bluuebunny/crossref_metadata_2025 huggingface dataset:
Idiopathic Hyperphosphatasia and TNFRSF11B Mutations: Relationships Between Phenotype and Genotype
Authors: Chong Belinda , Hegde Madhuri , Fawkner Matthew , Simonet Scott , Cassinelli Hamilton , Coker Mahmut , Kanis John , Seidel Joerg , Tau Cristina , Tüysüz Beyhan , Yüksel Bilgin , Love Donald , Cundy Tim
Published: April 2006

Homozygous mutations in TNFRSF11B, the gene encoding osteoprotegerin, were found in affected members from six of nine families with idiopathic hyperphosphatasia. The severity of the phenotype was related to the predicted effects of the mutations on osteoprotegerin function.

Introduction: Idiopathic hyperphosphatasia (IH) is a rare high bone turnover congenital bone disease in which affected children are normal at birth but develop progressive long bone deformities, fractures, vertebral collapse, skull enlargement, and deafness. There is, however, considerable phenotypic variation from presentation in infancy with severe progressive deformity through to presentation in late childhood with minimal deformity. Two recent reports have linked idiopathic hyperphosphatasia with deletion of, or mutation in, the TNFRSF11B gene that encodes osteoprotegerin (OPG), an important paracrine modulator of RANKL-mediated bone resorption.

Materials and Methods: We studied subjects with a clinical diagnosis of IH and unaffected family members from nine unrelated families. Clinical, biochemical, and radiographic data were collected, and genomic DNA examined for mutations in TNFRSF11B. The relationship between the mutations, their predicted effects on OPG function, and the phenotype were then examined.

Results: Of the nine families studied, affected subjects from six were homozygous for novel mutations in TNFRSF11B. Their parents were heterozygous, consistent with autosomal recessive inheritance. Four of the six mutations occurred in the cysteine-rich ligand-binding domain and are predicted to disrupt binding of OPG to RANKL. Missense mutations in the cysteine residues, predicted to cause major disruption to the ligand-binding region, were associated with a severe phenotype (deformity developing before 18 months age and severe disability), as was a large deletion mutation. Non-cysteine missense mutations in the ligand-binding domain were associated with an intermediate phenotype (deformity recognized around the age of 5 years and an increased rate of long bone fracture). An insertion/deletion mutation at the C-terminal end of the protein was associated with the mildest phenotype.

Conclusion: Mutations in TNFRSF11B account for the majority of, but not all, cases of IH, and there are distinct genotype-phenotype relationships.
12-O-tetradecanoylphorbol-13-acetate (TPA) Inhibits Osteoclastogenesis by Suppressing RANKL-Induced NF-κB Activation
Authors: Wang Cathy , Steer James H , Joyce David A , Yip Kirk HM , Zheng Ming H , XU Jiake
Published: April 2006

The mechanism by which TPA-induced PKC activity modulates osteoclastogenesis is not clear. Using a RAW264.7 cell culture system and assays for NF-κB nuclear translocation, NF-κB reporter gene activity, and MAPK assays, we demonstrated that TPA inhibits osteoclastogenesis through the suppression of RANKL-induced NF-κβ activation.

Introduction: The protein kinase C (PKC) pathway has been suggested to be an important regulator of osteoclastic bone resorption. The role of PKC in RANKL-induced osteoclastogenesis, however, is not clear. In this study, we examined the effects of 12-O-tetradecanoylphorbol-13-acetate (TPA), a PKC activator, on osteoclastogenesis and studied its role in RANKL-induced signaling.

Materials and Methods: RANKL-induced RAW264.7 cell differentiation into osteoclast-like cells was used to assess the effect of TPA on osteoclastogenesis. Assays for NF-κB nuclear translocation, NF-κB reporter gene activity, protein kinase activity, and Western blotting were used to examine the effects of TPA on RANKL-induced NF-κβ, c-Jun N-terminal kinase (JNK), and MEK/ERK and p38 signal transduction pathways.

Results: We found that TPA inhibited RANKL-induced RAW264.7 cell differentiation into osteoclasts in a dose-dependent manner. Time course analysis showed that the inhibitory effect of TPA on RANKL-induced osteoclastogenesis occurs predominantly at an early stage of osteoclast differentiation. TPA alone had little effect on NF-κβ activation in RAW264.7 cells, but it suppresses the RANKL-induced NF-κβ activation in a dose-dependent fashion. Interestingly, the suppressive effect of TPA on RANKL-induced NF-κβ activation was prevented by a conventional PKC inhibitor, Go6976. Supershift studies revealed that the RANKL-induced DNA binding of NF-κβ complexes consisted of C-Rel, NF-κB1 (p50), and RelA (p65). In addition, TPA induced the activation of JNK in RAW264.7 cells but had little effect on RANKL-induced activation of JNK. TPA also inhibited RANKL-induced activation of ERK but had little effect on p38 activation.

Conclusion: Given that NF-κB activation is obligatory for osteoclast differentiation, our studies imply that inhibition of osteoclastogenesis by TPA is, at least in part, caused by the suppression of RANKL-induced activation of NF-κβ during an early stage of osteoclastogenesis. Selective modulation of RANKL signaling pathways by PKC activators may have important therapeutic implications for the treatment of bone diseases associated with enhanced bone resorption.
Quantitative Trait Loci on Chromosomes 2p, 4p, and 13q Influence Bone Mineral Density of the Forearm and Hip in Mexican Americans
Authors: Kammerer Candace M , Schneider Jennifer L , Cole Shelley A , Hixson James E , Samollow Paul B , O'Connell Jeffrey R , Perez Reina , Dyer Thomas D , Almasy Laura , Blangero John , Bauer Richard L , Mitchell Braxton D
Published: April 2006

We performed a genome scan using BMD data of the forearm and hip on 664 individuals in 29 Mexican-American families. We obtained evidence for QTL on chromosome 4p, affecting forearm BMD overall, and on chromosomes 2p and 13q, affecting hip BMD in men.

Introduction: The San Antonio Family Osteoporosis Study (SAFOS) was designed to identify genes and environmental factors that influence bone mineral density (BMD) using data from large Mexican-American families.

Materials and Methods: We performed a genome-wide linkage analysis using 416 highly polymorphic microsatellite markers spaced approximately 9.5 cM apart to locate and identify quantitative trait loci (QTL) that affect BMD of the forearm and hip. Multipoint variance components linkage analyses were done using data on all 664 subjects, as well as two subgroups of 259 men and 261 premenopausal women, from 29 families for which genotypic and phenotypic data were available.

Results: We obtained significant evidence for a QTL affecting forearm (radius midpoint) BMD in men and women combined on chromosome 4p near D4S2639 (maximum LOD = 4.33, genomic p = 0.006) and suggestive evidence for a QTL on chromosome 12q near locus D12S2070 (maximum conditional LOD = 2.35). We found suggestive evidence for a QTL influencing trochanter BMD on chromosome 6 (maximum LOD = 2.27), but no evidence for QTL affecting the femoral neck in men and women combined. In men, we obtained evidence for QTL affecting neck and trochanter BMD on chromosomes 2p near D2S1780 (maximum LOD = 3.98, genomic p = 0.013) and 13q near D13S788 (maximum LOD = 3.46, genomic p = 0.039), respectively. We found no evidence for QTL affecting forearm or hip BMD in premenopausal women.

Conclusion: These results provide strong evidence that a QTL on chromosome 4p affects radius BMD in Mexican-American men and women, as well as evidence that QTL on chromosomes 2p and 13q affect hip BMD in men. Our results are consistent with some reports in humans and mice.
Congenic Strains of Mice for Verification and Genetic Decomposition of Quantitative Trait Loci for Femoral Bone Mineral Density
Authors: Shultz Kathryn L , Donahue Leah Rae , Bouxsein Mary L , Baylink David J , Rosen Clifford J , Beamer Wesley G
Published: April 2006

Peak femoral volumetric bone mineral density (femoral bone mineral density) in C57BL/6J (B6) 4-month-old female mice is 50% lower than in C3H/HeJ (C3H) and 34% lower than in CAST/EiJ (CAST) females. Genome-wide analyses of (B6 × C3H)F2 and (B6 × CAST)F2 4-month-old female progeny demonstrated that peak femoral bone mineral density is a complex quantitative trait associated with genetic loci (QTL) on numerous chromosomes (Chrs) and with trait heritabilities of 83% (C3H) and 57% (CAST). To test the effect of each QTL on femoral bone mineral density, two sets of loci (six each from C3H and CAST) were selected to make congenic strains by repeated backcrossing of donor mice carrying a given QTL-containing chromosomal region to recipient mice of the B6 progenitor strain. At the N6F1 generation, each B6.C3H and B6.CAST congenic strain (statistically 98% B6-like in genomic composition) was intercrossed to obtain N6F2 progeny for testing the effect of each QTL on femoral bone mineral density. In addition, the femoral bone mineral density QTL region on Chr 1 of C3H was selected for congenic subline development to facilitate fine mapping of this strong femoral bone mineral density locus. In 11 of 12 congenic strains, 6 B6.C3H and 5 B6.CAST, femoral bone mineral density in mice carrying c3h or cast alleles in the QTL regions was significantly different from that of littermates carrying b6 alleles. Differences also were observed in body weight, femoral length, and mid-diaphyseal periosteal circumference among these 11 congenic strains when compared with control littermates; however, these latter three phenotypes were not consistently correlated with femoral bone mineral density. Analyses of eight sublines derived from the B6.C3H-1T congenic region revealed two QTLs: one located between 36.9 and 49.7 centiMorgans (cM) and the other located between 73.2 and 100.0 cM distal to the centromere. In conclusion, these congenic strains provide proof of principle that many QTLs identified in the F2 analyses for femoral bone mineral density exert independent effects when transferred and expressed in a common genetic background. Furthermore, significant differences in femoral bone mineral density among the congenic strains were not consistently accompanied by changes in body weight, femur length, or periosteal circumference. Finally, decomposition of QTL regions by congenic sublines can reveal additional loci for phenotypes assigned to a QTL region and can markedly refine genomic locations of quantitative trait loci, providing the opportunity for candidate gene testing.
Vitamin D-Mediated Hypercalcemia in Lymphoma: Evidence for Hormone Production by Tumor-Adjacent Macrophages
Authors: Hewison Martin , Kantorovich Vitaly , Liker Harley R , Van Herle Andre J , Cohan Pejman , Zehnder Daniel , Adams John S
Published: April 2006

Nearly one-half of all hypercalcemic patients with lymphoma present with inappropriately elevated circulating concentrations of the active vitamin D metabolite 1,25-dihydroxyvitamin D (1,25(OH)2D3). However, the cellular source of the vitamin D hormone in lymphomas remains unclear. To address this, we report the case of a 75-year-old man with hypercalcemia associated with raised circulating concentrations of 1,25(OH)2D3 and suppressed parathyroid hormone (PTH) levels. Positron emission tomographic (PET) and computed tomographic (CT) imaging revealed the presence of a large lymphoma that was confined to the spleen; subsequent pathological analysis showed that this was an intermediate grade B-cell lymphoma. After surgical removal of the spleen, serum calcium and 1,25(OH)2D3 levels became normalized within 24 h. Immunolocalization of the vitamin D-activating enzyme 25-hydroxyvitamin D3-1α-hydroxylase (1α-hydroxylase) in sections of resected spleen showed that staining was negative in the lymphoma cells but positive in neighboring macrophages. This case study indicates that the hypercalcemia associated with lymphomas may be due, in some instances, to excessive extrarenal production of 1,25(OH)2D3. Furthermore, by using immunohistochemistry to assess the distribution of 1α-hydroxylase, we have been able to show for the first time that tissue macrophages, rather than actual tumor cells, are the most likely ectopic source of this enzyme. Based on this case study, we propose that the abnormal synthesis of 1,25(OH)2D3 associated with some lymphomas is because of paracrine regulation of tumor-associated macrophages.
Phosphate Is a Specific Signal for ATDC5 Chondrocyte Maturation and Apoptosis-Associated Mineralization: Possible Implication of Apoptosis in the Regulation of Endochondral Ossification
Authors: Magne D , Bluteau G , Faucheux C , Palmer G , Vignes-Colombeix C , Pilet P , Rouillon T , Caverzasio J , Weiss P , Daculsi G , Guicheux J
Published: April 2006

Involvement of Pi and Ca in chondrocyte maturation was studied because their levels increase in cartilage growth plate. In vitro results showed that Pi increases type X collagen expression, and together with Ca, induces apoptosis-associated mineralization, which is similar to that analyzed in vivo, thus suggesting a role for both ions and apoptosis during endochondral ossification.

Introduction: During endochondral ossification, regulation of chondrocyte maturation governs the growth of the cartilage plate. The role of inorganic phosphate (Pi), whose levels strongly increase in the hypertrophic zone of the growth plate both in intra- and extracellular compartments, on chondrocyte maturation and mineralization of the extracellular matrix has not yet been deciphered.

Materials and Methods: The murine chondrogenic cell line ATDC5 was used. Various Pi and calcium concentrations were obtained by adding NaH2PO4/Na2HPO4 and CaCl2, respectively. Mineralization was investigated by measuring calcium content in cell layer by atomic absorption spectroscopy and by analyzing crystals with transmission electron microscopy and Fourier transform infrared microspectroscopy. Cell differentiation was investigated at the mRNA level (reverse transcriptase-polymerase chain reaction [RT-PCR] analysis). Cell viability was assessed by methyl tetrazolium salt (MTS) assay and staining with cell tracker green (CTG) and ethidium homodimer-1 (EthD-1). Apoptosis was evidenced by DNA fragmentation and caspase activation observed in confocal microscopy, as well as Bcl-2/Bax mRNA ratio (RT-PCR analysis).

Results: We showed that Pi increases expression of the hypertrophic marker, type X collagen. When calcium concentration is slightly increased (like in cartilage growth plate), Pi also induces matrix mineralization that seems identical to that observed in murine growth plate cartilage and stimulates apoptosis of differentiated ATDC5 cells, with a decrease in Bcl-2/Bax mRNA ratio, DNA fragmentation, characteristic morphological features, and caspase-3 activation. In addition, the use of a competitive inhibitor of phosphate transport showed that these effects are likely dependent on Pi entry into cells through phosphate transporters. Finally, inhibition of apoptosis with ZVAD-fmk reduces π-induced mineralization.

Conclusions: These findings suggest that Pi regulates chondrocyte maturation and apoptosis-associated mineralization, highlighting a possible role for Pi in the control of skeletal development.
Decreased AP-1 Activity and Interleukin-11 Expression by Bone Marrow Stromal Cells May Be Associated With Impaired Bone Formation in Aged Mice
Authors: Tohjima Emiko , Inoue Daisuke , Yamamoto Nobuchika , Kido Shinsuke , Ito Yuji , Kato Shuji , Takeuchi Yasuhiro , Fukumoto Seiji , Matsumoto Toshio
Published: April 2006

Expression of an osteogenic cytokine, IL-11, is decreased in SAMP6. We show here that IL-11 transcription largely depends on AP-1 transcription factors, activities of which are decreased in SAMP6 as well as aged ICR mice. Therefore, diminished AP-1 activity and the resultant decline in IL-11 expression may play a role in impaired bone formation in the aged.

Introduction: Evidence suggests that impaired osteoblastogenesis contributes to aging-associated osteopenia. The P6 strain of senescence-accelerated mice (SAM) is an animal model of senile osteoporosis, which exhibits low bone mass caused by impaired bone formation. Bone marrow stromal cells from SAMP6 show decreased osteoblastogenesis and increased adipogenesis. We previously demonstrated that these abnormalities of SAMP6 stromal cells may be attributed to decreased expression of interleukin (IL)-11.

Methods: In this study, we attempted to determine the molecular mechanism of decreased IL-11 expression by SAMP6 stromal cells by cloning and analyzing the mouse IL-11 gene promoter.

Results and Conclusions: We found that two tandem activating protein-1 (AP-1) sites that reside immediately upstream of TATA box play critical roles in IL-11 gene transcription. Gel shift analysis showed that binding activity to the IL-11 AP-1 sites was reduced in SAMP6 stromal cell nuclear extracts. Among multiple components of AP-1 transcription factors, Jun D binding was particularly decreased. Furthermore, decreased Jun D binding and IL-11 expression by stromal cells was also observed in aged mice of the ICR strain. Therefore, decreased AP-1 activity and a resultant decline in IL-11 expression by bone marrow stromal cells may play a role in impaired bone formation in the aged.
Quantitative Trait Loci for Femoral Size and Shape in a Genetically Heterogeneous Mouse Population
Authors: Volkman Suzanne K , Galecki Andrzej T , Burke David T , Paczas Michael R , Moalli Maria R , Miller Richard A , Goldstein Steven A
Published: April 2006

The aim of this study was to examine the genetic effects on cortical bone geometry. Genotypes from 487 mice were compared with geometric traits obtained from μCT. We found 14 genetic markers that associate with geometric traits, showing the complexity of genetic control over bone geometry.

Introduction: Previous studies have shown that genetic background affects bone characteristics, particularly bone mineral density, in both mouse and human populations. Much less is known, however, about the effects of polymorphic genes on bone size, shape, and mechanical integrity. In this study, we investigated the genetic determinants of geometric properties of cortical bone in mice.

Materials and Methods: This study used a genetically heterogeneous mouse population, which is denoted UM-HET3 stock and is derived as the progeny of (BALB/cJ × C57BL/6J) F1 females and (C3H/HeJ × DBA/2J) F1 males. The experimental group consisted of 487 female UM-HET3 mice. Genotypic data from 99 polymorphic genetic loci was obtained from the mice at 4 weeks of age. At 18 months of age, the mice were humanely killed, and the right femurs were scanned with microcomputed tomography to assess geometric properties of cortical bone. A permutation-based test was used to detect significant associations between genetic markers and geometric traits. This test generates experimentwise p values, which account for the effect of testing multiple hypotheses. An experiment-wise p ≤ 0.05 was considered statistically significant.

Results: Fourteen genetic markers were found to significantly associate with one or more geometric traits. Two markers (D3Mit62 and D4Mit155) were associated with traits describing bone size; 2 (D12Mit167 and D14Mit170) were linked with traits describing bone shape; and 10 (D1Nds2, D5Mit95, D6Mit216, D7Mit91, D8Mit51, D9Mit110, D11Mit83, D15Mit100, D15Mit171, and D17Mit46) were associated with both size and shape.

Conclusions: Our results indicate that the genetic control of cortical bone geometry is complex and that femoral size and shape may be influenced by different, although overlapping, groups of polymorphic loci.
Adrenarche and Bone Modeling and Remodeling at the Proximal Radius: Weak Androgens Make Stronger Cortical Bone in Healthy Children
Authors: Remer Thomas , Boye Kai R , Hartmann Michaela , Neu Christina M , Schoenau Eckhard , Manz Friedrich , Wudy Stefan A
Published: April 2006

Adrenarche, the physiological increase in adrenal androgen secretion, may contribute to better bone status. Proximal radial bone and 24-h urinary steroid hormones were analyzed cross-sectionally in 205 healthy children and adolescents. Positive adrenarchal effects on radial diaphyseal bone were observed. Obviously, adrenarche is one determinant of bone mineral status in children.

Introduction: Increased bone mass has been reported in several conditions with supraphysiological adrenal androgen secretion during growth. However, no data are available for normal children. Therefore, our aim was to examine whether adrenal androgens within their physiological ranges may be involved in the strengthening of diaphyseal bone during growth.

Methods: Periosteal circumference (PC), cortical density, cortical area, bone mineral content, bone strength strain index (SSI), and forearm cross-sectional muscle area were determined with peripheral quantitative computed tomography (pQCT) at the proximal radial diaphysis in healthy children and adolescents. All subjects, aged 6–18 years, who collected a 24-h urine sample around the time of their pQCT analysis (100 boys, 105 girls), were included in the present study, and major urinary glucocorticoid (C21) and androgen (C19) metabolites were quantified using gas chromatography-mass spectrometry.

Results and Conclusions: We found a significant influence of muscularity, but not of hormones, on periosteal modeling (PC) before the appearance of pubic hair (prepubarche). Similarly, no influence of total cortisol secretion (C21) was seen on the other bone variables. However, positive effects of C19 on cortical density (p < 0.01), cortical area (p < 0.001), bone mineral content (p < 0.001), and SSI (p < 0.001)—reflecting, at least in part, reduction in intracortical remodeling—were observed in prepubarchal children after muscularity or age had been adjusted for. This early adrenarchal contribution to proximal radial diaphyseal bone strength was further confirmed for all cortical variables (except PC) when, instead of C19 and C21, specific dehydroepiandrosterone metabolites were included as independent variables in the multiple regression model. During development of pubic hair (pubarche), muscularity and pubertal stage rather than adrenarchal hormones seemed to influence bone variables. Our study shows that especially the prepubarchal increase in adrenal androgen secretion plays an independent role in the accretion of proximal radial diaphyseal bone strength in healthy children.
Periprosthetic Osteolysis: Induction of Vascular Endothelial Growth Factor From Human Monocyte/Macrophages by Orthopaedic Biomaterial Particles
Authors: Miyanishi Keita , Trindade Michael CD , Ma Ting , Goodman Stuart B , Schurman David J , Smith R Lane
Published: April 2006

VEGF and VEGF receptor, Flt-1, expression was observed in periprosthetic tissues surrounding loosened total joint implants. Exposure of monocyte/macrophages to titanium particles resulted in increased VEGF expression, p44/42 MAPK activation, and VEGF-dependent macrophage chemotaxis. Increased levels of angiogenic factors, such as VEGF, may be critically important in wear debris-induced implant loosening after total joint arthroplasty.

Introduction: Periprosthetic osteolysis after total hip arthroplasty occurs in association with formation of a vascularized granulomatous tissue in response to particulate debris.

Materials and Methods: This study examined expression of vascular endothelial growth factor (VEGF) and the VEGF receptor in 10 periprosthetic tissues from loosened prostheses and quantified effects of titanium particles on VEGF release, intracellular signaling, and VEGF-dependent chemotaxis in primary cultures of human monocyte/macrophages.

Results: Double immunofluorescent staining showed that VEGF and Flt-1 co-localized with cells positive for the macrophage marker, CD11b, in the periprosthetic tissues. Monocyte/macrophages challenged with titanium particles showed a dose- and time-dependent release of VEGF ranging from 2.8- to 3.1-fold and exhibited increased expression of VEGF121 and VEGF165 mRNAs, reaching levels up to 5.0- and 8.6-fold, respectively, by 48 h (p < 0.01). Exposure of monocyte/macrophages to titanium particles upregulated phosphorylated-p44/42 mitogen-activated protein kinase (MAPK) within 30 minutes. Particle-induced activation of p44/42 MAPK and release of VEGF were dose-dependently suppressed by pretreatment of cells with PD98059, a specific inhibitor of p44/42 MAPK. Monocyte/macrophages challenged with titanium particles also showed a time-dependent activation of AP-1, a transcription factor associated with VEGF expression (p < 0.01). Supernatants from particle-challenged monocyte/macrophages increased macrophage chemotactic activity by 30%, which was significantly inhibited by anti-VEGF neutralizing antibody (p < 0.01).

Conclusions: This study suggests that induction of VEGF release from monocyte/macrophages in response to orthopaedic biomaterial wear debris may contribute to periprosthetic osteolysis and implant loosening.
ALK2 Functions as a BMP Type I Receptor and Induces Indian Hedgehog in Chondrocytes During Skeletal Development
Authors: Zhang Donghui , Schwarz Edward M , Rosier Randy N , Zuscik Michael J , Puzas J Edward , O'Keefe Regis J
Published: April 2006

Growth plate chondrocytes integrate multiple signals during normal development. The type I BMP receptor ALK2 is expressed in cartilage and expression of constitutively active (CA) ALK2 and other activated type I BMP receptors results in maturation-independent expression of Ihh in chondrocytes in vitro and in vivo. The findings suggest that BMP signaling modulates the Ihh/PTHrP signaling pathway that regulates the rate of chondrocyte differentiation.

Introduction: Bone morphogenetic proteins (BMPs) have an important role in vertebrate limb development. The expression of the BMP type I receptors BMPR-IA (ALK3) and BMPR-IB (ALK6) have been more completely characterized in skeletal development than ALK2.

Methods: ALK2 expression was examined in vitro in isolated chick chondrocytes and osteoblasts and in vivo in the developing chick limb bud. The effect of overexpression of CA ALK2 and the other type I BMP receptors on the expression of genes involved in chondrocyte maturation was determined.

Results: ALK2 was expressed in isolated chick osteoblasts and chondrocytes and specifically mediated BMP signaling. In the developing chick limb bud, ALK2 was highly expressed in mesenchymal soft tissues. In skeletal elements, expression was higher in less mature chondrocytes than in chondrocytes undergoing terminal differentiation. CA ALK2 misexpression in vitro enhanced chondrocyte maturation and induced Ihh. Surprisingly, although parathyroid hormone-related peptide (PTHrP) strongly inhibited CA ALK2 mediated chondrocyte differentiation, Ihh expression was minimally decreased. CA ALK2 viral infection in stage 19–23 limbs resulted in cartilage expansion with joint fusion. Enhanced periarticular expression of PTHrP and delayed maturation of the cartilage elements were observed. In the cartilage element, CA ALK2 misexpression precisely colocalized with the expression with Ihh. These findings were most evident in partially infected limbs where normal morphology was maintained. In contrast, BMP-6 had a normal pattern of differentiation-related expression. CA BMPR-IA and CA BMPR-IB overexpression similarly induced Ihh and PTHrP.

Conclusions: The findings show that BMP signaling induces Ihh. Although the colocalization of the activated type I receptors and Ihh suggests a direct BMP-mediated signaling event, other indirect mechanisms may also be involved. Thus, while BMPs act directly on chondrocytes to induce maturation, this effect is counterbalanced in vivo by induction of the Ihh/PTHrP signaling loop. The findings suggest that BMPs are integrated into the Ihh/PTHrP signaling loop and that a fine balance of BMP signaling is essential for normal chondrocyte maturation and skeletal development.
Effect of Hyper- and Microgravity on Collagen Post-Translational Controls of MC3T3-E1 Osteoblasts
Authors: Saito Mitsuru , Soshi Shigeru , Fujii Katsuyuki
Published: April 2006

We attempted to study the effects of microgravity (by clinostat) and hypergravity (using centrifugation) on collagen metabolism using murine MC3T3-E1 osteoblasts, especially focusing on collagen cross-link formation. We found that altered gravitational load affected the post-translational modification of collagen, particularly the collagen maturation pathway, through altered expression of enzymes involved in cross-link formation.

Introduction: Gravitational loading plays important roles in the stimulation of differentiated osteoblast function and in the maintenance of skeletal tissues, whereas microgravity seems to result in osteopenia caused by impaired osteoblast differentiation. The aim of our study was to clarify the effects of altered gravitational environments on collagen metabolism, particularly the relationship between post-translational collagen quality and enzymes involved in cross-link formation, using murine osteoblastic MC3T3-E1 cells.

Materials and Methods: Cells were cultured under vector-averaged microgravity (1 × 10−3g) using a clinostat or under conventional centrifugation techniques to generate hypergravity (20g and 40g) for 72 h. We then examined the expression patterns of lysyl oxidase and the two lysyl hydroxylase isoforms telopeptidyl lysyl hydroxylase (TLH; procollagen-lysine, 2-oxyglutarate, 5-dioxigenase 2 [PLOD2]) and helical lysyl hydroxylase (HLH; [PLOD1]) by quantitative real time polymerase chain reaction (PCR) analysis. Quantitative analysis of reducible immature (dihydroxylysinonorleucine, hydroxylysinonorleucine, and lysinonorleucine) and nonreducible mature (pyridinoline and deoxypyridinoline) cross-links, and maturation rate analysis of immature to mature cross-links by conventional metabolic labeling using tritium lysine were also performed.

Results: Hypergravity upregulated both TLH mRNA expression and enzyme activity compared with stationary cultures, whereas microgravity stimulated both HLH mRNA expression and enzyme activity. These results were consistent with increased relative occupancy rates of telopeptidyl hydroxylysine-derived cross-links and helical hydroxylysine-derived forms observed under hypergravity and microgravity, respectively. Hypergravity stimulated not only lysyl oxidase mRNA expression but also increased enzyme activity and the sum of immature and mature cross-links. Furthermore, the conversion rate of immature cross-links to mature compounds was markedly increased under hypergravity but decreased under microgravity.

Conclusion: Altered gravitational loading may affect the post-translational modification of collagen through altered expression of enzymes involved in cross-link formation. These observations may be important in elucidating the mechanisms of osteopenia during space flight.
Cis-Acting Intronic Elements That Regulate Cartilage-Specific Alternative Splicing of the Type II Collagen (Col2) Pre-mRNA Lie at or Near Splice Site Junction Sequences Flanking Exon 2 of the Gene
Authors: Nishiyama Takayuki , Hatano Hiroshi , Kurosaka Masahiro , Bolander Mark E , Sarkar Gobinda
Published: April 2006

Knowledge of the cis-acting elements is required for identifying trans-acting splicing factors underlying cartilage-specific alternative splicing of Col2 pre-mRNA. By performing desired deletions in the mouse Col2 pre-mRNA, location of the intronic cis-acting elements was narrowed down to be at or near splice-junction sequences flanking exon 2 of the gene.

Introduction: Type II collagen (Col2) pre-mRNA undergoes cartilage-specific alternative splicing involving exon 2 during chondrocyte differentiation. Thus, the trans-acting protein factors that regulate the splicing are associated with the differentiation of chondrocytes. Knowledge of the cognate cis-acting elements is necessary to eventually identify the trans-acting factors.

Materials and Methods: To localize the cis-acting sequences, we created several deletions within a minigene containing exon 1 to exon 4 of mouse Col 2 gene and evaluated alternative splicing of the resulting pre-mRNAs in ATDC5 cells, a model of insulin-stimulated chondrocyte differentiation. The first deletion reduced intron 1 from 3799 to 259 bp, the second reduced intron 2 from 1108 to 94 bp, the third combined the above two deletions, and the fourth was derived from the third by removing intron 3 and exon 4. ATDC5 cells harboring these constructs were cultured for up to 21 days with or without insulin. Alternative splicing was evaluated by determining the ratio of Col2B (lacks exon 2) to Col2A (has exon 2) RNAs by reverse transcription-polymerase chain reaction.

Results: The deletion in intron 1 had no effect on the alternative splicing while other deletions affected splicing (demonstrated by the presence of splicing intermediates) in cells cultured without insulin or with insulin for 1 week. The splicing intermediates were not seen from any construct when cells were cultured longer (14–21 days) with insulin.

Conclusion: These results show that the 259-bp intron 1, the 94-bp intron 2, and exon 2 sequences retained in the fourth construct provide cis-acting signal sufficient for insulin-induced cartilage-specific alternative splicing of Col2 pre-mRNA.
The Nitrogen-Containing Bisphosphonate, Zoledronic Acid, Influences RANKL Expression in Human Osteoblast-Like Cells by Activating TNF-α Converting Enzyme (TACE)
Authors: Pan Beiqing , Farrugia Amanda N , To Luen Bik , Findlay David M , Green Jonathan , Lynch Kevin , Zannettino Andrew CW
Published: April 2006

Bisphosphonates are used to prevent osteoclast-mediated bone loss. Zoledronic acid inhibits osteoclast maturation indirectly by increasing OPG protein secretion and decreasing transmembrane RANKL expression in human osteoblasts. The decreased transmembrane RANKL expression seems to be related to the upregulation of the RANKL sheddase, TACE.

Introduction: Bisphosphonates (BPs) exhibit high affinity for hydroxyapatite mineral in bone and are used extensively to treat malignancy-associated bone disease and postmenopausal bone loss by inhibiting osteoclast (OC)-mediated bone resorption.

Materials and Methods: We examined the effect of the most potent nitrogen-containing BP available, zoledronic acid (ZOL), on the expression of RANKL and osteoprotegerin (OPG), critical factors in the regulation of OC formation and activation, in primary osteoblast (OB)-like cells derived from human bone, using flow cytometry, ELISA, semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR), in situ immunofluorescence staining, and Western blotting.

Results: Our studies show that ZOL, while not significantly affecting RANKL or OPG gene expression, markedly increased OPG protein secretion and reduced transmembrane RANKL protein expression in OB-like cells. The reduction in transmembrane RANKL expression was preceded by a marked increase in the expression of the metalloprotease-disintegrin, TNF-α converting enzyme (TACE). In addition, the decreased transmembrane expression of RANKL could be partially reversed by a TACE inhibitor, TAPI-2.

Conclusions: Our studies indicate that ZOL, in addition to its direct effects on mature OCs, may inhibit the recruitment and differentiation of OCs by cleavage of transmembrane RANKL in OB-like cells by upregulating the sheddase, TACE.
Identification of Promoter Regions Involved in Cell- and Developmental Stage-Specific Osteopontin Expression in Bone, Kidney, Placenta, and Mammary Gland: An Analysis of Transgenic Mice
Authors: Higashibata Yuji , Sakuma Takahiko , Kawahata Hirohisa , Fujihara Shinji , Moriyama Keiji , Okada Atsushi , Yasui Takahiro , Kohri Kenjiro , Kitamura Yukihiko , Nomura Shintaro
Published: April 2006

Cell-specific expression of GFP under the control of different lengths of the osteopontin promoter in transgenic mice identified the positive and negative regulatory regions for respective cell types. The results provide new insights for physiological and pathological expression of the osteopontin gene.

Introduction: Osteopontin (OPN) is a major non-collagenous bone matrix protein that is involved in normal and pathological calcification and is expressed in a tissue-specific manner. To investigate how such tissue-specific OPN gene expression is regulated in vivo, transgenic mice expressing the green fluorescent protein (GFP) reporter gene controlled by different lengths of the OPN promoter were generated.

Materials and Methods: Cell- and developmental stage-specific osteopontin expression in transgenic mice was examined by Northern blotting, immunoblotting, fluorescence examination, and in situ hybridization and compared with those of OPN.

Results and Conclusions: The line bearing the −5505 to +14 region of the OPN promoter was shown by Northern blotting and immunoblotting to express GFP in the same cells that express endogenous OPN (osteoblasts, hypertrophic chondrocytes, renal and mammary gland epithelial cells, and granulated metrial gland [GMG] placental cells) at the same stage in development. Thus, the 5.5-kb −5505 to +14 promoter region is sufficient for proper tissue-specific OPN expression. The lines carrying shorter segments of the OPN promoter showed different expression patterns. These patterns revealed a putative cis-acting element in the −5269 to −5263 region that restricts OPN expression to hypertrophic chondrocytes and a mammary gland-specific expressing element and a GMG cell-specific enhancing element in the −5505 to −3156 region. Furthermore, the −3155 to −1576 region seems to contain positive renal epithelial cell- and GMG cell-specific expression motif(s) as well as a negative regulatory element that prevents OPN expression in fibroblasts. Moreover, the −1576 to −910 region seems to contain a positive osteoblast-specific-expressing element. Thus, the 5.5-kb OPN promoter contains multiple cis-acting elements encoding positive and negative cell-specific regulatory systems.
Optimization of BMD Measurements to Identify High Risk Groups for Treatment—A Test Analysis
Authors: Johansson Helena , Oden Anders , Johnell Olof , Jonsson Bengt , de Laet Chris , Oglesby Alan , McCloskey Eugene V , Kayan Karthik , Jalava Tarja , Kanis John A
Published: April 2006

The aim of this study was to optimize the use of BMD measurements in case finding strategies. The use of clinical risk factors with and without BMD was explored in a random sample of the Sheffield female population ≥75 years of age. The use of clinical risk factors alone could identify women well above or well below a threshold of fracture risk. BMD assessment can be confined to a minority of women (∼20%) in whom the measurement aids in prognostication of fracture.

Introduction: The aim of this study was to develop a methodology to optimize the role of BMD measurements in a case finding strategy. We studied 2113 women ≥75 years of age randomly selected from Sheffield, UK, and adjacent regions. Baseline assessment included hip BMD and clinical risk factors. Outcomes included death and fracture in women followed for 6723 person-years.

Materials and Methods: Poisson models were used to identify significant risk factors for all fractures and for death with and without BMD and the hazard functions were used to compute fracture probabilities. Women were categorized by fracture probability with and without a BMD assessment. A 10-year fracture probability threshold of 35% was taken as an intervention threshold. Discordance in categorization of risk (i.e., above or below the threshold probability) between assessment with and without BMD was examined by logistic regression as probabilities of re-classification. Age, prior fracture, use of corticosteroids, and low body mass index were identified as significant clinical risk factors.

Results: A total of 16.8% of women were classified as high risk based on these clinical risk factors. The average BMD in these patients was ∼1 SD lower than in low-risk women; 21.5% of women were designated to be at high risk with the addition of BMD. Fifteen percent of all women were reclassified after adding BMD to clinical risk factors, most of whom lay near the intervention threshold. When a high probability of reclassification was accepted (without a BMD test) for high risk to low risk (p1 ≤ 0.8) and a low probability accepted for low to high risk (p2 ≤ 0.2), BMD tests would be required in only 21% of the population.

Conclusion: We conclude that the use of clinical risk factors can identify elderly women at high fracture risk and that such patients have a low average BMD. BMD testing is required, however, in a minority of women—a fraction that depends on the probabilities accepted for classification and the thresholds of risk chosen. These findings need to be validated in other cohorts at different ages and from different regions of the world.
How Many Women Have Osteoporosis?
Authors: Melton L. Joseph , Chrischilles Elizabeth A. , Cooper Cyrus , Lane Ann W. , Riggs B. Lawrence
Published: April 2006

Osteoporosis is widely viewed as a major public health concern, but the exact magnitude of the problem is uncertain and likely to depend on how the condition is defined. Noninvasive bone mineral measurements can be used to define a state of heightened fracture risk (osteopenia), or the ultimate clinical manifestation of fracture can be assessed (established osteoporosis). If bone mineral measurements more than 2 standard deviations below the mean of young normal women represent osteopenia, then 45% of white women aged 50 years and over have the condition at one or more sites in the hip, spine, or forearm on the basis of population-based data from Rochester, Minnesota. A smaller proportion is affected at each specific skeletal site: 32% have bone mineral values this low in the lumbar spine, 29% in either of two regions in the proximal femur, and 26% in the midradius. Although this overall estimate is substantial, some other serious chronic diseases are almost as common. More importantly, low bone mass is associated with adverse health outcomes, especially fractures. The lifetime risk of any fracture of the hip, spine, or distal forearm is almost 40% in white women and 13% in white men from age 50 years onward. If the enormous costs associated with these fractures are to be reduced, increased attention must be given to the design and implementation of control programs directed at this major health problem.
Desmoid Tumor With Ossification in Chest Wall: Possible Involvement of BAMBI Promoter Hypermethylation in Metaplastic Bone Formation
Authors: Kitazawa Sohei , Kitazawa Riko , Obayashi Chiho , Yamamoto Tetsuji
Published: April 2006

A rare case of desmoid-type fibromatosis with focal metaplastic bone in the chest wall suggested that enhanced responsiveness to BMP signaling by decreasing BAMBI expression through promoter hypermethylation plays a crucial role in the formation of metaplastic bone.

Introduction: Desmoid-type fibromatosis, originating from mesenchymal cells with myofibroblastic features, is a locally aggressive and frequently recurring infiltrative lesion. One such sporadic case with metaplastic ossification in the chest wall is presented.

Materials and Methods: A 43-year-old man was referred to the hospital with a gradually enlarging hard mass in the left anterolateral chest wall. A thoracotomy was carried out, and histopathological specimens were used for immunohistochemical, genetic, and methylation studies.

Results: Accumulation of altered β-catenin associated with a somatic heterozygous activating mutation in codon 41 was detected in the typical desmoid-type fibromatosis and at the ossifying focus. Among factors related to bone formation and the classical wnt-β-catenin signaling pathway, BMP and activin membrane-bound inhibitor (BAMBI) expression was specifically downregulated at the ossifying focus. Hypermethylation of the BAMBI promoter was observed in microdissected tissue from the ossifying focus but not in that from the typical desmoid-type fibromatosis.

Conclusions: Because both BMP and classical Wnt/β-catenin/LEF1 signaling cooperatively and mutually induce differentiation of mesenchymal cells into osteoblastic cells and promote bone formation, the epigenetic event leading to the enhanced responsiveness to BMP signaling may play a crucial role in the formation of metaplastic bone.
The theory of newsworthiness applied to Mexico's press. How the news factors influence foreign news coverage in a transitional country
Authors: Schwarz Andreas
Published: April 2006

Empirical studies which examine the theory of newsworthiness and the predictability of news coverage in transnational or developing countries still remain on the agenda of journalism research. Therefore, this study examines the influence of news factors on the foreign news coverage of three Mexican newspapers. Two main questions guide the research. First, is the theory of newsworthiness a valid approach for predicting news selection in a cultural context that is significantly different from western industrialized countries? Secondly, which are the relevant news factors that predict foreign news coverage in the Mexican press and thus shape the image of foreign nations? The results of a quantitative content analysis confirm the hypotheses of selection, additivity, and replication as they were originally postulated by Galtung and Ruge and thus prove the external validity of the theory of newsworthiness beyond western journalism.
Design eines Mobiltelefons für Senioren (Design of a Mobile Phone for Senior Citizens)
Authors: Baumann Konrad
Published: April 2006

Zu Beginn des Jahres 2005 können wir auf zehn Jahre Mobiltelefonie als Massenphänomen zurückblicken. Gleichzeitig gibt es immer noch zu wenige Mobiltelefone, die den Bedürfnissen der in der westlichen Welt anwachsenden Zielgruppe der Senioren entgegenkommen. Ein neuartiges Konzept von hoher Einfachheit führte zur Entwicklung eines Mobiltelefons bestehend aus Hardware, Software und Serviceangebot. Im Zuge des Designprojekts wurde eine Befragung der Zielgruppe mit interessanten Ergebnissen durchgeführt.
Opposite associations of age-dependent insulin-like growth factor-I standard deviation scores with nutritional state in normal weight and obese subjects
Authors: Schneider Harald Jörn , Saller Bernhard , Klotsche Jens , März Winfried , Erwa Wolfgang , Wittchen Hans-Ullrich , Stalla Günter Karl
Published: April 2006

Objective: Insulin-like growth factor-I (IGF-I) has been suggested to be a prognostic marker for the development of cancer and, more recently, cardiovascular disease. These diseases are closely linked to obesity, but reports of the association of IGF-I with measures of obesity are divergent. In this study, we assessed the association of age-dependent IGF-I standard deviation scores with body mass index (BMI) and intra-abdominal fat accumulation in a large population.

Design: A cross-sectional, epidemiological study.

Methods: IGF-I levels were measured with an automated chemiluminescence assay system in 6282 patients from the DETECT study. Weight, height, and waist and hip circumference were measured according to the written instructions. Standard deviation scores (SDS), correcting IGF-I levels for age, were calculated and were used for further analyses.

Results: An inverse U-shaped association of IGF-I SDS with BMI, waist circumference, and the ratio of waist circumference to height was found. BMI was positively associated with IGF-I SDS in normal weight subjects, and negatively associated in obese subjects. The highest mean IGF-I SDS were seen at a BMI of 22.5–25 kg/m2 in men (+0.08), and at a BMI of 27.5–30 kg/m2 in women (+0.21). Multiple linear regression models, controlling for different diseases, medications and risk conditions, revealed a significant negative association of BMI with IGF-I SDS. BMI contributed most to the additional explained variance to the other health conditions.

Conclusions: IGF-I standard deviation scores are decreased in obesity and underweight subjects. These interactions should be taken into account when analyzing the association of IGF-I with diseases and risk conditions.