bluuebunny/crossref_metadata_2025_split huggingface dataset:
Peptide Aldehyde Inhibitors of Cathepsin K Inhibit Bone Resorption Both In Vitro and In Vivo
Authors: Votta Bartholomew J. , Levy Mark A. , Badger Alison , Bradbeer Jeremy , Dodds Robert A. , James Ian E. , Thompson Scott , Bossard Mary J. , Carr Thomas , Connor Janice R. , Tomaszek Thaddeus A. , Szewczuk Lawrence , Drake Fred H. , Veber Daniel F. , Gowen Maxine 
Published: April 2006

Abstract
We have shown previously that cathepsin K, a recently identified member of the papain superfamily of cysteine proteases, is expressed selectively in osteoclasts and is the predominant cysteine protease in these cells. Based upon its abundant cell type-selective expression, potent endoprotease activity at low pH and cellular localization at the bone interface, cathepsin K has been proposed to play a specialized role in osteoclast-mediated bone resorption. In this study, we evaluated a series of peptide aldehydes and demonstrated that they are potent cathepsin K inhibitors. These compounds inhibited osteoclast-mediated bone resorption in fetal rat long bone (FRLB) organ cultures in vitro in a concentration-dependent manner. Selected compounds were also shown to inhibit bone resorption in a human osteoclast-mediated assay in vitro. Cbz-Leu-Leu-Leu-H (in vitro enzyme inhibition Ki,app = 1.4 nM) inhibited parathyroid hormone (PTH)-stimulated resorption in the FRLB assay with an IC-50 of 20 nM and inhibited resorption by isolated human osteoclasts cultured on bovine cortical bone slices with an IC-50 of 100 nM. In the adjuvant-arthritic (AA) rat model, in situ hybridization studies demonstrated high levels of cathepsin K expression in osteoclasts at sites of extensive bone loss in the distal tibia. Cbz-Leu-Leu-Leu-H (30 mg/kg, intraperitoneally) significantly reduced this bone loss, as well as the associated hind paw edema. In the thyroparathyriodectomized rat model, Cbz-Leu-Leu-Leu-H inhibited the increase in blood ionized calcium induced by a 6 h infusion of PTH. These data indicate that inhibitors of cathepsin K are effective at reducing osteoclast-mediated bone resorption and may have therapeutic potential in diseases of excessive bone resorption such as rheumatoid arthritis or osteoporosis.
Histomorphometry of Distraction Osteogenesis in a Caprine Tibial Lengthening Model
Authors: Dr. Welch Robert D. , Birch John G. , Makarov Marina R. , Samchukov Mikhail L. 
Published: April 2006

Abstract
Standardized histomorphometry of bone formation and remodeling during distraction osteogenesis (DO) has not been well characterized. Increasing the rhythm or number of incremental lengthenings performed per day is reported to enhance bone formation during limb lengthening. In 17 skeletally immature goats, unilateral tibial lengthenings to 20 or 30% of original length were performed at a rate of 0.75 mm/day and rhythms of 1, 4, or 720 times per day using standard Ilizarov external fixation and an autodistractor system. Two additional animals underwent frame application and osteotomy without lengthening and served as osteotomy healing controls. Histomorphometric indices were measured at predetermined regions from undecalcified tibial specimens. Within the distraction region, bone formation and remodeling activity were location dependent. Intramembranous bone formed linearly oriented columns of interconnecting trabecular plates of woven and lamellar type bone. Total new bone volume and bone formation indices were significantly increased within the distraction and osteotomy callus regions (Tb.BV/TV, 226% [p &lt; 0.05]; BFR/BS, 235–650% [p &lt; 0.01]) respectively, compared with control metaphyseal bone. Bone formation indices were greatest adjacent to the mineralization zones at the center of the distraction gap; mineral apposition rate 96% (p &lt; 0.01); mineralized bone surfaces 277% [p &lt; 0.001]); osteoblast surfaces 359% [p &lt; 0.001]); and bone formation rate (650% [p &lt; 0.01]). There was no significant difference (p &lt; 0.14; R = 0.4) in the bone formation rate of the distracted callus compared with the osteotomy control callus. Within the original cortices of the lengthened tibiae, bone remodeling indices were significantly increased compared with osteotomy controls; activation frequency (200% [p &lt; 0.05]); osteoclast surfaces (295% [p &lt; 0.01]); erosion period (75%); porosity (240% [p &lt; 0.001]). Neither the rhythm of distraction nor the percent lengthening appeared to significantly influence any morphometric parameter evaluated. Distraction osteogenesis shares many features of normal fracture gap healing. The enhanced bone formation and remodeling appeared to result more from increased recruitment and activation of bone forming and resorbing cells rather than from an increased level of individual cellular activity.
Induction of Human Osteoclast-like Cells by Treatment of Blood Monocytes with Anti-Fusion Regulatory Protein-1/CD98 Monoclonal Antibodies
Authors: Higuchi Shigeomi , Tabata Nobutada , Tajima Masatoshi , Ito Morihiro , Tsurudome Masato , Sudo Akihiro , Uchida Atsumasa , Ito Yasuhiko 
Published: April 2006

Abstract
We have developed a new and simple system of human osteoclast formation by fusing peripheral blood monocytes with anti-Fusion Regulatory Protein-1 (anti–FRP-1) monoclonal antibody (mAb). When human blood monocytes were cultured in the presence of anti-FRP–1/CD98 mAbs, polykaryocytes began to appear at approximately 15 h and increased in size with time until 3–4 days of incubation with anti–FRP-1 mAb. These fused cells showed positive staining in tartrate-resistant acid phosphatase, possessed numerous calcitonin receptors, and were capable of bone resorption. These results strongly suggest that anti–FRP-1 antibody-induced multinucleated cells are osteoclasts. Furthermore, FRP-1 antigens were detected in osteoclasts isolated from human bone and in the osteoclast-like cells obtained from human giant cell tumors of bone.
The Migration of Purified Osteoclasts Through Collagen Is Inhibited by Matrix Metalloproteinase Inhibitors
Authors: Sato Takuya , Foged Niels Tækker , Delaissé Jean-Marie 
Published: April 2006

Abstract
The most obvious proteolytic event controlled by the osteoclast is bone matrix removal in the resorption compartment. Here, however, we investigated whether matrix metalloproteinase (MMP) activity of the osteoclast might be involved in its migration to its future bone resorption site. We seeded either nonpurified or purified osteoclasts onto either uncoated or collagen-coated dentine slices and cultured them in the presence or absence of specific MMP inhibitors. When nonpurified osteoclasts were cultured on uncoated dentine, MMP inhibitors did not prevent pit formation, as previously reported. However, when collagen-coated dentine was used, pit formation was strongly inhibited by MMP inhibitors. The same results were obtained when performing these experiments with purified osteoclasts, thus demonstrating the ability of osteoclasts by themselves to migrate through collagen via an MMP-dependent pathway. This demonstration was confirmed by using collagen-coated invasion chambers. In addition, the invasions were not, or only slightly, inhibited by inhibitors of serine proteinases, cysteine proteinases, and carbonic anhydrase, though the latter two are well established bone resorption inhibitors that strongly inhibited pit formation. It is concluded that osteoclasts can migrate through collagen in the absence of other cells and that this migration relies on MMP activity, whereas other enzymes typically required for bone removal in the resorption compartment are not essential for migration. Some of the osteoclast MMPs might thus be relevant to the migratory/invasive activity of the osteoclast, rather than to its bone resorptive activity itself.
High Fatty Acid Content in Rabbit Serum Is Responsible for the Differentiation of Osteoblasts Into Adipocyte-like Cells
Authors: Diascro Dominic D. , Vogel Robert L. , Johnson Timothy E. , Witherup Keith M. , Pitzenberger Steven M. , Rutledge Su Jane , Prescott David J. , Rodan Gideon A. , Schmidt Azriel 
Published: April 2006

Abstract
Osteoblasts and adipocytes originate from common mesenchymal precursors. With aging, there is a decrease in osteoprogenitor cells that parallels an increase of adipocytes in bone marrow. We observed that rabbit serum (RS) induces adipocyte-like differentiation in human osteosarcoma SaOS-2/B10 and MG-63 cell lines, in rat ROS17/2.8 cells, and in mouse calvaria-derived osteoblastic MB1.8 cells, as evidenced by the accumulation of Oil Red O positive lipid vesicles and the decrease in alkaline phosphatase expression. Both SaOS-2/B10 and MG-63 cells, but not ROS17/2.8 nor MB1.8 cells, express significant levels of PPARγ mRNA, a member of the peroxisome proliferator activated receptor (PPAR) family that has been implicated in the control of adipocyte differentiation. However, both ROS17/2.8 and MG-63 cells express significant levels of the adipocyte selective marker, aP2 fatty acid binding mRNA, which can be further increased by RS. These cell types express PPARδ/NUC-1 but not PPARα, indicating that cells that do not express either PPARγ or PPARα are capable of differentiating into adipocyte-like cells. Transfection experiments in COS cells showed that compared with fetal bovine serum (FBS), RS is rich in agents that stimulate PPAR-dependent transcription. The stimulatory activity was ethyl acetate extractable and was 35-fold more abundant in RS than in FBS. Purification and analysis revealed that the major components of this extract are free fatty acids. Furthermore, the same fatty acids, a mixture of palmitic, oleic, and linoleic acids, activate the PPARs and induce adipocyte-like differentiation of both ROS17/2.8 and SaOS-2/B10 cells. These findings suggest that fatty acids or their metabolites can initiate the switch from osteoblasts to adipocyte-like cells.
A BMP Responsive Transcriptional Region in the Chicken Type X Collagen Gene
Authors: Volk Susan W. , Luvalle Phyllis , Leask Todd , Leboy Phoebe S. 
Published: April 2006

Abstract
Bone morphogenetic proteins (BMPs) were originally identified by their ability to induce ectopic bone formation and have been shown to promote both chondrogenesis and chondrocyte hypertrophy. BMPs have recently been found to activate a membrane serine/threonine kinase signaling mechanism in a variety of cell types, but the downstream effectors of BMP signaling in chondrocyte differentiation remain unidentified. We have previously reported that BMP-2 markedly stimulates type X collagen expression in prehypertrophic chick sternal chondrocytes, and that type X collagen mRNA levels in chondrocytes cultured under serum-free (SF) conditions are elevated 3- to 5-fold within 24 h. To better define the molecular mechanisms of induction of chondrocyte hypertrophy by BMPs, we examined the effect of BMPs on type X collagen production by 15-day chick embryo sternal chondrocytes cultured under SF conditions in the presence or absence of 30 ng/ml BMP-2, BMP-4, or BMP-7. Two populations of chondrocytes were used: one representing resting cartilage isolated from the caudal third of the sterna and the second representing prehypertrophic cartilage from the cephalic third of the sterna. BMP-2, BMP-4, and BMP-7 all effectively promoted chondrocyte maturation of cephalic sternal chondrocytes as measured by high levels of alkaline phosphatase, diminished levels of type II collagen, and induction of the hypertrophic chondrocyte-specific marker, type X collagen. To test whether BMP control of type X collagen expression occurs at the transcriptional level, we utilized plasmid constructs containing the chicken collagen X promoter and 5′ flanking regions fused to a reporter gene. Constructs were transiently transfected into sternal chondrocytes cultured under SF conditions in the presence or absence of 30 ng/ml BMP-2, BMP-4, or BMP-7. A 533 bp region located 2.4–2.9 kb upstream from the type X collagen transcriptional start site was both necessary and sufficient for strong BMP responsiveness in cells destined for hypertrophy, but not in chondrocytes derived from the lower sterna.
Mouse Osteoblastic Cell Line (MC3T3-E1) Expresses Extracellular Calcium (Ca2+o)–Sensing Receptor and Its Agonists Stimulate Chemotaxis and Proliferation of MC3T3-E1 Cells
Authors: Yamaguchi Toru , Chattopadhyay Naibedya , Kifor Olga , Butters Robert R. , Sugimoto Toshitsugu , Brown Edward M. 
Published: April 2006

Abstract
The calcium-sensing receptor (CaR) is a G protein-coupled receptor that plays key roles in extracellular calcium ion (Ca2+o) homeostasis in parathyroid gland and kidney. Osteoblasts appear at sites of osteoclastic bone resorption during bone remodeling in the “reversal” phase following osteoclastic resorption and preceding bone formation. Bone resorption produces substantial local increases in Ca2+o that could provide a signal for osteoblasts in the vicinity, leading us to determine whether such osteoblasts express the CaR. In this study, we used the mouse osteoblastic, clonal cell line MC3T3-E1. Both immunocytochemistry and Western blot analysis, using an antiserum specific for the CaR, detected CaR protein in MC3T3-E1 cells. We also identified CaR transcripts in MC3T3-E1 cells by Northern analysis using a CaR-specific riboprobe and by reverse transcription-polymerase chain reaction with CaR-specific primers, followed by nucleotide sequencing of the amplified products. Exposure of MC3T3-E1 cells to high Ca2+o (up to 4.8 mM) or the polycationic CaR agonists, neomycin and gadolinium (Gd3+), stimulated both chemotaxis and DNA synthesis in MC3T3-E1 cells. Therefore, taken together, our data strongly suggest that the osteoblastic cell line MC3T3-E1 possesses both CaR protein and mRNA very similar, if not identical, to those in parathyroid and kidney. Furthermore, the CaR in these osteoblasts could play a key role in regulating bone turnover by stimulating the proliferation and migration of such cells to sites of bone resorption as a result of local release of Ca2+o.
17β-Hydroxysteroid Dehydrogenases in Human Bone Cells
Authors: Dong Yu , Qiu Qing Qing , Debear Joanna , Lathrop William F. , Bertolini Donald R. , Tamburini Paul P. 
Published: April 2006

Abstract
Interconversion of estrogens by osteoblasts may play a role in regulating bone mass. As a first step toward exploring this possibility, we investigated the expression and activity of 17β-hydroxysteroid dehydrogenases (17β-HSDs) in cultured human osteoblasts (HOB) and osteoblast-like osteosarcoma cells (MG63, TE85, and SaOS-2). Significant 17β-HSD activity was detected in cell-free extracts of all bone cells with oxidation of estradiol to estrone predominating over reduction. Reverse transcription-polymerase chain reaction (RT-PCR) experiments showed that the mRNA for 17β-HSD I was detectable only in MG63 cells, albeit at low levels, while 17β-HSD II was present in MG63, TE85, and HOB, but not SaOS-2, and 17β-HSD III was absent from each bone cell type. 17β-HSD IV was the only isoform present in all bone cells analyzed. Further analysis of the expression of 17β-HSD IV in these bone cells by immunoblotting revealed both the full-length 83 kDa protein and the proteolytic 38 kDa form. The kinetic parameters for estradiol oxidation by purified recombinant 17β-HSD IV (Km = 49.7 μM, Vmax = 79.4 nmol/minute/mg of protein) and its HSD-domain (Km = 79.4 μM, Vmax = 476 nmol/minute/mg of protein) were significantly higher than previously reported, but consistent with the values obtained with crude cell-free extracts of SaOS-2 cells (Km = 98.8 μM, Vmax = 0.07 nmol/minute/mg of protein) which contain only 17β-HSD IV based on RT-PCR. These studies show that bone cells have the capacity to interconvert circulating estrogens and suggest that bone cell 17β-HSDs serve primarily to attenuate the continuing actions of estradiol through conversion to its less potent form, estrone, under certain conditions.
Osteocyte Shape Is Dependent on Actin Filaments and Osteocyte Processes Are Unique Actin-Rich Projections
Authors: Tanaka-Kamioka Kayo , Kamioka Hiroshi , Ris Hans , Lim Soo-Siang 
Published: April 2006

Abstract
Osteocytes are derived from a select group of osteoblasts that have undergone a final differentiation. Due to their inaccessibility when embedded in the bone matrix, very little is known about the osteocyte cytoskeleton. This study provides an extensive analysis of the osteocyte cytoskeleton, based on the successful isolation of osteocytes from 16-day embryonic chick calvariae. We used OB7.3, a chicken osteocyte-specific monoclonal antibody, to confirm the osteocytic phenotype of the isolated cells and established culture conditions to promote growth of cells that most resemble osteocytes in vivo. Immunofluorescence staining with antitubulin, antivimentin, and antiactin showed the relative distribution of the microtubules, intermediate filaments, and actin filaments in both osteocyte cell body and processes. Field emission scanning electron microscopy revealed the three-dimensional relationships of the cytoskeletal elements and a unique organization of actin bundles that spanned the cell body and osteocyte processes. When combined with drug studies, these experiments demonstrate that actin filaments are crucial for the maintenance of osteocyte shape. Furthermore, we identified two actin-bundling proteins, alpha-actinin and fimbrin, in osteocyte processes. The prominence and unique distribution of fimbrin in osteocyte processes provides the possibility of its use as an intracellular marker to distinguish osteocytes from osteoblasts.
A Novel Polymorphism in the Promoter Region for the Human Osteocalcin Gene: The Possibility of a Correlation with Bone Mineral Density in Postmenopausal Japanese Women
Authors: Dohi Yoshiko , Iki Masayuki , Ohgushi Hajime , Gojo Satoshi , Tabata Shiro , Kajita Etsuko , Nishino Harumi , Yonemasu Kunio 
Published: April 2006

Abstract
We present a polymorphism of the human osteocalcin gene (also known as BGP, for bone Gla protein) due to a 1 base pair (bp) substitution from cytosine to thymine at position 298 nucleotides (nt), which is at position 198 nt upstream from the BGP exon 1. This mutation was detected by single-strand conformation polymorphism analysis after polymerase chain reaction for the osteocalcin gene fragment (326 bp) and sequencing analysis. The cytosine/thymine polymorphism can be defined by restriction fragment length polymorphism analysis using a modified primer pair and the restriction endonuclease HindIII. The osteocalcin genotype was determined in 160 postmenopausal Japanese women (age 48–80 years). Osteocalcin alleles were designated according to the absence (H) or presence (h) of the HindIII restriction site. There were 12 HH, 49 Hh, and 99 hh individuals, and the allele frequencies were 22.8% for H and 77.2% for h. To determine if genetic variation influences bone mineral density (BMD) and thus can be a determinant of susceptibility to osteoporosis in older women, we examined the association of BMD with the osteocalcin genotypes found in the present study. The subjects with genotype HH had the smallest BMD and those with hh had the greatest BMD among subjects, but these differences did not reach statistical significance. The HindIII genotype showed a significant effect on the prevalence of osteopenia in the subjects, that is, women with genotype HH had a 5.74 times greater risk for osteopenia (p &lt; 0.05) and those with genotype Hh had a 1.59 times greater risk than women with genotype hh. We identified the osteocalcin gene polymorphism, detected with the HindIII genotype, which was suggested to influence bone density and is a possible genetic marker for bone metabolism.
Activation of Phospholipase D Signaling Pathway by Epidermal Growth Factor in Osteoblastic Cells
Authors: Carpio Lillian Carol , Dziak Rosemary 
Published: April 2006

Abstract
The receptor-mediated activation of phospholipase D (PLD) is a major signaling pathway in several cell systems. This study determined the effects of epidermal growth factor (EGF) on PLD activity in normal rat osteoblastic cells. Primary cultures were obtained from fetal rat calvaria by sequential collagenase digestion and seeded in BGJb media supplemented with 10% fetal calf serum. PLD activity was assayed by the transphosphatidylation reaction in [H3]myristic acid (5 μCi/ml)-labeled cells treated with EGF in the presence of 5% ethanol and measuring the production of phosphatidylethanol (PEtOH). Lipids were extracted and separated by thin-layer chromatography, detected by iodine staining, and the areas of interest were scraped off and transferred to vials for scintillation counting. EGF significantly increased PEtOH production in a dose-dependent manner and at short (10–60 s) and long (up to 30 minutes) incubation periods (p &lt; 0.05). Phosphatidic acid levels were also significantly increased (p &lt; 0.05) compared with unstimulated controls, but the levels were ∼60% less than those of PEtOH. 4b-phorbol 12-myristate, 13-acetate (PMA) also produced a significant increase in PEtOH levels when compared with unstimulated control cultures, but when PMA was added together with EGF, the production of PEtOH was reduced about 30%. Pretreatment of cells with the protein kinase C (PKC) inhibitor H-7 caused a significant increase in PEtOH levels, compared with cells stimulated with EGF alone. Preincubation of cells with pertussis toxin produced a partial decrease in PEtOH levels. This study demonstrates that EGF activates the PLD signaling cascade in normal rat osteoblastic cells and that the pathway appears to involve, at least in part, a PKC- and Gi protein-dependent mechanism.
Trabecular Bone Mineral and Calculated Structure of Human Bone Specimens Scanned by Peripheral Quantitative Computed Tomography: Relation to Biomechanical Properties
Authors: Jiang Yebin , Zhao Jenny , Augat Peter , Ouyang Xiaolong , Lu Ying , Majumdar Sharmila , Genant Harry K. 
Published: April 2006

Abstract
The relationship of cortical bone mineral density (BMD), and geometry to bone strength has been well documented. In this study, we used peripheral quantitative computerized tomography (pQCT) to acquire trabecular BMD and high-resolution images of trabeculae from specimens to determine their relationship with biomechanical properties. Fifty-eight human cubic trabecular bone specimens, including 26 from the vertebral bodies, were scanned in water and air. Trabecular structure was quantitated using software developed with Advanced Visual Systems interfaced on a Sun/Sparc Workstation. BMD was also obtained using a whole-body computerized tomography scanner (QCT). Nondestructive testing of the specimens was performed to assess their elastic modulus. QCT and pQCT measurements of BMD of specimens in water were strongly correlated (r2 = 0.95, p &lt; 0.0001), with a slope (0.96) statistically not significantly different from 1. Strong correlations were found between pQCT measurements of specimens in water and in air, for BMD (r2 = 0.96, p &lt; 0.0001), and for apparent trabecular structural parameters (r2 = 0.89–0.93, p &lt; 0.0001). Correlations were moderate between BMD and apparent trabecular structural parameters (r2 = 0.37–0.64, p &lt; 0.0001). Precision as coefficient of variation (CV) and standardized coefficient of variation (SCV) for these measurements was &lt; 5%. For the vertebral specimens, the correlation was higher between elastic modulus and BMD (r2 = 0.76, p &lt; 0.0001) than between elastic modulus and apparent trabecular structural parameters (r2 = 0.58–0.72, p &lt; 0.0001), while the addition of apparent trabecular nodes and branches to BMD in a multivariate regression model significantly increased the correlation with the elastic modulus (r2 = 0.86, p &lt; 0.01). Thus, pQCT can comparably and reproducibly measure trabecular bone mineral in water or air, and trabecular structure can be quantitated from pQCT images. The combination of volumetric BMD with trabecular structural parameters rather than either alone improves the prediction of biomechanical properties. Such a noninvasive approach may be useful for the preclinical study of osteoporosis.