bluuebunny/crossref_metadata_2025 huggingface dataset:
Suggestive Linkage of the Parathyroid Receptor Type 1 to Osteoporosis
Authors: Duncan Emma L. , Brown Matthew A. , Sinsheimer Janet , Bell John , Carr Andrew J. , Wordsworth B. Paul , Wass John A. H.
Published: April 2006

We have investigated the role of 23 candidate genes in the control of bone mineral density (BMD) by linkage studies in families of probands with osteoporosis (lumbar spine [LS] or femoral neck [FN] BMD T score < −2.5) and low BMD relative to an age- and gender-matched cohort (Z score < −2.0). One hundred and fifteen probands (35 male, 80 female) and 499 of their first- or second-degree relatives (223 males and 276 females) were recruited for the study. BMD was measured at the LS and FN using dual-energy X-ray absorptiometry and expressed as age- and gender-matched Z scores corrected for body mass index. The candidate genes studied were the androgen receptor, type I collagen A1 (COLIA1), COLIA2, COLIIA1, vitamin D receptor (VDR), colony-stimulating factor 1, calcium-sensing receptor, epidermal growth factor (EGF), estrogen receptor 1 (ESR1), fibrillin type 1, insulin-like growth factor 1, interleukin-1 alpha (IL-1α), interleukin-4 (IL-4), interleukin-6 (IL-6), interleukin-11 (IL-11), osteopontin, parathyroid hormone (PTH), PTH-related peptide, PTH receptor type 1 (PTHR1), transforming growth factor-beta 1, and tumor necrosis factors alpha and beta. Sixty-four microsatellites lying close to or within these genes were investigated for linkage with BMD. Using the program MapMaker/Sibs there was suggestive evidence of linkage between BMD and PTHR1 (maximum LOD score obtained [MLS] 2.7–3.5). Moderate evidence of linkage was also observed with EGF (MLS 1.8), COLIA1 (MLS 1.7), COLIIA1/VDR (MLS 1.7), ESR1 (MLS 1.4), IL-1α (MLS 1.4), IL-4 (MLS 1.2), and IL-6 (MLS 1.2). Variance components analysis using the program ACT, correcting for proband-wise ascertainment, also showed evidence of linkage (p ≤ 0.05) at markers close to or within the candidate genes IL-1α, PTHR1, IL-6, and COLIIA1/VDR. Further studies will be required to confirm these findings, to refine the location of gene responsible for the observed linkage, and to screen the candidate genes targeted at these loci for mutations.
Alkaline Phosphatase Knock-Out Mice Recapitulate the Metabolic and Skeletal Defects of Infantile Hypophosphatasia
Authors: Fedde Kenton N. , Blair Libby , Silverstein Julie , Coburn Stephen P. , Ryan Lawrence M. , Weinstein Robert S. , Waymire Katrina , Narisawa Sonoko , Millán José L. , Macgregor Grant R. , Whyte Michael P.
Published: April 2006

Hypophosphatasia is an inborn error of metabolism characterized by deficient activity of the tissue-nonspecific isoenzyme of alkaline phosphatase (TNSALP) and skeletal disease due to impaired mineralization of cartilage and bone matrix. We investigated two independently generated TNSALP gene knock-out mouse strains as potential models for hypophosphatasia. Homozygous mice (–/–) had < 1% of wild-type plasma TNSALP activity; heterozygotes had the predicted mean of ∼50%. Phosphoethanolamine, inorganic pyrophosphate, and pyridoxal 5′-phosphate are putative natural substrates for TNSALP and all were increased endogenously in the knock-out mice. Skeletal disease first appeared radiographically at ∼10 days of age and featured worsening rachitic changes, osteopenia, and fracture. Histologic studies revealed developmental arrest of chondrocyte differentiation in epiphyses and in growth plates with diminished or absent hypertrophic zones. Progressive osteoidosis from defective skeletal matrix mineralization was noted but not associated with features of secondary hyperparathyroidism. Plasma and urine calcium and phosphate levels were unremarkable. Our findings demonstrate that TNSALP knock-out mice are a good model for the infantile form of hypophosphatasia and provide compelling evidence for an important role for TNSALP in postnatal development and mineralization of the murine skeleton.
Fluid Shear Stress Increases Interleukin-11 Expression in Human Osteoblast-like Cells: Its Role in Osteoclast Induction
Authors: Sakai Kenji , Mohtai Masaaki , Shida Jun-Ichi , Harimaya Katsumi , Benvenuti Susanna , Brandi Maria L. , Kukita Toshio , Iwamoto Yukihide
Published: April 2006

It is unclear how mechanical stress influences bone cells. Mechanical stress causes fluid shear stress (FSS) in the bone. Osteoblast lineage cells are thought to sense FSS and regulate bone remodeling. We therefore investigated the effects of FSS on human osteoblast-like osteosarcoma cells: SaOS-2 cells in vitro. The conditioned medium of the SaOS-2 cells after 24 h of FSS (24 h-FSS CM) showed such osteoclastic phenotype inductions as significantly increasing the number of tartrate-resistant acid phosphatase (TRAP) positive multinuclear cells in rat bone marrow cells and TRAP-positive cells in human preosteoclastic cells: FLG 29.1 cells. An enzyme-linked immunosorbent assay showed interleukin-11 (IL-11) protein to increase 7-fold in the 24 h-FSS CM. A Northern analysis showed that IL-11 mRNA increased 4-fold in the SaOS-2 cells after 6 h-FSS; however, no IL-6 mRNA expression was detected. Furthermore, the anti-human IL-11 antibody significantly neutralized the osteoclastic phenotype induction of the 24 h-FSS CM. The IL-11 mRNA up-regulation in SaOS-2 cells by the 6 h-FSS was not inhibited by the anti-human transforming growth factor-β1 antibody, but it was significantly inhibited by indomethacin. An enzymeimmunoassay showed prostaglandin E2 to increase 7-fold in the 1 h-FSS CM. These findings thus suggest that FSS induces osteoblasts to produce IL-11 (mediated by prostaglandins) and thus stimulates bone remodeling.
Bone Mineral Density Testing and Osteoporosis Education Improve Lifestyle Behaviors in Premenopausal Women: A Prospective Study
Authors: Jamal Sophie A. , Ridout Rowena , Chase Cheryl , Fielding Lynda , Rubin Laurence A. , Hawker Gillian A.
Published: April 2006

One way to decrease the risk of osteoporosis is to maximize peak bone mass. Peak bone mass may be moderately influenced by lifestyle behaviors: increasing calcium and exercise, decreasing alcohol intake and smoking may increase peak bone mass. We examined the effects of osteoporosis education and bone mineral density (BMD) testing on self-reported lifestyle behaviors in 669 premenopausal women enrolled in a prospective study to assess determinants of peak bone mass. Study participants completed a questionnaire that assessed lifestyle behaviors, received pamphlets about osteoporosis, and had BMD testing. One year later, the women completed a similar questionnaire. After education about osteoporosis and BMD testing, women reported that they were less likely to smoke (odds ratio [OR] = 0.55; 95% confidence interval [95% CI]: 0.28–1.0), consume alcohol (OR = 0.13; 95% CI: 0.04–0.34), and caffeinated beverages (OR = 0.43; 95% CI: 0.27–0.68). Women were more likely to use calcium supplements (OR = 4.3; 95% CI: 3.04–6.2), vitamin D supplements (OR = 12.6; 95% CI: 7.4–22.9), and drink at least one glass of milk a day (OR = 13.3; 95% CI: 7.8–23.9). Further, women with low bone mass were more likely to use calcium supplements (OR = 1.7; 95% CI: 1.2–2.3) and vitamin D supplements (OR = 1.6; 95% CI: 1.1–2.2) compared with women who had normal bone mass. Thus, our intervention improved self-reported lifestyle behaviors in premenopausal women. Such behaviors may ultimately increase peak bone mass and decrease the risk of developing osteoporosis.
Growing C57Bl/6 Mice Increase Whole Bone Mechanical Properties by Increasing Geometric and Material Properties
Authors: Brodt Michael D. , Ellis Cara B. , Silva Matthew J.
Published: April 2006

In vivo murine models are becoming increasingly important in bone research. To establish baseline data for researchers using these models, we studied the long bones from C57BL/6 female mice, a strain that is widely used in bone research. We determined the femoral structural and material properties in both torsion and four-point bending for mice at ages 4–24 weeks. Measurements of femoral cross-sectional geometry and tibial densitometric properties were also obtained. Results indicated that all structural properties (except ultimate energy), changed significantly with age (p < 0.001). Ultimate torque, ultimate moment, torsional rigidity, and bending rigidity all increased to peak values at 20 weeks, whereas ultimate rotation and ultimate displacement decreased to minimum values at 20 weeks. Our data indicate that increases in the material properties contributed more than increases in cross-sectional geometry to the changes in structural rigidity and ultimate load. For example, from 4–20 weeks torsional rigidity increased 1030%, while shear modulus increased 610% and the polar moment of inertia (a measure of the geometric resistance to rotation) increased by only 85%. Changes in the cross-sectional geometry with age were due to increases in periosteal diameter and decreases in endosteal diameter. In general, both torsion and bending techniques revealed large changes in structural and material properties with age. We conclude that peak bone strength is not achieved before 20 weeks in C57BL/6 female mice, and that torsion and four-point bending tests are equally well suited for evaluating mechanical properties of murine long bones.
Molecular Characterization of the α1 Subunit of the L Type Voltage Calcium Channel Expressed in Rat Calvarial Osteoblasts
Authors: Loza Juan C. , Carpio Lillian C. , Bradford Peter G. , Dziak Rosemary
Published: April 2006

Voltage-activated calcium channels (VACCs) regulate extracellular calcium influx in many cells. VACCs are composed of five subunits. The α1 subunit is considered the most important in regulating channel function. Three isoforms of this subunit have been described: skeletal, cardiac, and neuroendocrine. It was the purpose of the present study to determine the molecular identity of the α1 subunit of the VACCs in rat calvarial osteoblasts and to study the nature of the regulation of these channels as a function of cellular growth. We also attempted to identify which isoform of the α1 subunit of the VACCs mediates the effects of epidermal growth factor (EGF) on osteoblastic cell proliferation. Reverse transcription-polymerase chain reaction was used to detect the isoforms of the VACCs that are expressed in osteoblastic cells. These analyses showed that the proliferative state of the cell and the time in culture influence RNA expression. The only α1 subunit detected in osteoblasts corresponds to the cardiac isoform. In additional experiments, the effects of EGF on cytosolic calcium and osteoblast proliferation were determined. For these experiments, the synthesis of the different isoforms of the VACCs was selectively blocked by antisense oligonucleotides prior to EGF stimulation. These studies showed that the cardiac isoform mediates the effects of EGF on cytosolic calcium and cellular proliferation in rat calvarial osteoblasts.
Cloning of a 2.5 kb Murine Bone Sialoprotein Promoter Fragment and Functional Analysis of Putative Osf2 Binding Sites
Authors: Benson M. Douglas , Aubin Jane E. , Xiao Guozhi , Thomas Peedikayil E. , Dr. Franceschi Renny T.
Published: April 2006

Bone sialoprotein (BSP) is an extracellular matrix protein that is intimately associated with the process of biomineralization. Osf2, a member of the Cbf/runt family of transcription factors, is required for the development of osteoblasts in vivo and has been reported to stimulate the transcription of BSP when overexpressed in mesenchymal cell lines. To investigate the role of Osf2 in BSP expression, we cloned a 2.5 kb fragment of a 5′ untranscribed sequence from the murine BSP gene and evaluated it for putative Osf2 binding sites. This promoter, which was able to direct 5- to 10-fold higher levels of luciferase reporter expression in osteoblastic cells than in nonbone cell lines, contains two consensus core binding sites for members of the Cbf/runt family. One, at –61 relative to the start of transcription, is within a region having 75% overall sequence identity with the rat and human BSP promoters. The other is located at −1335, outside this highly conserved region. Neither site is completely conserved in the rat or human sequences. Only the −1335 site was able to bind a protein in nuclear extracts of osteoblastic cells, and this protein was identified as Osf2. Despite this in vitro binding ability, we detected no significant enhancer activity in the −1335 element when placed in front of a minimal osteocalcin promoter driving a luciferase reporter gene in osteoblastic cells nor any loss in transcriptional activity of a 5′ promoter deletion which eliminated this element as compared with the full-length 2.5 kb promoter. These results suggest that Osf2 binding to the BSP promoter is not essential for its osteoblast-selective expression.
The Parathyroid Hormone–Related Protein (PTHrP) Gene: Use of Downstream TATA Promotor and PTHrP 1–139 Coding Pathways in Primary Breast Cancers Vary with the Occurrence of Bone Metastasis
Authors: Dr. Bouizar Zhor , Spyratos Frederique , De vernejoul Marie-Christine
Published: April 2006

We analyzed the use of different promoters and the splicing patterns of the exons encoding 5′- and 3′-untranslated sequence amounts of parathyroid hormone–related protein (PTHrP) gene products in breast cancers. Tumor samples from 74 cases of primary breast cancer that had been followed from 1 to 14 years were selected retrospectively according to the occurrence of metastasis: 18 patients developed no metastasis (NM), 56 developed metastases (M), 22 of whom developed metastases in soft tissues (MB−) and 34 of whom developed bone metastases (MB+). The amount of the 1–139 isoform mRNA was much higher in the tumors of patients who later developed metastases (M: 0.29 ± 0.03) than in those of patients who developed no metastases (NM, 0.13 ± 0.03; p < 0.01). This isoform mRNA was also more abundant in breast tumors from patients who developed bone metastases (MB+, 0.39 ± 0.04) than in those of patients who developed metastases in soft tissues (MB−, 0.15 ± 0.03; p < 0.0001). By contrast, the amounts of the 1–141 isoform mRNA in these three groups of tumors were similar, but its concentration was higher in the tumors of premenopausal women than in those of postmenopausal women (p < 0.05). Analysis with 5′ untranslated regions-specific primers showed transcription from all three putative transcription start sites of PTHrP (P1, P2, and P3). The P3-initiated transcripts were more abundant in patients who developed metastases (M, 0.31 ± 0.03) than in the nonmetastatic tumors (NM, 0.13 ± 0.03; p < 0.01). The amount of P3 element did not differ with the site of metastasis (BM+, 0.32 ± 0.05; BM−, 0.28 ± 0.05; NS). The same trend was observed for the P2 element. However, the use of P2-initiated messages was strongly associated with the absence of estrogen receptors from the breast tumors (p < 0.01). We thus find a close association between the pattern of PTHrP gene expression and the outcome of breast cancer. The P3-initiated start site and the presence of PTHrP 139 mRNA could help identify patients at risk of developing metastases.
Profound Hypogonadism Has Significant Negative Effects on Calcium Balance in Males: A Calcium Kinetic Study
Authors: Mauras Nelly , Hayes Valerie Y. , Vieira Nancy E. , Yergey Alfred L. , O'brien Kimberly O.
Published: April 2006

The impact of estrogen deficiency on bone has been extensively studied in the female; however, the effects of androgen deficiency on calcium fluxes in males have been less well characterized. We investigated the effect of short-term, severe androgen deficiency on measures of calcium absorption and kinetics as well as on markers of bone turnover in males. To accomplish this, 11 healthy male volunteers were recruited (mean age 23.3 ± 0.5 years [SEM], body mass index 25.3 ± 0.8 kg/m2). They consumed a weight maintenance diet for at least 3 days prior to admission to our Research Unit, with a calcium intake of ∼1200 mg/day. At baseline (D1), subjects received42Ca intravenously as well as44Ca PO mixed with milk or juice. A 29-h urine collection was begun and blood samples collected at frequent intervals for the measurement of the isotopic enrichment of42Ca and44Ca using thermal ionization mass spectrometry. Twice daily urine samples were collected for 5 days after the administration of the isotopes. A gonadotropin-releasing hormone agonist (Lupron) was given after D1, again 3 weeks later, and studies repeated identically 4 weeks (D2, n = 6) and 10 weeks from baseline (D3, n = 7) (two subjects completed three studies). Testosterone concentrations were markedly suppressed on both D2 and D3 (−95%, p < 0.006), whereas there were no detectable changes in growth hormone and insulin-like growth factor-1 concentrations. Urinary calcium excretion increased significantly after 4 weeks (43%, p = 0.0007) and 10 weeks (73%, p = 0.003) of sustained hypogonadism. Using a multicompartmental kinetic model, the contribution of oral calcium to the urinary losses was decreased by D3 (−41%, p = 0.01), yet the contribution of bone calcium to urine losses increased by 10 weeks (+11%, p = 0.01). There was a 21% decrease in bone calcium deposition (Vo+) by D3 (p < 0.05) with no significant change in bone resorption rates (Vo−). There was a significant correlation between the decrease in testosterone concentration and the increase in urinary calcium excretion, especially at 10 weeks (R2 = 0.84, p = 0.004). These kinetic changes were accompanied by a decrease in osteocalcin concentrations on D2, with improvements by D3. Urinary N telopeptide, a measure of bone resorption, also increased during the studies. In summary, profound hypogonadism in young males is associated with marked increases in urinary calcium losses, with a greater contribution of bone calcium to those losses and decreased kinetic markers of bone calcium deposition. We conclude that even short-term, severe deficiency in gonadal steroids can have profound negative effects on calcium and bone metabolism in males.
Monitoring Alendronate Therapy for Osteoporosis
Authors: De castro machado Adriana Braga , Hannon Rosemary , Prof. Eastell Richard
Published: April 2006

Alendronate is an antiresorptive therapy for osteoporosis and results in a decrease in bone turnover. To choose the optimal measurement for monitoring this therapy, the size of the change needs to be compared with the variability of the measurement. We studied 26 women with postmenopausal osteoporosis (bone mineral density [BMD] T score < –2.5), who were randomized in a 2:1 ratio to receive alendronate (10 mg/day) and calcium carbonate (500 mg/day) or calcium carbonate alone for 6 months. We measured serum markers of bone formation (osteocalcin [OC], bone isoform of alkaline phosphatase [BAP], and collagen type I C-terminal propeptide [CICP]) and urinary markers of bone resorption (cross-linked N-telopeptide [NTx], free deoxypyridinoline [iFDpd], and free pyridinolines). All subjects had two measurements 1 week apart at baseline to calculate the short-term variability. Biochemical measurements were then made at 4, 8, 12, 24, and 25 weeks. Measurements of bone mass were made by dual-energy X-ray absorptiometry (DXA) at the lumbar spine and proximal femur twice at baseline and then at 24 and 25 weeks. The mean difference in change in BMD and markers between both groups at the end of the study that were significant were (short-term variability in brackets): DXA total hip 4.3% (2.5%), NTX 49% (10%), iFDpd 22% (12%), OC 28% (13%), BAP 31% (13%), and CICP 31% (11%). Five of the six markers showed significant responses to alendronate therapy, but they differed in the relationship between size of response and variability. These biochemical markers performed better than DXA for monitoring alendronate therapy over 6 months.
Alteration in the Mechanical Competence and Structural Properties in the Femoral Neck and Vertebrae of Ovariectomized Rats
Authors: Peng Zhiqi , Tuukkanen Juha , Zhang Hongxue , Väänänen H. Kalervo
Published: April 2006

The structural and mechanical properties of bone in the femoral neck and various other sites were investigated in intact (INT), sham-operated (Sham), and ovariectomized (OVX) rats. Six weeks after operation, the maximal load and energy absorption of the femoral neck were significantly lower in the OVX than in the INT or Sham groups, being 73.2 ± 1.4 (SE) N, 86.3 ± 4.1 N, and 87.1 ± 3.2 N, respectively (p < 0.01) for load. The total cross-sectional area of the femoral neck did not change after OVX, but the marrow cavity area was enlarged, leading to a reduced bone area (including both cortical and trabecular bone) (p < 0.01). Histomorphometric analysis showed that new bone formation could not be detected at the periosteum of the femoral neck below the femoral head, but at the endocortical surfaces the double tetracycline labeling revealed an increased mineral apposition rate (MAR) and bone formation rate (BFR) in OVX animals (p < 0.001). In contrast, MAR and BFR were significantly increased in both periosteal and endocortical surfaces of the tibia, humerus, and femoral shaft, thus preventing a decrease in cortical bone area. The maximal bending loads of the tibia and humerus were not different in the various groups of animals. The correlation coefficient between maximal load and bone area revealed positive relationships in the femoral neck (r = 0.54, p < 0.01), tibia (r = 0.46, p < 0.01), and humerus (r = 0.51, p < 0.01). Ovariectomy resulted in a decreased trabecular bone volume of lumbar vertebra VI (L6) decreased compressive loads of lumbar vertebrae I, III, and IV. These lumbar bone loads were positively related to their L6 bone area (L4/L6: r = 0.66, p < 0.001). Element analyses (energy dispersion spectrometer) from trabecular and cortical areas of bone showed some changes related to aging but not to OVX. These results indicate that ovariectomy influences the biomechanical properties of rat bone by changing structural properties rather than material ones.
Determinants of Peak Bone Mass: Clinical and Genetic Analyses in a Young Female Canadian Cohort
Authors: Dr. Rubin Laurence A. , Hawker Gillian A. , Peltekova Vanya D. , Fielding Lynda J. , Ridout Rowena , Cole David E. C.
Published: April 2006

Peak bone mass has been shown to be a significant predictor of risk for osteoporosis. Previous studies have demonstrated that skeletal mass accumulation is under strong genetic control, and efforts have been made to identify candidate loci. Determinants of peak bone mass also include diet, physical activity, hormonal status, and other clinical factors. The overall contribution of these factors, genetic and nongenetic, and their interaction in determining peak bone density status have not been delineated. Six hundred and seventy-seven healthy unrelated Caucasian women ages 18–35 years were assessed. A detailed, standardized interview was conducted to evaluate lifestyle factors, menstrual and reproductive history, medical conditions, medication use, and family history of osteoporosis. Bone mineral density (BMD) was measured at the lumbar spine (L2–L4) and the femoral neck (hip) using dual-energy X-ray absorptiometry. Genotyping of the vitamin D receptor (VDR) locus at three polymorphic sites (BsmI, ApaI, and TaqI) was performed. In bivariate analyses, BMD at the lumbar spine and hip was positively correlated with weight, height, body mass index (BMI), and level of physical activity, both now and during adolescence, but negatively correlated with a family history of osteoporosis. Hip, but not spine BMD, correlated positively with dietary intake of calcium, and negatively with amenorrhea of more than 3 months, with caffeine intake, and with age. Spine, but not hip BMD, correlated positively with age and with number of pregnancies. VDR haplotype demonstrated significant associations with BMD at the hip, level of physical activity currently, and BMI. In multivariate analysis, independent predictors of greater BMD (at the hip or spine) were: age (younger for the hip, older for the spine), greater body weight, greater height (hip only), higher level of physical activity now and during adolescence, no family history of osteoporosis, and VDR genotype (hip only). Weight, age, level of physical activity, and family history are independent predictors of peak BMD. Of these factors, weight accounts for over half the explained variability in BMD. VDR alleles are significant independent predictors of peak femoral neck, but not lumbar spine BMD, even after adjusting for family history of osteoporosis, weight, age, and exercise. However, the overall contribution of this genetic determinant is modest. Taken together, these factors explained ∼17% and 21% of the variability in peak spine and hip BMD, respectively, in our cohort. Future research should be aimed at further evaluation of genetic determinants of BMD. Most importantly, understanding the critical interactive nature between genes and the environment will facilitate development of targeted strategies directed at modifying lifestyle factors as well as earlier intervention in the most susceptible individuals.
Developmental Regulation of Nitric Oxide Synthase Expression in Rat Skeletal Bone
Authors: Hukkanen Mika V. J. , Platts Louise A. M. , De Marticorena Iñigo Fernandez , O'Shaughnessy Meg , Macintyre Iain , Polak Julia M.
Published: April 2006

Nitric oxide (NO) has been implicated in bone growth and remodeling by studies showing that inhibition of NO-synthase (NOS) activity retards normal gain in bone mineral density both during skeletal development and after sexual maturity. In the present study, we aimed to assess the level of expression and cellular localization of the three NOS isoforms during skeletal bone development from neonatal to sexual maturity in female Wistar rats. Reverse transcription polymerase chain reaction (RT-PCR) was used to analyze the presence of NOS1 (neuronal), NOS2 (inducible), and NOS3 (endothelial) transcripts in femoral bone from neonatal, 4-, 8-, and 12-week-old rats. RT-PCR amplified NOS1, NOS2, and NOS3 transcripts of 472-, 807-, and 289-bp, respectively. There were no detectable differences in the levels of NOS1 mRNA between the groups; however, NOS2 mRNA was more abundant in the neonatal group compared with 4-, 8-, and 12-week groups. Expression of NOS1 protein could not be detected in bones by either Western blotting or immunocytochemistry in any of the age groups investigated. Western blots for NOS2 revealed expression in the neonatal group only and it was not detected in any of the older age groups. Immunostaining for NOS2 was also most evident in the neonatal group and was localized specifically to trabecular osteoblasts and osteoclasts. In all age groups studied, NOS3 mRNA and protein were found in bone-resorbing osteoclasts, cuboidal active osteoblasts, and osteocytes. Semiquantitative RT-PCR provided evidence of down-regulation of NOS3 transcripts during the skeletal development. This was confirmed using in situ hybridization, which showed higher expression in neonatal and 4-week groups than in other groups. Western blots and counting the ratio of trabecular osteoblasts that were NOS3 immunoreactive showed parallel down-regulation of NOS3 protein during skeletal development. Taken together, these data show that there is regulation of NOS2 and in particular NOS3 expression during skeletal development and this may be significant to trabecular bone growth and remodeling.
The Role of the Plasminogen System in Bone Resorption In Vitro
Authors: Daci E. , Udagawa N. , Martin T. J. , Bouillon R. , Carmeliet G.
Published: April 2006

The plasminogen/plasmin proteolytic cascade plays an important role in extracellular matrix remodeling. The presence of the two plasminogen activators (PAs), tissue-type plasminogen activator (tPA), and urokinase-type plasminogen activator (uPA), and their inhibitor type 1 (PAI-1) in bone cells, suggests a role in one or more aspects of bone resorption such as osteoclast formation, mineral dissolution, and degradation of the organic matrix. These different processes were assayed in vitro using cells derived from mice with either tPA (tPA−/−), uPA (uPA−/−), PAI-1 (PAI-1−/−) inactivation or with a combined inactivation (tPA−/−:uPA−/−) and compared with wild-type mice (WT). First, osteoclast formation, assessed by investigating the number and characteristics of tartrate-resistant acid phosphatase–positive multinucleated cells formed in cocultures of primary osteoblasts and bone marrow cells treated with 1α,25-dihydroxyvitamin D3, was not different between the different cell types. Second, dentine resorption, an assay for osteoclast activity, was not affected by the combined deficiency of both tPA and uPA. Finally, the ability to degrade nonmineralized bone-like matrix was however, significantly reduced in tPA−/−:uPA−/− cells compared with WT cells (28.1 ± 0.6%, n = 6 vs. 56.4 ± 3.1%, n = 6, respectively, p < 0.0001). Surprisingly, collagen proteolysis by bone cells was not dependent on the presence of plasmin as suggested by degradation assays performed on type I3H-collagen films. Taken together, these data suggest that the plasminogen activator/plasmin system is not required for osteoclast formation, nor for the resorption of the mineral phase, but is involved in the removal of noncollagenous proteins present in the nonmineralized bone matrix.
Systemic Administration of Acidic Fibroblast Growth Factor (FGF-1) Prevents Bone Loss and Increases New Bone Formation in Ovariectomized Rats
Authors: Dunstan C. R. , Boyce R. , Boyce B. F. , Garrett I. R. , Izbicka E. , Burgess W. H. , Mundy G. R.
Published: April 2006

There are no universally accepted agents that will substantially increase bone mass in osteoporotic patients. A number of peptides important in normal bone formation, such as members of the transforming growth factor-β superfamily, are not satisfactory for this purpose either because their beneficial effects are predominantly local or there is systemic toxicity associated with their administration. We have examined the effects of exogenous fibroblast growth factor-1 and -2 (FGF-1 and FGF-2) on bone in vivo, since FGFs have been shown recently to be essential for normal skeletal development. FGF-1 was injected daily (0.2 mg/kg intravenously) for 28 days into the tail vein of adult female rats immediately following and 6 months after sham operation or ovariectomy (OVX). In rats treated immediately post-OVX, OVX produced more than a 30% decrease in tibial bone density, which was prevented by FGF-1 and estrogen. However, FGF-1 also had an anabolic effect. In sham-operated rats, FGF-1 increased bone density to 2-fold, whereas estrogen had no effect. In rats 6 months post-OVX, severe bone loss and disruption of trabecular microarchitecture occurred similar to that seen in patients with severe osteoporosis. In these rats, administration of FGF-1 induced extensive new woven bone formation with new trabecular-like structures filling much of the marrow spaces, and bone density in the tibial metaphysis increased 3-fold. FGF-1 and FGF-2 were also administered subcutaneously over the calvaria of mice in doses of 2–2000 μg/day for 3 days and shown to produce substantial increases in bone formation when examined morphologically. Thus, we conclude that both local and systemic FGF-1 increases new bone formation and bone density, and systemic FGF-1 also appears to restore bone microarchitecture and prevent bone loss associated with estrogen-withdrawal.
Midkine Is Expressed During Repair of Bone Fracture and Promotes Chondrogenesis
Authors: Ohta Susumu , Muramatsu Hisako , Senda Takao , Zou Kun , Iwata Hisashi , Muramatsu Takashi
Published: April 2006

Midkine (MK) is a heparin-binding growth/differentiation factor implicated in the control of development and repair of various tissues. Upon fracture of the murine tibia, MK was found to be transiently expressed during bone repair. MK was immunohistochemically detected in spindle-shaped mesenchymal cells at the fracture site on day 4 after fracture and in chondrocytes in the area of endochondral ossification on day 7. MK expression was decreased on day 14 and scarcely seen on day 28 when bone repair was completed. This mode of MK expression is reminiscent of MK expression during development. MK was expressed in hypertrophic chondrocytes of the prebone cartilage rudiments on embryonic day 14 in mouse embryos. MK was also strongly expressed in the epiphyseal growth plate. MK was localized intracellularly during both bone repair and development, and this localization was confirmed by immunoelectron microscopy for embryonic chondrocytes. When MK cDNA was transfected into ATDC5 chondrogenic cells and overexpressed, the majority of transfected cells with strong MK expression showed enhanced chondrogenesis as revealed by increased synthesis of sulfated glycosaminoglycans, aggrecan, and type II collagen. These results suggest that MK plays important roles in chondrogenesis and contributes to bone formation and repair.
Dietary Conjugated Linoleic Acids Alter Serum IGF-I and IGF Binding Protein Concentrations and Reduce Bone Formation in Rats Fed (n-6) or (n-3) Fatty Acids
Authors: Li Yong , Seifert Mark F. , Ney Denise M. , Grahn Mike , Grant Alan L. , Allen Kenneth G. D. , Watkins Bruce A.
Published: April 2006

A study was designed to examine the effects of dietary conjugated linoleic acid (CLA) on serum concentrations of insulin-like growth factor-I (IGF-I) and IGF binding proteins (IGFBP) and the relationship of these factors to bone metabolism. Weanling male rats were fed AIN-93G diet containing 70 g/kg of added fat for 42 days. Treatments included 0 g/kg or 10 g/kg of CLA and soybean oil (SBO) or menhaden oil + safflower oil (MSO) following a 2 × 2 factorial design. Serum IGFBP was influenced by dietary polyunsaturated fatty acid (PUFA) type ((n-6) and (n-3)) and CLA (p = 0.01 for 38–43 kDa bands corresponding to IGFBP-3). CLA increased IGFBP level in rats fed SBO (p = 0.05) but reduced it in those fed MSO (p = 0.01). Rats fed MSO had the highest serum IGFBP-3 level. Both (n-3) fatty acids and CLA lowered ex vivo prostaglandin E2 production in bone organ culture. In tibia, rats given CLA had reduced mineral apposition rate (3.69 vs. 2.79 μm/day) and bone formation rate (BFR) (0.96 vs. 0.65 μm3/μm2/day); however, the BFR tended to be higher with MSO. Dietary lipid treatments did not affect serum intact osteocalcin or bone mineral content. These results showed that dietary PUFA type and CLA modulate local factors that regulate bone metabolism.
Oral Administration of the Growth Hormone Secretagogue MK-677 Increases Markers of Bone Turnover in Healthy and Functionally Impaired Elderly Adults
Authors: Murphy M. G. , Bach M. A. , Plotkin D. , Bolognese J. , Ng J. , Krupa D. , Cerchio K. , Gertz B. J.
Published: April 2006

Growth hormone (GH) stimulates osteoblasts in vitro and increases bone turnover and stimulates osteoblast activity when given to elderly subjects. Probably a major effect of GH on bone is mediated through stimulation of either circulating or locally produced insulin-like growth factor I (IGF-I). We determined the effect of chronic administration of the GH secretagogue, MK-677, on serum IGF-I and markers of bone turnover in 187 elderly adults (65 years or older) enrolled in three randomized, double-blind, placebo-controlled clinical studies lasting 2–9 weeks. Urine was collected for determination of N-telopeptide cross-links (NTXs), a marker of bone resorption, and blood was collected for determination of serum osteocalcin and bone-specific alkaline phosphatase (BSAP), as bone formation markers, and serum IGF-I levels pre- and post-treatment. Dose response data were initially obtained in healthy elderly subjects who received oral doses of 10 mg or 25 mg of MK-677 or placebo for 2 weeks (n = 10–12/group). Treatment with 10 mg and 25 mg of MK-677 for 2 weeks increased mean urine NTXs 10% and 17%, respectively (p < 0.05 vs. placebo). Additionally, 50 healthy elderly subjects received either placebo (n = 20) for 4 weeks or 25 mg of MK-677 (n = 30) daily for 2 weeks followed by 50 mg daily for 2 weeks. MK-677 increased mean serum osteocalcin by 8% (p < 0.05 vs. placebo). In both studies, MK-677 increased serum IGF-I levels significantly (55–94%). Subsequently, the biological effects of MK-677 were studied in 105 elderly subjects who met objective criteria for functional impairment. Subjects were randomized to receive oral doses of placebo for 9 weeks or either 5, 10, or 25 mg of MK-677 daily for an initial 2 weeks followed by 25 mg of MK-677 daily for the next 7 weeks(n = 63 on MK-677 and n = 28 on placebo completed 9 weeks of therapy). Treatment with MK-677 (all MK-677 groups combined) for 9 weeks increased mean serum osteocalcin by 29.4% and BSAP by 10.4% (p < 0.001 vs. placebo) and mean urinary NTX excretion by 22.6% (p < 0.05 vs. placebo). The change from baseline serum osteocalcin correlated with the change from baseline serum IGF-I in the MK-677 group (r = 0.37; p < 0.01). In conclusion, once daily dosing with MK-677, an orally active GH secretagogue, stimulates bone turnover in elderly subjects based on elevations in biochemical markers of bone resorption and formation.
A PCR Analysis of ERα and ERβ mRNA Abundance in Rats and the Effect of Ovariectomy
Authors: Dr. Lim S. K. , Won Y. J. , Lee H. C. , Huh K. B. , Park Y. S.
Published: April 2006

To study the relative abundance and the changes of both estrogen receptor alpha (ERα) and ERβ mRNA before and after ovariectomy in major organs important to the regulation of calcium homeostasis, we compared the degree of mRNA expression of ERα to that of ERβ in rat tissues by performing competitive reverse transcription polymerase chain reaction (RT-PCR) with internal standards. Both ERα and ERβ were highly expressed in the ovary {ERα[(2.2 ± 0.33) × 107 copies/μg of total RNA] > ERβ[(1.2 ± 0.33) × 105 copies/μg of total RNA]} as we expected. The bone marrow and renal cortex were very important target organs of estrogen because ERα was highly expressed ∼2 × 105 copies/μg of total RNA, but marrow cells revealed only a very weak expression of ERβ [(0.7 ± 0.21) × 102 copies/μg of total RNA]. Both ERα and ERβ were expressed in the trabecular bone [(3.2 ± 0.56) × 103 copy/μg of RNA] and [(2.8 ± 0.21) × 102 copy/μg of RNA], respectively. However, they were not detected in the cortical bone. In the jejunum, the expression of ERα was not detectable, while ERβ was expressed very weakly [(1.1 ± 0.24) × 102 copies/μg of total RNA]. The thyroid gland expressed low copy numbers of ERβ [(6.0 ± 0.23) × 102 copies/μg of total RNA], but the parathyroid gland was negative for both ERα and ERβ mRNA. In cultured stromal cells, ERα and ERβ mRNAs were not detected after a 24-h culture; however, the rates of mRNA expression of ERα and ERβ reached ∼105 copies/μg of total RNA and ∼102 copies/μg of total RNA, respectively, after 9-, 11-, and 13-day cultures. After ovariectomy, the expression of ERα mRNA decreased abruptly in the bone marrow and renal cortex, and both ERα and ERβ were barely detected in the trabecular bone. In conclusion, ERα might be the main ER in organs important for calcium homeostasis, except in the jejunum. The mRNA expression of ERα in the bone marrow and renal cortex decreased abruptly after ovariectomy, which may partially explain why the effect of estrogen deficiency can be amplified and why trabecular bone loss is more predominant than cortical bone loss shortly after surgical or natural menopause.
Estrogen Regulation of Intestinal Calcium Absorption in the Intact and Ovariectomized Adult Rat
Authors: ten Bolscher Marieke , Netelenbos J. Coen , Barto Rob , van Buuren Lotti M. , van der vijgh Wim J. F.
Published: April 2006

Studies were carried out to examine the mechanism of action of estrogen on intestinal calcium absorption in the rat. Three-month-old Wistar rats were sham-operated or ovariectomized (OVX). They were fed a diet containing 0.4% Ca, 0.4% P, and 2000 IU vitamin D3/kg. Eight weeks after operation, both OVX and sham-operated rats were randomly assigned to eight treatment groups. Five groups received per 100 g of body weight 12.5 ng calcitriol (1,25-dihydroxyvitamin D3); 7.5 μg of estradiol-benzoate; 7.5 μg of estradiol-benzoate and 0.1 mg of ICI 182780; 12.5 ng of calcitriol and 0.1 mg of ICI 182780; and 0.1 mg of ICI 182780, respectively. Three groups received the various vehicles used. Intestinal calcium absorption was measured in vivo using single pass perfusion of the duodenum. OVX did not change intestinal calcium absorption. A pharmacological dose of estradiol-benzoate caused a significant increase in intestinal absorption of calcium, which was comparable to that of a pharmacological dose of calcitriol in both OVX and sham-operated rats. Estrogen-induced rise in intestinal calcium absorption was completely blocked to basal level by the pure estrogen receptor (ER) antagonist ICI 182780. In contrast, ICI 182780 did not antagonize calcitriol-enhanced intestinal calcium absorption. Our findings suggest that estrogen stimulates intestinal calcium absorption via an ER.
Age-Related Changes in Bone Turnover in Men
Authors: Fatayerji D. , Dr. Eastell R.
Published: April 2006

Biochemical markers of bone turnover can be used to study the pathophysiology of osteoporosis. So far there have been few such studies in men. The aims of this study were to determine the effect of aging on bone turnover and to identify which hormones might regulate bone turnover in men. We studied 178 healthy Caucasian men, ages 20–79 years (30 per decade). The data for the effect of age on bone turnover was best fit by a quadratic function (nadirs at age 56, 57, 53, 39, and 58 years for intact propeptide of type I procollagen, osteocalcin, bone alkaline phosphatase, free deoxypyridinoline, and cross-linked N-telopeptides of type I collagen, respectively). For most markers, bone turnover tended to be highest in the third decade, lowest in the fifth and sixth decade, with a small increase in some markers in the eighth decade. Insulin-like growth factor-I (IGF-I), insulin-like growth factor binding protein-3, dehydroepiandrosterone sulfate, testosterone, estradiol, and free androgen index all decreased significantly with age (54, 17, 76, 26, 33, and 57%, respectively), while sex hormone binding globulin and parathyroid hormone increased significantly with age (62% and 43%). IGF-I and sex hormones were positively correlated with bone turnover, and this association was stronger in young men than older men. In conclusion, increased IGF-I and sex hormones may be associated with increased bone turnover in young men, with less influence on bone turnover in older men.
Interleukin-17: A New Bone Acting Cytokine In Vitro
Authors: Van Bezooijen Rutger L. , Farih-Sips Hetty C. M. , Papapoulos Socrates E. , Löwik Clemens W. G. M.
Published: April 2006

Interleukin-17 (IL-17) is a recently cloned cytokine that is exclusively produced by activated T cells, but its receptor has been found on several cells and tissues. Like other proinflammatory cytokines produced by activated T cells, IL-17 may affect osteoclastic resorption and thereby mediate bone destruction accompanying some inflammatory diseases. In the present study, we investigated whether osteogenic cells possess the receptor for IL-17 (IL-17R) and whether IL-17 affects osteoclastic resorption. We found that IL-17R mRNA is expressed both in mouse MC3T3-E1 osteoblastic cells and fetal mouse long bones, suggesting that osteogenic cells may be responsive to IL-17. In fetal mouse long bones, IL-17 had no effect on basal and IL-1β–stimulated osteoclastic bone resorption, but when given together with tumor necrosis factor-α (TNF-α) it increased bone resorption dose dependently in serum-free conditions. In addition, IL-17 increased TNF-α–induced IL-1α, IL-1β, and IL-6 mRNA expression in fetal mouse metatarsals and IL-1α and IL-6 mRNA expression in MC3T3-E1 cells. In conclusion, IL-17R mRNA was expressed by mouse osteoblastic cells and fetal mouse long bones, and IL-17 in combination with TNF-α, but not IL-1β, increased osteoclastic resorption in vitro. IL-17 may therefore affect bone metabolism in pathological conditions characterized by the presence of activated T cells and TNF-α production such as rheumatoid arthritis and loosening of bone implants.
Differentiation of Human Marrow Stromal Precursor Cells: Bone Morphogenetic Protein-2 Increases OSF2/CBFA1, Enhances Osteoblast Commitment, and Inhibits Late Adipocyte Maturation
Authors: Gori Francesca , Thomas Thierry , Hicok Kevin C. , Spelsberg Thomas C. , Riggs B. Lawrence
Published: April 2006

Because regulation of the differentiation to osteoblasts and adipocytes from a common progenitor in bone marrow stroma is poorly understood, we assessed effects of bone morphogenetic protein-2 (BMP-2) on a conditionally immortalized human marrow stromal cell line, hMS(2–6), which is capable of differentiation to either lineage. BMP-2 did not affect hMS(2–6) cell proliferation but enhanced osteoblast differentiation as assessed by a 1.8-fold increase in expression of OSF2/CBFA1 (a gene involved in commitment to the osteoblast pathway), by increased mRNA expression and protein secretion for alkaline phosphatase (ALP), type I procollagen and osteocalcin (OC) (except for OC protein), and by increased mineralized nodule formation. Transient transfection with Osf2/Cbfa1 antisense oligonucleotide substantially reduced BMP-2–stimulated expression of ALP mRNA and protein. The effects of BMP-2 on adipocyte differentiation varied: expression of peroxisome proliferator-activated receptor γ2 (a gene involved in commitment to the adipocyte pathway) was unchanged, mRNA expression of the early differentiation marker, lipoprotein lipase, was increased, and mRNA and protein levels of the late differentiation marker, leptin, and the formation of cytoplasmic lipid droplets were decreased. Thus, by enhancing osteoblast commitment and by inhibiting late adipocyte maturation, BMP-2 acts to shunt uncommitted marrow stromal precursor cells from the adipocyte to the osteoblast differentiation pathway.
Renal Chloride Channel, CLCN5, Mutations in Dent's Disease
Authors: Cox Jeremy P. D. , Yamamoto Katsusuke , Christie Paul T. , Wooding Carol , Feest Terry , Flinter Frances A. , Goodyer Paul R. , Leumann Ernst , Neuhaus Thomas , Reid Christopher , Williams Paul F. , Wrong Oliver , Thakker Rajesh V.
Published: April 2006

Dent's disease is an X-linked renal tubular disorder characterized by low-molecular-weight proteinuria, hypercalciuria, nephrocalcinosis, nephrolithiasis, and renal failure. Patients with Dent's disease may also suffer from rickets and other features of the renal Fanconi Syndrome. Patients may have mutations in the X-linked renal chloride channel gene, CLCN5, which encodes a 746-amino-acid protein with 12–13 transmembrane domains. We have investigated the 11 coding exons of CLCN5 for mutations in eight unrelated patients with Dent's disease. Leukocyte DNA was used for the polymerase chain reaction amplification of CLCN5 and the products analyzed for single-stranded conformational polymorphisms (SSCPs). Abnormal SSCPs were sequenced and revealed eight mutations. These consisted of three nonsense mutations (Arg34Stop, Arg648Stop, Arg704Stop), four deletions involving codons 40, 86, 157, and 241, and one acceptor splice consensus sequence mutation tgcag → tgaag. The mutations were confirmed either by restriction endonuclease or sequence-specific oligonucleotide hybridization analysis. In addition, an analysis of 110 alleles from 74 unrelated normal individuals demonstrated that the DNA sequence changes were not common polymorphisms. All of the mutations predict truncated chloride channels that are likely to result in a functional loss. Thus, our findings expand the spectrum of CLCN5 mutations associated with Dent's disease and the results will help to elucidate further the functional domains of this novel chloride channel.