O-1
IS GEOMETRICAL INSTABILITY OF THE PROXIMAL FEMUR A CRITICAL FACTOR IN THE
PATHOPHYSIOLOGY OF MINIMALLY TRAUMATIC HIP FRACTURES?
M. J. Walters1,2*, J. Meyer2
1Division Of Veterinary and Biomedical Sciences, Murdoch
University, Perth, Western Australia
2Department of Anatomy and Human Biology, University of
Western Australia, Perth, Western Australia
Low bone mineral density (BMD) is a known factor in the pathophysiology
of minimally traumatic hip fracture (MTHF). Another variable that appears to contribute to
fracture risk is the geometry of the proximal femur. Hip axis length (HAL) has been shown
to predict hip fracture in prospective studies, but in retrospective studies femoral neck
and shaft widths, and not HAL, vary with fracture incidence. This study sought an
explanation of this conundrum through a detailed comparison of the variations in the
components of HAL and other aspects of the geometry of the proximal femur in normal aging
and in cases of hip fracture.
The projected geometry of the proximal femurs of 1132 women aged 18 to 90
years without hip fractures and of the unfractured limbs of 140 women was assessed from
dual energy x-ray absorptiometry printouts within 9 days of a MTHF. Decade-by-decade
differences in the widths of the femoral neck (FNW) and proximal shaft (PFSW), as well as
HAL and its components (femoral head + acetabular breadth (AcH), femoral neck length (FNL)
and greater trochanteric width) were evaluated by Analysis of Variance (ANOVA) and those
between women with and without fractures <65 years (N=222) by Student's T-test.
Amongst women without hip fractures most geometric measures varied
significantly and directly with age and inversely with BMD. The exceptions were HAL and
FNW. The proportion of HAL made up by the femoral head and acetabulum (AcH/HAL) did
demonstrate some age-dependency. In contrast, in women with fractures, geometric
dimensions tended to decline directly with BMD and no systematic variation in AcH/HAL was
seen with age. There were no statistically significant differences in HAL between women of
similar age with and without fractures, but the AcH/HAL ratio was significantly lower in
women with fractures. The dimensions of geometric features on the contralateral limbs
adjacent to common fracture sites, as well as BMD, differentiated significantly between
women with different types of fracture and those without fractures.
The results of this study are discussed in the light of possible
instabilities of structure and variations in the projected geometry of the proximal femur
arising from age, stress and osteoporosis-related remodelling.
[Programme]
O-2
PEAK BONE MASS IN THE CALCANEUS IN MEN AND THE INFLUENCE OF LIFESTYLE
FACTORS
U. Pettersson1*, C. Nyman2, R. Lorentzon1,
K. Landin-Wilhelmson2, L. Hulthén2, O. Johnell3, R.
Kullenberg4, E. Norjavaara5, L. Samuelsson5, D. Mellstrom2
1Sports Medicine Unit, Umeå University, Umeå, Sweden
2Department of Geriatrics and Internal Medicine, Goteborg
University, Goteborg, Sweden
3Department of Orthopaedics, Malmo University, Malmo, Sweden
4Department of Radiophysics, Goteborg University, Goteborg,
Sweden
5The National Service Administration, Goteborg, Sweden
Aims: The aim of this study was to evaluate the time of attaining peak
bone mass in the calcaneus in men. The present study is part of an ongoing study
investigating relationships between anthropometric parameters, muscle strength, lifestyle
factors including physical activity and calcium intake and genetic analysis and BMD in
12000 male military recruits.
Methods: Since 1998, 2805 men (age 17.3-19.9 years) have been recruited
from the compulsory military service in Gothenburg, Sweden. Bone mineral density (BMD,
g/cm2) of the calcaneus was measured with DEXA, Calscan. Isometric muscle
strength of the total body was measured in Newton meters (Nm) using Isokai and physical
capacity was estimated from a maximal stress test. Lifestyle factors including smoking,
use of corticosteroids, training habits and calcium intake were evaluated by a
questionnaire.
Results: ANCOVA analysis revealed that peak BMD of the calcaneus
(0.65±0.10 g/cm2) occurred at the age of 18.3 years. BMD was significantly
associated with body weight (r=0.14), BMI (r=0.14), physical capacity (r=0.15), muscle
strength (r=0.24), calcium intake (r=0.08), and years of regular physical activity
(r=0.30). Smoking was a significant negative predictor (r=-0.06). Stepwise regression
analysis revealed that years of regular physical activity were the strongest predictor,
explaining 8% of the variation in BMD. Other independent predictors were muscle strength
(beta=0.13) and BMI (beta=0.08). All these factors totally explained 11% of the variation
of BMD.
Conclusion: This study suggests that peak bone mass in the calcaneus in
men is attained around 18 years. Years of regular physical activity were the strongest
predictor of BMD, indicating the sensitivity of the calcaneus bone for mechanical loading.
[Programme]
O-3
PEAK BONE MASS: A STUDY OF THE GENETIC AND ENVIRONMENTAL DETERMINANTS IN
YOUNG MEN AND WOMEN
F. E. McGuigan1*, L. Murray2, A. Gallagher3,
G. Davey-Smith4, C. E. Neville3, R. van't Hof1, C.
Boreham3, S. H. Ralston1
1Aberdeen University, Aberdeen, Scotland
2Queens University Belfast, N. Ireland
3University of Ulster, N. Ireland
4Department of Social Medicine, University of Bristol,
Bristol, UK
Attainment of peak bone mass is an important risk factor for the
development of osteoporosis in later life. Although it is believed that genetic,
intrauterine and environmental factors all contribute to the regulation of bone mass, how
they interact to determine peak bone mass is not yet fully understood.
In this study we investigated the role of polymorphisms of the vitamin D
receptor (VDR), estrogen receptor alpha (ESR) and collagen type I alpha 1 (COLIA1) genes
and the contribution of birth weight, diet, exercise and other environmental variables to
the regulation of peak bone mass in a population based cohort of 460 men and women whose
average age was 22 years.
Stepwise multiple regression analysis showed that body weight was the
strongest predictor of BMD in women accounting for 16.4% of the variance in spine BMD and
8.4% of the variance in hip BMD. Other predictors at the spine were VDR genotype (3.8%)
and carbohydrate intake (1.6%) whereas other predictors at the hip were vitamin D intake
(3.4%) and ER genotype (3.4%). In men, physical activity was the strongest predictor of
BMD, accounting for 6.7% of the variance at the spine and 5.1% at the hip. Other
significant predictors at the spine were body weight (5%), and ER PvuII genotype (2.8%)
whereas other predictors at the hip were weight (3.4%), age (2.9%) alcohol intake (2%) and
ER PvuII allele (4.1%).
Birthweight was not found to be a significant predictor of BMD at either
the spine or hip. Interestingly, of all the polymorphisms studied, only COLIA1 genotype
was found to be a significant predictor of birthweight in women accounting for 4% of the
variance.
We conclude that peak bone mass is regulated by an overlapping, but
distinct set of environmental and genetic influences which differ in men and women. Much
of the variance in peak bone mass was unexplained by the variables studied here however,
suggesting either that most of the genes which regulate BMD remain to be discovered, or
that major environmental influences on BMD exist that have not yet been identified.
[Programme]
O-4
BONE REMODELLING IN CELIAC DISEASE: AN IN VITRO STUDY
M. Longo1*, A. Taranta1, M. L. Bianchi2,
S. Saraifoger2, M. T. Bardella3, A. Teti4
1Department of Histology and Medical Embryology, University of
Rome 'La Sapienza', Rome, Italy
2Bone Metabolic Unit, Istituto Auxologico Italiano - IRCCS
3Cattedra di Gastroenterologia - IRCCS Ospedale Maggiore,
Milan, Italy
4Department of Experimental Medicine, University of L'Aquila,
Italy
Celiac disease (CD), an inflammatory disorder of the small bowel due to
gluten intolerance, is often associated with low bone mass. The mechanisms altering bone
remodelling in celiac patients are still poorly understood. To explore whether CD affects
bone formation, sera from patients were tested for their ability to influence human
osteoblasts. Three groups of subjects were selected: (1) CD patients on gluten-free diet
(GFD) for at least 2 years, in stable conditions, with no sign of disease activity and
negative to anti-gliadin and anti-endomysium antibodies; (2) untreated, newly diagnosed CD
patients; and (3) age-matched healthy donors. Human osteoblasts were obtained from
surgical specimens of healthy subjects and exposed to 5% sera.
3H-thymidine incorporation revealed that sera from both groups
of CD patients stimulated osteoblast proliferation (p<0.01 vs. controls). Histochemical
and biochemical analyses showed significantly higher alkaline phosphatase activity upon
exposition to sera from untreated patients (p<0.005). Consistently, matrix
mineralization was also increased, in a semiquantitative analysis, by the sera of CD
patients, with the highest levels in the untreated patient group. These data hint to
osteoblast stimulation rather than inhibition in CD subjects, possibly due to released
factors enhancing their function. On the basis of these observations, we analysed whether
CD-sera - treated osteoblasts stimulated bone resorption through the release of cytokines
or other growth factors influencing osteoclast function.
Transcriptional expression of the osteoclast stimulating cytokines IL-1
and IL-6, and of the specific osteoclast inhibitory factor, osteoprotegerin (OPG), was
evaluated, and showed similar mRNA levels for IL-1 and IL-6 in all groups, while OPG mRNA
was significantly reduced in the osteoblast exposed to sera of untreated patients
(p<0.01). This suggests a partial suppression of the OPG-mediated block of osteoclast
function, which may increase bone resorption. We therefore tested the sera for altered
osteoclast formation in cultures of human peripheral blood monocytes and observed,
consistently, that sera from CD patients increased osteoclastogenesis relative to healthy
donors.
We conclude that systemic factors may contribute to bone loss in CD
patients, partly due to osteoblast-mediated enhancement of osteoclast bone resorption.
[Programme]
O-5
WOMEN WITH ANTIBODIES AGAINST HELICOBACTER PYLORI HAVE INCREASED RISK OF
OSTEOPOROTIC FRACTURES
B. L. Langdahl1*, J. M. Hansen2, M. K. Møller1,
H. Nielsen2, L. Østergaard3
1Dept. of Endocrinology, Aarhus University Hospital, Aarhus,
Denmark
2Dept. of Infectious Diseases, Aalborg Hospital, Aalborg,
Denmark
3Dept. of Infectious Diseases, Aarhus University Hospital,
Aarhus, Denmark
Osteoporosis is a multifactorial disease. From epidemiological studies it
is known that thin women have an increased risk of developing osteoporotic fractures.
Chronic infection with Helicobacter pylori (H. Pylori) has been associated with various
extra-gastrointestinal disorders, such as growth retardation in children and
cardiovascular disease. We therefore wanted to investigate if prevalence of antibodies
against H. Pylori is associated with osteoporotic fractures.
We examined 79 female patients (mean age: 65.4±6.3 years) with
osteoporosis defined as having at least one low-energy vertebral fracture and low bone
mineral density (BMD) and 79 healthy female controls (mean age: 64.2±7.0 years). Serum
samples from each individual were examined for antibodies against H. Pylori by a latex
agglutination test. BMD was examined by DXA at the lumbar spine and at the femoral neck. A
total of 38.0 % (30/79) osteoporotic women had antibodies against H. Pylori compared with
22.5 % (17/79) of agematched normal women (chi-sq=5.12, p=0.02). The oddsratio for having
an osteoporotic fracture if seropositive for H. Pylori is 2.23 (1.11-4.51). BMD corrected
for age was 0.821±0.158 g/cm2 (mean±SD) at the lumbar spine in individuals
with antibodies against H. Pylori (n=47) compared with 0.861±0.162 g/cm2 in
individuals without (n=111), p=0.16. At the femoral neck the corresponding values were
0.683±0.098 g/cm2 and 0.709±0.107 g/cm2, respectively (p=0.16).
In conclusion, seropositivity for H. Pylori infection is associated with
increased risk of osteoporotic fractures and a tendency towards reduced bone mass in the
spine and the hip.
[Programme]
O-6
PREGNANCY AND LACTATION HAVE NO LONG TERM EFFECTS UPON BONE DENSITY IN A
HEALTHY POPULATION: A TWIN STUDY
J. D. Wark1*, L. Paton1, J. Alexander1,
C. Margerison1, M. Frame1, B. Kaymakci1, C. Nowson2
1Department of Medicine, Royal Melbourne Hospital, University
of Melbourne, Australia
2School of Health Sciences, Deakin University, Australia
Pregnancy and lactation impose a high maternal calcium demand. To
determine whether these stresses have long-term effects upon bone, we sought differences
in bone mineral density (BMD) in healthy female twin pairs, (1) who differed in their
absolute number of pregnancies beyond 20 weeks, and (2) in a cross- sectional analysis of
1354 individuals twins and their relatives.
Study 1 comprised 498 female twin pairs, mean age 42(15.0) years of whom
83 pairs were discordant for ever being pregnant. Subjects in study 2 were grouped
according to the number of pregnancies: group A, no pregnancy (n=426), group B, 1 or 2
pregnancies (n=455) and group C, 3 or more pregnancies (n=473). BMD at the lumbar spine
(LS) and total hip (HP) and total body (TB) bone mineral content (BMC) were measured.
Where appropriate, BMD was adjusted for age, height and fat mass. Study 1: No significant
within-pair differences were observed in BMD at the LS, HP or in TB BMC in twins
discordant for ever being pregnant (paired t-tests). Pairs were grouped according to the
difference in number of pregnancies (range:0-6). Subjects were also divided into three
groups: equal number of pregnancies, different by 1 or 2 pregnancies, or different by 3 or
more. No within-pair difference in unadjusted/adjusted HP BMD, LS BMD or TBBMC was
observed with the difference in number of pregnancies. Study 2: Cross-sectionally, group B
and group C subjects had higher adjusted LS BMD (3.0% and 3.2% respectively, both p=0.001)
and adjusted TB BMC (2.7% and 3.1%, respectively, both p<0.001) compared with
non-parous subjects (group A). Adjusted HP BMD was greater in group C compared with group
A (2%, p=0.018). Parous women who breastfed had higher adjusted TB BMC (3.1%, p=0.005) and
a marginally higher adjusted HP BMD (2.5%, p=0.04) than parous non-breastfeeders.
There was some evidence from the cross-sectional analysis for a positive
association between parity and BMD, but there was no observed within-pair difference in
bone mineral measures in twins discordant for ever being pregnant or for the number of
pregnancies. We detected no long-term detrimental effect of pregnancy or breastfeeding on
BMD.
[Programme]
O-7
A PHOSPHONOCARBOXYLATE ANALOGUE OF RISEDRONATE (NE10790) INHIBITS RAB
PRENYLATION IN OSTEOCLASTS IN VIVO AND DISRUPTS THE MEMBRANE LOCALIZATION OF RAB PROTEINS
IN OSTEOCLASTS IN VITRO
F. P. Coxon1*, E. Mules2, M. C. Seabra2,
F. H. Ebetino3, M. J. Rogers1
1Department of Medicine & Therapeutics, University of
Aberdeen, Aberdeen, UK
2Department of Cell & Molecular Biology, Imperial College,
London, UK
3Procter & Gamble Pharmaceuticals, Mason, Ohio, USA
Nitrogen-containing bisphosphonates such as risedronate act by inhibiting
farnesyl diphosphate (FPP) synthase, thereby preventing the synthesis of isoprenoid lipids
required for the prenylation of small GTPases in osteoclasts. We recently found that a
weakly anti-resorptive analogue of risedronate, NE10790, selectively prevents prenylation
of Rab GTPases in cells in vitro by inhibiting Rab geranylgeranyl transferase (Rab
GGTase). NE10790 has no effect on FPP synthase, farnesyl transferase (FTase) or GGTase I
and therefore does not affect the prenylation of other proteins.
We have now investigated the effects of NE10790 on rabbit osteoclasts in
vivo. Rabbits were subcutaneously injected with 2.1 mg P/kg NE10790. After 24 hours,
osteoclasts were purified from a crude preparation of bone cells by immunomagnetic bead
separation with an anti-vitronectin receptor (VNR) antibody. Using an in vitro prenylation
assay, we found that NE10790 caused the accumulation of unprenylated Rabs in VNR-positive
osteoclasts, but not in VNR-negative cells.
We have also investigated the effect of NE10790 on subcellular
localization of Rab6 by immunostaining and western blotting. Rab6 localized to the Golgi,
seen as distinct perinuclear staining in osteoclasts and punctate staining in J774 cells.
Treatment of osteoclasts or J774 cells with either NE10790 or risedronate disrupted this
localization, resulting in diffuse staining for Rab6 throughout the cell. By contrast,
FTI-277 and GGTI-298 (inhibitors of FTase and GGTase I, respectively) had no effect. For
western blotting, lysates were fractionated by ultracentrifugation or Triton X-114
separation. Rab6 was almost completely localized to the membrane fraction in untreated
J774 cells and osteoclasts, but accumulated in the cytoplasmic fraction in cells treated
with NE10790 and risedronate. By contrast, only risedronate caused the accumulation of Rac
(which is prenylated by GGTase I) in the cytosol.
These data suggest that the anti-resorptive effects of NE10790 are likely
due to inhibition of Rab prenylation in osteoclasts, resulting in the cytoplasmic
accumulation of unprenylated Rab proteins.
[Programme]
O-8
TGF-BETA INDUCES HUMAN OSTEOCLAST FORMATION AND BONE RESORPTION IN THE
ABSENCE OF SOLUBLE RANKL
I. Itonaga, A. Sabokbar, O. Kudo, N. A. Athanasou*
Nuffield Department of Orthopaedic Surgery, Nuffield Orthopaedic Centre,
University of Oxford, Oxford, UK
Osteoclast progenitors can differentiate into mature bone resorbing
osteoclasts in the presence of macrophage colony stimulating factor (M-CSF) and RANK
ligand (RANKL), which is expressed on bone stromal/osteoblastic cells. Osteoprotegerin
(OPG) inhibits RANKL-induced osteoclast formation and bone resorption. It has been
reported that tumour necrosis factor-alpha (TNF-alpha), a potent cytokine involved in
regulation of osteoclast activity via a primary effect on osteoblasts, can directly (in
the presence of M-CSF) induce the differentiation of osteoclast progenitors into mature
osteoclasts. These studies revealed that TNF- alpha-induced osteoclast formation is
independent of RANK/RANKL interaction. In the present study we sought to determine whether
transforming growth factor beta (TGF-beta), another powerful modifier of bone resorption,
can similarly induce osteoclast formation and bone resorption in vitro. To address this,
mononuclear cells were isolated from peripheral blood of healthy volunteers and cultured
for up to 24 days on glass coverslips and dentine slices in the presence of: (i) RANKL and
M-CSF; (ii) TGF-beta, M-CSF±OPG; (iii) TGF-beta, M-CSF±anti TNF-alpha antibody. In some
experiments, CD14+ mononuclear cells isolated using Mini MACS CD14 Micro Beads cell sorter
were cultured. The extent of osteoclast formation and bone resorption was determined by
generation of TRAP- positive multinucleated cells, actin ring formation and lacunar
resorption on dentine slices. We noted that addition of TGF-beta, in the presence of
M-CSF, but in the absence of RANKL, was sufficient to induce the formation of
TRAP-positive multinucleated osteoclast-like cells which were capable of forming actin
ring and lacunar resorption on dentine slice in vitro. This osteoclast formation was also
induced in CD14-sorted mononuclear cell cultures. The addition of OPG or anti TNF-alpha
antibody to the cultures containing TGF-beta did not inhibit osteoclast formation and bone
resorption, thus suggesting that TGF-beta induces osteoclast formation in manner
independent of the RANK/RANKL or TNF-alpha mechanism. Our results indicate that TGF-beta,
which is abundant in bone and thought to have potent effects on bone metabolism, can
directly induce osteoclast precursors to differentiate into active bone resorbing
osteoclasts.
[Programme]
O-9
PATTERNS OF EXPRESSION OF CHEMOKINES AND THEIR RECEPTORS IN OSTEOCLASTS
J. M. Lean*, C. Murphy, K. Fuller, T. J. Chambers
St George's Hospital Medical School, London, UK
Although much has been learned recently of the mechanisms by which
osteoclast differentiation and function are regulated, much less is known of the factors
that regulate their migration and localisation. In related cell types, chemokines play a
major role in these processes. We therefore systematically tested the expression of RNA
for chemokine receptors and their ligands by osteoclasts. Because bone is the natural
substrate for osteoclasts and may influence osteoclast behaviour, we also tested
expression on bone slices. Quantitative RT-PCR using real time analysis with sybr green
was therefore performed on RNA isolated from bone marrow cells after incubation with M-CSF
with/without RANKL, on bone or plastic, and assayed for known murine chemokines and their
receptors. As a measure of osteoclast differentiation TRAP expression was also measured.
We found that RANKL induced a 100-fold increase in TRAP in cells grown on plastic. This
was increased a further 10-fold when grown on bone. RANKL reduced the expression of RNA
for the Fc receptor (CD16) and c-fms. The chemokines most highly expressed by bone marrow
cells incubated on tissue culture plastic with M-CSF and RANKL were: CX3CL
(neurotactin/fractalkine), CCL2(MCP-1), CCL3(MIP1-alpha), CCL6(MRP-1), CCL7(MCP3),
CCL9(MIP1-gamma), CCL12(MCP-5), CXCL4(PF4) and CXCL6. The most abundant chemokine receptor
was CCR1, followed by CX3CR, CCR3, CCR5 and CXCR4. However, we found that the major effect
of RANKL was to reduce RNA for chemokines most strongly associated with inflammation.
Thus, CCL2(MCP-1), CCL3(MIP1-alpha) and CCL6(MRP-1) were strongly inhibited by RANKL.
Cells incubated on bone showed even greater inhibition, with expression of CCL7(MCP3) and
CXCL9(MIG) becoming undetectable.
RANKL increased expression of CCL9(MIP1-gamma) (up to 50-fold),
CCL22(MDC) (up to 10-fold) and CXCL13(BCA-1) (from undetectable). RANKL also upregulated
the expression of receptors CCR1 and CCR3. There was a highly significant correlation
between the number of copies of mRNA for TRAP and CCL9(MIP1-gamma).
The expression of CCL9(MIP1-gamma) and CCL22(MDC) and their corresponding
receptors suggest an autocrine or paracrine role for these chemokines in osteoclasts.
Initial studies have shown no effect of these agents on osteoclast differentiation or
resorption, but both agents stimulate cytoplasmic motility, suggesting that they may play
a role in osteoclastic migration/localisation.
[Programme]
O-10
STIMULATION OF RANKL-DEPENDENT OSTEOCLAST FORMATION BY INFLAMMATORY
(RHEUMATOID/CRYSTAL) ARTHRITIS SYNOVIAL FLUID
L. Danks, A. Sabokbar, I. Itonaga, N. A. Athanasou*
Nuffield Department of Orthopaedic Surgery, Nuffield Orthopaedic Centre,
University of Oxford, Oxford, UK
Periarticular bone resorption is a feature of arthritis due to crystal
deposition and rheumatoid disease. The inflammatory synovial fluid in these conditions
contains numerous inflammatory cells and humoral factors which promote osteoclast
formation. In order to determine the effect of inflammatory synovial fluid (SF) on human
osteoclast formation in this study, SF from rheumatoid arthritis (RA), pyrophosphate
crystal arthritis (PPA) and osteoarthritis (OA) patients was added to cultures of
peripheral blood mononuclear cells (PBMCs) in the presence and absence of RANKL (30ng/ml)
and M-CSF (25ng/ml). The extent of osteoclast formation and lacunar resorption was
assessed by tartrate resistant acid phosphatase (TRAP), vitronectin receptor (VNR) and a
lacunar resorption assay. The number of TRAP and VNR positive cells was dose dependently
increased following the addition of 1, 5 and 10% SF to PBMC cultures. Mononuclear TRAP and
VNR positive cells also were noted in PBMC cultures incubated with 10% SF. The mean
percentage area of lacunar resorption was significantly increased in cultures incubated
with 10 % RA and PPA SF compared to OA SF (p=0.03 and p=0.02, respectively). SF alone was
incapable of supporting osteoclast formation from PBMCs. These results indicate that
inflammatory synovial fluid stimulates RANKL-induced osteoclast formation and lacunar
resorption. Some of these cultures consisted entirely of mononuclear TRAP and VNR positive
cells, indicating that multinuclearity is not a sufficient marker for osteoclast
formation. The extent of osteoclast formation and lacunar resorption was consistently
extensive in these cultures (i.e. 90-100% area resorption). Our findings suggest that
factors present in inflammatory synovial fluid of RA and PPA patients is likely to be of
pathogetic significance in periarticular bone resorption.
[Programme]
O-11
MET, A NOVEL MODULATOR OF ESTROGEN INDUCED TRANSCRIPTION
S. M. Colley1*, A. Flynn2, M. Norman2,
D. Wynick2, J. H. Tobias1
1Rheumatology Unit, Division of Medicine, University of
Bristol, Bristol, UK
2University Research Centre for Neuroendocrinology, University
of Bristol, Bristol, UK
High-dose estrogen is known to stimulate osteoblast activity in
postmenopausal women as well as rodent models. To investigate the molecular mechanisms
underpinning this effect, we compared the gene expression profiles of mRNA isolated from
the tibiae of adult mice treated with either estrogen or vehicle for 4 days by subtractive
hybridisation analysis. During the course of this investigation, a gene fragment showing
no significant homology with previously characterised sequences was isolated and found to
detect a single 3.8Kb transcript by northern analysis. A full length cDNA homologous to
this gene was generated by EST directed Rt-PCR using bone marrow cDNA as the source of
template. Sequence analysis of the cloned products revealed it coded for a 1031 amino acid
protein. This peptide contains a SAF Box DNA binding motif, an RNA binding domain and
shares an overall identity of 34% with the estrogen suppresser SAF-B/HET/ HAP. SET has
previously been reported bind the estrogen receptor directly, act as a repressor both in
breast and bone cell lines and augment the anti-estrogen effects of the SERM Tamoxifen.
When the cDNA we have isolated was expressed as a fusion product with enhanced yellow
fluorescent protein, it was found to localise exclusively to the nucleus. It was further
observed that its expression in MCF-7 cells resulted in a dose dependent reduction by up
to 50% in estrogen induced ERE luciferase reporter gene expression. From these data we
conclude that this novel sequence represents a previously undescribed Modulator of
Estrogen induced Transcription, which we refer to as MET, that is likely to play a
significant role in modulating estrogen responses in bone and other tissues.
[Programme]
O-12
MODULATION OF HUMAN ER ALPHA F PROMOTER ACTIVITY BY A PROTEIN KINASE C -
DEPENDENT MECHANISM IN HUMAN DIFFERENTIATED OSTEOBLAST-LIKE CELLS
M. Longo1,3*, V. De Luca1,3, S. Denger2,
G. F. Caselli3, F. Gannon2, A. Teti3, S. Migliaccio3,4
1Department of Histology & Medical Embryology, University
'La Sapienza', Rome, Italy
2EMBL, Heidelberg, Germany
3Department of Experimental Medicine, University of L'Aquila,
Italy
4Department of Medical Physiopathology, University 'La
Sapienza', Rome, Italy
The gene for human estrogen receptor alpha (ERalpha) gives rise to six
different mRNAs driven in a tissue-specific manner by six different promoters (A-F) and
translated into a single protein. The F-promoter has been described as specific for the
bone tissue, but its modulation has not been clarified yet.
To elucidate the role of ERalpha promoters in bone physiology and its
regulation, we generated human osteoblast-like cells (SAOS-2) stably transfected with
expression vectors carrying human ERalpha A, B, and F promoter sequences upstream of the
luciferase reporter gene. No luciferase activity was detected in cells containing
promoters A and B relative to cells transfected with empty vectors. In contrast, high
basal luciferase activity was found in two clones carrying the F promoter.
Our previous results have evidenced significant modulations in ERalpha
levels and activity upon confluence-induced differentiation in osteoblasts, involving
close interaction with PKC isoforms. This prompted us to analyse F promoter activity
during osteoblast differentiation and its possible dependence on PKC isoform expression
and/or activity.
F promoter activity was evaluated in sub-confluent (proliferating) and
post-confluent (differentiated) cells, and found to be constantly up-regulated in
differentiated versus proliferating osteoblasts, in identical serum conditions. A
two/three-fold higher luciferase activity was detectable 24 hr after confluence, with no
significant increase up to 96 hr thereafter.
Overnight treatment of post-confluent cells with 10-8M
12-O-tetradecanoylphorbol-13-acetate (TPA), known to abolish PKC isoform expression,
resulted in a marked decrease of F promoter activity up to the level detected in
proliferating counterparts, which strongly suggests a PKC-dependent up-regulation of the
promoter upon differentiation.
We also observed that in proliferating cells identical TPA treatments
induced a slight increase of promoter activity. In contrast, during proliferation the
promoter was unaffected by treatment with calphostin C, a compound inhibiting the
activity, but not the expression, of most PKCs, while decreased luciferase activity
relative to untreated counterparts was again observed in differentiated cells, but to a
lesser extent.
These data strongly suggest tight dependence on intact PKC pathways for F
promoter regulation, possibly involving both kinase activity -independent and - dependent
PKC functions.
[Programme]
O-13
ACTIVATION OF THE BMP-6 PROMOTER BY THE ESTROGEN RECEPTOR
S. M. Colley1*, D. B. Ong1, S. Kitazawa2,
M. Norman3, D. Wynick3, J. H. Tobias1
1Rheumatology Unit, Division of Medicine, University of
Bristol, Bristol, UK
2Division of Molecular Pathology, Kobe University Graduate
School of Medicine, Kobe, Japan
3University Research Centre for Neuroendocrinology, University
of Bristol, England
High dose estrogen is known to induce osteogenesis in the long bones of
adult mice. In searching for factors that play a role in this process, we observed a
significant increase in Bone Morphogenetic Protein 6 (BMP-6) transcripts in mRNA isolated
from femora of animals treated with 17 beta-estradiol (E2) compared to vehicle (Plant et
al. 2001). Based on evidence that E2 increases BMP-6 expression in osteoblasts in a cell
autonomous manner in vitro (Rickard et al 1998), we explored the mechanisms by which
estrogen induces osteogenesis by studying the regulation of BMP-6 expression in
osteoblasts. Initially, the activity of a 1.2Kb BMP-6 promoter sequence coupled to a
luciferase reporter (Tamada et al 1998) was compared between ROS and SMER osteosarcoma
cells, of which the latter represent ROS cells stably transfected with estrogen receptor
(ER) alpha. Basal BMP-6 reporter activity was found to be considerably higher in SMER
versus ROS cells, with no further increase after the addition of E2. This suggestion that
BMP-6 promoter activity is influenced by ER in a non-ligand dependent manner was confirmed
by comparing reporter activity in ROS cells transiently transfected with the BMP-6
reporter and an ER alpha expression vector; co-transfection with ER alpha stimulated BMP-6
reporter activity by four-fold irrespective of the presence of E2. Equivalent findings
were observed in liver and renal cell lines (HepG2 and COS cells respectively). Taken
together, these results indicate that ER acts to increase the level of BMP-6 promoter
activity in a non ligand dependent manner irrespective of cell background, as assessed
using a construct based on the 1.2Kb proximal sequence. This suggests that
ligand-dependent activation of BMP-6, as observed in previous in vitro and in vivo studies
of the effects of estrogen on BMP-6 mRNA, involves an interaction between the proximal
portion of the BMP-6 promoter and more distal sites yet to be identified.
Plant A. et al (2001) In press: J. Bone Miner. Res
Rickard D. (1998) J. Clin. Invest. 101, 413-22.
Tamada H. (1998) Biochim. Biophys. Acta 1395, 247-51.
[Programme]
O-14
MICE DEFICIENT IN 11BETA-HYDROXYSTEROID DEHYDROGENASE TYPE 1 LACK BONE
MARROW ADIPOCYTES AND MAINTAIN NORMAL BONE FORMATION
J. Justesen1*, L. I. Mosekilde3, K. Stenderup1,
M. Holmes4, J. J. Mullins4, J. R. Seckl4, E. F. Eriksen1,
M. Kassem1,2
1University Department of Endocrinology and Metabolism,
University Hospital of Aarhus, Denmark
2University Department of Endocrinology and Metabolism,
University Hospital of Odense, Denmark
3Department of Cell Biology, Institute of Anatomy, University
of Aarhus, Denmark
4Molecular Medicine Centre, Western General Hospital,
Edinburgh, UK
The physiological effects of glucocorticoids (GCs) on the skeleton are
complex and poorly understood. The cellular activity of GCs is regulated at a pre-
receptor level by 11beta-hydroxysteroid dehydrogenases (HSDs). The type 1 isozyme
catalyses the formation of active cortisol (corticosterone) within cells from circulating
inert 11-keto forms. This isoform is expressed in bone. Thus, we studied the bone
phenotype of HSD1 deficient (HSD1-/-) mice and wild type (Wt) animals using
bone histomorphometry applied to proximal tibial metaphysis and vertebrae. In addition, we
studied the cellular characteristics of cultured osteoblastic cells obtained from femurs,
calvariae and bone marrow from both groups in vitro.
See table for histomorphometric results. Osteoblastic cells from both
HSD1-/- and Wt mice exhibited similar growth characteristics, alkaline
phosphatase activity and in vitro mineralization. They were able to form adipocytes in
presence of the dexamethasone, indomethacin, and IBMX. We are currently investigating the
in vivo bone formation potential of the cells.
Parameter |
HSD-/- |
Wt |
p |
Number of mice |
6 |
6 |
|
Trabecular bone volume (%) |
27±3 |
23±4 |
n.s. |
Adipose tissue volume (%) |
0 |
3±2 |
<0.05 |
Volume of vascular sinusoids (%) |
25±5 |
29±4 |
n.s. |
Bone formation/bone surface (%) |
5±1 |
3±1 |
n.s. |
Bone resorption/bone surface (%) |
8±1 |
8±1 |
n.s. |
Bone formation rate/bone surface
(microm/day) |
0.87±0.09 |
0.88±0.15 |
n.s. |
Mineral apposition rate (microm/day) |
4.45±0.34 |
4.55±0.57 |
n.s. |
Conclusions: (1) HSD1 amplification of intracellular GC actions affects bone marrow
adipocyte formation but not bone formation, (2) adipocyte and osteoblast differentiation
can be regulated independently, (3) the reported negative effects of GC treatment on the
skeleton may represent pharmacological rather than physiological effects.
[Programme]
O-15
ROLE OF BETA-ARRESTINS IN THE REGULATION OF PARATHYROID HORMONE (PTH)
ACTIVITY IN VITRO AND IN VIVO
S. L. Ferrari1*, D. Sternlight2, D. Pierroz1,
R. Lefkowitz3, M. Bouxsein2
1Div. of Bone Diseases, University Hospital, Geneva,
Switzerland
2Orthopedic Biomechanics Laboratory, Beth Israel Deaconess
Medical Center, Boston, MA, USA
3Howard Hughes Medical Institute Laboratories, Duke University
Medical Center, Durham, NC, USA
Beta-arrestin 1 and 2 (b-arr1 and b-arr2) are cytoplasmic adaptor
molecules involved in the regulation of signaling and intracellular trafficking of many G
protein-coupled receptors. We have previously reported that recruitment of b-arr2 to the
cell membrane upon activation of human PTH/PTH-related protein (PTHrP) receptor (PTH1Rc)
is responsible for desensitization of PTH (and PTHrP)-stimulated cAMP signaling and for
internalization of ligand-receptor complexes. Using a fully automated fluorescence
microscope coupled to an ultrasensitive digital CCD camera, we have now generated a movie
showing PTH- stimulated internalization and fusion of membrane vesicles (endosomes)
containing the PTH1Rc, b-arr2 and b-arr1 in an isolated living cell. The importance of
this phenomenon in the regulation of cAMP signaling is further demonstrated by the
development of PTHrP-derived agonists that don't mobilize arrestins nor internalize
PTH1Rc, thereby triggering prolonged activation of adenylyl cyclase in vitro.
In order to evaluate the influence of beta-arrestins on PTH biological
activity in vivo, we have started to investigate mice null for beta-arrestin 2. Adult
b-arr2 KO mice and their wild-type (wt) littermates show no gross differences in their
apparent phenotype nor in skeletal morphology on conventional radiographs. However,
evaluation of bone mineral density (BMD) by pDXA (PIXImus) and of skeletal
microarchitecture by high resolution micro-computed tomography and histomorphometry
suggests that, compared to wild type littermates, six-months-old b-arr2 KO mice have lower
BMD (-9%) and altered trabecular microarchitecture (BV/TV, -17%, connectivity, -55%).
These osteoporotic features are compatible with a sustained (continuous) and catabolic
effect of endogenous PTH in b-arr2 KO mice. In order to further examine this possibility,
the development of the bone phenotype in b-arr2 KO mice is currently being surveyed by
longitudinal assessment of BMD at 4, 8, 12 16 and 20 weeks. Moreover, to directly evaluate
whether PTH has a prolonged and sustained biological activity in these mice, the calcemic
response (at 2, 6, 12 and 24 hours) to acute PTH administration (500microg/kg) is
currently being measured.
Altogether, these studies will provide further evidence for the role of
beta-arrestins in the regulation of PTH biological activity in vitro and in vivo.
[Programme]
O-16
BONE MORPHOGENETIC PROTEIN-7 PROMOTES TENDON GRAFT INTEGRATION IN THE
RECONSTRUCTION OF THE ANTERIOR CRUCIATE LIGAMENT
R. Mihelic1*, V. Kusec2, D. Bobinac3, S.
Zoricic3, M. Jelic4,5, M. Pecina5, S. Vukicevic4
1Orthopaedic Hospital, Lovran, Croatia
2Clinical Institute of Laboratory Diagnosis, Clinical Hospital
Centre, Zagreb, Croatia
3Department of Anatomy, School of Medicine, University of
Rijeka, Croatia
4Department of Anatomy, School of Medicine, University of
Zagreb, Croatia
5Department of Orthopaedic Surgery, School of Medicine,
University of Zagreb, Croatia
Reconstruction of the anterior cruciate ligament requires an early
integration of the tendon graft in the bone tunnel. However, an undesired outcome, as
widening of the bone tunnel (whipe-screen effect) has been described. It has been well
documented that bone morphogenetic proteins (BMP) induce bone formation and regeneration
of other skeletal-related tissues when applied locally. In this study BMP-7 was injected
with the tendon graft in the bone tunnel of the ovine femur and tibia in order to
investigate its effect upon graft integration. Ten sheep (1 year old, male, 37 kg) were
operated under general anaesthesia, tunnels of 4.5 mm in diameter were drilled in the
right femur and tibia, and approximately 5 cm of the m. peroneus tertius tendon inserted
and fixed. In the experimental group 25 microg of BMP-7 (OP-1) in 0.2 mL buffer was
applied in the bone tunnel of 5 sheep. Helistat was used as carrier in three animals. In
the control group (n=5) the entire procedure was carried out identically but without the
addition of BMP. Animals were sacrified after three weeks, operated knees were excised and
processed for histologic analysis.
The outcome was satisfactory in all experimental animals, which was also
assessed by X-rays. In all specimens the tendon fibres of the epitendineum invaded the
surrounding bone marrow space. Bone formation and remodelling was enhanced with thicker
trabeculae on the bone-tendon interface. In the experimental group areas of woven bone
with large lacunae and dense trabecular structure were present. This was less pronounced
in the specimen without Helistat. These areas of newly formed bone differed in size and
were located at different sites along the bone tunnel. This was either not observed or
present in a much less extent in control specimen. Invasion of the tendon fibrous tissue
into the bone marrow space was significantly (p<0.01) greater for the experimental
group (approx. 600- 1800 mm) than in the control animals (approx. 400-800 mm). These
results suggest that BMP-7 contributes to better integration of the tendon possibly by
stimulating outgrowth of tendon fibers into the marrow space and by inducing rapid new
bone formation.
[Programme]
O-17
THE MULTIPLE FUNCTIONS OF OSTEOBLAST-STIMULATING FACTOR-1 IN BONE
DEVELOPMENT
H. I. Roach*, R. S. Tare, R. O. C. Oreffo, N. M. P. Clarke
University Orthopaedics, University of Southampton, General Hospital,
Southampton, UK
In search for anabolic agents that increase osteoblast activity, we
examined the functions of osteoblast-stimulating factor-1 (OSF-1), an extracellular
matrix- associated growth/ differentiation factor. We studied bone development up to 30
weeks of age in transgenic mice, which carried six extra copies of the human OSF- 1 gene,
and also examined the effects of OSF-1 on osteogenic cells and cartilage explants in
vitro. OSF-1 was synthesized by osteoblasts at an early stage of differentiation, prior to
late markers of the osteoblast phenotype, i.e. bone sialoprotein and matrix
mineralization. In vivo, OSF-1 was localized (using immunocytochemistry) to sites of new
bone formation, i.e. the periosteum, at the groove of Ranvier, and in the primary
spongiosa. The factor was secreted and stored in the bone matrix, presumably for future
use during the remodeling process. In vitro, OSF-1 increased the osteogenic
differentiation of mesenchymal stem cells in mouse bone marrow cultures by ~50% at a
concentration of only 10 pg/ml, which was considerably lower than the concentrations of
BMP-2 required to achieve a similar effect. However, OSF-1 did not have BMP-like
osteoinductive capacity, since it was unable to convert the differentiation pathway of
C2C12 pre- myoblastic cells into the osteoblast lineage, whereas BMP-2 was able to do so.
Overexpression of OSF-1 in transgenic mice did not result in larger animals. However, the
calcium content/ mg bone was ~10% higher in the male transgenic mice. In the transgenic
(but not control) mice, OSF-1 was localized in the growth plates and in articular
chondrocytes, some of which also synthesized type I collagen. To confirm that excess OSF-1
had provided the stimulus for synthesis of this bone-type collagen, cartilage explants
were cultured with exogenous OSF-1 (50ng/ml). This also induced chondrocytes to synthesize
type I collagen.
Thus the functions of OSF-1 in bone development range from enhancing
osteogenic differentiation and increasing bone mineral content to, perhaps, inducing
osteogenic differentiation of chondrocytes. The results suggest that OSF-1 is a factor
important for bone formation and remodelling.
[Programme]
O-18
PROTEIN KINASE D MEDIATES ACTIVATION OF MAP KINASES P38 AND JNK INDUCED
BY Gq PROTEIN-COUPLED RECEPTORS IN OSTEOBLAST-LIKE CELLS
J. Caverzasio*, J. Lemonnier, Ch. Gayor
Dept of Medicine, University Hospital, Geneva, Switzerland
G protein-coupled receptors (GPCRs) are transducers of the anabolic
effect of potent osteotropic factors such as parathyroid hormone and fluoride. Recent
studies suggest the implication of several mitogen-activated protein kinases (MAPKs) in
the regulation of osteoblastic cell activity by GPCRs.
In this study, we investigated the effect of PGF2alpha (PGF2), a GqPCR
agonist, on activation of MAPKs in MC3T3-E1 (E1) osteoblast-like cells and the molecular
mechanism involved in activation p38 and JNK by PGF2.
PGF2 (1 microM) activated the three MAPKs Erk, JNK and p38 with different
kinetics. Erk activation was rapid, maximal at 5 min and lasted about 10 min whereas JNK
and p38 (JNK/p38) activation was apparent after 15 min, maximal at one hour and lasted
about 3 h. Two PKC inhibitors, Go6983 and Go6976 differentially blunted activation of
MAPKs induced by PGF2. G06983, an inhibitor of typical PKCs, completely blocked Erk
activation without influencing activation of JNK and p38 whereas Go6976 had the opposite
effect. Go6976 is a potent inhibitor of protein kinase D (PKD) (also called PKCmu) and the
role of this kinase in mediating activation of JNK/p38 by PGF2 was therefore investigated.
Kinetic analysis indicated that PGF2 induced a rapid translocation of PKD from the cytosol
to a membrane fraction wherein it became phosphorylated on several serine residues (p-PKD)
before returning in the cytosol. Interestingly, the return of p-PKD in the cytosol
correlated with activation of JNK/p38 by PGF2 and this return was blocked by Go6976.
Moreover, in E1 cells stably transfected with a kinase dead PKD mutant (K612W) and having
a normal growth, activation of JNK/p38 by PGF2 was impaired (80-90% inhibition).
In conclusion, GqPCRs in osteoblast-like cells can stimulate the three
MAPKs Erk, JNK and p38 by different mechanisms. Erk activation is mediated by a typical
PKC isoform whereas the newly described PKD mediates activation of JNK and p38. The
molecular mechanism by which PKD activates JNK and p38 is complex. Indeed, it involves the
translocation of PKD from the cytosol to a membrane fraction for its phosphorylation and
activation before returning in the cytosol for the stimulation of the JNK and p38
pathways.
[Programme]
O-19
FIBROBLAST GROWTH FACTOR-2 INHIBITS APOPTOSIS IN HUMAN CALVARIA
OSTEOBLASTS THROUGH A PKC- DEPENDENT PATHWAY
F. Debiais, E. Hay, P. J. Marie*
INSERM U349 Affiliated CNRS, Paris, France
We previously showed that Fibroblast Growth Factor-2 (FGF-2) controls
osteoblast human calvaria cells through protein kinase C (PKC) and src signalling
pathways. In this study, we determined the effects of FGF-2 on apoptosis in human calvaria
osteoblasts and we analysed the underlying signaling pathways. Apoptosis was determined in
normal human calvaria osteoblasts and in immortalized human neonatal calvaria (IHNC) cells
by terminal deoxynucleotidyl transferase-mediated nick-end labeling (TUNEL) or trypan blue
staining. Treatment with rhFGF-2 (50 ng/ml) for 96 h increased the number of
TUNEL-positive cells in primary human calvaria osteoblasts cultured in serum-deprived
medium (P<0.02). In contrast, rhFGF-2 decreased the number of apoptotic cells at early
time-points, from 6 to 24 h of treatment (P<0.05). A similar dual effect of FGF-2 on
apoptosis was found in IHNC cells. The analysis of mechanisms involved in this dual effect
showed that rhFGF-2 (50 ng/ml) decreased caspase-2 activity at 15-48 h and increased this
activity at 96 h in serum-deprived IHNC cells (P<0.05). Consequently, rhFGF-2
significantly reduced effector caspase (-3,-6,-7) activity at 15-48 h and increased this
activity at 96 h (P<0.05). In contrast, rhFGF-2 did not affect caspase-8 and caspase-9
activities in IHNC cells at any time point. This shows that FGF-2 induced a biphasic
effect on apoptosis in human osteoblasts by modulating caspase-2 activity. We then
analysed the signaling pathway involved in the anti-apoptotic effect of FGF-2. Treatment
of IHNC cells with the PKC inhibitor calphostin C (1 µM) abolished the inhibitory effect
of rhFGF-2 on both caspase-2 and caspase-3 activities at 24 h (P<0.05). In contrast,
the MAP kinases inhibitors PD 98059 and SB 203580, that inhibit Erk1,2, and p38
respectively, did not suppress the inhibitory effect of FGF-2 on caspase-2 and caspase-3
activities. These results show that 1) FGF-2 induces a dual effect on apoptosis in human
osteoblasts through a selective modulation of caspase-2 activity which regulates effector
caspases and DNA fragmentation, and 2) the early anti-apoptotic effect of FGF-2 on human
calvaria osteoblasts is mediated by PKC signaling. This identifies a novel PKC-dependent
anti-apoptotic effect of FGF-2 in human osteoblasts.
[Programme]
O-20
INHIBITION OF OSTEOBLAST APOPTOSIS BY THROMBIN
C. N. Pagel, C. Chinni, L. A. Abraham, E. J. Mackie*
School of Veterinary Science, University of Melbourne, Parkville,
Australia
Maintenance of the skeleton is critically dependent on the availability
of osteoblasts, which itself is determined by rates of osteoblast proliferation and
apoptosis. The recent observation that steroid-induced osteoporosis is associated with
osteoblast apoptosis has highlighted the importance of understanding the factors
regulating this process. We demonstrated recently that thrombin is a survival factor for
skeletal myoblasts, and instigated the current study to determine whether thrombin also
inhibits osteoblast apoptosis. Primary cultures of neonatal rat calvarial osteoblasts and
human osteoblast-like (Saos-2) cells were deprived of serum for 24 h, then incubated in
the presence or absence of thrombin for 36 h. The percentage of cells undergoing apoptosis
was quantitated on the basis of nuclear morphology and the presence of DNA strand breaks
(detected by the TUNEL method). In both cell types, thrombin caused a significant
reduction in the proportion of cells undergoing apoptosis. The effect was
concentration-dependent with a maximal effect (>75% reduction below control levels) at
100 nM. The effect was dependent on thrombin's proteolytic activity, but could not be
mimicked by urokinase or factor Xa. Three members of the protease-activated receptor (PAR)
family, PARs-1, -3 and -4, have been identified as thrombin receptors. Although PAR-1 is
known to be expressed by osteoblasts, thrombin's effect was not mimicked by a peptide
activator of PAR-1. In osteoblasts from PAR-1-null mice, thrombin inhibited apoptosis as
effectively as in osteoblasts from wildtype mice. In RT-PCR studies, Saos-2 cells were
shown to express PAR-3 but not PAR-4, whereas primary osteoblast-like cells were shown to
express PAR-4 but not PAR-3. Serum-deprived Saos-2 cells were treated with thrombin for 1
h, then the medium was replaced with fresh medium (without thrombin) which was collected
20 h later. This conditioned medium when applied to serum-deprived osteoblasts caused a
significant inhibition of apoptosis. Medium from cells treated concurrently with thrombin
and cycloheximide contained no thrombin-induced apoptosis inhibitory activity. These
results indicate that thrombin inhibits osteoblast apoptosis through induction of a
secreted inhibitor of apoptosis. The receptor mediating thrombin's effect has not yet been
identified, but is clearly not PAR-1.
[Programme]
O-21
COEXPRESSION OF BSP AND CBFA1/RUNX2 IN HUMAN MELANOMA: AN INCOMPLETE
EPITHELIAL TO OSTEOGENIC CONVERSION AS THE LINK BETWEEN BSP EXPRESSION AND TUMOR
INVASIVENESS?
M. Riminucci1,2*, A. Corsi1,2, K. Peris3,
L. W. Fisher4, S. Chimenti5, S. Licci1, P. Bianco1
1Department of Experimental Medicine and Pathology, La
Sapienza University, Rome, Italy
2Department of Experimental Medicine, Laboratory of Pathology,
Univeristy of L'Aquila, Italy
3Department of Biomedical Sciences, Division of Dermatology,
University of L'Aquila, Italy
4Craniofacial and Skeletal Diseases Branch, National Institute
of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD, USA
5Department of Dermatology, Tor Vergata University, Rome,
Italy
Ectopic expression of the osteoblast-specific protein Bone Sialoprotein
(BSP) occurs in some malignant epithelial tumors capable of metastatic growth in the
skeleton. In breast and prostate cancer,production of BSP has also been positively
correlated with tumor progression. For this reason, the expression of BSP as a potential
predictive marker is under intensive investigation in early, localized malignant tumors.
However, the biological significance of BSP expression in cancer and its underlying
mechanisms have remained elusive.
We analyzed the expression of BSP in 17 human melanocytic tumors in vivo
by immunolocalization. The expression of BSP was detected in 5/5 Clark's level IV
melanomas and related lymph node metastasis and in one level III lesion with histological
evidence of vascular invasion. By contrast, neither 2 level I nor 1 level II and 1 level
III (without vascular invasion) melanomas expressed BSP. In cultures of two human melanoma
cell lines (MeWo, SK) the pattern of BSP localization was determined by confocal
immunofluorescence and compared to that observed in a rat osteosarcoma cell line (UMR
106-01-BSP) and in normal primary rat osteoblats. In all systems, BSP was immunolocalized
to the Golgi apparatus and to secretory vesicles. By contrast, BSP-labelled focal
adhesions were obvious in primary osteoblasts and absent both in osteosarcoma and melanoma
cells. BSP mRNA was demonstrated in both melanoma cell lines by RT-PCR. These data
demonstrated that human melanoma cells express BSP mRNA and protein as a reflection of an
invasive behavior both in vivo and in vitro.
We then showed that in both melanoma cell lines BSP expression was
associated with the expression of CBFA1/Runx2, a pivotal transcriptional regulator of
osteoblast - specific genes. This suggests that the expression of BSP in melanoma (and
perhaps in other epithelial cancers) may represent an incomplete 'epithelial - to -
osteogenic conversion' dictated by the ectopic expression of the CBFA1/Runx2 gene. Since
CBFA1/Runx2 is involved in the transcriptional regulation of some matrix degrading enzymes
involved in tumor invasion, the production of BSP (and other bone matrix proteins) by some
tumors, and its unexplained link to tumor invasiveness would then be traced to a common
regulatory mechanism.
[Programme]
O-22
PROTEIN KINASE C PLAYS AN ESSENTIAL ROLE IN THE REGULATION OF HUMAN
CALVARIA OSTEOBLAST PHENOTYPE BY FGF-2, FGFR-2 AND BMP-2
P. J. Marie*, F. Debiais, J. Lemonnier, Ph Delannoy, E. Hay
INSERM U349 affiliated CNRS, Paris, France
The formation of cranial bone requires the differentiation of osteoblasts
from indifferentiated mesenchymal cells. The balance between osteoblast recruitment,
proliferation, differentiation and apoptosis is essential for calvarial bone formation.
However, the mechanisms controlling the human osteoblast phenotype during normal calvarial
bone formation are poorly understood. Using human calvarial osteoblasts, we determined the
mechanisms that regulate the differentiated osteoblast phenotype in human calvaria. We
first showed that Fibroblast Growth Factor-2 (FGF-2) promotes osteoblast differentiation
in human calvaria osteoblasts. Consistently, we found that constitutive activation of FGF
receptor-2 (FGFR-2) activates the expression of alkaline phosphatase (ALP), type I
collagen and osteocalcin gene expression in human calvaria cells. Using specific PKC
inhibitors, we showed that this effect is mediated mainly by activation of protein kinase
C (PKC) by FGFR-2 signaling. Bone Morphogenetic Protein-2 (BMP-2) is another factor
involved in cranial bone development. We found that BMP-2 activates PKC activity and
osteoblast gene expression in human calvaria cells. In addition, we showed that direct
activation of PKC by phorbol ester promotes ALP activity, a marker of the osteoblast
phenotype, in human calvaria cells. Finally, we investigated the implication of PKC in
osteoblast apoptosis, a phenomenon which plays an important role in bone formation. We
found that both FGFR-2 activation and BMP-2 increased apoptosis in human calvaria
osteoblasts. Moreover, functional studies in human calvaria osteoblasts showed that
apoptosis induced by both effectors is mediated by PKC activity, leading to the downstream
activation of specific caspases. These studies indicate that PKC is an essential signaling
molecule involved in the regulation of the human calvaria osteoblast phenotype by FGF-2,
FGFR-2 and BMP-2.
[Programme]
O-23
MUTATIONS IN TRANSFORMING GROWTH FACTOR-BETA1 CAUSE CAMURATI-ENGELMANN
DISEASE BY AFFECTING EITHER SECRETION OR ACTIVATION OF THE PROTEIN
K. Janssens1*, S. H. Ralston2, W. Van Hul1
1Center of Medical Genetics, University of Antwerp, Antwerp,
Belgium
2Department of Medicine and Therapeutics, University of
Aberdeen Medical School, Aberdeen, UK
Camurati-Engelmann disease (CED) or Progressive Diaphyseal Dysplasia is a
rare bone disorder, inherited in an autosomal dominant mode. Its main clinical symptoms
are a waddling gait, pain in the lower and upper limbs, fatigue and reduced muscle mass.
Radiologically, CED is characterised by hyperostosis of the diaphyses and metaphyses of
the long bones. Sclerosis can also be seen at the base of the skull.
In 2000, the gene responsible for CED was located on the chromosomal
region 19q13.2 and shortly afterwards, the gene was identified as being TGFB1. So far, 7
mutations have been reported in 17 CED families. With only one exception - a small
duplication in the signal peptide - all mutations are missense mutations, located in the
latency associated peptide (LAP), which confers latency to the mature TGF-beta1.
In order to unravel the underlying mechanism, we performed several
functional studies using conditioned medium and/or lysates of cells transfected with
overexpression constructs of control and mutant TGFB1.
ELISA of conditioned media showed a marked difference between controls
and CED-patients. Patients with missense mutation R218C or C225R secrete a normal amount
of TGF-beta1, but have a two- to threefold higher amount of active TGF-beta1; the
mutations in the LAP apparently influence the ease with which the mature TGF-beta1 is
being activated. The duplication in the signal peptide on the contrary decreases TGF-beta1
secretion. ELISA of lysates shows that this diminished secretion is reflected by a
retention of TGF-beta1 intracellularly. The results of the ELISA were confirmed with a
luciferase reporter assay. The diminished secretion and increased retention of TGF-beta1
by the duplication in the signal peptide could also be confirmed by Western blotting.
In conclusion, we were able to illustrate that the mutations found in
CED-patients have a profound effect on the function of TGF-beta1, by affecting either the
secretion or the activation of the protein. Further experiments are however necessary to
reveal how mutations with an apparently opposing effect can give rise to the same
disorder.
[Programme]
O-24
MUTATED SUBUNIT A3 OF VACUOLAR PROTON PUMP IN PATIENTS WITH AUTOSOMAL
RECESSIVE OSTEOPETROSIS: GENOTYPE-PHENOTYPE CORRELATION
A. Taranta1*, A. Ricci1, S. Migliaccio1,
I. Recchia1, G. De Rossi2, E. Lanino3, S. H. Ralston4,
A. Villa5, P. Vezzoni5, A. Teti1
1Department of Experimental Medicine, University of L'Aquila,
Italy
2Bambino Gesù Hospital, Rome, Italy
3Gaslini Hospital, Genoa, Italy
4Dept. of Medicine and Therapeutics, University of Aberdeen,
Scotland
5Istituto di Tecnologìe Biomediche Avanzate, Consiglio
Nazionale delle Ricerche, Segrate, Italy
Autosomal recessive osteopetrosis (arOP) is a severe genetic disease
characterised by dense and fragile bones due to impaired osteoclast bone resorption.
Genotyping of arOP patients analysed so far has allowed the identification of ATP6i gene
mutation in 50% of the cases. This gene encodes the osteoclast specific a3 V-ATPase
subunit which contributes to proton transport and acidification of the osteoclast
resorbing compartment. The aim of our study was to identify genotype-phenotype
relationship in our ATP6i-dependent arOP patients. Clinical follow-up showed diffuse
osteosclerosis and endobone appearance, macro- and hydro-cephaly, pale optic nerve
papillae, anaemia, low neutrophil counts, elevated alkaline phosphatase and, remarkably
high PTH (parathyroid hormone) levels. The iliac crest biopsies were characterized by
osteosclerotic pattern, massive and irregular bone trabeculae and unresorbed mineralized
cartilage. Excess of normally polarised osteoclasts and scarce haematopoietic tissue,
against a background of diffuse fibrosis, were observed. Osteoclasts obtained from
peripheral blood monocytes showed no morphological differences relative to normal
osteoclasts and their TRAP (tartrate-resistent acid phosphatase) activity was several fold
higher than average. No immunostain was observed with an antibody raised against peptide
811-829 localized in the C-terminus of the a3 subunit, downstream of the mutation. These
osteoclasts resorbed bone, but their lacunae were very shallow. In contrast to other forms
of OP, cytoskeletal arrangement, podosome pattern and alphaVbeta3 integrin distribution
were unremarkable. Pyk2 and c-src appeared normally distributed at the paramarginal area
where these tyrosonine kinases contributed to integrin-mediated osteoclast adhesion and
outside-in signalling.
In conclusion, this is the first genotype-phenotype correlation in
ATP6i-dependent osteopetrosis, and shows that mutations of the ATP6i gene increases TRAP
activity but do not affect osteoclast morphology and adhesion structures, and that bone
resorption is severely impaired but not completely abolished. We hypothesize that the
elevated serum PTH levels found in these patients may likely account, at least in part,
for the high numbers of osteoclasts observed in bone biopsies.
[Programme]
O-25
ALBERS-SCHÖNBERG DISEASE (AUTOSOMAL DOMINANT OSTEOPETROSIS, TYPE II) IS
CAUSED BY MUTATIONS IN THE CLCN7 CHLORIDE CHANNEL GENE
E. Cleiren1, O. Bénichou2, E. Van Hul1,
J. Gram3, J. Bollerslev4, F. R. Singer5, K. Beaverson6,
M. P. Whyte7, M. C. de Vernejoul2, W. Van Hul1*
1Dept Medical Genetics, University of Antwerp, Belgium
2Laboratoire INSERM U 349, Hôpital Lariboisière, Paris,
France
3Dept. Medicine, Ribe County Hospital, Esbjerg, Denmark
4Dept. Endocrinology, Rikshospitalet, Oslo, Norway
5John Wayne Cancer Institute, Santa Monica, CA, USA
6Weill Medical College of Cornell University, New York, NY,
USA
7Center for Metabolic Bone Disease and Molecular Research,
Shriners Hospitals for Children, St. Louis, MO, USA
Albers-Schönberg disease, or autosomal dominant osteopetrosis, type II
(ADO II), is the most common form of osteopetrosis, a group of conditions characterised by
an increased skeletal mass due to impaired bone and cartilage resorption. However, due to
its relatively benign clinical picture, with many patients being asymptomatic and only
detected by coincidental radiographic examination, the prevalence of ADO is
underestimated. Among families with ADO, two subtypes are generally reported based
primarily on radiographic features. Type I (ADOI) features a generalised, diffuse
osteosclerosis affecting especially the cranial vault. Clinical manifestations of ADO II
include non-traumatic fractures, especially of long bones, cranial nerve palsies,
osteoarthritis of the hip and mandibular osteomyelitis. ADO II manifests radiographically
with a segmentary osteosclerosis, predominately at the vertebral endplates ('rugger jersey
spine'), iliac wings ('bone within bone' sign), and skull base.
Recently, a genome wide search led us to assign a gene underlying ADO II
to chromosome 16p13.3. Following this gene assignment, we now report seven different
mutations in the gene encoding the ClCN7 chloride channel in all 12 ADO II families
analysed. This chloride channel is essential for the acidification of the extracellular
resorption lacuna necessary for the osteoclast-mediated degradation of bone tissue.
Additionally, a patient with the severe, autosomal recessive, infantile form of
osteopetrosis (ARO) was identified as being homozygous for a ClCN7 mutation. From
genotype-phenotype correlations, it seems that ADO II reflects a dominant negative effect,
whereas loss-of-function mutations in ClCN7 do not cause abnormalities in heterozygous
individuals. Because some ARO patients have mutations in both copies of the ClCN7 gene,
ADO II is allelic with a subset of ARO cases.
[Programme]
O-26
HARDNESS AND COMPOSITION OF CANCELLOUS BONE IN OSTEOPOROSIS AND
OSTEOARTHRITIS
A. M. Coats1*, P. Zioupos2, R. M. Aspden3
1Department of Chemistry, University of Aberdeen, Meston Walk,
Aberdeen AB24 3UE, UK
2Materials and Medical Sciences, Cranfield University,
Shrivenham SN6 8LA, UK
3Orthopaedic Surgery, University of Aberdeen, Polworth
Building, Foresterhill, Aberdeen AB25 2ZD, UK
Cancellous bone from patients with osteoarthritis (OA) has a reduced
material density and appears to be undermineralized. It is hypothesized that this will
result in a reduction in the mechanical stiffness and strength of the bone matrix. In this
study, bone was obtained from superior and inferior sites, subjected to relatively high
and low loads respectively, from human femoral heads retrieved after surgery for
osteoporotic hip fracture (OP) or for hip arthroplasty due to osteoarthritis (OA).
Microindentation testing and electron probe microanalysis (EPMA) were used to measure the
hardness and the elemental composition of cancellous bone from immediately adjacent
microscopic sites at various depths from the subchondral bone plate. Overall, OA bone was
found to have hardness values that were 7% lower than those from OP bone. Bone from the
inferior site was harder than that from the superior in both diseases. There was no
variation with depth below the subchondral plate and no difference between sexes. Using
EPMA, only the Ca/P ratio was significantly different between disease groups (OA: 1.715,
OP: 1.694, P=0.015) and no correlation was found between hardness and any of the
composition measurements. Though only an indirect measurement of stiffness, the reduction
in hardness values support the hypothesis that OA bone would have reduced mechanical
properties.
[Programme]
O-27
COLIA1 SP1 BINDING SITE POLYMORPHISM PREDISPOSES TO OSTEOPOROTIC
FRACTURES BY ALTERING COLLAGEN PRODUCTION AND IMPAIRING BONE MINERALISATION
T. L. Stewart1*, P. Roschger2, B. Grabner2,
P. Fratzl3, V. Mann1, R. M. Aspden1, K. Klaushofer2,
S. H. Ralston1
1Bone Research Group, University of Aberdeen, UK
2Ludwig Boltzman Institute of Osteology, Vienna, Austria
3Erich Schmid Institute of Material Science, Leoben, Austria
A polymorphic Sp1 binding site has been identified in the COL1A1 gene
which predicts the risk of osteoporotic fractures, independent of differences in bone
mineral density. In this study, we examined the functional mechanisms responsible for this
observation. Ex-vivo mechanical testing of bone cores from patients of different genotype
showed reduced yield strength (adjusted for bone density) in G/T heterozygotes (n=7) when
compared with G/G homozygotes (n=10) (4.60±SD 0.3 MPa vs 3.55±SD 0.2 MPa; p=0.034).
Further analysis, using quantitative backscattered electron imaging (qBEI), showed reduced
mineralisation of bone in G/T heterozygotes (n=7) compared with G/G homozygotes (n=6)
(20.2±SD 1.2 % vs 21.5±SD 0.5 %; p=0.049) and increased heterogeneity of mineralisation,
as reflected by broadening of the bone mineralisation density distribution peak (4.5±SD
0.5 % vs 3.5±SD 0.5 % p=0.038). Further studies have used an in-vitro model of bone
mineralisation, in which calcified bone nodules were generated from long term cultures of
primary human osteoblasts from patients of different genotype, and calcium deposition was
quantified by Alizarin Red S staining. Cultures from G/T heterozygotes produced an
abnormally increased ratio of the collagen type I alpha (1) chain to the alpha I (2) chain
(G/T=2.3:1.0) in vitro, compared with G/G homozygotes (1.99:1.0; p=0.007), and formed
significantly less mineralised bone nodules than G/G homozygotes (mean±SD 1650 + 140 vs
690±SD 130 microM Alizarin Red S /105 cells; p<0.0001), even though total
cell number, collagen production and alkaline phosphatase activity were similar in both
genotype groups. Our data indicate that the COLIA1 'T' allele influences the ratio, but
not the total amount of collagen type I alpha chains produced by bone cells, leading to
abnormal mineralisation of bone both in vivo and in vitro and reduced bone strength. The
studies define a novel genetic mechanism of osteoporosis which predisposes to fracture by
affecting bone quality rather than bone quantity.
[Programme]
O-28
INTERACTION BETWEEN POLYMORPHISMS IN THE METHYLENETETRAHYDROFOLATE
REDUCTASE (MTHFR) GENE AND THE COLLAGEN TYPE I ALPHA 1 (COLIA1) GENE IN BONE MINERAL
DENSITY AND FRACTURE RISK
J. B. J. van Meurs1*, P. Arp1, M. van der Klift2,
A. Hofman2, H. A. P. Pols1,2, A. G. Uitterlinden1,2
1Department of Internal Medicine, Erasmus University Centre,
Rotterdam, The Netherlands
2Department of Epidemiology and Biostatistics, Erasmus
University Centre, Rotterdam, The Netherlands
Homocystinuria, characterised by high levels of homocysteine, is
associated with osteoporosis, possibly due to interference of homocysteine with collagen
metabolism. We found earlier that mildly elevated levels of serum homocysteine are
associated with increased risk for osteoporotic fractures. We here examined possible
genetic influences on this relation. We studied a functional variant of MTHFR (C677T), an
essential enzyme in homocysteine metabolism, and interaction between this variant and the
Sp1 G/T polymorphism in the COLIA1 gene in a population-based sample of 1532
postmenopausal women in relation to BMD and incident non-vertebral fractures.
Homozygote MTHFR 677T-carriers had a 2.5% lower BMD (p=0.01) than
homozygote 677C-carriers, but no association was found of this polymorphism with fracture
risk.
To study interaction between MTHFR and COLIA1 polymorphisms, subjects
were divided our population into four groups based on presence of risk allele(s) (see
Table).
Number of subjects |
Presence of risk
allele |
BMD |
fracture risk |
|
MTHFR |
COLIA1 |
FN |
LS |
OR [95%CI] |
461 |
- |
- |
0.83 |
1.04 |
1.0 |
576 |
+ |
- |
0.81 |
1.02 |
1.2 [0.8-1.8] |
214 |
- |
+ |
0.81 |
1.03 |
1.3 [0.8-2.2] |
281 |
+ |
+ |
0.79 |
0.99 |
1.9 [1.2-2.9] |
BMD measures and fracture risk
expressed as odds ratios (OR) according to combined MTHFR and COLIA1 genotype, 95% CI=95%
confidence intervals, FN=femoral neck, LS=lumbar spine. |
Compared with the [--] group, the [++] group had on average a 5% lower BMD at the femoral
neck and lumbar spine (p<0.001). This effect was larger then the effect of MTHFR or
COL1A1 alone. The BMD-differences between [++] and [--] showed an age-dependent trend
(p=0.02) with larger differences between the extreme groups at older ages (>70 years).
Women with the [++] genotype had an almost 2 times increased fracture risk, which was
significant and larger then the effects of the [+-] and [-+] groups alone. The risk in the
[++] group increased further to 2.5 (95% CI: 1.5-4.2) for older women, and this risk was
largely independent of BMD differences.
We conclude that the combination of COLIA1 and MTHFR polymorphisms
predisposes women to osteoporotic fractures. This risk is only partly explained by
differences in bone mineral density, suggesting that factors, other then those reflected
by BMD, determine the increased fracture risk.
[Programme]
O-29
ASSOCIATION STUDY OF THE CDX-2 POLYMORPHISM IN THE 1A PROMOTER REGION OF
THE HUMAN VITAMIN D RECEPTOR GENE AND FRACTURE
Y. Fang1*, J. B. J. van Meurs1, A. Hofman2,
H. P. T. M. van Leeuwen1, C. M. van Duijn2, H. A. P. Pols1,2,
A. G. Uitterlinden1,2
1Department of Internal Medicine, Erasmus University Medical
Center, Rotterdam, The Netherlands
2Department of epidemiology, Erasmus University Medical
Center, Rotterdam, The Netherlands
Polymorphisms of the human Vitamin D Receptor (hVDR) gene are reported to
be associated with several clinical endpoints including osteoporosis. Recently, a single
nucleotide polymorphism (SNP) was discovered in the 1a promoter region of the VDR gene.
This G to A substitution in a binding site of the intestinal- specific transcription
factor Cdx-2, was found to modulate the transcription of hVDR gene and to be associated
with decreased BMD in a small group of postmenopausal Japanese women. We here present a
comparison of the frequency of the A allele in the three major human races, and observed a
negative correlation between prevalence of the A allele and hip fracture incidence in
these ethnic groups, suggesting a protective effect of this allele on fracture risk. To
study this putative relation in more detail, we performed an association study of this SNP
in a large cohort of Dutch Caucasian elderly. First, we developed an allele-specific
multiplex PCR test and then determined the Cdx-2 genotype in 2447 men and women aged 55
year or older. We found the frequency of the G- and the A-allele to be 81% and 19%,
respectively. The A allele had a protective effect on occurrence of osteoporotic
fractures, especially for non-vertebral fracture in women (OR of AA vs. GG genotype is
0.2, 95%CI is 0.05-0.8). This effect did not vary by adjustment for age, weight and bone
minal density (BMD). We conclude that the A allele of the VDR Cdx-2 polymorphism is
present in Caucasians, albeit at low frequency. We demonstrate a protective effect of this
allele on non-vertebral fracture, which is not explained by differences in BMD in this
population.
[Programme]
O-30
POLYMORPHISMS OF THE TNFR2 GENE ARE ASSOCIATED WITH BONE MINERAL DENSITY
IN WOMEN FROM THE UK
O. M. E. Albagha*, P. N. Tasker, F. E. A. Mcguigan, D. M. Reid, S. H.
Ralston
Dept of Medicine and Therapeutics, University of Aberdeen, Aberdeen, UK
Genetic factors are important in the pathogenesis of osteoporosis and
linkage analysis has identified a candidate locus for hip bone mass on chromosome 1p36
(Devoto et al. Eur J Hum Genet 1998). One of the positional candidate genes within this
region is tumour necrosis factor receptor 2 (TNFR2). We investigated the relationship
between TNFR2 gene polymorphisms and bone mineral density in 1246 perimenopausal women
randomly selected from the local population in Scotland. A single nucleotide polymorphism
(SNP) located in exon 6 along with 3 SNPs located in the 3'UTR were studied. The exon 6
genotypes were determined by standard PCR based methods. The 3'UTR polymorphisms, which
are located at nucleotides (nt.) 593, 598, and 620 of the TNFR2 gene (Genbank Accession
U52165) were typed by DNA sequencing of PCR products spanning the region of interest.
Genotype frequencies were similar to those previously reported and we found that women
with the genotype A-T-C (corresponding to nt593-A/A, nt598-T/T, and nt620-C/C) had
significantly lower femoral neck (FN) BMD (mean Z-score±sem=-0.441±0.084; n=86) than
those without this genotype (0.050±0.031; n=1160; p-value<0.00001). A similar trend
was observed for LS-BMD but it did not reach statistical significance (p-value=0.39). The
A-T-C haplotype also significantly predicted FN BMD (p-value<0.0001) in a multiple
linear regression model which included other possible predictors of BMD (such as age,
height, weight, years since menopause, and duration of HRT use) with evidence for a gene
dose effect. No association was found between exon 6 polymorphism and BMD in the
population studied. In summary, we have confirmed that allelic variation in the 3'UTR of
the TNFR2 gene is associated with BMD but in contrast with previous work we found an
association between hip rather than LS- BMD. These data suggest that the 3'UTR
polymorphisms are strongly related to FN-BMD, however further studies are required to
investigate whether the 3'UTR polymorphisms directly influence the TNFR2 gene function or
whether they are in linkage disequilibrium with causal polymorphisms elsewhere in the
TNFR2 gene or in other genes nearby.
[Programme]
O-31
CONGENITAL PSEUDARTHROSIS OF THE TIBIA: TREATMENT WITH A BONE
MORPHOGENETIC PROTEIN (OP-1 DEVICE)
D. Anticevic1*, Z. Stanec2, M. Jelic1,
S. Vukicevic3
1Department of Orthopedic Surgery, Medical School, Zagreb,
Croatia
2Department of Plastic Surgery, Medical School, Zagreb,
Croatia
3Department of Anatomy, Medical School, Zagreb, Croatia
PURPOSE: To evaluate locally administered osteogenic protein-1 device
(OP-1, bone morphogenetic protein-7, BMP-7) on bone regeneration in congenital
pseudarthrosis of the tibia (CPT).
METHODS (Case report): A six-year old girl with Type III (Crawford)
cystic CPT anteriorly angulated for 80 degrees and two inch limb shortening was initially
treated with resection of pseudarthrotic and cystic bony lesion. A free vascularized
fibular 1.5 inch long graft was then transferred into the defect area. The proximal and
distal tibial ends were cut in a step-like fashion to increase contact area with the
graft. OP-1 device was carefully placed on the contact area between the tibia and the
fibula graft. The bones were fixed with an intramedullary rod and the Ilizarov device that
were removed after three and six months, respectively. Gradual weight bearing started
three months after surgery and the leg protection with bivalved splint started six months
after surgery.
RESULTS: Three weeks following surgery a significant amount of new bone
was observed along the graft and at both proximal and distal contact surfaces. At four
months the proximal part of the graft was fully incorporated with the tibia while the
contact area between the tibia and fibula at the distal end was significantly smaller. At
seven months following surgery both proximal cortices and medullary canal between the
tibial and fibular graft were fused and already remodeled, while distally a much wider
contact area between the graft and the tibia was formed. One year following surgery lower
limb was one and a half-inch shorter. No valgus ankle deformity was detected.
CONCLUSION: The time to bone union was four months on the proximal and
seven months on the distal graft contact surface. In patients with CPT adjuvant local
application of OP-1 could thus significantly stimulate the tibial bone union and restore a
fully weight-bearing lower limb.
SIGNIFICANCE: The first child with highly angulated CPT was treated
successfully with a recombinant bone morphogenetic protein to enhance bone regeneration
and repair.
[Programme]
O-32
OSTEOPETROSIS WITH SPARSE HAIR AND DENTAL DYSPLASIA - AN UNUSUAL
PRESENTATION CASE REPORT
I. Baric1*, V. Kusec2, V. Sarnavka3, M.
Cuk1, D. Krpan4, S. Mutar-Susic5, I. Skrinjaric5,
D. Begovic1
1Department of Pediatrics, Clinical Hospital Centre, Zagreb,
Croatia
2Clinical Institute of Laboratory Diagnosis, Clinical Hospital
Centre, Zagreb, Croatia
3Department of Dermatology, Clinical Hospital Centre, Zagreb,
Croatia
4General Hospital 'Sveti Duh', Zagreb, Croatia
5School of Dentistry, Zagreb, Croatia
Increased bone density was diagnosed in the age of 2 years after
observing radiodense appearance of skull X-ray. Other clinical features of the 6 year old
girl include: radiodense appearance of almost the entire skeleton, dental dysplasia,
compression of cranial nerves, occasional seizures, brittle and sparse hair, absence of
eyebrows and eyelashes. No fracture has occurred so far despite her lively behaviour. No
abnormalities of the internal organs or nail dysplasia are present. No haematological
abnormalities or impairment of haematopoiesis have developed. Measurements of markers of
bone turnover indicate increased bone formation and resorption, most probably appropriate
for age and growing skeleton. Densitometry performed at 3 and 5 years indicated a dramatic
increase in bone density Z-score being plus 2.3 and 14.4, respectively. Bone biopsy at the
iliac crest revealed increased bone volume (93 percent), almost resembling cortical bone
but without cartilage residues. Bone marrow spaces were grossly reduced and contained no
haematopoiesis. Bone formation was increased. ATP6i gene mutation was not confirmed in
this girl. No treatment has been attempted so far and her general condition has not
changed considerably. Despite all efforts a definite diagnosis has not been established.
[Programme]
O-33
OSTEOPETROSIS - PLAIN FILM RADIOGRAPHS VS MRI - CASE REPORT OF ADOP II
TYPE
P. Marusic1*, L. J. Vojnic1, H. Landeka2,
B. Brkljacic1
1Department of Radiology, University Hospital 'Dubrava',
Zagreb, Croatia
2Department of Neurology,University Hospital 'Dubrava',
Zagreb, Croatia
Osteopetrosis (marble bones, Albers-Schenborg disease) is a group of
inherited bone disorders characterized radiographically by variable generalized symetrical
skeletal sclerosis due to defective osteoclast function. Autosomal dominant (benign)
osteopetrosis - ADOP may be asymptomatic and discovered incidentally on plain radiographs.
On the basis of radiographic criteria ADOP has been differentited in two types. Type I is
characterized by sclerotic thickening of the calvarium, most prominent in the vault, and
litle sclerosis of the spine. Type II is characterized by sclerotic thickening of the base
of the skull while the calvarium is nearly normal. The other radiographic findings include
hyperostotic vertebral endplates (sandwich sign, 'rugger-jersey spine') and endobones in
other bones.
We would like to present a case of a 75-year-old woman who was admitted
to the hospital, for the first time, because of difficult walking, and pain in her left
leg and lumbar region.
Plain film of the cranium revealed sclerotic thickening of the basis of
the skull and obliteration of the mastoid air cells. The calvarium was normal. Computed
tomography of the brain confirmed sclerotic areas on the basis of the skull. Radiographs
of the lumbar spine revealed narrowing of the L4/L5 and L5/S1 intervertebral space and
impressive endplates sclerotic thickening of the vertebraes' bodies (sandwich sign). MRI
of this region revealed disc protrusion at L4/L5 and L5/S1 with decreased bone marrow
signal intensity. Plain film of the foot revealed sclerotic changes of the all tarsal
bones with forming endobones (bone within bone sign) and scattared increase in bone
density of the metetarsal bones. MRI of this region also revealed 'bone within bone sign'
especially in calcaneus, talus and cuboid bone.
The aim is to compare plain films of the spine and foot with MRI of these
regions. The conclusion is that plain film is better in showing the changes of the
vertebraes' bodies and MRI of this region should be used only when patient has spinal cord
symptoms. MRI gives better results in examination of the foot compared to the plain films
because of multiplanar projections used in MRI analysis.
[Programme]
O-34
NEUROENDOCRINE TUMOR CAUSING FGF23-INDEPENDENT ONCOGENIC HYPOPHOSPHATEMIC
OSTEOMALACIA
F. Paglia1*, A. Corsi2,3, S. Minisola1,
S. Dionisi1, S. De Geronimo1, S. Licci2, A. Marzullo2,
F. Malchiodi4, M. Riminucci2,3, P. Bianco2
1Department of Clinical Science, La Sapienza University, Rome,
Italy
2Department of Eperimental Medicine and Pathology, La Sapienza
University, Rome, Italy
3Department of Experimental Medicine, Laboratory of Pathology,
Univeristy of L'Aquila, Italy
4Laboratory of Ultrastructure, ISS, Rome, Italy
Oncogenic Hypophosphatemic Osteomalacia (OHO) is a paraneoplastic
syndrome characterized by osteomalacia, muscle weakness, phosphate wasting,
hypophosphatemia and low 1,25-dihydroxyvitamin D3 (1,25-D3). We describe a 65 years-old
woman with OHO due to a soft tissue tumor diagnosed 16 years after the onset of symptoms.
The patient presented at the age of 49 with weakness and back pain. Spontaneous fractures
of pelvis and right hip developed in the following three years. Biochemical abnormalities
included hypophosphatemia and low tubular maximum transport of phosphate. Iliac crest
biopsy revealed osteomalacia as the basis for the tendency to fractures. The patient was
periodically treated with calcium carbonate, vitamin D and phosphate without improvement.
During the following years, the patient underwent other spontaneous fractures of left hip,
humeri and vertebras until immobilisation. Biochemical studies performed at our hospital
revealed hypophosphatemia, increased total and bone-specific alkaline phosphatase, ionised
calcium and intact parathyroid hormone, low 1,25-D3 and a selective defect of tubular
phosphate resorption. Although total body computed tomography (CT) failed to demonstrate
any focal lesion, an increased uptake was detected at the left groin by whole-body
Indium-111 octreotide scintigraphy. A spiral CT scan performed specifically at this site
revealed a soft tissue mass 4 cm in largest dimension. The excision of the mass improved
clinical symptoms and biochemical values, even though serum calcium and parathyroid
hormone were still high. Histology of the mass was consistent with the mixed connective
tissue variant of phosphaturic mesenchymal tumor. Extensive abnormal mineral deposition,
shown by electron probe microanalysis to consist of calcium phosphate, occurred within the
tumor in the face of the hypophosphatemia. Immunoreactivity for chromogranin-A and
electron microscopy evidence of neurosecretory granules in a proportion of neoplastic
cells indicated the neuroendocrine nature of the tumor. Although the expression of FGF23,
one of the putative factors involved in OHO, has been previously reported in tumors
causing OHO, RT-PCR analysis on frozen tumor samples demonstrated no FGF23 mRNA expression
in our case. While studies on the potential involvement of other factors (MEPE, PHEX) in
this case are in progress, these data highlight the occurrence of non-FGF23 producing,
neuroendocrine tumors as a cause of OHO.
[Programme]
O-35
VARIATION IN HUMAN BONE CELL RESPONSE TO STIMULATION BY FLUID FLOW OR
CYCLIC STRAIN
M. Mullender1*, A. J. El Haj2, Y. Yang2,
J. Magnay2, M. A. van Duin1, J. Klein-Nulend1
1Dept. Oral Cell Biology, ACTA-Vrije Universiteit, Amsterdam,
The Netherlands
2Centre for Science and Technology in Medicine, Dept.
Biomedical Eng., Stoke-on-Trent, UK
The aim of this study was to compare the response of human bone cells to
two different mechanical stimuli in vitro, pulsating fluid flow and cyclic strain.
Comparisons were measured in the production of nitric oxide (NO) and prostaglandin E2
(PGE2), both early mediators in mechanically induced bone adaptation.
Primary human bone cells were cultured from tibia fragments of a healthy
16-year-old male. Second-passage primary cultures were used for stimulation by either
pulsating fluid flow (PFF) or cyclic strain (CS) by four-point bending. Cells were plated
on polylysine coated glass slides and incubated for 24 h in DMEM with 2% FBS. The cells
were then loaded for 1 h by PFF (0.6±0.3 Pa, 8.5 Pa/sec, 5 Hz) or CS (~1000 microstrain,
1 Hz). Control cultures were kept accordingly, only without undergoing loading. All cell
cultures were post-incubated until 24 hours after loading. The conditioned medium was
assayed for NO using Griess reagent and PGE2 by enzyme-immunoassay. Cells were
harvested for measurement of total protein and DNA.
The DNA content did not differ between treatment and control groups.
Treatment with PFF caused an increase of NO and PGE2 (p<0.05). CS did not
stimulate the release of NO or PGE2 significantly. The release of NO was
increased up to 2-fold after 10 to 30 minutes of stimulation with PFF. The total amount of
PGE2 increased from 5-fold after 10 minutes to 15-fold after 1 hour of
treatment with PFF, measured after 24 h post incubation.
Fluid flow through the lacuno-canalicular system is generally accepted to
be a key mechanism by which bone senses mechanical loading. In this study we compared the
response of primary human bone cells to stimulation by pulsating fluid flow and four-point
bending of the cell substrate. Both PFF and CS cause cell deformation. Yet, the response
of the cells to these types of mechanical loading differed. NO and PGE2 release
increased after PFF, however, CS did not evoke this response. This suggests that bone
cells may differentiate between strain directly applied through the substrate and strain
induced by flow-derived shear stress, by differing signaling pathways.
[Programme]
O-36
THE BONE-SPECIFIC TRANSCRIPTIONAL REGULATOR CBFA1 IS A TARGET OF
MECHANICAL SIGNALS IN OSTEOBLASTIC CELLS
P. G. Ziros1, A-P. Rojas Gil1, T. Georgakopoulos1,
E. K. Basdra2, A. G. Papavassiliou1*
1Department of Biochemistry, School of Medicine, University of
Patras, GR-26110 Patras, Greece
2Department of Orthodontics, Heidelberg University, D-69120
Heidelberg, Germany
A primary goal of bone research is to understand the mechanism(s) by
which mechanical forces dictate the cellular and metabolic activities of osteoblasts, the
bone-forming cells. Several studies indicate that osteoblastic cells respond to physical
loading by transducing signals that alter gene expression patterns. Accumulated data have
documented the fundamental role of the osteoblast-specific transcription factor Cbfa1
(core binding factor) in osteoblast differentiation and function.
In the present study we demonstrate that low-level mechanical deformation
(stretching) of human osteoblastic cells directly up-regulates the expression and
DNA-binding activity of Cbfa1. This effect seems to be fine-tuned by stretch-triggered
induction of distinct MAPK (mitogen-activated protein kinase) cascades. Our novel finding
that activated ERK (extracellular signal-regulated kinase) MAPK physically interacts and
phosphorylates endogenous Cbfa1 in vivo (ultimately potentiating this transcription
factor), provides a molecular link between mechanostressing and stimulation of osteoblast
differentiation.
Elucidation of the specific modifiers and cofactors that operate in this
mechanotranscription circuitry will contribute to a better understanding of mechanical
load-induced bone formation that may set the basis for non-pharmacological intervention in
bone loss pathologies.
[Programme]
O-37
PROTEIN KINASE C ALPHA / ESTROGEN RECEPTOR ALPHA INTERACTION
IN OSTEOBLAST DIFFERENTIATION
M. Longo1*, M. Brama1, M. Marino2, K. S.
Korach3, W. C. Wetsel4, R. Scandurra2, T. Faraggiana2,
R. Baron5, A. Teti1, S. Migliaccio1,2
1Department of Experimental Medicine, University of L'Aquila,
Italy
2University 'La Sapienza', Rome, Italy
3NIEHS, Research Triangle Park, NC, USA
4Duke University Medical Center, Durham, NC, USA
5Dept. Orthop. & Cell Biol., Yale Univ., New Haven, CT,
USA
In osteoblastic cells undergoing confluence-induced differentiation,
protein kinase C (PKC) activity increases while estrogen receptor alpha (ERalpha) binding
and responsiveness to estrogens decrease. We tested the hypothesis that physical
interaction occurs between ERalpha and PKC isoforms as part of the underlying
mechanism(s).
Sub- and post-confluent rat primary osteoblasts were used for
anti-ERalpha and anti-PKCalpha immunoprecipitations. Both PKCalpha and ERalpha were
detected in either immunoprecipitate, but PKCalpha was much higher in anti-ERalpha samples
from post-confluent cells, which expressed higher levels of the differentiation marker
alkaline phosphatase. Furthermore, only the 66 KDa ERalpha variant was present in
sub-confluent cells, whereas its newly described 46 KDa form became detectable upon
differentiation. In rat osteosarcoma cells ROS.SMER 14, expressing murine ERalpha
constitutively, we found no significant variation of co-immunoprecipitable PKCalpha
levels, despite much lower ERalpha in cytosolic fractions from post-confluent cells. Not
too surprisingly, all of our anti-ERalpha immunprecipitates contained a single PKCalpha
post-translational variant, i.e., its well-known 40 KDa cleavage product encompassing the
catalytic site. This was most evident in the case of ROS.SMER 14, where both 80 and 40 KDa
forms were readily detectable, but the former appeared only in the anti- PKCalpha
immunoprecipitates.
Like calcium- and lipid-dependent PKCalpha, calcium-independent but
lipid-dependent PKCepsilon also increased to some extent in these cells upon
differentiation, whereas the atypical PKCzeta did not. Consistently, inositol-phospholipid
turnover was higher in differentiated cells.
As evidenced by our previous results, c-Src plays an important role in
confluence-induced differentiation in osteoblastic cells, and has been found to interact
with PKC isoforms. This prompted us to analyse c-Src expression and activity in our cells.
In ROS.SMER 14 we observed a significant, but transient, increase in expression and
activity of c-Src preceding maximal PKC increase. PKCalpha from differentiated osteoblasts
induced c-Src phosphorylation in vitro. In contrast, c- Src was unable to phosphorylate
PKCalpha, although tyrosine phosphorylation was apparent in PKCalpha and increased in
differentiated cells.
These data suggest close interactions between PKCalpha, c-Src and ERalpha
in osteoblastic cells, which may be crucial for the differentiation process and the
related decrease in estrogen responsiveness.
[Programme]
O-38
METALLOPROTEINASE-14 IS INDUCED AT BEGINNING OF OSTEOGENESIS IN VITRO AND
ITS DOWN-REGULATION IS REQUIRED FOR OSTEOGENIC PROGRESSION
D. Palmieri, S. Poggi, V. Ulivi, P. Manduca*
Dobig, University of Genova, Italy
We have reported the up-modulation of Metalloproteinase-14 (MMP-14) mRNA,
synthesis and function at the beginning of osteogenic differentiation of rat osteoblasts
and association of its expression with the mature phenotype in vitro, and with active
osteogenic cells in vivo. We also reported that MMP-14 is down- modulated in osteoblast
cultures before mineralization and not detected in osteocytes in vivo.
We here report that the expression of MMP-14 in pre-osteoblasts is
induced, in a time dependent fashion, by adhesion to the endogenous extracellular matrix
(ECM), which can be substituted by Fibronectin and Collagen type I, but not Collagen type
III, polilysin or VN. MMP-14 up-modulation requires mRNA and protein synthesis and is
inhibited by inhibitors of phosphorylation. The induction of MMP-14 is accompanied by the
activation of pro-MMP-2, and the active forms are recovered in the conditioned medium.
The persistence of MMP-14 function, obtained in differentiating cultures
with the use of a specific activating antibody, inhibits the decline of Alkaline
phosphatase (AP) activity, the morphogenesis of nodules and the incorporation of Calcium.
Two clones of rat osteoblasts, constitutive for AP, which did not form nodules and had a
very low level of Ca incorporation in comparison with the control cells, when tested,
showed constitutive expression of MMP-14 for up to 40 days in differentiation culture
conditions. These data suggest that down modulation of MMP-14 is required for the
progression of the osteogenic phenotype and is associated to the down modulation of AP.
[Programme]
O-39
DIFFERENTAL EXPRESSION OF CCN-FAMILY MEMBERS DURING OSTEOGENESIS,
CHONDROGENESIS AND ADIPOGENESIS USING PRIMARY HUMAN MESENCHYMAL STEM CELLS
N. Schütze*, U. Nöth, J. Schneidereit, C. Hendrich, J. Eulert, F. Jakob
Orthopädische UniversitätsklinikClinic, Labor für Molekulare
Experimentelle Orthopädie, Würzburg, Germany
Members of the family of connective tissue growth factor (CTGF), the
cystein-rich protein 61 (CYR61) and the nephroblastoma overexpressed protein (nov)
(CCN-family) function in processes such as differentiation and tissue regeneration mainly
as growth factors and matrix associated signal molecules. Previously, we have shown that
the expression of human CYR61 (hCYR61) in vivo was associated with conditions of enhanced
bone formation and fracture healing. Further results in the literature point towards the
expression of other CCN proteins in bone such as CTGF, CTGF-L and WISP-3.
Primary cultures of human mesenchymal stem cells were derived from the
femoral head of patients undergoing total hip arthroplasty. Differentiation into
adipocytes and osteoblasts was done in monolayer culture, differentiation into
chondrocytes was induced in pellet culture. For either pathway established differentiation
markers and CCN-members were analyzed at the mRNA level by RT-PCR and at the protein level
by immunocytochemistry.
RNA or protein levels of established markers revealed the appropriate
phenotype of differentiated cells (alizarin-red, alkaline phosphatase, osteocalcin,
collagens, cbfa1, PPARgamma, oil-red-o, aggrecan). Mesenchymal stem cells expressed nestin
mRNA and all CCN-family members. The hCYR61 expression was high in stem cells and
decreased 7-fold during osteogenesis and chondrogenesis and more than 10-fold during
adipogenesis. These results were confirmed by immuncytochemical analyses. CTGF-L and
WISP-3 RNA expression were enhanced slightly during osteogenesis and decreased in
chondrogenesis and adipogenesis (3- and 10-fold, respectively). The CTGF-L expression was
decreased >10-fold in adipogenesis.
The expression of CCN-family members was dependent on the differentiation
status of human mesenchymal stem cells in vitro. hCYR61, which plays a role as a signal
molecule of the extracellular matrix, is important in the early nestin positive stem cells
used in this study. The marked decrease in hCYR61 expression during all differentiation
pathways suggests a specific role of hCYR61 for maintenance of the stem cell phenotype.
The differential expression of WISP-3, CTGF, CTGF-L and mainly hCYR61 indicates, that the
CCN-family might be important for the function of mesenchymal stem cells as well as the
regulation of proliferation and specific differentiation pathways of human mesenchymal
stem cells from bone marrow.
[Programme]
O-40
OP-1 DEVICE IN SURGICAL TREATMENT OF THE SCAPHOID BONE NON-UNION
R. Bilic1*, M. Jelic1,2, M. Pecina1, S.
Vukicevic2
1Department of Orthopedic Surgery, School of Medicine,
University of Zagreb, Croatia
2Departments of Anatomy, School of Medicine, University of
Zagreb, Croatia
Osteosynthesis with autogenous corticocancellous graft is a widely used
operative treatment for the scaphoid non-unions. Long period of 14 weeks postoperative
immobilization, leads eventually to arthrotic changes in the radioscaphoid or radiocarpal
joint (n=180). Recently, we demonstrated that the scaphoid union and postoperative
immobilization can be reduced to 9 weeks (n=130) if adding the compressed cancellous bone
graft to the corticocancellous graft. When nesting site in the scaphoid bone and the
autogenous graft with compressed cancellous bone were covered with OP-1 putty in a
prospective randomized study, the new bone formation at the operation site was observed at
4 weeks (4 patients). OP-1 device can, thus, significantly reduce the immobilization time
for achieving scaphoid union when using autogenous corticocancellous graft. In an attempt
to avoid the second surgical procedure at the autogenous corticocancellous graft donor
site, we treated 4 patients with allogeneic corticocancellous graft and compressed
cancellous bone from the iliac crest of a 25 y donor. The scaphoid bone nesting site and
the allograft were covered with OP-1 putty. The scaphoid union was achieved at 8 weeks
following surgery. Trabecular bone union evidenced by plain x-rays and CT scans correlated
well with the disappearance of pain and with the improvement of functional tests. This is
the first evidence that scaphoid non- union osteosynthesis could be performed in 8 weeks
using an allograft and OP-1 device.
[Programme]
O-41
CP-533,536, A NON-PROSTANOID EP2 RECEPTOR AGONIST, STIMULATES PERIOSTEAL
BONE FORMATION AND ENHANCES FRACTURE HEALING IN RAT MODEL
M. Li*, D. R. Healy, Y. Li, H. Z. Ke, V. M. Paralkar, T. A. Owen, L. C.
Pan, K. Cameron, B. A. Lefker, P. DaSilva-Jardine, F. Dumont, R. Korsmeyer, D. D. Thompson
Pfizer Global Research and Development, Groton, CT, USA
Prostaglandin E2 (PGE2) is known to stimulate bone formation when
administered systemically or locally, but its side effects limit its utilization for
enhancing bone formation. We have found that the EP2 receptor subtype plays an important
role in the local bone anabolism of PGE2. CP-533,536 is a new non-prostanoid EP2 receptor
selective agonist. Utilizing rat-models, we evaluated the potential beneficial effects of
this compound on bone healing upon local administration. In the first study, CP-533,536 at
0.4 mg/rat/day or vehicle was injected onto the periosteum of the right femurs of
3-week-old male rats for 14 consecutive days. Radiography showed an increased local bone
formation on femurs injected with CP-533,536 as compared with vehicle injection. Total
bone area and bone mineral content of the injected site with CP-533,536 were significantly
increased by 11% and 19%, respectively. In the same animal model, a single dose (0.3 mg)
of CP- 533,536 in poly-lactide-co-glycolide (PLGH) matrix significantly increased total
bone area and bone mineral content by 19% and 14%, respectively, as compared with PLGH
alone. In the second study, the right femurs of male rats were stabilized with
intramedullary pins and were subjected to closed transverse fracture (under anesthesia).
On days 3, 4, and 5 post-surgery, the rats were percutaneously injected with 0 or 5 mg of
CP-533,536 to the fracture site. At 3 weeks post- fracture, the femurs treated with
CP-533,536 had larger and denser calluses than those in controls as assessed by
radiography. Histological measurements of fracture calluses showed significant increases
in total callus area (29%), bony callus area (14%) and cartilaginous callus area (47%) in
the rats treated with the compounds as compared with controls. Biomechanical test showed
that maximal load and stiffness were significantly increased by 12 and 29%, respectively,
for the femurs treated with CP-533,536 compared with those treated with vehicle. In the
same animal model, fracture healing was significantly improved by a single dose (15 mg) of
CP-533,536 in PLGH matrix. These data demonstrated that CP-533,536 stimulated periosteal
bone formation and callus formation in rats. These results suggested that non-prostanoid
EP2 receptor agonists are effective in enhancing bone healing.
[Programme]
O-42
VERTEBRAL FRACTURE EFFICACY OF ONCE-A-WEEK RISEDRONATE
R. Eastell1*, N. B. Watts2, Z. Li3, M.
Hoseyni3, E. Seeman4, R. Lindsay5
1University of Sheffield, Sheffield, UK
2University of Cincinnati, Cincinnati, USA
3Procter & Gamble Pharmaceuticals, Mason, USA
4University of Melbourne, Melbourne, Australia
5Helen Hayes Hospital, West Haverstraw, USA
A recent randomized, active-controlled trial demonstrated that
risedronate (35 mg) once-a-week (OaW) and 5 mg daily produce comparable increases in BMD
and reductions in bone remodeling. Although morphometric vertebral fractures were assessed
in this trial, antifracture efficacy was not evaluated because of the absence of a placebo
group. The objective of our analysis was to estimate the efficacy of OaW risedronate in
reducing the risk of new vertebral fractures at 1 year, by using the OaW trial's
enrollment criteria to construct matching control and risedronate 5 mg groups from
patients enrolled in the risedronate Phase III fracture studies. Comparison of baseline
characteristics indicated a high degree of similarity between the constructed placebo and
5 mg groups and the 35 mg weekly and 5 mg daily groups as enrolled in the OaW study (mean
lumbar spine T-score: -3.17 to -3.03; mean age: 67.6 to 68.1; incidence of prevalent
vertebral fracture: 34.8% to 35.7%).
No statistically significant difference was observed in fracture
incidence for the 5 mg groups in the Phase III and OaW studies. Compared with matched
placebo, weekly treatment reduced the risk of new vertebral fractures by 77% within 1
year. This reduction is consistent with the previously reported 1-year fracture efficacy
of daily risedronate.
Group |
Incidence |
RR (95%C.I.) |
P-value |
5mg daily (phase III) |
1.7% |
0.88 |
|
5mg daily (OaW) |
1.5% |
(0.16, 9.0) |
1.00 |
Placebo (phase III) |
5.0% |
0.23 |
|
35mg weekly (OaW) |
1.2% |
(0.05, 0.91) |
0.018 |
[Programme]
O-43
EFFECTS OF RISEDRONATE (RIS) ON TRABECULAR ARCHITECTURE IN EARLY
POSTMENOPAUSAL WOMEN AS MEASURED BY MICROCOMPUTED TOMOGRAPHY
A. Grauer1*, B. Borah2, T. E. Dufresnes2,
P. A. Chmielewski2, M. C. Prenger2, M. D. Manhart2
1Procter and Gamble Pharmaceuticals, Geneva, Switzerland
2Procter & Gamble Pharmaceuticals, Cincinnati, Ohio, USA
RIS treatment reduces the risk of radiographic vertebral fracture by up
to 65% within the first year of treatment. Increases in bone mineral density (BMD) explain
only about 1 third of the fracture risk reduction, suggesting that other factors, such as
the preservation of bone architecture, are responsible for most of the antifracture
efficacy. We used three-dimensional microcomputed tomography (3D microCT) to examine
paired (baseline and 1 year) iliac crest bone biopsy samples in order to determine the
effects of RIS on trabecular architecture. Biopsy samples were obtained from women (6-60
months postmenopausal) who were enrolled in a double-blind, placebo-controlled study
evaluating the effects of RIS (5 mg daily) on lumbar spine (LS) BMD. The mean percent
change from baseline in LS BMD after 1 year was - 2.9% in the placebo group and +1.4% in
the RIS group. Compared with the RIS group, the placebo group (n=12 pairs) showed
decreases in bone volume (BV/TV) (p=0.032), trabecular thickness (p=0.061), and trabecular
number (p=0.025), and increases in trabecular separation (p=0.014) and bone surface/bone
volume (p=0.051), indicating a deterioration of trabecular architecture in the placebo
group. There were no significant changes in bone volume or architectural parameters vs.
baseline in the RIS group (n=14 pairs).
These data demonstrate that RIS preserves trabecular architecture in
postmenopausal women within 1 year, which may partly explain RIS's rapid anti-fracture
benefit.
[Programme]
O-44
COMPARATIVE MINERAL BINDING AFFINITIES OF SELECTED BISPHOSPHONATES
R. G. Russell1*, A. H. Mangood2, E. M. Gaafar2,
W. Wu2, F. H. Ebetino3, D. J. White3, R. J. Phipps3,
G. H. Nancollas2
1Oxford University, Oxford, UK
2SUNY Buffalo, Buffalo, NY 14260, USA
3Procter & Gamble Pharmaceuticals, Mason, OH 45040, USA
Both clinical and experimental data indicate that bisphosphonates (BPs)
differ in terms of their interactions with bone, and that these differences may result in
subtle differences in their pharmacological behaviour. The current study further examines
the potential contribution of mineral binding to the potency and duration of action of
individual BPs. We investigated the effects the BPs alendronate, risedronate, and
etidronate on hydroxyapatite (HAP) and octa calcium phosphate (OCP) crystal growth and
dissolution, using the Constant Composition method at ionic strength 0.15 M and 37 deg C.
Adsorption affinity constants (KL) were calculated from the kinetic results.
Significant differences in KL (P<0.001) were observed among the three BPs
for HAP growth (pH 7.4) and OCP growth (pH 6.5), resulting in a rank order alendronate
> risedronate > etidronate. These conditions probably simulate the rate and extent
of BP binding onto bone. Binding affinities were lower for crystal dissolution under
conditions likely to prevail during osteoclastic resorption (HAP at pH 4.5 and OCP at pH
6.0), but the order alendronate > risedronate > etidronate was the same. Preliminary
results from other BPs suggest that ibandronate and zoledronate have higher KL
than alendronate and risedronate, at least for HAP dissolution.
In conclusion, risedronate has lower kinetic binding affinity than
alendronate for the mineral substrates HAP and OCP. This difference may contribute to the
apparent shorter terminal (bone) half-life of risedronate, and to a faster clinical
on-response (fracture reduction) and off-response (recovery of bone turnover after
cessation of dosing) seen with risedronate compared with alendronate.
BP |
KL HAP Growth
(L/mole)
pH 7.4 |
KL OCP Growth
(L/mole)
pH 6.5 |
KL HAP Dissolution
(L/mole)
pH 4.5 |
Risedronate |
4.46x106 |
5.30x106 |
1.39x105 |
Alendronate |
11.67x106 |
8.00x106 |
1.97x105 |
Etidronate |
1.17x106 |
4.00x106 |
0.97x105 |
[Programme]
O-45
THE REPAIR OF AN ULNAR CRITICAL DEFECT WITH A SELECTIVE PROSTAGLANDIN E2
(PGE2) EP-2 RECEPTOR AGONIST
V. M. Paralkar1*, F. Borovecki2, F. Dumont1,
R. Korsmeyer1, D. Plowchauk1, H. Z. Ke1, K. O. Cameron1,
B. A. Lefker1, T. A. Owen1, M. Li1, P. DaSilva-Jardine1,
S. Vukicevic2, D. D. Thompson1,
1Pfizer Global Research and Development, Groton, CT, USA
2University of Zagreb School of Medicine, Zagreb, Croatia
In spite of significant advances in our understanding of the biology of
fracture healing, the morbidity and mortality associated with impaired/delayed fracture
healing remains high and a significant medical need exists to ensure rapid bone healing.
Significant improvements in bone repair have been demonstrated with biological products
such as Bone Morphogenetic Proteins. However, problems remain with the use of these
proteins. Prostaglandin E2 is known to increase bone formation and enhance bone healing.
However, due to side effects including diarrhea, PGE2 is an unacceptable
therapeutic option. PGE2 mitigates its pharmacological activity via four
receptor subtypes, EP1, EP2, EP3 and EP4. We have discovered that the EP2 receptor subtype
is responsible for the local bone anabolic activity of PGE2. In this abstract
we report the ability of CP-533,536 a selective EP2 agonist to heal ulnar critical size
defect in one year old male beagle dogs 11±1 kg in weight. A 1.5 cm segmental defect was
made in the mid-ulna. The dogs were divided in to the following groups.
A 1 ml of PLGH
B 50 mg of CP-533,536 (1 ml PLGH formulation).
C 10 mg of CP-533,536 ( 1 ml PLGH formulation).
D 10 mg of CP-533,536 ( 0.2 ml PLGH formulation).
It was observed that a single application of CP-533,536 induced full
rebridgement in dogs present in groups B, C and D. The newly formed bone remodeled back to
the same shape and size as the contralateral bone by week 24. In comparison ulnae treated
with the vehicle did not show any significant healing as assessed by radiography and
histology. The overall complete bone regeneration and repair was observed in more than 70%
of animals tested. Since healing was observed at both 10 mg and 50 mg of CP-533,536 we do
not know the lowest efficacious dose nor a dose volume co-relationship. In conclusion, a
single administration at the time of surgery with CP-533, 536 (a non-prostanoidal EP-2
agonist) in PLGH matrix healed a critical sized defect in the canine ulna. These data
indicate that EP2 receptor selective agonists may have therapeutic potential for bone
healing in humans.
[Programme]
O-46
ALLELE OF RUNX2/CBFA1 INCREASES ADULT BONE MINERAL DENSITY AND PROTECTS
AGAINST COLLES' FRACTURE OF THE WRIST
T. Vaughan1*, J. A. Pasco2, M. A. Kotowicz2,
G. C. Nicholson2, N. A. Morrison2
1Genomics Research Centre, School of Health Science, Griffith
University, Gold Coast, Australia
2Department of Medicine, The University of Melbourne, Barwon
Health, Geelong Hospital, Geelong, Australia
The aim of this study was to determine if DNA polymorphism within
runt-related gene 2 (RUNX2)/core binding factor A1 (CBFA1) is related to bone mineral
density (BMD). RUNX2 contains a glutamine-alanine repeat where mutations causing
cleidocranial dysplasia have been observed. Two common variants were detected within the
alanine repeat: an 18 base pair deletion and synonymous alanine codon polymorphism with
alleles, GCA and GCG. Rare mutations that may be related to low BMD were observed within
the glutamine repeat. Subjects of the Geelong Osteoporosis Survey (GOS) were ranked
according to age and weight adjusted femoral neck BMD. The GCA allele was over represented
(p=0.019) in the highest decile (n=130) compared to the lowest decile (n=135) of BMD. In
575 randomly selected subjects of GOS, the GCA allele was associated with higher BMD at
all sites tested. The effect was maximal at the ultra-distal radius in postmenopausal
women (p=0.006). In a separate fracture study, the GCA was significantly protective
against Colles' fracture in elderly women but not spine and hip fracture. The GCA allele
confers increased BMD and is protective against a common form of osteoporotic fracture,
suggesting that RUNX2 variants may be related to genetic effects on BMD and osteoporosis.
[Programme]
O-47
OSTEOPROTEGERIN ALLELES PREDISPOSE TO PAGET'S DISEASE
A. Daroszewska1*, L. J. Hocking1, B. L. Langdahl2,
F. E. A. McGuigan1, W. D. Fraser3, S. H. Ralston1
1Bone Research Group, University of Aberdeen, Aberdeen, UK
2Department of Endocrinology, Arhus Amtssygehus, Arhus,
Denmark
3Department of Clinical Chemistry, University of Liverpool,
Liverpool, UK
Paget's disease of bone (PDB) is a common condition with a strong genetic
component, but the genes responsible are poorly defined. Recent work has shown that
mutations in the RANK gene cause familial expansile osteolysis (FEO) - a rare bone
dysplasia with similarities to PDB. In view of this, we investigated the hypothesis that
allelic variation in other components of the RANK pathway might predispose to PDB. In this
study, we conducted mutation screening of the Osteoprotegerin (OPG) gene and looked for
evidence of an allelic association with Paget's disease in a series of 181 UK Pagetic
patients and 184 non Pagetic UK controls. We identified several single nucleotide
polymorphisms (SNP) in the OPG gene and investigated seven of these in an association
study. We studied 3 SNP's in the OPG promoter; one in exon 1 (a C/G substitution resulting
in a Lys-Asn amino acid change), one close to the splice site in intron 2 (IVS2 +4C/T) a
conservative change in exon 4 (A/G; Leu-Leu) and a change close to the splice site in
intron 4 (IVS4 +8A/C). All SNP's were in linkage disequilibium (p<0.0001; data not
shown). Two SNP's showed a significant association with Paget's disease; an A/G change
situated at position -943 in the gene promoter at a binding site for the transcription
factor Oct-3 and another C/T change situated 4 bases from the splice donor site in intron
2 (IVS2 +4C/T). The odds ratio for Paget's disease in 'GG' homozygotes at the -943A/G site
was 6.46 [1.42-29.4], when compared with AA homozygotes (p=0.016) and the odds ratio for
CC homozygotes at the IVS2 +4C/T site was 1.77 [1.14-2.74] when compared with carriers of
the 'T' allele (p=0.010). These data indicate that common allelic variants in the OPG gene
predispose to Paget's disease. Further work is in progress to define the functional
effects of the polymorphisms we have described and to extend these observations to
patients with familial Paget's disease and to other populations.
[Programme]
O-48
COMMON HERBS, ESSENTIAL OILS AND MONOTERPENES POTENTLY MODULATE BONE
METABOLISM
R. C. Mühlbauer*, R. Felix, A. Lozano, S. Palacio, A. Reinli
Bone Biology Group, Dept. Clinical Research, University of Bern, Bern,
Switzerland
Osteoporosis is a major health issue in aging populations. It has been
suggested that vegetable components in the food inhibit bone resorption and may help to
prevent bone loss. Therefore, we investigated the antiosteoporotic potential of herbs,
essential oils (EO) and monoterpenes (MT).
Herbs (1g/day), EO and MT (100 mg/day) were added to the food of 250 g
rats. Bone resorption was assessed with the urinary excretion of tritiated tetracycline
from prelabeled rats. Bone mineral density (BMD) was measured in the proximal tibia by
computer tomography. In vitro resorptive activity (pits/osteoclast) was assessed in
osteoclasts isolated from long bones of 1-2 day old rats cultured on ivory slices.
Herbs rich in EO (sage, rosemary and thyme), EO extracted from these
herbs and other plants (oils of sage, rosemary, juniper, pine and eucalyptus) as well as
their main components (the MT thujone, eucalyptol, camphor, borneol, thymol, alpha-pinene,
beta-pinene, bornylacetate and menthol) inhibit bone resorption. Pine oil, eucalyptol and
camphor are also inhibitory at doses of 20, 24 and 14 mg/day respectively. Pine oil, used
as a representative EO, protects from loss of trabecular BMD in aged ovariectomized rats.
Borneol, thymol and camphor ([1 mM]) inhibit resorption activity of
osteoclasts. Non-polar MT require metabolism to be active in vitro, as demonstrated for
cis-verbenol, a metabolite of alpha-pinene occurring in human urine, inhibiting osteoclast
activity in contrast to the parent compound. Borneol inhibits within 30 minutes the
formation of actin rings which indicate cell polarization, a prerequisite for resorbing
osteoclasts. Thus, MT and/or their metabolites inhibit resorption also in vitro.
Our study demonstrates for the first time that EO and MT are efficient
inhibitors of bone resorption in the rat. Thus, a generous consumption by man of herbs as
well as of food products flavored with these compounds may decrease bone resorption.
Furthermore, there is a long history of use of EO and MT taking advantage of the
transdermal and pulmonary absorption for medical applications for relief of colds and
muscle pain. Thus, the external application of EO and MT may offer an additional
possibility to decrease the incidence of osteoporosis.
[Programme]
O-49
HIP FRACTURES, MORPHOMETRY AND GEOMETRY
J. S. Gregory1,2*, D. Testi3, P. E. Undrill2,
R. M. Aspden1
1Department of Orthopaedics, University of Aberdeen, UK
2Department of Bio-Medical Physics and Bio-Engineering,
University of Aberdeen, UK
3Istituti Ortopedici Rizzoli, University of Bologna, Italy
A new method has been developed for measuring the morphometry of the
femur which provides a powerful tool for discriminating between Osteoporotic- fracture and
control subjects. We will show that a mathematical description of the shape of the
proximal femur is more effective than both individual geometric and most BMD measurements.
By combining BMD and morphometric measurements, we were able to discriminate between the
two groups with almost 90% accuracy.
Active shape modelling (ASM) is an image-processing technique that can be
used to build a model of a shape. Shapes that have common features but are subject to
natural variation, such as leaves are described by a series of orthogonal (and therefore
independent) descriptors. After building a model, new images can be analysed by examining
how the location of landmark-points deviate from the mean co-ordinates. Geometric
measurements, such as neck length, width and neck- shaft angle have previously been shown
to be significantly related to fracture risk. However such measurements are highly
correlated and, therefore, of limited usefulness.
A set of 74 images was used for this study (46 control, 28 fracture). All
subjects underwent DXA and standard radiographic examination. The following BMD
measurements were recorded: total body BMD, total body BMC, femoral neck BMD, trochanter
BMD, and Ward's triangle BMD. ASM and geometric measurements were calculated from
radiographs.
Discriminant analysis was applied to find out which variable or
combination of variables was best able to discriminate between the two groups. Using a
single variable, ASM (77% correct) was more accurate than 4 out of 5 of the BMD
measurements, being beaten only by Ward's triangle BMD (81%), whilst the geometric
classifier was the weakest. By combining Ward's triangle, with the ASM data,
classification improved to 89%.
This study shows that morphological analysis alone may be a more powerful
discriminator than most BMD measurements and, in combination with BMD data, can create an
accurate classification method. These initial results are taken from a relatively small
data set and further studies are being undertaken to confirm these promising results.
[Programme] |