bluuebunny/crossref_metadata_2025 huggingface dataset:
The Nucleolar Targeting Signal (NTS) of Parathyroid Hormone Related Protein Mediates Endocytosis and Nucleolar Translocation
Authors: Aarts M. M. , Rix A. , Guo J. , Bringhurst R. , Henderson J. E.
Published: April 2006

Previous work has identified the parathyroid hormone–related protein (PTHrP) nucleolar targeting signal (NTS) as both necessary and sufficient for localization of PTHrP to the nucleus and nucleolus of a variety of cells where it is believed to participate in the regulation of cell proliferation, differentiation, and apoptotic cell death. The mechanism whereby a secreted peptide, such as PTHrP, gains access to the nuclear compartment remains a question of debate. The current work examines the possibility that exogenous PTHrP is internalized and transported to the nuclear compartment by a mechanism that is dependent on preservation of the PTHrP NTS. Transiently expressed, PTHrP(1–141) was detected at the cell surface as well as in the cytoplasmic and nuclear compartments of COS-1 cells. Deletion of the NTS, or mutation of the conserved GxKKxxK motif within the NTS, effectively prevented both cell-surface binding and nuclear/nucleolar accumulation of PTHrP(1–141). A biotinylated peptide corresponding to the PTHrP NTS (PTHrP-NTS-biotin) was internalized and translocated to the nucleus and nucleolus in a time-, temperature-, and concentration-dependent manner, whereas a peptide representing a similar bipartite NTS from Nucleolin was not. Internalization and nucleolar targeting of PTHrP-NTS-biotin were indistinguishable in CFK2 cells, which express the common PTH/PTHrP receptor, and in 27m21 cells, which do not. In addition, pretreatment with a saturating dose of synthetic PTHrP(74–113) was capable of abrogating nucleolar accumulation of the PTHrP-NTS peptide, whereas pretreatment with PTHrP(1–34) or PTHrP(67–86) was not. These observations demonstrate that binding of exogenous, full-length PTHrP to the cell surface is mediated through a conserved motif embedded in the NTS and suggest that internalization and nucleolar targeting of an NTS peptide are mediated through binding to a cell surface protein distinct from the PTH/PTHrP receptor. In total, the data support the hypothesis that secreted PTHrP(1–141) can be endocytosed and targeted to the nucleolus through a mechanism that is dependent on preservation of a core motif within the PTHrP NTS.
24,25-Dihydroxyvitamin D3 Suppresses the Rapid Actions of 1,25-Dihydroxyvitamin D3 and Parathyroid Hormone on Calcium Transport in Chick Intestine
Authors: Nemere Ilka
Published: April 2006

Studies were undertaken to determine whether 24,25-dihydroxyvitamin D3 (24,25(OH)2D3) modulates the rapid effects of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) and parathyroid hormone (PTH) on calcium transport in the perfused chick intestine. Perfusion with control media resulted in a transport ratio (treated/average basal) of 1.07 ± 0.06 at t = 40 minutes, while perfusion with 65, 130, 300, or 650 pM 1,25(OH)2D3 yielded ratios of 1.92 ± 0.23, 2.6 ± 0.4, 2.8 ± 0.08, and 3.34 ± 0.37, respectively. Simultaneous perfusion with each of these doses and 6.5 nM 24,25(OH)2D3 reduced treated/average basal ratios to ∼1.4 after 40 minutes of perfusion. Vascular perfusion with 65 pM bovine PTH [bPTH(1–34)] stimulated intestinal calcium transport ratios to 3.0 ± 0.5 after 40 minutes, while the inclusion of 6.5 nM 24,25(OH)2D3 reduced ratios at this time point to 0.56 ± 0.19. To investigate the effect of these agents on signal transduction, isolated intestinal cells were monitored for intracellular calcium changes using the indicator dye fura-2. After establishing a stable baseline, addition of 130 pM 1,25(OH)2D3 induced rapid calcium oscillations. Intestinal cells exposed to 6.5 nM 24,25(OH)2D3 also exhibited rapid oscillations in fluorescence, which were not further altered by subsequent addition of 1,25(OH)2D3. Incubation of isolated cells with 130 pM 1,25(OH)2D3 was found to increase protein kinase C (PKC) activity within 5 minutes, and protein kinase A (PKA) activity within 7 minutes. Exposure of cells to 65 pM bPTH(1–34) had minimal effect on PKC activity, but resulted in pronounced increases in PKA activity. Stimulation of protein kinases by either secosteroid or peptide hormone was inhibited in the presence of 6.5 nM 24,25(OH)2D3. It is concluded that 24,25(OH)2D3 may exert endocrine actions on intestine.
Expression of Thyroid Hormone Receptor Isoforms in Rat Growth Plate Cartilage In Vivo
Authors: Ballock R. Tracy , Mita Barry C. , Zhou Xiaolan , Chen Daniel H.-C. , Mink Lynn M.
Published: April 2006

Although thyroid hormone has been known for many years to be a potent regulator of skeletal maturation in vivo, it has not definitively been determined whether this effect is a result of a direct or indirect action of the hormone. Previous in vivo studies have suggested that thyroid hormone may stimulate longitudinal bone growth by increasing the secretion of growth hormone; however, growth hormone alone is unable to stimulate cartilage maturation. There are also indications that thyroid hormone is able to act directly on growth plate chondrocytes through growth hormone–independent mechanisms. In this study, we demonstrate that rat growth plate chondrocytes in vivo express genes encoding three of the four isoforms of the thyroid hormone receptors described to date, but the corresponding protein can only be detected for the TRα1 and TRβ1 isoforms of the receptor. As has been noted in other tissues, there is generally poor correlation between the mRNA levels for each isoform and the relative amount of corresponding protein as measured by immunoblotting, suggesting the possibility that receptor expression may be regulated by post-transcriptional mechanisms.
Markers of Bone Turnover Predict Postmenopausal Forearm Bone Loss Over 4 Years: The OFELY Study
Authors: Garnero Patrick , Sornay-Rendu Elisabeth , Duboeuf François , Delmas Pierre D.
Published: April 2006

The ability of biochemical markers to predict the rate of postmenopausal bone loss is still controversial. To investigate this issue further, baseline levels of a panel of specific and sensitive biochemical bone markers were correlated to the rate of change of forearm bone mineral density (BMD) assessed by four measurements over a 4-year period using dual-energy X-ray absorptiometry in a large population-based prospective cohort of 305 women aged 50–88 years (mean 64 years), 1–38 years postmenopausal. In the whole population, higher baseline levels of bone formation (serum osteocalcin and serum type I collagen N-terminal propeptide) and bone resorption markers (urinary N-telopeptides; urinary and serum C-telopeptides) were significantly associated with faster BMD loss (r = −0.19 to −0.30, p < 0.001), independently of age. In women within 5 years of menopause that have the highest rate of bone loss, the predictive value of bone markers was increased with correlation coefficients reaching 0.53. Women with an abnormally high bone turnover, i.e., with levels of bone markers at baseline 2 SD above the mean of premenopausal women, had a rate of bone loss that was 2- to 6-fold higher than women with a low turnover (p = 0.01–0.0001) according to the marker. When the population was categorized according to quartiles of bone markers at baseline, a similar relationship between increased levels of bone markers and faster rate of bone loss was found (p = 0.008–0.0001). In the logistic regression model, the odds-ratio of fast bone loss, defined as the rate of bone loss in the upper tertile of the population, was increased by 1.8- to 3.2-fold for levels of biochemical markers in the high turnover group compared with levels within the premenopausal range, with, however, a limited value for identifying individual fast bone losers. We conclude that increased levels of some of the new biochemical markers of bone turnover are associated with greater radial bone loss. Because increased bone loss is associated with an increased risk of fracture, bone turnover markers may be useful to improve the prediction of the risk of osteoporosis in postmenopausal women.
Transgenic Mice Overexpressing Tartrate-Resistant Acid Phosphatase Exhibit an Increased Rate of Bone Turnover
Authors: Angel Nicola Z. , Walsh Nicole , Forwood Mark R. , Ostrowski Michael C. , Cassady A. Ian , Hume David A.
Published: April 2006

Tartrate-resistant acid phosphatase (TRAP) is a secreted product of osteoclasts and a lysosomal hydrolase of some tissue macrophages. To determine whether TRAP expression is rate-limiting in bone resorption, we overexpressed TRAP in transgenic mice by introducing additional copies of the TRAP gene that contained the SV40 enhancer. In multiple independent mouse lines, the transgene gave a copy number–dependent increase in TRAP mRNA levels and TRAP activity in osteoclasts, macrophages, serum, and other sites of normal low-level expression (notably, liver parenchymal cells, kidney mesangial cells, and pancreatic secretory acinar cells). Transgenic mice had decreased trabecular bone consistent with mild osteoporosis. Measurements of the bone formation rate suggest that the animals compensate for the increased resorption by increasing bone synthesis, which partly ameliorates the phenotype. These mice provide evidence that inclusion of an irrelevant enhancer does not necessarily override a tissue-specific promoter.
Trabecular Bone Microarchitecture, Bone Mineral Density, and Vertebral Fractures in Male Osteoporosis
Authors: Legrand Erick , Chappard Daniel , Pascaretti Christian , Duquenne Marc , Krebs Stéphanie , Rohmer Vincent , Basle Michel-Félix , Audran Maurice
Published: April 2006

Some studies have indicated that the risk of fragility fractures in men increases as bone mineral levels decrease, but there is an overlap in the bone mineral density (BMD) measurements between patients with or without fractures. Furthermore, it has been suggested that the biomechanical competence of trabecular bone is dependent not only on the absolute amount of bone present but also on the trabecular microarchitecture. In the present study, 108 men (mean age 52.1 years) with lumbar osteopenia (T score <−2.5) were recruited to examine the relationships between BMD, architectural changes in trabecular bone, and the presence of vertebral fractures. Lumbar BMD was assessed from L2 to L4 in the anteroposterior view with dual-energy X-ray absorptiometry. At the upper left femur, hip BMD was measured at the transcervical site. Spinal X-ray films were analyzed independently by two trained investigators, and vertebral fracture was defined as a reduction of at least 20% in the anterior, middle, or posterior vertebral height. Transiliac bone biopsy specimens were obtained for all patients. Histomorphometric studies were performed on an image analyzer, and the following parameters were determined: trabecular bone volume (BV/TV), trabecular thickness (Tb.Th), number (Tb.N), and separation (Tb.Sp), interconnectivity index (ICI), characterization of the trabecular network (node count and strut analysis), and star volume of the marrow spaces. Spinal radiographs evidenced at least one vertebral crush fracture in 62 patients (group II) and none in 46 patients (group I). After adjusting for age, body mass index, and BMD, there were no significant differences between the two groups in BV/TV, Tb.Th, or star volume. In contrast, the mean values of ICI, free end–to–free end struts (FF/TSL), and Tb.Sp were significantly higher, whereas Tb.N and node-to-node struts (NN/TSL) were lower in patients with at least one vertebral fracture. Logistic regression analysis showed that only ICI, FF/TSL, NN/TSL, and Tb.N were significant predictors of the presence of vertebral fracture: odds ratios for an alteration of 1 SD ranged from 1.7 (1.0–3.2) for NN/TSL to 3.2 (1.1–10.1) for ICI. Patients with at least three vertebral fractures (n = 23) were categorized as “multiple fractures.” The results of logistic regression showed that spine BMD, BV/TV, and all architectural parameters were significant predictors of multiple vertebral fractures: odds ratios for an alteration of 1 SD ranged from 2.2 (1.1–4.6) for star volume to 3.7 (1.4–9.7) for ICI. These results strongly suggest that bone trabecular microarchitecture is a major and independent determinant of vertebral fractures in middle-aged men with osteopenia.
Analysis of Ovariectomy and Estrogen Effects on Body Composition in Rats by X-Ray and Magnetic Resonance Imaging Techniques
Authors: Sharp J. C. , Copps J. C. , Liu Q. , Ryner L. N. , Sebastian R. A. , Zeng G. Q. , Smith S. , Niere J. O. , Tomanek B. , Sato M.
Published: April 2006

Resistance of bone to fracture—bone strength—has been shown to depend on both the amount of bone and its architectural spatial organization. In vivo magnetic resonance (MR) techniques have the capability of imaging bone tissue, including the trabecular microarchitecture and the marrow composition. We have applied in vivo and ex vivo MR methods to the tibia in an ovariectomized rat model of osteoporosis. Specifically, in vivo high-resolution three-dimensional MR imaging and localized MRS were facilitated by specialized coils and high field magnets, resulting in enhanced sensitivity of detection. As a result, in vivo and ex vivo differences in marrow composition were found between sham-ovariectomized, ovariectomized, and ovariectomized animals treated with 17-β-estradiol. Estrogen effects were detected in vivo 7 days after surgery (3 days into treatment) as a decrease in the tibial fat signal level. The in vivo effects of ovariectomy were observed 56 days after surgery as an increase in MR image fat signal level and spectral fat/water ratio in the proximal tibia. Ex vivo measurements of tibial marrow water signal discriminated clearly between the sham and ovariectomized groups and showed increased individual variations in the treatment group. Imaging further showed that the highest fat content is observed in the epiphysis. Computed tomography confirmed ovariectomy-induced loss of bone in the proximal tibial metaphysis compared with the sham group. This loss of cancellous bone with ovariectomy is consistent with the MR observations of increases in both fat and water in the metaphysis. These data showed that MR techniques complement X-ray techniques in the bone, water, and fat compositional analysis of the appendicular skeleton in response to ovariectomy and pharmacological treatment.
Effect of First Treatment with Aminobisphosphonates Pamidronate and Ibandronate on Circulating Lymphocyte Subpopulations
Authors: Pecherstorfer Martin , Jilch Ruth , Sauty Alain , Horn Eva , Keck Andrea Vera , Zimmer-Roth Ilse , Thiebaud Daniel
Published: April 2006

Up to 60% of patients receiving their first infusion of the bisphosphonate pamidronate experience an acute-phase reaction. In this study, we used flow cytometry to determine the effects of pamidronate treatment on circulating lymphocyte subpopulations, and we investigated whether pamidronate and ibandronate treatment affect lymphocyte subpopulations differently. Twenty patients received a pamidronate infusion, 20 patients received intravenously injected ibandronate, and 10 controls received a clodronate infusion. Pamidronate treatment was followed by a significant increase in median body temperature at the 10-hour measurement and a significant decrease in counts of circulating lymphocytes, natural killer cells, T cells, and CD4+ and CD8+ T-cell subsets. Ibandronate treatment did not affect median body temperature, and it was associated at the 10-hour measurement with maximum increases in total lymphocyte count, B cells, T cells, and CD4+ and CD8+ T-cell subsets. Thus, there is a substantial difference in the hematologic response to initial treatments with pamidronate and ibandronate. Clodronate treatment did not induce changes in body temperature or significantly affect the number of circulating T cells and NK cells. The reduction in lymphocyte subsets after initial pamidronate therapy might be mediated by the release of tumor necrosis factor α, whose source in the acute-phase reaction could be T cells.
Expression of FGFR3 with the G380R Achondroplasia Mutation Inhibits Proliferation and Maturation of CFK2 Chondrocytic Cells
Authors: Henderson Janet E. , Naski Michael C. , Aarts Michelle M. , Wang Dashen , Cheng Li , Goltzman David , Ornitz David M.
Published: April 2006

A G380R substitution in the transmembrane-spanning region of FGFR3 (FGFR3Ach) results in constitutive receptor kinase activity and is the most common cause of achondroplastic dwarfism in humans. The epiphyseal growth plates of affected individuals are disorganized and hypocellular and show aberrant chondrocyte maturation. To examine the molecular basis of these abnormalities, we used a chondrocytic cell line, CFK2, to stably express the b variant of wild-type FGFR3 or the the constitutively active FGFR3Ach. Overexpression of FGFR3 had minimal effects on CFK2 proliferation and maturation compared with the severe growth retardation found in cells expressing FGFR3Ach. Cells expressing the mutant receptor also showed an abnormal apoptotic response to serum deprivation and failed to undergo differentiation under appropriate culture conditions. These changes were associated with altered expression of integrin subunits, which effectively led to a switch in substrate preference of the immature cell from fibronectin to type II collagen. These in vitro observations support those from in vivo studies indicating that FGFR3 mediates an inhibitory influence on chondrocyte proliferation. We now suggest that the mechanism is related to altered integrin expression.
The Roles of Osteoprotegerin and Osteoprotegerin Ligand in the Paracrine Regulation of Bone Resorption
Authors: Hofbauer Lorenz C. , Khosla Sundeep , Dunstan Colin R. , Lacey David L. , Boyle William J. , Riggs B. Lawrence
Published: April 2006

Although multiple hormones and cytokines regulate various aspects of osteoclast formation, the final two effectors are osteoprotegerin ligand (OPG-L)/osteoclast differentiation factor (ODF), a recently cloned member of the tumor necrosis factor superfamily, and macrophage colony–stimulating factor. OPG-L/ODF is produced by osteoblast lineage cells and exerts its biological effects through binding to its receptor, osteoclast differentiation and activation receptor (ODAR)/receptor activator of NF-κB (RANK), on osteoclast lineage cells, in either a soluble or a membrane-bound form, the latter of which requires cell-to-cell contact. Binding results in rapid differentiation of osteoclast precursors in bone marrow to mature osteoclasts and, at higher concentrations, in increased functional activity and reduced apoptosis of mature osteoclasts. The biological activity of OPG-L/ODF is neutralized by binding to osteoprotegerin (OPG)/osteoclastogenesis inhibitory factor (OCIF), a member of the TNF-receptor superfamily that also is secreted by osteoblast lineage cells. The biological importance of this system is underscored by the induction in mice of severe osteoporosis by targeted ablation of OPG/OCIF and by the induction of osteopetrosis by targeted ablation of OPG-L/ODF or overexpression of OPG/OCIF. Thus, osteoclast formation may be determined principally by the relative ratio of OPG-L/ODF to OPG/OCIF in the bone marrow microenvironment, and alterations in this ratio may be a major cause of bone loss in many metabolic disorders, including estrogen deficiency and glucocorticoid excess. That changes in but two downstream cytokines mediate the effects of large numbers of upstream hormones and cytokines suggests a regulatory mechanism for osteoclastogenesis of great efficiency and elegance.
Determinants of Bone Loss from the Femoral Neck in Women of Different Ages
Authors: Melton L. Joseph , Atkinson Elizabeth J. , O'Connor Michael K. , O'Fallon W. Michael , Riggs B. Lawrence
Published: April 2006

An age-stratified sample of 304 women from Rochester, Minnesota, aged 30–94 years (median 60 years) at baseline underwent measurement of femoral neck bone mineral density (BMD) over a follow-up period extending to 16 years. The average rate of change in femoral neck BMD was −1.0% per year (range −10.0% to +13.4%) and did not vary significantly with age. Because there was no marked increase in the rate of loss around the time of menopause, nor convincing evidence of there being a subset of fast losers, there was fairly good tracking of individual values over time; the correlation of baseline with femoral neck BMD values 16 years later was 0.83. Although a large number of potential determinants was assessed, the only consistent predictor of femoral neck bone loss in women of different ages was baseline femoral neck BMD (r = −0.15; p = 0.023). Otherwise, different sets of risk factors were identified for premenopausal women, women within 20 years of menopause, and women 20 years or more postmenopausal, but the predictive power of these different multivariate models was modest. Nonetheless, these data indicate that femoral neck BMD is quite predictable for extended periods of time. This is reassuring with respect to the use of statistical models that incorporate such data to estimate future fracture risk.
In Vitro and In Vivo Suppression of Osteoclast Function by Adenovirus Vector–Induced csk Gene
Authors: Miyazaki Tsuyoshi , Takayanagi Hiroshi , Isshiki Masashi , Takahashi Tokiharu , Okada Masato , Fukui Yasuhisa , Oda Hiromi , Nakamura Kozo , Hirai Hisamaru , Kurokawa Takahide , Tanaka Sakae
Published: April 2006

The proto-oncogene c-src, which encodes a non–receptor-type tyrosine kinase c-Src, has been shown to be essential for osteoclastic bone resorption by the finding that the targeted disruption of the c-src gene induced osteopetrosis in mice. The csk (C-terminal Src family kinase) gene encodes a cytoplasmic protein-tyrosine kinase that specifically phosphorylates the negative regulatory site of c-Src (Tyr-527), thereby inhibiting its kinase activity. To regulate osteoclast function by modulating the kinase activity of c-Src, we constructed an adenovirus vector that carries this gene. The recombinant adenovirus vector carrying csk cDNA induced Csk expression in mouse osteoclast-like cells formed in vitro and clearly reduced c-Src kinase activity in a dose-dependent manner. The expression of Csk caused cytoskeletal disorganization of osteoclast-like cells and strongly suppressed pit-forming activity of the cells in vitro. In addition, the viral vector carrying csk gene dramatically suppressed interleukin-1α–induced bone resorption in vivo. Conversely, kinase-inactive Csk caused an increase in c-Src kinase activity and bone resorbing activity of the cells both in vitro and in vivo, acting as a dominant negative molecule against intrinsic Csk. These findings indicate that the inhibition of c-Src activity by adenovirus vector-mediated csk expression offers an efficient means for inhibiting pathological bone resorption by suppressing osteoclast function.
Loss of Osteocyte Integrity in Association with Microdamage and Bone Remodeling After Fatigue In Vivo
Authors: Verborgt Olivier , Gibson Gary J. , Schaffler Mitchell B.
Published: April 2006

As a result of fatigue, bone sustains microdamage, which is then repaired by bone-remodeling processes. How osteoclastic activity is targeted at the removal of microdamaged regions of bone matrix is unknown. In the current studies, we tested the hypothesis that changes in osteocyte integrity, through the initiation of regulated cell death (apoptosis), are associated with fatigue-related microdamage and bone resorption. Ulnae of adult rats were fatigue-loaded to produce a known degree of matrix damage. Osteocyte integrity was then assessed histomorphometrically from terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate–nick end labeling (TUNEL)–stained sections to detect cells undergoing DNA fragmentation associated with apoptosis; toluidine blue–stained sections were used for secondary morphological confirmation. Ten days after loading, large numbers of TUNEL-positive osteocytes were found in bone surrounding microcracks and in bone surrounding intracortical resorption spaces (∼300% increases over controls, p < 0.005). TUNEL labeling in loaded ulnae at sites distant from microcracks or resorption foci did not differ from that in control bone. Osteocytes in toluidine blue–stained sections showed equivalent trends to TUNEL-stained sections, with significant increases in pyknotic nuclei and empty lacunae associated with microcracks and intracortical resorption spaces. TUNEL-positive osteocytes were observed around bone microdamage by 1 day after loading (p < 0.01 relative to baseline), and their number remained elevated throughout the entire experimental period. Increases in empty lacunae and decreases in normal osteocyte numbers were observed over time as well. These studies show that (1) osteocyte apoptosis is induced by bone fatigue, (2) this apoptosis is localized to regions of bone that contain microcracks, and (3) osteoclastic resorption after fatigue also coincides with regions of osteocyte apoptosis. The strong associations between microdamage, osteocyte apoptosis, and subsequent bone remodeling support the hypothesis that osteocyte apoptosis provides a key part of the activation or signaling mechanisms by which osteoclasts target bone for removal after fatigue-induced matrix injury.
Role of the Latent Transforming Growth Factor β–Binding Protein 1 in Fibrillin-Containing Microfibrils in Bone Cells In Vitro and In Vivo
Authors: Dallas Sarah L. , Keene Douglas R. , Bruder Scott P. , Saharinen Juha , Sakai Lynn Y. , Mundy Gregory R. , Bonewald Lynda F.
Published: April 2006

Latent transforming growth factor β–binding proteins (LTBPs) are extracellular matrix (ECM) proteins that bind latent transforming growth factor β (TGF-β) and influence its availability in bone and other connective tissues. LTBPs have homology with fibrillins and may have related functions as microfibrillar proteins. However, at present little is known about their structural arrangement in the ECM. By using antibodies against purified LTBP1, against a short peptide in LTBP1, and against epitope-tagged LTBP1 constructs, we have shown colocalization of LTBP1 and fibrillin 1 in microfibrillar structures in the ECM of cultured primary osteoblasts. Immunoelectron microscopy confirmed localization of LTBP1 to 10- to 12-nm microfibrils and suggested an ordered aggregation of LTBP1 into these structures. Early colocalization of LTBP1 with fibronectin suggested a role for fibronectin in the initial assembly of LTBP1 into the matrix; however, in more differentiated osteoblast cultures, LTBP1 and fibronectin 1 were found in distinct fibrillar networks. Overexpression of LTBP1 deletion constructs in osteoblast-like cells showed that N-terminal amino acids 67–467 were sufficient for incorporation into fibrillin-containing microfibrils and suggested that LTBP1 can be produced by cells distant from the site of fibril formation. In embryonic long bones in vivo, LTBP1 and fibrillin 1 colocalized at the surface of newly forming osteoid and bone. However, LTBP1-positive fibrils, which did not contain fibrillin 1, were present in cartilage matrix. These studies show that in addition to regulating TGFβ1, LTBP1 may function as a structural component of connective tissue microfibrils. LTBP1 may therefore be a candidate gene for Marfan-related connective tissue disorders in which linkage to fibrillins has been excluded.
Different Bone Growth Rates Are Associated With Changes in the Expression Pattern of Types II and X Collagens and Collagenase 3 in Proximal Growth Plates of the Rat Tibia
Authors: Álvarez Jesús , Balbín Milagros , Santos Fernando , Fernández Marta , Ferrando Susana , López José M.
Published: April 2006

Skeletal growth depends on endochondral ossification in growth plate cartilage, where proliferation of chondrocytes, matrix synthesis, and increases in chondrocyte size all contribute to the final length of a bone. To learn more about the potential role of matrix synthesis/degradation dynamics in the determination of bone growth rate, we investigated the expression of matrix collagens and collagenase 3 in tibial growth plates in three age groups of rats (21, 35, and 80 days after birth), each characterized by specific growth rates. By combining stereological and in situ hybridization techniques, it was found that the expression of matrix collagens and collagenase 3 was specifically turned on or off at specific stages of the chondrocyte-differentiation cycle, and these changes occurred as a temporal sequence that varied depending of animal growth rate. Furthermore, the expression of these matrix proteins by a growth plate chondrocyte was found to be sped up or slowed down depending of the growth rate. In addition to expression of types II and X collagen, collagenase-3 expression was found to constitute a constant event in the series of changes in gene expression that takes place during the chondrocyte-differentiation process. Collagenase-3 expression was found to show a biphasic pattern: it was intermittently expressed at the proliferative phase and uniformly expressed at the hypertrophic stage. An intimate relationship between morphological and kinetic changes associated with chondrocyte hypertrophy and changes in the expression pattern of matrix collagens and collagenase 3 was observed. Present data prove that the matrix synthesis/degradation dynamics of the growth plate cartilage varied depending on growth rate; these results support the hypothesis that changes in matrix degradation and synthesis are a critical link in the sequence of tightly regulated events that lead to chondrocytic differentiation.
The Bone Morphogenetic Protein 2 Signaling Mediator Smad1 Participates Predominantly in Osteogenic and not in Chondrogenic Differentiation in Mesenchymal Progenitors C3H10T½
Authors: Ju Wenjun , Hoffmann Andrea , Verschueren Kristin , Tylzanowski Przemko , Kaps Christian , Gross Gerhard , Huylebroeck Danny
Published: April 2006

The role of the bone morphogenetic protein (BMP)-signaling mediator Smad1 in osteogenic or chondrogenic differentiation was investigated in murine parental mesenchymal progenitors C3H10T½ and its derivatives constitutively expressing BMP-2 (C3H10T½-BMP-2) and, therefore, undergo BMP-mediated osteogenic/chondrogenic development. The functions of the three Smad1 domains, that is, the N-terminal (MH1) domain, the C-terminal (MH2) domain, and the midregional proline-rich linker domain, were documented and compared with full-length Smad1. We showed that expression of the MH2 domain in parental C3H10T½ cells was sufficient to initiate osteogenic differentiation. Interestingly, MH1 was sufficient to initiate transcription of osteogenic marker genes like the osteocalcin or parathyroid hormone/parathyroid hormone-related protein (PTH/PTHrP) receptor. However, MH1 interfered with the histologically distinct formation of osteoblast-like cells. A dominant-negative effect on MH2-mediated osteogenic development in C3H10T½ cells was observed by the dose-dependent trans-expression of the midregional linker domain. Importantly, in contrast to osteogenic differentiation, Smad1 and its domains do not mimic or interfere with BMP-2-dependent chondrogenic development as monitored by the inability of MH2 to give rise to histologically distinct chondrocytes in parental C3H10T½ cells and by the inefficiency of the MH1 or linker domain to interfere with BMP-2-mediated chondrogenic differentiation.
Force-Induced Osteoclast Apoptosis In Vivo Is Accompanied by Elevation in Transforming Growth Factor β and Osteoprotegerin Expression
Authors: Kobayashi Yasuhiro , Hashimoto Fumio , Miyamoto Hisatsugu , Kanaoka Kazuhiro , Miyazaki-Kawashita Yumiko , Nakashima Tomoki , Shibata Mitsue , Kobayashi Kazuhide , Kato Yuzo , Sakai Hideaki
Published: April 2006

The mechanism controlling the disappearance of osteoclasts from bone surfaces after bone resorption in vivo is largely unknown. This is because there is no suitable experimental system to trace the final fate of osteoclasts. Here, we used an experimental model of tooth movement in rats to show that preexisting osteoclasts disappeared from the bone surface through apoptosis during a force-induced rapid shift from bone resorption to formation. On the distal alveolar bone surface of the maxillary molar in growing rats, many mature osteoclasts were present. When light tensional force was applied to the bone surface through an orthodontic appliance, these preexisting osteoclasts gradually disappeared. One day after the application of force, about 24% of the osteoclasts exhibited apoptotic morphology and the proportion of apoptotic cells was increased to 41% by day 2, then decreased afterward. These changes were undetectable on the control distal alveolar bone surface, which is free from tensional force. As shown by in situ hybridization, a marked increase in transforming growth factor β1 (TGF-β1) and osteoprotegerin (OPG) messenger RNA (mRNA) was observed in the stretched cells on the tensioned distal bone surface, simultaneously with the loss of osteoclasts. Both of these factors are known to have a negative effect on osteoclast recruitment and survival. As early as 2 days after force application, some of these stretched cells were identified as cuboidal osteoblasts showing intense signals for both factors. Our data suggest there may be a sequential link in tensional force applied on the bone lining cells, up-regulation of TGF-β1/OPG, and disappearance of osteoclasts.
Three-Dimensional Morphometry of the L6 Vertebra in the Ovariectomized Rat Model of Osteoporosis: Biomechanical Implications
Authors: Kinney J. H. , Haupt D. L. , Balooch M. , Ladd A. J. C. , Ryaby J. T. , Lane N. E.
Published: April 2006

This article summarizes the results of a three-dimensional study of changes in the morphology of the L6 rat vertebra at 120 days after ovariectomy (OVX), with estrogen replacement therapy used as a positive control. Synchrotron radiation microtomography was used to quantify the structural parameters defining trabecular bone architecture, while finite-element methods were used to explore the relationships between these parameters and the compressive elastic behavior of the vertebrae. There was a 22% decrease in trabecular bone volume (TBV) and a 19% decline in mean trabecular thickness (Tb.Th) with OVX. This was accompanied by a 150% increase in trabecular connectivity, a result of the perforation of trabecular plates. Finite-element analysis of the trabecular bone removed from the cortical shell showed a 37% decline in the Young's modulus in compression after OVX with no appreciable change in the estrogen-treated group. The intact vertebrae (containing its trabecular bone) exhibited a 15% decrease in modulus with OVX, but this decline lacked statistical significance. OVX-induced changes in the trabecular architecture were different from those that have been observed in the proximal tibia. This difference was a consequence of the much more platelike structure of the trabecular bone in the vertebra.
Cross-Sectional Geometry, Bone Strength, and Bone Mass in the Proximal Femur in Black and White Postmenopausal Women
Authors: Nelson D. A. , Barondess D. A. , Hendrix S. L. , Beck T. J.
Published: April 2006

Osteoporosis is characterized by both a low bone mass and a disruption of the architectural arrangement of bone tissue, leading to decreased skeletal strength and increased fracture risk. Although there are well-known ethnic differences in bone mass and fracture risk, little is known about possible ethnic differences in bone structure. Therefore, we studied cross-sectional geometry in the hip in a sample of postmenopausal black and white women in order to investigate ethnic differences that might contribute to differences in bone strength and ultimately hip fracture risk. We recruited 371 postmenopausal black and white women who were entering the Women's Health Initiative (WHI) clinical trials in Detroit. Bone density measurements of the proximal femur were done by dual-energy X-ray absorptiometry (DXA) using a Hologic 1000 Plus bone densitometer. The DXA data were used for hip structure analysis, which treats the entire proximal femur as a continuous curved beam from the proximal shaft to the femoral neck. This permits the analysis of cross-sectional geometric properties in two narrow regions corresponding to thin (5 mm) cross-sectional slabs seen on edge. The results indicate significant ethnic differences in bone density, cross-sectional geometry, and dimensional variables. Specifically, the black women have a significantly higher bone density in both locations (10.1% and 4.1% for the neck and shaft, respectively); greater cross-sectional geometric properties in the neck (ranging from 6.1% to 11.6%), but a smaller endocortical diameter in the neck (3.6%). There are fewer significant differences in cross-sectional geometry in the shaft location. Our data suggest that the spatial distribution of bone is arranged in the femoral neck to resist greater loading in black women compared with white women.
Associations Between Maternal Peak Bone Mass and Bone Mass in Prepubertal Male and Female Children
Authors: Jones Graeme , Nguyen Tuan V.
Published: April 2006

The aim of this study was to estimate heritability of bone density in premenopausal women, prepubertal male, and prepubertal female child pairs. We studied 291 pairs (mothers, mean age, 33 years, range 22–45 years; children, mean age, 7.92 years, range 7.32-8.92 years). Bone density and body composition were assessed by dual-energy X-ray absorptiometry. Height and weight were measured in both mother and child. Body size-adjusted heritability estimates for areal bone density (g/cm2) were all statistically significant (femoral neck, 59%; lumbar spine, 38%; total body, 41%) and were consistently and significantly higher in mother-daughter pairs (n = 105) as compared with mother-son pairs (n = 186). Heritability estimates for bone mineral apparent density (BMAD; g/cm3) were marginally lower but remained statistically significant at all sites (femoral neck, 51%; lumbar spine, 32%; total body, 38%). Maternal osteopenia was associated with significant reductions in bone mass at all sites in the children (femoral neck, 0.75 SD and p < 0.0001; lumbar spine, 0.61 SD and p < 0.0001; total body, 0.43 SD and p = 0.012). Mother-child bone areal bone density correlation coefficients and prediction of low bone mass in the child were greater (but this did not reach statistical significance) if the corresponding anatomical site in the mother was used for prediction with the exception of the total body. These data confirm that heritability of bone mass extends to prepubertal children and is gender- and possibly site-specific as well as under separate genetic control to growth. Furthermore, the strength of the mother-child association is such that bone density screening of mothers would make it possible to identify most prepubertal children at higher risk of osteoporosis in later life.
Vertebral Fractures in Beijing, China: The Beijing Osteoporosis Project
Authors: Ling Xu , Cummings Steven R. , Mingwei Qin , Xihe Zhao , Xioashu Chen , Nevitt Michael , Stone Katie
Published: April 2006

Women in China have much lower risk of hip fracture than women in Europe or North America but their risk of vertebral fractures is not known. Lateral spine radiographs, hip and lumbar spine bone density, and potential risk factors for and consequences of vertebral fractures were assessed in a random sample of 402 women age 50 years or older living in Beijing, China. The prevalence of vertebral fractures, defined by vertebral morphometry, increased from 5% (95% CI, 1–9%) in 50- to 59-year olds to 37% (27-46%) among women age 80 years or older. The age-standardized prevalence of vertebral fractures was 5.5% lower than found by similar methods for women in Rochester, MN, U.S.A. Each SD lower spine bone mineral density (BMD) was associated with a 2.4-fold (1.7-3.5) increased odds of having a vertebral fracture. Women with a history of heavy physical labor had a lower risk of vertebral fractures. Vertebral fractures were associated with decreased height loss and limited physical function but not chronic back pain. Women in Beijing, China have lower bone density and a slightly lower rate of vertebral fracture than white women in the United States. Low bone density and more sedentary occupations increase the risk of fracture in women living in urban China.
Alendronate Administration and Skeletal Response During Chronic Alcohol Intake in the Adolescent Male Rat
Authors: Wezeman Frederick H. , Emanuele Mary Ann , Moskal Stanley F. , Steiner Jennifer , Lapaglia Nancy
Published: April 2006

Alendronate is an aminobisphosphonate that inhibits bone resorption in osteoporotic humans and rats but does not induce osteomalacia. Several bisphosphonates, including alendronate, also have direct positive actions on osteoblasts, bone formation, and mineralization. We studied the effects of alendronate on skeletal development in adolescent male rats during chronic alcohol intake. Four groups of age- and weight-matched male Sprague-Dawley rats (35 days of age) were fed the Lieber-DeCarli diet containing 36% of calories as EtOH (E), the EtOH diet plus 60 mg/kg alendronate (EA) every other day intraperitoneally (ip), an isocaloric diet (I), or the isocaloric diet plus 60 mg/kg alendronate (IA) every other day ip. Body weight, femur length, serum levels of osteocalcin (OC), insulin-like growth factor 1 (IGF-1), testosterone, and luteinizing hormone (LH); femur distal metaphyseal and middiaphyseal bone mineral density (BMD) and tibial metaphyseal gene expression for α-1-type I collagen (Col I), OC, and bone alkaline phosphatase (AP); and femur strength by four-point bending to failure were measured after 28 days of feeding and alendronate injections. Serum alcohol levels at death were 156 ± 13 mg/dl (E) and 203 ± 40 mg/dl (EA). Alendronate given to alcohol-fed rats increased metaphyseal BMD by more than 3-fold over rats fed alcohol alone. Alendronate given to isocaloric pair-fed rats increased metaphyseal BMD by more than 2.5-fold over rats fed the isocaloric diet alone. Cortical BMD was reduced by alcohol but was increased by alendronate. Alcohol consumption reduced serum IGF-1 levels, and alendronate increased IGF-1 levels in alcohol-fed rats. Serum OC, testosterone, and LH were unaffected by alcohol and alendronate. Quantitative dot blot hybridization using rat complementary DNA (cDNA) probes and normalization against 18S subunit ribosomal RNA (rRNA) levels revealed no changes in tibial metaphyseal gene expression for type I collagen, osteocalcin, or alkaline phosphatase. Alcohol significantly reduced the biomechanical properties of the femurs that were partially compensated by alendronate. Chronic alcohol consumption uncouples formation from ongoing resorption, and resorption is inhibited by alendronate. However, alendronate's positive effects on osteoblast-mediated mineralization during chronic alcohol consumption point to the potential use of bisphosphonates in the treatment of decreased bone formation secondary to alcohol-induced diminished osteoblast function.
Concentration of Bisphosphonate (Incadronate) in Callus Area and Its Effects on Fracture Healing in Rats
Authors: Li Jiliang , Mori Satoshi , Kaji Yoshio , Kawanishi Jun , Akiyama Tomoyuki , Norimatsu Hiromichi
Published: April 2006

The aim of the present study was to investigate effects of incadronate on early stages of fracture healing and to detect its concentration in callus area (Ca.Ar). Rats were injected three times per week with either two doses of incadronate (10 μg/kg and 100 μg/kg) or vehicle for 2 weeks. Femora were then fractured and fixed and animals were divided into pretreatment (P-10 and P-100) and continuous treatment (C-10 and C-100) groups. Incadronate treatment was stopped in P-10 and P-100 groups but continued in C-10 and C-100 groups. Animals were killed at 2 weeks and 4 weeks after fracture. Results showed significantly large callus, compared with the control, only in C-100 group at 4 weeks but not at 2 weeks. Both linear labeled surface (LS) and eroded surface (ES) decreased significantly in C-10 and C-100 groups at 2 weeks and 4 weeks. Osteoclast number (N.Oc) decreased significantly in C-10 and C-100 groups at 2 weeks but increased slightly at 4 weeks. However, there was no significant difference in the above parameters in P-10 and P-100 groups at 4 weeks. Apoptotic osteoclasts were observed only in the C-100 group at 4 weeks. A time-course decrease in incadronate concentration was detected in P-10 and P-100 groups whereas an increase was observed in C-10 and C-100 groups. These findings suggest that larger callus under incadronate treatment may result from the inhibition of bone resorption, histological characteristics of callus may be correlated with incadronate concentration, and metabolism of incadronate in bone may be related to the rate of bone turnover.
GNAS1 Mutation and Cbfa1 Misexpression in a Child with Severe Congenital Platelike Osteoma Cutis
Authors: Yeh George L. , Mathur Sameer , Wivel Ashley , Li Ming , Gannon Francis H. , Ulied Angels , Audi Laura , Olmsted Elizabeth A. , Kaplan Frederick S. , Shore Eileen M.
Published: April 2006

We evaluated a 7-year-old girl with severe platelike osteoma cutis (POC), a variant of progressive osseous heteroplasia (POH). The child had congenital heterotopic ossification of dermis and subcutaneous fat that progressed to involve deep skeletal muscles of the face, scalp, and eyes. Although involvement of skeletal muscle is a prominent feature of POH, heterotopic ossification has not been observed in the head, face, or extraocular muscles. The cutaneous ossification in this patient was suggestive of Albright hereditary osteodystrophy (AHO); however, none of the other characteristic features of AHO were expressed. Inactivating mutations of the GNAS1 gene, which encodes the α-subunit of the stimulatory G protein of adenylyl cyclase, is the cause of AHO. Mutational analysis of GNAS1 using genomic DNA of peripheral blood and of lesional and nonlesional tissue from our patient revealed a heterozygous 4-base pair (bp) deletion in exon 7, identical to mutations that have been found in some AHO patients. This 4-bp deletion in GNAS1 predicts a protein reading frameshift leading to 13 incorrect amino acids followed by a premature stop codon. To investigate pathways of osteogenesis by which GNAS1 may mediate its effects, we examined the expression of the obligate osteogenic transcription factor Cbfa1/RUNX2 in lesional and uninvolved dermal fibroblasts from our patient and discovered expression of bone-specific Cbfa1 messenger RNA (mRNA) in both cell types. These findings document severe heterotopic ossification in the absence of AHO features caused by an inactivating GNAS1 mutation and establish the GNAS1 gene as the leading candidate gene for POH.