bluuebunny/crossref_metadata_2025 huggingface dataset:
Interactions of Interleukin-6 Promoter Polymorphisms With Dietary and Lifestyle Factors and Their Association With Bone Mass in Men and Women From the Framingham Osteoporosis Study
Authors: Ferrari Serge L , Karasik David , Liu Jun , Karamohamed Samev , Herbert Alan G , Cupples L Adrienne , Kiel Douglas P
Published: April 2006

Lifestyle and dietary factors may influence the association of IL-6 polymorphisms with bone mass. In 1574 unrelated men and women from the Framingham Offspring Cohort, we observed significant hip BMD differences between IL-6 −174 genotypes only in older women, those without estrogens, and those with a poor calcium intake. Hence, association of IL-6 polymorphisms with BMD may be limited to discrete population subgroups.

Introduction: Interleukin (IL)-6 plays a central role in the pathogenesis of osteoporosis. Two functional variants in the IL-6 promoter have previously been associated with IL-6 expression, bone resorption levels, and BMD in late postmenopausal women, but results were conflicting in different populations. We hypothesized that the association between IL-6 promoter alleles and BMD may be affected by interactions with lifestyle and dietary factors known to influence bone turnover.

Materials and Methods: Among the Offspring Cohort of the Framingham Heart Study, 1574 unrelated men and women were genotyped for IL-6 −572 and −174 alleles. Interaction analyses with years since menopause, estrogen status, physical activity, smoking, dietary calcium, vitamin D, and alcohol intake were based on BMD measurements at the hip.

Results and Conclusions: In models that considered only the main effects of IL-6 polymorphisms, no significant association with BMD was observed in either gender. In contrast, p values (0.003–0.096 by ANOVA) suggestive of an interaction between IL-6 −174 genotypes and years since menopause, estrogen status, dietary calcium, and vitamin D intake were observed in women (n = 819). In turn, BMD was significantly lower with genotype −174 GG compared with CC, and intermediate with GC, in women who were more than 15 years past menopause and in those without estrogens or with calcium intake <940 mg/day. In estrogen-deficient women with poor calcium intake, BMD differences between genotypes CC and GG were 10.2% at femoral neck (p = 0.012), 12.0% at trochanter (p = 0.012), and 16.8% at Ward's area (p = 0.0014). In contrast, no such interactions were observed in men (n = 755). In conclusion, IL-6 genetic variation was prominently associated with hip BMD in late postmenopausal women, those without estrogen replacement therapy, and those with inadequate calcium intake. In contrast, IL-6 polymorphisms are unlikely to be significant determinants of bone mass in other women or men.
Extracellular Nucleotides Act Through P2X7 Receptors to Activate NF-κB in Osteoclasts
Authors: Korcok Jasminka , Raimundo Lin N , Ke Hua Z , Sims Stephen M , Dixon S Jeffrey
Published: April 2006

Nucleotides, released in response to mechanical and other stimuli, act on P2 receptors in osteoclasts and other cell types. In vitro studies of osteoclasts from rabbits and P2X7 receptor-deficient mice revealed that P2X7 receptors couple to activation of the key transcription factor NF-κB.

Introduction: Osteoclasts express functional P2X4 and P2X7 receptors, which are ATP-gated cation channels. Knockout (KO) of the P2X7 receptor has revealed its role in regulating bone formation and resorption, but the underlying signals are not known. The transcription factor NF-κB plays a key role in the response of osteoclasts to RANKL and other cytokines. The aim of this study was to examine whether P2X receptors on osteoclasts signal through NF-κB.

Materials and Methods: Osteoclasts were isolated from neonatal rabbits or wildtype (WT) and P2X7 receptor KO mice. Immunofluorescence was used to detect the p65 subunit of NF-κB, which, on activation, translocates from the cytosol to the nuclei. The concentration of cytosolic free Ca2+ ([Ca2+]i) was monitored in single osteoclasts loaded with fura-2.

Results: In control samples, few rabbit osteoclasts demonstrated nuclear localization of NF-κB. Benzoyl-benzoyl-ATP (BzATP, a P2X7 agonist, 300 μM) induced nuclear translocation of NF-κB after 3 h in ∼45% of rabbit osteoclasts. In contrast, a low concentration of ATP (10 μM, sufficient to activate P2X4 and P2Y2, but not P2X7 receptors) did not induce nuclear translocation of NF-κB. Because BzATP activates multiple P2 receptors, we examined responses of osteoclasts derived from WT and P2X7 receptor KO mice. Treatment with BzATP for 30 minutes increased nuclear localization of NF-κB in osteoclasts from WT but not KO mice, showing involvement of P2X7 receptors. Both ATP (10 μM) and BzATP (300 μM) caused transient elevation of [Ca2+]i, indicating that rise of calcium alone is not sufficient to activate NF-κB. Pretreatment of rabbit osteoclasts with osteoprotegerin inhibited translocation of NF-κB induced by RANKL but not by BzATP, establishing that the effects of BzATP are independent of RANKL signaling.

Conclusion: These findings show that P2X7 nucleotide receptors couple to activation of NF-κB in osteoclasts. Thus, nucleotides, released at sites of inflammation or in response to mechanical stimuli, may act through NF-κB to regulate osteoclast formation and activity.
Genetically Linked Site-Specificity of Disuse Osteoporosis
Authors: Judex Stefan , Garman Russell , Squire Maria , Busa Bhavin , Donahue Leah-Rae , Rubin Clinton
Published: April 2006

The genetic influence on bone loss in response to mechanical unloading was investigated within diaphyseal and distal femoral regions in three genetically distinct strains of mice. One mouse strain failed to lose bone after removal of function, whereas osteopenia was evident in multiple regions of the remaining two strains but in different areas of the bone.

Introduction: It is well recognized that susceptibility to osteoporosis is, in large measure, determined by the genome, but whether this influence is systemic or site-specific is not yet known. Here, the extent to which genetic variations influence regional bone loss caused by disuse was studied in the femora of adult female mice from three inbred strains.

Materials and Methods: Adult C57BL/6J (B6), C3H/HeJ (C3H), and BALB/cByJ (BALB) mice were subjected to 15–21 days of disuse, achieved by hindlimb suspension, and six distinct anatomical regions of the femur were analyzed by high-resolution μCT.

Results and Conclusions: In B6 mice, the amount of disuse stimulated bone loss was relatively uniform across all regions, with 20% loss of trabecular bone and 10% loss of cortical bone. The degree of bone loss in BALB mice varied greatly, ranging from 59% in the metaphysis to 3% in the proximal diaphysis. In this strain, the nonuniformity of bone loss was directly related to the nonuniform distribution of baseline bone morphology (r2 = 0.94). In direct contrast with BALB and B6, disuse failed to produce significant losses of bone in any of the analyzed regions of the C3H mice. Instead, these animals displayed a unique compensatory mechanism to disuse, where the large loss of calcified tissue from the endocortical surface (−24%) was compensated for by an expansion of the periosteal envelope (10%). These data indicate a strong, yet complex, genetic dependence of the site-specific regulation of bone remodeling in response to a powerful catabolic signal. Consequently, the skeletal region of interest and the genetic make-up of the individual may have to be considered interdependently when considering the pathogenesis of osteoporosis or the efficacy of an intervention to prevent or recover bone loss.
Osteoprotegerin and RANKL in the Pathogenesis of Thalassemia-Induced Osteoporosis: New Pieces of the Puzzle
Authors: Morabito Nunziata , Gaudio Agostino , Lasco Antonino , Atteritano Marco , Pizzoleo Maria Antonia , Cincotta Maria , La Rosa Mariangela , Guarino Roberta , Meo Anna , Frisina Nicola
Published: April 2006

Osteoporosis represents an important cause of morbidity in adult thalassemic patients, and its pathogenesis has not, as yet, been completely clarified. In our study, we observed that thalassemic patients showed a significantly lower OPG/RANKL ratio than normal subjects. These data are extremely important for the possible therapeutic use of RANKL antagonists such as OPG in thalassemia-induced osteoporosis.

Introduction: Osteoporosis represents an important cause of morbidity in adult thalassemic patients who display increased fracture risk. The etiology of this bone disease is multifactorial, but it is thought that the main role is played by hypogonadism. The mechanisms by which the skeletal effects of sex steroids are mediated are still not fully understood. Recently, two new cytokines, osteoprotegerin (OPG) and RANKL, have been implicated in the pathogenesis of postmenopausal osteoporosis and other metabolic bone diseases. Thus, the aim of this study was to characterize the possible role of the OPG/RANKL system in thalassemia-related bone loss.

Materials and Methods: We measured, in 30 thalassemic patients and in 20 healthy control subjects, serum OPG and RANKL levels, and determined their correlations with bone turnover markers, BMD, sex steroid levels, erythropoietin, and hemoglobin.

Results: Thalassemic patients had an unbalanced bone turnover with an increased resorption phase (shown by high levels of pyridinium cross-links) and a decreased neoformation phase (shown by the slightly low levels of osteocalcin). Moreover, they displayed lower BMD values than controls both at the lumbar and femoral level. As far as the OPG/RANKL system is concerned, thalassemic patients showed no differences in plasma levels of OPG compared with controls, and significantly higher plasma levels of RANKL, with a consequent significantly lower OPG/RANKL ratio.

Conclusions: Our data suggest that, in thalassemic patients, an altered modulation of the OPG/RANKL system, resulting in increased expression of RANKL by stromal or osteoblastic cells, could contribute to the enhanced osteoclastic bone resorption and bone loss characteristic of these patients.
Regulation of Human Skeletal Stem Cells Differentiation by Dlk1/Pref-1
Authors: Abdallah Basem M , Jensen Charlotte H , Gutierrez Gloria , Leslie Robert GQ , Jensen Thomas G , Kassem Moustapha
Published: April 2006

Dlk-1/Pref-1 was identified as a novel regulator of human skeletal stem cell differentiation. Dlk1/Pref-1 is expressed in bone and cultured osteoblasts, and its constitutive overexpression led to inhibition of osteoblast and adipocyte differentiation of human marrow stromal cells.

Introduction: Molecular control of human mesenchymal stem cell (hMSC) differentiation into osteoblasts and adipocytes is not known. In this study, we examined the role of delta-like 1/preadipocyte factor-1 (Dlk1/Pref-1) in regulating the differentiation of hMSCs.

Materials and Methods: As a model for hMSCs, we have stably transduced telomerase-immortalized hMSC (hMSC-TERT) with the full length of human Dlk1/Pref-1 cDNA and tested its effect on hMSC growth and differentiation into osteoblasts or adipocytes as assessed by cytochemical staining, FACS analysis, and real time PCR. Ex vivo calvaria organ cultures assay was used to confirm the in vitro effect of Dlk/Pref-1 on bone formation.

Results: Dlk1/Pref-1 was found to be expressed in fetal and adult bone, hMSCs, and some osteoblastic cell lines. A retroviral vector containing the human Dlk1/Pref-1 cDNA was used to create a cell line (hMSC-dlk1) expressing high levels of Dlk1/Pref-1 protein. Overexpression of Dlk1/Pref-1 did not affect the proliferation rate of hMSC, but the ability to form mature adipocytes, mineralized matrix in vitro, and new bone formation in neonatal murine calvariae organ cultures was reduced. These effects were associated with inhibition of gene expression markers of late stages of adipocyte (adipocyte fatty acid-binding protein [aP2], peroxisome proliferator-activated receptor-gamma2 [PPARγ2], and adiponectin [APM1]) and osteoblast differentiation (alkaline phosphatase [ALP], collagen type I [Col1], and osteocalcin [OC]). Lineage commitment markers for adipocytes (adipocyte determination and differentiation factor −1 [ADD1]) and osteoblasts (core binding factor/runt-related binding factor 2 [Cbfa1/Runx2]) were not affected.

Conclusion: During hMSC differentiation, Dlk1/Pref-1 maintains the size of the bipotential progenitor cell pool by inhibiting the formation of mature osteoblasts and adipocytes.
In Vivo Inhibition of Osteoblastic Metalloproteinases Leads to Increased Trabecular Bone Mass
Authors: Geoffroy Valérie , Marty-Morieux Caroline , Le Goupil Nathalie , Clement-Lacroix Phillippe , Terraz Catherine , Frain Monique , Roux Sophie , Rossert Jérome , de Vernejoul Marie Christine
Published: April 2006

Mice specifically overexpressing TIMP-1 in osteoblasts have been generated to investigate the role of MMPs in bone in vivo. These mice displayed increased trabecular bone volume and decreased bone turnover. This model provides evidence of the role played by the MMPs in bone remodeling and balance.

Introduction: Although it has been suggested that the matrix metalloproteinases (MMPs) may play a role in initiating the bone resorption process in vitro, there is no evidence that they play any role in in vivo bone maintenance.

Materials and Methods: We used an artificial promoter specifically driving cells of the osteoblastic lineage to overexpress the tissue inhibitor of MMPs (TIMP-1) cDNA in mice. Densitometric analysis, using DXA and pQCT, and static and dynamic histomorphometry were used to evaluate the bone phenotype both in male and female transgenic mice. We evaluated osteoblastic differentiation using a primary osteoblast culture and osteoclast activity using an ex vivo organ culture.

Results and Conclusion: We showed that at 1 and 2.5 months of age, only the female mice exhibited a bone phenotype. These mice displayed specific increases in the BMD and bone volume of trabecular bone. This increase was accompanied by decreased trabecular separation, suggesting a decrease in bone resorption. Using an ex vivo resorption assay, we demonstrated that parathyroid hormone (PTH)-stimulated bone resorption was reduced in these mice. Evaluation of the bone histomorphometric dynamic parameters showed that the mineralizing surfaces and bone formation rate were both reduced. There was no change in the mineralization lag time or number of osteocyte lacunae. Using primary osteoblast culture and molecular analysis, we showed that the differentiation and function of osteoblasts from transgenic mice were normal, but that the ex vivo formation of mineralized nodules was delayed. This model is the first to show that in vivo MMPs play a role in bone remodeling and bone balance. Moreover, our data suggest that MMP activity could be involved in the hormonal regulation of bone resorption by osteoblasts.
The Balance Between Concurrent Activation of ERs and PPARs Determines Daidzein-Induced Osteogenesis and Adipogenesis
Authors: Dang ZhiChao , Löwik Clemens WGM
Published: April 2006

The soy phytoestrogen daidzein has biphasic dose responses, but the underlying mechanisms are not yet clear. Transcriptional and biochemical data show that PPARs, in addition to ERs, are molecular targets of daidzein, which divergently regulates osteogenesis and adipogenesis. Dose responses are the result of a balance among PPARs and between ERs and PPARs.

Introduction: Soy phytoestrogens have been used for the purposes of treatment and prevention of osteoporosis. Biphasic dose responses of daidzein, one of the main soy phytoestrogens, have long been recognized, but the underlying molecular mechanisms of action are not yet clear.

Materials and Methods: Mouse bone marrow cells and mouse osteoprogenitor KS483 cells that concurrently differentiate into osteoblasts and adipocytes were cultured. Biochemical measurement of alkaline phosphatase (ALP) activity, RT-PCR, and gene reporter assays were used in this study.

Results: Daidzein, one of the major soy phytoestrogens, had biphasic effects on osteogenesis and adipogenesis. Daidzein stimulated osteogenesis (ALP activity and nodule formation) and decreased adipogenesis (the number of adipocytes) at concentrations below 20 μM, whereas it inhibited osteogenesis and stimulated adipogenesis at concentrations higher than 30 μM. When estrogen receptors (ERs) were blocked by ICI182,780, daidzein-induced effects were not biphasic. A decrease in osteogenesis and an increase in adipogenesis were observed at the concentrations higher than 20 and 10 μM, respectively. In addition to ERs, daidzein transactivated not only peroxisome proliferator-activate receptor γ (PPARγ), but also PPARα and PPARδ at micromolar concentrations. Activation of PPARα had no direct effects on osteogenesis and adipogenesis. In contrast, activation of PPARδ stimulated osteogenesis but had no effects on adipogenesis, whereas PPARγ inhibited osteogenesis and stimulated adipogenesis. Transfection experiments show that an activation of PPARα or PPARγ by daidzein downregulated its estrogenic transcriptional activity, whereas activation of PPARδ upregulated its estrogenic transcriptional activity. Activation of ERα or ERβ by daidzein downregulated PPARγ transcriptional activity but had no influence on PPARα or PPARδ transcriptional activity.

Conclusions: Daidzein at micromolar concentrations concurrently activates different amounts of ERs and PPARs, and the balance of the divergent actions of ERs and PPARs determines daidzein-induced osteogenesis and adipogenesis.
Maintenance of Increased Bone Mass After Recombinant Human Parathyroid Hormone (1-84) With Sequential Zoledronate Treatment in Ovariectomized Rats
Authors: Rhee Yumie , Won Ye-Yeon , Baek Myong-Hyun , Lim Sung-Kil
Published: April 2006

The concept of lose, restore, maintain (LRM) for reversing existing osteoporosis was tested in rats. The withdrawal of PTH results in the loss of the acquired bone mass, but sequential therapy with zoledronate quite effectively maintained the PTH(1-84)-acquired bone quantity and quality.

Introduction: Because antiresorptive agents against osteoporosis are presently quite limited, strong anabolic agents such as human parathyroid hormone (hPTH) are quite helpful. However, because hPTH(1-34) is available only through injection and has a critical side effect of causing bone tumors during life-long administration in the rat, it would be practical to use PTH for the shortest possible duration to obtain the maximal effect. To determine the effectiveness of the osteoporosis-reversing concept of lose, restore, and maintain (LRM), recombinant hPTH(1-84) [rhPTH(1-84)] and the respective antiresorptive agents were sequentially studied.

Materials and Methods: Thirty-six, 20-week-old Sprague-Dawley rats were used. Treatment started at the 25th week after ovariectomy, which was performed at 20 weeks of age, with 5 weeks of rhPTH(1-84) 100 μg/kg/day, 5 days/week, followed by the respective sequential therapies for 5 weeks as follows: (1) ovariectomized rats (OVX; n = 6), (2) sham-operated rats (SHAM; n = 6), (3) OVX rats with PTH maintenance (PTH-M; n = 6), (4) OVX rats treated with PTH and then PTH was withdrawn (PTH-W; n = 6), (5) PTH-treated OVX rats treated with 17β-estradiol (PTH-E; 10 μg/day SC, 5 days/week; n = 6), and (6) PTH-treated OVX rats treated with zoledronate (PTH-Z; 12.5 μg/kg SC weekly; n = 6). BMD of the right femora was measured by DXA. μCT was used to measure the structural parameters of the second lumbar vertebrae. Three-point bending test of the femora and compressive tests of vertebrae were also performed.

Results: Bone quantity data showed that the BMD and most of the microstructural parameters were significantly higher in the PTH-M and PTH-Z groups than in the OVX and PTH-W groups (p < 0.05). Measurement of the cortical thickness revealed that only the PTH-M group showed a significant increase (p = 0.001). The ultimate force (Fu) at the midshaft of the femora was similar in the treated groups and stronger than in the OVX group (p < 0.05). However, in the vertebrae, the Fu of the PTH-M and PTH-Z groups was significantly higher, by ∼44-47%, than in the OVX and PTH-E groups and showed a higher tendency than in the PTH-W group.

Conclusion: PTH withdrawal resulted in the loss of acquired BMD, and sequential therapy with antiresorptives prevented further loss (17β-estradiol versus zoledronate). The zoledronate after rhPTH(1-84) as a sequential regimen was quite consistently effective.
Seasonal Periodicity of Serum Vitamin D and Parathyroid Hormone, Bone Resorption, and Fractures: The Geelong Osteoporosis Study
Authors: Pasco Julie A , Henry Margaret J , Kotowicz Mark A , Sanders Kerrie M , Seeman Ego , Pasco John R , Schneider Hans G , Nicholson Geoffrey C
Published: April 2006

In this population-based study, seasonal periodicity was seen with reduced serum vitamin D, increased serum PTH, and increased bone resorption in winter. This was associated with an increased proportion of falls resulting in fracture and an increased risk of wrist and hip fractures.

Introduction: In a population of women who reside in a temperate climate and do not generally receive dietary vitamin D supplementation, we investigated whether seasonal vitamin D insufficiency is associated with increased risk of fracture.

Materials and Methods: An observational, cross-sectional, population-based study set in southeastern Australia (latitude 38–39° S). Participants were drawn from a well-defined community of 27,203 women ≥55 years old: 287 randomly selected from electoral rolls, 1635 with incident fractures, and 1358 presenting to a university hospital with falls. The main outcome measures were annual periodicities of ultraviolet radiation, serum 25-hydroxyvitamin D [25(OH)D], serum parathyroid hormone (PTH), serum C-telopeptide (CTx), BMD, falls, and fractures.

Results: Cyclic variations in serum 25(OH)D lagged 1 month behind ultraviolet radiation, peaking in summer and dipping in winter (p < 0.001). Periodicity of serum PTH was the inverse of serum 25(OH)D, with a phase shift delay of 1 month (p = 0.004). Peak serum CTx lagged peak serum PTH by 1–2 months. In late winter, a greater proportion of falls resulted in fracture (p < 0.001). Seasonal periodicity in 439 hip and 307 wrist fractures also followed a simple harmonic model (p = 0.078 and 0.002, respectively), peaking 1.5–3 months after the trough in 25(OH)D.

Conclusions: A fall in 25(OH)D in winter is accompanied by increases in (1) PTH levels, (2) bone resorption, (3) the proportion of falls resulting in fracture, and (4) the frequency of hip and wrist fracture. Whether vitamin D supplementation in winter can reduce the population burden of fractures requires further investigation.
NF1 Tumor Suppressor Protein and mRNA in Skeletal Tissues of Developing and Adult Normal Mouse and NF1-Deficient Embryos
Authors: Kuorilehto Tommi , Nissinen Marja , Koivunen Jussi , Benson M Douglas , Peltonen Juha
Published: April 2006

NF1 is a heritable disease with multiple osseous lesions. The expression of the NF1 gene was studied in embryonic and adult rodent skeleton and in NF1-deficient embryos. The NF1 gene was expressed intensely in the cartilage and the periosteum. Impaired NF1 expression may lead to inappropriate development and dynamics of bones and ultimately to the osseous manifestations of the disease.

Introduction: Neurofibromatosis type 1 is caused by mutations in the NF1 gene encoding the Ras GTPase activating protein (Ras-GAP) neurofibromin. Skeletal ailments such as short stature, kyphoscoliosis, and tibial bowing and pseudarthrosis are common osseous manifestations of NF1. These symptoms are congenital, implying a role for neurofibromin in proper bone growth. However, little is known about its expression in skeletal tissues during their development.

Materials and Methods: The expression of the NF1 gene was studied in normal and NF1+/− mouse fetuses at embryonic days 12.5-15.5 and in skeletal tissues of adult mice and rats. In situ hybridization, immunohistochemistry, and Western blot analysis were used to identify the NF1 gene expression profile.

Results: NF1 mRNA and protein were elevated in resting, maturation, and hypertrophic chondrocytes at the growth plate. Parallel studies on NF1+/− embryos showed expression patterns identical to wildtype. The periosteum, including osteoblasts and osteoclasts, and osteocytes of the cortical bone of adult mice were also intensely labeled for NF1 protein and mRNA. Western transfer analysis detected NF1 protein in the respective rat tissues. Phosphorylation of p42 and p44 MAP kinases, the downstream consequence of Ras activation, was elevated in hypertrophic chondrocytes of NF1+/− embryos.

Conclusions: The results suggest that neurofibromin may act as a Ras-GAP in skeletal cells to attenuate Ras transduced growth signals and thus play a role during ossification and dynamics of bone. Loss of NF1 function may therefore lead to dysplastic bone growth, thereby causing the debilitating osseous symptoms of NF1.
Cadmium Exposure and Distal Forearm Fractures
Authors: Alfvén Tobias , Elinder Carl-Gustaf , Hellström Lennart , Lagarde Frédéric , Järup Lars
Published: April 2006

The aim of this study was to analyze the relationship between low-level cadmium exposure and distal forearm fractures. Altogether, 1021 men and women exposed to cadmium in Sweden were included. The study indicates that cadmium exposure is associated with increased risk of forearm fractures in people over the age of 50.

Introduction: Very few studies have been performed on environmental risk factors for fractures. Cadmium is known to cause damage to the kidneys and in high doses to the bone. The aim of this study was to analyze the relationship between low-level cadmium exposure and distal forearm fractures.

Materials and Methods: A total of 479 men and 542 women, 16-81 years of age, that were environmentally or occupationally exposed to cadmium were examined in 1997. Cadmium in urine was used to estimate dose, and information about previous fractures and risk factors for fractures was obtained from questionnaires. Fractures were validated using medical records. The association between cadmium dose and risk of forearm fracture was evaluated using Cox proportional hazard regression analysis.

Results and Conclusion: The mean urinary cadmium in the study population was 0.74 nmol cadmium/mmol creatinine (10% and 90% percentiles are 0.19 and 1.42, respectively). For fractures occurring after the age of 50 years (n = 558, 32 forearm fractures), the fracture hazard ratio, adjusted for gender and other relevant co-variates, increased by 18% (95% CI, 1.0-38%) per unit urinary cadmium (nmol cadmium/mmol creatinine). When subjects were grouped in exposure categories, the hazard ratio reached 3.5 (90% CI, 1.1, 11) in the group of subjects with urinary cadmium between 2 and 4 nmol/mmol creatinine and 8.8 (90% CI, 2.6, 30) in the group of subjects with ≥4 nmol/mmol creatinine. Associations between cadmium and fracture risk were absent before the age of 50. Cadmium exposure is associated with increased risk of forearm fractures in people over 50 years of age.
Two Novel Mutations at Exon 8 of the Sequestosome 1 (SQSTM1) Gene in an Italian Series of Patients Affected by Paget's Disease of Bone (PDB)
Authors: Falchetti Alberto , Di Stefano Marco , Marini Francesca , Del Monte Francesca , Mavilia Carmelo , Strigoli Debora , De Feo Maria L , Isaia Giovan , Masi Laura , Amedei Antonietta , Cioppi Federica , Ghinoi Valentina , Bongi Susanna Maddali , Di Fede Giuseppina , Sferrazza Carmela , Rini Giovan B , Melchiorre Daniela , Matucci-Cerinic Marco , Brandi Maria L
Published: April 2006

PDB is genetically heterogeneous. Mutations of the sequestosome1 gene have been reported in sporadic and familial forms of Paget's in patients of French Canadian and British descent. Mutational analyses in different ethnic groups are needed to accurately investigate hereditary diseases. We describe two novel mutations of sequestosome1 in 62 Italian sporadic patients, confirming the role of the encoded protein in this disorder.

Introduction: Paget's disease of bone (PDB) is a relatively common disease of bone metabolism reported to affect up to 3% of whites over 55 years of age. The disorder is genetically heterogeneous, and at present, there is scientific evidence that at least eight different human chromosomal loci are correlated with its pathogenesis. Mutations of the sequestosome1 (SQSTM1) gene were identified as responsible for most of the sporadic and familial forms of Paget in patients of French Canadian and British descent. Such mutations were located at exon 7 and 8 levels, encoding for the ubiquitin protein-binding domain (UBA) and representing a mutational hot spot area.

Materials and Methods: To verify the involvement of this gene in Italian subjects affected by PDB, we performed mutational analysis in 62 sporadic PDB cases.

Results: We described three different mutations at exon 8 level: P392L, already described in the French Canadian population and families predominantly of British descendent, and two novel mutations consisting of the amino acid substitutions M404V and G425R. No significant differences in the clinical history of PDB have been observed in patients with SQSTM1 mutations in respect to those without.

Conclusions: Even though our findings suggest a minor involvement of the SQSTM1 gene in the pathogenesis of sporadic Italian Paget's cases, the identification of different significant mutations within the SQSTM1 gene in unrelated, but clinically similar individuals, offers extremely convincing evidence for a causal relationship between this gene and PDB. Longitudinal studies are needed to assess the penetrance of genotype/phenotype correlations. Our findings confirm the evidence of a clustered mutation area at this level in this disorder.
Genetically Modified Animal Models as Tools for Studying Bone and Mineral Metabolism
Authors: Davey Rachel A , MacLean Helen E , McManus Julie F , Findlay David M , Zajac Jeffrey D
Published: April 2006

Genetic modification of mice is a powerful tool for the study of bone development and metabolism. This review discusses the advantages and disadvantages of various approaches used in bone-related research and the contributions these studies have made to bone biology.

Genetic modification of mice is a powerful tool for the study of bone development and metabolism. This review discusses the advantages and disadvantages of various approaches used in bone-related research and the contributions these studies have made to bone biology. The approaches to genetic modification included in this review are (1) overexpression of genes, (2) global gene knockouts, (3) tissue-specific gene deletion, and (4) gene knock-in models. This review also highlights issues that should be considered when using genetically modified animal models, including the rigorous control of genetic background, use of appropriate control lines, and confirmation of tissue specificity of gene expression where appropriate. This technology provides a unique and powerful way to probe the function of genes and is already revolutionizing our approach to understanding the physiology of bone development and metabolism.
Associations Between Baseline Risk Factors and Vertebral Fracture Risk in the Multiple Outcomes of Raloxifene Evaluation (MORE) Study
Authors: Johnell Olof , Kanis John A , Black Dennis M , Balogh Adam , Poor Gyula , Sarkar Somnath , Zhou Chunmei , Pavo Imre
Published: April 2006

Different risk factors may influence the effectiveness of osteoporosis therapies. The interaction of 30 baseline risk factors and the effectiveness of raloxifene in the MORE study were assessed. The efficacy of raloxifene in reducing vertebral fractures is largely independent of the presence of clinical risk factors for osteoporotic fractures.

Introduction: The aim of this analysis was to determine the effect of different risk factors on the effectiveness of raloxifene to reduce vertebral fractures in the Multiple Outcomes of Raloxifene Evaluation (MORE) study using logistic regression models.

Materials and Methods: The association was assessed using univariate analyses and a multivariate model between 30 potential risk factors at baseline and the risk of vertebral fractures after 3 years in the placebo group, as well as the interaction of risk factors with raloxifene therapy (at a dose of 60 or 120 mg/day).

Results and Conclusions: In the univariate analysis of the placebo group, after adjusting for baseline lumbar spine BMD (LS BMD), short stature (odds ratio [OR] = 1.18), age (OR = 1.38), years since menopause (OR = 1.38), impaired cognitive function, visuospatial capabilities (OR = 1.19), impaired musculoskeletal strength (OR = 1.23), low femoral neck BMD (OR = 1.21), and prior vertebral fracture (OR = 4.95) were significantly associated with the incidence of new vertebral fractures. In the univariate analysis, significant interactions were observed between raloxifene treatment and age (p = 0.04), serum triglycerides (p = 0.03), LS BMD (p = 0.08), and diabetes mellitus (p = 0.04). In the multivariate analysis, the effectiveness of raloxifene was independent of almost all risk factors, with the exception of baseline serum triglyceride level and LS BMD, suggesting an increased efficacy of raloxifene in patients with increased triglyceride levels (p = 0.006) and lower LS BMD values (p = 0.008) at baseline. These data suggest that the efficacy of raloxifene in reducing vertebral fractures is largely independent of the presence of clinical risk factors for osteoporotic fractures.
Risk Factors for Incident Vertebral Fractures in Men and Women: The Rotterdam Study
Authors: van der Klift Marjolein , de Laet Chris EDH , McCloskey Eugene V , Johnell Olof , Kanis John A , Hofman Albert , Pols Huibert AP
Published: April 2006

Low BMD and prevalent vertebral fractures are known risk factors for incident vertebral fractures. In 3001 men and women from the Rotterdam Study, prevalent nonvertebral fractures, early menopause, current smoking, and walking aid use were also strong risk factors for incident vertebral fractures.

Introduction: Thus far, age, low BMD, and prevalent vertebral fractures are the only well-known risk factors for incident vertebral fractures. Therefore, our aim was to investigate other potential risk factors for incident vertebral fractures in the elderly.

Materials and Methods: This study was based on the Rotterdam Study, a large prospective population-based cohort study among men and women ≥55 years of age. For 3001 subjects, spinal radiographs were obtained at baseline and again ∼6.3 years later. These follow-up radiographs were scored for vertebral fractures using the McCloskey-Kanis method. Whenever a vertebral fracture was detected, the radiograph was compared with the baseline radiograph. If this fracture was not already present at baseline, it was considered incident. At baseline, information on potential risk factors was obtained.

Results: Low BMD and prevalent vertebral fractures were strong risk factors for incident vertebral fractures in both men and women (RR 2.3 [1.6-3.3] and 2.2 [0.9-5.0] for men and RR 2.1 [1.6-2.6] and 4.1 [2.5-6.7] for women, respectively). For women, age, early menopause (≤45 years of age; RR 1.0 [1.1-3.5]), current smoking (2.1 [1.2-3.5]), and walking aid use (2.5 [1.1-5.5]) were additional independent risk factors. For men, only a history of nonvertebral fractures was a significant independent risk factor (OR 2.4 [1.2-4.8]).

Conclusion: Apart from low BMD and prevalent vertebral fractures, prevalent nonvertebral fractures are associated with an increased incident vertebral fracture risk in men. In women, early menopause, current smoking, and walking aid use are additional independent risk factors for incident vertebral fractures.
Novel UBA Domain Mutations of SQSTM1 in Paget's Disease of Bone: Genotype Phenotype Correlation, Functional Analysis, and Structural Consequences
Authors: Hocking Lynne J , Lucas Gavin JA , Daroszewska Anna , Cundy Tim , Nicholson Geoff C , Donath Judit , Walsh John P , Finlayson Catriona , Cavey James R , Ciani Barbara , Sheppard Paul W , Searle Mark S , Layfield Robert , Ralston Stuart H
Published: April 2006

Three novel missense mutations of SQSTM1 were identified in familial PDB, all affecting the UBA domain. Functional and structural analysis showed that disease severity was related to the type of mutation but was unrelated to the polyubiquitin-binding properties of the mutant UBA domain peptides.

Introduction: Mutations affecting the ubiquitin-associated (UBA) domain of Sequestosome 1 (SQSTM1) gene have recently been identified as a common cause of familial Paget's disease of bone (PDB), but the mechanisms responsible are unclear. We identified three novel SQSTM1 mutations in PDB, conducted functional and structural analyses of all PDB-causing mutations, and studied the relationship between genotype and phenotype.

Materials and Methods: Mutation screening of the SQSTM1 gene was conducted in 70 kindreds with familial PDB. We characterized the effect of the mutations on structure of the UBA domain by protein NMR, studied the effects of the mutant UBA domains on ubiquitin binding, and looked at genotype-phenotype correlations.

Results and Conclusions: Three novel missense mutations affecting the SQSTM1 UBA domain were identified, including a missense mutation at codon 411 (G411S), a missense mutation at codon 404 (M404V), and a missense mutation at codon 425 (G425R). We also identified a deletion leading to a premature stop codon at 394 (L394X). None of the mutations were found in controls. Structural analysis showed that M404V and G425R involved residues on the hydrophobic surface patch implicated in ubiquitin binding, and consistent with this, the G425R and M404V mutants abolished the ability of mutant UBA domains to bind polyubiquitin chains. In contrast, the G411S and P392L mutants bound polyubiquitin chains normally. Genotype-phenotype analysis showed that patients with truncating mutations had more extensive PDB than those with missense mutations (bones involved = 6.05 ± 2.71 versus 3.45 ± 2.46; p < 0.0001). This work confirms the importance of UBA domain mutations of SQSTM1 as a cause of PDB but shows that there is no correlation between the ubiquitin-binding properties of the different mutant UBA domains and disease occurrence or extent. This indicates that the mechanism of action most probably involves an interaction between SQSTM1 and a hitherto unidentified protein that modulates bone turnover.
Association of Five Quantitative Ultrasound Devices and Bone Densitometry With Osteoporotic Vertebral Fractures in a Population-Based Sample: The OPUS Study
Authors: Dr Glüer Claus C , Eastell Richard , Reid David M , Felsenberg Dieter , Roux Christian , Barkmann Reinhard , Timm Wolfram , Blenk Tilo , Armbrecht Gabi , Stewart Alison , Clowes Jackie , Thomasius Friederike E , Kolta Sami
Published: April 2006

We compared the performance of five QUS devices with DXA in a population-based sample of 2837 women. All QUS approaches discriminated women with and without osteoporotic vertebral fractures. QUS of the calcaneus performed as well as central DXA.

Introduction: Quantitative ultrasound (QUS) methods have found widespread use for the assessment of bone status in osteoporosis, but their optimal use remains to be established. To determine QUS performance for current devices in direct comparison with central DXA, we initiated a large population-based investigation, the Osteoporosis and Ultrasound Study (OPUS).

Materials and Methods: A total of 463 women 20–39 years of age and 2374 women 55–79 years of age were measured on five different QUS devices along with DXA of the spine and the proximal femur. Their vertebral fracture status was evaluated radiographically. The association of QUS and DXA with vertebral fracture status was evaluated using logistic regression.

Results: All QUS approaches tested discriminated women with and without osteoporotic vertebral fractures (20% height reduction), with age-adjusted standardized odds ratios ranging 1.2–1.3 for amplitude-dependent speed of sound (AD-SOS) at the finger phalanges, 1.2–1.4 for broadband ultrasound attenuation (BUA) at the calcaneus, and 1.4–1.5 for speed of sound (SOS) at the calcaneus, 1.4–1.6 for DXA of the total femur, and 1.5–1.6 for DXA at the spine. For more severe fractures (40% height reduction), age-adjusted standardized odds ratios increased to up to 1.9 for DXA of the spine and 2.3 for SOS of the calcaneus.

Conclusions: In conclusion, all five QUS devices tested showed significant age-adjusted differences between subjects with and without vertebral fracture. When selecting the strongest variable, QUS of the calcaneus worked as well as central DXA for identification of women at high risk for prevalent osteoporotic vertebral fractures. QUS-based case-finding strategies would allow halving the number of radiographs in high-risk populations, and this strategy works increasingly well for women with more severe vertebral fractures. It is likely that the good performance of QUS was in part achieved by rigorous quality assurance measures that should also be used in clinical practice.
A Single-Dose Placebo-Controlled Study of AMG 162, a Fully Human Monoclonal Antibody to RANKL, in Postmenopausal Women
Authors: Bekker Pirow J , Holloway Donna L , Rasmussen Amy S , Murphy Robyn , Martin Steven W , Leese Philip T , Holmes Gregory B , Dunstan Colin R , DePaoli Alex M
Published: April 2006

The safety and bone antiresorptive effect of a single subcutaneous dose of AMG 162, a human monoclonal antibody to RANKL, was investigated in 49 postmenopausal women. AMG 162 is a potent antiresorptive agent for diseases such as osteoporosis.

Introduction: RANKL is an essential osteoclastic differentiation and activation factor.

Materials and Methods: The bone antiresorptive activity and safety of AMG 162, a fully human monoclonal antibody to RANKL, were evaluated in postmenopausal women in this randomized, double-blind, placebo-controlled, single-dose, dose escalation study. Six cohorts of eight to nine women were randomly assigned to receive a single subcutaneous injection of either AMG 162 or placebo (3:1 ratio). AMG 162 doses were 0.01, 0.03, 0.1, 0.3, 1.0, and 3.0 mg/kg. Subjects were followed up to 6 months in all cohorts and 9 months in the three highest dose cohorts. Second morning void urinary N-telopeptide/creatinine (NTX; Osteomark), serum NTX, and serum bone-specific alkaline phosphatase (BALP, Ostase) were assessed as bone turnover markers.

Results and Conclusions: Forty-nine women were enrolled. A single subcutaneous dose of AMG 162 resulted in a dose-dependent, rapid (within 12 h), profound (up to 84%), and sustained (up to 6 months) decrease in urinary NTX. At 6 months, there was a mean change from baseline of −81% in the 3.0 mg/kg AMG 162 group compared with −10% in the placebo group; serum NTX changes were −56% and 2%, respectively. BALP levels did not decrease remarkably until after 1 month, indicating that the effect of AMG 162 is primarily antiresorptive. Intact parathyroid hormone (PTH) levels increased up to ∼3-fold after 4 days in the 3.0 mg/kg dose group, but returned toward baseline with follow-up. Albumin-adjusted serum calcium did not decrease >10% on average in any group, and no subject had values below 2 mmol/liter. AMG 162 was well tolerated. No related serious adverse events occurred. No clinically meaningful laboratory changes, other than those described above, were observed. In summary, a single subcutaneous dose of AMG 162 resulted in a dose-dependent rapid and sustained decrease from baseline in bone turnover and could be an effective and convenient treatment for osteoporosis.
Effects of Estrogen Replacement Therapy on Bone Turnover in Subchondral Bone and Epiphyseal Metaphyseal Cancellous Bone of Ovariectomized Cynomolgus Monkeys
Authors: Ham Kimberley D , Carlson Cathy S
Published: April 2006

ERT decreases the severity of OA in OVX cynomolgus monkeys. We show that bone formation is greater in subchondral bone compared with epiphyseal/metaphyseal cancellous bone of the proximal tibia in these animals and that ERT decreases bone formation in both sites. ERT may decrease the risk of OA by decreasing bone formation in the SC bone.

Introduction: Estrogen replacement therapy (ERT) decreases the risk of osteoporosis and osteoarthritis (OA) in postmenopausal women and has been shown to have direct effects on cells of the bone and cartilage. The effects of ERT have been studied extensively in cancellous bone, but subchondral (SC) bone directly beneath the articular cartilage has not been specifically evaluated.

Materials and Methods: Adult feral female cynomolgus monkeys were bilaterally ovariectomized (OVX) to simulate menopause; treated with ERT, soy phytoestrogens (SPE), or no hormones (OVX control group) for 3 years; and labeled with calcein before necropsy. At necropsy, the proximal tibias of 20 randomly selected animals from each treatment group were embedded in bioplastic and sectioned. Areas and labels were measured in a carefully defined region of the SC bone and epiphyseal/metaphyseal cancellous (EMC) bone, and derived dynamic and static indices were compared between the SC and EMC bone and among the three treatment groups. Student's t-tests and ANOVA were used to compare the data.

Results and Conclusions: In both the SC and EMC bone, most of the values for the dynamic indices were highest in the OVX control group, intermediate in the SPE group, and lowest in the ERT group. The mineralizing surface, double-labeled surface, and bone formation rate (surface referent) were significantly higher in the SC bone compared with the EMC bone in the OVX control group. The trabecular bone volume was higher in the SPE-treated group compared with the OVX control group. In conclusion, the bone turnover indices were higher in the SC bone compared with the EMC bone, and ERT decreased these indices in both sites. In addition, SPE was protective against loss of bone volume.
Lifestyle Influences on 9-Year Changes in BMD in Young Women
Authors: Mein Anna L , Briffa N Kathryn , Dhaliwal Satvinder S , Price Roger I
Published: April 2006

The effects of dietary calcium intake and physical activity on longitudinal changes in BMD over a mean of 9.4 years were examined in 62 healthy young women. Proximal femur BMD declined, lumbar spine BMD increased, and physical activity was associated with BMD change at intertrochanter and total hip sites.

Introduction: Maximizing premenopausal BMD is an important strategy for the prevention of osteoporosis and resultant fractures later in life.

Materials and Methods: Young women who previously participated in a placebo-controlled 2-year calcium intervention study at a mean age of 18.5 ± 0.3 years were remeasured at 27.8 ± 1.0 years of age. DXA (Hologic QDR 1000W) was used to measure changes in BMD, and lifestyle factors were ascertained by questionnaire.

Results and Conclusions: Early decline in BMD at the neck of femur (−3.3%/decade) and the converse gain in BMD at the lumbar spine (+4.3%/decade) and intertrochanter (+1.9%/decade) suggest site-specific changes in BMD in young premenopausal women. No effect of previous calcium supplementation was seen on current BMD or changes in BMD (p > 0.10). Lifestyle predictors of change in BMD were determined using hierarchical regression analysis after forced correction for the covariates baseline BMD and previous calcium supplementation. Physical activity was positively associated with change in BMD at total hip and intertrochanter sites (β-coefficients, β = 0.26 and 0.26 respectively; p < 0.05). Calcium intake was negatively associated with change in BMD at the lumbar spine (β = −0.27, p < 0.05). Parity was negatively associated with change in BMD at all sites (β = −0.40 to −0.26, p < 0.05). These data show that BMD is already declining at the proximal femur in these healthy young women. Physical activity assists in maintenance of BMD at some sites and thus may contribute to lifelong fracture prevention. There was no positive association between calcium intake and change in BMD.