bluuebunny/crossref_metadata_2025 huggingface dataset:
Altered Expression of Bone Sialoproteins in Vitamin D–Deficient rBSP2.7Luc Transgenic Mice
Authors: Dr. Chen Jake (Jinkun) , Jin Hangqing , Ranly Don M. , Sodek Jaro , Boyan Barbara D.
Published: April 2006

Bone sialoprotein (BSP) and osteopontin (OPN) are two major noncollagenous matrix proteins in mineralized connective tissue that have discrete roles in bone matrix formation, mineralization, and remodeling. The osteotropic secosteroid, 1,25-dihydroxyvitamin D3, a potent regulator of bone remodeling required for normal bone development, has been shown to exert differential effects on OPN and BSP expression by bone cells in vitro. To investigate these effects in vivo, we induced vitamin D3 deficiency in a transgenic mouse line (rBSP2.7Luc) that has a 2.7 kb rat BSP promoter linked to a luciferase reporter gene in its genome. Pregnant rBSP2.7Luc mice were fed vitamin D3–deficient food and demineralized water for 6 weeks. Their offspring were weaned at 3 weeks of age and then fed vitamin D–deficient food for an additional week. The control group were fed normal rodent pellets and water during the entire experimental procedure. Bone tissues from 40, 4-week-old offspring in each group were analyzed for BSP, OPN and luciferase expression. Vitamin D3–deficient mice displayed a rachitic phenotype that included reduced size and malformation of bones. Assays of the BSP promoter transgene in calvariae, mandibles, and tibiae of the rachitic mice showed increases in luciferase activity of 3.1-, 1.9-, and 4.6-fold, respectively, when compared with control littermates. Semiquantitative reverse transcriptase polymerase chain reaction assays of BSP mRNA revealed increases of 7-, 74-, and 66-fold, respectively, in the same rachitic bones, while OPN mRNA was reduced 12.5-fold in calvariae and 2-fold in tibiae and mandibles. In situ hybridization using mouse cRNA probes revealed that the increased BSP expression and decreased OPN expression in the vitamin D3–deficient mice was primarily in osteoblastic cells on the surface of calvariae and endosteal spaces of alveolar bone, on newly formed epiphyseal bone, and in cementoblasts and in hypertrophic chondrocytes. These studies are the first to show that BSP and OPN are differentially regulated by vitamin D3 in vivo, reflecting the diverse roles of these protein in bone remodeling. Moreover, the increased expression of the BSP transgene in the rachitic mice demonstrates that vitamin D3 regulation of BSP expression is mediated, in part, by element(s) within the 2.7 kb promoter region.
Mapping the Gene Causing Hereditary Primary Hyperparathyroidism in a Portuguese Kindred to Chromosome 1q22-q31
Authors: Williamson C. , Cavaco B. M. , Jauch A. , Dixon P. H. , Forbes S. , Harding B. , Holtgreve-Grez H. , Schoell B. , Pereira M. C. , Font A. P. , Loureiro M. M. , Sobrinho L. G. , Santos M. A. , Prof. Thakker R. V.
Published: April 2006

A Portuguese kindred with autosomal dominant isolated primary hyperparathyroidism (HPT) that was associated with parathyroid adenomas and carcinomas was investigated with the aim of determining the chromosomal location of this gene, designated HPTPort. Leukocyte DNA from 9 affected and 16 unaffected members and 7 parathyroid tumors from 4 patients was used in comparative genomic hybridization (CGH), tumor loss of heterozygosity (LOH), and family linkage studies. The CGH studies revealed abnormalities of chromosomes 1 and 13, and the results of LOH studies were consistent with the involvements of tumor suppressor genes from these regions. Family segregation studies mapped HPTPort to chromosome 1q22-q31 by establishing linkage with eight loci (D1S254, D1S222, D1S202, D1S238, D1S428, D1S2877, D1S422, and D1S412) (peak two-point LOD scores = 3. 46–5.14 at 0% recombination), and defined the location of HPT Port to a 21 cM region flanked centromerically by D1S215 and telomerically by D1S306. Thus, HPTPort has been mapped to chromosome 1q22-q31, and a characterization of this gene will help to elucidate further the mechanisms that are involved in the development of parathyroid tumors.
Magnesium Deficiency and Bone Loss After Cardiac Transplantation
Authors: Boncimino K. , Mcmahon D.J. , Addesso V. , Bilezikian J.P. , Shane Elizabeth
Published: April 2006

Magnesium depletion adversely affects many phases of skeletal metabolism and has been implicated as a risk factor in several forms of osteoporosis. Magnesium deficiency has also been reported after cardiac transplantation. To evaluate whether altered magnesium homeostasis could be related to the pathogenesis of early bone loss after cardiac transplantation, we prospectively measured serum and urinary magnesium and evaluated them with respect to biochemical indices of mineral metabolism and rates of bone loss. The study population included 60 patients (45 men, 15 women) aged 53 ± 11 years (SD) with measurements of biochemistries and bone mineral density by dual-energy X-ray absorptiometry before and 3 months after transplantation. All received prednisone, cyclosporine A, and azathioprine, plus calcium (1000 mg) and vitamin D (400 IU). After transplantation, serum magnesium decreased by 16 ± 15% (SD) from 2.0 ± 0.3 mg/dl to 1.6 ± 0.2 mg/dl (normal 1.8–2.2 mg/dl; p < 0.0001), accompanied by an increase in the fractional excretion of magnesium (7.1 ± 3.9% to 13.3 ± 5.6%; p < 0.0017). Forty-three patients with low 3-month serum magnesium levels (≤ 1.7 mg/dl) sustained 41% less bone loss at the lumbar spine (4.0 ± 4.6% vs. 6.8 ± 5.3%; p = 0.051) and 58% less bone loss at the femoral neck (3.1 ± 5.9% vs. 7.3 ± 5.6%; p < 0.01), despite receiving similar doses of prednisone and cyclosporine A. Of the biochemical parameters measured (including renal function, serum calcium, phosphorus, vitamin D metabolites, osteocalcin, urinary calcium, and markers of bone resorption), only intact parathyroid hormone (PTH) levels, which were 40% lower in patients with low serum magnesium levels differed significantly (45 ± 23 pg/ml vs. 74 ± 60 pg/ml; p = 0.019). Patients with low serum magnesium levels also had evidence of lower bone turnover. In summary, a highly significant decline in serum magnesium concentrations, associated with continued urinary magnesium losses, occurs after cardiac transplantation. Patients with low serum magnesium levels had significantly lower rates of bone loss, lower serum PTH concentrations, and lower bone turnover. Although the precise mechanisms by which the reduction in serum magnesium tends to protect against bone loss in the early post-transplantation period remain to be elucidated, reduced serum PTH and lower rates of bone turnover are likely to be important factors.
Preparation of a cDNA Library and Preliminary Assessment of 1400 Genes from Mouse Growth Cartilage
Authors: Okihana H. , Yamada K.
Published: April 2006

Cartilage is an inconvenient tissue for the isolation of mRNA, and this has hampered studies of its component mRNAs conducted to date. Here, we describe the preparation of a good quality cDNA library from mouse growth cartilage (mGC). A total of 1.7 μg of poly(A)+ RNA was obtained from about 1200 pieces of the mGC zone of 60 young mice (BALB/c, 4 weeks old). Using this poly(A)+ RNA, we constructed a cDNA library using the pAP3neo vector by the linker-primer method. The complexity of the cDNA library was 2.6 × 106 colony-forming units (cfu), which signified that almost all of the mRNA components in the mGC were present in this cDNA library. From this library, 1401 clones were randomly selected and their insert sizes were examined. Of these clones, 166 (12%) had no inserts, 466 (33%) had inserts ranging in size from 0–0.9 kbp, 480 (34%) had inserts of 1.0–1.9 kbp, 162 (12%) had inserts of 2.0–2.9 kbp, and 127 (9%) had sizes greater than 3.0 kbp. The average insert size was 1.45 kbp. The number of cfu and the insert size data qualified this library as of reasonably good quality. Clones with an insert size greater than 1 kbp (769 clones) were sequenced from their 5′ ends. Among the 769 clones examined, 608 gave sequence data. Among these, 196 (32%) were unknown, 2 were only poly A, and 410 (67%) coded for known proteins. Of these, 55 clones coded for type II (pro)collagen, 54 for osteonectin, and 22 for other cartilage collagens (type IX, type X, and type XI). The rest included cartilage extracellular matrix genes, general cellular genes, and others. To judge further the quality of the library, 45 species coding for type II collagen chain were aligned based on their 5′ end sequences. Three species (7%) contained almost the full-length insert, and the shortest one was 1.5 kbp in length (full-length 5.6 kbp). These data show that this cDNA library is of reasonably good quality, making it likely that the large number of unknown inserts (32%) will provide a suitable pool for the identification and functional determination of new GC genes.
Tartrate-Resistant Acid Phosphatase Forms Complexes with α2-Macroglobulin in Serum
Authors: Brehme Cheryl Shaffer , Roman Steven , Shaffer Jennifer , Wolfert Robert
Published: April 2006

Tartrate-resistant acid phosphatase (TRAP) is a standard histochemical marker of differentiated osteoclasts and has been proposed as a serum/plasma marker for osteoclast activity. Enzyme assays have been described that show elevated TRAP enzyme activity in the serum or plasma of patient groups known to have increased bone metabolism. However, the poor stability of the enzyme and potential contribution from nonosteoclastic sources make it problematic to measure in patient samples. Immunoassays developed to measure TRAP in serum and plasma have yielded widely varying TRAP levels in both normal and disease states. It is not clear if this variability is caused by differences in assay calibration, antibody specificity, and/or TRAP instability. In this paper, we report that purified TRAP spiked into serum forms high molecular weight complexes. Complex formation results in greatly decreased TRAP enzyme activity and immunoreactivity. The complexing protein in serum has been identified as α2-macroglobulin (α2M). Similar complexes are observed in stored patient samples. In vitro studies with purified components show that TRAP binds to α2M primarily through noncovalent ionic interactions. Our results demonstrate that one mechanism of TRAP instability in serum is complex formation with α2M and suggest further that current TRAP enzyme and immunoassays may not accurately measure the circulating level of TRAP.
The Addition of a Raloxifene Analog (LY117018) Allows for Reduced PTH(1–34) Dosing During Reversal of Osteopenia in Ovariectomized Rats
Authors: Dr. Hodsman A. B. , Drost D. , Fraher L. J. , Holdsworth D. , Thornton M. , Hock J. , Bryant H. , Watson P. H.
Published: April 2006

To test the hypothesis that an antiresorptive agent might reduce the dosing requirement for an anabolic drug during reversal of osteopenia due to estrogen deficiency, the following experiment was conducted in 6-month-old female rats. Ovariectomy or sham surgery was performed and the following six experimental groups were studied. Untreated (SHAM) or ovariectomized (OVX) animals served as control groups. Four weeks post-OVX, osteopenic rats (now 7 months old), were treated in one of four experimental protocols: human parathyroid hormone (hPTH(1–34), 80 μg/kg/day, given by subcutaneous injection 5 days/week; a selective estrogen receptor modulator (SERM), raloxifene analog LY117018 (RA), 3 mg/kg/day, given by gavage 5 days/week; and two combinations of LY117018 at the same dose and frequency with hPTH(1–34) (same dose, 5 times/week) and a reduced dosing interval of hPTH(1–34) (same dose, 2 times/week). After 12 weeks of treatment, the four experimental groups were sacrificed at age 10 months. SHAM and OVX controls were also studied at 7 and 10 months of age. Bone mineral density (BMD) was measured by dual-energy X-ray absorptiometry at four skeletal sites: two mixed cortical/trabecular sites (femur and tibia) and two predominantly trabecular sites (lumbar spine and pelvis). The differences in BMD were consistent at all four sites. RA alone maintained BMD at all skeletal sites, but the results were not significantly improved over OVX controls, at age 10 months. hPTH(1–34) injections given 5 days/week resulted in BMD increments significantly higher than in either OVX or SHAM controls (p < 0.001). While the RA did not enhance the anabolic effects of full doses of hPTH(1–34), the addition of RA treatment to twice-weekly hPTH(1–34) dosing resulted in BMD increments at all four skeletal sites that were similar to the more intensive anabolic regimen of hPTH(1–34) therapy given 5 times/week. Therefore, an antiresorptive agent such as SERMs may potentially reduce the pharmacologic doses of PTH needed to reverse estrogen deficiency-induced osteopenia.
A Quadripotential Mesenchymal Progenitor Cell Isolated from the Marrow of an Adult Mouse
Authors: Dennis James E. , Merriam Anita , Awadallah Amad , Yoo Jung U. , Johnstone Brian , Caplan Arnold I.
Published: April 2006

Adult marrow contains mesenchymal progenitor cells (MPCs) that have multiple differentiation potentials. A conditionally immortalized MPC clone, BMC9, has been identified that exhibits four mesenchymal cell phenotypes: chondrocyte, adipocyte, stromal (support osteoclast formation), and osteoblast. The BMC9 clone, control brain fibroblasts and another marrow-derived clone, BMC10, were isolated from a transgenic mouse (H-2Kb-tsA58) containing a gene for conditional immortality. To test for chondrogenic potential, cells were cultured in defined medium containing 10 ng/ml transforming growth factor β and 10−7 M dexamethasone in 15-ml polypropylene tubes (“aggregate cultures”). Adipogenic potential was quantitated by flow cytometry of Nile Red–stained cells cultured for 1 and 2 weeks in medium containing isobutyl methylxanthine, indomethacin, insulin, and dexamethasone. Support of osteoclast formation was measured by quantitating multinucleated tartrate-resistant acid phosphatase–positive cells in spleen cell cocultures of test clones (immortomouse clones and positive control ST2 cells) cultured in the presence of 10−7 M vitamin D3 and 150 mM ascorbate-2-phosphate. In vivo osteogenic potential was assayed by histologic examination of bone formation in subcutaneous implants, into athymic mouse hosts, of a composite of cells combined with porous calcium phosphate ceramics. The bone marrow–derived clone BMC9 has the potential to express each of the four mesenchymal characteristics tested, while brain fibroblasts, tested under identical conditions, did not exhibit any of these four mesenchymal characteristics. BMC10 cells exhibited osteogenic and chondrogenic phenotypes, but showed only minimal expression of adipocytic or osteoclast-supportive phenotypes. Clone BMC9 is, minimally, a quadripotential MPC isolated from the marrow of an adult mouse that can differentiate into cartilage and adipose, support osteoclast formation, and form bone. The BMC9 clone is an example of an adult-derived multipotential progenitor cell that is situated early in the mesenchymal lineage.
The Role of Geranylgeranylation in Bone Resorption and Its Suppression by Bisphosphonates in Fetal Bone Explants In Vitro: A Clue to the Mechanism of Action of Nitrogen-Containing Bisphosphonates
Authors: Van Beek Ermond , Löwik Clemens , Van Der Pluijm Gabri , Papapoulos Socrates
Published: April 2006

Bisphosphonates, synthetic compounds used in the treatment of skeletal disorders, suppress osteoclast-mediated bone resorption by a yet unidentified mechanism. Previous studies showed that some bisphosphonates can inhibit enzymes of the mevalonate pathway, and nitrogen-containing bisphosphonates inhibit protein prenylation in mouse macrophages. In the present study, we examined the involvement of the mevalonate pathway in basal and bisphosphonate-inhibited osteoclastic resorption in fetal mouse long bone explants, an experimental model representative of the in vivo action of bisphosphonates. Mevastatin inhibited bone resorption at concentrations similar to those of the potent bisphosphonate ibandronate. This effect could be totally reversed by the addition of mevalnate and geranylgeraniol but not farnesol. The first two intermediates but not the latter could also stimulate basal bone resorption. The inhibitory effect of ibandronate on bone resorption could be totally reversed by the addition of geranylgeraniol and to a small extent only by mevalonate and farnesol, indicating that the bisphosphonate acts at a level of the mevalonate pathway different from that of mevastatin. Histologic sections of ibandronate-treated bone explants showed further rescue of functioning osteoclasts during concomitant treatment with geranylgeraniol. Finally, the reversibility of bisphosphonate inhibited osteoclastic resorption by geranylgeraniol was also demonstrated for the potent nitrogen-containing bisphosphonates alendronate, olpadronate, and risedronate but not for the non–nitrogen-containing bisphosphonates clodronate and etidronate. These studies demonstrate that protein geranylgeranylation but not farnesylation is important for osteoclast-mediated bone resorption and that nitrogen-containing bisphosphonates exert their antiresorptive action probably by affecting enzymes of the mevalonate pathway involved in the generation of geranylgeranyl pyrophosphate.
Expression and Function of the Splice Variant of the Human Cadherin-11 Gene in Subordination to Intact Cadherin-11
Authors: Kawaguchi Jitsutaro , Takeshita Sunao , Kashima Takeshi , Imai Tatsuya , Machinami Rikuo , Dr. Kudo Akira
Published: April 2006

Cadherin-11, a member of the type II classic cadherin subfamily, differs from type I family molecules such as P-, E-, and N-cadherins. An isoform of the human cadherin-11 gene, termed the variant form, encodes a truncated protein with a different cytoplasmic domain. The resulting protein does not possess any part of the cytoplasmic domain common to other cadherins. In the present study, analysis of the genomic organization of the cadherin-11 gene revealed that an insertion of 179 bp in an intron generates an alternatively spliced form. The mRNA expression of the variant form of cadherin-11 was examined in normal tissues by reverse transcription-polymerase chain reaction and/or Northern blot analyses. The variant form was expressed in the heart, brain, placenta, lung, and bone, but not in the kidney, skeletal muscle, pancreas, and liver. Western blot analyses revealed that the variant form is expressed as an 85 kDa protein, and that an additional secreted form also exists as an 80 kDa protein originated from cleavage of the intact form. Gene transfer of the variant form into L cells demonstrated that it lacked the adhesion properties characteristic of the intact form of cadherin-11 but enhanced the activity of Ca2+-dependent adhesion of the intact form of cadherin-11. The variant was expressed on the surface together with the intact form and stabilized the interaction between the intact form and β-catenin. These findings suggest that expression of the variant form of human cadherin-11 may regulate the intact cadherin-11–mediated adhesion and alter the morphogenetic processes during mesenchymal cell differentiation including osteoblasts.
The Effects of Nitrogen-Containing Bisphosphonates on Human Epithelial (Caco-2) Cells, an In Vitro Model for Intestinal Epithelium
Authors: Twiss I. M. , Pas O. , Ramp-Koopmanschap W. , Den Hartigh J. , Vermeij P.
Published: April 2006

Nitrogen-containing bisphosphonates (N-PCP) are bisphosphonates with an increased antiresorptive potency. Aminobisphosphonates, N-PCPs with an amino group, can cause nonspecific gastrointestinal complaints. It is not known whether these side effects are specific for these bisphosphonates or for the whole class of N-PCPs. In this study, we investigated the effects of two aminobisphosphonates (pamidronate and alendronate) and a structurally similar N-PCP (olpadronate) and their three respective calcium complexes on the viability and the intracellular calcium concentration ([Ca2+]i) of cultured Caco-2 cells a model for intestinal epithelium. These cells were also examined for apoptosis or necrosis. In the presence of calcium, pamidronate and alendronate were toxic to the cells, with pamidronate being more toxic than alendronate. Olpadronate induced toxicity only at concentrations more than ten times higher than the toxic concentrations of pamidronate. In the absence of calcium definite signs of toxicity were observed only with pamidronate at clinically relevant concentrations. The complexes of pamidronate and alendronate with calcium were considerably less soluble than the olpadronate calcium complex. There were no signs of apoptosis. [Ca2+]i was transiently raised after treatment with the N-PCPs. Doses at which responses were seen were, respectively, 0.02 mM (pamidronate), 0.3 mM (alendronate), and 2 mM (olpadronate). The peak of response was slightly greater after pamidronate treatment than after alendronate or olpadronate, respectively. In conclusion pamidronate, either as an ion or as a calcium complex, is the most toxic of the bisphosphonates tested for Caco-2 cells. Alendronate was less toxic while olpadronate was the least toxic in presence of calcium. The solubility of the bisphosphonate complexes with calcium may account for these differences in toxicity.
Polycystic Bone Disease: A New, Autosomal Dominant Disorder
Authors: Dr. Whyte Michael P. , Eddy Mark C. , Podgornik Michelle N. , Mcalister William H.
Published: April 2006

We describe a new heritable bone disease characterized radiographically by increasingly numerous and enlarging cyst-like lesions throughout the skeleton. Beginning in early childhood, a father, son, and daughter all suffered from progressively frequent pathological fractures involving such radiolucencies. Healing occurred uneventfully and with little residual pain or deformity. Biochemical parameters of mineral homeostasis and skeletal turnover were normal. Bone scanning showed increased radioisotope uptake primarily in fractures and in the largest collections of the lesions. The histopathology is uncertain, but may reflect a form of intraosseous lipomatosis. This unique condition, which we have provisionally named polycystic bone disease, is inherited as an autosomal dominant trait with a high degree of penetrance.
Differential Expression of Human Lysyl Hydroxylase Genes, Lysine Hydroxylation, and Cross-Linking of Type I Collagen During Osteoblastic Differentiation In Vitro
Authors: Uzawa Katsuhiro , Grzesik Wojciech J. , Nishiura Teruhiro , Kuznetsov Sergei A. , Robey Pamela Gehron , Brenner David A. , Yamauchi Mitsuo
Published: April 2006

The pattern of lysyl hydroxylation in the nontriple helical domains of collagen is critical in determining the cross-linking pathways that are tissue specific. We hypothesized that the tissue specificity of type I collagen cross-linking is, in part, due to the differential expression of lysyl hydroxylase genes (Procollagen-lysine,2-oxyglutarate,5-dioxygenase 1, 2, and 3 [PLOD1, PLOD2, and PLOD3]). In this study, we have examined the expression patterns of these three genes during the course of in vitro differentiation of human osteoprogenitor cells (bone marrow stromal cells [BMSCs]) and normal skin fibroblasts (NSFs). In addition, using the medium and cell layer/matrix fractions in these cultures, lysine hydroxylation of type I collagen α chains and collagen cross-linking chemistries have been characterized. High levels of PLOD1 and PLOD3 genes were expressed in both BMSCs and NSFs, and the expression levels did not change in the course of differentiation. In contrast to the PLOD1 and PLOD3 genes, both cell types showed low PLOD2 gene expression in undifferentiated and early differentiated conditions. However, fully differentiated BMSCs, but not NSFs, exhibited a significantly elevated level (6-fold increase) of PLOD2 mRNA. This increase coincided with the onset of matrix mineralization and with the increase in lysyl hydroxylation in the nontriple helical domains of α chains of type I collagen molecule. Furthermore, the collagen cross-links that are derived from the nontriple helical hydroxylysine-aldehyde were found only in fully differentiated BMSC cultures. The data suggests that PLOD2 expression is associated with lysine hydroxylation in the nontriple helical domains of collagen and, thus, could be partially responsible for the tissue-specific collagen cross-linking pattern.
Modulation of Osteoblast-like Cell Behavior by Activation of Protease-Activated Receptor-1
Authors: Abraham Linda A. , Dr. Mackie Eleanor J.
Published: April 2006

In addition to playing a central role in thrombosis and hemostasis, the serine protease thrombin is a specific agonist for a variety of functional responses in cells including osteoblast-like cells. Many of the cellular responses to thrombin are mediated by protease-activated receptor-1 (PAR-1). Since osteoblasts express PAR-1 in vivo during development, the effect of PAR-1 activation on proliferation and differentiation in primary rat osteoblast-like cells was investigated. Thrombin or the rat PAR-1–activating peptide SFFLRNPSENTFELVPL (SFFL) stimulated cell proliferation (as assessed by3H- thymidine incorporation) of primary osteoblast-like cells derived from long bone or calvaria, and treatment with antibodies to PAR-1 abolished the proliferative response to thrombin. Activation of PAR-1 by thrombin or SFFL inhibited endogenous alkaline phosphatase (ALP) activity and caused a transient elevation of intracellular calcium in the osteoblast-like cells. Calcium mobilization was not, however, required for thrombin's effect on proliferation or ALP activity. The ability of a number of growth factors and hormones to regulate expression of PAR-1 in osteoblast-like cells was investigated. Expression of PAR-1 transcript and protein by osteoblast-like cells in vitro was markedly increased by treatment with transforming growth factor-β (TGF-β), and the proliferative response to thrombin was enhanced by TGF-β pretreatment. Platelet-derived growth factor-BB caused a slight but significant down-regulation of PAR-1 mRNA expression. Thrombin caused a transient increase in PAR-1 expression, whereas neither parathyroid hormone-related peptide nor 1,25-dihydroxyvitamin D3 had any effect. The observations described here suggest that PAR-1 mediates thrombin-induced osteoblast proliferation, which in turn may contribute to responses of osteoblasts to osteogenic growth factors.
FokI Polymorphism at Translation Initiation Site of the Vitamin D Receptor Gene Predicts Bone Mineral Density and Vertebral Fractures in Postmenopausal Italian Women
Authors: Gennari Luigi , Becherini Lucia , Mansani Riccardo , Masi Laura , Falchetti Alberto , Morelli Annamaria , Colli Emanuela , Gonnelli Stefano , Cepollaro Chiara , Brandi Maria Luisa
Published: April 2006

A novel T/C polymorphism (ATG to ACG) at the translation initiation site of the vitamin D receptor (VDR) gene, defined by FokI restriction endonuclease, has been recently associated with variation in bone mineral density (BMD) and rates of bone loss in a group of postmenopausal Mexican-American women. The presence of the restriction site, designated as f, allows protein translation to initiate from the first ATG, while the allele lacking the site, indicated as F, initiates translation at a second ATG. In this study, we investigated the role of FokI polymorphism in a group of 400 postmenopausal women of Italian descent stratified for BMD into osteoporotic (n = 164), osteopenic (n = 117), and normal (n = 119) groups. There were 159 (41%) FF homozygotes, 55 (14%) ff homozygotes, and 186 (45%) Ff heterozygotes. In the whole population, we observed a weak association between FokI polymorphism and lumbar BMD (p = 0.06, analysis of covariance [ANCOVA]) but not with femoral neck BMD (p = 0.5, ANCOVA). Interestingly, the effect of FokI genotypes on lumbar BMD was influenced by the years since menopause such that differences in BMD related to different VDR allelic variants were greater among women in the first 5 years of menopause (p = 0.04, ANCOVA), progressively declining afterward. In addition, a significantly higher prevalence of ff genotype in osteoporotic than in osteopenic and normal women was observed (p = 0.04, Chi-square test). Finally, ff genotype resulted significantly over-represented in the group of women with a vertebral fracture as compared with controls (p = 0.003, Chi-square test), equivalent to a relative risk of 2.58 (95% confidence intervals 1.36–4.91). We conclude that in this population, FokI polymorphism at the VDR gene locus accounts for a part of the heritable component of BMD at the lumbar spine.
Alendronate Increases Bone Density and Bone Strength at the Distal Radius in Postmenopausal Women
Authors: Schneider Peter F. , Fischer Manfred , Allolio Bruno , Felsenberg Dieter , Schröder Ulf , Semler Jutta , Dr. Ittner Joachim R.
Published: April 2006

In addition to the alendronate Osteoporosis Intervention Trial (FOSIT) core protocol 901–0A of 1908 enrolled patients, the use of peripheral quantitative computed tomography (pQCT) was explored for the assessment of response to therapy. Bone mineral and strength related parameters at two different sites at the distal radius were explored in a subset of the multicenter core study. One hundred and three patients were entered into the substudy and given either a daily dose of 10 mg of alendronate or placebo for 1 year. Measurements were done at months 0, 3, 6, and 12. Inclusion criteria were bone mineral density (BMD) measurements at the lumbar spine of −2 SD. The response to therapy was assessed by dual-energy X-ray absorptiometry in the lumbar spine and the hip, and by pQCT in the ultradistal and the shaft sites of the radius. In line with the FOSIT core study, alendronate increased BMD at the lumbar spine and the hip, and it decreased the serum biochemical markers of bone turnover. The substudy showed differences between the therapy and placebo group in trabecular bone density (8.4%, p = 0.095), in total density (6.8%, p = 0.009), and in the bone strength index (BSI) (15.6 mm3, p = 0.037) at the ultradistal site due to treatment and no changes at the radius shaft. A significant correlation was observed between percentage changes from baseline in BMD of the lumbar spine, and in total density and bone strength at the ultradistal radius site in the treatment group, but not in the placebo group. Thus, the ultradistal radius site did respond to alendronate therapy. The increased bone density accompanied a significant gain in the BSI at the ultradistal site, a finding that might help explain the reduced wrist fractures in the alendronate Fracture Intervention Trial.
The Monoclonal Antibodies 18d7/91f2 Recognize a Receptor Regulatory Protein on Mouse Bone Marrow Stromal Cells
Authors: Weng Lin , Falla Nancy , van den Heuvel Rosette , Raymackers Jos , Karperien Marcel , van Bezooijen Rutger , van Vlasselaer Peter , Löwik Clemens , Dr. Merregaert Jozef
Published: April 2006

Two monoclonal antibodies 18D7 and 91F2 were developed by immunizing rats with the mouse bone marrow-derived osteogenic cell line MN7. Hybridomas secreting rat antibodies against MN7 cell surface markers were selected by flow cytometry analysis. Both the monoclonal antibody 18D7 and the monoclonal antibody 91F2 are directed against the same cell surface antigen present on MN7 cells. Here, we report on the immunopurification of the 18D7/91F2 antigen and its identification as the prostaglandin F2α receptor regulatory protein (FPRP). FPRP is expressed as a single messenger RNA (mRNA) of approximately 6 kilobases (kb) in MN7 cells and is differentially expressed in developing osteogenic cultures of bone marrow cells of the mouse. However, addition of the monoclonal antibodies 18D7 and 91F2 to these cultures did not inhibit bone formation in vitro. Both monoclonal antibodies reacted with mouse stromal cell lines established from bone marrow, thymus, spleen, and mandibular condyles. Immunohistochemical analysis of mature tibia of mice using the monoclonal antibody 18D7 revealed the presence of a distinct population of bone marrow cells close to trabecular and endosteal bone surfaces. In the central bone marrow, hardly any positive cells were found. In 17-day-old fetal mouse radius 18D7 immunoreactivity was restricted to cells in the periosteum in close vicinity to the bone collar. Mature osteoblasts, osteoclasts, osteocytes, growth plate chondrocytes, and mature macrophages were all negative. Taken together, these results suggest that FPRP plays a role in the osteogenic differentiation process.
An In Vivo Model for Investigations of Mechanical Signal Transduction in Trabecular Bone
Authors: Moalli Maria R. , Caldwell Nancy J. , Patil Pravin V. , Goldstein Steven A.
Published: April 2006

The premise that bone cells are able to perceive and respond to mechanical forces is well accepted. This article describes the use of an in vivo hydraulic bone chamber for investigations of mechanical signal transduction. The servohydraulic loading mechanism was activated to apply a controlled compressive load to the woven trabecular bone that formed in one chamber, while the contralateral chamber served as an unloaded control. Specimens were harvested at a series of postload time points, and the cellular response to loading was evaluated by cytochemical, histomorphometric, and Northern blot analysis. A repetitive daily load stimulus elicited osteoblast biosynthetic activity characterized by an initial increase in type I procollagen by day 3 and a subsequent rise in alkaline phosphatase (ALP) activity after the sixth daily load episode. Application of a single load episode induced a biphasic pattern of c-fos and zif-268 gene expression with up-regulation at 30 minutes, down-regulation at 12 h, and up-regulation 24 h after the mechanical stimulus. The results show that a synchronized pattern of bone cell activity and gene expression occurs in response to controlled mechanical stimulation and that candidate load-responsive molecular mediators can be evaluated easily by this model.
Health-Related Quality of Life in Postmenopausal Women With Low BMD With or Without Prevalent Vertebral Fractures
Authors: Oleksik Anna , Lips Paul , Dawson Alison , Minshall Michael E. , Shen Wei , Cooper Cyrus , Kanis John
Published: April 2006

Fractures and subsequent morbidity determine the impact of established postmenopausal osteoporosis. Health-related quality of life (HRQOL) has become an important outcome criterion in the assessment and follow-up of osteoporotic patients. As part of the baseline measurements of the Multiple Outcomes of Raloxifene Evaluation (MORE) study, HRQOL was assessed in 751 osteoporotic (bone mineral density [BMD] T score ≥ −2.5) women from Europe with or without vertebral fractures (VFX). This was done using the quality of life questionnaire of the European Foundation for Osteoporosis (QUALEFFO), Nottingham Health Profile (NHP) and the EQ-5D (former EuroQol). QUALEFFO contains questions in five domains: pain, physical function, social function, general health perception, and mental function. Each domain score and QUALEFFO total scores are expressed on a 100-point scale, with 0 corresponding to the best HRQOL. In comparison with patients without VFX, those with VFX were older (66.2 ± 5.9 years vs. 68.8 ± 6.3 years; p < 0.001), had higher prevalence of nonvertebral fractures (25% vs. 36%; p = 0.002), and higher QUALEFFO scores (worse HRQOL; total score, 26 ± 14 vs. 36 ± 17; p < 0.001). QUALEFFO scores increased progressively with increasing number of VFX, especially lumbar fractures (p < 0.001). Patients with a single VFX already had a significant increase in QUALEFFO scores (p < 0.05). Similar, though weaker, associations were seen for NHP and EQ-5D scores. This study confirms decreased HRQOL for patients with prevalent VFX. In osteoporotic patients, QUALEFFO scores change in relation to the number of VFX. QUALEFFO is suitable for clinical studies in patients with postmenopausal osteoporosis.
Serial Changes in Bone Mineral Density and Bone Turnover After Correction of Secondary Hyperparathyroidism in a Patient with Pseudohypoparathyroidism Type Ib
Authors: Tollin Steven R. , Perlmutter Steven , Aloia John F.
Published: April 2006

Pseudohypoparathyroidism (PHP) is a disorder characterized by hypocalcemia and secondary hyperparathyroidism caused by primarily renal resistance to the effects of parathyroid hormone (PTH). However, as an indication of normal PTH responsiveness in bone, some patients with PHP develop skeletal disease because of longstanding secondary hyperparathyroidism. A patient is described with hypocalcemia, hyperphosphatemia, marked secondary hyperparathyroidism, and an increased alkaline phosphatase level. Subsequent evaluation revealed a diagnosis of PHP type Ib. The patient had radiographic evidence of skeletal disease caused by secondary hyperparathyroidism. A urinary level of N-telopeptide cross-links of type I collagen (NTX) was elevated markedly. Bone mineral density (BMD) was in the normal range at all measured sites, with BMD at the spine being higher than at the femur and distal radius. Treatment was initiated with calcium and calcitriol. Seven months later, calcium and PTH levels had normalized. The level of urinary NTX fell by 83%. Spinal BMD improved by 15%, and BMD at the femoral neck improved by 11%. Radial BMD was unchanged. This case emphasizes the importance of evaluating patients with PHP for hyperparathyroid bone disease and shows that correction of secondary hyperparathyroidism in patients with PHP can result in a significant suppression of previously accelerated bone turnover and to substantial gains in BMD at sites containing a major percentage of cancellous bone. The case also implies that assessment of bone turnover with urinary NTX and measurement of BMD with dual-energy X-ray absorptiometry (DEXA) may be useful in following the response of the skeleton to therapy in these patients and suggests the need for more studies of both NTX and BMD in patients with PHP.
Differential Expression Patterns of Runx2 Isoforms in Cranial Suture Morphogenesis
Authors: Park Mi-Hyun , Shin Hong-In , Choi Je-Yong , Nam Soon-Hyeun , Kim Young-Jin , Kim Hyun-Jung , Ryoo Hyun-Mo
Published: April 2006

Runx2 (previously known as Cbfa1/Pebp2αA/AML3), a key transcription factor in osteoblast differentiation, has at least two different isoforms using alternative promoters, which suggests that the isoforms might be expressed differentially. Haploinsufficiency of the Runx2 gene is associated with cleidocranial dysplasia (CCD), the main phenotype of which is inadequate development of calvaria. In spite of the biological relevance, Runx2 gene expression patterns in developing calvaria has not been explored previously, and toward this aim we developed three probes: pRunx2, which comprises the common coding sequence of Runx2 and hybridizes with all isoforms; pPebp2αA, which specifically hybridizes with the isoform transcribed with the proximal promoter; and pOsf2, which hybridizes with the isoform transcribed with the distal promoter. These probes were hybridized with tissue sections of mouse calvaria taken at various time points in development. Runx2 expression was localized to the critical area of cranial suture closure, being found in parietal bones, osteogenic fronts, and sutural mesenchyme. Pebp2αA and Osf2 showed tissue-specific expression patterns. The sites of Pebp2αA expression were almost identical to that of pRunx2 hybridization but expression was most intense in the sutural mesenchyme, where undifferentiated mesenchymal cells reside. The Osf2 isoform was strongly expressed in the osteogenic fronts, as well as in developing parietal bones, where osteopontin (OP) and osteocalcin (OC) also were expressed. However, in contrast to Pebp2αA, Osf2 expression did not occur in sutural mesenchyme. Pebp2αA also was expressed prominently in primordial cartilage that is found under the sutural mesenchyme and is not destined to be mineralized. Thus, Osf2 isoforms contribute to events later in osteoblast differentiation whereas the Pebp2αA isoform participates in a wide variety of cellular activities ranging from early stages of osteoblast differentiation to the final differentiation of osteoblasts.
Infrared Microscopic Imaging of Bone: Spatial Distribution of CO32−
Authors: Ou-Yang H. , Paschalis E. P. , Mayo W. E. , Boskey A. L. , Mendelsohn R.
Published: April 2006

This article describes a novel technology for quantitative determination of the spatial distribution of CO32− substitution in bone mineral using infrared (IR) imaging at ∼6 μm spatial resolution. This novel technology consists of an IR array detector of 64 × 64 elements mapped to a 400 μm × 400 μm spot at the focal plane of an IR microscope. During each scan, a complete IR spectrum is acquired from each element in the array. The variation of any IR parameter across the array may be mapped. In the current study, a linear relationship was observed between the band area or the peak height ratio of the CO32− v3 contour at 1415 cm−1 to the PO43− v1,v3 contour in a series of synthetic carbonated apatites. The correlation coefficient between the spectroscopically and analytically determined ratios (R2 = 0.989) attests to the practical utility of this IR area ratio for determination of bone CO32− levels. The relationship forms the basis for the determination of CO32− in tissue sections using IR imaging. In four images of trabecular bone the average CO32− levels were 5.95 wt% (2298 data points), 6.67% (2040 data points), 6.66% (1176 data points), and 6.73% (2256 data points) with an overall average of 6.38 ± 0.14% (7770 data points). The highest levels of CO32− were found at the edge of the trabeculae and immediately adjacent to the Haversian canal. Examination of parameters derived from the phosphate v1,v3 contour of the synthetic apatites revealed that the crystallinity/perfection of the hydroxyapatite (HA) crystals was diminished as CO32− levels increased. The methodology described will permit evaluation of the spatial distribution of CO32− levels in diseased and normal mineralized tissues.
Breast Cancer Cells Release Factors That Induce Apoptosis in Human Bone Marrow Stromal Cells
Authors: Fromigué Olivia , Kheddoumi Naïma , Lomri Abderrahim , Marie Pierre J. , Body Jean-Jacques
Published: April 2006

Breast cancer is associated frequently with skeletal metastases, which cause significant morbidity. The main mechanism is an increase in osteoclast-mediated bone resorption. We postulated that osteoblasts could be other essential target cells and previously showed that conditioned medium (CM) of breast cancer cells (BCCs) inhibits the proliferation of osteoblast-like cells. In this study, we investigated the effects of BCC-secreted products on osteoprogenitor cells using a clonal fetal human bone marrow stromal preosteoblastic cell line (FHSO-6) that expresses alkaline phosphatase (ALP) activity, type I collagen (COLI), and increased osteocalcin (OC) and osteopontin under treatment with dexamethasone (Dex), 1,25-dihydroxyvitamin D [1,25(OH)2D], or recombinant human bone morphogenetic protein 2 (rhBMP-2). Treatment with MCF-7 CM inhibited FHSO-6 cell survival in a dose-dependent and irreversible manner. Morphological investigation indicated that MCF-7 CM increased both apoptotic and necrotic cell number. MCF-7 CM increased caspases activity and a broad inhibitor of caspase activity (benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethyl ketone [z-VAD-fmk]) partly reversed the CM-induced inhibition of FHSO-6 cell survival. Western blot analyses revealed an increased bax/bcl-2 ratio in MCF-7 CM-treated FHSO-6 cells. MCF-7 cells exhibit FasLigand as membrane-bound protein and as a soluble cytokine in the CM. Deprivation of MCF-7 CM from active FasLigand by saturation with a soluble Fas molecule suppressed the induction of FHSO-6 apoptosis, whereas fibroblast CM, which did not contain FasLigand, only weakly modified FHSO-6 cell survival because of increased cell necrosis. These data indicate that FasLigand secreted by BCCs induces apoptosis and necrosis of human preosteoblastic stromal cells through caspase cascade modulated by the bax and bcl-2 protein level. The induction of apoptosis in human bone marrow stromal cells by BCCs may contribute to the inappropriately low osteoblast reaction and bone formation during tumor-induced osteolysis in bone metastases.
Matrix Gla Protein in Xenopus laevis: Molecular Cloning, Tissue Distribution, and Evolutionary Considerations
Authors: Cancela M. L. , Ohresser M. C. P. , Reia J. P. , Viegas C. S. B. , Williamson M. K. , Price P. A.
Published: April 2006

Matrix Gla protein (MGP) belongs to the family of vitamin K-dependent, Gla-containing proteins and in higher vertebrates, is found in the extracellular matrix of mineralized tissues and soft tissues. MGP synthesis is highly regulated at the transcription and posttranscription levels and is now known to be involved in the regulation of extracellular matrix calcification and maintenance of cartilage and soft tissue integrity during growth and development. However, its mode of action at the molecular level remains unknown. Because there is a large degree of conservation between amino acid sequences of shark and human MGP, the function of MGP probably has been conserved throughout evolution. Given the complexity of the mammalian system, the study of MGP in a lower vertebrate might be advantageous to relate the onset of MGP expression with specific events during development. Toward this goal, MGP was purified from Xenopus long bones and its N-terminal amino acid sequence was determined and used to clone the Xenopus MGP complementary DNA (cDNA) by a mixture of reverse-transcription (RT)- and 5′- rapid amplification of cDNA ends (RACE)-polymerase chain reaction (PCR). MGP messenger RNA (mRNA) was present in all tissues analyzed although predominantly expressed in Xenopus bone and heart and its presence was detected early in development at the onset of chondrocranium development and long before the appearance of the first calcified structures and metamorphosis. These results show that in this system, as in mammals, MGP may be required to delay or prevent mineralization of cartilage and soft tissues during the early stages of development and indicate that Xenopus is an adequate model organism to further study MGP function during growth and development.
Validation of Peripheral Dual-Energy X-Ray Absorptiometry for the Measurement of Bone Mineral in Intact and Excised Long Bones of Rats
Authors: Nagy Tim R. , Prince Charles W. , Li Jing
Published: April 2006

We evaluated the precision and accuracy of peripheral dual-energy X-ray absorptiometry (DXA) for the measurement of bone mineral density (BMD) and bone mineral content (BMC) in intact and excised femurs and tibias from rats. Thirty-one Sprague-Dawley rats (18F/13M; 114–360 g) were used in the study. Precision and accuracy were determined in 23 rats and prediction equations were evaluated in an independent sample of 8 animals. Precision was determined by measuring the right hindquarter three times with repositioning between scans. The femur and tibia were then excised, cleaned, and scanned in triplicate, with repositioning. CVs ranged from 0.66 to 2.24%. Accuracy of BMC was determined by comparison to bone ash values. BMC values for the intact and excised femur significantly overestimated bone ash (p < 0.001) by 33% and 5.5%, respectively. BMC for the intact tibia overestimated ash by 37% (p < 0.001), whereas BMC for the excised tibia underestimated ash by 1% (p < 0.05). However, BMC and bone ash were highly related for both bones, whether BMC was measured in the intact animal or after excision (r2 > 0.99). Cross-validation of prediction equations in an independent sample showed that there were no significant differences between predicted ash (based on BMC from DXA) and measured bone ash. These results suggest the peripheral DXA is a useful tool for measuring intact and excised rat leg bones.