RANKL from bone marrow adipose lineage cells promotes osteoclast formation and bone loss

Receptor activator of NF‐κB ligand (RANKL) is essential for osteoclast formation and bone remodeling. Nevertheless, the cellular source of RANKL for osteoclastogenesis has not been fully uncovered. Different from peripheral adipose tissue, bone marrow (BM) adipose lineage cells originate from bone marrow mesenchymal stromal cells (BMSCs). Here, we demonstrate that adiponectin promoter‐driven Cre expression (Adipoq^Cre ) can target bone marrow adipose lineage cells. We cross the Adipoq^Cre mice with rankl^fl/fl mice to conditionally delete RANKL from BM adipose lineage cells. Conditional deletion of RANKL increases cancellous bone mass of long bones in mice by reducing the formation of trabecular osteoclasts and inhibiting bone resorption but does not affect cortical bone thickness or resorption of calcified cartilage. Adipoq^Cre; rankl^fl/fl mice exhibit resistance to estrogen deficiency and rosiglitazone (ROS)‐induced trabecular bone loss but show bone loss induced by unloading. BM adipose lineage cells therefore represent an essential source of RANKL for the formation of trabecula osteoclasts and resorption of cancellous bone during remodeling under physiological and pathological conditions. Targeting bone marrow adiposity is a promising way of preventing pathological bone loss.     

Inhibition of Osteocyte Apoptosis Prevents the Increase in Osteocytic Receptor Activator of Nuclear Factor κB Ligand (RANKL) but Does Not Stop Bone Resorption or the Loss of Bone Induced by Unloading

Apoptosis of osteocytes and osteoblasts precedes bone resorption and bone loss with reduced mechanical stimulation, and receptor activator of NF-κB ligand (RANKL) expression is increased with unloading in mice. Because osteocytes are major RANKL producers, we hypothesized that apoptotic osteocytes signal to neighboring osteocytes to increase RANKL expression, which, in turn, increases osteoclastogenesis and bone resorption. The traditional bisphosphonate (BP) alendronate (Aln) or IG9402, a BP analog that does not inhibit resorption, prevented the increase in osteocyte apoptosis and osteocytic RANKL expression. The BPs also inhibited osteoblast apoptosis but did not prevent the increase in osteoblastic RANKL. Unloaded mice exhibited high serum levels of the bone resorption marker C-telopeptide fragments of type I collagen (CTX), elevated osteoclastogenesis, and increased osteoclasts in bone. Aln, but not IG9402, prevented all of these effects. In addition, Aln prevented the reduction in spinal and femoral bone mineral density, spinal bone volume/tissue volume, trabecular thickness, mechanical strength, and material strength induced by unloading. Although IG9402 did not prevent the loss of bone mass, it partially prevented the loss of strength, suggesting a contribution of osteocyte viability to strength independent of bone mass. These results demonstrate that osteocyte apoptosis leads to increased osteocytic RANKL. However, blockade of these events is not sufficient to restrain osteoclast formation, inhibit resorption, or stop bone loss induced by skeletal unloading.     

Receptor Activator of Nuclear Factor κB Ligand (RANKL) Protein Expression by B Lymphocytes Contributes to Ovariectomy-induced Bone Loss

Production of the cytokine receptor activator of NFκB ligand (RANKL) by lymphocytes has been proposed as a mechanism by which sex steroid deficiency causes bone loss. However, there have been no studies that functionally link RANKL expression in lymphocytes with bone loss in this condition. Herein, we examined whether RANKL expression in either B or T lymphocytes contributes to ovariectomy-induced bone loss in mice. Mice harboring a conditional RANKL allele were crossed with CD19-Cre or Lck-Cre mice to delete RANKL in B or T lymphocytes, respectively. Deletion of RANKL from either cell type had no impact on bone mass in estrogen-replete mice up to 7 months of age. However, mice lacking RANKL in B lymphocytes were partially protected from the bone loss caused by ovariectomy. This protection occurred in cancellous, but not cortical, bone and was associated with a failure to increase osteoclast numbers in the conditional knock-out mice. Deletion of RANKL from T lymphocytes had no impact on ovariectomy-induced bone loss. These results demonstrate that lymphocyte RANKL is not involved in basal bone remodeling, but B cell RANKL does contribute to the increase in osteoclasts and cancellous bone loss that occurs after loss of estrogen.     

Targeted disruption of ephrin B1 in cells of myeloid lineage increases osteoclast differentiation and bone resorption in mice

Disruption of ephrin B1 in collagen I producing cells in mice results in severe skull defects and reduced bone formation. Because ephrin B1 is also expressed during osteoclast differentiation and because little is known on the role of ephrin B1 reverse signaling in bone resorption, we examined the bone phenotypes in ephrin B1 conditional knockout mice, and studied the function of ephrin B1 reverse signaling on osteoclast differentiation and resorptive activity. Targeted deletion of ephrin B1 gene in myeloid lineage cells resulted in reduced trabecular bone volume, trabecular number and trabecular thickness caused by increased TRAP positive osteoclasts and bone resorption. Histomorphometric analyses found bone formation parameters were not changed in ephrin B1 knockout mice. Treatment of wild-type precursors with clustered soluble EphB2-Fc inhibited RANKL induced formation of multinucleated osteoclasts, and bone resorption pits. The same treatment of ephrin B1 deficient precursors had little effect on osteoclast differentiation and pit formation. Similarly, activation of ephrin B1 reverse signaling by EphB2-Fc treatment led to inhibition of TRAP, cathepsin K and NFATc1 mRNA expression in osteoclasts derived from wild-type mice but not conditional knockout mice. Immunoprecipitation with NHERF1 antibody revealed ephrin B1 interacted with NHERF1 in differentiated osteoclasts. Treatment of osteoclasts with exogenous EphB2-Fc resulted in reduced phosphorylation of ezrin/radixin/moesin. We conclude that myeloid lineage produced ephrin B1 is a negative regulator of bone resorption in vivo, and that activation of ephrin B1 reverse signaling inhibits osteoclast differentiation in vitro in part via a mechanism that involves inhibition of NFATc1 expression and modulation of phosphorylation status of ezrin/radixin/moesin.     

Perforation of cancellous bone trabeculae by damage-stimulated remodelling at resorption pits: a computational analysis

Loss of trabeculae in cancellous bone is often attributed to a general decline in the bone mass leading to fracture of the thin trabeculae. It has never been investigated whether trabecular perforation may have any other biomechanical mechanism. In this paper, an alternative hypothesis is proposed and tested using a computational model. Taking it as given that osteoclastic resorption is targeted to microdamage, it is hypothesised that the creation of a resorption cavity during normal bone remodelling could cause a stress-concentration in the bone tissue. If the resorption cavities were excessively deep, as is seen during osteoporosis, then this stress concentration may be sufficient to generate more microdamage so that osteoclasts "chase" newly formed damage leading to perforation. If this were true then we should find that, for a given trabecular thickness, there is a critical depth of resorption cavity such that smaller cavities refill whereas deeper cavities cause microdamage accumulation, continued osteoclast activity, and eventual trabecular perforation. Computer simulation is used to test this hypothesis. Using a remodelling stimulus calculated from both strain and damage and a simplified finite element model of a trabeculum with cavities of different sizes, it is predicted that such a critical depth of resorption cavity does indeed exist. Therefore we suggest that an increase in resorption depth relative to the thickness of trabeculae may be responsible for trabecular perforation during osteoporosis, rather than simply trabecular fracture due to insufficient strength.     

The role of estrogen receptor-beta, in the early age-related bone gain and later age-related bone loss in female mice

The molecular and cellular mechanism of estrogen action in skeletal tissue remains unclear. The purpose of this study was to understand the role of estrogen receptor-beta, (ERbeta) on cortical and cancellous bone during growth and aging by comparing the bone phenotype of 6- and 13-month-old female mice with or without ERbeta. Groups of 11-14 wild-type (WT) controls and ERbeta knockout (BERKO) female mice were necropsied at 6 and 13 months of age. At both ages, BERKO mice did not differ significantly from WT controls in uterine weight and uterine epithelial thickness, indicating that ERbeta does not regulate the growth of uterine tissue. Femoral length increased significantly by 5.5% at 6 months of age in BERKO mice compared with WT controls. At 6 months of age, peripheral quantitative computerized tomography (pQCT) analysis of the distal femoral metaphysis (DFM) and femoral shafts showed that BERKO mice had significantly higher cortical bone content and periosteal circumference as compared with WT controls at both sites. In contrast to the findings in cortical bone, at 6 months of age, there was no difference between BERKO and WT mice in trabecular density, trabecular bone volume (TBV), or formation and resorption indices at the DFM. In 13-month-old WT mice, TBV (-41%), trabecular density (-27%) and cortical thickness decreased significantly. while marrow cavity and endocortical circumference increased significantly compared with 6-month-old WT mice. These age-related decreases in cancellous and endocortical bone did not occur in BERKO mice. At 13 months of age, BERKO mice had significantly higher total, trabecular and cortical bone, while having significantly lower bone resorption, bone formation and bone turnover in DFM compared with WT mice. These results indicate that deleting ERbeta protected against age-related bone loss in both the cancellous and endocortical compartments by decreasing bone resorption and bone turnover in aged female mice. These data demonstrate that in female mice, ERbeta plays a role in inhibiting periosteal bone formation, longitudinal and radial bone growth during the growth period, while it plays a role in stimulating bone resorption, bone turnover and bone loss on cancellous and endocortical bone surfaces during the aging process.     

Continuous RANKL inhibition in osteoprotegerin transgenic mice and rats suppresses bone resorption without impairing lymphorganogenesis or functional immune responses

Receptor activator of NF-kappaB ligand (RANKL) is an essential mediator of osteoclast formation, function, and survival. The effects of RANKL are inhibited by a soluble decoy receptor called osteoprotegerin (OPG). Total ablation of RANKL in knockout mice leads to high bone mass, lymph node agenesis, and altered lymphocyte differentiation. In contrast, RANKL inhibition via OPG suppresses bone resorption but not inflammation in animal models of inflammatory bone loss. This suggests that the immune phenotype of RANKL knockout mice is related to total RANKL ablation. We hypothesized that prenatal RANKL inhibition via OPG overexpression would suppress bone resorption without influencing lymph node formation or subsequent immune responses. Transgenic rats were created, wherein soluble OPG was overexpressed by 100-fold vs wild type (WT) controls, by gestational day 11 (i.e., before lymph node formation). The structure of lymph nodes, spleen, and thymus of OPG-transgenic (OPG-Tg) animals were comparable to those of age-matched WT rats at gestational day 19 and in adulthood. The OPG-Tg neonates had elevated bone mass, confirming the prenatal inhibition of RANKL. Adult OPG-Tg rats and OPG-Tg mice exhibited no significant functional alterations relative to WT controls when subjected to immune challenges to test for altered innate and humoral responses (e.g., contact hypersensitivity to oxazolone, IgM response to Pneumovax, IgG response to keyhole limpet hemocyanin, or cytokine response to LPS). In summary, prenatal RANKL inhibition did not impair lymph node development, nor did continuous life-long RANKL inhibition cause obvious changes in innate or humoral immune responses in mice or rats.     

Increased formation and decreased resorption of bone in mice with elevated vitamin D receptor in mature cells of the osteoblastic lineage.

The microarchitecture of bone is regulated by complex interactions between the bone-forming and resorbing cells, and several compounds regulate both actions. For example, vitamin D, which is required for bone mineralization, also stimulates bone resorption. Transgenic mice overexpressing the vitamin D receptor solely in mature cells of the osteoblastic bone-forming lineage were generated to test the potential therapeutic value of shifting the balance of vitamin D activity in favor of bone formation. Cortical bone was 5% wider and 15% stronger in these mice due to a doubling of periosteal mineral apposition rate without altered body weight or calcium homeostatic hormone levels. A 20% increase in trabecular bone volume in transgenic vertebrae was also observed, unexpectedly associated with a 30% reduction in resorption surface rather than greater bone formation. These findings indicate anabolic vitamin D activity in bone and identify a previously unknown pathway from mature osteoblastic cells to inhibit osteoclastic bone resorption, counterbalancing the known stimulatory action through immature osteoblastic cells. A therapeutic approach that both stimulates cortical anabolic and inhibits trabecular resorptive pathways would be ideal for treatment of osteoporosis and other osteopenic disorders.     

Soluble rank ligand produced by myeloma cells causes generalised bone loss in multiple myeloma

Patients with multiple myeloma commonly develop focal osteolytic bone disease, as well as generalised osteoporosis. The mechanisms underlying the development of osteoporosis in patients with myeloma are poorly understood. Although disruption of the RANKL/OPG pathway has been shown to underlie formation of focal osteolytic lesions, its role in the development of osteoporosis in myeloma remains unclear. Increased soluble RANKL in serum from patients with myeloma raises the possibility that this molecule plays a key role. The aim of the present study was to establish whether sRANKL produced by myeloma cells contributes directly to osteoporosis. C57BL/KaLwRij mice were injected with either 5T2MM or 5T33MM murine myeloma cells. 5T2MM-bearing mice developed osteolytic bone lesions (p<0.05) with increased osteoclast surface (p<0.01) and reduced trabecular bone volume (p<0.05). Bone volume was also reduced at sites where 5T2MM cells were not present (p<0.05). In 5T2MM-bearing mice soluble mRANKL was increased (p<0.05), whereas OPG was not altered. In contrast, 5T33MM-bearing mice had no changes in osteoclast surface or trabecular bone volume and did not develop osteolytic lesions. Soluble mRANKL was undetectable in serum from 5T33MM-bearing mice. In separate experiments, RPMI-8226 human myeloma cells were transduced with an human RANKL/eGFP construct, or eGFP alone. RPMI-8226/hRANKL/eGFP cells, but not RPMI-8226/eGFP cells, stimulated osteoclastic bone resorption (p<0.05) in vitro. Sub-cutaneous injection of NOD/SCID mice with RPMI-8226/hRANKL/eGFP or RPMI-8226/eGFP cells resulted in tumour development in all mice. RPMI-8226/hRANKL/eGFP-bearing mice exhibited increased serum soluble hRANKL (p<0.05) and a three-fold increase in osteoclast number (p<0.05) compared to RPMI-8226/eGFP-bearing mice. This was associated with reduced trabecular bone volume (27%, p<0.05), decreased trabecular number (29%, p<0.05) and increased trabecular thickness (8%, p<0.05). Our findings demonstrate that soluble RANKL produced by myeloma cells causes generalised bone loss, suggesting that targeting RANKL may prevent osteoporosis in patients with myeloma.     

Receptor activator of NF-[kappa]B ligand arrests bone growth and promotes cortical bone resorption in growing rats.

Receptor activator of NF-kappaB ligand (RANKL), produced by osteoblastic lineage cells and activated T cells, is an essential factor for osteoclast differentiation, activation, and survival. Therefore, RANKL is a focal point of therapies targeting bone diseases where there is an imbalance of bone metabolism in favor of bone resorption. The present study assesses the effects of exogenous RANKL on growing bone. RANKL (100 microg x kg-1x day-1 for 7 days) administered to Sprague-Dawley weanling rats caused major deficits in growth, appearance, and bone mineral densities (BMD). Urinary deoxypyridinoline crosslinks, a measure of bone turnover, were higher in the RANKL-treated rats (P = 0.031), and the bone mineral content was lower (P < 0.001). The final BMD in the RANKL-treated rats was lower (P = 0.039) than in the control rats (19 +/- 7 vs. 38 +/- 5 mg/cm3). Moreover, calculated cortical bone density in each bone slice (total BMD - trabecular BMD) indicated there was only 5% cortical bone remaining in RANKL-treated rats. We conclude that therapies targeting RANKL are likely to have effects on cortical as well as trabecular bone density.     

Antioxidant alpha-lipoic acid inhibits osteoclast differentiation by reducing nuclear factor-kappaB DNA binding and prevents in vivo bone resorption induced by receptor activator of nuclear factor-kappaB ligand and tumor necrosis factor-alpha

The relationship between oxidative stress and bone mineral density or osteoporosis has recently been reported. As bone loss occurring in osteoporosis and inflammatory diseases is primarily due to increases in osteoclast number, reactive oxygen species (ROS) may be relevant to osteoclast differentiation, which requires receptor activator of nuclear factor-kappaB ligand (RANKL). Tumor necrosis factor-alpha (TNF-alpha) frequently present in inflammatory conditions has a profound synergy with RANKL in osteoclastogenesis. In this study, we investigated the effects of alpha-lipoic acid (alpha-LA), a strong antioxidant clinically used for some time, on osteoclast differentiation and bone resorption. At concentrations showing no growth inhibition, alpha-LA potently suppressed osteoclastogenesis from bone marrow-derived precursor cells driven either by a high-dose RANKL alone or by a low-dose RANKL plus TNF-alpha (RANKL/TNF-alpha). alpha-LA abolished ROS elevation by RANKL or RANKL/TNF-alpha and inhibited NF-kappaB activation in osteoclast precursor cells. Specifically, alpha-LA reduced DNA binding of NF-kappaB but did not inhibit IKK activation. Furthermore, alpha-LA greatly suppressed in vivo bone loss induced by RANKL or TNF-alpha in a calvarial remodeling model. Therefore, our data provide evidence that ROS plays an important role in osteoclast differentiation through NF-kappaB regulation and the antioxidant alpha-lipoic acid has a therapeutic potential for bone erosive diseases.     

Effect of gender on bone turnover in adult rats during simulated weightlessness.

Prologned spaceflight results in bone loss in astronauts, but there is considerable individual variation. The goal of this rat study was to determine whether gender influences bone loss during simulated weightlessness. Six-month-old Fisher 344 rats were hindlimb unweighted for 2 wk, after which the proximal tibiae were evaluated by histomorphometry. There were gender differences in tibia length, bone area, cancellous bone architecture, and bone formation. Compared with female rats, male rats had an 11.6% longer tibiae, a 27.8% greater cortical bone area, and a 37.6% greater trabecular separation. Conversely, female rats had greater cortical (316%) and cancellous (145%) bone formation rates, 28.6% more cancellous bone, and 30% greater trabecular number. Hindlimb unweighting resulted in large reductions in periosteal bone formation and mineral apposition rate in both genders. Unweighting also caused cancellous bone loss in both genders; trabecular number was decreased, and trabecular separation was increased. There was, however, no change in trabecular thickness in either gender. These architectural changes in cancellous bone were associated with decreases in bone formation and steady-state mRNA levels for bone matrix proteins and cancellous bone resorption. In conclusion, there are major gender-related differences in bone mass and turnover; however, the bone loss in hindlimb unweighted adult male and female rats appears to be due to similar mechanisms.     

Impaired bone formation and osteopenia in heterozygous β<Superscript>IVSII-654</Superscript> knockin thalassemic mice

β-thalassemia caused by the C→T mutation at nucleotide 654 of the intron 2 (β^IVSII-654) results in aberrant splicing of β-globin RNA, leading to an almost absence of β-globin synthesis. Although trabecular and cortical bone loss was previously reported in β-thalassemic mice with deletion of β-globin gene, the microscopic changes in trabecular structure in β^IVSII-654 thalassemic mice remained elusive. Here, we investigated the macroscopic and microscopic bone changes in 12-week-old β^IVSII-654 knockin thalassemic mice by dual-energy X-ray absorptiometry (DXA) and histomorphometric analysis, respectively. DXA revealed a decrease in bone mineral density in the lumbar vertebrae and tibial metaphysis, but not in the femoral diaphysis, suggesting that β^IVSII-654 thalassemia predominantly led to osteopenia at the trabecular site, but not the cortical site. Further histomorphometric analysis of the tibial secondary spongiosa showed that trabecular bone volume was significantly decreased with the expansion of marrow cavity. Decreases in osteoblast surface, osteoid surface, mineral apposition rate, mineralizing surface, and mineralized volume were also observed. Moreover, trabecular bone resorption was markedly enhanced as indicated by increases in the osteoclast surface and eroded surface. It could be concluded that β^IVSII-654 thalassemia impaired bone formation and enhanced bone resorption, thereby leading to osteopenia especially at the trabecular sites, such as the tibial metaphysis.     

Inhibition of TGF-beta receptor signaling in osteoblasts leads to decreased bone remodeling and increased trabecular bone mass.

Transforming growth factor-beta (TGF-beta) is abundant in bone matrix and has been shown to regulate the activity of osteoblasts and osteoclasts in vitro. To explore the role of endogenous TGF-(beta) in osteoblast function in vivo, we have inhibited osteoblastic responsiveness to TGF-beta in transgenic mice by expressing a cytoplasmically truncated type II TGF-beta receptor from the osteocalcin promoter. These transgenic mice develop an age-dependent increase in trabecular bone mass, which progresses up to the age of 6 months, due to an imbalance between bone formation and resorption during bone remodeling. Since the rate of osteoblastic bone formation was not altered, their increased trabecular bone mass is likely due to decreased bone resorption by osteoclasts. Accordingly, direct evidence of reduced osteoclast activity was found in transgenic mouse skulls, which had less cavitation and fewer mature osteoclasts relative to skulls of wild-type mice. These bone remodeling defects resulted in altered biomechanical properties. The femurs of transgenic mice were tougher, and their vertebral bodies were stiffer and stronger than those of wild-type mice. Lastly, osteocyte density was decreased in transgenic mice, suggesting that TGF-beta signaling in osteoblasts is required for normal osteoblast differentiation in vivo. Our results demonstrate that endogenous TGF-beta acts directly on osteoblasts to regulate bone remodeling, structure and biomechanical properties.     

Microfibril-associated Glycoprotein-1, an Extracellular Matrix Regulator of Bone Remodeling

MAGP1 is an extracellular matrix protein that, in vertebrates, is a ubiquitous component of fibrillin-rich microfibrils. We previously reported that aged MAGP1-deficient mice (MAGP1Δ) develop lesions that are the consequence of spontaneous bone fracture. We now present a more defined bone phenotype found in MAGP1Δ mice. A longitudinal DEXA study demonstrated age-associated osteopenia in MAGP1Δ animals and μCT confirmed reduced bone mineral density in the trabecular and cortical bone. Further, MAGP1Δ mice have significantly less trabecular bone, the trabecular microarchitecture is more fragmented, and the diaphyseal cross-sectional area is significantly reduced. The remodeling defect seen in MAGP1Δ mice is likely not due to an osteoblast defect, because MAGP1Δ bone marrow stromal cells undergo osteoblastogenesis and form mineralized nodules. In vivo, MAGP1Δ mice exhibit normal osteoblast number, mineralized bone surface, and bone formation rate. Instead, our findings suggest increased bone resorption is responsible for the osteopenia. The number of osteoclasts derived from MAGP1Δ bone marrow macrophage cells is increased relative to the wild type, and osteoclast differentiation markers are expressed at earlier time points in MAGP1Δ cells. In vivo, MAGP1Δ mice have more osteoclasts lining the bone surface. RANKL (receptor activator of NF-κB ligand) expression is significantly higher in MAGP1Δ bone, and likely contributes to enhanced osteoclastogenesis. However, bone marrow macrophage cells from MAGP1Δ mice show a higher propensity than do wild-type cells to differentiate to osteoclasts in response to RANKL, suggesting that they are also primed to respond to osteoclast-promoting signals. Together, our findings suggest that MAGP1 is a regulator of bone remodeling, and its absence results in osteopenia associated with an increase in osteoclast number.