Quiescent Osteoclast Precursors Research Articles

Silberline & Shinemax sign technical collaboration agreement

Osteoclasts are components of hematopoietic stem cell (HSC) niches, but their role as contributors to the HSC homeostasis and release are still controversial. We aimed to investigate whether an acute blood loss of 10% of total blood content, along with the consequent intense hematopoiesis, would affect osteoclast differentiation and activity. Isolated peripheral blood, spleen, and bone marrow (BM) cells from bones of hind limbs were investigated for the presence of specific subpopulations of osteoclast precursors: B220-CD3-NK1.1-CD11b-/lowCD115+CD117+ cells in BM, and B220-CD3-NK1.1-Gr-1-CD11b+CD115+ cells in peripheral blood and spleen as well as the receptor activator of nuclear factor κ-B+ cycle-arrested quiescent osteoclast precursors. Expression of osteoclastogenesis-related genes CD115, receptor activator of nuclear factor κ-B, and cathepsin K, the potential of BM cells to form osteoclast-like cells in vitro, and osteoclast activity in vivo were also evaluated. We observed an increase in spleen cellularity and myelopoiesis during week 1 following blood loss, without any significant effects on BM cellularity or BM myeloid precursors, including cells with high osteoclastogenic potential. However, at 1 week postbleeding, hematopoiesis significantly promoted the expression of cathepsin K, interleukin-34, and bone morphogenetic protein-6. Quiescent osteoclast precursors increased significantly in spleen 2 days following bleeding, whereas osteoclast activity remained unchanged up to 2 weeks postbleeding. Osteoclast-dependent B-cell differentiation was affected at the pre-B stage of maturation in BM, whereas the Lin-Sca-1+c-kit+ population expanded in BM and spleen after 2 days postbleeding. Our data demonstrate that an acute blood loss promotes differentiation and maturation of osteoclasts at 1 week but does not enhance osteoresorption at 2 weeks postbleeding. Our data also identify osteoclast differentiation as a consequent and important event in establishing HSC homeostasis following hematopoietic stress.

Huber buys Martinswerk (ATH & magnesium hydroxide) from Albemarle

Osteoclasts are components of hematopoietic stem cell (HSC) niches, but their role as contributors to the HSC homeostasis and release are still controversial. We aimed to investigate whether an acute blood loss of 10% of total blood content, along with the consequent intense hematopoiesis, would affect osteoclast differentiation and activity. Isolated peripheral blood, spleen, and bone marrow (BM) cells from bones of hind limbs were investigated for the presence of specific subpopulations of osteoclast precursors: B220-CD3-NK1.1-CD11b-/lowCD115+CD117+ cells in BM, and B220-CD3-NK1.1-Gr-1-CD11b+CD115+ cells in peripheral blood and spleen as well as the receptor activator of nuclear factor κ-B+ cycle-arrested quiescent osteoclast precursors. Expression of osteoclastogenesis-related genes CD115, receptor activator of nuclear factor κ-B, and cathepsin K, the potential of BM cells to form osteoclast-like cells in vitro, and osteoclast activity in vivo were also evaluated. We observed an increase in spleen cellularity and myelopoiesis during week 1 following blood loss, without any significant effects on BM cellularity or BM myeloid precursors, including cells with high osteoclastogenic potential. However, at 1 week postbleeding, hematopoiesis significantly promoted the expression of cathepsin K, interleukin-34, and bone morphogenetic protein-6. Quiescent osteoclast precursors increased significantly in spleen 2 days following bleeding, whereas osteoclast activity remained unchanged up to 2 weeks postbleeding. Osteoclast-dependent B-cell differentiation was affected at the pre-B stage of maturation in BM, whereas the Lin-Sca-1+c-kit+ population expanded in BM and spleen after 2 days postbleeding. Our data demonstrate that an acute blood loss promotes differentiation and maturation of osteoclasts at 1 week but does not enhance osteoresorption at 2 weeks postbleeding. Our data also identify osteoclast differentiation as a consequent and important event in establishing HSC homeostasis following hematopoietic stress.

Bone Cell Biology Assessed by Microscopic Approach. Mechanisms of in vivo osteoclastogenesis

Many new findings about osteoclastogenesis have been provided by in vitro osteoclast culture methods. However, it is necessary to identify genuine in vivo osteoclast precursors and analyze their dynamics in order to completely understand in vivo osteoclastogenesis. Previously, we identified an in vivo osteoclast precursor (qOP : quiescent osteoclast precursors). In this review, the differentiation and movement of qOP will be described based on recent experimental data. Moreover, I will show evidence that the expression level of RANK in qOP is increased in bone tissue, which is an important event for in vivo osteoclastogenesis.

Acute hematopoietic stress in mice is followed by enhanced osteoclast maturation in the bone marrow microenvironment

Osteoclasts are components of hematopoietic stem cell (HSC) niches, but their role as contributors to the HSC homeostasis and release are still controversial. We aimed to investigate whether an acute blood loss of 10% of total blood content, along with the consequent intense hematopoiesis, would affect osteoclast differentiation and activity. Isolated peripheral blood, spleen, and bone marrow (BM) cells from bones of hind limbs were investigated for the presence of specific subpopulations of osteoclast precursors: B220(-)CD3(-)NK1.1(-)CD11b(-/low)CD115(+)CD117(+) cells in BM, and B220(-)CD3(-)NK1.1(-)Gr-1(-)CD11b(+)CD115(+) cells in peripheral blood and spleen as well as the receptor activator of nuclear factor κ-B(+) cycle-arrested quiescent osteoclast precursors. Expression of osteoclastogenesis-related genes CD115, receptor activator of nuclear factor κ-B, and cathepsin K, the potential of BM cells to form osteoclast-like cells in vitro, and osteoclast activity in vivo were also evaluated. We observed an increase in spleen cellularity and myelopoiesis during week 1 following blood loss, without any significant effects on BM cellularity or BM myeloid precursors, including cells with high osteoclastogenic potential. However, at 1 week postbleeding, hematopoiesis significantly promoted the expression of cathepsin K, interleukin-34, and bone morphogenetic protein-6. Quiescent osteoclast precursors increased significantly in spleen 2 days following bleeding, whereas osteoclast activity remained unchanged up to 2 weeks postbleeding. Osteoclast-dependent B-cell differentiation was affected at the pre-B stage of maturation in BM, whereas the Lin(-)Sca-1(+)c-kit(+) population expanded in BM and spleen after 2 days postbleeding. Our data demonstrate that an acute blood loss promotes differentiation and maturation of osteoclasts at 1 week but does not enhance osteoresorption at 2 weeks postbleeding. Our data also identify osteoclast differentiation as a consequent and important event in establishing HSC homeostasis following hematopoietic stress.

Periodontitis mainly increases osteoclast formation via enhancing the differentiation of quiescent osteoclast precursors into osteoclasts.

Tartrate-resistant acid phosphatase (TRAP)-positive multinucleated osteoclasts are formed in sequential steps: proliferation and differentiation of hematopoietic progenitors into quiescent osteoclast precursors (QOPs), followed by fusion of QOPs. In this study, we investigated whether enhancement of osteoclast formation by periodontitis is derived from the stimulation of proliferation of hematopoietic progenitors or the differentiation of QOPs into osteoclasts. Ligatures were placed around the first molars in the left mandibles of Fischer 344 inbred rats. The rats received drinking water containing bromodeoxyuridine (BrdU) (which can be incorporated into dividing nuclei) after ligation during the experimental period. The number of inflammatory cells in the distal area was counted. Alveolar bone loss was histologically estimated by measuring the distance from the cementoenamel junction to the alveolar bone crest in the distal area and determining the percentage of periodontal ligament area in the furcation. The number of osteoclasts and percentage of BrdU(+) nuclei in total osteoclasts nuclei were counted after TRAP and BrdU double labeling. The number of polymorphonuclear cells increased on day 1 and then rapidly decreased. The number of mononuclear cells increased in a time-dependent manner up to day 5 and remained the same until day 10. Alveolar bone loss of ligatured teeth increased in a time-dependent manner. The number of osteoclasts peaked on day 3 then gradually decreased. At peak, the percentage of BrdU(+) nuclei in total osteoclasts nuclei in the distal and furcation areas were 7.9% and 4.1%, respectively. These results indicate that most of the osteoclasts formed after periodontitis induction are derived from preformed QOPs, suggesting that enhancement of osteoclast formation by periodontitis might be mainly caused by stimulating the differentiation of QOPs into osteoclasts.

Osteoclastic bone resorption directly activates osteoblast function

Osteoclasts, the multinucleated cells that resorb bone, originate from cell cycle-arrested quiescent osteoclast precursors [1]. Mesenchymal osteoblastic cells are involved in osteoclast differentiation. Osteoclast precursors express RANK (a receptor of RANKL), recognize RANKL expressed by osteoblasts through cell-cell interaction and differentiate into osteoclasts in the presence of M-CSF. OPG, produced mainly by osteoblasts, is a soluble decoy receptor for RANKL. Deficiency of OPG in mice induces osteoporosis caused enhanced bone resorption. Elevated osteoblastic activity was suppressed by bisphosphonate administration in OPG-deficient mice. These results suggest that bone formation is accurately coupled with bone resorption. Collagen sponge disks containing BMP-2 were implanted into the dorsal muscle pouches in OPG-deficient mice. TRAP-positive osteoclasts and ALP-positive osteoblasts were observed in BMP-2-disks preceding the onset of calcification for one week. OPG and soluble RANK inhibited BMP-2-induced osteoclast formation but not the appearance of ALP-positive cells in OPG-deficient mice. We then examined how osteoblasts are involved in osteoclastogenesis other than RANKL expression, using RANKL-deficient mice. RANKL-deficient mice showed severe osteopetrosis due to loss of osteoclasts. Injection of RANKL into RANKL-deficient mice induced many osteoclasts in bone but not soft tissues [2]. These results suggest that osteoblasts determine the place of osteoclastogenesis from haemopoietic stem cells in bone. We next explored roles of osteoclasts in ectopic bone formation induced by BMP using op/op and c-fos-deficient osteopetrotic mice. The ectopic bones formed in op/op mice showed extremely rough surfaces, whereas those in wild-type mice showed smooth ones. Bone mineral density of BMP-induced ectopic bone in op/op mice was about 2-times higher than that in wild-type mice. TRAP-positive osteoclasts exhibit in outer of the ectopic bone in the wild-type mice. In op/op mice, although osteoclasts strongly exhibit in inside of the BMP-induced ectopic bone, TRAP-positive osteoclasts did not exhibit in outer of the BMP-induced ectopic bone. Furthermore, the accentuation of the BMP-induced ectopic bone formation did not exist in osteopetrotic c-Fos-deficient mice. In c-Fos-deficient mice, which are completely osteoclasts deficiency, the accentuation of the BMP-induced ectopic bone formation did not exist. Furthermore, there is no RANK-positive osteoclast progenitors in bone derived from c-Fos-deficient mice. These results suggest that RANK-positive osteoclast progenitors are positively regulate the signal of bone formation. In summary, osteoclastic bone resorption directly activates osteoblast function and osteoclasts are involved in normal bone morphogenesis.

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.

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.

RANKL signaling and bone diseases. Quiescent osteoclast precursors and RANKL signaling

It is well known that bone marrow macrophages differentiate into osteoclasts in response to M-CSF and RANKL in culture. However, the characteristics and dynamics of osteoclast precursors in vivo are not clear. Cell cycle arrest in osteoclast precursors is a prerequisite step for their differentiation into osteoclasts. We named such precursors "QOPs (cell cycle-arrested quiescent osteoclast precursors) " . Injection of 2MD [a potent analogue of 1α, 25 (OH) (2)D(3)] , RANKL, or M-CSF to mice induced osteoclast differentiation from QOPs. Moreover, osteoclasts appearing in BMP (bone morphogenetic protein) -induced ectopic bone were also differentiated from QOPs without cell proliferation. These results indicate that (1)osteoclasts are formed from QOPs in response to bone resorbing stimuli in vivo, (2) QOPs circulate in the bloodstream and settle in the right place for osteoclastogenesis. Here I review recent advances in our understanding of osteoclast precursors in vivo.

Identification of cell cycle–arrested quiescent osteoclast precursors in vivo

Identification of cell cycle–arrested quiescent osteoclast precursors in vivo

Identification of cell cycle–arrested quiescent osteoclast precursors in vivo

Osteoclasts are multinucleated cells that resorb bone. Although osteoclasts originate from the monocyte/macrophage lineage, osteoclast precursors are not well characterized in vivo. The relationship between proliferation and differentiation of osteoclast precursors is examined in this study using murine macrophage cultures treated with macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-κB (RANK) ligand (RANKL). Cell cycle–arrested quiescent osteoclast precursors (QuOPs) were identified as the committed osteoclast precursors in vitro. In vivo experiments show that QuOPs survive for several weeks and differentiate into osteoclasts in response to M-CSF and RANKL. Administration of 5-fluorouracil to mice induces myelosuppression, but QuOPs survive and differentiate into osteoclasts in response to an active vitamin D3 analogue given to those mice. Mononuclear cells expressing c-Fms and RANK but not Ki67 are detected along bone surfaces in the vicinity of osteoblasts in RANKL-deficient mice. These results suggest that QuOPs preexist at the site of osteoclastogenesis and that osteoblasts are important for maintenance of QuOPs.