bluuebunny/crossref_metadata_2025_split huggingface dataset:
Two Human Osteoblast-like Osteosarcoma Cell Lines Show Distinct Expression and Differential Regulation of Parathyroid Hormone–Related Protein
Authors: Jemtland Rune , Rian Edith , Olstad Ole Kristoffer , Haug Egil , Bruland Øyvind S. , Bucht Elisabet , Gautvik Kaare M. 
Published: April 2006

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

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

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

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

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

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

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

Abstract
Previous studies have indicated that physiological levels of dynamic mechanical strain produce rapid increases in nitric oxide (NO) release from rat ulna explants and primary cultures of osteoblast-like cells and embryonic chick osteocytes derived from long bones. To establish the mechanism by which loading-induced NO production may be regulated, we have examined: nitric oxide synthase (NOS) isoform mRNA and protein expression, the effect of mechanical loading in vivo on NOS mRNA expression, and the effect of mechanical strain on NO production by bone cells in culture. Using Northern blot analyses, in situ hybridization, and immunocytochemistry we have established that the predominant NOS isoform expressed in rat long bone periosteal osteoblasts and in a distinct population of cortical bone osteocytes is the endothelial form of NOS (eNOS), with little or no expression of the inducible NOS or neuronal NOS isoforms. In contrast, in non–load-bearing calvariae there are no detectable levels of eNOS in osteocytes and little in osteoblasts. Consistent with these observations, ulnar explants release NO rapidly in response to loading in vitro, presumably through the activation of eNOS, whereas calvarial explants do not. The relative contribution of different bone cells to these rapid increases in strain-induced NO release was established by assessment of medium nitrite (stable NO metabolite) concentration, which showed that purified populations of osteocytes produce significantly greater quantities of NO per cell in response to mechanical strain than osteoblast-like cells derived from the same bones. Using Northern blot hybridization, we have also shown that neither a single nor five consecutive daily periods of in vivo mechanical loading produced any significant effect on different NOS isoform mRNA expression in rat ulnae. In conclusion, our results indicate that eNOS is the prevailing isoform expressed by cells of the osteoblast/osteocyte lineage and that strain produces increases in the activity of eNOS without apparently altering the levels of eNOS mRNA.
The Human Vitamin D Receptor Gene (VDR) Is Localized to Region 12cen-q12 by Fluorescent In Situ Hybridization and Radiation Hybrid Mapping: Genetic and Physical VDR Map
Authors: Taymans Susan E. , Pack Svetlana , Pak Evgenia , Orban Zsolt , Barsony Julia , Zhuang Zhengping , Stratakis Constantine A. 
Published: April 2006

Abstract
The vitamin D receptor (VDR) is a member of the steroid hormone receptor superfamily of ligand-activated transcription factors. The VDR gene was previously mapped to human chromosome 12q13–12q14, but its precise physical and genetic localization are unknown. The present study reports the mapping of the human VDR gene by radiation hybrid (RH) analysis, the isolation of a bacterial artificial chromosome (BAC) containing this gene, and physical mapping of the VDR gene by fluorescent in situ hybridization (FISH). RH analysis placed the VDR gene locus at chromosome 12cen-q12, flanked by Stanford Human Genome Center (SHGC) 30216 and SHGC 9798 (D12S1892) markers. FISH analysis of a BAC containing the VDR gene confirmed its centromeric location. Thus, we have identified a BAC and genetic markers which can be used in the genetic analysis of the VDR gene and investigation of its involvement in osteoporosis and related disorders. We conclude that the VDR gene is centromeric to its previously reported locus on chromosome 12.
Direct Three-Dimensional Morphometric Analysis of Human Cancellous Bone: Microstructural Data from Spine, Femur, Iliac Crest, and Calcaneus
Authors: Hildebrand Tor , Laib Andres , Müller Ralph , Dequeker Jan , Rüegsegger Peter 
Published: April 2006

Abstract
The appearance of cancellous bone architecture is different for various skeletal sites and various disease states. During aging and disease, plates are perforated and connecting rods are dissolved. There is a continuous shift from one structural type to the other. So traditional histomorphometric procedures, which are based on a fixed model type, will lead to questionable results. The introduction of three-dimensional (3D) measuring techniques in bone research makes it possible to capture the actual architecture of cancellous bone without assumptions of the structure type. This requires, however, new methods that make direct use of the 3D information. Within the framework of a BIOMED I project of the European Union, we analyzed a total of 260 human bone biopsies taken from five different skeletal sites (femoral head, vertebral bodies L2 and L4, iliac crest, and calcaneus) from 52 donors. The samples were measured three-dimensionally with a microcomputed tomography scanner and subsequently evaluated with both traditional indirect histomorphometric methods and newly developed direct ones. The results show significant differences between the methods and in their relation to the bone volume fraction. Based on the direct 3D analysis of human bone biopsies, it appears that samples with a lower bone mass are primarily characterized by a smaller plate-to-rod ratio, and to a lesser extent by thinner trabecular elements.
Lactulose Stimulates Calcium Absorption in Postmenopausal Women
Authors: van den heuvel Ellen G. H. M. , Muijs Theo , van dokkum Wim , Schaafsma Gertjan 
Published: April 2006

Abstract
Animal studies have indicated that calcium absorption is increased by lactulose, a synthetic disaccharide. Therefore, the influence of lactulose on calcium absorption was measured in postmenopausal women who may benefit from the possible enhancing effect of lactulose on calcium absorption. Twelve postmenopausal women drank 100 ml of water containing 5 or 10 g of lactulose or a reference substance at breakfast for 9 days. The three treatments were given according to a randomized, double-blind, cross-over design, separated by two 19-day wash-out periods. On the 8th day of each treatment period,44Ca dissolved in orange juice was drunk immediately after the solution with the study substance and just before a standard breakfast with 162 mg of carrier calcium. Within half an hour,48Ca was given intravenously. Based on isotope ratios measured in urine collected before and until 36 h after isotope administration, true fractional calcium absorption was calculated. Calcium absorption during the treatments with the reference substance, 5 g and 10 g of lactulose was (mean ± SD) 27.7 ± 7.7, 30.0 ± 7.6, and 32.2 ± 7.0, respectively. A significant difference in calcium absorption was found between the highest dose of lactulose and the reference treatment (p &lt; 0.01). A significant linear trend was found between the dose of lactulose and its positive effect on calcium absorption. In conclusion, in postmenopausal women a 9-day consumption of lactulose increases calcium absorption in a dose-response way. More research is warranted to explore how lactulose stimulates calcium absorption and whether it is able to improve calcium balance and/or to attenuate the rate of aging bone loss.