bluuebunny/crossref_metadata_2025 huggingface dataset:
Rho and Rho Kinase Are Involved in Parathyroid Hormone-Stimulated Protein Kinase C α Translocation and IL-6 Promoter Activity in Osteoblastic Cells
Authors: Radeff Julie M , Nagy Zsolt , Stern Paula H
Published: April 2006

The role of small G-proteins in PTH-stimulated PKC translocation and IL-6 promoter expression in UMR-106 cells was determined. The effects of PTH(1–34) and PTH(3–34) in stimulating PKCα translocation and IL-6 were inhibited by agents that interfere with the activity of small G-proteins of the Rho family and with the downstream kinase Rho kinase.

Introduction: Activation of protein kinase C (PKC) is a signaling mechanism by which parathyroid hormone (PTH) modulates interleukin-6 (IL-6) in osteoblasts, leading to osteoclastogenesis and bone resorption. PKCα and PKCβI are translocated after treatment with PTH in UMR-106 osteoblastic cells; however, the pathway leading to PKC isozyme translocation is not established. Diacylglycerol (DAG) generation from phospholipase D (PLD) is one pathway of PKC activation, and PTH-mediated PLD activity is dependent on small G-proteins of the Rho family. This study investigated whether Rho proteins modulate the PKCα translocation and IL-6 promoter activity stimulated by PTH in UMR-106 cells.

Materials and Methods: UMR-106 cells were treated with PTH(1–34) or PTH(3–34). PKC translocation was determined by immunofluorescence, Rho A activation by Rhotekin assay and by translocation assessed by Western blotting in membrane and cytosol fractions, and IL-6 promoter expression by luciferase assay.

Results and Conclusions: Inhibition of Rho proteins with Clostridium difficile toxin B or inhibition of Rho prenylation with GGTI attenuated PTH(1–34)- and PTH(3–34)-stimulated translocation of endogenous PKCα and IL-6 promoter activity. Expression of a constitutively active RhoA (RhoA63L) mimicked the effect of PTH(1–34) or PTH(3–34) to promote membrane localization of PKCα, whereas cells expressing a dominant negative RhoA (RhoA19N) did not respond to PTH(1–34) or PTH(3–34). The Rho kinase inhibitor Y27632 attenuated PTH(1–34)- and PTH(3–34)-stimulated PKCα translocation and IL-6 promoter activation. Rho seemed to be acting at a step before production of diacylglycerol (DAG), because the stimulation of PKCα translocation by the DAG mimetic phorbol 12,13 dibutyrate (PDBu) was unaffected by C. difficile toxin B or Y27632. These results indicate that Rho proteins are an important component of PTH signaling in osteoblastic cells and provide further demonstration of convergence between PKC and small G-protein signaling pathways.
Intravenous Pamidronate Prevents Femoral Bone Loss and Renal Stone Formation During 90-Day Bed Rest
Authors: Watanabe Yukiko , Ohshima Hiroshi , Mizuno Koh , Sekiguchi Chiharu , Fukunaga Masao , Kohri Kenjiro , Rittweger Jörn , Felsenberg Dieter , Matsumoto Toshio , Nakamura Toshitaka
Published: April 2006

Long-term bed rest has potential risks of bone loss and renal stone formation. We examined the effects of resistive exercise and intravenous pamidronate on BMD, bone turnover, urinary calcium, and renal stone formation in 25 healthy males during 90-day bed rest. Pamidronate prevented femoral bone loss and renal stone formation, but resistive exercise showed little effects.

Introduction: Long-term bed rest increases the risks of bone loss and urinary stone formation. Resistive exercise increases bone formation, and bisphosphonates reduce bone resorption. However, the effects of muscle exercise and bisphosphonates have not been examined side-by-side. The objectives of this study are to compare the effects of pamidronate with resistive exercise on BMD and renal stone formation during prolonged bed rest.

Materials and Methods: Twenty-five male white volunteers, 26–45 years of age, were randomly assigned to the control (n = 9), exercise (n = 9), and pamidronate (n = 7) groups and underwent 90-day 6° head-down tilt bed rest. Exercise group performed squats and heel raises on a flywheel device for 30 minutes every 3 days. Pamidronate (60 mg) was administered intravenously 14 days before bed rest. BMD of the head, forearm, lumbar spine, and proximal femur; biochemical bone markers; calcium (Ca) metabolism; and abdominal radiographs were examined during 90 days of bed rest and 360 days of reloading.

Results: In controls, proximal femoral BMD decreased, and bone resorption markers and urinary Ca increased during bed rest, along with development of renal stones in two of nine subjects. Resistive exercise increased bone formation but was unable to prevent femoral BMD decrease and increases in bone resorption and urinary Ca during bed rest, with formation of renal stones in four of nine subjects. Pamidronate maintained femoral BMD, reduced bone resorption and urinary Ca, and completely prevented renal stone formation.

Conclusions: Resistive exercise increased bone formation but could not reduce bone resorption and the risk of renal stones. In contrast, inhibition of bone resorption by pamidronate could preserve bone mineral and reduce the risk of renal stone formation during prolonged bed rest.
Additive Protective Effects of Estrogen and Androgen Treatment on Trabecular Bone in Ovariectomized Rats
Authors: Tivesten Åsa , Movérare-Skrtic Sofia , Chagin Andrei , Venken Katrien , Salmon Phil , Vanderschueren Dirk , Sävendahl Lars , Holmäng Agneta , Ohlsson Claes
Published: April 2006

Both ER and AR activation regulates trabecular bone mass. We show that combined estrogen and androgen treatment results in additive protection of trabecular bone in OVX rats. This may in part be attributable to the effect of AR activation to attenuate the inhibitory effect of ER activation on bone formation.

Introduction: Sex steroids are important regulators of trabecular bone mass. Both estrogen receptor (ER) and androgen receptor (AR) activation results in increased trabecular bone mass. The aim of this study was to investigate if combined estrogen and androgen treatment might be beneficial in the treatment of trabecular bone loss.

Materials and Methods: Twelve-week-old female rats were ovariectomized (OVX) and treated with vehicle (V), 17β-estradiol (E2; ER activation), dihydrotestosterone (DHT; AR activation), or the combination (E2 + DHT) for 6 weeks. The skeletal phenotype was analyzed by pQCT, μCT, histomorphometry of growth plates, and serum levels of biochemical bone markers.

Results: Both E2 (+121% over V) and DHT (+34%) preserved the trabecular volumetric BMD (tvBMD) in OVX rats. The effect of E2 and DHT on tvBMD was additive, resulting in a 182% increase over V in the rats given E2 + DHT. μCT analyses of the trabecular bone microstructure revealed that the effect of E2 and DHT was additive on the number of trabeculae. E2 treatment reduced serum markers of both bone resorption (collagen C-terminal telopeptide) and bone formation (osteocalcin), indicating reduced bone turnover. Addition of DHT to E2 treatment did not modulate the effects of E2 on the marker of bone resorption, whereas it attenuated the inhibitory effect of E2 on the bone formation marker, which might explain the additive protective effect of E2 and DHT on trabecular bone mass. In contrast, DHT partially counteracted the suppressive effect of E2 on longitudinal bone growth and the E2-induced alterations in growth plate morphology.

Conclusions: These findings show that combined estrogen and androgen treatment results in additive protective effects on trabecular bone in OVX rats. Our data suggest that a combined treatment with selective ER and AR modulators might be beneficial in the treatment of osteoporosis.
Sesquiterpene Lactone Parthenolide Blocks Lipopolysaccharide-Induced Osteolysis Through the Suppression of NF-κB Activity
Authors: Yip Kirk Ho Man , Zheng Ming H , Feng Hao Tian , Steer James H , Joyce David A , Xu Jiake
Published: April 2006

Effective treatment for bacteria-induced bone lytic diseases is not yet available. In this study, we showed that PAR, an NF-κB inhibitor found in medicinal herbs, can block LPS-induced osteolysis. PAR does this by inhibiting osteoclastogenesis and bone resorption and promoting apoptosis of osteoclasts through the suppression of NF-κB activity.

Introduction: Osteolysis induced by chronic gram-negative bacterial infection underlies many bone diseases such as osteomyelitis, septic arthritis, and periodontitis. Drugs that inhibit lipopolysaccharide (LPS)-induced osteolysis are critically needed for the prevention of bone destruction in infective bone diseases. In this study, we investigated the effect of parthenolide (PAR) on LPS-induced osteolysis in vivo and studied its role in osteoclastogenesis, bone resorption, apoptosis, and NF-κB activity.

Materials and Methods: The LPS-induced osteolysis in the mouse calvarium model was used to examine the effect of PAR in vivo. RANKL-induced osteoclast differentiation from RAW264.7 cells and bone resorption assays were used to assess the effect of PAR in vitro. Assays for NF-κB activation, p65 translocation, and IκB-α degradation were used to determine the mechanism of action of PAR in osteoclasts and their precursors. Flow cytometry and confocal microscopic analysis were used to examine cell apoptosis. Semiquantitative RT-PCR was performed to examine the effect of PAR on gene expression of RANK and TRAF6.

Results: We found that PAR (0.5 and 1 mg/kg), injected simultaneously with LPS (25 mg/kg) or 3 days later, blocked the LPS-induced osteolysis in the mouse calvarium model. In vitro studies showed that low concentrations of PAR (<1 μM) inhibited in vitro osteoclastogenesis and osteoclastic bone resorption, whereas higher concentrations (>5 μM) triggered apoptotic cell death of osteoclasts and their precursor cells in a dose-dependent manner. Furthermore, PAR inhibited LPS-induced NF-κB activation, p65 translocation, and IκB-α degradation both in mature osteoclasts and their precursors in a time- and dose-dependent manner. In addition, PAR inhibited NF-κB activation induced by osteoclastogenic factors RANKL, interleukin (IL)-1β, or TNF-α to varying degrees and reduced the gene expression of RANK and TRAF6.

Conclusion: The NF-κB pathway is known to mediate both osteoclast differentiation and survival. These findings indicate that PAR blocks LPS-induced osteolysis through the suppression of NF-κB activity and suggest that it might have therapeutic value in bacteria-induced bone destruction.
Multiple Genetic Loci From CAST/EiJ Chromosome 1 Affect vBMD Either Positively or Negatively in a C57BL/6J Background
Authors: Edderkaoui Bouchra , Baylink David J , Beamer Wesley G , Wergedal Jon E , Dunn Nancy R , Shultz Kathryn L , Mohan Subburaman
Published: April 2006

Skeletal phenotype analyses of 10 B6.CAST-1 congenic sublines of mice have revealed evidence for the presence of three closely linked QTLs in Chr 1 that influence femoral vBMD both positively and negatively.

Introduction: BMD is an important component of bone strength and a recognized predictor of risk for osteoporotic fracture. Our goal in this study was to fine map the chromosomal location of volumetric BMD (vBMD) quantitative trait loci (QTLs) in mouse distal chromosome 1 (Chr 1).

Materials and Methods: After several backcrosses of the B6.CAST-1T congenic strain, which carried the initial BMD QTL in Chr 1 with B6 mice, the N10F1 generation mice were intercrossed to obtain recombinations that yielded different regions of the QTL. Thirty-eight polymorphic markers were used to fine map the initial 1T QTL region (100-192 Mb). Different skeletal parameters were compared between the 10 sublines and B6 female mice at 16 weeks of age. A t-test was used to determine the significant difference between sublines and B6 control mice, whereas one-way ANOVA and posthoc (Newman-Keuls) tests were performed to compare the phenotype between the sublines.

Results: Significantly higher femur vBMD was found in sublines that carried cast alleles from 100 to 169 and 172 to 185 Mb of the centromere compared with the B6 control mice (10-12%, p < 0.001). However, sublines that carried cast alleles from 185 to 192 Mb showed significantly lower femur vBMD compared with the control mice (−6%, p < 0.05). Furthermore, femur vBMD phenotype showed a negative correlation with endosteal circumference (r = −0.8, p = 0.003), and a strong correlation with cortical thickness for combined data from the 10 sublines (r = 0.97, p < 0.001). Moreover, a high correlation was found between body weight and both periosteal and endosteal circumferences for sublines carrying cast alleles from 167 to 175, 168 to 185, and 169 to 185 Mb, whereas no significant correlation was found between these parameters for sublines carrying cast alleles from 172 to 185 Mb.

Conclusions: Genetic analysis using congenic sublines revealed that the initial BMD QTL on Chr 1 is a complex site with multiple loci affecting bone phenotypes, showing the value of the congenic approach in clearly identifying loci that control specific traits.
Effects of Cyclic Versus Daily hPTH(1-34) Regimens on Bone Strength in Association With BMD, Biochemical Markers, and Bone Structure in Mice
Authors: Iida-Klein Akiko , Hughes Christine , Lu Shi Shou , Moreno Annette , Shen Victor , Dempster David W , Cosman Felicia , Lindsay Robert
Published: April 2006

We developed a cyclic PTH regimen with repeated cycles of 1-week on and off daily PTH injection and explored its effects on bone strength, BMD, bone markers, and bone structure in mice. Cyclic protocols produced 60–85% of the effects achieved by daily protocols with 57% of the total PTH given, indicating more economic use of PTH. The study supports further exploration of cyclic PTH regimens for the treatment of osteoporosis.

Introduction: To minimize the cost and the catabolic action of hPTH(1-34), a cyclic PTH regimen with repeated 3-month cycles of on-and-off daily injection of hPTH(1-34) was developed in humans and shown to be as effective as a daily regimen in increasing vertebral BMD. However, changes in BMD may not adequately predict changes in bone strength. A murine model was developed to explore the efficacy of a cyclic PTH regimen on bone strength in association with other bone variables.

Materials and Methods: Twenty-week-old, intact, female C57BL/J6 mice (n = 7/group) were treated with (1) daily injection with vehicle for 7 weeks (control); (2) daily injection with hPTH(1-34) (40 μg/kg/day) for 7 weeks (daily PTH); and (3) daily injection with hPTH(1-34) and vehicle alternating weekly for 7 weeks (cyclic PTH). BMD was measured weekly by DXA, and serum bone markers, bone structure, and strength were measured at 7 weeks.

Results: Daily and cyclic PTH regimens increased BMD at all sites by 16–17% and 9–12%, respectively (all p < 0.01). The most dramatic effect of cyclic PTH occurred during the second week of treatment when PTH was off, with femoral and tibial BMD continuing to increase to the same extent as that produced by daily PTH. Both daily and cyclic PTH regimens significantly increased osteocalcin (daily, 330%; cyclic, 260%), mTRACP (daily, 145%; cyclic, 70%), femoral cortical width (daily, 23%; cyclic, 13%), periosteal circumference (daily, 5%; cyclic, 3.5%), and bone strength (max load: daily, 48%; cyclic, 28%; energy absorbed: daily, 103%; cyclic, 61%), respectively. Femoral bone strength was positively correlated with BMD, bone markers, and cortical structure. Neither regimen had an effect on vertebral bone strength. Although actual effects of cyclic PTH were 60–85% of those produced by daily PTH, the effects of cyclic PTH per unit amount administered were slightly greater than those of daily PTH for most measures.

Conclusions: PTH-enhanced femoral bone strength is positively correlated with its effects on femoral BMD, bone markers, and bone structure. Cyclic PTH regimens represent a potential economic use of PTH and warrant further study.
Pharmacological Sequestration of Intracellular Cholesterol in Late Endosomes Disrupts Ruffled Border Formation in Osteoclasts
Authors: Zhao Haibo , Väänänen H Kalervo
Published: April 2006

We showed that the ruffled border lacks a late endosomal lipid, LBPA, but is enriched in cholesterol. A hydrophobic amine, U18666A, causes cholesterol accumulation in LBPA+ late endosomes in osteoclasts. Specific targeting of cathepsin K and the vacuolar H+-ATPase at the ruffled border is blocked by U18666A. A membrane trafficking pathway from baso-lateral membrane toward the resorptive organelle is also arrested by the inhibitor. These results indicate cholesterol homeostasis regulates late endosomal/lysosomal trafficking and polarized secretion in resorbing osteoclasts.

Introduction: Protons and acidic proteases are secreted into the resorption lacuna through the ruffled border to solubilize bone mineral and digest the organic bone matrix, respectively. Whereas evidence suggests this event occurs through a vesicular trafficking mechanism, this issue remains unresolved.

Materials and Methods: The distribution of lysobisphosphatidic acid (LBPA) and cholesterol in resorbing osteoclasts was examined by laser scanning confocal microscopy. The effects of U18666A on ruffled border formation were observed by electron microscopy.

Results and Conclusions: The ruffled border does not contain LBPA but is enriched in cholesterol. We found a hydrophobic amine, U18666A, which blocks the efflux of cholesterol from late endosomes in other cells, causes cholesterol accumulation in LBPA-containing late endosomes in osteoclasts, leading to diminished cholesterol at the ruffled border. Reflecting the U18666A-mediated inhibition of late endosome/lysosome transport, the resorptive membrane is disrupted and contains a paucity of cathepsin K and the vacuolar H+-ATPase. These results indicate that the ruffled border is formed by the fusion of lysosomes with the plasma membrane in osteoclasts through a process that is cholesterol regulated.
Correlation of Trabecular Bone Structure with Age, Bone Mineral Density, and Osteoporotic Status: In Vivo Studies in the Distal Radius Using High Resolution Magnetic Resonance Imaging
Authors: Majumdar S. , Genant H. K. , Grampp S. , Newitt D. C. , Truong V.-H. , Lin J. C. , Mathur A.
Published: April 2006

High resolution magnetic resonance (MR) images of the distal radius were obtained at 1.5 Tesla in premenopausal normal, postmenopausal normal, and postmenopausal osteoporotic women. The image resolution was 156 μm in plane and 700 μm in the slice direction; the total imaging time was ∼16 minutes. An intensity-based thresholding technique was used to segment the images into trabecular bone and marrow, respectively. Extensions of standard stereological techniques were used to derive measures of trabecular bone structure from these segmented images. The parameters calculated included apparent measures of trabecular bone volume fraction, trabecular thickness, trabecular spacing, and trabecular number. Fractal-based texture parameters, such as the box-counting dimension, were also derived. Trabecular bone mineral density (BMD) and cortical bone mineral content (BMC) were measured in the distal radius using peripheral quantitative computed tomography (pQCT). In a subset of patients, spinal trabecular BMD was measured using quantitative computed tomography (QCT). Correlations between the indices of trabecular bone structure measured from these high-resolution MR images, age, BMD, and osteoporotic fracture status were examined. Cortical BMC and trabecular BMD at the distal radius, spinal BMD, trabecular bone volume fraction, trabecular thickness, trabecular number, and fractal dimension all decreased with age. Trabecular spacing showed the greatest percentage change and increased with age. In addition, significant differences were evident in spinal BMD, radial trabecular BMD, trabecular bone volume fraction, trabecular spacing, and trabecular number between the postmenopausal nonfracture and the postmenopausal osteoporotic subjects. Trabecular spacing and trabecular number showed moderate correlation with radial trabecular BMD but correlated poorly with radial cortical BMC. High resolution MR imaging, a potentially useful tool for quantifying trabecular structure in vivo, may have applications for understanding and evaluating skeletal changes related to age and osteoporosis.
A Preliminary Evaluation of the Lunar Expert-XL for Bone Densitometry and Vertebral Morphometry
Authors: Lang Thomas , Takada Masahiko , Gee Richard , Wu Chun , Li Jiao , Hayashi-Clark Cynthia , Schoen Susan , March Vesta , Genant Harry K.
Published: April 2006

We have evaluated the Lunar Expert-XL for standard bone mineral densitometry (BMD) and for morphometric imaging of the spine in the lateral projection. The short-term precision in vitro of the Expert-XL for BMD measurements was 0.7% for the Hologic and European spine phantoms, and the long-term stability (15-month) measurements had a 1.1% coefficient of variation. The precision in vivo for three operators examining a group of 10 premenopausal women was 0.9–1.5% for lumbar spine (L2–L4), 1.7–2.2% for the femoral neck, and 0.9% for the total hip region of interest. For a group of nine postmenopausal women, the lumbar spine, femoral neck, and total femur precision ranges were 2.0–2.4, 1.2–2.9, and 1.6–1.7% respectively. For L2–L4, BMD comparison between the Expert-XL and DPX-L yielded a correlation coefficient of r = 0.98, a slope of 0.86, and an intercept of 0.139 g/cm2. The femoral neck results were r = 0.92, slope = 1.00, and intercept = 0.03 g/cm2. In an evaluation of the Expert-XL for lateral morphometry, we employed a group of 16 postmenopausal women. Comparison of vertebral dimensions between the Expert-XL and radiographic morphometry showed strong agreement (r = 0.97), but the interobserver variability for vertebral height was higher for the Expert-XL than for radiographs (3–5% vs. 1–2%). In a subset of four women who had repeat scans, the interscan precision for measuring vertebral dimensions was 1.9, 4.1, and 4.3% for the L1–L4, T12–T8, and T7–T5 levels respectively. In the Expert-XL images, it was possible to identify clearly L4–T4 in 10 of 16 patients and L4–T6 in 15 of 16, indicating potential utility for vertebral fracture prescreening.
Pulsating Fluid Flow Stimulates Prostaglandin Release and Inducible Prostaglandin G/H Synthase mRNA Expression in Primary Mouse Bone Cells
Authors: Klein-Nulend Jenneke , Burger Elisabeth H. , Semeins Cornelis M. , Raisz Lawrence G. , Pilbeam Carol C.
Published: April 2006

Bone tissue responds to mechanical stress with adaptive changes in mass and structure. Mechanical stress produces flow of fluid in the osteocyte lacunar-canalicular network, which is likely the physiological signal for bone cell adaptive responses. We examined the effects of 1 h pulsating fluid flow (PFF; 0.7 ± 0.02 Pa, 5 Hz) on prostaglandin (PG) E2, PGI2, and PGF2α production and on the expression of the constitutive and inducible prostaglandin G/H synthases, PGHS-1, and PGHS-2, the major enzymes in the conversion of arachidonic acid to prostaglandins, using mouse calvarial bone cell cultures. PFF treatment stimulated the release of all three prostaglandins under 2% serum conditions, but with a different time course and to a different extent. PGF2α was rapidly increased 5–10 minutes after the onset of PFF. PGE2 release increased somewhat more slowly (significant after 10 minutes), but continued throughout 60 minutes of treatment. The response of PGI2 was the slowest, and only significant after 30 and 60 minutes of treatment. In addition, PFF induced the expression of PGHS-2 but not PGHS-1. One hour of PFF treatment increased PGHS-2 mRNA expression about 2-fold relative to the induction by 2% fresh serum given at the start of PFF. When the addition of fresh serum was reduced to 0.1%, the induction of PGHS-2 was 8- to 9-fold in PFF-treated cells relative to controls. This up-regulation continued for at least 1 h after PFF removal. PFF also markedly increased PGHS activity, measured as the conversion of arachidonic acid into PGE2. One hour after PFF removal, the production of all three prostaglandins was still enhanced. These results suggest that prostaglandins are important early mediators of the response of bone cells to mechanical stress. Prostaglandin up-regulation is associated with an induction of PGHS-2 enzyme mRNA, which may subsequently provide a means for amplifying the cellular response to mechanical stress.
Strain Gradients Correlate with Sites of Exercise-Induced Bone-Forming Surfaces in the Adult Skeleton
Authors: Judex Stefan , Gross Ted S. , Zernicke Ronald F.
Published: April 2006

Physical activity is capable of increasing adult bone mass. The specific osteogenic component of the mechanical stimulus is, however, unknown. Using an exogenous loading model, it was recently reported that circumferential gradients of longitudinal normal strain are strongly associated with the specific sites of periosteal bone formation. Here, we used high-speed running to test this proposed relation in an exercise model of bone adaptation. The strain environment generated during running in a mid-diaphyseal tarsometatarsal section was determined from triple-rosette strain gages in six adult roosters (>1 year). A second group of roosters was run at a high speed (1500 loading cycles/day) on a treadmill for 3 weeks. Periosteal surfaces were activated in five out of eight animals. Mechanical parameters as well as periosteal activation (as measured by incorporated fluorescent labels) were quantified site-specifically in 12 30° sectors subdividing a mid-diaphyseal section. The amount of periosteal mineralizing surface per sector correlated strongly (R2 = 0.63) with the induced peak circumferential strain gradients. Conversely, peak strain magnitude and peak strain rate were only weakly associated with the sites of periosteal activation. The unique feature of this study is that a specific mechanical stimulus (peak circumferential strain gradients) was successfully correlated with specific sites of periosteal bone activation induced in a noninvasive bone adaptation model. The knowledge of this mechanical parameter may help to design exercise regimens that are able to deposit bone at sites where increased structural strength is most needed.
Mechanism of the Mitogenic Effect of Fluoride on Osteoblast-like Cells: Evidences for a G Protein–Dependent Tyrosine Phosphorylation Process
Authors: Caverzasio Joseph , Palmer Gaby , Suzuki Atsushi , Bonjour Jean-Philippe
Published: April 2006

Recent results indicate that a fluoroalumino complex (AlFx) is probably the molecule responsible for the mitogenic effect of fluoride in MC3T3-E1 osteoblast-like cells. Initial analysis suggested that a tyrosine phosphorylation (tyr phos) process similar to that induced by thrombin and activation of the p42 MAP kinase (ERK 2) mediate this cellular response. In the present study, the signaling mechanism activated by AlFx was further investigated. The results indicated that AlFx dose-dependently enhanced the tyr phos of the cell adhesion proteins FAK and paxillin, as well as of the adaptor molecules p46shc, p52shc, and p66shc and their association with GRB2. Pretreatment of MC3T3-E1 cells with cytochalasin D completely prevented FAK and paxillin tyr phos without any alteration in the tyr phos of Shc proteins and activation of ERK2 induced by AlFx. This observation suggests that in confluent MC3T3-E1 cells, there is no link between the activation of FAK induced by AlFx and the stimulation of ERK2. Pretreatment of the cells with pertussis toxin inhibited Shc phosphorylation, activation of ERK2, and markedly reduced cell replication induced by AlFx. This toxin also significantly reduced the stimulation of Pi transport activity induced by AlFx in these cells. Alteration in tyr phos induced by AlFx was not associated with any detectable inhibition of tyrosine phosphatase activity in MC3T3-E1 cell homogenates, suggesting that enhanced tyr phos induced by AlFx probably resulted from activation of a tyrosine kinase. In conclusion, the results of this study suggest that the mitogenic effect of fluoride in MC3T3-E1 osteoblast-like cells is mediated by the activation of a pertussis toxin–sensitive Gi/o protein and suggest an important role for these heterotrimeric G proteins in controlling the growth and differentiation of bone-forming cells.
Parathyroid Hormone Up-Regulation of Connexin 43 Gene Expression in Osteoblasts Depends on Cell Phenotype
Authors: Schiller Paul C. , Roos Bernard A. , Howard Guy A.
Published: April 2006

Accumulating evidence indicates that gap junctions, primarily composed of connexin 43 (Cx43), are distributed extensively throughout bone. We have previously reported that in osteoblastic cells parathyroid hormone (PTH) increases both the steady-state levels of transcripts for Cx43 and gap-junctional intercellular communication in a process involving cyclic adenosine monophosphate (cAMP). We now present data showing that the mechanism of stimulation of Cx43 gene expression by PTH involves an increased rate of Cx43 gene transcription without affecting Cx43 transcript stability in UMR 106 osteoblastic cells. Activation of the protein kinase C pathway is not involved in this process. Inhibiting translation consistently decreases the PTH-mediated stimulation of Cx43 gene expression at all the times we tested (1–3 h). However, this effect is only partial, demonstrating that de novo protein synthesis is required for full stimulation. PTH increases the steady-state levels of Cx43 mRNA in several osteoblastic cell lines, albeit to different levels. We were unable to detect PTH stimulation in ROS 17/2.8 osteoblastic cells, suggesting that the effect of PTH on Cx43 gene expression may depend on the developmental state of the cell along the osteoblastic differentiation pathway. In the MC3T3-E1 preosteoblastic cell line, we find that PTH increases Cx43 gene expression in proliferating and maturing osteoblastic cells, but not in nondividing, differentiated osteoblasts, where the basal level of Cx43 gene expression is elevated. Unlike PTH, the osteotropic hormones 1,25-dihydroxyvitamin D3 and 17β-estradiol do not appear to affect Cx43 gene expression in UMR 106 osteoblastic cells.
Establishment of an Osteocyte-like Cell Line, MLO-Y4
Authors: Kato Yoichi , Windle Jolene J. , Koop Barbara A. , Mundy Gregory R. , Bonewald Lynda F.
Published: April 2006

Although osteocytes are the most abundant cells in bone, their functional role remains unclear. In part, this is due to lack of availability of osteocyte cell lines which can be studied in vitro. Since others have shown that cell lines can be readily developed from transgenic mice in which the SV40 large T-antigen oncogene is expressed under the control of a promoter which targets the cells of interest, we used this approach to develop an osteocyte cell line. We chose as a promoter osteocalcin, whose expression is essentially limited to bone cells and which is expressed more abundantly in osteocytes than in osteoblasts. From these transgenic mice, we isolated cells from the long bones using sequential collagenase digestion and maintained these cells on collagen-coated surfaces which are optimal for osteocyte maintenance and growth. We describe here the properties of a cell line cloned from these cultures, called MLO-Y4 (for murine long bone osteocyte Y4). The properties of MLO-Y4 cells are very similar to primary osteocytes. Like primary osteocytes and unlike primary osteoblasts, the cell line produces large amounts of osteocalcin but low amounts of alkaline-phosphatase. The cells produce extensive, complex dendritic processes and are positive for T-antigen, for osteopontin, for the neural antigen CD44, and for connexin 43, a protein found in gap junctions. This cell line also produces very small amounts of type I collagen mRNA compared with primary osteoblasts. MLO-Y4 cells lack detectable mRNA for osteoblast-specific factor 2, which appears to be a positive marker for osteoblasts but may be a negative marker for osteocytes. This newly established cell line should prove useful for studying the effects of mechanical stress on osteocyte function and for determining the means whereby osteocytes communicate with other bone cells such as osteoblasts and osteoclasts.
Unweighting Accelerates Tidemark Advancement in Articular Cartilage at the Knee Joint of Rats
Authors: Dr. O'Connor Kim M.
Published: April 2006

Morphologic development of articular cartilage is influenced by biologic adaptation to functional demands. It has been theorized that intermittent stresses generated by load bearing and motion determine cartilage thickness by controlling advancement of the subchondral mineralization front. The mineralization front is comprised of two interfaces, the tidemark and chondroosseus junction, each of which advances through a different biologic process. This study was designed to evaluate the influence of one month of hind limb unweighting, with and without concurrent restriction of joint motion, on mineral apposition at the tidemark and vascular invasion at the chondroosseus junction in the knee joints of young adult rats. In mobile joints, hind limb unweighting induced a 2-fold increase in the tidemark mineral apposition rate (p = 0.0001) at the primary weight bearing region, resulting in a thinning of the uncalcified cartilage layer and a concurrent thickening of the calcified layer. Cast immobilization negated the effect of unweighting at the tidemark while it activated subchondral vascular encroachment into the calcified cartilage (p = 0.001). These findings suggest that cartilage thinning associated with the elimination of weight bearing is mediated through a different biological mechanism than cartilage loss associated with restriction of joint motion.
The Usefulness of Bone Turnover in Predicting the Response to Transdermal Estrogen Therapy in Postmenopausal Osteoporosis
Authors: Dr. Gonnelli S. , Cepollaro C. , Pondrelli C. , Martini S. , Monaco R. , Gennari C.
Published: April 2006

Transdermal estrogen therapy is now an accepted form of treatment for postmenopausal osteoporosis. Ninety postmenopausal osteoporotic women were randomized to receive either transdermal estrogen (0.05 mg/day 17β-estradiol) and calcium (n = 45) or calcium alone (n = 45). The study period was 2 years. Bone mineral density (BMD) at the lumbar spine (by dual-energy X-ray absorptiometry [DXA]) and markers of bone turnover (alkaline phosphatase, osteocalcin, hydroxyproline, pyridinoline cross-links) were assessed at baseline and after 1 and 2 years. In the estrogen-treated group, BMD showed a significant increase (p < 0.001) both after 1 and 2 years, with a reduction in biochemical markers. To investigate the effectiveness of estrogen treatment of postmenopausal osteoporosis in relation to bone turnover, we also divided the patients on the basis of bone turnover, as assessed by measurement of whole body retention (WBR) of99mTc-methylene diphosphonate. WBR revealed that 26 patients had high bone turnover (HT) and 55 had low bone turnover (LT). The response to estrogen was greater in the HT patients than in the LT patients; in fact BMD increased by 5.7 and 6.6% in HT patients and by 2.6 and 2.7% in LT patients after 1 and 2 years, respectively. In conclusion, the present study demonstrates that, while the BMD decreases in the patients treated with calcium alone, 2-year treatment with transdermal estrogen increases axial BMD and that the response to estrogen treatment is influenced by bone turnover. Therefore, the evaluation of bone turnover may be useful to identify those postmenopausal osteoporotic women who may especially benefit from treatment with estrogen.
New Aspects of Endochondral Ossification in the Chick: Chondrocyte Apoptosis, Bone Formation by Former Chondrocytes, and Acid Phosphatase Activity in the Endochondral Bone Matrix
Authors: Roach Helmtrud I.
Published: April 2006

A detailed histological study of the growth plates from 9− to 20-day-old embryonic chick long bones was carried out with the aim of clarifying the long-debated question of the fate of the hypertrophic chondrocytes. Since resorption in chick bones does not occur synchronously across the plate as it does in mammals, specialized regions develop and the fate of the chondrocyte depends on its location within the growth plate. Where resorption took place, as at the sites of primary vascular invasion or at the main cartilage/marrow interface, chondrocytes underwent apoptosis before the lacunae were opened. In addition, spontaneous apoptosis of chondrocytes occurred at apparently random sites throughout all stages of chondrocyte differentiation. In older chick bones, a thick layer of endochondral bone matrix covered the cartilage edge. This consisted of type I collagen and the typical noncollagenous bone proteins but, in addition, contained tartrate-resistant acid phosphatase in the mineralized matrix. Where such matrix temporarily protected the subjacent cartilage from resorption, chondrocytes differentiated to bone-forming cells and deposited bone matrix inside their lacunae. At sites of first endochondral bone formation, some chondrocytes underwent an asymmetric cell division resulting in one daughter cell which underwent apoptosis, while the other cell remained viable and re-entered the cell cycle. This provided further support for the notion that chondrocytes as well as marrow stromal cells give rise to endochondral osteoblasts.
Influence of Physical Activity on Vertebral Deformity in Men and Women: Results from the European Vertebral Osteoporosis Study
Authors: Silman A. J. , O'Neill T. W. , Cooper C. , Kanis J. , Felsenberg D.
Published: April 2006

Physical activity is associated with an increased bone mass and a reduced risk of hip fracture. There are, however, no data from population samples of men and women concerning the effect of regular levels of physical activity on the risk of vertebral deformity. The aim of this study was to determine the relationship between regular physical activity and vertebral deformity in European men and women. A population survey method was used. Thirty-six centers from 19 European countries participated. Each center recruited a population sample of men and women aged 50 years and over. Those who took part received an interviewer-administered questionnaire and lateral thoracolumbar radiographs. Subjects were asked about two dimensions of physical activity: (1) the level of physical activity undertaken either at work or at home on a daily basis at three different age periods: 15–25 years, 25–50 years, and 50+ years; and (2) the amount of time spent walking or cycling out of doors each day. Spinal radiographs were evaluated morphometrically and the presence of vertebral deformity was defined according to the McCloskey method. In total, 14,261 subjects, aged 50–79 years, from 30 centers were studied, of whom 809 (12.0%) men and 884 (11.7%) women had one or more deformities. After adjusting for age, center, smoking, and body mass index, very heavy levels of activity in all three age groups were associated with an increased risk of vertebral deformity in men (odds ratios, age adjusted [OR], 1.5–1.7; with all 95% confidence intervals [CI] excluding unity). No increased risk was observed in women. Current walking or cycling more than ½ h/day was associated with a reduced risk of vertebral deformity in women (OR 0.8; 95% CI 0.7–1.0). We conclude that regular walking in middle-aged and elderly women is associated with a reduced risk of vertebral deformity. By contrast, heavy levels of physical activity in early and middle adult life are associated with an increased risk in men. These differences are of relevance in understanding the epidemiology of vertebral deformity and planning programs of prevention.
Involvement of Different Second Messengers in Parathyroid Hormone– and Interleukin-1–Induced Interleukin-6 and Interleukin-11 Production in Human Bone Marrow Stromal Cells
Authors: Kim Ghi S. , Kim Chul H. , Choi Cheol S. , Park Joong Y. , Lee Ki-Up
Published: April 2006

Previous studies have suggested that increased secretion of bone active cytokines, such as interleukin-6 (IL-6) and interleukin-11 (IL-11), from osteoblasts and stromal cells play a pivotal role in the activation of osteoclasts and the genesis of osteoporosis. Various systemic and local factors can stimulate IL-6/IL-11 production, but the intracellular mechanism for such stimulation is largely unknown. In this study, we characterized the second messenger signaling in parathyroid hormone (PTH)- and IL-1–induced production of IL-6/IL-11 and studied the possible modulating effects of estrogen. rhPTH(1–34) and rhIL-1α dose-dependently stimulated IL-6 and IL-11 production from human bone marrow stromal cells (hBMSCs). Agonists for protein kinase A (PKA) (forskolin), and protein kinase C (PKC) (phorbol 12-myristate 13-acetate; PMA) also stimulated IL-6/IL-11 production. Rp-diastereoisomer of adenosine cyclic 3′,5′-phosphorothioate (Rp-cAMPS) and H-8, inhibitors of PKA, significantly inhibited PTH-stimulated IL-6/IL-11 production, but did not inhibit IL-1–stimulated IL-6/IL-11 production. In contrast, staurosporine and calphostin C, inhibitors of PKC, suppressed IL-1–stimulated, but not PTH-stimulated, IL-6/IL-11 production. Pretreatment of cells with 17β-estradiol (17β-E2) antagonized IL-1–stimulated IL-6 production. However, PTH-stimulated IL-6 production and IL-1– and PTH-stimulated IL-11 production were not affected by 17β-E2. Similarly, 17β-E2 inhibited PMA-stimulated IL-6 production, whereas neither forskolin-stimulated IL-6/IL-11 production nor PMA-stimulated IL-11 production was affected by 17β-E2. These results indicate that different second messengers are involved in PTH- and IL-1–induced IL-6 and IL-11 production by hBMSCs: PTH and IL-1 stimulate IL-6/IL-11 production via a PKA-dependent and PKC-dependent pathway, respectively. Furthermore, our results suggest that regulation of cytokine production by estrogen in hBMSCs is selective; only the IL-1–induced IL-6 production, which is mediated by PKC pathway, is inhibited, but PTH-induced IL-6 production and PTH/IL-1–induced IL-11 production are not inhibited by estrogen.
Association Between Intestinal Vitamin D Receptor, Calcium Absorption, and Serum 1,25 Dihydroxyvitamin D in Normal Young and Elderly Women
Authors: Kinyamu H. Karimi , Gallagher J. Christopher , Prahl Jean M. , Deluca Hector F. , Petranick Kimberly M. , Lanspa Stephen J.
Published: April 2006

The exact mechanism for the decrease in intestinal calcium absorption with age is not yet understood. A decrease with age in serum 1,25-dihydroxyvitamin D (1,25(OH)2D) or a decrease in the intestinal vitamin D receptor (VDR) protein concentration are possible causes. The objective of this study was to examine the effect of age on these factors. Fifty-nine young women age 25–35 years were compared with 41 elderly women age 65–83 years who underwent measurements of VDR, calcium absorption using a 20 mg and 100 mg calcium carrier, and calciotropic hormones. Calcium absorption by both tests was lower in the elderly women compared with the young women (p < 0.05). Serum 1,25(OH)2D and duodenal VDR protein concentration were not significantly different between the two age groups. Serum 1,25(OH)2D correlated with the 20 mg calcium absorption test in both young (r = 0.35, p < 0.007) and elderly women (r = 0.58, p < 0.0001) and with the 100 mg calcium absorption in the elderly (r = 0.32; p < 0.05). VDR did not correlate with calcium absorption in young women or elderly women, nor did VDR correlate with serum 1,25(OH)2D and serum 25-hydroxyvitamin D. In summary, the decrease in calcium absorption cannot be explained by a decrease in intestinal VDR. The correlation between serum 1,25(OH)2D and both calcium absorption tests only accounts for 12–30% of the variance in the age-related change in the calcium absorption tests. Other factors, not yet understood, are responsible for the decline in calcium absorption with age.
Calcaneal Bone Mineral Density Predicts Fracture Occurrence: A Five-Year Follow-up Study in Elderly People
Authors: Cheng Sulin , Suominen Harri , Sakari-Rantala Ritva , Laukkanen Pia , Avikainen Veikko , Heikkinen Eino
Published: April 2006

A 5-year follow-up study investigated calcaneal bone mineral density (BMD) and changes in BMD in relation to fracture occurrence. The subjects comprised two cohorts born in 1914 and 1910 living in the city of Jyväskylä in central Finland. One hundred and three men (82%) and 188 women (73%), aged 75, and 57 men (74%) and 136 women (65%), aged 80, of the eligible population participated in the baseline bone measurements. The follow-up bone measurements were obtained for 59 men (68%) and 119 women (66%), aged 80 years, and for 21 men (53%) and 61 women (48%), aged 85 years. During the follow-up period, 8 men and 36 women from the younger and 11 men and 24 women from the older cohort sustained at least one fracture. When the baseline levels of BMD were related to fracture occurrence, the results clearly showed that with increased BMD values the probability of fracture decreased. Where men and women had similar BMD values, they also had a similar fracture probability. Except for one woman in the older cohort, none of those who had initial BMD values more than 1 standard deviation above the mean for their age developed a fracture during the follow-up period. The mean annual decrease in BMD was greater in the women (2.5–2.7%) than in the men (0.8–1.0%). The BMD change tended to associate with fracture occurrence only in the 75-year-old women (p = 0.075). The results suggest that calcaneus BMD can be used as a predictor of fracture occurrence in 75− to 80-year-old men and women. However, associating fractures with the change in BMD was difficult due to the limited number of survivors and initial differences in BMD values.
Down-Regulation of Gap Junctional Intercellular Communication Between Osteoblastic MC3T3-E1 Cells by Basic Fibroblast Growth Factor and a Phorbol Ester (12-O-Tetradecanoylphorbol-13-Acetate)
Authors: Shiokawa-Sawada Miho , Mano Hiroshi , Hanada Kooji , Kakudo Shinji , Kameda Takashi , Miyazawa Koshi , Nakamaru Yukiya , Yuasa Tatsuhisa , Mori Yoshihisa , Kumegawa Masayoshi , Hakeda Yoshiyuki
Published: April 2006

To address the relation between osteoblast growth and cell-to-cell communication, we examined the effects of basic fibroblast growth factor (bFGF) and 12-O-tetradecanoylphorbol-13-acetate (TPA), both potent stimulators of osteoblastic proliferation, on gap junctional intercellular communication between osteoblastic MC3T3-E1 cells. The level of intercellular communication was estimated by a photobleaching method. TPA inhibited the degree of intercellular communication in two different time-dependent manners. The early (<1 h) inhibition by TPA was consistent with an increase in the phosphorylation of connexin 43 (Cx43). The later inhibition was caused by reduction in the total amount of Cx43 on the plasma membrane, due to the decrease in the level of Cx43 transcripts. These qualitative and quantitative modulations by TPA were inhibited by a selective inhibitor of protein kinase C, GF109203X. bFGF also attenuated the gap junctional intercellular communication. However, short exposure (<5 h) to bFGF did not affect the communication. The fact that the growth factor immediately stimulated the phosphorylation of Cx43 indicates that the phosphorylation site(s) affected by bFGF was not involved in the inhibition of communication. The decrease in the intercellular communication level was detected by the longer exposure (>8 h) to bFGF and paralleled the decline in the Cx-mRNA level. This inhibitory effect of bFGF was abolished by the addition of a tyrosine kinase inhibitor, herbimycin A. Thus, gap junctional intercellular communication between osteoblasts was down-regulated by osteoblastic mitogens through different mechanisms of the modulation of Cx43.