia (OHO) is a paraneoplastic syndrome characterized by osteomalacia, muscle weakness, phosphate wasting, hypophosphatemia and low 1,25-dihydroxyvitamin D3 (1,25-D3). We describe a 65 years-old woman with OHO due to a soft tissue tumor diagnosed 16 years after the onset of symptoms. The patient presented at the age of 49 with weakness and back pain. Spontaneous fractures of pelvis and right hip developed in the following three years. Biochemical abnormalities included hypophosphatemia and low tubular maximum transport of phosphate. Iliac crest biopsy revealed osteomalacia as the basis for the tendency to fractures. The patient was periodically treated with calcium carbonate, vitamin D and phosphate without improvement. During the following years, the patient underwent other spontaneous fractures of left hip, humeri and vertebras until immobilisation. Biochemical studies performed at our hospital revealed hypophosphatemia, increased total and bone-specific alkaline phosphatase, ionised calcium and intact parathyroid hormone, low 1,25-D3 and a selective defect of tubular phosphate resorption. Although total body computed tomography (CT) failed to demonstrate any focal lesion, an increased uptake was detected at the left groin by whole-body Indium-111 octreotide scintigraphy. A spiral CT scan performed specifically at this site revealed a soft tissue mass 4 cm in largest dimension. The excision of the mass improved clinical symptoms and biochemical values, even though serum calcium and parathyroid hormone were still high. Histology of the mass was consistent with the mixed connective tissue variant of phosphaturic mesenchymal tumor. Extensive abnormal mineral deposition, shown by electron probe microanalysis to consist of calcium phosphate, occurred within the tumor in the face of the hypophosphatemia. Immunoreactivity for chromogranin-A and electron microscopy evidence of neurosecretory granules in a proportion of neoplastic cells indicated the neuroendocrine nature of the tumor. Although the expression of FGF23, one of the putative factors involved in OHO, has been previously reported in tumors causing OHO, RT-PCR analysis on frozen tumor samples demonstrated no FGF23 mRNA expression in our case. While studies on the potential involvement of other factors (MEPE, PHEX) in this case are in progress, these data highlight the occurrence of non-FGF23 producing, neuroendocrine tumors as a cause of OHO. VARIATION IN HUMAN BONE CELL RESPONSE TO STIMULATION BY FLUID FLOW OR CYCLIC STRAIN M. Mullender1*, A. J. El Haj2, Y. Yang2, J. Magnay2, M. A. van Duin1, J. Klein-Nulend1 1Dept. Oral Cell Biology, ACTA-Vrije Universiteit, Amsterdam, The Netherlands 2Centre for Science and Technology in Medicine, Dept. Biomedical Eng., Stoke-on-Trent, UK The aim of this study was to compare the response of human bone cells to two different mechanical stimuli in vitro, pulsating fluid flow and cyclic strain. Comparisons were measured in the production of nitric oxide (NO) and prostaglandin E2 (PGE2), both early mediators in mechanically induced bone adaptation. Primary human bone cells were cultured from tibia fragments of a healthy 16-year-old male. Second-passage primary cultures were used for stimulation by either pulsating fluid flow (PFF) or cyclic strain (CS) by four-point bending. Cells were plated on polylysine coated glass slides and incubated for 24 h in DMEM with 2% FBS. The cells were then loaded for 1 h by PFF (0.6±0.3 Pa, 8.5 Pa/sec, 5 Hz) or CS (~1000 microstrain, 1 Hz). Control cultures were kept accordingly, only without undergoing loading. All cell cultures were post-incubated until 24 hours after loading. The conditioned medium was assayed for NO using Griess reagent and PGE2 by enzyme-immunoassay. Cells were harvested for measurement of total protein and DNA. The DNA content did not differ between treatment and control groups. Treatment with PFF caused an increase of NO and PGE2 (p<0.05). CS did not stimulate the release of NO or PGE2 significantly. The release of NO was increased up to 2-fold after 10 to 30 minutes of stimulation with PFF. The total amount of PGE2 increased from 5-fold after 10 minutes to 15-fold after 1 hour of treatment with PFF, measured after 24 h post incubation. Fluid flow through the lacuno-canalicular system is generally accepted to be a key mechanism by which bone senses mechanical loading. In this study we compared the response of primary human bone cells to stimulation by pulsating fluid flow and four-point bending of the cell substrate. Both PFF and CS cause cell deformation. Yet, the response of the cells to these types of mechanical loading differed. NO and PGE2 release increased after PFF, however, CS did not evoke this response. This suggests that bone cells may differentiate between strain directly applied through the substrate and strain induced by flow-derived shear stress, by differing signaling pathways. THE BONE-SPECIFIC TRANSCRIPTIONAL REGULATOR CBFA1 IS A TARGET OF MECHANICAL SIGNALS IN OSTEOBLASTIC CELLS P. G. Ziros1, A-P. Rojas Gil1, T. Georgakopoulos1, E. K. Basdra2, A. G. Papavassiliou1* 1Department of Biochemistry, School of Medicine, University of Patras, GR-26110 Patras, Greece 2Department of Orthodontics, Heidelberg University, D-69120 Heidelberg, Germany A primary goal of bone research is to understand the mechanism(s) by which mechanical forces dictate the cellular and metabolic activities of osteoblasts, the bone-forming cells. Several studies indicate that osteoblastic cells respond to physical loading by transducing signals that alter gene expression patterns. Accumulated data have documented the fundamental role of the osteoblast-specific transcription factor Cbfa1 (core binding factor) in osteoblast differentiation and function. In the present study we demonstrate that low-level mechanical deformation (stretching) of human osteoblastic cells directly up-regulates the expression and DNA-binding activity of Cbfa1. This effect seems to be fine-tuned by stretch-triggered induction of distinct MAPK (mitogen-activated protein kinase) cascades. Our novel finding that activated ERK (extracellular signal-regulated kinase) MAPK physically interacts and phosphorylates endogenous Cbfa1 in vivo (ultimately potentiating this transcription factor), provides a molecular link between mechanostressing and stimulation of osteoblast differentiation. Elucidation of the specific modifiers and cofactors that operate in this mechanotranscription circuitry will contribute to a better understanding of mechanical load-induced bone formation that may set the basis for non-pharmacological intervention in bone loss pathologies. PROTEIN KINASE C ALPHA / ESTROGEN RECEPTOR ALPHA INTERACTION IN OSTEOBLAST DIFFERENTIATION M. Longo1*, M. Brama1, M. Marino2, K. S. Korach3, W. C. Wetsel4, R. Scandurra2, T. Faraggiana2, R. Baron5, A. Teti1, S. Migliaccio1,2 1Department of Experimental Medicine, University of L'Aquila, Italy 2University 'La Sapienza', Rome, Italy 3NIEHS, Research Triangle Park, NC, USA 4Duke University Medical Center, Durham, NC, USA 5Dept. Orthop. & Cell Biol., Yale Univ., New Haven, CT, USA In osteoblastic cells undergoing confluence-induced differentiation, protein kinase C (PKC) activity increases while estrogen receptor alpha (ERalpha) binding and responsiveness to estrogens decrease. We tested the hypothesis that physical interaction occurs between ERalpha and PKC isoforms as part of the underlying mechanism(s). Sub- and post-confluent rat primary osteoblasts were used for anti-ERalpha and anti-PKCalpha immunoprecipitations. Both PKCalpha and ERalpha were detected in either immunoprecipitate, but PKCalpha was much higher in anti-ERalpha samples from post-confluent cells, which expressed higher levels of the differentiation marker alkaline phosphatase. Furthermore, only the 66 KDa ERalpha variant was present in sub-confluent cells, whereas its newly described 46 KDa form became detectable upon differentiation. In rat osteosarcoma cells ROS.SMER 14, expressing murine ERalpha constitutively, we found no significant variation of co-immunoprecipitable PKCalpha levels, despite much lower ERalpha in cytosolic fractions from post-confluent cells. Not too surprisingly, all of our anti-ERalpha immunprecipitates contained a single PKCalpha post-translational variant, i.e., its well-known 40 KDa cleavage product encompassing the catalytic site. This was most evident in the case of ROS.SMER 14, where both 80 and 40 KDa forms were readily detectable, but the former appeared only in the anti- PKCalpha immunoprecipitates. Like calcium- and lipid-dependent PKCalpha, calcium-independent but lipid-dependent PKCepsilon also increased to some extent in these cells upon differentiation, whereas the atypical PKCzeta did not. Consistently, inositol-phospholipid turnover was higher in differentiated cells. As evidenced by our previous results, c-Src plays an important role in confluence-induced differentiation in osteoblastic cells, and has been found to interact with PKC isoforms. This prompted us to analyse c-Src expression and activity in our cells. In ROS.SMER 14 we observed a significant, but transient, increase in expression and activity of c-Src preceding maximal PKC increase. PKCalpha from differentiated osteoblasts induced c-Src phosphorylation in vitro. In contrast, c- Src was unable to phosphorylate PKCalpha, although tyrosine phosphorylation was apparent in PKCalpha and increased in differentiated cells. These data suggest close interactions between PKCalpha, c-Src and ERalpha in osteoblastic cells, which may be crucial for the differentiation process and the related decrease in estrogen responsiveness. METALLOPROTEINASE-14 IS INDUCED AT BEGINNING OF OSTEOGENESIS IN VITRO AND ITS DOWN-REGULATION IS REQUIRED FOR OSTEOGENIC PROGRESSION D. Palmieri, S. Poggi, V. Ulivi, P. Manduca* Dobig, University of Genova, Italy We have reported the up-modulation of Metalloproteinase-14 (MMP-14) mRNA, synthesis and function at the beginning of osteogenic differentiation of rat osteoblasts and association of its expression with the mature phenotype in vitro, and with active osteogenic cells in vivo. We also reported that MMP-14 is down- modulated in osteoblast cultures before mineralization and not detected in osteocytes in vivo. We here report that the expression of MMP-14 in pre-osteoblasts is induced, in a time dependent fashion, by adhesion to the endogenous extracellular matrix (ECM), which can be substituted by Fibronectin and Collagen type I, but not Collagen type III, polilysin or VN. MMP-14 up-modulation requires mRNA and protein synthesis and is inhibited by inhibitors of phosphorylation. The induction of MMP-14 is accompanied by the activation of pro-MMP-2, and the active forms are recovered in the conditioned medium. The persistence of MMP-14 function, obtained in differentiating cultures with the use of a specific activating antibody, inhibits the decline of Alkaline phosphatase (AP) activity, the morphogenesis of nodules and the incorporation of Calcium. Two clones of rat osteoblasts, constitutive for AP, which did not form nodules and had a very low level of Ca incorporation in comparison with the control cells, when tested, showed constitutive expression of MMP-14 for up to 40 days in differentiation culture conditions. These data suggest that down modulation of MMP-14 is required for the progression of the osteogenic phenotype and is associated to the down modulation of AP. DIFFERENTAL EXPRESSION OF CCN-FAMILY MEMBERS DURING OSTEOGENESIS, CHONDROGENESIS AND ADIPOGENESIS USING PRIMARY HUMAN MESENCHYMAL STEM CELLS N. Schütze*, U. Nöth, J. Schneidereit, C. Hendrich, J. Eulert, F. Jakob Orthopädische UniversitätsklinikClinic, Labor für Molekulare Experimentelle Orthopädie, Würzburg, Germany Members of the family of connective tissue growth factor (CTGF), the cystein-rich protein 61 (CYR61) and the nephroblastoma overexpressed protein (nov) (CCN-family) function in processes such as differentiation and tissue regeneration mainly as growth factors and matrix associated signal molecules. Previously, we have shown that the expression of human CYR61 (hCYR61) in vivo was associated with conditions of enhanced bone formation and fracture healing. Further results in the literature point towards the expression of other CCN proteins in bone such as CTGF, CTGF-L and WISP-3. Primary cultures of human mesenchymal stem cells were derived from the femoral head of patients undergoing total hip arthroplasty. Differentiation into adipocytes and osteoblasts was done in monolayer culture, differentiation into chondrocytes was induced in pellet culture. For either pathway established differentiation markers and CCN-members were analyzed at the mRNA level by RT-PCR and at the protein level by immunocytochemistry. RNA or protein levels of established markers revealed the appropriate phenotype of differentiated cells (alizarin-red, alkaline phosphatase, osteocalcin, collagens, cbfa1, PPARgamma, oil-red-o, aggrecan). Mesenchymal stem cells expressed nestin mRNA and all CCN-family members. The hCYR61 expression was high in stem cells and decreased 7-fold during osteogenesis and chondrogenesis and more than 10-fold during adipogenesis. These results were confirmed by immuncytochemical analyses. CTGF-L and WISP-3 RNA expression were enhanced slightly during osteogenesis and decreased in chondrogenesis and adipogenesis (3- and 10-fold, respectively). The CTGF-L expression was decreased >10-fold in adipogenesis. The expression of CCN-family members was dependent on the differentiation status of human mesenchymal stem cells in vitro. hCYR61, which plays a role as a signal molecule of the extracellular matrix, is important in the early nestin positive stem cells used in this study. The marked decrease in hCYR61 expression during all differentiation pathways suggests a specific role of hCYR61 for maintenance of the stem cell phenotype. The differential expression of WISP-3, CTGF, CTGF-L and mainly hCYR61 indicates, that the CCN-family might be important for the function of mesenchymal stem cells as well as the regulation of proliferation and specific differentiation pathways of human mesenchymal stem cells from bone marrow. OP-1 DEVICE IN SURGICAL TREATMENT OF THE SCAPHOID BONE NON-UNION R. Bilic1*, M. Jelic1,2, M. Pecina1, S. Vukicevic2 1Department of Orthopedic Surgery, School of Medicine, University of Zagreb, Croatia 2Departments of Anatomy, School of Medicine, University of Zagreb, Croatia Osteosynthesis with autogenous corticocancellous graft is a widely used operative treatment for the scaphoid non-unions. Long period of 14 weeks postoperative immobilization, leads eventually to arthrotic changes in the radioscaphoid or radiocarpal joint (n=180). Recently, we demonstrated that the scaphoid union and postoperative immobilization can be reduced to 9 weeks (n=130) if adding the compressed cancellous bone graft to the corticocancellous graft. When nesting site in the scaphoid bone and the autogenous graft with compressed cancellous bone were covered with OP-1 putty in a prospective randomized study, the new bone formation at the operation site was observed at 4 weeks (4 patients). OP-1 device can, thus, significantly reduce the immobilization time for achieving scaphoid union when using autogenous corticocancellous graft. In an attempt to avoid the second surgical procedure at the autogenous corticocancellous graft donor site, we treated 4 patients with allogeneic corticocancellous graft and compressed cancellous bone from the iliac crest of a 25 y donor. The scaphoid bone nesting site and the allograft were covered with OP-1 putty. The scaphoid union was achieved at 8 weeks following surgery. Trabecular bone union evidenced by plain x-rays and CT scans correlated well with the disappearance of pain and with the improvement of functional tests. This is the first evidence that scaphoid non- union osteosynthesis could be performed in 8 weeks using an allograft and OP-1 device. CP-533,536, A NON-PROSTANOID EP2 RECEPTOR AGONIST, STIMULATES PERIOSTEAL BONE FORMATION AND ENHANCES FRACTURE HEALING IN RAT MODEL M. Li*, D. R. Healy, Y. Li, H. Z. Ke, V. M. Paralkar, T. A. Owen, L. C. Pan, K. Cameron, B. A. Lefker, P. DaSilva-Jardine, F. Dumont, R. Korsmeyer, D. D. Thompson Pfizer Global Research and Development, Groton, CT, USA Prostaglandin E2 (PGE2) is known to stimulate bone formation when administered systemically or locally, but its side effects limit its utilization for enhancing bone formation. We have found that the EP2 receptor subtype plays an important role in the local bone anabolism of PGE2. CP-533,536 is a new non-prostanoid EP2 receptor selective agonist. Utilizing rat-models, we evaluated the potential beneficial effects of this compound on bone healing upon local administration. In the first study, CP-533,536 at 0.4 mg/rat/day or vehicle was injected onto the periosteum of the right femurs of 3-week-old male rats for 14 consecutive days. Radiography showed an increased local bone formation on femurs injected with CP-533,536 as compared with vehicle injection. Total bone area and bone mineral content of the injected site with CP-533,536 were significantly increased by 11% and 19%, respectively. In the same animal model, a single dose (0.3 mg) of CP- 533,536 in poly-lactide-co-glycolide (PLGH) matrix significantly increased total bone area and bone mineral content by 19% and 14%, respectively, as compared with PLGH alone. In the second study, the right femurs of male rats were stabilized with intramedullary pins and were subjected to closed transverse fracture (under anesthesia). On days 3, 4, and 5 post-surgery, the rats were percutaneously injected with 0 or 5 mg of CP-533,536 to the fracture site. At 3 weeks post- fracture, the femurs treated with CP-533,536 had larger and denser calluses than those in controls as assessed by radiography. Histological measurements of fracture calluses showed significant increases in total callus area (29%), bony callus area (14%) and cartilaginous callus area (47%) in the rats treated with the compounds as compared with controls. Biomechanical test showed that maximal load and stiffness were significantly increased by 12 and 29%, respectively, for the femurs treated with CP-533,536 compared with those treated with vehicle. In the same animal model, fracture healing was significantly improved by a single dose (15 mg) of CP-533,536 in PLGH matrix. These data demonstrated that CP-533,536 stimulated periosteal bone formation and callus formation in rats. These results suggested that non-prostanoid EP2 receptor agonists are effective in enhancing bone healing. VERTEBRAL FRACTURE EFFICACY OF ONCE-A-WEEK RISEDRONATE R. Eastell1*, N. B. Watts2, Z. Li3, M. Hoseyni3, E. Seeman4, R. Lindsay5 1University of Sheffield, Sheffield, UK 2University of Cincinnati, Cincinnati, USA 3Procter & Gamble Pharmaceuticals, Mason, USA 4University of Melbourne, Melbourne, Australia 5Helen Hayes Hospital, West Haverstraw, USA A recent randomized, active-controlled trial demonstrated that risedronate (35 mg) once-a-week (OaW) and 5 mg daily produce comparable increases in BMD and reductions in bone remodeling. Although morphometric vertebral fractures were assessed in this trial, antifracture efficacy was not evaluated because of the absence of a placebo group. The objective of our analysis was to estimate the efficacy of OaW risedronate in reducing the risk of new vertebral fractures at 1 year, by using the OaW trial's enrollment criteria to construct matching control and risedronate 5 mg groups from patients enrolled in the risedronate Phase III fracture studies. Comparison of baseline characteristics indicated a high degree of similarity between the constructed placebo and 5 mg groups and the 35 mg weekly and 5 mg daily groups as enrolled in the OaW study (mean lumbar spine T-score: -3.17 to -3.03; mean age: 67.6 to 68.1; incidence of prevalent vertebral fracture: 34.8% to 35.7%). No statistically significant difference was observed in fracture
incidence for the 5 mg groups in the Phase III and OaW studies. Compared with matched
placebo, weekly treatment reduced the risk of new vertebral fractures by 77% within 1
year. This reduction is consistent with the previously reported 1-year fracture efficacy
of daily risedronate.
EFFECTS OF RISEDRONATE (RIS) ON TRABECULAR ARCHITECTURE IN EARLY POSTMENOPAUSAL WOMEN AS MEASURED BY MICROCOMPUTED TOMOGRAPHY A. Grauer1*, B. Borah2, T. E. Dufresnes2, P. A. Chmielewski2, M. C. Prenger2, M. D. Manhart2 1Procter and Gamble Pharmaceuticals, Geneva, Switzerland 2Procter & Gamble Pharmaceuticals, Cincinnati, Ohio, USA RIS treatment reduces the risk of radiographic vertebral fracture by up to 65% within the first year of treatment. Increases in bone mineral density (BMD) explain only about 1 third of the fracture risk reduction, suggesting that other factors, such as the preservation of bone architecture, are responsible for most of the antifracture efficacy. We used three-dimensional microcomputed tomography (3D microCT) to examine paired (baseline and 1 year) iliac crest bone biopsy samples in order to determine the effects of RIS on trabecular architecture. Biopsy samples were obtained from women (6-60 months postmenopausal) who were enrolled in a double-blind, placebo-controlled study evaluating the effects of RIS (5 mg daily) on lumbar spine (LS) BMD. The mean percent change from baseline in LS BMD after 1 year was - 2.9% in the placebo group and +1.4% in the RIS group. Compared with the RIS group, the placebo group (n=12 pairs) showed decreases in bone volume (BV/TV) (p=0.032), trabecular thickness (p=0.061), and trabecular number (p=0.025), and increases in trabecular separation (p=0.014) and bone surface/bone volume (p=0.051), indicating a deterioration of trabecular architecture in the placebo group. There were no significant changes in bone volume or architectural parameters vs. baseline in the RIS group (n=14 pairs). These data demonstrate that RIS preserves trabecular architecture in postmenopausal women within 1 year, which may partly explain RIS's rapid anti-fracture benefit. COMPARATIVE MINERAL BINDING AFFINITIES OF SELECTED BISPHOSPHONATES R. G. Russell1*, A. H. Mangood2, E. M. Gaafar2, W. Wu2, F. H. Ebetino3, D. J. White3, R. J. Phipps3, G. H. Nancollas2 1Oxford University, Oxford, UK 2SUNY Buffalo, Buffalo, NY 14260, USA 3Procter & Gamble Pharmaceuticals, Mason, OH 45040, USA Both clinical and experimental data indicate that bisphosphonates (BPs) differ in terms of their interactions with bone, and that these differences may result in subtle differences in their pharmacological behaviour. The current study further examines the potential contribution of mineral binding to the potency and duration of action of individual BPs. We investigated the effects the BPs alendronate, risedronate, and etidronate on hydroxyapatite (HAP) and octa calcium phosphate (OCP) crystal growth and dissolution, using the Constant Composition method at ionic strength 0.15 M and 37 deg C. Adsorption affinity constants (KL) were calculated from the kinetic results. Significant differences in KL (P<0.001) were observed among the three BPs for HAP growth (pH 7.4) and OCP growth (pH 6.5), resulting in a rank order alendronate > risedronate > etidronate. These conditions probably simulate the rate and extent of BP binding onto bone. Binding affinities were lower for crystal dissolution under conditions likely to prevail during osteoclastic resorption (HAP at pH 4.5 and OCP at pH 6.0), but the order alendronate > risedronate > etidronate was the same. Preliminary results from other BPs suggest that ibandronate and zoledronate have higher KL than alendronate and risedronate, at least for HAP dissolution. In conclusion, risedronate has lower kinetic binding affinity than
alendronate for the mineral substrates HAP and OCP. This difference may contribute to the
apparent shorter terminal (bone) half-life of risedronate, and to a faster clinical
on-response (fracture reduction) and off-response (recovery of bone turnover after
cessation of dosing) seen with risedronate compared with alendronate.
THE REPAIR OF AN ULNAR CRITICAL DEFECT WITH A SELECTIVE PROSTAGLANDIN E2 (PGE2) EP-2 RECEPTOR AGONIST V. M. Paralkar1*, F. Borovecki2, F. Dumont1, R. Korsmeyer1, D. Plowchauk1, H. Z. Ke1, K. O. Cameron1, B. A. Lefker1, T. A. Owen1, M. Li1, P. DaSilva-Jardine1, S. Vukicevic2, D. D. Thompson1, 1Pfizer Global Research and Development, Groton, CT, USA 2University of Zagreb School of Medicine, Zagreb, Croatia In spite of significant advances in our understanding of the biology of fracture healing, the morbidity and mortality associated with impaired/delayed fracture healing remains high and a significant medical need exists to ensure rapid bone healing. Significant improvements in bone repair have been demonstrated with biological products such as Bone Morphogenetic Proteins. However, problems remain with the use of these proteins. Prostaglandin E2 is known to increase bone formation and enhance bone healing. However, due to side effects including diarrhea, PGE2 is an unacceptable therapeutic option. PGE2 mitigates its pharmacological activity via four receptor subtypes, EP1, EP2, EP3 and EP4. We have discovered that the EP2 receptor subtype is responsible for the local bone anabolic activity of PGE2. In this abstract we report the ability of CP-533,536 a selective EP2 agonist to heal ulnar critical size defect in one year old male beagle dogs 11±1 kg in weight. A 1.5 cm segmental defect was made in the mid-ulna. The dogs were divided in to the following groups. A 1 ml of PLGH B 50 mg of CP-533,536 (1 ml PLGH formulation). C 10 mg of CP-533,536 ( 1 ml PLGH formulation). D 10 mg of CP-533,536 ( 0.2 ml PLGH formulation). It was observed that a single application of CP-533,536 induced full rebridgement in dogs present in groups B, C and D. The newly formed bone remodeled back to the same shape and size as the contralateral bone by week 24. In comparison ulnae treated with the vehicle did not show any significant healing as assessed by radiography and histology. The overall complete bone regeneration and repair was observed in more than 70% of animals tested. Since healing was observed at both 10 mg and 50 mg of CP-533,536 we do not know the lowest efficacious dose nor a dose volume co-relationship. In conclusion, a single administration at the time of surgery with CP-533, 536 (a non-prostanoidal EP-2 agonist) in PLGH matrix healed a critical sized defect in the canine ulna. These data indicate that EP2 receptor selective agonists may have therapeutic potential for bone healing in humans. ALLELE OF RUNX2/CBFA1 INCREASES ADULT BONE MINERAL DENSITY AND PROTECTS AGAINST COLLES' FRACTURE OF THE WRIST T. Vaughan1*, J. A. Pasco2, M. A. Kotowicz2, G. C. Nicholson2, N. A. Morrison2 1Genomics Research Centre, School of Health Science, Griffith University, Gold Coast, Australia 2Department of Medicine, The University of Melbourne, Barwon Health, Geelong Hospital, Geelong, Australia The aim of this study was to determine if DNA polymorphism within runt-related gene 2 (RUNX2)/core binding factor A1 (CBFA1) is related to bone mineral density (BMD). RUNX2 contains a glutamine-alanine repeat where mutations causing cleidocranial dysplasia have been observed. Two common variants were detected within the alanine repeat: an 18 base pair deletion and synonymous alanine codon polymorphism with alleles, GCA and GCG. Rare mutations that may be related to low BMD were observed within the glutamine repeat. Subjects of the Geelong Osteoporosis Survey (GOS) were ranked according to age and weight adjusted femoral neck BMD. The GCA allele was over represented (p=0.019) in the highest decile (n=130) compared to the lowest decile (n=135) of BMD. In 575 randomly selected subjects of GOS, the GCA allele was associated with higher BMD at all sites tested. The effect was maximal at the ultra-distal radius in postmenopausal women (p=0.006). In a separate fracture study, the GCA was significantly protective against Colles' fracture in elderly women but not spine and hip fracture. The GCA allele confers increased BMD and is protective against a common form of osteoporotic fracture, suggesting that RUNX2 variants may be related to genetic effects on BMD and osteoporosis. OSTEOPROTEGERIN ALLELES PREDISPOSE TO PAGET'S DISEASE A. Daroszewska1*, L. J. Hocking1, B. L. Langdahl2, F. E. A. McGuigan1, W. D. Fraser3, S. H. Ralston1 1Bone Research Group, University of Aberdeen, Aberdeen, UK 2Department of Endocrinology, Arhus Amtssygehus, Arhus, Denmark 3Department of Clinical Chemistry, University of Liverpool, Liverpool, UK Paget's disease of bone (PDB) is a common condition with a strong genetic component, but the genes responsible are poorly defined. Recent work has shown that mutations in the RANK gene cause familial expansile osteolysis (FEO) - a rare bone dysplasia with similarities to PDB. In view of this, we investigated the hypothesis that allelic variation in other components of the RANK pathway might predispose to PDB. In this study, we conducted mutation screening of the Osteoprotegerin (OPG) gene and looked for evidence of an allelic association with Paget's disease in a series of 181 UK Pagetic patients and 184 non Pagetic UK controls. We identified several single nucleotide polymorphisms (SNP) in the OPG gene and investigated seven of these in an association study. We studied 3 SNP's in the OPG promoter; one in exon 1 (a C/G substitution resulting in a Lys-Asn amino acid change), one close to the splice site in intron 2 (IVS2 +4C/T) a conservative change in exon 4 (A/G; Leu-Leu) and a change close to the splice site in intron 4 (IVS4 +8A/C). All SNP's were in linkage disequilibium (p<0.0001; data not shown). Two SNP's showed a significant association with Paget's disease; an A/G change situated at position -943 in the gene promoter at a binding site for the transcription factor Oct-3 and another C/T change situated 4 bases from the splice donor site in intron 2 (IVS2 +4C/T). The odds ratio for Paget's disease in 'GG' homozygotes at the -943A/G site was 6.46 [1.42-29.4], when compared with AA homozygotes (p=0.016) and the odds ratio for CC homozygotes at the IVS2 +4C/T site was 1.77 [1.14-2.74] when compared with carriers of the 'T' allele (p=0.010). These data indicate that common allelic variants in the OPG gene predispose to Paget's disease. Further work is in progress to define the functional effects of the polymorphisms we have described and to extend these observations to patients with familial Paget's disease and to other populations. COMMON HERBS, ESSENTIAL OILS AND MONOTERPENES POTENTLY MODULATE BONE METABOLISM R. C. Mühlbauer*, R. Felix, A. Lozano, S. Palacio, A. Reinli Bone Biology Group, Dept. Clinical Research, University of Bern, Bern, Switzerland Osteoporosis is a major health issue in aging populations. It has been suggested that vegetable components in the food inhibit bone resorption and may help to prevent bone loss. Therefore, we investigated the antiosteoporotic potential of herbs, essential oils (EO) and monoterpenes (MT). Herbs (1g/day), EO and MT (100 mg/day) were added to the food of 250 g rats. Bone resorption was assessed with the urinary excretion of tritiated tetracycline from prelabeled rats. Bone mineral density (BMD) was measured in the proximal tibia by computer tomography. In vitro resorptive activity (pits/osteoclast) was assessed in osteoclasts isolated from long bones of 1-2 day old rats cultured on ivory slices. Herbs rich in EO (sage, rosemary and thyme), EO extracted from these herbs and other plants (oils of sage, rosemary, juniper, pine and eucalyptus) as well as their main components (the MT thujone, eucalyptol, camphor, borneol, thymol, alpha-pinene, beta-pinene, bornylacetate and menthol) inhibit bone resorption. Pine oil, eucalyptol and camphor are also inhibitory at doses of 20, 24 and 14 mg/day respectively. Pine oil, used as a representative EO, protects from loss of trabecular BMD in aged ovariectomized rats. Borneol, thymol and camphor ([1 mM]) inhibit resorption activity of osteoclasts. Non-polar MT require metabolism to be active in vitro, as demonstrated for cis-verbenol, a metabolite of alpha-pinene occurring in human urine, inhibiting osteoclast activity in contrast to the parent compound. Borneol inhibits within 30 minutes the formation of actin rings which indicate cell polarization, a prerequisite for resorbing osteoclasts. Thus, MT and/or their metabolites inhibit resorption also in vitro. Our study demonstrates for the first time that EO and MT are efficient inhibitors of bone resorption in the rat. Thus, a generous consumption by man of herbs as well as of food products flavored with these compounds may decrease bone resorption. Furthermore, there is a long history of use of EO and MT taking advantage of the transdermal and pulmonary absorption for medical applications for relief of colds and muscle pain. Thus, the external application of EO and MT may offer an additional possibility to decrease the incidence of osteoporosis. HIP FRACTURES, MORPHOMETRY AND GEOMETRY J. S. Gregory1,2*, D. Testi3, P. E. Undrill2, R. M. Aspden1 1Department of Orthopaedics, University of Aberdeen, UK 2Department of Bio-Medical Physics and Bio-Engineering, University of Aberdeen, UK 3Istituti Ortopedici Rizzoli, University of Bologna, Italy A new method has been developed for measuring the morphometry of the femur which provides a powerful tool for discriminating between Osteoporotic- fracture and control subjects. We will show that a mathematical description of the shape of the proximal femur is more effective than both individual geometric and most BMD measurements. By combining BMD and morphometric measurements, we were able to discriminate between the two groups with almost 90% accuracy. Active shape modelling (ASM) is an image-processing technique that can be used to build a model of a shape. Shapes that have common features but are subject to natural variation, such as leaves are described by a series of orthogonal (and therefore independent) descriptors. After building a model, new images can be analysed by examining how the location of landmark-points deviate from the mean co-ordinates. Geometric measurements, such as neck length, width and neck- shaft angle have previously been shown to be significantly related to fracture risk. However such measurements are highly correlated and, therefore, of limited usefulness. A set of 74 images was used for this study (46 control, 28 fracture). All subjects underwent DXA and standard radiographic examination. The following BMD measurements were recorded: total body BMD, total body BMC, femoral neck BMD, trochanter BMD, and Ward's triangle BMD. ASM and geometric measurements were calculated from radiographs. Discriminant analysis was applied to find out which variable or combination of variables was best able to discriminate between the two groups. Using a single variable, ASM (77% correct) was more accurate than 4 out of 5 of the BMD measurements, being beaten only by Ward's triangle BMD (81%), whilst the geometric classifier was the weakest. By combining Ward's triangle, with the ASM data, classification improved to 89%. This study shows that morphological analysis alone may be a more powerful discriminator than most BMD measurements and, in combination with BMD data, can create an accurate classification method. These initial results are taken from a relatively small data set and further studies are being undertaken to confirm these promising results. |
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