bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of a New Selective Estrogen Receptor Modulator (MDL 103,323) on Cancellous and Cortical Bone in Ovariectomized Ewes: A Biochemical, Histomorphometric, and Densitometric Study
Authors: Chavassieux Pascale , Garnero Patrick , Duboeuf Francois , Vergnaud Philippe , Brunner-Ferber Francoise , Delmas Pierre D. , Meunier Pierre J.
Published: April 2006

The aims of this study performed in ewes were: (1) to confirm in this animal model the effects on bone of ovariectomy (OVX) alone or associated with Lentaron (L), a potent peripheral aromatase inhibitor, used to amplify the effects of OVX and (2) to evaluate the effects of a new selective estrogen receptor modulator (SERM; MDL 103,323) on bone remodeling. Thirty-nine old ewes were divided into five groups: sham (n = 7); OVX (n = 8); OVX + L (n = 8); OVX + L + MDL; 0.1 mg/kg per day (n = 8); and OVX + L + MDL 1 mg/kg per day (n = 8). The animals were treated for 6 months. Biochemical markers of bone turnover (urinary excretion of type 1 collagen C-telopeptide [CTX], serum osteocalcin [OC], and bone alkaline phosphatase [BAP]) were measured each month. Bone biopsy specimens were taken at the beginning and after death at the end of the experiment. Bone mineral density (BMD) was measured by dual-energy X-ray absorptiometry (DXA) on the lumbar spine and femur. OVX induced a significant increase in biochemical markers. This effect was the highest after 3 months for CTX (+156% vs. sham) and after 4 months for OC and BAP (+74% and +53% vs. sham, respectively). L tended to amplify the effect of OVX on OC and BAP. OVX induced significant increases in the porosity, eroded, and osteoid surfaces in cortical bone but no effect was observed in cancellous bone. MDL treatment reduced the bone turnover as assessed by bone markers, which returned to sham levels as well as histomorphometry both in cortical and in cancellous bone. Cancellous osteoid thickness decreased by 27% (p < 0.05), mineralizing perimeter by 81% (p < 0.05), and activation frequency by 84% (p < 0.02) versus OVX + L. Femoral and spinal BMD were increased by MDL and tended to return to the sham values. The effects of OVX on bone turnover were different on cortical and cancellous bone. These effects on cortical bone were reflected by changes in biochemical markers. MDL markedly reduces bone turnover and increases BMD suggesting that this new agent may prevent postmenopausal bone loss.
Comparison of Trabecular Bone Microarchitecture and Remodeling in Glucocorticoid-Induced and Postmenopausal Osteoporosis
Authors: Carbonare L. Dalle , Arlot M. E. , Chavassieux P. M. , Roux J. P. , Portero N. R. , Meunier P. J.
Published: April 2006

Long-term treatment with glucocorticoids (GCs) leads to a rapid bone loss and to a greater risk of fractures. To evaluate the specific effects of this treatment on cancellous bone remodeling, structure, and microarchitecture, we compared 22 transiliac biopsy specimens taken in postmenopausal women (65 ± 6 years) receiving GCs (≥7.5 mg/day, for at least 6 months) and 22 biopsy specimens taken in age-matched women with postmenopausal osteoporosis (PMOP), all untreated and having either at least one vertebral fracture or a T score < −2.5 SD. On these biopsy specimens, we measured static and dynamic parameters reflecting trabecular bone formation and resorption. Also, we performed the strut analysis and evaluated the trabecular bone pattern factor (TBPf), Euler number/tissue volume (E/TV), interconnectivity index (ICI), and marrow star volume (MaSV). Glucocorticoid-induced osteoporosis (GIOP), when compared with PMOP, was characterized by lower bone volume (BV/TV), trabecular thickness (Tb.Th), wall thickness (W.Th), osteoid thickness (O.Th), bone formation rate/bone surface (BFR/BS), adjusted mineral apposition rate/bone surface (Aj.AR/BS), and higher ICI and resorption parameters. After adjustment for BV/TV, the W.Th remained significantly lower in GIOP (p < 0.0001). The active formation period [FP(a+)] was not different. Patients with GIOP were divided into two groups: high cumulative dose GCs (HGCs; 23.7 ± 9.7 g) and low cumulative dose GCs (LGCs; 2.7 ± 1.2 g). HGC when compared with LGC was characterized by lower W.Th (p < 0.05), BV/TV (p < 0.001), Tb.Th (p < 0.05), trabecular number (Tb.N; p < 0.05), FP(a+) (p < 0.05), and nodes (p < 0.05), and higher E/TV (p < 0.05), ICI (p < 0.005), and TBPf (p < 0.05). When HGC was compared with PMOP, the results were similar except for the MaSV, which was significantly higher (p < 0.005). In summary, GIOP was characterized by lower formation and higher resorption than in PMOP, already present after LGC. With HGCs, these changes were associated with a more dramatic bone loss caused by a major loss of trabecular connectivity.
The Loss of Smad3 Results in a Lower Rate of Bone Formation and Osteopenia Through Dysregulation of Osteoblast Differentiation and Apoptosis
Authors: Borton Anita J. , Frederick Joshua P. , Datto Michael B. , Wang Xiao-Fan , Weinstein Robert S.
Published: April 2006

Smad3 is a well-characterized intracellular effector of the transforming growth factor β (TGF-β) signaling pathway and was implicated recently in the potentiation of vitamin D receptor (VDR)-mediated signaling. Given that both TGF-β and vitamin D are important regulators of bone remodeling, it is expected that Smad3 plays an integral role in normal maintenance of bone. However, the exact mechanisms by which Smad3 functions in bone remodeling are unknown. Here, we show that mice with targeted deletion of Smad3 are osteopenic with less cortical and cancellous bone compared with wild-type littermates. Decreases in bone mineral density (BMD) in Smad3 null mice reflect the inability of osteoblasts to balance osteoclast activity, although osteoclast numbers are normal and vitamin D mediated serum calcium homeostasis is maintained. The osteopenia of Smad3 null mice is attributed to a decreased rate of bone formation associated with increased osteocyte number and apoptosis. These findings are supported by studies with isolated primary osteoblasts that show TGF-β can no longer inhibit the differentiation of osteoblasts in the absence of Smad3; yet, TGF-β-stimulated proliferation remains intact. Together these data support a model that a loss of Smad3 increases the osteocyte fate of the osteoblast and decreases the duration of osteoblast function by shortening lifespan, ultimately resulting in osteopenia.
Focal Adhesion Kinase Activity Is Required for Bone Morphogenetic Protein—Smad1 Signaling and Osteoblastic Differentiation in Murine MC3T3-E1 Cells
Authors: Tamura Yasuhiro , Takeuchi Yasuhiro , Suzawa Miyuki , Fukumoto Seiji , Kato Mitsuyasu , Miyazono Kohei , Fujita Toshiro
Published: April 2006

Cell-matrix interactions via integrins are essential for osteoblastic differentiation. We have shown that signals activated by aggregation of α2β1-integrin with type I collagen are involved in the differentiation of osteoblastic MC3T3-E1 cells. Focal adhesion kinase (FAK) is an immediate downstream signal of the β1-integrin, and inactivation of FAK has been shown to disrupt osteoblastic differentiation. To elucidate roles of FAK in osteoblastic cells, we examined MC3T3-E1 cells stably expressing antisense FAK (asFAK) messenger RNA (mRNA). Alkaline phosphatase (ALP) activity, an osteoblastic marker, did not increase in asFAK cells with a long-term culture until 21 days or in response to bone morphogenetic protein 2 (BMP-2). Treatment with BMP-2 also failed to stimulate the expression of osteocalcin in asFAK cells. In control MC3T3-E1 cells, BMP-2 induced translocation of Smad1 into nuclei to stimulate transcriptional activity of the Smad6 promoter gene that contains a Smad1 response element. In contrast, BMP-2 failed to increase transcriptional activity of Smad6 promoter gene in asFAK cells, although it induced nuclear translocation of Smad1. These results indicate that FAK was involved in Smad1-dependent transcriptional activity but not in nuclear translocation of Smad1 in osteoblastic cells. Hence, FAK activation by integrins might converge transcriptional activation by BMP of its target genes in osteoblastic cells. These observations suggest that FAK activity is essential for BMP-Smad signaling to stimulate osteoblastic differentiation.
Transforming Growth Factor β Affects Osteoclast Differentiation via Direct and Indirect Actions
Authors: Quinn Julian M. W. , Itoh Kanami , Udagawa Nobuyuki , Häusler Karl , Yasuda Hisataka , Shima Nobuyuki , Mizuno Atsuko , Higashio Kanji , Takahashi Naoyuki , Suda Tatsuo , Martin T. John , Gillespie Matthew T.
Published: April 2006

Transforming growth factor β (TGF-β) is abundant in bone and has complex effects on osteolysis, with both positive and negative effects on osteoclast differentiation, suggesting that it acts via more than one mechanism. Osteoclastogenesis is determined primarily by osteoblast (OB) expression of the tumor necrosis factor (TNF)-related molecule receptor activator of NF-κB ligand (RANKL) and its decoy receptor osteoprotegerin (OPG), which are increased and decreased, respectively, by osteolytic factors. A RANKL-independent osteoclastogenic mechanism mediated by TNF-α has also been shown. Therefore, we investigated TGF-β effects on osteoclast formation in culture systems in which osteoclastogenic stimulus is dependent on OBs and culture systems where it was provided by exogenously added RANKL or TNF-α. Both OPG and TGF-β inhibited osteoclast formation in hemopoietic cell/OB cocultures, but the kinetics of their action differed. TGF-β also inhibited osteoclastogenesis in cocultures of cells derived from OPG null (opg−/−) mice. TGF-β strongly decreased RANKL messenger RNA (mRNA) expression in cultured osteoblasts, and addition of exogenous RANKL to TGFβ-inhibited cocultures of opg−/− cells partially restored osteoclastogenesis. Combined, these data indicate that the inhibitory actions of TGF-β were mediated mainly by decreased OB production of RANKL. In contrast, in the absence of OBs, TGF-β greatly increased osteoclast formation in recombinant RANKL- or TNF-α-stimulated cultures of hemopoietic cells or RAW 264.7 macrophage-like cells to levels several-fold greater than attainable by maximal stimulation by RANKL or TNF-α. These data suggest that TGF-β may increase osteoclast formation via action on osteoclast precursors. Therefore, although RANKL (or TNF-α) is essential for osteoclast formation, factors such as TGF-β may powerfully modify these osteoclastogenic stimuli. Such actions may be critical to the control of physiological and pathophysiological osteolysis.
Antisense Oligodeoxynucleotide Evidence That a Unique Osteoclastic Protein-Tyrosine Phosphatase Is Essential for Osteoclastic Resorption
Authors: Suhr Sung Min , Pamula Sujatha , Baylink David J. , Lau K.-H. William
Published: April 2006

This study tested the hypothesis that a unique osteoclastic transmembrane protein tyrosine phosphatase (PTP-oc) is involved in osteoclastic resorption by determining whether suppression of PTP-oc expression with a specific phosphorothioated 20-mer PTP-oc antisense oligodeoxynucleotide (oligo) would inhibit basal, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]-stimulated, and PTH-stimulated osteoclastic resorption. Treatment of rabbit osteoclasts with 1 μM of the antisense oligo for up to 4 days showed a time-dependent reduction in PTP-oc protein level, indicating that this PTP-oc antisense oligo was effective. To assess the effect of PTP-oc antisense oligo on osteoclastic resorption, rabbit osteoclasts were pretreated for 3 days with 1 μM of the antisense, a scramble oligo, or vehicle, respectively, followed by a 3-day treatment with vehicle, 10 nM of 1,25(OH)2D3, or 10 nM of parathyroid hormone (PTH). 1,25(OH)2D3 and PTH each alone increased PTP-oc cellular level and stimulated resorptive activity of rabbit osteoclasts. The antisense oligo treatment, but not the scramble oligo, decreased the basal and the stimulated resorption activity and reduced the PTP-oc protein level. Treatment with the PTP-oc antisense oligo, but not the scramble oligo, also markedly increased the Y527 phosphorylation level of c-src in rabbit osteoclasts. In conclusion, these results provide the first antisense oligo evidence that PTP-oc plays an essential role in osteoclastic resorption.
Spectroscopic Characterization of Collagen Cross-Links in Bone
Authors: Paschalis E. P. , Verdelis K. , Doty S. B. , Boskey A. L. , Mendelsohn R. , Yamauchi M.
Published: April 2006

Collagen is the most abundant protein of the organic matrix in mineralizing tissues. One of its most critical properties is its cross-linking pattern. The intermolecular cross-linking provides the fibrillar matrices with mechanical properties such as tensile strength and viscoelasticity. In this study, Fourier transform infrared (FTIR) spectroscopy and FTIR imaging (FTIRI) analyses were performed in a series of biochemically characterized samples including purified collagen cross-linked peptides, demineralized bovine bone collagen from animals of different ages, collagen from vitamin B6-deficient chick homogenized bone and their age- and sex-matched controls, and histologically stained thin sections from normal human iliac crest biopsy specimens. One region of the FTIR spectrum of particular interest (the amide I spectral region) was resolved into its underlying components. Of these components, the relative percent area ratio of two subbands at ∼1660 cm−1 and ∼1690 cm−1 was related to collagen cross-links that are abundant in mineralized tissues (i.e., pyridinoline [Pyr] and dehydrodihydroxylysinonorleucine [deH-DHLNL]). This study shows that it is feasible to monitor Pyr and DHLNL collagen cross-links spatial distribution in mineralized tissues. The spectroscopic parameter established in this study may be used in FTIRI analyses, thus enabling the calculation of relative Pyr/DHLNL amounts in thin (∼5 μm) calcified tissue sections with a spatial resolution of ∼7 μm.
Bone Mineral Density and Fracture Among Prevalent Kidney Stone Cases in the Third National Health and Nutrition Examination Survey
Authors: Lauderdale Diane S. , Thisted Ronald A. , Wen Ming , Favus Murray J.
Published: April 2006

Concern that people who form kidney stones may have reduced bone mineral density (BMD) and increased fracture risk has motivated clinical and population-based studies, but findings are inconsistent. In this cross-sectional study, we use the Third National Health and Nutrition Examination Survey (NHANES III) to determine whether a history of kidney stones (n = 793) is associated with lower femoral neck BMD and whether the association is similar for men and women. We further ask whether dietary calcium modifies the association between kidney stone history and BMD and whether there is an association between kidney stone history and prevalent spine or wrist fracture. We find that men with kidney stone history have lower femoral neck BMD than men without kidney stone history after adjusting for age, body mass index (BMI), race/ethnicity, and other potential confounders. The effect of kidney stone history on BMD is weaker for women. Men with kidney stone history also are more likely to report prevalent wrist and spine fractures. Dietary calcium, represented by usual milk consumption, is associated positively with BMD for both men and women and modifies the effect of kidney stone history on BMD for men. For men who form kidney stones, milk consumption is associated more strongly with femoral neck BMD than for men without such a history. The effect modification is such that the difference in BMD between men with and without kidney stone history is observed only at lower levels of milk consumption.
Regional and Hormone-Dependent Effects of Apolipoprotein E Genotype on Changes in Bone Mineral in Perimenopausal Women
Authors: Gerdes Lars Ulrik , Vestergaard Peter , Hermann Anne Pernille , Mosekilde Leif
Published: April 2006

We studied 479 perimenopausal Danish women aged 45–58 years to examine differences between APOE genotypes with respect to (1) baseline total body bone mineral density (BMD) and densities measured in five different regions (ultradistal forearm, proximal forearm, lumbar spine, femoral neck, and total hip region); (2) serum levels of alkaline phosphatase, bone isoenzyme alkaline phosphatase, osteocalcin, parathyroid hormone (PTH), 25-hydroxyvitamin D, and urine hydroxyproline/creatinine excretion ratio; and (3) changes in bone mineral during 5 years of follow-up. Baseline BMDs were identical, whereas serum levels of alkaline phosphatase and its bone isoenzyme were higher in women with APOE 2–2 and APOE 2–3 than in women with APOE 3–3 and APOE 3–4 and lower in women with APOE 4–4. Among women not receiving hormonal-replacement therapy (HRT; n = 262), those with APOE 2–2 and APOE 2–3 had 30–40% lower rates of femoral neck and total hip bone mineral loss than women with APOE 3–3 and APOE 3–4, whereas the rates of mineral loss in other skeletal regions did not differ between these APOE genotypes. Women with APOE 4–4 appeared to have lower rates of bone mineral loss in all regions. Women treated with hormones throughout the follow-up period (n = 113) gained bone mineral, and women with APOE 3–4 and APOE 4–4 gained relatively more mineral than other women. A comparison of untreated and treated women with APOE 2–3, APOE 3–3, and APOE 3–4 suggests a possible modification of the effect of APOE genotype by HRT. In conclusion, the common APOE polymorphism has a complex effect on bone metabolism in perimenopausal Danish women including possible modification by hormone use: (1) among women not receiving HRT, those with APOE2 have lower bone mineral losses in the femoral neck and hip region than other women, and (2) among women receiving HRT, those with APOE4 gain more bone mineral than other women.
Perspective: Postmenopausal Osteoporosis as a Failure of Bone's Adaptation to Functional Loading: A Hypothesis
Authors: Lanyon Lance , Skerry Tim
Published: April 2006

There is substantial evidence that bones' ability to withstand functional loading without damage depends on the processes of bone modeling and remodeling, which are responsible for establishing and maintaining bone architecture, being influenced by a feedback mechanism related to the control of functional strains. It is probably useful to consider the diminished ability to maintain bone strength in postmenopausal osteoporosis as a failure of this mechanism. Acceptance of this approach would not only increase understanding of the etiology of postmenopausal osteoporosis but also significantly influence the ways in which it is investigated and treated. This would not mean that the many other factors affecting bone mass and bone cell activity will be ignored, but rather these factors will be put in perspective. Research to prevent or treat osteoporosis could be directed usefully to understanding how osteoblasts, lining cells, and osteocytes respond to mechanically derived information and how these responses are converted into stimuli controlling structurally appropriate modeling and remodeling. Evidence suggesting that early strain-related responses of bone cells in males and females involve the estrogen receptor (ER) could explain decreased effectiveness of this pathway when ER levels are low.
Chromosome 13 Locus, Pbd2, Regulates Bone Density in Mice
Authors: Shimizu Motoyuki , Higuchi Keiichi , Kasai Soichiro , Tsuboyama Tadao , Matsushita Mutsumi , Mori Masayuki , Shimizu Yasuhiko , Nakamura Takashi , Hosokawa Masanori
Published: April 2006

Bone density is inherited as a complex polygenic trait. Previously, we identified two quantitative trait loci (QTLs) specifying the peak relative bone mass (bone mass corrected by bone size) on chromosomes (Chrs) 11 and 13 by interval mapping in two mouse strains: SAMP2 and SAMP6. The latter strain is an established murine model of senile osteoporosis and exhibits a significantly lower peak relative bone mass than SAMP2 mice. In this study, we report the effects of the Chr 13 QTL on peak bone density (Pbd2). First, we constructed a congenic strain P6.P2-Pbd2b, which carried a single genomic interval from the Chr 13 of SAMP2 on an SAMP6-derived osteoporotic background, to dissect this polygenic trait into single gene factors. This congenic strain had a higher bone density than the background strain using three measurement methods with different principles for bone density. Next, we measured the peak relative bone mass of the AKR/J strain and the 13 senescence-accelerated mouse (SAM) strains, which are considered to be a series of recombinant-like inbred (RI) strains derived from the AKR/J strain and other unspecified strains. We then determined the microsatellite marker haplotypes of these strains around the Pbd2 locus, in which three strains with a high relative bone mass shared the same haplotype over the 26-centimorgan (cM) region. In the Pbd2 locus, a high relative bone mass was associated with alleles of the unknown strain, whereas a low relative bone mass was associated with the alleles from the AKR/J strain. These results confirmed the existence of a Pbd2 locus regulating bone density in the SAM strains.
The Frequency of Common Progenitors for Adipocytes and Osteoblasts and of Committed and Restricted Adipocyte and Osteoblast Progenitors in Fetal Rat Calvaria Cell Populations
Authors: Bellows C. G. , Heersche J. N. M.
Published: April 2006

In fetal rat calvaria (RC) cell populations, adipocyte differentiation is stimulated by both dexamethasone (Dex) and 1,25 dihydroxyvitamin D3 [1,25(OH)2D3], whereas osteoblast differentiation is stimulated by Dex but inhibited by 1,25(OH)2D3. We examined whether the osteoblastic and adipocytic colonies were derived from a common progenitor, from committed and restricted adipocyte and osteoblast progenitors, or from both and whether the adipocyte progenitors stimulated by 1,25(OH)2D3 constitute a population of progenitors that is different from that stimulated by Dex. RC cells were isolated by sequential enzyme digestion yielding five populations designated I-V. In population I the effect of Dex on adipocyte formation was greater than that of 1,25(OH)2D3, whereas the effect of 1,25(OH)2D3 was greater than that of Dex in populations III-V. We next applied replica plating techniques to further investigate the response characteristics of individual osteoprogenitors and adipocyte progenitors by looking at the fate of duplicate colonies derived from the same progenitor under different culture conditions. RC cells were plated at 1000–1500 cells/100 mm culture dish and a 17-μm mesh polyester membrane overlaid onto master dishes on day 4 or day 5 and removed on day 11 or day 12. Then, replicas and master dishes were cultured separately in medium containing either Dex, 1,25(OH)2D3, or Dex plus 1,25(OH)2D3 for a further 17–21 days and then fixed and stained with both Sudan IV and the von Kossa technique. Nine hundred twenty-seven matched colonies present on both master dishes and replica membranes were screened and colonies were classified as either adipocytic, osteoblastic (bone or osteoid), or fibroblastic. Results show convincingly that most of the osteoprogenitors present in fetal RC cells are committed and restricted to the osteoblastic cell lineage (95.29%); that the 1,25(OH)2D3-responsive adipocyte progenitors are different from the Dex-responsive adipocyte progenitors, but both are restricted to form adipocytes and finally; and that a common osteoblast/adipocyte progenitor is present in a low frequency (4.71% of osteoprogenitors).
Early Detection of Bone Metastases in a Murine Model Using Fluorescent Human Breast Cancer Cells: Application to the Use of the Bisphosphonate Zoledronic Acid in the Treatment of Osteolytic Lesions
Authors: Peyruchaud Olivier , Winding Bent , Pécheur Isabelle , Serre Claire-Marie , Delmas Pierre , Clézardin Philippe
Published: April 2006

A very common metastatic site for human breast cancer is bone. The traditional bone metastasis model requires human MDA-MB-231 breast carcinoma cell inoculation into the left heart ventricle of nude mice. MDA-MB-231 cells usually develop osteolytic lesions 3–4 weeks after intracardiac inoculation in these animals. Here, we report a new approach to study the formation of bone metastasis in animals using breast carcinoma cells expressing the bioluminescent jellyfish protein (green fluorescent protein [GFP]). We first established a subclone of MDA-MB-231 cells by repeated in vivo passages in bone using the heart injection model. On stable transfection of this subclone with an expression vector for GFP and subsequent inoculation of GFP-expressing tumor cells (B02/GFP.2) in the mouse tail vein, B02/GFP.2 cells displayed a unique predilection for dissemination to bone. Externally fluorescence imaging of live animals allowed the detection of fluorescent bone metastases approximately 1 week before the occurrence of radiologically distinctive osteolytic lesions. The number, size, and intensity of fluorescent bone metastases increased progressively with time and was indicative of breast cancer cell progression within bone. Histological examination of fluorescent long bones from B02/GFP.2-bearing mice revealed the occurrence of profound bone destruction. Treatment of B02/GFP.2-bearing mice with the bisphosphonate zoledronic acid markedly inhibited the progression of established osteolytic lesions and the expansion of breast cancer cells within bone. Overall, this new bone metastasis model of breast cancer combining both fluorescence imaging and radiography should provide an invaluable tool to study the effectiveness of pharmaceutical agents that could suppress cancer colonization in bone.
A Role for G Protein-Coupled Lysophospholipid Receptors in Sphingolipid-Induced Ca2+ Signaling in MC3T3-E1 Osteoblastic Cells
Authors: Lyons Jeremy M. , Karin Norman J.
Published: April 2006

Sphingolipids have been proposed to modulate cell function by acting as intracellular second messengers and through binding to plasma membrane receptors. Exposure of MC3T3-E1 osteoblastic cells to sphingosine (SPH), sphingosine-1-phosphate (SPP), or sphingosylphosphorylcholine (SPC) led to the release of Ca2+ from the endoplasmic reticulum (ER) and acute elevations in cytosolic-free Ca2+ ([Ca2+]i). Desensitization studies suggest that SPP and SPC bind plasma membrane endothelial differentiation gene (Edg) receptors for lysophosphatidic acid (LPA). Consistent with the coupling of Edg receptors to G proteins, SPP- and SPC-induced Ca2+ signaling was inhibited by pretreatment of the cells with pertussis toxin (PTx). Of the Edg receptors known to bind SPH derivatives in other cell types, MC3T3-E1 cells were found to express transcripts encoding Edg-1 and Edg-5 but not Edg-3, Edg-6, or Edg-8. In contrast to SPP and SPC, the ability of SPH to elicit [Ca2+]i elevations was affected neither by prior exposure of cells to LPA nor by PTx treatment. However, LPA-induced Ca2+ signaling was blocked in MC3T3-E1 cells previously exposed to SPH. Elevations in [Ca2+]i were not evoked by SPP or SPC in cells treated with 2-aminoethoxydiphenylborate (2-APB), an inhibitor of inositol 1,4,5-trisphosphate (IP3)-gated Ca2+ channels in the ER. No effect of 2-APB was observed on SPH- or LPA-induced [Ca2+]i elevations. The data support a model in which SPP and SPC bind Edg-1 and/or Edg-5 receptors in osteoblasts leading to the release of Ca2+ from the ER through IP3-gated channels.
Extracellular Signal-Regulated Kinases and Calcium Channels Are Involved in the Proliferative Effect of Bisphosphonates on Osteoblastic Cells In Vitro
Authors: Mathov Irina , Plotkin Lilian I. , Sgarlata Cecilia L. , Leoni Juliana , Bellido Teresita
Published: April 2006

Bisphosphonates (BPs) are analogues of pyrophosphate, which are widely used for the treatment of different pathologies associated with imbalances in bone turnover. Recent evidence suggested that cells of the osteoblastic lineage might be targets of the action of BPs. The objective of this work was to determine whether BPs induce proliferation of osteoblasts and whether this action involves activation of the extracellular signal-regulated kinases (ERKs). We have shown that three different BPs (olpadronate, pamidronate, and etidronate) induce proliferation in calvaria-derived osteoblasts and ROS 17/2.8 as measured by cell count and by [3H]thymidine uptake. Osteoblast proliferation induced by all BPs diminished to control levels in the presence of U0126, a specific inhibitor of the upstream kinase MEK 1 responsible for ERK phosphorylation. Consistent with this, BPs induced ERK activation as assessed by in-gel kinase assays. Phosphorylation of ERK1/2 was induced by the BPs olpadronate and pamidronate within 30 s, followed by rapid dephosphorylation, whereas etidronate induced phosphorylation of ERKs only after 90 s of incubation and returned to basal levels within 15–30 minutes. In addition, both BP-induced cell proliferation and ERK phosphorylation were reduced to basal levels in the presence of nifedipine, an L-type voltage-sensitive calcium channel (VSCC) inhibitor. These results show that BP-induced proliferation of osteoblastic cells is mediated by activation of ERKs and suggest that this effect requires influx of Ca2+ from the extracellular space through calcium channels.
In Vitro and In Vivo Analysis of the Immune System of Vitamin D Receptor Knockout Mice
Authors: Mathieu Chantal , Van Etten Evelyne , Gysemans Conny , Decallonne Brigitte , Kato Shigeaki , Laureys Jos , Depovere JOS , Valckx Dirk , Verstuyf Annemieke , Bouillon Roger
Published: April 2006

Immune cells carry receptors for 1,25-dihydroxyvitamin D3 [1,25(OH)2D3; vitamin D receptor (VDR)] and individuals with severe vitamin D deficiency have immune abnormalities. The aim of this study was to investigate the role of vitamin D in the immune system by studying VDR-knockout (VDR-KO) mice. VDR-KO mice had the same metabolic phenotype as rachitic animals with severe hypocalcemia. Leukocytosis, lymphocyte subset composition in different immune organs, and splenocyte proliferation to several stimuli were normal, except for a lower response to anti-CD3 stimulation (simulation index [SI] of 13 ± 4 vs. 24 ± 9 in wild-type mice; p < 0.01). Macrophage chemotaxis was impaired (41 ± 19% vs. 60 ± 18% in wild-type mice; p < 0.01) but phagocytosis and killing were normal. In vivo rejection of allogeneic (31 ± 12 days vs. 45 ± 26 days of survival in wild-type mice, NS) or xenogeneic (10 ± 2 days vs. 16 ± 9 days of survival in wild-type mice, NS) islet grafts was comparable with wild-type mice. Surprisingly, VDR-KO mice were protected from low-dose streptozotocin-induced diabetes mellitus (LDSDM; 5% vs. 65% in wild-type mice; p < 0.001). Correcting hypocalcemia by use of lactose-rich or polyunsaturated fat-rich diets fully restored the immune abnormalities in vitro and the sensitivity to diabetes in vivo. On the other hand, treatment with 1,25(OH)2D3 protected wild-type mice against diabetes but did not protect normocalcemic VDR-KO mice. We conclude that immune defects observed in VDR-KO mice are an indirect consequence of VDR disruption because they can be restored by calcium homeostasis normalization. This study proves that although 1,25(OH)2D3 is a pharmacologic and probably a physiological immunomodulator, its immune function is redundant. Moreover, we confirm the essential role of calcium in the immune system.
Effects of Current and Discontinued Estrogen Replacement Therapy on Hip Structural Geometry: The Study of Osteoporotic Fractures
Authors: Beck Thomas J. , Stone Katie L. , Oreskovic Tammy L. , Hochberg Marc C. , Nevitt Michael C. , Genant Harry K. , Cummings Steven R.
Published: April 2006

It is assumed that estrogen influences bone strength and risk of fractures by affecting bone mineral density (BMD). However, estrogen may influence the mechanical strength of bones by altering the structural geometry in ways that may not be apparent in the density. Repeated dual energy X-ray absorptiometry (DXA) hip scan data were analyzed for bone density and structural geometry in elderly women participating in the Study of Osteoporotic Fractures (SOF). Scans were studied with a hip structural analysis program for the effects of estrogen replacement therapy (ERT) on BMD and structural geometry. Of the 3964 women with ERT-use data, 588 used ERT at both the start and end of the ∼3.5-year study, 1203 had past use which was discontinued by clinic visit 4, and 2163 women had never used ERT. All groups lost BMD at the femoral neck, but the reduced BMD among users of ERT was entirely due to subperiosteal expansion and not bone loss, whereas both bone loss and expansion occurred in past or nonusers. BMD increased 0.8%/year at the femoral shaft among ERT users but decreased 0.8%/year among nonusers. Section moduli increased at both the neck and shaft among ERT users but remained unchanged in past and nonusers. Current, but not past, use of estrogen therapy in elderly women seems to increase mechanical strength of the proximal femur by improving its geometric properties. These effects are not evident from changes in femoral neck BMD.
Alendronate Prevents Further Bone Loss in Renal Transplant Recipients
Authors: Giannini Sandro , D'Angelo Angela , Carraro Gianni , Nobile Martino , Rigotti Paolo , Bonfante Luciana , Marchini Francesco , Zaninotto Martina , Carbonare Luca Dalle , Sartori Leonardo , Crepaldi Gaetano
Published: April 2006

The aim of this study was to investigate the effects of alendronate, calcitriol, and calcium in bone loss after kidney transplantation. We enrolled 40 patients (27 men and 13 women, aged 44.2 ± 11.6 years) who had received renal allograft at least 6 months before (time since transplant, 61.2 ± 44.6 months). At baseline, parathyroid hormone (PTH) was elevated in 53% of the patients and the Z scores for bone alkaline phosphatase (b-ALP) and urinary type I collagen cross-linked N-telopeptide (u-NTX) were higher than expected (p < 0.001). T scores for the lumbar spine (−2.4 ± 1.0), total femur (−2.0 ± 0.7), and femoral neck (−2.2 ± 0.6) were reduced (p < 0.001). After the first observation, patients were advised to adhere to a diet containing 980 mg of calcium daily and their clinical, biochemical, and densitometric parameters were reassessed 1 year later. During this period, bone density decreased at the spine (−2.6 ± 5.7%; p < 0.01), total femur (−1.4 ± 4.2%; p < 0.05), and femoral neck (−2.0 ± 3.0%; p < 0.001). Then, the patients were randomized into two groups: (1) group A—10 mg/day of alendronate, 0.50 μg/day of calcitriol, and 500 mg/day of calcium carbonate; and (2) group B—0.50 μg/day of calcitriol and 500 mg/day of calcium carbonate. A further metabolic and densitometric reevaluation was performed after the 12-month treatment period. At the randomization time, group A and group B patients did not differ as to the main demographic and clinical variables. After treatment, bone turnover markers showed a nonsignificant fall in group B patients, while both b-ALP and u-NTX decreased significantly in alendronate-treated patients. Bone density of the spine (+5.0 ± 4.4%), femoral neck (+4.5 ± 4.9%), and total femur (+3.9 ± 2.8%) increased significantly only in the alendronate-treated patients. However, no trend toward further bone loss was noticed in calcitriol and calcium only treated subjects. No drug-related major adverse effect was recorded in the two groups. We conclude that renal transplanted patients continue to loose bone even in the long-term after the graft. Alendronate normalizes bone turnover and increases bone density. The association of calcitriol to this therapy seems to be advantageous for better controlling the complex abnormalities of skeletal metabolism encountered in these subjects.
Alendronate Reduces Periprosthetic Bone Loss After Uncemented Primary Total Hip Arthroplasty: A Prospective Randomized Study
Authors: Venesmaa Petri K. , Kröger Heikki P. J. , Miettinen Hannu J. A. , Jurvelin Jukka S. , Suomalainen Olavi T. , Alhava Esko M.
Published: April 2006

Periprosthetic bone loss, especially in the proximal part of the femur, is common after cemented and uncemented total hip arthroplasty (THA). Bone loss can be progressive and, in the extreme, may threaten survival of the prosthesis. To study whether alendronate therapy can reduce bone loss adjacent to prostheses, 13 uncemented primary THA patients were randomized to the study. They received 10 mg alendronate + 500 mg calcium (n = 8) or 500 mg calcium only (n = 5) daily for 6 months follow-up after THA. Periprosthetic bone mineral density (BMD) was measured with dual energy X-ray absorptiometry (DXA). Decreases in periprosthetic BMD in the alendronate-treated group were lower compared with the changes in the calcium-only group in the same regions of interest at the same follow-up time. In the proximal femur, the mean BMD decrease was 17.1% in the calcium-only group, whereas in the alendronate-treated group the decrease was only 0.9% (p = 0.019). The mean periprosthetic BMD change was also significantly different in the total periprosthetic area between the study groups at the end of the follow-up (calcium-only group −9.9% vs. alendronate-treated group −2.6%; p = 0.019). Alendronate therapy led to a significant reduction in periprosthetic bone loss after primary uncemented THA compared with the changes found in patients without therapy. This kind of bone response may improve the support of the prosthesis and may result in better survival of the prosthesis. However, in this study the follow-up time was too short and the study population was too small to make any long-term conclusions as to the prognosis for THA patients treated with alendronate.
Bone Loss in Patients with Untreated Chronic Obstructive Pulmonary Disease Is Mediated by an Increase in Bone Resorption Associated with Hypercapnia
Authors: Dimai Hans P. , Domej Wolfgang , Leb Georg , Lau K.-H. William
Published: April 2006

This study sought to determine whether the bone loss in untreated chronic obstructive pulmonary disease (COPD) is associated with hypercapnia and/or respiratory acidosis. Bone mineral density (BMD) measured at the distal forearm of the nondominant arm (with peripheral quantitative computed tomography [pQCT]) and serum markers of bone turnover were determined in 71 male patients with untreated COPD and 40 healthy male subjects who matched the patients in age, weight, and body mass index (BMI). The COPD patients, compared with controls, had reduced pulmonary functions, lower arterial pH, and elevated arterial partial pressure of CO2 (P CO 2). The BMD (in T score) was significantly lower in COPD patients than that in control subjects (−1.628 ± 0.168 vs. −0.058 ± 0.157; p < 0.001). The BMD of COPD patients correlated positively with arterial pH (r = 0.582; p < 0.001), negatively with P CO 2 (r = −0.442; p < 0.001), and negatively with serum cross-linked telopeptide of type I collagen (ICTP), a bone resorption marker (r = −0.444; p < 0.001) but not with serum osteocalcin, a bone formation marker. Serum ICTP, but not osteocalcin, correlated with P CO 2 (r = 0.593; p < 0.001) and arterial pH (r = −0.415; p < 0.001). To assess the role of hypercapnia, COPD patients were divided into the hypercapnic (P CO 2 > 45 mm Hg; n = 35) and eucapnic (P CO 2 = 35–45 mm Hg) group (n = 36). Patients with hypercapnia had lower BMD, lower arterial pH, and higher serum ICTP than did patients with eucapnia. Arterial pH and serum ICTP of eucapnic patients were not different from those of controls. To evaluate the role of uncompensated respiratory acidosis, COPD patients with hypercapnia were subdivided into those with compensatory respiratory acidosis (pH ≥ 7.35; n = 20) and those with uncompensated respiratory acidosis (pH < 7.35; n = 15). The BMD and serum ICTP were not different among the two subgroups. In conclusion, this study presents the first associative evidence that the bone loss in COPD is at least in part attributed to an increased bone resorption that is associated primarily with hypercapnia rather than uncompensated respiratory acidosis.
Five-Week Intake of Short-Chain Fructo-Oligosaccharides Increases Intestinal Absorption and Status of Magnesium in Postmenopausal Women
Authors: Tahiri Maha , Tressol Jean C. , Arnaud Josiane , Bornet Francis , Bouteloup-Demange Corinne , Feillet-Coudray Christine , Ducros Véronique , Pépin Denise , Brouns Fred , Roussel Anne M. , Rayssiguier Yves , Coudray Charles
Published: April 2006

Fermentable carbohydrates have been shown to be nondigestible by human enzymes in the small intestine but are fermented extensively in the large bowel to short-chain fatty acids (SCFAs), which can increase mineral absorption. It has been shown that feeding such carbohydrates including short-chain fructo-oligosaccharides (sc-FOSs) increases intestinal magnesium (Mg) absorption in animals, but their beneficial impact on Mg absorption in humans still remains to be established. Therefore, this work aimed to investigate the effect of moderate daily doses of sc-FOSs (10 g/day) on the intestinal absorption and status of Mg in postmenopausal women without hormone replacement therapy (HRT). Eleven healthy postmenopausal women aged 59 ± 6 years (mean ± SD) received for 5 weeks sc-FOS or sucrose (placebo) treatments according to a randomized, double-blind, crossover design separated by a washout period of at least 3 weeks. Subjects ingested 87.5 mg of stable isotope25Mg together with a fecal marker. Subsequently, feces were collected for 5–7 days. An inductively coupled plasma mass spectrometer (ICP/MS) was used for25Mg stable isotope measurements in feces, urine, and blood. Mg levels were assessed also at the beginning and at the end of each treatment in plasma, erythrocytes, and urine. These measurements allowed for the determination of net intestinal Mg absorption and Mg status. The results show that the addition of 10 g sc-FOS to the diet increased Mg absorption by 12.3%, from 30.2 ± 5.0% (placebo treatment) to 33.9 ± 7.2% (sc-FOS treatment; mean ± SD; p < 0.02). This increase in intestinal Mg absorption was accompanied by an increase in plasma25Mg level and led to a higher urinary25Mg excretion. This is the first time that such an effect is shown in humans. The overall conclusion of this work is that the ingestion of moderate doses of sc-FOS did improve intestinal Mg absorption and status in postmenopausal women. Because of the important role of Mg in many cellular functions, such Mg absorption improvement may be particularly interesting when the dietary intake of Mg is limited.
Perspective: Reconsidering the Effects of Antiresorptive Therapies in Reducing Osteoporotic Fracture
Authors: Chesnut C. H. , Rosen C. J.
Published: April 2006

Concepts of what constitutes osteoporosis have evolved from the single criterion of low bone mass to a more inclusive consideration of bone strength, based on both quantity and quality. The evidence driving this shift is drawn from many sources. For example, recent studies of bone geometry have shown what engineers have always known: material properties and structural strength are inseparable. Genetic factors also argue against a one-dimensional (1D) view of osteoporosis. Large-scale family studies present a strong case for genetic influences on bone mass and predisposition to fracture. The contribution of aging to fracture risk has long been known, but we are only now beginning to understand what happens to bone remodeling and microstructure in an aging skeleton. The recognition that osteoporosis is far more complex than previously thought suggests that factors in addition to bone mineral density (BMD) may be useful for evaluating bone fragility and therapeutic effectiveness. Although assessment of BMD is noninvasive and widely available, the degree of increase in BMD alone fails to account for the broader effectiveness of antiresorptive agents in reducing the risk of fractures related to osteoporosis. Indeed, the very multiplicity of factors that determine fracture risk implies that response to therapy may be equally complex. Studies of response to antiresorptive agents and the cellular processes they induce are at best preliminary at this time. Although new technologies have been applied to studying bone microarchitecture, their invasive nature limits wide use. New methods are needed to provide insight into the causes and effects of bone fragility. The definition of osteoporosis, meanwhile, must still be considered a work in progress.
Parathyroid Hormone-Related Protein Is Required for Normal Intramembranous Bone Development
Authors: Suda Naoto , Baba Otto , Udagawa Nobuyuki , Terashima Tatsuo , Kitahara Yutaka , Takano Yoshiro , Kuroda Takayuki , Senior Paul V. , Beck Felix , Hammond Vicki E.
Published: April 2006

It is well established that parathyroid hormone-related protein (PTHrP) regulates chondrocytic differentiation and endochondral bone formation. Besides its effect on cartilage, PTHrP and its major receptor (type I PTH/PTHrP receptor) have been found in osteoblasts, suggesting an important role of PTHrP during the process of intramembranous bone formation. To clarify this issue, we examined intramembranous ossification in homozygous PTHrP-knockout mice histologically. We also analyzed phenotypic markers of osteoblasts and osteoclasts in vitro and in vivo. A well-organized branching and anastomosing pattern was seen in the wild-type mice. In contrast, marked disorganization of the branching pattern of bone trabeculae and irregularly aligned osteoblasts were recognized in the mandible and in the bone collar of the femur of neonatal homozygous mutant mice. In situ hybridization showed that most of the osteoblasts along the bone surfaces of the wild-type mice and some of the irregularly aligned osteoblastic cells in the homozygous mice expressed osteocalcin. Alkaline phosphatase (ALP) activity and expression of osteopontin messenger RNA (mRNA) in primary osteoblastic cells did not show significant differences between cultures derived from the mixture of heterozygous mutant and wild-type mice (+/? mice) and those from homozygous mutant mice. However, both mRNA and protein levels of osteocalcin in the osteoblastic cells of homozygous mutant mice were lower than those of +/? mice, and exogenous PTHrP treatment corrected this suppression. Immunohistochemical localization of characteristic markers of osteoclasts and ruffled border formation did not differ between genotypes. Cocultures of calvarial osteoblastic cells and spleen cells of homozygous mutant mice generated an equivalent number of tartrate-resistant acid phosphatase-positive (TRAP+) mononuclear and multinucleated cells and of pit formation to that of +/? mice, suggesting that osteoclast differentiation is not impaired in the homozygous mutant mice. These results suggest that PTHrP is required not only for the regulation of cartilage formation but also for the normal intramembranous bone development.