bluuebunny/crossref_metadata_2025 huggingface dataset:
Progressive Osseous Heteroplasia
Authors: Kaplan Frederick S. , Shore Eileen M.
Published: April 2006

Progressive osseous heteroplasia (POH) is a recently described genetic disorder of mesenchymal differentiation characterized by dermal ossification during infancy and progressive heterotopic ossification of cutaneous, subcutaneous, and deep connective tissues during childhood. The disorder can be distinguished from fibrodysplasia ossificans progressiva (FOP) by the presence of cutaneous ossification, the absence of congenital malformations of the skeleton, the absence of inflammatory tumorlike swellings, the asymmetric mosaic distribution of lesions, the absence of predictable regional patterns of heterotopic ossification, and the predominance of intramembranous rather than endochondral ossification. POH can be distinguished from Albright hereditary osteodystrophy (AHO) by the progression of heterotopic ossification from skin and subcutaneous tissue into skeletal muscle, the presence of normal endocrine function, and the absence of a distinctive habitus associated with AHO. Although the genetic basis of POH is unknown, inactivating mutations of the GNAS1 gene are associated with AHO. The report in this issue of the JBMR of 2 patients with combined features of POH and AHO—one with classic AHO, severe POH-like features, and reduced levels of Gsα protein and one with mild AHO, severe POH-like features, reduced levels of Gsα protein, and a mutation in GNAS1—suggests that classic POH also could be caused by GNAS1 mutations. This possibility is further supported by the identification of a patient with atypical but severe platelike osteoma cutis (POC) and a mutation in GNAS1, indicating that inactivating mutations in GNAS1 may lead to severe progressive heterotopic ossification of skeletal muscle and deep connective tissue independently of AHO characteristics. These observations suggest that POH may lie at one end of a clinical spectrum of ossification disorders mediated by abnormalities in GNAS1 expression and impaired activation of adenylyl cyclase. Analysis of patients with classic POH (with no AHO features) is necessary to determine whether the molecular basis of POH is caused by inactivating mutations in the GNAS1 gene.
Parathyroid Hormone-Related Peptide Stimulates Proliferation of Highly Tumorigenic Human SV40-Immortalized Breast Epithelial Cells
Authors: Cataisson Christophe , Lieberherr Michele , Cros Magali , Gauville Christiane , Graulet Anne-Marie , Cotton Joel , Calvo Fabien , de Vernejoul Marie-Christine , Foley John , Bouizar Zhor
Published: April 2006

Parathyroid hormone-related protein (PTHrP) is the main mediator of humoral hypercalcemia of malignancy (HHM) and it is produced by many tumors, including breast cancers. Breast epithelial cells as well as breast cancer tumors and cell lines have been reported as expressing PTHrP and the PTH/PTHrP receptor, suggesting that PTHrP may act as an autocrine factor influencing proliferation or differentiation of these cell types. We investigated PTHrP gene expression, PTH/PTHrP receptor signaling, and PTHrP-induced mitogenesis in three immortalized human mammary epithelial cell lines that exhibit differential tumorigenicity. The most tumorigenic cells expressed the highest levels of PTHrP messenger RNA (mRNA) and protein. We used reverse-transcription polymerase chain reaction (RT-PCR) and immunoblotting to detect the PTH/PTHrP receptor transcripts and proteins in all of the three cell lines. Treatment with human PTHrP(1-34) [hPTHrP(1-34)] and hPTH(1-34) increased intracellular cyclic adenosine monophosphate (cAMP) but not free Ca2+ in the nontumorigenic line. These agonists increased both cAMP and free Ca2+ levels in the moderately tumorigenic line, but only increased free Ca2+ in the highly tumorigenic line. Application of the PTH/PTHrP receptor antagonist [Asn10,Leu11,D Trp12]PTHrP(7-34) or PTHrP antibodies reduced [3H]thymidine incorporation in a dose-dependent fashion in the highly tumorigenic cell line but did not affect the other lines. Thus, treatment with a PTH/PTHrP receptor antagonist reduced cell proliferation, suggesting that PTHrP signaling mediated by the phospholipase C (PLC) pathway stimulates proliferation of a highly tumorigenic immortalized breast epithelial cell line.
Mechanical Strain Stimulates Osteoblast Proliferation Through the Estrogen Receptor in Males as Well as Females
Authors: Damien E. , Price J. S. , Lanyon L. E.
Published: April 2006

Mechanical strain, testosterone, and estrogen all stimulate proliferation of primary cultures of male rat long bone (LOB)-derived osteoblast-like cells as determined by [3H]thymidine incorporation. The maximum proliferative effect of a single period of mechanical strain (3400 με, 1 Hz, and 600 cycles) is additional to that of testosterone (10−8 M) or estrogen (10−8 M). The cells' proliferative response to strain is abolished both by concentrations of tamoxifen that cause proliferation (10−8 M) and by those that have no effect (10−6 M). Strain-related proliferation also is reduced by the estrogen antagonist ICI 182,780 (10−8 M) but is unaffected by the androgen receptor antagonist hydroxyflutamide (10−7 M). Tamoxifen, ICI 182,780, and the aromatase inhibitor 4-dihydroandrostenedione, at concentrations that have no effect on basal proliferation, significantly reduce the proliferative effect of the aromatizable androgen testosterone but not that of the nonaromatizable androgen 5α-dihydrotestosterone. Hydroxyflutamide, at a concentration that has no effect on basal proliferation (10−7 M), eliminates the proliferative effect of 5α-dihydro-testosterone but had no significant effect on that caused by testosterone. Proliferation associated with strain is blocked by neutralizing antibody to insulin-like growth factor II (IGF-II) but not by antibody to IGF-I. Proliferation associated with testosterone is blocked by neutralizing antibody to IGF-I but is unaffected by antibody to IGF-II. These data suggest that in rat osteoblast-like cells from males, as from females, strain-related proliferation is mediated through the estrogen receptor (ER) in a manner that does not compete with estrogen but that can be blocked by ER modulators. Proliferation associated with testosterone appears to follow its aromatization to estrogen and is mediated through the ER, whereas proliferation associated with 5α-dihydrotestosterone is mediated by the androgen receptor. Strain-related proliferation in males, as in females, is mediated by IGF-II, whereas proliferation associated with estrogen and testosterone is mediated by IGF-I.
Cloning, Sequencing, and Functional Characterization of the Rat Homologue of Receptor Activator of NF-κB Ligand
Authors: Xu Jiake , Tan Jamie Wy , Huang Lin , Gao Xiu-Hui , Laird Rebecca , Liu Dan , Wysocki Stan , Zheng Ming H.
Published: April 2006

A complementary DNA (cDNA) encoding the rat homologue of receptor activator of NF-κB ligand/osteoprotegerin ligand/osteoclast differentiation factor/tumor necrosis factor (TNF)-related activation-induced cytokine (RANKL/OPGL/ODF/TRANCE) was cloned and sequenced from tibias of ovariectomized (OVX) rats. The predicted amino acid sequence of rat RANKL (rRANKL) has 84% and 96% identity to that of human and mouse RANKL, respectively, and 35% and 37% similarity to that of human and mouse TNF-related apoptosis-inducing ligand (TRAIL), respectively. RANKL transcripts were expressed abundantly in the thymus and bone tissues of OVX rats. rRANKL has a single hydrophobic region between residues 53 and 69, which is most likely to serve as a transmembrane domain. The long C-terminal region containing β-sheet-forming sequences of the TNF-like core is considered the extracellular region. Three truncated domains within the TNF-like core region were expressed as glutathione S-transferase (GST) fusion proteins and investigated for their ability to induce osteoclastogenesis. The results showed that GST-rRANKL (aa160-318) containing the full TNF-like core region had the highest capability to induce the formation of osteoclast-like cells from RAW264.7 cells. GST-rRANKL (aa239-318 and aa160-268) had lesser degrees of osteoclast inductivity. Furthermore, the GST-rRANKL (aa160-318) is capable of (1) inducing osteoclast formation from rat spleen cells in the presence of macrophage colony-stimulating factor (M-CSF), (2) stimulating mature rat osteoclast polarization and bone resorption ex vivo, and (3) inducing systemic hypercalcemia in vivo; thus the full TNF-like core region of rRANKL is an important regulator of calcium homeostasis and osteoclastic function.
Protein Kinase A Regulates Chondrogenesis of Mesenchymal Cells at the Post-Precartilage Condensation Stage via Protein Kinase C-α Signaling
Authors: Yoon Young-Mee , Oh Chun-Do , Kang Shin-Sung , Chun Jang-Soo
Published: April 2006

Chondrogenesis of mesenchymal cells during in vitro micromass culture requires the generation of cyclic adenosine monophosphate (cAMP) and subsequent activation of cAMP-dependent protein kinase A (PKA). In this study, we investigated the regulatory activity of PKA during chondrogenesis of chick limb bud mesenchymal cells. PKA activity was high in 1-day and 2-day cultures, which was followed by a slight decrease in 4-day and 5-day old cultures. Inhibition of PKA blocked chondrogenesis. It did not affect precartilage condensation, but it blocked the progression from the precartilage condensation stage to cartilage nodule formation. The PKA inhibition-induced blockage of chondrogenesis was accompanied by an altered expression of N-cadherin. Although expression of N-cadherin was detected during the early period of chondrogenesis, it became reduced as chondrogenesis proceeded. Still, inhibition of PKA maintained expression of N-cadherin throughout the micromass culture period. The inhibition of PKA did not affect expression of protein kinase C-α (PKCα), PKCϵ, PKCζ, and PKCλ/ι, which are the isoforms expressed in differentiating mesenchymal cells. However, PKA inhibition completely blocked activation of PKCα. Because PKC activity regulates N-cadherin expression and chondrogenesis, the PKA-mediated regulation of PKCα appears to be responsible for the PKA regulation of N-cadherin expression and chondrogenesis. Taken together, our results suggest that PKA regulates chondrogenesis by activating PKCα at the stage of post-precartilage condensation.
Expansile Skeletal Hyperphosphatasia: A New Familial Metabolic Bone Disease
Authors: Whyte Michael P. , Mills Barbara G. , Reinus William R. , Podgornik Michelle N. , Roodman G. David , Gannon Francis H. , Eddy Mark C. , Mcalister William H.
Published: April 2006

We describe a new familial metabolic bone disease characterized by expanding hyperostotic long bones, early onset deafness, premature tooth loss, and episodic hypercalcemia. The condition affects a mother and daughter studied at the age of 36 years and 11 years, respectively. Both individuals lost all hearing in early childhood and suffered premature shedding of teeth. Skeletal pains began just before puberty. Swelling and aching of most middle phalanges in the hands is an especially troublesome manifestation. The mother also had episodes of symptomatic hypercalcemia first documented in late childhood and subsequently during intercurrent illness and postpartum lactation. Radiographs show hyperostosis and/or osteosclerosis predominantly in the skull and appendicular skeleton. Long bones also are expanded considerably, especially the middle phalanges in the fingers. The mother's skeletal abnormalities are more severe. Biochemical parameters of bone turnover, including serum alkaline phosphatase (ALP) activity, are elevated substantially. In the proposita, dynamic histomorphometry of nondecalcified sections of iliac crest revealed rapid skeletal remodeling. In the mother, who had been treated with bisphosphonates, electron microscopy (EM) showed disorganized collagen bundles as well as necrotic and apoptotic bone cells but no osteocytic osteolysis. Measles virus gene transcripts were not detected in peripheral blood monocytes. Karyotyping was normal, 46,XX. Hyperphosphatasia with bone disease previously has been reported as either a sporadic or autosomal recessive condition. Expansile skeletal hyperphosphatasia (ESH) is probably inherited as an autosomal dominant trait with a high degree of penetrance.
Basic Fibroblast Growth Factor Stimulates Vascular Endothelial Growth Factor Release in Osteoblasts: Divergent Regulation by p42/p44 Mitogen-Activated Protein Kinase and p38 Mitogen-Activated Protein Kinase
Authors: Tokuda Haruhiko , Kozawa Osamu , Uematsu Toshihiko
Published: April 2006

We previously showed that basic fibroblast growth factor (bFGF) activates p38 mitogen-activated protein (MAP) kinase via Ca2+ mobilization, resulting in interleukin-6 (IL-6) synthesis in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the effect of bFGF on the release of vascular endothelial growth factor (VEGF) in these cells. bFGF stimulated VEGF release dose dependently in the range between 10 and 100 ng/ml. SB203580, an inhibitor of p38 MAP kinase, markedly enhanced the bFGF-induced VEGF release. bFGF induced the phosphorylation of both p42/p44 MAP kinase and p38 MAP kinase. PD98059, an inhibitor of upstream kinase of p42/p44 MAP kinase, reduced the VEGF release. SB203580 enhanced the phosphorylation of p42/p44 MAP kinase induced by bFGF. The enhancement by SB203580 of the bFGF-stimulated VEGF release was suppressed by PD98059. The depletion of extracellular Ca2+ by [ethylenebis-(oxyethylenenitrilo)]tetracetic acid (EGTA) or 1,2-bis-(O-aminophinoxy)-ethane-N,N,N,N-tetracetic acid tetracetoxymethyl ester (BAPTA/AM), a chelator of intracellular Ca2+, suppressed the bFGF-induced VEGF release. A23187, a Ca ionophore, or thapsigargin, known to induce Ca2+ release from intracellular Ca2+ store, stimulated the release of VEGF by itself. A23187 induced the phosphorylation of p42/p44 MAP kinase and p38 MAP kinase. PD98059 suppressed the VEGF release induced by A23187. SB203580 had little effect on either A23187-induced VEGF release or the phosphorylation of p42/p44 MAP kinase by A23187. These results strongly suggest that bFGF stimulates VEGF release through p42/p44 MAP kinase in osteoblasts and that the VEGF release is negatively regulated by bFGF-activated p38 MAP kinase.
Caveolin-Enriched Membrane Signaling Complexes in Human and Murine Osteoblasts
Authors: Solomon Keith R. , Danciu Theodora E. , Adolphson Layla D. , Hecht Leah E. , Hauschka Peter V.
Published: April 2006

Osteoblasts receive regulatory signals from hormones, growth factors, calcium, extracellular matrix, and other cells through a variety of receptors that utilize an array of signaling pathways and cytoplasmic messengers. This article addresses the nonuniform distribution of important signaling molecules (platelet-derived growth factor receptors [PDGFRs], nonreceptor tyrosine kinases, tyrosine kinase adaptor proteins, G proteins, and nitric oxide synthases [NOSs]) in the surface membranes of human and murine osteoblasts. We show that particular inner leaflet signaling molecules (e.g., heterotrimeric G proteins and Src family tyrosine kinases) are clustered and concentrated in Triton X-100-insoluble membranes that are enriched in caveolin, the major structural component of caveolae (50- to 100-nm flask-shaped invaginations of the plasma membrane that apparently are organized by oligomers of the protein caveolin). In addition, we show that a subset of highly ligand-responsive PDGFRs and mitogen-activated protein (MAP) kinase pathway effectors are present in the caveolin-enriched membrane fraction of osteoblasts.
Sox-4 Messenger RNA Is Expressed in the Embryonic Growth Plate and Regulated via the Parathyroid Hormone/Parathyroid Hormone-Related Protein Receptor in Osteoblast-like Cells
Authors: Reppe Sjur , Rian Edith , Jemtland Rune , Olstad Ole K. , Gautvik Vigdis T. , Gautvik Kaare M.
Published: April 2006

Parathyroid hormone (PTH) and PTH-related protein (PTHrP) exert potent and diverse effects in cells of the osteoblastic and chondrocytic lineages. However, downstream mediators of these effects are characterized inadequately. We identified a complementary DNA (cDNA) clone encoding the 5′ end of the transcription factor Sox-4, using a subtracted cDNA library enriched in PTH-stimulated genes from the human osteoblast-like cell line OHS. The SOX-4 gene is a member of a gene family (SOX and SRY) comprising transcription factors that bind to DNA through their high mobility group (HMG)-type binding domain, and previous reports have implicated Sox proteins in various developmental processes. In situ hybridization of fetal and neonatal mouse hindlimbs showed that Sox-4 messenger RNA (mRNA) was expressed most intensely in the zone of mineralizing cartilage where chondrocytes undergo hypertrophy, and by embryonic day 17 (ED17), after the primary ossification center was formed, its expression was detected only in the region of hypertrophic chondrocytes. Sox-4 mRNA was detected in osteoblast-like cells of both human and rodent origin. In OHS cells, physiological concentrations (10−10–10−9 M) of human PTH 1-84 [hPTH(1-84)] and hPTH(1-34), but not hPTH(3-84), stimulated Sox-4 mRNA expression in a time-dependent manner, indicating involvement of the PTH/PTHrP receptor. Sox-4 transcripts also were detected in various nonosteoblastic human cell lines and tissues, in a pattern similar to that previously reported in mice. The presence of Sox-4 mRNA in hypertrophic chondrocytes within the mouse epiphyseal growth plate at sites that overlap or are adjacent to target cells for PTH and PTHrP, and its strong up-regulation via activated PTH/PTHrP receptors in OHS cells, makes it a promising candidate for mediating downstream effects of PTH and PTHrP in bone.
The Protective Effect of Hormone-Replacement Therapy on Fracture Risk Is Modulated by Estrogen Receptor α Genotype in Early Postmenopausal Women
Authors: Salmén Timo , Heikkinen Anna-Mari , Mahonen Anitta , Kröger Heikki , Komulainen Marja , Saarikoski Seppo , Honkanen Risto , Mäenpää Pekka H.
Published: April 2006

Genetic factors regulate bone mineral density (BMD) and possibly development of osteoporosis. It has been suggested that estrogen receptor α (ERα) genotype is associated with BMD, but the association between ERα genotype, fracture risk, and postmenopausal hormone replacement therapy (HRT) has not been studied. Therefore, we evaluated whether ERα polymorphism is associated with fracture risk in a 5-year trial with HRT in a population-based, randomized group of 331 early postmenopausal women. The participants consisted of two treatment groups: the HRT group (n = 151) received a sequential combination of 2 mg of estradiol valerate (E2Val) and 1 mg of cyproterone acetate with or without vitamin D3, 100-300 IU + 93 mg calcium as lactate per day; and the non-HRT group (n = 180) received 93 mg of calcium alone or in combination with vitamin D3, 100-300 IU/day. All new symptomatic, radiographically defined fractures were recorded. Pvu II restriction fragment length polymorphism of the ERα was determined using polymerase chain reaction (PCR). In all, 28 women sustained 33 fractures during the approximately 5.1-year follow-up. In the HRT group, the ERα genotype (PP, Pp, and pp) was not significantly associated with fracture risk (p = 0.138; Cox proportional hazards model). When the genotype was dichotomized (PP + Pp vs. pp), the incidence of new fractures in the HRT group was significantly reduced in women with the P allele (p = 0.046) with the relative risk (HR) of 0.25 (95% CI, 0.07-0.98), in comparison with the non-P allele group. After adjustment for time since menopause and previous fracture, the association between the dichotomous genotype and fracture risk persisted with HR of 0.24 (95% CI, 0.06-0.95; p = 0.042). In the non-HRT group, the ERα genotype was not significantly associated with fracture risk. During HRT, women with the pp genotype have a greater fracture risk than those with the P allele. The results suggest that the pp genotype is a relatively hormone-insensitive genotype, and it appears that women with the P allele may benefit more from the protective effect of HRT on fracture risk than women with the pp genotype.
Detraining Reverses Positive Effects of Exercise on the Musculoskeletal System in Premenopausal Women
Authors: Winters Kerri M. , Snow Christine M.
Published: April 2006

We studied the effects of a 6-month withdrawal of exercise after 12 months of progressive impact (jump) plus lower body resistance training on risk factors for hip fracture in premenopausal women (age, 30-45 years). Twenty-nine women completed the 12-month training and detraining programs and were compared with 22 matched controls. Bone mineral density (BMD) at the greater trochanter, femoral neck, lumbar spine, and whole body and body composition (% body fat) were measured by dual energy X-ray absorptiometry (DXA; Hologic QDR-1000/W). Knee extensor and hip abductor strength were assessed via isokinetic dynamometry (Kin-Com 500H); maximum leg power was tested using a Wingate Anaerobic Power test; and dynamic postural stability was measured on a stabilimeter (Biodex). All measurements were conducted at baseline, 12 months and 18 months with an additional midtraining measurement of BMD. Exercisers trained three times per week in a program of 100 jumps and 100 repetitions of resistance exercises at each session. Intensity was increased using weighted vests to final values of 10% and 13% of body weight (BW) for jump and resistance exercises, respectively. Differences between groups from training were analyzed by repeated measures analysis of covariance (ANCOVA), adjusted for baseline values. Detraining effects were analyzed by comparing the changes from training with the changes from detraining using repeated measures analysis of variance (ANOVA). Baseline values were not significantly different between exercisers and controls. Percent change over the training period was significantly greater in the exercise group than in the control group at the greater trochanter (2.7 ± 2.5% vs. 0.8 ± 0.8%, respectively; p < 0.01) and approached significance at the femoral neck (1.2 ± 3.2% vs. −0.3 ± 1.9%, respectively; p = 0.06). Significant improvements also were observed in exercisers versus controls for strength and power with exercisers increasing 13-15% above controls, whereas stability was not different between groups. After 6 months of detraining, BMD and muscle strength and power decreased significantly toward baseline values, whereas control values did not change. We conclude that the positive benefits of impact plus resistance training on the musculoskeletal system in premenopausal women reverse when training is withdrawn. Therefore, continued training, perhaps at a reduced frequency and intensity, is required to maintain the musculoskeletal benefit from exercise that may lower fracture risk in later life.
Effect of Dietary Protein on Bone Loss in Elderly Men and Women: The Framingham Osteoporosis Study
Authors: Hannan Marian T. , Tucker Katherine L. , Dawson-Hughes Bess , Cupples L. Adrienne , Felson David T. , Kiel Douglas P.
Published: April 2006

Few studies have evaluated protein intake and bone loss in elders. Excess protein may be associated with negative calcium balance, whereas low protein intake has been associated with fracture. We examined the relation between baseline dietary protein and subsequent 4-year change in bone mineral density (BMD) for 391 women and 224 men from the population-based Framingham Osteoporosis Study. BMD (g/cm2) was assessed in 1988-1989 and in 1992-1993 at the femur, spine, and radius. Usual dietary protein intake was determined using a semiquantitative food frequency questionnaire (FFQ) and expressed as percent of energy from protein intake. BMD loss over 4 years was regressed on percent protein intake, simultaneously adjusting for other baseline factors: age, weight, height, weight change, total energy intake, smoking, alcohol intake, caffeine, physical activity, calcium intake, and, for women, current estrogen use. Effects of animal protein on bone loss also were examined. Mean age at baseline (±SD) of 615 participants was 75 years (±4.4; range, 68-91 years). Mean protein intake was 68 g/day (±24.0; range, 14-175 g/day), and mean percent of energy from protein was 16% (±3.4; range, 7-30%). Proportional protein intakes were similar for men and women. Lower protein intake was significantly related to bone loss at femoral and spine sites (p ≤ 0.04) with effects similar to 10 lb of weight. Persons in the lowest quartile of protein intake showed the greatest bone loss. Similar to the overall protein effect, lower percent animal protein also was significantly related to bone loss at femoral and spine BMD sites (all p < 0.01) but not the radial shaft (p = 0.23). Even after controlling for known confounders including weight loss, women and men with relatively lower protein intake had increased bone loss, suggesting that protein intake is important in maintaining bone or minimizing bone loss in elderly persons. Further, higher intake of animal protein does not appear to affect the skeleton adversely in this elderly population.
A Role for N-Cadherin in the Development of the Differentiated Osteoblastic Phenotype
Authors: Ferrari Serge L. , Traianedes Kathy , Thorne Marielle , Lafage-Proust Marie-Helene , Genever Paul , Cecchini Marco G. , Behar Vered , Bisello Alessandro , Chorev Michael , Rosenblatt Michael , Suva Larry J.
Published: April 2006

Cadherins are a family of cell surface adhesion molecules that play an important role in tissue differentiation. A limited repertoire of cadherins has been identified in osteoblasts, and the role of these molecules in osteoblast function remains to be elucidated. We recently cloned an osteoblast-derived N-cadherin gene from a rat osteoblast complementary DNA library. After in situ hybridization of rat bone and immunohistochemistry of human osteophytes, N-cadherin expression was localized prominently in well-differentiated (lining) osteoblasts. Northern blot hybridization in primary cultures of fetal rat calvaria and in human SaOS-2 and rat ROS osteoblast-like cells showed a relationship between N-cadherin messenger RNA expression and cell-to-cell adhesion, morphological differentiation, and alkaline phosphatase and osteocalcin gene expression. Treatment with a synthetic peptide containing the His-Ala-Val (HAV) adhesion motif of N-cadherin significantly decreased bone nodule formation in primary cultures of fetal rat calvaria and inhibited cell-to-cell contact in rat osteoblastic TRAB-11 cells. HAV peptide also regulated the expression of specific genes such as alkaline phosphatase and the immediate early gene zif268 in SaOS-2 cells. Transient transfection of SaOS-2 cells with a dominant-negative N-cadherin mutant (NCADΔC) significantly inhibited their morphological differentiation. In addition, aggregation of NCTC cells derived from mouse connective tissue stably transfected with osteoblast-derived N-cadherin was inhibited by either treatment with HAV or transfection with NCADΔC. Together, these results strongly support a role for N-cadherin, in concert with other previously identified osteoblast cadherins, in the late stages of osteoblast differentiation. (J Bone Miner Res 2000;15:198–208)
Functional Gap Junctions Between Osteocytic and Osteoblastic Cells
Authors: Yellowley Clare E. , Li Zhongyong , Zhou Zhiyi , Jacobs Christopher R. , Donahue Henry J.
Published: April 2006

Morphological evidence shows that osteocytes, bone cells that exist enclosed within bone matrix, are connected to one another and to surface osteoblasts via gap junctions; however, it is unknown whether these gap junctions are functional. Using a newly established murine osteocytic cell line MLO-Y4, we have examined functional gap junctional intercellular communication (GJIC) between osteocytic cells and between osteocytic and osteoblastic cells. In our hands, MLO-Y4 cells express phenotypic characteristics of osteocytic cells including a stellate morphology, low alkaline phosphatase activity, and increased osteocalcin messenger RNA (mRNA) compared with osteoblastic cells. Northern and Western blot analysis revealed that MLO-Y4 cells express abundant connexin 43 (Cx43) mRNA and protein, respectively. Lucifer yellow dye transferred from injected to adjacent cells suggesting that osteocytic cells were functionally coupled via gap junctions. Functional GJIC between osteocytic and osteoblastic (MC3T3-E1) cells was determined by monitoring the passage of calcein dye between the two cell types using a double labeling technique. The ability of bone cells to communicate a mechanical signal was assessed by mechanically deforming the cell membrane of single MLO-Y4 cells, cocultured with MC3T3-E1 cells. Deformation induced calcium signals in MLO-Y4 cells and those elicited in neighboring MC3T3-E1 cells were monitored with the calcium sensitive dye Fura-2. Our results suggest that osteocytic MLO-Y4 cells express functional gap junctions most likely composed of Cx43. Furthermore, osteocytic and osteoblastic cells are functionally coupled to one another via gap junctions as shown by the ability of calcein to pass between cells and the ability of cells to communicate a mechanically induced calcium response. (J Bone Miner Res 2000;15:209–217)
1,25-Dihydroxyvitamin D3 Hypersensitivity of Osteoclast Precursors from Patients with Paget's Disease
Authors: Menaa Cheikh , Barsony Julia , Reddy Sakamuri V. , Cornish Jillian , Cundy Tim , Roodman G. David
Published: April 2006

Our previous studies suggested that increased osteoclast formation and activity in Paget's disease may be related in part to increased responsiveness of highly purified osteoclast precursors to 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. However, the basis for this enhanced sensitivity to 1,25-(OH)2D3 is unclear. To address this question, we examined 24-hydroxylase and 1,25-(OH)2D3 receptor (VDR) messenger RNA (mRNA) expression during human osteoclast differentiation from normal subjects and patients with Paget's disease in response to 1,25-(OH)2D3 as well as VDR content and affinity. Reverse-transcription polymerase chain reaction (RT-PCR) analysis of granulocyte–macrophage colony–forming unit (GM-CFU), the earliest identifiable osteoclast precursor, derived from patients with Paget's disease demonstrated 24-hydroxylase mRNA expression in response to 1,25-(OH)2D3 was induced at concentrations of 1,25-(OH)2D3 that were at least one log less than that required for normal GM-CFU. VDR mRNA and VDR protein were detected in both immature and more differentiated osteoclast precursors, as well as in osteoclast-like multinucleated cells (MNCs). However, VDR expression was lower in MNCs than the mononuclear precursor cells. Osteoclast precursors and MNCs from patients with Paget's disease had levels of VDR expression similar to those of normal subjects but showed increased VDR affinity for 1,25-(OH)2D3. Because the effects of 1,25-(OH)2D3 are in part mediated by induction of expression of RANK ligand on marrow stromal cells, which in turn stimulates osteoclast formation, we examined expression of RANK ligand mRNA by marrow stromal cell lines derived from patients with Paget's disease and normal subjects in response to 1,25-(OH)2D3. RT-PCR analysis showed no difference in sensitivity of marrow stromal cells to 1,25-(OH)2D3 from normal subjects or patients with Paget's disease although the Paget's stromal cells expressed increased basal levels of RANK ligand mRNA. These results show that VDR protein is expressed in early and more differentiated osteoclast precursors, that expression levels of VDR decline with osteoclast differentiation, and that 1,25-(OH)2D3 has direct effects on osteoclast precursors. The enhanced sensitivity to 1,25-(OH)2D3 is an intrinsic property of osteoclast precursors from patients with Paget's disease that distinguishes them from normal osteoclast precursors. Furthermore, our results suggest that an increased affinity of VDR for 1,25-(OH)2D3 may be responsible for the enhanced 1,25-(OH)2D3 sensitivity of osteoclast precursors in patients with Paget's disease compared with normal subjects. (J Bone Miner Res 2000;15:228–236)
Biological Activity of CD-Ring Modified 1α,25-Dihydroxyvitamin D Analogues: C-Ring and Five-Membered D-Ring Analogues
Authors: Verstuyf Annemieke , Verlinden Lieve , Van Etten Evelyne , Shi Ling , Wu Yusheng , D'Halleweyn Chris , Van Haver Dirk , Zhu Gui-Dong , Chen Yong-Jun , Zhou Xiaoming , Haussler Mark R. , De Clercq Pierre , Vandewalle Maurits , Van Baelen Hugo , Mathieu Chantal , Bouillon Roger
Published: April 2006

Nonsteroidal analogues of 1α,25(OH)2D3, lacking either the full five-membered D ring (C-ring analogues) or the full six-membered C ring (D-ring analogues) are more potent inhibitors of cell proliferation or inducers of cell differentiation than is 1α,25(OH)2D3. Maximal superagonistic activity was seen for the C-ring analogue with a 24(R)-hydroxyl group in the side chain [30- to 60-fold the activity of 1α,25(OH)2D3]. The 19-nor-16-ene-26,27-bishomo C-ring analogue showed the best ratio of antiproliferative to calcemic effects (1275-fold better than 1α,25(OH)2D3 and severalfold better than all vitamin D analogues so far described). The analogues are able to stimulate specific vitamin D-dependent genes and are active in transfection assays using an osteocalcin promoter VDRE. Low binding affinity to the vitamin D binding protein, differences in metabolism, or affinity for the vitamin D receptor (VDR) are not the most important explanations for the enhanced intrinsic activity. However, the analogues are able to induce conformational changes in the VDR, which makes the VDR-ligand complex more resistant against protease digestion than is 1α,25(OH)2D3. In contrast to 20-epimer steroidal vitamin D analogues, 20-epimer C-ring analogues were less potent than analogues with a natural C-20 configuration. In conclusion, several nonsteroidal vitamin D analogues are superagonists of 1α,25(OH)2D3 despite lower receptor affinity and, for the C-ring analogues, higher flexibility of the side chain; moreover, they have a better selectivity profile than all analogues yet published. (J Bone Miner Res 2000;15;237–252)
Inhibition of Chondrogenesis by Parathyroid Hormone In Vivo During Repair of Full-Thickness Defects of Articular Cartilage
Authors: Kudo Satoshi , Mizuta Hiroshi , Otsuka Yutaka , Takagi Katsumasa , Hiraki Yuji
Published: April 2006

We studied the effects of parathyroid hormone (PTH) on differentiation of chondroprogenitor cells during the repair of full-thickness articular cartilage defects. Three-millimeter cylindrical full-thickness articular cartilage defects, which are small enough to be resurfaced spontaneously by hyaline cartilage, were created in the femoral trochlea of the rabbit knee. Recombinant human PTH(1–84) (hPTH[1–84]) (25 ng/h) then was administered into the joint cavity with an osmotic pump, or in control animals, saline alone was administered. The animals were killed at 1, 2, 4, and 8 weeks. At 1 week, the defects were filled with undifferentiated cells, regardless of the PTH treatments. By 8 weeks, well-developed cartilage covered the defects with reconstitution of subchondral bone up to the original bone–articular cartilage junction. In contrast, no evidence of chondrogenic differentiation was seen at any time during the experimental period in the defects treated with PTH. The reparative tissues also were examined immunohistochemically using anti-proliferating cell nuclear antigen (PCNA) and anti-PTH/PTH–related peptide (PTHrP) receptor antibodies. Interestingly, the chondro-progenitor cells that filled the defects expressed PTH/PTHrP receptor, suggesting that these cells are capable of responding to PTH/PTHrP signaling before overt chondrogenesis. Application of PTH did not interfere with proliferation but inhibited chondrogenic differentiation of the cells resulting in the formation of fibrous tissue that lost the expression of PTH/PTHrP receptor within 4 weeks. (J Bone Miner Res 2000;15:253–260)
Regulatory Roles of Zinc in Matrix Vesicle-Mediated Mineralization of Growth Plate Cartilage
Authors: Kirsch Thorsten , Harrison Gerald , Worch Kai P. , Golub Ellis E.
Published: April 2006

Zinc (Zn2+) has long been known to play important roles in mineralization and ossification of skeletal tissues, but the mechanisms of Zn2+ action are not well understood. In this study we investigated the effects of Zn2+ on mineralization in a cell culture system in which terminal differentiation and mineralization of hypertrophic growth plate chondrocytes was induced by retinoic acid (RA) treatment. Addition of Zn2+ to RA-treated cultures decreased mineralization in a dose-dependent manner without affecting alkaline phosphatase (APase) activity. Characterization of matrix vesicles (MVs), particles that initiate the mineralization process, revealed that vesicles isolated from RA-treated and RA/Zn2+-treated cultures showed similar APase activity, but vesicles from RA/Zn2+-treated cultures contained significantly less Ca2+ and Pi. MVs isolated from RA-treated cultures were able to take up Ca2+ and mineralize in vitro, whereas vesicles isolated from RA/Zn2+-treated cultures were not able to do so. Detergent treatment, which ruptures the MV membrane and exposes preformed intravesicular Ca2+-Pi-phospholipid complexes, did not restore the Ca2+ uptake abilities of MVs isolated from RA/Zn2+-treated cultures, suggesting that vesicles from RA/Zn2+-treated cultures did not contain functional Ca2+-Pi− phospholipid complexes. Zn2+ treatment did not affect the content of annexins II, V, and VI in MVs or the Ca2+-dependent, EDTA-reversible binding of these molecules to the membrane surface. However, Zn2+ treatment did affect the EDTA-nonreversible binding of these molecules to the MV membrane, suggesting that Zn2+ interferes with the assembly of annexins in the MV membrane. In addition, Zn2+ inhibited annexin II-, V-, and VI-mediated Ca2+ influx into liposomes. In conclusion, Zn2+ inhibits the mineralizing competence of intravesicular Ca2+-Pi–phospholipid complexes and function of annexin channels, thereby controlling Ca2+ influx into MVs, the formation of the first crystal phase inside the vesicles and initiation of mineralization. (J Bone Miner Res 2000;15:261–270)
Bone Structural and Mechanical Properties Are Affected by Hypotransferrinemia But Not by Iron Deficiency in Mice
Authors: Malecki Elise A. , Buhl Kathleen M. , Beard John L. , Jacobs Christopher R. , Connor James R. , Donahue Henry J.
Published: April 2006

Hypotransferrinemia is a genetic defect in mice resulting in <1% of normal plasma transferrin (Tf) concentrations; heterozygotes for this mutation (+/hpx) have low circulating Tf concentrations. We used this mutant mouse in conjunction with dietary iron deficiency to study the influence of Tf and iron on bone structural and mechanical properties. Twenty-one weanling wild-type BALB/cj +/+ mice and 21 weanling +/hpx mice were fed iron-deficient or iron-adequate diets for 8 weeks. Twelve hpx/hpx mice were fed the iron-adequate diet. Hypotransferrinemia resulted in increased tibia iron and calcium concentrations, lower femur failure load, and extrinsic stiffness. Because the femurs of the hpx/hpx mice were disproportionately small, these bones actually had increased tissue material properties (ultimate stress [US] and modulus of elasticity) than those of wild-type mice. This is the first report on the effect of dietary iron deficiency on bone structural and mechanical properties. Dietary iron deficiency in +/+ and +/hpx mice decreased tibia iron concentrations but had no effect on tibia calcium and phosphorus concentrations or femur structural or mechanical properties. Because the bones of the hpx/hpx mice were small, but had superior tissue mechanical properties, we conclude that Tf is important for normal bone mineralization. (J Bone Miner Res 2000; 15: 271–277)
Is BMU-Coupling a Strain-Regulated Phenomenon? A Finite Element Analysis
Authors: Smit Theo H. , Burger Elisabeth H.
Published: April 2006

Histologically, two types of bone reconstruction are distinguished: modeling and remodeling. Modeling changes the amount of bone and determines its geometrical form in relation to the prevailing mechanical loads and their resulting deformation (strain). Remodeling renews existing bone in a sequence of resorption and formation. However, in both processes the cells responsible for resorption and formation are the same: osteoclasts and osteoblasts. We studied if there is a relation between the activity of these cells and the deformation of the local bone tissue during remodeling. Two finite element models were built on a microscopic, supracellular level: (1) a secondary osteon in cortical bone and (2) a Howship's lacuna in a trabecula. Both models were loaded in the “natural,” that is, longitudinal direction. Equivalent strains were determined as a measure for the deformation of the bone tissue. In the first model, the strain field around the osteon showed a region of decreased deformation in front of the tunnel, just where osteoclasts excavate cortical bone tissue. Behind the cutting cone, elevated strain levels appear in the tunnel wall at locations where osteoblasts are active. The second model showed that a local excavation of a loaded trabecula leads to higher strains at the bottom of the lacuna, where resorption is stopped and osteoblasts are recruited to refill the gap. However, in the direction of loading reduced strain levels appear, just where resorption continues to proceed along the trabecular surface. We conclude that at the tissue level, strain distributions occur during the remodeling process that show a relationship to the activity of osteoblasts and osteoclasts. This suggests that BMU coupling, that is, the subsequent activation of osteoclasts and osteoblasts during remodeling, is a strain-regulated phenomenon. (J Bone Miner Res 2000;15: 301–307)
Apolipoprotein E Gene Polymorphism and Bone Loss: Estrogen Status Modifies the Influence of Apolipoprotein E on Bone Loss
Authors: Salamone Loran M. , Cauley Jane A. , Zmuda Joseph , Pasagian-Macaulay Araxi , Epstein Robert S. , Ferrell Robert E. , Black Dennis M. , Kuller Lewis H.
Published: April 2006

The identification of genes that contribute to bone mineral density (BMD) and bone loss has widespread implications for the understanding and prevention of osteoporosis. The objective of this study was to examine the relationship between the presence and absence of the apolipoprotein E*4 (APOE*4) allele and both BMD and annualized percentage rate of change in BMD at the lumbar spine and hip in a population of 392 healthy, pre-, peri-, and postmenopausal white women participating in the Women's Healthy Lifestyle Project. APOE genotype was analyzed by restriction enzyme analysis from genomic DNA. BMD at the lumbar spine and hip was measured at baseline and after a mean of 2.5 years using dual-energy X-ray absorptiometry (DXA). In premenopausal women, there were no significant differences in BMD or in the annualized percentage rate of change in BMD at the spine or hip when comparing women with and without the APOE*4 allele. In contrast, spine bone loss was significantly greater in peri- and postmenopausal women having an APOE*4 allele than in women without this allele (−1.75 + 1.5% per year vs. −0.98 ± 1.4% per year, respectively, p = 0.018). Among peri- and postmenopausal women currently using hormone replacement therapy (HRT), there were no differences in the annualized percentage rate of change in spine BMD; whereas, among non-HRT users, there was a 2-fold higher rate of spine bone loss in women with an APOE*4 allele compared with women without this allele (−2.31 ± 1.5% per year vs. −1.27 ± 1.3% per year, respectively, p = 0.033; APOE*4 ×; HRT interaction, p = 0.076). In conclusion, this study shows the importance of APOE*4 allele in spine bone loss in peri- and postmenopausal women and, more importantly, it provides evidence for a genetic and lifestyle interaction in modulating spine bone loss. (J Bone Miner Res 2000;15:308–314)
Nitric Oxide Modulates Fracture Healing
Authors: Diwan Ashish D. , Wang Min X. , Jang Daniel , Zhu Wei , Murrell George A. C.
Published: April 2006

The role of the messenger molecule nitric oxide has not been evaluated in fracture healing. NO is synthesized by three kinds of nitric oxide synthase (NOS): inducible NOS (iNOS), endothelial (eNOS), and neuronal (bNOS). We evaluated the role of these enzymes in a rat femur fracture-healing model. There was no messenger RNA (mRNA) expression, immunoreactivity, or enzymatic activity for NOS in unfractured femoral cortex. After fracture, however, mRNA, protein, and enzymatic activity for iNOS were identified in the healing rat femoral fracture callus, with maximum activity on day 15. The mRNA expression for eNOS and bNOS was induced slightly later than for iNOS, consistent with a temporal increase in calcium-dependent NOS activity that gradually increased up to day 30. mRNA expression for the three NOS isoforms also was found in six of six human fracture callus samples. To study the effect of suppression of NO synthesis on fracture healing, an experimental group of rats was fed an NOS inhibitor, l-nitroso-arginine methyl ester (l-NAME), and the control group was fed its inactive enantiomer, d-nitroso-arginine methyl ester (d-NAME). An 18% (p ≤ 0.01) decrease in cross-sectional area and a 45% (p ≤ 0.05) decrease in failure load were observed in the NOS-inhibited group on day 24 after fracture. Furthermore, the effect of NO supplementation to fracture healing was studied by delivering NO to the fracture site using carboxybutyl chitosan NONOate locally. On day 17 after fracture, there was a 30% (p ≤ 0.05) increase in cross-sectional area in the NO-donor group compared with the NOS inhibition group. These results show for the first time that NO is expressed during fracture healing in rats and in humans, that suppression of NOS impairs fracture healing, and that supplementation of NO can reverse the inhibition of healing produced by NOS inhibitors. (J Bone Miner Res 2000;15:342–351)
Platelet-Osteosarcoma Cell Interaction is Mediated Through a Specific Fibrinogen-Binding Sequence Located Within the N-Terminal Domain of Thrombospondin 1
Authors: Voland Carole , Serre Claire-Marie , Delmas Pierre , Clézardin Philippe
Published: April 2006

Approximately 20% of patients with osteosarcoma have metastatic disease in lungs or bones at diagnosis. The requirement of platelets in hematogenous dissemination of metastatic cells is now well established. Tumor cells interact with platelets and induce platelet aggregation. In this respect, metastatic potential of tumor cells correlates with their capacity to aggregate platelets in vitro. We have previously shown that thrombospondin 1 (TSP-1) is synthesized and expressed on the surface of MG-63 osteosarcoma cells and mediates platelet-osteosarcoma cell interaction. However, active sites mimicking the function of TSP-1 during platelet-osteosarcoma cell interaction are not known. In this study, a panel of antibodies directed against the N-terminal and C-terminal domains and type 1, type 2, and type 3 repeats of TSP-1 were first used to delineate the structural requirement for the binding of osteosarcoma cell surface–associated TSP-1 to platelets. A drastic inhibition of the platelet-aggregating activity of MG-63 cells was obtained in the presence of a monoclonal antibody directed against the N-terminal domain of TSP-1. Among a series of 16 synthetic peptides spanning the whole N-terminal domain of TSP-1, only synthetic peptide N12/I encompassing amino acid residues 151–164 of the N-terminal domain of TSP-1 inhibited the platelet-aggregating activity of MG-63 cells. Electron microscopy studies showed that peptide N12/I strongly inhibited platelet-osteosarcoma cell interaction. A polyclonal antibody directed against peptide N12/I specifically bound to the surface of MG-63 cells, recognized TSP-1 and drastically inhibited the platelet-aggregating activity of MG-63 cells. In addition, peptide N12/I specifically bound to fibrinogen and inhibited TSP-1/fibrinogen interaction. Overall, our results provide evidence that a fibrinogen-binding sequence located within the N-terminal domain of TSP-1 mediates the binding of osteosarcoma cell surface–associated TSP-1 to platelet-bound fibrinogen. (J Bone Miner Res 2000;15:361–368)