bluuebunny/crossref_metadata_2025 huggingface dataset:
The Effect of Vitamin D Supplementation on the Bone Mineral Density of the Femoral Neck Is Associated with Vitamin D Receptor Genotype
Authors: Graafmans W. C. , Lips P. , Ooms M. E. , Van Leeuwen J. P. T. M. , Pols H. A. P. , Uitterlinden A. G.
Published: April 2006

Recent studies suggest that variations of the vitamin D receptor (VDR) gene are related to bone mineral density (BMD). In this study, we examined the effect of vitamin D3 supplementation on BMD at the femoral neck in relation to VDR genotype. We analyzed 81 women, age 70 years and over, who participated in a placebo-controlled clinical trial on the effect of vitamin D3 supplementation (400 IU daily for at least 2 years) on BMD and fracture incidence. VDR genotype was based on the presence (b) or absence (B) of the BsmI restriction site. Mean BMD of the right and left femoral neck was measured at baseline and after 1 and 2 years. Dietary calcium, body mass index, and years since menopause were assessed at baseline while biochemical markers were measured at baseline and after 1 year. There was no difference among the BB, Bb, and bb genotype for baseline measurements of BMD at the femoral neck (mean and SD, g/cm2: 0.70 (0.10), 0.71 (0.12), and 0.69 (0.10), respectively), nor for any of the biochemical indices. The mean increase of BMD in the vitamin D group relative to the placebo group, expressed as percentage of baseline BMD, was significantly higher (p = 0.03) in the BB (ΔBMD: 4.4%, p = 0.04) and Bb genotype (ΔBMD: 4.2%, p = 0.007) compared with the bb genotype (ΔBMD: −0.3%, p = 0.61). No significant changes were found for any of the other measured parameters. The VDR genotype-dependent effect of vitamin D supplementation in these elderly subjects suggest a functional involvement of VDR gene variants in determining BMD.
Parathyroid Tumor Suppressor on 1p: Analysis of the p18 Cyclin-Dependent Kinase Inhibitor Gene As a Candidate
Authors: Tahara Hideki , Smith Alison P. , Gaz Randall D. , Zariwala Maimoona , Xiong Yue , Arnold Andrew
Published: April 2006

Loss of chromosome arm 1p DNA is the most common molecular defect thus far observed in human parathyroid adenomas, suggesting that 1p is the location of a putative tumor suppressor gene (or genes) whose inactivation contributes frequently to parathyroid tumorigenesis. To narrow the genomic location of this tumor suppressor gene, we analyzed 25 sporadic parathyroid adenomas for allelic loss of polymorphic DNA loci on chromosome 1 using 11 microsatellite markers not previously scored for this set of tumors. Allelic loss on chromosome arm 1p DNA was observed in 8 of 25 adenomas. Marker deletion patterns showed some complexity, with the regions most commonly deleted in these tumors being 1p36 and 1p35–p31. The 1p35–p31 region contains an excellent candidate tumor suppressor gene, p18, whose product is a cell cycle regulator that inhibits the cyclin D1-associated kinase CDK6. Given that cyclin D1 is a parathyroid oncogene, inactivation of an inhibitor of cyclin D1 function, like p18, might also cause excessive parathyroid growth. To examine the involvement of p18 in parathyroid tumorigenesis, we analyzed 25 parathyroid adenomas for mutations of the p18 coding exons by single strand conformational polymorphism analysis and sequencing. No point mutations were found in any of the 25 adenomas. These observations indicate that inactivating mutation of the p18 gene occurs uncommonly, if at all, in parathyroid adenomas. In addition, the data raise the important possibility that more than a single tumor suppressor gene on 1p could contribute to parathyroid neoplasia.
Enhancement by Sex Hormones of the Osteoregulatory Effects of Mechanical Loading and Prostaglandins in Explants of Rat Ulnae
Authors: Cheng Ming Zhao , Zaman Gul , Rawlinson Simon C. F. , Pitsillides Andrew A. , Suswillo Rosemary F. L. , Lanyon Lance E.
Published: April 2006

Explants of ulnae from 5-week-old male and female rats were cleaned of marrow and soft tissue and, in the presence and absence of 10−8 M 17β-estradiol (E2) or 5α-dihydrotestosterone (DHT), mechanically loaded or treated with exogenous prostanoids previously shown to be produced during loading. Over an 18-h period, mechanical loading (peak strain 1300 μϵ, 1 Hz, 8 minutes, maximum strain rate 25,000 μϵ/s), prostaglandin E2 (PGE2) and prostacyclin (PGI2) (10−6 M), each separately produced quantitatively similar increases in cell proliferation and matrix production in bones from males and females, as indicated by incorporation of [3H]thymidine into DNA and [3H]proline into collagen. E2 and DHT both increased [3H]thymidine and [3H]proline incorporations, E2 producing greater increases in females than in males. Indomethacin abrogated the effects of loading, but had no effects on those of sex hormones. Loading, or prostanoids, together with sex hormones, produced responses generally equal to or greater than the addition of the individual influences acting independently. In females there was a synergistic response in [3H]thymidine incorporation between loading and E2, which was quantitatively similar to the interaction between E2 and PGE2 or PGI2. The interaction between loading and E2 for [3H]proline incorporation was not mimicked by these prostanoids. In males the synergism in [3H]proline incorporation seen between loading and DHT was mimicked by that between PGI2 and DHT. We conclude that loading stimulates increased bone cell proliferation and matrix production in situ through a prostanoid-dependent mechanism. This response is equal in size in males and females. Estrogen and testosterone increase proliferation and matrix production through a mechanism independent of prostanoid production. The interactions between loading and hormones are reproduced in some but not all cases by E2 and prostaglandins. E2 with loading and prostaglandins has greater effects in female bones, while DHT with loading and prostaglandins has greater effects in males.
Prospective Ten-Month Exercise Intervention in Premenarcheal Girls: Positive Effects on Bone and Lean Mass
Authors: Morris Fiona L. , Naughton Geraldine A. , Gibbs Joanne L. , Carlson John S. , Wark John D.
Published: April 2006

Enhancement of bone mineral acquisition during growth may be a useful preventive strategy against osteoporosis. The aim of this study was to explore the lean mass, strength, and bone mineral response to a 10-month, high-impact, strength-building exercise program in 71 premenarcheal girls, aged 9–10 years. Lean body mass, total body (TB), lumbar spine (LS), proximal femur (PF), and femoral neck (FN) bone mineral were measured using the Hologic QDR 2000+ bone densitometer. Strength was assessed using a grip dynamometer and the Cybex isokinetic dynamometer (Cybex II). At baseline, no significant difference in body composition, pubertal development, calcium intake, physical activity, strength, or bone mineral existed between groups. At completion, there were again no differences in height, total body mass, pubertal development, calcium intake, or external physical activity. In contrast, the exercise group gained significantly more lean mass, less body fat content, greater shoulder, knee and grip strength, and greater TB, LS, PF, and FN BMD (exercise: TB 3.5%, LS 4.8%, PF 4.5%, and FN 12.0%) compared with the controls (controls: TB 1.2%, LS 1.2%, PF 1.3%, and FN 1.7%). TB bone mineral content (BMC), LS BMC, PF BMC, FN BMC, LS bone mineral apparent density (BMAD), and FN bone area also increased at a significantly greater rate in the exercise group compared with the controls. In multiple regression analysis, change in lean mass was the primary determinant of TB, FN, PF, and LS BMD accrual. Although a large proportion of bone mineral accrual in the premenarcheal skeleton was related to growth, an osteogenic effect was associated with exercise. These results suggest that high-impact, strength building exercise is beneficial for premenarcheal strength, lean mass gains, and bone mineral acquisition.
Five Jumps per Day Increase Bone Mass and Breaking Force in Rats
Authors: Umemura Yoshihisa , Ishiko Toshihiro , Yamauchi Takeshi , Kurono Masashi , Mashiko Shoji
Published: April 2006

The effects of jump training on bone morphological and mechanical properties were investigated in immature bones of female Fischer 344 rats. Five-week-old rats were divided into control or five jump-trained groups comprised of 5-, 10-, 20-, 40-, and 100-jump groups, representing the number of jumps per day. The rats were jump-trained 5 days/week for 8 weeks, and the height of jump was increased to 40 cm progressively. The femur and tibia in the 5-jump group had significantly greater fat-free dry weights per body weight and maximum loads at the fracture tests than those in the control group. The tibia in the 5-jump group also had significantly larger cortical area at the cross-sectional analysis. Although a slight tendency toward increase according to the number of jumps per day was observed, there were few differences in bone morphological and mechanical parameters among the 10-, 20-, and 40-jump groups. The present results indicate that a large number of strains per day is not necessary for bone hypertrophy to develop in rats.
The Relationship Between Magnesium and Calcium Kinetics in 9- to 14-Year-Old Children
Authors: Abrams Steven A.
Published: April 2006

Few measurements of magnesium (Mg) kinetics have been performed, especially in children and adolescents. Simultaneous Mg and calcium (Ca) kinetics following intravenous administration of42Ca and25Mg were measured in 22 children (10 boys and 12 girls) 9–14 years of age. Kinetic values, including the body pool masses (MgComp and CaComp for the Mg and Ca compartment masses, respectively) and bone/tissue (hard and soft tissue) Ca and Mg deposition rates (Vo+Ca and Vo+Mg, respectively) were calculated using the Simulation, Analysis, and Modeling (SAAM) program. No significant differences were found between males and females (p > 0.3 for each comparison) for pool masses or deposition rates. Vo+Ca and Vo+Mg were highly correlated (r = 0.78, p < 0.001). Vo+Mg, but not Vo+Ca, was closely correlated with body weight (r = 0.55, p < 0.01 and r = 0.16, p = 0.47, respectively). Similarly, MgComp was more closely correlated with body weight (r = 0.76, p < 0.01) than CaComp (r = 0.37, p = 0.10). Neither Vo+Ca nor CaComp increased significantly over the age range studied. However, Vo+Mg and MgComp were significantly positively correlated with age. These findings demonstrate differences in regulation of Ca versus Mg kinetics by young adolescents. These differences may be related to the greater utilization of Mg for muscle and soft tissue growth. The close relationship between Vo+Mg and body weight suggests that the current practice of relating Mg intake requirements to body weight, as well as age, is appropriate for young adolescents.
A Longitudinal Study of Bone Gain in Pubertal Girls: Anthropometric and Biochemical Correlates
Authors: Cadogan Joanna , Blumsohn Aubrey , Barker Margo E. , Eastell Richard
Published: April 2006

The aim of this longitudinal study was to investigate the factors associated with bone mineral acquisition in pubertal girls. Subjects were 37 healthy, Caucasian girls aged 12.1 years (SD 0.3). Measurements were made at 6-month intervals over a period of 18 months and included total body bone mineral content (TBBMC), total body bone mineral density (TBBMD), lean mass, and fat mass by dual-energy X-ray absorptiometry, anthropometry, lifestyle factors, four biochemical markers of bone turnover, hormonal status, and fractional calcium absorption. In multiple regression analysis, correlates of relative gain in TBBMC were gain in lean mass (p < 0.001) and estradiol (p = 0.008). For TBBMD, correlates were gain in lean (p < 0.001) and fat mass (p = 0.003), estradiol (p < 0.001), dietary energy intake (p = 0.003), and parathyroid hormone (p = 0.023). Statural growth and gain in bone mass were unrelated; both height velocity and bone turnover peaked ∼20 months prior to menarche, whereas gain in bone mass peaked at menarche. Bone turnover markers correlated with height velocity (0.40 < r < 0.62), but not with bone gain. Estradiol was independently and negatively associated with all markers of bone turnover (−0.67 < r < −0.80). We conclude that estradiol is an important determinant of bone mineral gain in pubertal girls and is probably responsible for the reduction in bone turnover in late puberty; lean mass was the body composition parameter most closely associated with bone gain; height gain and bone gain are dissociated during the period of rapid growth at puberty; and bone turnover markers are modestly related to height gain, but are not predictive of bone gain.
Bone Mineral and Body Composition Measurements: Cross-Calibration of Pencil-Beam and Fan-Beam Dual-Energy X-Ray Absorptiometers
Authors: Ellis Kenneth J. , Shypailo Roman J.
Published: April 2006

Pencil-beam dual-energy X-ray absorptiometers (DXA) are being replaced with instruments that rely solely on fan-beam technology. However, information has been lacking regarding the translation of bone mineral and body composition data between the two devices. We have compared total body scans using pencil-beam (Hologic QDR-2000W) and fan-beam (Hologic QDR-4500A) instruments for 33 children (ages 3–18 years) and 14 adults. Bone mineral content (BMC), bone mineral density (BMD), fat, lean, and body fatness (%fat) values were highly correlated (r2 = 0.984–0.998) between the two DXA instruments. The mean differences between the paired measurements were: ΔBMC = 7.5 ± 73.6 g, ΔBMD = 0.0074 ± 0.0252 g/cm2, Δlean = 1.05 ± 1.8 kg, Δfat = −0.77 ± 1.7 kg, and Δ%fat = −0.94% ± 2.5%. The BMC and BMD values were not statistically different, whereas the differences for the body composition values were significant (p < 0.02–0.005). Regression equations are provided for conversion of bone and body composition data between pencil-beam and fan-beam values for the whole body. To test the performance of these equations for a second group (23 subjects), predicted values were compared with the measured data obtained using the fan-beam instrument. The mean differences were −1.0% to 1.4%, except for body fat mass, where the difference was 6.4%. For cross-sectional studies, the two DXA technologies can be considered equivalent after using the translational equations provided. For longitudinal studies in which small changes in body composition for the individual are to be detected, we recommend that the same DXA instrument be used whenever possible. For example, transition from a pencil-beam to a fan-beam instrument could, in extreme cases, result in differences as large as 19% for the estimate of body fat mass.
Quantitative Trait Loci Affecting Peak Bone Mineral Density in Mice
Authors: Klein Robert F. , Mitchell Steve R. , Phillips Tamara J. , Belknap John K. , Orwoll Eric S.
Published: April 2006

Peak bone mass is a major determinant of risk of osteoporotic fracture. Family and twin studies have found a strong genetic component to the determination of bone mineral density (BMD). However, BMD is a complex trait whose expression is confounded by environmental influences and polygenic inheritance. The number, locations, and effects of the individual genes contributing to natural variation in this trait are all unknown. Experimental animal models provide a means to circumvent complicating environmental factors, and the development of dense genetic maps based on molecular markers now provides opportunities to resolve quantitative genetic variation into individual regions of the genome influencing a given trait (quantitative trait loci, QTL). To begin to identify the heritable determinants of BMD, we have examined genetically distinct laboratory mouse strains raised under strict environmental control. Mouse whole-body bone mineral content by dual-energy X-ray absorptiometry (DXA) correlated strongly with skeletal calcium content by ashing, and peak whole-body BMD by DXA in female mice occurred at ∼80–90 days of age. We therefore determined mean body weight and peak whole body BMD values in 12-week-old female mice from a panel of 24 recombinant inbred (RI) BXD strains, derived from a cross between C57BL/6 and DBA/2 progenitors. The distribution of body weight and BMD values among the strains clearly indicated the presence of strong genetic influences on both of these traits, with an estimated narrow sense heritability of 60% and 35%, respectively. The patterns of differences in body weight and peak whole body BMD in the BXD strains were then integrated with a large database of genetic markers previously defined in the RI BXD strains to generate chromosome map sites for QTL. After correction for redundancy among the significant correlations, QTL analysis of the BXD RI strain series provisionally identified 10 chromosomal sites linked to peak bone mass development in the female. Several of the identified sites map near genes encoding hormones, structural proteins, and cell surface receptors that are intricately involved in skeletal homeostasis. Four QTL for body weight were also identified. One of these loci was also strongly linked to inherited variation in BMD. This finding suggests that body weight and peak BMD may be influenced by linked genes or perhaps by common genes with pleiotropic effects. Our phenotyping in the RI BXD strains has allowed us to map a number of specific genetic loci strongly related to the acquisition of peak BMD. Confirmation of these findings will likely result in the understanding of which genes control skeletal health.
Molecular Mechanism of Action of Fluoride on Bone Cells
Authors: Lau K.-H. William , Baylink David J.
Published: April 2006

Fluoride is an effective anabolic agent to increase spinal bone density by increasing bone formation, and at therapeutically relevant (i.e., micromolar) concentrations, it stimulates bone cell proliferation and activities in vitro and in vivo. However, the fluoride therapy of osteoporosis has been controversial, in large part because of a lack of consistent antifracture efficacy. However, information regarding the molecular mechanism of action of fluoride may improve its optimum and correct usage and may disclose potential targets for the development of new second generation drugs that might have a better efficacy and safety profile. Accordingly, this review will address the molecular mechanisms of the osteogenic action of fluoride. In this regard, we and other workers have proposed two competing models, both of which involve the mitogen activated protein kinase (MAPK) mitogenic signal transduction pathway. Our model involves a fluoride inhibition of a unique fluoride-sensitive phosphotyrosine phosphatase (PTP) in osteoblasts, which results in a sustained increase in the tyrosine phosphorylation level of the key signaling proteins of the MAPK mitogenic transduction pathway, leading to the potentiation of the bone cell proliferation initiated by growth factors. The competing model proposes that fluoride acts in coordination with aluminum to form fluoroaluminate, which activates a pertussis toxin-sensitive Gi/o protein on bone cell membrane, leading to an activation of cellular protein tyrosine kinases (PTKs), which in turn leads to increases in the tyrosine phosphorylation of signaling proteins of the MAPK mitogenic signal transduction pathway, ultimately leading to a stimulation of cell proliferation. A benefit of our model, but not the other model, is that it accounts for all the unique properties of the osteogenic action of fluoride. These include the low effective fluoride dose, the skeletal tissue specificity, the requirement of PTK-activating growth factors, the sensitivity to changes in medium phosphate concentration, the preference for undifferentiated osteoblasts, and the involvement of the MAPK. Unlike fluoride, the mitogenic action of fluoroaluminate is not specific for skeletal cells. Moreover, the mitogenic action of fluoroaluminate shows several important, different characteristics than that of fluoride. Thus, it is likely that our model of a fluoride-sensitive PTP represents the actual molecular mechanism of the osteogenic action of fluoride.
Conditionally Immortalized Murine Osteoblasts Lacking the Type 1 PTH/PTHrP Receptor
Authors: Divieti P. , Lanske B. , Kronenberg H. M. , Bringhurst F. R.
Published: April 2006

Osteoblasts synthesize and mineralize bone matrix and are principal target cells for parathyroid hormone (PTH). The type 1 PTH/PTH-related protein (PTHrP) receptor (PTH1R), cloned from rat osteoblastic cells, activates multiple intracellular signaling mechanisms. The specific roles of these PTH1R signals, or of responses to other types of PTH receptors that may be expressed, in regulating osteoblast function are incompletely understood. Use of established mammalian osteoblastic cell lines has led to much understanding of PTH action in bone, although such cells are of neoplastic origin or have other characteristics that compromise their validity as models of normal osteoblasts. To examine the role of the PTH1R in osteoblast biology, we have isolated a series of clonal murine calvarial osteoblastic cell lines that are only conditionally immortalized, via expression of a transgene encoding the tsA58 temperature-sensitive SV40 large T antigen, and that lack both functional alleles of the PTH1R gene. When cultured under nontransforming conditions, these cells stopped proliferating, expressed a series of characteristic osteoblastic genes (including the nonfunctional remnant of the PTH1R gene), and, after 3–4 weeks, produced mineralized bone nodules in a manner that was regulated by 1,25-dihydroxyvitamin D3 but not by PTH(1–84). Cyclic AMP measurements revealed no evidence of expression of alternate species of Gs-linked PTH receptors. Stable transfection with PTH1R cDNA reconstituted both PTH binding and adenylyl cyclase activation, increased basal osteocalcin expression, and supported PTH stimulation of c-Fos expression and matrix mineralization. These conditionally transformed, PTH1R(−/−) clonal osteoblastic cell lines should prove useful for studies of the regulation of osteoblast differentiation and function by both endogenous nonclassical species of PTH (or PTHrP) receptors and mutant signal-selective PTH1Rs.
Linkage of a QTL Contributing to Normal Variation in Bone Mineral Density to Chromosome 11q12–13
Authors: Koller D. L. , Rodriguez L. A. , Christian J. C. , Slemenda C. W. , Econs M. J. , Hui S. L. , Morin P. , Conneally P. M. , Joslyn G. , Curran M. E. , Peacock M. , Johnston C. C. , Dr. Foroud T.
Published: April 2006

Osteoporosis is a leading public health problem that is responsible for substantial morbidity and mortality. A major determinant of the risk for osteoporosis in later life is bone mineral density (BMD) attained during early adulthood. BMD is a complex trait that presumably is influenced by multiple genes. Recent linkage of three Mendelian BMD-related phenotypes, autosomal dominant high bone mass, autosomal recessive osteoporosis-pseudoglioma, and autosomal recessive osteopetrosis to chromosome 11q12–13 led us to evaluate this region to determine if the underlying gene(s) could also contribute to variation in BMD in the normal population. We performed a linkage study in a sample of 835 premenopausal Caucasian and African–American sisters to identify genes underlying BMD variation. A maximum multipoint LOD score of 3.50 with femoral neck BMD was obtained near the marker D11S987, in the same chromosomal region as the three Mendelian traits mentioned above. Our results suggest that the gene(s) underlying these Mendelian phenotypes also play a role in determining peak BMD in the normal population and are the first using linkage methods to establish a chromosomal location for a gene important in determining peak BMD. These findings support the hypothesis that a gene responsible for one or more of the rare Mendelian BMD traits linked to chromosome 11q12–13 has an important role in osteoporosis in the general population.
Effect on the Femur of a New Hip Fracture Preventive System Using Dropped-Weight Impact Testing
Authors: Okuizumi Hiroyasu , Harada Atsushi , Iwata Hisashi , Konishi Nobuo
Published: April 2006

To reduce hip fractures during falls, we devised a new hip fracture preventive system to attenuate impact on the greater trochanteric region and studied the effects of the system on the femur. Twelve coupled, embalmed, cadaveric femora were used. Right femora were fractured without protection as a control and compared with left femora covered by the protection system, which consisted of a silicone gel pad or the silicone gel pad combined with a resin cover. The impact of a fall was simulated by mounting a femur in the horizontal plane and dropping an 8.4 kg mass on its greater trochanteric region. The impact load and time were measured using a load cell within the mass. The maximum strain during impact at the inferior side of the femoral neck was determined from an attached strain gauge. Trochanteric fractures were produced in 18 of the 24 femora (75%). The mean impact load for a drop height of 25 cm was reduced from 3117 N to 2176 N by silicone gel (p < 0.01) and to 1681 N by the addition of the resin cover (p < 0.01). The mean maximum strain was similarly reduced from 2276 με to 1872 με (p = 0.15) and to a mean 1559 με (p < 0.05). The mean impact time was prolonged from 13 ms to 20 ms (p < 0.01) and 22 ms (p < 0.01), respectively. The effect of the cover became more conspicuous as height increased. We concluded that the silicone gel pad provided effective impact attenuation, and the addition of the rigid cover was even more effective for impact reduction. This system was thought to be clinically useful in preventing hip fractures.
The Mouse Mammary Tumor Cell Line, MMT060562, Produces Prostaglandin E2 and Leukemia Inhibitory Factor and Supports Osteoclast Formation In Vitro Via a Stromal Cell–Dependent Pathway
Authors: Akatsu Takuhiko , Ono Katsuhiro , Katayama Yasuyuki , Tamura Tatsuya , Nishikawa Miyuki , Kugai Nobuo , Yamamoto Michiko , Nagata Naokazu
Published: April 2006

Osteoclastic bone resorption increases at the site of bone metastasis, but little is known about how tumor cells induce osteoclast (OC) recruitment in the bone marrow microenvironment. To clarify this point, we examined the effects of various mouse tumor cells on OC recruitment using cocultures of tumor cells and mouse marrow cells. The mouse mammary tumor cell lines, MMT060562 (MMT), BALB/c-MC, Jyg-MC(A), or other nonmammary tumor cell lines, LLC and B16, were cocultured with mouse marrow cells, and OC recruitment from marrow cells was determined by counting the number of tartrate-resistant acid phosphatase–positive multinucleated cells (TRAP(+) MNCs) formed. Of the tumor cells examined, MMT and BALB/c-MC stimulated OC formation, but other tumor cells did not. OC formation with MMT was dependent on the number of MMTs inoculated, and only ten cells per well were sufficient to induce OC development. OCs appeared on day 4, and the number reached a maximum on days 5–8 and decreased thereafter. TRAP(+) MNCs induced by MMT satisfied the major criteria of OCs, such as the presence of calcitonin receptors and the ability to resorb calcified tissues. The majority of OCs were formed adjacent to the stromal cells, which were positive for alkaline phosphatase. When spleen cells were cocultured with MMT, no OCs were formed. In contrast, when osteoblastic cells were added to cocultures of spleen cells and MMT, many OCs were formed. The cultured media (CM) of MMT induced OC formation in mouse marrow cultures. Neither parathyroid hormone–like nor interleukin 1-like activity was present in the CM. MMT constitutively produced prostaglandin E2 (PGE2) and OC formation in cocultures was completely inhibited by indomethacin. Fractionation of the CM of MMT by ultrafiltration indicated that the OC-inducing activities were present not only in the fraction with molecular weight below 3 kDa but also in the fraction with molecular weight above 3 kDa. OC-inducing activity with high molecular weight was eluted around 50 kDa by Bio-Gel P-60 column chromatography. The active fractions also possessed leukemia inhibitory factor (LIF) activity, and OC-inducing activity of the peak fraction was inhibited in the presence of anti-LIF neutralizing antibody. The results of this study indicated that MMTs release PGE2 and LIF, which in turn stimulate OC formation via a stromal cell–dependent pathway. These culture systems will help to clarify the mechanisms by which tumor cells induce OC formation in a bone marrow microenvironment.
The Preventive and Interventional Effects of Raloxifene Analog (LY117018 HCL) on Osteopenia in Ovariectomized Rats
Authors: Li Xiaodong , Takahashi Masaaki , Kushida Kazuhiro , Inoue Tetsuo
Published: April 2006

The effects of LY117018 HCL (LY) treatment on bone metabolism, spine bone mineral density (BMD), bone mineral content (BMC), and serum cholesterol were studied in ovariectomized (OVX) rats. Experiment 1 was designed to observe the preventive effects of LY on bone loss due to ovariectomy (OVX; prevention study). The rats were divided into three groups: sham group, OVX + vehicle, and OVX + LY. LY was administrated at the same time of OVX. Experiment 2 was designed to investigate the interventional effects of LY on OVX rats with osteopenia (intervention study). The rats were divided into the sham and OVX groups, first. The OVX rats were allowed to lose bone for 6 weeks. At 6 weeks post-OVX, the OVX rats were divided into two groups: OVX + vehicle and OVX + LY. The longitudinal effects of LY on bone were studied by dual-energy X-ray absorptiometry and biochemical markers including urinary pyridinoline (Pyr), deoxypyridinoline (Dpyr), and serum osteocalcin. Urinary Pyr and Dpyr increased maximally at 6 weeks post-OVX, decreased at 12 and 18 weeks post-OVX, although the OVX rats had significantly higher levels of Pyr and Dpyr than the sham group during the experiment. LY was a very potent inhibitor of Pyr and Dpyr excretion while at the same time only partially reducing the bone loss in the high turnover phase at 6 weeks postovariectomy. However, at the later time points at 12 and 18 weeks, no further bone loss occurred in rats treated with LY, while the vehicle-treated group lost another 10% in spine BMD and BMC. LY also completely blocked further bone loss when used in an intervention protocol. LY significantly reduced serum cholesterol levels in OVX rats. The results suggest that LY is not fully protective during the early rapid bone loss phase, but the compound is fully protective during the later slow phase of bone loss in both the protocols.
The Chemokine Interleukin-8 Regulates Parathyroid Secretion
Authors: Angeletti Ruth Hogue , D'Amico Thomas , Ashok Sheela , Russell John
Published: April 2006

Interleukin-8 (IL-8) is a chemokine important in inflammatory processes. Homology cloning experiments performed using bovine parathyroid cDNA and degenerate primers encoding transmembrane regions III and VI of peptide and protein hormone G-protein coupled receptors identified a set of known receptors not previously identified in the parathyroid. Among these was the IL-8 type B receptor. Incubation of freshly isolated bovine parathyroid cells with recombinant IL-8 for 6–48 h produced an increase in the levels of mRNA for parathyroid hormone (PTH). The levels of PTH secreted in response to nanomolar amounts of IL-8 were also elevated in cells incubated for 1 h with IL-8. Differential display analysis of mRNA from parathyroid cells, incubated in the presence and absence of IL-8, permitted the identification of cDNA clones for RNA species whose expression was either elevated or suppressed. These experiments suggest that IL-8 and inflammatory events play a role in bone homeostasis through actions on the parathyroid gland.
Chinese Hamster Ovary Cells Expressing α4β1 Integrin Stimulate Osteoclast Formation In Vitro
Authors: Akatsu Takuhiko , Ono Katsuhiro , Murakami Takehiko , Katayama Yasuyuki , Nishikawa Miyuki , Wada Seiki , Yamamoto Michiko , Kugai Nobuo , Matsuura Nariaki , Takada Yoshikazu , Nagata Naokazu
Published: April 2006

It is reported that Chinese hamster ovary cells transfected with human α4 cDNA (α4CHOs) and expressing functional α4β1 integrin developed bone metasasis in nude mice. To clarify the role of α4β1 integrin in bone metastasis, in terms of tumor-mediated bone destruction, we examined whether α4CHOs stimulate osteoclast formation in cocultures with mouse bone marrow cells. The number of osteoclast-like cells identified as tartrate-resistant acid phosphatase positive multinucleated cells (TRAP(+) MNCs) formed from bone marrow cells increased with the increasing number of α4CHOs cocultured. The effects of 1,25-dihydroxyvitamin D3(1,25(OH)2D3) and prostaglandin E2 (PGE2) on TRAP(+) MNC formation were enhanced in cocultures with α4CHOs. TRAP(+) MNCs induced by α4CHOs possessed calcitonin receptors and resorbed calcified tissues. In cocultures, α4CHOs and bone marrow stromal cells were in contact with each other and bone marrow stromal cells expressed vascular cell adhesion molecule-1 (VCAM-1), which is one of the ligands for α4β1 integrin. TRAP(+) MNC formation was not stimulated in cocultures where direct contact between α4CHOs and bone marrow cells was inhibited by membrane filters. α4CHOs do not support TRAP(+) MNC formation in cocultures with spleen cells but do support TRAP(+) mononuclear cell and MNC formation from spleen cells in the presence of osteoblastic cells. Cultured media from α4CHOs, bone marrow cells, and cocultures of α4CHOs and bone marrow cells did not stimulate TRAP(+) MNC formation or enhance the effects of 1,25(OH)2D3 and PGE2 in bone marrow cultures. The concentrations of PGE2 and interleukin-6 (IL-6) in cultured media were not different between the cultures of bone marrow cells and the cocultures of bone marrow cells and α4CHOs. Anti-human α4 and anti-mouse VCAM-1 antibodies inhibited TRAP(+) MNC formation induced by α4CHOs. These results indicate that α4CHOs stimulated TRAP(+) MNC formation through direct cell-to-cell interaction between α4β1 and VCAM-1. It is suggested that in addition to various soluble factors regulating osteoclast formation, cell-to-cell interaction between tumor cells and bone marrow cells is important for inducing osteoclasts at the site of bone metastasis and leading to bone destruction.
The Escherichia coli Chaperonin 60 (groEL) Is a Potent Stimulator of Osteoclast Formation
Authors: Reddi Krisanavane , Meghji Sajeda , Nair Sean P. , Arnett Timothy R. , Miller Andrew D. , Preuss Monika , Wilson Michael , Henderson Brian , Hill Peter
Published: April 2006

Chaperonins (cpns) are intracellular oligomeric protein complexes that fold and refold proteins in a catalytic manner and aid in the transmembrane transport of cellular proteins. We reported previously that the lipopolysaccharide-free recombinant cpn60 of Escherichia coli (groEL) is able to stimulate the breakdown of murine calvarial bone in culture and showed that such resorption is potently inhibited by an inhibitor of the enzyme cyclo-oxygenase and to a lesser extent by inhibitors of 5-lipoxygenase. In this study, we have investigated the effects of groEL on the resorptive activity and formation of osteoclasts in culture. In low density, osteoclast-containing cultures from neonatal rats incubated for 24 or 96 h on dentine discs, groEL (1–1000 ng/ml) stimulated resorption pit formation up to 4-fold, but this effect was essentially dependent on cell number. Using 12-day cultures of mouse bone marrow to assess osteoclast recruitment, groEL (1–1000 ng/ml) caused a dramatic dose-dependent stimulation of the formation of tartrate-resistant acid phosphatase-positive multinucleated cells and the resorption of the dentine on which bone marrow cells were cultured. Osteoclast formation elicited by groEL was almost completely abolished by indomethacin, an inhibitor of cyclo-oxygenase, but was unaffected by inhibitors of 5-lipoxygenase, suggesting that prostaglandins but not leukotrienes may mediate the action of groEL on osteoclastogenesis. It is possible that bacterial cpn60s such as groEL may play a role in the osteolysis associated with bone infections. Whether endogenous (“self”) chaperonins have a role in other bone loss disorders, such as osteoporosis, is an intriguing possibility.
Bone Mineral Density and Bone Turnover in Asthmatics Treated with Long-Term Inhaled or Oral Glucocorticoids
Authors: Ebeling Peter R. , Erbas Bircan , Hopper John L. , Wark John D. , Rubinfeld A. Roman
Published: April 2006

Inhaled glucocorticoids are pivotal in maintenance therapy of chronic bronchial asthma; however, conflict exists over their effects on bone and mineral metabolism. We measured bone mineral density (BMD), bone turnover markers, and adrenal steroid hormones in 53 patients (34 female, 19 male) with chronic bronchial asthma who had taken either inhaled beclomethasone or budesonide in doses of ≥ 1500 μg/day for at least 12 months to determine pathogenetic mechanisms of bone loss. To account for the effect of prior oral glucocorticoid exposure we divided patients into two groups: one with (OG) and the other without (IG) a past history of maintenance (> 1 month) oral glucocorticoid therapy. Lumbar spine (LS) and proximal femur BMDs were ∼1 SD lower in men and women taking OG or high-dose IG for chronic bronchial asthma, potentially equivalent to a doubling of the risk of fracture at these sites. Prior exposure to OG in women was also associated with lower LS and proximal femur BMDs, while men were more sensitive to the adverse effects of IG on LS and Ward's triangle BMDs. Bone formation markers were decreased; however, bone resorption marker concentrations were normal. All patients had evidence of suppression of both endogenous glucocorticoid and adrenal androgen production. Both total duration of OG and biochemical bone turnover marker concentrations were negatively related to proximal femur and rib BMDs and total body bone mineral content, but not to LS BMD. These were stronger for bone resorption markers. Uncoupling of ongoing normal bone resorption from suppressed bone formation may therefore contribute to glucocorticoid-associated bone loss in asthma. Adrenal androgen suppression may also increase the susceptibility of postmenopausal women in particular to bone loss with OG. Although the effects of high-dose IG on BMD are associated with lower LS BMD in men, this observation should now be investigated further in prospective studies.
Establishing an Immortalized Human Osteoprecursor Cell Line: OPC1
Authors: Winn Shelley R. , Randolph Gannon , Uludag Hasan , Wong Shou C. , Hair Gregory A. , Hollinger Jeffrey O.
Published: April 2006

The present studies evaluated the feasibility of establishing a conditionally immortalized osteoprecursor cell line derived from human fetal bone tissue. Primary cultures were transfected with a plasmid in which the Mx-1 promoter drives the expression of SV40 T-antigen when activated by human A/D interferon. Several neomycin (G418)-resistant colonies were characterized for cell growth and alkaline phosphatase (ALP) enzyme activity. The clone, designated OPC1 (osteoblastic precursor cell line 1), which exhibited the highest ALP enzyme activity at passage 10 (P10), was selected for additional osteogenic phenotypic characterization. Reverse transcription-polymerase chain reaction (RT-PCR) phenotyping revealed abundant mRNA for osteocalcin (OC), osteonectin (ON), osteopontin (OP), parathyroid hormone receptor (PTHr), ALP, and procollagen type I (ProI). In addition, the levels of quantitative RT-PCR product of ON, OP, PTHr, and ProI mRNAs exhibited a marked up-regulation when maintained in medium containing an osteogenic supplement (OS). The ability to stimulate osteogenic differentiation was characterized in postconfluent OPC1 cells maintained in tissue culture medium supplemented with recombinant human bone morphogenetic protein-2 (rhBMP-2) either with or without an OS. All treatment groups exhibited a striking up-regulation of ALP enzyme activity that coincided with ALP histochemical observations. Postconfluent cells also exhibited the ability to form mineralized nodules under all treatments (confirmed by von Kossa histochemical staining and calcium deposition). An enzyme immunosorbent assay (EIA) was utilized to measure intact human OC from the OPC1 line under the various treatments. Abundant OC was evident in the tissue culture medium indicating de novo sythesis and release from the OPC1 line under appropriate conditions. The clonal human-derived OPC1 line represents a homogeneous osteogenic cell line that not only has maintained a consistent bone phenotype from P10 to at least P30, but has also exhibited the capacity to generate programmed differentiation in the presence of low dose rhBMP-2 (10 ng/ml). Thus, the OPC1 line is a human-derived osteoprecursor that provides a sensitive in vitro cell culture system to evaluate bone development, cell/biomaterial interactions, and may be a useful screen for putative bone differentiating factors.
The Transcription Factor Sox9 Is Involved in BMP-2 Signaling
Authors: Zehentner Barbara K. , Dony Carola , Burtscher Helmut
Published: April 2006

We investigated the regulation of Sox9, a transcription factor known to play a role in chondrogenesis, by bone morphogenetic protein-2 (BMP-2) and hedgehog proteins in order to better understand their signaling function in endochondral bone formation. The mesenchymal progenitor cell line C3H10T1/2 was stimulated with BMP-2. Sox9 expression levels were measured by quantitative reverse transcriptase-polymerase chain reaction and Northern analysis. We found that Sox9 was up-regulated by BMP-2 in a dose-dependent manner. The expression of Col2a1, a downstream response gene of Sox9, was also significantly increased upon BMP-2 addition. We also monitored Sox9 expression after the addition of BMP-2 to osteosarcoma cell lines; BMP-2 treatment increased Sox9 mRNA levels in MG63, considered to be early osteoblast-like, but not in human osteogenic sarcoma (HOS) cells, which are thought to be more advanced in the osteoblastic lineage. This response seems to be influenced by differences in BMP receptor expression; MG63 cells express BMP receptor IA (BMPR-IA), whereas HOS cells express BMPR-IA and BMPR-IB. We also saw an increase in Sox9 mRNA levels in BMP-2–treated primary human bone cells (HBCs) derived from femoral heads. We found that in addition to BMP-2, Sonic and Indian hedgehog can increase Sox9 expression in C3H10T1/2 and primary HBCs. Time course studies with C3H10T1/2 cells after BMP-2 stimulation showed increasing expression of cartilage markers, decrease of collagen I mRNA, and a late induction of osteocalcin expression. Moreover, the treatment of C3H10T1/2 cells with Sox9 antisense oligonucleotides revealed that Sox9 is a downstream mediator of BMP-2 affecting the expression of chondrocyte and osteoblast marker genes. Our data show that Sox9 is an important downstream mediator of the BMP-2 and hedgehog signaling pathways in osteogenic cells.
Mechanical Strain Stimulates ROS Cell Proliferation Through IGF-II and Estrogen Through IGF-I
Authors: Cheng Ming Zhao , Zaman Gul , Rawlinson Simon C. F. , Mohan Subburaman , Baylink David J. , Lanyon Lance E.
Published: April 2006

The mechanism by which mechanical strain stimulates bone cell proliferation was investigated and compared with that of estrogen in ROS 17/2 .8 cells. Similarity of strain-related responses between ROS cells and osteoblasts was established by demonstrating that ROS cells respond to a short single period of strain in their substrate (1000–3500 μϵ, 600 cycles, 1 Hz) by a similar strain magnitude-related increase in glucose 6-phosphate dehydrogenase activity as rat osteoblasts and osteocytes in explants in situ. ROS17/2 .8 cells also showed similar proliferative responses to strain and 17β-estradiol, as assessed by [3H]thymidine incorporation and cell counting, as primary cultures of long bone-derived osteoblast-like cells. Strain-related increase in proliferation in ROS cells was accompanied by a 4-fold increase in levels of insulin-like growth factor-II (IGF-II) in conditioned medium. Neither strain nor estrogen had an effect on the conditioned medium levels of IGF-I. Exogenous truncated IGFs tIGF-I and tIGF-II both increased proliferation in a dose-dependent manner. The neutralizing monoclonal antibody (nMAb) to IGF-I blocked proliferation stimulated by tIGF-I but not that due to tIGF-II and vice versa. IGF-I receptor blocking antibody (IGF-IRBAb) blocked the proliferative effect of tIGF-I but not that to tIGF-II. The proliferative effect of estrogen was abolished by IGF-I nMAb and IGF-IRBAb, but these antibodies had no effect on the proliferative response to strain. In contrast IGF-II nMAb abolished the proliferative effect of strain but had no effect on that of estrogen. These data show that ROS17/2 .8 cells have similar responses to strain and estrogen qualitatively and quantitatively as rat osteoblasts in situ and rat long bone–derived osteoblast-like cells in primary culture. Estrogen-related proliferation in ROS17/2 .8 cells appears to be mediated by IGF-I acting through the IGF-I receptor and does not involve IGF-II. In contrast, strain-related proliferation appears to be mediated by IGF-II and does not involve either IGF-I or the IGF-I receptor.
Bone Physiology During Pregnancy and Lactation in Young Macaques
Authors: Ott Susan M. , Lipkin Edward W. , Newell-Morris Laura
Published: April 2006

We used a nonhuman primate model (Macaca nemestrina) of adolescent human pregnancy to characterize bone remodeling at midpregnancy and at weaning and the associated changes in bone mass. In this longitudinal study, 125 nulliparous females were followed through pregnancy, 6 months of lactation, and 3 months postweaning; 13 nonpregnant females served as controls. Between early pregnancy and midpregnancy, the whole body bone mineral increased. There was no significant change between midpregnancy and parturition. Between parturition and 3 months lactation, the animals lost 3.0% of their bone mineral (p < 0.01), which was regained by 3 months after weaning. The vertebral bone mineral apparent density decreased during pregnancy and 6 months of lactation, followed by an increase during the 3 months after weaning. Calcium, phosphate, 25-hydroxyvitamin D, and osteocalcin increased significantly from midpregnancy to weaning whereas 1,25-dihydroxyvitamin D values showed significant decreases. Histomorphometric measurements from bone biopsies showed significant increases in most parameters of bone formation between pregnancy and weaning. These results are consistent with the hypothesis that at midpregnancy bone formation is decreased and cancellous bone resorption may have increased. During lactation, losses occur in both cortical and cancellous bone, partially depleting the maternal reservoir of calcium, but a subsequent increase in bone formation enables restoration of bone mineral after weaning to values similar to those in the control group.