bluuebunny/crossref_metadata_2025 huggingface dataset:
Cloning and Functional Analysis of a Family of Nuclear Matrix Transcription Factors (NP/NMP4) that Regulate Type I Collagen Expression in Osteoblasts
Authors: Thunyakitpisal Pasutha , Alvarez Marta , Tokunaga Kunihiko , Onyia Jude E. , Hock Janet , Ohashi Naoko , Feister Hilary , Rhodes Simon J. , Bidwell Joseph P.
Published: April 2006

Collagen expression is coupled to cell structure in connective tissue. We propose that nuclear matrix architectural transcription factors link cell shape with collagen promoter geometry and activity. We previously indicated that nuclear matrix proteins (NP/NMP4) interact with the rat type I collagen α1(I) polypeptide chain (COL1A1) promoter at two poly(dT) sequences (sites A and B) and bend the DNA. Here, our objective was to determine whether NP/NMP4-COL1A1 binding influences promoter activity and to clone NP/NMP4. Promoter-reporter constructs containing 3.5 kilobases (kb) of COL1A1 5′ flanking sequence were fused to a reporter gene. Mutation of site A or site B increased promoter activity in rat UMR-106 osteoblast-like cells. Several full-length complementary DNAs (cDNAs) were isolated from an expression library using site B as a probe. These clones expressed proteins with molecular weights and COL1A1 binding activity similar to NP/NMP4. Antibodies to these proteins disrupted native NP/NMP4-COL1A1 binding activity. Overexpression of specific clones in UMR-106 cells repressed COL1A1 promoter activity. The isolated cDNAs encode isoforms of Cys2His2 zinc finger proteins that contain an AT-hook, a motif found in architectural transcription factors. Some of these isoforms recently have been identified as Cas-interacting zinc finger proteins (CIZ) that localize to fibroblast focal adhesions and enhance metalloproteinase gene expression. We observed NP/NMP4/CIZ expression in osteocytes, osteoblasts, and chondrocytes in rat bone. We conclude that NP/NMP4/CIZ is a novel family of nuclear matrix transcription factors that may be part of a general mechanical pathway that couples cell structure and function during extracellular matrix remodeling.
Fracture Site-Specific Deficits in Bone Size and Volumetric Density in Men with Spine or Hip Fractures
Authors: Seeman Ego , Duan Yunbo , Fong Christopher , Edmonds Jan
Published: April 2006

To study the structural basis of bone fragility in men, we compared bone size and volumetric bone mineral density (vBMD) of the third lumbar vertebra and femoral neck in 95 men with spine fractures, 127 men with hip fractures, and 395 healthy controls using dual-energy X-ray absorptiometry (DXA). The results were expressed in absolute terms and age-specific SD scores (mean ± SEM). In controls, vertebral body and femoral neck width increased across age, being 0.46 ± 0.11 SD and 0.91 ± 0.08 SD higher in elderly men than in young men, respectively (both, p < 0.001). Men with spine fractures had reduced vertebral body width (−0.45 ± 0.10 SD; p < 0.01) but not femoral neck width (−0.15 ± 0.10 SD, NS). Men with hip fractures had reduced femoral neck width (−0.45 ± 0.11 SD; p < 0.01) and vertebral body width (−0.25 ± 0.10 SD; p < 0.05). The deficits in bone volume (BV) exaggerated the deficits in bone mineral content (BMC) by 40% at the vertebrae in men with spine fractures and by 9% at the femoral neck in men with hip fractures. vBMD deficits were greater at the vertebrae in men with spine fractures than in men with hip fractures (−1.37 ± 0.08 SD vs.−0.70 ± 0.10 SD, respectively; p < 0.01) but were similar at the femoral neck (−0.93 ± 0.10 SD and −0.76 ± 0.11 SD, respectively, NS), despite the men with spine fracture being 10 years younger. Bone fragility leading to spine or hip fractures in men may be the result of fracture site-specific deficits in bone size and vBMD that have their origins in growth, aging, or both.
Estrogen Receptor α Gene Polymorphisms and Bone Mineral Density: Haplotype Analysis in Women from the United Kingdom
Authors: Albagha Omar M. E. , McGuigan Fiona E. A. , Reid David M. , Ralston Stuart H.
Published: April 2006

Genetic factors are important in the pathogenesis of osteoporosis and the estrogen receptor has been suggested as a possible candidate gene for regulation of bone mineral density (BMD). We investigated the relationship between PvuII, XbaI, and dinucleotide (TA)n repeat polymorphisms of the estrogen receptor α (ER-α) gene and BMD in a study of women from northeast Scotland in the United Kingdom. No significant association was observed between BMD values at the lumbar spine (LS) and femoral neck (FN) in relation to PvuII and XbaI polymorphisms individually, but haplotype analysis showed that BMD values (Z score) were significantly lower in those who carried the Px haplotype (n = 36) compared with those who did not (n = 170) at both the LS (mean ± SEM; −0.775 ± 0.125 vs. −0.285 ± 0.082; p = 0.002) and the FN (−0.888 ± 0.130 vs. −0.335 ± 0.083; p = 0.0006). In keeping with this, the Px haplotype also was found to be an independent predictor of LS BMD (p = 0.019) and FN BMD (p = 0.005) in a multiple regression analysis model that included other possible predictors of BMD including age, years since menopause (YSM), hormone-replacement therapy (HRT) use, weight, and height. This model explained 15.7% and 23.4% of the total observed variance in LS and FN BMD, respectively, with the Px haplotype accounting for ∼3% of the variance at both sites. Although the TA repeat polymorphism was in strong linkage disequilibrium (LD) with the PvuII (χ2 = 109.8; p < 0.0001) and XbaI (χ2 = 97.2; p < 0.0001) polymorphisms, there was no overall association between TA repeat number and BMD. We conclude that polymorphisms of the ER-α gene are significantly related to BMD in our population and that this association is dependent on the Px haplotype, suggesting that it is the Px haplotype, or a linked polymorphism, that confers risk.
The Unique Tryptophan Residue of the Vitamin D Receptor Is Critical for Ligand Binding and Transcriptional Activation
Authors: Solomon Cynthia , Macoritto Michael , Gao Xiao-Ling , White John H. , Kremer Richard
Published: April 2006

The human vitamin D receptor (hVDR) is a member of the nuclear receptor superfamily of transcriptional regulators. Here we show that tryptophan 286 of the hVDR is critical for ligand binding and transactivation of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] target genes. Two mutants of the hVDR were produced, W286A and W286F, in which the tryptophan was replaced with an alanine or a phenylalanine, respectively. The W286A mutant did not bind 1,25(OH)2D3, interact with steroid receptor coactivator 1 (SRC-1) in vitro, or activate transcription. Moreover, the W286A receptor did not heterodimerize in a ligand-dependent manner with the human retinoid X receptor α (hRXRα). Although the W286F receptor heterodimerized with hRXRα, interacted with SRC-1, and bound 1,25(OH)2D3, its capacity to transactivate was attenuated severely. Thus, tryptophan 286 of hVDR plays an important role in specific 1,25(OH)2D3 ligand interaction and subsequently in hVDR/RXR interaction, SRC-1 binding, and ligand-dependent transactivation of 1,25(OH)2D3 target genes. These results identify the first amino acid that is absolutely required for ligand binding in the VDR and further define the structure-function relationship of 1,25(OH)2D3 interaction with its receptor.
25-Hydroxyvitamin D 1α-Hydroxylase: Structure of the Mouse Gene, Chromosomal Assignment, and Developmental Expression
Authors: Panda Dibyendu K. , Kawas Sausan Al , Seldin Michael F. , Hendy Geoffrey N. , Goltzman David
Published: April 2006

The murine homologue of the 25-hydroxyvitamin D [25(OH)D] 1α-hydroxylase gene [1α(OH)ase; Cyp27b1], which is mutated in humans with vitamin D-dependent rickets type I (VDDR-I; also known as pseudovitamin D-deficiency rickets [PDDR]) was cloned and characterized. Like the human, the mouse gene has nine exons, and the exon-intron organization is well conserved. By interspecific backcross analysis, the Cyp27b1 gene was mapped to 70.5 cM on mouse Chr 10. This is in a region syntenic with human Chr 12q13.1-q13.3 to which the human 1α(OH)ase gene was previously mapped. Kidney expression of the 1α(OH)ase was localized to cortical tubules and was higher in the adult mouse than in the fetus, consistent with the increased role of its product as a circulating hormone postnatally. Prenatally, the 1α(OH)ase gene, together with the vitamin D receptor (VDR) gene, was expressed in embryonic stem cells, and expression of 1α(OH)ase in bone and intestine was higher in the fetus than in the adult. These observations suggest that 1,25-dihydroxyvitamin D [1,25(OH)2D] plays a role in fetal development. In view of the fact that humans lacking 1α(OH)ase have apparently normal prenatal development, this may point to functional redundancy in the fetal vitamin D system, which now can be explored further in mouse models in which the 1α(OH)ase gene has been deleted.
High-Turnover Periprosthetic Bone Remodeling and Immature Bone Formation Around Loose Cemented Total Hip Joints
Authors: Takagi Michiaki , Santavirta Seppo , Ida Hideo , Ishii Masaji , Takei Isao , Niissalo Sirkku , Ogino Toshihiko , Konttinen Yrjö T.
Published: April 2006

Aseptic loosening and periprosthetic osteolysis are the major problems awaiting solution in total hip surgery. The clinical investigation focused on the analysis of periprosthetic bone remodeling to clarify one important key event in the cascade of periprosthetic connective tissue weakening and osteolysis around loose artificial hip joints. Twelve acetabular bone samples adjacent to granulomatous synovial-like membrane of loose hip prosthesis were retrieved at revision surgery and processed for Villanueva bone staining for morphological observation and bone histomorphometric analysis. Eight well-fixed bony samples were used as control. Although osteoclastic surface and eroded surface by osteoclasts were evident in the periprosthetic bone from loose hip joints (p = 0.003 and p = 0.027), increased osteoid/low-mineralized bone matrix (p < 0.001) and osteoid width (p < 0.001) also were significant findings in structural analysis. In addition, not only elevated mineral apposition rate (MAR; p = 0.044) but also increased mineralizing surface (p = 0.044) and bone formation rate (BFR; p = 0.002) in loose periprosthetic bones were shown in dynamic data analysis. These results were confirmed by precise morphological observation by confocal laser scanning microscopy. Active coupling of bone formation and resorption and increased osteocytes with abundant bone canalicular projections were found in combined with the presence of immature bone matrices (osteoid and low-mineralized bone areas) in periprosthetic bones from loose hip joints. These results indicated that active osteoclastic bone resorption and/or defective bone formation are coupled with monocyte/macrophage-mediated foreign body-type granuloma in the synovial-like interface membrane of loose hip joints. Thus, this unique high-turnover periprosthetic bone remodeling with bad bone quality probably is caused by the result of cellular host response combined with inappropriate cyclic mechanical loading. The fragile periprosthetic bone may contribute to hip prosthesis loosening.
Bone Morphogenetic Protein Regulation of Forkhead/Winged Helix Transcription Factor Foxc2 (Mfh1) in a Murine Mesodermal Cell Line C1 and in Skeletal Precursor Cells
Authors: Nifuji Akira , Miura Naoyuki , Kato Naoko , Kellermann Odile , Noda Masaki
Published: April 2006

Mfh1/Foxc2 is a member of forkhead/winged helix transcription factor family in which its members serve as key regulators in embryogenesis and cell differentiation in various species. Mutant mice null for Mfh1 show defects in axial and cranial skeletogenesis, suggesting requirement of Mfh1 for skeletal tissue development. However, the roles of Mfh1 and its regulation during early skeletogenesis have not been understood fully yet. In this study, we investigated developmental regulation of Mfh1 expression during embryonic skeletogenesis in vivo and in vitro chondrogenic cell differentiation using a mesodermal progenitor-like cell line C1. We first examined expression patterns of Mfh1 in relation to the cartilage phenotype-related molecules including bone morphogenetic proteins (BMPs) during mouse embryogenesis by in situ hybridization. In 10.5 days postcoitum (dpc) mouse limb, Mfh1 messenger RNA (mRNA) was expressed in the mesenchymal cells in the tissues that later give rise to skeleton. In 11.5 dpc embryos, Mfh1 transcripts were expressed in the cell condensation of skeletal blastemas. BMP2 transcripts were expressed in the cell condensation proximal to the Mfh1-expressing cells in the limbs and those of BMP-7 were expressed in the mesenchymal tissue surrounding the Mfh1-positive cell condensation. In 12.5 dpc and 13.5 dpc embryos, the expression of Mfh1 was localized to the perichondrium, which surrounds cells that express noggin and SOX9 mRNA. BMP-2 expression was overlapped with that of Mfh1 in the peripheral layer of 12.5 dpc and 13.5 dpc limb skeletal blastemas. Mfh1 expression persisted in the perichondrium of 15.5 dpc embryos though its level was reduced. We then examined the expression of Mfh1 in the mouse mesodermal cell line C1 that differentiates into chondrocytes in vitro. Mfh1 mRNA was expressed constitutively at low levels in C1 cells before the induction of its differentiation. On the differentiation of C1 cells into chondrocytes by the treatment with dexamethasone (Dex), Mfh1 expression was increased and peaked on day 4 of Dex treatment. Treatment with BMP-4/7 and BMP-7 protein also enhanced Mfh1 expression in C1 cells. To further examine the causative relationship between BMP and Mfh1 in mesenchymal tissue, we performed a mouse limb bud organ culture to implant BMP proteins with carriers into the mesenchymal tissue of the limb bud. Implantation of BMP-7 protein in the limb bud of 11.5 dpc embryos induced Mfh1 expression, suggesting that BMP regulates Mfh1 expression in limb mesenchyme. These results indicate that Mfh1 expression is associated with the early stage of chondrogenic differentiation both in vivo and in vitro and that BMPs regulate Mfh1 expression in skeletal precursor cells.
Osteoclast Inhibitory Peptide 2 Inhibits Osteoclast Formation via Its C-Terminal Fragment
Authors: Choi Sun Jin , Kurihara Noriyoshi , Oba Yasuo , Roodman G. David
Published: April 2006

Osteoclast inhibitory peptide 2 (OIP-2) is a novel autocrine/paracrine factor produced by osteoclasts (OCLs) that inhibits bone resorption and OCL formation in vitro and in vivo. It is identical to the asparaginyl endopeptidase legumain. During maturation of OIP-2, a signal peptide and a 17-kDa C-terminal fragment (CTF) are cleaved to produce the mature enzyme. To determine if enzyme activity is required for inhibition of OCL formation or if only the CTF is responsible for these effects, we synthesized His-tagged complementary DNA (cDNA) constructs for the CTF of OIP-2, the proform of OIP-2, and the “mature enzyme” form of OIP-2. The proform or the CTF portion of OIP-2 inhibited OCL formation in a dose-dependent manner in murine bone marrow cultures stimulated with 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. The mature form of OIP-2, which was enzymatically active, did not inhibit OCL formation. In addition, OIP-2 inhibited OCL formation in cultures of highly purified human OCL precursor cells or RAW264.7 cells stimulated with 10 ng/ml of receptor activator of NF-κB (RANK) ligand. Binding studies with His-tagged OIP-2 showed expression of a putative OIP-2 receptor on RAW264.7 cells treated with RANK ligand for 4 days and human marrow cultures treated with 1,25(OH)2D3 for 3 weeks. These data show that the CTF of OIP-2, rather than the mature enzyme, mediates the inhibitory effects of OIP-2 through a putative receptor on OCL precursors.
Effects of 2,3,7,8-Tetrachlorodibenzo-p-Dioxin on Bone in Two Rat Strains with Different Aryl Hydrocarbon Receptor Structures
Authors: Jämsä Timo , Viluksela Matti , Tuomisto Jouni T. , Tuomisto Jouko , Tuukkanen Juha
Published: April 2006

Polychlorinated dibenzo-p-dioxins (PCDDs) are highly toxic environmental contaminants, and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is the most potent dioxin. Here, we studied the effects of TCDD on bone. Two rat strains, Han/Wistar (H/W) and Long-Evans (L-E), were used because they exhibit a 1000-fold sensitivity difference in acute lethality of TCDD, which difference is related to the aryl hydrocarbon receptor (AHR). TCDD inhibited the tibial growth dose dependently, the effect being manifested at lower doses in the more sensitive L-E strain. In H/W rats the effect of TCDD was seen only at the high dose of 170 μg/kg (p < 0.05), whereas in the sensitive L-E rats a significant reduction of bone growth was already seen at 1.7 μg/kg (p < 0.01). This reduction was caused by the smaller tibial size because the diaphyseal bone mineral density (BMD) did not change. The three-point bending breaking force of the tibia was significantly reduced in H/W rats at 170 μg/kg (p < 0.05), but tibial stiffness was lower already at the dose of 17 μg/kg (p < 0.05). In the sensitive L-E strain, both breaking force and stiffness were reduced at the dose of 17 μg/kg (p < 0.001). These results indicate that TCDD dose-dependently interferes with bone growth, modeling, and mechanical strength. The altered transactivation domain of AHR is associated with a lower sensitivity of bone to TCDD in H/W rats, suggesting that AHR plays a role in modulating the effects of dioxins on bone.
Bone Brittleness Varies with Genetic Background in A/J and C57BL/6J Inbred Mice
Authors: Jepsen Karl J. , Pennington Douglas E. , Lee Ying-Lung , Warman Matthew , Nadeau Joseph
Published: April 2006

The contribution of genetic and environmental factors to variations in bone quality are understood poorly. We tested whether bone brittleness varies with genetic background using the A/J and C57BL/6J inbred mouse strains. Whole bone four-point bending tests revealed a 70% decrease in postyield deflection of A/J femurs compared with C57BL/6J, indicating that A/J femurs failed in a significantly more brittle manner. Cyclic loading studies indicated that A/J femurs accumulated damage differently than C57BL/6J femurs, consistent with their increased brittleness. Differences in matrix composition also were observed between the two mouse strains. A/J femurs had a 4.5% increase in ash content and an 11.8% decrease in collagen content. Interestingly, a reciprocal relationship was observed between femoral geometry and material stiffness; this relationship may have contributed to the brittle phenotype of A/J femurs. A/J femurs are more slender than those of C57BL/6J femurs; however, their 47% smaller moment of inertia appeared to be compensated by an increased tissue stiffness at the expense of altered tissue damageability. Importantly, these differences in whole bone mechanical properties between A/J and C57BL/6J femurs could not have been predicted from bone mass or density measures alone. The results indicated that bone brittleness is a genetically influenced trait and that it is associated with genetically determined differences in whole bone architecture, bone matrix composition, and mechanisms of cyclical damage accumulation.
Confirmation and Fine Mapping of Chromosomal Regions Influencing Peak Bone Mass in Mice
Authors: Klein Robert F. , Carlos Amy S. , Vartanian Kristina A. , Chambers Virginia K. , Turner Renn J. , Phillips Tamara J. , Belknap John K. , Orwoll Eric S.
Published: April 2006

Bone mineral density (BMD) is determined by both environmental influences and polygenic inheritance. The extreme difficulty of dissecting out environmental factors from genetic ones in humans has motivated the investigation of animal models. Previously, we used quantitative trait locus (QTL) analysis to examine peak BMD in 24 recombinant inbred (RI) mouse strains, derived from a cross between C57BL/6 (B6) and DBA/2 (D2) progenitors (RI-BXD). The distribution of BMD values among these strains indicated strong genetic influences and a number of chromosomal sites linked to BMD were identified provisionally. Using three additional independent mapping populations derived from the same progenitors, we have confirmed loci on chromosomes 1, 2, and 4, and 11 that contain genes that influence peak BMD. Using a novel fine-mapping approach (RI segregation testing [RIST]), we have substantially narrowed two of the BMD-related chromosomal regions and in the process eliminated a number of candidate genes. The homologous regions in the human genome for each of these murine QTLs have been identified in recent human genetic studies. In light of this, we believe that findings in mice should aid in the identification of specific candidate genes for study in humans.
Gender Specificity in the Genetic Determinants of Peak Bone Mass
Authors: Orwoll Eric S. , Belknap John K. , Klein Robert F.
Published: April 2006

Peak bone mass is a major determinant of osteoporotic fracture risk. Gender differences in peak bone mass acquisition are well recognized in humans and may account for a substantial share of the increased prevalence of fragility fractures in women compared with men. Skeletal development is regulated by both heritable and environmental factors. Experimental animal models provide a means to circumvent complicating environmental factors. In this study we examined the heritability of peak bone mineral density (BMD) in genetically distinct laboratory mouse strains raised under strict environmental control and sought to identify genetic loci that may contribute to gender differences in this skeletal phenotype. Peak whole body BMD of male and female mice from a panel of 18 recombinant inbred (RI) strains derived from a cross between C57BL/6 and DBA/2 progenitors (BXD) was measured by dual-energy X-ray absorptiometry (DXA). A highly significant relationship existed between body weight and BMD in the BXD RI mice (r2 = 0.25; p = 1 × 10−43). To allow for comparison between male and female RI strains, whole body BMD values were corrected for the influence of body weight. The distribution of weight-corrected BMD (WC-BMD) values among the strains indicated the presence of strong genetic influences in both genders, with an estimated narrow sense heritability of 45% and 22% in male and female mice, respectively. Comparison of RI strain results by two-way analysis of variance (ANOVA) revealed a significant strain-by-gender interaction (F1,17,479 = 6.13; p < 0.0001). Quantitative trait locus (QTL) analysis of the BXD RI strain series provisionally identified nine chromosomal sites linked to peak bone mass development in males and seven regions in females. In two cases, the provisional chromosomal loci were shared between genders, but in most cases they were distinct (five female-specific QTLs and six male-specific QTLs). QTL analysis of a genetically heterogeneous F2 population derived from the B6 and D2 progenitor strains provided additional support for the gender specificity of two loci. A significant phenotype-genotype correlation was only observed in male F2 mice at microsatellite marker D7Mit114 on chromosome 7, and a correlation at D2Mit94 on chromosome 2 was only observed in female F2 mice. The present data highlight the important role of gender in the genetic basis of peak bone mass in laboratory mice. Because the male phenotype is associated with considerable fracture risk reduction, an elucidation of the nature of that effect could provide the basis for novel diagnostic, preventative, or therapeutic approaches.
Differential Expression of Neuroleukin in Osseous Tissues and Its Involvement in Mineralization During Osteoblast Differentiation
Authors: Zhi Jizu , Sommerfeldt Dirk W. , Rubin Clinton T. , Hadjiargyrou Michael
Published: April 2006

Osteoblast differentiation is a multistep process that involves critical spatial and temporal regulation of cellular processes marked by the presence of a large number of differentially expressed molecules. To identify key functional molecules, we used differential messenger RNA (mRNA) display and compared RNA populations isolated from the defined transition phases (proliferation, matrix formation, and mineralization) of the MC3T3-E1 osteoblast-like cell line. Using this approach, a complementary DNA (cDNA) fragment was isolated and identified as neuroleukin (NLK), a multifunctional cytokine also known as autocrine motility factor (AMF), phosphoglucose isomerase (PGI; phosphohexose isomerase [PHI]), and maturation factor (MF). Northern analysis showed NLK temporal expression during MC3T3-E1 cell differentiation with a 3.5-fold increase during matrix formation and mineralization. Immunocytochemical studies revealed the presence of NLK in MC3T3-E1 cells as well as in the surrounding matrix, consistent with a secreted molecule. In contrast, the NLK receptor protein was detected primarily on the cell membrane. In subsequent studies, a high level of NLK expression was identified in osteoblasts and superficial articular chondrocytes in bone of 1-, 4-, and 8-month-old normal mice, as well as in fibroblasts, proliferating chondrocytes, and osteoblasts within a fracture callus. However, NLK was not evident in hypertrophic chondrocytes or osteocytes. In addition, treatment of MC3T3 cells with 6-phosphogluconic acid (6PGA; a NLK inhibitor) resulted in diminishing alkaline phosphatase (ALP) activity and mineralization in MC3T3-E1 cells, especially during the matrix formation stage of differentiating cells. Taken together, these data show specific expression of NLK in discrete populations of bone and cartilage cells and suggest a possible role for this secreted protein in bone development and regeneration.
Expression of Matrix Metalloproteinases During Ascorbate-Induced Differentiation of Osteoblastic MC3T3-E1 Cells
Authors: Mizutani Akifumi , Sugiyama Ikuko , Kuno Eriko , Matsunaga Sachiko , Tsukagoshi Norihiro
Published: April 2006

The mouse calvarial osteoblast MC3T3-E1 cells released 92 kDa and 68 kDa of gelatinase activities into the conditioned media (CMs) from undifferentiated cells. When differentiation was induced by cultivating cells with ascorbate-2-phosphate (AscP), 68-kDa activity increased significantly in parallel with production of 60-kDa activity. These enzymes required Ca2+ and Zn2+ ions for their proteolytic activities. The 68-kDa activity was immunologically identified as latent matrix metalloproteinase 2 (MMP-2). The 92-kDa activity was deduced to be latent MMP-9 based on its molecular mass. The 60-kDa activity band was found to possess both gelatin and β-casein hydrolyzing activities, indicating that this activity band might comprise the active form of MMP-2 and latent MMP-13. MC3T3-E1 cells were found to express MMP-2, MMP-13, and membrane type (MT)1-MMP genes by Northern blotting. MMP-2 was expressed constitutively. MMP-13 was up-regulated during the growth with AscP. MT1-MMP expression also was modulated by AscP; at the early stage of differentiation, its messenger RNA (mRNA) level increased and then decreased gradually to the control level. These changes in the profiles of MMPs observed here could be attributed to the maturation of collagenous extracellular matrix (ECM) induced by AscP.
Midregion Parathyroid Hormone-Related Protein Inhibits Growth and Invasion In Vitro and Tumorigenesis In Vivo of Human Breast Cancer Cells
Authors: Luparello Claudio , Romanotto Rita , Tipa Annalisa , Sirchia Rosalia , Olmo Nieves , López de Silanes Isabel , Turnay Javier , Lizarbe M. Antonia , Stewart Andrew F.
Published: April 2006

Parathyroid hormone-related protein (PTHrP) is critical for normal mammary development and is overexpressed by breast cancers. PTHrP is a peptide hormone that undergoes extensive post-translational processing, and PTHrP(38–94)-amide is one of the mature secretory forms of the peptide. In this study, we explored the effect of PTHrP(38–94)-amide in a panel of six breast cancer cell lines “in vitro” and in MDA-MB231 cells “in vivo” specifically examining cell viability, proliferation, invasiveness, and growth in nude mice. PTHrP(38–94)-amide markedly inhibited proliferation and also caused striking toxicity and accelerated cell death in breast cancer cells. In addition, direct injection of PTHrP(38–94)-amide into MDA-MB231 breast cancer cells passaged in immunodeficient mice produced a marked reduction in tumor growth. These studies (i) indicate breast cancer cells are one of the few tissues in which specific effects of midregion PTHrP have been established to date, (ii) support a role for midregion secretory forms of PTHrP in modulating not only normal but also pathological mammary growth and differentiation, (iii) add further evidence for the existence of a specific midregion PTHrP receptor, and (iv) provide a novel molecule for modeling of small molecule analogues that may have anti-breast cancer effects.
Preonset Studies of Spondyloepiphyseal Dysplasia Tarda Caused by a Novel 2-Base Pair Deletion in SEDL Encoding Sedlin
Authors: Mumm Steven , Zhang Xiafang , Gottesman Gary S. , McAlister William H. , Whyte Michael P.
Published: April 2006

Spondyloepiphyseal dysplasia tarda (SEDT), an X-linked recessive skeletal disorder, presents with disproportionate short stature and “barrel-chest” deformity in affected (hemizygous) adolescent boys. In four reported families to date, mutations in a gene designated SEDL (spondyloepiphyseal dysplasia late) cosegregate with SEDT. We diagnosed SEDT in a short-stature, kyphotic 15-year-old boy because of his characteristic vertebral malformations. Clinical manifestations of SEDT were evident in at least four previous generations. A novel 2-base pair (bp) deletion in exon 5 of SEDL was found in the propositus by polymerase chain reaction (PCR) amplification and sequencing of all four coding exons. The mutation ATdel241-242 cosegregated with the kindred's skeletal disease. The deletion is adjacent to a noncanonical splice site for exon 5 but does not alter splicing. Instead, it deletes 2 bp from the coding sequence, causing a frameshift. A maternal aunt and her three young sons were investigated subsequently. Radiographs showed subtle shaping abnormalities of her pelvis and knees, suggesting heterozygosity. X-rays of the spine and pelvis of her 8-year-old son revealed characteristic changes of SEDT, but her younger sons (aged 6 years and 3 years) showed no abnormalities. SEDL analysis confirmed that she and only her eldest boy had the 2-bp deletion. Molecular testing of SEDL enables carrier detection and definitive diagnosis before clinical or radiographic expression of SEDT. Although there is no specific treatment for SEDT, preexpression molecular testing of SEDL could be helpful if avoiding physical activities potentially injurious to the spine and the joints proves beneficial.
Growth Hormone Is Permissive for Skeletal Adaptation to Mechanical Loading
Authors: Forwood Mark R. , Li Li , Kelly Wendy L. , Bennett Michael B.
Published: April 2006

The Lewis dwarf (DW) rat was used as a model to test the hypothesis that growth hormone (GH) is permissive for new bone formation induced by mechanical loading in vivo. Adult female Lewis DW rats aged 6.2 ± 0.1 months (187 ± 18 g) were allocated to four vehicle groups (DW), four GH treatment groups at 32.5 μg/100 g body mass (DWGH1), and four GH treatment groups at 65 μg/100 g (DWGH2). Saline vehicle or GH was injected intraperitoneally (ip) at 6:30 p.m. and 6:30 a.m. before mechanical loading of tibias at 7:30 a.m. A single period of 300 cycles of four-point bending was applied to right tibias at 2.0 Hz, and magnitudes of 24, 29, 38, or 48N were applied. Separate strain gauge analyses in 5 DW rats validated the selection of loading magnitudes. After loading, double-label histomorphometry was used to assess bone formation at the periosteal surface (Ps.S) and endocortical surface (Ec.S) of tibias. Comparing left (unloaded) tibias among groups, GH treatment had no effect on bone formation. Bone formation in tibias in DW rats was insensitive to mechanical loading. At the Ec.S, mechanically induced lamellar bone formation increased in the DWGH2 group loaded at 48N (p < 0.05), and no significant increases in bone formation were observed among other groups. The percentage of tibias expressing woven bone formation (Wo.B) at the Ps.S was significantly greater in the DWGH groups compared with controls (p < 0.05). We concluded that GH influences loading-related bone formation in a permissive manner and modulates the responsiveness of bone tissue to mechanical stimuli by changing thresholds for bone formation.
Total Body Bone Mineral Content and Tibial Cortical Bone Measures in Preschool Children
Authors: Specker Bonny L. , Johannsen Neil , Binkley Teresa , Finn Kevin
Published: April 2006

This study was undertaken to identify factors that influence total body bone area (TBBA), total body bone mineral content (TBBMC), and tibial cortical bone measures in 239 children aged 3–5 years. We obtained information on demographic and anthropometric characteristics and measurements of diet, physical activity, and strength. In multiple regression analysis, TBBA correlated with height (p < 0.001), weight (p < 0.001), percent body fat (p < 0.001), and calcium intake (p = 0.02). TBBMC correlated with TBBA (p < 0.001), age (p = 0.001), and weight (p = 0.02) and inversely correlated with height (p < 0.001) and percent body fat (p < 0.001). Children born preterm had lower TBBMC compared with children born at term (p = 0.02). Both periosteal and endosteal circumferences were correlated with weight (both, p < 0.001) and inversely correlated with age (p = 0.006 and p = 0.003, respectively) and percent body fat (p = 0.002 and p = 0.005 respectively). Endosteal circumference was greater and cortical bone area was lower in children born preterm compared with those born at term (both, p = 0.04). Findings of higher TBBA and lower TBBMC in children with high percent body fat indicate undermineralization of bone and suggest that obesity in preschool children may have detrimental effects on total body bone mass accretion. A smaller tibial periosteal circumference and thus cross-sectional area in children with the same weight but higher percent body fat also would lead to a biomechanical disadvantage in these children. Findings of low TBBMC and cortical bone area among children born preterm need to be confirmed in other populations. We speculate that differences in these measurements between children born preterm and at term may be caused by differences in activity.
Variation in Bone Biomechanical Properties, Microstructure, and Density in BXH Recombinant Inbred Mice
Authors: Turner Charles H. , Hsieh Yeou-Fang , Müller Ralph , Bouxsein Mary L. , Rosen Clifford J. , McCrann Margaret E. , Donahue Leah Rae , Beamer Wesley G.
Published: April 2006

To test the hypothesis that factors associated with bone strength (i.e., volumetric bone mineral density [vBMD], geometry, and microstructure) have heritable components, we exploited the 12 BXH recombinant inbred (RI) strains of mice derived from C57BL/6J (B6; low bone mass) and C3H/HeJ (C3H; high bone mass) progenitor strains. The femurs and lumbar vertebrae from each BXH RI strain were characterized for phenotypes of vBMD, microstructural, biomechanical, and geometrical properties. Methods included bending (femur) and compression (vertebra) testing, peripheral quantitative computed tomography (pQCT), and microcomputed tomography (μCT). Segregation patterns of femoral and vertebral biomechanical properties among the BXH RI strains suggested polygenic regulation. Femoral biomechanical properties were strongly associated with femoral width in the anteroposterior (AP) direction and cortical thickness-geometric properties with complex genetic regulation. Vertebral vBMD and biomechanical properties measured in BXH RI strains showed a greater variability than either B6 or C3H progenitors, suggesting both progenitor strains have independent subsets of genes that yield similar vBMD and strength. The μCT and pQCT data suggested that the distribution of vertebral mineral into cortical and trabecular compartments is regulated genetically. Although the B6 and C3H progenitors had similar vertebral strength, their vertebral structures were markedly different: B6 had good trabecular bone structure and modest cortical bone mineral content (BMC), whereas C3H had high cortical BMC combined with a deficiency in trabecular structure. These structural traits segregated independently in the BXH RI strains. Finally, vertebral strength was not correlated consistently with femoral strength among the BXH RI strains, suggesting genetic regulation of bone strength is site specific.
Expression of Functional Gap Junctions and Regulation by Fluid Flow in Osteocyte-Like MLO-Y4 Cells
Authors: Cheng Benxu , Zhao Shujie , Luo Jian , Sprague Eugene , Bonewald Lynda F. , Jiang Jean X.
Published: April 2006

Osteocytes are thought to be mechanosensory cells that respond to mechanical stress by sending signals to other bone cells to initiate bone remodeling. An osteocyte-like cell line MLO-Y4 provides a model system to examine whether gap junctions participate in the regulation of osteocyte function and signaling by mechanical stress. In this study, we show that MLO-Y4 cells are coupled and that gap junction channels mediate this coupling. Biochemical analyses show that connexin 43 (Cx43) is a major gap junction protein expressed in MLO-Y4 cells and approximately 5% of Cx43 protein is phosphorylated. MLO-Y4 cells were exposed to mechanical stress using a parallel plate flow chamber to model bone fluid flow shear stress. Fluid flow increased significantly the length of the dendritic processes, a morphological characteristic of osteocytes. A redistribution of the gap junction protein, Cx43 also was observed from a location circling the nucleus to punctate spots in the cytoplasm and in the dendritic processes. “Scrape-loading” dye transfer analyses showed that fluid flow increased intercellular coupling and increased the number of cells coupled immediately after fluid flow treatment, in direct proportion to shear stress magnitude. Although intercellular coupling continued to increase, stimulation of Cx43 protein expression during the poststress period was found to be biphasic. Cx43 protein was elevated 30 minutes after application of stress but decreased at 24 h poststress. Pulsating fluid flow had a similar stimulatory effect as steady fluid flow on gap junctions. However, this stimulatory effect in osteocyte-like cells was not observed in osteoblast-like 2T3 cells. Together, these results show that fluid flow has stimulatory effects on osteocyte-like MLO-Y4 cells with early effects on cellular morphology, opening of gap junctions, and redistribution of Cx43 protein and delayed effects on Cx43 protein expression. The high expression of Cx43 and its location in the cytoplasm also suggest that Cx43 may have unknown functions in addition to forming gap junctions. These studies indicate that gap junctions may serve as channels for signals generated by osteocytes in response to mechanical loading.
Indapamide, a Thiazide-Like Diuretic, Decreases Bone Resorption In Vitro
Authors: Lalande Agnes , Roux Sophie , Denne Marie-Annick , Stanley E. Richard , Schiavi Pierre , Guez David , De Vernejoul Marie-Christine
Published: April 2006

We recently showed that indapamide (IDP), a thiazide-related diuretic, increases bone mass and decreases bone resorption in spontaneously hypertensive rats supplemented with sodium. In the present study, we evaluated the in vitro effects of this diuretic on bone cells, as well as those of hydrochlorothiazide (HCTZ), the reference thiazide, and acetazolamide (AZ), a carbonic anhydrase (CA) inhibitor. We showed that 10−4 M IDP and 10−4 M AZ, as well as 10−5 M pamidronate (APD), decreased bone resorption in organ cultures and in cocultures of osteoblast-like cells and bone marrow cells in the presence of 10−8 M 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. We investigated the mechanism of this antiresorptive effect of IDP; IDP decreased osteoclast differentiation as the number of osteoclasts developing in coculture of marrow and osteoblast-like cells was decreased markedly. We then investigated whether IDP affected osteoblast-like cells because these cells are involved in the osteoclast differentiation. Indeed, IDP increased osteoblast-like cell proliferation and alkaline phosphatase (ALP) expression. Nevertheless, it did not modify the colony-stimulating factor 1 (CSF-1) production by these cells. In addition, osteoblast-like cells expressed the Na+/Cl− cotransporter that is necessary for the renal action of thiazide diuretics, but IDP inhibited bone resorption in mice lacking this cotransporter, so the inhibition of bone resorption and osteoclast differentiation did not involve this pathway. Thus, we hypothesized that IDP may act directly on cells of the osteoclast lineage. We observed that resorption pits produced by spleen cells cultured in the presence of soluble osteoclast differentiation factor (sODF) and CSF-1 were decreased by 10−4 M IDP as well as 10−5 M APD. In conclusion, in vitro IDP increased osteoblast proliferation and decreased bone resorption at least in part by decreasing osteoclast differentiation via a direct effect on hematopoietic precursors.
Stimulation of Protein Kinase C Activity in Cells Expressing Human Parathyroid Hormone Receptors by C- and N-Terminally Truncated Fragments of Parathyroid Hormone 1–34
Authors: Whitfield J. F. , Isaacs R. J. , Chakravarthy B. , Maclean S. , Morley P. , Willick G. , Divieti P. , Bringhurst F. R.
Published: April 2006

The parathyroid hormone (PTH) fragment PTH(1–34) stimulates adenylyl cyclase, phospholipase C (PLC), and protein kinase C's (PKCs) in cells that express human, opossum, or rodent type 1 PTH/PTH-related protein (PTHrP) receptors (PTHR1s). Certain carboxyl (C)-terminally truncated fragments of PTH(1–34), such as human PTH(1–31) [hPTH-(1–31)NH2], stimulate adenylyl cyclase but not PKCs in rat osteoblasts or PLC and PKCs in mouse kidney cells. The hPTH(1–31)NH2 peptide does fully stimulate PLC in HKRK B7 porcine renal epithelial cells that express 950,000 transfected hPTHR1s per cell. Amino (N)-terminally truncated fragments, such as bovine PTH(3–34) [bPTH(3–34)], hPTH(3–34)NH2, and hPTH(13–34), stimulate PKCs in Chinese hamster ovary (CHO) cells expressing transfected rat receptors, opossum kidney cells, and rat osteoblasts, but an intact N terminus is needed to stimulate PLC via human PTHR1s in HKRK B7 cells. We now report that the N-terminally truncated analogs bPTH(3–34)NH2 and hPTH(13–34)OH do activate PKC via human PTHR1s in HKRK B7 cells, although less effectively than hPTH(1–34)NH2 and hPTH(1–31)NH2. Moreover, in a homologous human cell system (normal foreskin fibroblasts), these N-terminally truncated fragments stimulate PKC activity as strongly as hPTH(1–34)NH2 and hPTH(1–31)NH2. Thus, it appears that unlike their opossum and rodent equivalents, hPTHR1s can stimulate both PLC and PKCs when activated by C-terminally truncated fragments of PTH(1–34). Furthermore, hPTHR1s, like the PTHR1s in rat osteoblasts, opossum kidney cells, and rat PTHR1-transfected CHO cells also can stimulate PKC activity by a mechanism that is independent of PLC. The efficiency with which the N-terminally truncated PTH peptides stimulate PKC activity depends on the cellular context in which the PTHR1s are expressed.
Down-Regulation of Procollagen α1[I] Messenger RNA by Titanium Particles Correlates with Nuclear Factor κB (NF-κB) Activation and Increased Rel A and NF-κB1 Binding to the Collagen Promoter
Authors: Roebuck Kenneth A. , Vermes Csaba , Carpenter Laura R. , Fritz Elizabeth A. , Narayanan Ramesh , Glant Tibor T.
Published: April 2006

Previously, we showed that exposure of human osteoblasts to titanium particles stimulates protein tyrosine phosphorylation (PTP), activates the transcription factor nuclear factor κB (NF-κB), and causes an approximately 50% decrease in the steady-state messenger RNA (mRNA) level of procollagen α1[I]. In this study, we identify three NF-κB binding sites within the human procollagen α1[I] gene promoter, show that titanium particles stimulate their binding of the NF-κB subunits Rel A (p65) and NF-κB1 (p50), and find NF-κB activation correlates with collagen gene suppression by titanium particles in osteoblasts. Protein tyrosine kinase (PTK) inhibitors, which significantly reduce the suppressive effect of titanium particles on collagen gene expression, inhibited NF-κB binding activity showing that titanium particle stimulation of PTK signals in osteoblasts are critical for both NF-κB activation and collagen gene expression. The antioxidant pyrrolidine dithiocarbamate (PDTC), which also inhibits the titanium particle suppression of collagen, abrogated the titanium particle activation of NF-κB, suggesting the involvement of redox signals in NF-κB-mediated collagen gene expression. The RNA polymerase II inhibitor actinomycin D (Act D) decreased procollagen α1[I] mRNA expression and effectively blocked the titanium-induced suppressive effect, suggesting that titanium particles activate a cascade of signals in osteoblasts, which result in a suppression of procollagen α1[I] mRNA. Collectively, these results show that titanium particles can activate NF-κB signaling in osteoblasts and suggest that NF-κB binding to the collagen gene promoter has a functional role in the down-regulation of procollagen α1[I] gene transcription.
1α-Hydroxyvitamin D2 Is Less Toxic but Not Bone Selective Relative to 1α-Hydroxyvitamin D3 in Ovariectomized Rats
Authors: Weber Karin , Goldberg Michel , Stangassinger Manfred , Erben Reinhold G.
Published: April 2006

Identification of bone selective vitamin D analogues would provide an interesting substance class for the treatment of osteoporosis. The synthetic prodrug 1α-hydroxyvitamin D2 [1α(OH)D2] has been shown to combine equal bone-preserving activity with distinctly reduced calcemic effects relative to 1α-hydroxyvitamin D3 [1α(OH)D3] in 3-month-old ovariectomized (OVX) rats. Therefore, 1α(OH)D2 may be a bone-selective compound. The aim of this study was to compare the bone protective and the calcemic activities of chronically administered 1α(OH)D2 and 1α(OH)D3 in 6-month-old OVX rats over a broad dose range from ineffective to toxic doses. Ninety-six female 6-month-old Fischer-344 rats were used for this experiment. Eighty rats were bilaterally OVX, 8 rats were sham-operated (SHAM), and 8 rats were killed at the time of surgery as a baseline control. Groups of OVX rats received vehicle alone (n = 16) or daily doses in the diet of 0.025, 0.05, 0.1, and 0.2 μg of 1α(OH)D2 or 1α(OH)D3 per kg body weight (BW) per day (n = 8 each). After calcein double-labeling, all animals were killed 3 months post-OVX. Orally administered 1α(OH)D2 was significantly less toxic compared with 1α(OH)D3 in terms of BW gain and kidney calcium content. The effects of 1α(OH)D2 and 1α(OH)D3 on serum calcium and urinary calcium excretion were generally similar at all doses in this study. Both 1α(OH)D2 and 1α(OH)D3 prevented the estrogen deficiency-induced bone loss in OVX rats, and induced profound bone anabolic effects at high dosages. 1α(OH)D3 and 1α(OH)D2 also dose-dependently increased total bone mineral density (BMD), cortical area, and cortical thickness in the tibial diaphysis of OVX rats. Bone resorption as assessed by osteoclast numbers (Oc.Ns) in vertebral cancellous bone and urinary excretion of deoxypyridinoline (DPD) was dose-dependently suppressed by 1α(OH)D2 and 1α(OH)D3. These data show that although 1α(OH)D2 was slightly but significantly less toxic compared with 1α(OH)D3, it did not have increased skeletal effects at any dose. Taken together, our findings argue against selective metabolic activation of 1α(OH)D2 in bone.