bluuebunny/crossref_metadata_2025 huggingface dataset:
1,25(OH)2D3 Regulates Protein Kinase C Activity Through Two Phospholipid-Dependent Pathways Involving Phospholipase A2 and Phospholipase C in Growth Zone Chondrocytes
Authors: Sylvia V. L. , Schwartz Z. , Curry D. B. , Chang Z. , Dean D. D. , Boyan B. D.
Published: April 2006

We have previously shown that 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) plays a major role in growth zone chondrocyte (GC) differentiation and that this effect is mediated by protein kinase C (PKC). The aim of the present study was to identify the signal transduction pathway used by 1,25(OH)2D3 to stimulate PKC activation. Confluent, fourth passage GC cells from costochondral cartilage were used to evaluate the mechanism of PKC activation. Treatment of GC cultures with 1,25(OH)2D3 elicited a dose-dependent increase in both inositol-1,4,5-trisphosphate and diacylglycerol (DAG) production, suggesting a role for phospholipase C and potentially for phospholipase D. Addition of dioctanoylglycerol to plasma membranes isolated from GCs increased PKC activity. Neither pertussis toxin nor choleratoxin had an inhibitory effect on PKC activity in control or 1,25(OH)2D3-treated GCs, indicating that neither Gi nor Gs proteins were involved. Phospholipase A2 inhibitors, quinacrine, OEPC (selective for secretory phospholipase A2), and AACOCF3 (selective for cytosolic phospholipase A2), and the cyclooxygenase inhibitor indomethacin decreased PKC activity, while the phospholipase A2 activators melittin and mastoparan increased PKC activity in GC cultures. Arachidonic acid and prostaglandin E2, two downstream products of phospholipase A2 action, also increased PKC activity. These results indicate that 1,25(OH)2D3-dependent stimulation of PKC activity is regulated by two distinct phospholipase-dependent mechanisms: production of DAG, primarily via phospholipase C and production of arachidonic acid via phospholipase A2.
Cathepsin K mRNA Detection Is Restricted to Osteoclasts During Fetal Mouse Development
Authors: Dr. Dodds R. A. , Connor J. R. , Drake F. , Feild J. , Gowen Maxine
Published: April 2006

We recently identified a novel cysteine protease, cathepsin K, by random sequencing of an osteoclast cDNA library, and in situ hybridization studies in adult human tissues demonstrated high and specific expression in osteoclasts. To determine whether the expression of cathepsin K mRNA during mouse embryogenesis was more widespread, cryostat sections of early (day 11–13) and late (day 15–17) mouse fetuses were analyzed by in situ hybridization. Serial cross-sections were collected through each fetus, and co-reacted for tartrate-resistant acid phosphatase (TRAP) and nonspecific esterase (NSE), selective markers for the osteoclast, and precursor cells derived from the macrophage/monocyte lineage, respectively. In the 11–13 day fetuses, cathepsin K mRNA was not expressed in any extraskeletal tissue; at this stage of embryogenesis, no osteoclasts are present. However, in the 15–17 day fetuses, a distinctive, developmental stage-dependent pattern of cathepsin K expression was observed in osteoclasts and preosteoclasts at sites of cartilage and bone modeling. Cathepsin K positive osteoclasts differentiated within a peripheral zone of the osteogenic stacked cell layer of the cartilage rudiments (prior to ossification), migrated and/or resorbed the bone collar, and invaded the cartilage core. Furthermore, following the invasive penetration of vasculature into the degenerating cartilage core, the calcified cartilage was resorbed by cathepsin K positive mononuclear osteoclast precursors (NSE+ve, negligible TRAP); cells positive for both enzymes were identified indicative of osteoclast differentiation. The deposition of bone by osteoblasts onto the cartilage remnants is followed by mononucleated and multinucleated osteoclastic resorption; these osteoclasts demonstrated intense cathepsin K expression. Similar expression patterns were observed at sites of intramembranous ossification. No expression was observed in chondrocytes, osteoblasts, marrow, or in any other nonskeletal tissue at these time points. These data indicated that cathepsin K expression during embryogenesis occurred only following the onset of osteoclast differentiation.
Wortmannin Inhibits Spreading and Chemotaxis of Rat Osteoclasts In Vitro
Authors: Pilkington Mary F. , Sims Stephen M. , Dr. Dixon S. Jeffrey
Published: April 2006

Wortmannin (WT) and 17β–hydroxywortmannin (HWT), which are inhibitors of phosphatidylinositol-3(OH)-kinase (PI3K), have been shown previously to inhibit bone resorption in vitro and in vivo, possibly by interfering with formation of the osteoclast ruffled border. Since migration of osteoclasts also plays an important role in the process of bone resorption, we investigated the effects of these inhibitors on osteoclast morphology and motility. Both HWT and WT caused a sustained decrease in the planar area of osteoclasts in vitro (half maximal effect at 25 and 165 nM, respectively), with the effect of HWT on cell area more readily reversible than WT. These agents also caused accumulation of intracellular vesicles. Time-lapse video microscopy was used to record the migration of osteoclasts in response to macrophage colony-stimulating factor (M-CSF) or vehicle, flowing passively from a micropipette positioned 200–400 μm from the cell. M-CSF caused directed migration of osteoclasts, indicating chemotaxis (over 3 h osteoclasts migrated 96 ± 14 μm in response to M-CSF vs. 11 ± 2 μm in control experiments). Both WT (100 or 500 nM) and LY294002 (100 μM), a specific PI3K inhibitor structurally unrelated to WT, significantly inhibited osteoclast chemotaxis in response to M-CSF. Taken together, these effects of WT, HWT, and LY294002 are consistent with an important role for PI3K in regulating cytoskeletal function in osteoclasts. The inhibitory effects of WT and HWT on bone resorption may be due, in part, to impairment of osteoclast motility.
Bone Mineral Density and Its Change in White Women: Estrogen and Vitamin D Receptor Genotypes and Their Interaction
Authors: Willing Marcia , Sowers Maryfran , Aron David , Clark M. K. , Burns Trudy , Bunten Carol , Crutchfield Mary , D'Agostino Danielle , Jannausch Mary
Published: April 2006

Low bone mineral density (BMD) is a major risk factor for development of osteoporosis; increasing evidence suggests that attainment and maintenance of peak bone mass as well as bone turnover and bone loss have strong genetic determinants. We examined the association of BMD levels and their change over a 3-year period, and polymorphisms of the estrogen receptor (ER), vitamin D receptor (VDR), type I collagen, osteonectin, osteopontin, and osteocalcin genes in pre- and perimenopausal women who were part of the Michigan Bone Health Study, a population-based longitudinal study of BMD. Body composition measurements, reproductive hormone profiles, bone-related serum protein measurements, and life-style characteristics were also available on each woman. Based on evaluation of women, ER genotypes (identified by PvuII [n = 253] and XbaI [n = 248]) were significantly predictive of both lumbar spine (p < 0.05) and total body BMD level, but not their change over the 3-year period examined. The VDR BsmI restriction fragment length polymorphism was not associated with baseline BMD, change in BMD over time, or any of the bone-related serum and body composition measurements in the 372 women in whom it was evaluated. Likewise, none of the other polymorphic markers was associated with BMD measurements. However, we identified a significant gene × gene interaction effect (p < 0.05) for the VDR locus and PvuII (p < 0.005) and XbaI (p < 0.05) polymorphisms, which impacted BMD levels. Women who had the (−/−) PvuII ER and bb VDR genotype combination had a very high average BMD, while individuals with the (−/−) PvuII ER and BB VDR genotype had significantly lower BMD levels. This contrast was not explained by differences in serum levels of osteocalcin, parathyroid hormone, 1,25-dihydroxyvitamin D, or 25-dihydroxyvitamin D. These data suggest that genetic variation at the ER locus, singly and in relation to the vitamin D receptor gene, influences attainment and maintenance of peak bone mass in younger women, which in turn may render some individuals more susceptible to osteoporosis than others.
Systemic Over-Secretion of Growth Hormone in Transgenic Mice Results in a Specific Pattern of Skeletal Modeling and Adaptation
Authors: Tseng Kuo-Fung , Goldstein Steven A.
Published: April 2006

The effects of growth hormone (GH) over-secretion on bone modeling and remodeling in transgenic mice were investigated using systematic and hierarchical methods. The results showed that continual systemic stimulation of GH over-secretion in mice resulted in an initial increase in bone mass, but its bone quality was not comparable with that of the littermate controls. Further continual over-stimulation of GH resulted in an increase in bone resorption leading to thinner cortices with inferior tissue mechanical properties. Three biological mechanisms following GH over-production are formulated to explain the observed results: GH over-secretion stimulated the production of inferior bone matrices, i.e., woven bone and cartilage, in addition to the production of normal lamellar bone; the increased bone formation stimulated by GH was followed by an exaggerated bone resorption process, resulting in cortical tissue with inferior mechanical properties; and a cortical shift occurred following GH over-stimulation that appeared to be caused by the combined effects of GH over-secretion and a variation in mechanical stimuli in the metallothionein-1 growth hormone regulating hormone transgenic mice. An “Optimal Strain Environment” model is proposed to help explain the observed results. From the results of the present and previous studies, we believe that the primary goal of bone adaptation to a biological perturbation, at least in mice, is to ensure proper mechanical function by maintaining structural integrity. In the process of maintaining a targeted whole bone structural integrity, the mechanical integrity of tissue may be sacrificed.
Collagen-Related Markers of Bone Turnover Reflect the Severity of Liver Fibrosis in Patients with Primary Biliary Cirrhosis
Authors: Guañabens Nuria , Parés Albert , Alvarez Luisa , De Osaba Ma Jesús Martínez , Monegal Ana , Peris Pilar , Ballesta Antonio M. , Rodés Joan
Published: April 2006

The influence of a nonskeletal disease with increased connective tissue synthesis or degradation in the collagen-related markers of bone turnover has been evaluated in 34 women with primary biliary cirrhosis (PBC; age range 41–81 years), a disease with increased hepatic fibrosis, often associated with osteoporosis. Serum osteocalcin (BGP), and carboxy-terminal (PICP) and amino-terminal (PINP) propeptides of type I collagen were assessed as indexes of bone formation, whereas serum tartrate-resistant acid phosphatase (TRAP), and cross-linked carboxy-terminal telopeptide of type I collagen (ICTP), and urinary hydroxyproline (HYP), pyridinoline (PYR), deoxypyridinoline (DPYR), and type I collagen cross-linked N- (NTX) and C-telopeptide (CTX) were measured as markers of bone resorption. The histologic stage of the disease and serum amino-terminal propeptide of type III collagen (PIIINP) as an index of liver fibrogenesis were also evaluated. BGP levels were significantly lower, whereas PICP and PINP levels were higher in patients than in controls. Among the bone resorption markers, serum ICTP and urinary PYR, DPYR, HYP, NTX, and CTX levels were significantly higher in patients than in controls. Serum PIIINP levels were also increased in PBC patients. BGP did not correlate with PICP and PINP, but these markers of bone formation as well as ICTP, PYR, DPYR, and NTX correlated with serum PIIINP levels. Serum TRAP did not correlate with collagen-related markers of bone resorption. Moreover, patients with PIIINP and bilirubin above normal levels had higher PICP, PINP, ICTP PYR, DPYR, CTX, and NTX. These markers correlated with the histologic stage of the disease, but not with osteopenia measured by densitometric procedures in 22 patients. In conclusion, collagen-related markers of bone turnover do not reflect bone remodeling in PBC. The close association of these markers with PIIINP and the clinical and histologic stage of the liver disease suggests that they are influenced by liver collagen metabolism.
Collagen Iα1 Sp1 Polymorphism, Bone Mass, and Bone Turnover in Healthy French Premenopausal Women: The OFELY Study
Authors: Garnero P. , Borel O. , Grant S. F. A. , Ralston S. H. , Delmas P. D.
Published: April 2006

Bone mineral density (BMD) is under strong genetic control. Recent work has suggested that a polymorphism affecting an Sp1 binding site in the collagen I (COLI) A1 gene is associated with BMD and vertebral fracture in postmenopausal women. We analyzed this polymorphism in relation to BMD and bone turnover in 220 healthy premenopausal women aged 31–57 years. There were 61% SS homozygotes, 35% Ss heterozygotes, and 4% ss homozygotes, genotype frequencies similar to those previously reported in other Caucasian populations. Women in the three genotype groups were matched for age, body weight, physical activity, smoking habits, and oral contraceptive use, but height was greatest in the SS group and lowest in the ss group (p = 0.03). Between-group comparisons by analysis of variance (ANOVA) showed that COLI A1 genotype was significantly associated with spine BMD (p = 0.05), total body BMD (p = 0.046), and total body bone mineral content (BMC) (p = 0.02), but the differences between extreme genotypes were small (4, 5, and 10%, for spine BMD, total body BMD, and total body BMC, respectively). After adjustment for height, the differences between genotypes decreased and were no longer significant by ANOVA (p = 0.08, 0.17, and 0.33 for spine BMD, total body BMD, and total body BMC). Furthermore, no significant difference between genotypes was observed for femoral neck, trochanter, Ward's triangle, or forearm BMD. COLI A1 genotype was associated with serum C-terminal extension propeptide of type I collagen (p = 0.04), with lowest levels in ss individuals, but not with any other marker of bone formation (osteocalcin, alkaline phosphatase, and type I collagen N-terminal extension propeptide) or bone resorption (urinary excretion of type I collagen C and N telopeptide breakdown products). The COLI A1 Sp1 polymorphism is associated with height, peak total body BMD and BMC, and spine BMD. The genotype-specific differences account for only a small proportion of variance in BMD at these sites and are not significant after adjustment for height, suggesting that part of the effect on bone mass may be due to differences in body size. Our data support the view that COLI A1 may be a candidate gene for regulation of bone mass, but our results must be treated with caution, in view of the small number of ss individuals, and will require confirmation in larger studies.
The Relationship Between Three-Dimensional Connectivity and the Elastic Properties of Trabecular Bone
Authors: Kinney J. H. , Ladd A. J. C.
Published: April 2006

A finite-element model was used to explore the relationship between connectivity density and the elastic modulus of trabecular bone. Six cubic specimens of trabecular bone, three prepared from human distal radii and three from L1 vertebrae, were imaged with synchrotron microtomography. The three-dimensional images were reconstructed into binary volumes of mineralized bone and soft tissue, and incorporated into the finite-element model. The relationship between three-dimensional connectivity and elastic modulus was explored by uniform thinning (atrophy) and thickening (recovery) of the trabecular bone. Though no functional relationship was found between connectivity and elastic modulus, there was a linear relationship, after a full cycle of atrophy and recovery, between the loss of elastic modulus and the overall loss of connectivity. The results indicate that recovery of mechanical function depends on preserving or restoring trabecular connectivity.
Evidence That Ibuprofen Antagonizes Selective Actions of Estrogen and Tamoxifen on Rat Bone
Authors: Sibonga Jean D. , Bell Norman H. , Turner Russell T.
Published: April 2006

Studies were performed to determine if the nonsteroidal anti-inflammatory drug ibuprofen alters bone and mineral metabolism in female rats. In experiment 1, four groups of growing rats underwent either sham operation or ovariectomy (OVX). One week later, controlled-release pellets with ibuprofen or placebo were implanted subcutaneously at the back of the neck. Following 3 weeks of treatment, rats were sacrificed and blood and bone samples were removed for serum assays and histomorphometric analysis. Body growth rate and the static cortical bone measurements made at the tibial diaphysis did not change in response to OVX. OVX, however, did increase radial bone growth, lowered serum 17β-estradiol, reduced uterine weight, and decreased the cancellous bone area of the tibial metaphysis in the rats. Ibuprofen did not alter serum 17β-estradiol or uterine weight but reduced radial bone growth as well as cancellous bone area of the tibial metaphysis in both sham-operated and OVX animals. In experiments 2 and 3, we tested the influence of ibuprofen on the effects of the tissue-selective estrogen agonist tamoxifen and of exogenous 17β-estradiol in the OVX rat. Ibuprofen completely blocked the effects of tamoxifen and partially blocked the effects of 17β-estradiol to prevent cancellous osteopenia. In contrast, ibuprofen did not influence the effects of tamoxifen and 17β-estradiol to reduce radial bone growth. Besides the skeletal effects, ibuprofen suppressed estrogen-induced uterine growth. Our data suggest that ibuprofen blocks selective estrogen receptor-mediated activities in the rat.
Accretion of Bone Mass and Strength with Parathyroid Hormone Prior to the Onset of Estrogen Deficiency Can Provide Temporary Beneficial Effects in Skeletally Mature Rats
Authors: Shen V. , Birchman R. , Liang X. G. , Wu D. D. , Dempster D. W. , Lindsay R.
Published: April 2006

Intermittent administration of parathyroid hormone (PTH) has been shown to be an anabolic agent for animal and human skeletons. In previous studies, PTH has been used concurrent with, or subsequent to, the onset of bone loss. However, it is entirely possible that PTH may be used as an anabolic agent in a situation where there is stable skeletal remodeling. Increasing bone mass at this time might confer long-lasting beneficial effects when bone loss begins, for example, subsequent to the loss of ovarian function. To test this hypothesis, we evaluated the effects of administering rat PTH(1–34) (80 μg/kg/day, subucutaneously [sc]) to 6-month-old rats for a 2-week period prior to ovariectomy, and followed the natural occurrence of bone loss over a 14-week period. To determine the effects of estrogen intervention on bone gained by PTH treatment, one group was repleted with 17β-estradiol (10 μg/kg/day via sc implant). Serial measurements of bone mass in vivo at the distal femur were obtained at 2-week intervals using dual-energy X-ray absorptiometry, while histologic and mechanical strength data were obtained from excised proximal tibiae and distal femurs after sacrifice. Two weeks of PTH treatment resulted in an increase of bone mineral density (BMD), mechanical strength, and cancellous bone volume (CnBV/TV). Four weeks after PTH withdrawal, significant residual beneficial effects on BMD and strength, irrespective of ovarian status, were observed. However, 14 weeks after PTH withdrawal, although there were still residual effects on CnBV/TV in ovariectomized animals pretreated with PTH, the PTH effects on BMD and mechanical strength had been lost. Estradiol repletion during the rapid bone loss phase following ovariectomy prevented the reduction in BMD associated with either ovariectomy or PTH withdrawal. Our results suggest that: treatment of rats with PTH prior to ovariectomy produces an increase in BMD and strength, these beneficial effects extend for a period of at least three times the treatment duration, the BMD that is lost when PTH is discontinued equates to the amount accrued during the PTH treatment, estrogen replacement can be used to maintain the bone gained as a result of PTH treatment.
The Side-to-Side Differences of Bone Mass at Proximal Femur in Female Rhythmic Sports Gymnasts
Authors: Wu J. , Ishizaki S. , Kato Y. , Kuroda Y. , Fukashiro S.
Published: April 2006

This cross-sectional study examined the side-to-side differences of the bone mineral density (BMD) at proximal femora in female rhythmic sports gymnasts (RSGs). The hypothesis on which the study is based is that gymnasts use a different leg in take-off (left leg) and in landing (right leg) and therefore differ in the loading for the left and right legs. The gymnasts made up two groups: the regular group, which consisted of 15 regular players who had trained for about 28 h/week, and the substitutes group, which consisted of 8 substitute players who had trained for about 12 h/week. The control group consisted of 10 nonathletic college women who had not participated in any kind of regular sports activity. BMD (g/cm2) was measured in three hip sites using the XR-26 dual-energy X-ray absorptiometer scanner. Muscle strength at knee extensors (EXT) and flexors (FLX) was examined using an isokinetic dynamometer (CYBEX6000), and the vertical ground reaction force was determined with a force platform during take-off and landing movements. In the regular players, the BMDs of the left leg were significantly higher than those of the right leg at the femoral necks, greater trochanters, and Ward's triangles (p < 0.01 ∼ 0.005). The side-to-side differences were 4.7 ∼ 9.6%. Regarding the strength parameters, the left side was greater than the right side significantly at EXT 60°/s (p < 0.01), although the overall side-to-side difference was small. In the substitutes, the BMDs at the three sites mentioned above were also higher in the left leg than the right, but the side-to-side difference was statistically significant only at Ward's triangles (9.3%, p < 0.05). The side-to-side difference of strength was not significant. In the controls, there were small left-to-right differences of the BMDs, ranging from −1.8 to 0.5%, which was significantly lower than in the regular players at each site. The overall average strength measurements were larger in the right leg than in the left except at the 120°/s. The side-to-side difference was statistically significant at EXT 30°/s and 60°/s (p < 0.05). The peak force was greater in take-off than in landing, and the unit time force during take-off was significantly greater than that during landing (p < 0.001). In conclusion, regarding the side-to-side difference of the BMD at proximal femora, our results demonstrate: that the left leg for take-off had higher measurements than the right leg for landing in both gymnasts' groups, which accounts for the vertical ground reaction force during take-off being greater than that during landing; that the difference in the regular players group was greater than that in the substitute group, which can be explained because the regular players practiced much more than the substitutes did; and that there was no difference in the control group.
Interleukin-1 Represses COLIA1 Promoter Activity in Calvarial Bones of Transgenic ColCAT Mice In Vitro and In Vivo
Authors: Harrison John R. , Kleinert Lisa M. , Kelly Penny L. , Krebsbach Paul H. , Woody Charles , Clark Stephen , Rowe David W. , Lichtler Alexander C. , Kream Barbara E.
Published: April 2006

Interleukin-1 (IL-1) inhibits collagen synthesis in osteoblastic cell lines and primary osteoblast-like cells. However, promoter elements regulating type I collagen A1 (COLIA1) expression in vivo and in organ culture may differ from those regulating expression in cell culture. We have examined the effects of IL-1 on reporter gene activity in neonatal transgenic mouse calvariae bearing COLIA1 promoter-chloramphenicol acetyltransferase (ColCAT) fusion genes. The parent construct, ColCAT 3.6, contains 3.5 kb of 5′ flanking sequence and 115 bp of 5′ untranslated region fused to the CAT reporter. In 48-h calvarial organ cultures, IL-1 repressed ColCAT 3.6 promoter activity and collagen synthesis in a dose-related manner, with a maximal inhibition of 40–65%. This repression was retained in 5′ deletion constructs truncated to −1719 bp. The inhibition of transgene mRNA was blocked by cycloheximide, indicating a requirement for new protein synthesis. Pretreatment with indomethacin diminished the inhibitory effect of IL-1 on CAT activity and collagen synthesis, suggesting partial mediation by prostaglandins. Local in vivo injection of IL-1 (500 ng) decreased calvarial transgene mRNA after 8 h, an effect that was partially blocked by indomethacin. ColCAT transgenic mice represent a useful model for in vitro and in vivo assessment of COLIA1 promoter regulation by cytokines and other factors.
Increased Interleukin-6 Production in Mouse Osteoblastic MC3T3-E1 Cells Expressing Activating Mutant of the Stimulatory G Protein
Authors: Motomura Takashi , Kasayama Soji , Takagi Miki , Kurebayashi Shogo , Matsui Hideo , Hirose Takahisa , Miyashita Yoshihiro , Yamauchi-Takihara Keik , Yamamoto Takehisa , Okada Shintaro , Kishimoto Tadamitsu
Published: April 2006

The McCune–Albright syndrome (MAS) is characterized by polyostotic fibrous dysplasia, café-au-lait spots, and multiple endocrine hyperfunction. An activating missense mutation of the α subunit of the Gs protein (Gsα) was found in several affected tissues, resulting in prolonged stimulation of adenylate cyclase. Our recent study has indicated that the cells derived from the fibrous bone dysplasia tissues in MAS patients produced increased levels of interleukin-6 (IL-6), which may be responsible for the increased bone resorption in this disease. In the present investigation, to analyze the molecular mechanism of the increased IL-6 production by activating mutant Gsα in bone cells, we established mouse osteoblastic MC3T3-E1 cells stably transfected with the activating mutant Gsα expression vector. These cells showed a significant increase of intracellular cAMP levels and produced a higher amount of IL-6 than the cells transfected with control vector or wild-type Gsα expression vector. Analysis of the IL-6 promoter revealed that any of the AP-1, nuclear factor (NF)-IL6, and NF-κB binding elements are important for the activating mutant Gsα-induced gene expression. Electrophoretic mobility-shift assays using nuclear extracts of the mutant Gsα-expressing cells showed that phospho(Ser133)-cAMP-responsive element binding protein (CREB), AP-1, NF-IL6, and NF-κB were increased, compared with the control cells or the wild-type Gsα-expressing cells. These results indicate that activating mutant Gsα increases the transcriptional factors binding to CRE, AP-1, NF-IL6, and NF-κB elements to induce IL-6 gene expression in the osteoblastic cells.
Mice Lacking Osteopontin Show Normal Development and Bone Structure but Display Altered Osteoclast Formation In Vitro
Authors: Rittling Susan R. , Matsumoto Hiroko N. , Mckee Marc D. , Nanci Antonio , An Xiao-Rong , Novick Kristine E. , Kowalski Aaron J. , Noda Masaki , Denhardt David T.
Published: April 2006

We have used homologous recombination in embryonic stem cells to generate mice with a targeted disruption of the osteopontin (Opn, or Spp1, for secreted phosphoprotein 1) gene. Mice homozygous for this disruption fail to express osteopontin (OPN) as assessed at both the mRNA and protein level, although an N-terminal fragment of OPN is detectable at extremely low levels in the bones of −/− animals. The Opn−/− mice are fertile, their litter size is normal, and they develop normally. The bones and teeth of animals not expressing OPN are morphologically normal at the level of light and electron microscopy, and the skeletal structure of young animals is normal as assessed by radiography. Ultrastructurally, proteinaceous structures normally rich in OPN, such as cement lines, persist in the bones of the Opn−/− animals. Osteoclastogenesis was assessed in vitro in cocultures with a feeder layer of calvarial osteoblast cells from wild-type mice. Spleen cells from Opn−/− mice cells formed osteoclasts 3- to 13-fold more frequently than did control Opn+/+ cells, while the extent of osteoclast development from Opn−/− bone marrow cells was about 2- to 4-fold more than from the corresponding wild-type cells. Osteoclast development occurred when Opn−/− spleen cells were differentiated in the presence of Opn−/− osteoblasts, indicating that endogenous OPN is not required for this process. These results suggest that OPN is not essential for normal mouse development and osteogenesis, but can modulate osteoclast differentiation.
Response of Biochemical Markers of Bone Turnover to Hormone Replacement Therapy: Impact of Biological Variability
Authors: Hannon Rosemary , Blumsohn Aubrey , Naylor Kim , Eastell Richard
Published: April 2006

Biochemical markers of bone turnover may be useful to monitor patients taking hormone replacement therapy (HRT). The aim of this study was to assess the utility of markers in monitoring HRT by comparing the response of a large panel of markers to HRT with their within subject variability. We measured the response of markers to transdermal estradiol in 11 postmenopausal women over 24 weeks. We measured the within subject variability of markers in 11 untreated healthy postmenopausal women over the same period. The mean decrease in markers of bone formation after 24 weeks treatment ranged from 19% for procollagen type I C-terminal propeptide (PICP) to 40% for procollagen type I N-terminal propeptide (PINP). The mean decrease in markers of bone resorption ranged from 10% for tartrate-resistant acid phosphatase (TRAP) to 67% for C-terminal cross-linked telopeptide. The least significant change (LSC at p < 0.05), calculated from the within subject variability in the untreated group, was used to define response. LSC for osteocalcin was 21%, bone alkaline phosphatase 28%, PICP 24%, PINP 21%, type I collagen telopeptide 28%, TRAP 17%, urinary calcium 90%, hydroxyproline 75%, total deoxypyridinoline 47%, free pyridinoline 36%, free deoxypyridinoline 26%, N-terminal cross-linked telopeptide 70%, and C-terminal cross-linked telopeptide 132%. The greatest number of responders after 24 weeks of treatment were found using PINP and osteocalcin (9 each), and free deoxypyridinoline (8 each) and total deoxypyridinoline (7 each). Lumbar spine bone mineral density defined four patients as responders. The ability to detect a response differs between markers and is not dependent on the magnitude of response to therapy.
In Vivo High Resolution MRI of the Calcaneus: Differences in Trabecular Structure in Osteoporosis Patients
Authors: Link Thomas M. , Majumdar Sharmila , Augat Peter , Lin John C. , Newitt David , Lu Ying , Lane Nancy E. , Genant Harry K.
Published: April 2006

The purpose of this study was to use high resolution (HR) magnetic resonance (MR) images of the calcaneus to investigate the trabecular structure of patients with and without osteoporotic hip fractures and to compare these techniques with bone mineral density (BMD) in differentiating fracture and nonfracture patients. Axial and sagittal HR MR images of the calcaneus were obtained in 50 females (23 postmenopausal patients with osteoporotic hip fractures and 27 postmenopausal controls). A three-dimensional gradient–echo sequence was used with a slice thickness of 500 μm and in plane resolution of 195 × 195 μm. Texture analysis was performed using morphological features, analogous to standard histomorphometry and fractal dimension. Additionally, BMD measurements of the hip (dual-energy X-ray absorptiometry) were obtained in all patients. Significant differences between both patient groups were obtained using morphological parameters and fractal dimension as well as hip BMD (p < 0.05). Odds ratios for the texture parameters apparent (app.) bone volume/total volume and app. trabecular separation were higher than for hip BMD. Receiver operator characteristic values of texture measures and hip BMD were comparable. In conclusion, trabecular structure measures derived from HR MR images of the calcaneus can differentiate between postmenopausal women with and without osteoporotic hip fractures.
Camurati-Engelmann Disease: Two Case Reports Describing Metadiaphyseal Dysplasia Associated with Cerebellar Ataxia
Authors: Kormas Nic , Diamond Terry , Shnier Ronnie
Published: April 2006

This clinical report describes two patients presenting with progressive diaphyseal dysplasia (Camurati–Engelmann Disease) and cerebellar ataxia. The clinical and magnetic resonance imaging findings of the bony and cerebellar lesions are presented.
Identification of a Membrane Receptor for 1,25-Dihydroxyvitamin D3 Which Mediates Rapid Activation of Protein Kinase C
Authors: Nemere I. , Schwartz Z. , Pedrozo H. , Sylvia V. L. , Dean D. D. , Boyan B. D.
Published: April 2006

This paper is the first definitive report demonstrating a unique membrane receptor for 1,25-dihydroxyvitamin D3(1,25(OH)2D3) which mediates the rapid and nongenomic regulation of protein kinase C (PKC). Previous studies have shown that 1,25(OH)2D3 exerts rapid effects on chondrocyte membranes which are cell maturation-specific, do not require new gene expression, and do not appear to act via the traditional vitamin D receptor. We used antiserum generated to a [3H]1,25(OH)2D3 binding protein isolated from the basal lateral membrane of chick intestinal epithelium (Ab99) to determine if rat costochondral resting zone (RC) or growth zone (GC) cartilage cells contain a similar protein and if cell maturation-dependent differences exist. Immunohistochemistry demonstrated that both RC and GC cells express the protein, but levels are highest in GC. The binding protein is present in both plasma membranes and matrix vesicles and has a molecular weight of 66,000 Da. The 66 kDa protein in GC matrix vesicles has a Kd of 17.2 fmol/ml and Bmax of 124 fmol/mg of protein for [3H]1,25(OH)2D3. In contrast, the 66 kDa protein in RC matrix vesicles has a Kd of 27.7 fmol/ml and a Bmax of 100 fmol/mg of protein. Ab99 blocks the 1,25(OH)2D3-dependent increase in PKC activity in GC chondrocytes, indicating that the 1,25(OH)2D3-binding protein is indeed a receptor, linking ligand recognition to biologic function.
Reduced Expression of Interleukin-11 in Bone Marrow Stromal Cells of Senescence-Accelerated Mice (SAMP6): Relationship to Osteopenia with Enhanced Adipogenesis
Authors: Kodama Yoshiaki , Takeuchi Yasuhiro , Suzawa Miyuki , Fukumoto Seiji , Murayama Hisashi , Yamato Hideyuki , Fujita Toshiro , Kurokawa Takahide , Matsumoto Toshio
Published: April 2006

Aging is associated with an increase in bone marrow adipose tissue and a reduction in bone turnover. The P6 strain of senescence-accelerated mice (SAM) exhibit an early decrease in bone mass with a reduction in bone remodeling. In the bone marrow, suppressed osteoblastogenesis and osteoclastogenesis with enhanced adipogenesis are observed. The present study was undertaken to clarify the mechanism of age-related changes in bone turnover using bone marrow cells from SAMP6 mice. Because interleukin (IL)-11 has been shown to potently inhibit adipogenesis and to stimulate osteoclast formation, the effect of IL-11 on the differentiation of bone marrow cells was examined. The impaired formation of both osteoblasts and osteoclasts was restored and the enhanced formation of adipocytes was suppressed by the addition of 10 pM recombinant human IL-11. Other cytokines that activate gp130 as a common signal transducer, IL-6 and leukemia inhibitory factor, did not have such effects. Sequence analysis of the entire coding region of IL-11 cDNA obtained from SAMP6 stromal cells revealed no mutations. Constitutively secreted IL-11 protein into culture media, and its mRNA expression stimulated by transforming growth factor β were reduced in stromal cells from SAMP6 compared with those in control mice. These results demonstrate that the expression of IL-11 is reduced in bone marrow cells of SAMP6 and suggest that the reduction in IL-11 actions is involved in the impairment of both osteoblastogenesis and osteoclastogenesis in these mice. There is a possibility that alterations in IL-11 actions may be associated with the age-related impairment in bone metabolism.
Calcitonin Receptor Binding Properties in Bone and Kidney of the Chicken During the Oviposition Cycle
Authors: Yasuoka Tadashi , Kawashima Mitsuo , Takahashi Tetsuya , Tatematsu Norichika , Tanaka Katuhide
Published: April 2006

The binding property of calcitonin (CT) in the membrane fraction of calvaria and kidney of egg-laying and nonlaying hens was analyzed using a [125I]CT binding assay system. Binding properties of CT receptors in both tissues satisfy the authentic criteria of a receptor-ligand interaction in terms of specificity, reversibility, and saturation. Scatchard plots revealed a single class of binding sites. Values of the equilibrium dissociation constant (Kd) and binding capacity (Bmax) in laying hens showed a decrease during the period between 3 h before and 2 h after oviposition. No change was observed in nonlaying hens. In vivo administration of 17β-estradiol or progesterone caused the decrease in Kd and Bmax values. The results suggest that the binding affinity and capacity of the CT receptor in the calvaria and the kidney of the hen may be modulated by the ovarian steroid hormone.
Variation in the Short-Term Changes in Bone Cell Activity in Three Regions of the Distal Femur Immediately Following Ovariectomy
Authors: Baldock Paul A. J. , Morris Howard A. , Need Allan G. , Moore Robert J. , Durbridge Timothy C.
Published: April 2006

The effect of ovariectomy (OVX) on cancellous bone in the rat is not uniform at all sites of the skeleton. We report variation in the short-term effects of adult OVX in three regions of the distal femur: the diaphysis (DIA), the metaphysis (META), and the epiphysis (EPI). Cancellous bone parameters were estimated in the three separate zones of the femora and compared with changes in bone cell activity, as estimated by osteoclast surface (Oc.S) and bone formation rate (BFR). Changes were studied for 30 days in a series of rats either sham-operated (Sham) or ovariectomized (OVX) at 7 months of age. Oc.S and BFR were elevated following OVX in all regions. The time course for the OVX-induced changes differed between regions: DIA, both Oc.S and BFR were elevated at day 9; META, Oc.S was also elevated at day 9, while the rise in BFR was delayed until day 21; EPI, Oc.S remained stable but increased relative to ovary-intact rats by day 18 due to reduced levels in the latter, but BFR did not rise until day 28. These changes in bone cell activity following OVX produced a 71% reduction of cancellous bone in the DIA and a 35% reduction in the META. In contrast, no OVX-induced bone loss was observed in the EPI. This study shows that bone cell activity increases in each region of the distal femur within the first 30 days following OVX, independent of bone loss. However, the time course of increased bone cell activity is not uniform. These data highlight the role of local factors in the response to ovarian hormone deficiency.
Changes in Bone Turnover Markers and Menstrual Function After Short-term Oral DHEA in Young Women with Anorexia Nervosa
Authors: Gordon Catherine M. , Grace Estherann , Jean Emans S. , Goodman Elizabeth , Crawford Margaret H. , Leboff Meryl S.
Published: April 2006

Bone loss is a serious consequence of anorexia nervosa (AN). Subnormal levels of serum dehydroepiandrosterone (DHEA) are seen in patients with AN and may be causally linked to their low bone density. We hypothesized that oral DHEA would decrease markers of bone resorption (urinary N-telopeptides [NTx]), and increase markers of bone formation (serum bone-specific alkaline phosphatase and osteocalcin [OC]). Fifteen young women (age 15–22 years) with AN were enrolled in a 3-month, randomized, double-blinded trial of 50, 100, or 200 mg of daily micronized DHEA. Blood and urinary levels of adrenal and gonadal steroids and bone turnover markers were measured. No adverse clinical side effects of DHEA were noted, and a 50 mg daily dose restored physiologic hormonal levels. At 3 months, NTx levels had decreased significantly in both the 50 mg (p = 0.018) and the 200 mg (p = 0.016) subgroups. OC levels simultaneously increased within treatment groups over time (p = 0.002). Eight out of 15 (53%) subjects had at least one menstrual cycle while on therapy. Short-term DHEA was well-tolerated and appears to normalize bone turnover in young women with AN. Resumption of menses in over half of subjects suggests that DHEA therapy may also lead to estradiol levels sufficient to stimulate the endometrium in this group of patients.
Maternal Smoking During Pregnancy, Growth, and Bone Mass in Prepubertal Children
Authors: Jones Graeme , Riley Malcolm , Dwyer Terence
Published: April 2006

There have been no studies of smoking during pregnancy and bone mineralization in children. The objective of this population-based longitudinal study was to determine whether maternal smoking during pregnancy is associated with bone mass and other growth variables in prepubertal children. We studied 330 8-year-old male and female children representing 47% of those who originally took part in a study of risk factors for Sudden Infant Death Syndrome in 1988. The main outcome measures were bone mineral density measured by a Hologic QDR2000 densitometer: birth weight, placental weight, height, and weight. Maternal smoking during pregnancy was associated with deficits in growth with these children having lower height (−1.53 cm, 95% confidence interval [CI] −3.03 to −0.03) and a trend to lower weight (−1.35 kg, 95% CI −2.75 to 0.11) at age 8. Furthermore, there was a disproportionate deficit in bone mass such that those children whose mothers smoked during pregnancy had lower size adjusted bone mass at the lumbar spine (−0.019 g/cm2, 95% CI −0.033 to −0.005) and femoral neck (−0.018 g/cm2, 95%CI −0.034 to −0.002) but not total body (−0.005 g/cm2, 95% CI −0.015 to 0.005). This association was only present for children born at term. Mothers who smoked during pregnancy also had lower placental weight (− 56 g, 95% CI −95 to −17), and further adjustment for placental weight led to nonsignificant results for smoking with both growth and bone parameters, suggesting that these associations may be mediated through placental size and function. Maternal smoking habit in 1996 was not significantly associated with bone mass at any site. In conclusion, this study has demonstrated a long-term negative association between maternal smoking during pregnancy and both growth and bone mass in children born at term, and suggests that the timing of exposure rather than the dose or duration is critical. If these associations are present in other populations and they persist until the attainment of peak bone mass, then our findings suggest that osteoporosis prevention programs should start very early in the life cycle.
Neurokinin-A in Bone and Joint Tissues: Changes in Adjuvant Arthritis
Authors: Elhassan Adlan M. , Lindgren J. U. , Hultenby K. , Adem Abdu
Published: April 2006

The localization of neurokinin A (NK-A) in the normal ankle joint of rats was investigated by an immunoelectron microscopic technique with specific antisera to NK-A. Immunoreactivity was detected in bone matrix, myelinated nerve fiber in the periosteum, and bone marrow and synovial cells. No immunoreactivity was observed in osteoblasts, osteocytes, and osteoclasts. Using radioimmunoassay (RIA), a detectable concentration of NK-A was observed in the bone marrow, periosteum, cortical bone, and ankle of normal rats. In rats with chronic adjuvant arthritis, induced by intradermal injection of mycobacterium butyricum in paraffin oil into the base of the tail, the concentrations of NK-A using RIA in ankles and spinal cords were found to be significantly increased compared with acute or control rats. There were no significant differences between the latter two. Similarly, increased NK-A labeling was observed using immunoelectron microscopy in bone matrix and bone marrow monocyte cells of the chronic arthritic rats. These findings indicate the existence of as well as a biological role of NK-A in bone and joint tissues.
Cartilage-Derived Retinoic Acid–Sensitive Protein and Type II Collagen Expression During Fracture Healing Are Potential Targets for Sox9 Regulation
Authors: Sakano Shinji , Zhu Yong , Sandell Linda J.
Published: April 2006

Cartilage-derived retinoic acid-sensitive protein (CD-RAP) and mRNA were examined in the mouse fracture model by immunohistochemistry and Northern blot analysis and compared with the expression of type II collagen. We also studied the expression of the transcription factor Sox9, reported to enhance type II collagen and CD-RAP gene expression in vitro. CD-RAP was first detected in immature chondrocytes on day 5. Intense signals for CD-RAP were found in fracture cartilage on days 7 and 9. CD-RAP decreased at the phase of endochondral ossification. Throughout fracture healing, CD-RAP was detected in cartilage and not in bone or fibrous tissue, thus CD-RAP may be a molecular marker of cartilage formation during fracture healing. Northern blot analysis revealed similar changes in CD-RAP and type II collagen mRNA levels. However, with respect to protein levels, CD-RAP decreased faster than type II collagen implying the stability is lower than type II collagen. Increased levels of Sox9 mRNA and protein were detected on day 5 and coincided with the initial increase of CD-RAP and type II collagen mRNAs. Sox9 mRNA levels declined with the progress of chondrocyte hypertrophy, followed by a concomitant decrease in CD-RAP and type II collagen mRNA levels. These changes in Sox9 expression compared with the cartilage-specific genes (CD-RAP and type II collagen) suggest that cell differentiation during fracture healing may be controlled by specific transcriptional factors which regulate phenotypic changes of the cells.
Temporal and Spatial Characterization of Regenerate Bone in the Lengthened Rabbit Tibia
Authors: Richards Mark , Goulet James A. , Schaffler Mitchell B. , Goldstein Steven A.
Published: April 2006

A rabbit model of bilateral tibial lengthening was used to investigate temporal and spatial changes in new bone volume and architecture during regenerate bone formation. Tibiae were lengthened 9.0 mm at 0.75 mm/day after a 6-day latency period. Animals were euthanized at four time points, and new bone volume and architecture within the distraction gap were assessed by microcomputed tomography and histomorphometry. New bone formation began before day 18 postsurgery and increased markedly between day 18 (completion of distraction) and day 24. This period of high bone formation activity might therefore be optimal for biologic and mechanical interventions aimed at enhancing bone regeneration. Regions of both endochondral and intramembranous bone formation were observed throughout the consolidation period. Significant increases in bone volume fraction were observed early in the consolidation period and were attributed to significant increases in trabecular thickness. This suggested that increased mineral density in the gap tissue with time was a consequence of increased osteoblast activity and associated trabecular thickening. New bone formation was shown to be highly oriented toward the distraction axis throughout lengthening. More bone formed consistently in lateral and proximal regions of the distraction gap, perhaps due to improved blood supply or progenitor cell availability in these areas. No differences in trabecular architecture were detected between regions having more or less bone volume, suggesting that bony tissue differentiation in all regions of the distraction gap was similar. Homotypical variations in measures of bone architecture were small; thus, these outcome variables seem appropriate for determining the effects of biological and mechanical interventions on bone regeneration in this animal model.