bluuebunny/crossref_metadata_2025 huggingface dataset:
Two-Site Immunoassays for Osteoclastic Tartrate-Resistant Acid Phosphatase Based on Characterization of Six Monoclonal Antibodies
Authors: Halleen Jussi M. , Karp Matti , Viloma Sari , Laaksonen Pirjo , Hellman Jukka , Käkönen Sanna-Maria , Stepan Jan J. , Holmes Stephen , Väänänen H. Kalervo , Pettersson Kim
Published: April 2006

Tartrate-resistant acid phosphatase (TRAP), an enzyme expressed in bone-resorbing osteoclasts, is secreted into the circulation during bone resorption. We used six monoclonal antibodies (MAbs) to optimize direct two-site fluoroimmunoassays for determining serum TRAP concentrations. Four of the MABs, 1F1, 2H1, 4E6, and 5C1, were raised against recombinant human TRAP, and the other two, O1A and J1B, against human bone TRAP. 2H1, J1B, and O1A appeared to be highly specific for TRAP. 1F1 and 4E6 were poor in recognizing bone TRAP and were not useful in the assay. 5C1, while having a good affinity for the bone enzyme, was not specific. Serum TRAP is relatively stable, because 7 days of storage of serum samples at 4°C and −20°C or five thawing-freezing cycles, did not change the TRAP concentration detected using the two-site assays. All studied assays detected an increase in serum TRAP concentrations of postmenopausal women compared with premenopausal women, the difference being highest with MAB pairs 2H1–5C1 and O1A–J1B. These results suggest that serum TRAP may be a useful bone resorption marker, and the MAB pairs 2H1–5C1 and O1A–J1B may be useful in determining the bone resorption rate.
Human Retinoic X Receptor β: Complete Genomic Sequence and Mutation Search for Ossification of Posterior Longitudinal Ligament of the Spine
Authors: Numasawa Takuya , Koga Hiroaki , Ueyama Kazumasa , Maeda Shingo , Sakou Takashi , Harata Seiko , Leppert Mark , Dr. Inoue Ituro
Published: April 2006

Ossification of the posterior longitudinal ligament of the spine (OPLL) is characterized by ectopic bone formation in the ligament. OPLL is a very common disorder, in fact it constitutes the leading cause of myelopathy among Japanese. In the previous report, we provided the genetic linkage evidence that the genetic susceptibility of OPLL mapped to HLA complex of chromosome 6. As a candidate gene approach, retinoic X receptor β (RXRβ), assigned to chromosome 6p21.3 adjacent to HLA class II, was analyzed for a possible causality. To start screening for the molecular variants of RXRβ in OPLL subjects, we first obtained P1 phage genomic clones containing the entire human RXRβ and elucidated the genomic organization of the gene. The human RXRβ is composed of 10 exons spanning over 6.2 kb of genomic DNA. Sequence analysis of the promoter region revealed a GC-rich sequence without TATA motif. We have identified three distinct molecular variants, one was in exon 10 and two were in the intergenic region between RXRβ and collagen 11A2 (COL11A2). Two variants in the intergenic region, 3′ end + 140 and 3′ end + 561, exhibit statistically significant associations with OPLL in case-control study (p = 0.0028 for 3′ end + 140 and p = 0.034 for 3′ end + 561). These results indicate that the genetic causality of OPLL lies within or close to the RXRβ/COL11A2 locus.
Immunological Characterization of Circulating Osteoprotegerin/Osteoclastogenesis Inhibitory Factor: Increased Serum Concentrations in Postmenopausal Women with Osteoporosis
Authors: Yano Kazuki , Tsuda Eisuke , Washida Naohiro , Kobayashi Fumie , Goto Masaaki , Harada Atsushi , Ikeda Kyoji , Higashio Kanji , Yamada Yoshiji
Published: April 2006

Osteoprotegerin (OPG)/osteoclastogenesis inhibitory factor (OCIF) is a soluble member of the tumor necrosis factor receptor family of proteins and plays an important role in the negative regulation of osteoclastic bone resorption. Whether OPG/OCIF circulates in human blood and how its level changes under pathological conditions is not known. To address these issues, a panel of monoclonal antibodies was generated against recombinant OPG/OCIF and screened for reactivity with solid-phase monomeric and homodimeric forms of the recombinant protein. Antibodies that showed high affinity for both forms of OPG/OCIF and those that selectively recognized the homodimer were identified, enabling development of two types of sensitive enzyme-linked immunosorbent assay (ELISA): one that detects both forms of OPG/OCIF equally and one specific for the homodimer. Characterization of circulating OPG/OCIF with these ELISAs revealed that the protein exists in human serum mainly in the monomeric form. The serum concentration of OPG/OCIF increased with age in both healthy Japanese men and women, and was significantly higher in postmenopausal women with osteoporosis than in age-matched controls. Within the osteoporotic group, serum OPG/OCIF concentrations were higher in patients with low bone mass. Serum OPG/OCIF concentrations were also significantly increased in those postmenopausal women with a high rate of bone turnover, as determined by increased serum bone-specific alkaline phosphatase and urinary excretion of pyridinoline and deoxypyridinoline. The results suggested that circulating OPG/OCIF levels are regulated by an age-related factor(s) and that the increased serum concentration may reflect a compensative response to enhanced osteoclastic bone resorption and the resultant bone loss rather than a cause of osteoporosis.
ERK Pathway Mediates the Activation of Cdk2 in IGF-1–Induced Proliferation of Human Osteosarcoma MG-63 Cells
Authors: Zhang Wenyu , Lee John C. , Kumar Sanjay , Gowen Maxine
Published: April 2006

Insulin-like growth factor-1 (IGF-1) is a potent mitogen for osteoblasts. The primary signaling mechanism involved in mediating this proliferative effect of IGF-1 is not well defined. The roles of extracellular signal-regulated kinase 1 (ERK1) and cyclin-dependent kinase 2 (Cdk2) kinases in the IGF-1–induced proliferative signaling pathway of human osteosarcoma MG63 cells were investigated using a selective inhibitor of MEK, PD98059, and a Cdk inhibitor, olomoucine. Treatment of MG63 cells with PD98059 and olomoucine inhibited IGF-1–stimulated proliferation of these cells and induced cell cycle arrest at G0/G1. PD98059 significantly abolished IGF-1–stimulated kinase activity of ERK1 in a dose-dependent manner. PD98059 also inhibited the kinase activity of Cdk2 in IGF-1–stimulated cells, although the inhibition by olomoucine was much greater. The extent of inhibition of Cdk2 activity by PD98059 and olomoucine was consistent with their effects on cell proliferation and cell cycle. Cyclin A was complexed with Cdk2 in unstimulated MG63 cells, but Cdk2 kinase activity in the complex was up-regulated only in IGF-1–treated cells. This was consistent with an observed IGF-1–stimulated hyperphosphorylation of retinoblastoma protein (pRb) with the possibility that the activated Cdk2 kinase is involved in phosphorylation of pRb in IGF-1–induced cell proliferation. Taken together, these results suggest that the MEK/ERK pathway act in a positive regulatory fashion to activate Cdk2 in IGF-1–induced mitogenesis in osteoblasts.
Anabolic Effects of Human Biosynthetic Parathyroid Hormone Fragment (1–34), LY333334, on Remodeling and Mechanical Properties of Cortical Bone in Rabbits
Authors: Hirano Toru , Burr David B. , Turner Charles H. , Sato Masahiko , Cain Rick L. , Hock Janet M.
Published: April 2006

Intermittent administration of parathyroid hormone (PTH) has an anabolic effect in cancellous bone of osteoporotic humans. However, the effect of PTH on cortical bone with Haversian remodeling remains controversial. The aim of this study was to determine the effects of biosynthetic human PTH(1–34) on the histology and mechanical properties of cortical bone in rabbits, which exhibit Haversian remodeling. Mature New Zealand white rabbits were treated with once daily injections of vehicle, or PTH(1–34), LY333334, at 10 μg/kg/day or 40 μg/kg/day for 140 days. Body weight in rabbits treated with PTH did not change significantly over the experimental period. Serum calcium and phosphate were within the normal range, but a 1 mg/ml increase in serum calcium was observed in rabbits given the higher dose of PTH. Histomorphometry of cortical bone in the midshaft of the tibia showed significant increases in periosteal and endocortical bone formation in these rabbits. Intracortical bone remodeling in the tibia was activated and cortical porosity increased by PTH. Cross-sectional bone area and bone mass of the midshaft of the femur increased significantly after PTH treatment. Ultimate force, stiffness, and work to failure of the midshaft of the femur of rabbits given the 40 μg dose of PTH were significantly greater than those in the control group, whereas elastic modulus was significantly lower than that in the rabbits given the 10 μg dose of PTH, but not different from controls. In the third lumbar vertebra, PTH increased both formation and resorption without increasing cancellous bone volume. The increases in bone turnover and cortical porosity were accompanied by concurrent increases in bone at the periosteal and endocortical surfaces. The combination of these phenomena resulted in an enhancement of the ultimate stress, stiffness, and work to failure of the femur.
Effects of Continuous Infusion of PTH on Experimental Tooth Movement in Rats
Authors: Soma S. , Iwamoto M. , Higuchi Y. , Kurisu K.
Published: April 2006

Development of new methods for accelerating orthodontic tooth movement has been strongly desired for shortening of the treatment period. The rate of orthodontic tooth movement is dependent on the rate of bone resorption occurring in the compressed periodontium in the direction of orthodontic force applied to the tooth. In the present study, we examined the effects of continuous infusion of parathyroid hormone (PTH) on tooth movement. Male rats weighing 350–400 g were treated with subcutaneous infusion of vehicle or hPTH(1–84) at 1–10 μg/100 g of body weight/day. When the upper right first molar (M1) was moved mesially for 72 h by the insertion of an elastic band between the first and second molars, M1 movement was accelerated by PTH infusion at 10 μg. PTH infusion caused a 2- to 3-fold increase in the number of osteoclasts in the compressed periodontium of M1, indicating that such treatment accelerated tooth movement by enhancing bone resorptive activity induced in the compressed periodontium. When M1 was moved mesially by an orthodontic coil spring ligated between upper incisors and M1 for 12 days, PTH(1–84) infusion at 10 μg caused a 2-fold increase in the rate of M1 movement. PTH(1–34) infusion at 4 μg had an effect comparable to that of PTH(1–84). However, intermittent injection of PTH(1–34) did not accelerate M1 movement. PTH infusion for 13 days did not affect either bone mineral measurements or the serum calcium level. These findings suggest that continuous administration of PTH is applicable to accelerate orthodontic tooth movement.
The Proportion of Carboxylated to Total or Intact Osteocalcin in Serum Discriminates Warfarin-Treated Patients from Control Subjects
Authors: Obrant Karl J. , Käkönen Sanna-Maria , Astermark Jan , Lilja Hans , Lövgren Timo , Åkesson Kristina , Pettersson Kim
Published: April 2006

We assessed the serum concentration of γ-carboxylated osteocalcin (OC), total OC, and full-length OC in a clinical setting of 37 patients on continuous warfarin treatment (international normalized ratio 2.0–3.8). A comparison was done with the results from 30 untreated age-matched controls. Four monoclonal antibodies, previously generated and characterized as to their ability to recognize different human OC forms and fragments, were used in three two-site immunofluorometric assays. The warfarin-treated patients had significantly lower levels of carboxylated OC 4.9 ± 3.8 (± 1 SD) ng/ml compared with the controls 13.1 ± 9.7 (p < 0.0001). There was no difference in the levels of total OC or full-length OC between the two groups of patients. A strong correlation was found between the serum concentration of carboxylated OC and total OC, both for the warfarin-treated patients (r = 0.98) and for the controls (r = 0.99). There was a distinct cut-off level at 0.80, in the quotient carboxylated OC/total OC, at which all warfarin-treated patients fell below and all controls above this level. Hence, the concentration or ratio of serum γ-carboxylated OC in clinical settings such as warfarin-treated patients could be measured using two-site immunoassays.
Longitudinal Study of Calcium Intake, Physical Activity, and Bone Mineral Content in Infants 6–18 Months of Age
Authors: Specker Bonny L. , Mulligan Lizanne , Ho Mona
Published: April 2006

Although increased physical activity early in life is recommended for optimizing bone health, no controlled trials on the effect of activity on bone mass accretion during periods of rapid growth have been reported. The purpose of this study was to determine whether infants randomized to a 1 year gross motor activity program had a greater bone mass accretion than infants randomized to a fine motor activity program. The gross motor program included activities that focused on loading the skeleton and were performed for 15–20 minutes/day, 5 days/week by study personnel. Infants (n = 72) were enrolled at 6 months of age, and total body bone mineral content (BMC), 3-day diet records, and activity levels were obtained at 6, 9, 12, 15, and 18 months. BMC was associated with weight, length, and bone area at all ages and correlated with earlier calcium intakes. Calcium intake appeared to modify the effect of gross motor activity on bone mass accretion; infants in both groups had similar bone accretion at moderately high calcium intakes, but at low calcium intakes infants in the gross motor program had less bone accretion than infants in the fine motor program. Compliant infants in the gross motor group had lower BMC at 18 months compared with noncompliant infants. These results indicate that BMC in infants is related to calcium intake, and we speculate that participation in a gross motor program during rapid bone growth may lead to reduced bone accretion in the presence of a moderate to moderately low calcium intake.
Dietary Calcium and Bone Density in Adolescent Girls and Young Women in Europe
Authors: Kardinaal A. F. M. , Ando S. , Charles P. , Charzewska J. , Rotily M. , Väänänen K. , Van erp-baart A. M. J. , Heikkinen J. , Thomsen J. , Maggiolini M. , Deloraine A. , Chabros E. , Juvin R. , Schaafsma G.
Published: April 2006

The objective of this study was to investigate the association between dietary calcium intake and radial bone density among young women, over the whole range of intake and at different levels of calcium intake. The study design was a cross-sectional, observational multicenter investigation in six European countries. One thousand one hundred and sixteen healthy Caucasian girls aged 11–15 years and 526 women aged 20–23 years participated, after having been selected from larger population samples to represent a large range in calcium intake. Bone mineral density (BMD) was measured with dual-energy X-ray absorptiometry at the ultradistal and middistal radius. Calcium intake was assessed with 3-day food records. Other potential determinants of BMD were measured by anthropometry or questionnaires. Mean calcium intake among the girls varied between 609 mg/day in Italy and 1267 mg/day in Finland; intakes for women were in a similar range. After adjustment for height, weight, and age at menarche for the women, and adjustment for age, height, weight, Tanner stage, and bone area for the girls, radial BMD at both sites did not significantly vary among quartiles of calcium intakes for both age groups. In multivariate linear regression, calcium was weakly positively associated with BMD at both sites in the girls (per 100 mg of calcium: β = 0.57 mg/cm2, p = 0.03 for middistal BMD and β = 0.56 mg/cm2, p = 0.01 for ultradistal BMD). For middistal BMD, the association was observed predominantly in premenarcheal girls. The associations were no longer statistically significant after full adjustment for all determinants of BMD, except again in pre-menarcheal girls. Radial BMD in the women was not associated with calcium intake, except after full adjustment for determinants of BMD, when ultradistal BMD became inversely associated with calcium intake (per 100 mg β = −1.02, p = 0.03); this finding was due to results in one of the countries and not found in other countries. There was no evidence for a different relation between calcium and BMD at different levels of intake; although there was a positive association at calcium intake levels <600 mg/day, the interaction was not significant and there was no consistent trend over intake categories. These results do not support the hypothesis that dietary calcium is a determinant of peak BMD in European women, for a wide range of intake. This study does not provide evidence that Recommended Dietary Allowances for calcium should be increased.
Ovariectomized Murine Model of Postmenopausal Calcium Malabsorption
Authors: Kalu Dike N. , Chen Cang
Published: April 2006

Two experiments were carried out to examine whether the ovariectomized mouse is a potential in vivo model of intestinal calcium malabsorption as occurs in postmenopausal osteoporosis. In the first experiment, we compared the effects of ovariectomy and 17β-estradiol (E2) therapy on calcium absorption in C3H/HeJ (C3H) and C57BL/6J (C57BL) mice, which have high and low peak bone mass, respectively. For each strain of mice, three groups were studied: sham operated, ovariectomized, (OVX), and OVX + E2 (60 μg/kg of body weight [bw]/day). Therapy was continued for 35 days and calcium absorption measured. In the C3H mice, ovariectomy caused an increase in fecal calcium (17.6%), and a decrease in the amount (12.8%) and percentage (12.5%) of calcium absorbed. The decrease was prevented by E2 therapy, but the differences in the calcium absorption parameters among the groups were not statistically significant. In contrast, in the C57BL mice, ovariectomy caused a marked increase in fecal calcium (84.5%, p < 0.01), and a marked decrease in the amount (55%, p < 0.01) and percentage (34.8%, p < 0.001) of calcium absorbed, and the decrease in the percentage of calcium absorbed was partially prevented by E2 therapy (p < 0.05). In experiment 2, a dose-response study of the effects of E2 therapy on calcium absorption in OVX C57BL mice was carried out. Five groups of mice were studied: Group 1, sham operated; Group 2, OVX; Group 3, OVX + 60 μg of E2/kg of bw/day; Group 4, OVX + 120 μg of E2/kg of bw/day; Group 5, OVX + 240 μg of E2/kg of bw/day. Therapy was continued for 28 days and calcium absorption measured. Ovariectomy caused a marked increase in fecal calcium (50%, p < 0.0001) and urinary calcium (31%, p < 0.0001) and a marked decrease in calcium absorption (44.9%, p < 0.0001), and these changes were prevented by E2 therapy. The highest level of calcium absorption (109%, p < 0.0001, vs. OVX) was observed in the 120 μg of E2 group. Ovariectomy and E2 slightly increased plasma 1,25-dihydroxyvitamin D (1,25(OH)2D) levels but there was no significant correlation between 1,25(OH)2D levels and calcium absorption. The findings in the C57BL mice suggest that estrogen is a physiological regulator of calcium absorption in this strain of mice. Furthermore, these findings share many characteristics with intestinal calcium malabsorption and its reversal by estrogen therapy in hypoestrogenic women. We propose that the OVX C57BL mice warrants further characterization as a potential animal model for investigating issues related to calcium malabsorption in postmenopausal women.
Changes in Bone Resorption During the Menstrual Cycle
Authors: Chiu Kit Mui , Ju Julia , Mayes Darrel , Bacchetti Peter , Weitz Steve , Arnaud Claude D.
Published: April 2006

To determine if the cyclic changes of female sex hormones during the menstrual cycle are related to changes in bone formation and resorption, we measured serum bone-specific alkaline phosphatase (BAP) and osteocalcin (OC) and bone resorption markers, serum and urine deoxypyridinoline (Dpyr), three times per week during one menstrual cycle in 20 healthy premenopausal women. Serum estradiol (E2) and progesterone (P) showed characteristic cyclic fluctuations. Serum Dpyr was higher during the follicular phase (FP) than in the luteal phase (p = 0.027). Serum BAP, OC, and urine Dpyr levels did not change substantially across the cycle. Serum Dpyr correlated negatively with serum E2 values measured 6 (p = 0.011) and 8 (p = 0.001) days earlier and with P measured concurrently (p = 0.033) 2 (p = 0.002), 4 (p = 0.003), and 6 (p = 0.014) days earlier. BAP correlated negatively with E2 measured 6 days earlier (p = 0.006). We found no statistically significant correlations of E2 or P with OC or urine Dpyr within women over their cycles. BAP was positively correlated with concurrent serum Dpyr (p = 0.015) during the menstrual cycle. Serum OC levels correlated inversely with age (rs = −0.48, p = 0.036). Women with higher mean urine Dpyr levels had higher mean serum OC levels (rs = 0.49, p = 0.033) and showed a trend toward lower hip bone mineral density (rs = −0.40, p = 0.078). We conclude that the low level of E2 and/or P observed during the FP of the normal menstrual cycle is associated with increased bone resorption. These relationships suggest that normal women experience monthly episodes of increased bone resorption from menarche to menopause.
Does Combining the Results from Multiple Bone Sites Measured by a New Quantitative Ultrasound Device Improve Discrimination of Hip Fracture?
Authors: Dr. Hans D. , Srivastav S. K. , Singal C. , Barkmann R. , Njeh C. F. , Kantorovich E. , Glüer C. C. , Genant H. K.
Published: April 2006

There is a growing interest in the use of quantitative ultrasound (QUS) measurements as an alternative to current radiation-based bone densitometry techniques for the noninvasive assessment of fracture risk. While most of the commercialized ultrasound devices measure only single predefined peripheral skeletal sites, the Omnisense prototype (Sunlight Ltd., Israel) can be used on multiple bones, including the spinous processes. In this study, we examined the ability of speed of sound measured at the calcaneus, distal third and ultradistal radius, proximal third phalanx, metacarpal, capitate, patella, and the posterior process of the thoracic spine to differentiate subjects with hip fractures from normal controls. Seventy-nine postmenopausal Caucasian Israeli women who had sustained an atraumatic fracture of the proximal femur within the last 6 months were recruited from the local population (mean age 80 ± 8.9 years). As controls, 295 postmenopausal Caucasian Israeli women without osteoporotic fractures were also included (mean age 70 ± 8.7 years). Discrimination of hip fractures with QUS at all ultrasound sites was highly statistically significant (p < 0.01) (odds ratios [ORs] = 1.4–3.0; area under the ROC curve [AUC] 77–92%), except for the hand metacarpal. Distal radius and calcaneus measurements (ORs = 2.4 and 3.0) were the best discriminators of hip fracture patients from controls. Using a forward selective linear regression model, the discriminator values of combined assessment at two sites were investigated. There was moderate improvement in diagnostic value, but the best combination was the calcaneus with the distal radius, which improved the AUC by 3% and raised both the sensitivity and specificity to 94%. These data demonstrate the encouraging potential of improving discrimination of hip fracture by using multiple-site ultrasonic measurements.
Role of Osteopontin in Bone Remodeling Caused by Mechanical Stress
Authors: Terai Kunihiro , Takano-Yamamoto Teruko , Ohba Yasuo , Hiura Kenji , Sugimoto Mizuo , Sato Motohiko , Kawahata Hirohisa , Inaguma Naohiro , Kitamura Yukihiko , Nomura Shintaro
Published: April 2006

Changes in the number and proportion of osteopontin mRNA (Opn) expressing osteocytes and osteoclasts caused by the mechanical stress applied during experimental tooth movement were examined in the present study. Opn expression was detected in the osteocytes on the pressure side at the early stage, and gradually spread to those on the tension side and also to the osteoblasts and bone-lining cells in the alveolar bone. Only 3.3% of the osteocytes located on the pressure side expressed Opn in the interradicular septum of control rats; in contrast, the value was increased to 87.5% at 48 h after the initiation of tooth movement. These results indicate that these cells responded to mechanical stress loaded on the bone with expression of the osteopontin gene. Following the increased expression of Opn in these cells, a 17-fold greater number of osteoclasts compared with the control and numerous resorption pits were observed on the pressure side of the alveolar bone. Injection of arginine-glycine-aspartic acid-serine peptide but not that of arginine-glycine-glutamic acid-serine peptide strongly inhibited the increase in the number of osteoclasts. Furthermore, an in vitro migration assay demonstrated the chemotactic activity of osteopontin (OPN) on the precursor of osteoclasts. Our study strongly suggests that OPN is an important factor triggering bone remodeling caused by mechanical stress.
No Evidence for Mutations in the Calcium-Sensing Receptor Gene in Sporadic Parathyroid Adenomas
Authors: Cetani Filomena , Pinchera Aldo , Pardi Elena , Cianferotti Luisella , Vignali Edda , Picone Antonella , Miccoli Paolo , Viacava Paolo , Marcocci Claudio
Published: April 2006

Inactivating mutations of the calcium-sensing receptor gene (CaR) might explain abnormalities in the regulation of both parathyroid cell proliferation and parathyroid hormone secretion. In a previous study, using RNAse A protection assay, no mutations were identified in a series of parathyroid specimens from patients with primary and secondary hyperparathyroidism, but the analysis was incomplete, since part of exon 6 could not be analyzed. In the present study, we examined the presence of mutations in the CaR gene in 20 parathyroid adenomas using direct sequencing. The entire coding region of the CaR gene was successfully amplified by polymerase chain reaction and directly sequenced. This analysis did not identify CaR gene mutations in any tumors studied. A polymorphism that encoded a single amino acid change (Ala826Thr) was identified in 4 parathyroid adenomas and in 8 of 50 normal unrelated subjects. Loss of heterozygosity studies were also performed on adenomas using markers for the locus of the CaR gene on chromosome 3q. No allelic loss was demonstrated. In conclusion, our results extend previous observation and suggest that clonal somatic mutations of the CaR gene and allelic loss at the CaR locus on chromosome 3q do not play a major role in the pathogenesis of sporadic parathyroid tumors.
Matrix Vesicle Plasma Cell Membrane Glycoprotein-1 Regulates Mineralization by Murine Osteoblastic MC3T3 Cells
Authors: Johnson Kristen , Moffa Allison , Chen Ying , Pritzker Kenneth , Goding James , Terkeltaub Robert
Published: April 2006

A naturally occurring nonsense truncation mutation of the inorganic pyrophosphate (PPi)-generating nucleoside triphosphate pyrophosphohydrolase (NTPPPH) PC-1 is associated with spinal and periarticular ligament hyperostosis and cartilage calcification in “tiptoe walking” (ttw) mice. Thus, we tested the hypothesis that PC-1 acts directly in the extracellular matrix to restrain mineralization. Cultured osteoblastic MC3T3 cells expressed PC-1 mRNA and produced hydroxyapatite deposits at 12–14 days. NTPPPH activity increased steadily over 14 days. Transforming growth factor-β and 1,25-dihydroxyvitamin D3 increased PC-1 and NTPPPH in matrix vesicles (MVs). Because PC-1/NTPPPH was regulated in mineralizing MC3T3 cells, we stably transfected or infected cells with recombinant adenovirus, in order to express 2- to 6-fold more PC-1. PC-1/NTPPPH and PPi content increased severalfold in MVs derived from cells transfected with PC-1. Furthermore, MC3T3 cells transfected with PC-1 deposited ∼80–90% less hydroxyapatite (by weight) than cells transfected with empty plasmid or enzymatically inactive PC-1. ATP-dependent45Ca precipitation by MVs from cells overexpressing active PC-1 was comparably diminished. Thus, regulation of PC-1 controls the PPi content and function of osteoblast-derived MVs and matrix hydroxyapatite deposition. PC-1 may provide a novel therapeutic target in certain disorders of bone mineralization.
Isolation and Characterization of MC3T3-E1 Preosteoblast Subclones with Distinct In Vitro and In Vivo Differentiation/Mineralization Potential
Authors: Wang Dian , Christensen Kurt , Chawla Kanwal , Xiao Guozhi , Krebsbach Paul H. , Dr. Franceschi Renny T.
Published: April 2006

A series of subclonal cell lines with high or low differentiation/mineralization potential after growth in the presence of ascorbic acid (AA) were derived from murine MC3T3-E1 cells. Subclones were characterized in terms of their ability to mineralize a collagenous extracellular matrix both in vitro and in vivo and express osteoblast-related genes. When compared with nonmineralizing cells, mineralizing subclones selectively expressed mRNAs for the osteoblast markers, bone sialoprotein (BSP), osteocalcin (OCN), and the parathyroid hormone (PTH)/parathyroid hormone-related protein (PTHrP) receptor. In contrast, alkaline phosphatase mRNA was present in certain nonmineralizing as well as mineralizing subclones, suggesting that its expression may be subject to different controls from other osteoblast markers. Only highly differentiating subclones exhibited strong AA-dependent induction of a transiently transfected OCN promoter-luciferase reporter gene, indicating that there was a good correlation between mRNA levels and transcriptional activity. Consistent with its postulated role in biomineralization, BSP as measured by Western blotting was only present in mineralizing subclones. After implantation into immunodeficient mice, highly differentiating subclones formed bone-like ossicles resembling woven bone, while poorly differentiating cells only produced fibrous tissue. Interestingly, subclones with both high and low differentiation potential produced similar amounts of collagen in culture and expressed comparable basal levels of mRNA encoding Osf2/Cbfa1, an osteoblast-related transcription factor. Although some strongly differentiating cells exhibited a modest AA-dependent up-regulation of Osf2/Cbfa1 mRNA, there was no clear relationship between levels of this message and induction of mRNAs for other differentiation markers. Thus, the mere presence of Osf2/Cbfa1 in a subclone was not sufficient for osteoblast differentiation. These subclones will be very useful for studying critical events in osteoblast differentiation and mineralization.
Two Human Osteoblast-like Osteosarcoma Cell Lines Show Distinct Expression and Differential Regulation of Parathyroid Hormone–Related Protein
Authors: Jemtland Rune , Rian Edith , Olstad Ole Kristoffer , Haug Egil , Bruland Øyvind S. , Bucht Elisabet , Gautvik Kaare M.
Published: April 2006

Parathyroid hormone (PTH)-related protein (PTHrP) acts as a local regulator of osteoblast function via mechanisms that involve PTH/PTHrP receptors linked to protein kinase A (PKA) and C (PKC). However, the regulation of PTHrP production and mRNA expression in human osteoblasts is poorly understood. Here we have characterized alternative PTHrP mRNA 3′ splicing variants, encoding PTHrP isoforms of 139, 141, and 173 amino acids, and studied the regulation of PTHrP and its mRNAs by activated PKA and PKC in two human osteoblast-like cell lines (KPDXM and TPXM). Using exon-specific Northern analysis and reverse transcriptase-coupled polymerase chain reaction, we identified mRNAs encoding PTHrP(1–139) and PTHrP(1–141) in both cell lines. PTHrP(1–139) mRNAs predominated in TPXM cells and PTHrP(1–173) mRNAs were only detected in TPXM cells. Activation of PKA or PKC resulted in different effects on PTHrP and its mRNAs in the two cell lines. In TPXM cells, peptide-specific immunoassays detected high basal levels of PTHrP, increasing by 2-fold in cell extracts and 4-fold in culture media at 7 h and 24 h after exposure to forskolin, respectively, paralleling changes in PTHrP mRNA expression. Phorbol ester 12-O-tetradecanoyl-phorbol 13-acetate (TPA), a PKC activator, had no effect. In KPDXM cells, PTHrP was not detected in culture media under basal experimental conditions, and barely detectable amounts were present in cell extracts of TPA-treated cells, although the mRNA levels increased substantially in response to TPA. In the responsive cell lines, the effects on mRNA levels were dose dependent, and increased by 6.9- to 10.5-fold and 2.0- to 4.1-fold at 4 h in TPXM and KPDXM cells after exposure to 10 μM forskolin and 150 nM TPA, respectively. PTHrP mRNA levels then declined but were sustained above controls also at 12 h in both cell lines, albeit at considerably higher levels in TPXM cells. The different responsiveness to agents activating PKA- and PKC-dependent pathways may depend on the cellular state of differentiation, or alternatively, cancer cell line–specific defects. Our data demonstrating distinct differences in mRNA species and the amounts of PTHrP produced by the two cell lines as compared with roughly equivalent overall mRNA levels may suggest that post-transcriptional mechanisms play an important role in limiting the production of intracellular and secreted PTHrPs in human osteoblastic cells.
Stimulation of Osteoblast Proliferation by C-Terminal Fragments of Parathyroid Hormone–Related Protein
Authors: Dr. Cornish Jillian , Callon Karen E. , Lin Cindy , Xiao Conrad , Moseley Jane M. , Reid Ian R.
Published: April 2006

Parathyroid hormone (PTH)-related protein (107–139) (PTHrP(107–139)) and PTHrP(107–111) have been reported to be potent inhibitors of isolated osteoclast activity, and inhibition of bone resorption by PTHrP(107–139) occurs in vivo. However, the actions of C-terminal PTHrP on osteoblast activity has not been studied much. The present study addresses this issue by examining the effect of PTHrP(107–139), PTHrP(107–119), PTHrP(120–139), and PTHrP(107–111) on the proliferation of fetal rat osteoblasts. Treatment with PTHrP(107–139) for 24 h caused a dose-dependent increase in cell number, [3H]thymidine and [3H]phenylalanine incorporation in cultured osteoblasts. The effect was apparent at concentrations of 10−10 M and greater and was sustained over time. PTHrP(107–119) and PTHrP(107–111) had effects on cell number, DNA, and protein synthesis which were comparable to those of PTHrP(107–139), whereas PTHrP(120–139) was without effect. Retroverted PTHrP(107–111) also stimulated all three activities but was only one tenth as potent as PTHrP(107–139). PTHrP(107–139) had no effect on osteoblast apoptosis. It is concluded that PTHrP(107–139) is not only an inhibitor of osteoclastic bone resorption but that it also stimulates osteoblast growth. This activity resides within the pentapeptide fragment PTHrP(107–111). These findings support a possible role for C-terminal fragments of PTHrP in the normal regulation of bone cell function and, possibly, bone mass.
Steady and Transient Fluid Shear Stress Stimulate NO Release in Osteoblasts Through Distinct Biochemical Pathways
Authors: McAllister T. N. , Frangos J. A.
Published: April 2006

Fluid flow has been shown to be a potent stimulus in osteoblasts and osteocytes and may therefore play an important role in load-induced bone remodeling. The objective of this study was to investigate the characteristics of flow-activated pathways. Previously we reported that fluid flow stimulates rapid and continuous release of nitric oxide (NO) in primary rat calvarial osteoblasts. Here we demonstrate that flow-induced NO release is mediated by shear stress and that this response is distinctly biphasic. Transients in shear stress associated with the onset of flow stimulated a burst in NO production (8.2 nmol/mg of protein/h), while steady flow stimulated sustained NO production (2.2 nmol/mg of protein/h). Both G-protein inhibition and calcium chelation abolished the burst phase but had no effect on sustained production. Activation of G-proteins stimulated dose-dependent NO release in static cultures of both calvarial osteoblasts and UMR-106 osteoblast-like cells. Pertussis toxin had no effect on NO release. Calcium ionophore stimulated low levels of NO production within 15 minutes but had no effect on sustained production. Taken together, these data suggest that fluid shear stress stimulates NO release by two distinct pathways: a G-protein and calcium-dependent phase sensitive to flow transients, and a G-protein and calcium-independent pathway stimulated by sustained flow.
Macrophage Colony Stimulating Factor Increases Bone Resorption in Dispersed Osteoclast Cultures by Increasing Osteoclast Size
Authors: Lees Rita L. , Dr. Heersche Johan N. M.
Published: April 2006

Several reports indicate that macrophage colony stimulating factor (MCSF) is one of the major factors required for osteoclast proliferation and differentiation. Paradoxically, it has also been reported that MCSF inhibits osteoclastic activity. We therefore decided to investigate in detail the effects of MCSF on resorption and osteoclast formation to try and clarify this issue. Osteoclast-containing cultures were obtained from rabbit long bones and cultured on plastic culture dishes or devitalized bovine bone slices. MCSF (4–400 ng/ml) stimulated osteoclastic bone resorption in a time-dependent manner and at all doses examined. After 48 h of culture in the presence of MCSF, we observed a 2-fold increase in the total area of bone resorbed, as well as a significant increase in the area of bone resorbed per osteoclast and the number of resorption pits per osteoclast. This effect was paralleled by an increase in the number of larger osteoclasts (as determined by the number of nuclei per cell) and an increase in the size and depth of the resorption pits. Since the total number of osteoclasts remained the same, the MCSF-induced increase in resorptive activity appeared to be related to an increase in the average size of the osteoclasts. When resorption was expressed as the amount of bone resorbed per osteoclast nucleus, larger osteoclasts resorbed more per nucleus, suggesting that large osteoclasts, as a population, are more effective resorbers than small osteoclasts. Interestingly, when osteoclasts were plated at one-fifth the standard density, the amount of bone resorbed per osteoclast decreased considerably, indicating that resorptive activity is also affected by cell density of osteoclasts and/or of other cells present. However, at this lower density MCSF still increased osteoclast size and resorption by the same fold increase over control, suggesting that the effect of MCSF was independent of factors related to cell density.
Intermittent Parathyroid Hormone (1–34) Treatment Increases Callus Formation and Mechanical Strength of Healing Rat Fractures
Authors: Andreassen Troels T. , Ejersted Charlotte , Oxlund Hans
Published: April 2006

The influence of intermittent parathyroid hormone (PTH(1–34)) administration on callus formation and mechanical strength of tibial fractures in rats was investigated after 20 and 40 days of healing. A dose of 60 μg of PTH(1–34)/kg/day and 200 μg of PTH(1–34)/kg/day, respectively, was administered during the entire periods of healing, and control animals with fractures were given vehicle. The dose of 200 μg of PTH(1–34)/kg/day increased the ultimate load and the external callus volume of the fractures by 75% and 99%, respectively, after 20 days of healing and by 175% and 72%, respectively, after 40 days of healing. The dose of 60 μg of PTH(1–34)/kg/day did not influence either ultimate load or external callus volume of the fractures after 20 days of healing, but the ultimate load was increased by 132% and the external callus volume was increased by 42% after 40 days of healing. During the healing period, the callus bone mineral content (BMC) increased in all groups. After 40 days of healing, the callus BMC was increased by 108% in the 200 μg of PTH(1–34)/kg/day group and by 76% in the 60 μg of PTH(1–34)/kg/day group. Both doses of PTH(1–34) steadily augmented the contralateral intact tibia BMC (20 days and 40 days: 60 μg of PTH (1–34)/kg/day 9% and 19%, respectively; 200 μg of PTH (1–34)/kg/day 12% and 27%, respectively) and bone mineral density (20 days and 40 days: 60 μg of PTH(1–34)/kg/day 11% and 12%, respectively; 200 μg of PTH(1–34)/kg/day 11% and 15%, respectively).
A Tibial Shaft Fracture Sustained in Childhood or Adolescence Does Not Seem to Interfere with Attainment of Peak Bone Density
Authors: Leppälä Jari , Kannus Pekka , Sievänen Harri , Vuori Ilkka , Järvinen Markku
Published: April 2006

High peak bone mass or density in early adulthood is an important protective factor against osteoporotic fractures in later life, but it is not known whether injuries on growing bones affect the attainment of peak bone mass and density. The purpose of this study was therefore to examine with dual-energy X-ray absorptiometry the areal bone mineral density (BMD) of the injured and uninjured extremity (the femoral neck, trochanter area of the femur, distal femur, patella, proximal tibia, and distal tibia), lumbar spine, and distal radius of young adults with a history of early life tibial shaft fracture and to find out whether the fracture had affected the attainment of peak bone density of these patients. The second objective was to clarify whether any background or clinical follow-up variable would predict the BMD difference between the affected and unaffected extremity. Thus, the BMD and clinical status of 45 patients (34 men and 11 women), who had sustained a tibial shaft fracture in childhood or in adolescence (between 7 and 15 years of age) an average 11 years before the study, were examined. The results showed that the fracture had created a small but statistically significant injured-to-uninjured side BMD difference (proximal tibia −1.7%; p = 0.011, and distal tibia 2.6%; p = 0.014), while the other sites showed no significant side-to-side differences. There were neither significant differences in the spinal or radial BMDs between the patients and their age-, height-, and weight-matched healthy controls. A further analysis of the data showed that the better the muscle strength in the injured lower limb, the lower the side-to-side BMD deficit in the proximal tibia of the same limb (r = 0.51; p < 0.001). Smoking had a significant association with the relative BMD in the injured distal tibia (mean injured-to-uninjured side BMD difference: smokers 6.1% vs. nonsmokers −0.6%, p = 0.016). Also patient's age at the time of the injury showed an association: the younger the patient at the time of the injury, the lower the side-to-side BMD deficit in the injured distal tibia (r = −0.35; p = 0.048). In conclusion, this study indicates that early life tibial fracture leads to a small long-term BMD deficit in the fractured bone while the other sites of the skeleton seem not to be affected. Thus, a tibial shaft fracture sustained in childhood or adolescence seems to only marginally interfere the attainment of peak bone density, the important predictor of the osteoporotic fractures in later life.
Collagen Integrin Receptors Regulate Early Osteoblast Differentiation Induced by BMP-2
Authors: Jikko Akitoshi , Harris Stephen E. , Chen Di , Mendrick Donna L. , Damsky Caroline H.
Published: April 2006

Studies in several cell types indicate that the actions of integrin receptors for extracellular matrix and receptors for growth factors are synergistic in regulating cellular differentiation and function. We studied the roles of the α1β1 and α2β1 integrin collagen receptors in regulating the differentiation of 2T3 osteoblastic cells in response to bone morphogenetic protein (BMP)-2. The immortalized 2T3 cell line was established from the calvaria of mice transgenic for a BMP-2 promoter driving SV40 T-antigen. These cells require exogenous BMP-2, as well as ascorbic acid and β-glycerolphosphate, for expression of a mature osteoblast phenotype and formation of a mineralized matrix. To determine how integrin receptors for collagen-I affect BMP-2 signaling, function-perturbing anti-rat α1 and/or α2 integrin subunit, or anti-type I collagen (Col-I), antibodies were added to human recombinant (hr)BMP-2–treated 2T3 cultures at confluence (C0) or at 4 or 8 days postconfluence (C4, C8). After 4 days of exposure to the antibodies, cultures were assayed for alkaline phosphatase (ALP) mRNA levels and enzyme activity and for cAMP production in response to parathyroid hormone. Addition of anti-Collagen-I or both anti–integrin-α1 and -α2 antibodies to C0 cultures blocked expression of these early osteoblast markers by more than 90%, and also blocked mineralization (0.5–1.8% control) of these cells. In all cases, adding anti-α1 or anti-α2 antibodies separately produced partial effects, while their combined effect approached that of anti-Collagen-I. When antibodies were added to more differentiated 2T3 cells, the inhibitory effects decreased. 2T3 cells carrying constitutively active BMP receptor (caBMPR-IB) showed elevated ALP activity without hrBMP-2; this constitutive activity was also suppressed by α1 and α2 integrin antibodies and by anti-Col-I antibody. Together, our data suggest that a signal(s) from collagen integrin receptors regulates the response to BMP downstream of BMPR-IB and upstream of the regulation of ALP mRNA and other early markers of osteoblast differentiation.
Mechanical Strain Stimulates Nitric Oxide Production by Rapid Activation of Endothelial Nitric Oxide Synthase in Osteocytes
Authors: Dr. Zaman G. , Pitsillides A. A. , Rawlinson S. C. F. , Suswillo R. F. L. , Mosley J. R. , Cheng M. Z. , Platts L. A. M. , Hukkanen M. , Polak J. M. , Lanyon L. E.
Published: April 2006

Previous studies have indicated that physiological levels of dynamic mechanical strain produce rapid increases in nitric oxide (NO) release from rat ulna explants and primary cultures of osteoblast-like cells and embryonic chick osteocytes derived from long bones. To establish the mechanism by which loading-induced NO production may be regulated, we have examined: nitric oxide synthase (NOS) isoform mRNA and protein expression, the effect of mechanical loading in vivo on NOS mRNA expression, and the effect of mechanical strain on NO production by bone cells in culture. Using Northern blot analyses, in situ hybridization, and immunocytochemistry we have established that the predominant NOS isoform expressed in rat long bone periosteal osteoblasts and in a distinct population of cortical bone osteocytes is the endothelial form of NOS (eNOS), with little or no expression of the inducible NOS or neuronal NOS isoforms. In contrast, in non–load-bearing calvariae there are no detectable levels of eNOS in osteocytes and little in osteoblasts. Consistent with these observations, ulnar explants release NO rapidly in response to loading in vitro, presumably through the activation of eNOS, whereas calvarial explants do not. The relative contribution of different bone cells to these rapid increases in strain-induced NO release was established by assessment of medium nitrite (stable NO metabolite) concentration, which showed that purified populations of osteocytes produce significantly greater quantities of NO per cell in response to mechanical strain than osteoblast-like cells derived from the same bones. Using Northern blot hybridization, we have also shown that neither a single nor five consecutive daily periods of in vivo mechanical loading produced any significant effect on different NOS isoform mRNA expression in rat ulnae. In conclusion, our results indicate that eNOS is the prevailing isoform expressed by cells of the osteoblast/osteocyte lineage and that strain produces increases in the activity of eNOS without apparently altering the levels of eNOS mRNA.