bluuebunny/crossref_metadata_2025 huggingface dataset:
Race and Sex Effects on the Association Between Muscle Strength, Soft Tissue, and Bone Mineral Density in Healthy Elders: The Health, Aging, and Body Composition Study
Authors: Taaffe Dennis R. , Cauley Jane A. , Danielson Michelle , Nevitt Michael C. , Lang Thomas F. , Bauer Douglas C. , Harris Tamara B.
Published: April 2006

Two factors generally reported to influence bone density are body composition and muscle strength. However, it is unclear if these relationships are consistent across race and sex, especially in older persons. If differences do exist by race and/or sex, then strategies to maintain bone mass or minimize bone loss in older adults may need to be modified accordingly. Therefore, we examined the independent effects of bone mineral-free lean mass (LM), fat mass (FM), and muscle strength on regional and whole body bone mineral density (BMD) in a cohort of 2619 well-functioning older adults participating in the Health, Aging, and Body Composition (Health ABC) Study with complete measures. Participants included 738 white women, 599 black women, 827 white men, and 455 black men aged 70-79 years. BMD (g/cm2) of the femoral neck, whole body, upper and lower limb, and whole body and upper limb bone mineral-free LM and FM was assessed by dual-energy X-ray absorptiometry (DXA). Handgrip strength and knee extensor torque were determined by dynamometry. In analyses stratified by race and sex and adjusted for a number of confounders, LM was a significant (p < 0.001) determinant of BMD, except in white women for the lower limb and whole body. In women, FM also was an independent contributor to BMD at the femoral neck, and both FM and muscle strength contributed to limb BMD. The following were the respective β-weights (regression coefficients for standardized data, Std β) and percent difference in BMD per unit (7.5 kg) LM: femoral neck, 0.202-0.386 and 4.7-5.9%; lower limb, 0.209-0.357 and 2.9-3.5%; whole body, 0.239-0.484 and 3.0-4.7%; and upper limb (unit = 0.5 kg), 0.231-0.407 and 3.1-3.4%. Adjusting for bone size (bone mineral apparent density [BMAD]) or body size BMD/height) diminished the importance of LM, and the contributory effect of FM became more pronounced. These results indicate that LM and FM were associated with bone mineral depending on the bone site and bone index used. Where differences did occur, they were primarily by sex not race. To preserve BMD, maintaining or increasing LM in the elderly would appear to be an appropriate strategy, regardless of race or sex.
Soy Intake and the Maintenance of Peak Bone Mass in Hong Kong Chinese Women
Authors: Ho Suzanne C. , Chan Sieu Gaen , Yi Qilong , Wong Eric , Leung P. C.
Published: April 2006

Our previous study on bone health among premenopausal women showed that bone mass consolidation is attained by the early 30s, and small loss of spinal bone mineral density (SBMD) occurs soon after peak bone mass attainment. Recent interest has been shown in the potential beneficial effects of phytoestrogens on bone health. However, data are lacking, particularly in Asian women. This study aims to investigate the effect of soy isoflavones intake on the maintenance of peak bone mass in a cohort of 132 women aged 30-40 years who were followed up for 3 years. Baseline measurements of SBMD (L2-L4) were obtained using dual-energy X-ray densitometry, and dietary intake of soy foods and other key nutrients, including dietary calcium, were obtained through a quantitative food frequency method. Information on body measurements; physical activity (PA), weight-bearing activity in particular; age of menarche; and number of pregnancies were obtained at baseline. Repeated measurements of SBMD were obtained yearly for a further 3 years with an average follow-up time of 38 months. Analyses were performed on 116 subjects with at least three SBMD measurements (at baseline, 3-year follow-up, and at least one measurement during follow-up). The individual SBMD regression slope was computed for each of the subjects. Soy isoflavones consumption was categorized as quartiles of intake. We observed a significant difference in the SBMD individual regression slopes between women belonging to the fourth and first soy isoflavones intake quartiles. The positive effect of soy isoflavones on SBMD remained after adjusting for age and body size (height, weight, and bone area). Multiple linear regression analysis including the other known covariates (lean body mass, PA, energy adjusted calcium, and follow-up time) showed that soy isoflavones, together with these variables, accounted for 24% of the variances of the SBMD individual regression slope. This longitudinal study shows that soy intake had a significant effect on the maintenance of SBMD in women aged 30-40 years. The effects of phytoestrogens on bone health should be explored further in a population with habitual dietary soy but low calcium intake.
Female Estrogen Receptor β−/− Mice Are Partially Protected Against Age-Related Trabecular Bone Loss
Authors: Windahl Sara H. , Hollberg Karin , Vidal Olle , Gustafsson Jan-Åke , Ohlsson Claes , Andersson Göran
Published: April 2006

Recently, it has been shown that inactivation of estrogen receptor β (ER-β) by gene targeting results in increased cortical bone formation in adolescent female mice. To study the possible involvement of ER-β in the regulation of the mature skeleton, we have extended the analyses to include 1-year-old ER-β knockout mice (ER-β−/−). Male ER-β−/− mice did not express any significant bone phenotypic alterations at this developmental stage. However, the increase in cortical bone parameters seen already in the adolescent female ER-β−/− mice was maintained in the older females. The aged female ER-β−/− mice further exhibited a significantly higher trabecular bone mineral density (BMD) as well as increased bone volume/total volume (BV/TV) compared with wild-type (wt) mice. This was caused by a less pronounced loss of trabecular bone during adulthood in female ER-β−/− mice. The growth plate width was unaltered in the female ER-β−/− mice. Judged by the expression of the osteoclast marker tartrate-resistant acid phosphatase (TRAP) and cathepsin K (cat K; reverse-transcription-polymerase chain reaction [RT-PCR]) as well as the serum levels of C-terminal type I collagen cross-linked peptide, bone resorption appeared unaffected. However, an increase in the messenger RNA (mRNA) expression levels of the osteoblast marker core-binding factor α1 (Cbfa1) suggested an anabolic effect in bones of old female ER-β−/− mice. In addition, the mRNA expression of ER-α was augmented, indicating a role for ER-α in the development of this phenotype. Taken together, the results show that ER-β is involved in the regulation of trabecular bone during adulthood in female mice and suggest that ER-β acts in a repressive manner, possibly by counteracting the stimulatory action of ER-α on bone formation.
Inhibition of Na+/Ca2+ Exchange with KB-R7943 or Bepridil Diminishes Mineral Deposition by Osteoblasts
Authors: Stains Joseph P. , Gay Carol V.
Published: April 2006

Osteoblasts form new bone by secreting a complex extracellular matrix that has the capacity to mineralize when adequate amounts of calcium and phosphate are supplied. The studies reported here show that long-term treatment of cultured, primary osteoblasts with Na+/Ca2+ exchanger (NCX) inhibitors, bepridil and KB-R7943, impacts in a dose-dependent manner the ability of the cells to form a calcified matrix. Treatment of confluent osteoblast cultures for 14 days with low levels of bepridil (3.0 μM) or KB-R7943 (1.0 μM and 0.1 μM) resulted in a significantly diminished capacity of these cells to mineralize bone matrix, without significantly altering cell morphology, viability, or cell differentiation. The data indicate that inhibition of NCX reduces mineral accumulation in the bone matrix by blocking the efflux of Ca2+ from the osteoblast into the bone fluid. In addition, immunocytochemistry of type I collagen (COLI) and bone sialoprotein (BSP) suggests that inhibition of NCX by 1.0 μM KB-R7943 also may impair the secretion of bone matrix proteins by the osteoblasts. This study is the first to show that NCX is an important regulator of the bone fluid microenvironment and that NCX appears critical to the mineralization process.
Accelerated Turnover of Metaphyseal Trabecular Bone in Mice Overexpressing Cathepsin K
Authors: Kiviranta Riku , Morko Jukka , Uusitalo Hannele , Aro Hannu T. , Vuorio Eero , Rantakokko Juho
Published: April 2006

This study is based on a hypothesis that overexpression of an osteoclast enzyme, cathepsin K, causes an imbalance in bone remodeling toward bone loss. The hypothesis was tested in transgenic (TG) mice harboring additional copies of the murine cathepsin K gene (Ctsk) identifiable by a silent mutation engineered into the construct. For this study, three TG mouse lines harboring 3-25 copies of the transgene were selected. Tissue specificity of transgene expression was determined by Northern analysis, which revealed up to 6-fold increases in the levels of cathepsin K messenger RNA (mRNA) in calvarial and long bone samples of the three TG lines. No changes were seen in the mRNA levels of other osteoclast enzymes, indicating that the increase in cathepsin K mRNA was not a reflection of activation of all osteoclast enzymes. Immunohistochemistry confirmed that cathepsin K expression in the TG mice was confined to osteoclasts and chondroclasts. Histomorphometry revealed a significantly decreased trabecular bone volume (BV), but, surprisingly, also a marked increase in the number of osteoblasts, the rate of bone turnover, and the amount of mineralizing surface (MS). However, monitoring of bone density in the proximal tibias of the TG mice with peripheral quantitative computed tomography (pQCT) failed to reveal statistically significant changes in bone density. Similarly, no statistically significant alterations were observed in biomechanical testing at the age of 7 months. The increases in parameters of bone formation triggered by increased cathepsin K expression is an example of the tight coupling of bone resorption and formation during the bone-remodeling cycle.
Reconstituting Telomerase Activity Using the Telomerase Catalytic Subunit Prevents the Telomere Shorting and Replicative Senescence in Human Osteoblasts
Authors: Yudoh Kazuo , Matsuno Hiroaki , Nakazawa Fujio , Katayama Rie , Kimura Tomoatsu
Published: April 2006

The rate of bone formation is largely determined by the number of osteoblasts, which in turn is determined by the rate of replication of progenitors and the life span of mature cells, reflecting the timing of death by apoptosis. However, the exact age-dependent changes of the cellular activity, replicative potential, and life span of osteoblasts have not been investigated to date. Here, we present evidence that the cellular activity, telomere lengths, and replicative life span of osteoblastic cells obtained from juxta-articular bone marrow gradually decrease with the advance of donor age. Recently, telomerase reverse transcriptase (hTERT) has been identified as a human telomerase catalytic subunit. We transfected the gene encoding hTERT into telomerase-negative human osteoblastic cells from donors and osteoblastic cell strain NHOst 54881 cells and showed that expression of hTERT induces telomerase activity in these osteoblastic cells. In contrast to telomerase-negative control cells, which exhibited telomere shortening and senescence after 10-15 population doublings, telomerase-expressing osteoblastic cells had elongated telomere lengths and showed continued alkaline phosphatase activity and procollagen I C-terminal propeptide (PICP) secretion for more than 30 population doublings. These results indicate that osteoblasts with forced expression of hTERT may be used in cell-based therapies such as ex vivo gene therapy, tissue engineering, and transplantation of osteoblasts to correct bone loss or osteopenia in age-related osteoporotic diseases.
Dual Mechanism of Intercellular Communication in HOBIT Osteoblastic Cells: A Role for Gap-Junctional Hemichannels
Authors: Romanello Milena , D'Andrea Paola
Published: April 2006

Intercellular communication allows tissue coordination of cell metabolism and sensitivity to extracellular stimuli. Paracrine stimulation and cell-to-cell coupling through gap junctions induce the formation of complex cellular networks, which favors the intercellular exchange of nutrients and second messengers. Intercellular Ca2+ signaling was investigated in human osteoblast-like initial transfectant (HOBIT) cells, a human osteoblastic cell line in which cells retain most of the osteoblastic differentiation markers. HOBIT cells express connexin43 (Cx43) clustered at the cell-to-cell boundary and display functional intercellular coupling as assessed by the intercellular transfer of Lucifer yellow. Mechanical stimulation of a single cell induced a wave of increased Ca2+ that was radially propagated to surrounding cells. Treatment of cells with thapsigargin blocked mechanically induced signal propagation. Intercellular Ca2+ spreading and dye transfer were inhibited by 18α-glycyrrhetinic acid (18-GA), showing the involvement of gap junctions in signal propagation. Pretreatment of cells with suramin or with apyrase decreased the extent of wave propagation, suggesting that ATP-mediated paracrine stimulation contribute to cell-to-cell signaling. The functional expression of gap-junctional hemichannels was evidenced in experiments of Mn2+ quenching, extracellular dye uptake, and intracellular Ca2+ release, activated by uptake of inositol 1,4,5-trisphosphate (InsP3) from the external medium. Gap-junctional hemichannels were activated by low extracellular Ca2+ concentrations and inhibited by 18-GA. A role for Cx hemichannels in adenosine triphosphate (ATP) release and paracrine stimulation is suggested.
Characterization of Tissue Transglutaminase in Human Osteoblast-like Cells
Authors: Heath Deborah J. , Downes Sandra , Verderio Elisabetta , Griffin Martin
Published: April 2006

Tissue transglutaminase (tTG) is a calcium-dependent and guanosine 5′-triphosphate (GTP) binding enzyme, which catalyzes the post-translational modification of proteins by forming intermolecular ϵ(γ-glutamyl)lysine cross-links. In this study, human osteoblasts (HOBs) isolated from femoral head trabecular bone and two osteosarcoma cell lines (HOS and MG-63) were studied for their expression and localization of tTG. Quantitative evaluation of transglutaminase (TG) activity determined using the [1,414C]-putrescine incorporation assay showed that the enzyme was active in all cell types. However, there was a significantly higher activity in the cell homogenates of MG-63 cells as compared with HOB and HOS cells (p < 0.001). There was no significant difference between the activity of the enzyme in HOB and HOS cells. All three cell types also have a small amount of active TG on their surface as determined by the incorporation of biotinylated cadaverine into fibronectin. Cell surface-related tTG was further shown by preincubation of cells with tTG antibody, which led to inhibition of cell attachment. Western blot analysis clearly indicated that the active TG was tTG and immunocytochemistry showed it be situated in the cytosol of the cells. In situ extracellular enzyme activity also was shown by the cell-mediated incorporation of fluorescein cadaverine into extracellular matrix (ECM) proteins. These results clearly showed that MG-63 cells have high extracellular activity, which colocalized with the ECM protein fibronectin and could be inhibited by the competitive primary amine substrate putrescine. The contribution of tTG to cell surface/matrix interactions and to the stabilization of the ECM of osteoblast cells therefore could by an important factor in the cascade of events leading to bone differentiation and mineralization.
A Bone-Seeking Clone Exhibits Different Biological Properties from the MDA-MB-231 Parental Human Breast Cancer Cells and a Brain-Seeking Clone In Vivo and In Vitro
Authors: Yoneda Toshiyuki , Williams Paul J. , Hiraga Toru , Niewolna Maria , Nishimura Riko
Published: April 2006

Breast cancer has a predilection for spreading to bone. The mechanism of preferential metastasis of breast cancer to bone is unknown. We hypothesize that breast cancer cells that develop bone metastases have the capacity to facilitate their colonization in bone. To examine this hypothesis, we established bone-seeking (MDA-231BO) and brain-seeking (MDA-231BR) clones of the human breast cancer cell line MDA-MB-231 by repeated sequential passages in nude mice and in vitro of metastatic cells obtained from bone and brain metastases, respectively. These clones were examined for distinguishing biological characteristics and compared with the MDA-231 parental cells (MDA-231P) in vivo and in vitro. Both the MDA-231BR and the MDA-231BO showed identical tumorigenicity to MDA-231P at the orthotopic site. MDA-231P that was inoculated into the heart developed metastases in bone, brain, ovary, and adrenal glands. On the other hand, MDA-231BO exclusively metastasized to bone with larger osteolytic lesions than MDA-231P. MDA-231BR exclusively disseminated to brain and failed to develop bone metastases. In culture, MDA-231BO produced greater amounts of parathyroid hormone-related protein (PTH-rP) than MDA-231BR and MDA-231P in the absence or presence of transforming growth factor β (TGF-β). Furthermore, the anchorage-independent growth of MDA- 231BO in soft agar was not inhibited by TGF-β, whereas TGF-β profoundly inhibited the growth of MDA-231P and MDA-231BR. Insulin-like growth factor I (IGF-I) markedly promoted the anchorage-independent growth of MDA-231BO, whereas marginal or no stimulation was observed in MDA-231BR or MDA-231P, respectively. Our data suggest that these phenotypic changes allow breast cancer cells to promote osteoclastic bone resorption, survive, and proliferate in bone, which consequently leads to the establishment of bone metastases.
Trabecular Structure Assessment in Lumbar Vertebrae Specimens Using Quantitative Magnetic Resonance Imaging and Relationship with Mechanical Competence
Authors: Beuf Olivier , Newitt David C. , Mosekilde Lis , Majumdar Sharmila
Published: April 2006

The purpose of this study was to use quantitative magnetic resonance imaging (MRI; high-resolution [HR] and relaxometry) to assess trabecular bone structure in lumbar vertebrae specimens and to compare these techniques with bone mineral density (BMD) in predicting stress values obtained from mechanical tests. Fourteen vertebral midsagittal sections from lumbar vertebrae L3 were obtained from cadavers (aged 22-76 years). HR images with a slice thickness of 300 μm and an in-plane spatial resolution of 117 μm2 × 117 μm2 were obtained. Transverse relaxation time T2′ distribution was measured by using an asymmetric spin-echo (ASE) sequence. Traditional morphometric measures of bone structure such as apparent trabecular bone fraction (app. BV/TV), apparent trabecular bone number (app. Tb.N), apparent trabecular bone separation (app. Tb.Sp), and apparent trabecular bone thickness (app. Tb.Th) as well as the directional mean intercept length (MIL) were calculated. Additionally, BMD measurements of these sections were obtained by dual-energy X-ray absorptiometry (DXA) and biomechanical properties such as directional stress values (to fracture) were determined on adjacent specimens. With the exception of T2′, all morphological parameters correlated very well with age, BMD, and stress values (|R| between 0.79 and 0.92). However, in the direction perpendicular to the magnetic field, T2′ values enhanced the adjusted R2 correlation value with horizontal (M/L) stress values in addition to BMD from 0.70 to 0.91 (p < 0.05).
Variability in Skeletal Mass, Structure, and Biomechanical Properties Among Inbred Strains of Rats
Authors: Turner Charles H. , Roeder Ryan K. , Wieczorek Arlo , Foroud Tatiana , Liu Guangda , Peacock Munro
Published: April 2006

The aim of this study was to assess the usefulness of the inbred rat model for studies of genetic influences on skeletal fragility. We characterized bone mass, geometry, and skeletal biomechanics in 11 inbred strains of rats. This study showed that considerable variation exists in bone structure, areal bone mineral density (aBMD), and fragility phenotypes among inbred strains of rats. Interestingly, the variability in skeletal phenotypes in rats was site specific, suggesting that no single gene regulates skeletal fragility at all sites. For instance, the Copenhagen 2331 (COP) strain had the greatest biomechanical properties in the femoral neck but only modest bone strength at the femoral midshaft, compared with other strains. Consequently, COP rats appear to have alleles that specifically enhance femoral neck biomechanical properties and may serve as a model for studying genetic influences on hip strength. The Brown Norway (BN) and Fischer 344 (F344) strains may provide models for vertebral fragility because each has relatively fragile lumbar vertebrae. The F344 rats also had the most fragile femora and, thus, appear to carry alleles that cause overall skeletal fragility. We identified two inbred rat crosses that will facilitate the study of genetic influences on skeletal fragility at clinically relevant skeletal sites: Lewis (LEW) with F344 (primarily for vertebral fragility) and COP with DA (primarily for femoral neck fragility). The results strongly suggest that selected crosses of inbred strains of rats will provide useful models for studying genetic influences on bone strength and structure.
The Development of Metaphyseal Cortex—Implications for Distal Radius Fractures During Growth
Authors: Rauch Frank , Neu Christina , Manz Friedrich , Schoenau Eckhard
Published: April 2006

Fractures of the distal radial metaphysis are very common in otherwise healthy children. The reasons for this high fracture incidence are not entirely clear. To address this problem, we undertook a detailed analysis of distal radius development using peripheral quantitative computed tomography (pQCT) at a site 4% proximal to the radial articular surface. The study population comprised 337 healthy children and adolescents (aged 6‐18 years; 171 girls) and 107 adults (aged 29‐40 years; 88 women). Total volumetric bone mineral density (vBMD) remained stable at about 70% of the adult value between the ages of 6‐7 years and 14‐15 years in both genders. Cortical thickness increased little between 6‐7 years and 12‐13 years in girls and 14‐15 years in boys. Strength‐Strain Index (SSI; a parameter combining geometry and density) was still at only 20% of the adult value in girls aged 10‐11 years and at 21% of the adult level in boys aged 12‐13 years. At these ages, factors that contribute to the mechanical challenge to the distal radius in case of a fall (forearm length and body weight) had already reached 49% and 36% of the adult value in girls and boys, respectively. The shaping of the distal radius cortex (metaphyseal inwaisting) was assessed by analyzing the decrease in cross‐sectional bone size between adjacent bone slices in a separate population of 44 children (aged 8‐19 years; 26 girls). The rates of periosteal resorption and endocortical apposition were estimated to average 8 μm/day and 10 μm/day, respectively, during the growth period. In conclusion, during growth the increase in distal radius strength lags behind the increase in mechanical challenges caused by a fall, because metaphyseal cortical thickness does not increase sufficiently. The endocortical apposition rate is already very high at that site and apparently cannot be further increased to levels that would be necessary to keep bone strength adapted to the mechanical requirements.
Bone Morphogenetic Proteins, Extracellular Matrix, and Mitogen-Activated Protein Kinase Signaling Pathways Are Required for Osteoblast-Specific Gene Expression and Differentiation in MC3T3-E1 Cells
Authors: Xiao Guozhi , Gopalakrishnan Rajaram , Jiang Di , Reith Elizabeth , Benson M. Douglas , Franceschi Renny T.
Published: April 2006

Osteoblasts secrete a complex extracellular matrix (ECM) containing collagenous and noncollagenous proteins, bone morphogenetic proteins (BMPs), and growth factors. Osteoblast-specific gene expression requires ascorbic acid (AA)-dependent assembly of a collagenous ECM. Matrix responsiveness requires an α2β1 integrin-collagen interaction and mitogen-activated protein kinase (MAPK) activity, which phosphorylates and activates the osteoblast-specific transcription factor Cbfa1. This study examines interactions between this integrin/MAPK-mediated pathway and signals initiated by BMPs contained in the osteoblast matrix. MC3T3-E1 cells were shown to constitutively express BMP-2, BMP-4, and BMP-7. Noggin, a specific BMP inhibitor, reversibly blocked AA-induced gene expression, indicating that BMP production by MC3T3-E1 cells was necessary for differentiation. The ability of exogenously added BMP-2, BMP-4, or BMP-7 to stimulate osteocalcin (OCN) and bone sialoprotein (BSP) mRNAs or OCN promoter activity was synergistically increased in cells that were actively synthesizing an ECM (i.e., were grown in the presence of AA). A minimum of 4 days of ECM accumulation was required for this synergistic response to be observed. Neither BMP-7, AA, nor a combination of these two treatments had major effects on Cbfa1 messenger RNA (mRNA) or protein levels, as would be expected if regulation was mainly at the posttranscriptional level. U0126, a specific inhibitor of MAPK/extracellular signal-regulated kinase (MEK), blocked AA- or BMP-7/AA-dependent gene expression in a time- and dose-dependent manner that was closely correlated with inhibition of extracellular signal-regulated kinase (ERK) phosphorylation. This work establishes that autocrine BMP production as well as integrin-mediated cell-collagen interactions are both required for osteoblast differentiation, and both these pathways require MAP kinase activity.
Nucleotide Pyrophosphatase Gene Polymorphism Associated With Ossification of the Posterior Longitudinal Ligament of the Spine
Authors: Koshizuka Yu , Kawaguchi Hiroshi , Ogata Naoshi , Ikeda Toshiyuki , Mabuchi Akihiko , Seichi Atsushi , Nakamura Yusuke , Nakamura Kozo , Ikegawa Shiro
Published: April 2006

Ossification of the posterior longitudinal ligament (OPLL) of the spine is a disease that causes paralysis by compressing the spinal cord. Based on the fact that the nucleotide pyrophosphatase (Npps) gene is responsible for ectopic ossification in ttw, an OPLL model mouse, the possibility was explored whether the human NPPS gene is associated with susceptibility to and severity of OPLL. First, we screened for single-nucleotide polymorphisms (SNPs) in the human NPPS locus using selected 25 OPLL patients with young onset (<35 years old) or severe ossification (>10 ossified vertebrae), and identified three novel SNPs in the locus. A case-control association study between 180 OPLL patients and 265 non-OPLL controls showed that one of these SNPs, IVS15-14T → C substitution, was more frequently observed in OPLL patients (p = 0.022), especially in those with severe ossification (p < 0.0001) and young onset (p = 0.002), than in controls. A stratified study with the number of ossified vertebrae in OPLL patients revealed that IVS15-14T → C substitution (p = 0.013) as well as young onset (p = 0.046) and female sex (p = 0.006) were associated with severe ossification. We conclude that the IVS15-14T → C substitution in the human NPPS gene is associated not only with susceptibility to, but also with severity of OPLL.
Role of Low Levels of Endogenous Estrogen in Regulation of Bone Resorption in Late Postmenopausal Women
Authors: Heshmati Hassan M. , Khosla Sundeep , Robins Simon P. , O'Fallon W. Michael , Melton L. Joseph , Riggs B. Lawrence
Published: April 2006

Although median levels of bone turnover are increased in postmenopausal women, it is unclear whether the low circulating levels of endogenous estrogen exert a regulatory role on these levels. This issue was evaluated by assessing the effect of a blockade of estrogen synthesis on bone turnover markers in 42 normal women (mean age ± SD, 69 ± 5 years) randomly assigned to groups receiving the potent aromatase inhibitor letrozole or placebo for 6 months. Letrozole treatment reduced serum estrone (E1) and estradiol (E2) to near undetectable levels (p < 0.0001). This treatment did not affect bone formation markers but, as compared with the placebo group, increased bone resorption markers (urine 24-h pyridinoline [PYD] by 13.3% [p < 0.05] and 24-h urine deoxypyridinoline [DPD] by 14.2% [p < 0.05]) and decreased serum parathyroid hormone (PTH) by 22% (p = 0.002). These data indicate that in late postmenopausal women even the low serum estrogen levels present exert a restraining effect on bone turnover and support the concept that variations in these low levels may contribute to differences in their rate of bone loss.
Osteogenesis Imperfecta Type VI: A Form of Brittle Bone Disease with a Mineralization Defect
Authors: Glorieux Francis H. , Ward Leanne M. , Rauch Frank , Lalic Ljiljana , Roughley Peter J. , Travers Rose
Published: April 2006

Osteogenesis imperfecta (OI) is a heritable disease of bone in which the hallmark is bone fragility. Usually, the disorder is divided into four groups on clinical grounds. We previously described a group of patients initially classified with OI type IV who had a discrete phenotype including hyperplastic callus formation without evidence of mutations in type I collagen. We called that disease entity OI type V. In this study, we describe another group of 8 patients initially diagnosed with OI type IV who share unique, common characteristics. We propose to name this disorder “OI type VI.” Fractures were first documented between 4 and 18 months of age. Patients with OI type VI sustained more frequent fractures than patients with OI type IV. Sclerae were white or faintly blue and dentinogenesis imperfecta was uniformly absent. All patients had vertebral compression fractures. No patients showed radiological signs of rickets. Lumbar spine areal bone mineral density (aBMD) was low and similar to age-matched patients with OI type IV. Serum alkaline phosphatase levels were elevated compared with age-matched patients with type IV OI (409 ± 145 U/liter vs. 295 ± 95 U/liter; p < 0.03 by t-test). Other biochemical parameters of bone and mineral metabolism were within the reference range. Mutation screening of the coding regions and exon/intron boundaries of both collagen type I genes did not reveal any mutations, and type I collagen protein analyses were normal. Qualitative histology of iliac crest bone biopsy specimens showed an absence of the birefringent pattern of normal lamellar bone under polarized light, often with a “fish-scale” pattern. Quantitative histomorphometry revealed thin cortices, hyperosteoidosis, and a prolonged mineralization lag time in the presence of a decreased mineral apposition rate. We conclude that type VI OI is a moderate to severe form of brittle bone disease with accumulation of osteoid due to a mineralization defect, in the absence of a disturbance of mineral metabolism. The underlying genetic defect remains to be elucidated.
Matrix Regulation of Skeletal Cell Apoptosis II: Role of Arg-Gly-Asp-Containing Peptides
Authors: Perlot Robert L. , Shapiro Irving M. , Mansfield Kyle , Adams Christopher S.
Published: April 2006

This investigation was based on the assumption that arg-gly-asp (RGD)-containing peptides are released from the extracellular matrix of bone and cartilage during the remodeling cycle. We asked the question: Can RGD peptides influence skeletal cell viability? Primary human osteoblasts, mouse MC-3T3-E1 cells, and chick chondrocytes were incubated with purified RGD-containing peptides and cell viability was determined. The RGD peptide did not kill osteoblasts, chondrocytes, or MC-3T3-E1 cells. In contrast, RGDS and GRGDSP peptides killed all three cell types. Osteoblast death was quite rapid, occurring within 6 h of treatment. transferase uridyl mediated nick end labeling (TUNEL) and transmission electron microscopy (TEM) analysis indicated that death was mediated by apoptosis. To learn if mitochondria transduced the death signal, cells were treated with RGDS and organelle function was evaluated using a voltage-sensitive fluorescent probe. It was observed that there was no net loss of fluorescence and, hence, it was concluded that mitochondria were not the primary effectors of the apoptotic response. Experiments were performed with enzyme inhibitors to determine the import of the caspase pathway on RGDS-mediated osteoblast apoptosis. Results of these studies, as well as a study conducted using a fluorescent substrate, pointed to caspase 3 mediating the effector stage of the apoptotic process. Finally, using a purified labeled-RGDS peptide, we showed that the molecule was not restricted by the plasma membrane because it was accumulated in the cytosolic compartment. Results of the investigation support the view that resorption of the extracellular matrix generates peptide products that can induce apoptosis of vicinal cells.
Effects of Geranylgeranoic Acid in Bone: Induction of Osteoblast Differentiation and Inhibition of Osteoclast Formation
Authors: Wang Xinxiang , Wu Jian , Shidoji Yoshihiro , Muto Yasutoshi , Ohishi Nobuko , Yagi Kunio , Ikegami Sachie , Shinki Toshimasa , Udagawa Nobuyuki , Suda Tatsuo , Ishimi Yoshiko
Published: April 2006

Retinoids are known to be of special importance for normal bone growth and development. Recently, we reported that retinoids not only induced osteoblast differentiation, but also inhibited osteoclast formation in vitro. In this study, we examined the osteogenic effects of geranylgeranoic acid (GGA), a chemically synthesized acyclic retinoid, in bone in vitro and in vivo. GGA not only suppressed proliferation of osteoblastic MC3T3-E1 cells, but also up-regulated differentiation markers of osteoblasts such as alkaline phosphatase (ALP) activity and expression of osteopontin (OP) messenger RNA (mRNA). In contrast, GGA inhibited osteoclast formation induced by 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3] in cocultures of mouse bone marrow cells and primary osteoblasts. Treatment of stromal ST2 cells with GGA restored the 1α,25(OH)2D3- or prostaglandin E2 (PGE2)-induced suppression of osteoprotegerin (OPG) mRNA expression. GGA inhibited osteoclast formation induced by macrophage colony-stimulating factor (M-CSF) and soluble receptor activator of nuclear factor κB ligand (sRANKL) in the culture of bone marrow macrophages. Thus, it is likely that GGA inhibits osteoclast formation by affecting both osteoblasts and osteoclast progenitors in the coculture system. Furthermore, in vivo, GGA increased bone mineral density (BMD) of total as well as distal femur in a P6 strain of senescence-accelerated mice (SAMP6). These results indicate that GGA increases bone mass by maintaining a positive balance of bone turnover by inducing osteoblast differentiation and suppressing osteoclast formation.
Mapping Quantitative Trait Loci That Influence Femoral Cross-sectional Area in Mice
Authors: Klein Robert F. , Turner Renn J. , Skinner Lisa D. , Vartanian Kristina A. , Serang Maqsood , Carlos Amy S. , Shea Marie , Belknap John K. , Orwoll Eric S.
Published: April 2006

Size and shape are critical determinants of the mechanical properties of skeletal elements and can be anticipated to be highly heritable. Moreover, the genes responsible may be independent of those that regulate bone mineral density (BMD). To begin to identify the heritable determinants of skeletal geometry, we have examined femoral cross-sectional area (FCSA) in male and female mice from two inbred strains of mice with divergent FCSA (C57BL/6 [B6] and DBA/2 [D2]), a large genetically heterogeneous population (n = 964) of B6D2F2 mice and 18 BXD recombinant inbred (RI) strains derived from their F2 cross. Femora were harvested from 16-week-old mice and FCSA (bone and marrow space enclosed within the periosteum) was measured at the midshaft by digital image analysis. In all mouse populations examined, FCSA was positively correlated with body weight and weight-corrected FCSA (WC-FCSA) values were normally distributed in the BXD-RI and F2 populations, suggesting polygenic control of this trait. Genome-wide quantitative trait locus (QTL) analysis of the B6D2F2 population revealed regions on four different chromosomes that were very strongly linked to WC-FCSA (chromosomes 6, 8, 10, and X) in both genders. Evidence of gender-specific genetic influences on femoral geometry was also identified at three other chromosomal sites (chromosomes 2, 7, and 12). Supporting evidence for the WC-FCSA QTLs on chromosomes 2, 7, 8, 10, and 12 also was present in the RI strains. Interestingly, none of these WC-FCSA QTLs were identified in our previous QTL analysis of whole body BMD in the same B6D2F2 population. Thus, the genetic determinants of bone size appear to be largely, if not entirely, distinct from those that regulate BMD attainment. The identification of the genes responsible for geometric differences in bone development should reveal fundamentally important processes in the control of skeletal integrity.
Localization of the Mutation Responsible for Osteopetrosis in the op Rat to a 1.5-cM Genetic Interval on Rat Chromosome 10: Identification of Positional Candidate Genes by Radiation Hybrid Mapping
Authors: Dobbins David E. , Joe Bina , Hashiramoto Akira , Salstrom Jennifer L. , Dracheva Svetlana , Ge Lynn , Wilder Ronald L. , Remmers Elaine F.
Published: April 2006

Osteopetrosis is caused by a heterogenous group of bone diseases that result in an increase in skeletal mass because of inadequate osteoclastic bone resorption. In the op osteopetrotic rat, the disease has been linked to a single genetic locus located at the proximal end of rat chromosome 10. In this study, we identified a 1.5-cM genetic interval that contains the mutation. We then generated an improved radiation hybrid (RH) map of this region to identify potential functional and positional candidates for the op gene. Using the rat genome radiation hybrid panel, we mapped 57 markers including 24 genes (14 that have not yet been mapped in the rat) and 10 expressed sequence tag markers. Included in the mapped genes are several candidate genes that might significantly influence the biochemical pathways involved in osteopetrosis. These include genes involved in osteoclast differentiation, apoptosis, and the functional capabilities of mature osteoclasts to resorb bone. Further analysis of the genes and expressed transcripts mapped to this region may yield important insights into the multifactorial control of osteoclast function and the mechanisms of failed bone homeostasis in diseases such as osteopetrosis, osteoporosis, and rheumatoid arthritis in which failed bone homeostasis is an instigating or exacerbating circumstance of the disease process.
Involvement of Rho and p38 MAPK in Endothelin-1-Induced Expression of PGHS-2 mRNA in Osteoblast-Like Cells
Authors: Windischhofer W. , Zach D. , Fauler G. , Raspotnig G. , Köfeler H. , Leis H. J.
Published: April 2006

Prostaglandins (PGs) play an important role in bone remodeling because eicosanoids are local mediators of bone metabolism, which can induce physiological and pathological responses of bone tissue. Biosynthesis of PGs is catalyzed by constitutively expressed PG endoperoxide G/H synthase (PGHS) 1 and by the inducible isoform PGHS-2. In MC3T3-E1 osteoblast-like cells, expression of PGHS-2 was shown by mechanical forces, cytokines, growth factors, and hormones. Recently, endothelin (ET) 1-stimulated PGHS-2 mRNA expression was described, leading to a burst in prostaglandin E2 (PGE2) production. In this study, we investigated ET-1-induced signal transduction pathway(s) involved in the PGHS-2 mRNA production. Time course of PGHS-2 mRNA expression reaching the maximum within 45 minutes is in good agreement with the concept of an immediate early gene product. Inhibition of phospholipase C (PLC), phospholipase D (PLD), phosphatidylinositol-3 kinase (PI-3-kinase), and protein kinase C (PKC) had no influence on PGHS-2 synthesis. Using specific blockers of tyrosine kinases indicated involvement of p38 MAPK but not p42/44 MAPK. By preloading cells with exoenzyme C3, we were able to show requirement of the Rho family of G proteins for p38 MAPK phosphorylation and PGHS-2 mRNA synthesis, whereas pertussis toxin (PTX) and cholera toxin (CTX) had no remarkable effect.
Phosphate and Calcium Uptake by Rat Odontoblast-Like MRPC-1 Cells Concomitant With Mineralization
Authors: Lundquist P. , Ritchie H. H. , Moore K. , Lundgren T. , Linde A.
Published: April 2006

It has been suggested that odontoblasts are instrumental in translocating Ca2+ and inorganic phosphate (Pi) ions during the mineralization of dentin. The aim of this study was to characterize cellular Pi and Ca2+ uptake in the novel rat odontoblast-like cell line mineralizing rat pulpal cell line (MRPC) 1 during mineralization to see if changes in the ion transport activity would occur as the cultures develop and begin forming a mineralized matrix. MRPC-1 cells were cultured in chemically defined medium containing ascorbate and Pi, and cultures were specifically analyzed for cellular Pi and Ca2+ uptake activities and expression of type II high-capacity Na+-Pi cotransporters. The odontoblast-like phenotype of the cell line was ascertained by monitoring the expression of collagen type I and dentin phosphopoprotein (DPP). Mineralized nodule formation started at day 9 after confluency and then rapidly increased. Ca2+ uptake by the cells showed a maximum during the end of the proliferative phase (days 5–7). Pi uptake declined to a basal level during proliferation and then was up-regulated simultaneously with the onset of mineralization to a level fourfold of the basal uptake, suggesting an initiating and regulatory role for cellular Pi uptake in mineral formation. This up-regulation coincided with a conspicuously increased glycosylation of NaPi-2a, indicating an activation of this Na+-Pi cotransporter. The study showed that MRPC-1 cells express an odontoblast-like phenotype already at the onset of culture, but that to mineralize the collagenous extracellular matrix (ECM) that formed, a further differentiation involving their ion transporters is necessary.
Relationship of Serum Leptin Concentration With Bone Mineral Density in the United States Population
Authors: Ruhl Constance E. , Everhart James E.
Published: April 2006

Overweight is associated with both higher bone mineral density (BMD) and higher serum leptin concentrations. In humans, little is known about the relationship of leptin concentration and bone density. We studied this relationship in a large, national population-based sample. Participants included 5815 adults in the Third U.S. National Health and Nutrition Examination Survey (NHANES III; 1988–1994) who underwent DXA of the proximal femur and measurement of fasting serum leptin. Mean ± SE BMD (gm/cm2) of the total hip was 1.01 ± 0.005 in men, 0.94 ± 0.004 in premenopausal women, and 0.78 ± 0.007 in postmenopausal women. Bone density increased with increasing leptin concentration in men (p = 0.003), premenopausal women (p < 0.001), and postmenopausal women (p < 0.001). However, after adjusting for body mass index (BMI) and other bone density-related factors, an inverse association emerged in men (p < 0.001), being most evident among men <60 years old. There was no association of leptin and BMD in premenopausal women (p = 0.66) or postmenopausal women (p = 0.69) in multivariate analysis. Controlling for leptin had no effect on the strong positive association of BMI and BMD in either men or women. Serum leptin concentration did not appear to affect directly BMD. If present, the association appeared to be limited to younger men who are at lower risk of osteoporosis.
Cell-Type-Dependent Up-Regulation of In Vitro Mineralization After Overexpression of the Osteoblast-Specific Transcription Factor Runx2/Cbfa1
Authors: Byers Benjamin A. , Pavlath Grace K. , Murphy T. J. , Karsenty Gerard , García Andrés J.
Published: April 2006

Functional expression of the transcriptional activator Runx2/Cbfa1 is essential for osteoblastic differentiation and bone formation and maintenance. Forced expression of Runx2 in nonosteoblastic cells induces expression of osteoblast-specific genes, but the effects of Runx2 overexpression on in vitro matrix mineralization have not been determined. To examine whether exogenous Runx2 expression is sufficient to direct in vitro mineralization, we investigated sustained expression of Runx2 in nonosteoblastic and osteoblast-like cell lines using retroviral gene delivery. As expected, forced expression of Runx2 induced several osteoblast-specific genes in NIH3T3 and C3H10T1/2 fibroblasts and up-regulated expression in MC3T3-E1 immature osteoblast-like cells. However, Runx2 expression enhanced matrix mineralization in a cell-type-dependent manner. NIH3T3 and IMR-90 fibroblasts overexpressing Runx2 did not produce a mineralized matrix, indicating that forced expression of Runx2 in these nonosteogenic cell lines is not sufficient to direct in vitro mineralization. Consistent with the pluripotent nature of the cell line, a fraction (25%) of Runx2-expressing C3H10T1/2 fibroblast cultures produced mineralized nodules in a viral supernatant-dependent manner. Notably, bone sialoprotein (BSP) gene expression was detected at significantly higher levels in mineralizing Runx2-infected C3H10T1/2 cells compared with Runx2-expressing cultures which did not mineralize. Treatment of Runx2-infected C3H10T1/2 cultures with dexamethasone enhanced osteoblastic phenotype expression, inducing low levels of mineralization independent of viral supernatant. Finally, Runx2 overexpression in immature osteoblast-like MC3T3-E1 cells resulted in acceleration and robust up-regulation of matrix mineralization compared with controls. These results suggest that, although functional Runx2 is essential to multiple osteoblast-specific activities, in vitro matrix mineralization requires additional tissue-specific cofactors, which supplement Runx2 activity.
Function of Mutant (G1144A) Tissue-Nonspecific ALP Gene From Hypophosphatasia
Authors: Watanabe Hisashi , Goseki-Sone Masae , Orimo Hideo , Hamatani Ryoko , Takinami Hiroyuki , Ishikawa Isao
Published: April 2006

Hypophosphatasia (HOPS) is a clinically heterogeneous heritable disorder characterized by defective skeletal mineralization, deficiency of tissue-nonspecific alkaline phosphatase (TNSALP) activity, and premature loss of deciduous teeth. The gene for TNSALP is located on chromosome 1p34–36.1 and consists of 12 exons and 11 introns. In our previous study, we found the novel point mutations (G1144A and T979C) from the genomic TNSALP gene of a patient with childhood HOPS. In this study, we have characterized the protein translated from the mutant G1144A gene. Wild-type and G1144A mutant-type TNSALP cDNA expression vector pcDNA3 have been constructed and transfected to COS-1 cells by lipofectin technique. After 48-h or 72-h transfection, cells were collected and homogenized using polytron homogenizer. After centrifugation at 10,000g for 10 minutes, the supernatant was assayed. ALP activity was determined with 10 mM of p-nitrophenylphosphate as a substrate in 100 mM of 2-amino-2-methyl-1,3-propanediol-HCl buffer containing 5 mM of MgCl2. ALP activity of cells transfected with the mutant cDNA (G1144A) plasmid after 48-h or 72-h transfection exhibited 0.063 ± 0.012 U/mg and 0.054 ± 0.012 U/mg, respectively. As the enzymatic activity of the wild type was taken as 100%, the value of the mutant was estimated as 2.7% and 1.7%, respectively. These values were not significantly different from those found with mock-transfected cells, that is, 2.5% and 1.5%, respectively. This study indicated that the mutation (G1144A) produced the inactive ALP enzyme and would be a disease-causing mutation of the childhood-type HOPS.