bluuebunny/crossref_metadata_2025 huggingface dataset:
Limited Rescue of Osteoclast-Poor Osteopetrosis After Successful Engraftment by Cord Blood From an Unrelated Donor
Authors: Nicholls Brian M , Bredius Robbert GM , Hamdy Neveen AT , Gerritsen Egbert JA , Lankester Arjan C , Hogendoorn Pancras CW , Nesbitt Stephen A , Horton Michael A , Flanagan Adrienne M
Published: April 2006

We report on a case of osteoclast-poor osteopetrosis who received a hematopoietic stem cell graft and, despite hematological engraftment, showed little signs of response in the skeletal defect. Clinical and laboratory studies supported the concept that the bone microenvironment remained abnormal, thus reducing the clinical response to transplantation.

Introduction: Osteopetrosis is a rare genetic disorder characterized by severely reduced bone resorption resulting from a defect in either osteoclast development (osteoclast-poor osteopetrosis) or activation (osteoclast-rich osteopetrosis). Patients with osteoclast-rich osteopetrosis can be rescued by allogenic hematopoietic stem cell transplantation; however, little information exists concerning the success of transplantation as a treatment for osteoclast-poor osteopetrosis. We report on a child with osteoclast-poor osteopetrosis whose diagnosis was delayed, consequently receiving a cord blood transplant from an unrelated donor at the age of 8 years. Engraftment was deemed successful by peripheral blood genotyping, although >3 years after transplantation there was little rescue of the skeletal defect and anemia, and extramedullary hematopoiesis persisted.

Materials and Methods: Peripheral blood mononuclear cells from the osteopetrosis patient, before and after transplantation, were used to generate osteoclasts in vitro in the presence of macrophage colony-stimulating factor (M-CSF) and RANKL.

Results: Before transplantation few, small mononuclear osteoclasts formed (F-actin ring-positive cells, co-localizing with vitronectin receptor [αvβ3 integrin] and TRACP) associated with occasional, small resorption lacunae. Low levels of collagen C-terminal telopeptide (CTx) fragments were released from these cultures as assessed by ELISA (CrossLaps; patient, 12.85 nM; control, 448.6 nM). In contrast, osteoclasts formed in cultures after transplantation formed to a similar degree to control cultures from healthy individuals: large numbers of osteoclasts containing numerous nuclei were present, and approximately 50% of the surface of bone slices was resorbed, associated with intermediate levels of collagen fragment release (116.48 nM). The culture data reflect the histopathology and radiological findings and also support previous studies showing that neither M-CSF nor RANKL rescues osteoclast-poor osteopetrosis.

Conclusions: This is the first case reported in which a successful hematopoietic engraftment failed to correct an osteopetrotic skeletal defect, and this finding may be credited to the age at which the child was transplanted.
Dominant Negative N-Cadherin Inhibits Osteoclast Differentiation by Interfering With β-Catenin Regulation of RANKL, Independent of Cell-Cell Adhesion
Authors: Shin Chan Soo , Her Sun-Ju , Kim Jeong-Ah , Kim Do Hee , Kim Sang Wan , Kim Seong Yeon , Kim Hyo-Soo , Park Ki Ho , Kim Jung Gu , Kitazawa Riko , Cheng Su-Li , Civitelli Roberto
Published: April 2006

We studied the effects of dominant negative N-cadherin (NCadΔC) expression in ST2 cells on their ability to support osteoclastogenesis. Expression of NCadΔC in ST2 cells did not decrease cell-to-cell adhesion but significantly reduced osteoclast formation when co-cultured with BMMs. NCadΔC inhibited β-catenin/TCF signaling, resulting in decreased RANKL expression, which could contribute to the reduced osteoclast formation.

Introduction: Cadherin is a calcium-dependent cell adhesion molecule that plays major roles during embryonic development and morphogenesis. Classic cadherins interact with β-catenin, which is also involved in the Wnt signaling pathway. We tested whether disruption of N-cadherin function in stromal cells by dominant negative N-cadherin affects their ability to support osteoclastogenesis by altering heterotypic interaction with osteoclast precursors.

Materials and Methods: ST2 cells were transduced with retrovirus encoding extracellular domain-truncated, dominant negative N-cadherin (NCadΔC) and co-cultured with bone marrow macrophages (BMMs) to study the ability to support osteoclastogenesis. As a downstream target of NCadΔC, β-catenin/T-cell factor (TCF) transcriptional activity was analyzed using TOPflash reporter construct. Real-time RT-PCR analysis and RANKL-luciferase reporter assays were performed to study the effects of NCadΔC on the osteoprotegerin (OPG)/RANKL system.

Results: Immunoblotting analysis showed that primary bone marrow stromal cells, ST2 cells, and BMMs expressed N-cadherin. Retroviral expression of NCadΔC in ST2 cells did not significantly inhibit cell adhesion but markedly impaired the formation of TRACP+ osteoclasts (>40%) when co-cultured with BMMs. However, the inhibition of osteoclastogenesis was not reproduced by neutralizing antibody against N-cadherin. Expression of NCadΔC, however, strongly suppressed β-catenin/TCF transcriptional activity in ST2 cells, which was rescued by constitutively active β-catenin adenovirus (Ad ΔN46 β-catenin) or constitutively active TCF mutant (pCS2-VP16ΔβXTCF-3). As a potential downstream target of Wnt signaling, we found that the expression of RANKL was reduced in ST2 cells expressing NCadΔC. Moreover, Wnt-3A, Ad ΔN46 β-catenin, and VP16ΔβXTCF-3 increased the expression of RANKL and enhanced the transcriptional activity of mouse RANKL promoter in ST2 cells.

Conclusions: Our data suggest that expression of dominant negative N-cadherin in ST2 cells suppressed osteoclastogenesis by interfering with β-catenin regulation of RANKL independent of cell-cell adhesion.
Growth Without Growth Hormone Receptor: Estradiol Is a Major Growth Hormone-Independent Regulator of Hepatic IGF-I Synthesis
Authors: Venken Katrien , Schuit Frans , Van Lommel Leentje , Tsukamoto Katsura , Kopchick John J , Coschigano Karen , Ohlsson Claes , Movérare Sofia , Boonen Steven , Bouillon Roger , Vanderschueren Dirk
Published: April 2006

The role of estrogens in the regulation of pubertal growth independently of GH and its receptor was studied in male mice with disrupted GHRKO. E2 rescued skeletal growth rates in GHRKO associated with an increase in hepatic and serum IGF-I. These data show that E2 rescues pubertal growth during GH resistance through a novel mechanism of GHR-independent stimulation of hepatic IGF-I production.

Introduction: Growth hormone (GH) and estrogen play a pivotal role in pubertal growth and bone mineral acquisition. Estrogens can affect GH secretion and thereby provide a GH-dependent mechanism for their effects on skeletal growth. It is presently unclear if or to what extent estrogens are able to regulate pubertal growth and bone mineral accrual independently of GH and its receptor.

Materials and Methods: Estradiol (E2; 0.03 μg/day by subcutaneous silastic implants) was administered to orchidectomized (ORX) male mice with disrupted GHR (GHRKO) and corresponding WTs during late puberty (6–10 weeks). Longitudinal and radial bone growth, IGF-I in serum and its expression in liver, muscle, and bone, and liver gene expression were studied by histomorphometry, RIA, RT-PCR, microarrays, and Western blotting, respectively.

Results: E2 stimulated not only longitudinal (femur length and growth plate thickness) and radial growth (cortical thickness and periosteal perimeter), but also rescued longitudinal and periosteal growth rates in ORX GHRKO, whereas no significant changes occurred in WT. E2 thereby upregulated serum IGF-I and liver IGF-I synthesis (+21% and +52%, respectively) in ORX GHRKO, whereas IGF-I synthesis in femur or muscle was unaffected. Study of the underlying mechanism of the stimulation of hepatic IGF-I expression showed that E2 restored downregulated receptor signaling systems, such as the estrogen receptor α and the prolactin receptor. E2 thereby recovered the Janus kinase (JAK)/signal transducers and activators of transcription (STAT) pathway as evidenced by a significantly increased activation of the transcription factor STAT5 in ORX GHRKO.

Conclusions: Our data show a stimulation of skeletal growth through upregulation of hepatic IGF-I by a hormone other than GH. E2 rescues pubertal skeletal growth during GH resistance through a novel mechanism of GHR-independent stimulation of IGF-I synthesis in the liver.
Histone Deacetylase Inhibitors Promote Osteoblast Maturation
Authors: Schroeder Tania M , Westendorf Jennifer J
Published: April 2006

HDIs are potential therapeutic agents for cancer and neurological diseases because of their abilities to alter gene expression, induce growth arrest or apoptosis of tumors cells, and stimulate differentiation. In this report, we show that several HDIs promote osteoblast maturation in vitro and in calvarial organ cultures.

Introduction: Histone deacetylase inhibitors (HDIs) are currently in phase I and II clinical trials as anticancer agents. Some HDIs are also commonly prescribed treatments for epilepsy and bipolar disorders. Although administered systemically, the effects of HDIs on osteoblasts and bone formation have not been extensively examined. In this study, we investigated the effect of histone deacetylase inhibition on osteoblast proliferation and differentiation.

Materials and Methods: MC3T3-E1 cells, calvarial-derived primary osteoblasts, and calvarial organ cultures were treated with various commercially available HDIs (trichostatin A [TSA], sodium butyrate [NaB], valproic acid [VPA], or MS-275). The effects of these inhibitors on cell proliferation, viability, cell cycle progression, Runx2 transcriptional activity, alkaline phosphatase production, and matrix mineralization were determined. Expression levels of osteoblast maturation genes, type I collagen, osteopontin, bone sialoprotein, and osteocalcin in response to TSA were measured by quantitative PCR.

Results: Concentrations of HDIs that caused hyperacetylation of histone H3 induced transient increases in osteoblast proliferation and viability but did not alter cell cycle profiles. These concentrations of HDIs also increased the transcriptional activity of Runx2. TSA accelerated alkaline phosphatase production in MC3T3-E1 cells and calvarial organ cultures. In addition, TSA accelerated matrix mineralization and the expression of osteoblast genes, type I collagen, osteopontin, bone sialoprotein, and osteocalcin in MC3T3-E1 cells.

Conclusions: These studies show that histone deacetylase activity regulates osteoblast differentiation and bone formation at least in part by enhancing Runx2-dependent transcriptional activation. Therefore, HDIs are a potentially new class of bone anabolic agents that may be useful in the treatment of diseases that are associated with bone loss such as osteoporosis and cancer.
Regulation of Osteoclast Apoptosis and Motility by Small GTPase Binding Protein Rac1
Authors: Fukuda Akira , Hikita Atsuhiko , Wakeyama Hidetoshi , Akiyama Toru , Oda Hiromi , Nakamura Kozo , Tanaka Sakae
Published: April 2006

The role of Rac1 in osteoclast survival and bone-resorbing activity was examined using adenovirus vector expression systems. Rac1 is critically involved in M-CSF receptor signaling and mediates survival signaling primarily through PI3K/Akt pathways. Rac1 also plays a significant role in bone resorptive activity, probably by regulating the motility of osteoclasts.

Introduction: Rac1 is a member of Rho family small G-proteins, and recent studies have revealed that it mediates anti-apoptotic signals in some types of cells. Rac1 is reported to be required for the cytoskeletal organization and bone-resorbing activity of osteoclasts, but their roles in osteoclast survival and function are not fully elucidated.

Materials and Methods: We constructed the adenovirus vector carrying cDNA of either the dominant negative Rac1 (Rac1DN) or constitutively active Rac1 (Rac1CA) gene, and osteoclast-like cells (OCLs) generated in mouse co-culture system were infected with these viruses. To examine the role of Rac1 in osteoclast survival and function, we performed pit formation assays, survival assays, and Western blotting, including an activated-Rac1 pull-down assay using adenovirus-infected OCLs. To further clarify the mechanism of Rac1 regulation in osteoclast survival, some specific inhibitors and adenovirus vectors of signal transduction molecules were used. To quantify membrane movement before and after macrophage colony-stimulating factor (M-CSF) treatment, OCLs expressing either enhanced green fluorescent protein (EGFP) or Rac1DN were recorded with a time-lapse video microscope.

Results: Adenovirus vector-mediated dominant negative Rac1 (Rac1DN) expression significantly reduced pit formation, and promoted their apoptosis. M-CSF rapidly activated Rac1, and the prosurvival effect of M-CSF for OCLs was abrogated by Rac1DN overexpression. Constitutively active Rac1 enhanced OCL survival, which was completely suppressed by phosphatidylinositol 3′-kinase (PI3K) inhibitors, whereas a Mek inhibitor had only partial effect. Rac1DN also partially blocked the activation of Akt induced by the overexpressing catalytic subunit of PI3K. Using time-lapse video microscopy, we found that Rac1DN expression reduced membrane ruffling and the spreading of OCLs in response to M-CSF.

Conclusions: Small guanosine triphosphatase (GTPase) Rac1 is critically involved in M-CSF receptor signaling and mediates survival signaling of osteoclasts primarily by modulating PI3K/Akt pathways. Rac1 also plays a significant role in the bone resorptive activity of cells, probably by regulating the motility of osteoclasts.
Genetic Background Influences Cortical Bone Response to Ovariectomy
Authors: Li Chao Yang , Schaffler Mitchell B , Wolde-Semait Henock T , Hernandez Christopher J , Jepsen Karl J
Published: April 2006

Peak bone mass is genetically determined, but little is known about the heritability of bone loss. Inbred mice were ovariectomized at 16 weeks of age and killed at three time-points after surgery. We found that the variation in estrogen deficit-related cortical bone loss is genetically determined.

Introduction: Variability in adult bone morphology and composition among three inbred mouse strains—A/J, C57BL/6J (B6), and C3H/HeJ (C3H)—suggests that they gain bone in different ways during growth. In this study, we tested the hypothesis that these strains would also lose bone differently after estrogen deprivation.

Materials and Methods: Female A/J, B6, and C3H mice (N = 70/strain) were either ovariectomized (OVX) or sham-operated at 16 weeks of age and killed at 4, 8, and 16 weeks after surgery. Cortical bone histomorphometry was performed on right femoral mid-diaphyseal cross-sections. Mechanical properties were determined by loading left femoral mid-diaphyses to failure in four-point bending.

Results: Both OVX-A/J and OVX-B6 mice showed a 7–8% decrease in cortical area and width because of an 8–10% marrow expansion at 16 weeks after OVX. This bone loss did not affect mechanical properties in OVX-A/J femurs, but maximum load and stiffness in OVX-B6 decreased slightly (9%) at 4 and 8 weeks, and markedly (14–19%) at 16 weeks after OVX. In contrast, OVX-C3H showed a significant decrease in cortical area and width (6–7%) at 4 weeks after OVX and a slight decrease in the subperiosteal area (4%) at 8 weeks after OVX, although marrow area remained unchanged. Surprisingly, intracortical resorption spaces, which were present in sham-C3H mice, were greatly increased (+195%) in OVX-C3H mice at 8 weeks after OVX. Bone strength and stiffness in OVX-C3H mice decreased markedly (12–14%) at 4 weeks but slightly (8–10%) at 8 weeks after OVX. All indices except intracortical pore area in OVX-C3H mice returned to sham levels at 16 weeks after OVX.

Conclusions: The magnitude, timing, and location of cortical bone loss after OVX varied significantly among A/J, B6, and C3H mice. The subsequent changes in mechanical properties after OVX depended on the variable bone patterns as well as the size and shape of the adult bone. Our results suggest that patterns of estrogen deficit-associated cortical bone loss are genetically determined.
Bone and Body Composition of Children and Adolescents With Repeated Forearm Fractures
Authors: Goulding Ailsa , Grant Andrea M , Williams Sheila M
Published: April 2006

DXA measurements in 90 children and adolescents with repeated forearm fractures showed reduced ultradistal radius BMC and BMD values and elevated adiposity, suggesting site-specific bone weakness and high body weight increase fracture risk. Symptoms to cow milk, low calcium intakes, early age of first fracture, and overweight were over-represented in the sample.

Introduction: Although many apparently healthy children fracture their forearms repeatedly during growth, no previous studies of their bone health and body composition have been undertaken. Nor has the prevalence of established risk factors for fracture in such a population been assessed.

Materials and Methods: Ninety children and adolescents (47 girls and 43 boys) 5–19 years of age, who had experienced at least two fractures of the forearm, were studied. Bone size and mineralization were assessed using DXA at the ultradistal radius, one-third radius, neck of femur, hip trochanter, lumbar spine, and total body. Total body lean mass and fat mass were also determined. The prevalence of six risk factors for fracture were also examined, and their influence on ultradistal BMC Z scores was assessed.

Results: Participants experienced 295 fractures (74.9% forearm). Children with an early age of first fracture had higher rates of fracture per l00 years of exposure than those fracturing later. Four risk factors for fracture were over-represented in observed versus expected percentages: early age of first fracture (27.7% versus 11.3%), adverse symptoms to cow milk (22.2% versus 6.7%), low dietary calcium intake (20% versus 4.5%), and overweight (33.3% versus 15.5%). However, physical activity levels were similar to the reference population. Z scores for BMC and BMD were reduced, particularly at the ultradistal radius, whereas Z scores for weight, body mass index, fat mass, and body fat percentage were increased. Mean (SD) BMC Z scores were lowest at the ultradistal radius, −0.66 (1.22), where symptoms to milk were associated with reduced values (p < 0.009) and overweight with increased values (p < 0.003).

Conclusions: Our results suggest site-specific weakness and high body weight contribute to fracture risk in children and adolescents who fracture their forearms repeatedly. These findings are consonant with work showing adult Colles fractures increase as ultradistal radius BMD falls and with evidence that overweight children and adolescents are fracture prone.
Multiple Mechanisms Are Involved in Inhibition of Osteoblast Differentiation by PTHrP and PTH in KS483 Cells
Authors: van der Horst Geertje , Farih-Sips Hetty , Löwik Clemens WGM , Karperien Marcel
Published: April 2006

We examined the mechanism by which PTHrP and PTH inhibit KS483 osteoblastic differentiation. We show that PTHrP and PTH inhibit differentiation downstream of early BMP signaling and downregulated components of the hedgehog (Hh) signaling cascade. In addition, PTHrP and PTH repressed RunX2 and osx expression. Overexpression of either gene, however, could not relieve PTHrP and PTH's inhibitory actions. Our data suggest that multiple parallel mechanisms are involved in the inhibition of osteoblast differentiation and matrix mineralization by PTHrP and PTH.

Introduction: PTH-related peptide (PTHrP) and PTH are potent inhibitors of osteoblast differentiation in vitro by as yet unexplained mechanisms.

Materials and Methods: We treated murine bone marrow stromal cells and the mesenchymal progenitor cell line KS483 with PTHrP and PTH in combination with either BMPs or hedgehog (Hh) and measured early and late markers of osteoblast differentiation and studied the expression of RunX2 and Osterix (osx). In addition, we examined the PTHrP and PTH response in stable KS483 cells overexpressing either RunX2 or osx.

Results: PTHrP and PTH inhibited BMP- and Hh-induced osteogenesis downstream of early BMP signaling and by downregulation of components of the Hh signaling cascade. PTHrP and PTH prevented the upregulation of RunX2 expression associated with osteoblast differentiation in an indirect response. However, PTHrP and PTH could still inhibit differentiation, and particularly matrix mineralization, of cells expressing RunX2. In addition, PTHrP and PTH potently downregulated osx expression only in mature osteoblasts in an intermediate early response, but osx overexpression could not relieve the inhibitory effects of PTHrP and PTH on matrix mineralization.

Conclusions: Our data suggest that, besides transcriptional repression of RunX2 and osx, other mechanisms in parallel with or downstream of RunX2 and osx are involved in the inhibition of osteoblast differentiation and matrix mineralization by PTHrP and PTH in vitro.
A Nonprostanoid EP4 Receptor Selective Prostaglandin E2 Agonist Restores Bone Mass and Strength in Aged, Ovariectomized Rats
Authors: Ke Hua Zhu , Crawford D Todd , Qi Hong , Simmons Hollis A , Owen Thomas A , Paralkar Vishwas M , Li Mei , Lu Bihong , Grasser William A , Cameron Kimberly O , Lefker Bruce A , DaSilva-Jardine Paul , Scott Dennis O , Zhang Qing , Tian Xiao Yan , Jee Webster SS , Brown Thomas A , Thompson David D
Published: April 2006

CP432 is a newly discovered, nonprostanoid EP4 receptor selective prostaglandin E2 agonist. CP432 stimulates trabecular and cortical bone formation and restores bone mass and bone strength in aged ovariectomized rats with established osteopenia.

Introduction: The purpose of this study was to determine whether a newly discovered, nonprostanoid EP4 receptor selective prostaglandin E2 (PGE2) agonist, CP432, could produce bone anabolic effects in aged, ovariectomized (OVX) rats with established osteopenia.

Materials and Methods: CP432 at 0.3, 1, or 3 mg/kg/day was given for 6 weeks by subcutaneous injection to 12-month-old rats that had been OVX for 8.5 months. The effects on bone mass, bone formation, bone resorption, and bone strength were determined.

Results: Total femoral BMD increased significantly in OVX rats treated with CP432 at all doses. CP432 completely restored trabecular bone volume of the third lumbar vertebral body accompanied with a dose-dependent decrease in osteoclast number and osteoclast surface and a dose-dependent increase in mineralizing surface, mineral apposition rate, and bone formation rate-tissue reference in OVX rats. CP432 at 1 and 3 mg/kg/day significantly increased total tissue area, cortical bone area, and periosteal and endocortical bone formation in the tibial shafts compared with both sham and OVX controls. CP432 at all doses significantly and dose-dependently increased ultimate strength in the fifth lumber vertebral body compared with both sham and OVX controls. At 1 and 3 mg/kg/day, CP432 significantly increased maximal load in a three-point bending test of femoral shaft compared with both sham and OVX controls.

Conclusions: CP432 completely restored trabecular and cortical bone mass and strength in established osteopenic, aged OVX rats by stimulating bone formation and inhibiting bone resorption on trabecular and cortical surfaces.
Infant Growth Influences Proximal Femoral Geometry in Adulthood
Authors: Javaid M Kassim , Lekamwasam Sarath , Clark Judith , Dennison Elaine M , Syddall Holly E , Loveridge Nigel , Reeve Jonathan , Beck Tom J , Cooper Cyrus
Published: April 2006

The relationship between early growth and adult femoral geometry has not been studied previously. In 333 adults, we were able to show that infant weight predicts femoral width and cross-sectional moment of inertia but not femoral neck length. These results support the hypothesis that growth in early life leads to persisting differences in proximal femoral geometry.

Introduction: Both femoral geometry and bone mass have been shown independently to predict both hip strength and fracture risk. Whereas growth during intrauterine and early postnatal life has been shown to influence adult bone mass, the relationship between growth in early life and adult femoral geometry has not been described previously.

Materials and Methods: We studied the relationship between growth during early life, adult hip geometry, and proximal femur bone mass in a sample of 333 men and women (60–75 years of age), for whom birth weight and weight at 1 year of age were recorded. Hip geometry was derived using Hip Structure Analysis software from proximal femur DXA scans (Hologic QDR 1000).

Results: There were significant (p < 0.002) relationships between weight at age 1 year and measures of femoral width as well as intertrochanteric (IT) cross-sectional moment of inertia (CSMI), but not with femoral neck length. The relationships with measures of femoral width but not CSMI remained after adjusting for adult body weight and were independent of proximal femoral BMC.

Conclusions: These results support the hypothesis that different patterns of growth in utero and during the first year of life lead to persisting differences in proximal femoral geometry, thereby mediating in part the effects of early growth on risk of hip fracture in adulthood.
Relative Impact of Androgen and Estrogen Receptor Activation in the Effects of Androgens on Trabecular and Cortical Bone in Growing Male Mice: A Study in the Androgen Receptor Knockout Mouse Model
Authors: Venken Katrien , De Gendt Karel , Boonen Steven , Ophoff Jill , Bouillon Roger , Swinnen Johannes V , Verhoeven Guido , Vanderschueren Dirk
Published: April 2006

The relative importance of AR and ER activation has been studied in pubertal male AR knockout and WT mice after orchidectomy and androgen replacement therapy, either with or without an aromatase inhibitor. AR activation dominates normal trabecular bone development and cortical bone modeling in male mice. Moreover, optimal periosteal bone expansion is only observed in the presence of both AR and ER activation.

Introduction: Androgen receptor (AR)–mediated androgen action has traditionally been considered a key determinant of male skeletal growth. Increasing evidence, however, suggests that estrogens are also essential for normal male bone growth. Therefore, the relative importance of AR-mediated and estrogen receptor (ER)–mediated androgen action after aromatization remains to be clarified.

Materials and Methods: Trabecular and cortical bone was studied in intact or orchidectomized pubertal AR knockout (ARKO) and male wildtype (WT) mice, with or without replacement therapy (3–8 weeks of age). Nonaromatizable (dihydrotestosterone [DHT]) and aromatizable (testosterone [T]) androgens and T plus an aromatase inhibitor (anastrazole) were administered to orchidectomized ARKO and WT mice. Trabecular and cortical bone modeling were evaluated by static and dynamic histomorphometry, respectively.

Results: AR inactivation or orchidectomy induced a similar degree of trabecular bone loss (−68% and −71%, respectively). Both DHT and T prevented orchidectomy-induced bone loss in WT mice but not in ARKO mice. Administration of an aromatase inhibitor did not affect T action on trabecular bone. AR inactivation and orchidectomy had similar negative effects on cortical thickness (−13% and −8%, respectively) and periosteal bone formation (−50% and −26%, respectively). In orchidectomized WT mice, both DHT and T were found to stimulate periosteal bone formation and, as a result, to increase cortical thickness. In contrast, the periosteum of ARKO mice remained unresponsive to either DHT or T. Interestingly, administration of an aromatase inhibitor partly reduced T action on periosteal bone formation in orchidectomized WT mice (−34% versus orchidectomized WT mice on T), but not in ARKO mice. This effect was associated with a significant decrease in serum IGF-I (−21% versus orchidectomized WT mice on T).

Conclusions: These findings suggest a major role for AR activation in normal development of trabecular bone and periosteal bone growth in male mice. Moreover, optimal stimulation of periosteal growth is only obtained in the presence of both AR and ER activation.
BMP Signaling Is Required for RUNX2-Dependent Induction of the Osteoblast Phenotype
Authors: Phimphilai Mattabhorn , Zhao Zhouran , Boules Heidi , Roca Hernan , Franceschi Renny T
Published: April 2006

RUNX2 expression in mesenchymal cells induces osteoblast differentiation and bone formation. BMP blocking agents were used to show that RUNX2-dependent osteoblast differentiation and transactivation activity both require BMP signaling and, further, that RUNX2 enhances the responsiveness of cells to BMPs.

Introduction: BMPs and the RUNX2 transcription factor are both able to stimulate osteoblast differentiation and bone formation. BMPs function by activating SMAD proteins and other signal transduction pathways to stimulate expression of many target genes including RUNX2. In contrast, RUNX2 induces osteoblast-specific gene expression by directly binding to enhancer regions in target genes. In this study, we examine the interdependence of these two factors in controlling osteoblast differentiation in mesenchymal progenitor cells.

Materials and Methods: C3H10T1/2 mesenchymal cells and primary cultures of marrow stromal cells were transduced with a RUNX2 adenovirus and treated with BMP blocking antibodies or the natural antagonist, NOGGIN. Osteoblast differentiation was determined by assaying alkaline phosphatase and measuring osteoblast-related mRNA using quantitative RT/PCR. Activation of BMP-responsive signal transduction pathways (SMAD, extracellular signal-regulated kinase [ERK], p38, and c-jun-N-terminal kinase [JNK]) was assessed on Western blots.

Results and Conclusions: C3H10T1/2 cells constitutively synthesize BMP2 and 4 mRNA and protein, and this BMP activity is sufficient to activate basal levels of SMAD phosphorylation. Inhibition of BMP signaling was shown to disrupt the ability of RUNX2 to stimulate osteoblast differentiation and transactivate an osteocalcin gene promoter-luciferase reporter in C3H10T1/2 cells. BMP blocking antibodies also inhibited RUNX2-dependent osteoblast differentiation in primary cultures of murine marrow stromal cells. Conversely, RUNX2 expression synergistically stimulated BMP2 signaling in C3H10T1/2 cells. However, RUNX2 did not increase the ability of this BMP to activate SMAD, ERK, p38, and JNK pathways. This study shows that autocrine BMP production is necessary for the RUNX2 transcription factor to be active and that BMPs and RUNX2 cooperatively interact to stimulate osteoblast gene expression.
Effect of Monoterpenes on the Formation and Activation of Osteoclasts In Vitro
Authors: Dolder Silvia , Hofstetter Willy , Wetterwald Antoinette , Mühlbauer Roman C , Felix Rolf
Published: April 2006

Monoterpenes, present in aromatic plants, are known to inhibit bone resorption in vivo. In this in vitro study, they inhibited the activation of osteoclasts only at high concentrations but inhibited the formation at much lower concentrations. Therefore, monoterpenes may act in vivo directly on osteoclastogenesis.

Introduction: Monoterpenes are the major components of essential oils, which are formed in many plants. Typically, they are found in herbs and certain fruits. When fed to rats, they inhibit bone resorption by an unknown mechanism. In this study, their effect on the activity and formation of osteoclasts in vitro was studied.

Materials and Methods: The effect of monoterpenes on the development of osteoclasts was studied in co-cultures of bone marrow cells and osteoblasts and in cultures of spleen cells grown with colony stimulating factor (CSF)-1 and RANKL. In cultures of primary osteoblasts, alkaline phosphatase activity and levels of mRNA encoding RANKL and osteoprotegerin (OPG) mRNA (RT-PCR), and in osteoblast and spleen cell cultures, lactate dehydrogenase activity, a measure of toxicity, were determined. The activity of isolated rat osteoclasts was determined by counting the osteoclasts with actin rings using histofluorometry.

Results: The monoterpenes inhibited the formation of osteoclasts more strongly in co-cultures (≥1 μM) than in cultures of spleen cells (≥10 μM). They had a minor effect on osteoblasts. Toxic effects were not observed. The inhibition of the formation of osteoclasts was not reversed by the addition of farnesol and geranylgeraniol, excluding an effect of the monoterpenes through the mevalonate pathway. A high concentration of 1 mM was required to inhibit the activation of osteoclasts. This effect, shown for menthol and borneol, was reversible.

Conclusions: The results suggest that the monoterpenes inhibit bone resorption in vivo through a direct effect on the formation of osteoclasts acting mainly on the hemopoietic cells.
Long-Term Effects of Calcium Supplementation on Serum Parathyroid Hormone Level, Bone Turnover, and Bone Loss in Elderly Women
Authors: Riggs B. Lawrence , O'Fallon W. Michael , Muhs Joan , O'Connor Michael K. , Kumar Rajiv , Melton L. Joseph
Published: April 2006

We report a 4-year randomized, double-blind, placebo-controlled clinical trial in 236 normal postmenopausal women (mean age ± SE, 66.3 ± 0.2 years) who were randomized to a calcium (1600 mg/day as the citrate) or placebo group. The women were seen every 6 months; 177 completed the trial. Net percentage changes in each group are given relative to baseline. The differences in net percentage changes (calcium group minus placebo group) in medians were: for lumbar spine bone density, 2.0% (p < 0.001) at year 1 and 0.3% (not significant) at year 4; for proximal femur bone density, 1.3% (p = 0.003) at year 1 and 1.3% (p = 0.015) at year 4; and for total body bone mineral, 0.4% (p = 0.002) at year 1 and 0.9% (p = 0.017) at year 4. Similar differences at year 4 were: −18.9% (p = 0.002) for parathyroid hormone (PTH), −11.9% (p = 0.026) for serum osteocalcin, and −32.2% (p = 0.003) for urine free pyridinoline. We conclude that long-term administration of calcium supplements to elderly women partially reverses age-related increases in serum PTH level and bone resorption and decreases bone loss. However, the effects on bone loss were weaker than those reported for estrogen, bisphosphonates, or calcitonin therapy, indicating that calcium supplements alone cannot substitute for these in treating established osteoporosis. Nonetheless, because of their safety, high tolerance, and low expense, calcium supplements may be a useful preventive measure for elderly postmenopausal women whose bone mineral density values are normal for their age.
LIF, but Not IL-6, Regulates Osteoprogenitor Differentiation in Rat Calvaria Cell Cultures: Modulation by Dexamethasone
Authors: Dr. Malaval Luc , Gupta Ashwani K. , Liu Fina , Delmas Pierre D. , Aubin Jane E.
Published: April 2006

Cytokines of the interleukin 6 (IL-6) subfamily are a group of factors produced by osteoblasts and acting through the same transducing element, membrane protein gp130. We have previously shown that exogenous (added to the culture medium) leukemia inhibitory factor (LIF) inhibits bone nodule formation and expression of osteoblast-associated genes in fetal rat calvaria (RC) cell cultures and that dexamethasone (Dex) increases the ID50 of LIF. To investigate the respective roles of IL-6–related cytokines and receptors in osteprogenitor differentiation, and their regulatory interplay with Dex, we used reverse transcribed polymerase chain reaction, bioassay, and blocking antibody techniques to assess the time courses of LIF, IL-6, LIF transmembrane receptor, IL-6 receptor, and gp130 expression in RC cell cultures grown with and without Dex. The levels of the mRNAs for IL-6, LIF, and gp130 decreased concomitantly with the formation of bone nodules. Dex treatment, which stimulates bone nodule formation, reduced the expression of LIF and IL-6 mRNAs and IL-6 bioactivity in the culture medium. LIF treatment strongly stimulated the expression of IL-6. Incubation with anti-LIF antibodies increased the number of nodules, while an antibody blocking IL-6 activity had little or no effect on nodule numbers and did not antagonize the action of exogenous LIF, indicating that IL-6 does not mediate the action of LIF in this system. Moreover, although exogenously added IL-6 was active in the cultures as noted by a reduction of nodule mineralization, it had no effect on nodule numbers, i.e., on osteoprogenitor differentiation, in the presence or absence of Dex. In conclusion, IL-6, LIF, and their receptors are expressed throughout the time-course of osteogenesis in RC cell cultures. However, only LIF, but not IL-6, appears to play a significant role in autocrine regulation of osteoblastic differentiation in this system. The antagonist action of Dex on the effects of exogenously added LIF, as well as the bone-promoting action of Dex in RC cell cultures, could be exerted partly through the down-regulation of the expression of endogenous LIF.
IL-1α, IL-1β, IL-6, and TNF-α Steady-State mRNA Levels Analyzed by Reverse Transcription-Competitive PCR in Bone Marrow of Gonadectomized Mice
Authors: Van Bezooijen Rutger L. , Farih-Sips Hetty C. M. , Papapoulos Socrates E. , Löwik Clemens W. G. M.
Published: April 2006

Loss of gonadal function in both females and males is associated with increased rates of bone loss by a yet unidentified mechanism. There is ample evidence that cytokines that are produced in the bone microenvironment and stimulate the activity and/or formation of osteoclasts are involved. In the present study, we examined whether gonadectomy increases cytokine production via increased transcription in the bone marrow of mice. For this, the in vivo steady-state mRNA levels of multiple cytokines were determined in the central bone marrow compartment of mice at different time points following ovariectomy or orchidectomy by reverse transcription-competitive polymerase chain reaction. The limit of detectable differences in mRNA expression was approximately 2-fold. Bone marrow mRNA levels of the cytokines interleukin-1α (IL-1α), interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) were elevated up to 30-fold after treatment of mice with lipopolysaccharide. Following gonadectomy, there were no differences in the mRNA expression of these cytokines in bone marrow of female and male mice 4, 7, and 14 days after surgery. Gender steroid deficiency does not, therefore, increase steady-state mRNA levels of IL-1α, IL-1β, IL-6, and TNF-α in cells of the central bone marrow compartment in mice. If changes have occurred these should have been less than 2-fold or in a small cell population. These results do not preclude an important role of these cytokines in the induction of bone loss after gonadectomy. For example, bone marrow cells situated close to the bone surface or bone cells may be responsible for increased cytokine synthesis. Alternatively, the loss of gender steroids may alter post-transcriptional events in cytokine synthesis and activity or may modify the responsiveness of target cells.
The Derivation and Characterization of Stromal Cell Lines from the Bone Marrow of p53−/− Mice: New Insights into Osteoblast and Adipocyte Differentiation
Authors: Thompson Deborah L. , Lum Karen D. , Nygaard Sean C. , Kuestner Rolf E. , Kelly Katherine A. , Gimble Jeffrey M. , Moore Emma E.
Published: April 2006

We have derived a series of clonal cell lines from the bone marrow of p53−/− mice that represent different stages of osteoblast and adipocyte differentiation. All cell lines show indefinite growth potential (>300 population doublings) and have generation times of 12–20 h. These cell lines have been grouped into three categories. The least mature clones are heterogeneous and appear to contain a subpopulation of stem cells, which can spontaneously generate foci that contain either adipocytes or mineralizing osteoblasts. The second category of clones are homogenous and clearly correspond to mature osteoblasts because they express high levels of the anticipated osteoblastic markers in a stable fashion and cannot differentiate into adipocytes even in the presence of inducers. The clones in the third category are the most unique. Initially they appeared to correspond to mature osteoblasts because they express alkaline phosphatase in a homogenous manner, secrete type I collagen, show a significant cyclic adenosine monophosphate response to parathyroid hormone, secrete osteocalcin, and mineralize extensively after only 4–7 days. However, in contrast to the mature osteoblasts, these clones can be induced to undergo massive adipocyte differentiation, and this differentiation is accompanied by the complete loss of expression of all osteoblastic markers except alkaline phosphatase. These observations indicate that some cells that have acquired all of the characteristics of mature osteoblasts can be diverted to the adipocyte pathway. Further characterization of these clones may be particularly relevant to osteoporotic conditions where increased adipocyte formation appears to occur at the expense of osteoblast formation.
Acute Changes in Trabecular Bone Connectivity and Osteoclast Activity in the Ovariectomized Rat In Vivo
Authors: Lane Nancy E. , Thompson Jennifer M. , Haupt David , Kimmel Donald B. , Modin Gunnar , Kinney John H.
Published: April 2006

Estrogen deficiency results in a loss of trabecular bone mass and structure that leads to an increased incidence of osteoporotic fractures. The purpose of this study was to determine the time course for trabecular structure deterioration and changes in bone turnover just after ovariectomy in the rat. Six-month-old female virgin Sprague-Dawley rats had their right proximal tibia scanned by X-ray tomographic microscopy (XTM) at baseline (day 0). Animals were then randomized into two groups, and in each group 9 were sham-operated and 11 were ovariectomized and had repeat XTM scans on days 5, 13, 29, and 42 postovariectomy in group 1 and on days 8, 13, 33, and 50 postovariectomy in group 2. Urine was collected for deoxypyridinoline (DPD) cross-link measurements 24 h before each XTM scan and analyzed by ELISA. Trabecular bone structural variables and bone turnover endpoints were calculated from XTM data and standard histomorphometry. Trabecular connectivity decreased 27% by days 5 and 8 postovariectomy (p < 0.01) and continued to decrease up to day 50 postovariectomy (p < 0.01). The trabecular bone volume decreased 25% by 8 days postovariectomy (p < 0.01), and it continued to decrease through day 50. DPD cross-link excretion had increased 37% on day 13 (p < 0.01) and by over 100% of baseline by day 50 postovariectomy. Trabecular bone connectivity and volume deteriorate rapidly while DPD cross-link excretion increased more slowly in acute estrogen deficiency. These data suggest that if an agent is to preserve fully trabecular bone structure, it must be instituted very early in the estrogen-deficient state. They also suggest that a lag time exists before DPD excretion properly mirrors newly induced conditions of high bone turnover in this rat model.
Differential Effects of Aminosubstituted Analogs of Hydroxy Bisphosphonates on the Growth of Dictyostelium discoideum
Authors: Brown R. J. , Van Beek E. , Watts D. J. , Löwik C. W. G. M. , Papapoulos S. E.
Published: April 2006

Replacing the hydroxyl group in the bone-binding site of three clinically useful bisphosphonates (etidronate, pamidronate, and olpadronate) by an amino group resulted in great differences in their antiresorptive potencies in vitro. In the present study, this is also shown in vivo in mice treated with the six bisphosphonates at doses of up to 16 μM/kg/day for 12 days. Because binding to bone mineral is nearly the same for all tested bisphosphonates, these findings suggest that the aminosubstitution affects the cellular action of the bisphosphonates. This was tested in the cellular slime mould Dictyostelium discoideum in which cellular effects of bisphosphonates can be examined independently of binding to bone mineral. Etidronate and its aminosubstituted analog were equipotent in inhibiting amebal growth, while pamidronate was somewhat more potent than its analog. Whereas olpadronate was a potent inhibitor of axenic growth of Dictyostelium amebae, the aminosubstitution reduced its potency drastically (IC50 12 μM and 700 μM, respectively). The similarities between the inhibitory effects of the bisphosphonates tested on bone resorption in vitro and in vivo and on the growth of Dictyostelium amebae confirm that the differences in antiresorptive potencies found reflect differences in cellular effects and suggest that bisphosphonates may bind to more than one intracellular target.
Hereditary 1,25-Dihydroxyvitamin D–Resistant Rickets Due to an Opal Mutation Causing Premature Termination of the Vitamin D Receptor
Authors: Zhu Wenjing , Malloy Peter J. , Delvin Edgard , Chabot Gilles , Feldman David
Published: April 2006

Mutations in the vitamin D receptor (VDR) gene have been shown to cause hereditary vitamin D–resistant rickets (HVDRR). The patient in this study is a young French-Canadian boy with no known consanguinity in his family. The child exhibited the clinical characteristics of HVDRR with early onset rickets, hypocalcemia, secondary hyperparathyroidism, and elevated 1,25-dihydroxyvitamin D (1,25(OH)2D) levels as well as total alopecia. Fibroblasts were cultured from a skin biopsy of the patient and used to assess the VDR. Northern blot analysis showed that a normal size VDR transcript was expressed; however, [3H]1,25(OH)2D3-binding levels were very low and Western blot analysis failed to detect any VDR protein. Total resistance to 1,25(OH)2D3 was demonstrated by the failure of the cultured fibroblasts to induce the transcription of the 25-hydroxyvitamin D-24-hydroxylase gene when treated with high concentrations of 1,25(OH)2D3. Analysis of the DNA sequence revealed a unique C to T base change corresponding to nucleotide 218 of the VDR cDNA. This single base substitution changes the codon for arginine (CGA) to an opal stop codon (TGA), resulting in the truncation of the VDR at amino acid 30. The Arg30stop mutation prematurely terminates translation and deletes 398 amino acids including most of the zinc fingers as well as the entire ligand-binding domain. Restriction fragment length polymorphism analysis of a DdeI restriction site created by the mutation showed that the parents were heterozygous for the mutant allele. In conclusion, the Arg30stop mutation truncates the VDR and leads to a hormone-resistant condition which is the molecular basis of HVDRR in this patient.