bluuebunny/crossref_metadata_2025 huggingface dataset:
Gender Differences in the Genetic Factors Responsible for Variation in Bone Density and Ultrasound
Authors: Naganathan Vasi , Macgregor Alex , Snieder Harold , Nguyen Tuan , Spector Tim , Sambrook Philip
Published: April 2006

Although genetic factors are thought to explain a large proportion of the variation in bone density in women, few studies have been conducted in men. Therefore, it is unclear whether the individual differences in bone strength between men and women are a reflection of gender differences in the relative influence of genetic and environmental factors on bone density variance. The aim of this study was to determine if there were gender differences in the genetic components of variance for bone density and ultrasound. In addition, the study aimed to explore the hypothesis that there are unique gender-specific genetic determinants of these traits. Bone mineral density (BMD) of the hip, distal forearm, and lumbar spine were measured by dual-energy X-ray absorptiometry (DXA) as well as quantitative ultrasound (QUS) at the calcaneus in healthy female twin pairs (286 identical [MZ] and 265 nonidentical [DZ]), male twin pairs (72 MZ and 65 DZ), and 82 opposite-sex (OS) pairs aged between 18 and 80 years. For hip BMD, distal forearm, and QUS measurements, the differences between MZ correlations and like-sex DZ correlations were similar for both sexes, suggesting little difference in the component of total variance explained by genetic factors between male and female twin pairs. However, correlations between OS twin pairs were lower than that of like-sex twin pairs, suggesting the possibility of unique gender-specific genetic effects. At the forearm, model fitting suggested a small gender difference in the magnitude of genetic variance as well as the presence of a unique gender-specific genetic variance component. Hip, lumbar spine, and QUS measurements were better explained by models that assumed no gender differences in genetic variance between the sexes, but the study had insufficient power to detect small differences in the genetic components of variance. The results of this study suggest that the proportion of bone strength variance explained by genetic factors is similar for men and women. However, at some regions there is evidence to suggest a gender-specific genetic component to the overall genetic variance.
Retinol Intake and Bone Mineral Density in the Elderly: The Rancho Bernardo Study
Authors: Promislow Joanne H. E. , Goodman-Gruen Deborah , Slymen Donald J. , Barrett-Connor Elizabeth
Published: April 2006

Retinol is involved in bone remodeling, and excessive intake has been linked to bone demineralization, yet its role in osteoporosis has received little evaluation. We studied the associations of retinol intake with bone mineral density (BMD) and bone maintenance in an ambulatory community-dwelling cohort of 570 women and 388 men, aged 55–92 years at baseline. Regression analyses, adjusted for standard osteoporosis covariates, showed an inverse U-shaped association of retinol, assessed by food-frequency questionnaires in 1988–1992, with baseline BMD, BMD measured 4 years later, and BMD change. Supplemental retinol use, reported by 50% of women and 39% of men, was an effect modifier in women; the associations of log retinol with BMD and BMD change were negative for supplement users and positive for nonusers at the hip, femoral neck, and spine. At the femoral neck, for every unit increase in log retinol intake, supplement users had 0.02 g/cm2 (p = 0.02) lower BMD and 0.23% (p = 0.05) greater annual bone loss, and nonusers had 0.02 g/cm2 (p = 0.04) greater BMD and 0.22% (p = 0.19) greater bone retention. However, among supplement users, retinol from dietary and supplement sources had similar associations with BMD, suggesting total intake is more important than source. In both sexes, increasing retinol became negatively associated with skeletal health at intakes not far beyond the recommended daily allowance (RDA), intakes reached predominately by supplement users. This study suggests there is a delicate balance between ensuring that the elderly consume sufficient vitamin A and simultaneously cautioning against excessive retinol supplementation.
Radioguided Parathyroidectomy via VATS Combined With Intraoperative Parathyroid Hormone Testing: The Surgical Approach of Choice for Patients With Mediastinal Parathyroid Adenomas?
Authors: O'Herrin Jacquelyn K. , Weigel Tracey , Wilson Michael , Chen Herbert
Published: April 2006

Despite the excellent results with bilateral exploration, minimally invasive parathyroidectomy has become the procedure of choice for patients with hyperparathyroidism in which a single parathyroid lesion can be localized preoperatively. In this article, we discuss a patient who presented with primary hyperparathyroidism for the first time and had a Tc-99m sestamibi scan to localize a single parathyroid lesion in the left, anterior mid-mediastinum. We subsequently performed a radioguided parathyroidectomy via video-assisted thoracoscopic surgery (VATS) to resect this parathyroid adenoma and used intraoperative parathyroid hormone (PTH) testing to confirm cure and avoid neck exploration. We concluded that radioguided parathyroidectomy via VATS combined with intraoperative PTH testing is an effective approach for patients with primary hyperparathyroidism and mediastinal parathyroid lesions, and perhaps should be the technique of choice.
Induction of Transcriptional Activity of the Cyclic Adenosine Monophosphate Response Element Binding Protein by Parathyroid Hormone and Epidermal Growth Factor in Osteoblastic Cells
Authors: Swarthout John T. , Tyson Darren R. , Jefcoat Stephen C. , Partridge Nicola C.
Published: April 2006

Previously, we have shown that parathyroid hormone (PTH) transactivation of cyclic adenosine monophosphate (cAMP) response element binding protein (CREB) requires both serine 129 (S129) and serine 133 (S133) in rat osteosarcoma cells UMR 106-01 (UMR) cells. Furthermore, although protein kinase A (PKA) is responsible for phosphorylation at S133, glycogen synthase kinase 3β (GSK-3β) activity is required and may be responsible for phosphorylation of CREB at S129. Here, we show, using the GAL4-CREB reporter system, that epidermal growth factor (EGF) can transactivate CREB in UMR cells in addition to PTH. Additionally, treatment of UMR cells with both PTH and EGF results in greater than additive transactivation of CREB. Furthermore, using mutational analysis we show that S129 and S133 are required for EGF-induced transcriptional activity. EGF activates members of the MAPK family including p38 and extracellular signal—activated kinases (ERKs), and treatment of UMR cells with either the p38 inhibitor (SB203580) or the MEK inhibitor (PD98059) prevents phosphorylation of CREB at S133 by EGF but not by PTH. Treatment of cells with either SB203580 or PD98059 alone or together significantly inhibits transactivation of CREB by EGF but not by PTH, indicating that EGF regulates CREB phosphorylation and transactivation through p38 and ERKs and PTH does not. Finally, the greater than additive transactivation of CREB by PTH and EGF is significantly inhibited by the PKA inhibitor H-89 or by cotreatment with SB203580 and PD98059. Thus, several different signaling pathways in osteoblastic cells can converge on and regulate CREB activity. This suggests, in vivo, that circulating agents such as PTH and EGF are acting in concert to exert their effects.
Localization and Regulation of the Growth Hormone Receptor and Growth Hormone-Binding Protein in the Rat Growth Plate
Authors: Gevers Evelien F. , Van Der Eerden Bram C. J. , Karperien Marcel , Raap Anton K. , Robinson Iain C. A. F. , Wit Jan-Maarten
Published: April 2006

Growth hormone (GH) has direct effects on the growth plate to stimulate longitudinal growth, but it is not clear which chondrocyte populations GH acts on. The dual effector theory suggests that GH would act primarily on the “stem cells.” However, staining with a GH receptor (GHR) antibody is found in all layers of the growth plate in rabbits and humans. We now have investigated the localization and regulation of GHR and the related GH binding protein (GHBP) in the rat growth plate using a sensitive immunohistochemical method involving tyramide signal amplification (TSA) and antibodies specific for GHR or GHBP. Both GHR and GHBP were shown in the germinal and proliferative chondrocytes, but most clearly in early maturing chondrocytes at the interface between proliferative and hypertrophic cells. Staining for GHR and GHBP was located in both the cytoplasm and the nucleus. Expression of GHR mRNA and GHBP mRNA in the growth plate was confirmed by reverse-transcription polymerase chain reaction (RT-PCR). Immunohistochemical staining for GHR and GHBP decreased with age; in 12-week-old normal rats, only the early maturing chondrocytes were stained. In GH-deficient dwarf rats, staining seemed less than in normal rats, and in hypophysectomized (Hx) rats, staining for GHBP was clearly reduced. Treatment of Hx rats with thyroid hormones (T3 + T4), via subcutaneously (sc) implanted osmotic minipumps, induced little growth and induced a small layer of GHR-positive and GHBP-positive early maturing chondrocytes. Treatment with GH and thyroid hormones (TH) resulted in greater growth and a broader layer of GHR-positive and GHBP-positive cells, indistinguishable from normal rats. In contrast, dexamethasone treatment of normal rats inhibited their growth and reduced GHR and GHBP staining in the growth plate. These results show that GHR and GHBP in the growth plate are under hormonal control. The localization of GHR/GHBP suggests that in addition to actions on germinal and proliferative cells in young rats, GH also has effects on early maturing chondrocytes and may be involved in their differentiation to a fully hypertrophic chondrocyte.
Bone Morphogenetic Protein 2 Induces Dental Follicle Cells to Differentiate Toward a Cementoblast/Osteoblast Phenotype
Authors: Zhao Ming , Xiao Guozhi , Berry Janice E. , Franceschi Renny T. , Reddi Anand , Somerman Martha J.
Published: April 2006

When triggered appropriately, dental follicle cells are considered to be able to differentiate toward a cementoblast/osteoblast phenotype. However, factors and mechanisms regulating follicle cell differentiation remain undefined. This study focused on determining the ability of bone morphogenetic protein (BMP) 2 to promote the differentiation of follicle cells and periodontal ligament (PDL) cells along a cementoblast/osteoblast pathway. Follicle cells and PDL cells were isolated from the first molar region of CD-1 mice and immortalized with SV40. Both cell types expressed BMP-4 and BMP receptors (BMPR) IA and II, but only follicle cells expressed BMP-2 mRNA. Cells were exposed to recombinant human BMP (rhBMP)-2 (0–100 ng/ml) and Northern blots were used to determine the expression of mineral-associated markers. BMP-2, in a dose- and time-dependent manner, induced cementoblast/osteoblast differentiation of follicle cells, as reflected by enhanced core binding factor α1 (Cbfa1), bone sialoprotein (BSP), and osteocalcin (OCN) mRNA expression and enhanced mineral formation. U0126, a specific inhibitor of MEK-1/2 members of the MAPK family, abolished BMP-2-mediated expression of BSP and OCN. In contrast, exposure of PDL cells to BMP-2 resulted in modest expression of OCN and minimal promotion of mineralization. These results suggest that BMP-2 triggers follicle cells to differentiate toward a cementoblast/osteoblast phenotype and that the MAPK pathway is involved.
Activation of Extracellular Signal–Regulated Kinase Is Involved in Mechanical Strain Inhibition of RANKL Expression in Bone Stromal Cells
Authors: Rubin Janet , Murphy Tamara C. , Fan Xian , Goldschmidt Mark , Taylor W. Robert
Published: April 2006

Mechanical input is known to regulate skeletal mass. In vitro, application of strain inhibits osteoclast formation by decreasing expression of the ligand RANKL in bone stromal cells, but the mechanism responsible for this down-regulation is unknown. In experiments here, application of 1.8% equibiaxial strain for 6 h reduced vitamin D-stimulated RANKL mRNA expression by nearly one-half in primary bone stromal cells. Application of strain caused a rapid activation of ERK1/2, which returned to baseline by 60 minutes. Adding the ERK1/2 inhibitor PD98059 30 minutes before strain delivery prevented the strain effect on RANKL mRNA expression, suggesting that activation of ERK1/2 was required for transduction of the mechanical force. Mechanical strain also activated N-terminal Jun kinase (JNK) that, in contrast, did not return to baseline during 24 h of continuous strain. This suggests that JNK may represent an accessory pathway for mechanical transduction in bone cells. Our data indicate that strain modulation of RANKL expression involves activation of MAPK pathways.
Bioavailability and Biological Efficacy of a New Oral Formulation of Salmon Calcitonin in Healthy Volunteers
Authors: Buclin Thierry , Rochat Monica Cosma , Burckhardt Peter , Azria Moïse , Attinger Martine
Published: April 2006

Salmon calcitonin (SCT) is a well-tolerated peptide drug with a wide therapeutic margin and is administered parenterally for long-term treatments of bone diseases. Its clinical usefulness would be enhanced by the development of an orally active formulation. In this randomized crossover double-blinded phase I trial, controlled by both a placebo and a parenteral verum, we have tested a new oral formulation of SCT associated with a caprylic acid derivative as carrier. Eight healthy volunteers received single doses of 400, 800, and 1200 μg of SCT orally, a placebo, and a 10-μg (50 IU) SCT intravenous infusion. SCT was reliably absorbed from the oral formulation, with an absolute bioavailability of 0.5–1.4%, depending on the dose. It induced a marked, dose-dependent drop in blood and urine C-terminal telopeptide of type I collagen (CTX), a sensitive and specific bone resorption marker, with the effects of 1200 μg exceeding those of 10 μg intravenously. It also decreased blood calcium and phosphate, and increased the circulating levels of parathyroid hormone (PTH) and, transiently, the urinary excretion of calcium. It was well-tolerated, with some subjects presenting mild and transient nausea, abdominal cramps, diarrheic stools, and headaches. This study shows that oral delivery of SCT is feasible with reproducible absorption and systemic biological efficacy. Such an oral formulation could facilitate the use of SCT in the treatment of osteoporosis and other bone diseases.
Alleles of RUNX2/CBFA1 Gene Are Associated With Differences in Bone Mineral Density and Risk of Fracture
Authors: Vaughan Tanya , Pasco Julie A. , Kotowicz Mark A. , Nicholson Geoff C. , Morrison Nigel A.
Published: April 2006

The aim of this study was to determine if DNA polymorphism within runt-related gene 2 (RUNX2)/core binding factor A1 (CBFA1) is related to bone mineral density (BMD). RUNX2 contains a glutamine-alanine repeat where mutations causing cleidocranial dysplasia (CCD) have been observed. Two common variants were detected within the alanine repeat: an 18-bp deletion and a synonymous alanine codon polymorphism with alleles GCA and GCG (noted as A and G alleles, respectively). In addition, rare mutations that may be related to low BMD were observed within the glutamine repeat. In 495 randomly selected women of the Geelong Osteoporosis Study (GOS), the A allele was associated with higher BMD at all sites tested. The effect was maximal at the ultradistal (UD) radius (p = 0.001). In a separate fracture study, the A allele was significantly protective against Colles' fracture in elderly women but not spine and hip fracture. The A allele was associated with increased BMD and was protective against a common form of osteoporotic fracture, suggesting that RUNX2 variants may be related to genetic effects on BMD and osteoporosis.
Two Polymorphisms in the Vitamin D Receptor Gene—Association With Bone Mass and 5-Year Change in Bone Mass With or Without Hormone-Replacement Therapy in Postmenopausal Women: The Danish Osteoporosis Prevention Study
Authors: Tofteng C. L. , Jensen J. E. B. , Abrahamsen B. , Odum L. , Brot C.
Published: April 2006

The significance of an interrelation between nongenetic factors and genotype effects in the regulation of bone mass is not clear. In this prospective study of 429 healthy early postmenopausal Danish women, we investigated the association between bone mineral density (BMD) and the FokI and BsmI polymorphisms in the vitamin D receptor (VDR) gene. Participants were allocated to either hormone-replacement therapy (HRT) or no treatment by randomization or personal choice. After 5 years, 332 women with unchanged treatment status were available for analyses, 98 of these women were still on HRT. No association with initial BMD or 5-year change in BMD was found for either polymorphism. In women with body mass index (BMI) < 25 (n = 282), the f allele was associated with lower BMD of the hip (p < 0.001) and forearm (p = 0.001), and the b allele was associated with lower spine BMD (p = 0.02). Comparing thin/normal weight women with overweight/obese women of the same genotype, FF women had similar BMD at all measured sites in contrast to Ff andff women in whom BMD, as expected, was higher in the overweight/obese women. Similar results were found for the BsmI polymorphism with no difference in BMD between BMI groups in BB women. Segregation into groups according to dietary calcium intake did not reveal any genotype association with BMD. These results provide some evidence of a modifying effect of nongenetic factors, specifically BMI, on the association between VDR genotype and BMD. High BMI may protect against lower BMD seen in association with the f or b alleles. In some genotypes (FF and BB), BMI had relatively little effect on BMD.
Improved Bone Structure and Strength After Long-Term Mechanical Loading Is Greatest if Loading Is Separated Into Short Bouts
Authors: Robling Alexander G. , Hinant Felicia M. , Burr David B. , Turner Charles H.
Published: April 2006

Mechanical loading presents a potent osteogenic stimulus to bone cells, but bone cells desensitize rapidly to mechanical stimulation. Resensitization must occur before the cells can transduce future mechanical signals effectively. Previous experiments show that mechanical loading protocols are more osteogenic if the load cycles are divided into several discrete bouts, separated by several hours, than if the cycles are applied in a single uninterrupted bout. We investigated the effect of discrete mechanical loading bouts on structure and biomechanical properties of the rat ulna after 16 weeks of loading. The right ulnas of 26 adult female rats were subjected to 360 load cycles/day, delivered in a haversine waveform at 17 N peak force, 3 days/week for 16 weeks. One-half of the animals (n = 13) were administered all 360 daily cycles in a single uninterrupted bout (360 × 1); the other half were administered 90 cycles four times per day (90 × 4), with 3 h between bouts. A nonloaded baseline control (BLC) group and an age-matched control (AMC) group (n = 9/group) were included in the experiment. The following measurements were collected after death: in situ mechanical strain at the ulna midshaft; ulnar length; maximum and minimum second moments of area (IMAX and IMIN) along the entire length of the ulnas (1-mm increments); and ultimate force, energy to failure, and stiffness of whole ulnas. Qualitative observations of bone morphology were made from whole bone images reconstructed from microcomputed tomography (μCT) slices. Loading according to the 360 × 1 and 90 × 4 schedules improved ultimate force by 64% and 87%, energy to failure by 94% and 165%, IMAX by 13% and 26% (in the middistal diaphysis), IMIN by 69% and 96% (in the middistal diaphysis), and reduced peak mechanical strain by 40% and 36%, respectively. The large increases in biomechanical properties occurred despite very low 5–12% gains in areal bone mineral density (aBMD) and bone mineral content (BMC). Mechanical loading is more effective in enhancing bone biomechanical and structural properties if the loads are applied in discrete bouts, separated by recovery periods (90 × 4 schedule), than if the loads are applied in a single session (360 × 1). Modest increases in aBMD and BMC can improve biomechanical properties substantially if the new bone formation is localized to the most biomechanically relevant sites, as occurs during load-induced bone formation.
Insulin‐Like Growth Factor I Is Required for the Anabolic Actions of Parathyroid Hormone on Mouse Bone
Authors: Bikle Daniel D. , Sakata Takeshi , Leary Colin , Elalieh Hashem , Ginzinger David , Rosen Clifford J. , Beamer Wesley , Majumdar Sharmila , Halloran Bernard P.
Published: April 2006

Parathyroid hormone (PTH) is a potent anabolic agent for bone, but the mechanism(s) by which it works remains imperfectly understood. Previous studies have indicated that PTH stimulates insulin‐like growth factor (IGF) I production, but it remains uncertain whether IGF‐I mediates some or all of the skeletal actions of PTH. To address this question, we examined the skeletal response to PTH in IGF‐I‐deficient (knockout [k/o]) mice. These mice and their normal littermates (NLMs) were given daily injections of PTH (80 μg/kg) or vehicle for 2 weeks after which their tibias were examined for fat‐free weight (FFW), bone mineral content, bone structure, and bone formation rate (BFR), and their femurs were assessed for mRNA levels of osteoblast differentiation markers. In wild‐type mice, PTH increased FFW, periosteal BFR, and cortical thickness (C.Th) of the proximal tibia while reducing trabecular bone volume (BV); these responses were not seen in the k/o mice. The k/o mice had normal mRNA levels of the PTH receptor and increased mRNA levels of the IGF‐I receptor but markedly reduced basal mRNA levels of the osteoblast markers. Surprisingly, these mRNAs in the k/o bones increased several‐fold more in response to PTH than the mRNAs in the bones from their wild‐type littermates. These results indicate that IGF‐I is required for the anabolic actions of PTH on bone formation, but the defect lies distal to the initial response of the osteoblast to PTH.
1α,25‐Dihydroxyvitamin D3 Promotes Vascularization of the Chondro‐osseous Junction by Stimulating Expression of Vascular Endothelial Growth Factor and Matrix Metalloproteinase 9
Authors: Lin Roberto , Amizuka Norio , Sasaki Tomoyo , Aarts Michelle M. , Ozawa Hideharo , Goltzman David , Henderson Janet E. , White John H.
Published: April 2006

Vitamin D deficiency results in defects in endochondral bone development characteristic of rickets, which include elongation of the cartilaginous growth plates and disorganization of the primary spongiosa. These defects are caused in part by impaired cartilage mineralization and vascularization of the chondro‐osseous junction. Blood vessel invasion of mineralized cartilage is an essential step in endochondral ossification, providing access for cells that degrade cartilage as well as those that form bone. Vascular endothelial growth factor (VEGF) was shown to be a key regulator of this process when infusion of a dominant negative VEGF receptor effectively blocked vascular invasion and endochondral ossification in the growth plates of juvenile mice. Here, we show that the active metabolite of vitamin D 1α,25‐dihydroxyvitamin D3 [1α,25(OH)2D3] directly stimulates transcription of mRNAs encoding VEGF121 and −165 isoforms in the CFK2 chondrogenic cell line. Enhanced VEGF expression also was evident in growth plate chondrocytes and osteoblasts in the tibia of juvenile mice treated systemically with 1α,25(OH)2D3. This was seen in conjunction with enhanced expression of matrix metalloproteinase (MMP) 9, which activates VEGF stored in the cartilage matrix, in osteoclastic cells adjacent to the chondro‐osseous junction. The alterations in VEGF and MMP‐9 expression were accompanied by enhanced vascular invasion of mineralized cartilage, as assessed by CD31 immunoreactivity. These results provide evidence that 1α,25(OH)2D3 signaling stimulates VEGF and MMP‐9 gene expression and promotes neovascularization of the epiphyseal growth plate in vivo through increased availability of active growth factor.
Mechanical Properties of Adult Vertebral Cancellous Bone: Correlation With Collagen Intermolecular Cross‐Links
Authors: Banse X. , Sims T. J. , Bailey A. J.
Published: April 2006

Although the mechanical strength of cancellous bone is well known to depend on its apparent density, little is known about the influence of other structural or biochemical parameters. This study specifically investigates the cross‐linking of the collagen in human vertebral bone samples and its potential influence on their mechanical behavior. Multiple cylindrical samples were cored vertically in the vertebral bodies of nine subjects (aged 44–88 years). Three spinal levels (T9, T12 or L1, and L4) and three sample sites within a vertebral body (anterior, posterior, and lateral) were used, for a total of 68 samples. The density was measured with peripheral quantitative computed tomography (pQCT) and all cylinders were mechanically tested in compression. After mechanical testing, they were unmounted and used for biochemical analysis. The amount of collagen (wt/wt of bone) and its content in reduced immature cross‐links, that is, hydroxylysinonorleucine (HLNL, mol/mol of collagen) and dihydroxylysinornorleucine (DHLNL), as well as stable mature cross‐links, that is, hydroxylysyl‐pyridinoline (HP), lysyl‐pyridinoline (LP), and pyrrole cross‐link were determined for each cylinder. None of the biochemical parameters correlated to the density. On multiple linear regression, the prediction of the mechanical properties was improved by combining density data with direct collagen cross‐link assessment. The HP/LP ratio appeared as a significant predictor to the strength (r = 0.40; p = 0.001) and stiffness (r = 0.47; p < 0.001) samples with a high HP/LP ratio being stronger and stiffer. Additionally, the ultimate strain correlated to the HP or LP concentration (r = 0.38 or 0.49; p < 0.01). Different subjects had different HP/LP ratios and different HP or LP concentrations in their vertebral bone samples, and the location of origin within a subject had no influence on the concentration. These observations suggest that the nature of the organic matrix in adult vertebral bone is variable and that these variations influence its mechanical competence.
Regional Alterations of Type I Collagen in Rat Tibia Induced by Skeletal Unloading
Authors: Shiiba Masashi , Arnaud Sara B. , Tanzawa Hideki , Kitamura Eiji , Yamauchi Mitsuo
Published: April 2006

Skeletal unloading induces loss of mineral density in weight‐bearing bones that leads to inferior bone mechanical strength. This appears to be caused by a failure of bone formation; however, its mechanisms still are not well understood. The objective of this study was to characterize collagen, the predominant matrix protein in bone, in various regions of tibia of rats that were subjected to skeletal unloading by 4 weeks tail suspension. Sixteen male Sprague‐Dawley rats (4 months old) were divided into tail suspension and ambulatory controls (eight rats each). After the tail suspension, tibias from each animal were collected and divided into five regions and collagen was analyzed. The collagen cross‐linking and the extent of lysine (Lys) hydroxylation in unloaded bones were significantly altered in proximal epiphysis, diaphysis, and, in particular, proximal metaphysis but not in distal regions. The pool of immature/nonmineralized collagen measured by its extractability with a chaotropic solvent was significantly increased in proximal metaphysis. These results suggest that skeletal unloading induced an accumulation of post‐translationally altered nonmineralized collagen and that these changes are bone region specific. These alterations might be caused by impaired osteoblastic function/differentiation resulting in a mineralization defect.
The Effect of In Vivo Mechanical Loading on Estrogen Receptor α Expression in Rat Ulnar Osteocytes
Authors: Ehrlich P. J. , Noble B. S. , Jessop H. L. , Stevens H. Y. , Mosley J. R. , Lanyon L. E.
Published: April 2006

The presence of estrogen receptor α (ERα) in osteocytes was identified immunocytochemically in transverse sections from 560 to 860 μm distal to the midshaft of normal neonatal and adult male and female rat ulnas (n = 3 of each) and from adult male rat ulnas that had been exposed to 10 days of in vivo daily 10‐minute periods of cyclic loading producing peak strains of either −3000 (n = 3) or −4000 microstrain (n = 5). Each animal ambulated normally between loading periods, and its contralateral ulna was used as a control. In animals in which limbs were subject to normal locomotor loading alone, 14 ±1.2% SEM of all osteocytes in each bone section were ERα positive. There was no influence of either gender (p = 0.725) or age (p = 0.577) and no interaction between them (p = 0.658). In bones in which normal locomotion was supplemented by short periods of artificial loading, fewer osteocytes expressed ERα (7.5 ± 0.91% SEM) than in contralateral control limbs, which received locomotor loading alone (14 ± 1.68% SEM; p = 0.01; median difference, 6.43; 95% CI, 2.60, 10.25). The distribution of osteocytes expressing ERα was uniform across all sections and thus did not reflect local peak strain magnitude. This suggests that osteocytes respond to strain as a population, rather than as individual strain‐responsive cells. These data are consistent with the hypothesis that ERα is involved in bone cells' responses to mechanical strain. High strains appear to decrease ERα expression. In osteoporotic bone, the high strains assumed to accompany postmenopausal bone loss may reduce ERα levels and therefore impair the capacity for appropriate adaptive remodeling.
Transcription From the Tartrate‐resistant Acid Phosphatase Promoter Is Negatively Regulated by the Myc Oncoprotein
Authors: Daumer Kathleen M. , Taparowsky Elizabeth J. , Hall David J. , Steinbeck Marla J.
Published: April 2006

TRAP, a characteristic marker of osteoclast differentiation, is an enzyme that plays an active role in the process of bone resorption. Despite the importance of TRAP in osteoclast biology, the components involved in the transcriptional regulation of this gene are largely unknown. This study investigated the regulation of TRAP transcription by the Myc oncoprotein in three different cell types. A series of nested TRAP promoter deletion constructs were cotransfected into P388D1 murine macrophages and C3H10T1/2 murine embryonic fibroblasts along with a backbone plasmid control or expression plasmids containing v‐Myc, c‐Myc, or an inactive v‐Myc protein construct (Δ84/NLS). Both v‐Myc and c‐Myc negatively regulated transcription from the TRAP promoter in P388D1 and C3H10T1/2 cells, 90% and 50%, respective to cell type and amount of endogenous Myc protein, and Δ84/NLS had no effect. The functional Myc‐responsive element(s) within the TRAP promoter was localized to a region between −436 and +1 bp, which contains two putative Myc‐inhibitory binding sites coincident with an initiator element (Inr) at −116 bp and −18 bp. Conversely, in the HD‐11EM chicken v‐Myc transformed preosteoclast cell line, the full‐length TRAP promoter transcription was increased when endogenous v‐Myc levels were decreased in response to pretreatment of these cells with 1α,25‐dihydroxyvitamin D3 [1α,25(OH)2D3]. This report provides the first evidence of the specific regulation of TRAP at the transcriptional level by Myc, a transcription factor that is normally expressed at relatively high levels in preosteoclasts and other myelomonocytic cells and suggests that Myc plays an active role in suppressing the transcription of a mature osteoclast selective gene.
Transforming Growth Factor‐β1 Negatively Regulates Crystallin Expression in Teeth
Authors: Thyagarajan Tamizchelvi , Kulkarni Ashok B.
Published: April 2006

Previously, we have reported that targeted overexpression of transforming growth factor (TGF) β1 in the teeth of the transgenic mice (dTGF‐β1) results in a novel tooth phenotype phenomimicking the most prevalent tooth disorders in human. This phenotype was associated with discoloration and attrition of teeth due to defective mineralization. Here, we report a novel expression of crystallin family members in developing mouse teeth and its regulation by TGF‐β1 in these transgenic mice. αB‐ and β‐crystallins were found to be elevated in dTGF‐β1 mouse teeth, whereas γ‐crystallin (γB, γC, and γF), a marker of cell differentiation, was significantly reduced. Because crystallins are believed to be stress‐related proteins, their expression in teeth implicates them in a similar role because teeth are constantly subjected to physical friction and temperature fluctuations.
Genome Screen for Quantitative Trait Loci Contributing to Normal Variation in Bone Mineral Density: The Framingham Study
Authors: Karasik D. , Myers R. H. , Cupples L. A. , Hannan M. T. , Gagnon D. R. , Herbert A. , Kiel D. P.
Published: April 2006

A genome‐wide scan was performed in a randomly ascertained set of 330 extended families from the population‐based Framingham Study to identify chromosomal regions possibly linked to bone mineral density (BMD). A set of 401 microsatellite markers was typed at a 10‐centimorgan (cM) average density throughout the genome. BMD was measured at the femoral neck, trochanter, Ward's area, and lumbar spine in 1557 participants of both Framingham cohorts. BMDs were adjusted for age, body mass index (BMI), height, alcohol, caffeine, calcium and vitamin D intakes, smoking, physical activity, and estrogen use in women within each sex and cohort. Strong heritabilities (values between 0.543 and 0.633) were found for the adjusted BMD at all sites. Two‐point and multipoint quantitative linkage analyses were performed for each BMD site using the maximum likelihood variance components method. By two‐point screening, loci of suggestive linkage were identified on chromosomes 6 and 21, with the maximum log10 of the odds ratio (LOD) scores of 2.34 for the trochanter at D21S1446 and 2.93 for the femoral neck at D6S2427. Lumbar spine BMD had maxima at D6S2427 (LOD = 1.88) and at D12S395 (LOD = 2.08). Multipoint linkage analysis revealed suggestive linkage of trochanteric BMD at a broad (∼20 cM) interval on chromosome 21q, with the peak linkage close to D21S1446 (LOD = 3.14). LOD scores were 2.13 at 8q24 with Ward's BMD and 1.92 at 14q21.3 with lumbar spine BMD. This largest genome screen to date for genes underlying normal variation in BMD, adjusted for a large number of covariates, will help to identify new positional candidate genes, otherwise unrecognized.
Disorders Associated With Acute Rapid and Severe Bone Loss
Authors: Epstein Solomon , Inzerillo Angela M , Caminis John , Zaidi Mone
Published: April 2006

We describe a constellation of bone diseases characterized by the common feature of acute, rapid, and severe bone loss accompanied by dramatic fracture rates. These disorders are poorly recognized, resulting mainly from systemic diseases, frailty, immobilization, and immunosuppressive drugs, such as glucocorticoids and the calcineurin inhibitors. The opportunity to prevent or treat fractures is commonly missed because they are often not detected. Ideally, patients need to be identified early and preventative therapy initiated promptly to avoid the rapid bone loss and fractures. The most effective therapy at present seems to be the bisphosphonates, particularly when bone resorption is predominant. However, more severe forms of bone loss that result from an osteoblastic defect and reduced bone formation may benefit potentially more from newer anabolic agents, such as recombinant human parathyroid hormone (rhPTH).
Trabecular Bone Response to Mechanical and Parathyroid Hormone Stimulation: The Role of Mechanical Microenvironment
Authors: Kim Chi Hyun , Takai Erica , Zhou Hua , Von Stechow Dietrich , Müller Ralph , Dempster David W , Guo X Edward
Published: April 2006

Bone response under combined mechanical and PTH stimuli is important in osteoporosis. A rat tail animal model with computer modeling was used to examine bone response to loading and PTH. PTH enhances and sustains increased bone formation rate, which directly correlates to mechanical microenvironment, suggesting beneficial effects of combined PTH treatment and exercise in preventing osteoporosis.

Introduction: Using an in vivo rat tail vertebra model combined with a specimen-specific, high-resolution microcomputed tomography (μCT)-based finite element analysis (FEA) technique, trabecular bone response to combined dynamic compressive loading and parathyroid hormone (PTH) stimulation was characterized.

Materials and Methods: Two hundred twenty-four male Sprague-Dawley rats were randomly divided into seven treatment groups: (1) Control, (2) vehicle + 0N, (3) PTH + 0N, (4) vehicle + 50N, (5) PTH + 50N, (6) vehicle + 100N, and (7) PTH + 100N, with three treatment durations (1, 2, or 4 weeks). Rat PTH(1–34) was administered daily in the PTH-stimulated groups approximately 3 h before daily mechanical stimulation with 0, 50, or 100N dynamic compressive loading. μCT-based FEA was performed for each loaded vertebra after death. Bone histomorphometry was performed on trabecular bone with double fluorochrome labeling to assess bone formation.

Results: Daily mechanical loading or PTH administration significantly increased bone formation rate (BFR) compared with control or V + 0N with significant increases in both mineral apposition rate (MAR) and labeled bone surface (LS/BS). PTH, when combined with mechanical loading, enhanced BFR mainly through a significant increase in MAR after the first week and through a significant increase in LS/BS after 2 and 4 weeks. Synergistic effects in BFR were present when PTH was combined with mechanical loading, especially after 2 and 4 weeks, where the increase in BFR was sustained. However, when either PTH or mechanical loading was the only stimulus, the bone formation response diminished to the level of Control animals after 4 weeks. Furthermore, significant correlations were observed between the bone formation indices and trabecular bone tissue mechanical microenvironments at 1 and 2 weeks, with PTH administration enhancing and sustaining these correlations into 4 weeks.

Conclusions: The synergistic effects of combined PTH and mechanical stimulation on trabecular bone formation rate suggest a potential benefit for combined PTH administration and exercise in the treatment of osteoporosis.
Overexpression of Nell-1, a Craniosynostosis-Associated Gene, Induces Apoptosis in Osteoblasts During Craniofacial Development
Authors: Zhang Xinli , Carpenter Dale , Bokui Nobuyuki , Soo Chia , Miao Steve , Truong Thien , WU Benjamin , Chen Ingrid , Vastardis Heleni , Tanizawa Katsuyuki , Kuroda Shun″Ichi , Ting Kang
Published: April 2006

We studied the cellular function of Nell-1, a craniosynostosis-related gene, in craniofacial development. Nell-1 modulates calvarial osteoblast differentiation and apoptosis pathways. Nell-1 overexpression disrupts these pathways resulting in craniofacial anomalies such as premature suture closure.

Introduction: Craniosynostosis (CS), one of the most common congenital craniofacial deformities, is the premature closure of cranial sutures. Previously, we reported NELL-1 as a novel molecule overexpressed during premature cranial suture closure in patients with CS. Nell-1 overexpression induced calvarial overgrowth and resulted in premature suture closure in a rodent model. On a cellular level, Nell-1 is suggested to promote osteoblast differentiation.

Materials and Methods: Different levels of Nell-1 were introduced into osteoblastic cells by viral infection and recombinant protein. Apoptosis and gene expression assays were performed. Mice overexpressing Nell-1 were examined for apoptosis.

Results: In this report, we further showed that overexpression of Nell-1 induced apoptosis along with modulation of apoptosis-related genes. The induction of apoptosis by Nell-1 was observed only in osteoblastic cells and not in NIH3T3 or primary fibroblasts. The CS mouse model overexpressing Nell-1 showed increased levels of apoptosis in the calvaria.

Conclusion: We show that Nell-1 expression modulates calvarial osteoblast differentiation and apoptosis pathways. Nell-1 overexpression disrupts these pathways resulting in craniofacial anomalies such as premature suture closure.
Functional Characterization of Two Naturally Occurring Mutations in the Human Sodium-Phosphate Cotransporter Type IIa
Authors: Virkki Leila V , Forster Ian C , Hernando Nati , Biber JÜRg , Murer Heini
Published: April 2006

Mutations in the gene encoding the human sodium-phosphate cotransporter (NPT2), causing reduced phosphate affinity and dominant-negative behavior, were described.(1) We found no evidence of altered kinetics or dominant-negative effects. Thus, the mutations cannot account for the clinical phenotype.

Introduction: Mutations in NPT2a, the gene encoding the sodium-phosphate cotransporter NaPi-IIa, were for the first time linked to human disease by Priè and colleagues. Two patients are described with renal phosphate wasting who were heterozygous for either the A48F or V147M mutation. Expressed in Xenopus oocytes, both mutants showed reduced phosphate affinity. Furthermore, coexpression of mutants with wildtype (WT) NaPi-IIa resulted in reduced cotransport function, explaining the mutants' dominant-negative effect in the patients. Intrigued by the implications of these findings on transporter kinetics, we decided to examine the transport characteristics of the two mutants in more detail.

Materials and Methods: We recreated the two mutants, expressed them in Xenopus oocytes, and analyzed their kinetic behavior by two-electrode voltage clamp. We also performed coexpression experiments where we injected mRNA for WT and mutants containing an additional S462C mutation, enabling complete inhibition of cotransport function with cysteine-modifying reagents. Finally, we expressed WT and mutant NaPi-IIa as C-terminal fusions to green fluorescent protein (GFP) in opossum kidney (OK) cells.

Results and Conclusions: We found in our oocyte expression experiments that Pi-induced currents were reduced in both mutants, whereas Pi and Na affinities and other transport characteristics were not affected. The amount of cotransport activity remaining after cysteine modification, corresponding to WT activity, was not affected by coexpression of either mutant. Finally, GFP-tagged WT and mutants were expressed at the apical membrane in OK cells, showing that both mutants are correctly targeted in a mammalian cell. In conclusion, our data from oocyte and OK cell expression studies suggest that the heterozygous A48F and V147M mutations cannot explain the pathological phenotype observed by Priè and colleagues.
Deletion Mutants of BMP Folding Variants Act as BMP Antagonists and Are Efficient Inhibitors for Heterotopic Ossification
Authors: Weber Franz E , Schmökel Hugo , Oelgeschläger Michael , Nickel Joachim , Maly Friedrich E , Hortschansky Peter , Grätz Klaus W
Published: April 2006

Heterotopic ossification is a frequent complication in patients who have suffered head and neck traumas or have undergone total hip replacement. In this report, stable folding variants of the natural occurring osteoinductive BMPs were shown to act as inhibitors for heterotopic ossification. The most effective BMP folding variant construct performed even better than the natural occurring BMP antagonist Noggin because it also inhibited calcium deposition of pre-osteoblastic cells.

Introduction: Signal transduction through receptor and ligand binding depends on the proper folding of all partners, especially when it involves the formation of a heterotetramer. In the case, the receptor binding of the ligand can be uncoupled from signal transduction, and folding variants of a ligand can be developed into antagonists of the natural bioactivity of the ligand. Here we present a deletion mutant of a bone morphogenetic protein (BMP) folding variant capable of inhibiting the bone-inducing action of natural occurring BMPs.

Materials and Methods: Deletion mutants and site-directed mutants of BMP folding variants were generated and tested for their ability to reduce alkaline phosphatase activity and mineralization in a pre-osteoblastic cell line. In vivo activity of the optimized folding variant was determined in a heterotopic ossification model in rodents and in two Xenopus laevis model systems. Biosensor interaction analysis was used to determine the affinity of the optimized BMP folding variant to the extracellular domain of BMP receptors.

Results: In vitro and in vivo tests in rodents revealed that the structural elements of the wrist epitope combined with finger 2 and a positive charge proximal to the tip of this finger are sufficient to induce osteoinhibition with deletion mutants and folding variants of mature BMP-4. The inhibitor designed to suppress heterotopic ossification showed BMP antagonist activity in embryos and animal caps of X. laevis. Binding studies of the inhibitor to ectodomains of type I and type II BMP receptors revealed a concentration-dependent binding, especially to the high-affinity BMP receptor.

Conclusions: Deletion mutants of BMP folding variants are a new form of BMP antagonists and act through competition with osteoinductive BMP for BMP receptor binding. The excellent in vivo performance of the optimized folding variant is because of its ability to block signaling of endogenous BMPs deposited in the extracellular matrix even more effectively than the natural occurring BMP antagonist Noggin.
Role of Gastrointestinal Hormones in Postprandial Reduction of Bone Resorption
Authors: Henriksen Dennis B , Alexandersen Peter , Bjarnason Nina H , Vilsbøll Tina , Hartmann Bolette , Henriksen Eva EG , Byrjalsen Inger , Krarup Thure , Holst Jens J , Christiansen Claus
Published: April 2006

Collagen type I fragments, reflecting bone resorption, and release of gut hormones were investigated after a meal. Investigations led to a dose escalation study with glucagon like peptide-2 (GLP-2) in postmenopausal women. We found a dose-dependent effect of GLP-2 on the reduction of bone resorption.

Introduction: The C-terminal telopeptide region of type I collagen as measured in serum (s-CTX) can be used to assess bone resorption. This marker of bone resorption has a significant circadian variation that is influenced by food intake. However, the mediator of this variation has not been identified.

Materials and Methods: We studied the release of the gut hormones glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-2 (GLP-2; a representative of the intestinal proglucagon-derived peptides) after ingestion of glucose, fat, protein, and fructose, as well as their effects after parenteral administration in relation to bone turnover processes in healthy volunteers. Furthermore, we studied the effect on bone turnover of a single subcutaneous injection of GLP-2 in four different dosages (100, 200, 400, or 800 μg GLP-2) or placebo in 60 postmenopausal women (mean age, 61 ± 5 years).

Results: All macronutrients significantly (p < 0.05) reduced bone resorption as assessed by s-CTX (39–52% from baseline), and only the glucagon-like peptides were secreted in parallel. Parenteral administration of GIP and GLP-1 did not result in a reduction of the s-CTX level, whereas GLP-2 caused a statistically significant and dose-dependent reduction in the s-CTX level from baseline compared with placebo (p < 0.05). Urine DPD/creatinine, a marker of bone resorption, was significantly reduced by 25% from baseline in the 800-μg GLP-2 group (p < 0.01). An area under the curve (AUC0–8h) analysis for s-CTX after GLP-2 injection confirmed the dose-dependent decrease (ANOVA, p = 0.05). The s-osteocalcin level was unaffected by the GLP-2 treatment.

Conclusion: These studies exclude both GIP and GLP-1 as key mediators for the immediate reduction in bone resorption seen after a meal. The dose-dependent reduction of bone resorption markers found after subcutaneous injection of GLP-2 warrants further investigation into the mechanism and importance of GLP-2 for the bone turnover processes.