Yuko Nakamichi

Suppressive function of androgen receptor in bone resorption.

As locally converted estrogen from testicular testosterone contributes to apparent androgen activity, the physiological significance of androgen receptor (AR) function in the beneficial effects of androgens on skeletal tissues has remained unclear. We show here that inactivation of AR in mice using a Cre-loxP system-mediated gene-targeting technique caused bone loss in males but not in females. Histomorphometric analyses of 8-week-old male AR knockout (ARKO) mice showed high bone turnover with increased bone resorption that resulted in reduced trabecular and cortical bone mass without affecting bone shape. Bone loss in orchidectomized male ARKO mice was only partially prevented by treatment with aromatizable testosterone. Analysis of primary osteoblasts and osteoclasts from ARKO mice revealed that AR function was required for the suppressive effects of androgens on osteoclastogenesis supporting activity of osteoblasts but not on osteoclasts. Furthermore, expression of the receptor activator of NF-κB ligand (RANKL) gene, which encodes a major osteoclastogenesis inducer, was found to be up-regulated in osteoblasts from AR-deficient mice. Our results indicate that AR function is indispensable for male-type bone formation and remodeling.

Evaluation of Pharmaceuticals With a Novel 50-Hour Animal Model of Bone Loss

Osteoporosis remains a major public health problem through its associated fragility fractures. Several animal models for the study of osteoporotic bone loss, such as ovariectomy (OVX) and denervation, require surgical skills and several weeks to establish. Osteoclast differentiation and activation is mediated by RANKL. Here we report the establishment of a novel and rapid bone loss model by the administration of soluble RANKL (sRANKL) to mice. Mice were injected intraperitoneally with sRANKL and used to evaluate existing anti‐osteoporosis drugs. sRANKL decreased BMD within 50 h in a dose‐dependent manner. The marked decrease in femoral trabecular BMD shown by pQCT and the 3D images obtained by μCT were indistinguishable from those observed in the OVX model. Histomorphometry showed that osteoclastic activity was significantly increased in the sRANKL‐injected mice. In addition, serum biochemical markers of bone turnover such as Ca, C‐telopeptide of type 1 collagen (CTX), and TRACP5b were also significantly increased in the sRANKL‐injected mice in a dose‐dependent manner. Bisphosphonates (BPs), selective estrogen receptor modulators (SERMs), and PTH are commonly used for the treatment of osteoporosis. We successfully evaluated the effects of anti–bone‐resorbing agents such as BPs, a SERM, and anti–RANKL‐neutralizing antibody on bone resorption in a couple of weeks. We also evaluated the effects of PTH on bone formation in 2 wk. A combination of sRANKL injections and OVX made it possible to evaluate a SERM. The sRANKL model is the simplest, fastest, and easiest of all osteoporosis models and could be useful in the evaluation of drug candidates for osteoporosis.

Muramyl Dipeptide Enhances Osteoclast Formation Induced by Lipopolysaccharide, IL-1α, and TNF-α through Nucleotide-Binding Oligomerization Domain 2-Mediated Signaling in Osteoblasts

Muramyl dipeptide (MDP) is the minimal essential structural unit responsible for the immunoadjuvant activity of peptidoglycan. As well as bone-resorbing factors such as 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3) and PGE2, LPS and IL-1α stimulate osteoclast formation in mouse cocultures of primary osteoblasts and hemopoietic cells. MDP alone could not induce osteoclast formation in the coculture, but enhanced osteoclast formation induced by LPS, IL-1α, or TNF-α but not 1α,25(OH)2D3 or PGE2. MDP failed to enhance osteoclast formation from osteoclast progenitors induced by receptor activator of NF-κB ligand (RANKL) or TNF-α. MDP up-regulated RANKL expression in osteoblasts treated with LPS or TNF-α but not 1α,25(OH)2D3. Osteoblasts expressed mRNA of nucleotide-binding oligomerization domain 2 (Nod2), an intracellular sensor of MDP, in response to LPS, IL-1α, or TNF-α but not 1α,25(OH)2D3. Induction of Nod2 mRNA expression by LPS but not by TNF-α in osteoblasts was dependent on TLR4 and MyD88. MDP also enhanced TNF-α-induced osteoclast formation in cocultures prepared from Toll/IL-1R domain-containing adapter protein (TIRAP)-deficient mice through the up-regulation of RANKL mRNA expression in osteoblasts, suggesting that TLR2 is not involved in the MDP-induced osteoclast formation. The depletion of intracellular Nod2 by small interfering RNA blocked MDP-induced up-regulation of RANKL mRNA in osteoblasts. LPS and RANKL stimulated the survival of osteoclasts, and this effect was not enhanced by MDP. These results suggest that MDP synergistically enhances osteoclast formation induced by LPS, IL-1α, and TNF-α through RANKL expression in osteoblasts, and that Nod2-mediated signals are involved in the MDP-induced RANKL expression in osteoblasts.

Lineage‐committed osteoclast precursors circulate in blood and settle down into bone

Osteoclasts are derived from the monocyte/macrophage lineage, but little is known about osteoclast precursors in circulation. We previously showed that cell cycle–arrested quiescent osteoclast precursors (QOPs) were detected along bone surfaces as direct osteoclast precursors. Here we show that receptor activator of NF‐κB (RANK)‐positive cells isolated from bone marrow and peripheral blood possess characteristics of QOPs in mice. RANK‐positive cells expressed c‐Fms (receptors of macrophage colony‐stimulating factor) at various levels, but scarcely expressed other monocyte/granulocyte markers. RANK‐positive cells failed to exert phagocytic and proliferating activities, and differentiated into osteoclasts but not into dendritic cells. To identify circulating QOPs, collagen disks containing bone morphogenetic protein‐2 (BMP disks) were implanted into mice, which were administered bromodeoxyuridine daily. Most nuclei of osteoclasts detected in BMP‐2–induced ectopic bone were bromodeoxyuridine‐negative. RANK‐positive cells in peripheral blood proliferated more slowly and had a much longer lifespan than F4/80 (a macrophage marker)‐positive macrophages. When BMP disks and control disks were implanted in RANK ligand‐deficient mice, RANK‐positive cells were observed in the BMP disks but not in the controls. F4/80‐positive cells were distributed in both disks. Administration of FYT720, a sphingosine 1‐phosphate agonist, promoted the egress of RANK‐positive cells from hematopoietic tissues into bloodstream. These results suggest that lineage‐determined QOPs circulate in the blood and settle in the bone.

Daily administration of eldecalcitol (ED-71), an active vitamin D analog, increases bone mineral density by suppressing RANKL expression in mouse trabecular bone.

Eldecalcitol (ED‐71) is a new vitamin D3 derivative recently approved for the treatment of osteoporosis in Japan. Previous studies have shown that the daily administration of ED‐71 increases bone mineral density (BMD) by suppressing bone resorption in various animal models. In this study, we examined how ED‐71 suppresses bone resorption in vivo, by analyzing bone histomorphometry and ex vivo osteoclastogenesis assays. Daily administration of ED‐71 (50 ng/kg body weight) to 8‐week‐old male mice for 2 and 4 weeks increased BMD in the femoral metaphysis without causing hypercalcemia. Bone and serum analyses revealed that ED‐71 inhibited bone resorption and formation, indicating that the increase in BMD is the result of the suppression of bone resorption. This suppression was associated with a decrease in the number of osteoclasts in trabecular bone. We previously identified cell cycle‐arrested receptor activator of NF‐κB (RANK)‐positive bone marrow cells as quiescent osteoclast precursors (QOPs) in vivo. Daily administration of ED‐71 affected neither the number of RANK‐positive cells in vivo nor the number of osteoclasts formed from QOPs in ex vivo cultures. In contrast, ED‐71 suppressed the expression of RANK ligand (RANKL) mRNA in femurs. Immunohistochemical experiments also showed that the perimeter of the RANKL‐positive cell surface around the trabecular bone was significantly reduced in ED‐71‐treated mice than in the control mice. ED‐71 administration also increased BMD in 12‐week‐old ovariectomized mice, through the suppression of RANKL expression in the trabecular bone. These results suggest that the daily administration of ED‐71 increases BMD by suppressing RANKL expression in trabecular bone in vivo.

Noncanonical Wnt5a enhances Wnt/β-catenin signaling during osteoblastogenesis

Wnt regulates bone formation through β-catenin-dependent canonical and -independent noncanonical signaling pathways. However, the cooperation that exists between the two signaling pathways during osteoblastogenesis remains to be elucidated. Here, we showed that the lack of Wnt5a in osteoblast-lineage cells impaired Wnt/β-catenin signaling due to the reduced expression of Lrp5 and Lrp6. Pretreatment of ST2 cells, a stromal cell line, with Wnt5a enhanced canonical Wnt ligand-induced Tcf/Lef transcription activity. Short hairpin RNA-mediated knockdown of Wnt5a, but not treatment with Dkk1, an antagonist of Wnt/β-catenin signaling, reduced the expression of Lrp5 and Lrp6 in osteoblast-lineage cells under osteogenic culture conditions. Osteoblast-lineage cells from Wnt5a-deficient mice exhibited reduced Wnt/β-catenin signaling, which impaired osteoblast differentiation and enhanced adipocyte differentiation. Adenovirus-mediated gene transfer of Lrp5 into Wnt5a-deficient osteoblast-lineage cells rescued their phenotypic features. Therefore, Wnt5a-induced noncanonical signaling cooperates with Wnt/β-catenin signaling to achieve proper bone formation.

Chondromodulin I Is a Bone Remodeling Factor

ABSTRACT Chondromodulin I (ChM-I) was supposed from its limited expression in cartilage and its functions in cultured chondrocytes as a major regulator in cartilage development. Here, we generated mice deficient in ChM-I by targeted disruption of the ChM-I gene. No overt abnormality was detected in endochondral bone formation during embryogenesis and cartilage development during growth stages of ChM-I−/− mice. However, a significant increase in bone mineral density with lowered bone resorption with respect to formation was unexpectedly found in adult ChM-I−/− mice. Thus, the present study established that ChM-I is a bone remodeling factor.

IL-34 and CSF-1: similarities and differences

Colony-stimulating factor-1 (CSF-1) is widely expressed and considered to regulate the development, maintenance, and function of mononuclear phagocyte lineage cells such as monocytes, macrophages, dendritic cells (DCs), Langerhans cells (LCs), microglia, and osteoclasts. Interleukin-34 (IL-34) was recently identified as an alternative ligand for the CSF-1 receptor (CSF-1R) through functional proteomics experiments. It is well established that the phenotype of CSF-1R-deficient (CSF-1R−/−) mice is more severe than that of mice bearing a spontaneous null mutation in CSF-1 (CSF-1op/op). CSF-1R−/− mice are severely depleted of macrophages and completely lack LCs, microglia, and osteoclasts during their lifetime. In contrast, CSF-1op/op mice exhibit late-onset macrophage development and osteoclastogenesis, whereas they show modestly reduced numbers of microglia and a relatively normal LC development. In contrast, IL-34-deficient (IL-34−/−) mice show a marked reduction of LCs and a decrease in microglia. IL-34 and CSF-1 display different spatiotemporal expression patterns and have distinct biological functions. In this review, we focus on the functional similarities and differences between IL-34 and CSF-1 in vivo.

Interleukin‐4 inhibition of osteoclast differentiation is stronger than that of interleukin‐13 and they are equivalent for induction of osteoprotegerin production from osteoblasts

Interleukin (IL)‐4 and IL‐13 are closely related cytokines known to inhibit osteoclast formation by targeting osteoblasts to produce an inhibitor, osteoprotegerin (OPG), as well as by directly targeting osteoclast precursors. However, whether their inhibitory actions are the same remains unclear. The inhibitory effect of IL‐4 was stronger than that of IL‐13 in an osteoclast‐differentiation culture system containing mouse osteoblasts and osteoclast precursors. Both cytokines induced OPG production by osteoblasts in similar time‐ and dose‐dependent manners. However, IL‐4 was stronger in direct inhibition that targeted osteoclast precursors. Furthermore, IL‐4 induced phosphorylation of signal transducer and activator of transcription‐6 (STAT6) at lower concentrations than those of IL‐13 in osteoclast precursors. IL‐4 but not IL‐13 strongly inhibited the expression of nuclear factor of activated T‐cells, cytoplasmic 1 (nuclear factor‐ATc1), a key factor of osteoclast differentiation, by those precursors. Thus, the activities of IL‐4 and IL‐13 toward osteoclast precursors were shown to be different in regards to inhibition of osteoclast differentiation, whereas those toward osteoblasts for inducing OPG expression were equivalent.

Spleen serves as a reservoir of osteoclast precursors through vitamin D-induced IL-34 expression in osteopetrotic op/op mice.

Osteoclasts are generated from monocyte/macrophage-lineage precursors in response to colony-stimulating factor 1 (CSF-1) and receptor activator of nuclear factor-κB ligand (RANKL). CSF-1–mutated CSF-1op/op mice as well as RANKL−/− mice exhibit osteopetrosis (OP) caused by osteoclast deficiency. We previously identified RANKL receptor (RANK)/CSF-1 receptor (CSF-1R) double-positive cells as osteoclast precursors (OCPs), which existed in bone in RANKL−/− mice. Here we show that OCPs do not exist in bone but in spleen in CSF-1op/op mice, and spleen acts as their reservoir. IL-34, a newly discovered CSF-1R ligand, was highly expressed in vascular endothelial cells in spleen in CSF-1op/op mice. Vascular endothelial cells in bone also expressed IL-34, but its expression level was much lower than in spleen, suggesting a role of IL-34 in the splenic generation of OCPs. Splenectomy (SPX) blocked CSF-1–induced osteoclastogenesis in CSF-1op/op mice. Osteoclasts appeared in aged CSF-1op/op mice with up-regulation of IL-34 expression in spleen and bone. Splenectomy blocked the age-associated appearance of osteoclasts. The injection of 2-methylene-19-nor-(20S)-1α,25(OH)2D3 (2MD), a potent analog of 1α,25-dihidroxyvitamin D3, into CSF-1op/op mice induced both hypercalcemia and osteoclastogenesis. Administration of 2MD enhanced IL-34 expression not only in spleen but also in bone through a vitamin D receptor-mediated mechanism. Either splenectomy or siRNA-mediated knockdown of IL-34 suppressed 2MD-induced osteoclastogenesis. These results suggest that IL-34 plays a pivotal role in maintaining the splenic reservoir of OCPs, which are transferred to bone in response to diverse stimuli, in CSF-1op/op mice. The present study also suggests that the IL-34 gene in vascular endothelial cells is a unique target of vitamin D.