Stimulate Osteoclast Formation Research Articles

Reduced expression of interleukin-11 in bone marrow stromal cells of senescence-accelerated mice (SAMP6): relationship to osteopenia with enhanced adipogenesis.

Aging is associated with an increase in bone marrow adipose tissue and a reduction in bone turnover. The P6 strain of senescence-accelerated mice (SAM) exhibit an early decrease in bone mass with a reduction in bone remodeling. In the bone marrow, suppressed osteoblastogenesis and osteoclastogenesis with enhanced adipogenesis are observed. The present study was undertaken to clarify the mechanism of age-related changes in bone turnover using bone marrow cells from SAMP6 mice. Because interleukin (IL)-11 has been shown to potently inhibit adipogenesis and to stimulate osteoclast formation, the effect of IL-11 on the differentiation of bone marrow cells was examined. The impaired formation of both osteoblasts and osteoclasts was restored and the enhanced formation of adipocytes was suppressed by the addition of 10 pM recombinant human IL-11. Other cytokines that activate gp130 as a common signal transducer, IL-6 and leukemia inhibitory factor, did not have such effects. Sequence analysis of the entire coding region of IL-11 cDNA obtained from SAMP6 stromal cells revealed no mutations. Constitutively secreted IL-11 protein into culture media, and its mRNA expression stimulated by transforming growth factor beta were reduced in stromal cells from SAMP6 compared with those in control mice. These results demonstrate that the expression of IL-11 is reduced in bone marrow cells of SAMP6 and suggest that the reduction in IL-11 actions is involved in the impairment of both osteoblastogenesis and osteoclastogenesis in these mice. There is a possibility that alterations in IL-11 actions may be associated with the age-related impairment in bone metabolism.

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.

Estrogen via the estrogen receptor blocks cAMP-mediated parathyroid hormone (PTH)-stimulated osteoclast formation.

Several lines of evidence indicate that estrogen inhibits parathyroid hormone (PTH)-induced bone resorption in vivo and in vitro. However, its precise mechanism remains unknown. The present study was performed to investigate whether osteoclast precursor cells possess the receptors for PTH/PTH-related protein (PTHrP) and/or estrogen and to clarify the mechanism by which estrogen affects PTH-induced osteoclast-like cell (Ocl) formation. The polymerase chain reaction (PCR) product corresponding in size to the mouse PTH/PTHrP receptor cDNA was detected in mouse hemopoietic blast cells supported by granulocyte-macrophage colony-stimulating factor (GM-CSF) as well as in osteoblastic MC3T3-E1 cells. The nucleotide sequence of the PTH/PTHrP receptor PCR product of hemopoietic blast cells was found to be 95.4% identical to that of PTH/PTHrP receptor cDNA of rat osteoblastic ROS cells. The PCR product corresponding in size to the mouse estrogen receptor cDNA was detected in mouse hemopoietic blast cells supported by GM-CSF as well as in MC3T3-E1 cells. The nucleotide sequence of the estrogen receptor PCR product of hemopoietic blast cells was completely identical to that of mouse estrogen receptor cDNA. 17Beta-estradiol (17beta-E2) but not 17alpha-E2 dose dependently antagonized Ocl formation stimulated by human (h) PTH(1-34) at a minimal effective concentration of 10(-10) M in the hemopoietic blast cell culture. 17Beta-E2 also significantly inhibited Ocl formation stimulated by 10(-8) M hPTHrP(1-34), while it did not affect 1,25-dihydroxyvitamin D3-induced Ocl formation. However, 10(-8) M 17beta-E2 significantly inhibited Ocl formation stimulated by dibutyryladenosine cAMP (10(-4) M) and Sp-cAMPS (10(-4) M), an activator of cAMP-dependent protein kinase (PKA) as well as forskolin (10(-5) M). In contrast, 17beta-E2 did not affect Ocl formation by either phorbol myristate acetate (10(-7) M), an activator of protein kinase C (PKC), or A23187 (10(-7) M), a calcium ionophore. The pretreatment with 17beta-E2 significantly inhibited Ocl formation induced by the combined treatment with PTH and PKC inhibitors (H7 or staurosporine), while it did not affect Ocl formation stimulated by the combined treatment with PTH and Rp-cAMPS, a PKA inhibitor. The present data indicate that estrogen inhibits PTH-stimulated Ocl formation by directly acting on hemopoietic blast cells, possibly through blocking a PKA pathway but not a calcium/PKC pathway.

The cellular actions of interleukin-11 on bone resorption in vitro.

The pleiotropic cytokine interleukin-11 (IL-11) stimulates osteoclast formation in vitro, but it is not known whether it influences other steps in the bone-resorptive cascade. Using a variety of in vitro model systems for studying bone resorption we have investigated the effects of IL-11 on 1) osteoclast formation, fusion, migration, and activity; and 2) osteoblast-mediated osteoid degradation. The involvement of matrix metalloproteinases (MMPs) and products of arachidonic acid metabolism in IL-11-mediated resorption were also assessed. We first examined the bone-resorptive effects of IL-11 by assessing 45Ca release from neonatal mouse calvarial bones. IL-11 dose-dependently stimulated bone resorption with an EC50 of 10(-10) M. The kinetics of IL-11-mediated 45Ca release demonstrated that it was without effect for the first 48 h of culture, but by 96 h, it stimulated 45Ca release to the same level as that produced by 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] (a hormone that stimulates osteoclast formation and activity). IL-11 also produced a dose-dependent increase in osteoblast-mediated type I collagen degradation with a maximum of 58.0 +/- 6.2% at 5 x 10(-9) M; this effect of IL-11 was less than that produced by 1,25-(OH)2D3 (76.5 +/- 7.1%) and was prevented by an inhibitor of MMPs, but not those blocking arachidonic acid metabolism. We then tested the effects of IL-11 on isolated mouse osteoclasts cultured on ivory slices in the presence and absence of primary mouse osteoblasts. IL-11 had no effect on isolated osteoclast activity even in coculture with primary osteoblasts. We then examined the effects of IL-11 on the formation of osteoclast-like multinucleate cells in mouse bone marrow cultures and the resorptive activity of such cultures using ivory as a substrate. IL-11 dose-dependently increased 1) the number of tartrate-resistant acid phosphatase-positive osteoclast-like multinucleate cells and 2) the surface area of lacunar resorption, although the effects were less than that of 1,25-(OH)2D3. The effect of IL-11 on bone marrow lacunar resorption was prevented by a combination of inhibitors of 5-lipoxygenase and cyclooxygenase. In 17-day-old metatarsal bones, IL-11 prevented the migration of (pre)osteoclasts to future resorption sites, whereas their fusion was unaffected. These results provide strong evidence that IL-11 stimulates bone resorption by enhancing osteoclast formation and osteoblast-mediated osteoid degradation rather than stimulating osteoclast migration and activity. Our data also suggest that the stimulatory effects of IL-11 involve both MMPs and products of arachidonic acid metabolism.

The mouse mammary tumor cell line, MMT060562, produces prostaglandin E2 and leukemia inhibitory factor and supports osteoclast formation in vitro via a stromal cell-dependent pathway.

Osteoclastic bone resorption increases at the site of bone metastasis, but little is known about how tumor cells induce osteoclast (OC) recruitment in the bone marrow microenvironment. To clarify this point, we examined the effects of various mouse tumor cells on OC recruitment using cocultures of tumor cells and mouse marrow cells. The mouse mammary tumor cell lines, MMT060562 (MMT), BALB/c-MC, Jyg-MC(A), or other nonmammary tumor cell lines, LLC and B16, were cocultured with mouse marrow cells, and OC recruitment from marrow cells was determined by counting the number of tartrate-resistant acid phosphatase-positive multinucleated cells (TRAP(+) MNCs) formed. Of the tumor cells examined, MMT and BALB/c-MC stimulated OC formation, but other tumor cells did not. OC formation with MMT was dependent on the number of MMTs inoculated, and only ten cells per well were sufficient to induce OC development. OCs appeared on day 4, and the number reached a maximum on days 5-8 and decreased thereafter. TRAP(+) MNCs induced by MMT satisfied the major criteria of OCs, such as the presence of calcitonin receptors and the ability to resorb calcified tissues. The majority of OCs were formed adjacent to the stromal cells, which were positive for alkaline phosphatase. When spleen cells were cocultured with MMT, no OCs were formed. In contrast, when osteoblastic cells were added to cocultures of spleen cells and MMT, many OCs were formed. The cultured media (CM) of MMT induced OC formation in mouse marrow cultures. Neither parathyroid hormone-like nor interleukin 1-like activity was present in the CM. MMT constitutively produced prostaglandin E2 (PGE2) and OC formation in cocultures was completely inhibited by indomethacin. Fractionation of the CM of MMT by ultrafiltration indicated that the OC-inducing activities were present not only in the fraction with molecular weight below 3 kDa but also in the fraction with molecular weight above 3 kDa. OC-inducing activity with high molecular weight was eluted around 50 kDa by Bio-Gel P-60 column chromatography. The active fractions also possessed leukemia inhibitory factor (LIF) activity, and OC-inducing activity of the peak fraction was inhibited in the presence of anti-LIF neutralizing antibody. The results of this study indicated that MMTs release PGE2 and LIF, which in turn stimulate OC formation via a stromal cell-dependent pathway. These culture systems will help to clarify the mechanisms by which tumor cells induce OC formation in a bone marrow microenvironment.

The Cell-surface Form of Colony-stimulating Factor-1 Is Regulated by Osteotropic Agents and Supports Formation of Multinucleated Osteoclast-like Cells

Colony-stimulating factor-1 (CSF-1) is a hematopoietic growth factor that is released by osteoblasts and is recognized to play a critical role in bone remodeling in vivo and in vitro. CSF-1 is synthesized as a soluble or cell-surface protein. It is unclear, however, whether human osteoblasts express both molecular forms of CSF-1, and whether these isoforms can independently mediate osteoclastogenesis. In the present study, using a combination of quantitative reverse transcriptase polymerase chain reaction, flow cytometry, and Western immunoblot analysis, we have demonstrated that human osteoblast-like cells as well as primary human osteoblasts express the cell-surface form of CSF-1 both constitutively and in response to parathyroid hormone and tumor necrosis factor. Furthermore, using an in vitro co-culture system, we have shown that cell-surface CSF-1 alone is sufficient to support osteoclast formation. These findings may be especially significant in view of evidence that direct cell-to-cell contact is critical for osteoclast formation, and suggest that differential regulation of expression of the CSF-1 isoforms may influence osteoclast function modulated by osteotropic hormones.

Insulin-like growth factor-I mediates osteoclast-like cell formation stimulated by parathyroid hormone.

There have been several lines of evidence that parathyroid hormone (PTH) stimulates production of insulinlike growth factor I (IGF-I) in bone and that IGF-I stimulates osteoclast formation. Thus, the present study was performed to clarify the possible role of IGF-I in PTH-stimulated osteoclastlike cell formation and the role of PTH-responsive dual signal transduction systems (cyclic [c] AMP-dependent protein kinase [PKA] and calcium/protein kinase C [PKC]) in its mechanism. Treatment with anti-IGF-I antibody (1-10 micrograms/ml) partially but significantly blocked hPTH-(1-34)-stimulated osteoclastlike cell formation in unfractionated mouse bone cell cultures, although it did not affect osteoclastlike cell formation stimulated by 1,25-dihydroxyvitamin D3. Rp-cAMP5 (10(-4) M), a direct PKA inhibitor, as well as two types of PKC inhibitors, H-7 (10 microM) and staurosporine (3 nM), and dantrolene (10(-5) M), an inhibitor of calcium mobilization from intracellular calcium stores, all significantly blocked PTH-stimulated osteoclastlike cell formation. Anti-IGF-I antibody (3 micrograms/ml) significantly blocked osteoclastlike cell formation stimulated by 10(-4) M dbcAMP, 10(-4) M Sp-cAMPS, a direct PKA activator, and 10(-5) M forskolin in mouse bone cell cultures. Dibutyryl cAMP, forskolin, and hPTH-(1-34) significantly stimulated mRNA expression of both IGF-I and IGF-binding protein 5 (IGFBP-5) in these cultures, but neither 10(-7) M PMA, a PKC activator, nor 10(-7) M A23187 did. Moreover, anti-IGF-I antibody significantly blocked osteoclastlike cell formation stimulated by the conditioned medium from MC3T3-E1 cells pretreated with 10(-8) PTH-(1-34), which induced IGF-I and IGFBP-5 mRNA expression in these cells. In conclusion, the present study indicates that IGF-I mediates osteoclastlike cell formation stimulated by PTH and that the PKA pathway is involved in its mechanism. However, IGF-I does not seem to be the sole effector molecule to be active in this system.

Insulin‐like growth factor‐I mediates osteoclast‐like cell formation stimulated by parathyroid hormone

There have been several lines of evidence that parathyroid hormone (PTH) stimulates production of insulinlike growth factor I (IGF-I) in bone and that IGF-I stimulates osteoclast formation. Thus, the present study was performed to clarify the possible role of IGF-I in PTH-stimulated osteoclastlike cell formation and the role of PTH-responsive dual signal transduction systems (cyclic [c] AMP-dependent protein kinase [PKA] and calcium/protein kinase C [PKC]) in its mechanism. Treatment with anti-IGF-I antibody (1–10 μg/ml) partially but significantly blocked hPTH-(1-34)-stimulated osteoclastlike cell formation in unfractionated mouse bone cell cultures, although it did not affect osteoclastlike cell formation stimulated by 1,25-dihydroxyvitamin D3. Rp-cAMPS (10-4 M), a direct PKA inhibitor, as well as two types of PKC inhibitors, H-7 (10 μM) and staurosporine (3 nM), and dantrolene (10-5 M), an inhibitor of calcium mobilization from intracellular calcium stores, all significantly blocked PTH-stimulated osteoclastlike cell formation. Anti-IGF-I antibody (3 μg/ml) significantly blocked osteoclastlike cell formation stimulated by 10-4 M dbcAMP, 10-4 M Sp-cAMPS, a direct PKA activator, and 10-5 M forskolin in mouse bone cell cultures. Dibutyryl cAMP, forskolin, and hPTH-(1-34) significantly stimulated mRNA expression of both IGF-I and IGF-binding protein 5 (IGFBP-5) in these cultures, but neither 10-7 M PMA, a PKC activator, nor 10-7 M A23187 did. Moreover, anti-IGF-I antibody significantly blocked osteoclastlike cell formation stimulated by the conditioned medium from MC3T3-E1 cells pretreated with 10-8 PTH-(1-34), which induced IGF-I and IGFBP-5 mRNA expression in these cells. In conclusion, the present study indicates that IGF-I mediates osteoclastlike cell formation stimulated by PTH and that the PKA pathway is involved in its mechanism. However, IGF-I does not seem to be the sole effector molecule to be active in this system. J. Cell. Physiol. 172:55–62, 1997. © 1997 Wiley-Liss, Inc.

Mice lacking 5-lipoxygenase have increased cortical bone thickness.

5-lipoxygenase (5-LO) metabolites have complex effects on bone cells. We have found that the leukotrine LTB4 and the peptido leukotrienes stimulate osteoclast formation in vitro and in vivo, and are likely involved in the generation of active osteoclast-like cells in giant cell tumors of bone. Recent preliminary observation in mice lacking the gene for 5-LO have not as yet demonstrated any effect on osteoclast function, but these mice have increased cortical bone, suggesting that 5-LO is a negative regulator of bone formation. 5-LO metabolites thus have unique effects on bone cell function, and clarifying their role may be important for the understanding of physiological as well as pathological bone remodeling.

Tumor necrosis factor-α induces transcription of the colony-stimulating factor-1 gene in murine osteoblasts

Tumor necrosis factor-alpha (TNF-alpha) stimulates bone resorption both in vitro and in vivo. The cellular mechanisms for this effect are not known but one pathway may be via release of osteoblast derived factors which stimulate osteoclast formation. Because colony-stimulating factor-1 (CSF-1) is essential for osteoclast progenitor proliferation, we examined the effect of TNF-alpha on osteoblast expression of CSF-1. TNF-alpha treatment of MC3T3-E1 or primary mouse osteoblasts stimulated the secretion of an activity that was mitogenic for a CSF-1 responsive cell line and was completely neutralized by antiserum to CSF-1. By Northern analysis, TNF-alpha caused a dose and time (3 to 24 h) dependent increase in CSF-1 transcript expression in MC3T3-E1 cells. mRNA stability studies using actinomycin D revealed that TNF-alpha does not affect CSF-1 mRNA half-life in MC3T3-E1 cells, while nuclear-run off analysis demonstrated that TNF-alpha increases CSF-1 gene transcription. Cycloheximide treatment of MC3T3-E1 cells up-regulated CSF-1 mRNA, and compared to either agent alone, cycloheximide and TNF-alpha in combination resulted in augmentation of CSF-1 expression. A series of studies using both agonists and inhibitors indicated that TNF-alpha-induced CSF-1 expression did not involve the arachidonic acid, PKC, or cAMP pathways. These results suggest that TNF-alpha induces CSF-1 expression in osteoblasts by a transcriptional mechanism which is largely independent of new protein synthesis and of the second messenger pathways examined.

Prostaglandin E2 stimulates osteoclast formation via endogenous IL-1 beta expressed through protein kinase A.

The present study was conducted to determine whether endogenous IL-1 is involved as a potent mediator of PGE2-stimulated osteoclast formation in 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25-(OH)2D3)-primed calvarial cells from mouse embryos. PGE2 induced IL-1 beta gene expression in the primed calvarial cells. IL-1 beta gene expression was also induced in a dose-dependent manner by forskolin and dibutyryl cAMP. PGE2-induced IL-1 beta gene expression was markedly inhibited by H-89, a potent inhibitor of protein kinase A. On the other hand, osteoclast formation in 1 alpha,25-(OH)2D3-primed calvarial cells was also stimulated by forskolin and dibutyryl cAMP, and their stimulatory effects were dose dependent. H-89 also inhibited PGE2-stimulated osteoclast formation. The presence of the IL-1 beta gene product in the conditioned medium of 1 alpha,25-(OH)2D3-primed calvarial cells treated with PGE2 was proved by the results of an immunoprecipitation assay using anti-mouse IL-1 beta Ab. The addition of anti-mouse IL-1 beta Ab to 1 alpha,25-(OH)2D3 primed calvarial cell cultures markedly inhibited PGE2-stimulated osteoclast formation. The stimulatory effect of conditioned medium of primed calvarial cells treated with PGE2 on osteoclast formation was also inhibited by anti-IL-1 beta Ab pretreatment. Furthermore, we found that endogenous IL-6 is partially involved in PGE2-stimulated osteoclast formation.

Interleukin (IL)-6 induction of osteoclast differentiation depends on IL-6 receptors expressed on osteoblastic cells but not on osteoclast progenitors.

We reported that interleukin (IL) 6 alone cannot induce osteoclast formation in cocultures of mouse bone marrow and osteoblastic cells, but soluble IL-6 receptor (IL-6R) strikingly triggered osteoclast formation induced by IL-6. In this study, we examined the mechanism of osteoclast formation by IL-6 and related cytokines through the interaction between osteoblastic cells and osteoclast progenitors. When dexamethasone was added to the cocultures, IL-6 could stimulate osteoclast formation without the help of soluble IL-6R. Osteoblastic cells expressed a very low level of IL-6R mRNA, whereas fresh mouse spleen and bone marrow cells, both of which are considered to be osteoclast progenitors, constitutively expressed relatively high levels of IL-6R mRNA. Treatment of osteoblastic cells with dexamethasone induced a marked increase in the expression of IL-6R mRNA. By immunoblotting with antiphosphotyrosine antibody, IL-6 did not tyrosine-phosphorylate a protein with a molecular mass of 130 kD in osteoblastic cells but did so in dexamethasone-pretreated osteoblastic cells. Osteoblastic cells from transgenic mice constitutively expressing human IL-6R could support osteoclast development in the presence of human IL-6 alone in cocultures with normal spleen cells. In contrast, osteoclast progenitors in spleen cells from transgenic mice overexpressing human IL-6R were not able to differentiate into osteoclasts in response to IL-6 in cocultures with normal osteoblastic cells. These results clearly indicate that the ability of IL-6 to induce osteoclast differentiation depends on signal transduction mediated by IL-6R expressed on osteoblastic cells but not on osteoclast progenitors.

Osteoclast function in Paget's disease and multiple myeloma

Paget's disease of bone and multiple myeloma are characterized by increased numbers of osteoclasts and markedly increased bone resorption at the sites of the disease. In Paget's disease the osteoclasts are abnormal morphologically and contain viral-like nuclear inclusions, but in multiple myeloma the osteoclasts are normal. The bone lesions in both Paget's disease and multiple myeloma appear to be due to local stimulation of osteoclast formation and bone resorption. In situ hybridization techniques, bone marrow cultures, and cytokine assays have been used to examine osteoclast function in Paget's disease and multiple myeloma. Interleukin-6 (IL-6) has been implicated as a potential mediator for the increased osteoclast activity in both diseases. In Paget's disease, IL-6 is produced by the osteoclasts, the osteoclasts express IL-6 receptors and IL-6 mRNA, and increased levels of IL-6 are present in the marrow plasma and serum of these patients. Similarly, increased levels of IL-6 have been detected in sera from some patients with multiple myeloma. Multiple myeloma cells do not produce IL-6 in vivo but marrow stromal cells or the osteoclasts may be the source of IL-6 in multiple myeloma. IL-6 is a growth factor for multiple myeloma cells, and treating patients with anti-IL-6 decreases the tumor burden in some patients. Thus, IL-6 may be an autocrine/paracrine factor in both Paget's disease and in multiple myeloma. Multiple myeloma cells also produce osteoclast activating factors (OAFs) that can stimulate osteoclast formation and activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Interleukin-6 enhances hypercalcemia and bone resorption mediated by parathyroid hormone-related protein in vivo.

Tumors frequently induce the multifunctional cytokine IL-6, which has been linked to several paraneoplastic syndromes, most notably cachexia. IL-6 stimulates osteoclast formation, causes mild hypercalcemia, and is produced by bone cells in vitro upon exposure to systemic hormones. Since IL-6 is produced together with parathyroid hormone-related protein (PTH-rP) in some patients with cancer, we tested the hypothesis that production of IL-6 potentiates the effects of PTH-rP on Ca2+ homeostasis and osteoclastic bone resorption and examined potential mechanisms for these interactions in vivo. Chinese hamster ovarian (CHO) cells stably transfected with cDNAs for IL-6 (CHO/IL-6) and PTH-rP sense (CHO/PTH-rP) or antisense (CHO/PTH-rP AS) were inoculated intramuscularly into nude mice. Experimental groups included CHO/IL-6 plus CHO/PTH-rP; CHO/IL-6 plus CHO/PTH-rP AS; CHO/IL-6 alone; and CHO/PTH-rP alone. Blood ionized Ca2+ was measured on days 0, 7, 10, 12, and 13. Three different developmental stages in the osteoclast lineage were examined at day 13: the early multipotential precursor, granulocyte macrophage colony-forming units (CFU-GM); more mature mononuclear osteoclast precursors, assessed by their capacity to form tartrate-resistant acid phosphatase-positive multinucleated cells in marrow cultures; and mature osteoclasts, assessed by histomorphometry. IL-6 increased CFU-GM but not bone resorption or Ca2+. In contrast, PTH-rP induced hypercalcemia and bone resorption and increased multinucleated osteoclasts and more mature precursors cells, but not CFU-GM. However, mice treated with both IL-6 and PTH-rP had very marked hypercalcemia and osteoclastosis as well as an increase in the number of both CFU-GM and mature osteoclast precursors. These data demonstrate that IL-6 enhances PTH-rP-mediated hypercalcemia and bone resorption, most likely by increasing the pool of early osteoclast precursors that in turn can differentiate to mature osteoclasts. We conclude that IL-6 stimulatory effects on osteoclast precursors may enhance the effects of other bone resorption factors that act at later stages in the osteoclast lineage.

Effects of transforming growth factor-β1 on formation and activation of osteoclasts

Transforming growth factor-β1 (TGF-β1) has biological functions in various types of cells. However, its roles in the regulation of osteoclast formation and function are unclear. To examine them, we employed a culture system in which unfractionated cells obtained from long bones of 13-day-old mice were cultured on a dentine slice. We found that TGF-β1 has a potent inhibitory effect on osteoclastic bone resorption at a dose of 0.2–5 ng/ml. By electron microscopy the osteoclasts appeared to have fewer mitochondria and ruffled borders than those in control cultures. But in the presence of 1,25-dihydroxyvitamin D3, [1,25-(OH)2D3], TGF-β1 at a dose of 0.2–1 ng/ml stimulated the formation of osteoclasts from unfractionated bone cell cultures in which preexistent osteoclasts had degenerated. Thus, using stromal cell-free he-mopoietic blast cells, we examined the direct action of TGF-β1 on osteoclast precursors. Although TGF-β1 inhibited tartrate-resistant acid phosphatase-positive (TRAP) multinucleate cell (MNC) formation induced by 1,25-(OH)2D3, the conditioned medium (CM) of TGF-β1-treated MC3T3-E1 cells stimulated such formation. These results suggest that TGF-β1 inhibits osteoclastic bone resorption but stimulates osteoclast formation via the action of factor(s) produced by TGF-β1-treated osteoblasts in the presence of 1,25-(OH)2D3.

Osteoblasts mediate insulin-like growth factor-I and -II stimulation of osteoclast formation and function

Osteoblasts mediate insulin-like growth factor-I and -II stimulation of osteoclast formation and function

Interleukin-4 inhibits spontaneous and parathyroid hormone-related protein-stimulated osteoclast formation in mice

We examined the in vivo effects of recombinant murine IL-4 (rmIL-4) on spontaneous and stimulated mouse osteoclast formation. EC-GI cells, which produce PThrP and IL-1 alpha, were explanted in nude mice. These EC-GI cell-bearing nude mice developed hypercalcemia (4.90 +/- 0.68 mM), and the calcium levels were decreased to near normal (3.48 +/- 0.73 mM, p < 0.05) at day 3 by continuous infusion of rmIL-4 at a dose of 7 micrograms/day. When infused with 0.6 nmol/day of PTHrP(1-34) in ICR mice, rmIL-4 at a dose of 1 or 5 micrograms/day for 3 days caused a marked inhibitory effect on hypercalcemia induced by PTHrP(1-34) (3.73 +/- 0.56-2.54 +/- 0.14 mM, p < 0.01). However, rmIL-4 alone did not change the serum calcium in mice. Histomorphometric analysis revealed that rmIL-4 inhibits both spontaneous and PTHrP(1-34)-stimulated osteoclast formation in mice, with a decrease in osteoclastic surface and in the number of osteoclasts per mm bone surface, respectively. We conclude that IL-4 inhibits spontaneous and stimulated bone resorption resulting from inhibition of osteoclast formation and modulates the development of humoral hypercalcemia of malignancy.

Phorbol myristate acetate stimulates osteoclast formation in 1α, 25-dihydroxyvitamin D3-primed mouse embryonic calvarial cells by a Prostaglandin-dependent mechanism

Our previous study provided a novel assay system utilizing devitalized bone slices for study of the differentiation of osteoclast progenitors into preosteoclasts and mature osteoclasts among calvarial cells of mouse embryos. Using this assay system, we examined the effect of phorbol myristate acetate (PMA) on osteoclast formation as assessed by the appearance of tartrate-resistant acid phosphatase (TRAP)-positive cells and bone resorption lacunae. PMA alone was directly unable to induce the appearance of TRAP-positive cells and bone resorption lacunae of calvarial bone cells of mouse embryos. However, PMA markedly stimulated increases in the number of TRAP-positive cells and area of the resorption lacunae of the calvarial cells when the bone cells were primed by 1 alpha,25-(OH)2D3. This stimulatory effect of PMA was dose dependent. H-7, having relatively high affinity for protein kinase C, strongly inhibited in a dose-dependent fashion the stimulatory effect of PMA on the bone resorption of the hormone-primed calvarial cells. We also examined the involvement of prostaglandin in this stimulatory effect of PMA. Indomethacin, a cyclooxygenase inhibitor, markedly abolished the stimulatory effect of PMA on the bone resorption of the calvarial cells. PMA stimulated prostaglandin E2 (PGE2) production by the calvarial cells primed with 1 alpha,25-(OH)2D3 in a dose-dependent fashion. However, the PMA stimulation of the PGE2 production was significantly inhibited by H-7 and also by indomethacin. Furthermore, we observed that the addition of PGE2 to the calvarial cells primed with 1 alpha,25-(OH)2D3 for 1 or 3 days resulted in an increased number of TRAP-positive cells and increased bone resorption.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of 1,25-dihydroxyvitamin D 3 on osteoclast formation in fetal mouse metatarsal organ cultures

We have previously described a model system, using 15-day fetal mouse metatarsals cultured in serumless medium, in which osteoclasts and their precursors develop from in situ progenitors in a mariner which is similar, both temporally and spatially, to that which occurs in vivo. In this report we evaluate the role of the osteotropic hormone 1,25-dihydroxyvitamin D 3 (1,25-D3) on osteoclast formation in this model system by characterizing its effects on proliferation, differentiation, and fusion of cells of the osteoclast lineage. Morphologic evaluation was used to enumerate osteoclast precursors, mono- and multinucleate osteoclasts, and osteoclast nuclei in serial paraffin sections. Dose response data reveal a significant stimulation of osteoclast formation by 1,25-D3 in the range of 0.6 nM to 40 nM, and kinetic analyses suggest that these effects are on proliferation of osteoclast progenitors as well as on differentiation of precursors to form osteoclasts. A single 48 h exposure between day 4 and 6 of culture is necessary and sufficient to induce maximal osteoclast formation, while continuous exposure beyond this “critical period” inhibits multinucleate osteoclast formation. Simultaneous treatment with indomethacin inhibits the effects of 1,25-D3, while treatment with PGE 2 stimulates osteoclast formation without significantly increasing precursor numbers, or inhibiting multinucleate osteoclast formation. These results suggest that the effect of 1,25-D3 to induce differentiation of precursors to form mono- and multinucleate osteoclasts is mediated by endogenous prostaglandin synthesis. On the other hand, the inhibition of polykaryon formation observed with continuous 1,25-D3 treatment, does not appear to be a prostaglandin mediated phenomena. However, the difference between the effects of exogenous PGE 2 compared to that produced endogenously in response to 1,25-D3, suggest that endogenous prostaglandins other than PGE 2 may mediate some of the effects of 1,25-D3 on osteoclast formation.

Prostaglandins: Mechanisms of action and regulation of production in bone

Prostaglandins (PGs), particularly PGE2, are produced by bone and have powerful effects on bone metabolism. PGs have an initial, transient, direct inhibitory effect on osteoclast function. However, the major long-term effect in bone organ culture is to stimulate bone resorption by increasing the replication and differentiation of new osteoclasts. PGs also stimulate osteoclast formation in cell culture systems. Stimulation of osteoclastic bone resorption may be important in mediating bone loss in response to mechanical forces and inflammation. PGs have a biphasic effect on bone formation. At relatively low concentrations or in the presence of glucocorticoids, the replication and differentiation of osteoblasts is stimulated and bone formation is increased. This increase is associated with an increase in production of insulin-like growth factor-I (IGF-I). However, at high concentrations or in the presence of IGF-I, PGE2 inhibits collagen synthesis. In osteoblastic cell lines this inhibition can be shown to occur at the level of transcription of the collagen gene. The stimulatory effect on bone formation has been demonstrated when PGs are administered exogenously, but it is not clear how endogenous PG production affects bone formation in physiological or pathologic circumstances. The production of PGs in bone is highly regulated. The major source appears to be cells of the osteoblast lineage. A major site of regulation is at the level of the enzyme PG endoperoxide synthase (cyclooxygenase or PGH synthase). PGE2 production and PGH synthase mRNA are increased by PTH and interleukin-1 and decreased by estrogen. Glucocorticoids probably act by a different mechanism, decreasing either arachidonic acid or PGH synthase activity.(ABSTRACT TRUNCATED AT 250 WORDS)