ORAL PRESENTATIONS

O-1

SIGNALING VIA BETA-CATENIN DIRECTS MESENCHYMAL LINEAGE ALLOCATION TOWARDS OSTEOGENESIS

G. Mbalaviele^1,2, S. Sheikh¹, C. Castro¹, S-L. Cheng¹, J. Stains¹, R. Civitelli¹*

¹Div. of Bone and Mineral Diseases, Washington University in St. Louis, Missouri, USA

²Pharmacia Corp., St. Louis, Missouri, USA

Loss of function mutations of some wnt family members, as well as Lef1, Tcf1 or LRP-5 genes deletion cause abnormal development or lack of skeletal elements, whereas transcriptionally active beta-catenin prevents adipogenesis and stimulates chondrogenesis and bone growth in limb development. We tested the hypothesis that beta-catenin is involved in mesenchymal lineage allocation to osteogenic cells. A beta-catenin mutant with constitutive transcriptional activity (deltaN151) had no significant effects on markers of osteogenic differentiation in uncommitted, multipotential C3H10T1/2 and C2C12 cells. However, active beta-catenin synergized with BMP-2 resulting in dramatic stimulation of alkaline phosphatase activity, osteocalcin gene expression and matrix mineralization, whereas the abundance of the osteoblast-specific transcription factor, Cbfa1 was unaffected. Likewise, deltaN151 facilitated differentiation of immature but committed MC3T3-E1 osteoblastic cells. By contrast, deltaN151 prevented adipogenic differentiation from pre-adipocytic 3T3- L1 cells and uncommitted C3H10T1/2 cells. BMP-2 did not significantly stimulate Tcf/Lef dependent transactivation, and expression of a dominant-negative Tcf-3 construct did not inhibit BMP-2 induced alkaline phosphatase stimulation in C3H10T1/2 cells, indicating that BMP-2 action is independent of Tcf/Lef mediated transactivation. Thus, beta-catenin directs osteogenic lineage allocation by enhancing mesenchymal cell responsiveness to osteogenic factors, such as BMP-2, while inhibiting adipogenesis. In further support of this hypothesis, 3-month-old transgenic mice expressing a truncated, dominant negative cadherin in osteoblasts exhibited 10% lower total body bone mass and 15% higher fat body mass relative to wild type littermate. Accordingly, bone marrow stromal cells from these animals contained 40% less osteoblast precursors and 2-3-fold more adipocyte precursors, and the ratio of membrane to cytoplasm-nucleus beta-catenin was higher in transgenic relative to wild type cells. Viral transduction of deltaN151 in bone marrow stromal cells from transgenic mice rescued this phenotype, restoring the number of osteogenic and adipogenic precursors to normal levels. Thus, depletion of transcriptionally active beta-catenin pools by the dominant-negative cadherin may cause a shift from osteogenesis to adipogenesis in vitro and in vivo. We propose that beta-catenin signaling directs mesenchymal cell differentiation towards the osteogenic lineage. In the absence of beta-catenin signaling, differentiation defaults to the adipogenic pathway.

[Programme]

 
O-2

FUNCTIONAL ROLE OF CANONICAL WNT SIGNALLING DURING OSTEOGENIC DIFFERENTIATION OF MESENCHYMAL STEM CELLS

S. L. Etheridge¹*, G. J. Spencer¹, D. Heath², P. G. Genever¹

¹Biomedical Tissue Research, University of York, York, UK

²Smith and Nephew GRC, York, UK

Mesenchymal stem cells (MSCs) differentiate into specialised cell types, including osteoblasts, adipocytes, myoblasts and stromal fibroblasts. They are potential candidates for a range of therapeutic applications but the precise signalling pathways that determine the differentiated fate of MSCs are not fully understood. Recent evidence suggests that the Wnt signalling pathway may be instrumental in regulating osteogenesis. Wnts are secreted proteins that bind to transmembrane Frizzled (Fz) receptors and downstream canonical signalling results in the inhibition of GSK-3beta causing the translocation of beta-catenin into the nucleus to induce expression of Wnt target genes. To identify potential roles for Wnt signalling in mesenchymal fate determination, we determined expression profiles and functionality of key components of the Wnt pathway in primary human MSCs and mesenchymal cell lines in the presence and absence of osteogenic stimuli.

Primary human bone marrow stromal cells were isolated from femoral heads and maintained in culture. Flow cytometric analysis revealed that repeat passaging generated a discrete population of cells (designated MSCs) that expressed the mesenchymal markers CD29, CD44, CD105 and CD166. Using RT-PCR and western blot analysis, we identified that MSCs expressed Wnt2b, Wnt4, Wnt5a, Fz3, Fz4, and that levels of expression were dependent on the extent of osteogenic induction. By quantitative immunofluorescent image analysis, we identified nuclear beta-catenin in 20-30% of total DAPI-positive nuclei within these cells, indicative of endogenous Wnt signalling. Exposure to LiCl, which promotes pan-specific canonical Wnt signalling by inhibiting GSK-3beta, increased beta-catenin nuclear translocation to around 90% within 2 hours. LiCl application to MSCs and primitive osteoblasts grown under osteogenic conditions, also caused a dose-dependent decreased in alkaline phosphatase activity and von Kossa-positive bone nodule formation, compared to untreated or NaCl-treated controls.

Therefore a tightly controlled level of Wnt signalling appears to be fundamental to the maintenance and differentiation of mesenchymal progenitor cells. Whilst endogenous Wnt/beta-catenin signalling operates in MSCs, macro-activation of canonical Wnt pathways through Li-induced inhibition of GSK-3beta appears to significantly retard osteoblast differentiation. These data indicate that the identification specific Wnt/Fz pairs that influence mesenchymal differentiation will contribute significantly to our understanding of the molecular mechanisms that regulate bone formation.

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O-3

PLATELET-DERIVED GROWTH FACTOR-D, A NOVEL BONE- ANABOLIC GROWTH FACTOR

S. Topouzis*, K. Lum, M. S. Holdren, J. Shin, C. Birks, D. Dong, S. D. Hughes, H. P. Ren, T. Palmer, E. E. Moore

ZymoGenetics Inc., Seattle, USA

We report on the biochemical characterization and bone anabolic properties of a new member of the PDGF family, PDGF-D. PDGF-D is secreted as an inactive precursor dimer, PDGF-DD-Full-Length (PDGF-DD-FL). Proteolytic cleavage at the Arginine 249 residue (human sequence) releases the bioactive C-terminal moiety, PDGF-DD-Growth-Factor-Domain (PDGF-DD-GFD). PDGF-DD-GFD binds only to PDGF receptor-beta and mediates its biological effects through stimulation of both PDGF receptor beta-beta or alpha-beta dimers. PDGF-B has been previously reported to possess bone-anabolic properties. For this reason, we compared the effects of PDGF-B and PDGF-D on the skeleton by bolus delivery of adenoviral constructs expressing recombinant human PDGF-BB (rhPDGF-BB) or rhPDGF-DD-FL into two-month-old female mice. ELISA analysis of sera showed that circulating PDGF- BB and PDGF-DD protein levels peaked around ten to fifteen days post virus treatment and had returned to basal by four weeks. Histopathological evaluation of multiple bones including the femur, humerus and vertebrae revealed extensive formation of mineralized endosteal bone within the marrow cavity as early as two weeks after treatment with either rhPDGF-BB or rhPDGF-DD-FL adenoviral constructs but not with a parental adenoviral construct. The newly formed bone persisted with some signs of remodeling for at least eight weeks post-treatment. In order to elucidate the underlying mechanism of action, we tested the effect of recombinant factors on marrow-derived human Mesenchymal Stem Cells (huMSC) in vitro. Both rhPDGF-BB (expressed in E.coli) and Glu-Glu-tagged rhPDGF-DD-GFD (expressed using baculovirus system technology) induced eight- to ten-fold increases in thymidine incorporation in huMSC. Furthermore, when huMSC were maintained in an osteogenic medium, treatment with rhPDGF-BB or rhPDGF-DD-GFD for the initial 6 days resulted in increased mineralization at 3 weeks, as assessed by von Kossa staining and calcium content. In addition, immunocytochemical analysis showed that treatment with either protein enhanced the nuclear accumulation of the osteoblast-specific marker Osf2/CBFA-1. These observations are consistent with a positive effect of PDGF-B and PDGF-D on osteoblast differentiation from multipotential precursors and may explain their dramatic effects on trabecular bone formation in vivo. In combination, the present results support the notion that PDGF- D, like PDGF-B, has bone anabolic potential and may be clinically useful in bone regeneration.

[Programme]

 
O-4

GENETIC ABLATION OF PROLINE-RICH TRANSCRIPT OF THE BRAIN IS ASSOCIATED WITH INCREASED BONE MASS IN MICE

D. W. Sommerfeldt¹*, M. Priemel¹, X. Wang², T. Schinke¹, M. Amling¹, S. Mansour², J. M. Rueger¹

¹Dept. of Trauma and Reconstructive Surgery, Hamburg University Hospital, Hamburg, Germany

²Dept. of Human Genetics, University of Utah, Salt Lake City, USA

Using differential display-PCR we have previously shown that proline-rich transcript of the brain (prtb) is also expressed in osteoblasts where its expression is upregulated during serum exposure and adhesion to various substrates. In addition, an increased expression during osteoblast differentiation in vitro was observed. To determine the effect of prtb during bone remodeling in vivo, we analyzed the bones of prtb-deficient mice using static and dynamic histomorphometry.

Initial contact radiographies of 10 week-old prtb-/- mice, which have no gross pathological abnormalities, demonstrated an increase in bone density. Static histomorphometry confirmed a 41%-increase in bone mass within the spine of 10- week-old prtb-/- mice as compared to wildtype controls (17.85 ± 1.12 % vs. 12.7 ± 2.01 %, p<0.001). A similar phenotype was observed in mature 6-month-old animals (16.68 ± 2.25 % vs. 12.79 ± 3.36 %). Surprisingly, bone cell number and bone cell

surface indices were nearly identical between prtb-/- and wildtype mice. In contrast, dynamic histomorphometry after fluorochrome labeling revealed a significant increase in bone formation rate in prtb-/- mice at 10 weeks and 6 months of age compared to wildtype littermates (223.79 ± 76.42 mm3/mm2/y vs. 144.61 ± 39.08 mm3/mm2/y, p<0.05). These findings suggest that the bone phenotype in prtb- deficient mice is due to a functional change of osteoblast activity and not due to an effect on bone cell differentiation and/or survival. Taken together, our data demonstrate that in adult mice the prtb gene is selectively expressed in brain and bone and that prtb-deficiency leads to an increased bone mass in mice due to increased bone formation.

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O-5

DLK1 (EGF-LIKE HOMEOTIC PROTEIN) OVEREXPRESSION INHIBITS MATURE ADIPOCYTE AND OSTEOBLAST FORMATION AND MAINTAINS THE BIOPOTENTIAL DIFFERENTIATION CAPACITY OF HUMAN BONE MARROW STROMAL CELLS

B. M. Abdallah¹*, C. H. Jensen², G. Leslie², T. B. Jensen³, M. Kassem^1,4

¹Clinic for Molecular Endocrine Treatment KMEB, Odense University Hospital, Odense, Denmark

²Institute of Medical Immunology, Univeristy of Southern Denmark, Odense, Denmark

³Department of Human Genetics, University of Aarhus, Aarhus, Denmark

⁴Dept of Endocrinology M, Odense University Hospital, Odense, Denmark

The molecular mechanisms of marrow stromal cells (also called mesenchymal stem cells, MSC) differentiation into specific lineages are poorly understood. Dlk1 (delta like, also named FA1, Pref-1) is an imprinted gene, that is paternally expressed and encodes for cell-surface transmembrane protein containing six epidermal growth factor (EGF) repeat motifs homologues to those present in delta/notch/serrate family. Mice deficient for dlk1 are small with increase fat deposition and skeletal abnormalities suggesting that dlk1 is important for mesecnhymal stem cell differentiation into adipocytes and osteoblasts, Thus, we examined the role of dlk1 during bone development and MSC differentiation, Using real time PCR, we found that dlk1 is highly expressed in human fatal bone with maximal expression at week 11 of gestational age and that its expression declines in adult bone. Osteoblastic cells derived from human fetal bone, adult bone and MSC expressed dlk1. Full length human dlk1 was cloned in a retroviral vector and employed to transduce a multipotential human MSC. The resultant cell line (hMSC-dlk1+) expressed high levels of dlk1 protein in the conditioned media (more than 1.2 microg/ml) as determined by ELISA and more than 90 per cent of the cells expressed dlk1 as shown by immunostaining. Growth of hMSC-dlk1+ cells did not differ from wild type cell. Under culture conditions that promote adipocyte formation, hMSC-dlk1+ cells formed significantly fewer mature adipocytes quantitated by flow cytometry and Oil red staining as compared with wild type MSC. Similarly, when the hMSC-dlk+ cells were cultured in osteoblast medium, the cells formed less mineralized bone matrix compared with wild type MSC. Analysis of gene expression revealed that gene markers of late stages of adipocyte differentiation (aP2, adiponectin) and osteoblast differentiation (osteocalcin, collagen type I) were inhibited. However, the lineage commitment markers of adipocytes (PPARã2, ADD1) and osteoblasts (Runx2/Cbfa1) were not affected. Our results suggest that dlk1 plays an important role in expanding the pool of bipotential precursor cells during mesenchymal stem cell differentiation.

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O-6

SKELETAL CHANGES IN TRANSGENIC MALE MICE OVEREXPRESSING HUMAN AROMATASE

Z. Q. Peng¹*, X. D. Li², M. Poutanen², R. Santti¹, H. K. Väänänen¹

¹Dept. of Anatomy, Univ. of Turku, Finland

²Dept. of Physiology, Univ. of Turku, Finland

Aromatase enzyme catalysing the conversion of androgens to estrogens is expressed in many tissues and participates to the regulation of steroid homeostasis. In order to understand the skeletal responses to testosterone/estrogen and especially to the excess of estrogen produced by extragonadal biosynthesis, we investigated the bone changes in human aromatase over-expressing transgenic (AROM+) mice. In the young animals body weight gain of AROM+ mice was somewhat slower, and lengths of long bones were significantly shorter in AROM+ than in wild type (WT) animals (3%-5.2%, p<0.05). However, the ash weight of those bones was not different between groups. pQCT measurements showed that AROM+ animals had a much higher density of the trabecular bone in the proximal tibia and distal femur compared to the WT animals. Histomophometric data was consistent with pQCT results, and further demonstrated that the longitudinal growth rate of tibia was decreased in AROM+ mice, and bone formation rate (BFR) decreased in trabecular bone as well as on periosteum. All these changes were more striking in males than in females. Aged male AROM+ mice mainly showed similar changes in trabecular bone than young animals, but their BFR was significantly increased compared to age matched WT animals. Length of femur was 3% shorter in old AROM+ than WT mice. In contrast, the tibia was much shorter (23.2%), while its ash weight was much heavier (24.0%), and bending strength was markedly higher (21.2%) in old AROM+ when compared to WT mice. Concentration of testosterone was decreased in both serum and interstitial fluid of testis in young AROM+ than in WT mice, and the concentration of estradiol was only increased in testis of AROM+. However, in aged AROM+ mice the testosterone and estradiol were both increased in serum and testis fluid. These results are in agreement with the suggestion that enhanced production of estrogen from testosterone in the peripheral tissues by aromatase enzyme can affect the skeletal growth in male. These results also suggest a marked difference in response between femur and tibia.

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O-7

RALOXIFENE INCREASES SERUM ESTRADIOL AND TESTOSTERONE AND DECREASES BIOCHEMICAL MARKERS OF BONE TURNOVER IN MIDDLE AGED HEALTHY MEN WITH LOW BASELINE SEX STEROID SERUM VALUES

B. Uebelhart¹*, F. Herrmann², I. Pavo³, R. Rizzoli¹

¹Division of Bone Diseases, University Hospital, Geneva, Switzerland

²Department of Geriatrics, University Hospital, Geneva, Switzerland

³Eli Lilly, Vienna, Austria

Raloxifene (RLX) is a SERM which acts as an estrogen-agonist in bone of postmenopausal women. To investigate the effects of estrogen on bone remodeling in men without the risk of its feminizing action, we conducted a randomized RLX vs placebo (PBO) two-sequence crossover study in 43 healthy eugonadal men (mean age:56 years, range 49-70). Subjects were randomly assigned to receive either RLX 120 mg/day or PBO for a 6-week duration followed by a 2-month wash-out period, before crossing-over. Biological bone markers, osteocalcin (OC), total alkaline phosphatase (Alk.Ph.), urinary total deoxypyridinoline/creatinine (d-Pyr) and serum sex steroid hormones, total and bioavailable testosterone (T) and estradiol (E2) were measured before and after PBO and RLX periods. To test the hypothesis whether serum E2 and T levels could influence the bone response to RLX, we used a logistic regression model to predict the decrease of d-Pyr in relation to baseline E2 and T as the independent variables. We thus determined thresholds of 101.8pmol/l and 4.79nmol/l for total and bioavailable E2, and of 19.4nmol/l and 0.35nmol/l for total and bioavailable T, respectively which maximized both sensitivity and specificity of the predictive models. In patients below the thresholds, RLX increased total (+13%) and bioavailable (+19%) T and total (+16%) and bioavailable (+16%) E2 without any significant changes in the patients with the highest baseline sex steroid values. In patients below the E2 thresholds (total, bioavailable), RLX decreased OC (-15%, - 18%), Alk.Ph. (-7%, -9%) and d-Pyr (-10%, -12%) without affecting the values in patients above these thresholds. In patients below the T thresholds (total, bioavailable), RLX decreased OC (-11%, -14%), Alk.Ph. (-5%, -4%) and d-Pyr (-6%, -4.5%) without any significant changes in patients above these thresholds. In conclusion, RLX 120 mg/day for 6 weeks increased serum total and bioavailable E2 and T and decreased biochemical markers of bone turnover in middle-aged healthy men with low baseline total and bioavailable E2 and T values without any significant changes in subjects with higher values. This suggest that RLX action on bone remodeling is dependent on serum sex hormones levels.

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O-8

SHORT COURSES OF HORMONE REPLACEMENT TREATMENT IN HEALTHY WOMEN HAVE LONG-TERM PREVENTIVE EFFECTS ON BONE MASS AND OSTEOPOROTIC FRACTURES

Y. Z. Bagger*, L. B. Tanko, P. Alexandersen, C. Christiansen

Center for Clinical and Basic Research, Ballerup, Denmark

Background: Hormone replacement therapy (HRT) is the agent of first choice for the prevention of postmenopausal bone loss, but is commonly given for only a few years. Randomized studies indicate that HRT can increase bone mineral density and reduce the incidence of fractures by about 50%. Some studies have indicated that a decrease risk of fractures is evident only in women who have taken HRT for at least 5 years. However, few studies showed the controversial results regarding withdrawal effect on bone mass

Aim: To investigate the long-term effects of short-term HRT on bone loss and incidence of osteoporotic fractures assessed 5-15 years after stopping treatment.

Methods: We studied a group of 347 early postmenopausal women who had previously participated in 4 placebo-controlled HRT trials and were re-examined 5, 11, or 15 years after stopping HRT. Of those women, 263 received 2-3 years of HRT or placebo with no further treatment, and 84 women took HRT for more than 3 years. Spine BMD and forearm BMC were assessed at baseline, at the end of HRT and at the time of follow-up. Lateral X-ray of the thoracic and lumbar spine was performed. Vertebral deformities from T4 to L4 were assessed by digital measurement. A more than 20% reduction of the vertebral height was considered as a fracture. Non-vertebral fractures were recorded by standard questionnaire.

Results: The residual effects of 2-3 years of HRT on bone mass (>4%) compared to placebo were evident up to 15 years after stopping treatment. After treatment, the rate of bone loss returned to normal postmenopausal rates. Long-term use of HRT showed more pronounced ultimate effect on bone mass. The risk of all-osteoporotic fractures was significantly decreased by previous short-term HRT (OR=0.42 [95% confidence

interval 0.24-0.75]). 'Fast loser' subjects who never received HRT had more than four- fold higher incidence of osteoporotic fractures compared to women who received short-term HRT and had normal rate of bone loss after stopping treatment.

Conclusions: Short-term HRT administered in the early postmenopausal years offers long-lasting benefits for the prevention of postmenopausal bone loss and osteoporortic fracture.

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O-9

POLYMORPHISMS IN THE CYP19 AND ANDROGEN RECEPTOR GENES AND BONE MASS IN WOMEN WITH OR WITHOUT HORMONE REPLACEMENT THERAPY. THE DANISH OSTEOPOROSIS PREVENTION STUDY

C. L. Tofteng¹*, A. Kindmark², H. Brandstrom², B. Abrahamsen³, S. Petersen¹, F. Stiger², L. S. Stilgren³, J. E. B. Jensen¹, P. Vestergaard, B. I. Langdahl⁴, L. Mosekilde⁴

¹Osteoporosis Research Clinic, Hvidovre Hospital, Hvidovre, Denmark

²Department of Medical Sciences, Uppsala University Hospital, Uppsala, Sweden

³Department of Endocrinology, Odense University Hospital, Odense, Denmark

⁴Department of Endocrinology, Aarhus County Hospital, Aarhus, Denmark

Polymorphisms in the androgen receptor (AR) gene and genes encoding enzymes involved in synthesis of sex steroids (e.g the CYP19 gene encoding aromatase) have recently received attention in osteoporosis research. In the Danish Osteoporosis Prevention Study, recent postmenopausal women were allocated to either hormone replacement therapy (HRT) or no treatment. We genotyped 1792 women for the CYP19 (TTTA)n repeat (short (TTTA)n<or=7, long (TTTA)n>7), the CYP19 C1558- T, and the AR (CAG)n repeat polymorphism (short (CAG)n<22, long (CAG)n>or=22), and investigated associations with bone mineral density (BMD) and 5-year change in BMD.

The CYP19 polymorphisms were in strong linkage disequilibrium. Perimenopausal bone mass or bone loss in untreated women was not associated with the CYP19 polymorphisms. In hormone treated women, BMD increase in the femoral neck was highest (0.3 pct/year) for long CYP19 alleles, lowest (-0.09 pct/year) for short alleles, and intermediate (-0.002 pct/year) in heterozygous women, P=0.015. Differences were also significant in the lumbar spine, total hip, and ultradistal forearm. The C1558-T T-allele was associated with a more pronounced response to HRT (P=0.04, total hip).

AR genotype was not related to BMD, but a modifying effect of sex hormone binding globulin (SHBG) was present. In the highest SHBG quartile (SHBG>95 nmol/l, n=222), AR genoype was associated with baseline BMD (femoral neck: P<0.001, total hip: P=0.008), but without a clear gene dosage effect.

In conclusion, we have demonstrated that polymorphisms in the CYP19 gene are associated with the magnitude of bone gain in response to HRT and that the (CAG)n repeat polymorphism in the AR gene is associated with bone mass in women with high levels of SHBG. These findings emphasize the complexity of the genetics of bone mass and bone loss.

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O-10

GENDER DIFFERENCES IN ASSOCIATION OF 5' ESTROGEN RECEPTOR ALPHA GENE POLYMORPHISMS WITH BONE PARAMETERS, HEIGHT AND FRACTURE RISK

S. C. E. Schuit^1,2*, J. B. J. van Meurs^1,2, A. E. A. M. Weel¹, M. van der Klift², A. P. Bergink^1,2, Y. Fang¹, P. P. Arp¹, J. P. T. M. van Leeuwen¹, H. A. P. Pols^1,2, A. G. Uitterlinden¹

¹Department of Internal Medicine, Erasmus MC, Rotterdam, The Netherlands

²Department of Epidemiology & Biostatistics, Erasmus MC, Rotterdam, The Netherlands

We investigated the influence of genetic variation of the estrogen receptor alpha (ESR1) gene on bone parameters in two population-based cohort studies: 1500 pre- menopausal women of The Eindhoven Study and 1276 post-menopausal women and 1125 men (age 55-80) of The Rotterdam Study. We analysed haplotypes of the PvuII and XbaI RFLPs in intron 1 of the ESR1gene and observed 3 haplotype alleles: 1=px, 53%; 2=PX, 35%, 3=Px, 12%. In post-menopausal women, without vertebral fractures, as well as in pre-menopausal women, we observed an allele-dose effect of the haplotype 1 allele leading to decreased stature (allele dose = 0.8 cm, p-trend = 0.02; allele dose = 0.5 cm, p-trend = 0.04, respectively). In post-menopausal women, we observed decreased lumbar spine BMD (allele dose = 0.03 g/cm²; p-trend < 0.01). and a 3-fold (95% CI 1.5-6.2) increased risk of vertebral fracture risk in individuals homozygous for haplotype 1. However, in the pre-menopausal women of the Eindhoven Study we found no association between haplotype 1 and lumbar spine BMD. In men, no association between the ESR1 haplotypes and BMD or vertebral fracture risk was observed. Interestingly, in a subset of 439 men and 379 women from the Rotterdam study, we observed a borderline significant association between haplotype 1 and E2 levels only in men (allele dose=3.3 pmol/l, p-trend = 0.06), but not in the (postmenopausal) women.

We conclude that the ESR1 gene PvuII-XbaI haplotype 1 is associated with lumbar spine BMD, vertebral fracture risk and height in post-menopausal women, while in pre-menopausal women haplotype 1 is associated with stature but not lumbar spine BMD.

We hypothesize that ESR1 genotype-dependent differences in stature are determined at puberty and remain throughout life. However, genotype-dependent differences in lumbar spine BMD materialize in women only after menopause. In this relatively estrogen-deficient state, genotype-dependent differences in ESR1 'sensitivity' lead to differences in fracture risk (and BMD) in women, but not in men in whom E2 levels are on average 3-fold higher. We hypothesize that, in contrast to post- menopausal women, men have an intact estrogen feedback mechanism and can counter-balance genotype-dependent differences in ESR1 'sensitivity'.

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O-11

NEW INSIGHTS INTO CALCINEURIN ALPHA FUNCTION AND SIGNALING

E. Abe¹, X. B. Wu¹, M. Lu¹, X. Zhang¹, B. S. Moonga¹, S. Epstein¹, H. C. Blair², L. Sun¹, M. Zaidi¹*

¹The Mount Sinai Bone Program and Bronx VA GRECC, New York, NY. USA

²University of Pittsburgh, Pittsburgh, PA. USA

Calcineurin Aalpha, a ubiquitously expressed Ca²⁺/calmodulin-sensitive phosphatase, is a target for two of the most widely used immunosuppressants, cyclosporine (CsA) and tacrolimus(FK506). We found that calcineurin Aalpha and Abeta are expressed in both osteoblasts and osteoclasts. Deletion of the Calcineurin Aalpha gene resulted in dramatic osteoporosis evident from a decrease in bone mineral density at the femur, tibia and lumbar spine. This was associated with reduced cortical thickness and a ~40% reduction in bone formation in tetracycline double labeling studies. Osteoblast progenitors, measured as colony-forming units-fibroblasts (CFU-Fs), in bone mineral cultures of calcineurin Aalpha^-/- mice showed no decrease when compared to wild type littermates. Likewise, the inhibition of calcineurin Aalpha by CsA and FK506 resulted in unchanged CFU-F counts. In contrast, there was a marked reduction in osteoclasts formation from hematopoetic progenitor cells in calcineurin Aalpha^-/- mice, as well as in wild type cultures treated with CsA or FK506. An action of calcineurin Aalpha on osteoblast differentiation was evident from a dramatic increase in alkaline phosphatase activity of MC3T3.E1 cells transduced with a TAT-calcineurin Aalpha fusion protein. Together, the results provide compelling evidence that calcineurin Aalpha has a key role in bone formation without affecting the osteoblast progenitor pool. In parallel, chemical cross-linking studies, immunoprecipitation experiments, and phosphatase activity assays revealed that calcineurin Aalpha bound to and dephosphorylated not only the transcription factor, NFATc, but also the transcription inhibitor, IkappaBbeta. This interaction resulted in the modulation of a variety of genes. Most notably, cells transfected with calcineurin Aalpha displayed a dramatic increase in NFATc1 and type 1 ryanodine receptor expression and a decrease in the phosphorylated form of IkapaBbeta. Likewise, deletion of the calcineurin Aalpha gene in the Aalph^-/- mouse resulted in a marked reduction in NFATc and NFkappaB (p50, p65 and c-Rel) expression and an increased expression of phosphorylated IkappaBbeta. The results suggest that calcineurin, through its phosphatase activity, not only dephosphorylates NFATc, but also dephosphorylates IkappaBbeta to enhance its binding to the NFkappaB members thus preventing their nuclear localization. Taken together, our results demonstrate that calcineurin Aalpha is a key regulator of osteoblastic bone formation, and that this effect may be exerted not only through the traditional NFATc pathway, but also via the dephosphorylation of IkappaBbeta.

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O-12

WNT-MEDIATED TCF GENE TRANSCRIPTION IN OSTEOBLASTS: A NOVEL EFFECTOR OF OSTEOTROPIC HORMONES AND CYTOKINES?

G. J. Spencer*, S. L. Etheridge, P. G. Genever

Biomedical Tissue Research, University of York, York, UK

Understanding communication between bone cells is essential to the development of novel therapeutic strategies to target diseases such as osteoporosis that result from aberrant bone mass homeostasis. Over recent years Wnt/beta-catenin signalling has emerged as a candidate pathway involved in bone growth and the regulatory control of bone mass although their exact cellular mechanism of action remains poorly understood. We have demonstrated that canonical Wnt/beta-catenin signalling operates in osteoblasts, which culminates in the activation of TCF/LEF-1 dependent gene transcription, the end-point effector of signalling. In this study we have identified TCF/LEF-1 responsive target genes and investigated the effects of osteotropic hormones and cytokines on canonical Wnt/beta-catenin mediated TCF/LEF-1 dependent transcription in osteoblasts. Using a specific luciferase reporter gene containing TCF consensus sites, we demonstrated that the Wnt mimetic LiCl and the cytokines IL-6, TNF-alpha and oestrogen caused a time and dose-dependent increase in basal osteoblastic TCF/LEF-1 dependent gene transcription. In contrast, vitamin D3 had an opposing effect, attenuating transcription. Transcriptional responses of osteoblasts correlated with nuclear translocation and were potently inhibited by overexpression of dominant negative beta-catenin. The effects of these

cytokines on TCF/LEF-1 gene transcription raise the intriguing possibility that the classic actions of these osteotropic agents may be mediated, at least in part, by cross talk or convergence with the osteoblastic Wnt signalling pathway on common transcription targets. By western blot analysis we demonstrated that LiCl inhibited RANKL expression and potentiated osteoprotogerin expression by osteoblasts. As such, osteoblastic Wnt signalling may control osteoblast-mediated osteoclastogenesis through direct TCF/LEF-1 mediated transcription from the RANKL and OPG promoters, both of which contain multiple consensus TCF binding sites. Using co- culture assays of mouse primary osteoblasts and bone marrow mononuclear cells, LiCl potently inhibited the formation of TRAP-positive osteoclastic cells and markedly attenuated in vitro bone resorption. Taken together, these data provide compelling evidence to support a role for Wnt signalling and TCF/LEF-1 dependent gene transcription in bone cell responses to osteotropic cytokines, the manipulation of which could have pervasive clinical applications in diseases characterised by abnormal bone remodelling.

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O-13

FIBROBLAST GROWTH FACTOR-2 ENHANCES HUMAN OSTEOBLAST SURVIVAL THROUGH ACTIVATION OF THE PI3K AND GSK-3 SIGNALING PATHWAY

F. Debiais¹*, S. Lemée¹, G. Lefèvre², F. Mascarelli², P. J. Marie¹

¹Inserm U349, Paris, France

²Inserm U403, Paris, France

Fibroblast growth factor (FGF-2) plays an important role in the regulation of osteoblast function and bone formation. However, its mechanism of action on osteoblast survival is not known. We investigated the mechanism of FGF-2 action and signal transduction pathways involved in osteoblast apoptosis in primary and immortalized (IHNC) human osteoblasts. We showed that FGF-2 (50 ng/ml) induced a time-dependent biphasic effect on human osteoblast apoptosis. FGF-2 first inhibited at 24h and then increased apoptosis at 96h by downregulating and upregulating caspases-2 and -3 activity, respectively. We then investigated the signaling pathways involved in osteoblast survival induced by FGF-2. The addition of the ERK1/2 inhibitor PD98059 or the p38 MAPK inhibitor SB203580 had no effect on the early inhibition of caspases-2 and -3 activity induced by rhFGF-2 at 24h. In contrast, LY294002, a selective inhibitor of phosphatidylinositol-3 kinase (P13K) reversed the inhibitory effect of FGF-2 on caspase activity, indicating that FGF-2-mediated osteoblast survival requires P13K, but not Erk1/2 or p38 MAPK. We then looked for phosphorylation of downstream substrates of PI3K that may be involved in the effect of FGF-2 on cell survival. The time course study showed that rhFGF-2 did not specifically increased Akt phosphorylation at serine 473 or threonine 308 in IHNC cells. Consistently, rhFGF-2 did not induce phosphorylation of the downstream protein kinase p70 S6 kinase. In contrast, FGF-2 rapidly induced phosphorylation of glycogen synthase kinase (GSK-3) alpha (serine 21) and beta (serine 9) during the time course study (10 min-24h), resulting in GSK-3 inactivation. Treatment with rhFGF-2 in the presence of LiCl, a potent non-competitive inhibitor of GSK-3, or SB216763, a highly selective inhibitor of GSK-3 enhanced GSK-3 phosphorylation induced by FGF-2, further indicating that GSK-3 is a target kinase for FGF-2 signaling in osteoblasts. Furthermore, inhibition of PI3K with LY294002 (5-50 µM) completely suppressed GSK-3 phosphorylation induced by FGF-2, showing that GSK-3 inactivation by FGF-2 is dependent on PI3K activation in osteoblasts. Together, these data provide the first evidence that FGF-2 stimulates the PI3K/GSK-3 pathway in human osteoblasts and identifies a role for this signaling pathway in osteoblast survival induced by FGF-2.

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O-14

ACTIVATION OF PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR-GAMMA INHIBITS THE RUNX2-MEDIATED TRANSCRIPTION OF OSTEOCALCIN IN OSTEOBLASTS

C. S. Shin^1,2*, M. J. Jeon², S. H. Kwon¹, J. A. Kim², S. W. Kim¹, S. Y. Kim^1,2

¹Department of Internal Medicine, Seoul National University College of Medicine, Seoul, Korea

²Seoul National University Hospital Clinical Research Institute, Seoul, Korea

Mesenchymal cells are able to differentiate into several distinct cell types, including osteoblasts and adipocytes. The commitment to a particular lineage may be regulated by specific transcription factors. Peroxisome proliferator activated receptor- gamma(PPAR-gamma) acting in conjunction with C/EBP-alpha has been suggested as a key regulator of adipogenic differentiation. Previous studies have shown that the activation of PPAR-gamma in osteoblasts suppresses osteoblast differentiation and the expression of osteocalcin, one of the osteoblast-specific proteins. However, the mechanism of this inhibition remains unclear. We investigated the effect of PPAR- gamma activation on the expression of osteocalcin and analyzed the underlying molecular mechanism. Mouse osteoblastic cell line MC3T3-E1 cells express PPAR- gamma, which is transcriptionally active, whereas rat osteosarcoma cell line, ROS 17/2.8 does not. Treatment of MC3T3-E1 osteoblasts and ROS 17/2.8 cells stably transfected with PPAR-gamma2 with the PPAR-gamma activator, 15-deoxy-delta- 12,14 prostaglandin J2 (15d-PGJ2), inhibited the mRNA expression of osteocalcin and Runx2, the latter of which is a key transcription factor in the osteoblastic differentiation. These decreased expressions of osteocalcin and Runx2 were partly explained by the decreased abundance of Runx2 resulting from the suppressed transcription from Runx2 promoter. However, in addition to this indirect effect, the activation of PPAR-gamma by 15d-PGJ2 directly suppressed the Runx2-mediated induction of osteocalcin promoter activity and of the activity of the artificial promoter p6OSE2, which contained six tandem copies of the OSE2 element, the Runx2-binding promoter sequence. This inhibition was found to be mediated by a physical interaction between PPAR-gamma and Runx2 and the subsequent repression of the transcriptional activity of the OSE2 sequence. Thus, this study demonstrates that the activation of PPAR-gamma inhibits osteocalcin expression both by suppressing the expression of Runx2 and by interfering with the transactivation ability of Runx2.

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O-15

IDENTIFICATION OF TWO EPITHELIAL CALCIUM CHANNELS, TRPV5 AND 6, ALONG WITH OTHER PROTEINS INVOLVED IN CALCIUM TRANSPORT IN HUMAN OSTEOBLASTS, IN VITRO AND VIVO

B. C. J. van der Eerden¹*, M. Eijken¹, H. Jahr^1,2, J. G. J. Hoenderop³, H. A. P. Pols¹, R. J. M. Bindels³, J. P. T. M. van Leeuwen¹

¹Department of Internal Medicine, Erasmus MC, Rotterdam, The Netherlands

²Department of Orthopedics, Erasmus MC, Rotterdam, The Netherlands

³Department of Cell Physiology, University Medical Center, Nijmegen, The Netherlands

Recently, a new member of the transient receptor cation channel (TRP) family was identified in rabbit kidney, known as TRPV5 (formerly known as ECaC1) and was recognized as the rate limiting step of calcium reabsorption in the distal nephron. In analogy to uptake of calcium in kidney, calcium transport is also crucial for bone formation. Therefore, we were interested whether TRPV5 and its closely related family member, TRPV6 (ECaC2) are expressed in osteoblasts.

Human osteoblasts (SV-HFO) were cultured for 23 days in the absence or presence of 1 microM dexamethasone (dex), which is required for mineralization. At day 5, 7, 9, 12, 14, 16, 19, 21 and 23, total RNA was isolated and reverse-transcribed into cDNA. Real-time PCR was performed to study the expression of TRPV5 and 6, calbindin-D_9K and -D_28K, the plasma membrane calcium ATPase 1 (PMCA1) and the thiazide-sensitive sodium-chloride cotransporter (NCC) during SV-HFO differentiation. We also studied TRPV6 in SV-HFO and MG63 (osteosarcoma) cells by immunohistochemistry.

All six genes were expressed in SV-HFO throughout differentiation in a stage and condition dependent fashion. The abundancies varied between the genes: TRPV5, 9.3x10³; TRPV6, 5.3x10⁴; calbindin-D_9K, 0.4x10³; calbindin-D_28K, 1.3x10⁴; NCC, 1.0x10⁵ and PMCA1, 4.6x10⁷ copies/microg cDNA). From day 14 onwards, when mineralization starts, TRPV6, Calbindin-D_28K and NCC were upregulated. In a cDNA sample obtained from a biopsy of a human femoral head, all 6 genes were also expressed but at different abundancies compared to the SV-HFO cells (TRPV5, 1.2x10⁶; TRPV6, 3.2x10⁷; Calbindin-D_9K, 2.0x10³; Calbindin-D_28K, 3.5x10⁶; NCC, 3.3x10⁶ and PMCA1, 1.8x10⁷ copies/mg cDNA). In addition, TRPV6 immunofluorescence was detected in SV-HFO and MG63 cells.

In conclusion, this is the first report to demonstrate TRPV5 and 6 in human bone, both in vitro and in vivo. All calcium channels and transport proteins shown to be crucial for calcium absorption in kidney and intestine are also expressed in human osteoblasts. Interestingly, expression of some of the genes studied is coupled to the presence of dexamethasone and mineralisation of the matrix.

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O-16

PKCALPHA IS DOWNSTREAM OF ALPHAVBETA3 INTEGRIN AND CONTRIBUTES TO ADHESION-INDUCED ERK1/2 ACTIVATION IN OSTEOCLASTS AND IN CHINESE HAMSTER OVARY (CHO) CELLS

N. Rucci¹*, L. Orrù¹, C. DiGiacinto¹, S. Migliaccio¹, M. Longo¹, R. Baron², A. Teti¹

¹Department of Experimental Medicine, University of L'Aquila, L'Aquila, Italy

²Department of Cell Biology and Orthopaedics, Yale University School of Medicine, New Haven, CT., USA

The alphaVbeta3 integrin is central to osteoclast activity. It mediates cell adhesion and triggers intracellular pathways, which are, at present, not yet fully elucidated. The protein kinase C (PKC) family is thought to be involved in integrin signalling, but its relation with the alphaVbeta3 and the specific isoforms engaged are still unknown. We investigated the signalling pathways activated by the alphaVbeta3 integrin in primary mouse osteoclast-like cells upon adhesion to immobilized alphaVbeta3 monoclonal antibody LM609, and observed that osteoclast interaction with this substrate induced translocation of the PKCalpha isoform from the cytosolic to the TritonX-insoluble, presumably cytoskeletal, compartment. CHO cells expressing the alphaV, but not the beta3, integrin subunit failed to adhere to LM609 substrate. In contrast, CHO cells stably transfected with the beta3 subunit, thus carrying the alphaVbeta3 receptor (CHOalphaVbeta3), readily attached to LM609 and this adhesion also triggered PKCalpha translocation to the insoluble fraction. A similar response was observed for PKCbeta1, but not for the -delta, -epsilon and -zeta isoforms. PKCalpha translocation was independent of PLCgamma activation, as this

enzyme was not phosphorylated upon adhesion to LM609. Immunoprecipitation assays revealed that PKCalpha, but not beta-1, was engaged in a complex with the alphaVbeta3 integrin upon adhesion of CHOalphaVbeta3 to LM609. This complex did not apparently associate with the adapter protein Shc, assumed to contribute to integrin signalling. Adhesion of CHOalphaVbeta3 cells to LM609 triggered ERK1/2 phosphorylation. Pretreatment of CHOalphaVbeta3 cells with EGTA was unremarkable. The intracellular calcium chelator BAPTA inhibited PKCalpha activation, its recruitment by the alphaVbeta3 integrin, and ERK1/2 phosphorylation, suggesting dependence of these events on intracellular calcium concentration. Moreover, PKCalpha translocation to the insoluble fraction was blocked by inhibition of the PI3K. Pretreatment of cells with the selective PKCalpha inhibitor G06976, or PKC downregulation by long-term treatment with phorbol esters, did not affect cell adhesion, but inhibited ERK1/2 activation. In conclusion, we demonstrated that PKCalpha mediates the alphavbeta3 integrin-mediated downstream molecular cascade and ERK1/2 phosphorylation in an intracellular calcium-dependent manner. We believe that this pathway may contribute to the molecular mechanism underlying alphaVbeta3 intracellular signalling in osteoclasts and in other cell types.

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O-17

TSH NEGATIVELY REGULATES BOTH OSTEOCLAST AND OSTEOBLAST FORMATION AND SURVIVAL: EFFECTS ON JNK SIGNALING AND C-JUN TRANSLOCATION

E. Abe¹*, R. Marians¹, T. Ando¹, Y. Li², H. Blair², T. F. Davies¹, M. Zaidi¹

¹The Mount Sinai Bone Program and Bronx VA GRECC, New York, NY, USA

²University of Pittsburgh, Pittsburgh, PA, USA

We report for the first time, that TSH receptors (TSHRs) negatively regulate bone remodeling. TSH null mice generated by disrupting exon 1 of the TSHR gene including the euthyroid heterozygotes were profoundly osteoporotic with a marked reduction in bone mineral density at the femur, tibia and lumbar spine. Histological analysis revealed focal sclerosis, woven bone, and disorganized collagen: hallmarks of rapid bone turnover that are reminiscent of pagetic bone. Tetracycline double labeling showed an increased bone formation rate. Osteoblast progenitors in bone marrow cell cultures, CFU-F and CFU-OB, were also markedly elevated. Likewise, hematopoietic stem cell cultures showed enhanced TRAP-positive osteoclast formation. TSH inhibited both the formation and survival of TRAP-positive osteoclasts. Together, the results argue strongly for the TSHR as a negative regulator of osteoblast and osteoclast formation, function and survival. We next explored the localization of TSHRs to bone cells by following the expression of GFP integrated at the TSHR deletion site. Both CFU-Fs and osteoclast precursors in bone marrow cell cultures derived from TSHR deficient mice showed intense GFP fluorescence. Dual photon confocal scanning microscopy of the inner table of the skull likewise confirmed GFP localization to osteoblasts in situ. That TSHRs were expressed in wild type bone tissue and marrow cell cultures was further confirmed by real time RT-PCR and (or) immunoblotting. Expression of the TSHR coincided with RANK and calcitonin receptor expression at day 2, and preceded TRAP expression. The functional significance of the TSHR in negatively regulating osteoclast formation was further confirmed by demonstrating a marked inhibition of TRAP-positive osteoclast formation from TSHR expressing RAW264-C3 cells. In these cells, TSH inhibited JNK signaling and c-jun nuclear translocation achieved by exposure to RANK-ligand. Overall, therefore, the localization of the TSHR to bone cells, its direct regulation of osteoclast and osteoblast formation and function, and the significant impact on bone of its absence reveal a hitherto yet uncharacterized role for the TSHR and its ligand, TSH, in the negative regulation of bone remodeling.

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O-18

INHIBITION OF OSTEOCLAST DIFFERENTIATION BY PROTEASE- ACTIVATED RECEPTOR-2

R. Smith¹, M. Ransjö², R. N. Pike¹, E. J. Mackie³*

¹Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia

²Department of Oral Cell Biology, Umeå University, Umeå, Sweden

³School of Veterinary Science, University of Melbourne, Parkville, Victoria, Australia

Protease-activated receptor-2 (PAR-2), which is expressed by osteoblasts, is activated specifically by a small number of tissue proteases. PAR-2 is also activated by a peptide (RAP) which corresponds to the 'tethered ligand' created by cleavage of the extracellular N-terminal domain. The effect of RAP on osteoclast differentiation has been investigated in mouse bone marrow cultures. RAP inhibited osteoclast differentiation induced by parathyroid hormone (PTH), 1,25 dihydroxyvitamin D3 (1,25D3) or interleukin-11. Semi-quantitative RT-PCR was used to investigate expression of mediators of osteoclast differentiation in mouse bone marrow cultures and primary calvarial osteoblast cultures treated with RAP. In bone marrow and osteoblast cultures treated with PTH or 1,25D3, RAP inhibited expression of RANKL; in osteoblast cultures, RAP also stimulated osteoprotegerin expression. In bone marrow cultures treated with PTH or 1,25D3, activation of PAR-2 led to reduced expression of both the constitutive and inducible prostaglandin H synthases (PGHS-1 and -2). In osteoblasts treated with PTH, activation of PAR-2 caused a suppression of PGHS-2 expression. RAP inhibited PTH- or 1,25D3-induced expression of interleukin-6 in bone marrow cultures but not in osteoblast cultures. PTH-induced cAMP production was inhibited by RAP in bone marrow and osteoblast cultures. RAP also inhibited expression of RANK in PTH- or 1,25D3-treated bone marrow cells, which may be mediated by a direct effect on osteoclast precursors, since the RAW264.7 osteoclastogenic macrophage cell line was shown to express PAR-2. These observations indicate that PAR-2 activation inhibits osteoclast differentiation by affecting multiple mediators of the effects of osteoclastogenic hormones.

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O-19

CYTOKINE-ACTIVATED T-CELLS SUPPORT OSTEOCLASTOGENESIS FROM PERIPHERAL BLOOD MONONUCLEAR CELL PRECURSORS

D. O'Gradaigh*, D. Ireland, J. E. Compston

Bone Research Group, University of Cambridge School of Clinical Medicine, Addenbrooke's Hospital, Cambridge, UK

Osteoclasts, implicated in joint erosion in rheumatoid arthritis (RA), require RANKL and M-CSF for differentiation and activation. Synovial T-cells are a potential source of RANKL in RA. T-cells activated by PHA or anti-CD3 antibodies express RANKL but T-cells activated by anti-CD3 antibodies may also suppress osteoclastogenesis, mediated by interferon gamma (IFNg). As cytokine-activated T- cells may mimic RA synovial T-cells (Brennan, 2001), we postulated that cytokine- activated T-cells express a balance of IFNg and RANKL which favours osteoclastogenesis.

CD14+ monocytes (pre-osteoclasts) and CD4+ T-cells were isolated from human peripheral blood mononuclear cells by magnetic cell sorting. T-cells were treated with IL-2 (unstimulated controls, Tns) or with cytokines IL-6, IL-15 and TNF-alpha (Tck) or PMA+ionomycin (Tpi). Monocytes from the same donor were cultured on dentine slices with M-CSF+5ng/mL RANKL (control) or co-cultured with T-cells from each condition and M-CSF + RANKL (5ng/mL) or M-CSF alone. Osteoclastogenesis was determined by area of resorption of dentine slices after 14 days, expressed as percent of control values. IFNg and RANKL mRNA expression was analysed by RT-PCR.

In co-cultures, addition of cytokine-activated T-cells significantly increased area of resorption over control (157% [sd 33.6], p=0.03). Tpi did not significantly increase resorption area (127.9% of control, [sd 21.1]). Of interest, addition of unstimulated T- cells resulted in the greatest increase in resorption area (184%, [sd 34.5]). Small areas of resorption (1-3%) were seen when RANKL was omitted from co-cultures, but control cultures did not generate osteoclasts without adding RANKL. mRNA for RANKL (normalised to GAPDH, compared with Tns) was increased 5-fold in Tck and over 25-fold in Tpi. IFNg mRNA expression increased over 1000-fold in Tpi, but only 10-fold in Tck.

In conclusion, cytokine-activated T-cells increase RANKL-driven osteoclastogenesis from CD14+ precursors and can also generate osteoclasts without exogenous RANKL. Lower expression of IFNg may be an important factor in the net effect of cytokine-activated T-cells on in vitro osteoclastogenesis, suggesting that the deficiency of IFNg in RA may be relevant in the pathogenesis of periarticular erosion.

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O-20

THE ABSENCE OF CBL-B CAUSES INCREASED OSTEOCLAST ACTIVITY AND OSTEOPENIA

R. Chiusaroli¹*, A. Sanjay¹, H. Gu², R. Baron¹

¹School of Medicine, Yale University, New Haven, CT, USA

²NIH, Bethesda, MD, USA

c-Cbl and Cbl-b, members of the Cbl protein family that are predominantly expressed in hematopoietic cells, mediate the down-regulation of both receptor and non-receptor tyrosine kinases by functioning as ubiquitin ligases. We have found that c-Cbl also forms a trimolecular complex with Src and Pyk2 in a signaling pathway downstream of the vitronectin receptor that regulates cell adhesion and motility, and that deletion of the c-Cbl gene altered the ability of osteoclasts (OCs) to migrate in vitro and in vivo during development, delaying bone resorption and cartilage ossification in long bones. However, detailed histomorphometric analysis of the bones from adult c-Cbl mice failed to detect any changes in bone volume or in bone resorption parameters. While both c-Cbl and Cbl-b have homologous phosphotyrosine binding domains and RING fingers, some of the protein binding domains in the C- terminal half of the molecule are specific to one or the other protein, suggesting that the two Cbl proteins have some unique functions. To further investigate the role of these Cbl proteins in osteoclast function, we performed a detailed histomorphometric analysis of adult Cbl-b-/- mice. In contrast to c-Cbl-/- mice, the trabecular bone volume of 12 wks old Cbl-b-/- mice was markedly decreased (4.6 ±1.6 for Cbl-b-/-vs. 7.7 ±1.1 for wt, mean ±SD, p <0.01), while no changes in osteoblast as well as in osteoclast surface and number were observed. Dynamic parameters of bone formation were also unaffected. In vitro pit assay revealed that the area resorbed by Cbl-b-/- OCs was 2.5 fold higher than with wt control OCs (wt, 0.044 ±0.017; c-Cbl-/-, 0.039 ±0.009; Cbl-b-/-, 0.110 ±0.02, p<0.01 vs. wt; pit area/total area/number of OCs, mean ±SD), in contrast to the bone resorbing activity of OCs derived from c-Cbl-/- mice,

which was similar to wt . Thus, Cbl-b-/- osteoclasts display a cell autonomous defect which results in increased bone resorption activity, and consequently an osteopenic phenotype observed in Cbl-b-/- mice.

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O-21

THE LRP5 GENE IS INVOLVED IN DIFFERENT CONDITIONS WITH AN INCREASED BONE DENSITY AS ILLUSTRATED BY THE IDENTIFICATION OF SIX NOVEL MISSENSE MUTATIONS

L. Van Wesenbeeck¹*, E. Cleiren¹, J. Gram², R. K. Beals³, M. L. Warman⁴, M. C. deVernejoul⁵, J. Bollerslev², W. Van Hul¹

¹Dept. Medical Genetics, University of Antwerp, Belgium

²Dept. Endocrinology, Rikshospitalet, Oslo, Norway

³Dept. Orthopaedics and Rehabilitation, Oregon Health Sciences University, Portland, Oregon, USA

⁴Dept Genetics, Case Western Reserve Univ, Cleveland, OH, USA

⁵Laboratoire INSERM U 349, Hôpital Laribosisière, Paris, France

The LDL receptor-related protein 5 (LRP5) acts as a coreceptor for Wnt proteins in osteoblasts and is required for the osteoblast proliferation and function. Recently, it has been shown that the LRP5 is involved in both the osteoporosis-pseudoglioma syndrome and the High Bone Mass phenotype and that it turned out to be an important regulator of peak bone mass in vertebrates. We now performed mutation analysis of the LRP5 gene in 10 families or isolated patients with different conditions with an increased bone density diagnosed with endosteal hyperostosis, Van Buchem disease, autosomal dominant osteosclerosis or osteopetrosis type I. Direct sequencing of the LRP5 gene revealed 19 sequence variants. 13 of these were confirmed as polymorphisms but six novel missense mutations (D111Y, G171R, A214T, A214V, A242T, T253I) are most likely disease causing. As the previously reported mutation (G171V) causing the High Bone Mass phenotype, all mutations are located in the aminoterminal part of the gene before the first epidermal growth factor-like domain. These results indicate that different conditions with an increased bone density affecting mainly the cortices of the long bones and the skull are often caused by missense mutations in the LRP5 gene. Functional analysis of the effects of the different mutations will be of interest to evaluate whether all the mutations give rise to the same pathogenic mechanism.

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O-22

ASSOCIATION OF LRP5 POLYMORPHISMS WITH BONE MINERAL DENSITY IN ELDERLY MEN AND WOMEN

J. B. J. van Meurs^1,2*, W. Hugens¹, P. P. Arp¹, C. Bartels³, M. Ai³, A. Hofman², M. Warman³, H. A. P. Pols^1,2, A. G. Uitterlinden^1,2

¹Department of Internal Medicine, Erasmus MC, Rotterdam, The Netherlands

²Department of Epidemiology and Biostatistics, Erasmus MC, Rotterdam, The Netherlands

³Department of Genetics, Case Western Reserve University, Cleveland, USA

It was recently demonstrated that mutations in the low-density lipoprotein receptor-related protein 5 (LRP5; 11q12) gene give rise to the Mendelian disease Osteoporosis-Pseudoglioma Syndrome (OPS) or a trait known as High Bone Mass. Polymorphic variations in LRP5 might therefore contribute to population variance in BMD and/or osteoporotic fracture risk. We therefore examined four polymorphisms in the LRP5 gene: T-4C-exon 5 (intron 4), Val667Met (exon 9), C-63T-exon18 (intron 17), and Ala1330Val (exon 18), which were identified by sequencing LRP5 in OPS patients and from database searches. We genotyped these variants by a multiplex Single Base Extension assay (SnaPshot; ABI) to analyse 1979 elderly men and women (aged 55-80) from the Rotterdam Study, a large population-based prospective cohort study.

Frequencies of the alleles were int4C=38%, ex9Met=6%, int17T=6%, ex18Val=15%. All genotype distributions were in Hardy-Weinberg Equilibrium and frequencies were similar in men and women and stable with age.We compared allele frequencies of all polymorphisms in two groups with extreme BMD (n=695). The 1330Val-allele was over-represented in the lowest quintile of FN-BMD (16.7% versus 11.5%, p=0.02 Chi-square), for both men and women while the same trend was seen for lumbar spine BMD. We then analysed mean BMD by genotype in the whole study population and found carriers of the 1330Val-allele to have 0.2 SD (or 3%) decreased BMD at femoral neck (p=0.001 for men and p=0.03 for women) and at lumbar spine (p=0.009 for men and p=0.0003 for women) with evidence for an allele-dose effect. The effect-size we observed on BMD was too small to be detected as genotype- dependent differences in fracture risk in an analysis of 190 non-vertebral and 141 vertebral fractures.

In conclusion, the relatively rare Val-1330 variant of the LRP5-gene (15%) is associated with BMD in both men and women and at both the femoral neck and the lumbar spine, while no association with fracture risk was observed. The 1330 amino- acid variation is situated in one of the putative LDL-receptor ligand binding domains of the LRP5 protein, which makes functionality of this variation plausible. The contribution of this variation to risk for osteoporosis is likely to be modest.

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O-23

CIRCULATING AMOUNTS OF OSTEOPROTEGERIN AND RANK LIGAND: GENETIC INFLUENCE AND RELATIONSHIP WITH BONE MINERAL DENSITY ASSESSED IN FEMALE TWINS

B. Abrahamsen¹*, P. Kostenuik², L. S. Stilgren¹, K. Kyvik³, S. Adamu², K. Brixen¹, B. L. Langdahl⁴

¹Dept of Endocrinology, Odense Univ. Hospital, DK-5000 Odense, Denmark

²Amgen, Metabolic Disorders Research, Thousand Oaks, CA 91320, USA

³The Danish Twin Registry, Institute of Public Health, University of Southern Denmark, DK-5000 Odense, Denmark

⁴Univ. Department of Endocrinology C, Aarhus Amtssygehus, DK-8000 Aarhus C, Denmark

Osteoprotegerin (OPG) is a circulating receptor, which inhibits osteoclastogenesis by binding to RANK ligand (RANK-L). OPG knock-out animals develop severe osteoporosis. Treatment with OPG lowers bone resorption and increases BMD. OPG production is affected by a wide range of hormones and cytokines. The influence of genetic factors on circulating amounts of OPG and RANK-L is not known. BMD has been demonstrated to have a high heritability and there is evidence also that bone turnover and bone loss rates are controlled at least in part by genetic factors.

Objective: Assessing the genetic impact of serum OPG and RANK-L in women and estimation of the relative contribution of OPG inheritance to the total heritability of BMD.

Methods: Serum levels of OPG and RANK-L were measured by ELISA (Biomedica, Vienna, Austria) in a cohort of healthy female twins (N=182: 50 DZ and 41 MZ pairs, age mean 35y, range 19-64, spine BMD 1.04 ±0.11) from the Danish Twin Registry. This register covers twins born in this country since 1870. Heritability was estimated by means of the classical model.

Results: Serum RANK-L is highly genetically determined (h² = 0.82) whereas the genetic influence on OPG is minor (h² = 0.06). In accordance with other studies, BMD is under strong genetic regulation (h² = 0.68 to 0.96).

The RANK-L/OPG ratio is significantly associated with BMD of the spine (r=0.20, p<0.01) and hip (r=0.16, p<0.05). However, the heritability of BMD is only slightly reduced (from 98% to 94%) when adjusted for variation in RANK-L/OPG ratio.

Conclusions: Serum levels of RANK-L are under strong genetic influence, while the impact of heredity on OPG is low. BMD is also strongly heritable, but only a small proportion of this inheritance is accounted for by RANK-L and OPG.

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O-24

BONE FRACTURES AND AGE AT FRACTURE TIME IS ASSOCIATED WITH ESTROGEN RECEPTOR ALPHA1 GENE POLYMORPHISMS IN INSTITUTIONALIZED PATIENTS

B. M. Obermayer-Pietsch*, U. Hartl, C. M. Bonelli, A. Fahrleitner-Pammer, J. C. Piswanger-Sölkner, P. Maritschnegg, G. Leb, H. Dobnig

Dep. of Internal Medicine, Karl-Franzens-University, Graz, Austria

Background

Bone mineral density and bone fractures have been shown to be influenced by genetic factors such as estrogen receptor alpha 1 (ER) gene alleles. Osteoporosis and concomitant bone fractures are known major problems in elderly people in nursing homes. In this study, we evaluated XbaI and PvuII polymorphisms of the ER gene and parameters of bone and mineral metabolism in order to define associations between bone fracture status and risk parameters for osteoporosis.

Methods

In a cross-sectional study, we investigated 1254 elderly Caucasian persons (mean age 83 ± 6 years) living in 95 Austrian nursing homes. Medical history and past fractures were obtained from review of medical records. Bone ultrasound (at the radius, proximal phalanx and calcaneus), neurophysiological parameters (e.g. bio- impedance, muscle strength) and serum parameters (vitamin D, crosslaps, PTH, osteocalcin, routine lab) were measured in all of them. DNA probes were genotyped for the ER XbaI and PvuII polymorphisms.

Results

67% of the residents showed vitamin D deficiency and a high prevalence for secondary hyperparathyroidism and increased bone turnover. ER genotype frequencies were normal (XX 12.6%, Xx 47.1%, xx 40.3% for XbaI alleles, PP 21.3%, Pp 49.4%, pp 29.2% for PvuII alleles). Both genotypes showed a significant

association to hip fractures (e.g. 22% PP vs. 12% pp, p=0.001), to the age at fracture time (mean age 78 vs. 83 years, p=0.02) and to age-adjusted bone ultrasound values at the calcaneus.

Conclusion

ER PvuII and XbaI genotypes were significantly associated with fractures and age at fracture time in our cohort of elderly residents, who are at high risk for osteoporosis. In context with clinical data, these genotypes may therefore be useful for the identification of patients at high risk for fractures. Therapeutic and diagnostic implications warrants to be further elucidated.

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O-25

INFLUENCE OF SERUM VITAMIN D LEVEL ON EFFECT OF CDX-2 POLYMORPHISM IN THE 1E PROMOTER OF THE VDR GENE IN DETERMINING BMD

Y. Fang¹*, N. F. Barley², M. Khanji², J. B. J. van Meurs¹, A. P. Bergink^1,3, J. P. T. M. van Leeuwen¹, H. A. P. Pols^1,3, J. R. F. Walters², A. G. Uitterlinden^1,3

¹Department of Internal Medicine, Erasmus MC, Rotterdam, The Netherlands

²Gastroenterology Section, Faculty of Medicine, Imperial College London, UK

³Department of Epidemiology, Erasmus MC, Rotterdam, The Netherlands

Vitamin D regulates, through the vitamin D receptor (VDR), the expression of genes related to intestinal calcium absorption, e.g., Calbindin 9k and ECAC2/CAT1. Recently, a G to A polymorphism was found at a binding site for the intestinal- specific caudal-related homeobox transcription factor Cdx2, in the 1e promoter region of the VDR gene. The G-allele of was shown to result in lower VDR expression in intestinal cells. Consequently, by interaction with vitamin D, the Cdx2 polymorphism could influence intestinal calcium-absorption, bone mineral density (BMD), and, ultimately, fracture risk. We previously reported the G-allele to be associated with increased fracture risk.We therefore investigated the influence of serum vitamin D level on this association in 1024 elderly subjects (aged 55-80) from the Rotterdam study for whom we obtained data on serum levels of 25-hydroxy vitamin D3 (25(OH)D₃) and 1, 25(OH)₂D₃. Furthermore, we measured mRNA expression of Calbindin D9k and ECAC2/CAT1 in duodenal biopsies from 32 subjects with normal endoscopies.

In the association study, the effect of the VDR Cdx2 polymorphism on BMD was strongly modified by circulating 25(OH)D₃ level. In the GG genotype group (frequency=70%), BMD increased by increasing serum 25(OH)D₃ level, but we did not observe such an increase for A-allele carriers (30%). This greater sensitivity of the GG genotype group was observed when 25(OH)D₃ serum levels were stratified in tertiles (p = 0.001 at femoral neck, and p = 0.004 at lumbar spine) and when stratified in quartiles. Similar trends were observed for 1, 25(OH)₂D₃ but these failed to reach significance. In the mRNA expression analysis of the 32 duodenal biopsies, we observed a greater sensitivity to 1, 25(OH)₂D₃ in the GG genotype group, where the relationship with ECAC2/CAT1 and calbindin-D9k was significantly stronger (p = 0.02) but not in A-carrier group.

In conclusion, we found the VDR Cdx2 GG genotype to be more sensitive to serum vitamin D level, by analysis of (1) vitamin D-responsive calcium transport gene expression in the intestine and (2) of BMD. Since vitamin D levels are lower in elderly subjects, our findings could contribute to explain the VDR Cdx2 genotype dependent differences in risk of fracture.

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O-26

CHARACTERISATION OF AN INACTIVATING MUTATION IN OSTEOPROTEGERIN

C. A. Middleton-Hardie*, T. Cundy, I. R. Reid, J. Cornish, D. Naot

University of Auckland, New Zealand

Genetic analysis of a large family with Familial Idiopathic Hyperphosphatasia identified linkage to a region on chromosome 8q, containing the gene for osteoprotegerin (OPG), a key regulator of bone remodelling. Sequencing of the OPG gene in members of the family identified an in-frame 3 base pair deletion, resulting in the loss of an aspartate residue at position 182. To determine whether this mutation affected the function of OPG, wild-type and mutant OPG cDNA were cloned and expressed in HEK293 cells. We have previously reported that unlike wild-type OPG, the mutant protein was unable to inhibit bone resorption in neonatal murine calvarial organ cultures, indicating that the deletion of asp182 impairs the ability of OPG to inhibit osteoclast activity (1). Subsequently, we tested the peptides in a murine bone marrow osteoclastogenesis system. In this assay the wild-type OPG inhibited osteoclast formation (p<0.0001) whereas the mutant OPG did not. We therefore conclude that the deletion of aspartate 182 is an inactivating mutation that affects the ability of OPG to inhibit osteoclast formation and activation.

Further to our report that the mutant OPG is hyperglycosylated (1), we have investigated the importance of glycosylation to the function of wild-type OPG. Cells expressing wild-type OPG were treated with tunicamycin, a glycosylation inhibitor. Conditioned media samples containing the non-glycosylated OPG were tested in murine neonatal calvarial organ culture. Unlike glycosylated OPG, the non- glycosylated OPG could not inhibit bone resorption, suggesting that glycosylation is important for the function of full-length OPG. At present we are using site-directed mutagenesis to try and elucidate the sites of hyperglycosylation in the mutant OPG. We have also conducted a preliminary study to investigate the binding ability of wild- type and mutant OPG to RANKL. Using the surface plasmon resonance technology of BIAcore we have determined that the mutant OPG cannot bind with RANKL to the same extent as the wild-type OPG.

Our study highlights the effects of a single amino acid deletion on the structure and function of OPG. Also, it confirms the crucial role of OPG in normal bone physiology in humans.

(1) Cundy et al. Hum Mol Genet 2002; 11(18):2119-2127

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O-27

INTRON 8 POLYMORPHISM IN CHLORIDE CHANNEL 7 GENE (CLCN7) AND ASSOCIATION WITH BONE MINERAL DENSITY

A. Taranta¹*, M. Mirolo², A. Frattini², O. M. E. Albagha³, F. E. A. McGuigan³, D. M. Reid³, P. Vezzoni², A. Villa², A. Teti¹, S. H. Ralston³

¹Dept of Experimental Medicine, University of L'Aquila, L'Aquila, Italy

²Institute of Biomedical Technology, CNR, Milan, Italy

³Dept of Medicine and Therapeutics, University of Aberdeen, Aberdeen, Scotland, UK

The CLCN7 gene encodes an endosomal/lysosomal ion channel which is highly expressed in the osteoclast ruffled border. The CLCN7 gene product is involved, along with the vacuolar proton pump, in acidification of the bone resorption compartment. Missense mutations of CLCN7 gene cause a mild type of osteopetrosis, termed autosomal dominant osteopetrosis type II. These studies identify CLCN7 as a genetic regulator of BMD but the role of CLCN7 polymorphisms in regulating BMD in normal subjects remains unclear. The CLCN7 gene maps to human chromosome 16p13, and comprises 25 exons and contains a 50bp tandem repeat polymorphism within intron 8. The aim of this study was to look for evidence of an association between the intron 8 polymorphism and Bone Mineral Density (BMD) in normal subjects. We carried out genotyping for the intron 8 polymorphism by PCR and agarose gel electrophoresis and the number of repeats was related to BMD in a population based sample of 1023 normal women aged 45-50. We observed 2 to 9 tandem repeats in 23 different allelic combinations. Subjects harbouring 4 tandem repeats were found to have lower BMD values at the lumbar spine that other genotype groups and a similar, but non-significant trend was observed at the femoral neck (Table). Analysis of the data by GLM ANOVA showed that presence or absence of the intron 8 '4' allele was an independent predictor of spine BMD after correcting for age, weight and height (p=0.034). We conclude that the intron 8 tandem repeat polymorphism of CLCN7 is associated with spine BMD in this relatively large cohort of women. Further studies are in progress to determine if this association is directly mediated through the effect of the intron 8 polymorphism or other polymorphisms in the CLCN7 gene, which are in linkage disequilibrium with alleles containg 4 tandem repeats.

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O-28

ASSOCIATION OF GENETIC DISPOSITION FOR ADULT LACTOSE INTOLERANCE TO CALCIUM INTAKE, BONE DENSITY AND BONE FRACTURES IN POSTMENOPAUSAL WOMEN

B. M. Obermayer-Pietsch¹*, C. M. Bonelli¹, D. E. Walter¹, A. Fahrleitner-Pammer¹, A. Berghold², W. Goessler³, V. Stepan¹, H. Dobnig¹, G. Leb¹, W. Renner⁴

¹Dep. of Internal Medicine, Karl-Franzens-University, Graz, Austria

²Insitute for Medical Informatics, Statistics and Documentation, Karl-Franzens- University, Graz, Austria

³Institute of Chemistry - Analytical Chemistry, Karl-Franzens-University, Graz, Austria

⁴Institute of Molecularbiological Analytics, Graz, Austria

Background

Lactase deficiency is a frequent autosomal recessive condition leading to adult lactose intolerance and calcium malabsorption. A recently discovered -13910 T/C polymorphism nearby the lactase-phlorizin hydrolase (LCT) gene has been reported to be strongly associated with adult lactase non persistence, and may have an impact on calcium supply, bone density and risk for osteoporotic fractures in the elderly.

Methods

We determined LCT genotypes TT, TC and CC in 258 postmenopausal women using a polymerase-chain-reaction-based assay. Genotypes were related to self- reported milk intolerance, nutritional calcium intake, intestinal calcium absorption, bone mineral density and the occurrence of nonvertebral fractures.

Results

Women with CC genotype had significantly lower milk calcium intake (-55%, p=0.004) and significantly higher aversion to milk consumption (+166%, p=0.01) as compared to the other genotypes, but there were no differences in overall dietary

calcium intake or in intestinal calcium absorption test values. The frequency of CC genotypes and well as H2-positive lactose intolerance tests in our population is both about 25%.

They showed significantly lower age-adjusted bone density at the hip (-7% at the femoral neck, p=0.04, -8% at the total hip, p=0.02, and -11% at Ward's triangle, p=0.04). Bone density at the lumbar spine was reduced by -8% in individuals with genotypes CC and TC as compared to TT genotypes (p=0.02). LCT(T/C-13910) polymorphisms alone accounted for 2-4% of bone mineral density in a multiple regression model. Increased nonvertebral bone fracture incidence was significantly associated with CC genotypes (p=0.001).

Conclusions

CC genotypes of the LCT(T/C-13910) polymorphism are associated with individual milk intolerance, reduced milk calcium intake and reduced bone density at the hip and the lumbar spine and may predispose to bone fractures resulting from differences in intestinal calcium absorption by lactase deficient individuals. Genetic testing for lactase deficiency may complement indirect methods in the detection of individuals at risk for both lactose malabsorption and osteoporosis.

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O-29

LOSS OF FRA-2 EXPRESSION LEADS TO OSTEOPOROSIS

A. Hoebertz¹*, R. Eferl¹, F. Karreth¹, A. F. Schilling², M. Priemel², M. Amling², E. F. Wagner¹

¹Research Institute of Molecular Pathology (IMP), Vienna, Austria

²Department of Trauma and Reconstructive Surgery, University School of Medicine, Hamburg, Germany

The three Fos proteins c-Fos, FosB, Fra-1 have been shown to play crucial roles in bone biology, but little is known about the fourth Fos protein, Fra-2. To study its role in development and bone remodelling, we generated both Fra-2 knockout mice and mice overexpressing Fra-2.

Fra-2 knockout mice die postnatally between day 1-5 and display severe osteoporosis. Histomorphometrical analysis showed that the bone volume is reduced by 50% and that both number and size of osteoclasts were dramatically increased, whereas the number of osteoblasts was unchanged. To study the mechanism leading to the bone loss and 'giant' osteoclast appearance, we performed in vitro cultures of both fetal liver-derived osteoclasts and primary calvarial osteoblasts. Primary osteoblasts showed a severe differentiation defect, as assessed by bone nodule formation with a parallel increase in adipocyte differentiation. This suggests that decreased bone formation by osteoblasts is also contributing to the bone loss in vivo, in addition to enhanced bone resorption. Unexpectedly, primary osteoclast cultures showed a differentiation and fusion defect; the numbers but also the size of TRAP- positive osteoclasts were smaller compared to controls. However, this defect could be rescued by addition of TGFbeta to the cultures, but not by increasing RANKL or M- CSF concentrations. Reciprocal co-cultures of Fra-2 -/- osteoclasts and wildtype or Fra-2 -/- osteoblasts could not rescue the osteoclast differentiation defect, indicating that other systemic or paracrine signals must be responsible for the 'giant' osteoclast phenotype.

Transgenic mice overexpressing Fra-2 (Fra-2 Tg mice) showed initially no overt phenotype. However, histomorphometrical analysis of 3 month old Fra-2 Tg mice revealed that these mice had a 2 times increase in bone volume. Primary Fra-2 Tg osteoblast cultures displayed enhanced differentiation potential, whereas in vitro osteoclast differentiation was not affected. This gain-of-function approach supports our data from the Fra-2 knockout model. In conclusion, we provide first evidence that Fra-2 plays important roles in both osteoclast and osteoblast differentiation and that loss or overexpression of Fra-2 result in imbalanced bone remodelling leading to bone diseases.

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O-30

TRANSGENIC OVEREXPRESSION OF IGFBP-2 PRODUCES SEX- AND SITE-SPECIFIC INHIBITORY EFFECTS ON GH-INDUCED SKELETAL CHANGES IN MICE

F. Eckstein¹*, D. Bürklein¹, T. Fisch², M. Priemel³, R. Wanke², A. Hoeflich⁴, M. Amling³, S. Mohan⁵, E. Wolf⁴

¹Intitute of Anatomy, LMU München, Germany

²Institute of Veterinary Pathology, LMU München, Germany

³Department for Trauma and Reconstructive Surgery, Experimental Surgery, Hamburg University, Germany

⁴Institute of Molecular Animal Breeding, LMU München, Germany

⁵Jerry L Petttis VA Medical Center, Loma Linda, CA, USA

The growth hormone (GH) insulin-like growth factor (IGF) system plays a critical role in the development and maintenance of the skeleton. Accordingly, we have recently shown that transgenic mice overexpressing growth hormone (GH) exhibit a significant increase in BMC in both female and male mice. However, an increase in trabecular bone density of the femoral metaphysis was only observed in female, but not in male mice. Based on the findings that bone cells produce IGFBP-2 and that IGFBP-2 exerts inhibitory effects on IGF effects in variety of cell types, we proposed the hypothesis that IGFBP-2 overexpression in GH transgenic mice acts to inhibit the anabolic effects of GH on bone. To test this hypothesis, we measured skeletal changes in mice overexpressing GH and/or IGFBP-2 and corresponding control mice. We examined 82 mice from 8 genetic groups (controls, GH transgenic, IGFBP-2 transgenic, double transgenic, male and female) aged 18 weeks. Skeletal changes were examined by histology, DEXA, pQCT, and by micro CT. IGFBP-2 overexpression alone caused a small reduction (2-10%) in various skeletal parameters compared to control mice, however, these changes were not always significant. IGFBP-2 overexpression on the other hand nearly completely abolished the anabolic effects of GH (e.g. total content, periosteal circumference) at the mid diaphysis of femur and tibia. In contrast to the mid diaphysis, IGFBP-2 overexpression only partially (30-50%) abolished the anabolic effects of GH in the femoralö metaphysis and vertebra. Furthermore, the inhibitory effect of IGFBP-2 on GH anabolic effect was greater in male mice compared to female mice in the vertebra. Accordingly, total content and total area of vertebra were reduced by 40-50% in male transgenic mice overexpressing GH and IGFBP-2 compared to GH while they were reduced by 20- 25% in female transgenic overexpressing GH and IGFBP-2 compared to GH. Summary: 1) The anabolic effects of GH on the skeleton are inhibited by IGFBP-2 overexpression in mice. 2) The magnitude of inhibitory effects of IGFBP-2 on GH anabolic effects is both skeletal site- and sex-dependent.

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O-31

PARATHYROID HORMONE EXERTS ANABOLIC EFFECTS ON HUMAN BONE MARROW MESENCHYMAL STEM CELLS

C. Clausen*, B. M. Abdallah, M. Kassem

Department of Endocrinology, Odense University Hospital, Odense, Denmark

Parathyroid hormone (PTH) has been recognised as an anabolic agent in bone for many years and has recently proved its efficacy in treatment of osteoporosis. However the cellular mechanisms underlying its action are not known in details. Thus, we examined the direct effects of PTH on cell growth and osteogenic differentiation markers in an in vitro model for human mesenchymal stem cells (hMSC) stably transfected with human telomerase gene (TERT), which express the PTH/PTHrp receptor.

Two different fragments of PTH (1-34) and (3-84) that mediates different signal transduction pathways were employed. Both fragments were capable of stimulating cell proliferation in a dose dependent pattern with maximal stimulation of 45% in PTH (3-84) treated cultures compared to 35% PTH (1-34) treated cultures. Using H- 89 and GF109203X that blocks the PKA/AC and PKC/PLC pathway respectively, we found that blocking PKC/PLC pathway resulted in abolishing the PTH-dependent increased cell proliferation. Furthermore, we examined PTH effects on cell apoptosis using TUNNEL-assay and FACS analysis that allows quantification of the number of apoptotic cells. PTH treatment resulted in 50% reduction in the number of apoptotic cells that were induced by dexamethasone treatment. Using real time PCR analysis of osteoblastic gene marker expression (Cbfa1/Runx2, collagen type 1 and osteocalcin), we examined the effects of treatment of cells with PTH fragments (1-34) and (3-84). Both fragments induced a dose-dependent increase in all markers, where the highest responsiveness was seen for Cbfa1/Runx2 expression.

In conclusion our results suggest that the in vivo observed anabolic effects of PTH on bone are mediated by increased osteogenic cell recruitment and differentiation and these effects are mediated mainly through the activation of the PKC/PLC pathway.

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O-32

TERIPARATIDE INCREASES THE WIDTH OF MODELING AND REMODELING OSTEONS AT THE TRABECULAR AND ENDOSTEAL ENVELOPE

E. F. Eriksen*, D. W. Donley, Y. L. Ma

Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN, USA

Teriparatide [rhPTH(1-34), TPTD], a new bone formation agent for osteoporosis, reverses osteoporotic changes in bone structure and decreases vertebral and nonvertebral fracture rates (Neer et al. N. Eng. J. Med. 2001). A significant proportion of new bone formed during teriparatide treatment seems to be formed via modeling, i.e. formation of new bone on quiescent bone surfaces without previous resorption. We analyzed the occurrence and dimensions of modeling and remodeling osteons in iliac crest biopsies obtained from patients treated with placebo or teriparatide (20 and 40 ug/day s.c., duration 12-24 months) in a large randomized, placebo-controlled trial. A total of 49 biopsies (placebo (n=19), TPTD20 (n=17), TPTD40 (n=13)) were studied. Active bone forming, tetracycline-labeled osteons on trabecular and endocortical surfaces were studied using polarized light for collagen orientation and cement line stains. Trabecular and endosteal osteons were classified according to the presence of smooth or scalloped cement lines and collagen orientation (i.e. modeling and remodeling osteons, respectively). Remodeling wall width was also quantified. In patients treated with teriparatide, a dose-dependent increase in modeling osteons was seen for TPTD20 (0.5%) and TPTD40 (3.9%) (P<0.0001). Also a group of trabecular osteons classified as mixed remodeling/modeling osteons showed a dose dependent increase (TPTD20 (2.3%) and TPTD40 (4.0%) (P<0.0001)). In the post-treatment biopsies, the remodeling wall width of trabecular packets was increased by 15% in both TPTD groups compared with placebo (P<0.05). Even more pronounced increases in wall width were noted on endosteal packets for TPTD20 (22%) and TPTD40 (19%) when compared with placebo (P<0.05).

In conclusion, our studies suggest that teriparatide induces pure modeling bone formation at quiescent surfaces, and increases bone formation at remodeling sites. This leads to increased thickness of completed bone structural units at both the trabecular and endosteal envelope. These mechanisms may all contribute to the improvement of trabecular and cortical architecture demonstrated after teriparatide treatment.

[Programme]

 
O-33

GENE EXPRESSION ANALYSIS OF WILD TYPE AND VDR KO- MICE CHALLENGED WITH 1ALPHA,25-DIHYDROXY-VITAMIN D3 AND 24R,25-DIHYDROXY-VITAMIN D3

R. Scott¹, H. L. Henry², M. Heim¹, J. E. Bishop², T. Pennimpede¹, G. Kampmann¹, W. Hunziker¹, P. Weber¹, A. W. Norman², I. Bendik¹*

¹Roche Vitamins Ltd, Research and Development, Human Nutrition & Health, Basel, Switzerland

²University of California, Riverside, CA, USA

1alpha,25-dihydroxy vitamin D3, the active form of vitamin D, has roles in many biological processes such as calcium homeostasis and bone formation. Vitamin D3 can be also hydroxylated to 24R,25-dihydroxy vitamin D3. In contrast to 1a,25- dihydroxy vitamin D3, less is known about the biological function of 24R,25- dihydroxy vitamin D3. Wild type (WT) and vitamin D receptor (VDR) knock-out (VDR-KO) mice (6 animals per group) that were fed a vitamin D deficient diet were given an intraperitoneal dose of either 1alpha,25-dihydroxy vitamin D3 (250 ng), 24R,25-dihydroxy vitamin D3 (500 ng), or vehicle (ethanol/propanediol, 1:1) 8 hours prior to tissue harvest. Total RNA from the small intestine was isolated from each individual animal and biotin-labeled cRNA was synthesized for hybridization to mouse Affymetrix GeneChip arrays (U74Av2). The expression levels entitled maximal mean were determined using standard Affymetrix MAS 5.0 analysis software. Transcripts having a maximal mean above 20 were considered to be those present and were used for further analysis. Using a p-value less than 0.05 we identified 209 genes (out of more than 12'400 probe sets) that were more than 2-fold down or upregulated. The number of regulated transcripts for the WT mice treated with either 1alpha,25-dihydroxy vitamin D3 or 24R,25-dihydroxy vitamin D3 were 67 and 38 respectively. Thirty-two transcripts were upregulated by 1a,25-dihydroxy vitamin D3 and 26 transcripts were upregulated by 24R,25-dihydroxy vitamin D3. In the VDR-KO animals 1alpha,25-dihydroxy vitamin D3 and 24R,25-dihydroxy vitamin D3 regulated 50 and 25 mRNAs respectively. Within the VDR-KO group 16 transcripts were increased by 1alpha,25-dihydroxy vitamin D3 and 7 by 24R,25- dihydroxy vitamin D3. Using this GeneChip approach we could show that, in the absence of the liganded VDR complex, 1alpha,25-dihydroxy vitamin D3 and 24R,25- dihydroxy vitamin D3 can independently regulate genes in the intestine. These candidate genes will be further analyzed for their role in calcium homeostasis.

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O-34

SIGNALLING NETWORKS IN THE RAPID ACTION OF ANDROGENS IN MALE OSTEOBLASTS. FROM G BETA 4 TO ELK1 ACTIVATION

Y. Zagar, G. Chaumaz, M. Lieberherr*

LNSA, INRA, Jouy-en-Josas, France

Androgens elicit genomic effects on transcription through intracellular androgen receptors (AR), and rapid AR-independent effects. We therefore characterized the multi-step processes involved in the rapid actions of androgens in male osteoblasts, which possess the classical androgen and estrogen receptors. The 5alpha- dihydroxytestosterone (100 pM, DHT) increased within 1 min the phosphorylation of the transcription factor Elk1 connected to early genes controlling proliferation and differentiation. The activation of Elk1 was inhibited by pertussis toxin (PTX). DHT activated within 15 s ERK1/2, a substrate for Elk1, but had no effect on p38MAPK or JNK/SAPK. MEK1/2 inhibitor (PD98059), PKC inhibitors (Go6976, Go6983, chelerythrine), phosphatidylinositol 3-kinase (PI3K) inhibitors (wortmannin, LY294002), PP1 (Src inhibitor) and PTX diminished the DHT-increased phosphorylation of ERK1/2. DHT increased within 5 s the phosphorylation of cRaf-1, which was sequentially blocked by cPKC and PIK3 inhibitors and PTX. The first protein responsible for this cascade of events was the PTX-sensitive G beta 4 sub-unit coupled to PLC-beta2, which triggered a rapid (5 s) increase in intracellular calcium, and diacylglycerol formation. Cyproterone acetate, a nuclear antagonist, did not modify the responses to DHT. These effects were gender-specific since 17beta- estradiol had no effect and ICI 182,780 , an estrogen antagonist, did not block the effects of DHT. They were also stereo-specific since 5beta-DHT has no effect. Lastly, an anti-AR antibody, directed against the ligand binding domain, recognized a protein located at the plasma membrane. The cascade of rapid effects induced by androgens may involve the classical AR located at the plasma membrane or an uncharacterized form of AR insensitive to nuclear antagonists.

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O-35

OSTEOBLAST-LIKE CELLS FROM ESTROGEN RECEPTOR ALPHA KNOCKOUT (ERKO) MICE HAVE DEFICIENT RESPONSES TO MECHANICAL STRAIN

H. L. Jessop*, R. F. L. Suswillo, S. C. F. Rawlinson, G. Zaman, K. Lee, V. Das- Gupta, A. A. Pitsillides, L. E. Lanyon

Department of Veterinary Basic Sciences, The Royal Veterinary College, London, UK

Bone formation induced by mechanical loading in vivo has been shown to be three fold lower in homozygous Estrogen Receptor alpha knockout (ERKO) mice compared to wildtype littermates. To investigate the mechanisms of ER involvement in the early responses of bone cells to strain we now compare the responses of primary cultures of osteoblasts derived from homozygous ERKO mice with those from their wildtype littermates and from ER beta knockout (BERKO) mice.

We have shown previously that osteoblast-like cells respond to mechanical strain in vitro by release of nitric oxide and prostaglandins, ERK-mediated phosphorylation of ER alpha, upregulation of AP-1 and Estrogen Response Element activity and stimulation of proliferation. Osteoblasts of all genotypes derived from the ERKO and BERKO colonies proliferated in response to strain except ER alpha -/- osteoblasts. Transfection of fully functional, but not mutated, ER alpha restores the proliferative response to strain in these cells. The strain-related response of ER alpha -/- cells is also deficient in that they show no increased activity of an AP-1 driven reporter vector and no strain-related nitric oxide production. Also, they do not proliferate in response to exogenous IGF-I or IGF-II. However, the strain-related increase in prostaglandin production is retained.

These data support the obligatory involvement of ER alpha in a number of early responses to mechanical strain in osteoblasts, including those resulting in proliferation. Lack of osteoblast proliferation in response to mechanical loading may help explain the low loading-related bone formation observed in ERKO homozygous mice in vivo. These data are also consistent with the hypothesis that less effective processing of loading-related information, due to a reduction in ER alpha expression (and/or function) following estrogen withdrawal, could be a major factor in the aetiology of post-menopausal osteoporosis.

[Programme]

 
O-36

INTRAVENOUS NERIDRONATE IN ADULTS WITH OSTEOGENESIS IMPERFECTA

F. Colapietro*, S. Adami, R. Prizzi, D. Gatti, V. Braga, D. Gerardi, D. Righetti, M. Rossini

Rheumatological Rehabilitation, University Hospital Valeggio S/M, Italy

Osteogenesis Imperfecta (OI) is a heritable disease of connective tissue, characterized by increased bone fragility. Bisphosphonates currently seems to be the most promising therapy at least in children. We tested intravenous neridronate, an amino-bisphosphonate structurally similar to alendronate and pamidronate in adults with OI. Twenty-three men and 23 premenopausal women with OI were randomized to either I.V. neridronate (100 mg infused I.V. in 30 minutes every 3 months) or no treatment with a ratio of 2 to 1. Control patients were given the same bisphosphonate therapy at the end of the first year. Clinical evaluation included bone densitometry measurements using Dual Energy X-ray Absortiometry (DXA), fasting serum and urinary biochemistry every 6 months, and radiographs of the spine taken at baseline and after 12 and 24 months of follow-up. Spine and hip BMD rose by 3.0, 4.6SD % and by 4.3, 3.9SD%, respectively, within the first 12 months of treatment whereas small insignificant changes were observed in the control group. During the second year of follow-up an additional 3.91% and an 1.49% increases were observed at the spine and hip respectively. Markers of skeletal turnover significantly fell during neridronate treatment. The difference in fracture incidence during treatment (1/77 patient years) and during pooled pre-recruitment and control time (18/199 patient years) was at the limit of significance (p=0.03, Fisher's exact t-test, RR 0.14, 95% CI 0.02 to 1.09).

In conclusion: Neridronate I.V. infusions, administered quarterly, significantly increase BMD and lowered the risk of clinical fracture in adults with OI. Bisphosphonate therapy seems to provide clinical benefits not only to children with OI, but also to adult patients.

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O-37

TREATMENT OF ESTABLISHED GLUCOCORTICOID-INDUCED OSTEOPOROSIS WITH ALFACALCIDOL OR PLAIN VITAMIN D

J. D. Ringe¹*, A. Dorst¹, H. Faber¹, E. Schacht²

¹Klinikum Leverkusen, Medizinische Klinik 4, University of Cologne, Germany

²University Clinic Balgrist, Zurich, Switzerland

A supplementation with plain vitamin D plus calcium is in general regarded as an effective prevention or first step treatment for glucocorticoid-induced osteoporosis (GIOP). The aim of our study was to compare the therapeutic efficacy of the vitamin D analogue alfacalcidol to plain vitamin D in patients with established GIOP with or without vertebral fractures.

Patients on long-term glucocorticoid therapy were included as matched-pairs to receive randomly either 1microg alfacalcidol plus 500 mg calcium per day (group A, n=103) or 1000 IU vitamin D3 plus 500 mg calcium (group B, n=101). Corrrespondingly the two groups were not different in terms of mean age, sex ratio, average height and weight and the percentages of the three included underlying diseases (chronic obstructive pulmonary disease, rheumatoid arthritis, polymyalgia rheumatica) The average initial bone mineral density (BMD) values for the two groups were at the lumbar spine of a T-score of -3.26 and -3.25 and at the femoral neck -2.81 and -2.84 resp.. Rates of prevalent vertebral and non-vertebral fractures were not different between groups.

During the three year study we observed in group A significant increases of BMD at the lumbar spine (+2.4%) and at the femoral neck (+1.2%). In group B there were no significant changes at both sites (-0.7% and +0.7% resp.). At the end of the study 16 new vertebral fractures had occurred in 10 patients of group A and 35 in 25 patients of group B. There was a statistically significant difference between the number of patients with at least one new vertebral fracture (p= 0,005). Between the rates of patients with new non-vertebral fractures however (15 vs. 25) no significant difference could be proved (p=0.081). In accordance with the observed fracture rates the alfacalcidol group showed a significant decrease in back pain whereas no change was registered in the vitamin D group (p < 0,0001).

We conclude that alfacalcidol plus calcium is highly superior to plain vitamin D3 plus calcium in the treatment of established GIOP.

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O-38

TRAIL IS EXPRESSED IN QUANTITIES SIMILAR TO RANKL IN HUMAN PERIPROSTHETIC PSEUDOMEMBRANE, OSTEOARTHRITIC STROMAL CELLS AND OSTEOBLASTS

A. Sabokbar¹*, H. C. Blair², S. Sun¹, J. Edwards¹, L. Danks¹, N. A. Athanasou¹

¹University of Oxford, UK

²University of Pittsburgh, USA

Apoptosis is critical in bone development and in turnover of mature bone. Members of the TNF cytokine family play important roles in bone cell survival and apoptosis. One of the TNF family members, the Apo2 ligand, TRAIL (TNF-related apoptosis inducing ligand), mediates apoptosis in a wide variety of cells. As well as binding to its apoptosis-inducing receptor, TRAIL binds to soluble 'decoy' receptors that regulate the activity of TNF-family proteins. One of these decoy receptors, osteoprotegerin (OPG) is an important regulator of bone turnover via interactions with the ligand for the receptor-activator of nuclear factor kB (RANK). Because of the importance of TRAIL in regulating apoptosis, and because of the interaction of TRAIL and OPG, we hypothesized that TRAIL is expressed in bone and co-regulated, along with RANKL, by OPG. The initial phase of investigation of this hypothesis has been a survey of TRAIL occurrence in human osteoblasts and bone-associated stromal cells in normal and pathological conditions. Human osteoblasts and stromal cells were obtained by enzymatic digestion of excess bone and joint tissue and grown in vitro. MG-63 osteosarcoma cells were also studied. Semi-quantitative RT-PCR amplification of 300 bp segments of TRAIL or RANKL from uniform 5 micro gram isolates of RNA and Western analysis on aliquots of proteins from 2 x 10⁶ cells, were performed. We found that TRAIL mRNA is expressed in MG63 cells and stromal cells from normal bone, arthritic bone and synovial fibroblasts. A liposarcoma from bone, did not express measurable quantities of TRAIL. Analysis by immunoprecipitation, using rabbit anti TRAIL or semi-quantitative PCR, suggested that TRAIL production and expression is often similar to RANKL. Western blot results showed 20 kD mature TRAIL in all bone cell culture extracts. Recombinant human TRAIL (19.6 kD) was used as a positive control. Controls for antibody specificity included reaction with RANKL which were non-reactive with the antibody. Thus, our data indicate that the TRAIL system is highly likely to be important in bone apoptosis and expression of OPG is certainly important not only in regulation of osteoclasts but in regulation of apoptosis via the Apo2-TRAIL system.

[Programme]

 
O-39

OPG AND CELLULAR GENE THERAPY WITH IL-4 DECREASE BONE RESORPTION-RELATED INFLAMMATION IN COLLAGEN- INDUCED ARTHRITIS

N. Saidenberg-Kermanac'h¹*, N. Bessis¹, M. C. de Vernejoul², M. C. Boissier¹, M. E. Cohen-Solal²

¹EA-3408, University Paris XIII, Bobigny, France

²INSERM U349, Paris, France

Interleukin-4 (IL-4) has anti-inflammatory effects in collagen-induced arthritis (CIA), a murine model of rheumatoid arthritis (RA). RANK-L secreted by osteoblasts and activated lymphocytes promotes focal and general osteolysis observed in RA. Osteoprotegerin (OPG) inhibits bone resorption in osteoclast precursors. The effects of OPG on inflammation and on bone resorption-induced inflammation remain to be defined. The aim of this study is to evaluate the respective and combined effect of OPG and IL-4 on inflammation and on bone resorption. CIA was induced in DBA/1 mice by immunization (bovine type II collagen, CII). Mice were treated with :1) sc injections of OPG-Fc or 2) with IL-4 DBA/1 transfected-fibroblasts, a cellular gene therapy with homologous DBA/1 fibroblasts transfected by a plasmid encoding IL-4, or 3) both OPG-Fc + Il-4. Control groups received 4) DBA/Tst (non IL-4 producing fibroblasts), 5) saline (placebo) or 6) naive mice. Bone mineral density (BMD) was measured at the total body (Piximus Lunar) at baseline before immunization and at sacrifice allowing to measure bone gain (Delta BMD) and Deoxypyridinolin levels were measured in the urines. Spleen cells were cultured after CII stimulation and IFN- gamma levels were measured in the supernatants. CIA significantly improved in IL-4 groups (p<0.01 vs Tst), whereas OPG had no effect on arthritis clinical scores. Histological scores were reduced in OPG, IL-4 and OPG+IL-4 groups vs placebo- treated CIA mice. OPG resulted in increased BMD (28 ± 3% vs 9 ±3% vs placebo- treated CIA, p<0.01) but IL-4 has no effect (17 ± 2% vs 17 ± 2% in Tst). Moreover, combination of OPG and IL-4 gene exerted an additive effect on BMD (42 ±7% vs placebo and tst, p<0.01). In addition, D-pyridinolin levels decreased by 43% in OPG- treated mice (p<0.001 vs control) and 91% when mice were treated with both OPG and IL-4. IFN-gamma secretion was inhibited in IL-4 treated-mice compared with controls. Interestingly, OPG had a moderate effect on IFN-gamma but potentiated the inhibitory effect observed in IL-4 treated-mice. In conclusion, systemic administration of OPG prevent bone loss in CIA-mice model. OPG and IL-4 gene therapy could have additive effects on bone mineral density.

[Programme]

 
O-40

DEVELOPMENT OF NOVEL DUAL-ACTION SRC KINASE INHIBITORS PREVENTING BONE RESORPTION AND STIMULATING BONE FORMATION IN VITRO AND IN VIVO

B. F. Boyce¹*, L. Xing¹, Y. Wang², R. Sundaramoorthi², T. Keenan², T. Yamashita1¹, W. Shakespeare², D. Dalgarno², J. Iuliucci², T. Sawyer²

¹Department of Pathology, University of Rochester, Rochester, NY, USA

²ARIAD Pharmaceuticals, Cambridge, MA, USA

Expression of the non-receptor tyrosine kinase, Src, is required for osteoclast activation and bone resorption. In addition to this role in osteoclasts, Src also negatively regulates osteoblast function, src-/- osteoblasts having enhanced function in vitro and in vivo. Thus, Src is a well-validated therapeutic target for the prevention and treatment of diseases characterized by bone loss, such as postmenopausal and other forms of osteoporosis. To this end, we have developed a series of novel, potent (low nM IC50s), non-peptide Src tyrosine kinase inhibitors (MW <500) using structure-based drug design. We targeted these inhibitors to bone by incorporating chemical moieties that confer them with high affinity for hydroxyapatite as well as cellular selectivity (osteoclasts and osteoblasts). Furthermore, we demonstrate that they have significant selectivity (>100-1000 fold) to inhibit Src relative to a panel of more than 30 protein kinases. AP23588 is a promising lead compound from this series of bone-targeted Src tyrosine kinase inhibitors, which possess dual action properties with respect to osteoclasts and osteoblasts. These include: 1) inhibition of osteoclastic resorption in vitro (pit area and number reduced by 55-97% at 1-10 microM), effects comparable to alendronate; 2) inhibition of PTH-induced hypercalcemia (50-100% inhibition) and bone resorption in vivo; 3) dose-dependent stimulation of alkaline phosphatase activity and mineraliz