TNFalpha receptor knockout in mice reduces adverse effects of magnesium deficiency on bone.

Epidemiologic studies have linked low dietary magnesium (Mg) intake to osteoporosis. Dietary Mg restriction in animal models has demonstrated a decrease in bone mass and an increase in skeletal fragility. The exact mechanism for the decrease in bone mass is not clear but a decrease in osteoblast number and an increase in osteoclast number (Oc.No/B.Pm) suggests an uncoupling of bone formation and bone resorption favoring skeletal loss. Mg depletion results in an increase in inflammatory cytokines, which could explain the increase in bone resorption. We have previously demonstrated an increase in TNFalpha in bone from Mg deficient rodents. Here we report results of a 3 week study of a low magnesium (LM) diet and normal Mg diet in 35-day-old TNFalpha receptor knockout mice (TNF-r-KO) versus wild type (WT) control mice. Our results indicated that a LM diet resulted in a greater increase in Oc.No/B.Pm in the WT mice, with a trend toward greater eroded bone perimeter, as compared to TNF-r-KO. These findings suggest that TNFalpha may play a role in Mg deficiency-induced bone loss.

Effects of cyclic vs. daily treatment with human parathyroid hormone (1-34) on murine bone structure and cellular activity.

Previously, we demonstrated that the human parathyroid hormone (1-34) fragment (hPTH(1-34)) increased bone strength in proportion to its effects on BMD and cortical bone structure in the murine femur by comparing cyclic vs. daily administration of hPTH(1-34). Both cyclic and daily regimens increased vertebral BMD similarly at 7 weeks. Here, we have examined the effects of daily and cyclic PTH regimens on bone structure and cellular activity by static and dynamic histomorphometry. Twenty-week-old, intact female C57BL/J6 mice were treated with the following regimens (n=7 for each group): daily injection with vehicle for 7 weeks [control]; daily injection with hPTH(1-34) (40 microg/kg/day) for 7 weeks [daily PTH]; and daily injection with hPTH(1-34) (40 microg/kg/day) and vehicle alternating weekly for 7 weeks [cyclic PTH]. At days 9 and 10, and 2 and 3 prior to euthanasia, calcein (10 mg/kg) was injected subcutaneously. At the end of study, the lumbar vertebrae 1-3 and the left femora were excised, cleaned, and processed for histomorphometry. In the lumbar vertebrae, daily and cyclic PTH regimens significantly increased cancellous bone volume (BV/TV), trabecular number, trabecular osteoclast and osteoblast perimeters, trabecular mineral apposition rate (MAR) and bone formation rate (BFR), and periosteal MAR and BFR compared to control, with no significant difference between the two PTH-treated groups. Increased trabecular tunneling was observed in both PTH-treated groups. Both regimens tended to increase vertebral cortical bone formation parameters with the effects at the periosteum site being more marked than those at the endosteum site, resulting in a significant increase in cortical width. In the femur, the effects of cyclic PTH on BV/TV, trabecular width and number, trabecular and endocortical osteoblast and osteoclast perimeters, cortical width, and trabecular and periosteal BFR were less marked than those of daily PTH. A cyclic PTH regimen was as effective as a daily regimen in improving cancellous and cortical bone microarchitecture and cellular activity in the murine vertebra.

Effects of cyclic versus daily hPTH(1-34) regimens on bone strength in association with BMD, biochemical markers, and bone structure in mice.

UNLABELLED: 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 microg/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.

Anabolic action of parathyroid hormone is skeletal site specific at the tissue and cellular levels in mice.

The cellular and molecular events triggering the anabolic response of the skeleton to exogenous parathyroid hormone (PTH) are not well understood. Despite the numerous bone mass studies in rats, few data are available for mice. Therefore, we treated 10-week-old female intact C57BL/6J mice with human PTH(1-34) delivered subcutaneously at a dose of 40 microg/kg per day 5 days a week for 3 weeks and 7 weeks. Bone mineral density (BMD) of total bone, femur, tibia, and lumbar vertebrae was measured weekly by PIXImus. Bone turnover was examined by histomorphometry, and gene expression of bone formation and resorption markers and osteoclastogenesis regulators in the excised femur and tibia was assessed by reverse-transcription polymerase chain reaction (RT-PCR) at 3 weeks and 7 weeks. The PTH-stimulated increase in BMD was more prominent in the tibia and femur than in the lumbar vertebrae, with an anabolic effect detected within 1-2 weeks and BMD continuing to increase. The appearance of a detectable PTH-stimulated increase in BMD was slower in the lumbar vertebrae where the increase was only significant after 7 weeks of treatment. Histomorphometric analysis of the proximal tibia at both 3 weeks and 7 weeks indicated significant time-dependent increases in trabecular area, trabecular number, trabecular and cortical widths, and osteoblast and osteoid perimeters. In the lumbar vertebrae, these stimulatory effects of PTH on trabecular area, trabecular number, and cortical width were smaller and not detected until 7 weeks. PTH-stimulated increases in bone turnover were evident by increased gene expression of osteocalcin (OC), tartrate-resistant acid phosphatase (TRAP), and receptor of activator nuclear factor kappaB (NF-kappaB) ligand (RANKL) in the tibia and femur. No significant difference in gene expression was observed between the two long bone sites. In conclusion, PTH exerts an anabolic action at the tissue and cellular levels in intact mice and the magnitude and temporal pattern of this anabolic action, as assessed by densitometry and histomorphometry, are skeletal site specific.

Precision, accuracy, and reproducibility of dual X-ray absorptiometry measurements in mice in vivo.

Dual X-ray absorptiometry (DXA) has currently become a clinical standard for the assessment of bone mass and bone mineral density (BMD) at multiple sites for the diagnosis and follow-up assessment of osteoporosis in humans. The precision of DXA measurement in human studies has been well documented during the last two decades. However, there have been no systematic reports on the precision and accuracy of BMD measurements in mice using DXA, although mice have proven to be useful models for the study of osteoporosis. Accordingly, BMD of total body as well as regions of interest (ROIs) was measured twice in mice in vivo after a short (10-min) and long (16-hr) interval between scans by DXA, and scanning variations were calculated. Inter- and intra-analyzer variations from the same scans were also determined. The percent coefficients (%CVs) of short-interval scanning variation and inter- and intra-analyzer variations for total body and regional BMDs were less than 2% at sites, demonstrating high precision of in vivo BMD measurements in mice. Moreover, the BMD values comparing in vivo and ex vivo samples from the same animals were of %CV less than 10% at all sites. The correlation of bone mineral content (BMC) to bone ash was further examined, and the correlation between ROI BMC and bone ash was relatively high at all sites both in vivo and ex vivo, with the latter higher. We conclude that in vivo DXA BMD measurements in mice are very reliable with high precision and acceptable accuracy, and therefore useful for longitudinal studies of the mouse skeleton.

Autophagy in osteoblasts is involved in mineralization and bone homeostasis.

Bone remodeling is a tightly controlled mechanism in which osteoblasts (OB), the cells responsible for bone formation, osteoclasts (OC), the cells specialized for bone resorption, and osteocytes, the multifunctional mechanosensing cells embedded in the bone matrix, are the main actors. Increased oxidative stress in OB, the cells producing and mineralizing bone matrix, has been associated with osteoporosis development but the role of autophagy in OB has not yet been addressed. This is the goal of the present study. We first show that the autophagic process is induced in OB during mineralization. Then, using knockdown of autophagy-essential genes and OB-specific autophagy-deficient mice, we demonstrate that autophagy deficiency reduces mineralization capacity. Moreover, our data suggest that autophagic vacuoles could be used as vehicles in OB to secrete apatite crystals. In addition, autophagy-deficient OB exhibit increased oxidative stress and secretion of the receptor activator of NFKB1 (TNFSF11/RANKL), favoring generation of OC, the cells specialized in bone resorption. In vivo, we observed a 50% reduction in trabecular bone mass in OB-specific autophagy-deficient mice. Taken together, our results show for the first time that autophagy in OB is involved both in the mineralization process and in bone homeostasis. These findings are of importance for mineralized tissues which extend from corals to vertebrates and uncover new therapeutic targets for calcified tissue-related metabolic pathologies.

Normal human osteoclasts formed from peripheral blood monocytes express PTH type 1 receptors and are stimulated by PTH in the absence of osteoblasts.

The prevailing view for many years has been that osteoclasts do not express parathyroid hormone (PTH) receptors and that PTH's effects on osteoclasts are mediated indirectly via osteoblasts. However, several recent reports suggest that osteoclasts express PTH receptors. In this study, we tested the hypothesis that human osteoclasts formed in vitro express functional PTH type 1 receptors (PTH1R). Peripheral blood monocytes (PBMC) were cultured on bone slices or plastic culture dishes with human recombinant RANK ligand (RANKL) and recombinant human macrophage colony-stimulating factor (M-CSF) for 16-21 days. This resulted in a mixed population of mono- and multi-nucleated cells, all of which stained positively for the human calcitonin receptor. The cells actively resorbed bone, as assessed by release of C-terminal telopeptide of type I collagen and the formation of abundant resorption pits. We obtained evidence for the presence of PTH1R in these cells by four independent techniques. First, using immunocytochemistry, positive staining for PTH1R was observed in both mono- and multi-nucleated cells intimately associated with resorption cavities. Second, PTH1R protein expression was demonstrated by Western blot analysis. Third, the cells expressed PTH1R mRNA at 21 days and treatment with 10(-7) M hPTH (1-34) reduced PTH1R mRNA expression by 35%. Finally, bone resorption was reproducibly increased by two to threefold when PTH (1-34) was added to the cultures. These findings provide strong support for a direct stimulatory action of PTH on human osteoclasts mediated by PTH1R. This suggests a dual regulatory mechanism, whereby PTH acts both directly on osteoclasts and also, indirectly, via osteoblasts.