MNC Formation Research Articles

The extra domain A of fibronectin facilitates osteoclastogenesis in radicular cysts through vascular endothelial growth factor.

To analyse the effects of the alternatively spliced fibronectin (FN) gene and its isoforms on osteoclastogenesis in radicular cysts. Specimens of radicular cysts were collected surgically from 22 patients whose radiolucent periapical areas were measured on digital panoramic radiographs before surgery. The associations between the radiolucent areas and FN isoforms, vascular endothelial growth factor (VEGF) expression or micro-vessel density, as well as the relationships amongst them, were analysed by immunohistochemical staining using the antibodies IST-9, BC-1, P1F11, VEGF and CD34. Fibroblasts isolated from those specimens were used to induce Trap+MNCs, and the effects of induction were assessed by blocking FN containing extra domain A (EDA+FN), COX-2 or VEGF in vitro. The effects of EDA exon knockout using CRISPR/Cas system were also assessed. Quantitative PCR was used to analyse relative expression of FN isoforms and osteoclastogenic genes. Data were analysed using linear regression, Spearman's rank correlation analysis, chi-square test and Student's t-test; P<0.05 was considered significant. Micro-vessel density and EDA+FN staining were positively associated with the size of radiolucent periapical areas (mm2 ; P<0.05), consistent with a positive association between Trap+MNCs and VEGF expression in fibroblasts (P<0.05). Blocking the interaction between EDA+FN and fibroblasts inhibited Trap+MNC formation. In addition, EDA exon knockout decreased VEGF expression and inhibited Trap+MNC formation to the extent of blocking VEGF by bevacizumab, but osteoclastogenic induction was restored by recombinant VEGF. Using retrospective clinicopathological data, VEGF staining was shown to be positively associated with EDA+FN staining, micro-vessel density and the size of radiolucent areas (P<0.05). In fibrous capsules of radicular cysts, the alternatively spliced isoform EDA+FN generated by fibroblasts stimulated VEGF expression via an autocrine effect and then facilitated osteoclastogenesis. Both blockage of VEGF and EDA exon knockout could be used to inhibit bone destruction.

First-Principle Calculations for Magnetism of Mn-Doped Graphene

Mn doped graphene-based dilute magnetic semiconductors (DMS) are investigated using the first-principle calculation based on density functional theory. In this paper, the Mn-C bond length, formation energy and magnetic moment are calculated in different doping systems and their density of states is made a detailed analysis. It is found that Mn-doped graphene has strong ferromagnetic properties and the magnetic moments of graphene supercells are different with the impurity concentrations. These supercells of a Mn atom substituting a C atom are increasingly stable with extending cells and the 11×11 supercell possesses the biggest magnetic moment of 3.8μB in these systems. The analysis of the density of states indicates the magnetic properties of Mn-doped graphene derive from the p-d exchange mechanism.

Effects of selective prostaglandin EP4 receptor antagonist on osteoclast formation and bone resorption in vitro

Prostaglandin estradiol (PGE2) stimulates bone resorption by a cyclic AMP (cAMP)-dependent mechanism that involves prostaglandin E receptors of the EP2 and EP4 subtypes. We tested a potent selective EP4 antagonist (EP4RA), which blocks PGE2 binding to EP4 receptors. We examined the effects of EP4RA on osteoclastogenesis in murine marrow cultures, on cAMP production in primary osteoblastic (POb) cell cultures, and on bone resorption in organ cultures. EP4RA (1 μmol/L) decreased the number of tartrate-resistant acid phosphatase-positive multinucleated cells (TRAP+ MNC) by 46%–48% in cultures treated with 0.1–1.0 μmol/L PGE2 and by 96% in cultures treated with 0.01 μmol/L PGE2. EP4RA also decreased TRAP+ MNC formation by 60% in 1,25-dihydroxyvitamin D (1,25D)-treated cultures and by 62% in parathyroid hormone (PTH)-treated cultures. A chemically related analog of EP4RA that lacks antagonist activity did not inhibit TRAP+ MNC formation. EP4RA decreased cAMP production in PGE2-treated POb by 44% but did not block cAMP response to PTH. EP4RA inhibited the increase in receptor activator of NF-κB ligand (RANKL) mRNA levels produced by PGE2. In fetal rat long bone cultures, EP4RA decreased 45Ca release from control, unstimulated cultures by 12%–25% and from PGE2-stimulated cultures by 22%–37%. Because EP4RA partially inhibited osteoclastogenesis not only in response to PGE2 but also in response to 1,25D and PTH, these results suggest that activation of the EP4 receptor may play a general role in osteoclastic bone resorption. EP4RA showed partial inhibition of PGE2-stimulated osteoclastogenesis at 1 μmol/L, but almost complete inhibition at 0.01 μmol/L PGE2. This could be due to the limited efficacy of the antagonist at high concentrations of PGE2, or an alternative pathway, such as activation of the EP2 receptor.

Cell-to-Cell adhesion via intercellular adhesion molecule-1 and leukocyte function-associated antigen-1 pathway is involved in 1alpha,25(OH)2D3, PTH and IL-1alpha-induced osteoclast differentiation and bone resorption.

Cell-to-cell interaction is required for the differentiation of osteoclast precursors as well as for osteoclast function. The present study was undertaken to determine whether 1,25 dihydroxyvitamin D3 (1,25D), PTH, IL-1alpha and PGE2 depend on cell-to-cell interactions through the intercellular adhesion molecule (ICAM)-1/leukocyte function-associated antigen (LFA)-1 pathway in osteoclast formation and bone resorption. We found that mouse osteoblasts expressed ICAM-1 and that the expression was increased by treatment with PTH, IL-1alpha or 1,25D, but not by PGE2. In resorption assays measuring either 45Ca release from bone organ cultures or pit formation in bone cell cultures, 1,25D-, PTH- and IL-1alpha-stimulated resorption was inhibited by anti-ICAM-1 monoclonal antibody (mAb) and/or anti-LFA-1 mAb, while basal and PGE2-stimulated bone resorbing activities were not affected by these mAbs. Furthermore, in a mouse bone marrow culture system, stimulation of osteoclast-like (OCL) cell formation by 1,25D (10 nM), PTH (10 ng/ml) or IL-1alpha (10 ng/ml) was inhibited by the addition of anti-ICAM-1 mAb and/or anti-LFA-1 mAb. In a coculture system of murine spleen cells and osteoblasts, the ICAM-1/LFA-1 interaction was also involved in 1,25D-, PTH- and IL-1alpha-stimulated TRAP-positive MNC formation. However, anti-ICAM-1 mAb and anti-LFA-1 mAb did not alter either 1,25D- or PTH-stimulated receptor activator of NF-kappaB ligand (RANKL) mRNA transcription in bone marrow cultures. Taken together, we here propose that ICAM-1-mediated cell-to-cell adhesion of osteoblasts and osteoclast precursors is involved in RANKL-dependent osteoclast maturation stimulated by 1,25D, PTH, and IL-1alpha.

Knockout of the Murine Prostaglandin EP2 Receptor Impairs Osteoclastogenesis in Vitro*

Prostaglandin E2 (PGE2) stimulates the formation of osteoclast-like tartrate-resistant acid phosphatase-positive multinucleated cells (TRAP + MNC) in vitro. This effect likely results from stimulation of adenylyl cyclase, which is mediated by two PGE2 receptors, designated EP2 and EP4. We used cells from mice in which the EP2 receptor had been disrupted to test its role in the formation of TRAP + MNC. EP2 heterozygous (+/-) mice in a C57BL/6 x 129/SvEv background were bred to produce homozygous null (EP2 -/-) and wild-type (EP2 +/+) mice. PGE2, PTH, or 1,25 dihydroxyvitamin D increased TRAP+ MNC in 7-day cultures of bone marrow cells from EP2 +/+ mice. In cultures from EP2 -/- animals, responses to PGE2, PTH, and 1,25 dihydroxyvitamin D were reduced by 86%, 58%, and 50%, respectively. A selective EP4 receptor antagonist (EP4RA) further inhibited TRAP+ MNC formation in both EP2 +/+ and EP2 -/- cultures. In cocultures of spleen and calvarial osteoblastic cells, the response to PGE2 or PTH was reduced by 92% or 85% when both osteoblastic cells and spleen cells were from EP2 -/- mice, by 88% or 68% when only osteoblastic cells were from EP2 -/- mice and by 58% or 35% when only spleen cells were from EP2 -/- mice. PGE2 increased receptor activator of nuclear factor (NF)-kappaB ligand (RANKL) messenger RNA expression in osteoblastic and bone marrow cell cultures from EP2 +/+ mice 2-fold but had little effect on cells from EP2 -/- mice. Spleen cells cultured with RANKL and macrophage colony stimulating factor produced TRAP+ MNC. PGE2 increased the number of TRAP+ MNC in spleen cell cultures from EP2 +/+ mice but not in cultures from EP2 -/- mice. EP4RA had no effect on the PGE2 response in spleen cell cultures. PGE2 decreased the expression of messenger RNA for granulocyte-macrophage colony stimulating factor in spleen cell cultures from EP2 +/+ mice but had little effect on cells from EP2 -/- mice. These data demonstrate that the prostaglandin EP2 receptor plays a role in the formation of osteoclast-like cells in vitro. A major defect in EP2 -/- mice appears to be in the capacity of osteoblastic cells to stimulate osteoclast formation. In addition, there appears to be a defect in the response of cells of the osteoclastic lineage to PGE2 in EP2 -/- mice.

Chinese hamster ovary cells expressing alpha4beta1 integrin stimulate osteoclast formation in vitro.

It is reported that Chinese hamster ovary cells transfected with human alpha4 cDNA (alpha4CHOs) and expressing functional alpha4beta1 integrin developed bone metasasis in nude mice. To clarify the role of alpha4beta1 integrin in bone metastasis, in terms of tumor-mediated bone destruction, we examined whether alpha4CHOs stimulate osteoclast formation in cocultures with mouse bone marrow cells. The number of osteoclast-like cells identified as tartrate-resistant acid phosphatase positive multinucleated cells (TRAP(+) MNCs) formed from bone marrow cells increased with the increasing number of alpha4CHOs cocultured. The effects of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) and prostaglandin E2 (PGE2) on TRAP(+) MNC formation were enhanced in cocultures with alpha4CHOs. TRAP(+) MNCs induced by alpha4CHOs possessed calcitonin receptors and resorbed calcified tissues. In cocultures, alpha4CHOs and bone marrow stromal cells were in contact with each other and bone marrow stromal cells expressed vascular cell adhesion molecule-1 (VCAM-1), which is one of the ligands for alpha4beta1 integrin. TRAP(+) MNC formation was not stimulated in cocultures where direct contact between alpha4CHOs and bone marrow cells was inhibited by membrane filters. Alpha4CHOs do not support TRAP(+) MNC formation in cocultures with spleen cells but do support TRAP(+) mononuclear cell and MNC formation from spleen cells in the presence of osteoblastic cells. Cultured media from alpha4CHOs, bone marrow cells, and cocultures of alpha4CHOs and bone marrow cells did not stimulate TRAP(+) MNC formation or enhance the effects of 1,25(OH)2D3 and PGE2 in bone marrow cultures. The concentrations of PGE2 and interleukin-6 (IL-6) in cultured media were not different between the cultures of bone marrow cells and the cocultures of bone marrow cells and alpha4CHOs. Anti-human alpha4 and anti-mouse VCAM-1 antibodies inhibited TRAP(+) MNC formation induced by alpha4CHOs. These results indicate that alpha4CHOs stimulated TRAP(+) MNC formation through direct cell-to-cell interaction between alpha4beta1 and VCAM-1. It is suggested that in addition to various soluble factors regulating osteoclast formation, cell-to-cell interaction between tumor cells and bone marrow cells is important for inducing osteoclasts at the site of bone metastasis and leading to bone destruction.

Conditionally Immortalized Murine Bone Marrow Stromal Cells Mediate Parathyroid Hormone-Dependent Osteoclastogenesis in Vitro

PTH recruits and activates osteoclasts to cause bone resorption. These actions of PTH are thought to be mediated indirectly via type 1 PTH/PTH-related peptide receptors (PTH1Rs) expressed by adjacent marrow stromal or osteoblastic cells, although some evidence suggests that PTH may act directly on early hematopoietic osteoclast progenitors. We have established clonal, conditionally immortalized, PTH-responsive, bone marrow stromal cell lines from mice that harbor both a transgene encoding a temperature-sensitive mutant of the simian virus 40 large T antigen and deletion of a single allele of the PTH1R gene. Of 60 stromal cell lines isolated, 45 expressed functional PTH1Rs. During coculture with normal murine spleen cells, 5 of 42 such cell lines could support formation of tartrate-resistant acid phosphatase-positive, multinucleated cells (TRAP+ MNCs) in response to 1,25-dihydroxyvitamin D3, but only 2 of these did so in response to PTH. One of these, MS1 cells, expressed numerous cytokines and proteins characteristic of the osteogenic lineage and showed increased production of interleukin-6 in response to PTH. MS1 cells supported dose-dependent induction by rat (r) PTH-(1–34) (0.1–100 nm) of TRAP+ MNCs that expressed calcitonin receptors and formed resorption lacunae on dentine slices. This effect of PTH, which required cell to cell contact between MS1 and spleen cells, was mimicked by coadministration of cAMP analog and phorbol ester but only partially by either agent alone. The carboxyl-terminal fragment rPTH-(53–84) also induced osteoclast-like cell formation, but the maximal effect was only 30% as great as that of rPTH-(1–34). Importantly, rPTH-(1–34) induced TRAP+ MNC formation even when PTH1R−/− osteoclast progenitors (from fetal liver of mice homozygous for ablation of the PTH1R gene) were cocultured with MS1 cells. We conclude that activation of PTH1Rs on cells of the osteoclast lineage is not required for PTH-(1–34)-induced osteoclast formation in the presence of appropriate PTH-responsive marrow stromal cells. MS1 cells provide a useful model for further study of PTH regulation of osteoclastogenesis.

Conditionally immortalized murine bone marrow stromal cells mediate parathyroid hormone-dependent osteoclastogenesis in vitro.

PTH recruits and activates osteoclasts to cause bone resorption. These actions of PTH are thought to be mediated indirectly via type 1 PTH/PTH-related peptide receptors (PTH1Rs) expressed by adjacent marrow stromal or osteoblastic cells, although some evidence suggests that PTH may act directly on early hematopoietic osteoclast progenitors. We have established clonal, conditionally immortalized, PTH-responsive, bone marrow stromal cell lines from mice that harbor both a transgene encoding a temperature-sensitive mutant of the simian virus 40 large T antigen and deletion of a single allele of the PTH1R gene. Of 60 stromal cell lines isolated, 45 expressed functional PTH1Rs. During coculture with normal murine spleen cells, 5 of 42 such cell lines could support formation of tartrate-resistant acid phosphatase-positive, multinucleated cells (TRAP+ MNCs) in response to 1,25-dihydroxyvitamin D3, but only 2 of these did so in response to PTH. One of these, MS1 cells, expressed numerous cytokines and proteins characteristic of the osteogenic lineage and showed increased production of interleukin-6 in response to PTH. MS1 cells supported dose-dependent induction by rat (r) PTH-(1-34) (0.1-100 nM) of TRAP+ MNCs that expressed calcitonin receptors and formed resorption lacunae on dentine slices. This effect of PTH, which required cell to cell contact between MS1 and spleen cells, was mimicked by coadministration of cAMP analog and phorbol ester but only partially by either agent alone. The carboxyl-terminal fragment rPTH-(53-84) also induced osteoclast-like cell formation, but the maximal effect was only 30% as great as that of rPTH-(1-34). Importantly, rPTH-(1-34) induced TRAP+ MNC formation even when PTH1R-/- osteoclast progenitors (from fetal liver of mice homozygous for ablation of the PTH1R gene) were cocultured with MS1 cells. We conclude that activation of PTH1Rs on cells of the osteoclast lineage is not required for PTH-(1-34)-induced osteoclast formation in the presence of appropriate PTH-responsive marrow stromal cells. MS1 cells provide a useful model for further study of PTH regulation of osteoclastogenesis.

Macrophage colony stimulating factor down-regulates MCSF-receptor expression and entry of progenitors into the osteoclast lineage.

Macrophage colony-stimulating factor (MCSF), although necessary for entry of precursors into the early preosteoclast pathway, inhibits osteoclastogenesis at high doses. To clarify the relationship between MCSF and osteoclast formation, we investigated the effect of exogenous MCSF in murine bone marrow culture. Precursor proliferation and the expression of MCSF-receptor were examined after 4 days of culture in the presence or absence of accessory stromal cells. In both mixed marrow and destromalized cell cultures, exogenous MCSF dose-dependently decreased 125I-MCSF binding (by 65 +/- 5.0% at 3500 and 87 +/- 16.7% at-7000 U/ml, respectively) while enhancing mononuclear cell proliferation after 3 days of exposure (by 2.8- and 6.3-fold, respectively). These effects were maintained 24 h after removal of exogenous MCSF and, as such, likely represented an MCSF-induced change in MCSF receptor-bearing cells. Exposure to exogenous MCSF (3500 U/ml) days 2-4 dose-dependently inhibited tartrate resistant acid phosphatase positive multinuclear cell (TRAP+ MNC) formation counted at the end of day 7, by 64.3 +/- 4.1%. This inhibition of TRAP+ MNC formation was preceded by a 92 +/- 9% decrease in the expression of carbonic anhydrase II mRNA measurable at 4 days. These results indicate that MCSF promotes proliferation of a population of cells expressing lower cognate receptor sites. Changes in MCSF-receptor expression appear to modulate the final lineage selection of the pluripotent monoblastic progenitor.

Bisphosphonates act on osteoblastic cells and inhibit osteoclast formation in mouse marrow cultures

We examined the mode of action of bisphosphonates on osteoclastic cell recruitment using mouse marrow cultures with or without osteoblastic cells. Tartrate-resistant acid phosphatase-positive multinucleated cells [TRAP(+)MNC] formed in cultures were determined to be osteoclastic cells. In marrow cultures, TRAP(+) MNC formation in the presence of 10(-8) mol/L 1,25(OH)2D3 was not affected by the addition of 10(-6) mol/L dihydrogen (cycloheptylamino)-methylenebisphosphonate monohydrate (YM175). However, it was inhibited in cocultures of marrow cells with osteoblastic cells. The inhibitory effect was evident throughout the entire culture period. YM175 dose dependently inhibited TRAP(+) MNC formation, and other bisphosphonates--pamidronate and alendronate--also inhibited TRAP(+) MNC formation in the coculture. Similar observations were also made in the coculture of spleen cells with osteoblastic cells. The conditioned media of osteoblastic cells treated with 10(-6) mol/L YM175 inhibited TRAP(+) MNC formation in marrow cultures. The presence of YM175 in methylcellulose cultures affected neither the colony formation of monocyte-macrophage lineage, nor TRAP(+) MNC formation in the succeeding cocultures of recovered cells with osteoblastic cells. These results indicate that YM175 and probably other bisphosphonates as well preferentially inhibit the later stage of osteoclastogenesis through its action on osteoblastic cells. Our findings suggest that part of the inhibitory action by osteoblastic cells in the presence of bisphosphonates is mediated through soluble factor(s).

Expression of the calcitonin receptor in bone marrow cell cultures and in bone: a specific marker of the differentiated osteoclast that is regulated by calcitonin.

We studied the temporal sequence of osteoclast (OC) differentiation from precursor cells in murine marrow cultures. Two markers of the OC phenotype, calcitonin (CT) receptor (CTR) and tartrate resistant acid phosphatase (TRAP), were assessed. Marrow cells from C57BL/6 mice were cultured for 3, 5, 7, and 9 days with or without 1,25-(OH)2vitamin D3 (10(-8) M). In controls only small numbers of osteoclastic multinucleated cells 9MNCs) formed per well (< 15 per well). In contrast, 1,25-(OH)2D3 strongly stimulated MNC formation (> 80 per well on day 7). Messenger RNA (mRNA) for TRAP was detectable by reverse transcription-polymerase chain reaction amplification in both control and 1,25-(OH)2D3 treated groups at all times. However, TRAP mRNA was detectable in MNCs by the less sensitive in situ hybridization only on days 5, 7, and 9 and only in 1,25-(OH)2D3 treated cells. In control cultures, CTR mRNA was present on day 3 only in nonadherent cells and was not present in adherent cells (where MNCs formed) at any time point. In 1,25-(OH)2D3 treated cultures CTR mRNA was detectable in nonadherent cells on day 3 and in adherent cells on day 5 and thereafter. Peak levels of CTR mRNA were seen in adherent cells on day 7 (15-fold more than day 5 and 4-fold more than day 9). CT (10(-7) M) treatment of 7 day cultures, which had been stimulated to express the osteoclastic phenotype, caused a marked decrease in CTR mRNA expression at 24 h. There was no effect of CT treatment on CTR mRNA expression at 3 h or on TRAP mRNA expression at 3 or 24 h. In neonatal mouse calvaria cultures, CTR mRNA expression was constitutively present and was markedly decreased by 48 h of CT treatment. Similarly, bone resorption in these cultures was inhibited at 24 h by CT treatment, but at 48 and 72 h there was escape from the inhibitory effects of CT on resorption. In the marrow cultures, MNCs were greater than 98% positive for [125I]-salmon calcitonin (sCT) binding and this binding was completely competed away by excess cold sCT (10(-7) M). All primary isolated osteoclasts from 1- to 3-day-old mouse long bones exhibited [125I]-sCT binding and TRAP activity and were strongly positive for CTR and TRAP mRNA by in situ hybridization. Both MNCs that formed in bone marrow cultures and isolated primary osteoclasts formed resorption pits on bone slices.(ABSTRACT TRUNCATED AT 400 WORDS)

Establishment and characterization of bone marrow stromal cell lines that support osteoclastogenesis

We established bone marrow stromal cell lines that support tartrate-resistant acid phosphatase-positive multinucleated cell [TRAcP(+)MNC] formation by using transgenic mice harboring simian virus 40 large T antigen gene. The morphology of these TM cell lines (large T-immortalized marrow cells) was spindle-like at sparse cell density, whereas it became smaller and cuboidal at confluence. The TM cell lines showed diverse ranges of activity in supporting TRAcP(+)MNC formation when they were examined in the cocultures with spleen cells in the presence of 1 alpha,25-dihydroxyvitamin D3. Among these cell lines, TM8 supported the TRAcP(+)MNC formation most efficiently (from 400-1500 cells/well) when cocultured with spleen cells. Another bone marrow-derived cell line, TM5, supported TRAcP(+)MNC formation in the coculture assay, whereas the efficiency was approximately one fifth that of TM8. Interestingly, TM8 cells also supported TRAcP(+)MNC formation even in the cocultures at low serum concentration (0.5% fetal bovine serum) with an efficiency yielding over 200 TRAcP(+)MNCs/well. TM8 cells expressed certain levels of macrophage colony-stimulating factor and stem cell factor messenger RNAs (mRNAs), but low levels of c-fms mRNA. Expression of c-kit mRNA in TM5 and TM8 cells was undetectable. 1 alpha,25-Dihydroxyvitamin D3 treatment enhanced the expression of osteopontin mRNA more than 10-fold in these cells, indicating the presence of the receptor for this steroid. These TRAcP(+)MNCs, which developed in the cocultures of the TM8 and spleen cells, formed pits when cultured on bone slices, indicating that they were capable of resorbing bone. The various levels of expression of these genes and the difference in the supporting activities for the TRAcP(+)MNC development in the diverse TM cell lines suggest the heterogeneity in the marrow cell populations in vivo regarding their activity in supporting osteoclastogenesis.

Establishment and characterization of bone marrow stromal cell lines that support osteoclastogenesis.

We established bone marrow stromal cell lines that support tartrate-resistant acid phosphatase-positive multinucleated cell [TRAcP(+)MNC] formation by using transgenic mice harboring simian virus 40 large T antigen gene. The morphology of these TM cell lines (large T-immortalized marrow cells) was spindle-like at sparse cell density, whereas it became smaller and cuboidal at confluence. The TM cell lines showed diverse ranges of activity in supporting TRAcP(+)MNC formation when they were examined in the cocultures with spleen cells in the presence of 1 alpha,25-dihydroxyvitamin D3. Among these cell lines, TM8 supported the TRAcP(+)MNC formation most efficiently (from 400-1500 cells/well) when cocultured with spleen cells. Another bone marrow-derived cell line, TM5, supported TRAcP(+)MNC formation in the coculture assay, whereas the efficiency was approximately one fifth that of TM8. Interestingly, TM8 cells also supported TRAcP(+)MNC formation even in the cocultures at low serum concentration (0.5% fetal bovine serum) with an efficiency yielding over 200 TRAcP(+)MNCs/well. TM8 cells expressed certain levels of macrophage colony-stimulating factor and stem cell factor messenger RNAs (mRNAs), but low levels of c-fms mRNA. Expression of c-kit mRNA in TM5 and TM8 cells was undetectable. 1 alpha,25-Dihydroxyvitamin D3 treatment enhanced the expression of osteopontin mRNA more than 10-fold in these cells, indicating the presence of the receptor for this steroid. These TRAcP(+)MNCs, which developed in the cocultures of the TM8 and spleen cells, formed pits when cultured on bone slices, indicating that they were capable of resorbing bone. The various levels of expression of these genes and the difference in the supporting activities for the TRAcP(+)MNC development in the diverse TM cell lines suggest the heterogeneity in the marrow cell populations in vivo regarding their activity in supporting osteoclastogenesis.

Tiludronate tablet (Skelid®) 400mg/d produces lengthy clinical remission of Paget's disease: follow up of a dose ranging study

Prostaglandin estradiol (PGE2) stimulates bone resorption by a cyclic AMP (cAMP)-dependent mechanism that involves prostaglandin E receptors of the EP2 and EP4 subtypes. We tested a potent selective EP4 antagonist (EP4RA), which blocks PGE2 binding to EP4 receptors. We examined the effects of EP4RA on osteoclastogenesis in murine marrow cultures, on cAMP production in primary osteoblastic (POb) cell cultures, and on bone resorption in organ cultures. EP4RA (1 μmol/L) decreased the number of tartrate-resistant acid phosphatase-positive multinucleated cells (TRAP+ MNC) by 46%–48% in cultures treated with 0.1–1.0 μmol/L PGE2 and by 96% in cultures treated with 0.01 μmol/L PGE2. EP4RA also decreased TRAP+ MNC formation by 60% in 1,25-dihydroxyvitamin D (1,25D)-treated cultures and by 62% in parathyroid hormone (PTH)-treated cultures. A chemically related analog of EP4RA that lacks antagonist activity did not inhibit TRAP+ MNC formation. EP4RA decreased cAMP production in PGE2-treated POb by 44% but did not block cAMP response to PTH. EP4RA inhibited the increase in receptor activator of NF-κB ligand (RANKL) mRNA levels produced by PGE2. In fetal rat long bone cultures, EP4RA decreased 45Ca release from control, unstimulated cultures by 12%–25% and from PGE2-stimulated cultures by 22%–37%. Because EP4RA partially inhibited osteoclastogenesis not only in response to PGE2 but also in response to 1,25D and PTH, these results suggest that activation of the EP4 receptor may play a general role in osteoclastic bone resorption. EP4RA showed partial inhibition of PGE2-stimulated osteoclastogenesis at 1 μmol/L, but almost complete inhibition at 0.01 μmol/L PGE2. This could be due to the limited efficacy of the antagonist at high concentrations of PGE2, or an alternative pathway, such as activation of the EP2 receptor.

Characterization of MCSF-induced proliferation and subsequent osteoclast formation in murine marrow culture.

To clarify events involved in 1,25(OH)2D3-stimulated osteoclast-like cell (OCLC) formation in primary murine marrow culture, we have characterized kinetics of precursor proliferation and fusion and their dependence on macrophage colony-stimulating factor (MCSF). 3H-thymidine nuclear incorporation in tartrate-resistant acid phosphatase positive multinucleated cells (TRAP+ MNCs) was assessed: 3H-thymidine incorporation was greatest when tracer was added during day 4 or 5, with labeled nuclei in 81% (day 4) and 90% (day 5) of the TRAP+ MNCs counted at the end of day 7. The percentage of total nuclei labeled was highest when 3H-thymidine was dosed on day 4 (58%), decreasing to 2% by day 7. Final TRAP+ MNC numbers were depleted by 80% when treated for 24 h with hydroxyurea on either day 3 or 4; this inhibition dropped to 57% and 12% when hydroxyurea was pulsed during days 5 or 6, respectively. The absence of 1,25(OH)2D3 during days 1-4 caused 70% attenuation of TRAP+ MNC formation; however, exposure to 3H-thymidine during day 4 in this experiment resulted in subsequent labeling of 81% of the TRAP+ MNCs formed, indicating that precursor proliferation occurred in the absence of 1,25(OH)2D3. To demonstrate that proliferation required MCSF, cultures were exposed to a monoclonal anti-MCSF antibody during days 3, 4, 5, 6, or 7. Inhibition of TRAP+ MNC formation was 85% when antibody was added during day 3. Antibody treatment after day 5 had little effect on the OCLC number. Fusion of precursors showed steady progression with OCLCs containing 4.8 +/- 0.3 nuclei at the end of day 4, 8.3 +/- 0.5 nuclei after day 5, 12.0 +/- 1.3 after day 6, and 13.7 +/- 1.5 at the end of day 7. This steady accretion of nuclei was unaffected by doses of MCSF antibody which blocked proliferation. In conclusion, we have shown that OCLCs arise from an MCSF-dependent expansion of the precursor pool occurring during days 3 and 4. Fusion of these precursors, which begins as proliferation diminishes, is able to progress in the presence of anti-MCSF antibody. These results should help refine the analysis of factors affecting proliferation and fusion of osteoclasts in murine marrow culture.

The role of cadherin in the generation of multinucleated osteoclasts from mononuclear precursors in murine marrow.

A critical step in bone resorption is the fusion of mononuclear osteoclast precursors to form multinucleated osteoclasts. However, little is known of the molecular mechanisms that are responsible for this important process. Since the expression of proteins in the cadherin family of homophilic calcium-dependent cell adhesion molecules is involved in the fusion process for certain other cells, we examined their role in osteoclast formation. Immunohistochemical examination of human and mouse bone using monoclonal antibodies to human and mouse E-cadherin clearly demonstrated positive staining in osteoclasts. N- and P-cadherin were not detected. In cultures of murine marrow mononuclear cells in which osteoclasts form by cell fusion, E-cadherin expression determined by Western blotting reached the highest levels as fusion was taking place. Expression of E-cadherin gene fragment was also detected in the marrow cultures by polymerase chain reaction. To study the functional role of E-cadherin expression in osteoclastic differentiation, neutralizing monoclonal antibodies were examined for their effects on osteoclast formation. The antibodies decreased the number of tartrate-resistant acid phosphatase (a marker of murine osteoclast)-positive multinucleated cell (TRAP-positive MNC) by inhibiting the fusion of mononuclear osteoclast precursors, but not proliferation of these cells or their attachment to plastic dish surfaces. This inhibitory effect was reversible. Furthermore, synthetic peptides containing the cell adhesion recognition sequence of cadherins also decreased TRAP-positive MNC formation. The antibodies and peptides inhibited not only osteoclast formation but also bone resorption. Antibodies to other types of cadherins and control rat IgG had no effects in these culture systems. Our findings suggest that E-cadherin expression may be involved in fusion (differentiation) of hemopoietic osteoclast precursors into mature multinucleated osteoclasts.

Inhibitory effect of interleukin-4 on osteoclast-like cell formation in mouse bone marrow culture

Recently, interleukin 4 (IL-4) was reported to inhibit bone resorption in mouse long bone culture. To test the effect of IL-4 on the formation of osteoclast-like cells, we used a mouse bone marrow culture system that formed mononuclear and multinucleated cells with osteoclast characteristics. Recombinant mouse IL-4 dose-dependently inhibited the formation of tartrate-resistant acid phosphatase-positive multinucleated cells [TRAP(+)MNC] induced by 1,25(OH)2D3, PTH(1-34) or Interleukin-1 alpha (IL-1 alpha). The minimum effective inhibitory concentration was 0.01 ng/ml, and 1 ng/ml IL-4 completely inhibited TRAP(+)MNC formation. IL-4 also dose-dependently inhibited the formation of tartrate-resistant acid phosphatase-positive mononuclear cells. Treatment of cultures with IL-4 for the first or last 48 h of an 8-day culture period inhibited TRAP(+)MNC formation to the same extent, whereas IFN-gamma and calcitonin suppressed TRAP(+)MNC formation mainly at the early and the late phase, respectively. IL-4, as a macrophage fusion factor, at higher concentrations (0.1-10 ng/ml), increased formation of tartrate-resistant acid phosphatase-negative multinucleated cells [TRAP(-)MNC] with giant macrophage characteristics. The half-maximal concentrations inhibiting TRAP(+)MNC formation and stimulating TRAP(-)MNC formation were 0.05 ng/ml and 2 ng/ml, respectively. These results demonstrate that IL-4 inhibits bone resorption by inhibiting the recruitment of osteoclast precursor and formation of multinucleated osteoclast-like cells, and by stimulating the formation of macrophage polykaryons.

Effects of 1 alpha, 25-dihydroxyvitamin D3-26,23-lactone and its intermediate metabolites on bone metabolism in vivo and in vitro.

Which intermediate metabolite of 23(S)25(R)-1 alpha,25-(OH)2D3-26,23-lactone causes the unique biological functions of 1 alpha,25-(OH)2D3-26,23-lactone different from that of 1 alpha,25-(OH)2D3 was examined comparatively. The three intermediate metabolites, 1 alpha,25(R)26-(OH)3D3, 1 alpha,23(S)25(R)26-(OH)4D3, and 23(S)25(R)-1 alpha,25-(OH)2D3-26,23-lactol stimulated bone resorption, as 1 alpha,25-(OH)2D3 did, in vitamin D-deficient rats. On the other hand, 1 alpha,25-(OH)2D3-26,23-lactone only inhibited bone resorption. 1 alpha,25-(OH)2D3, 1 alpha,25-(OH)2D3-26,23-lactone and the other three metabolites stimulated MNC formation from hematopoietic blast cells in a manner correlated with their binding affinities for the 1 alpha,25-(OH)2D3 receptor. However, CM obtained from 1 alpha,25-(OH)2D3-26,23-lactone-treated MC3T3-E1 cells inhibited MNC formation, probably by the action of some inhibitory factors elaborated by the cells treated with 1 alpha,25-(OH)2D3-26,23-lactone, whereas CM obtained from 1 alpha,25-(OH)2D3 or other metabolite-treated MC3T3-E1 cells stimulated MNC formation. These facts indicate that only 1 alpha,25-(OH)2D3-26,23-lactone has an inhibitory action of bone resorption and that the lactone ring plays a major part in its expression.

Existence of parathyroid hormone binding sites on murine hemopoietic blast cells

We demonstrated that 125I-labeled human parathyroid hormone (1–34;8,18-Nle, 34-Tyr)[[ 125I]hPTH(1–34)] bound specifically to hemopoietic blast cells supported by granulocyte-macrophage colony-stimulating factor. Half-maximal inhibition of binding was achieved at concentrations of unlabeled hPTH(1–34) of about 5 × 10 −9M. Insulin and hPTH(39–68) did not compete for PTH binding sites. Specific binding of hPTH(1–34) was detected in neither macrophages nor multinucleated cells (MNC's). Furthermore, treatment of hemopoietic blast cells with hPTH(1–34) stimulated MNC formation, and the range of concentrations (10 −10–10 −8M) over which hPTH(1–34) caused these effects was similar to that which inhibited the binding of [ 125I]hPTH(1–34). These findings suggest the presence of a PTH receptor on osteoclast precursors and the direct effect of PTH on them, resulting in osteoclast-mediated bone resorption.