bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

Abstract
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

Abstract
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

Abstract
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 &lt; 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

Abstract
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

Abstract
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 &lt; 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

Abstract
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

AbstractFractures 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

Abstract
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

Abstract
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 (&lt;35 years old) or severe ossification (&gt;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 &lt; 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

Abstract
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 &lt; 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 &lt; 0.05] and 24-h urine deoxypyridinoline [DPD] by 14.2% [p &lt; 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

Abstract
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 &lt; 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

Abstract
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.