bluuebunny/crossref_metadata_2025 huggingface dataset:
Osteoprotegerin Abrogates Chronic Alcohol Ingestion-Induced Bone Loss in Mice
Authors: Zhang Jian , Dai Jinlu , Lin Din-Lii , Habib Paula , Smith Peter , Murtha Jill , Fu Zheng , Yao Zhi , Qi Yinghua , Keller Evan T.
Published: April 2006

To investigate the role of osteoprotegerin (OPG) on alcohol (ethanol)-mediated osteoporosis, we measured a variety of bone remodeling parameters in mice that were either on a control diet, an ethanol (5%) diet, or an ethanol (5%) diet plus OPG administration. OPG diminished the ethanol-induced (1) decrease in bone mineral density (BMD) as determined by dual-energy densitometry, (2) decrease in cancellous bone volume and trabecular width and the increase of osteoclast surface as determined by histomorphometry of the femur, (3) increase in urinary deoxypyridinolines (Dpd's) as determined by ELISA, and (4) increase in colony-forming unit-granulocyte macrophage (CFU-GM) formation and osteoclastogenesis as determined by ex vivo bone marrow cell cultures. Additionally, OPG diminished the ethanol-induced decrease of several osteoblastic parameters including osteoblast formation and osteoblast culture calcium retention. These findings were supported by histomorphometric indices in the distal femur. Taken together, these data show that OPG diminishes ethanol-induced bone loss. Furthermore, they suggest that OPG achieves this through its ability to abrogate ethanol-induced promotion of osteoclastogenesis and promote osteoblast proliferation.
Genetic Effects for Femoral Biomechanics, Structure, and Density in C57BL/6J and C3H/HeJ Inbred Mouse Strains
Authors: Koller Daniel L , Schriefer Jennifer , Sun Qiwei , Shultz Kathryn L , Donahue Leah R , Rosen Clifford J , Foroud Tatiana , Beamer Wesley G , Turner Charles H
Published: April 2006

Genome-wide QTL analysis for bone density, structure, and biomechanical phenotypes was performed in 999 (B6xC3H)F2 mice. Multivariate phenotypes were also derived to test for pleiotropic QTL effects. Highly significant QTLs were detected with pleiotropic effects on many of these phenotypes, and QTLs with unique effects on specific phenotypes were found as well.

Introduction: The inbred C57BL/6J (B6) and C3H/HeJ (C3H) mouse strains were previously shown to segregate quantitative trait loci (QTLs) for femoral bone density.

Materials and Methods: The 999 s filial (F2) mouse progeny were further phenotyped for measures of femoral biomechanics (load to failure, Fu; work to failure, U; stiffness, S), structure (polar moment of inertia, Ip; moment of inertia ratio, Ir), and more specific femoral midshaft bone density measures (cortical and total vBMD). Two novel multivariate phenotypes were computed using principal component analysis, thus aiding in the exploration of pleiotropic effects of the QTLs detected.

Results and Conclusions: Results of a genome-wide analysis provided strong evidence of pleiotropic QTL effects on chromosome 4, with six of the seven primary phenotypic measures, representing femoral biomechanics, density, and structure, producing LOD scores greater than 8. Chromosomes 1, 8, 13, and 14 were also identified as harboring QTLs that affect phenotypes in two of the three aspects of bone properties. QTLs uniquely contributing to variability in biomechanical measures were identified on chromosomes 10 and 12, whereas a QTL solely affecting structure was found on chromosome 17. Analysis of the evidence for pleiotropic effects using principal component analysis revealed pleiotropic QTLs on chromosomes 4 and 14, influencing nearly all the bone phenotypes measured and revealed QTLs on chromosomes 1, 8, 13, and 17 with pleiotropic effects restricted to either density or the structure and stiffness phenotypes. The use of multivariate phenotypes has allowed us to identify pleiotropic effects of several QTLs previously linked in studies of other mouse strains and in human studies of bone mineral density and femoral structure, which will provide important insight regarding the importance of allelic variation on the entire skeleton.
Temporal and Spatial Expression Profiles of BMP Receptors and Noggin During BMP-2-Induced Ectopic Bone Formation
Authors: Nakamura Yukio , Wakitani Shigeyuki , Nakayama Jun , Wakabayashi Shinji , Horiuchi Hiroshi , Takaoka Kunio
Published: April 2006

The mechanism of ectopic bone formation has not been clear. After BMP-2 implantation into the back muscles of 198 mice, expression of BMPR-1A,–2, and Noggin was increased during the early phase of the reaction. The results suggest that positive and negative feedback mechanisms modulate ectopic osteogenesis induced by this growth factor.

Introduction: The expression of bone morphogenetic protein receptors (BMPRs) and Noggin during ectopic bone formation after implantation of BMP-2 into the back muscles of adult mice was investigated in this study.

Methods: One hundred ninety-eight male ddy mice were divided into groups and received either collagen disks containing BMP-2, collagen disks alone, or sham surgery with no disk implantation. Changes in the temporal and spatial expression profiles of BMPRs and Noggin were examined by Northern blotting, in situ hybridization, Western blotting, and immunohistochemistry.

Results and Conclusions: In the BMP group, expression of BMPR-1A,–2, and Noggin mRNA and protein was enhanced 2–4 days after implantation in undifferentiated mesenchymal cells and regenerating muscle fibers located close to the BMP-retaining implants. On day 7, the expression was also observed in cartilage cells, and after day 14, in the osteoblastic cells around bone tissue. The level of expression peaked at day 4 after implantation and persisted at a much lower level during the bone forming process. No significant expression of BMPR-1B was detected at the mRNA and protein levels during the bone-forming reaction. In the BMP free control groups, a mild enhancement of BMPR-2 expression was also noted around the implant, but this was not observed for BMPR-1A, -1B, or Noggin. Upregulated expression of BMPR-1A, -2, and Noggin in undifferentiated mesenchymal cells and regenerating muscle fibers occurs during the early phase of BMP-2-induced bone formation. The coordinate expression of these positive and negative regulators of BMP signaling points to a potential regulatory mechanism for bone induction.
Focal Adhesion Kinase pp125FAK Interacts With the Large Conductance Calcium-Activated hSlo Potassium Channel in Human Osteoblasts: Potential Role in Mechanotransduction
Authors: Rezzonico Roger , Cayatte Corinne , Bourget-Ponzio Isabelle , Romey Georges , Belhacene Nathalie , Loubat Agnès , Rocchi Stéphane , Van Obberghen Emmanuel , Girault Jean-Antoine , Rossi Bernard , Schmid-Antomarchi Heidy
Published: April 2006

Molecular events of mechanotransduction in osteoblasts are poorly defined. We show that the mechanosensitive BK channels open and recruit the focal adhesion kinase FAK in osteoblasts on hypotonic shock. This could convert mechanical signals in biochemical events, leading to osteoblast activation.

Introduction: Mechanical strains applied to the skeleton influence bone remodeling and architecture mainly through the osteoblast lineage. The molecular mechanisms involved in osteoblastic mechanotransduction include opening of mechanosensitive cation channels and the activation of protein tyrosine kinases, notably FAK, but their interplay remains poorly characterized. The large conductance K+ channel (BK) seems likely as a bone mechanoreceptor candidate because of its high expression in osteoblasts and its ability to open in response to membrane stretch or hypotonic shock. Propagation of the signals issued from the mechanosensitivity of BK channels inside the cell likely implies complex interactions with molecular partners involved in mechanotransduction, notably FAK.

Methods: Interaction of FAK with the C terminus of the hSlo α-subunit of BK was investigated using the yeast two-hybrid system as well as immunofluorescence microscopy and coimmunoprecipitation experiments with a rabbit anti-hslo antibody on MG63 and CAL72 human osteosarcoma cell lines and on normal human osteoblasts. Mapping of the FAK region interacting with hSlo was approached by testing the ability of hSlo to recruit mutated ot truncated FAK proteins.

Results: To the best of our knowledge, we provide the first evidence of the physical association of FAK with the intracellular part of hslo. We show that FAK/hSlo interaction likely takes place through the Pro-1-rich domain situated in the C-terminal region of the kinase. FAK/hSlo association occurs constitutively at a low, but appreciable, level in human osteosarcoma cells and normal human osteoblasts that express endogenous FAK and hSlo. In addition, we found that application of an hypo-osmotic shock to these cells induced a sustained activation of BK channels associated to a marked increase in the recruitment of FAK on hSlo.

Conclusions: Based on these data, we propose that BK channels might play a triggering role in the signaling cascade induced by mechanical strains in osteoblasts.
Characterization of C4–2 Prostate Cancer Bone Metastases and Their Response to Castration
Authors: Pfitzenmaier Jesco , Quinn Janna E , Odman Austin M , Zhang Jian , Keller Evan T , Vessella Robert L , Corey Eva
Published: April 2006

New well-characterized preclinical models of prostate cancer (CaP) bone metastases are needed to improve our understanding of the development of CaP-related bone disease in patients. Here we describe characterization of a model consisting of direct injection of C4–2 cells into tibias.

Introduction: Prostate cancer (CaP) has a high proclivity to metastasize to bone. Development and characterization of preclinical models of CaP bone metastases are of high interest. The objective of this study was to characterize C4–2 bone metastases and their response to castration.

Materials and Methods: Cell suspensions of C4-2, a subline of LNCaP, were injected directly into the tibias of intact male mice. In groups A (n = 7) and B (n = 5), animals were killed 3 and 8 weeks after injection of C4–2 cells, respectively. In group C (n = 7), animals were castrated 3 weeks after injection and killed 5 weeks after castration. Serum prostate-specific antigen (PSA) levels and bone mineral density (BMD) were measured, and bone histomorphometric analysis was performed.

Results: C4–2 cells decreased BMD of the injected tibias by 36.1% and bone volume by 74.1% versus normal tibias. Castration caused a 32.3% drop in serum PSA (p = 0.0438), with a nadir at day 14, after which it began to rise again. Bone destruction in the tumorous tibias of castrated animals was decreased by 15.9% versus tumorous tibias of intact animals (p = 0.0392). However, BMD in the tumorous tibias of castrated mice was still lower than in normal tibias of intact animals. Castration also decreased BMD and bone volume in nontumorous tibias (p = 0.0406 and 0.0232, respectively).

Conclusions: The C4–2 model of bone metastasis recapitulates the response to androgen deprivation observed in CaP patients with bone metastases and is suitable for study of interactions between tumor and bone cells and evaluation of new therapeutic modalities.
Television, Computer, and Video Viewing; Physical Activity; and Upper Limb Fracture Risk in Children: A Population-Based Case Control Study
Authors: MA Deqiong , Jones Graeme
Published: April 2006

The effect of physical activity on upper limb fractures was examined in this population-based case control study with 321 age- and gender-matched pairs. Sports participation increased fracture risk in boys and decreased risk in girls. Television viewing had a deleterious dose response association with wrist and forearm fractures while light physical activity was protective.

Introduction: The aim of this population-based case control study was to examine the association between television, computer, and video viewing; types and levels of physical activity; and upper limb fractures in children 9–16 years of age.

Materials and Methods: A total of 321 fracture cases and 321 randomly selected individually matched controls were studied. Television, computer, and video viewing and types and levels of physical activity were determined by interview-administered questionnaire. Bone strength was assessed by DXA and metacarpal morphometry.

Results: In general, sports participation increased total upper limb fracture risk in boys and decreased risk in girls. Gender-specific risk estimates were significantly different for total, contact, noncontact, and high-risk sports participation as well as four individual sports (soccer, cricket, surfing, and swimming). In multivariate analysis, time spent television, computer, and video viewing in both sexes was positively associated with wrist and forearm fracture risk (OR 1.6/category, 95% CI: 1.1–2.2), whereas days involved in light physical activity participation decreased fracture risk (OR 0.8/category, 95% CI: 0.7–1.0). Sports participation increased hand (OR 1.5/sport, 95% CI: 1.1–2.0) and upper arm (OR 29.8/sport, 95% CI: 1.7–535) fracture risk in boys only and decreased wrist and forearm fracture risk in girls only (OR 0.5/sport, 95% CI: 0.3–0.9). Adjustment for bone density and metacarpal morphometry did not alter these associations.

Conclusion: There is gender discordance with regard to sports participation and fracture risk in children, which may reflect different approaches to sport. Importantly, television, computer, and video viewing has a dose-dependent association with wrist and forearm fractures, whereas light physical activity is protective. The mechanism is unclear but may involve bone-independent factors, or less likely, changes in bone quality not detected by DXA or metacarpal morphometry.
A Novel, Non-Prostanoid EP2 Receptor-Selective Prostaglandin E2 Agonist Stimulates Local Bone Formation and Enhances Fracture Healing
Authors: LI Mei , KE Hua Zhu , QI Hong , Healy David R , LI Yan , Crawford D Todd , Paralkar Vishwas M , Owen Thomas A , Cameron Kimberly O , Lefker Bruce A , Brown Thomas A , Thompson David D
Published: April 2006

CP-533,536, a newly discovered, non-prostanoid EP2 receptor-selective PGE2 agonist, stimulates local bone formation and enhances fracture healing in rat models.

Introduction: There is a significant medical need for agents that can stimulate local bone formation and enhance fracture healing. We tested the effects of CP-533,536, a newly discovered, non-prostanoid EP2 receptor-selective prostaglandin E2 (PGE2) agonist, in stimulating local bone formation and enhancing fracture healing in rat models.

Materials and Methods: In the first model, a single injection of CP-533,536 at doses of 0.3, 1, or 3 mg/kg to the proximal tibial metaphysis of 6-week-old male rats was given on day 1, and the local bone anabolic effect was determined on day 7. We then tested the effects of this compound in inducing bone formation on rat periosteum of the femur. A single dose of 0.3 mg of CP-533,536 incorporated in a poly-(d,l-lactide-co-glycolide) (PLGH) matrix was injected onto the periosteum of the femur in 3-week-old male rats, and local bone formation was determined on day 14. Finally, the ability of CP-533,536 in PLGH matrix in enhancing fracture healing was tested using the rat femoral fracture model. CP-533,536 in PLGH matrix at doses of 0.05, 0.5, or 5 mg was delivered to the local fracture site on the same day of fracture, and its efficacy was evaluated on day 21.

Results and Conclusions: A single injection of CP-533,536 at doses of 0.3, 1, or 3 mg/kg to the proximal tibial metaphysis dose-dependently stimulated local lamellar bone formation on trabecular, endocortical, and periosteal surfaces, and thus increased bone mineral content and bone strength at the injected site. Similarly, a single injection of 0.3 mg of CP-533,536 incorporated in PLGH matrix onto the periosteum of the femur induced significantly local bone formation. In the rat femoral fracture model, CP-533,536 in PLGH matrix at doses of 0.05, 0.5, and 5 mg dose-dependently increased callus size, density, and strength compared with PLGH matrix alone. These results show that CP-533,536 stimulates new bone formation on trabecular, endocortical, and periosteal surfaces and enhances fracture healing. These data reveal that EP2 receptor-selective agonists provide therapeutic potential for local bone augmentation, bone repair, and bone healing in humans.
Regulatory Mechanisms and Physiological Relevance of a Voltage-Gated H+ Channel in Murine Osteoclasts: Phorbol Myristate Acetate Induces Cell Acidosis and the Channel Activation
Authors: Mori Hiroyuki , Sakai Hiromu , Morihata Hirokazu , Kawawaki Junko , Amano Hitoshi , Yamano Tsunekazu , Kuno Miyuki
Published: April 2006

The voltage-gated H+ channel is a powerful H+ extruding mechanism of osteoclasts, but its functional roles and regulatory mechanisms remain unclear. Electrophysiological recordings revealed that the H+ channel operated on activation of protein kinase C together with cell acidosis.

Introduction: H+ is a key signaling ion in bone resorption. In addition to H+ pumps and exchangers, osteoclasts are equipped with H+ conductive pathways to compensate rapidly for pH imbalance. The H+ channel is distinct in its strong H+ extrusion ability and voltage-dependent gatings.

Methods: To investigate how and when the H+ channel is available in functional osteoclasts, the effects of phorbol 12-myristate 13-acetate (PMA), an activator for protein kinase C, on the H+ channel were examined in murine osteoclasts generated in the presence of soluble RANKL (sRANKL) and macrophage-colony stimulating factor (M-CSF).

Results and Conclusions: Whole cell recordings clearly showed that the H+ current was enhanced by increasing the pH gradient across the plasma membrane (ΔpH), indicating that the H+ channel changed its activity by sensing ΔpH. The reversal potential (Vrev) was a valuable tool for the real-time monitoring of ΔpH in clamped cells. In the permeabilized patch, PMA (10 nM-1.6 μM) increased the current density and the activation rate, slowed decay of tail currents, and shifted the threshold toward more negative voltages. In addition, PMA caused a negative shift of Vrev, suggesting that intracellular acidification occurred. The PMA-induced cell acidosis was confirmed using a fluorescent pH indicator (BCECF), which recovered quickly in a K+-rich alkaline solution, probably through the activated H+ channel. Both cell acidosis and activation of the H+ channel by PMA were inhibited by staurosporine. In ∼80% of cells, the PMA-induced augmentation in the current activity remained after compensating for the ΔpH changes, implying that both ΔpH-dependent and -independent mechanisms mediated the channel activation. Activation of the H+ channel shifted the membrane potential toward Vrev. These data suggest that the H+ channel may contribute to regulation of the pH environments and the membrane potential in osteoclasts activated by protein kinase C.
Maturation-Dependent Thiol Loss Increases Chondrocyte Susceptibility to Apoptosis
Authors: Teixeira Cristina C , Rajpurohit Ramesh , Mansfield Kyle , Nemelivsky Yelena V , Shapiro Irving M
Published: April 2006

The major aim of the current investigation was to evaluate the role of thiols during chondrocyte maturation and apoptosis. Using a thiol-sensitive fluorescent probe, we found that in chick growth plate chondrocytes, hypertrophy is accompanied by a decrease in the glutathione content. In this study, we show that the maturation-dependent loss of thiol, although not causing death of maturing chondrocytes, drastically increases susceptibility to apoptosis by oxidative and nitrosoactive stress. To investigate how the loss of thiol content in cultured chondrocytes affects the expression of the hypertrophic phenotype, we chemically manipulated intracellular thiol levels and analyzed the expression of important maturation markers. We found that thiol depletion causes a decrease in the expression of osteopontin, type X and type II collagen and a significant loss of alkaline phosphatase activity, suggesting that the expression of the hypertrophic phenotype is tightly regulated by redox levels in chondrocytes. Furthermore, severe thiol depletion profoundly affected cell survival under oxidative and nitrosoactive stress. It was concluded that the loss of thiol reserve is not only linked to the expression of the hypertrophic phenotype but also influenced chondrocyte survival, linking chondrocyte maturation and the activation of the apoptotic pathway.
Osteoclastogenesis-Related Antigen, a Novel Molecule on Mouse Stromal Cells, Regulates Osteoclastogenesis
Authors: Arai Satoshi , Amizuka Norio , Azuma Yoshiaki , Takeshita Sunao , Kudo Akira
Published: April 2006

Osteoclastogenesis is regulated by RANKL expressed on stromal cells. In this study, we sought to isolate a new surface molecule regulating osteoclastogenesis on stromal cells by generating monoclonal antibodies. A rat was immunized with the mouse stromal cell line, TSB13, which can support osteoclastogenesis, and a monoclonal antibody, A15-1, was obtained. A15-1 bound to a surface antigen on TSB13 cells, termed osteoclastogenesis-related antigen (OCRA), and immunoprecipitation with this antibody revealed that OCRA was a 220-kDa molecule. By means of flow cytometry, the A15-1 antigen (OCRA) was found to be expressed on various mesenchymal cell lines but not on hematopoietic cell lines, and the expression level of OCRA on the TSB13 cells was slightly increased by treatment with 1α,25(OH)2D3. When osteoclast progenitors and TSB13 cells were co-cultured in the presence of 1α,25(OH)2D3, the addition of A15-1 inhibited osteoclast differentiation in a dose-dependent manner; however, no significant inhibition of soluble RANKL-induced osteoclastogenesis was observed, suggesting that A15-1 inhibited only stromal cell-dependent osteoclastogenesis. The same inhibitory effect of A15-1 was also observed when primary bone marrow-derived stromal cells were used. The osteoclastogenesis-promoting effects of other osteotropic factors, such as parathyroid hormone (PTH) and interleukin (IL)-1β, were also inhibited by A15-1. Time-course analysis of osteoclast differentiation in vitro indicated that the initial 2 days of treatment with A15-1 was sufficient for inhibition, suggesting that A15-1 inhibits the early stages of osteoclast differentiation. Finally, we investigated the in vivo effects of A15-1 on PTH-induced hypercalcemia in mice. Treatment with A15-1 significantly decreased the osteoclast surface in the PTH-administered mice. Taken together, our data indicate that OCRA, a novel A15-1-detected antigen, regulates stromal cell-dependent osteoclastogenesis.
Perivascular Niche of Postnatal Mesenchymal Stem Cells in Human Bone Marrow and Dental Pulp
Authors: Shi Songtao , Gronthos Stan
Published: April 2006

Mesenchymal stem cell populations have previously been identified in adult bone marrow and dental pulp that are capable of regenerating the bone marrow and dental pulp microenvironments, respectively. Here we show that these stem cell populations reside in the microvasculature of their tissue of origin. Human bone marrow stromal stem cells (BMSSCs) and dental pulp stem cells (DPSCs) were isolated by immunoselection using the antibody, STRO-1, which recognizes an antigen on perivascular cells in bone marrow and dental pulp tissue. Freshly isolated STRO-1 positive BMSSCs and DPSCs were tested for expression of vascular antigens known to be expressed by endothelial cells (von Willebrand factor, CD146), smooth muscle cells, and pericytes (α-smooth muscle actin, CD146), and a pericyte-associated antigen (3G5), by immunohistochemistry, fluorescence-activated cell sorting (FACS), and/or immunomagnetic bead selection. Both BMSSCs and DPSCs lacked expression of von Willebrand factor but were found to be positive for α-smooth muscle actin and CD146. Furthermore, the majority of DPSCs expressed the pericyte marker, 3G5, while only a minor population of BMSSCs were found to be positive for 3G5. The finding that BMSSCs and DPSCs both display phenotypes consistent with different perivascular cell populations, regardless of their diverse ontogeny and developmental potentials, may have further implications in understanding the factors that regulate the formation of mineralized matrices and other associated connective tissues.
A Common Methylenetetrahydrofolate Reductase (C677T) Polymorphism Is Associated With Low Bone Mineral Density and Increased Fracture Incidence After Menopause: Longitudinal Data From the Danish Osteoporosis Prevention Study
Authors: Abrahamsen BO , Madsen Jonna Skov , Landbo Tofteng Charlotte , Stilgren Lis , Bladbjerg Else Marie , Kristensen Søren Risom , Brixen Kim , Mosekilde Leif
Published: April 2006

A polymorphism in the gene encoding methylenetetrahydrofolate reductase (MTHFR) has recently been associated with bone mineral density (BMD) in postmenopausal Japanese women. It is not known whether this effect is also present in European populations and whether it is caused by lower peak bone mass or accelerated postmenopausal bone loss. MTHFR genotyping was done in 1748 healthy postmenopausal Danish women participating in a prospective study of risk factors for osteoporosis. At the time of enrollment, 3–24 months after last menstrual period, the less prevalent genotype (TT, 8.7% of the population) was associated with significantly lower BMD at the femoral neck (ANOVA, p < 0.05), total hip (p < 0.01), and spine (p < 0.05 adjusted for lifestyle covariates, p = 0.06 without adjustment). The mean difference was between 0.1 and 0.3 SD, depending on measurement site. MTHFR genotype added significantly to prediction of BMD by weight and age. Fracture incidence was increased more than 2-fold in subjects with the TT genotype (risk ratio [RR], 2.6; 95% CI 1.2–5.6). This remained significant when the Cox analysis was controlled for BMD (RR, 2.4; 95% CI 1.1–5.2). No differences in serum osteocalcin, bone-specific alkaline phosphatase, and 25-OH-vitamin D were found between genotypes. The response to hormone replacement therapy (HRT) did not differ, but the association of the TT genotype with reduced BMD was maintained at the total hip after 5 years of HRT. The MTHFR TT genotype is associated with low BMD and increased fracture incidence in early postmenopausal women.
Effect of Vitamin K2 on Cortical and Cancellous Bones in Orchidectomized and/or Sciatic Neurectomized Rats
Authors: Iwamoto Jun , Yeh James K , Takeda Tsuyoshi
Published: April 2006

We examined the effect of vitamin K2 on cortical and cancellous bones in orchidectomized and/or sciatic neurectomized rats. Ninety male Sprague-Dawley rats, 3 months of age, were randomized by stratified weight method into nine groups with 10 rats in each group: baseline control (BLC), age-matched intact control (IN), IN+vitamin K2 administration (K), orchidectomy (ORX), ORX+K, unilateral sciatic neurectomy (NX), NX+K, ORX+NX (ONX), and ONX+K. Vitamin K2 (menatetrenone) was administered orally twice a week at a dose of 30 mg/kg each. After 10 weeks of feeding, the tibial shaft and proximal tibia were processed for cortical and cancellous bone histomorphometric analyses, respectively. An ORX-induced reduction in maturation-related cortical bone gain and ORX-induced cancellous bone loss were attributable to increased endocortical and trabecular bone turnover, respectively. NX- and ONX-induced reductions in maturation-related cortical bone gain were attributable to decreased periosteal bone formation and increased endocortical bone turnover, while NX- and ONX-induced cancellous bone loss was attributable to increased bone resorption and decreased bone formation. ORX-induced cancellous bone loss was more pronounced when combined with immobilization. Vitamin K2 administration did not significantly alter any parameters in IN rats. Vitamin K2 administration in ORX rats suppressed endocortical bone resorption and trabecular bone turnover, retarding a reduction in maturation-related cortical bone gain and cancellous bone loss. This effect on cancellous bone loss was primarily because of prevention of a reduction of trabecular thickness. Vitamin K2 administration in NX and ONX rats suppressed bone resorption and stimulated bone formation (mineralization), with retardation of a reduction of trabecular thickness without any significant effect on cancellous bone mass, and suppressed endocortical bone resorption, retarding a reduction in maturation-related cortical bone gain. The present study provides evidence indicating that vitamin K2 has the potential to suppress bone resorption or bone turnover and/or stimulate bone formation in vivo in ORX and/or NX rats.
The Type I Collagen Fragments ICTP and CTX Reveal Distinct Enzymatic Pathways of Bone Collagen Degradation
Authors: Garnero P , Ferreras M , Karsdal MA , Nicamhlaoibh R , Risteli J , Borel O , Qvist P , Delmas PD , Foged NT , Delaissé JM
Published: April 2006

Bone resorption may generate collagen fragments such as ICTP and CTX, which can be quantified in serum and/or urine by using specific immunoassays, and which are used as clinical markers. However, the relative abundance of ICTP and CTX varies according to the type of bone pathology, suggesting that these two fragments are generated through distinct collagenolytic pathways. In this study, we analyzed the release of ICTP and CTX from bone collagen by the proteinases reported to play a role in the solubilization of bone matrix. Cathepsin K released large amounts of CTX, but did not allow a detectable release of ICTP. Conversely, the matrix metalloproteinases (MMPs) MMP-2, -9, -13, or -14 released ICTP, but did not allow a detectable release of CTX. Next we analyzed the release of ICTP and CTX from bone explants cultured in the presence of well-established inhibitors of these proteinases and of matrix solubilization. An inhibitor of cysteine proteinases including cathepsin K, inhibited the release of CTX, but not the release of ICTP. MMP inhibitors inhibited the release of ICTP, but also that of CTX, in agreement with the putative role of MMPs in the initiation of bone resorption in addition to matrix solubilization. Similarly the treatment of mice bearing bone metastasis with an MMP inhibitor led to a significant reduction of serum ICTP and CTX, and osteolytic lesions. We conclude that the generation of ICTP and CTX depends on different collagenolytic pathways. This finding may explain why these two markers may discriminate between different bone pathologies.
Prevention and Treatment of Glucocorticoid-Induced Osteoporosis: A Comparison of Calcitriol, Vitamin D Plus Calcium, and Alendronate Plus Calcium
Authors: Sambrook Philip N , Kotowicz Mark , Nash Peter , Styles Colin B , Naganathan Vasi , Henderson-Briffa Kathy N , Eisman John A , Nicholson Geoff C
Published: April 2006

High-dose corticosteroids, used for many medical conditions, are associated with rapid bone loss from sites such as the vertebrae, and compression fractures can be observed within months. Recent trials suggest treatment with bisphosphonates or active vitamin D analogs can reduce bone loss and the risk of fracture associated with glucocorticoids, but few studies have directly compared such agents. We conducted a randomized, multicenter, open-label trial to compare the efficacy of alendronate, calcitriol, and simple vitamin D in prevention and treatment of glucocorticoid-induced bone loss. A total of 195 subjects (134 females and 61 males) commencing or already taking glucocorticoids were randomized to one of three groups: calcitriol, 0.5 to 0.75 μg/day; simple vitamin D (ergocalciferol, 30,000 IU weekly) plus calcium carbonate (600 mg daily); or alendronate, 10 mg/day plus calcium carbonate (600 mg daily). Over 2 years, mean lumbar bone mineral density change was +5.9% with alendronate, −0.5% with ergocalciferol, and −0.7% with calcitriol (p < 0.001). At the femoral neck, there was no significant difference in bone mineral density change between the treatments over 2 years: alendronate (+0.9%), ergocalciferol (−3.2%), and calcitriol (−2.2%). Lumbar bone loss varied according to whether patients were starting or receiving chronic glucocorticoids, and there was a significant treatment × prior glucocorticoid use interaction effect. Six of 66 calcitriol subjects, 1 of 61 ergocalciferol subjects, and 0 of 64 alendronate subjects sustained new vertebral fractures. These data do not suggest any difference between simple vitamin D and calcitriol but do show that alendronate was superior to either treatment for glucocorticoid induced bone loss.
Transforming Growth Factor-β Regulation of the Insulin-Like Growth Factor Binding Protein-4 Protease System in Cultured Human Osteoblasts
Authors: Ortiz Christopher O , Chen Bing-Kun , Bale Laurie K , Overgaard Michael T , Oxvig Claus , Conover Cheryl A
Published: April 2006

IGFBP-4 is an inhibitor of IGF-I in bone. We show that TGF-β regulates IGFBP-4 and enhances IGF-I-stimulated growth of cultured human bone cells through increased expression of an IGFBP-4 protease, PAPP-A. This effect of TGF-β on IGF-I bioavailability may promote local bone formation.

Insulin-like growth factor binding protein (IGFBP-4) proteolysis is implicated in the regulation of local insulin-like growth factor (IGF)-I bioavailability during bone remodeling. The IGFBP-4 protease secreted by normal adult human osteoblastic (hOB) cells in culture is a novel metalloproteinase, pregnancy-associated plasma protein-A (PAPP-A). We have recently identified an inhibitor of PAPP-A, the precursor form of major basic protein (proMBP). Very little is known about the molecular regulation of this IGFBP-4 protease system. In the present study, we determined the effect of transforming growth factor (TGF)-β and IGF-II, the two most abundant growth factors in human bone, on PAPP-A and proMBP expression in primary cultures of hOB cells. Treatment with TGF-β resulted in time- and dose-dependent increases in PAPP-A mRNA expression, with a maximal 12-fold increase after 24 h of stimulation with 10 ng/ml TGF-β. Increased PAPP-A levels in hOB cell-conditioned medium paralleled PAPP-A gene expression. In addition, TGF-β completely suppressed proMBP expression. Treatment of hOB cells with IGF-II had no effect on PAPP-A or proMBP gene expression. However, IGFBP-4 proteolysis in cell-free assay was dependent on IGF-II, and there was increased IGF-II-dependent IGFBP-4 protease activity in conditioned medium from hOB cells that were treated with TGF-β. IGF-I stimulation of hOB cell proliferation was markedly enhanced by pretreatment with TGF-β and [Leu27]IGF-II, and this enhancement was prevented with protease-resistant IGFBP-4. In summary, TGF-β regulates IGFBP-4 proteolysis in hOB cells through increased expression of the protease, PAPP-A, and decreased expression of the inhibitor, proMBP. However, functional activation of the IGFBP-4 protease system is dependent on IGF-II, which acts at a post-translational level. These data support a model whereby local TGF-β and IGF-II in the bone microenvironment coordinately amplify IGF-I bioavailability through controlled IGFBP-4 proteolysis, which may be a means to promote bone formation.
Adverse Outcomes of Osteoporotic Fractures in the General Population
Authors: Melton L Joseph
Published: April 2006

Osteoporotic fractures exact a terrible toll on the population with respect to morbidity and cost, and to a lesser extent mortality, which will increase dramatically with the growing elderly population. Attention has focused on the 12-20% excess deaths after hip fracture, but most are caused by underlying medical conditions unrelated to osteoporosis. More important is fracture-related morbidity. An estimated 10% of patients are disabled by hip fracture, and 19% require institutionalization, accounting for almost 140,000 nursing home admissions annually in this country. Distal forearm and vertebral fractures less commonly result in nursing home placement, but about 10% of postmenopausal women have vertebral deformities that cause chronic pain, and a substantial minority have poor function after forearm fracture. These fractures interfere greatly with the activities of daily living, and all of them can have a substantial negative impact on quality of life. Annual expenditures for osteoporotic fracture care in the United States ($17.5 million in 2002 dollars) are dominated by hip fracture treatment, but vertebral fractures, distal forearm fractures, and importantly, the other fractures related to osteoporosis contribute one-third of the total. Although all fracture patients are at increased risk of future fractures, few of them are currently treated for osteoporosis, and only a subset (i.e., those with vertebral fractures) are considered candidates for many clinical trials. Eligibility criteria should be expanded and fracture end-points generalized to acknowledge the overall burden of osteoporotic fractures.
High Bone Mass in Mice Expressing a Mutant LRP5 Gene
Authors: Babij Philip , Zhao Weiguang , Small Clayton , Kharode Yogendra , Yaworsky Paul J , Bouxsein Mary L , Reddy Padmalatha S , Bodine Peter Vn , Robinson John A , Bhat Bheem , Marzolf James , Moran Robert A , Bex Frederick
Published: April 2006

A unique mutation in LRP5 is associated with high bone mass in man. Transgenic mice expressing this LRP5 mutation have a similar phenotype with high bone mass and enhanced strength. These results underscore the importance of LRP5 in skeletal regulation and suggest targets for therapies for bone disease.

A mutation (G171V) in the low-density lipoprotein receptor related protein 5 (LRP5) has been associated with high bone mass (HBM) in two independent human kindreds. To validate the role of the mutation, several lines of transgenic mice were created expressing either the human LRP5 G171V substitution or the wildtype LRP5 gene in bone. Volumetric bone mineral density (vBMD) analysis by pQCT showed dramatic increases in both total vBMD (30-55%) and trabecular vBMD (103-250%) of the distal femoral metaphysis and increased cortical size of the femoral diaphysis in mutant G171V transgenics at 5, 9, 17, 26, and 52 weeks of age (p < 0.01 for all). In addition, high-resolution microcomputed tomography (microCT) analysis of the distal femorae and lumbar vertebrae revealed an increase (110-232%) in trabecular bone volume fraction caused by both increased trabecular number (41-74%) and increased trabecular thickness (34-46%; p < 0.01 for all) in the mutant G171V mice. The increased bone mass was associated with significant increases in vertebral compressive strength (80-140%) and the increased cortical size with significant increases in femoral bending strength (50-130%). There were no differences in osteoclast number at 17 weeks of age. However, compared with littermate controls, the mutant G171V transgenic mice showed an increase in actively mineralizing bone surface, enhanced alkaline phosphatase staining in osteoblasts, and a significant reduction in the number of TUNEL-positive osteoblasts and osteocytes. These results suggest that the increased bone mineral density in mutant G171V mice was caused by increased numbers of active osteoblasts, which could in part be because of their increased functional lifespan. While slight bone anabolic activity was observed from overexpression of the wildtype LRP5 gene, it is clear that the G171V mutation, rather than overexpression of the receptor itself, is primarily responsible for the dramatic HBM bone effects. Together, these findings establish the importance of this novel and unexpected role of a lipoprotein receptor in regulating bone mass and afford a new model to explore LRP5 and its recent association with Wnt signaling in bone biology.
Role of Osteoclast Extracellular Signal-Regulated Kinase (ERK) in Cell Survival and Maintenance of Cell Polarity
Authors: Nakamura Hiroaki , Hirata Azumi , Tsuji Takehito , Yamamoto Toshio
Published: April 2006

Morphological changes of osteoclasts by a MEK1 inhibitor, PD98059, were investigated to clarify a role of ERK. PD98059 promoted apoptosis of osteoclasts and the loss of ruffled borders. This study supports the importance of ERK in survival and polarity of osteoclasts.

Introduction: Extracellular signal-regulated kinase (ERK) is a mitogen activated protein kinase (MAPK) that has been reported to play a role in the survival and apoptosis of osteoclasts. However, the precise signal transduction mechanism is not fully understood. The aim of this study was to clarify the role of ERK in osteoclasts by histological analysis.

Materials and Methods: Using a rat calvarial organ culture system, the inhibition of ERK phosphorylation by PD98059, a MAPK/ERK kinase 1 (MEK1) inhibitor, was assayed by immunoblotting. Morphological changes in osteoclasts induced by PD98059 were elucidated by light and electron microscopy. The cellular localization of ERK was also determined by immunoelectron microscopy.

Results: PD98059 inhibited phosphorylated ERK after a 1-h incubation. Ultrastructural study demonstrated that PD98059 induced the accumulation of vesicles and vacuoles in osteoclasts and the loss of ruffled border at 1 h. At 3 h, some osteoclasts showed apoptosis with nuclear condensation, and at 6 h after PD98059 treatment, many osteoclasts were detached from the bone surface and had lost their cell polarity. Electron microscopic immunohistochemistry revealed that ERK was mainly localized in the cytoplasm of clear zones in control osteoclasts, but apoptotic osteoclasts also showed immunoreactivity in clear zone-like structures in contact with osteoblast-lineage cells.

Conclusion: These findings indicate that ERK in osteoclasts is involved in their survival and may be involved in the formation of a ruffled border and the maintenance of cell polarity.
Osteomalacic and Hyperparathyroid Changes in Fibrous Dysplasia Of Bone: Core Biopsy Studies and Clinical Correlations
Authors: Corsi Alessandro , Collins Michael T , Riminucci Mara , Howell Peter GT , Boyde Alan , Robey Pamela Gehron , Bianco Paolo
Published: April 2006

Deposition, mineralization, and resorption of FD bone compared with unaffected bone from FD patients was investigated in iliac crest biopsy specimens from 13 patients. Compared with unaffected bone, lesional FD bone seemed to be very sensitive to the effects of PTH and renal phosphate wasting, which respectively bring about hyperparathyroid or osteomalacic changes in the lesional bone.

Introduction: Fibrous dysplasia is a genetic noninherited disease caused by activating mutations of the GNAS1 gene, resulting in the deposition of qualitatively abnormal bone and marrow. This study was designed to learn more about the local processes of bone deposition, mineralization, and resorption within lesional fibrous dysplasia (FD) bone compared with unaffected bone of FD patients.

Methods: Histology, histomorphometry, and quantitative back-scattered electron imaging (qBSE) analysis was conducted on affected and unaffected biopsy specimens from 13 patients and correlated to markers of bone metabolism.

Results and Conclusions: There was a marked excess of unmineralized osteoid with a nonlamellar structure and a reduced mineral content in mineralized bone within FD lesions (p < 0.001). A negative correlation (p = 0.05) between osteoid thickness (O.Th) and renal tubular phosphate reabsorption (measured as TmP/GFR) was observed for lesional bone, but not for unaffected bone, in which no histological or histomorphometric evidence of osteomalacia was observed in patients with renal phosphate wasting. Histological and histomorphometric evidence of increased bone resorption was variable in lesional bone and correlated with serum levels of parathyroid hormone (PTH). Hyperparathyroidism-related histological changes were observed in fibrous dysplastic bone, but not in the unaffected bone, of patients with elevated serum PTH secondary to vitamin D deficiency. Our data indicate that, compared with unaffected bone, lesional FD bone is very sensitive to the effects of PTH and renal phosphate wasting, which, respectively, bring about hyperparathyroid or osteomalacic changes in the lesional bone. Osteomalacic and hyperparathyroid changes, which emanate from distinct metabolic derangements (which superimpose on the local effects of GNAS1 mutations in bone), influence, in turn, the severity and type of skeletal morbidity in FD.
Identification of a Parathyroid Hormone in the Fish Fugu rubripes
Authors: Danks Janine A , Ho Patricia MW , Notini Amanda J , Katsis Frosa , Hoffmann Peter , Kemp Bruce E , Martin T John , Zajac Jeffrey D
Published: April 2006

A PTH gene has been isolated from the fish Fugu rubripes. The encoded protein of 80 amino acid has the lowest homology with any of the PTH family members. Fugu PTH(1–34) had 5-fold lower potency than human PTH(1–34) in a mammalian cell system.

Introduction: Parathyroid hormone (PTH) is the major hypercalcemic hormone in higher vertebrates. Fish lack parathyroid glands, but there have numerous attempts to identify and isolate PTH from fish.

Materials and Methods: Polymerase chain reaction (PCR) was performed with primers based on preliminary data from the Joint Genome Institute database. PCR amplification was performed on genomic DNA isolated from Fugu rubripes. PCR products were purified and DNA was sequenced. All sequence was confirmed from more than one independently amplified PCR product. Multiple sequence alignments were carried out, and the percentage of identities and similarities were calculated. An unrooted phylogenetic tree, using all the known PTH and PTH-related protein (PTHrP) amino acid sequences, was determined. Synthetic peptides were tested in a biological assay that measured cyclic adenosine 3′,5′-monophosphate formation in UMR106.1 cells. Rabbit polyclonal antisera specific for N-terminal human PTHrP and one rabbit polyclonal antiserum specific for N terminus hPTH were used to test the cross-reactivity with fPTH(1–34) in immunoblots.
A Role for Synaptotagmin VII-Regulated Exocytosis of Lysosomes in Neurite Outgrowth from Primary Sympathetic Neurons
Authors: Arantes Rosa M. E. , Andrews Norma W.
Published: April 2006

Neurite outgrowth is mediated by the exocytosis of intracellular vesicles at the tips of elongating neuronal processes. The lysosomal vesicle-associated solubleN-ethylmaleimide-sensitive factor attachment protein receptor tetanus neurotoxin insensitive vesicle-associated membrane protein (TI-VAMP)/VAMP7 was previously implicated in membrane fusion events mediating neurite outgrowth, but the participation of lysosomes in this exocytic process has remained unclear. Here, we show that VAMP7 and the lysosomal glycoprotein Lamp1 extensively colocalize in vesicles present throughout the soma and neurite outgrowths of primary sympathetic neurons. Synaptotagmin VII (Syt VII), a Ca2+-sensing synaptotagmin isoform previously shown to interact with VAMP7 during lysosomal exocytosis in fibroblasts, was detected on a subset of these lysosomal glycoprotein 1 (Lamp1)/VAMP7-positive neuronal vesicles. Ionophore-stimulated exocytosis triggered exposure of the luminal domains of both Lamp1 and Syt VII at overlapping sites on the neuronal surface, indicating that the Syt VII-containing lysosomal compartments fuse with the plasma membrane in response to [Ca2+]ielevation. To determine whether Syt VII was required for the exocytic events mediating neurite extension, we followed the development of superior cervical ganglion neurons explanted from Syt VII-deficient mice. The results revealed a marked defect in neurite outgrowth and arborization, suggesting that Ca2+-dependent, Syt VII-regulated exocytosis of late endosomes/lysosomes plays a role in the addition of new membrane to developing neurite extensions.