Expression of Kielin/chordin-like protein is regulated by BMP-2 in osteoblasts.

Bone morphogenetic protein (BMP), an osteoinductive factor, is a cytokine that induces osteoblast differentiation and mineralization, and expected to be applicable for hard tissue reconstruction. Kielin/chordin-like protein (Kcp), a member of the family of cysteine-rich proteins, enhances BMP signaling. The present study found that expression of Kcp in osteoblasts was induced by BMP-2 in a concentration- and time-dependent manner. Up-regulation of Kcp by BMP-2 was inhibited by Dorsomorphin, a SMAD signaling inhibitor. The involvement of up-regulation of Kcp by BMP-2 in induction of osteoblast differentiation by BMP-2 was also examined, which showed that suppression of Kcp expression by si Kcp partially inhibited induction of osteoblast differentiation and mineralization by BMP-2. Together, these results suggest that Kcp induced by BMP-2 functions to provide positive feedback for promotion of osteoblastic differentiation and mineralization by BMP-2 in osteoblasts.

Cathepsin K degrades osteoprotegerin to promote osteoclastogenesis in vitro.

Osteoblasts produce the receptor activator of nuclear factor-kappa B ligand (RANKL) and osteoprotegerin, the inducer and the suppressor of osteoclast differentiation and activation. We previously proposed that the degradation of osteoprotegerin by lysine-specific gingipain of Porphyromonas gingivalis and neutrophil elastase is one of the mechanisms of bone resorption associated with infection and inflammation. In the present study, we found that cathepsin K (CTSK) also degraded osteoprotegerin in an acidic milieu and the buffer with a pH of 7.4. The 37 k fragment of osteoprotegerin produced by the reaction with CTSK was further degraded into low molecular weight fragments, including a 13 k fragment, depending on the reaction time. The N-terminal amino acid sequence of the 37 k fragment matched that of the intact osteoprotegerin, indicating that CTSK preferentially hydrolyzes the death domain-like region of osteoprotegerin, not its RANKL-binding region. The 13 k fragment of osteoprotegerin was the C-terminal 13 k portion within the RANKL-binding region of the 37 k fragment. Finally, CTSK restored RANKL-dependent osteoclast differentiation that was suppressed by the addition of osteoprotegerin. Collectively, CTSK is a possible positive regulator of osteoclastogenesis.

8-Nitro-cGMP suppresses mineralization by mouse osteoblasts.

Nitric oxide and reactive oxygen species regulate bone remodeling, which occurs via bone formation and resorption by osteoblasts and osteoclasts, respectively. Recently, we found that 8-nitro-cGMP, a second messenger of nitric oxide and reactive oxygen species, promotes osteoclastogenesis. Here, we investigated the formation and function of 8-nitro-cGMP in osteoblasts. Mouse calvarial osteoblasts were found to produce 8-nitro-cGMP, which was augmented by tumor necrosis factor-α (10 ng/ml) and interleukin-1ß (1 ng/ml). These cytokines suppressed osteoblastic differentiation in a NO synthase activity-dependent manner. Exogenous 8-nitro-cGMP (30 µmol/L) suppressed expression of osteoblastic phenotypes, including mineralization, in clear contrast to the enhancement of mineralization by osteoblasts induced by 8-bromo-cGMP, a cell membrane-permeable analog of cGMP. It is known that reactive sulfur species denitrates and degrades 8-nitro-cGMP. Mitochondrial cysteinyl-tRNA synthetase plays a crucial role in the endogenous production of RSS. The expression of osteoblastic phenotypes was suppressed by not only exogenous 8-nitro-cGMP but also by silencing of the Cars2 gene, indicating a role of endogenous 8-nitro-cGMP in suppressing the expression of osteoblastic phenotypes. These results suggest that 8-nitro-cGMP is a negative regulator of osteoblastic differentiation.

Extracellular acidification augments sclerostin and osteoprotegerin production by Ocy454 mouse osteocytes.

Osteocytes sense the microenvironmental stimuli, including mechanical stress, and regulate bone resorption by osteoclasts and bone formation by osteoblasts. Diabetes and cancer metastasis to bone raise l-lactic acid in the bone tissue, causing acidification. Here, we investigated the effects of l-lactic acid and extracellular acidification on the function of mouse Ocy454 osteocytes. L- and d-lactic acid with low chiral selectivity and acidification of the medium raised the production of sclerostin and osteoprotegerin by Ocy454 cells. The mRNA expression of their genes increased after either treatment of L- and d-lactic acid or acidification of the medium. Furthermore, the conditioned medium of Ocy454 cells cultured in an acidic environment suppressed the induction of alkaline phosphatase activity in MC3T3-E1 cells, which was recovered by the anti-sclerostin antibody. While it is reported that HDAC5 inhibits the transcription of the sclerostin gene, extracellular acidification reduced the nuclear localization of HDAC5 in Ocy454 cells. While calmodulin kinase II (CaMKII) is known to phosphorylate and induce extranuclear translocation of HDAC5, KN-62, an inhibitor of CaMKII lowered the expression of the sclerostin gene in Ocy454 cells. Collectively, extracellular acidification is a microenvironmental factor that modulates osteocyte functions.

Establishment of Down's syndrome periodontal ligament cells by transfection with SV40T-Ag and hTERT.

Down's syndrome is one of the most common human congenital genetic diseases and affected patients have increased risk of periodontal disease. To examine involvement of the disease with periodontal disease development, we established immortalized periodontal ligament cells obtained from a Down's syndrome patient by use of SV40T-Ag and hTERT gene transfection. Expressions of SV40T-Ag and hTERT were observed in periodontal ligament cell-derived immortalized cells established from healthy (STPDL) and Down's syndrome patient (STPDLDS) samples. Primary cultured periodontal ligament cells obtained from a healthy subject (pPDL) had a limited number of population doublings (< 40), while STPDL and STPDLDS cells continued to grow with more than 80 population doublings. Primary cultured periodontal ligament cells obtained from the patient showed a chromosome pattern characteristic of Down's syndrome with trisomy 21, whereas STPDLDS samples showed a large number of abnormal chromosomes in those results. Gene expression analysis revealed that expression of DSCR-1 in STPDLDS is greater than that in STPDL. These results suggest that the newly established STPDLDS cell line may be a useful tool for study of periodontal disease in Down's syndrome patients.

Phorbol-12-myristate 13-acetate inhibits Nephronectin gene expression via Protein kinase C alpha and c-Jun/c-Fos transcription factors.

Nephronectin (Npnt) is an extracellular matrix protein and ligand of integrin α8ß1 known to promote differentiation of osteoblasts. A search for factors that regulate Npnt gene expression in osteoblasts revealed that phorbol 12-myristate 13-acetate (PMA), which activates protein kinase C (PKC), had a strong effect to suppress that expression. Research was then conducted to elucidate the signaling pathway responsible for regulation of Npnt gene expression by PMA in osteoblasts. Treatment of MC3T3-E1 cells with PMA suppressed cell differentiation and Npnt gene expression. Effects were noted at a low concentration of PMA, and were time- and dose-dependent. Furthermore, treatment with the PKC signal inhibitor Gö6983 inhibited down-regulation of Npnt expression, while transfection with small interfering RNA (siRNA) of PKCα, c-Jun, and c-Fos suppressed that down-regulation. The present results suggest regulation of Npnt gene expression via the PKCα and c-Jun/c-Fos pathway.

Neural crest-derived cells in nasal conchae of adult mice contribute to bone regeneration.

Neural crest-derived cells (NCDCs), a class of adult stem cells not restricted to embryonic tissues, are attractive tissue regenerative therapy candidates because of their ease of isolation, self-renewing properties, and multipotency. Although adult NCDCs can undergo osteogenic differentiation in vitro, whether they induce bone formation in vivo remains unclear. Previously, our group reported findings showing high amounts of NCDCs scattered throughout nasal concha tissues of adult mice. In the present study, NCDCs in nasal conchae labeled with enhanced green fluorescent protein (EGFP) were collected from adult P0-Cre/CAG-CAT-EGFP double transgenic mice, then cultured in serum-free medium to increase the number. Subsequently, NCDCs were harvested and suspended in type I atelocollagen gel, then an atelocollagen sponge was used as a scaffold for the cell suspension. Atelocollagen scaffolds with NCDCs were placed on bone defects created in a mouse calvarial bone defect model. Over the ensuing 12 weeks, micro-CT and histological analysis findings showed that mice with scaffolds containing NCDCs had slightly greater bone formation as compared to those with a scaffold alone. Furthermore, Raman spectroscopy revealed spectral properties of bone in mice that received scaffolds with NCDCs similar to those of native calvarial bone. Bone regeneration is important not only for gaining bone mass but also chemical properties. These results are the first to show the validity of biomolecule-free adult nasal concha-derived NCDCs for bone regeneration, including the chemical properties of regenerated bone tissue.

Zoledronate promotes inflammatory cytokine expression in human CD14-positive monocytes among peripheral mononuclear cells in the presence of γδ T cells.

Bisphosphonates distributed to bone exert toxic effects specifically towards osteoclasts. On the other hand, intravenous administration of a nitrogen-containing bisphosphonate (N-BP) such as zoledronate induces acute-phase reactions (APRs), including influenza-like fever 1 day later, indicating an interaction with immunocompetent cells circulating blood. Although it has been reported that activation of γδ T cells is pivotal to induce an APR following treatment with zoledronate, downstream events, including the production of inflammatory cytokines after activation of γδ T cells, remain obscure. We investigated the effects of zoledronate on inflammatory cytokine expression in human peripheral blood mononuclear cells (PBMCs) in vitro. While zoledronate induced mRNA expressions of tumour necrosis factor-α (TNF-α), interleukin (IL)-1ß, IL-6 and interferon-γ (IFN-γ) in PBMC, depletion of γδ T cells abolished that zoledronate-induced expression of those cytokines, indicating the necessity of γδ T cells for expression induction by zoledronate. However, which types of cells were responsible for the production of those cytokines in blood remained unclear. As it is generally accepted that monocytes and macrophages are primary sources of inflammatory cytokines, CD14+ cells from PBMC were exposed to zoledronate in the presence of PBMC, which resulted in induced expression of mRNAs for IL-1ß, IL-6 and IFN-γ, but not for TNF-α. These results indicate that CD14+ cells are responsible, at least in part, for the production of IL-1ß, IL-6 and IFN-γ in blood exposed to zoledronate. This suggests that CD14+ cells play an essential role in the occurrence of APRs following N-BP administration.

Osteoblastic differentiation of bone marrow mesenchymal stem cells in uremic rats.

Severe secondary hyperparathyroidism (SHPT) represents a high turnover bone disease, osteitis fibrosa, but the pathogenesis of osteitis fibrosa remains to be fully elucidated. We examined the characteristics of the differentiation of bone marrow mesenchymal stem cells (BMSCs) into osteoblasts in uremic rats. We bred 5/6 nephrectomized (Nx) rats with a high phosphorus (P) diet to induce SHPT (Nx + HP), or Nx (Nx + ND) and normal rats (Nc + ND) fed a standard diet (ND). After 8 weeks, BMSCs were isolated from the femur and serum were analyzed. BMSCs underwent flow cytometric examination for the expression patterns of cell surface markers (CD90+, CD29+, CD45-, and CD31-). Serum creatinine (Cre) levels were significantly elevated in the Nx + NP rats compared with the Nc + NP rats. Cre levels in the Nx + HP rats were levels to those in the Nx + ND rats. Serum P and PTH levels were significantly elevated in the Nx + HP rats compared with the Nx + ND rats. Bone morphometrical analysis showed increases in both osteoid volume and eroded surfaces in the Nx + HP but not in the Nx + ND rats. The populations of harvested BMSCs were similar between all three groups. Alp, Runx2, Pth1r and Cyclin D1 mRNA expression in the BMSCs from the Nx + ND rats were significantly suppressed compared with those isolated from the Nc + ND groups. Alizarin red staining tended to be similar to the expression of these mRNA. These results suggest that the BMSCs differentiation into osteoblasts was disturbed in the uremic rats.

Possible involvement of elastase in enhanced osteoclast differentiation by neutrophils through degradation of osteoprotegerin.

Neutrophils are one of the most abundant leukocytes in the sites of lesion of inflammatory diseases such as periodontitis and rheumatoid arthritis. These diseases are accompanied by bone loss, which worsens the quality of life of the patients. However, the role of neutrophils in the inflammatory bone loss has not been fully investigated. In the present study, we found that human neutrophils enhanced osteoclast differentiation from mouse bone marrow cells co-cultured with mouse osteoblasts in the presence of active vitamin D3. The enhanced osteoclast differentiation was significantly suppressed by elastatinal, a synthetic inhibitor of neutrophil elastase. Also, we found that human neutrophils degraded human recombinant osteoprotegerin (OPG), a decoy receptor for nuclear factor κB (RANK) ligand (RANKL), the essential osteoclast differentiation-inducing factor, expressed by osteoblasts. Degradation of OPG by neutrophils was suppressed by human α1-protease inhibitor, the major endogenous inhibitor of neutrophil elastase. Recombinant human neutrophil elastase degraded human OPG in its death domain-like region. These results indicated that the degradation of OPG by elastase contributed at least in part to the enhanced osteoclast differentiation by neutrophils. There is a possibility that neutrophils play an important role in inflammatory bone loss.

Roles of monocarboxylate transporter subtypes in promotion and suppression of osteoclast differentiation and survival on bone.

Monocarboxylate transporters (MCTs) provide transmembrane transport of monocarboxylates such as lactate and pyruvate. The present results showed that α-cyano-4-hydroxycinnamic acid (CHC), an inhibitor of MCTs, promoted osteoclast differentiation from macrophages at lower concentrations (0.1-0.3 mM) and suppressed that at a higher concentration (1.0 mM). On the other hand, CHC reduced the number of mature osteoclasts on the surface of dentin in a concentration-dependent manner. Additionally, macrophages and osteoclasts were found to express the Mct1, Mct2, and Mct4 genes, with Mct1 and Mct4 expression higher in macrophages, and that of Mct2 higher in osteoclasts. Although Mct1 gene knockdown in macrophages enhanced osteoclast formation induced by RANKL, Mct2 gene knockdown suppressed that. Finally, Mct2 gene silencing in mature osteoclasts decreased their number and, thereby, bone resorption. These results suggest that MCT1 is a negative regulator and MCT2 a positive regulator of osteoclast differentiation, while MCT2 is required for bone resorption by osteoclasts.

Visualization of junctional epithelial cell replacement by oral gingival epithelial cells over a life time and after gingivectomy.

Junctional epithelium (JE), which is derived from odontogenic epithelial cells immediately after eruption, is believed to be gradually replaced by oral gingival epithelium (OGE) over a lifetime. However, the detailed process of replacement remains unclear. The aim of the present study was to clarify the process of JE replacement by OGE cells using a green fluorescent protein (GFP)-positive tooth germ transplantation method. GFP-positive JE was partly replaced by OGE cells and completely replaced on day 200 after transplantation, whereas there was no difference in the expression of integrin ß4 (Itgb4) and laminin 5 (Lama5) between JE before and after replacement by OGE cells. Next, GFP-positive JE was partially resected. On day 14 after resection, the regenerated JE consisted of GFP-negative cells and also expressed both Itgb4 and Lama5. In addition, the gene expression profile of JE derived from odontogenic epithelium before gingivectomy was partly different from that of JE derived from OGE after gingivectomy. These results suggest that JE derived from the odontogenic epithelium is gradually replaced by OGE cells over time and JE derived from the odontogenic epithelium might have specific characteristics different to those of JE derived from OGE.

In Vitro Study of the Effects of Denosumab on Giant Cell Tumor of Bone: Comparison with Zoledronic Acid.

Giant cell tumor of bone (GCTB) is a locally aggressive primary bone tumor that contains numerous osteoclasts formed from marrow-derived precursors through receptor activator of nuclear factor κ-B ligand (RANKL), an osteoclast differentiation factor expressed in neoplastic cells of GCTB. Denosumab, a fully human monoclonal antibody targeting RANKL, has recently been used for the treatment of GCTB, and superior treatment effects have been reported. The aim of this work was to elucidate the mechanism of action of denosumab, and the differences between denosumab and zoledronic acid at the level of GCTB cells. We isolated GCTB cells from 3 patients and separated them into osteoclasts, osteoclast precursors and proliferating spindle-shaped stromal cells (the true neoplastic component), and examined the action of denosumab on differentiation, survival and bone resorption activity of osteoclasts. Denosumab and zoledronic acid inhibited osteoclast differentiation from mononuclear cells containing osteoclast precursors. Zoledronic acid inhibited osteoclast survival, whereas an inhibitory effect of denosumab on osteoclast survival was not observed. The inhibitory effect on bone resorption by both agents was confirmed in culture on dentin slices. Furthermore, zoledronic acid showed dose-dependent inhibition of cell growth of neoplastic cells whereas denosumab had no inhibitory effect on these cells. Denosumab has an inhibitory effect on osteoclast differentiation, but no inhibitory effects on survival of osteoclasts or growth of neoplastic cells in GCTBs.

Production of 8-nitro-cGMP in osteocytic cells and its upregulation by parathyroid hormone and prostaglandin E2.

Osteocytes regulate bone remodeling, especially in response to mechanical loading and unloading of bone, with nitric oxide reported to play an important role in that process. In the present study, we found that 8-nitroguanosine 3',5'-cyclic monophosphate (8-nitro-cGMP), a second messenger of nitric oxide in various types of cells, was produced by osteocytes in bone tissue as well as cultured osteocytic Ocy454 cells. The amount of 8-nitro-cGMP in Ocy454 cells increased during incubation with parathyroid hormone or prostaglandin E2, both of which are known to upregulate receptor activator of nuclear factor-κB ligand (RANKL) mRNA expression in osteocytes. On the other hand, exogenous 8-nitro-cGMP did not have effects on either the presence or absence of these bioactive substances. Furthermore, neither an inhibitor of nitric oxide synthase nor 8-bromo-cGMP, a cell-permeable analog of cGMP, showed remarkable effects on mRNA expression of sclerostin or RANKL. These results indicate that neither nitric oxide nor its downstream compounds, including 8-nitro-cGMP, alone are sufficient for induction of functional changes in osteocytes.

The inhibition of malignant melanoma cell invasion of bone by the TLR7 agonist R848 is dependent upon pro-inflammatory cytokines produced by bone marrow macrophages.

Distant metastasis remarkably worsens the prognoses of malignant melanoma patients. Toll-like receptors (TLRs) recognize molecules derived from many types of pathogens and activate the innate intravital immune system. In this study, we examined the effects of R848, a TLR7 ligand, on bone invasion by malignant melanoma cells. Mice underwent transplantation with cells of a malignant melanoma cell line B16F10, and were also administered R848 every three days. Hindlimbs were obtained 13 days after transplantation and invasion of bone marrow by B16F10 cells was evaluated. ELISA was used to determine the concentrations of cytokines in mouse serum and in the culture medium from bone marrow macrophages (BMMs) in the presence or absence of R848. In addition, MTS assays were used to examine the effects of media from BMM cultures on the proliferation of B16F10 cells. The rate of infiltration by B16F10 cells and the area of invasion were significantly reduced with R848 administration. Furthermore, serum levels of IL-6, IL-12, and IFN-γ were significantly increased in mice administered R848, with the same trend observed in the culture medium of BMMs treated with R848. In addition, B16F10 cell proliferation was suppressed by the addition of medium from cultured BMMs treated with R848. Neutralization by antibodies against IL-6, IL-12, and IFN-γ abrogated the suppression of proliferation of B16F10 cells by culture medium from BMMs treated with R848. Our results suggest that R848 drives the production of IL-6, IL-12, and IFN-γ in BMMs, which reduces proliferation and bone invasion by B16F10 cells.

Monocarboxylate transporter-1 promotes osteoblast differentiation via suppression of p53, a negative regulator of osteoblast differentiation.

Monocarboxylate transporter-1 (MCT-1) is a transmembrane transporter for monocarboxylates including lactate and pyruvate. Silencing Mct1 by its small interfering RNA (siRNA) suppressed the expression of marker genes for osteoblast differentiation, namely, Tnap, Runx2, and Sp7, induced by BMP-2 in mouse myoblastic C2C12 cells. Mct1 siRNA also suppressed alkaline phosphatase activity, as well as expressions of Tnap and Bglap mRNAs in mouse primary osteoblasts. On the other hand, Mct1 siRNA did not have effects on the Smad1/5 or ERK/JNK pathways in BMP-2-stimulated C2C12 cells, while it up-regulated the mRNA expression of p53 (Trp53) as well as nuclear accumulation of p53 in C2C12 cells in a BMP-2-independent manner. Suppression of osteoblastic differentiation by Mct1 siRNA in C2C12 cells was abolished by co-transfection of Trp53 siRNA. Together, these results suggest that MCT-1 functions as a positive regulator of osteoblast differentiation via suppression of p53.

Nanoindentation time-dependent deformation/recovery suggestive of methylglyoxal induced glycation in calcified nodules.

Although empirical findings have indicated increase in bone fracture risk in type 2 diabetes patients, that has yet to be proven by results obtained at the material level. Here, we report evidence showing nanoscale time-dependent deformation/recovery of in vitro calcified nodules mimicking bone turnover in type 2 diabetes in respect to methylglyoxal (MG)-induced glycation. Nanoindentation test results revealed that calcified nodules cultured with MG did not show adequate dimensional recovery, despite a large creep rate during constant load indentation testing. This lesser recovery is likely based on the linear matrix polymerization network formed by advanced glycation end products (AGEs) as a secondary product of MG. Since elevated serum MG and abnormal bone turnover related to the amount of AGEs are observed in cases of type 2 diabetes, this time-dependent behavior may be one of the factors of the bone fracture mechanism at the material level in affected patients.

Induction of osteoblastic differentiation of neural crest-derived stem cells from hair follicles.

The neural crest (NC) arises near the neural tube during embryo development. NC cells migrate throughout the embryo and have potential to differentiate into multiple cell types, such as peripheral nerves, glial, cardiac smooth muscle, endocrine, and pigment cells, and craniofacial bone. In the present study, we induced osteoblast-like cells using whisker follicles obtained from the NC of mice. Hair follicle cells derived from the NC labeled with enhanced green fluorescent protein (EGFP) were collected from protein zero-Cre/floxed-EGFP double transgenic mice and cultured, then treated and cultured in stem cell growth medium. After growth for 14 days, results of flow cytometry analysis showed that 95% of the EGFP-positive (EGFP+) hair follicle cells derived from the NC had proliferated and 76.2% of those expressed mesenchymal stem cells markers, such as platelet-derived growth factor α and stem cell antigen-1, and also showed constitutive expression of Runx2 mRNA. Cells stimulated with bone morphogenetic protein-2 expressed osteocalcin, osterix, and alkaline phosphatase mRNA, resulting in production of mineralized matrices, which were detected by von Kossa and alizarin red staining. Moreover, EGFP+ hair follicle cells consistently expressed macrophage colony-stimulating factor and osteoprotegerin (OPG). Addition of 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3] (10-8 M) to the cultures suppressed OPG expression and induced RANKL production in the cells. Furthermore, multinucleated osteoclasts appeared within 6 days after starting co-cultures of bone marrow cells with EGFP+ cells in the presence of 1,25(OH)2D3 and PGE2. These results suggest that NC-derived hair follicle cells possess a capacity for osteoblastic differentiation and may be useful for developing new bone regenerative medicine therapies.

Extracellular matrix loss in chondrocytes after exposure to interleukin-1ß in NADPH oxidase-dependent manner.

Osteoarthritis is a degenerative joint disease caused by excessive death of chondrocytes and loss of the extracellular matrix (ECM) in articular cartilage. We previously reported that reactive oxygen species (ROS) generated by the NADPH oxidase (NOX) isoform NOX-2 are involved in chondrocyte death induced by interleukin-1ß (IL-1ß). In this study, we investigate the role of NOX-2 in the production and degradation of ECM by chondrocytes. Although IL-1ß lowered the mRNA expression of type II collagen (Col2a1) and aggrecan (Acan) in mouse chondrocyte-like ATDC5 cells, RNA silencing of Nox2 did not change the mRNA expression of these major components of the ECM of cartilage. Hence, NOX-2 is not involved in the IL-1ß-induced suppression of ECM production. On the other hand, the NOX inhibitor 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF), the ROS scavenger N-acetylcysteine and an antisense oligodeoxynucleotide for Nox2 prevented the loss of proteoglycan induced by IL-1ß in highly differentiated ATDC5 cells. Furthermore, AEBSF did not affect the expression of hyaluronidase-1 and -2, whereas it suppressed hyaluronidase activity in culture medium. IL-1ß-induced intra- and extracellular acidification was also suppressed by AEBSF, as was the antisense oligodeoxynucleotide for Nox2. Since hyaluronidase activity is known to be higher under acidic conditions, NOX-2 probably contributes to ECM loss by the activation of hyaluronidase through acidification.

Down-regulation of Irf8 by Lyz2-cre/loxP accelerates osteoclast differentiation in vitro.

Interferon regulatory factor 8 (Irf8) is a transcription factor that negatively regulates osteoclast differentiation and Irf8 global knockout (Irf8 -/-) mice have been shown to have reduced bone volume resulting from increased osteoclast numbers. However, detailed analysis of the functions of Irf8 in osteoclast precursors with a monocyte/macrophage linage is difficult, because the population and properties of hematopoietic cells in Irf8 -/- mice are severely altered. Therefore, to clearly elucidate the functions of Irf8 during osteoclastogenesis, we established myeloid cell-specific Irf8 conditional knockout (Irf8 fl/fl ;Lyz2 cre/+) mice. We found that trabecular bone volume in the Irf8 fl/fl ;Lyz2 cre/+ mice was not significantly affected, while exposure to M-CSF and RANKL significantly increased TRAP activity in vitro in osteoclasts that underwent osteoclastogenesis from bone marrow-derived macrophages (BMMs) induced from bone marrow cells (BMCs) of those mice by addition of M-CSF. Our results also showed that expression of Irf8 mRNA and protein in BMMs obtained from Irf8 fl/fl ;Lyz2 cre/+ mice and cultured with M-CSF was reduced. These findings predicted that Lyz2/Lyz2-cre expression is induced when BMCs differentiate into BMMs in cultures with M-CSF. In osteoclast differentiation cultures, Lyz2 was gradually increased by M-CSF during the first 3 days of culture, then rapidly decreased by the addition of RANKL with M-CSF during the next 3 days. Furthermore, BMCs differentiated into osteoclasts while maintaining a low level of Lyz2 expression when cultured simultaneously with both M-CSF and RANKL from the initiation of culture. These findings suggest that Lyz2-cre expression is induced along with differentiation to BMMs by BMCs obtained from Irf8 fl/fl ;Lyz2 cre/+ mice and cultured with M-CSF. In addition, Irf8 was down-regulated by activation of the cre/loxP recombination system in BMMs and osteoclastogenesis was accelerated. Based on our results, we propose the existence in vivo of a new lineage of osteoclast precursors among BMCs, which differentiate into osteoclasts without up-regulation of Lyz2 expression.