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Bone loss associated with microgravity exposure poses a significant barrier to long-duration spaceflight. Osteoprotegerin-Fc (OPG-Fc) is a receptor activator of nuclear factor kappa-B ligand (RANKL) inhibitor that causes sustained inhibition of bone resorption after a single subcutaneous injection. We tested the ability of OPG-Fc to preserve bone mass during 12 days of spaceflight (SF). 64-day-old female C57BL/6J mice (n=12/group) were injected subcutaneously with OPG-Fc (20 mg/kg) or an inert vehicle (VEH), 24 hours prior to launch. Ground control (GC) mice (VEH or OPG-Fc) were maintained under environmental conditions that mimicked those in the space shuttle middeck. Age-matched baseline (BL) controls were sacrificed at launch. GC/VEH, but not SF/VEH mice, gained tibia BMD and trabecular volume fraction (BV/TV) during the mission (p

Bone is a complex tissue that provides mechanical support for muscles and joints, protection for vital organs, a mineral reservoir that is essential for calcium homeostasis, and the environment and niches required for haematopoiesis. The regulation of bone mass in mammals is governed by a complex interplay between bone-forming cells termed osteoblasts and bone-resorbing cells termed osteoclasts, and is guided physiologically by a diverse set of hormones, cytokines and growth factors. The balance between these processes changes over time, causing an elevated risk of fractures with age. Osteoclasts may also be activated in the cancer setting, leading to bone pain, fracture, spinal cord compression and other significant morbidities. This Review chronicles the events that led to an increased understanding of bone resorption, the elucidation of the signalling pathway mediated by osteoprotegerin, receptor activator of NF-κB (RANK) and RANK ligand (RANKL) and its role in osteoclast biology, as well as the evolution of recombinant RANKL antagonists, which culminated in the development of the therapeutic RANKL-targeted antibody denosumab.

The physiological role of Dickkopf-1 (Dkk1) during postnatal bone growth in rodents and in adult rodents was examined utilizing an antibody to Dkk1 (Dkk1-Ab) that blocked Dkk1 binding to both low density lipoprotein receptor-related protein 6 (LRP6) and Kremen2, thereby preventing the Wnt inhibitory activity of Dkk1. Treatment of growing mice and rats with Dkk1-Ab resulted in a significant increase in bone mineral density because of increased bone formation. In contrast, treatment of adult ovariectomized rats did not appreciably impact bone, an effect that was associated with decreased Dkk1 expression in the serum and bone of older rats. Finally, we showed that Dkk1 plays a prominent role in adult bone by mediating fracture healing in adult rodents. These data suggest that, whereas Dkk1 significantly regulates bone formation in younger animals, its role in older animals is limited to pathologies that lead to the induction of Dkk1 expression in bone and/or serum, such as traumatic injury. © 2011 American Society for Bone and Mineral Research

The development of bone-rebuilding anabolic agents for treating bone-related conditions has been a long-standing goal. Genetic studies in humans and mice have shown that the secreted protein sclerostin is a key negative regulator of bone formation. More recently, administration of sclerostin-neutralizing monoclonal antibodies in rodent studies has shown that pharmacologic inhibition of sclerostin results in increased bone formation, bone mass, and bone strength. To explore the effects of sclerostin inhibition in primates, we administered a humanized sclerostin-neutralizing monoclonal antibody (Scl-AbIV) to gonad-intact female cynomolgus monkeys. Two once-monthly subcutaneous injections of Scl-AbIV were administered at three dose levels (3, 10, and 30 mg/kg), with study termination at 2 months. Scl-AbIV treatment had clear anabolic effects, with marked dose-dependent increases in bone formation on trabecular, periosteal, endocortical, and intracortical surfaces. Bone densitometry showed that the increases in bone formation with Scl-AbIV treatment resulted in significant increases in bone mineral content (BMC) and/or bone mineral density (BMD) at several skeletal sites (ie, femoral neck, radial metaphysis, and tibial metaphysis). These increases, expressed as percent changes from baseline were 11 to 29 percentage points higher than those found in the vehicle-treated group. Additionally, significant increases in trabecular thickness and bone strength were found at the lumbar vertebrae in the highest-dose group. Taken together, the marked bone-building effects achieved in this short-term monkey study suggest that sclerostin inhibition represents a promising new therapeutic approach for medical conditions where increases in bone formation might be desirable, such as in fracture healing and osteoporosis.

Administration of active vitamin D sterols to treat secondary hyperparathyroidism in patients with chronic kidney disease receiving dialysis has been associated with elevated serum calcium and phosphorus levels, which may lead to increased risk of vascular calcification. However, calcimimetics, by binding to the parathyroid gland calcium-sensing receptors, reduce serum parathyroid hormone, calcium, phosphorus, and the calcium-phosphorus product. Using cultured bovine aorta vascular smooth muscle cells (BASMCs), an in vitro model of vascular calcification, we compared calcification levels and gene expression profiles after exposure to the phosphate source ss-glycerolphosphate (BGP), the active vitamin D sterols calcitriol and paricalcitol, the calcimimetic R-568, or BGP with the active vitamin D sterols or R-568. Cells exposed to BGP (10 mM) alone or with calcitriol or paricalcitol showed dose-dependent BASMC calcification. No change in calcification was observed in cultures exposed to BGP with R-568, consistent with the observed lack of calcium-sensing receptor expression. Microarray analysis using total cellular RNA from cultures exposed to vehicle or BGP in the absence and presence of 10(-8) M calcitriol or paricalcitol for 7 days showed that cells exposed to BGP with calcitriol or BGP with paricalcitol had virtually identical gene expression profiles, which differed from those of cells treated with BGP or vehicle alone. Several osteoblast- and chondrocyte-associated genes were modulated by BGP and vitamin D exposure. In this study, exposure of BASMCs to phosphate and active vitamin D sterols induced calcification and changes in expression of genes associated with mineralized tissue.

Osteoprotegerin (OPG) acts by neutralizing the receptor activator of nuclear factor-kappaB ligand (RANKL), the primary mediator of osteoclast differentiation, function, and survival. We examined whether OPG could affect the bone loss associated with chronic kidney disease (CKD) in a rodent model of CKD and secondary hyperparathyroidism (SHPT). SHPT was induced in rats by 5/6 nephrectomy (5/6 Nx) and a 1.2% P/0.6% Ca(2+) diet. Starting 1 week after 5/6 Nx, rats were treated with vehicle (veh) or OPG-Fc (3 mg/kg, intravenously) every 2 weeks for 9 weeks. At baseline, 3, 6, and 9 weeks, blood was taken and bone mineral density (BMD) and bone mineral content (BMC) were assessed by dual-energy X-ray absorptiometry. Serum parathyroid hormone (sPTH) levels reached 912 pg/ml in 5/6 Nx rats vs. 97 pg/ml in shams at 9 weeks. OPG-Fc had no effect on sPTH or Ca(2+) levels throughout the 9-week study, indicating that SHPT was a renal effect independent of bone changes. At 3 weeks, 5/6 Nx-veh rats had osteopenia compared with sham-veh rats and 5/6 Nx-OPG-Fc rats had significantly higher percent changes in whole-body BMC, leg BMD, and lumbar BMD versus 5/6 Nx-veh rats. By 6-9 weeks, elevated sPTH was associated with reversal of bone loss and osteitis fibrosa in the proximal tibial metaphysis. OPG-Fc decreased this sPTH-driven high bone turnover, resulting in augmented thickness of proximal tibial trabeculae in 5/6 Nx rats. Thus, RANKL inhibition with OPG-Fc can block the deleterious effects of continuously elevated sPTH on bone, suggesting that RANKL may be an important therapeutic target for protecting bone in patients with CKD and SHPT.

The progressive depletion of skeletal muscle is a hallmark of many types of advanced cancer and frequently is associated with debility, morbidity, and mortality. Muscle wasting is primarily mediated by the activation of the ubiquitin-proteasome system, which is responsible for degrading the bulk of intracellular proteins. E3 ubiquitin ligases control polyubiquitination, a rate-limiting step in the ubiquitin-proteasome system, but their direct involvement in muscle protein catabolism in cancer remains obscure. Here, we report the full-length cloning of E3α-II, a novel “N-end rule” ubiquitin ligase, and its functional involvement in cancer cachexia. E3α-II is highly enriched in skeletal muscle, and its expression is regulated by proinflammatory cytokines. In two different animal models of cancer cachexia, E3α-II was significantly induced at the onset and during the progression of muscle wasting. The E3α-II activation in skeletal muscle was accompanied by a sharp increase in protein ubiquitination, which could be blocked by arginine methylester, an E3α-selective inhibitor. Treatment of myotubes with tumor necrosis factor α or interleukin 6 elicited marked increases in E3α-II but not E3α-I expression and ubiquitin conjugation activity in parallel. E3α-II transfection markedly accelerated ubiquitin conjugation to endogenous cellular proteins in muscle cultures. These findings show that E3α-II plays an important role in muscle protein catabolism during cancer cachexia and suggest that E3α-II is a potential therapeutic target for muscle wasting.

Calcimimetic compounds, which activate the parathyroid cell Ca(2+) receptor (CaR) and inhibit parathyroid hormone (PTH) secretion, are under experimental study as a treatment for hyperparathyroidism. This report describes the salient pharmacodynamic properties, using several test systems, of a new calcimimetic compound, cinacalcet HCl. Cinacalcet HCl increased the concentration of cytoplasmic Ca(2+) ([Ca(2+)](i)) in human embryonic kidney 293 cells expressing the human parathyroid CaR. Cinacalcet HCl (EC(50) = 51 nM) in the presence of 0.5 mM extracellular Ca(2+) elicited increases in [Ca(2+)](i) in a dose- and calcium-dependent manner. Similarly, in the presence of 0.5 mM extracellular Ca(2+), cinacalcet HCl (IC(50) = 28 nM) produced a concentration-dependent decrease in PTH secretion from cultured bovine parathyroid cells. Using rat medullary thyroid carcinoma 6-23 cells expressing the CaR, cinacalcet HCl (EC(50) = 34 nM) produced a concentration-dependent increase in calcitonin secretion. In vivo studies in rats demonstrated cinacalcet HCl is orally bioavailable and displays approximately linear pharmacokinetics over the dose range of 1 to 36 mg/kg. Furthermore, this compound suppressed serum PTH and blood-ionized Ca(2+) levels and increased serum calcitonin levels in a dose-dependent manner. Cinacalcet was about 30-fold more potent at lowering serum levels of PTH than it was at increasing serum calcitonin levels. The S-enantiomer of cinacalcet (S-AMG 073) was at least 75-fold less active in these assay systems. The present findings provide compelling evidence that cinacalcet HCl is a potent and stereoselective activator of the parathyroid CaR and, as such, might be beneficial in the treatment of hyperparathyroidism.

This portion of the study quantified the effects of a 12-day space shuttle mission (Space Transport System-108/UF-1) on body and lymphoid organ masses, activation marker expression, cytokine secretion, and erythrocyte and thrombocyte characteristics in C57BL/6 mice. Animals in flight (Flt group) had 10–12% lower body mass compared with ground controls housed either in animal enclosure modules or under standard vivarium conditions ( P < 0.001) and the smallest thymus and spleen masses. Percentages of CD25+lymphocytes, CD3+/CD25+T cells, and NK1.1+/CD25+natural killer cells from Flt mice were higher compared with both controls ( P < 0.05). In contrast, CD71 expression was depressed in the Flt and animal enclosure module control mice compared with vivarium control animals ( P < 0.001). Secretion of interferon-γ, IL-2, and IL-4, but not tumor necrosis factor-α and IL-5, by splenocytes from Flt mice was decreased relative to either one or both ground controls ( P < 0.05). Flt mice also had high red blood cell and thrombocyte counts compared with both sets of controls; low red blood cell volume and distribution width, percentage of reticulocytes, and platelet volume were also noted ( P < 0.05) and were consistent with dehydration. These data indicate that relatively short exposure to the spaceflight environment can induce profound changes that may become significant during long-term space missions.

Background. Reactive oxygen species (ROS) and glutathione (GSH) depletion contribute to organ injury after bone marrow transplantation (BMT). Keratinocyte growth factor (KGF) ameliorates graft-versus-host disease (GVHD)-associated organ injury in murine BMT models. Methods. B10.BR mice received total body irradiation (TBI; day −1) ± cyclophosphamide (Cy; 120 mg/kg/day i.p., days −3 and −2), then were transplanted on day 0 with C57BL/6 bone marrow + spleen cells as a source of GVHD-causing T cells. KGF (5 mg/kg/day subcutaneously [s.c.]) or saline was given on days −6, −5, and −4. Lung and liver GSH and oxidized GSH disulfide (GSSG) levels were measured on days 0 and 5 and glutathione redox potential (Eh) calculated. Organ malondialdehyde (MDA) was determined on day 5 as an index of ROS-mediated lipid peroxidation. Results. In lung, TBI+BMT oxidized GSH Eh and increased MDA. Cy further oxidized lung GSH Eh. In liver, neither BMT regimen altered GSH redox status or MDA. KGF prevented the decrease in lung GSH after TBI+Cy and decreased lung MDA after both TBI and TBI+Cy. KGF increased liver GSH levels and GSH Eh after TBI and GSH Eh after TBI+Cy. Conclusions. In murine allogeneic BMT, TBI oxidizes the lung GSH redox pool and Cy exacerbates this response by 5 days post-BMT. In contrast, liver GSH redox status is maintained under these experimental conditions. KGF treatment attenuates the Cy-induced decrease in lung GSH, decreases post-BMT lung lipid peroxidation, and improves liver GSH redox indices. KGF may have a therapeutic role to prevent or attenuate GSH depletion and ROS-mediated organ injury in BMT.

SALL1 is a mammalian homolog of the Drosophila region-specific homeotic gene spalt ( sal ); heterozygous mutations in SALL1 in humans lead to Townes-Brocks syndrome. We have isolated a mouse homolog of SALL1 ( Sall1 ) and found that mice deficient in Sall1 die in the perinatal period and that kidney agenesis or severe dysgenesis are present. Sall1 is expressed in the metanephric mesenchyme surrounding ureteric bud; homozygous deletion of Sall1 results in an incomplete ureteric bud outgrowth, a failure of tubule formation in the mesenchyme and an apoptosis of the mesenchyme. This phenotype is likely to be primarily caused by the absence of the inductive signal from the ureter, as the Sall1 -deficient mesenchyme is competent with respect to epithelial differentiation. Sall1 is therefore essential for ureteric bud invasion, the initial key step for metanephros development.

This study examines the ability of osteoprotegerin (OPG) to prevent the local bone resorption caused by sciatic nerve damage. Sixty-five 18-week-old male mice were assigned to one of six groups (n = 10-11/group). A baseline control group was sacrificed on day zero of the 10-day study. The remaining groups were placebo sham operated, placebo nerve crush (Plac NC) operated, 0.1 mg/kg/day OPG + nerve crush (LOW), 0.3 mg/kg/day OPG + nerve crush (MED), and 1.0 mg/kg/day OPG + nerve crush (HI). Nerve crush or sham operations were performed on the right leg. The left leg served as a contralateral control to the nerve crushed (ipsilateral) leg. The difference in mass between the right and left femur and tibia was examined. Additionally, quantitative histomorphometry was performed on the right and left femur and tibia diaphyses. Nerve crush resulted in a significant loss of bone mass in the ipsilateral side compared to the contralateral side. Bone mass for the ipsilateral bones of the Plac NC group were significantly reduced by 3.8% in the femur and 3.5% in the tibia compared to the contralateral limb. The percent diminution was reduced for OPG treated mice compared to the Plac NC group for both the femur and tibia. In the femur, the percent reduction of ipsilateral bone mass was reduced to 1.0% (LOW), 1.3% (MED) and 1.6% (HI) compared to the contralateral limb. In the tibia, loss of bone mass in the ipsilateral limb was reduced to 1.4% (LOW), 1.4% (MED), and 2.4% (HI) compared to the contralateral. OPG also decreased the amount of tibial endocortical resorption compared to the Plac NC group. In summary, OPG mitigated bone loss caused by damage to the sciatic nerve.

Osteoprotegerin (OPG) is a naturally secreted protein that decreases bone resorption by inhibiting osteoclast differentiation and activation while promoting osteoclast apoptosis [8]. In this study, the effects of osteoprotegerin injections on long bone mechanical and material properties were investigated in young male Sprague-Dawley rats. OPG increased fracture strength at the femur mid-diaphysis in three-point bending by 30%, without affecting the elastic or maximum strength. At the femoral neck, OPG significantly increased the elastic (45%), maximum (15%), and fracture (35%) strengths. There was not a difference in microhardness at the femur mid-diaphysis in comparing the placebo and OPG groups. There were, however, significant increases in whole bone dry mass (25%), mineral mass (30%), organic mass (17%), and percent mineralization (4%); percent mineralization at the mid-diaphysis (3%); and percent mineralization at the distal epiphysis (6%) due to the OPG treatment. While OPG decreased endocortical bone formation (52%), total bone area, endocortical bone area, and periosteal bone formation were maintained with OPG treatment. A 30% increase in the X-ray opacity of the bone at the proximal metaphysis of the right tibiae was observed. Overall, OPG increased mineralization and strength indices in the rat femur. Its effects on strength were more pronounced in the femoral neck than at the mid-diaphysis.

Osteoprotegerin and osteoprotegerin ligand have recently been identified as novel proteins that inhibit and stimulate, respectively, osteoclast formation. We examined the possibility that osteoprotegerin would inhibit cancer-induced osteoclastogenesis and cancer growth in bone. An experimental model was used in which osteolytic tumors are known to stimulate osteoclastogenesis and grow in femora of osteoclast-deficient mice (op/op). Osteoprotegerin treatment decreased the number of osteoclasts by 90% (p < 0.0007) at sites of tumor in a dose-dependent manner and decreased bone tumor area by greater than 90% (p < 0.003). The mechanisms through which osteoprotegerin decreased osteoclast formation in tumor-bearing animals included (a) an osteoprotegerin-mediated, systemic reduction in the number of splenic and bone marrow-residing osteoclast precursor cells, (b) a decrease in the number of osteoclast precursor cells at sites of tumor as detected by cathepsin K and receptor activator of NFkappaB mRNA expression, and (c) a decrease in osteoprotegerin ligand mRNA at sites of tumor. These findings suggest that osteoprotegerin treatment, in addition to having direct antagonistic effects on endogenous osteoprotegerin ligand, decreases the number of osteoclast precursors and reduces production of osteoprotegerin ligand at sites of osteolytic tumor.

Osteoclast precursors (OCPs) circulate in the mononuclear fraction of peripheral blood (PB), but their abundance and surface characteristics are unknown. Previous studies suggest that the receptor activator for NF-kappaB (RANK) on cytokine-treated OCPs in mouse bone marrow interacts with osteoprotegerin ligand (OPGL/TRANCE/RANKL/ODF) to initiate osteoclast differentiation. Hence, we used a fluorescent form of human OPGL (Hu-OPGL-F) to identify possible RANK-expressing OCPs in untreated peripheral blood mononuclear cells (PBMCs) using fluorescence-activated cell sorting analysis. Monocytes [CD14-phycoerythrin (PE) antibody (Ab) positive (+) cells, 10-15% of PBMCs] all (98-100%) co-labelled with Hu-OPGL-F (n > 18). T lymphocytes (CD3-PE Ab+ cells, 66% of PBMCs) did not bind Hu-OPGL-F; however, B cells (CD19-PE Ab+ cells, 9% of PBMCs) were also positive for Hu-OPGL-F. All Hu-OPGL-F+ monocytes also co-labelled with CD33, CD61, CD11b, CD38, CD45 and CD54 Abs, but not CD34 or CD56 Abs. Hu-OPGL-F binding was dose dependent and competed with excess Hu-OPGL. When Hu-OPGL-F+, CD14-PE Ab+, CD33-PE Ab+, Hu-OPGL-F+/CD14-PE Ab+ or Hu-OPGL-F+/CD33-PE Ab+ cells were cultured with OPGL (20 ng/ml) and colony-stimulating factor (CSF)-1 (25 ng/ml), OC-like cells readily developed. Thus, all freshly isolated monocytes demonstrate displaceable Hu-OPGL-F binding, suggesting the presence of RANK on OCPs in PB; also, OCPs within a purified PB monocyte population form osteoclast-like cells in the complete absence of other cell types in OPGL and CSF-1 containing medium.

Osteoprotegerin-ligand (OPGL) is a key osteoclast differentiation/activation factor essential for bone remodeling. We report that mice lacking OPGL or its receptor RANK fail to form lobulo-alveolar mammary structures during pregnancy, resulting in death of newborns. Transplantation and OPGL-rescue experiments in opgl−/− and rank−/− pregnant females showed that OPGL acts directly on RANK-expressing mammary epithelial cells. The effects of OPGL are autonomous to epithelial cells. The mammary gland defect in female opgl−/− mice is characterized by enhanced apoptosis and failures in proliferation and PKB activation in lobulo-alveolar buds that can be reversed by recombinant OPGL treatment. These data provide a novel paradigm in mammary gland development and an evolutionary rationale for hormonal regulation and gender bias of osteoporosis in females.

High systemic levels of osteoprotegerin (OPG) in OPG transgenic mice cause osteopetrosis with normal tooth eruption and bone elongation and inhibit the development and activity of endosteal, but not periosteal, osteoclasts. We demonstrate that both intravenous injection of recombinant OPG protein and transgenic overexpression of OPG in OPG−/2 mice effectively rescue the osteoporotic bone phenotype observed in OPG-deficient mice. However, intravenous injection of recombinant OPG over a 4-wk period could not reverse the arterial calcification observed in OPG−/− mice. In contrast, transgenic OPG delivered from mid-gestation through adulthood does prevent the formation of arterial calcification in OPG−/− mice. Although OPG is normally expressed in arteries, OPG ligand (OPGL) and receptor activator of NF-κB (RANK) are not detected in the arterial walls of wild-type adult mice. Interestingly, OPGL and RANK transcripts are detected in the calcified arteries of OPG−/− mice. Furthermore, RANK transcript expression coincides with the presence of multinuclear osteoclast-like cells. These findings indicate that the OPG/OPGL/RANK signaling pathway may play an important role in both pathological and physiological calcification processes. Such findings may also explain the observed high clinical incidence of vascular calcification in the osteoporotic patient population.

We previously reported that keratinocyte growth factor (KGF) is up-regulated by the action of progesterone (P) in the primate endometrium, and we suggested that this protein is a likely mediator of P-dependent stromal-epithelial paracrine interactions in this tissue. At the end of the menstrual cycle, P levels fall, and the abundance of endometrial KGF transcripts decreases approximately 9-fold. In macaques, withdrawal of P induces the luteal-follicular transition (LFT), marked by menstrual sloughing of the functionalis zone and apoptotic regression of the basalis zone. Because KGF levels fall so dramatically during the LFT, we hypothesized that replacement with exogenous KGF during the LFT would prevent some of the endometrial changes seen after P withdrawal. Here we describe two studies of the effects of exogenously administered KGF during the LFT in rhesus macaques. In one experiment we administered KGF systemically to ovariectomized, juvenile rhesus macaques during an LFT induced by hormonal manipulations. KGF had dramatic proliferative effects on the bladder and salivary glands, known targets of KGF, but did not affect cell proliferation in the endometrium or block menstrual sloughing and bleeding. However, KGF strongly inhibited apoptosis in the basalis zone, increased glandular sacculation and folding in this zone, and had a marked trophic effect on the spiral arteries. In the second experiment we installed oviductal catheters in ovariectomized adult rhesus macaques and infused KGF directly into the uterine lumen during a hormonally induced LFT. Again, arteriotrophic, antiapoptotic, and basalis gland sacculation effects were observed in the absence of any effect on cell proliferation. We concluded that although KGF is mitogenic for many epithelial cell types, it does not play this role in the primate endometrium. Its most important roles may be to stimulate spiral artery growth and inhibit glandular apoptosis during the nonfertile menstrual cycle. Because its expression rises coincident with the time of implantation and because spiral arteries are essential to successful establishment of pregnancy, the role of KGF in the fertile menstrual cycle deserves further study.

Bone remodelling and bone loss are controlled by a balance between the tumour necrosis factor family molecule osteoprotegerin ligand (OPGL) and its decoy receptor osteoprotegerin (OPG)1,2,3. In addition, OPGL regulates lymph node organogenesis, lymphocyte development and interactions between T cells and dendritic cells in the immune system3,4,5. The OPGL receptor, RANK, is expressed on chondrocytes, osteoclast precursors and mature osteoclasts4,6. OPGL expression in T cells is induced by antigen receptor engagement7, which suggests that activated T cells may influence bone metabolism through OPGL and RANK. Here we report that activated T cells can directly trigger osteoclastogenesis through OPGL. Systemic activation of T cells in vivo leads to an OPGL-mediated increase in osteoclastogenesis and bone loss. In a T-cell-dependent model of rat adjuvant arthritis characterized by severe joint inflammation, bone and cartilage destruction and crippling, blocking of OPGL through osteoprotegerin treatment at the onset of disease prevents bone and cartilage destruction but not inflammation. These results show that both systemic and local T-cell activation can lead to OPGL production and subsequent bone loss, and they provide a novel paradigm for T cells as regulators of bone physiology.

Recent studies have identified osteoprotegerin ligand (OPG-L) as the essential factor required for osteoclastogenesis, and that the effects are prevented by its soluble receptor, osteoprotegerin (OPG). However, there are limited data at present on the regulation of OPG-L expression in human osteoblastic cells by other cytokines. Because interleukin (IL)-1beta, tumor necrosis factor (TNF)-alpha, and IL-6 all increase osteoclastogenesis, we assessed whether OPG-L mRNA steady-state levels were regulated by these cytokines in human osteoblastic cells. By northern analysis, IL-1beta (5 nmol/L) and TNF-alpha (9 nmol/L) increased OPG-L mRNA steady-state levels by up to two- to three-fold in normal marrow stromal cells (MS), an immortalized marrow stromal cell line (hMS), and the osteosarcoma cell line, MG-63, whereas IL-6 (2 nmol/L, with or without its soluble receptor) had no effect on OPG-L mRNA levels in any of these cells. IL-1beta and TNF-alpha increased OPG-L mRNA steady-state levels in the normal MS cells and the hMS cell line in a time- and dose-dependent fashion by up to 4.1-fold and up to 2.6-fold, respectively. Our data are thus consistent with the hypothesis that the proinflammatory and bone-resorbing cytokines, IL-1beta and TNF-alpha, but not IL-6, may stimulate osteoclastogenesis by inducing the expression of OPG-L.

Osteoprotegerin (OPG) and its ligand (OPGL) negatively and positively regulate osteoclastogenesis in the mouse. OPG inhibits osteoclastogenesis by sequestering its ligand, OPGL, the osteoclast differentiation and activation factor. This study demonstrates the effects of soluble muOPGL and huOPG on the developing human osteoclast phenotype, on bone slices, using peripheral blood mononuclear cells (PBMCs), cultured for 2 weeks, without stromal cells. OPGL (2-50 ng/ml), in combination with CSF-1, hydrocortisone (HC), and 1,25(OH)2D3, increases the size of osteoclast-like cells on bone, as defined by the acquisition of osteoclast markers: vitronectin receptor (VR), tartrate-resistant acid phosphatase (TRAP), multinuclearity, and bone resorption. By 14 days, with 20 ng/ml OPGL, the largest cells/10x field have achieved an average diameter of 163+/-38 microm, but only approximately 10-20 microm in its absence and the number of osteoclast-like cells/mm2 bone surface is about 128. By scanning electron microscopy, OPGL-treated (20-ng/ml) cultures contain small osteoclast-like cells on bone with ruffled "apical" surfaces by day 7; by day 15, large osteoclast-like cells are spread over resorption lacunae. At 15 ng/ml OPGL, about 37% of the bone slice area is covered by resorption lacunae. OPG (5-250 ng/ml) antagonizes the effects of OPGL on the morphology of the osteoclast-like cells that form, as well as bone erosion. For cells grown on plastic, Cathepsin K mRNA levels, which are barely detectable at plating, are elevated 7-fold, by 5 days, in the presence, not the absence, of OPGL (20 ng/ml) + CSF-1 (25 ng/ml). Similar findings are observed in experiments performed in the absence of HC and 1,25(OH)2D3, indicating that HC and 1,25(OH)2D3 are not needed for OPGL-induced osteoclast differentiation. In conclusion, this study confirms a pivotal role for OPGL and OPG in the modulation of human osteoclast differentiation and function, suggesting a use for OPG for treating osteoclast-mediated bone disease in humans.

To investigate the effects of keratinocyte growth factor (KGF) on the structure of the stratified squamous epithelium of the tongue, buccal mucosa and oesophagus of normal and irradiated mice.Female BDF1 mice were exposed to total body irradiation from a caesium source. The irradiated mice and normal, unirradiated mice were injected with 5 mg/kg per day KGF or vehicle. Thickness and proliferation in the epithelium were measured.KGF caused epithelial thickening of the non-keratinized layers in oral epithelium in normal mice. It increased the number of nucleated layers and influenced differentiation of post-mitotic cells in the upper layers by increasing the size and number of keratohyalin granules, and the number of desmosomes. Single and fractionated doses of radiation caused inhibition of proliferation as detected by markedly reduced BrdU incorporation following exposure, followed by epithelial atrophy. KGF treatment of mice reversed the inhibition of proliferation and atrophy that occurred in control irradiated mice.These data show that KGF reverses epithelial atrophy in mouse oral cavity caused by irradiation and suggest that KGF may be useful for the treatment of mucositis of the upper aerodigestive tract of patients treated with aggressive regimens of radiation therapy.

Purpose Predict the intravascular distribution of carbon dioxide during angiography. Materials and Methods Mathematical modeling was used to predict the flow pattern of CO 2 in a pulsatile system as a function of the CO 2 flow rate. Findings were validated in an in vitro pulsatile circuit. Results The annular flow pattern with filling of nearly the entire lumen with CO 2 is the most desirable, followed by intermittent bubble flow (provided individual bubbles are large). Stratified flow relates to a continuous floating CO 2 bubble. Configuration of the CO 2 bolus depends on fluid properties, fluid velocity, flow rates, mean intraluminal pressure, pressure amplitude, pulse rate, and vessel diameter. In vessels with less than 10-mm inner diameter, annular flow can be achieved relatively easily with injection rates above 20–30 mL/sec. Higher rates are not expected to produce superior results. When imaging a 2-cm artery, the best that can be realized clinically is intermittent flow with large bubbles. Bubbles size increases with increasing CO 2 flow rate. In aneurysms, only stratified flow can be achieved with reasonable injection rates. Periodicity of the flow patterns is determined by the pulsatile circuit and can produce indentations in the CO 2 bolus, which can be mistaken for stenoses. Conclusions Flow regime maps can be used to optimize bolus configuration during CO 2 angiography.

Keratinocyte growth factor (KGF) is important in tissue repair and wound healing and its administration can abrogate chemical- and radiation-induced tissue damage in rodents. We investigated KGF as a therapeutic agent for the prevention of graft-versus-host disease (GVHD)-induced tissue damage, morbidity, and mortality in an established murine allogeneic bone marrow transplantation (BMT) model. B10.BR (H2k) recipient mice were lethally irradiated and transplanted with C57BL/6 (H2b) bone marrow (BM) with spleen cells (BMS) as a source of GVHD-causing T cells. KGF-treated mice (5 mg/kg/d subcutaneously days −6, −5, and −4 pre-BMT) receiving BMS exhibited better survival than those not receiving KGF (P = .0027). Cyclophosphamide (Cy), a common component of total body irradiation (TBI)-containing regimens, was administered to other cohorts of mice at a dose of 120 mg/kg/d intraperitoneally on days −3 and −2 before BMT. KGF-treated mice again exhibited a better survival rate than those not receiving KGF (P = .00086). However, KGF-treated recipients receiving TBI or Cy/TBI BMS were not GVHD-free, as shown by lower body weights compared with BM groups. GVHD target tissues were assessed histologically during a 38-day post-BMT observation period. KGF ameliorated GVHD-induced tissue damage in the liver, skin, and lung (completely in some recipients) and moderately so in the spleen, colon, and ileum, even with Cy conditioning. These studies demonstrate that KGF administration, completed before conditioning, has potential as an anti-GVHD therapeutic agent.

Keratinocyte growth factor (KGF) promotes epithelial growth and differentiation and has potent effects on the liver. The coinjection of lipopolysaccharide (LPS) and d -galactosamine (GalN) results in hepatic failure in mice. Mechanistically, LPS-induced tumor necrosis factor (TNF) triggers hepatocyte apoptosis, which is enhanced by GalN-arrested transcription. Similarly, the combination of TNF and actinomycin D (ActD) causes hepatocyte apoptosis in vitro. We studied the effect of KGF on LPS and GalN-induced hepatic failure in vivo and on TNF- and ActD-induced hepatocyte apoptosis in vitro, where it was compared with those of hepatic growth factor (HGF) and epidermal growth factor (EGF). Mice treated with human recombinant KGF (1 mg/kg subcutaneously) 24 hours before intraperitoneal coinjection of LPS and GalN sustained prolonged survival compared with control mice, although overall mortality was not changed. The counts of apoptotic hepatocytes, serum alanine and aspartate transaminases, and DNA fragments in the cytosolic fraction of liver homogenates were higher in control mice than in treated mice 6 hours after LPS and GalN coinjection, before any mortality occurred. In vitro, hepatocytes pretreated with KGF exhibited reduced TNF- and ActD-induced cell damage and DNA fragmentation, similar to hepatocytes pretreated with HGF and EGF. In conclusion, KGF prolongs survival during LPS- and GalN-induced hepatic failure by temporarily protecting hepatocytes against apoptosis. It also protects hepatocytes in vitro against TNF- and ActD-induced apoptosis.

The ligand for osteoprotegerin has been identified, and it is a TNF-related cytokine that replaces the requirement for stromal cells, vitamin D3, and glucocorticoids in the coculture model of in vitro osteoclastogenesis. OPG ligand (OPGL) binds to a unique hematopoeitic progenitor cell that is committed to the osteoclast lineage and stimulates the rapid induction of genes that typify osteoclast development. OPGL directly activates isolated mature osteoclasts in vitro, and short-term administration into normal adult mice results in osteoclast activation associated with systemic hypercalcemia. These data suggest that OPGL is an osteoclast differentiation and activation factor. The effects of OPGL are blocked in vitro and in vivo by OPG, suggesting that OPGL and OPG are key extracellular regulators of osteoclast development.

The effect of chronic expression of flt3 ligand (FL) on in vivo hematopoiesis was studied. Retroviral vector-mediated gene transfer was used in a mouse model of bone marrow transplantation to enforce expression of mouse FL cDNA in hematopoietic tissues. As early as 2 weeks posttransplantation, peripheral blood white blood cell counts in FL-overexpressing recipients were significantly elevated compared with controls. With the exception of eosinophils, all nucleated cell lineages studied were similarly affected in these animals. Experimental animals also exhibited severe anemia and progressive loss of marrow-derived erythropoiesis. All of the FL-overexpressing animals, but none of the controls, died between 10 and 13 weeks posttransplantation. Upon histological examination, severe splenomegaly was noted, with progressive fibrosis and infiltration by abnormal lymphoreticular cells. Abnormal cell infiltration also occurred in other organ systems, including bone marrow and liver. In situ immunocytochemistry on liver sections showed that the cellular infiltrate was CD3+/NLDC145+/CD11c+, but B220− and F4/80−, suggestive of a mixed infiltrate of dendritic cells and activated T lymphocytes. Infiltration of splenic blood vessel perivascular spaces resulted in vascular compression and eventual occlusion, leading to splenic necrosis consistent with infarction. These results show that FL can affect both myeloid and lymphoid cell lineages in vivo and further demonstrate the potential toxicity of in vivo treatment with FL.

Megakaryocyte growth and development factor (MGDF) has recently been identified as a ligand for the c-mpl receptor. Using retroviral- mediated gene transfer, MGDF has been overexpressed in mice to evaluate the systematic effects due to chronic exposure to this growth factor. MGDF overexpressing mice had more rapid platelet recovery than control mice after transplantation. Following this recovery, the platelet levels continued increasing to fourfold to eightfold above normal baseline levels and remained elevated (five-fold above control mice) in these animals, which are alive and well at more than 4 months posttransplantation. Increased megakaryocyte numbers were detected in a number of organs in these mice including bone marrow, spleen, liver, and lymph nodes. Prolonged overexpression of MGDF led to decreased marrow hematopoiesis, especially erythropoiesis, with a shift to extramedullary hematopoiesis in the spleen and liver. All the MGDF overexpressing mice analyzed to date developed myelofibrosis and osteosclerosis, possibly induced by megakaryocyte and platelet produced cytokines. No significant effect on other hematopoietic lineages was seen in the MGDF overexpressing mice, showing that the stimulatory effect of MGDF in vivo is restricted to the megakaryocyte lineage.

Purpose: To determine the effect of keratinocyte growth factor (KGF), a mesenchymally derived epithelial growth factor that can cause proliferation of pulmonary, gastrointestinal and mammary epithelia, on urothelium.Materials and Methods: Recombinant human KGF was systemically administered to rats and Rhesus monkeys, and the proliferative effects on the bladder were evaluated.Results: Keratinocyte growth factor causes proliferation of transitional epithelial cells. Proliferating cell nuclear antigen (PCNA) expression in rat bladder is dramatically increased along the basal layer of urothelium 1, 3, 7 and 14 days after daily injections of KGF. Incorporation of 5-bromodeoxyuridine (BrdU) at 7 and 14 days in the urothelium of KGF-treated rats parallels PCNA immunoreactivity and confirms that KGF increases DNA synthesis in urothelial cells. Urothelial cell proliferation is accompanied histologically by an increase in mitotic activity. Keratinocyte growth factor-induced PCNA expression is reversible up...

Therapeutic enhancement of fracture healing would help to prevent the occurrence of orthopedic complications such as nonunion and revision surgery. Sclerostin is a negative regulator of bone formation, and treatment with a sclerostin monoclonal antibody (Scl-Ab) results in increased bone formation and bone mass in animal models. Our objective was to investigate the effects of systemic administration of Scl-Ab in two models of fracture healing. In both a closed femoral fracture model in rats and a fibular osteotomy model in cynomolgus monkeys, Scl-Ab significantly increased bone mass and bone strength at the site of fracture. After 10 weeks of healing in nonhuman primates, the fractures in the Scl-Ab group had less callus cartilage and smaller fracture gaps containing more bone and less fibrovascular tissue. These improvements at the fracture site corresponded with improvements in bone formation, bone mass, and bone strength at nonfractured cortical and trabecular sites in both studies. Thus the potent anabolic activity of Scl-Ab throughout the skeleton also was associated with an anabolic effect at the site of fracture. These results support the potential for systemic Scl-Ab administration to enhance fracture healing in patients.

The biologically active metabolite of vitamin D3, 1,25 (OH)2 D3, exerts important immunoregulatory effects in addition to being a central mediator of calcium/phosphate metabolism. Utilizing an interleukin 1 responsive murine T cell line and 125I-interleukin 1 alpha, we show that 1,25 (OH)2 D3 (5,50 nM) enhanced 125I-interleukin 1 alpha binding up to almost 2-fold over control. This 1,25 (OH)2 D3 effect occurred in a dose-dependent manner and was detectable after 24 h but not before 7 h of culture. Scatchard analysis of 125I-interleukin 1 alpha binding data demonstrated that 1,25 (OH)2 D3 enhanced interleukin 1 receptor number without a significant change in affinity. The biologically less potent metabolite of vitamin D3, 25 (OH) D3, also augmented 125I-interleukin 1 alpha binding but at steroid levels 2-3 log orders greater than 1,25 (OH)2 D3. This observation, combined with the presence of high-affinity 3H-1,25 (OH)2 D3 receptors (88 sites/cell, K = 0.45 nM) in cytosolic extracts, strongly suggests that the nuclear vitamin D receptor mediates this steroid's effect on interleukin 1 receptor expression. Based on the capacity of an anti-type 1 interleukin 1 receptor monoclonal antibody (35F5) to block 1,25 (OH)2 D3-enhanced 125I-interleukin 1 alpha binding, we conclude that this steroid augments type 1 interleukin 1 receptor expression. When combined with interleukin 1, a cytokine that also impacts MD10 interleukin 1 receptor expression, 1,25 (OH)2 D3 enhanced interleukin 1 receptor expression. Northern blots hybridized with a 32P-type 1 interleukin 1 receptor cDNA probe show that 1,25 (OH)2 D3 enhanced type 1 interleukin 1 receptor steady state mRNA levels. Functionally, 1,25 (OH)2 D3 pretreatment augmented the MD10 proliferative response to suboptimal levels of interleukin 1 (100 fM interleukin 1 alpha). These findings further support 1,25 (OH)2 D3's role as an immunoregulatory molecule and provides a possible mechanism by which this steroid could potentiate certain immune activities.

The development of bone-rebuilding anabolic agents for potential use in the treatment of bone loss conditions, such as osteoporosis, has been a long-standing goal. Genetic studies in humans and mice have shown that the secreted protein sclerostin is a key negative regulator of bone formation, although the magnitude and extent of sclerostin's role in the control of bone formation in the aging skeleton is still unclear. To study this unexplored area of sclerostin biology and to assess the pharmacologic effects of sclerostin inhibition, we used a cell culture model of bone formation to identify a sclerostin neutralizing monoclonal antibody (Scl-AbII) for testing in an aged ovariectomized rat model of postmenopausal osteoporosis. Six-month-old female rats were ovariectomized and left untreated for 1 yr to allow for significant estrogen deficiency-induced bone loss, at which point Scl-AbII was administered for 5 wk. Scl-AbII treatment in these animals had robust anabolic effects, with marked increases in bone formation on trabecular, periosteal, endocortical, and intracortical surfaces. This not only resulted in complete reversal, at several skeletal sites, of the 1 yr of estrogen deficiency-induced bone loss, but also further increased bone mass and bone strength to levels greater than those found in non-ovariectomized control rats. Taken together, these preclinical results establish sclerostin's role as a pivotal negative regulator of bone formation in the aging skeleton and, furthermore, suggest that antibody-mediated inhibition of sclerostin represents a promising new therapeutic approach for the anabolic treatment of bone-related disorders, such as postmenopausal osteoporosis.

Introduction: Sclerosteosis is a rare high bone mass genetic disorder in humans caused by inactivating mutations in SOST, the gene encoding sclerostin. Based on these data, sclerostin has emerged as a key negative regulator of bone mass. We generated SOST knockout (KO) mice to gain a more detailed understanding of the effects of sclerostin deficiency on bone. Materials and Methods: Gene targeting was used to inactivate SOST and generate a line of SOST KO mice. Radiography, densitometry, μCT, histomorphometry, and mechanical testing were used to characterize the impact of sclerostin deficiency on bone in male and female mice. Comparisons were made between same sex KO and wildtype (WT) mice. Results: The results for male and female SOST KO mice were similar, with differences only in the magnitude of some effects. SOST KO mice had increased radiodensity throughout the skeleton, with general skeletal morphology being normal in appearance. DXA analysis of lumbar vertebrae and whole leg showed that there was a significant increase in BMD (>50%) at both sites. μCT analysis of femur showed that bone volume was significantly increased in both the trabecular and cortical compartments. Histomorphometry of trabecular bone revealed a significant increase in osteoblast surface and no significant change in osteoclast surface in SOST KO mice. The bone formation rate in SOST KO mice was significantly increased for trabecular bone (>9-fold) at the distal femur, as well as for the endocortical and periosteal surfaces of the femur midshaft. Mechanical testing of lumbar vertebrae and femur showed that bone strength was significantly increased at both sites in SOST KO mice. Conclusions:SOST KO mice have a high bone mass phenotype characterized by marked increases in BMD, bone volume, bone formation, and bone strength. These results show that sclerostin is a key negative regulator of a powerful, evolutionarily conserved bone formation pathway that acts on both trabecular and cortical bone.

Skeletal anabolism with PTH is achieved through daily injections that result in brief exposure to the peptide. We hypothesized that similar anabolic effects could be achieved with less frequent but more sustained exposures to PTH. A PTH-Fc fusion protein with a longer half-life than PTH(1-34) increased cortical and cancellous BMD and bone strength with once- or twice-weekly injections. Introduction: The anabolic effects of PTH are currently achieved with, and thought to require, daily injections that result in brief exposure to the peptide. We hypothesized that less frequent but more sustained exposures to PTH could also be anabolic for bone, provided that serum levels of PTH were not constant. Materials and Methods: PTH(1-34) was fused to the Fc fragment of human IgG1 to increase the half-life of PTH. Skeletal anabolism was examined in mice and rats treated once or twice per week with this PTH-Fc fusion protein. Results: PTH-Fc and PTH(1-34) had similar effects on PTH/PTHrP receptor activation, internalization, and signaling in vitro. However, PTH-Fc had a 33-fold longer mean residence time in the circulation of rats compared with that of PTH(1-34). Subcutaneous injection of PTH-Fc once or twice per week resulted in significant increases in bone volume, density, and strength in osteopenic ovariectomized mice and rats. These anabolic effects occurred in association with hypercalcemia and were significantly greater than those achiev- able with high concentrations of daily PTH(1-34). PTH-Fc also significantly improved cortical bone volume and density under conditions where daily PTH(1-34) did not. Antiresorptive co-therapy with estrogen further enhanced the ability of PTH-Fc to increase bone mass and strength in ovariectomized rats. Conclusions: These results challenge the notion that brief daily exposure to PTH is essential for its anabolic effects on cortical and cancellous bone. PTH-derived molecules with a sustained circulating half-life may represent a powerful and previously undefined anabolic regimen for cortical and cancellous bone. J Bone Miner Res 2007;22:1534-1547. Published online on June 18, 2007; doi: 10.1359/JBMR.070616

Mutations affecting the activity of the Wnt co-receptors LRP5 and LRP6 that cause alterations in skeletal biology confirmed the involvement of Wnt signaling in bone formation. We evaluated the potential role of Dkk1, an inhibitor of LRP5/6 activity, in bone formation by examining the normal expression pattern of Dkk1 in normal young mice and by assessing the consequences of osteoblast overexpression of Dkk1 in transgenic mice. Endogenous Dkk1 expression was detected primarily in osteoblasts and osteocytes. Transgenic over-expression of Dkk1 using two different rat collagen 1A1 promoters resulted in distinct bone phenotypes. More widespread Dkk1 expression (driven by the Col1A1 3.6 kb promoter) yielded osteopenia with forelimb deformities and hairlessness, while expression restricted to osteoblasts (driven by the Col1A1 2.3 kb promoter) induced severe osteopenia without limb defects or alopecia. The decrease in bone mass in vivo resulted from a significant 49% reduction in osteoblast numbers and was reflected in a 45% reduction in serum osteocalcin concentration; an in vitro study revealed that Dkk1 caused a dose-dependent suppression of osteoblast matrix mineralization. These data indicate that Dkk1 may directly influence bone formation and suggest that osteopenia develops in mice over-expressing Dkk1 at least in part due to diminished bone formation resulting from reduced osteoblast numbers.

Background Calcitriol treatment of secondary hyperparathyroidism (HPT) in chronic kidney disease (CKD) patients can lead to increased serum calcium and phosphorus, which have been associated as risk factors for vascular calcification. Cinacalcet HCl (Sensipar/Mimpara) {(alphaR)-(-)-alpha-methyl-N-[3-[3-(trifluoromethylphenyl)propyl]-1-napthalenemethanamine hydrochloride} lowers serum parathyroid hormone (PTH), calcium, phosphorus and calcium-phosphorous (CaxP) product in stage 5 CKD dialysis patients; however, its effects on vascular calcification are unknown. Methods Cinacalcet HCl (10 or 1 mg/kg, p.o. gavage), 1,25-dihydroxyvitamin D(3) (0.1 microg, s.c, calcitriol) or the combination was administered daily for 26 days in a rat model of secondary HPT [5/6 nephrectomy]. After dosing, aortic calcification was determined using the von Kossa staining method. Serum PTH and blood chemistries were determined on days 0, 26 and 0, 14, 26, respectively, prior to and after dosing. Results Calcitriol-treated rats had moderate to marked aortic calcification, whereas no significant calcification was observed in vehicle- or cinacalcet HCl-only treated groups. Co-administration of cinacalcet HCl with calcitriol did not attenuate the calcitriol-mediated increase in CaxP product or calcitriol-mediated aortic calcification. Both calcitriol and cinacalcet HCl therapy significantly reduced serum PTH levels. Calcitriol significantly elevated serum calcium, serum phosphorous and CaxP product above pretreatment levels, or those seen with vehicle or cinacalcet HCl. Cinacalcet HCl (10 or 1 mg/kg) decreased serum ionized calcium and decreased calcitriol-induced hypercalcaemia. Conclusion Cinacalcet HCl and calcitriol both effectively reduce PTH, albeit via different mechanisms, but unlike calcitriol, cinacalcet HCl did not produce hypercalcaemia, an increased CaxP product or vascular calcification.

Cinacalcet HCl attenuates parathyroid hyperplasia in a rat model of secondary hyperparathyroidism . Background Secondary hyperparathyroidism (HPT) in chronic kidney disease (CKD) is a physiologic response to kidney failure characterized by elevated serum parathyroid hormone (PTH) levels and parathyroid gland enlargement. Calcimimetic agents acting through allosteric modification of the calcium-sensing receptor (CaR) can attenuate parathyroid hyperplasia in rats with secondary HPT. The present study explores the effects of the calcimimetic cinacalcet HCl on parathyroid hyperplasia, apoptosis, and PTH secretion in a rat model of secondary HPT. Methods Cinacalcet HCl was gavaged daily (1, 5, or 10 mg/kg) for 4 weeks starting 6 weeks post-5/6 nephrectomy. After dosing, hyperplasia was determined using parathyroid weight and proliferating cell nuclear antigen (PCNA) immunochemistry. Apoptosis was determined using in situ techniques. Serum PTH (1–34) and blood chemistries were determined throughout the course of the study. Results Administration of cinacalcet HCl (5 or 10 mg/kg) significantly reduced the number of PCNA-positive cells and decreased parathyroid weight compared with vehicle-treated 5/6 nephrectomized rats. There was no difference in apoptosis from cinacalcet HCl–treated or vehicle-treated animals. Serum PTH and blood ionized calcium levels decreased in cinacalcet HCl–treated animals compared with vehicle-treated controls. Conclusion The results confirm previous work demonstrating that calcimimetic agents attenuate the progression of parathyroid hyperplasia in subtotally nephrectomized rats, extending earlier observations to now include cinacalcet HCl. These results support a role for the CaR in regulating parathyroid cell proliferation. Therefore, cinacalcet HCl may represent a novel therapy for improving the management of secondary HPT.

Background and aims: Transfer of CD4 + CD45RB Hi T cells into semi syngeneic immunodeficient mice represents a model of inflammatory bowel disease (IBD). As patients with IBD often suffer from osteopenia, we studied if this T cell transfer in mice results in osteopenia in addition to colitis, and if treatment with osteoprotegerin (OPG) has effects on the bone mineral density of T cell transferred mice. We also investigated whether osteopenia was due to malabsorption as a result of a dysregulated digestive tract or as a consequence of the inflammatory process. Methods: CD4 + CD45RB Hi or CD4 + CD45RB Lo T cells (4×10 5 ) were sorted from CB6F1 and transferred into C.B.17 scid/scid mice. Recipient mice were treated with human IgG1 Fc (control) or Fc-OPG three times per week in a prophylactic regimen as well as a therapeutic regimen (after 10% body weight loss) and were evaluated for osteopenia and colitis. Results: Mice that received CD4 + CD45RB Hi T cells developed osteopenia (as indicated by decreased bone density accompanied by decreased osteoblasts and increased osteoclasts) and colitis (as indicated by histological changes in the large intestine). Mice that received CD4 + CD45RB Lo T cells developed neither osteopenia nor colitis. All animals consumed, on average, the same amount of food and water over the course of the study. Prophylactic treatment with Fc-OPG increased bone density in mice that received either CD4 + CD45RB Hi or CD4 + CD45RB Lo T cells but had no effects on the gastrointestinal tract. Fc-OPG treatment of osteopenic mice with established IBD caused the normalisation of bone density. Osteopenia in CD4 + CD45RB Hi T cell recipients was accompanied by hypoparathyroidism that was partially normalised by treatment with Fc-OPG. CD4 + CD45RB Hi T cell recipients also had a bone marrow inflammatory cell infiltrate expressing tumour necrosis factor α which was unaffected by treatment with Fc-OPG. Conclusions: CD4 + CD45RB Hi T cell transfer results in osteopenia in addition to colitis. Evidence suggests that this osteopenia was induced by inflammatory cell infiltration and not by malabsorption of calcium. Recombinant human osteoprotegerin effectively treated the osteopenia. OPG may be a useful therapeutic option for treating osteopenia in patients with IBD.

This study examined whether the calcium-sensing receptor (CaR) is expressed in normal adult human osteoblastic and osteoclastic cells in culture, and whether the calcimimetic, cinacalcet HCl (AMG 073), potentiates the effects of calcium (via CaR, or some other receptor/mechanism). When mouse or human osteoblastic cells were treated with higher concentrations of calcium (6.6 or 8.6 mM in alpha-MEM/10% FBS) than present in control cultures (1.6 mM), the previously well-documented increase in cell number was demonstrated. Cinacalcet HCl affected cell proliferation of CHO cells transfected with CaR, dose dependently, but had no effect on human or mouse osteoblastic cell proliferation in calcium-containing medium (1.6 or 8.6 mM). To test cinacalcet HCl and calcium on osteoclastic cells, peripheral blood mononuclear cells were cultured in medium containing RANK ligand and M-CSF, supplemented with calcium, and/or cinacalcet HCl. Tartrate-resistant acid phosphatase-positive multinucleated osteoclastic cells on plastic or bone were then counted at 11 and 21 days, respectively. Calcium (greater than 6.0 mM) inhibited osteoclast formation, but cinacalcet HCl (30-1000 nM) had no effect on osteoclastic formation or resorption in the presence of calcium (1.6 or 6.1 mM). RT-PCR did not detect CaR in human, rat, or mouse primary osteoblastic cells and cell lines or osteoclastic cells. In conclusion, these studies indicate that the calcium-induced increase in osteoblastic cell number, and the decrease in formation/function of osteoclastic cells, involves a mechanism or receptor other than CaR. In addition, the calcimimetic agent did not potentiate the effects of calcium on normal adult human bone cells in vitro.

To investigate the effects of microgravity on murine skeletal muscle fiber size, muscle contractile protein, and enzymatic activity, female C57BL/6J mice, aged 64 days, were divided into animal enclosure module (AEM) ground control and spaceflight (SF) treatment groups. SF animals were flown on the space shuttle Endeavour (STS-108/UF-1) and subjected to ∼11 days and 19 h of microgravity. Immunohistochemical analysis of muscle fiber cross-sectional area revealed that, in each of the muscles analyzed, mean muscle fiber cross-sectional area was significantly reduced ( P < 0.0001) for all fiber types for SF vs. AEM control. In the soleus, immunohistochemical analysis of myosin heavy chain (MHC) isoform expression revealed a significant increase in the percentage of muscle fibers expressing MHC IIx and MHC IIb ( P < 0.05). For the gastrocnemius and plantaris, no significant changes in MHC isoform expression were observed. For the muscles analyzed, no alterations in MHC I or MHC IIa protein expression were observed. Enzymatic analysis of the gastrocnemius revealed a significant decrease in citrate synthase activity in SF vs. AEM control.

We reported an association between the ability of recombinant human keratinocyte growth factor (rHuKGF) to upregulate the expression of surfactant protein A (SP-A) and to downregulate pulmonary inflammation that occurs after allogeneic bone marrow transplantation (BMT). To establish a causal relationship, rHuKGF (5 mg/kg) was administered subcutaneously for three consecutive days before irradiation to SP-A-sufficient and -deficient [SP-A(+/+) and SP-A(-/-), respectively] mice given inflammation-inducing allogeneic spleen T cells at the time of BMT. In contrast with SP-A(+/+) mice, rHuKGF failed to suppress the high levels of TNF-α, IFN-γ, and nitric oxide contained in bronchoalveolar lavage fluids collected on day 7 after BMT from SP-A(-/-) mice. Early post-BMT weight loss was attenuated by rHuKGF in both SP-A(+/+) and SP-A(-/-) recipients. In the absence of supportive respiratory care, however, SP-A deficiency eventually abolished the ability of rHuKGF to prevent weight loss and to improve survival monitored for 1 mo after allogeneic BMT. In further experiments, the addition of cyclophosphamide (which is known to cause severe injury to the alveolar epithelium in donor T cell-recipient mice) to the conditioning regimen prevented rHuKGF-induced upregulation of SP-A and suppression of lung inflammation in both SP-A(+/+) and SP-A(-/-) mice. We conclude that endogenous baseline SP-A levels and optimal upregulation of SP-A are required for the anti-inflammatory protective effects of KGF after allogeneic transplantation.

Osteoprotegerin (OPG) is a naturally occurring negative regulator of osteoclast differentiation, activation, and survival. We created a recombinant form of human OPG (rhOPG), with a sustained serum half-life, to achieve prolonged antiresorptive activity. This study describes the rapid and sustained antiresorptive effects that are achieved with a single treatment with rhOPG. Male Sprague-Dawley rats (10 weeks old) were given a single bolus intravenous injection of vehicle (PBS) or rhOPG (5 mg/kg). PBS- and rhOPG-treated rats (n = 6/group) were killed at 0, 0.5, 1, 2, 5, 10, 20, and 30 days post-treatment. rhOPG-treated rats were compared with their age-matched controls. The main pharmacologic effect of rhOPG was a rapid (24 h) reduction in osteoclast surface in the tibia, which reached a nadir on days 5 and 10 (95% reduction vs. vehicle controls). Osteoclast surface remained significantly reduced 30 days after the single treatment with rhOPG. Tibial cancellous bone volume was significantly increased within 5 days of rhOPG treatment (23%) and reached a peak increase of 58% on day 30. Femoral bone mineral density was significantly increased in rhOPG-treated rats on days 10 and 20. Pharmacokinetic analysis revealed that serum concentrations of rhOPG remained at measurable levels throughout the 30-day study. These data show that a single intravenous injection of rhOPG in young growing rats causes significant gains in bone volume and density, which are associated with rapid and sustained suppression of osteoclastic bone resorption.

PTH is a potent bone anabolic factor, and its combination with antiresorptive agents has been proposed as a therapy for osteoporosis. We tested the effects of PTH, alone and in combination with the novel antiresorptive agent OPG, in a rat model of severe osteopenia. Sprague Dawley rats were sham-operated or ovariectomized at 3 months of age. Rats were untreated for 15 months, at which time ovariectomy had caused significant decreases in bone mineral density in the lumbar vertebrae and femur. Rats were then treated for 5.5 months with vehicle (PBS), human PTH-(1-34) (80 microg/kg), rat OPG (10 mg/kg), or OPG plus PTH (all three times per wk, sc). Treatment of ovariectomized rats with OPG or PTH alone increased bone mineral density in the lumbar vertebrae and femur, whereas PTH plus OPG caused significantly greater and more rapid increases than either therapy alone (P0.05). OPG significantly reduced osteoclast surface in the lumbar vertebrae and femur (P0.05 vs. sham or ovariectomized), but had no effect on osteoblast surface at either site. Ovariectomy significantly decreased the mechanical strength of the lumbar vertebrae and femur. In the lumbar vertebrae, OPG plus PTH was significantly more effective than PTH alone at reversing ovariectomy-induced deficits in stiffness and elastic modulus. These data suggest that OPG plus PTH represent a potentially useful therapeutic option for patients with severe osteoporosis.

Osteoprotegerin (OPG) regulates bone resorption by inhibiting osteoclast formation, function, and survival. The current studies employed a mouse ovariectomy (OVX) model of estrogen deficiency to investigate gene therapy with OPG as a means of preventing osteoporosis. Young adult females injected with a recombinant adenoviral (Ad) vector carrying cDNA of either full-length OPG or a fusion protein combining the hOPG ligand-binding domain with the human immunoglobulin constant domain (Ad-hOPG-Fc) developed serum OPG concentrations exceeding the threshold needed for efficacy. However, elevated circulating OPG levels were sustained for up to 18 months only in mice given Ad-hOPG-Fc. Administration of Ad-hOPG-Fc titers between 10(7) and 10(9) pfu yielded dose-dependent increases in serum OPG. Mice subjected to OVX or sham surgery followed by immediate treatment with Ad-hOPG-Fc had significantly more bone volume with reduced osteoclast numbers in axial and appendicular bones after 4 weeks. In contrast, animals given OVX and either a control vector or vehicle had significantly less bone than did comparably treated sham-operated mice. This study demonstrates that a single adenoviral gene transfer can produce persistent high-level OPG expression and shows that gene therapy to provide sustained delivery of OPG may prove useful in treating osteoporosis.

Osteoprotegerin ligand (OPGL) targets osteoclast precursors and osteoclasts to enhance differentiation and activation, however, little is known about OPGL effects on osteoclast survival. In vitro, the combination of OPGL + colony-stimulating factor-1 (CSF-1) is required for optimal osteoclast survival. Ultrastructurally, apoptotic changes were observed in detached cells and culture lysates exhibited elevated caspase 3 activity, particularly in cultures lacking CSF-1. DEVD-FMK (caspase 3 inhibitor) partially protected cells when combined with OPGL, but not when used alone or in combination with CSF-1. CSF-1 maintained NF-kappaB activation and increased the expression of bcl-2 and bcl-X(L) mRNA, but had no effect on JNK activation. In contrast, OPGL enhanced both NF-kappaB and JNK kinase activation and increased the expression of c-src, but not bcl-2 and bcl-X(L) mRNA. These data suggest that aspects of both OPGL's and CSF-1's signaling/survival pathways are required for optimal osteoclast survival. In mice, a single dose of OPG, the OPGL decoy receptor, led to a90% loss of osteoclasts because of apoptosis within 48 hours of exposure without impacting osteoclast precursor cells. Therefore, OPGL is essential, but not sufficient, for osteoclast survival and endogenous CSF-1 levels are insufficient to maintain osteoclast viability in the absence of OPGL.

Osteoprotegerin (OPG) is a recently discovered protein related to the tumor necrosis factor receptor family. It has been shown to inhibit ovariectomy (ovx)-induced resorption in rats and increase bone mineral density in young mice. Tail suspension is a procedure that inhibits bone formation in maturing rodents. This study was designed to quantify OPG's effect on cortical bone formation. Fifty-four mice were assigned to one of five groups (n = 10-11/group). A baseline control group was killed on day 0 of the 10 day study. The remaining groups were: vivarium housed (nonsuspended) control mice receiving 0.3 mg/kg per day OPG; vivarium control mice receiving daily placebo injections; tail-suspended mice receiving 0. 3 mg/kg per day OPG; and tail-suspended mice receiving placebo injections. Tetracycline was administered on days 0 and 8. OPG treatment of tail-suspended mice produced mechanical properties similar to those of placebo-treated, vivarium-housed mice: structural stiffness (8.5%, 20.7%) and elastic (13.9%, 10.1%) and maximum (4.7%, 8.1%) force were increased compared with placebo controls (vivarium, suspended groups). Percent mineral composition was highly significantly greater (p < 0.001 for all comparisons) for OPG-treated mice in the femur, tibia, and humerus, relative to placebo treatment. Matrix mass was also significantly increased in the femur, although not to the same degree as mineral mass. OPG decreased the amount of femoral endocortical resorption compared with the placebo-treated groups for both vivarium (27%) and suspended (24%) mice. Administration of OPG significantly decreased endocortical formation of the tibia. Periosteal bone formation rates were not altered by OPG. OPG-mitigated tail suspension induced osteopenia not by returning bone formation to normal levels, but by inhibiting resorption and increasing percent mineral composition.

We have generated RANK (receptor activator of NF-κB) nullizygous mice to determine the molecular genetic interactions between osteoprotegerin, osteoprotegerin ligand, and RANK during bone resorption and remodeling processes. RANK −/− mice lack osteoclasts and have a profound defect in bone resorption and remodeling and in the development of the cartilaginous growth plates of endochondral bone. The osteopetrosis observed in these mice can be reversed by transplantation of bone marrow from rag1 −/− (recombinase activating gene 1) mice, indicating that RANK −/− mice have an intrinsic defect in osteoclast function. Calciotropic hormones and proresorptive cytokines that are known to induce bone resorption in mice and human were administered to RANK −/− mice without inducing hypercalcemia, although tumor necrosis factor α treatment leads to the rare appearance of osteoclast-like cells near the site of injection. Osteoclastogenesis can be initiated in RANK −/− mice by transfer of the RANK cDNA back into hematopoietic precursors, suggesting a means to critically evaluate RANK structural features required for bone resorption. Together these data indicate that RANK is the intrinsic cell surface determinant that mediates osteoprotegerin ligand effects on bone resorption and remodeling as well as the physiological and pathological effects of calciotropic hormones and proresorptive cytokines.

Although multiple hormones and cytokines regulate various aspects of osteoclast formation, the final two effectors are osteoprotegerin ligand (OPG-L)/osteoclast differentiation factor (ODF), a recently cloned member of the tumor necrosis factor superfamily, and macrophage colony-stimulating factor. OPG-L/ODF is produced by osteoblast lineage cells and exerts its biological effects through binding to its receptor, osteoclast differentiation and activation receptor (ODAR)/receptor activator of NF-kappa B (RANK), on osteoclast lineage cells, in either a soluble or a membrane-bound form, the latter of which requires cell-to-cell contact. Binding results in rapid differentiation of osteoclast precursors in bone marrow to mature osteoclasts and, at higher concentrations, in increased functional activity and reduced apoptosis of mature osteoclasts. The biological activity of OPG-L/ODF is neutralized by binding to osteoprotegerin (OPG)/osteoclastogenesis inhibitory factor (OCIF), a member of the TNF-receptor superfamily that also is secreted by osteoblast lineage cells. The biological importance of this system is underscored by the induction in mice of severe osteoporosis by targeted ablation of OPG/OCIF and by the induction of osteopetrosis by targeted ablation of OPG-L/ODF or overexpression of OPG/OCIF. Thus, osteoclast formation may be determined principally by the relative ratio of OPG-L/ODF to OPG/OCIF in the bone marrow microenvironment, and alterations in this ratio may be a major cause of bone loss in many metabolic disorders, including estrogen deficiency and glucocorticoid excess. That changes in but two downstream cytokines mediate the effects of large numbers of upstream hormones and cytokines suggests a regulatory mechanism for osteoclastogenesis of great efficiency and elegance.

Osteoprotegerin (OPG) is a secreted protein that inhibits osteoclast formation and activity and appears to be a critical regulator of bone mass and metabolism. In the current study, mice were challenged with various cytokines and hormones (interleukin-1beta, tumor necrosis factor-alpha, parathyroid hormone, parathyroid hormone-related protein, and 1alpha,25-dihydroxyvitamin D3) that are known to increase bone resorption and cause hypercalcemia and treated concurrently with either a recombinant chimeric Fc fusion form of human OPG, with enhanced biological activity (cOPG) (2.5 mg/kg/day) or vehicle. Mice receiving these bone-resorbing factors became hypercalcemic by day 3 after commencing treatment and had increased bone resorption as evidenced by elevated osteoclast numbers on day 5. Concurrent cOPG treatment prevented hypercalcemia (p < 0.05) and maintained osteoclast numbers in the normal range (p < 0.001). The demonstration that cOPG can inhibit bone resorption suggests that this molecule may be useful in the treatment of diseases including hyperparathyroidism, humoral hypercalcemia of malignancy, osteoporosis, and inflammatory bone disease, which are characterized, in part, by increases in osteoclastic bone resorption.

The identity of the paracrine mediator(s) of the antiresorptive action of estrogen on bone cells is controversial. Osteoprotegerin (OPG) was recently identified as a soluble member of the tumor necrosis factor (TNF) receptor (TNF-R) superfamily that is secreted by osteoblast lineage cells and acts by binding to and neutralizing its cognate ligand, OPG-L, a required factor for osteoclastogenesis. OPG prevents bone loss when administered to ovariectomized rats, induces osteoporosis when ablated in knock-out mice, and induces osteopetrosis when overexpressed in transgenic mice. In conditionally immortalized, human osteoblastic hFOB/ER-3 and hFOB/ER-9 cell lines containing physiological concentrations of approximately 800 and approximately 8,000 functional estrogen receptors (ER)/nucleus, respectively, we found that 17beta-estradiol dose- and time-dependently increased OPG mRNA and protein levels to maximal levels of 370% and 320%, respectively (P0.001); co-treatment with the "pure" antiestrogen ICI 182,780 abrogated these effects completely. 17beta-Estradiol also dose-dependently increased OPG mRNA and protein levels in normal human osteoblasts with approximately 400 ER/nucleus by 60% and 73%, respectively. Thus, estrogen enhancement of OPG secretion by osteoblastic cells may play a major role in the antiresorptive action of estrogen on bone.