bluuebunny/crossref_metadata_2025 huggingface dataset:
In Vivo Demonstration that Human Parathyroid Hormone 1–38 Inhibits the Expression of Osteoprotegerin in Bone with the Kinetics of an Immediate Early Gene
Authors: Onyia J. E. , Miles R. R. , Yang X. , Halladay D. L. , Hale J. , Glasebrook A. , McClure D. , Seno G. , Churgay L. , Chandrasekhar S. , Martin T. J.
Published: April 2006

Osteoprotegerin (OPG) is a potent inhibitor of osteoclast formation and function. To elucidate how OPG is regulated in bone, we examined (1) the expression and localization of OPG protein in bone tissue, (2) the effect of human parathyroid hormone 1–38 (hPTH 1–38) on OPG messenger RNA (mRNA) levels in rat femur metaphyseal and diaphyseal bone, and (3) the effect of hPTH(1–38) on expression of OPG mRNA in cultured osteoblast-like cells derived from the metaphysis and diaphysis, and in ROS 17/2.8 osteosarcoma cells. Because PTH has been shown to stimulate osteoblast activity via the cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) signal transduction pathway we also investigated whether PTH action on OPG in vivo is dependent on activation of cAMP/PKA pathway. Immunohistochemistry was used to evaluate OPG protein expression and Northern blot hybridization was used to analyze OPG mRNA expression both in vivo and in vitro. Immunohistochemistry of OPG protein expression in the rat distal femur metaphysis revealed that it was localized predominantly in preosteoblasts, osteoblasts, lining cells, and the osteoid layer, with occasional immunoreactivity in osteocytes and cells of the bone marrow. Subcutaneous (sc) administration of a single injection of hPTH(1–38) at 80 μg/kg induced a rapid and transient decrease in OPG mRNA expression in both metaphyseal and diaphyseal bone. The decrease in OPG message was evident by 1 h and mRNA levels returned to baseline after 3 h. PTH analog PTH(1–31), which stimulates intracellular cAMP accumulation, inhibited OPG expression, whereas PTH analogs (3–34 and 7–34) that do not stimulate cAMP production had no effect on expression. In contrast to PTH, prostaglandin E2 (PGE2) had no effect on OPG mRNA expression in vivo in the metaphyseal bone cells, under conditions in which PGE2 does promote expression of the c-fos gene. The in vivo effects of hPTH(1–38) on OPG mRNA were confirmed in isolated primary osteoblast cultures derived from either metaphyseal or diaphyseal bone as well as in ROS 17/2.8 osteosarcoma cells. We propose that the rapid and transient decrease in OPG expression may initiate a cascade of events resulting in the differentiation of osteoclast progenitor. Such a spatially and temporally programmed effect of PTH might contribute to bone turnover. (J Bone Miner Res 2000;15:863–871)
Protein Kinase C Involvement in Interleukin-6 Production by Parathyroid Hormone and Tumor Necrosis Factor-α in UMR-106 Osteoblastic Cells
Authors: Sanders Jennifer L. , Stern Paula H.
Published: April 2006

The cytokine interleukin-6 (IL-6) is increased in bone and bone cells by several resorptive stimuli, including parathyroid hormone (PTH), IL-1β, and tumor necrosis factor-α (TNF-α). The current studies were designed to determine the contribution of the protein kinase C (PKC) signaling pathway to the effects of these three agents to increase IL-6 in UMR-106 rat osteoblastic cells. Cells were pretreated with vehicle (dimethylsulf-oxide [DMSO]) or the phorbol ester, phorbol 12,13-dibutyrate (PDB; 300 nM) for 48 h to down-regulate phorbol-sensitive PKC isozymes. Either PTH (0.1–10 nM), IL-1β (0.1–10 nM), or TNF-α (5 nM and 10 nM) was then added for 24 h in the continued presence of vehicle or PDB. PKC isozymes were visualized by Western immunoblotting and IL-6 was determined by bioassay. PDB pretreatment caused a partial down-regulation of the conventional α-PKC and βI-PKC isozymes and complete down-regulation of the novel δ-isoenzyme and ϵ-isozymes but it had no effect on the atypical Ξ-PKC isozyme. PDB pretreatment reduced IL-6 responses to 5 nM and 10 nM PTH by 61% and 33%, respectively, reduced IL-6 responses to 5nM and 10 nM TNF-α by 54% and 42%, respectively, and failed to inhibit the IL-6 responses to 0.1–10 nM IL-1β. The PDB pretreatment protocol significantly enhanced PTH-stimulated cyclic adenosine monophosphate (cAMP) production. The PKC inhibitor calphostin C also decreased IL-6 responses to PTH. Thus, in this osteoblast cell line, the PKC pathway is an important component of the signaling pathway for the IL-6 production stimulated by PTH and TNF-α but not that from IL-1β. (J Bone Miner Res 2000;15:885–893)
Thiazide Diuretics Affect Osteocalcin Production in Human Osteoblasts at the Transcription Level Without Affecting Vitamin D3 Receptors
Authors: Lajeunesse D. , Delalandre A. , Guggino S. E.
Published: April 2006

Besides their natriuretic and calciuretic effect, thiazide diuretics have been shown to decrease bone loss rate and improve bone mineral density. Clinical evidence suggests a specific role of thiazides on osteoblasts, because it reduces serum osteocalcin (OC), an osteoblast-specific protein, yet the mechanisms implicated are unknown. We therefore investigated the role of hydrochlorothiazide (HCTZ) on OC production by the human osteoblast-like cell line MG-63. HCTZ dose-dependently (1–100 μM) inhibited 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]- induced OC release by these cells (maximal effect, −40–50% and p < 0.005 by analysis of variance [ANOVA]) as measured by ELISA. This effect of HCTZ on OC release was caused by a direct effect on OC gene expression because Northern blot analysis revealed that OC messenger RNA (mRNA) levels were reduced in the presence of increasing doses of the diuretic (–47.2 ± 4.0%; p < 0.0001 by paired ANOVA with 100 μM HCTZ). HCTZ (100 μM) also stimulated calcium (Ca2+) uptake (8.26 ± 1.78 pmol/mg protein/15 minutes vs. 13.6 ± 0.49 pmol/mg protein/15 minutes; p < 0.05) in MG-63 cells. Reducing extracellular Ca2+ concentration with 0.5 mM EDTA or 0.5 mM ethylene glycol-bis(β-amino ethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) only partly prevented the inhibitory effect of the diuretic on OC secretion (maximal effect, −22.5 ± 6.9%), suggesting that thiazide-dependent Ca2+ influx is not sufficient to elicit the inhibition of OC secretion. Because OC production is strictly dependent on the presence of 1,25(OH)2D3 in human osteoblasts, we next evaluated the possible role of HCTZ on vitamin D3 receptors (VDR) at the mRNA and protein levels. Both Northern and Western blot analyses showed no effect of HCTZ (1–100 μM) on VDR levels. The presence of EGTA in the culture media reduced slightly the VDR mRNA levels under basal condition but this was not modified in the presence of increasing levels of HCTZ. The OC gene promoter also is under the control of transcription factors such as Yin Yang 1 (YY1) and cFOS. Western blot analysis revealed no changes in YY1 levels in response to HCTZ either in the presence or in the absence of 0.5 mM EGTA in the culture media. In contrast, HCTZ induced a dose-dependent increase in cFOS levels (p < 0.002 by ANOVA), a situation prevented by incubation with EGTA. These studies indicate that HCTZ inhibits OC mRNA expression independently of an effect on VDR, YY1, or extracellular Ca2+ levels but involves changes in cFOS levels. As OC retards bone formation/mineralization, the inhibition of OC production by HCTZ could explain its preventive role in bone loss rate. (J Bone Miner Res 2000;15:894–901)
Stimulatory Effect of Insulin-Like Growth Factor Binding Protein-5 on Mouse Osteoclast Formation and Osteoclastic Bone-Resorbing Activity
Authors: Kanatani Masanori , Sugimoto Toshitsugu , Nishiyama Katsuhito , Chihara Kazuo
Published: April 2006

Insulin-like growth factor binding protein-5 (IGFBP-5) stimulates osteoblast proliferation directly or indirectly through IGF-I action, but its effects on osteoclast formation and osteoclastic activity are unknown. We tested the effects of IGFBP-5 on osteoclastic activity and osteoclast formation. IGFBP-5 significantly stimulated pit formation by pre-existent osteoclasts in mouse bone cell cultures and its stimulatory effect was completely blocked by IGF-I antibody (Ab). However, IGFBP-5 did not affect the bone-resorbing activity of isolated rabbit osteoclasts. When IGFBP-5 was added to unfractionated bone cells after degeneration of pre-existent osteoclasts, IGFBP-5 (77 pM–7.7 nM) dose-dependently stimulated osteoclast-like cell formation, irrespective of the presence of IGF-I Ab. Moreover, osteoclast-like cells newly formed by IGFBP-5 from unfractionated bone cells possessed the ability to form pits on dentine slices. We next examined the direct effect of IGFBP-5 on osteoclast precursors in the absence of stromal cells, using hemopoietic blast cells derived from spleen cells. IGFBP-5 dose-dependently stimulated osteoclast-like cell formation from osteoclast precursors, irrespective of the presence of IGF-I Ab. Growth hormone (GH) as well as IGF-I significantly stimulated bone resorption by pre-existent osteoclasts in mouse bone cell cultures and these stimulatory effects were completely blocked by IGF-I Ab. GH as well as IGF-I stimulated osteoclast-like cell formation from unfractionated bone cells and this stimulatory effect of GH was significantly but partially blocked by IGF-I Ab. The direct stimulatory effect of GH on osteoclast-like cell formation from hemopoietic blast cells was not affected by IGF-I Ab. The present data indicate that IGFBP-5 stimulates bone resorption both by stimulation of osteoclast formation in an IGF-I–independent fashion and by IGF-I–dependent activation of mature osteoclasts, possibly via osteoblasts, in vitro. (J Bone Miner Res 2000;15:902–910)
Effect of a Low Calcium Dialysate on Parathyroid Hormone Secretion in Diabetic Patients on Maintenance Hemodialysis
Authors: Holgado Roberto , Haire Henry , Ross Dennis , Sprague Stuart , Pahl Madeleine , Jara Aquiles , Martin-Malo Alejandro , Rodriguez Mariano , Almaden Yolanda , Felsenfeld Arnold J.
Published: April 2006

Diabetic patients on maintenance dialysis often are characterized by a relative parathyroid hormone (PTH) deficiency and a form of renal osteodystrophy with low bone turnover known as adynamic bone. The goal of the present study was to determine whether a reduction in the dialysate calcium concentration would increase the predialysis (basal) PTH and maximal PTH level. Thirty-three diabetic maintenance hemodialysis patients with basal PTH values less than 300 pg/ml were randomized to be dialyzed with either a regular (3.0 mEq/liter or 3.5 mEq/liter, group I) or low (2.25 mEq/liter or 2.5 mEq/liter, group II) calcium dialysate for 1 year. At baseline and after 6 months and 12 months of study, low (1 mEq/liter) and high (4 mEq/liter) calcium dialysis studies were performed to determine parathyroid function. At baseline, basal (I, 126 ± 20 vs. II, 108 ± 19 pg/ml) and maximal (I, 269 pg/ml ± 40 pg/ml vs. II, 342 pg/ml ± 65 pg/ml) PTH levels were not different. By 6 months, basal (I, 98 ± 18 vs. II, 200 ± 34 pg/ml, p = 0.02) and maximal (I, 276 pg/ml ± 37 pg/ml vs. II, 529 pg/ml ± 115 pg/ml; p = 0.05) PTH levels were greater in group II. Repeated measures analysis of variance (ANOVA) of the 20 patients who completed the entire 12-month study showed that only in group II patients were basal PTH (p = 0.01), maximal PTH (p = 0.01), and the basal/maximal PTH ratio (p = 0.03) different; by post hoc test, each was greater (p < 0.05) at 6 months and 12 months than at baseline. When study values at 0,6, and 12 months in all patients were combined, an inverse correlation was present between basal calcium and both the basal/maximal PTH ratio (r = −0.59; p < 0.001) and the basal PTH (r = −0.60; p < 0.001). In conclusion, in diabetic hemodialysis patients with a relative PTH deficiency (1) the use of a low calcium dialysate increases basal and maximal PTH levels, (2) the increased secretory capacity (maximal PTH) during treatment with a low calcium dialysate suggests the possibility of enhanced parathyroid gland growth, and (3) the inverse correlation between basal calcium and both the basal/maximal PTH ratio and the basal PTH suggests that the steady-state PTH level is largely determined by the prevailing serum calcium concentration. (J Bone Miner Res 2000;15:927–935)
Bone Mass Continues to Increase at the Hip After Parathyroid Hormone Treatment Is Discontinued in Glucocorticoid-Induced Osteoporosis: Results of a Randomized Controlled Clinical Trial
Authors: Lane Nancy E. , Sanchez Sarah , Modin Gunnar W. , Genant Harry K. , Pierini Elena , Arnaud Claude D.
Published: April 2006

Glucocorticoid-induced osteoporosis is the most common secondary cause of osteoporosis. In this 24-month study, we report changes in bone turnover and bone mass after 12 months of daily injections of human parathyroid hormone 1–34 [hPTH(1–34)] and 12 months off treatment in postmenopausal women (mean age, 63 years) with osteoporosis treated with glucocorticoid and hormone replacement therapy. Response to the treatment was assessed with bone mineral density (BMD) measurements of the lumbar spine by quantitative computed tomography (QCT); BMD measurements of the lumbar spine, hip, and forearm by dual-energy X-ray absorptiometry (DXA); and biochemical markers of bone turnover. The mean (± SEM) change in BMD of the lumbar spine by QCT and DXA in the PTH group at 24 months was 45.9 ± 6.4% and 12.6 ± 2.2% (p < 0.001). The change in total hip and femoral neck BMD was not significant at 12 months but increased to 4.7 ± 0.9% (p < 0.01) and 5.2 ± 1.3% at 24 months, respectively, as compared with a relatively small change of 1.3 ± 0.9% and 2.6 ± 1.7% in the estrogen-only group. The mean percent differences in BMD of the lumbar spine by QCT and DXA between the groups at 24 months were 43.1% and 11.9%, respectively (p < 0.001). The mean percent differences over the estrogen-only group in hip BMD were 3.4% for total hip (p < 0.01) and 2.6% for femoral neck at 24 months. Biochemical markers of bone turnover increased to more than 150% during the first 6 months of therapy, remained elevated throughout the 12-month treatment period, and returned to baseline values within 6 months of discontinuing the PTH treatment. These results suggest that PTH dramatically increases bone mass in the lumbar spine and hip in postmenopausal women with glucocorticoid-induced osteoporosis who are taking hormone replacement therapy. However, the maximum effect of this anabolic agent on bone mass at the hip after 12 months of treatment requires at least 6–12 months after the PTH treatment is discontinued (J Bone Miner Res 2000;15:944–951)
Corticosteroids Do Not Alter the Threshold for Vertebral Fracture
Authors: Selby P. L. , Halsey J. P. , Adams K. R. H. , Klimiuk P. , Knight S. M. , Pal B. , Stewart I. M. , Swinson D. R.
Published: April 2006

Corticosteroid use is one of the most important secondary causes of osteoporosis. Generally, it has been believed that in addition to its effect on bone mineral density (BMD), it also causes an alteration in bone quality that means that fractures occur at a lower BMD than might be expected. To establish if this is the case, we have compared the relationship between BMD and vertebral fracture in patients receiving corticosteroids with that in patients who had never received such therapy. Information was gathered on those patients who had been referred to the participating centers and had both BMD measurements and lateral thoracolumbar radiographs. In all, 452 patients (391 female) were identified; of these 82 (63 female) were receiving corticosteroids. There was no significant difference in BMD between the patients on corticosteroids and those with other suspected causes of osteoporosis. Vertebral fractures were present in 53% of patients on steroids compared with 35% of those who had no such treatment (p = 0.0035). The fractures were more likely to be multiple in patients on corticosteroids (p = 0.0042). However, if the relationship between bone density and fracture is investigated by plotting the cumulative prevalence of fracture against the bone density, measured by T score, the median BMD for fractures actually was marginally lower in patients on steroids, −2.74 (95% confidence interval [CI], −2.77 to −2.70) compared with −2.65 (95% CI, −2.66 to −2.65) in those who had not received steroids. Our results fail to support the notion that the fracture threshold is altered in patients on long-term steroids and suggest that the same diagnostic criteria should be used for osteoporosis in patients whether or not they are taking corticosteroid therapy. (J Bone Miner Res 2000;15:952–956)
Deficient Bone Formation in Idiopathic Juvenile Osteoporosis: A Histomorphometric Study of Cancellous Iliac Bone
Authors: Rauch Frank , Travers Rose , Norman Michael E. , Taylor Arlene , Parfitt A. Michael , Glorieux Francis H.
Published: April 2006

Idiopathic juvenile osteoporosis (IJO), a rare cause of osteoporosis in children, is characterized by the occurrence of vertebral and metaphyseal fractures. Little is known about the histopathogenesis of IJO. We analyzed by quantitative histomorphometry iliac crest biopsies from 9 IJO patients (age, 10.0–12.3 years; 7 girls) after tetracycline labeling. Results were compared with identically processed samples from 12 age-matched children without metabolic bone disease and 11 patients with osteogenesis imperfecta type I. Compared with healthy controls, cancellous bone volume (BV) was markedly decreased in IJO patients (mean [SD]: 10.0% [3.1%] vs. 24.4% [3.8%]), because of a 34% reduction in trabecular thickness (Tb.Th) and a 37% lower trabecular number (Tb.N; p < 0.0001 each; unpaired t-test). Bone formation rate (BFR) per bone surface was decreased to 38% of the level in controls (p = 0.0006). This was partly caused by decreased recruitment of remodeling units, as shown by a trend toward lower activation frequency (54% of the control value; p = 0.08). Importantly, osteoblast team performance also was impaired, as evidenced by a decreased wall thickness (W.Th; 70% of the control value; p < 0.0001). Reconstruction of the formative sites revealed that osteoblast team performance was abnormally low even before mineralization started at a given site. No evidence was found for increased bone resorption. Compared with children with osteogenesis imperfecta (OI), IJO patients had a similarly decreased cancellous BV but a much lower bone turnover. These results suggest a pathogenetic model for IJO, in which impaired osteoblast team performance decreases the ability of cancellous bone to adapt to the increasing mechanical needs during growth. This will finally result in load failure at sites where cancellous bone is essential for stability. (J Bone Miner Res 2000;15:957–963)
Increased Distraction Rates Influence Precursor Tissue Composition Without Affecting Bone Regeneration
Authors: Richards Mark , Kozloff Kenneth M. , Goulet James A. , Goldstein Steven A.
Published: April 2006

The effect of increased distraction rate on bony tissue differentiation was studied using a paired bilateral model of rat femur lengthening. After a 6-day latency period, one randomly selected femur for each rat was distracted at 0.5 mm/day (normal rate) for 12 days, and the contralateral femur was distracted at 1.5 mm/day (increased rate) for 4 days. Femoral lengthening for each side was 6.0 mm, leaving the increased rate leg with an extra 8 days of consolidation compared with the normal rate limb. Group I rats (n = 9) were killed at day 18 postsurgery and analyzed for cartilage tissue composition and distribution. Group II rats (n = 7) were killed on day 36 postsurgery and analyzed by three-dimensional microcomputed tomography (MCT) for changes in new bone volume. Digital color analysis of slides stained with type II collagen antibody showed increases in cartilaginous tissue formation on the increased rate side (1.51 mm2 vs. 0.83 mm2; p = 0.10). No differences in new bone volume were detected between increased rate limbs and their contralateral controls (46.13 mm3 vs. 42.69 mm3; p = 0.63). These findings suggest that intermediate distraction rates may influence precursor tissue composition without affecting the final amount of new bone formed. Because damage to the tissue was not detected at either time point, these changes in chondrogenesis may reflect sensitivity of the pluripotential gap tissue to tension accumulation during lengthening. Future work with this in vivo model is focused on improving our understanding of the mechanisms behind this strain sensitivity. (J Bone Miner Res 2000;15:982–989)
Efficacy and Safety of Daily Risedronate in the Treatment of Corticosteroid-Induced Osteoporosis in Men and Women: A Randomized Trial
Authors: Reid David M. , Hughes Rodney A. , Laan Roland F. J. M. , Sacco-Gibson Nancy A. , Wenderoth Dietrich H. , Adami Silvano , Eusebio Rachelle A. , Devogelaer Jean-Pierre
Published: April 2006

Long-term use of high-dose corticosteroids often results in bone loss, which may lead to osteoporosis-related fractures. This was a multicenter, double-blind study in which 290 ambulatory men and women receiving high-dose oral corticosteroid therapy (prednisone ≥ 7.5 mg/day or equivalent) for 6 or more months were randomized to receive placebo, risedronate 2.5 mg/day, or risedronate 5 mg/day for 12 months. All patients received calcium 1 g and vitamin D 400 IU daily. The primary endpoint was lumbar spine bone mineral density (BMD) at month 12. Additional measurements included BMD at the femoral neck and trochanter and the incidence of vertebral fractures. Overall, there were statistically significant treatment effects on BMD at 12 months at the lumbar spine (p < 0.001), femoral neck (p = 0.004), and trochanter (p = 0.010). Risedronate 5 mg increased BMD at 12 months by a mean (SEM) of 2.9% (0.49%) at the lumbar spine, 1.8% (0.46%) at the femoral neck, and 2.4% (0.54%) at the trochanter, whereas BMD was maintained only in the control group. Although not powered to show fracture efficacy, we observed a reduction in the incidence of vertebral fractures of 70% in the combined risedronate treatment groups, relative to placebo (p = 0.042). Risedronate was well tolerated, had a good safety profile, and was not associated with gastrointestinal adverse events. We conclude that risedronate increases BMD and potentially reduces the incidence of vertebral fractures in patients with corticosteroid-induced osteoporosis.
Human Osteoblastic Cells Propagate Intercellular Calcium Signals by Two Different Mechanisms
Authors: Jørgensen Niklas R. , Henriksen Zanne , Brot Christine , Eriksen Erik F. , Sørensen Ole H. , Civitelli Roberto , Steinberg Thomas H.
Published: April 2006

Effective bone remodeling requires the coordination of bone matrix deposition by osteoblastic cells, which may occur via soluble mediators or via direct intercellular communication. We have previously identified two mechanisms by which rat osteoblastic cell lines coordinate calcium signaling among cells: autocrine activation of P2 (purinergic) receptors leading to release of intracellular calcium stores, and gap junction-mediated communication resulting in influx of extracellular calcium. In the current work we asked whether human osteoblastic cells (HOB) were capable of mechanically induced intercellular calcium signaling, and if so, by which mechanisms. Upon mechanical stimulation, human osteoblasts propagated fast intercellular calcium waves, which required activation of P2 receptors and release of intracellular calcium stores but did not require calcium influx or gap junctional communication. After the fast intercellular calcium waves were blocked, we observed slower calcium waves that were dependent on gap junctional communication and influx of extracellular calcium. These results show that human osteoblastic cells can propagate calcium signals from cell to cell by two markedly different mechanisms and suggest that these two pathways may serve different purposes in coordinating osteoblast functions.
Nuclear Localization of the Type 1 PTH/PTHrP Receptor in Rat Tissues
Authors: Watson P. H. , Fraher L. J. , Hendy G. N. , Chung U.-I. , Kisiel M. , Natale B. V. , Hodsman A. B.
Published: April 2006

The localization of PTH/PTH-related peptide (PTHrP) receptor (PTHR) has traditionally been performed by autoradiography. Specific polyclonal antibodies to peptides unique to the PTHR are now available, which allow a more precise localization of the receptor in cells and tissues. We optimized the IHC procedure for the rat PTHR using 5-μm sections of paraffin-embedded rat kidney, liver, small intestine, uterus, and ovary. Adjacent sections were analyzed for the presence of PTHR mRNA (by in situ hybridization) and PTHrP peptide. A typical pattern of staining for both receptor protein and mRNA was observed in kidney in cells lining the proximal tubules and collecting ducts. In uterus and gut, the receptor and its mRNA are present in smooth muscle layers (PTHrP target) and in glandular cuboidal cells and surface columnar epithelium. This suggests that PTH, or more likely PTHrP, plays a role in surface/secretory epithelia that is as yet undefined. In the ovary, PTHR was readily detectable in the thecal layer of large antral follicles and oocytes, and was present in the cytoplasm and/or nucleus of granulosa cells, regions that also contained receptor transcripts. PTHR protein and mRNA were found in the liver in large hepatocytes radiating outward from central veins. Immunoreactive cells were also present around the periphery of the liver but not within two or three cell layers of the surface. Clear nuclear localization of the receptor protein was present in liver cells in addition to the expected cytoplasmic/peripheral staining. PTHR immunoreactivity was present in the nucleus of some cells in every tissue examined. RT-PCR confirmed the presence of PTHR transcripts in these same tissues. Examination of the hindlimbs of PTHR gene-ablated mice showed no reaction to this antibody, whereas hindlimbs from their wild-type littermates stained positively. The results emphasize that the PTHR is highly expressed in diverse tissues and, in addition, show that the receptor protein itself can be localized to the cell nucleus. Nuclear localization of the receptor suggests that there is a role for PTH and/or PTHrP in the regulation of nuclear events, either on the physical environment (nucleoskeleton) or directly on gene expression.
The Ribosomal Protein QM Is Expressed Differentially During Vertebrate Endochondral Bone Development
Authors: Green Helen , Canfield Ann E. , Hillarby M. Chantal , Grant Michael E. , Boot-Handford Raymond P. , Freemont Anthony J. , Dr. Wallis Gillian A.
Published: April 2006

Endochondral ossification is a carefully coordinated developmental process that converts the cartilaginous model of the embryonic skeleton to bone with accompanying long bone growth. To identify genes that regulate this process we performed a complementary DNA (cDNA) subtractive hybridization of fetal bovine proliferative chondrocyte cDNA from epiphyseal cartilage cDNA. The subtracted product was used to screen a fetal bovine cartilage cDNA library. Ten percent of the clones identified encoded the bovine orthologue of the human ribosomal protein “QM.” Northern and western blot analysis confirmed that QM was highly expressed by cells isolated from epiphyseal cartilage as opposed to proliferative chondrocytes. In contrast, no detectable difference in the expression of mRNA for the ribosomal protein S11 was detected. Immunohistochemical analysis of fetal bovine limb sections revealed that QM was not expressed by the majority of the epiphyseal chondrocytes but only by chondrocytes in close proximity to capillaries that had invaded the epiphyseal cartilage. Strongest QM expression was seen in osteoblasts in the diaphyseal region of the bone adjoining the growth plate, within the periosteum covering the growth plate and within secondary centers of ossification. Hypertrophic chondrocytes within the growth plate adjoining the periosteum also were positive for QM as were chondrocytes in the perichondrium adjoining the periosteum. In vitro investigation of the expression of QM revealed higher QM expression in nonmineralizing osteoblast and pericyte cultures as compared with mineralizing cultures. The in vivo and in vitro expression pattern of QM suggests that this protein may have a role in cell differentiation before mineralization.
The Association Between Heel Ultrasound and Hormone Replacement Therapy Is Modulated by a Two-Locus Vitamin D and Estrogen Receptor Genotype
Authors: Giguère Yves , Dodin Sylvie , Blanchet Claudine , Morgan Kenneth , Dr Rousseau François
Published: April 2006

Evidence supports the role of estrogen deprivation in the process of bone remodeling and increased risk of fracture in postmenopausal women but little is known about the genetic basis of individual differences in response to therapy. In a cross-sectional study, 425 ambulatory postmenopausal French-Canadian women from Quebec (age range, 42–85 years old) were genotyped for a common Bsm I polymorphism at the vitamin D receptor (VDR) gene as well as a Pvu II polymorphism in the estrogen receptor (ESR1) gene. Heel ultrasound was determined by right calcaneal quantitative ultrasound (QUS) and results were expressed as an age- and-weight-adjusted stiffness index (heel SI z score). Our aim was to investigate the interaction between hormone-replacement therapy (HRT) and receptor genotypes in an effect on heel SI. Notably, a two-locus genotype (VDR-bb/ESR-PP) present in 9.5% of women was responsible for over 30% of the total HRT-related heel SI difference in the whole sample. Women bearing this combined VDR/ESR1 genotype who received HRT for more than 5 years had a 21% (1.25 SD) greater heel SI (p = 0.002) than those bearing the same genotype but who received HRT for <5 years. This may translate into a 2- to 3-fold difference in the risk of fracture. Although follow-up studies are needed, our findings suggest that QUS of the heel in postmenopausal women taking HRT is affected by variation in VDR and ESR1 loci, jointly.
Evidence for a Major Gene for Bone Mineral Density in Idiopathic Osteoporotic Families
Authors: Dr. Cardon L. R. , Garner C. , Bennett S. T. , MacKay I. J. , Edwards R. M. , Cornish J. , Hegde M. , Murray M. A. F. , Reid I. R. , Cundy T.
Published: April 2006

Although there have been a number of studies indicating a heritable component for osteoporosis in middle to late adulthood, the etiology of osteoporosis in young people is uncertain. The present study aims to evaluate the extent to which genetic factors influence familial resemblance for bone mineral density (BMD) in families ascertained on the basis of young osteoporotic probands. The sample comprises eight families (74 total individuals) that were identified through a proband under the age of 35 years with a history of two or more fractures and a spinal bone density of at least 2.5 SDs below the mean for age and sex (Z score). Secondary causes of osteoporosis were excluded in the probands. In total, 27% (18/66) of the probands' relatives had osteoporosis and an additional 30% (20/66) had osteopenia. Classical segregation analysis was performed to evaluate the extent to which a genetic etiology could account for familial resemblance in these families. The results indicate a major gene of codominant inheritance for spinal BMD. Model-fitting comparisons revealed no support for environmental effects or for polygenic inheritance.
In Vivo Osteogenic Capability of Cultured Allogeneic Bone in Porous Hydroxyapatite: Immunosuppressive and Osteogenic Potential of FK506 In Vivo
Authors: Yoshikawa T. , Nakajima H. , Yamada E. , Akahane M. , Dohi Y. , Ohgushi H. , Tamai S. , Ichijima K.
Published: April 2006

Fischer or ACI rat marrow cells were obtained from femoral shafts and were cultured to confluence in Eagle's minimal essential medium (EMEM) supplemented with 15% fetal bovine serum. After trypsinization, the cells were subcultured on porous hydroxyapatite (HA; Interpore 500) blocks in the presence of β-glycerophosphate and 10 nM dexamethasone (Dex). After 2 weeks of subculture, a mineralized bone matrix with osteogenic cells developed on the HA pore surfaces. ACI or Fischer cultured bone tissue/HA constructs were implanted subcutaneously into the backs of Fischer rats and the immunosuppressant FK506 was given to the rats for 4 weeks. Implants were harvested 4 weeks and 8 weeks after insertion. At 4 weeks, the ACI constructs (allografts) showed high levels of osteogenic parameters (alkaline phosphatase [ALP] activity and osteocalcin content) and bone formation was observed together with active osteoblasts without obvious accumulation of inflammatory cells. At 8 weeks, active osteoblasts and progressive bone formation were still observed, while osteogenic parameters remained high and osteocalcin messenger RNA (mRNA) was detected. Without FK506 administration, the allografts showed neither bone formation nor osteocalcin mRNA and there were only trace levels of the osteogenic parameters. In the case of Fischer constructs (isografts), extensive bone formation was detected and all the osteogenic parameters were higher with FK506 than without FK506 at both 4 weeks and 8 weeks. These results indicate that cultured bone tissue/HA constructs possess a high osteogenic potential, even as allografts, and that FK506 not only has an immunosuppressive action, but also promotes bone formation.
Analysis of Proximal Femur DXA Scans in Growing Children: Comparisons of Different Protocols for Cross-Sectional 8-Month and 7-Year Longitudinal Data
Authors: Dr. McKay H. A. , Petit M. A. , Bailey D. A. , Wallace W. M. , Schutz R. W. , Khan K. M.
Published: April 2006

Dual-energy X-ray absorptiometry (DXA) is a widely used method for measuring bone mineral in the growing skeleton. Because scan analysis in children offers a number of challenges, we compared DXA results using six analysis methods at the total proximal femur (PF) and five methods at the femoral neck (FN). In total we assessed 50 scans (25 boys, 25 girls) from two separate studies for cross-sectional differences in bone area, bone mineral content (BMC), and areal bone mineral density (aBMD) and for percentage change over the short term (8 months) and long term (7 years). At the proximal femur for the short-term longitudinal analysis, there was an approximate 3.5% greater change in bone area and BMC when the global region of interest (ROI) was allowed to increase in size between years as compared with when the global ROI was held constant. Trend analysis showed a significant (p < 0.05) difference between scan analysis methods for bone area and BMC across 7 years. At the femoral neck, cross-sectional analysis using a narrower (from default) ROI, without change in location, resulted in a 12.9 and 12.6% smaller bone area and BMC, respectively (both p < 0.001). Changes in FN area and BMC over 8 months were significantly greater (2.3%, p < 0.05) using a narrower FN rather than the default ROI. Similarly, the 7-year longitudinal data revealed that differences between scan analysis methods were greatest when the narrower FN ROI was maintained across all years (p < 0.001). For aBMD there were no significant differences in group means between analysis methods at either the PF or FN. Our findings show the need to standardize the analysis of proximal femur DXA scans in growing children.
Molecular Events that Contribute to Lysyl Oxidase Enzyme Activity and Insoluble Collagen Accumulation in Osteosarcoma Cell Clones
Authors: Uzel Mehmet I. , Shih Sarah D. , Gross Howard , Kessler Efrat , Gerstenfeld Louis C. , Trackman Philip C.
Published: April 2006

Maximum collagen synthesis and maximum accumulation of insoluble collagen occur at different phenotypic stages in developing osteoblastic cell cultures. Insoluble collagen accumulation depends in part on the activity of extracellular enzymes including procollagen N-proteinases, procollagen C-proteinase (derived from the BMP1 gene), and lysyl oxidase. In addition to its action on procollagen, procollagen C-proteinase processes prolysyl oxidase to mature 32-kDa lysyl oxidase. The regulation of extracellular activities that control insoluble collagen accumulation has not been studied extensively. The present study compares molecular events that control production of a collagenous mineralized extracellular matrix in vitro among five different murine osteosarcoma cell clones derived from the same tumor, but which differ in their ability to produce an insoluble mineralized matrix. Levels of insoluble type I collagen, insoluble calcium, bone morphogenetic protein 1 (BMP-1), and lysyl oxidase expression, lysyl oxidase biosynthesis, lysyl oxidase activity, and prolysyl oxidase processing activity were determined. Results surprisingly indicate that lysyl oxidase activity is not related closely to lysyl oxidase messenger RNA (mRNA) levels among the different cell clones. However, it appears that BMP-1-dependent prolysyl oxidase processing could contribute to the observed lysyl oxidase activity. Highest collagen and BMP-1 mRNA levels, prolysyl oxidase processing activity, and lysyl oxidase activity occurred in a cell clone (K8) that showed the highest levels of insoluble collagen accumulation. Culture media from a cell clone (K37) that accumulates little insoluble collagen or calcium but expresses high levels of lysyl oxidase mRNA contained low molecular weight fragments of lysyl oxidase protein and showed low lysyl oxidase activity. By contrast the K14 cell line exhibits relatively high lysyl oxidase activity and collagen accumulation, but low levels of mature lysyl oxidase protein. Together, these studies indicate that catabolic as well as anabolic activities are important in regulating insoluble collagen accumulation in osteoblastic cells. In addition, results suggest that products of genes homologous to lysyl oxidase may contribute to observed lysyl oxidase activity.
Use of Oral Corticosteroids and Risk of Fractures
Authors: Van Staa T. P. , Leufkens H. G. M. , Abenhaim L. , Zhang B. , Cooper C.
Published: April 2006

Treatment with oral corticosteroids is known to decrease bone density but there are few data on the attendant risk of fracture and on the reversibility of this risk after cessation of therapy. A retrospective cohort study was conducted in a general medical practice setting in the United Kingdom (using data from the General Practice Research Database [GPRD]). For each oral corticosteroid user aged 18 years or older, a control patient was selected randomly, who was matched by age, sex, and medical practice. The study comprised 244,235 oral corticosteroid users and 244,235 controls. The average age was 57.1 years in the oral corticosteroid cohort and 56.9 years in the control cohort. In both cohorts 58.6% were female. The most frequent indication for treatment was respiratory disease (40%). The relative rate of nonvertebral fracture during oral corticosteroid treatment was 1.33 (95% confidence interval [CI], 1.29–1.38), that of hip fracture 1.61 (1.47–1.76), that of forearm fracture 1.09 (1.01–1.17), and that of vertebral fracture 2.60 (2.31–2.92). A dose dependence of fracture risk was observed. With a standardized daily dose of less than 2.5 mg prednisolone, hip fracture risk was 0.99 (0.82–1.20) relative to control, rising to 1.77 (1.55–2.02) at daily doses of 2.5–7.5 mg, and 2.27 (1.94–2.66) at doses of 7.5 mg or greater. For vertebral fracture, the relative rates were 1.55 (1.20–2.01), 2.59 (2.16–3.10), and 5.18 (4.25–6.31), respectively. All fracture risks declined toward baseline rapidly after cessation of oral corticosteroid treatment. These results quantify the increased fracture risk during oral corticosteroid therapy, with greater effects on the hip and spine than forearm. They also suggest a rapid offset of this increased fracture risk on cessation of therapy, which has implications for the use of preventative agents against bone loss in patients at highest risk.
High Bone Mass in a Female Hutterite Population
Authors: Wosje Karen S. , Binkley Teresa L. , Fahrenwald Nancy L. , Specker Bonny L.
Published: April 2006

We examined a Hutterite population (n = 243) to determine if their agriculturally diverse, self-sufficient communal lifestyle promotes optimal bone mass attainment because of adequate calcium intake and high physical activity levels during growth and young adulthood. We measured total body (TB) and lumbar bone mineral content (BMC) and bone mineral density (BMD) in 39 school-age (younger) females and 204 working (older) females. Forty-five percent of older females and 79% of younger females currently consumed ≥3 servings (svg) of dairy per day. Older females had lumbar (0.6 ± 1.3) and TB (1.1 ± 1.1) BMD Z scores greater than 0 (both, p < 0.001). The lumbar BMD Z score of younger females was not different from 0 (−0.1 ± 1.0; p = 0.5). Both lumbar (r = 0.46; p < 0.001) and TB (r = 0.20; p = 0.02) BMD Z scores increased with increasing age. In multiple regression analyses for older females, lumbar bone area (p < 0.001), weight (p < 0.001), current hours on feet per day (p = 0.01), colony workload (p < 0.01), and estrogen status (p = 0.06) predicted lumbar BMC. TB bone area (p < 0.001), current hours on feet per day (p < 0.01), and colony workload (p < 0.01) predicted TB BMC. For younger females, lumbar bone area (p < 0.001), weight (p < 0.01), years in present colony (p = 0.02), and menses (p < 0.001) predicted lumbar BMC. TB bone area (p < 0.001), height (p < 0.01), years in present colony (p = 0.03), and menses (p < 0.01) predicted TB BMC. The effect of colony workload could not be separated from other factors different by colony. A heritability estimate of 0.66 was calculated for lumbar BMD using mother and daughter Z scores. Adequate calcium intake during growth, high physical activity early in life, and genetic factors may be contributing to above normal BMD levels in adult female Hutterites.
Developmental Expression and Tissue Distribution of Phex Protein: Effect of the Hyp Mutation and Relationship to Bone Markers
Authors: Ruchon Andréa Frota , Tenenhouse Harriet S. , Marcinkiewicz Mieczyslaw , Siegfried Géraldine , Aubin Jane E. , Desgroseillers Luc , Crine Philippe , Boileau Guy
Published: April 2006

Mutations in PHEX, a phosphate-regulating gene with homology to endopeptidases on the X chromosome, are responsible for X-linked hypophosphatemia (XLH). The murine Hyp homologue has the phenotypic features of XLH and harbors a large deletion in the 3′ region of the Phex gene. We characterized the developmental expression and tissue distribution of Phex protein, using a monoclonal antibody against human PHEX, examined the effect of the Hyp mutation on Phex expression, and compared neprilysin (NEP), osteocalcin, and parathyroid hormone/parathyroid hormone-related protein (PTH/PTHrP) receptor gene expression in bone of normal and Hyp mice. Phex encodes a 100- to 105-kDa glycoprotein, which is present in bones and teeth of normal mice but not Hyp animals. These results were confirmed by in situ hybridization (ISH) and ribonuclease protection assay. Phex protein expression in femur and calvaria decreases with age, suggesting a correlation between Phex expression and bone formation. Immunohistochemical studies detected Phex protein in osteoblasts, osteocytes, and odontoblasts, but not in osteoblast precursors. In contrast to Phex, the abundance of NEP messenger RNA (mRNA) and protein is not significantly altered in Hyp bone. Similarly, osteocalcin and PTH/PTHrP receptor gene expression are not compromised in bone of Hyp mice. Our results are consistent with the hypothesis that loss of Phex function affects the mineralizing activity of osteoblasts rather than their differentiation.
Protein Geranylgeranylation Is Required for Osteoclast Formation, Function, and Survival: Inhibition by Bisphosphonates and GGTI-298
Authors: Dr. Coxon Fraser P. , Helfrich Miep H. , Van't Hof Robert , Sebti Saïd , Ralston Stuart H. , Hamilton Andrew , Rogers Michael J.
Published: April 2006

Bisphosphonates are the important class of antiresorptive drugs used in the treatment of metabolic bone diseases. Although their molecular mechanism of action has not been fully elucidated, recent studies have shown that the nitrogen-containing bisphosphonates can inhibit protein prenylation in macrophages in vitro. In this study, we show that the nitrogen-containing bisphosphonates risedronate, zoledronate, ibandronate, alendronate, and pamidronate (but not the non nitrogen-containing bisphosphonates clodronate, etidronate, and tiludronate) prevent the incorporation of [14C]mevalonate into prenylated (farnesylated and geranylgeranylated) proteins in purified rabbit osteoclasts. The inhibitory effect of nitrogen-containing bisphosphonates on bone resorption is likely to result largely from the loss of geranylgeranylated proteins rather than loss of farnesylated proteins in osteoclasts, because concentrations of GGTI-298 (a specific inhibitor of geranylgeranyl transferase I) that inhibited protein geranylgeranylation in purified rabbit osteoclasts prevented osteoclast formation in murine bone marrow cultures, disrupted the osteoclast cytoskeleton, inhibited bone resorption, and induced apoptosis in isolated chick and rabbit osteoclasts in vitro. By contrast, concentrations of FTI-277 (a specific inhibitor of farnesyl transferase) that prevented protein farnesylation in purified rabbit osteoclasts had little effect on osteoclast morphology or apoptosis and did not inhibit bone resorption. These results therefore show the molecular mechanism of action of nitrogen-containing bisphosphonate drugs in osteoclasts and highlight the fundamental importance of geranylgeranylated proteins in osteoclast formation and function.
EXT 1 Gene Mutation Induces Chondrocyte Cytoskeletal Abnormalities and Defective Collagen Expression in the Exostoses
Authors: Legeai-Mallet Laurence , Rossi Antonio , Benoist-Lasselin Catherine , Piazza Rocco , Mallet Jean-François , Delezoide Anne-Lise , Munnich Arnold , Bonaventure Jacky , Zylberberg Louise
Published: April 2006

Hereditary multiple exostoses (HME), an autosomal skeletal disorder characterized by cartilage-capped excrescences, has been ascribed to mutations in EXT 1 and EXT 2, two tumor suppressor-related genes encoding glycosyltransferases involved in the heparan sulfate proteoglycan (HSPG) biosynthesis. Taking advantage of the availability of three different exostoses from a patient with HME harboring a premature termination codon in the EXT 1 gene, morphological, immunologic, and biochemical analyses of the samples were carried out. The cartilaginous exostosis, when compared with control cartilage, exhibited alterations in the distribution and morphology of chondrocytes with abundant bundles of actin filaments indicative of cytoskeletal defects. Chondrocytes in the exostosis were surrounded by an extracellular matrix containing abnormally high amounts of collagen type X. The unexpected presence of collagen type I unevenly distributed in the cartilage matrix further suggested that some of the hypertrophic chondrocytes detected in the cartilaginous caps of the exostoses underwent accelerated differentiation. The two mineralized exostoses presented lamellar bone arrangement undergoing intense remodeling as evidenced by the presence of numerous reversal lines. The increased electrophoretic mobility of chondroitin sulfate and dermatan sulfate proteoglycans (PGs) extracted from the two bony exostoses was ascribed to an absence of the decorin core protein. Altogether, these data indicate that EXT mutations might induce a defective endochondral ossification process in exostoses by altering actin distribution and chondrocyte differentiation and by promoting primary calcification through decorin removal.