bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression of Measles Virus Nucleocapsid Protein in Osteoclasts Induces Paget's Disease-Like Bone Lesions in Mice
Authors: Kurihara Noriyoshi , Zhou Hua , Reddy Sakamuri V , Palacios Veronica Garcia , Subler Mark A , Dempster David W , Windle Jolene J , Roodman G David
Published: April 2006

We targeted the MVNP gene to the OCL lineage in transgenic mice. These mice developed abnormal OCLs and bone lesions similar to those found in Paget's patients. These results show that persistent expression of MVNP in OCLs can induce pagetic-like bone lesions in vivo.

Introduction: Paget's disease (PD) of bone is the second most common bone disease. Both genetic and viral factors have been implicated in its pathogenesis, but their exact roles in vivo are unclear. We previously reported that transfection of normal human osteoclast (OCL) precursors with the measles virus nucleocapsid (MVNP) or measles virus (MV) infection of bone marrow cells from transgenic mice expressing a MV receptor results in formation of pagetic-like OCLs.

Materials and Methods: Based on these in vitro studies, we determined if the MVNP gene from either an Edmonston-related strain of MV or a MVNP gene sequence derived from a patient with PD (P-MVNP), when targeted to cells in the OCL lineage of transgenic mice with the TRACP promoter (TRACP/MVNP mice), induced changes in bone similar to those found in PD.

Results: Bone marrow culture studies and histomorphometric analysis of bones from these mice showed that their OCLs displayed many of the features of pagetic OCLs and that they developed bone lesions that were similar to those in patients with PD. Furthermore, IL-6 seemed to be required for the development of the pagetic phenotype in OCLs from TRACP/MVNP mice.

Conclusions: These results show that persistent expression of the MVNP gene in cells of the OCL lineage can induce pagetic-like bone lesions in vivo.
Long-Term Fracture Prediction by DXA and QUS: A 10-Year Prospective Study
Authors: Stewart Alison , Kumar Vinod , Reid David M
Published: April 2006

This study investigated the ability of DXA and QUS to predict fractures long term when measured around the time of the menopause. We found both DXA and QUS are able to predict both any fracture and “osteoporotic” fractures and that QUS can predict independently of BMD.

Introduction: There are now many treatments available for prevention of osteoporotic fracture. To be cost-effective, we need to target those most at risk. This study examines the ability of DXA and QUS to predict fractures in an early postmenopausal population of women.

Materials and Methods: We prospectively measured 3883 women who had been randomly selected from a community-based register. At baseline, they were measured using DXA of spine and hip (Norland XR-26) and QUS of the heel (Walker Sonix UBA 575). Follow-up had a mean of 9.7 ± 1.1 (SD) years. All incident fractures were identified and validated by examination of X-ray reports, and these were compared with those without fracture in a Cox-regression model to calculate hazard ratios (HRs).

Results: We found adjusted HRs for any fracture per 1 SD reduction in spine BMD to be 1.61 (1.42-1.83), whereas neck of femur BMD was 1.54 (1.34-1.75). Areas under the curve (AUC) for a receiver operator characteristic (ROC) analysis were 0.62 for spine BMD and 0.59 for neck BMD. In a subgroup where QUS was also measured, the HR for a 1 SD reduction in BMD was 1.69 (1.29-2.22) for spine BMD and 1.55 (1.17-2.06) for neck BMD. The HR for a 1 SD reduction in broadband ultrasound attenuation (BUA) was 1.53 (1.19-1.96), and 1.44 (1.12-1.86) when further adjusted for neck BMD. The AUCs were 0.63 for spine BMD, 0.59 for neck BMD, and 0.62 for BUA. When only osteoporotic fractures were examined, the HRs increased in all situations. BUA showed the highest HR of 2.25 (1.51-3.34), and when further adjusted for neck BMD was 2.12 (1.38-3.28).

Conclusions: In conclusion, it may be possible to scan women around the time of the menopause to predict future fractures. It seems that, for “osteoporotic” fractures, BUA may be an improved predictor of fractures in comparison with DXA, because the relative risk is highest for BUA, and independent of BMD.
The Mitogenic Effect of Parathyroid Hormone Is Associated with E2F-Dependent Activation of Cyclin-Dependent Kinase 1 (cdc2) in Osteoblast Precursors
Authors: Onishi Takehisa , Zhang Wenyu , Cao Xu , Hruska Keith
Published: April 2006

Injections of parathyroid hormone (PTH) have been reported to stimulate skeletal accretion through increased bone formation in several species, and osteoblast proliferation is a critical component of bone formation. However, the biological mechanisms of PTH-stimulated bone cell proliferation are largely unknown. In this study, we demonstrated that PTH stimulates proliferation of the osteoblast precursor cell line, TE-85, in association with increasing cdc2 protein levels and its kinase activity. cdc2 antisense oligonucleotides blocked PTH-induced DNA synthesis and cell cycle progression. Analysis of the time course of PTH-stimulated cdc2 message levels demonstrated that cdc2 mRNA levels were increased 1.5- to 4-fold between 3–18 h following release from cell synchronization. Transfections of TE-85 cells with a series of cdc2 promoter-luciferase deletion constructs revealed PTH stimulation of the cdc2 promoter. Promoter constructs containing a mutation in the E2F binding site were not stimulated by PTH. Gel mobility shift assays demonstrated increased free E2F levels in TE-85 nuclear extracts in response to PTH. Furthermore, the ratios of hyperphosphorylated to hypophosphorylated forms of Rb protein were increased by PTH treatment. These results demonstrate that PTH-stimulated cdc2 expression was associated with TE-85 cell proliferation and that the mechanism of stimulating cdc2 gene expression involved increasing the levels of free E2F.
Further Observations on Programmed Cell Death in the Epiphyseal Growth Plate: Comparison of Normal and Dyschondroplastic Epiphyses
Authors: Ohyama Kazuhiko , Farquharson Colin , Whitehead Colin C. , Shapiro Irving M.
Published: April 2006

The objective of the investigation was to provide information on apoptosis in the normal epiphysis and to assess apoptosis in the plate of the dyschondroplastic chick. Apoptosis was evaluated using two terminal deoxynucleotide transferase end-labeling procedures, DNA fragmentation and nuclear morphology. We found that there was a minimal level of apoptosis in the dyschondroplastic cartilage. In the tibial dyschondroplastic (TD) lesion itself, only about 3% of cells are positive in the articular and proliferative regions; 11% of prehypertrophic chondrocytes are stained by the end-labeling procedure, and most of the cells are localized around vascular channels at the calcifying front. This finding suggests that dyschondroplasia is linked to impairment of apoptosis, and as a result the tissue contains immature cells that have outlived their normal life span. In contrast, in the normal plate, we noted that when the proliferative period was complete, the cells became terminal transferase positive; in addition, chondrocytes in the normal plate exhibited DNA fragmentation. Semiquantitative analysis of stained chondrocytes in the growth plate indicate that in the proliferative zone 15.5% of cells are terminal deoxynucleotidyl transferase (TUNEL) positive; in contrast, 44% of postmitotic chondrocytes are stained by the TUNEL procedure. The presence of a sharp border between the pre- and postmitotic zones suggests that the stimulus for apoptosis is maturation dependent and reflects local metabolic control. We also examined apoptosis in metaphyseal osteoblasts. We found that adjacent to the epiphysis, many osteoblasts were undergoing apoptosis. In more mature sites in the metaphysis, there was less cell death, indicating that osteoblast apoptosis was delayed and cells were completing their normal life cycle. Although terminal transferase end-labeled cells were not seen in articular cartilage, we noted that fibroblasts, in the perichondrial ligament surrounding the articular as well as the epiphyseal regions of the plate, were undergoing apoptosis. Apoptosis at this site may be related to lateral expansion of the cartilages, reflect a high cell turnover rate at the junction between the tissues, and result from paracrine signals received from the underlying cartilage.
Interaction Between Nitric Oxide Synthase and Cyclooxygenase Pathways in Osteoblastic MC3T3-E1 Cells
Authors: Kanematsu Masahiro , Ikeda Kyoji , Yamada Yoshiji
Published: April 2006

Interleukin 1 (IL-1) and tumor necrosis factor α (TNF-α) have been implicated in the pathogenesis of osteoporosis. These proinflammatory cytokines induce both cyclooxygenase (COX) and nitric oxide synthase (NOS) with the release of prostaglandin (PG) and NO, respectively. The present study was undertaken to examine the interaction between COX and NOS pathways and their role in the regulation of osteoblastic function in MC3T3-E1 cells. Addition of IL-1α and TNF-α induced a marked increase in the production of both NO and PGE2. Reverse transcription–polymerase chain reaction analysis showed that the increase in NO production was preceded by the expression of inducible NOS mRNA. The temporal profile of PGE2 production revealed a biphasic pattern: the first small peak at 3 h was caused by de novo synthesis of PGE2 through inducible COX (COX-2) mRNA, while the subsequent progressive accumulation of PGE2 was mediated through the activation of COX pathway by NO since (1) aminoguanidine (AG), a selective inhibitor of inducible NOS, significantly suppressed the PGE2 production by IL-1α and TNF-α, (2) NOC-18, an NO donor, reversed this suppression, and (3) NOC-18 increased PGE2 production by itself. The increase in NO production in response to IL-1α and TNF-α was further stimulated by aspirin and inhibited by exogenous addition of PGE2, suggesting that PGE2 produced by the cytokines, in turn, negatively modulates NO production. IL-1α and TNF-α inhibited alkaline phosphatase (ALP) activity, which was significantly reversed by AG. NOC-18 not only suppressed ALP activity by itself but also blocked the effect of AG, suggesting the role of NO in the inhibition of ALP activity. PGE2 decreased ALP activity, and the inhibitory effect of NOC-18 was attenuated in the presence of aspirin, suggesting the involvement of PGE2 in the negative modulation of ALP activity by NO. These results suggest that NO produced in response to proinflammatory cytokines participates in the modulation of ALP activity via the activation of COX pathway. The interaction between NO and the COX pathways may play an important role in the regulation of osteoblastic functions under physiologic as well as pathologic conditions.
Cytokine-Induced Nitric Oxide Inhibits Bone Resorption by Inducing Apoptosis of Osteoclast Progenitors and Suppressing Osteoclast Activity
Authors: Van 't Hof Robert J. , Ralston Stuart H.
Published: April 2006

Interferon-γ (IFN-γ) has been shown to inhibit interleukin-1 (IL-1) and tumor necrosis factor α (TNF-α) stimulated bone resorption by strongly stimulating nitric oxide (NO) synthesis. Here we studied the mechanisms underlying this inhibition. Osteoclasts were generated in 10-day cocultures of mouse osteoblasts and bone marrow cells and the effect of cytokine-induced NO on osteoclast formation and activity was determined. Stimulation of the cocultures with IL-1β, TNF-α and IFN-γ markedly enhanced NO production by 50- to 70-fold, and this was found to be derived predominantly from the osteoblast cell layer. When high levels of NO were induced by cytokines during early stages of the coculture, osteoclast formation was virtually abolished and bone resorption markedly inhibited. Cytokine stimulation during the latter stages of coculture also resulted in inhibition of bone resorption, but here the effects were mainly due to an inhibitory effect on osteoclast activity. At all stages, however, the inhibitory effects of cytokines on osteoclast formation and activity were blocked by the NO-synthase inhibitor L-NMMA. Further investigations suggested that the NO-mediated inhibition of osteoclast formation was due in part to apoptosis of osteoclast progenitors. Cytokine stimulation during the early stage of the culture caused a large increase in apoptosis of bone marrow cells, and these effects were blocked by L-NMMA and enhanced by NO donors. We found no evidence of apoptosis in osteoblasts exposed to high levels of cytokine-induced NO at any stage in the culture, however, or of apoptosis affecting mature osteoclasts exposed to high levels of NO, suggesting that immature cells in the bone marrow compartment are most sensitive to NO-induced apoptosis. In summary, these studies identify NO as a potentially important osteoblast-osteoclast coupling factor which has potent inhibitory effects on bone resorption. These actions, in turn, are mediated by inhibition of osteoclast formation probably due to NO-induced apoptosis of osteoclast progenitors and by inhibition of the resorptive activity of mature osteoclasts.
Endogenous Sex Steroids and Bone Mineral Density in Older Women and Men: The Rancho Bernardo Study
Authors: Greendale Gail A. , Edelstein Sharon , Barrett-Connor Elizabeth
Published: April 2006

This study examines the associations between endogenous sex steroids and bone mineral density (BMD), using data from a geographically defined cohort in Rancho Bernardo, California. Participants were community-dwelling women and men aged 50–89 years who took part in a study of endogenous sex steroid measurement between 1984–1987 and who had BMD measured in 1988–1991. Those taking corticosteroids or estrogen at the time of sex steroid determination were excluded. The main study outcomes were BMD of the ultradistal radius, midshaft radius, lumbar spine, and total hip by sex steroid level, adjusted for age, body mass index, cigarette smoking, alcohol consumption, leisure exercise, use of thiazides, thyroid hormones, and former estrogen use (women only). At the time of the hormone measurements, the mean age of the 457 women was 72.1 years and that of the 534 men was 68.6 years. A statistically significant positive relation was seen between bioavailable estradiol and BMD at all sites in women and men. Total estradiol was significantly associated with BMD at all sites in women and at all but the ultradistal radius in men. Estrone had a global effect on BMD in women and was not measured in men. Higher bioavailable (but not total) testosterone levels were associated with higher BMD of the ultradistal radius, spine, and hip in men and the ultradistal radius in women. Dehydroepiandrosterone was positively associated with BMD of the midradius, spine, and hip in women and was not associated with BMD at any site in men. Of the sex steroids tested, bioavailable estrogen was most strongly associated with BMD in both women and men. We conclude that endogenous sex steroid levels are significantly related to bone density in older women and men. Individual variation in age-related bone loss may be partially accounted for by alterations in sex steroid levels with aging. Further study to elucidate safe environmental and medical methods to maintain optimal sex steroid levels in old age is needed.
Bone Density Variation and Its Effects on Risk of Vertebral Deformity in Men and Women Studied in Thirteen European Centers: The EVOS Study
Authors: Lunt M. , Felsenberg D. , Reeve J. , Benevolenskaya L. , Cannata J. , Dequeker J. , Dodenhof C. , Falch J. A. , Masaryk P. , Pols H. A. P. , Poor G. , Reid D. M. , Scheidt-Nave C. , Weber K. , Varlow J. , Kanis J. A. , O'NEILL T. W. , Silman A. J.
Published: April 2006

In Europe there is a 3-fold variation, according to geographical center, in risk of vertebral deformity in men and women over the age of 50. We investigated the relationship between bone density, as assessed by dual-energy X-ray absorptiometry (DEXA) of the spine and hip and prevalent vertebral deformities in 13 of the 36 centers participating in the European Vertebral Osteoporosis Study (EVOS). Each center recruited an age-stratified sample of men and women aged 50 years and over, and of those who agreed to densitometry, 288/2088 women and 233/1908 men were found to have one or more deformities of the vertebrae between T4 and L4 as assessed by the McCloskey algorithm. DEXA was in each case performed on L2–L4, the proximal femur, or both. Bone densitometry results were cross-calibrated between centers using the European Spine Phantom prototype and results expressed as bone mineral density (BMD, g/cm2). In both genders, subjects with deformities involving loss of anterior vertebral body height alone comprised over 20% of the total with deformities and these related poorly to BMD. Other classes of deformity were found by logistic regression to relate significantly to BMD in one or both genders, with odds ratios for the risk of any of these ranging from 1.67 to 2.11 for a 1 SD reduction in bone density at spine, femoral neck, or trochanter (p < 0.001). Adjusting for anthropometric variables and BMD did not remove the effect of age on risk which rose 1.67- to 1.78-fold per decade according to gender. The greater unadjusted rate of increase in deformity risk with age in women was attributable to their faster rate of bone loss with age; after adjusting for age, body mass index (BMI), and BMD at the trochanter in grams per square centimeter, men had a 2-fold higher risk of deformity than women. Analysis of the relationship between mean bone density and the prevalence of deformity in each center demonstrated no significant differences between centers in either gender, after adjusting for BMD, age, and BMI together with an a posteriori statistical adjustment for imperfect cross-calibration of densitometers. It is concluded that BMD is an important determinant of deformity risk in both genders. Together with age, BMD explains much of the differences in risk both between the sexes and between individual geographical centers in Europe.
Functional Characterization of the Human Biglycan 5′-Flanking DNA and Binding of the Transcription Factor c-Krox
Authors: Heegaard Anne-Marie , Robey Pamela Gehron , Vogel Walther , Just Walter , Widom Russell L. , Schøller Jørgen , Fisher Larry W. , Young Marian F.
Published: April 2006

The transcriptional regulation of human biglycan expression under normal and pathological conditions was studied. The 5′-flanking regions of the human and mouse genes were isolated and analyzed; the two promoter regions share 81% identity. Both promoters are without a TATA and CAT box and contain multiple Sp1 sites. Human dermal fibroblasts were transiently transfected with progressive deletional human biglycan 5′-flanking DNA-CAT constructs, and a significant variation in activity among the individual constructs was found. A small deletion in several cases caused a more than 2-fold increase or decrease in promoter activity, thereby mapping the target sites for repressors or activators. Human biglycan expression is reduced in females with Ullrich-Turner syndrome (45,X) and increased in individuals with supernumerary sex chromosomes, and it has been speculated that biglycan plays a role in the short stature phenotype of Turner syndrome. Analysis of the transcriptional regulation of biglycan in individuals with sex chromosome anomalies showed that a −262 to −218 region of the biglycan promoter was differentially regulated. This region was extensively analyzed by DNAse footprinting and electrophoretic mobility shift assays, and a putative binding site for the transcription factor c-Krox was discovered. The binding of c-Krox to a site located at approximately −248 to −230 in the human biglycan promoter was confirmed by using extracts from COS cells expressing recombinant human c-Krox. The expression of c-Krox in bone was then examined by reverse-transcribed polymerase chain reaction and Northern blotting analysis; an ∼3.4 kb transcript was detected in primary osteoblastic cells, in MG–63 cells, and in human bone marrow stromal cells. This is the first detection of c-Krox in bone cells, and it suggests that c-Krox, like another member of the Krox family, Krox–20, might play a regulatory role in bone.
Quadriceps and Grip Strength Are Related to Vitamin D Receptor Genotype in Elderly Nonobese Women
Authors: Geusens Piet , Vandevyver Caroline , Vanhoof Johan , Cassiman Jean-Jacques , Boonen Steven , Raus Jef
Published: April 2006

Osteoporotic fragility fractures are related to bone density and injury, which are both related to muscle strength. The influence of genetic factors, such as the vitamin D receptor (VDR) polymorphism on bone mineral density (BMD), is documented but still controversial, and is not known for muscle strength. In the present study, we investigated the association between the VDR BsmI polymorphism and BMD (femoral neck [FN], lumbar spine [LS], and proximal forearm [FA]) and muscle strength (quadriceps and grip strength) in 501 healthy women older than 70 years. No association was found between the VDR genotypes and BMD in elderly women. However, in nonobese women (body mass index <30 kg/cm2), the BMD in the FN was 5% higher in women with the bb BsmI genotype than in women with the BB genotype (p < 0.05). After correction for muscle strength, no association was found. A significant association between the VDR genotypes and quadriceps and grip strength was observed. In nonobese women, a 23% difference in quadriceps strength (p < 0.01) and 7% in grip strength (NS) was observed between the bb and BB genotype of the VDR. After correction for confounding factors and BMD, this association was significant for quadriceps and grip strength. These results indicate a major association of an allelic variant at the VDR locus with muscle strength in elderly nonobese women, which could explain a small association between VDR polymorphism with BMD in the femoral neck in nonobese women. No such associations were found in obese women, suggesting that factors related to obesity obscure such an association.
A Comparison of Longitudinal Measurements in the Spine and Proximal Femur Using Lunar and Hologic Instruments
Authors: Pocock Nicholas A. , Noakes Kate A. , Griffiths Mathew , Bhalerao Narayan , Sambrook Philip N. , Eisman John A. , Freund Judith
Published: April 2006

Absolute values of bone mineral density (BMD), using dual-energy X-ray absorptiometry (DXA), differ between instruments from different manufacturers. Despite these differences, the rates of change calculated from serial measurements on different densitometers have been assumed to be comparable. We compared the change in BMD in 34 subjects in the lumbar spine and at the standard sites in the proximal femur, from measurements performed using a Lunar DPX-L and a Hologic QDR–1000. Measurements were obtained on the same day, and repeated on the same day, on both machines after a mean interval of 4.8 years (range 4.1–6.3 years). There were strong positive correlations between the percentage change in BMD calculated using the two machines in the lumbar spine, trochanteric region, and total proximal femur: r = 0.82, 0.84, and 0.73, respectively (p < 0.0001 at all sites). In the femoral neck and in Ward's triangle, the correlations were not as high: r = 0.55 (p = 0.003) and 0.43 (p = 0.028), respectively. At all sites, despite the significant correlations, the agreement between the two densitometers was not high and there may be significant errors in individual subjects if one uses measurements from one densitometer to predict the change in BMD using the scanner of the other manufacturer. There is less of a problem comparing group data in the lumbar spine and trochanteric region, although errors are still likely to occur in comparing group data of bone loss, calculated using different densitometers, in the other proximal femur sites. In conclusion, the study suggests that caution is necessary in combining bone loss data derived using densitometers from different manufacturers, particularly in the proximal femur. This has important implications for multicenter studies.
Vitamin D Receptor Polymorphisms, Bone Mineral Density, and Bone Metabolism in Postmenopausal Mexican–American Women
Authors: McClure Laurel , Eccleshall T. Ross , Gross Coleman , Villa Marie Luz , Lin Nancy , Ramaswamy Vyjayanti , Kohlmeier Lynn , Kelsey Jennifer L. , Marcus Robert , Feldman David
Published: April 2006

Common polymorphisms in the vitamin D receptor (VDR) gene have been shown to correlate with bone mineral density (BMD). However, attempts to replicate the original findings in other populations have yielded variable results. These disparities may reflect ethnic or environmental differences in the expression of the VDR effect upon BMD. We examined a relatively ethnically homogeneous group of 103 healthy postmenopausal Caucasian women of Mexican descent living in Northern California. We determined the VDR genotype and measured the BMD at the lumbar spine and femoral neck by dual-energy X-ray absorptiometry, as well as several biochemical indices of mineral metabolism. The prevalence of the BB genotype, associated in previous studies with the lowest BMD, was 8% and highly linked to the tt genotype. Absolute and age-adjusted BMD at both hip and spine showed a trend toward lower BMD in the BB, AA, and tt genotypes, but this trend did not achieve statistical significance. There were no consistent intergroup differences in change in BMD over 2 years of follow-up, nor in mean serum concentrations of 25-hydroxyvitamin D, 1,25-dihydroxyvitamin D, osteocalcin, or total urinary pyridinolines. Intact parathyroid hormone concentrations were significantly higher in subjects with the AA genotype, with a trend toward higher values in those with the BB and tt genotypes as well. Our data suggest that there may be a decrease in BMD associated with the B, A, and t alleles, but the intergroup difference in BMD is 0.2–0.5 standard deviations (SD) at the lumbar spine and 0.3 SD at the femoral neck, decreases that are smaller than previously reported. Given the relatively low prevalence of the BB/tt genotype in Mexican-American Caucasians, a larger sample would be required to detect a significant association between VDR alleles and differences in BMD of the magnitude suggested by our data. We conclude that a genotype effect of this magnitude, if present, would be clinically relevant, but the impact on BMD is too small to detect with statistical significance in a study of this size.
Composition and Mechanical Properties of Cancellous Bone from the Femoral Head of Patients with Osteoporosis or Osteoarthritis
Authors: Li Baohua , Dr. Aspden Richard M.
Published: April 2006

The material properties of cancellous bone from patients with osteoporosis (OP) or osteoarthritis (OA) were determined and compared with normal controls. Samples were selected from defined sites in human femoral heads which are subjected to different loads in vivo. Overall, OP bone had the lowest stiffness and OA the highest, and this same order was reflected in the apparent densities of the bone, with OA being the most dense and OP the least. Normal and OP bone were found to have very similar stiffness–density relationships and composition. However, OA bone differed significantly from normal. The stiffness of OA bone increased more slowly with apparent density and its material density was significantly reduced. These findings were due to an altered composition of the bone in which the mass fraction of mineral is 12% less than normal. There was also greater site variation of both apparent and material density, suggesting an altered sensitivity to applied load. These results support the concept that osteoporosis is a loss of normal bone. They also provide evidence for the hypothesis that osteoarthritis is, at least partly, a bone disease in which proliferation of defective bone results in an increase in bone stiffness.
Apparent Pre- and Postmenopausal Bone Loss Evaluated by DXA at Different Skeletal Sites in Women: The OFELY Cohort
Authors: Arlot Monique E. , Sornay-Rendu Elisabeth , Garnero Patrick , Vey-Marty Betty , Delmas Pierre D.
Published: April 2006

We measured the bone mineral density (BMD) at various skeletal sites (total body, hip, anteroposterior [AP] and lateral [lat] spine, and forearm) in a large population-based cohort of women aged 31–89 years (the OFELY cohort), and results were analyzed according to age and postmenopausal years. A significant apparent bone loss was found before the menopause in cancellous bone, i.e., at the lat spine and Ward's triangle (−10%; p < 0.05–0.001). Cross-sectional analysis indicated that, after the menopause, apparent bone loss was accelerated within the 10 years following menopause, continued thereafter at all sites except the AP spine, and was again accelerated in elderly menopausal for more than 25 years. Between 30 and 80 years, BMD decreased by 15 to 44% (T score −1.6 to −3.4) according to the site. The amount of apparent bone loss was highest at the Ward's triangle when expressed in percentage (44%) and at the mid- and distal radius when expressed in number of standard deviations from the peak bone mass (−3.4). As a result, the percentage of women classified as osteoporotic according to the World Heath Organization, i.e., with a T score ≤−2.5, varied substantially from site to site and was highest at the radius (37% and 46%) and lateral spine (25–31%), intermediate at the Ward's triangle, AP spine, and whole body BMD, and lowest at the whole body bone mineral content, femoral neck, and trochanter (10–12%). In conclusion, this cross-sectional but large study suggests that there is a moderate apparent premenopausal bone loss that occurs only at cancellous bone sites and that apparent bone loss is accelerated at most skeletal sites after the age of 75 years. Because of the highly variable coefficient of variation of the peak bone mass at various skeletal sites, the percentage of postmenopausal women identified as being osteoporotic varies widely according to the site of measurement.
Characterization and Cellular Distribution of the Osteoclast Ruffled Membrane Vacuolar H+-ATPase B-Subunit Using Isoform-Specific Antibodies
Authors: Mattsson Jan P. , Skyman Christine , Palokangas Harri , Väänänen Kalervo H. , Keeling David J.
Published: April 2006

Acidification of the bone surface, leading to bone resorption, is accomplished by a vacuolar-type H+-ATPase present in a specialized domain of the plasma membrane of the osteoclast known as the ruffled membrane. Structure and function appears to be highly conserved within this class of multisubunit enzymes. However, cloning and sequencing of complementary DNA has shown that one of the subunits in the catalytic domain, the B-subunit, exists in at least two forms, B1 and B2. B1 messenger RNA has been found almost exclusively in the kidney, whereas messenger RNA for B2 has been found in all tissues studied, including the kidney. It has been speculated that the B1 isoform might be involved in targeting to the plasma membrane. In the present study, we have characterized the B-subunit of the chicken osteoclast H+-ATPase using antibodies directed against peptides with isoform-specific or conserved sequences of the B-subunit. Western analysis was performed on chicken osteoclast membrane vesicles and on partially purified chicken osteoclast H+-ATPase and was compared with similar analysis of H+-ATPase isolated from bovine kidney and brain. The B1-specific antibody reacted with a polypeptide of approximately 56 kD on immunoblots of the renal H+-ATPase, whereas no reaction could be detected against the osteoclast H+-ATPase or the osteoclast membrane vesicle preparation. In contrast, the antibody against a B2-specific sequence reacted with a peptide of approximately 56 kD on immunoblots of the osteoclast H+-ATPase, the renal H+-ATPase, and the clathrin-coated vesicle H+-ATPase. The antibody against a conserved region of the B-subunit did not generate any evidence for the presence of isoforms other than B2 in the osteoclast. Immunocytochemistry of rat osteoclasts on bovine bone slices using the B2 antibody showed intense polarized staining along the plasma membrane facing the bone surface in actively resorbing osteoclasts whereas nonresorbing osteoclasts were diffusely stained throughout the cytoplasm. By confocal microscopy, the B2 staining was located to the level of the ruffled membrane and appeared to be concentrated to the peripheral areas of the membrane adjacent to the sealing zone. We conclude that the osteoclast vacuolar H+-ATPase contains the B2 isoform and suggest that upon initiation of resorption the pump is translocated from the cell interior to a special domain of the ruffled membrane close to the sealing zone.
A Vitamin D Receptor Gene Polymorphism in the Translation Initiation Codon: Effect on Protein Activity and Relation to Bone Mineral Density in Japanese Women
Authors: Arai Hidekazu , Miyamoto Ken-Ichi , Taketani Yutaka , Yamamoto Hironori , Iemori Yuka , Morita Kyoko , Tonai Takeharu , Nishisho Takehiko , Mori Shigenobu , Takeda Eiji
Published: April 2006

The effect of a T-C transition polymorphism at the translation initiation codon of the human vitamin D receptor (VDR) gene on the biological function of the encoded protein was investigated. Of 239 Japanese women volunteers subjected to genotype analysis for this polymorphism, 32 (13%) were genotype MM (the M allele is ATG at the putative translation start site), 75 (31%) were genotype mm (the m allele is ACG at the putative translation start site), and 132 (55%) were genotype Mm. The bone mineral density (BMD) in the lumbar spine (L2–L4) was determined for 110 healthy premenopausal women from the volunteers and was shown to be 12.0% greater (p < 0.05) for mm homozygotes than for MM homozygotes. Synthesis of the proteins by the M and m alleles from the cloned cDNAs in vitro and in transfected COS-7 cells revealed them to have a size of 50 and 49.5 kD, respectively, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. This size difference is consistent with initiation of translation of the M allele-encoded protein from an ATG codon located at nucleotides +10 to +12 in the conventional open reading frame. The extent of vitamin D–dependent transcriptional activation of a reporter construct under the control of a vitamin D response element in transfected HeLa cells was ∼1.7-fold greater for the m type VDR than for the M type protein. These results suggest that the polymorphism at the translation start site of the VDR gene may modulate BMD in premenopausal Japanese women.
Tartronates: A New Generation of Drugs Affecting Bone Metabolism
Authors: Caselli Gianfranco , Mantovanini Marco , Gandolfi Carmelo A. , Allegretti Marcello , Fiorentino Simonetta , Pellegrini Luigi , Melillo Gabriella , Bertini Riccardo , Sabbatini Wilma , Anacardio Roberto , Clavenna Gaetano , Sciortino Giancarlo , Teti Anna
Published: April 2006

In the search for a new class of bone-sparing agents for treating osteopenic disorders, we hypothesized that tartronic acid derivatives, sharing the chemical characteristics both of bisphosphonates and of Gla residues contained in matrix proteins such as osteocalcin, could positively affect bone metabolism. A series of tartronates was therefore tested for their ability to affect bone metabolism. In vitro resorption tests were performed examining pit formation by freshly isolated rat and rabbit osteoclasts plated onto bone slices and exposed to the drugs for 48 h. Tartronates bearing a linear side-chain (DF 1222 and DF 1363A) were the most effective in inhibiting pit excavation in the pM–nM range. Tartronates did not affect osteoclast viability, number, adhesion, or tartrate resistant acid phosphatase activity. Transient cell retraction was observed in osteoclasts plated onto glass and exposed to DF 1222. The maximal effect was seen in cells treated for 4 h at a concentration of 1 pM. DF 1222 accelerated mineralization in cultures of periosteal cells without affecting other osteoblast-like functions. This product was therefore tested in vivo in ovariectomized mice. Bone mass in femur was evaluated, by ash gravimetry, 21 days after ovariectomy. Unfortunately, DF 1222, the most active of tartronates in vitro, was inactive in this test because of its high hydrophilicity and the subsequent too short residence time. On the contrary, its tetrahydropyranyl ether derivative, DF 1363A, endowed with a significantly higher lipophilicity, showed a dose-dependent bone-sparing effect when administered subcutaneously at 10, 30, and 100 mg/kg/die, thus confirming the activity seen in in vitro tests. Because of their feasible parallel effect on both bone resorption and formation, tartronate derivatives may be tested to candidate this class of products for clinical studies.
Relationship of Bone Turnover to Bone Density and Fractures
Authors: Melton L. Joseph , Khosla Sundeep , Atkinson Elizabeth J. , O'Fallon W. Michael , Riggs B. Lawrence
Published: April 2006

To assess the influence of bone turnover on bone density and fracture risk, we measured serum levels of osteocalcin (OC), bone alkaline phosphatase (BAP), and carboxy-terminal propeptide of type I procollagen (PICP), as well as 24-h urine levels of cross-linked N-telopeptides of type I collagen (NTx) and the free pyridinium cross-links, pyridinoline (Pyd) and deoxypyridinoline (Dpd), among 351 subjects recruited from an age-stratified random sample of Rochester, Minnesota women. PICP, NTx, and Dpd were negatively associated with age among the 138 premenopausal women. All of the biochemical markers were positively associated with age among the 213 postmenopausal women, and the prevalence of elevated turnover (>1 standard deviation [SD] above the premenopausal mean) varied from 9% (PICP) to 42% (Pyd). After adjusting for age, most of the markers were negatively correlated with bone mineral density (BMD) of the hip, spine, or forearm as measured by dual-energy X-ray absorptiometry, and women with osteoporosis were more likely to have high bone turnover. A history of osteoporotic fractures of the hip, spine, or distal forearm was associated with reduced hip BMD and with elevated Pyd. After adjusting for lower BMD and increased bone resorption, reduced bone formation as assessed by OC was also associated with prior osteoporotic fractures. These data indicate that a substantial subset of elderly women has elevated bone turnover, which appears to adversely influence BMD and fracture risk. Combined biochemical and BMD screening may provide better prediction of future fracture risk than BMD alone.
Specific Inhibitors of Vacuolar H+-ATPase Trigger Apoptotic Cell Death of Osteoclasts
Authors: Okahashi Nobuo , Nakamura Ichiro , Jimi Eijiro , Koide Masanori , Suda Tatsuo , Nishihara Tatsuji
Published: April 2006

Osteoclasts are multinucleated bone-resorbing cells that play a critical role in bone remodeling. Specific inhibitors of vacuolar H+-ATPase (V-ATPase), concanamycin A and bafilomycin A1, abolish bone resorption by osteoclasts. In this study, we examined whether these V-ATPase inhibitors trigger apoptotic cell death in osteoclasts, using murine osteoclast-like multinucleated cells (OCLs) formed in vitro. Acridine orange staining revealed that the treatment of OCLs with concanamycin A resulted in chromatin condensation and alterations in nuclear morphology within a few hours. The TdT-mediated dUTP-nick-end labeling (TUNEL) reaction confirmed the apoptotic features of OCLs treated with concanamycin A. The accelerated apoptotic cell death induced by concanamycin A occurred in OCLs treated with interleukin-1α or macrophage colony-stimulating factor as well, which are known to elongate the survival time of osteoclasts. In contrast, these inhibitors did not induce cell death of osteoblastic cells isolated from mouse calvaria. These results suggest that functional impairment of V-ATPase triggers apoptotic cell death in osteoclasts.
Risk Factors for Hip Fracture in a Japanese Cohort
Authors: Fujiwara Saeko , Kasagi Fumiyoshi , Yamada Michiko , Kodama Kazunori
Published: April 2006

Risk factors for hip fracture were determined from a Japanese cohort. A cohort of 4573 people (mean age 58.5 ± 12.2) who participated in the Adult Health Study in 1978–1980 were subsequently followed by biennial examinations up to 1992. Fifty-five incident hip fractures not due to traffic accidents were identified by medical records during the follow-up period. Poisson regression analysis showed that baseline low body mass index (BMI), regular alcohol intake, prevalent vertebral fracture, and having five or more children significantly increased the risk of hip fracture, and low milk intake and later age at menarche were marginally associated with increased fracture risk, after multivariable adjustment. Regular alcohol intake doubled the risk of hip fracture (relative risk 1.91, 95% confidence interval 1.07–3.42). Those individuals who had a vertebral fracture had 2.6 times higher risk than those who did not. The risk was 2.5 times higher among women who had five or more children than women with one or two. Body height, health status, marital status, intake of fish, coffee, tea, Japanese tea, smoking, exposure to atomic bomb radiation, and age at menopause were not associated with hip fracture. Relative risk for hip fracture decreased with decreasing number of preventable risk factors (low BMI, low milk intake, and regular alcohol intake). We conclude that many factors, such as BMI, milk intake, alcohol intake, prevalent vertebral fracture, age at menarche, and number of children, are related to the risk of hip fracture, and prevention programs need to focus on reducing preventable risk factors.
Cellular Hypertrophy and Calcification of Embryonal Carcinoma-Derived Chondrogenic Cell Line ATDC5 In Vitro
Authors: Shukunami Chisa , Ishizeki Kiyoto , Atsumi Tadao , Ohta Yoshiyuki , Suzuki Fujio , Hiraki Yuji
Published: April 2006

During the process of endochondral bone formation, proliferating chondrocytes give rise to hypertrophic cells, which then deposit a mineralized matrix to form calcified cartilage prior to replacement by bone. Previously, we reported that a clonal cell line, ATDC5, undergoes efficient chondrogenic differentiation through a cellular condensation stage. Here we report that the differentiated ATDC5 cells became hypertrophic at the center of cartilage nodules, when the cells ceased to grow. Formation of hypertrophic chondrocytes took place in association with type X collagen gene expression and a dramatic elevation of alkaline phosphate (ALPase) activity. After 5 weeks of culture, mineralization of the culture could be discerned as Alizarin red-positive spots, which spread throughout the nodules even in the absence of β-glycerophosphate. Electron microscopy and electron probe microanalysis revealed that calcification was first initiated at matrix vesicles in the territorial matrix and that it advanced progressively along the collagen fibers in a manner similar to that which occurs in vivo. The infrared spectrum of the mineralized nodules indicated two absorption doublets around 1030 cm−1 and 600 cm−1, which are characteristic of apatitic mineral. Calcifying cultures of ATDC5 cells retained responsiveness to parathyroid hormone (PTH): PTH markedly inhibited elevation of ALPase activity and calcification in the culture in a dose-dependent manner. Thus, we demonstrated that ATDC5 cells keep track of the multistep differentiation process encompassing the stages from mesenchymal condensation to calcification in vitro. ATDC5 cells provide an excellent model to study the molecular mechanism underlying regulation of cartilage differentiation during endochondral bone formation.
Calcium for Prevention of Osteoporotic Fractures in Postmenopausal Women
Authors: Cumming Robert G. , Nevitt Michael C.
Published: April 2006

A systematic review of the literature was conducted to assess the effectiveness of calcium supplements and/or dietary calcium for the prevention of osteoporotic fractures in postmenopausal women. Studies were identified by conducting a Medline search using the text words “fracture” and “calcium” for the period 1966 to March 1997 and by reviewing articles known to the authors. Only studies with fracture outcomes were eligible. There were 14 studies of calcium supplements (including 4 randomized trials), 18 studies of dietary calcium and hip fracture (no randomized trials), and 5 studies of dietary calcium and other fracture sites (no randomized trials). The 4 randomized trials of calcium supplements (mean calcium dose: 1050 mg) found relative risk (RR) reductions between 25% and 70%. Meta-analytic techniques for dose-response data were used to investigate and pool the findings of 16 observational studies of dietary calcium and hip fracture. These hip fracture studies were not consistent and heterogeneity of study findings (p = 0.02) was not easily explained by subject characteristics or study design. Pooling study results gave an odds ratio (OR) of 0.96 (95% confidence interval, (CI) 0.93–0.99) per 300 mg/day increase in calcium intake (the equivalent of one glass of milk). This is likely to be an underestimate of calcium's true effect because of inaccurate measurement of dietary calcium in observational studies. This review supports the current clinical and public health policy of recommending increased calcium intake among older women for fracture prevention.
Decreased β-Isomerization of the C-Terminal Telopeptide of Type I Collagen α1 Chain in Paget's Disease of Bone
Authors: Garnero Patrick , Fledelius Christian , Gineyts Evelyne , Serre Claire-Marie , Vignot Emanuelle , Delmas Pierre D.
Published: April 2006

In Paget's disease of bone, the normal lamellar bone is replaced by a woven structure with an irregular arrangement of collagen fibers. In this study, we investigated whether the degree of β-isomerization within C-telopeptide of α1 chain of type I collagen was altered in Paget's disease compared with other bone diseases with no alteration of bone structure. In Paget's disease (n = 26), but not in patients with primary hyperparathyroidism (n = 6) or hyperthyroidism (n = 17), the urinary excretion of nonisomerized (α) fragments derived from degradation of type I collagen C-telopeptide (CTX) was markedly increased compared with β-isomerized CTX (+ 13-fold vs. + 3.5-fold over controls) resulting in an urinary α CTX/β CTX ratio 3-fold higher than in controls (2.6 ± 1.0 vs. 0.8 ± 0.3, p < 0.001). In five pagetic patients in complete remission, as demonstrated by normal total alkaline phosphatase activity, the α CTX/β CTX ratio was normal. The immunohistochemistry of normal and pagetic human bone sections showed a preferential distribution of α CTX within woven structure, while lamellar bone was intensely stained with an anti–β CTX antibody, suggesting a lower degree of β-isomerization of type I collagen in the woven pagetic bone. In collagenase digest of human bone specimens, we found a lower proportion of β-isomerized type I collagen molecules in pagetic bone (40% of β CTX) than in normal bone taken from trabecular (68%) and cortical compartments (71%). In conclusion, we found that in Paget's disease the α CTX/β CTX ratio in bone and in urine is markedly increased. This altered β isomerization can be accurately detected in vivo by measuring urinary degradation products arising from bone resorption.