bluuebunny/crossref_metadata_2025_split huggingface dataset:
Regulation of Osteoblast Differentiation by Pasteurella Multocida Toxin (PMT): A Role for Rho GTPase in Bone Formation
Authors: Harmey Dympna , Stenbeck Gudrun , Nobes Catherine D , Lax Alistair J , Grigoriadis Agamemnon E 
Published: April 2006

Abstract
The role of the Rho-Rho kinase signaling pathway on osteoblast differentiation was investigated using primary mouse calvarial cells. The bacterial toxin PMT inhibited, whereas Rho-ROK inhibitors stimulated, osteoblast differentiation and bone nodule formation. These effects correlated with altered BMP-2 and −4 expression. These data show the importance of Rho-ROK signaling in osteoblast differentiation and bone formation.
Introduction: The signal transduction pathways controlling osteoblast differentiation are not well understood. In this study, we used Pasteurella multocida toxin (PMT), a unique bacterial toxin that activates the small GTPase Rho, and specific Rho inhibitors to investigate the role of Rho in osteoblast differentiation and bone formation in vitro.
Materials and Methods: Primary mouse calvarial osteoblast cultures were used to investigate the effects of recombinant PMT and Rho-Rho kinase (ROK) inhibitors on osteoblast differentiation and bone nodule formation. Osteoblast gene expression was analyzed using Northern blot and RT-PCR, and actin rearrangements were visualized after phalloidin staining and confocal microscopy.
Results: PMT stimulated the proliferation of primary mouse calvarial cells and markedly inhibited the differentiation of osteoblast precursors to bone nodules with a concomitant inhibition of osteoblastic marker gene expression. There was no apparent causal relationship between the stimulation of proliferation and inhibition of differentiation. PMT caused cytoskeletal rearrangements because of activation of Rho, and the inhibition of bone nodules was completely reversed by the Rho inhibitor C3 transferase and partly reversed by inhibitors of the Rho effector, ROK. Interestingly, Rho and ROK inhibitors alone potently stimulated osteoblast differentiation, gene expression, and bone nodule formation. Finally, PMT inhibited, whereas ROK inhibitors stimulated, bone morphogenetic protein (BMP)-2 and −4 mRNA expression, providing a possible mechanism for their effects on bone nodule formation.
Conclusions: These results show that PMT inhibits osteoblast differentiation through a mechanism involving the Rho-ROK pathway and that this pathway is an important negative regulator of osteoblast differentiation. Conversely, ROK inhibitors stimulate osteoblast differentiation and may be potentially useful as anabolic agents for bone.
Autosomal Dominant Hypocalcemia in Monozygotic Twins Caused by a De Novo Germline Mutation Near the Amino-Terminus of the Human Calcium Receptor
Authors: Hu Jianxin , Mora Stefano , Weber Giovanna , Zamproni Ilaria , Proverbio Maria Carla , Spiegel Allen M 
Published: April 2006

Abstract
To define the molecular pathogenesis of severe postnatal hypocalcemia in monozygotic twin sisters, we sequenced their CaR gene and identified a missense mutation, K29E. Expression of the mutant receptor in vitro showed a marked increase in Ca2+ sensitivity explaining the observed phenotype. Additional mutagenesis studies lead us to speculate concerning a novel mechanism whereby the K29E mutation may lead to receptor activation.
Introduction: Activating mutations of the Ca2+-sensing receptor (CaR) gene have been identified in subjects with autosomal dominant hypocalcemia. Study of such mutations has provided insight into the mechanism of activation of the CaR.
Materials and Methods: We performed biochemical and molecular genetic studies on monozygotic twin sisters who presented with early postnatal hypocalcemia and on their unaffected sister and parents. Functional characterization of mutant CaRs transfected in HEK-293 cells included immunoblots to monitor protein expression and Ca2+ stimulation of phosphoinositide hydrolysis to measure Ca2+ sensitivity.
Results: We identified a K29E missense mutation in the twin sisters but not in their parents or unaffected sister. The K29E mutant CaR showed a marked increase in Ca2+ sensitivity, including when it was co-transfected with wildtype CaR cDNA, consistent with a dominant effect. Substitution of K29 by aspartate equivalently increased CaR sensitivity, whereas conservative substitution by arginine did not.
Conclusions: Severe postnatal hypocalcemia in the twin sisters was caused by a de novo germline activating mutation. In a model of the Venus flytrap-like domain of the extracellular amino-terminus of the CaR, K29 is located close to a peptide loop, “loop 2,” that forms part of the dimer interface and is the site of 10 of the previously reported naturally occurring activating CaR mutations. We speculate that K29E increases Ca2+ sensitivity of the CaR by disrupting a salt bridge between K29 and an acidic residue in loop 2 and thereby changes the normal structure of loop 2 that maintains the CaR in its inactive conformation.
Blood Leptin and Adiponectin as Possible Mediators of the Relation Between Fat Mass and BMD in Perimenopausal Women
Authors: Kontogianni Meropi D , Dafni Urania G , Routsias John G , Skopouli Fotini N 
Published: April 2006

Abstract
Fat mass is a predictor of BMD; however, the mechanisms involved remain uncertain. Two adipokines, leptin and adiponectin, were examined as potential mediators of this relation in 80 perimenopausal women. Adiponectin did not exert any effect on BMD, whereas leptin exerted a negative one, with insulin acting as a confounder to this relation.
Introduction: Fat mass is an important determinant of bone density, but the mechanism involved in this relation is uncertain. Leptin and adiponectin, as circulating peptides of adipocyte origin, are potential contributors to this relation. We investigated the role of leptin and adiponectin in mediating fat mass effects on the skeleton of perimenopausal women.
Materials and Methods: Twenty-five premenopausal and 55 postmenopausal, healthy women (42–68 years old) participated in our study. Lumbar spine BMD (BMDL2-L4) and total body BMC (TBBMC) were measured with DXA, leptin levels with ELISA, and adiponectin levels with radioimmunoassay (RIA). Additionally, body composition analysis was performed, as well as measurements of several hormones.
Results: It was shown that serum leptin levels were negatively correlated with BMD (β = −0.005, p = 0.027) and TBBMC (β = −14.32, p = 0.013). The above correlation was observed only when serum insulin levels were included, as an independent variable, in the regression analysis model. Adiponectin was not significantly correlated with BMDL2-L4 nor with TBBMC, either in the presence or absence of insulin.
Conclusion: Circulating adiponectin does not seem to exert any effect on bone mass. In contrast, circulating leptin showed a negative correlation with bone mass, dependent on serum insulin levels.
Brittle IV Mouse Model for Osteogenesis Imperfecta IV Demonstrates Postpubertal Adaptations to Improve Whole Bone Strength
Authors: Kozloff Kenneth M , Carden Angela , Bergwitz Clemens , Forlino Antonella , Uveges Thomas E , Morris Michael D , Marini Joan C , Goldstein Steven A 
Published: April 2006

Abstract
The Brtl mouse model for type IV osteogenesis imperfecta improves its whole bone strength and stiffness between 2 and 6 months of age. This adaptation is accomplished without a corresponding improvement in geometric resistance to bending, suggesting an improvement in matrix material properties.
Introduction: The Brittle IV (Brtl) mouse was developed as a knock-in model for osteogenesis imperfecta (OI) type IV. A Gly349Cys substitution was introduced into one col1a1 allele, resulting in a phenotype representative of the disease. In this study, we investigate the effect of the Brtl mutation on whole bone architecture, strength, and composition across a range of age groups.
Materials and Methods: One-, 2-, 6-, and 12-month-old Brtl and wildtype (WT) mice were analyzed. Femurs were assessed at the central diaphysis for cortical geometric parameters using μCT and were subsequently mechanically tested to failure by four-point bending. Matrix material properties were predicted using μCT data to normalize data from mechanical tests. Raman spectroscopy and DXA were used to assess matrix composition.
Results: Our findings show a postpubertal adaptation in which Brtl femoral strength and stiffness increase through a mechanism independent of changes in whole bone geometry. These findings suggest an improvement in the material properties of the bone matrix itself, rather than improvements in whole bone geometry, as seen in previous mouse models of OI. Raman spectroscopic results suggest these findings may be caused by changes in mineral/matrix balance rather than improvements in mineral crystallinity.
Conclusions: Our findings parallel the currently unexplained clinical observation of decreased fractures in human OI patients after puberty. The Brtl mouse remains an important tool for investigating therapeutic interventions for OI.
Shockwave Stimulates Oxygen Radical-Mediated Osteogenesis of the Mesenchymal Cells From Human Umbilical Cord Blood
Authors: Wang Feng-Sheng , Yang Kuender D , Wang Ching-Jen , Huang Hui-Cheng , Chio Chi-Chian , Hsu Te-Yao , Ou Chia-Yu 
Published: April 2006

Abstract
Human umbilical cord blood (HUCB) mesenchymal progenitor cells expressed stro-1 or CD44 or CD29, and subsequently, differentiated toward osteogenic lineage. Physical shockwave treatment increased osteogenic activity of HUCB mesenchymal progenitor cells through superoxide-mediated TGF-β1 induction. Transplantation of shockwave-treated HUCB mesenchymal progenitor cells enhanced healing of segmental femoral defect in severe combined immunodeficiency disease (SCID) mice.
Introduction: Mesenchymal progenitor cells (MPCs) in the bone marrow are precursors to bone development. It remains uncertain whether MPCs are present in human umbilical cord blood (HUCB) and are capable of differentiating into osteogenic cell lineage. Extending from a model of shockwave (SW) promotion of bone marrow stromal cell differentiation toward osteoprogenitors in rats, we further investigated how physical SW mediated biological responses in regulating osteogenic differentiation of HUCB MPCs.
Materials and Methods: HUCB was subjected to SW treatment at different energy flux densities and impulses. Colony-forming units-stroma (CFU-Stroma), osteogenic activities (Cbfa1/Runx2 expression, bone alkaline phosphatase activity, and bone nodule formation), and bone formation by heterologous transplantation into SCID mice were assessed.
Results: Few CD34+ stem cells (1.3%) and stro-1+ cells (1.0%) were present in the freshly prepared mononuclear cells (MNCs) from HUCB. The number of stro-1+ cells, but not CD34+, increased to 72.4% in the adherent cell culture over 6 days. Stro-1+ cells co-expressed CD44 and CD29 markers and grew into CFU-Stroma that matured into bone nodules. We found that the SW treatment (0.16 mJ/mm2 energy flux density, 200 impulses) elicited superoxide production and promoted formation of CFU-Stroma, but not of hematopoietic CFU-Mix. SW also enhanced the production of transforming growth factor (TGF)-β1, but not of interleukin (IL)-3 or granulocyte monocyte-colony stimulating factor (GM-CSF). Neutralization of TGF-β1 significantly reduced SW-promoted CFU-Stroma formation. Superoxide scavenging by superoxide dismutase blocked SW enhancement of TGF-β1 production and formation of CFU-Stroma. Administration of SW-treated HUCB MPCs to SCID mice with femoral segmental defects facilitated dense, bridging callus and gap closure.
Conclusion: HUCB MPCs subjected to SW treatment is a potential source for stem cells useful in the treatment of orthopedic disorders. An optimal physical SW treatment enhanced osteogenesis through superoxide-mediated TGF-β1 production. Physical stimulation is an alternative method for extending mesenchymal stem cells of HUCB.
Pubertal Girls Only Partially Adapt to Low Dietary Calcium Intakes
Authors: Abrams Steven A , Griffin Ian J , Hicks Penni D , Gunn Sheila K 
Published: April 2006

Abstract
We evaluated the effects of low calcium in the diets of young adolescent girls. We measured calcium absorption and excretion using stable isotopes. We found partial adaptation to low intakes but a persistent large deficit relative to recommended intakes. Low calcium intakes pose a substantial risk of inadequate calcium retention.
Introduction: A substantial number of adolescent girls in the United States have habitual calcium intakes &lt;500 mg/day (about 40% of the current recommended intake). The ability to adapt to these very low intakes by increasing calcium absorption and decreasing calcium excretion is not known. We sought to determine the effects of recommended (REC-Ca) versus very low (LO-Ca) calcium intakes on calcium absorption and excretion in white and black girls.
Materials and Methods: Pubertal, but premenarcheal girls, were adapted to low or recommended calcium intakes for at least 2 weeks before each study. Calcium absorption (n = 51) and endogenous fecal calcium excretion (n = 36 of the 51) were determined by dual-tracer stable isotope studies. Subjects were then switched to the other diet for at least 6 weeks, and the study was repeated.
Results: Calcium intake was 389 ± 10 mg/day on LO-Ca and 1259 ± 35 mg/day on REC-Ca diets. Fractional absorption increased from 44.9 ± 1.9% on REC-Ca to 63.4 ± 1.7% on LO-Ca (p &lt; 0.01), but the net calcium absorption remained less than one-half the value on LO-Ca as on REC-Ca. Despite decreases in both endogenous fecal calcium excretion and urinary calcium excretion, net calcium balance was much lower on LO-Ca compared with REC-Ca1 (131 ± 14 versus 349 ± 32 mg/day, respectively; p &lt; 0.001). We found significantly lower urinary calcium excretion but not calcium absorption in black girls compared with white girls.
Conclusions: Very low calcium intakes are only partially adapted to by increased absorption and decreased excretion. Very low calcium intakes place both white and black pubertal girls at substantial risk for inadequate calcium retention.
Terminal Differentiation of Osteoblasts to Osteocytes Is Accompanied by Dramatic Changes in the Distribution of Actin-Binding Proteins
Authors: Kamioka Hiroshi , Sugawara Yasuyo , Honjo Tadashi , Yamashiro Takashi , Takano-Yamamoto Teruko 
Published: April 2006

Abstract
Immunofluorescence staining of actin-binding proteins in osteoblasts and osteocytes was performed. α-Actinin, myosin, and tropomyosin showed similar organization in both osteoblastic stress fibers and osteocyte processes. However, fimbrin, villin, filamin, and spectrin showed dramatic differences in distribution between osteoblasts and osteocytes. This study suggested that terminal differentiation of osteoblasts to osteocytes is accompanied by highly dramatic changes in the distribution of actin-binding proteins.
Introduction: We previously reported that osteocyte shape is dependent on actin filaments. To analyze the terminal differentiation from osteoblasts to osteocytes, we investigated the actin-binding proteins, which are the control elements in the dynamic organization of the actin cytoskeleton.
Materials and Methods: We used primary chick osteocytes and osteoblasts, the phenotypes of which were confirmed by use of OB7.3, a chick osteocyte-specific monoclonal antibody and by detection of alkaline phosphatase activity, respectively. Immunofluorescence staining was performed for visualizing actin-binding proteins. Furthermore, we applied shear stress at 12 dyns/cm2 to the cells and compared the changes in fimbrin distribution.
Results: Immunofluorescence staining of fimbrin and α-actinin showed their presence in the processes of osteocytes, with especially strong signals of fimbrin at the sites of divarication of the processes. Anti-villin was reactive with the osteocyte cytoplasm but not with the processes. Interestingly, anti-villin immunoreactivity was much stronger in osteocytes than in osteoblasts. Filamin was localized along the stress fibers of osteoblasts but was seen only in those in the proximal base of osteocyte processes. Myosin and tropomyosin were found to have a similar pattern in both stress fibers of osteoblasts and osteocyte processes. The difference in the distribution of anti-spectrin staining was highly dramatic. Osteoblasts immunostained with anti-spectrin showed punctate signals on their cytoplasmic membranes, whereas anti-spectrin in osteocytes detected a filamentous organization; and the spectrin was totally colocalized with actin from the distal portion of the cytoplasmic processes to the cell center. In osteoblasts, shear stress induced recruitment of fimbrin to the end of stress fibers. However, fimbrin in the osteocyte processes did not change its localization.
Conclusion: We found that terminal differentiation of osteoblasts to osteocytes was accompanied by highly dramatic changes in the distribution of actin-binding proteins, changes of which may affect cellular function.
Effects of Simvastatin on Bone Turnover and BMD: A 1-Year Randomized Controlled Trial in Postmenopausal Osteopenic Women
Authors: Rejnmark Lars , Buus Henrik Niels , Vestergaard Peter , Heickendorff Lene , Andreasen Frederik , Larsen Lytken Mogens , Mosekilde Leif 
Published: April 2006

Abstract
To study effects of statins on human bone, 82 postmenopausal women were randomized to 1-year treatment with simvastatin 40 mg/day or placebo. The study showed no effect of simvastatin on biochemical bone markers or on BMD at the hip or spine. Thus, our results do not support a general beneficial effect of simvastatin on bone.
Introduction: Statins have been reported to cause bone anabolic as well as antiresorptive effects, and therefore statins have been suggested as potential agents in treatment of osteoporosis.
Materials and Methods: In a double-blinded design, 82 healthy postmenopausal women with osteopenia were randomized to 1-year simvastatin treatment 40 mg/day or placebo. BMD and plasma levels of cholesterol, parathyroid hormone (PTH), and biochemical bone markers were measured at baseline, after 1 year of treatment (week 52), and 26 weeks after withdrawal of treatment (week 78). Calcium supplements (400 mg/day) were administrated during the entire 1.5-year study period.
Results: Seventy-eight women completed the 1-year treatment. After 1 year, simvastatin but not placebo caused reduced plasma cholesterol (−27% versus +1%, p &lt; 0.001) and low-density lipoprotein (LDL) levels (−43% versus +1%, p &lt; 0.001). After withdrawal of treatment, cholesterol and LDL levels returned to baseline levels and no longer differed from the placebo group. However, plasma levels of PTH and biochemical bone markers did not differ between groups at week 52 or 78. Compared with placebo, simvastatin caused no changes in BMD at the lumbar spine, total hip, femoral neck, or whole body at week 52 or 78. However, a significant increase in BMD was found in response to simvastatin at the forearm. Within the simvastatin group, changes in cholesterol levels did not correlate to BMD changes at any site.
Conclusions: Our results do not support a general beneficial effect of simvastatin on bone.
Identification of Novel Regulators Associated With Early-Phase Osteoblast Differentiation
Authors: de Jong Diana S , Vaes Bart LT , Dechering Koen J , Feijen Alie , Hendriks Jose MA , Wehrens Ron , Mummery Christine L , van Zoelen Everardus JJ , Olijve Wiebe , Steegenga Wilma T 
Published: April 2006

Abstract
Key regulatory components of the BMP-induced osteoblast differentiation cascade remain to be established. Microarray and subsequent expression analyses in mice identified two transcription factors, Hey1 and Tcf7, with in vitro and in vivo expression characteristics very similar to Cbfa1. Transfection studies suggest that Tcf7 modulates BMP2-induced osteoblast differentiation. This study contributes to a better definition of the onset of BMP-induced osteoblast differentiation.
Introduction: Elucidation of the genetic cascade guiding mesenchymal stem cells to become osteoblasts is of extreme importance for improving the treatment of bone-related diseases such as osteoporosis. The aim of this study was to identify regulators of the early phases of bone morphogenetic protein (BMP)2-induced osteoblast differentiation.
Materials and Methods: Osteoblast differentiation of mouse C2C12 cells was induced by treatment with BMP2, and regulation of gene expression was studied during the subsequent 24 h using high-density microarrays. The regulated genes were grouped by means of model-based clustering, and protein functions were assigned. Real-time quantitative RT-PCR analysis was used to validate BMP2-induced gene expression patterns in C2C12 cells. Osteoblast specificity was studied by comparing these expression patterns with those in C3H10T1/2 and NIH3T3 cells under similar conditions. In situ hybridization of mRNA in embryos at embryonic day (E)14.5 and E16.5 of gestation and on newborn mouse tails were used to study in vivo expression patterns. Cells constitutively expressing the regulated gene Tcf7 were used to investigate its influence on BMP-induced osteoblast differentiation.
Results and Conclusions: A total of 184 genes and expressed sequence tags (ESTs) were differentially expressed in the first 24 h after BMP2 treatment and grouped in subsets of immediate early, intermediate early, and late early response genes. Signal transduction regulatory factors mainly represented the subset of immediate early genes. Regulation of expression of these genes was direct, independent of de novo protein synthesis and independent of the cell type studied. The intermediate early and late early genes consisted primarily of genes related to processes that modulate morphology, basement membrane formation, and synthesis of extracellular calcified matrix. The late early genes require de novo protein synthesis and show osteoblast specificity. In vivo and in vitro experiments showed that the transcription factors Hey1 and Tcf7 exhibited expression characteristics and cell type specificity very similar to those of the osteoblast specific transcription factor Cbfa1, and constitutive expression of Tcf7 in C2C12 cells differentially regulated osteoblast differentiation marker genes.