bluuebunny/crossref_metadata_2025 huggingface dataset:
F-18 NaF PET for Detection of Bone Metastases in Lung Cancer: Accuracy, Cost-Effectiveness, and Impact on Patient Management
Authors: Hetzel Martin , Arslandemir Coskun , König Hans-Helmut , Buck Andreas K , Nüssle Karin , Glatting Gerhard , Gabelmann Andreas , Hetzel JÜRgen , Hombach Vinzenz , Schirrmeister Holger
Published: April 2006

As bone metastases might be present in lung cancer despite a normal bone scan, we examined various alternatives prospectively. Positron emission tomography using F-18 sodium fluoride (PET) and single photon emission tomography (SPECT) were more sensitive than a planar bone scan. PET was more accurate with a shorter examination time than SPECT but had higher incremental costs.

Introduction: Previous studies have shown that vertebral bone metastases not seen on planar bone scans may be present on F-18 fluoride positron emission tomography (PET) scan or single photon emission computed tomography (SPECT). The purpose of this study was to measure the accuracy, clinical value and cost-effectiveness of tomographic bone imaging.

Materials and Methods: A total of 103 patients with initial diagnosis of lung cancer was prospectively examined with planar bone scintigraphy (BS), SPECT of the vertebral column and PET using F-18 sodium fluoride (F-18 PET). Receiver operating characteristic (ROC) curve analysis was used for determination of the diagnostic accuracy. A decision-analysis model and the national charge schedule of the German Hospital Association were used for determination of the cost-effectiveness.

Results: Thirteen of 33 patients with bone metastases were false negative on BS, 4 on SPECT, and 2 on F-18 PET. The area under the ROC curve was 0.771 for BS, 0.875 for SPECT, and 0.989 for F-18 PET (p < 0.05). As a result of SPECT and F-18 PET imaging, clinical management was changed in 8 (7.8%) and 10 (9.7%) patients. Compared with BS, the costs per additional correctly diagnosed patient were 1272 Euro with SPECT and 2861 Euro with F-18 PET. The threshold for the costs of F-18 PET being more cost-effective than SPECT was 345 EUR.

Conclusion: Routine performance of tomographic bone imaging improves the therapeutic strategy because of detection of otherwise missed metastases. F-18 PET is more effective than SPECT but is associated with higher incremental costs.
Evaluation of Treadmill Exercise in a Lower Body Negative Pressure Chamber as a Countermeasure for Weightlessness-Induced Bone Loss: A Bed Rest Study With Identical Twins
Authors: Smith Scott M , Davis-Street Janis E , Fesperman J Vernell , Calkins DS , Bawa Maneesh , Macias Brandon R , Meyer R Scott , Hargens Alan R
Published: April 2006

Counteracting bone loss is required for future space exploration. We evaluated the ability of treadmill exercise in a LBNP chamber to counteract bone loss in a 30-day bed rest study. Eight pairs of identical twins were randomly assigned to sedentary control or exercise groups. Exercise within LBNP decreased the bone resorption caused by bed rest and may provide a countermeasure for spaceflight.

Introduction: Bone loss is one of the greatest physiological challenges for extended-duration space missions. The ability of exercise to counteract weightlessness-induced bone loss has been studied extensively, but to date, it has proven ineffective. We evaluated the effectiveness of a combination of two countermeasures—treadmill exercise while inside a lower body negative pressure (LBNP) chamber—on bone loss during a 30-day bed rest study.

Materials and Methods: Eight pairs of identical twins were randomized into sedentary (SED) or exercise/LBNP (EX/LBNP) groups. Blood and urine samples were collected before, several times during, and after the 30-day bed rest period. These samples were analyzed for markers of bone and calcium metabolism. Repeated measures ANOVA was used to determine statistical significance. Because identical twins were used, both time and group were treated as repeated variables.

Results: Markers of bone resorption were increased during bed rest in samples from sedentary subjects, including the collagen cross-links and serum and urinary calcium concentrations. For N-telopeptide and deoxypyridinoline, there were significant (p < 0.05) interactions between group (SED versus EX/LBNP) and phase of the study (sample collection point). Pyridinium cross-links were increased above pre-bed rest levels in both groups, but the EX/LBNP group had a smaller increase than the SED group. Markers of bone formation were unchanged by bed rest in both groups.

Conclusions: These data show that this weight-bearing exercise combined with LBNP ameliorates some of the negative effects of simulated weightlessness on bone metabolism. This protocol may pave the way to counteracting bone loss during spaceflight and may provide valuable information about normal and abnormal bone physiology here on Earth.
Changes in Runx2/Cbfa1 Expression and Activity During Osteoblastic Differentiation of Human Bone Marrow Stromal Cells
Authors: Shui Chaoxiang , Spelsberg Thomas C , Riggs B Lawrence , Khosla Sundeep
Published: April 2006

Runx2/Cbfa1 has been identified as a “master gene” controlling osteoblast differentiation. However, its role in inducing the osteoblast phenotype has been characterized primarily in rodent systems. Thus, we examined Runx2/Cbfa1 messenger RNA, protein, and activity levels during osteoblastic differentiation of human bone marrow stromal (BMSC) cells. Semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis demonstrated that the expression of alkaline phosphatase and osteocalcin mRNAs increased in a time-dependent manner with the development of the osteoblast phenotype by these cells (hMS2–15). Type II Runx2/Cbfa1 messenger RNA was found to be constitutively expressed in hMS2–15 cells and not altered during differentiation; there was no detectable expression of the type I Runx2/Cbfa1 transcript. Interestingly, despite the absence of any change in Runx2/Cbfa1 messenger RNA levels during osteoblastic differentiation of these cells, the activity of Runx2/Cbfa1, as assessed by binding to the osteoblast-specific cis-acting element 2 (OSE2), increased markedly at all time-points examined, with the highest activity level seen at day 7. Similar results were observed in primary cultures of less differentiated human marrow-derived mesenchymal stem cells. Immunoprecipitation and Western blot analysis revealed that whereas there was no increase in Runx2/Cbfa1 protein levels with differentiation in hMS2–15 cells, there was an increase in Runx2/Cbfa1 phosphorylation. Thus, in contrast to rodent systems where osteoblast differentiation is associated with increased synthesis of Runx2/Cbfa1, we find that in human BMSC, osteoblastic differentiation is associated primarily with increases in Runx2/Cbfa1 activity, without a change in messenger RNA or protein levels. Our findings also show that the increase in Runx2/Cbfa1 activity occurs through a posttranslational mechanism involving phosphorylation of key residues.
Evaluation of Bone Mineral Density and Fat-Lean Distribution in Patients With Multiple Myeloma in Sustained Remission
Authors: Roux S , Bergot C , Fermand JP , Frija J , Brouet JC , Mariette X
Published: April 2006

To study the usefulness of bone mineral density (BMD) in the follow-up of myeloma (MM) patients, BMD was evaluated in 44 MM patients in sustained remission for at least 2 years (35.4 ± 10.5 months) after high-dose or conventional chemotherapy in a retrospective study. Patients never received bisphosphonates before or during the follow-up. Patients underwent lumbar spine (LS) BMD and a whole body (WB) BMD testing before therapy and at least once in the remission period. At baseline, mean LS BMD was 0.863 ± 0.026 g/cm2, mean lumbar Z-score was −1.45 SD. LS BMD significantly increased from baseline by 5 ± 1.8%, 9.3 ± 1.7%, and 14 ± 1.9% at 1, 2, and 3 years, respectively. The percentage of patients with a T-score below 2.5 SD decreased from 39% at baseline to 18.5% at 3 years. Compared with baseline, WB BMD decreased by −2.8 ± 0.5%, −2.6 ± 0.7%, and −1.7 ± 0.6% at 1, 2, and 3 years, respectively. Mean percentage change of the fat compartment increased from baseline by +28.4 ± 7.1% at the trunk, and + 17.1 ± 5% in peripheral areas at 3 years. In conclusion, in MM patients in remission after chemotherapy, LS BMD progressively increased after a mean follow-up of 3 years. These patients never received bisphosphonates, so this increase was related to the anti-myeloma treatment. The major effect on BMD was observed at the LS, which is primarily composed of trabecular bone containing the bone marrow. Interestingly, a drastic increase of the fat content was also observed. These results underlined that BMD and fat-lean evaluation could be of interest in the follow-up of MM patients.
Positive Linear Growth and Bone Responses to Growth Hormone Treatment in Children With Types III and IV Osteogenesis Imperfecta: High Predictive Value of the Carboxyterminal Propeptide of Type I Procollagen
Authors: Marini Joan C , Hopkins Elizabeth , Glorieux Francis H , Chrousos George P , Reynolds James C , Gundberg Caren M , Reing C Michael
Published: April 2006

Extreme short stature is a cardinal feature of severe osteogenesis imperfecta (OI), types III and IV. We conducted a treatment trial of growth hormone in children with OI and followed linear growth velocity, bone metabolism markers, histomorphometrics, and vertebral bone density. Twenty-six children with types III and IV OI, ages 4.5–12 years, were treated with recombinant growth hormone (rGH), 0.1–0.2 IU/kg per day for 6 days/week, for at least 1 year. Length, insulin-like growth factor (IGF-I), insulin-like growth factor binding protein (IGFBP-3), bone metabolic markers, and vertebral bone density by DXA were evaluated at 6-month intervals. An iliac crest biopsy was obtained at baseline and 12 months. Approximately one-half of the treated OI children sustained a 50% or more increase in linear growth over their baseline growth rate. Most responders (10 of 14) had moderate type IV OI. All participants had positive IGF-I, IGFBP-3, osteocalcin, and bone-specific alkaline phosphatase responses. Only the linear growth responders had a significant increase in vertebral DXA z-score and a significant decrease in long bone fractures. After 1 year of treatment, responders' iliac crest biopsy showed significant increases in cancellous bone volume, trabecular number, and bone formation rate. Responders were distinguished from nonresponders by higher baseline carboxyterminal propeptide (PICP) values (p < 0.05), suggesting they have an intrinsically higher capacity for collagen production. The results show that growth hormone can cause a sustained increase in the linear growth rate of children with OI, despite the abnormal collagen in their bone matrix. In the first year of treatment, growth responders achieve increased bone formation rate and density, and decreased fracture rates. The baseline plasma concentration of PICP was an excellent predictor of positive response.
Expression of Either NF-κB p50 or p52 in Osteoclast Precursors Is Required for IL-1-Induced Bone Resorption
Authors: Xing Lianping , Carlson Louise , Story Beryl , Tai Zhenxing , Keng Peter , Siebenlist Ulrich , Boyce Brendan F
Published: April 2006

Interleukin (IL)-1 is implicated in postmenopausal- and inflammation-mediated bone loss. Its expression is regulated by NF-κB and vice versa. To examine the role of NF-κB p50 and p52 (they are required for osteoclast formation during embryonic development) in IL-1-induced resorption, we used various NF-κB knockout (KO) mice, including p50−/− and p52−/− single KO, p50−/− and p52+/− (3/4KO), and p50−/− and p52−/− double KO (dKO) mice. IL-1 increased blood calcium and bone resorption in wild-type (wt), p50, and p52 single KO mice, but not in 3/4KO or dKO mice. Osteoclast formation was impaired in bone marrow cultures from 3/4KO compared with single KO and wt mice treated with IL-1. IL-1 receptor expression was similar in colony forming unit-granulocyte macrophage (CFU-GM) colony cells from wt and dKO mice. However, IL-1 promoted CFU-GM colony formation and survival as well as the formation, activity, and survival of osteoclasts generated from these colonies from wt mouse splenocytes, but not from dKO splenocytes. No difference in expression of the osteoclast regulatory cytokines, RANKL, and OPG, was observed in osteoblasts from wt and dKO mice. Thus, expression of either NF-κB p50 or p52 is required in osteoclasts and their precursors, rather than osteoblasts, for IL-1-mediated bone resorption.
Expression of RANKL and OPG Correlates With Age-Related Bone Loss in Male C57BL/6 Mice
Authors: Cao Jay , Venton Laura , Sakata Takeshi , Halloran Bernard P
Published: April 2006

Osteoblasts regulate the recruitment and activity of osteoclasts through expression of RANKL and osteoprotegerin (OPG). To determine whether expression of RANKL and OPG change with age and how these changes relate to the bone loss of aging, we measured bone mass and cancellous volume, and expression of RANKL, OPG, alkaline phosphatase (AP), osteocalcin (OC), and alpha I collagen (COLL) in whole bone and osteoblast-like cells in culture using 6-week- (young), 6-month- (adult), and 24-month-old (old) mice. Cancellous volume decreased by 20% from young to adult and by 52% from adult to old. RANKL mRNA levels in whole bone were 2.1-fold and 4.4-fold higher in adult and old mice, respectively, compared with young mice, whereas OPG mRNA levels decreased with age slightly. RANKL expression was negatively (r = −0.99) and OPG was positively (r = 0.92) correlated with cancellous bone volume. Expression of RANKL was higher and OPG lower in cells from older animals early in culture (day 7). With cell maturation, RANKL mRNA levels in cells from young and adult mice increased, whereas levels in cells from old animals decreased. By 21 and 28 days of culture, no differences were found in RANKL mRNA in osteoblast-like cells among different age groups. We conclude that expression of RANKL and OPG change with age in whole bone and in cultured osteoblast-like cells. These changes favor increased osteoclast over osteoblast activity, and may explain, in part, the imbalance in bone formation and resorption associated with aging.
Overexpression of RANKL Implicates IFN-β-Mediated Elimination of B-Cell Precursors in the Osteopetrotic Bone of Microphthalmic Mice
Authors: Roundy K , Smith R , Weis JJ , Weis JH
Published: April 2006

The microphthalmic (mi) mouse possesses a dominant negative mutation in the microphthalmia-associated transcript factor (MITF) transcription factor. These animals are characterized by reduced numbers of peripheral mast and natural killer (NK) cells, are osteopetrotic because of osteoclast reduction and malfunction, lack functional melanocytes, and are deficient for maturing B-cells within the bone marrow. Granulocyte precursor cells, however, are functionally maintained within the mi bone marrow. A central question has been whether the B-cell deficiency of the mi mouse marrow is caused by the absence of an MITF-controlled gene product or because of the compromised, osteopetrotic environment. In this report, we examined mi marrow by performing transcriptional mapping analyses of candidate genes whose products are instrumental for functional osteoclast and B-cell development. Surprisingly, the expression of a subset of such genes including RANKL, stromal-derived factor (SDF-1), B-cell lymphotactin chemokine (BLC), and RANK was dramatically enhanced in the mi marrow. Normal and mutant marrow were also analyzed by subtractive transcript cloning, which identified a number of known and unknown genes with altered transcriptional activity. One such unknown mouse gene possesses a human counterpart that is interferon-β (IFN-β) inducible, suggesting the osteopetrotic marrow is enriched for IFN-β, a cytokine that is known to eliminate B-cell precursors. A model is proposed suggesting excess RANKL sets off a cascade of cytokine production including IFN-β that leads to the preferential elimination of B-cell precursors in the marrow of osteopetrotic marrow.
Deficient Acquisition of Bone During Maturation Underlies Idiopathic Osteoporosis in Men: Evidence From a Three-Generation Family Study
Authors: Van Pottelbergh I , Goemaere S , Zmierczak H , De Bacquer D , Kaufman JM
Published: April 2006

To address the issue whether deficient acquisition of bone during maturation or adult-onset bone loss is primarily to blame for idiopathic osteoporosis in men, we assessed indices of bone mineral density and size, as well as biochemical markers of bone turnover in 61 probands (ages 20–65 years) with idiopathic osteoporosis (z-score ≤ −2.0 at the spine or hip), their first-degree relatives (n = 130), and age-matched controls. There was no indication of accelerated bone loss. Indeed, in probands, the observed bone deficit versus controls was unrelated to the age of probands, and indices of bone turnover were not significantly different from controls. On the other hand, a specific deficit in bone acquisition was suggested by findings of lower bone mineral density values in three generations of male and female relatives of the probands, including their offspring; bone turnover in relatives was not different from controls. The bone mineral density deficit was more pronounced in male compared with female relatives; approximately 60% of the sons had a spinal bone mineral density z-score of less than −2.0. There also was a skeletal site-specificity in probands and their male relatives with a larger areal bone mineral density deficit at the spine compared with the hip and the forearm. The deficit at the spine corresponded to a reduction of both volumetric bone mineral density and bone size; a similar less pronounced deficit in volumetric bone mineral density, but not in bone size, was observed at the femoral neck. These findings in probands and their first-degree relatives point toward a major contributory role of a genetically determined maturational defect in bone acquisition in the pathogenesis of idiopathic osteoporosis in men.
Relation of Androgen Receptor Gene Polymorphism to Bone Mineral Density and Fracture Risk in Early Postmenopausal Women During a 5-Year Randomized Hormone Replacement Therapy Trial
Authors: Salmén Timo , Heikkinen Anna-Mari , Mahonen Anitta , Kröger Heikki , Komulainen Marja , Pallonen Heli , Saarikoski Seppo , Honkanen Risto , Mäenpää Pekka H
Published: April 2006

In women, the influence of androgens on bone health is not clear. It has been suggested that the androgen receptor (AR) genotype is associated with bone mineral density and serum androgen levels in pre- and perimenopausal women, but the association between AR genotype, bone mineral density, and fracture risk has not been studied in postmenopausal women. Therefore, we studied whether AR polymorphism affects bone mineral density, bone mineral density change, or fracture risk in a 5-year randomized hormone replacement therapy (HRT) trial on 331 early postmenopausal women (mean baseline age, 52.7 ± 2.3 years). The participants consisted of two treatment groups: the HRT group (n = 151) received a sequential combination of 2 mg estradiol valerate and 1 mg cyproterone acetate with or without vitamin D3, 100–300 IU + 93 mg calcium as lactate/day, and the non-HRT group (n = 180) received 93 mg calcium alone or in combination with vitamin D3, 100–300 IU/day for 5 years. Bone mineral density was measured from lumbar spine and proximal femur (DXA) before and after the 5-year trial. All new symptomatic, radiographically defined fractures were recorded during the follow-up. The length of CAG repeat in exon 1 of AR gene was evaluated after polymerase chain reaction (PCR) amplification. The subjects were divided into three repeat groups according to AR alleles. None of the baseline characteristics were associated with AR gene polymorphism and HRT treatment. The polymorphism did not influence the calculated annual changes of lumbar or femoral neck bone mineral density during the 5-year follow-up in the HRT (p = 0.926 and 0.146, respectively) or non-HRT (p = 0.818 and 0.917, respectively) groups. In all, 28 women sustained 33 fractures during the follow-up. Thus, the numbers of fractures were limited. The AR repeat length variation was not significantly associated with fracture risk in the HRT or non-HRT groups (p = 0.632 and 0.459, respectively; Cox proportional hazards model). In conclusion, AR gene polymorphism was not associated with baseline bone mineral density, 5-year bone mineral density change, or fracture risk in early postmenopausal Finnish women.
Effects of Vitamin D and Calcium Supplementation on Falls: A Randomized Controlled Trial
Authors: Bischoff Heike A , Stähelin Hannes B , Dick Walter , Akos Regula , Knecht Margrith , Salis Christian , Nebiker Matthias , Theiler Robert , Pfeifer Michael , Begerow Bettina , Lew Robert A , Conzelmann Martin
Published: April 2006

Specific receptors for vitamin D have been identified in human muscle tissue. Cross-sectional studies show that elderly persons with higher vitamin D serum levels have increased muscle strength and a lower number of falls. We hypothesized that vitamin D and calcium supplementation would improve musculoskeletal function and decrease falls. In a double-blind randomized controlled trial, we studied 122 elderly women (mean age, 85.3 years; range, 63–99 years) in long-stay geriatric care. Participants received 1200 mg calcium plus 800 IU cholecalciferol (Cal+D-group; n = 62) or 1200 mg calcium (Cal-group; n = 60) per day over a 12-week treatment period. The number of falls per person (0, 1, 2–5, 6–7, >7 falls) was compared between the treatment groups. In an intention to treat analysis, a Poisson regression model was used to compare falls after controlling for age, number of falls in a 6-week pretreatment period, and baseline 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D serum concentrations. Among fallers in the treatment period, crude excessive fall rate (treatment − pretreatment falls) was compared between treatment groups. Change in musculoskeletal function (summed score of knee flexor and extensor strength, grip strength, and the timed up&go test) was measured as a secondary outcome. Among subjects in the Cal+D-group, there were significant increases in median serum 25-hydroxyvitamin D (+71%) and 1,25-dihydroxyvitamin D (+8%). Before treatment, mean observed number of falls per person per week was 0.059 in the Cal+D-group and 0.056 in the Cal-group. In the 12-week treatment period, mean number of falls per person per week was 0.034 in the Cal+D-group and 0.076 in the Cal-group. After adjustment, Cal+D-treatment accounted for a 49% reduction of falls (95% CI, 14–71%; p < 0.01) based on the fall categories stated above. Among fallers of the treatment period, the crude average number of excessive falls was significantly higher in the Cal-group (p = 0.045). Musculoskeletal function improved significantly in the Cal+D-group (p = 0.0094). A single intervention with vitamin D plus calcium over a 3-month period reduced the risk of falling by 49% compared with calcium alone. Over this short-term intervention, recurrent fallers seem to benefit most by the treatment. The impact of vitamin D on falls might be explained by the observed improvement in musculoskeletal function.
Identification of a Novel Tandem Duplication in Exon 1 of the TNFRSF11A Gene in Two Unrelated Patients With Familial Expansile Osteolysis
Authors: Johnson-Pais Teresa L , Singer Frederick R , Bone Henry G , McMurray Cynthia T , Hansen Marc F , Leach Robin J
Published: April 2006

Familial expansile osteolysis (FEO) is a rare autosomal dominant disorder characterized by striking focal expansile osteolytic bone lesions and generalized osteopenia, often accompanied by characteristic early hearing loss and dental disease. The TNFRSF11A gene encodes the receptor activator of nuclear factor-κB (RANK), which has been demonstrated to be essential in bone remodeling and osteoclast differentiation. Identical insertional mutations in the first exon of RANK have been identified in all published FEO kindreds. The mutation is an 18 base pair tandem duplication in the sequence coding for the signal peptide of RANK, which causes an increase in NF-κB signaling. We report the identification and mutational analysis of two unrelated FEO patients. One had no family history of FEO, but presented with bilateral hearing loss at an early age, deterioration of teeth, and severe pain and swelling in the distal tibia before the age of 20. The second patient had a family history of FEO and exhibited an extensive expansile tibial lesion and lesions in one humerus and a phalanx. She also had early hearing loss and dental disease. Mutational analysis of the TNFRSF11A gene in our patients demonstrated an 18 base pair tandem duplication, one base proximal to the duplications previously reported. This novel mutation results in addition of the same six amino acids to the RANK signal peptide that has been observed previously. Further analysis of the exon 1 sequence demonstrated that it has the ability to form a stable secondary structure that may facilitate the generation of tandem duplications.
Reduced Levels of MMP-2 and TIMP-1 in Dyssegmental Dysplasia
Authors: Uchide Kiyoshi , Ueno Hirohisa , Takizawa Noboru , Okada Yasunori
Published: April 2006

Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) were measured in a mild case of dyssegmental dysplasia. X-ray pictures of a female baby born vaginally at 39 weeks of gestation showed short, bent, dumbbell-shaped long bones of the limbs and profound dyssegmental ossification in the spine, findings characteristic of dyssegmental dysplasia. When the levels of MMP-1, MMP-2, MMP-9, TIMP-1, and TIMP-2 were measured, the levels of MMP-2 and TIMP-1 were significantly reduced. This case might provide a clue to disclose the etiology of dyssegmental dysplasia.
Autosomal Dominant Gnathodiaphyseal Dysplasia Maps to Chromosome 11p14.3-15.1
Authors: Tsutsumi Satoshi , Kamata Nobuyuki , Maruoka Yutaka , Ando Miki , Tezuka Osamu , Enomoto Shoji , Omura Ken , Nagayama Masaru , Kudo Eiji , Moritani Maki , Yamaoka Takashi , Itakura Mitsuo
Published: April 2006

Gnathodiaphyseal dysplasia (GDD) is a syndrome characterized by bone fragility, sclerosis of tubular bones, and cemento-osseous lesions of jawbones. Although some cases of this syndrome exist in families with autosomal dominant inheritance, the underlying gene has never been identified. We analyzed a large four-generation family with GDD by linkage analysis using genomic DNA from nine affected and six nonaffected family members. A genome-wide search using a set of highly polymorphic microsatellite markers showed evidence for linkage to chromosome 11p14.3-15.1. Two-point linkage analysis of microsatellite markers spanning this locus resulted in a maximum logarithm of odds (LOD) score of 2.70 with a recombination fraction (θ) of 0 at D11S1755, D11S1759, and D11S915, and a maximum LOD score of 3.01 at D11S4114 was obtained in multipoint linkage analysis. Haplotype analysis detected no recombination between GDD and six closely linked markers (D11S928, D11S1755, D11S4114, D11S1759, D11S915, and D11S929) and established the candidate interval of 8.7 cM on chromosome 11p for GDD. Although GDD has been considered to be a variation of osteogenesis imperfecta (MIM 166260), our results indicate that this syndrome is a new and distinct disease entity from other systemic bone diseases. Furthermore, these genetic markers are useful for presymptomatic diagnosis of GDD in some families and for identification of the GDD gene.
The Pseudohypoparathyroidism Type 1b Locus Is Linked to a Region Including GNAS1 at 20q13.3
Authors: Jan De Beur Suzanne M , O'Connell Jeffery R , Peila Rita , Cho Justin , Deng Zhichao , Kam Stephen , Levine Michael A
Published: April 2006

Pseudohypoparathyroidism (PHP) is characterized by biochemical hypoparathyroidism with elevated parathyroid hormone levels owing to reduced target tissue responsiveness to parathyroid hormone. Patients with PHP 1a have somatic defects termed Albright's hereditary osteodystrophy (AHO) and exhibit resistance to additional hormones because of heterozygous mutations in the GNAS1 gene that lead to a generalized deficiency of the α subunit of Gs, the heterotrimeric G protein that couples receptors to adenylyl cyclase. By contrast, patients with PHP 1b lack AHO and have selective parathyroid hormone (PTH) resistance, presumably because of an imprinting defect that impairs expression of Gsα in the proximal renal tubule. Although an epigenetic defect in GNAS1 has been identified in subjects with PHP1b, the genetic defect is unknown. To define the genetic defect in PHP 1b, we performed a genome-wide linkage analysis in five multi-generational PHP 1b families. Of the 408 polymorphic microsatellite markers examined, markers located on chromosome 20q13.3, the region containing GNAS1, demonstrated linkage to PHP 1b. Fine-mapping and multipoint linkage analysis of this region demonstrated linkage to a 5.7-cM region between 907rep2 and the telomere. Haplotype analysis established that affected individuals shared a 5-cM region including part of the GNAS1 gene to the telomere. Our data confirm that PHP1b is linked to a region that includes GNAS1, and further refine the locus, although the primary genetic mutation(s) that causes defective imprinting of GNAS1 remains undefined.
TRAF5 Functions in Both RANKL- and TNFα-Induced Osteoclastogenesis
Authors: Kanazawa Kiyoshi , Azuma Yoshiaki , Nakano Hiroyasu , Kudo Akira
Published: April 2006

Although TRAF6 is essential for both RANKL- and TNFα-induced osteoclastogenesis, it has remained unclear whether other members of the TRAF family are involved in osteoclastogenesis. We examined TRAF5 function in both RANKL- and TNFα-induced osteoclastogenesis by using osteoclast progenitor cells from TRAF5-deficient mice. The results demonstrated that RANKL or TNFα did not effectively induce osteoclast differentiation from osteoclast progenitor cells derived from these mice into mature multinucleated osteoclasts, although c-jun N-terminal kinase (JNK) and NF-κB activation was apparently observed in osteoclast progenitor cells. In the parathyroid hormone (PTH)-induced hypercalcemia model, calcium concentration peaked at day 3 after administration. However, in TRAF5-deficient mice, this peak was delayed and found at day 5, showing less effective osteoclast differentiation. Thus, we have provided the first evidence showing that TRAF5 is involved in osteoclastogenesis.
Cytokine Regulation and the Signaling Mechanism of Osteoclast Inhibitory Peptide-1 (OIP-1/hSca) to Inhibit Osteoclast Formation
Authors: Koide Masanori , Maeda Hidefumi , Roccisana Jennifer L , Kawanabe Noriaki , Reddy Sakamuri V
Published: April 2006

The osteoclast (OCL) is the primary bone resorbing cell. OCL formation and activity is regulated by local factors produced in the bone microenvironment. We recently identified OCL inhibitory peptide-1 (OIP-1/hSca) as a novel inhibitor of OCL formation and bone resorption that is produced by OCLs. OIP-1 is a glycosylphosphatidyl-inositol (GPI)-linked membrane protein (16 kDa) related to the mouse Ly-6 family of hematopoietic proteins. OIP-1 mRNA is expressed in human OCL precursors, granulocyte-macrophage colony-forming unit (GM-CFU), bone marrow cells, and osteoblast cells. We used cycle-dependent reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, which further demonstrated that interferon-γ (IFN-γ) strongly enhanced OIP-1/hSca mRNA expression in bone marrow cells and GM-CFU. Similarly, interleukin (IL)-1β also enhanced OIP-1 mRNA expression in GM-CFU. To determine the participation of OIP-1 in IFN-γ inhibition of OCL formation, we tested the capacity of a neutralizing antibody specific to OIP-1 c-peptide to inhibit IFN-γ's effects on OCL-like cell differentiation of mouse macrophages, RAW 264.7 cells. Anti-OIP-1 c-peptide specific antibody partially neutralized IFN-γ inhibition of OCL differentiation. Furthermore, OIP-1 inhibited phospho-c-Jun (p-c-Jun) kinase activity in RAW 264.7 cells. However, OIP-1/hSca did not affect NF-κB activation in these cells. Western blot analysis further demonstrated that OIP-1 significantly decreased TNF receptor associated factor 2 (TRAF-2) expression in RAW 264.7 cells. However, OIP-1 had no effect on TRAF-6 expression in these cells. These data show that IFN-γ enhances OIP-1/hSca expression in OCL precursors, GM-CFU, and that OIP-1 inhibits OCL formation through suppression of TRAF-2 and p-c-Jun kinase activity.
Functional Domains for Amelogenin Revealed by Compound Genetic Defects
Authors: Paine Michael L , Luo Wen , Zhu Dan-Hong , Bringas Pablo , Snead Malcolm L
Published: April 2006

We have previously used the yeast two-hybrid assay and multiple in vitro methodologies to show that amelogenin undergoes self-assembly involving two domains (A and B). Using transgenic animals, we show that unique enamel phenotypes result from disruptions to either the A- or B-domain, supporting the role of amelogenin in influencing enamel structural organization. By crossbreeding, animals bearing two defective amelogenin gene products have a more extreme enamel phenotype than the sum of the defects evident in the individual parental lines. At the nanoscale level, the forming matrix shows alteration in the size of the amelogenin nanospheres. At the mesoscale level of enamel structural hierarchy, 6-week-old enamel exhibits defects in enamel rod organization caused by perturbed organization of the precursor organic matrix. These studies reflect the critical dependency of amelogenin self-assembly to form a highly organized enamel organic matrix, and that amelogenins engineered to be defective in self-assembly produce compound defects in the structural organization of enamel.
The Effects of Tamoxifen and Toremifene on Bone Cells Involve Changes in Plasma Membrane Ion Conductance
Authors: Lehenkari Petri , Parikka Vilhelmiina , Rautiala Timo J , Weckström Matti , Dahllund Johanna , Härkönen Pirkko L , Väänänen H Kalervo
Published: April 2006

Selective estrogen receptor modulators (SERMs), tamoxifen (Tam) and toremifene (Tor), are widely used in the treatment of breast cancer. In addition, they have been demonstrated to prevent estrogen deficiency-induced bone loss in postmenopausal women. These effects are thought to be caused by the interaction of the SERMs with the estrogen receptor, although SERMs have also been shown to conduct non-receptor-mediated effects such as rapid changes in membrane functions. We compared the effects of Tam, Tor, and 17β-estradiol (E2) on the viability of rat osteoclasts and osteoblasts. Both Tam and Tor were found to cause osteoclast apoptosis in in vitro cultures, which was reversed by E2. In addition, at higher concentration (10 μM), both SERMs had an estrogen receptor-independent effect, which involved interaction with the plasma membrane as demonstrated with UMR-108 osteosarcoma cells by Tam and Tor, but not E2. A leak of protons leading to changes in intracellular pH was shown both in medullary bone derived membrane vesicles and in intact cells. These effects were followed by a rapid loss of cell viability and subsequent cell lysis. Our results show that both Tam and Tor have an ionophoric effect on the plasma membranes of bone cells and that these SERMs differed in this ability: Tor induced rapid membrane depolarization only in the presence of high concentration of potassium. These non-receptor-mediated effects may be involved in therapeutic responses and explain some clinical side effects associated with the treatment of patients with these SERMs.
Effects of Teriparatide [Recombinant Human Parathyroid Hormone (1–34)] on Cortical Bone in Postmenopausal Women With Osteoporosis
Authors: Zanchetta JR , Bogado CE , Ferretti JL , Wang O , Wilson MG , Sato M , Gaich GA , Dalsky GP , Myers SL
Published: April 2006

Treatment with teriparatide (rDNA origin) injection {teriparatide, recombinant human parathyroid hormone (1–34) [rhPTH(1–34)]}reduces the risk of vertebral and nonvertebral fragility fractures and increases cancellous bone mineral density in postmenopausal women with osteoporosis, but its effects on cortical bone are less well established. This cross-sectional study assessed parameters of cortical bone quality by peripheral quantitative computed tomography (pQCT) in the nondominant distal radius of 101 postmenopausal women with osteoporosis who were randomly allocated to once-daily, self-administered subcutaneous injections of placebo (n = 35) or teriparatide 20 μg (n = 38) or 40 μg (n = 28). We obtained measurements of moments of inertia, bone circumferences, bone mineral content, and bone area after a median of 18 months of treatment. The results were adjusted for age, height, and weight. Compared with placebo, patients treated with teriparatide 40 μg had significantly higher total bone mineral content, total and cortical bone areas, periosteal and endocortical circumferences, and axial and polar cross-sectional moments of inertia. Total bone mineral content, total and cortical bone areas, periosteal circumference, and polar cross-sectional moment of inertia were also significantly higher in the patients treated with teriparatide 20 μg compared with placebo. There were no differences in total bone mineral density, cortical thickness, cortical bone mineral density, or cortical bone mineral content among groups. In summary, once-daily administration of teriparatide induced beneficial changes in the structural architecture of the distal radial diaphysis consistent with increased mechanical strength without adverse effects on total bone mineral density or cortical bone mineral content.
Osteoblast-Specific Targeting of Soluble Colony-Stimulating Factor-1 Increases Cortical Bone Thickness in Mice
Authors: Abboud SL , Ghosh-Choudhury N , Liu C , Shen V , Woodruff K
Published: April 2006

The soluble and membrane-bound forms of CSF-1 are synthesized by osteoblasts and stromal cells in the bone microenvironment. Transgenic mice, generated to selectively express sCSF-1 in bone, showed increased cortical thickness in the femoral diaphysis caused by new bone formation along the endosteal surface. The ability of sCSF-1 to enhance bone cell activity in vivo is potentially relevant for increasing cortical bone in a variety of disorders.

Introduction: The soluble form of colony-stimulating factor-1 (sCSF-1) and the membrane-bound form of CSF-1 (mCSF-1) have been shown to support osteoclastogenesis in vitro; however, the effect of each peptide on bone remodeling in vivo is unclear. To determine the effect of sCSF-1, selectively expressed in bone, the skeletal phenotype of transgenic mice harboring the human sCSF-1 cDNA under the control of the osteocalcin promoter was assessed.

Methods: At 5 and 14 weeks, mice were analyzed for CSF-1 protein levels, weighed, and X-rayed, and femurs were removed for peripheral quantitative computed tomography, histology, and histomorphometry.

Results: High levels of human sCSF-1 were detected in bone extracts and, to a lesser extent, in plasma. Adult transgenic mice showed normal body weight and increased circulating monocytic cells. At 5 weeks, the femoral diaphysis was similar in CSF-1T and wt/wt littermates. However, by 14 weeks, the femoral diaphysis in CSF-1T mice showed increased cortical thickness and bone mineral density. In contrast to the diaphysis, the femoral metaphysis of CSF-1T mice showed normal cancellous bone comparable with wt/wt littermates at each time point. Histological sections demonstrated increased woven bone along the endosteal surface of the diaphysis and intracortical remodeling. Fluorochrome-labeling analysis confirmed endocortical bone formation in CSF-1T, with a 3.1-fold increase in the percentage of double-labeled surfaces and a 3.6-fold increase in the bone formation rate compared with wt/wt mice. Although remodeling resulted in a slightly porous cortex, sCSF-1 preferentially stimulated endocortical bone formation, leading to increased cortical thickness.

Conclusions: These findings indicate that sCSF-1 is a key determinant of bone cell activity in the corticoendosteal envelope.
p300/CBP Acts as a Coactivator to Cartilage Homeoprotein-1 (Cart1), Paired-Like Homeoprotein, Through Acetylation of the Conserved Lysine Residue Adjacent to the Homeodomain
Authors: Iioka Takashi , Furukawa Keizo , Yamaguchi Akira , Shindo Hiroyuki , Yamashita Shunichi , Tsukazaki Tomoo
Published: April 2006

The paired-like homeoprotein, Cart1, is involved in skeletal development. We describe here that the general coactivator p300/CBP controls the transcription activity of Cart1 through acetylation of a lysine residue that is highly conserved in other homeoproteins. Acetylation of this residue increases the interaction between p300/CBP and Cart1 and enhances its transcriptional activation.

Introduction: Cart1 encodes a paired-like homeoprotein expressed selectively in chondrocyte lineage during embryonic development. Although its target gene remains unknown, gene disruption studies have revealed that Cart1 plays an important role for craniofacial bone formation as well as limb development by cooperating with another homeoprotein, Alx4. In this report, we study the functional involvement of p300/CBP, coactivators with intrinsic histone acetyltransferase (HAT) activity, in the transcriptional control of Cart1.

Methods: To study the transcription activity of Cart1, a reporter construct containing a putative Cart1 binding site was transiently transfected with the expression vectors of each protein. The interaction between p300/CBP and Cart1 was investigated by glutathione S-transferase (GST) pull-down, yeast two-hybrid, and immunoprecipitation assays. In vitro acetylation assay was performed with the recombinant p300-HAT domain and Cart1 in the presence of acetyl-CoA.

Results and Conclusions: p300 and CBP stimulate Cart1-dependent transcription activity, and this transactivation is inhibited by E1A and Tax, oncoproteins that suppress the activity of p300/CBP. Cart1 binds to p300 in vivo and in vitro, and this requires the homeodomain of Cart1 and N-terminal 139 amino acids of p300. Confocal microscopy analysis shows that Cart1recruits overexpressed and endogenous p300 to a Cart1-specific subnuclear compartment. Cart1 is acetylated in vivo and sodium butyrate and trichostatin A, histone deacetylase inhibitors, markedly enhance the transcription activity of Cart1. Deletion and mutagenesis analysis identifies the 131st lysine that locates immediately adjacent to the homeodomain as a target of p300-HAT, and a point mutation to this residue attenuates the binding affinity to p300 as well as p300-dependent transcription activity. Together, these results indicate that p300/CBP acts as a cotransactivator to Cart1 through a direct interaction and specific lysine acetylation. In addition, because 131st lysine is highly conserved in other types of homeoprotein, this lysine may be a common target for HAT of p300/CBP for these proteins.
Regulation of Parathyroid Hormone-Stimulated Phospholipase D in UMR-106 Cells by Calcium, MAP Kinase, and Small G Proteins
Authors: Singh Amareshwar TK , Bhattacharyya Rumi S , Radeff Julie M , Stern Paula H
Published: April 2006

Signaling intermediates for PTH and phorbol activation of PLD in UMR-106 cells were determined. Calcium was required, and the effects of PTH, phorbol, and calcium were dependent on p42/44 MAP kinase and small G proteins, specifically RhoA, acting through Rho kinase.

Introduction: Phospholipase D (PLD) plays a key signaling role in numerous cellular processes. PLD-stimulated hydrolysis of phosphatidylcholine (PC) generates phosphatidic acid, a source of diacylglycerol (DAG). We previously reported that parathyroid hormone (PTH) stimulates PLD activity in UMR-106 osteoblastic cells by a protein kinase C (PKC)-independent mechanism. The current study investigated the roles of calcium, MAP kinase, and small G proteins in PTH- and phorbol-12,13-dibutyrate (PDBu)-stimulated transphosphatidylation of ethanol, a reaction catalyzed by PLD.

Methods: UMR-106 cells were labeled with3H-palmitic and treated in the presence of ethanol. Phosphatidylethanol was separated by thin-layer chromatography and detected by autoradiography, and the bands were scraped and counted. Statistical significance of the responses from three to nine replicates was determined by ANOVA and Tukey's post-test.

Results and Conclusions: PTH and PDBu effects were attenuated by EGTA, BAPTA, nifedipine, and dantrolene, whereas ionomycin or 2× calcium increased basal PLD activity. PTH activated p42/p44 MAP kinase, and the effects of PTH, PDBu, and ionomycin on PLD, but not on calcium influx, were prevented by the MEK inhibitors PD98059 and U0126. Small G proteins were shown to be involved in the effects of PTH, PDBu, and ionomycin on PLD. Inhibition of ARF by brefeldin prevented the PLD activation by all three agonists. A nonselective Rho/Rac/cdc-42 inhibitor, Clostridium difficile toxin B, also inhibited the effects of all three agonists on PLD. More selective inhibition of RhoA with a dominant negative RhoA construct or by inhibiting geranylgeranyltransferase I antagonized the effects of PTH, PDBu, and ionomycin, as did inhibiting the downstream kinase, Rho kinase. The current results reveal the importance of calcium, MAP kinase, and small G proteins in PTH and PDBu stimulation of PLD activity in UMR-106 cells.
Prostaglandin E2-Mediated Anabolic Effect of a Novel Inhibitor of Phosphodiesterase 4, XT-611, in the In Vitro Bone Marrow Culture
Authors: Miyamoto Ken-Ichi , Suzuki Hirokazu , Yamamoto Shinya , Saitoh Yukie , Ochiai Eiji , Moritani Shuzo , Yokogawa Koichi , Waki Yoshihiro , Kasugai Shohei , Sawanishi Hiroyuki , Yamagami Hideomi
Published: April 2006

The mechanism of osteoblast formation by a novel PDE4 inhibitor, XT-611, was studied in the in vitro bone marrow culture system. The compound potentiated the osteoblast differentiation through accumulation of cyclic AMP after autocrine stimulation of EP4 receptor by PGE2 in pro-osteoblastic cells.

Introduction: We previously reported that inhibitors of phosphodiesterase (PDE)4 isoenzyme increase osteoblast formation in an in vitro bone marrow culture system and inhibit bone loss in animal osteoporosis models. Here we investigated the mechanism of the effect of a novel PDE4 inhibitor, 3,4-dipropyl-4,5,7,8-tetrahydro-3H-imidazo[1,2-i]-purin-5-one (XT-611), on osteoblast formation in the in vitro bone marrow culture system.

Materials and Methods: Rodent bone marrow cells were cultured in the presence of 0.2 mM ascorbic acid phosphate ester, 1 mM β-glycerophosphate, and 10 nM dexamethasone for 10 days. Drug treatments were done for 24 h on day 3 of culture.

Results: PDE4 inhibitors, including XT-611, but not PDE3 and PDE5 inhibitors, increased mineralized nodule formation in rat and mouse bone marrow cell cultures. During culture of the bone marrow cells, prostaglandin E2 (PGE2) production increased with a peak on day 4, but the increase was completely inhibited by indomethacin, an unselective cyclo-oxygenase (COX) inhibitor. Spontaneous and XT-611-induced mineralized-nodule formation was also inhibited by indomethacin and COX-2 inhibitors, in a similar potential. Alkaline phosphatase-positive nodule formation in the absence or presence of XT-611 was inhibited by an antagonist of EP4 receptor, AH23848B, and synergistically potentiated by 11-deoxy-PGE1, but it was not influenced by other EP antagonists and agonists examined. The expression of PDE4 and EP4 mRNAs was observed in bone marrow cells. The effect of XT-611 was also confirmed to involve an increase of cyclic AMP and the cyclic AMP-dependent protein kinase pathway.

Conclusion: These results suggest that PGE2 stimulates differentiation of osteoblast progenitor cells through the EP4 receptor in an autocrine manner, and the PDE4 inhibitor potentiates the differentiation by inhibiting hydrolysis of cyclic AMP in the cells.