Osteoclastic Migration Research Articles

Mmu-miR-185 regulates osteoclasts differentiation and migration by targeting Btk.

Bones undergo a constant remodeling, a process involving osteoclast-mediated bone resorption and osteoblast-mediated bone formation, crucial for maintaining healthy bone mass. We previously observed that miR-185 depletion may promote bone formation by regulating Bgn expression and the BMP/Smad signaling pathway. However, the effects of miR-185-5p on the osteoclasts and bone remodeling have not been elucidated, warranting further exploration. Tartrate-resistant acid phosphatase staining was utilized to assess the differentiation ability of bone marrow mononuclear macrophages (BMMs) from mmu-miR-185 gene knockout (KO) mice and wild-type (WT) mice. A reverse transcriptase-quantitative PCR was conducted to compare differences in miR-185-5p and osteoclast marker molecules, including Trap, Dcstamp, Ctsk and Nfatc1, between the KO group and WT group BMMs. Western blot analysis was employed to observe the expression of osteoclast marker molecules. A cell-counting kit-8 was used to analyze cell proliferation ability. Transwell experiments were conducted to detect cell migration. Dual-luciferase reporter assays were employed to confirm whether Btk is a downstream target gene of miR-185-5p. miR-185 depletion promoted osteoclast differentiation in bone marrow-derived monocytes/macrophages. Overexpression of miR-185-5p in RAW264.7 cells inhibited differentiation and migration of osteoclasts. Furthermore, Btk was identified as a downstream target gene of miR-185-5p, suggesting that miR-185-5p may inhibit osteoclast differentiation and migration by targeting Btk. miR-185 regulates osteoclasts differentiation, with overexpression of miR-185-5p inhibiting osteoclast differentiation and migration in vitro. Additionally, miR-185-5p may modulate osteoclastic differentiation and migration by regulating Btk expression.

Phospholipase D2 controls bone homeostasis by modulating M-CSF-dependent osteoclastic cell migration and microtubule stability

Phospholipase D2 (PLD2), a signaling protein, plays a central role in cellular communication and various biological processes. Here, we show that PLD2 contributes to bone homeostasis by regulating bone resorption through osteoclastic cell migration and microtubule-dependent cytoskeletal organization. Pld2-deficient mice exhibited a low bone mass attributed to increased osteoclast function without altered osteoblast activity. While Pld2 deficiency did not affect osteoclast differentiation, its absence promoted the migration of osteoclast lineage cells through a mechanism involving M-CSF-induced activation of the PI3K–Akt–GSK3β signaling pathway. The absence of Pld2 also boosted osteoclast spreading and actin ring formation, resulting in elevated bone resorption. Furthermore, Pld2 deletion increased microtubule acetylation and stability, which were later restored by treatment with a specific inhibitor of Akt, an essential molecule for microtubule stabilization and osteoclast bone resorption activity. Interestingly, PLD2 interacted with the M-CSF receptor (c-Fms) and PI3K, and the association between PLD2 and c-Fms was reduced in response to M-CSF. Altogether, our findings indicate that PLD2 regulates bone homeostasis by modulating osteoclastic cell migration and microtubule stability via the M-CSF-dependent PI3K–Akt–GSK3β axis.

Why SNP rs3755955 is associated with human bone mineral density? A molecular and cellular study in bone cells.

SNP rs3755955 (major/minor allele: G/A) located in Iduronidase-Alpha-L- (IDUA) gene was reported to be significant for human bone mineral density (BMD). This follow-up study was to uncover the underlying association mechanism through molecular and cellular functional assays relevant to bone. We tested the effects of single nucleotide polymorphisms (SNP) rs3755955 (defined allele G as wild-type and allele A as variant-type) on osteoblastic and osteoclastic functions, as well as protein phosphorylation in stably transfected human fetal osteoblast (hFOB) cell and mononuclear-macrophage (RAW264.7) cell. In hFOB cells, transfection with variant-type IDUA significantly decreased osteoblastic gene expression (OPN, COL1A1 and RANKL) (p < 0.01), impeded cell proliferation (p < 0.05), stimulated cell apoptosis (p < 0.001) and decreased ALP enzyme activity, as compared with that of wild-type IDUA transfection. In RAW264.7 cells, transfection with variant-type IDUA significantly inhibited cell apoptosis (p < 0.01), promoted osteoclastic precursor cell migration (p < 0.0001), growth (p < 0.01), osteoclastic gene expression (TRAP, RANK, Inte-αv and Cath-K) (p < 0.05) and TRAP enzyme activity (p < 0.001), as compared with that of wild-type IDUA transfection. In both hFOB and RAW264.7 cells, the total protein and IDUA protein-specific phosphorylation levels were significantly reduced by variant IDUA transfection, as compared with that of wild-type IDUA transfection (p < 0.05). Variant allele A of phosSNP rs3755955 in IDUA gene regulates protein phosphorylation, inhibits osteoblast function and promotes osteoclastic activity. The SNP rs3755955 could alter IDUA protein phosphorylation, significantly regulates human osteoblastic and osteoclastic gene expression, and influences the growth, differentiation and activity of osteoblast and osteoclast, hence to affect BMD.

Roles of apoptotic chondrocyte-derived CXCL12 in the enhanced chondroclast recruitment following methotrexate and/or dexamethasone treatment.

Intensive use of methotrexate (MTX) and/or dexamethasone (DEX) for treating childhood malignancies is known to cause chondrocyte apoptosis and growth plate dysfunction leading to bone growth impairments. However, mechanisms remain vague and it is unclear whether MTX and DEX combination treatment could have additive effects in the growth plate defects. In this study, significant cell apoptosis was induced in mature ATDC5 chondrocytes after treatment for 48 h with 10-5 M MTX and/or 10-6 M DEX treatment. PCR array assays with treated cells plus messenger RNA and protein expression confirmation analyses identified chemokine CXCL12 having the most prominent induction in each treatment group. Conditioned medium from treated chondrocytes stimulated migration of RAW264.7 osteoclast precursor cells and formation of osteoclasts, and these stimulating effects were inhibited by the neutralizing antibody for CXCL12. Additionally, while MTX and DEX combination treatment showed some additive effects on apoptosis induction, it did not have additive or counteractive effects on CXCL12 expression and its functions in enhancing osteoclastic recruitment and formation. In young rats treated acutely with MTX, there was increased expression of CXCL12 in the tibial growth plate, and more resorbing chondroclasts were found present at the border between the hypertrophic growth plate and metaphysis bone. Thus, the present study showed an association between induced chondrocyte apoptosis and stimulated osteoclastic migration and formation following MTX and/or DEX treatment, which could be potentially or at least partially linked molecularly by CXCL12 induction. This finding may contribute to an enhanced mechanistic understanding of bone growth impairments following MTX and/or DEX therapy.

The Function of Wnt Ligands on Osteocyte and Bone Remodeling

Bone homeostasis is continually maintained by the process of bone remodeling throughout life. Recent studies have demonstrated that Wnt signaling pathways play a fundamental role in the process of bone homeostasis and remodeling. Intracellular Wnt signaling cascades are initially triggered by a Wnt ligand–receptor complex formation. In previous studies, the blocking of Wnt ligands from different osteoblastic differentiation stages could cause defective bone development at an early stage. Osteocytes, the most abundant and long-lived type of bone cell, are a crucial orchestrator of bone remodeling. However, the role of Wnt ligands on osteocyte and bone remodeling remains unclear. In our present study, we found that, besides osteoblasts, osteocytes also express multiple Wnt ligands in the bone environment. Then, we used a Dmp1-Cre mouse line, in which there is expression in a subset of osteoblasts but mainly osteocytes, to study the function of Wnt ligands on osteocyte and bone remodeling in vivo. Furthermore, we explored the role of Wnt ligands on osteocytic mineralization ability, as well as the regulatory function of osteocytes on the process of osteoblastic differentiation and osteoclastic migration and maturity in vitro. We concluded that Wnt proteins play an important regulatory role in 1) the process of perilacunar/canalicular remodeling, as mediated by osteocytes, and 2) the balance of osteogenesis and bone resorption at the bone surface, as mediated by osteoblasts and osteoclasts, at least partly through the canonical Wnt/β-catenin signaling pathway and the OPG/RANKL signaling pathway.

Distinctive and selective route of PI3K/PKCα-PKCδ/RhoA-Rac1 signaling in osteoclastic cell migration

Cell migration during specialized stages of osteoclast precursors, mononuclear preosteoclasts, and multinucleated mature osteoclasts remain uncertain. M-CSF- and osteopontin-induced osteoclastic cell migration was inhibited by function-blocking monoclonal antibodies specific to the integrin αv and β3 subunits, suggesting that integrin αvβ3 mediates migratory signaling induced by M-CSF and osteopontin. M-CSF and osteopontin stimulation was shown to regulate two branched signaling processes, PI3K/PKCα/RhoA axis and PI3K/PKCδ/Rac1 axis. Interestingly, inactivation of RhoA or Rac1 blocked preosteoclast and mature osteoclast migration but not osteoclast precursor migration in a transwell-based cell migration assay. Moreover, the inhibitory effect on preosteoclast and mature osteoclast migration induced by Rac1 inactivation was more effective than that by RhoA inactivation. Collectively, our findings suggest that osteoclast precursor migration depends on PI3K/PKCα-PKCδ signaling mediated via integrin αvβ3 bypassing RhoA and Rac1, whereas preosteoclast and mature osteoclast migration relies on PI3K/PKCα-PKCδ/RhoA-Rac1 axis signaling mediated via integrin αvβ3 with increased dependency on PKCδ/Rac1 signaling route as differentiation progresses.

MiR-29 Promotes Murine Osteoclastogenesis by Regulating Osteoclast Commitment and Migration

Osteoclast differentiation is regulated by transcriptional, post-transcriptional, and post-translational mechanisms. MicroRNAs are fundamental post-transcriptional regulators of gene expression. The function of the miR-29 (a/b/c) family in cells of the osteoclast lineage is not well understood. In primary cultures of mouse bone marrow-derived macrophages, inhibition of miR-29a, -29b, or -29c diminished formation of TRAP (tartrate-resistant acid phosphatase-positive) multinucleated osteoclasts, and the osteoclasts were smaller. Quantitative RT-PCR showed that all miR-29 family members increased during osteoclast differentiation, in concert with mRNAs for the osteoclast markers Trap (Acp5) and cathepsin K. Similar regulation was observed in the monocytic cell line RAW264.7. In stably transduced RAW264.7 cell lines expressing an inducible miR-29 competitive inhibitor (sponge construct), miR-29 knockdown impaired osteoclastic commitment and migration of pre-osteoclasts. However, miR-29 knockdown did not affect cell viability, actin ring formation, or apoptosis in mature osteoclasts. To better understand how miR-29 regulates osteoclast function, we validated miR-29 target genes using Luciferase 3'-UTR reporter assays and specific miR-29 inhibitors. We demonstrated that miR-29 negatively regulates RNAs critical for cytoskeletal organization, including Cdc42 (cell division control protein 42) and Srgap2 (SLIT-ROBO Rho GTPase-activating protein 2). Moreover, miR-29 targets RNAs associated with the macrophage lineage: Gpr85 (G protein-coupled receptor 85), Nfia (nuclear factor I/A), and Cd93. In addition, Calcr (calcitonin receptor), which regulates osteoclast survival and resorption, is a novel miR-29 target. Thus, miR-29 is a positive regulator of osteoclast formation and targets RNAs important for cytoskeletal organization, commitment, and osteoclast function. We hypothesize that miR-29 controls the tempo and amplitude of osteoclast differentiation.

Histochemical aspects of the vascular invasion at the erosion zone of the epiphyseal cartilage in MMP-9-deficient mice

We have histologically examined vascular invasion and calcification of the hypertrophic zone during endochondral ossification in matrix metalloproteinase (MMP)-9 deficient (MMP-9-/-) mice and in their littermates at 3 days, 3 weeks and 6 weeks after birth. Capillaries and osteoclasts at the chondro-osseous junction showed an intense MMP-9 immunopositivity, suggesting that they recognize chemical properties of cartilaginous matrices, and then release MMP-9 for cartilage degradation. CD31-positive capillaries and tartrate-resistant acid phosphatase-reactive osteoclasts could be found in the close proximity in the region of chondro-osseous junction in MMP-9-/- mice, while in wild-type mice, vascular invasion preceded osteoclastic migration into the epiphyseal cartilage. Although MMP-9-/- mice revealed larger hypertrophic zones, the index of calcified area was significantly smaller in MMP-9-/- mice. Interestingly, the lower layer of the MMP-9-/- hypertrophic zone showed intense MMP-13 staining, which could not be observed in wild-type mice. This indicates that MMP-13 may compensate for MMP-9 deficiency at that specific region, but not to a point at which the deficiency could be completely rescued. In conclusion, it seems that MMP-9 is the optimal enzyme for cartilage degradation during endochondral ossification by controlling vascular invasion and subsequent osteoclastic migration.

Histopathological observations on osteolytic bone metastasis

Three types of bone metastasis can be identified : the osteolytic, osteoblastic and the intertrabecular metastasis. Bone resorption is believed to be responsible for osteolytic metastasis. The early stage of metastatic lesion showed tumor cells loosely intermingled with osteoblasts, fibroblastic stromal cells, osteoclasts and endothelial cells. In the metastatic nest, many tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts accumulated in direct contact with alkaline phosphatase (ALP)--or receptor activator of NF-kappaB ligand (RANKL)-positive osteoblastic cells, indicating the osteoclastogenesis. On the other hand, osteoclasts express CD44 while osteopontin was abundant in the stromal tissue of tumor nests. Therefore, mediating its affinity for CD44, osteopontin may serve to facilitate osteoclastic migration. We review the histopathological microenvironment featuring osteoclastic bone resorption, angiogenesis and matrix degradation, which appears to facilitate proliferation of tumor cells after the onset of bone metastasis.

Histochemical evidence of osteoclastic degradation of extracellular matrix in osteolytic metastasis originating from human lung small carcinoma (SBC‐5) cells

The aim of this study was to assess the dynamics of osteoclast migration and the degradation of unmineralized extracellular matrix in an osteolytic metastasis by examining a well-standardized lung cancer metastasis model of nude mice. SBC-5 human lung small carcinoma cells were injected into the left cardiac ventricle of 6-week-old BALB/c nu/nu mice under anesthesia. At 25-30 days after injection, the animals were sacrificed and their femora and/or tibiae were removed for histochemical analyses. Metastatic lesions were shown to occupy a considerable area extending from the metaphyses to the bone marrow region. Tartrate resistant acid phosphatase (TRAPase)-positive osteoclasts were found in association with an alkaline phosphatase (ALPase)-positive osteoblastic layer lining the bone surface, but could also be localized in the ALPase-negative stromal tissues that border the tumor nodules. These stromal tissues were markedly positive for osteopontin, and contained a significant number of TRAPase-positive osteoclasts expressing immunoreactivity for CD44. We thus speculated that, mediating its affinity for CD44, osteopontin may serve to facilitate osteoclastic migration after their formation associated with ALPase-positive osteoblasts. We next examined the localization of cathepsin K and matrix metallo-proteinase-9 (MMP-9) in osteoclasts. Osteoclasts adjacent to the bone surfaces were positive for both proteins, whereas those in the stromal tissues in the tumor nests showed only MMP-9 immunoreactivity. Immunoelectron microscopy disclosed the presence of MMP-9 in the Golgi apparatus and in vesicular structures at the baso-lateral cytoplasmic region of the osteoclasts found in the stromal tissue. MMP-9-positive vesicular structures also contained fragmented extracellular materials. Thus, osteoclasts appear to either select an optimized function, namely secreting proteolytic enzymes from ruffled borders during bone resorption, or recognize the surrounding extracellular matrix by mediating osteopontin/CD44 interaction, and internalize the extracellular matrices. Microsc.

Many scattered bones do not a skeleton make

The vertebrate skeleton is an amazing organ system. It pleases one by its exquisite architectural and engineering beauty. It impresses by its strength and remarkable adaptability. It is a veritable beehive of cellular activities: it is constantly being remodeled by the concerted action of bone-

PYK2 and FAK in osteoclasts.

Integrin alpha(v)beta3 plays a pivotal role in osteoclastic bone resoprtion by mediating both osteoclast attachments on bone matrix and cytoskeleton reorganization. The focal adhesion kinase (FAK) family kinases including FAK and proline-rich tyrosine kinase 2 (PYK2) are major tyrosine kinases activated by integrin engagement. In osteoclasts, although FAK is expressed, PYK2 appears to be the predominant mediator of integrin alpha(v)beta3 signaling events that influence osteoclast physiology and pathology. Recent progress in the understanding of the role of PYK2/FAK kinases in the regulation of osteoclastic actin cytoskeletal organization, cell migration, and bone resorption will be discussed in this review.

Human breast-cancer cells stimulate the fusion, migration and resorptive activity of osteoclasts in bone explants.

A central event in bone resorption is the recruitment of osteoclasts to future resorption sites. Breast-cancer cells invariably metastasise to the skeleton and induce extensive bone destruction by osteoclasts. However, our understanding of the mechanisms by which cancer cells interact with osteoclasts remains unclear. Consequently, we compared the effects of conditioned medium (CM) from 2 human breast-cancer cell lines, MB-MDA-231 and MCF-7, with those of a normal human breast epithelial cell line, HME, on osteoclastic fusion, resorptive activity and migration from the periosteum to the developing marrow cavity of fetal mouse metatarsals in culture. Osteoclastic resorptive activity was assessed by pre-labelling 17-day-old fetal metatarsal explants with 45Ca, whilst fusion and migration were monitored by histomorphometry and osteoclasts were identified by their tartrate-resistant acid phosphatase activity. CM from TPA-stimulated breast-cancer cell lines produced a significant increase in osteoclastic resorptive activity, whilst the normal breast cell line produced a minimal increase. The breast-cancer cell lines also stimulated osteoclastic fusion and migration in the metatarsal explants, but the normal breast cell line was without effect. The stimulatory effect of CM from MDA-MB-231 cells on osteoclastic fusion, but not migration, was partially inhibited by preventing prostaglandin and leukotriene synthesis by cells within the bone explants. In contrast, a synthetic matrix metalloproteinase (MMP) inhibitor, but not a cysteine proteinase inhibitor, prevented the migration of osteoclasts to the calcified centre of the metatarsal explants in response to CM from MDA-MB-231 cells. MDA-MB-231 cells also induced an increase in the expression of MMP-9 by migrating osteoclasts. Fractionation of the TPA-stimulated breast cancer cell CM established that the resorptive activity was associated with factors of m.w. >3 kDa. We determined by immuno-assay that human breast-cancer cells secrete parathyroid hormone-related protein (PTH-rP), tumour necrosis factor-alpha (TNF-alpha) and interleukins (ILs) 6 and 11. Neutralizing experiments with human antibodies to these cytokines established that PTH-rP and TNF-alpha production by MDA-MB-231 cells were responsible for mediating their effects on osteoclastic migration and ultimately bone resorption in the metatarsal explants.

Macrophage colony-stimulating factor stimulates survival and chemotactic behavior in isolated osteoclasts.

Macrophage colony-stimulating factor (M-CSF) is known to play an important role in osteoclast formation. However, its actions on mature cells have not been fully characterized. We now report that M-CSF dramatically stimulates osteoclastic motility and spreading; osteoclasts responded to a gradient of M-CSF with orientation, and random cell polarization occurred after isotropic exposure. M-CSF also supported the survival of osteoclasts by preventing apoptosis. Paradoxically, M-CSF inhibits bone resorption by isolated osteoclasts. We found that this was effected predominantly by reduction in the number of excavations. Thus, M-CSF showed a propensity to suppress resorption through a reduction in the proportion of cells that were resorbing bone. Our data suggest that apart from the established role of M-CSF in the provision of precursors for osteoclastic induction, a major role for M-CSF in bone resorption is to enhance osteoclastic survival, migration, and chemotaxis. It seems appropriate that during these processes resorptive functions should be suppressed. We suggest that M-CSF continues to modulate osteoclastic activity once osteoclasts are on resorptive sites, through regulation of the balance between resorption and migration, such that not only the quantity, but the spatial pattern of resorption can be controlled by adjacent M-CSF-secreting cells of osteoblastic lineage.