bluuebunny/crossref_metadata_2025_split huggingface dataset:
Extracellular Nucleotides Act Through P2X7 Receptors to Activate NF-κB in Osteoclasts
Authors: Korcok Jasminka , Raimundo Lin N , Ke Hua Z , Sims Stephen M , Dixon S Jeffrey 
Published: April 2006

Abstract
Nucleotides, released in response to mechanical and other stimuli, act on P2 receptors in osteoclasts and other cell types. In vitro studies of osteoclasts from rabbits and P2X7 receptor-deficient mice revealed that P2X7 receptors couple to activation of the key transcription factor NF-κB.
Introduction: Osteoclasts express functional P2X4 and P2X7 receptors, which are ATP-gated cation channels. Knockout (KO) of the P2X7 receptor has revealed its role in regulating bone formation and resorption, but the underlying signals are not known. The transcription factor NF-κB plays a key role in the response of osteoclasts to RANKL and other cytokines. The aim of this study was to examine whether P2X receptors on osteoclasts signal through NF-κB.
Materials and Methods: Osteoclasts were isolated from neonatal rabbits or wildtype (WT) and P2X7 receptor KO mice. Immunofluorescence was used to detect the p65 subunit of NF-κB, which, on activation, translocates from the cytosol to the nuclei. The concentration of cytosolic free Ca2+ ([Ca2+]i) was monitored in single osteoclasts loaded with fura-2.
Results: In control samples, few rabbit osteoclasts demonstrated nuclear localization of NF-κB. Benzoyl-benzoyl-ATP (BzATP, a P2X7 agonist, 300 μM) induced nuclear translocation of NF-κB after 3 h in ∼45% of rabbit osteoclasts. In contrast, a low concentration of ATP (10 μM, sufficient to activate P2X4 and P2Y2, but not P2X7 receptors) did not induce nuclear translocation of NF-κB. Because BzATP activates multiple P2 receptors, we examined responses of osteoclasts derived from WT and P2X7 receptor KO mice. Treatment with BzATP for 30 minutes increased nuclear localization of NF-κB in osteoclasts from WT but not KO mice, showing involvement of P2X7 receptors. Both ATP (10 μM) and BzATP (300 μM) caused transient elevation of [Ca2+]i, indicating that rise of calcium alone is not sufficient to activate NF-κB. Pretreatment of rabbit osteoclasts with osteoprotegerin inhibited translocation of NF-κB induced by RANKL but not by BzATP, establishing that the effects of BzATP are independent of RANKL signaling.
Conclusion: These findings show that P2X7 nucleotide receptors couple to activation of NF-κB in osteoclasts. Thus, nucleotides, released at sites of inflammation or in response to mechanical stimuli, may act through NF-κB to regulate osteoclast formation and activity.
Genetically Linked Site-Specificity of Disuse Osteoporosis
Authors: Judex Stefan , Garman Russell , Squire Maria , Busa Bhavin , Donahue Leah-Rae , Rubin Clinton 
Published: April 2006

Abstract
The genetic influence on bone loss in response to mechanical unloading was investigated within diaphyseal and distal femoral regions in three genetically distinct strains of mice. One mouse strain failed to lose bone after removal of function, whereas osteopenia was evident in multiple regions of the remaining two strains but in different areas of the bone.
Introduction: It is well recognized that susceptibility to osteoporosis is, in large measure, determined by the genome, but whether this influence is systemic or site-specific is not yet known. Here, the extent to which genetic variations influence regional bone loss caused by disuse was studied in the femora of adult female mice from three inbred strains.
Materials and Methods: Adult C57BL/6J (B6), C3H/HeJ (C3H), and BALB/cByJ (BALB) mice were subjected to 15–21 days of disuse, achieved by hindlimb suspension, and six distinct anatomical regions of the femur were analyzed by high-resolution μCT.
Results and Conclusions: In B6 mice, the amount of disuse stimulated bone loss was relatively uniform across all regions, with 20% loss of trabecular bone and 10% loss of cortical bone. The degree of bone loss in BALB mice varied greatly, ranging from 59% in the metaphysis to 3% in the proximal diaphysis. In this strain, the nonuniformity of bone loss was directly related to the nonuniform distribution of baseline bone morphology (r2 = 0.94). In direct contrast with BALB and B6, disuse failed to produce significant losses of bone in any of the analyzed regions of the C3H mice. Instead, these animals displayed a unique compensatory mechanism to disuse, where the large loss of calcified tissue from the endocortical surface (−24%) was compensated for by an expansion of the periosteal envelope (10%). These data indicate a strong, yet complex, genetic dependence of the site-specific regulation of bone remodeling in response to a powerful catabolic signal. Consequently, the skeletal region of interest and the genetic make-up of the individual may have to be considered interdependently when considering the pathogenesis of osteoporosis or the efficacy of an intervention to prevent or recover bone loss.
Osteoprotegerin and RANKL in the Pathogenesis of Thalassemia-Induced Osteoporosis: New Pieces of the Puzzle
Authors: Morabito Nunziata , Gaudio Agostino , Lasco Antonino , Atteritano Marco , Pizzoleo Maria Antonia , Cincotta Maria , La Rosa Mariangela , Guarino Roberta , Meo Anna , Frisina Nicola 
Published: April 2006

Abstract
Osteoporosis represents an important cause of morbidity in adult thalassemic patients, and its pathogenesis has not, as yet, been completely clarified. In our study, we observed that thalassemic patients showed a significantly lower OPG/RANKL ratio than normal subjects. These data are extremely important for the possible therapeutic use of RANKL antagonists such as OPG in thalassemia-induced osteoporosis.
Introduction: Osteoporosis represents an important cause of morbidity in adult thalassemic patients who display increased fracture risk. The etiology of this bone disease is multifactorial, but it is thought that the main role is played by hypogonadism. The mechanisms by which the skeletal effects of sex steroids are mediated are still not fully understood. Recently, two new cytokines, osteoprotegerin (OPG) and RANKL, have been implicated in the pathogenesis of postmenopausal osteoporosis and other metabolic bone diseases. Thus, the aim of this study was to characterize the possible role of the OPG/RANKL system in thalassemia-related bone loss.
Materials and Methods: We measured, in 30 thalassemic patients and in 20 healthy control subjects, serum OPG and RANKL levels, and determined their correlations with bone turnover markers, BMD, sex steroid levels, erythropoietin, and hemoglobin.
Results: Thalassemic patients had an unbalanced bone turnover with an increased resorption phase (shown by high levels of pyridinium cross-links) and a decreased neoformation phase (shown by the slightly low levels of osteocalcin). Moreover, they displayed lower BMD values than controls both at the lumbar and femoral level. As far as the OPG/RANKL system is concerned, thalassemic patients showed no differences in plasma levels of OPG compared with controls, and significantly higher plasma levels of RANKL, with a consequent significantly lower OPG/RANKL ratio.
Conclusions: Our data suggest that, in thalassemic patients, an altered modulation of the OPG/RANKL system, resulting in increased expression of RANKL by stromal or osteoblastic cells, could contribute to the enhanced osteoclastic bone resorption and bone loss characteristic of these patients.
Regulation of Human Skeletal Stem Cells Differentiation by Dlk1/Pref-1
Authors: Abdallah Basem M , Jensen Charlotte H , Gutierrez Gloria , Leslie Robert GQ , Jensen Thomas G , Kassem Moustapha 
Published: April 2006

Abstract
Dlk-1/Pref-1 was identified as a novel regulator of human skeletal stem cell differentiation. Dlk1/Pref-1 is expressed in bone and cultured osteoblasts, and its constitutive overexpression led to inhibition of osteoblast and adipocyte differentiation of human marrow stromal cells.
Introduction: Molecular control of human mesenchymal stem cell (hMSC) differentiation into osteoblasts and adipocytes is not known. In this study, we examined the role of delta-like 1/preadipocyte factor-1 (Dlk1/Pref-1) in regulating the differentiation of hMSCs.
Materials and Methods: As a model for hMSCs, we have stably transduced telomerase-immortalized hMSC (hMSC-TERT) with the full length of human Dlk1/Pref-1 cDNA and tested its effect on hMSC growth and differentiation into osteoblasts or adipocytes as assessed by cytochemical staining, FACS analysis, and real time PCR. Ex vivo calvaria organ cultures assay was used to confirm the in vitro effect of Dlk/Pref-1 on bone formation.
Results: Dlk1/Pref-1 was found to be expressed in fetal and adult bone, hMSCs, and some osteoblastic cell lines. A retroviral vector containing the human Dlk1/Pref-1 cDNA was used to create a cell line (hMSC-dlk1) expressing high levels of Dlk1/Pref-1 protein. Overexpression of Dlk1/Pref-1 did not affect the proliferation rate of hMSC, but the ability to form mature adipocytes, mineralized matrix in vitro, and new bone formation in neonatal murine calvariae organ cultures was reduced. These effects were associated with inhibition of gene expression markers of late stages of adipocyte (adipocyte fatty acid-binding protein [aP2], peroxisome proliferator-activated receptor-gamma2 [PPARγ2], and adiponectin [APM1]) and osteoblast differentiation (alkaline phosphatase [ALP], collagen type I [Col1], and osteocalcin [OC]). Lineage commitment markers for adipocytes (adipocyte determination and differentiation factor −1 [ADD1]) and osteoblasts (core binding factor/runt-related binding factor 2 [Cbfa1/Runx2]) were not affected.
Conclusion: During hMSC differentiation, Dlk1/Pref-1 maintains the size of the bipotential progenitor cell pool by inhibiting the formation of mature osteoblasts and adipocytes.
In Vivo Inhibition of Osteoblastic Metalloproteinases Leads to Increased Trabecular Bone Mass
Authors: Geoffroy Valérie , Marty-Morieux Caroline , Le Goupil Nathalie , Clement-Lacroix Phillippe , Terraz Catherine , Frain Monique , Roux Sophie , Rossert Jérome , de Vernejoul Marie Christine 
Published: April 2006

Abstract
Mice specifically overexpressing TIMP-1 in osteoblasts have been generated to investigate the role of MMPs in bone in vivo. These mice displayed increased trabecular bone volume and decreased bone turnover. This model provides evidence of the role played by the MMPs in bone remodeling and balance.
Introduction: Although it has been suggested that the matrix metalloproteinases (MMPs) may play a role in initiating the bone resorption process in vitro, there is no evidence that they play any role in in vivo bone maintenance.
Materials and Methods: We used an artificial promoter specifically driving cells of the osteoblastic lineage to overexpress the tissue inhibitor of MMPs (TIMP-1) cDNA in mice. Densitometric analysis, using DXA and pQCT, and static and dynamic histomorphometry were used to evaluate the bone phenotype both in male and female transgenic mice. We evaluated osteoblastic differentiation using a primary osteoblast culture and osteoclast activity using an ex vivo organ culture.
Results and Conclusion: We showed that at 1 and 2.5 months of age, only the female mice exhibited a bone phenotype. These mice displayed specific increases in the BMD and bone volume of trabecular bone. This increase was accompanied by decreased trabecular separation, suggesting a decrease in bone resorption. Using an ex vivo resorption assay, we demonstrated that parathyroid hormone (PTH)-stimulated bone resorption was reduced in these mice. Evaluation of the bone histomorphometric dynamic parameters showed that the mineralizing surfaces and bone formation rate were both reduced. There was no change in the mineralization lag time or number of osteocyte lacunae. Using primary osteoblast culture and molecular analysis, we showed that the differentiation and function of osteoblasts from transgenic mice were normal, but that the ex vivo formation of mineralized nodules was delayed. This model is the first to show that in vivo MMPs play a role in bone remodeling and bone balance. Moreover, our data suggest that MMP activity could be involved in the hormonal regulation of bone resorption by osteoblasts.
The Balance Between Concurrent Activation of ERs and PPARs Determines Daidzein-Induced Osteogenesis and Adipogenesis
Authors: Dang ZhiChao , Löwik Clemens WGM 
Published: April 2006

Abstract
The soy phytoestrogen daidzein has biphasic dose responses, but the underlying mechanisms are not yet clear. Transcriptional and biochemical data show that PPARs, in addition to ERs, are molecular targets of daidzein, which divergently regulates osteogenesis and adipogenesis. Dose responses are the result of a balance among PPARs and between ERs and PPARs.
Introduction: Soy phytoestrogens have been used for the purposes of treatment and prevention of osteoporosis. Biphasic dose responses of daidzein, one of the main soy phytoestrogens, have long been recognized, but the underlying molecular mechanisms of action are not yet clear.
Materials and Methods: Mouse bone marrow cells and mouse osteoprogenitor KS483 cells that concurrently differentiate into osteoblasts and adipocytes were cultured. Biochemical measurement of alkaline phosphatase (ALP) activity, RT-PCR, and gene reporter assays were used in this study.
Results: Daidzein, one of the major soy phytoestrogens, had biphasic effects on osteogenesis and adipogenesis. Daidzein stimulated osteogenesis (ALP activity and nodule formation) and decreased adipogenesis (the number of adipocytes) at concentrations below 20 μM, whereas it inhibited osteogenesis and stimulated adipogenesis at concentrations higher than 30 μM. When estrogen receptors (ERs) were blocked by ICI182,780, daidzein-induced effects were not biphasic. A decrease in osteogenesis and an increase in adipogenesis were observed at the concentrations higher than 20 and 10 μM, respectively. In addition to ERs, daidzein transactivated not only peroxisome proliferator-activate receptor γ (PPARγ), but also PPARα and PPARδ at micromolar concentrations. Activation of PPARα had no direct effects on osteogenesis and adipogenesis. In contrast, activation of PPARδ stimulated osteogenesis but had no effects on adipogenesis, whereas PPARγ inhibited osteogenesis and stimulated adipogenesis. Transfection experiments show that an activation of PPARα or PPARγ by daidzein downregulated its estrogenic transcriptional activity, whereas activation of PPARδ upregulated its estrogenic transcriptional activity. Activation of ERα or ERβ by daidzein downregulated PPARγ transcriptional activity but had no influence on PPARα or PPARδ transcriptional activity.
Conclusions: Daidzein at micromolar concentrations concurrently activates different amounts of ERs and PPARs, and the balance of the divergent actions of ERs and PPARs determines daidzein-induced osteogenesis and adipogenesis.
Maintenance of Increased Bone Mass After Recombinant Human Parathyroid Hormone (1-84) With Sequential Zoledronate Treatment in Ovariectomized Rats
Authors: Rhee Yumie , Won Ye-Yeon , Baek Myong-Hyun , Lim Sung-Kil 
Published: April 2006

Abstract
The concept of lose, restore, maintain (LRM) for reversing existing osteoporosis was tested in rats. The withdrawal of PTH results in the loss of the acquired bone mass, but sequential therapy with zoledronate quite effectively maintained the PTH(1-84)-acquired bone quantity and quality.
Introduction: Because antiresorptive agents against osteoporosis are presently quite limited, strong anabolic agents such as human parathyroid hormone (hPTH) are quite helpful. However, because hPTH(1-34) is available only through injection and has a critical side effect of causing bone tumors during life-long administration in the rat, it would be practical to use PTH for the shortest possible duration to obtain the maximal effect. To determine the effectiveness of the osteoporosis-reversing concept of lose, restore, and maintain (LRM), recombinant hPTH(1-84) [rhPTH(1-84)] and the respective antiresorptive agents were sequentially studied.
Materials and Methods: Thirty-six, 20-week-old Sprague-Dawley rats were used. Treatment started at the 25th week after ovariectomy, which was performed at 20 weeks of age, with 5 weeks of rhPTH(1-84) 100 μg/kg/day, 5 days/week, followed by the respective sequential therapies for 5 weeks as follows: (1) ovariectomized rats (OVX; n = 6), (2) sham-operated rats (SHAM; n = 6), (3) OVX rats with PTH maintenance (PTH-M; n = 6), (4) OVX rats treated with PTH and then PTH was withdrawn (PTH-W; n = 6), (5) PTH-treated OVX rats treated with 17β-estradiol (PTH-E; 10 μg/day SC, 5 days/week; n = 6), and (6) PTH-treated OVX rats treated with zoledronate (PTH-Z; 12.5 μg/kg SC weekly; n = 6). BMD of the right femora was measured by DXA. μCT was used to measure the structural parameters of the second lumbar vertebrae. Three-point bending test of the femora and compressive tests of vertebrae were also performed.
Results: Bone quantity data showed that the BMD and most of the microstructural parameters were significantly higher in the PTH-M and PTH-Z groups than in the OVX and PTH-W groups (p &lt; 0.05). Measurement of the cortical thickness revealed that only the PTH-M group showed a significant increase (p = 0.001). The ultimate force (Fu) at the midshaft of the femora was similar in the treated groups and stronger than in the OVX group (p &lt; 0.05). However, in the vertebrae, the Fu of the PTH-M and PTH-Z groups was significantly higher, by ∼44-47%, than in the OVX and PTH-E groups and showed a higher tendency than in the PTH-W group.
Conclusion: PTH withdrawal resulted in the loss of acquired BMD, and sequential therapy with antiresorptives prevented further loss (17β-estradiol versus zoledronate). The zoledronate after rhPTH(1-84) as a sequential regimen was quite consistently effective.
Seasonal Periodicity of Serum Vitamin D and Parathyroid Hormone, Bone Resorption, and Fractures: The Geelong Osteoporosis Study
Authors: Pasco Julie A , Henry Margaret J , Kotowicz Mark A , Sanders Kerrie M , Seeman Ego , Pasco John R , Schneider Hans G , Nicholson Geoffrey C 
Published: April 2006

Abstract
In this population-based study, seasonal periodicity was seen with reduced serum vitamin D, increased serum PTH, and increased bone resorption in winter. This was associated with an increased proportion of falls resulting in fracture and an increased risk of wrist and hip fractures.
Introduction: In a population of women who reside in a temperate climate and do not generally receive dietary vitamin D supplementation, we investigated whether seasonal vitamin D insufficiency is associated with increased risk of fracture.
Materials and Methods: An observational, cross-sectional, population-based study set in southeastern Australia (latitude 38–39° S). Participants were drawn from a well-defined community of 27,203 women ≥55 years old: 287 randomly selected from electoral rolls, 1635 with incident fractures, and 1358 presenting to a university hospital with falls. The main outcome measures were annual periodicities of ultraviolet radiation, serum 25-hydroxyvitamin D [25(OH)D], serum parathyroid hormone (PTH), serum C-telopeptide (CTx), BMD, falls, and fractures.
Results: Cyclic variations in serum 25(OH)D lagged 1 month behind ultraviolet radiation, peaking in summer and dipping in winter (p &lt; 0.001). Periodicity of serum PTH was the inverse of serum 25(OH)D, with a phase shift delay of 1 month (p = 0.004). Peak serum CTx lagged peak serum PTH by 1–2 months. In late winter, a greater proportion of falls resulted in fracture (p &lt; 0.001). Seasonal periodicity in 439 hip and 307 wrist fractures also followed a simple harmonic model (p = 0.078 and 0.002, respectively), peaking 1.5–3 months after the trough in 25(OH)D.
Conclusions: A fall in 25(OH)D in winter is accompanied by increases in (1) PTH levels, (2) bone resorption, (3) the proportion of falls resulting in fracture, and (4) the frequency of hip and wrist fracture. Whether vitamin D supplementation in winter can reduce the population burden of fractures requires further investigation.
NF1 Tumor Suppressor Protein and mRNA in Skeletal Tissues of Developing and Adult Normal Mouse and NF1-Deficient Embryos
Authors: Kuorilehto Tommi , Nissinen Marja , Koivunen Jussi , Benson M Douglas , Peltonen Juha 
Published: April 2006

Abstract
NF1 is a heritable disease with multiple osseous lesions. The expression of the NF1 gene was studied in embryonic and adult rodent skeleton and in NF1-deficient embryos. The NF1 gene was expressed intensely in the cartilage and the periosteum. Impaired NF1 expression may lead to inappropriate development and dynamics of bones and ultimately to the osseous manifestations of the disease.
Introduction: Neurofibromatosis type 1 is caused by mutations in the NF1 gene encoding the Ras GTPase activating protein (Ras-GAP) neurofibromin. Skeletal ailments such as short stature, kyphoscoliosis, and tibial bowing and pseudarthrosis are common osseous manifestations of NF1. These symptoms are congenital, implying a role for neurofibromin in proper bone growth. However, little is known about its expression in skeletal tissues during their development.
Materials and Methods: The expression of the NF1 gene was studied in normal and NF1+/− mouse fetuses at embryonic days 12.5-15.5 and in skeletal tissues of adult mice and rats. In situ hybridization, immunohistochemistry, and Western blot analysis were used to identify the NF1 gene expression profile.
Results: NF1 mRNA and protein were elevated in resting, maturation, and hypertrophic chondrocytes at the growth plate. Parallel studies on NF1+/− embryos showed expression patterns identical to wildtype. The periosteum, including osteoblasts and osteoclasts, and osteocytes of the cortical bone of adult mice were also intensely labeled for NF1 protein and mRNA. Western transfer analysis detected NF1 protein in the respective rat tissues. Phosphorylation of p42 and p44 MAP kinases, the downstream consequence of Ras activation, was elevated in hypertrophic chondrocytes of NF1+/− embryos.
Conclusions: The results suggest that neurofibromin may act as a Ras-GAP in skeletal cells to attenuate Ras transduced growth signals and thus play a role during ossification and dynamics of bone. Loss of NF1 function may therefore lead to dysplastic bone growth, thereby causing the debilitating osseous symptoms of NF1.