bluuebunny/crossref_metadata_2025 huggingface dataset:
Evidence for Involvement of 17β-Estradiol in Intestinal Calcium Absorption Independent of 1,25-Dihydroxyvitamin D3 Level in the Rat
Authors: Colin E. M. , Van Den Bemd G. J. C. M. , Van Aken M. , Christakos S. , De Jonge H. R. , Deluca H. F. , Prahl J. M. , Birkenhäger J. C. , Buurman C. J. , Pols H. A. P. , van Leeuwen J. P. T. M.
Published: April 2006

The sex steroid 17β-estradiol (17β-E2) has a broad range of actions, including effects on calcium and bone metabolism. This study with 3-month-old Brown Norway rats was designed to investigate the role of 17β-E2 in the regulation of calcium homeostasis. Rats were divided in four groups, sham-operated, ovariectomized (OVX), and OVX supplemented with either a 0.025-mg or 0.05-mg 17β-E2 pellet implanted subcutaneously. After 4 weeks, in none of the groups was serum calcium, phosphate, or parathyroid hormone altered compared with the sham group, while only in the OVX rats was a significant reduction in urinary calcium found. Bone mineral density and osteocalcin were modified, as can be expected after OVX and 17β-E2 supplementation. OVX resulted in a nonsignificant increase in serum 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). Supplementation with either one of the 17β-E2 dosages resulted in an 80% reduction of 1,25(OH)2D3 and only a 20% reduction in 25-hydroxyvitamin D3 levels. OVX, as well as supplementation with 17β-E2, did not affect serum levels of vitamin D binding protein. As a consequence, the estimated free 1,25(OH)2D3 levels were also significantly decreased in the 17β-E2-supplemented group compared with the sham and OVX groups. Next, the consequences for intestinal calcium absorption were analyzed by the in situ intestinal loop technique. Although the 1,25(OH)2D3 serum level was increased, OVX resulted in a significant decrease in intestinal calcium absorption in the duodenum. Despite the strongly reduced 1,25(OH)2D3 levels (18.1 ± 2.1 and 16.4 ± 2.2 pmol/l compared with 143.5 ± 29 pmol/l for the OVX group), the OVX-induced decrease in calcium absorption could partially be restored by supplementation with either 0.025 mg or 0.05 mg of 17β-E2. None of the treatments resulted in a significant change in calcium handling in the jejunum, although the trends were similar as those observed in the duodenum. 17β-E2 did not change the VDR levels in both the intestine and the kidney. In conclusion, the present study demonstrates that 17β-E2 is positively involved in intestinal calcium absorption, and the data strengthen the assertion that 17β-E2 exerts this effect independent of 1,25(OH)2D3. In general, 17β-E2 not only affects bone turnover but also calcium homeostasis via an effect on intestinal calcium absorption.
Human NELL-1 Expressed in Unilateral Coronal Synostosis
Authors: Dr. Ting Kang , Vastardis Heleni , Mulliken John B. , Soo Chia , Tieu Andy , Do Huy , Kwong Emily , Bertolami Charles N. , Kawamoto Henry , Kuroda Shun'ichi , Longaker Michael T.
Published: April 2006

Surgical correction of unilateral coronal synostosis offers a unique opportunity to examine the molecular differences between an abnormal and a normal cranial suture. We isolated and identified a cDNA fragment whose expression was up-regulated in the premature fusing and fused coronal sutures, as compared with normal coronal sutures. The nucleotide sequence of the full-length cDNA of this gene, human NELL-1, has ∼61% homology with the chicken Nel gene. Both chicken Nel and human NELL-1 are comprised of six epidermal growth factor-like repeats. The human NELL-1 messages were localized primarily in the mesenchymal cells and osteoblasts at the osteogenic front, along the parasutural bone margins, and within the condensing mesenchymal cells of newly formed bone in sites of premature sutural fusion. Human multiorgan tissue mRNA blot showed that NELL-1 was specifically expressed in fetal brain but not in fetal kidney, liver, or lung. We also showed that Nell-1 was expressed in rat calvarial osteoprogenitor cells and was largely absent in rat tibiae and fibroblast cell cultures. In conclusion, our data suggest that the NELL-1 gene is preferentially expressed in cranial intramembranous bone and neural tissue (both of neural crest cell origin) and is up-regulated during unilateral premature closure of the coronal suture. The precise role of this gene is unknown.
1,25-Dihydroxyvitamin D3–Induced Calcium Efflux from Calvaria Is Mediated by Protein Kinase C
Authors: Dranitzki-Elhalel Michal , Wald Hanna , Popovtzer Mordecai M. , Sprague Stuart M.
Published: April 2006

1,25-dihydroxyvitamin D3 (1,25(OH)2D3) is an important regulator of bone metabolism involved in both formation and resorption. Traditionally it was assumed that vitamin D receptors are intracellular. Recent data indicate that vitamin D may also act through a membrane receptor, specifically raising intracellular calcium and inositol 1,4,5 triphosphate. The present study was undertaken to explore further the mechanism(s) of vitamin D–induced bone resorption in cultured bone. 1,25(OH)2D3 induced a dose-dependent increase of calcium efflux from cultured bone. This increase was completely obliterated by inhibition of protein kinase C (PKC) with either staurosporine or calphostin C. In cultured rat calvariae, 1,25(OH)2D3 also induced a dose-dependent translocation of PKC from cytosol to membrane. The activation of PKC by 1,25(OH)2D3 occurred following a 30-s incubation, peaked at 1 minute, and disappeared by 5 minutes. 1,25(OH)2D3 did not increase cAMP production in similarly cultured calvaria. These results suggest that the action of 1,25(OH)2D3 on calcium flux from cultured bone is mediated, in part, via activation of PKC.
Parathyroid Hormone–Related Protein Regulates Proliferation of Condylar Hypertrophic Chondrocytes
Authors: Suda Naoto , Shibata Shunichi , Yamazaki Kiyomi , Kuroda Takayuki , Senior Paul V. , Beck Felix , Hammond Vicki E.
Published: April 2006

The condylar cartilage, an important growth site in the mandible, shows characteristic modes of growth and differentiation, e.g., it shows delayed appearance in development relative to the limb bud cartilage, originates from the periosteum rather than from undifferentiated mesenchymal cells, and shows rapid differentiation into hypertrophic chondrocytes as opposed to the epiphyseal growth plate cartilage, which has resting and proliferative zones. Recently, attention has been focused on the role of parathyroid hormone–related protein (PTHrP) in modulating the proliferation and differentiation of chondrocytes. To investigate further the characteristic modes of growth and differentiation of this cartilage, we used mice with a disrupted PTHrP allele. Immunolocalization of type X collagen, the extracellular matrix specifically expressed by hypertrophic chondrocytes, was greatly reduced in the condylar cartilage of homozygous PTHrP-knockout mice compared with wild-type mice. In contrast, immunolocalization of type X collagen of the tibial cartilage did not differ. In wild-type mice, proliferative chondrocytes were mainly located in both the flattened cell layer and hypertrophic cell layer of the condylar cartilage, but were limited to the proliferative zone of the tibial cartilage. The number of proliferative chondrocytes was greatly reduced in both cartilages of homozygous PTHrP-knockout mice. Moreover, apoptotic chondrocytes were scarcely observed in the condylar hypertrophic cell layer, whereas a number of apoptotic chondrocytes were found in the tibial hypertrophic zone. Expression of the type I PTH/PTHrP receptor was localized in the flattened cell layer and hypertrophic cell layer of the condylar cartilage, but was absent from the tibial hypertrophic chondrocytes. It is therefore concluded that, unlike tibial hypertrophic chondrocytes, condylar hypertrophic chondrocytes have proliferative activity in the late embryonic stage, and PTHrP plays a pivotal role in regulating the proliferative capacity and differentiation of these cells.
Effect of Phosphate on Parathyroid Hormone Secretion In Vivo
Authors: Estepa Jose C. , Aguilera-Tejero Escolastico , Lopez Ignacio , Almaden Yolanda , Rodriguez Mariano , Felsenfeld Arnold J.
Published: April 2006

Alterations in phosphate homeostasis play an important role in the development of secondary hyperparathyroidism in renal failure. Until recently, it was accepted that phosphate retention only increased parathyroid hormone (PTH) secretion through indirect mechanisms affecting calcium regulation and calcitriol synthesis. However, recent in vitro studies have suggested that phosphate may directly affect PTH secretion. Our goal was to determine whether in vivo an intravenous phosphate infusion stimulated PTH secretion in the absence of changes in serum calcium. Three different doses of phosphate were infused intravenously during 120 minutes to increase the serum phosphate concentration in dogs. Sulfate was also infused intravenously as a separate experimental control. A simultaneous calcium clamp was performed to maintain a normal ionized calcium concentration throughout all studies. At the lowest dose of infused phosphate (1.2 mmol/kg), serum phosphate values increased to ∼3 mM, but PTH values did not increase. At higher doses of infused phosphate (1.6 mmol/kg and 2.4 mmol/kg), the increase in serum phosphate to values of ∼4 mM and 5 mM, respectively, was associated with increases in PTH, even though the ionized calcium concentration did not change. Increases in PTH were not observed until 30–60 minutes into the study. These increases were not sustained, since by 120 minutes PTH values were not different from baseline or controls despite the maintenance of marked hyperphosphatemia. During the sulfate infusion, serum sulfate values increased by ∼3-fold, but no change in PTH values were observed. In conclusion, an acute elevation in serum phosphate stimulated PTH secretion in the intact animal, but the magnitude of hyperphosphatemia exceeded the physiologic range. Future studies are needed to determine whether PTH stimulation is more sensitive to phosphate loading in states of chronic phosphate retention. Moreover, the mechanisms responsible for the delay in PTH stimulation and the failure to sustain the increased PTH secretion need further evaluation.
Depolarization of Osteoclast Plasma Membrane Potential by 17β-Estradiol
Authors: Brubaker Kristen D. , Gay Carol V.
Published: April 2006

The effect of estrogen on plasma membrane potential of isolated avian osteoclasts was examined through the use of a fluorescent potential-sensitive dye, bis-(1,3-dibutylbarbiturate) trimethine oxonol, also known as bis-oxonol. A decrease in potential was observed within seconds of addition of 17β-estradiol. Ouabain, a specific Na+K+-ATPase inhibitor, and BaCl2, an inhibitor of the inwardly rectifying K+ channel, blocked the estrogen response. Verapamil and lanthanum chloride (LaCl3), inhibitors of inward Ca2+ channels, and 4′4-diisothiocyanatostilbene-2′2-disulfonic acid (DIDS), an inhibitor of Cl− channels, did not affect the depolarization. Herbimycin A, a tyrosine kinase inhibitor, also had no effect on the decreased membrane potential. These data provide evidence which suggests that estrogen regulates osteoclasts through ion channel activities. The change in K+ channel activity was observed within seconds of addition of 17β-estradiol, indicating an action at the level of the plasma membrane.
Stromelysin (MMP-3) Synthesis Is Up-Regulated in Estrogen-Deficient Mouse Osteoblasts In Vivo and In Vitro
Authors: Breckon Jeremy J. W. , Papaioannou Stelios , Kon Luke W. M. , Tumber Anthony , Hembry Rosalind M. , Murphy Gillian , Reynolds John J. , Meikle Murray C.
Published: April 2006

Sex steroids are important regulators of bone cell function and osteoblast-derived matrix metalloproteinases (MMPs) are key mediators of bone resorption during the initial stage of osteoid removal prior to osteoclast attachment. To investigate the mechanism of bone loss following estrogen deficiency, we examined the effects of estrogen on osteoblast synthesis of MMPs and tissue inhibitor of metalloproteinases (TIMPs). Immunolocalization in mouse bone samples ex vivo and primary mouse osteoblast (MOB) cultures was used to document the synthesis of mouse interstitial collagenase (MMP-13), stromelysin-1 (MMP-3), gelatinase-A (MMP-2), and gelatinase-B (MMP-9). Endosteal bone lining cells from distal femoral head and lumbar vertebral body showed an increase in the pattern of synthesis of stromelysin-1 following ovariectomy, compared with sham-operated controls; the synthesis of other MMPs was unaffected. The expression of all classes of MMPs and TIMP-1 and TIMP-2 by MOB in culture was demonstrated by reverse transcriptase-polymerase chain reaction. Following the withdrawal of 17β-estradiol, MOB cultures showed a significant increase in the number of cells synthesizing stromelysin-1; this effect was enhanced by stimulation with either interleukin-1 or interleukin-6. Northern blot analysis showed only a slight increase in stromelysin-1 mRNA message following the withdrawal of 17β-estradiol. Our data show an unexpected up-regulation of stromelysin-1 synthesis by osteoblasts both in vivo and in vitro following estrogen withdrawal. Although this effect was not reflected in a significant change in stromelysin-1 mRNA expression in vitro, there is evidence to suggest a role for this enzyme in the early stages of bone loss during the pathogenesis of osteoporosis.
Skeletal Dysplasia and Defective Chondrocyte Differentiation by Targeted Overexpression of Fibroblast Growth Factor 9 in Transgenic Mice
Authors: Garofalo Silvio , Kliger-Spatz Michal , Cooke Jeremy L. , Wolstin Orit , Lunstrum Gregory P. , Moshkovitz Sharon M. , Horton William A. , Yayon Avner
Published: April 2006

Mutations in fibroblast growth factor receptor 3 (FGFR3) cause several human chondrodysplasias, including achondroplasia, the most common form of dwarfism in humans. From in vitro studies, the skeletal defects observed in these disorders have been attributed to constitutive activation of FGFR3. Here we show that FGF9 and FGFR3, a high-affinity receptor for this ligand, have similar developmental expression patterns, particularly in areas of active chondrogenesis. Targeted overexpression of FGF9 to cartilage of transgenic mice disturbs postnatal skeletal development and linear bone growth. The growth plate of these mice exhibits reduced proliferation and terminal differentiation of chondrocytes similar to that observed in the human disorders. The observations provide evidence that targeted, in vivo activation of endogenous FGFR3 inhibits bone growth and demonstrate that signals derived from FGF9–FGFR3 interactions can physiologically block endochondral ossification to produce a phenotype characteristic of the achondroplasia group of human chondrodysplasias.
Deep Zone Articular Chondrocytes In Vitro Express Genes That Show Specific Changes with Mineralization
Authors: Sun Yongliang , Kandel Rita
Published: April 2006

We have developed a method to form reconstituted mineralized articular cartilagenous tissue in vitro from isolated deep zone chondrocytes. The aim of this study was to characterize further these cultures prior to and during mineralization. Histologic examination of the cells up to 8 days in culture showed that the chondrocytes had formed cartilagenous tissue. Similar to the in vivo cartilage, the chondrocytes expressed aggrecan, types II, I, and X collagens, osteopontin, and alkaline phosphatase (ALP). No osteocalcin mRNA expression was detected in either the in vivo cartilage or in vitro–generated tissue. Addition of β-glycerophosphate (β-GP) to the medium on day 5 induced mineralization and changes in gene expression. Expression of type X collagen, type II collagen, aggrecan core protein, and ALP were inhibited significantly between 2 h and 24 h after the addition of β-GP. At 72 h, expression of these genes were still significantly depressed. These changes correlated with a decrease in collagen and proteoglycan synthesis, and ALP activity. Osteopontin expression increased within 8 h but returned to constitutive levels by 72 h. No change in type I collagen expression was detected. The changes in gene expression were not due to a direct effect of β-GP itself, because similar gene changes occurred in the presence of phosphoethanolamine, another agent which induces mineralization. No changes in gene expression were seen in nonmineralizing cultures. In summary, articular chondrocytes grown on filter culture show expression of similar genes to the chondrocytes in the deep zone of articular cartilage and that changes in expression of specific genes were observed during tissue mineralization, suggesting that it is a suitable model to use to study the mechanism(s) regulating the localized mineralization of articular cartilage.
Isoforms of Bone Alkaline Phosphatase: Characterization and Origin in Human Trabecular and Cortical Bone
Authors: Magnusson Per , Larsson Lasse , Magnusson Martin , Davie Michael W. J. , Sharp Christopher A.
Published: April 2006

Alkaline phosphatase (ALP) is a glycoprotein and functions as an ectoenzyme attached to the cell membrane by a hydrophobic glycosyl-phosphatidylinositol (GPI) anchor. Three bone ALP (BALP) isoforms in human serum were separated and quantitated by high-performance liquid chromatography. B/I, a minor fraction, is composed on average of bone (70%) and intestinal (30%) ALP, and two major isoforms, B1 and B2. Treatment with GPI-specific phospholipase C (GPI-PLC) did not influence the activities or retention times for B1 and B2, indicating that the biochemical differences between B1 and B2 are likely to be due to different glycosylation patterns. The B/I fraction in serum, on average 4% of total ALP, was found to be composed of B1 and B2 isoforms, each with an intact hydrophobic GPI cell membrane anchor. We investigated the origin of these three BALP isoforms and osteocalcin in human femora from five healthy individuals (four males), mean age 51 years, obtained from a tissue bank. Bone was sampled from three sites: cortical bone, trabecular bone from the diaphysis, and trabecular bone from the greater trochanter. Trabecular bone, from both sites, had higher BALP activities compared with cortical bone. Conversely, the osteocalcin content of cortical bone was more than 3-fold greater than that of trabecular bone. Cortical bone had approximately 2-fold higher activity of B1 compared with B2, whereas trabecular bone had ∼2-fold higher activity of B2 compared with B1. We observed a previously undescribed BALP isoform (B1x) in all bone samples. B1x was also observed in sera from some patients (60%) with severe renal insufficiency and on chronic dialysis therapy (n = 20). The isoforms of BALP may provide information relating to bone metabolism within specific bone compartments.
Tibolone, a Steroid with a Tissue-Specific Hormonal Profile, Completely Prevents Ovariectomy-Induced Bone Loss in Sexually Mature Rats
Authors: Ederveen A. G. H. , Kloosterboer H. J.
Published: April 2006

Tibolone (Org OD 14) is a synthetic steroid with combined estrogenic, progestagenic, and androgenic properties and behaves as a tissue-specific steroid. In the current study, we determined the effects of a 4-week treatment with different doses of tibolone on estrogen deficiency–induced bone loss in mature 3-month-old rats. As a reference, 17α-ethinyl estradiol (EE2) was used. The frequency of administration, once or twice a day, was also studied. Bone parameters were determined in sham operated controls, ovariectomized (OVX) controls and OVX-treated rats. Bone loss was assessed by peripheral quantitative computed tomography directly and by quantitative Roentgen densitometry after defatting to exclude influence of fat changes. Femoral bone geometric parameters, plasma osteocalcin level, and urinary deoxypyridinoline/creatinine ratio were also determined. Ovariectomy caused a significant decrease in trabecular bone mineral density in the distal metaphyseal part of the femur using both methods, whereas no change in cortical bone density was found. Trabecular bone loss was prevented in a dose-dependent manner by tibolone (250, 1000, and 4000 μg/rat/day) when given once or twice daily. EE2 also prevented trabecular bone loss but its efficacy was dependent upon the frequency of dosing. Both tibolone and EE2 induced a significant reduction in the urinary deoxypyridinoline/creatinine ratio and plasma osteocalcin level. Tibolone and EE2 had no effect on other femoral bone parameters except a reduction in femoral length. In conclusion, treatment with tibolone for 4 weeks prevented OVX-induced bone loss by suppressing both bone resorption and bone turnover in a similar way as EE2. However, the frequency of dosing is more important for EE2 than for tibolone. Tibolone acts in this animal model for postmenopausal bone loss as an estrogen agonist on bone.
Impaired Development of Bone Mineral Density During Chemotherapy: A Prospective Analysis of 46 Children Newly Diagnosed with Cancer
Authors: Arikoski Pekka , Komulainen Jorma , Riikonen Pekka , Parviainen Markku , Jurvelin Jukka S. , Voutilainen Raimo , Kröger Heikki
Published: April 2006

Osteopenia and osteoporosis are becoming increasingly recognized in children with cancer, though reasons for these changes are poorly understood. The purpose of the present study was to evaluate longitudinal changes in bone mineral density (BMD) and bone turnover in newly diagnosed children with a malignancy. Lumbar spine (L2–L4) and femoral neck bone mineral density (BMDareal, g/cm2) was measured by dual-energy X-ray absorptiometry in 46 children (age 2.9–16.0, median 8.0 years; 15 leukemias, 12 lymphomas, 19 solid tumors) at diagnosis, and after 6 months from the baseline. The apparent volumetric bone mineral density (BMDvol) was calculated to minimize the effect of bone size on BMD. Serum levels of osteocalcin (OC), type I collagen carboxy-terminal propeptide (PICP), and type I collagen carboxy-terminal telopeptide (ICTP) were analyzed at diagnosis, and during a 6-month follow-up. A significant decrease in lumbar BMDvol (–2.1%, p < 0.05), and in femoral BMDareal (–9.9%, p = 0.0001) and BMDvol (–8.5%, p = 0.0001) was observed after 6 months when compared with baseline measurements. The markers of bone formation (PICP, OC) were significantly decreased, and the marker of bone resorption (ICTP) was significantly increased at diagnosis as compared with normal values. By the end the follow-up, the levels of PICP and OC were normalized, whereas the level of ICTP continued to increase indicating that there was a negative balance in bone turnover. A deficient accumulation of bone mass might predispose children with a malignancy to impaired development of peak bone mass. A controlled study determining the benefits of an early intervention on bone turnover should be considered in these patients.
Severe Osteoporosis in Familial Hajdu–Cheney Syndrome: Progression of Acro-Osteolysis and Osteoporosis During Long-Term Follow-up
Authors: Leidig-Bruckner G. , Pfeilschifter J. , Penning N. , Limberg B. , Priemel M. , Delling G. , Ziegler R.
Published: April 2006

Hajdu–Cheney syndrome is an autosomal dominant inherited osteodysplastic bone disease with the hallmarks of acro-osteolysis, skull deformations, and generalized osteoporosis. Very few patients have been followed long-term with respect to the prognosis of acro-osteolysis and osteoporosis. Here we describe a 39-year-old woman and her 19-year-old daughter who are both affected with the Hajdu–Cheney syndrome. Skeletal lesions were followed in the mother between the ages of 22 and 39 years. The acro-osteolytic lesions progressed markedly and caused shortening of several fingers; some end phalanges had completely disappeared. Severe spinal osteoporosis with serial vertebral fractures was found at the age of 22 years. New vertebral fractures developed until the age of 33 years, but did not progress afterward. High turnover osteoporosis was found in the bone histology of iliac crest biopsies performed at the ages of 22 and 34 years. Bone mineral content (BMC) was strikingly decreased at the age of 34 years (T score –5.1 SD) and did not significantly change during further follow-up. In the daughter, BMC failed to increase between the ages of 12 and 19 years and was also markedly decreased (T score –4.4 SD). This suggests that osteoporosis in Hajdu–Cheney syndrome is related to a low peak bone mass and a high bone turnover, leading to insufficient bone formation compared with the increased bone resorption.
Short-Term Therapy with Oral Olpadronate in Active Paget's Disease of Bone
Authors: González Diana C. , Mautalen Carlos A.
Published: April 2006

One of the aims of the treatment of Paget's disease with bisphosphonates should be the normalization of the activity of the disease with the shortest possible exposure to the drug. Olpadronate (OPD) is a new bisphosphonate characterized by the dimethylation of the amino group, its potency is near to alendronate, and more soluble in the digestive media than other aminobisphosphonates. We treated 46 patients (28 men and 18 women, mean age 70 years) with active Paget's disease with oral OPD, 200 mg/day for 12 ± 2 days, except 2 patients who received 400 mg/day. Eight patients had never been treated before, and 38 had previously received antiosteolytic drugs. The period without treatment prior to OPD was (X ± 1 SD) 14 ± 12 months. Baseline bone alkaline phosphatase (BALP) (levels fell from (X ± 1 SD) 54.0 ± 62.7 IU/ml (range 22–396) to a lowest mean value of 16.2 ± 6.4 IU/ml (range 8–45) (normal range 5–21 IU/ml). Forty patients normalized BALP values, in most of the cases within the first 3 months after OPD treatment. Two patients showed partial response (> 50% decrease from baseline), three patients presented poor response (< 50% decrease from baseline), and one patient did not respond at all. Two patients complained of gastric discomfort, and one patient had diarrhea, which disappeared after discontinuation of the drug. Follow-up was carried out on 36 patients; 22 patients are still in remission, with an average length of 9.0 ± 2.6 months. Fourteen patients experienced relapse after 9 ± 2 months remission. In conclusion, a 12-day treatment with 200 mg/day of OPD proved to be a very effective and well tolerated therapy of Paget's disease and induced biochemical remissions in the vast majority of patients, even in those with very active disease.
Zoledronate Is a Potent Inhibitor of Myeloma Cell Growth and Secretion of IL-6 and MMP-1 by the Tumoral Environment
Authors: Derenne Sophie , Amiot Martine , Barillé Sophie , Collette Madeleine , Robillard Nelly , Berthaud Patrice , Harousseau Jean-LUC , Bataille Régis
Published: April 2006

Bisphosphonates have recently been introduced in the therapeutic armamentarium for the long-term treatment of patients with multiple myeloma (MM). These pyrophosphate analogs not only reduce the occurrence of skeletal-related events but also provide patients with a clinical benefit and improve the survival of some of them. We investigated the effects of two bisphosphonates, pamidronate and zoledronate, on both myeloma cells and bone marrow stromal cells (BMSCs). We show here that both bisphosphonates induce both myeloma cell and BMSC apoptosis. Furthermore, at lower concentrations, they induce a significant inhibition (40% and 60%, respectively) of the constitutive production of interleukin-6 (IL-6) by BMSCs. We have recently shown that BMSCs produce MMP-1, the major metalloproteinase involved in the initiation of bone resorption, production up-regulated by IL-1β. Here, we demonstrate that zoledronate significantly inhibits MMP-1 production by BMSCs stimulated with IL-1β more efficiently than pamidronate. However, zoledronate and to a lesser extent pamidronate are responsible for an up-regulation of MMP-2 secretion by BMSCs. MMP-2 is involved both in bone resorption and in the metastatic process. In conclusion, the apoptosis of myeloma cells and BMSCs and the inhibition of both IL-6 and MMP-1 production induced by bisphosphonates, mainly zoledronate, could have antitumoral effects in patients with MM. However, the up-regulation of MMP-2 secretion observed in vitro suggests a putative risk of tumor cell dissemination in vivo when using these new potent bisphosphonates. This potentially deleterious effect could be abolished by combining bisphosphonates with metalloproteinase inhibitors.
Coordinated Expression of Noggin and Bone Morphogenetic Proteins (BMPs) During Early Skeletogenesis and Induction of Noggin Expression by BMP-7
Authors: Nifuji Akira , Noda Masaki
Published: April 2006

Coordinated regulation of the activities of bone morphogenetic protein (BMP) and its inhibitors is essential for skeletal development since loss-of-function experiments show that both BMPs and BMP inhibitory signals, such as noggin, are required to establish proper formation of skeletal tissues. In this paper, we asked how and when noggin would be functional to interact with BMPs during skeletogenesis in mammals. For this purpose, we first analyzed the spatial and temporal patterns of noggin, BMP-2, BMP-4, and BMP-7 expression during early skeletogenesis in mouse embryos. In situ hybridization study revealed that noggin expression was detected at a low level in limb mesenchyme, whereas BMP-7 was expressed at a high level throughout limb mesenchyme 10.5 days postcoitum (dpc) in mouse embryos. One day later, noggin mRNA was expressed at a high level in the prechondrogenic condensations in appendicular and axial skeletal primordia, where sox9 transcripts were also expressed. At this stage, noggin-expressing cells were surrounded by those expressing BMP-7. The chondrogenic cell condensation continued to express noggin transcripts in 12.5 dpc and 13.5 dpc embryos, and again the noggin-expressing cells within the cartilaginous tissue were surrounded by those expressing BMP-7. We further examined interaction of noggin and BMPs by using organ cultures of 11.5 dpc mouse forelimbs and found that implantation of carriers containing BMP-7 protein into the forelimb explants induced noggin expression in the limb mesenchyme. BMP-7 also induced type II collagen and sox9 mRNAs in the same cell population, indicating that noggin induction occurred in the chondrogenic precursor cells. BMP-7 effects on noggin expression were observed in a dose-dependent manner within a dose range of 10–100 ng/μl. These results suggest that BMP-7 induced expression of noggin transcripts within skeletal cell condensation and that this noggin expression in turn could act antagonistically to attenuate BMP action in the early skeletogenesis.
Studies on the Role of Human Insulin-like Growth Factor-II (IGF-II)-Dependent IGF Binding Protein (hIGFBP)-4 Protease in Human Osteoblasts Using Protease-Resistant IGFBP-4 Analogs
Authors: Qin Xuezhong , Byun Dongwon , Strong Donna D. , Baylink David J. , Mohan Subburaman
Published: April 2006

To characterize the insulin-like growth factor binding protein-4 (IGFBP-4) protease produced by human osteoblasts (hOBs), we localized and determined the role of the proteolytic domains in human IGFBP-4 (hIGFBP-4) in modulating IGF-II actions. N-terminal amino acid sequence and mass spectrometric analyses of the 6xHis-tagged IGFBP-4 proteolytic fragments revealed that Met135-Lys136 was the only cleavage site recognized by the IGF-II–dependent IGFBP-4 protease produced by hOBs. This cleavage site was confirmed by the finding that deletion of His121 to Pro141 blocked proteolysis. However, unexpectedly, deletion of Pro94 to Gln119 containing no cleavage site had no effect on IGF-II binding activity but blocked proteolysis. Addition of the synthetic peptide corresponding to this region at concentrations of 250 or 1000 molar excess failed to block IGFBP-4 proteolysis. These data suggest that residues 94–119 may be involved in maintaining the IGFBP-4 conformation required to expose the cleavage site rather than being involved in direct protease-substrate binding. To determine the physiological significance of the IGF-II–dependent IGFBP-4 protease, we compared the effect of the wild-type IGFBP-4 and the protease-resistant IGFBP-4 analogs in blocking IGF-II–induced cell proliferation in normal hOBs, which produce IGFBP-4 protease, and MG63 cells, which do not produce IGFBP-4 protease. It was found that protease-resistant IGFBP-4 analogs were more potent than the wild-type protein in inhibiting IGF-II–induced cell proliferation in hOBs but not in MG63 cells. These data suggest that IGFBP-4 proteolytic fragments are not biologically active and that IGFBP-4 protease plays an important role in regulating IGFBP-4 bioavailability and consequently the mitogenic activity of IGFs in hOBs.
Medium Perfusion Enhances Osteogenesis by Murine Osteosarcoma Cells in Three-Dimensional Collagen Sponges
Authors: Mueller Stefan M. , Mizuno Shuichi , Gerstenfeld Louis C. , Glowacki Julie
Published: April 2006

In this study, we examined in vitro histogenesis by murine K8 osteosarcoma cells maintained in three-dimensional (3D) collagen sponges. We tested the hypothesis that perfusion of medium enhances cell viability and their biosynthetic activity as assessed by expression of the osteoblastic phenotype and mineral deposition. At intervals, samples were harvested and analyzed histologically, biochemically, and by Northern hybridization for type I collagen, osteopontin (OPN), osteocalcin (OC), and core binding factor alpha 1 (Cbfa1). Histologic evaluation showed greater viability, more alkaline phosphatase (ALP)-positive cells, and more mineralized tissue in the perfused sponges after 21 days. Immunohistological assessment of proliferating cell nuclear antigen revealed 5-fold more proliferating cells in the perfused sponges compared with the controls (p = 0.0201). There was 3-fold more ALP activity in the perfused sponges than the controls at 6 days and 14 days (p = 0.0053). The perfused sponges contained twice the DNA and eight times more calcium than the nonperfused controls after 21 days (p < 0.0001 for both). Northern hybridization analysis revealed more mRNA for collagen type I (2-fold) and 50% more for OC at 14 days and 21 days, whereas OPN and Cbfa1 mRNA expression remained unaffected by the medium perfusion. These results show that medium perfusion had beneficial effects on the proliferation and biosynthetic activity of this osteosarcoma cell line. This system mimics the 3D geometry of bone tissue and has the potential for revealing mechanisms of regulation of osteogenesis.
Evidence for a Pathogenic Role of Nitric Oxide in Inflammation-Induced Osteoporosis
Authors: Armour Katharine E. , Van 'T Hof Robert J. , Grabowski Peter S. , Reid David M. , Ralston Stuart H.
Published: April 2006

Inflammatory disease is associated with increased production of nitric oxide (NO) and activation of the inducible nitric oxide synthase (iNOS) pathway. Several studies have addressed the role of NO as a mediator of cytokine effects on bone cell activity in vitro. Stimulatory and inhibitory actions have been found, however, depending on the concentrations produced and model system used. In view of this, it has been difficult to predict whether increased production of NO during inflammation is likely to increase bone loss or prevent it. We have investigated the pathogenic role of NO in an animal model of inflammation-induced osteoporosis (IMO). NO production was increased in IMO when compared with controls (+344%; p < 0.01), and this was accompanied by activation of inducible NOS (iNOS) in the bone marrow space. Bone mineral density (BMD) was reduced in IMO when compared with controls (–64%; p < 0.01), and this was found to be associated with reduced osteoblast numbers (–44%; p < 0.05) and increased osteoclast numbers (+38%; p < 0.01). The NOS inhibitor L-NMMA reversed the deleterious effects of IMO on bone mass and bone turnover, but L-NMMA had no effect on bone mass in control animals. This study has important implications for many inflammatory diseases such as rheumatoid arthritis, ankylosing spondylitis, and inflammatory bowel disease which are associated with increased NO production and osteoporosis. Our data not only suggest that iNOS activation and increased NO production contribute to the pathogenesis of osteoporosis in these situations, but also suggest that NOS inhibitors could be of therapeutic value in the prevention and treatment of such bone loss.
Mechanisms of Bone Loss Following Allogeneic and Autologous Hemopoietic Stem Cell Transplantation
Authors: Ebeling Peter R. , Thomas David M. , Erbas Bircan , Hopper John L. , Szer Jeffrey , Grigg Andrew P.
Published: April 2006

A significant proportion of patients will be long-term survivors of bone marrow transplantation (BMT) and little is known about their risk of late bony complications. We therefore evaluated bone mineral density (BMD) prior to BMT, post-transplantation changes in BMD, and mechanisms of bone loss in long-term survivors. We performed two analyses. The first was a cross-sectional study of 83 consecutive BMT patients (38 F, 45 M), examining the relationship between BMD and bone turnover, measured immediately prior to transplantation, and a number of disease and patient variables. The second was a prospective study of 39 patients (19F, 20 M) followed for a median of 30 months (range 5–64 months) following either allogeneic (allo, n = 29) or autologous (auto, n = 10) BMT to determine if bone loss was related to treatment of graft versus host disease (GVHD) with glucocorticoids and cyclosporine A, high bone turnover rates, or hypogonadism. Auto BMT recipients acted as a control group for effects of GVHD therapy on BMD. Prior to BMT, spinal and femoral neck (FN) BMDs were 8.6% and 14% lower in female auto BMT recipients than in female allo BMT recipients, respectively (p = 0.12 and p = 0.003). Urinary bone resorption markers were higher than in normal gender- and age-matched control subjects. Patients treated previously with glucocorticoids also had 8% lower FN BMD. Glucocorticoid-pretreated women with amenorrhoea had lower lumbar spine (LS) and FN BMDs than eumenorrheic women and women receiving HRT. Post-allo BMT, patients lost 11.7% of FN BMD compared with a nonsignificant decrease of 1.1% post-auto BMT (p < 0.001). Spinal BMD and total body bone mineral content (TBBMC) decreased by 3.9% and 3.5%, respectively, post-allo, compared with an increase (1.5%, p = 0.03) or nonsignificant decrease (−3.7%, p = NS), respectively, post-auto BMT. Post-allo BMT bone loss correlated best with the cumulative prednisolone dose at the LS and FN, and with average daily prednisolone dose for TBBMC. At the spine, the rate of bone loss was 4%/10 g of prednisolone, while the rate of bone loss at the FN was greater (9%/10 g of prednisolone). Bone loss was also negatively related to the duration of cyclosporine therapy for GVHD and baseline deoxypyridinoline concentrations. Avascular necrosis of the femoral head occurred in four, and vertebral and rib fractures occurred in one of the allo BMT patients, but in no auto BMT patients. In conclusion, BMT recipients are at risk of osteoporosis secondary to bone loss associated with their underlying illness and/or chemotherapy, particularly in female autograft recipients, and in allograft recipients secondary to GVHD and its treatment.
Isolation and Characterization of Human Clonogenic Osteoblast Progenitors Immunoselected from Fetal Bone Marrow Stroma Using STRO-1 Monoclonal Antibody
Authors: Oyajobi Babatunde O. , Lomri Abderrahim , Hott Monique , Dr. Marie Pierre J.
Published: April 2006

Osteoprogenitor cells present in human fetal bone marrow (BM) stroma have not been characterized. We used density gradient centrifugation, aggregation on binding lectin, and enrichment by magnetic activated cell sorting with STRO-1 antibody to isolate STRO-1+ cells from nonadherent human fetal BM stromal cells. Immunoselected STRO-1+ cells were immortalized using SV-40 large T antigen and a clone, F/STRO-1+ A, with weak alkaline phosphatase (ALP) activity was selected. The cloned cells proliferated rapidly but were not tumorigenic. Preconfluent F/STRO-1+ A cells showed immunoreactivity for osteopontin, α1(I) procollagen, and parathyroid hormone–related peptide, but not for the late osteoblast differentiation markers, osteocalcin (OC), or bone sialoprotein. However, differentiation of F/STRO-1+ A cells was induced by dexamethasone and 1,25-dihydroxyvitamin D3, as shown by increased ALP activity. In addition, osteogenesis occurred in F/STRO-1+ A cells cultured in three-dimentional aggregates, as assessed morphologically, histologically, and biochemically. Moreover, reverse transcription-polymerase chain reaction analysis showed that OC expression was silent in exponentially growing cells and occurred when cell–cell contacts were established in monolayer and in aggregates, showing induction of mature osteoblast phenotype by cell–cell contacts. Thus, clonal F/STRO-1+ A cells immunoselected from human fetal BM stroma display features of immature osteoprogenitor cells which can differentiate into mature osteogenic cells by cell–cell interactions or inducing agents. The generation by immunoselection of an immortalized clonogenic human fetal BM stroma-derived cell line which behaves like an osteoprogenitor cell provides a novel model system for identifying the signals required for the commitment of osteoprogenitors in the human fetal BM stroma.
Thy-1 Antigen Expression by Cells in the Osteoblast Lineage
Authors: Chen Xiao-Dong , Qian He-Ying , Neff Lynn , Satomura Kazuhito , Dr. Horowitz Mark C.
Published: April 2006

Identification of surface markers involved in osteoblast differentiation provides a method to isolate osteoblasts at various stages of maturation. In this study, we examined expression of the T lymphocyte differentiation antigen, Thy-1, by osteoblastic cells from different species. Murine skeletal progenitor, neonatal calvarial, and adult bone cells (ABCs) were selected to represent osteoblasts at distinct stages of maturation. Flow cytometric analysis showed that Thy-1 expression was undetectable on the progenitor cells (mouse limb bud clones 14 and 17), appeared on calvarial cells (45%+), and was decreased on ABCs (< 10%+). Thy-1 was also detected in situ on osteoblastic cells in mouse calvariae. Thy-1 mRNA expression correlated with cell surface expression. Antigen expression was markedly increased during the cells' proliferative phase in culture. Furthermore, examination of primary rat and human osteoblast-like cells revealed that significant levels of Thy-1 were also expressed on those cells derived from subconfluent culture. This study indicates that osteoblasts express Thy-1 antigen and that its expression is maximal at their earliest stage of maturation, during the proliferative phase, and then declines as the cells mature. In a role similar to the one it plays in the hematopoietic system, Thy-1 antigen may be useful as a differentiation marker in following the development of the osteoblast.
Auditory Ossicle Abnormalities and Hearing Loss in the Toothless (Osteopetrotic) Mutation in the Rat and Their Improvement After Treatment with Colony-Stimulating Factor-1
Authors: Aharinejad S. , Grossschmidt K. , Franz P. , Streicher J. , Nourani F. , Mackay C. A. , Firbas W. , Plenk H. , Marks S. C.
Published: April 2006

Osteopetrosis describes a group of skeletal metabolic diseases of heterogeneous etiology and varied severity that produces a generalized accumulation of skeletal mass, the result of reduced bone resorption. Inherited in a variety of species including humans, the most severe forms are lethal. Among common features are progressive blindness and deafness of controversial etiologies for which there are no universally effective treatments. We have studied the auditory responsiveness and auditory ossicle quantitative histomorphology and temporal bone vasculature in the toothless (tl) rat, a lethal osteopetrotic mutation with few osteoclasts, very low bone turnover, and limited angiogenesis in the axial skeleton. Compared with normal littermates, 3-week-old mutants showed significantly reduced auditory responsiveness, a hearing loss due to abnormalities in both form and tissue composition of the stapes, and little capillary sprouting in the vascular bed of the temporal bone. Treatment of mutants with colony-stimulating factor 1 (CSF-1), known to greatly reduce sclerosis in the axial skeleton, significantly improved hearing, stapedial form and tissue composition, and angiogenesis in the temporal bone. In normal rats, the stapes consisted of 89.3% bone, 9.1% mineralized cartilage, and 0.8% porosity. In osteopetrotic rats, the stapes consisted of 48.3% bone, 35.9% mineralized cartilage, and 15.9% porosity, while after CSF-1 treatment, the bone content increased to 55.2%, cartilage was decreased to 21.7%, and porosity increased to 23.0%, respectively. This is the first demonstration of an auditory abnormality in an osteopetrotic animal mutation and shows that the hearing loss in tl rats can be significantly improved following treatment with CSF-1.
Increased Risk of Fracture in Patients Receiving Solid Organ Transplants
Authors: Ramsey-Goldman Rosalind , Dunn Julie E. , Dunlop Dorothy D. , Stuart Frank P. , Abecassis Michael M. , Kaufman Dixon B. , Langman Craig B. , Salinger Michael H. , Sprague Stuart M.
Published: April 2006

The success of organ transplantation is related to advances in immunosuppressive therapy. These medications are associated with medical complications including bone damage. The objective of this study was to estimate and compare age, gender-specific fracture incidence between transplant recipients, and a large sample representative of the civilian noninstitutionalized United States population using the 1994 National Health Interview Survey (NHIS). This was a cohort study set in tertiary care centers. Five hundred and thirty-nine individuals who received abdominal organ and 61 heart transplants surviving at least 30 days at our institution from 1986 to 1996 were included in the study. Incident fractures were ascertained by mail, in-person interview, telephone survey, or medical record review. All fractures were verified. Organ-, age-, and gender-specific fracture numbers and rates and person-years of observation, were calculated for the transplant patients. Weighted age- and gender-specific fracture rates from the 1994 NHIS were applied to the number of person-years of observation for each organ-specific age and gender category of transplant patients to generate an expected number of fractures. The ratio of observed to expected number of fractures was used to compare fracture experience of transplant patients to that of the national sample from the 1994 NHIS. Fifty-six of 600 (9.3%) patients had at least one fracture following 1221 person-years of observation. The sites of initial symptomatic fracture were as follows: foot (n = 22), arm (n = 8), leg (n = 7), ribs (n = 6), hip (n = 4), spine (n = 3), fingers (n = 3), pelvis (n = 2), and wrist (n = 1). Fracture incidence was 13 times higher than expected in male heart recipients age 45–64 years; nearly 5 times higher in male kidney recipients age 25–44 and age 45–64 years; and 18 times and 34 times higher in female kidney recipients age 25–44 years and 45–64 years compared with NHIS data. We have shown an increased incidence of fractures and estimated the magnitude of this problem in patients undergoing solid organ transplantation. Our work defines the need for a long-term prospective study of fracture risk in these patients.