Osteoclastogenesis In Bone Marrow Cells Research Articles

Modulating glycosphingolipid metabolism and autophagy improves outcomes in pre-clinical models of myeloma bone disease

Patients with multiple myeloma, an incurable malignancy of plasma cells, frequently develop osteolytic bone lesions that severely impact quality of life and clinical outcomes. Eliglustat, a U.S. Food and Drug Administration-approved glucosylceramide synthase inhibitor, reduced osteoclast-driven bone loss in preclinical in vivo models of myeloma. In combination with zoledronic acid, a bisphosphonate that treats myeloma bone disease, eliglustat provided further protection from bone loss. Autophagic degradation of TRAF3, a key step for osteoclast differentiation, was inhibited by eliglustat as evidenced by TRAF3 lysosomal and cytoplasmic accumulation. Eliglustat blocked autophagy by altering glycosphingolipid composition whilst restoration of missing glycosphingolipids rescued autophagy markers and TRAF3 degradation thus restoring osteoclastogenesis in bone marrow cells from myeloma patients. This work delineates both the mechanism by which glucosylceramide synthase inhibition prevents autophagic degradation of TRAF3 to reduce osteoclastogenesis as well as highlighting the clinical translational potential of eliglustat for the treatment of myeloma bone disease.

S100A8/A9 is not essential for the development of inflammation and joint pathology in interleukin-1 receptor antagonist knockout mice

BackgroundExcessive osteoclast activity, which is strongly stimulated by pro-inflammatory mediators, results in bone and cartilage degeneration as central features of many arthritides. Levels of the alarmin S100A8/A9 and interleukin (IL)-1β are both increased in arthritis patients and correlate with disease activity and progression of tissue erosion. We previously presented S100A8/A9 as a good biomarker for joint inflammation and arthritis pathology under circumstances of high IL-1 signaling in mice that lack the gene encoding IL-1 receptor antagonist (Il1rn−/− mice). Here, we investigated whether S100A8/A9 is also actively involved in the development of joint inflammation and both cartilage and bone pathology under these conditions by comparing Il1rn−/− mice with mice that have an additional deficiency for S100a9 (Il1rn−/−XS100a9−/−).MethodsIl1rn−/−XS100a9−/− on a BALB/c background were obtained by crossing S100a9−/− mice and Il1rn−/− mice. Arthritis incidence and severity were macroscopically scored. Myeloid cell populations in the bone marrow and spleen were determined using flow cytometry. In vitro osteoclastogenesis of bone marrow cells was evaluated with TRAP staining. Microscopic joint inflammation, cartilage degeneration, and bone destruction were evaluated using histology of ankle joints of 12- and 20-week-old mice.ResultsMacroscopically scored arthritis severity was comparable between Il1rn−/− and Il1rn−/−XS100a9−/− mice. Inflammation, cartilage erosion, and bone erosion were clearly present in 12-week-old mice of both strains lacking Il1rn−/−, but not significantly different between Il1rn−/−XS100a9−/− and Il1rn−/−. Moreover, we observed that the numbers of neutrophils and monocytes were increased by the absence of Il1rn, which was affected by the absence of S100a9 only in the spleen but not in the bone marrow. In line with our other findings, the absence of S100a9 did not affect the osteoclastogenic potential of osteoclast precursors in the absence of Il1rn. Finally, in agreement with the findings in early arthritis development in 12-week-old mice, cartilage and bone erosion in 20-week-old mice was significantly higher in both Il1rn−/− strains, but the additional absence of S100a9 did not further affect tissue pathology.ConclusionS100A8/A9 deficiency does not significantly affect inflammation and joint destruction in mice with high IL1β signaling suggesting that S100A8/A9 is not essential for the development of arthritis under these conditions.

The flavonoid p-hydroxycinnamic acid mediates anticancer effects on MDA-MB-231 human breast cancer cells in vitro: Implications for suppression of bone metastases.

Tumor invasion into bone tissues is associated with osteoclast and osteoblast recruitment, resulting in the liberation of growth factors from the bone matrix, which can feed back to enhance tumor growth resulting in the vicious cycle of bone metastasis. Activated nuclear factor-κB (NF-κB) in breast cancer cells has been shown to play a crucial role in the osteolytic bone metastasis of breast cancer in stimulating osteoclastogenesis. The flavonoid p-hydroxycinnamic acid (HCA) mediates bone anabolic and anti-catabolic effects by stimulating osteoblastic bone formation and suppressing osteoclastic bone resorption. However, the capacity of HCA to ameliorate the negative effects of breast cancer on bone cells has not been investigated. The present study was undertaken to determine the anticancer effects of HCA on MDA-MB-231 human breast cancer bone metastatic cells in vitro models. Proliferation of MDA-MB‑231 cells was suppressed by culture with HCA (10-1000 nM) due to G1 and G2/M phase cell cycle arrest. The suppressive effects of HCA were mediated through signaling pathways that are related to NF-κB, extracellular signal-regulated kinase (ERK), protein kinase C, calcium signaling, phosphatidylinositol 3-kinase (PI3K) and nuclear transcription activity. HCA was also found to induce death of confluent cancer cells. Furthermore, co-culture with MDA-MB-231 cells suppressed mineralization and stimulated osteoclastogenesis in bone marrow cells. These alterations were prevented by HCA (10-250 nM). The present study demonstrates that HCA possesses anticancer properties in MDA-MB-231 human breast cancer cells and alleviates the negative effects on osteoblastogenesis and osteoclastogenesis in vitro. HCA may have important applications in the treatment of breast cancer bone metastasis.

N-3 polyunsaturated fatty acids stimulate osteoclastogenesis through PPARγ-mediated enhancement of c-Fos expression, and suppress osteoclastogenesis through PPARγ-dependent inhibition of NFkB activation

n-3 polyunsaturated fatty acids (PUFAs), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have been reported to suppress osteoclastogenesis in vivo. In this study, the effect of PUFAs on receptor for activation of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis was examined using bone marrow-derived monocytes/macrophage precursor cells (BMMs) or bone marrow cells (BMCs) in vitro. EPA and DHA stimulated the osteoclastic differentiation of BMMs, but n-6 PUFAs, linoleic acid and arachidonic acid had no effect. The stimulation of osteoclastogenesis of BMMs by EPA and DHA was associated with enhancement of the gene expressions of c-Fos, tartrate-resistant acid phosphatase, cathepsin K and peroxisome proliferator-activated receptor-γ (PPARγ) and the protein levels of c-Fos, PPARγ and nuclear factor of activated T cells, cytoplasmic, calcineurin-dependent-1 (NFATc1). The PPARγ agonists, rosiglitazone and GW1929, also stimulated the osteoclastogenesis of BMMs. The PPARγ antagonists, T0070907 and GW9662, inhibited the stimulations of osteoclastogenesis and c-Fos expression by EPA or DHA. However, EPA and DHA inhibited the osteoclastogenesis in BMCs including BMMs and mesenchymal stem cells (MSCs). This inhibition was associated with suppression of the expression of RANKL and nuclear factor-κB (NFκB)-regulating genes, cyclooxygenase 2, TNFα and IL-6 in BMCs and MSCs. The agonists and antagonists of PPARγ showed that the inhibitions of NFκB transcriptional activity and osteoclastogenesis by EPA and DHA were PPARγ-dependent. These results suggest that EPA and DHA directly act on BMMs and stimulate osteoclastogenesis through enhancing c-Fos expression mediated by PPARγ but suppress osteoclastogenesis through the PPARγ-dependent inhibition of NFκB activation of MSCs in BMCs.

9-Demethoxy-medicarpin promotes peak bone mass achievement and has bone conserving effect in ovariectomized mice: Positively regulates osteoblast functions and suppresses osteoclastogenesis

We report a new bone anabolic and anti-catabolic pterocarpan 9-demethoxy-medicarpin (DMM) for the management of postmenopausal osteoporosis. DMM promoted osteoblast functions via activation of P38MAPK/BMP-2 pathway and suppressed osteoclastogenesis in bone marrow cells (BMCs). In calvarial osteoblasts, DMM blocked nuclear factor kappaB (NFκB) signaling and inhibited the mRNA levels of pro-inflammatory cytokines. DMM treatment led to increased OPG (osteoprotegrin) and decreased transcript levels of TRAP (tartarate resistant acid phosphatase), RANK (receptor activator of NFκB) and RANKL (RANK ligand) in osteoblast–osteoclast co-cultures. Immature female SD rats administered with DMM exhibited increased bone mineral density, bone biomechanical strength, new bone formation and cortical bone parameters. Ovx mice administered with DMM led to significant restoration of trabecular microarchitecture and had reduced formation of osteoclasts and increased formation of osteoprogenitor cells in BMCs. DMM exhibited no uterine estrogenicity. Overall, these results demonstrate the therapeutic potential of DMM for the management of postmenopausal osteoporosis.

Curcumin analogue UBS109 prevents bone loss in breast cancer bone metastasis mouse model: involvement in osteoblastogenesis and osteoclastogenesis.

Bone metastasis of breast cancer typically leads to osteolysis, which causes severe pathological bone fractures and hypercalcemia. Bone homeostasis is skillfully regulated through osteoblasts and osteoclasts. Bone loss with bone metastasis of breast cancer may be due to both activation of osteoclastic bone resorption and suppression of osteoblastic bone formation. This study was undertaken to determine whether the novel curcumin analogue UBS109 has preventive effects on bone loss induced by breast cancer cell bone metastasis. Nude mice were inoculated with breast cancer MDA-MB-231 bone metastatic cells (10(6) cells/mouse) into the head of the right and left tibia. One week after inoculation, the mice were treated with control (vehicle), oral administration (p.o.) of UBS109 (50 or 150mg/kg body weight), or intraperitoneal administration (i.p.) of UBS109 (10 or 20mg/kg body weight) once daily for 5days per week for 7weeks. After UBS109 administration for 7weeks, hind limbs were assessed using an X-ray diagnosis system and hematoxylin and eosion staining to determine osteolytic destruction. Bone marrow cells obtained from the femurs and tibias were cultured to estimate osteoblastic mineralization and osteoclastogenesis ex vivo and in vitro. Remarkable bone loss was demonstrated in the tibias of mice inoculated with breast cancer MDA-MB-231 bone metastatic cells. This bone loss was prevented by p.o. administration of UBS109 (50 and 150mg/kg body weight) and i.p. treatment of UBS109 (10 and 20mg/kg) in vivo. Culture of bone marrow cells obtained from the bone tissues of mice with breast cancer cell bone metastasis showed suppressed osteoblastic mineralization and stimulated osteoclastogenesis ex vivo. These changes were not seen after culture of the bone marrow cells obtained from mice treated with UBS109. Moreover, UBS109 was found to stimulate osteoblastic mineralization and suppress lipopolysaccharide (LPS)-induced osteoclastogenesis in bone marrow cells obtained from normal nude mice in vitro. These findings suggest that the novel curcumin analogue UBS109 prevents breast cancer cell bone metastasis-induced bone loss by stimulating osteoblastic mineralization and suppressing osteoclastogenesis.

Lysophosphatidic Acid Receptor Type 1 (LPA1) Plays a Functional Role in Osteoclast Differentiation and Bone Resorption Activity

Lysophosphatidic acid (LPA) is a natural bioactive lipid that acts through six different G protein-coupled receptors (LPA1-6) with pleiotropic activities on multiple cell types. We have previously demonstrated that LPA is necessary for successful in vitro osteoclastogenesis of bone marrow cells. Bone cells controlling bone remodeling (i.e. osteoblasts, osteoclasts, and osteocytes) express LPA1, but delineating the role of this receptor in bone remodeling is still pending. Despite Lpar1(-/-) mice displaying a low bone mass phenotype, we demonstrated that bone marrow cell-induced osteoclastogenesis was reduced in Lpar1(-/-) mice but not in Lpar2(-/-) and Lpar3(-/-) animals. Expression of LPA1 was up-regulated during osteoclastogenesis, and LPA1 antagonists (Ki16425, Debio0719, and VPC12249) inhibited osteoclast differentiation. Blocking LPA1 activity with Ki16425 inhibited expression of nuclear factor of activated T-cell cytoplasmic 1 (NFATc1) and dendritic cell-specific transmembrane protein and interfered with the fusion but not the proliferation of osteoclast precursors. Similar to wild type osteoclasts treated with Ki16425, mature Lpar1(-/-) osteoclasts had reduced podosome belt and sealing zone resulting in reduced mineralized matrix resorption. Additionally, LPA1 expression markedly increased in the bone of ovariectomized mice, which was blocked by bisphosphonate treatment. Conversely, systemic treatment with Debio0719 prevented ovariectomy-induced cancellous bone loss. Moreover, intravital multiphoton microscopy revealed that Debio0719 reduced the retention of CX3CR1-EGFP(+) osteoclast precursors in bone by increasing their mobility in the bone marrow cavity. Overall, our results demonstrate that LPA1 is essential for in vitro and in vivo osteoclast activities. Therefore, LPA1 emerges as a new target for the treatment of diseases associated with excess bone loss.

Effect of the interaction between periodontitis and type 1 diabetes mellitus on alveolar bone, mandibular condyle and tibia

Objective. This study examined the effect of the interaction between periodontitis and type 1 diabetes mellitus on alveolar bone, mandibular condyle and tibia in animal models. Materials and methods.Rats were divided into normal, periodontitis, diabetic and diabetic with periodontitis groups. After injection of streptozotocin to induce diabetes, periodontitis was induced by ligation of both lower-side first molars for 30 days. Alveolar bone loss and trabecular bone volume fraction (BVF) of the mandibular condyle and tibia were estimated via hematoxylin and eosin staining and micro-computed tomography, respectively. Osteoclastogenesis of bone marrow cells isolated from tibia and femur was assayed using tartrate-resistant acid phosphatase staining. Results. The cemento-enamel junction to the alveolar bone crest distance and ratio of periodontal ligament area in the diabetic with periodontitis group were significantly increased compared to those of the periodontitis group. Mandibular condyle BVF did not differ among groups. The BVF of tibia in the diabetic and diabetic with periodontitis groups was lower than that of the normal and periodontitis groups. Osteoclastogenesis of bone marrow cells in the diabetic groups was higher than that in the non-diabetic groups. However, the BVF of tibia and osteoclastogenesis in the diabetic with periodontitis group were not significantly different than those in the diabetic group. Conclusions. Type 1 diabetes mellitus aggravates alveolar bone loss induced by periodontitis, but periodontitis does not alter the mandibular condyle and tibia bone loss induced by diabetes. Alveolar bone, mandibular condyle and tibia may have different responses to bone loss stimuli in the diabetic environment.

IL-12- and IL-18-Mediated, Nitric Oxide-Induced Apoptosis in TNF-α-Mediated Osteoclastogenesis of Bone Marrow Cells

TNF-α has been recognized as an important factor for osteoclastogenesis and plays an important role in bone resorption under pathological conditions. IL-12 and IL-18, which are T-cell mediators, are also important inflammatory cytokines. We have reported that IL-12 and IL-18 induce apoptosis in bone marrow cells treated with TNF-α in vitro and that osteoclastogenesis is inhibited by the interaction of TNF-α-induced Fas and the IL-12-induced Fas ligand (FasL). However, the anti-FasL antibody could not completely inhibit apoptosis. Therefore, it is possible that IL-12 and IL-18 may also trigger some other apoptotic mechanisms. Nitric oxide (NO) may act as a mediator of the apoptotic effect. In this study, we examined whether NO causes the IL-12- and IL-18-induced apoptosis of bone marrow cells in TNF-α-mediated osteoclast formation. We found that NO production was induced in bone marrow cells cultured with IL-12 and IL-18 in the presence of TNF-α. When bone marrow cells were cultured with TNF-α, osteoclasts were formed. In contrast, when bone marrow cells were cultured with both TNF-α and IL-12 or IL-18, the adherent cells were induced to undergo apoptosis. Apoptosis was partially inhibited when bone marrow cells were treated with NO synthase inhibitors. Furthermore, IL-12 and IL-18 synergistically induced cell death and upregulated NO production in the presence of TNF-α. These results indicate that the simultaneous effects of TNF-α and IL-12 or IL-18 on bone marrow cells induce apoptosis and that apoptosis is induced by the production of NO.

Caffeine enhances osteoclast differentiation from bone marrow hematopoietic cells and reduces bone mineral density in growing rats

Caffeine-containing beverage consumption has been associated with low bone mass and increased fracture risk in some, but not most, observational studies. The effects of caffeine on bone metabolism are still controversial. We investigated the effects of caffeine on the differentiation of bone progenitor cells and bone mineral density (BMD) by in vitro and in vivo experiments. Low-concentration caffeine (0.005-0.1 mM) did not affect the bone marrow cell viability and alkaline phosphatase activity during osteoblast differentiation from bone marrow stromal cells, but it effectively enhanced the osteoclastogenesis from bone marrow hematopoietic cells and the bone resorption activity by pit formation assay. Moreover, caffeine effectively enhanced the receptor activator of NF-κB ligand (RANKL), but reduced the osteoprotegerin protein expressions in osteoblast MC3T3-E1 cells. Caffeine could also increase the cyclooxygenase-2 (COX-2) protein expression and prostaglandin (PG)E(2) production in cultured neonatal mouse calvariae. In animal study, BMD in lumbar vertebra, femur, or tibia was significantly lowered in growing rats supplemented with 0.2% caffeine in diets for 20 weeks compared with the control group. The calcium contents in tibia and femur of caffeine-treated rats were also lower than that in the control group. The osteoclastogenesis of bone marrow cells isolated from caffeine-treated rats was markedly enhanced as compared with the control group. Taken together, these results suggest that caffeine may reduce BMD in growing rats through the enhancement in osteoclastogenesis. Caffeine may possess the ability to enhance a COX-2/PGE(2) -regulated RANKL-mediated osteoclastogenesis.

Evaluation of Osteoclastogenesis via NFκB Decoy/mannosylated Cationic Liposome-Mediated Inhibition of Pro-inflammatory Cytokine Production from Primary Cultured Macrophages

To explore the effect of NFκB activation in macrophages on osteoclastogenesis of bone marrow cells for potential application as a new type of therapy for preventing bone loss. Primary cultured macrophages and bone marrow cells were prepared from mice. As macrophage-targeted carriers, Mannosylated cationic liposomes (Man-liposomes) were prepared and were allowed to form complexes with NFκB decoy (a double-stranded oligonucleotide). Cellular uptake, inhibition of NFκB activation, and cytokine production were evaluated using macrophages. Osteoclastogenesis was investigated using bone marrow cells, which were cultured in the conditioned medium prepared from macrophages with or without Man-liposome/NFκB decoy complexes treatment. Cellular accumulation of NFκB decoy was enhanced by Man-liposome. NFκB activation in macrophages and TNF-α production were suppressed in macrophages by Man-liposome/NFκB decoy complexes but not by the naked NFκB decoy, Gal-liposome/NFκB decoy complexes, or Man-liposome/random decoy complexes. Osteoclastogenesis of bone marrow cells was induced in the conditioned medium prepared from activated macrophages but not by activated macrophages treated with Man-liposome/NFκB decoy complexes. Osteoclastogenesis induced by activated macrophages could be suppressed by the treatment macrophages with Man-liposome/NFκB decoy complexes. Macrophage-targeted delivery of NFκB decoys using Man-liposomes may be promising in its use for the remediation of bone loss.

Medicarpin inhibits osteoclastogenesis and has nonestrogenic bone conserving effect in ovariectomized mice

Medicarpin, a pterocarpan class of naturally occurring benzopyran furanobenzene compound was synthesized in gram scale to investigate its effects on murine bone cells and in ovariectomized (OVx) mice. Medicarpin, at as low as 10 −10 M suppressed osteoclastogenesis in bone marrow cells (BMCs). Medicarpin-induced apoptosis of mature osteoclasts isolated from long bones. Effects of medicarpin in osteoclasts appear to be independent of estrogen receptor (ER) activation as ICI 180,782 failed to abrogate its effects on osteoclasts. In calvarial osteoblasts, medicarpin (10 −10 M) blocked nuclear factor kappaB (NF-κB) signaling assessed by tumor necrosis factor alpha (TNFα)-stimulated nuclear translocation of p65 subunit of NF-κB. Medicarpin also inhibited the expression of TNFα in mouse calvarial osteoblasts. This effect was ER dependent as ICI 180,782 reversed the suppressive effect of medicarpin on TNFα mRNA levels in osteoblasts. In addition, like 17β-estradiol, presence of medicarpin inhibited TNFα-induced upregulation of interleukin-1, and -6 mRNA levels in osteoblasts. In co-cultures consisting of calvarial osteoblasts and BMCs, presence of medicarpin increased osteoprotegerin (OPG)/receptor activator of NF-κB ligand (RANKL) ratio and reduced mRNA levels of osteoclast markers including tartrate-resistant acid phosphatase and RANK. OVx mice administered medicarpin (10.0 mg kg −1 day −1) orally for 30 days had reduced formation of osteoclasts but increased formation of osteoprogenitor cells in BMCs compared with OVx + vehicle group. Medicarpin treatment to OVx mice maintained parameters of trabecular microarchitecure. Medicarpin exhibited no uterine estrogenicity. Our findings point towards direct and indirect inhibitory effects of medicarpin on osteoclastogenesis in vitro that contribute to its bone sparing effect in OVx mice.

8,8″-Biapigeninyl stimulates osteoblast functions and inhibits osteoclast and adipocyte functions: Osteoprotective action of 8,8″-biapigeninyl in ovariectomized mice

8,8″-Biapigeninyl (BA), a condensation product of two apigenin molecules, is found abundantly in the nuts of Cupressus sempervirens. We investigated the effects of BA on murine bone cells in vitro and in ovariectomized (OVx) mice. BA at 10 −10 M and 10 −8 M, inhibited osteoclastogenesis of bone marrow cells (BMCs) and displayed concentration dependence. BA at 10 −8 M and 10 −6 M inhibited differentiation of 3T3-L1 and BMCs to mature adipocytes. BA (10 −10 M) stimulated osteoblast proliferation, differentiation and mineralization. In stimulating osteoblast function, BA was found to be 10 4-fold more potent than apigenin. The effect of BA in osteoblasts appeared to be mediated via estrogen receptors (ER) as antiestrogen, ICI-182780 abolished BA-stimulated osteoblast differentiation. In OVx mice BA treatment (at 1.0-, 5.0- and 10.0 mg kg −1 day −1 doses) given orally for 30 days dose-dependently inhibited mRNA levels of osteoclastic genes including tartrate-resistant acid phosphatase, receptor activator of nuclear factor (RANK), tumor necrosis factor alpha, interleukin-6 and the ratio of RANK ligand/osteoprotegerin ratio in bones compared with OVx mice treated with vehicle. In addition, BA treatment to OVx mice dose-dependently stimulated production of osteoprogenitor cells in the bone marrow and increased mRNA levels of osteogenic genes core binding factor alpha-1, type I collagen and bone morphogenic protein-2 in bones compared with OVx + vehicle group. Microcomputed tomography revealed that BA treatment to OVx mice improved parameters of trabecular and cortical architecture. BA exhibited no uterine estrogenicity. From these data, we conclude that BA exerts osteoprotective effect in OVx mice by multiple beneficial effects on bone cells.

Retinoids inhibit differentiation of hematopoetic osteoclast progenitors

Whether vitamin A promotes skeletal fragility, has no effect on fracture rate, or protects against bone loss is unclear. In the present study, effects of retinoids on osteoclast differentiation in cultured mouse bone marrow cells (BMCs), bone marrow macrophages (BMMs), spleen cells, and RAW264.7 cells were evaluated by analyzing osteoclast formation and expression of genes important in signal transduction and osteoclast function. All-trans-retinoic acid (ATRA) did not stimulate osteoclastogenesis in BMCs, but inhibited hormone and RANKL-induced gene expression and formation of osteoclasts. In BMMs, spleen cells, and RAW264.7 cells, osteoclast differentiation and formation stimulated by M-CSF/RANKL were inhibited (IC(50) = 0.3 nM) by ATRA. The effect was exerted at an early step of RANKL-induced differentiation. ATRA also abolished increases of the transcription factors c-Fos and NFAT2 stimulated by RANKL and suppressed down-regulation of the antiosteoclastogenic transcription factor MafB. By comparing effects of several compounds structurally related to ATRA, as well as by using receptor antagonists, evaluation pointed to inhibition being mediated by RARalpha, with no involvement of PPARbeta/delta. The results suggest that activation of RARalpha by retinoids in myeloid hematopoietic precursor cells decreases osteoclast formation by altering expression of the transcription factors c-Fos, NFAT2, and MafB.

Inhibition of Src Homology 2 Domain-Containing Protein Tyrosine Phosphatase Substrate-1 Reduces the Severity of Collagen-Induced Arthritis

To investigate whether the blockade of Src homology 2 domain-containing protein tyrosine phosphatase substrate-1 (SHPS-1) has any therapeutic effects on rheumatoid arthritis. A functional blocking monoclonal antibody for SHPS-1 (anti-SHPS-1 mAb) was administered at various doses to collagen-induced arthritis (CIA) mice, and severity of the arthritis was evaluated by clinical and histological scores of the limbs. To clarify the mechanisms of action of the antibody, the serum concentration of anti-type II collagen antibody was measured in those mice, and in vitro experiments were conducted to determine the effects of the antibody on the induction of osteoclasts and the release of cytokines from mouse spleen cells. Compared with mice given control IgG, the administration of anti-SHPS-1 mAb significantly reduced the severity of inflammation and destruction of bone and cartilage in CIA mice. This therapeutic effect was observed even when the antibody treatment was started after the onset of arthritis. The appearance of anti-type II collagen antibody in CIA mice was not altered by the antibody treatment. In in vitro experiments, the anti-SHPS-1 mAb significantly inhibited osteoclastogenesis of bone marrow cells, and significantly reduced the release of interleukin 1beta (IL-1beta), IL-2, IL-12, interferon-gamma, and tumor necrosis factor-alpha, but not that of IL-4 or IL-10, from the spleen cells after stimulation with concanavalin A. Administration of a monoclonal antibody for SHPS-1 reduced the severity of arthritis in CIA mice. Regulation of biological functions of SHPS-1 may be a novel and potent strategy to treat patients with rheumatoid arthritis.

Synthesis and biological evaluation of indolyl bisphosphonates as anti-bone resorptive and anti-leishmanial agents

A series of indole conjugated bisphosphonate derivatives have been synthesized and evaluated for their in vitro anti-bone resorptive activity using bone marrow osteoclast culture. Two bisphosphonates 23 and 24 significantly inhibited osteoclastogenesis, 23 showed inhibition at 10 and 100 pM which was lower than the concentration of standard drug alendronate, and 24 inhibited osteoclastogenesis at 100 nM which was comparable to alendronate. Two other compounds 13 and 14 also showed inhibition comparable to alendronate, but were cytotoxic in the osteoblast cells. The two active bisphosphonates 23 and 24 induced significant osteoclast apoptosis at concentrations 100 nM for compound 24 and at 10 pM for compound 23 compared to alendronate. In vivo effect of active bisphosphonates 23 and 24 resulted in osteoclastogenesis of bone marrow cells (BMCs) to almost 40–50% ( 23 showing 8.4% decrease and 24 showing 9.0%) compared to 16.5% of the ovariectomized group. Further, screening of anti-leishmanial activity, four compounds 24– 25 and 27– 28 showed more than 80% inhibition against both the promastigote and amastigote stages of the Leishmania parasite.

Osteoclast development in immobilized bone is suppressed by parathyroidectomy in mice

We tested the hypothesis that signaling of parathyroid hormone (PTH) facilitates osteoclastogenesis in bone marrow cells after immobilization, thereby reducing trabecular bone volume. We performed histomorphometric analyses in immobilized limbs after right sciatic neurectomy (IM) and in the contralateral limbs after sham surgery (M). Mice underwent thyroparathyroidectomy (TPTX) and then 0.2 microg/body of thyroxine was given three times a week, or the mice were subjected to sham surgery (sham). Six-week-old male ddY mice were assigned to four groups, as follows, after acclimatization for 1 week: M + sham, IM + sham; M + TPTX, and IM + TPTX. Bilateral tibial samples were used for analysis. Trabecular bone volume (BV/TV) in the secondary spongiosa of the proximal tibias in IM + sham was significantly reduced compared to that in M + sham. Osteoclast surface (Oc.S/BS) and number (Oc.N/BS) in IM + sham transiently increased at 3 and 4 weeks after IM. In contrast, TPTX partially prevented the IM-related reduction of BV/TV and completely suppressed the transient increases of Oc.S/BS and Oc.N/BS. In the bone marrow cells, the mRNA expression of RANKL was elevated in IM + sham, but not in IM + TPTX, compared to that in M + sham. The percentage of Mac-1-positive bone marrow cells, osteoclast precursors, was not altered after IM. There were no significant differences in the concentrations of interleukin (IL)-1alpha in the tibial bone marrow cell culture medium between M + sham and IM + sham. Our data demonstrated that significant increases in osteoclast surface and number after IM were suppressed in TPTX mice, closely associated with a reduction in the high expression of RANKL mRNA in the tibial bone marrow cells. We speculate that enhanced osteoclastogenesis due to limb immobilization may be related to the elevation of RANKL expression by the facilitation of parathyroid hormone signaling in bone marrow cells.

Possible involvement of MIP-1α in the recruitment of osteoclast progenitors to the distal tibia in rats with adjuvant-induced arthritis

In the rat model of rheumatoid arthritis, a marked formation of osteoclasts is found in the distal tibia and the metatarsal bone. It was therefore postulated that osteoclast progenitors would be increased in the bone marrow cavities of rats with adjuvant-induced arthritis (AA rats). Bone marrow cells obtained from tibia of AA rats were cultured to form cells in the osteoclast lineage to access the number of osteoclast progenitors. Unexpectedly, only a suppressed level of osteoclast progenitors was detected in the diaphyseal bone marrow of tibia in AA rats. Distribution of osteoclast progenitors in the bone marrow cavity was examined, and it was shown that osteoclast progenitors accumulated in the distal tibia. Macrophage inflammatory protein (MIP)-1α, an osteoclastogenic CC chemokine, was expressed in ED-1-positive macrophages localizing in the distal tibia with marked bone destruction. Chemotaxis studies showed that MIP-1α expressed significant activity towards bone marrow cells. The suppressed level of osteoclastogenesis in bone marrow cells of AA rats was restored to a normal level by the addition of MIP-1α. It was suggested that MIP-1α is involved in the migration of osteoclast progenitors to the distal tibia as well as in osteoclastogenesis in AA rats. In these rats, in situ hybridization of the distal tibia with a high level of bone destruction showed significant expression of Receptor activator nuclear factor κB ligand (RANKL) messenger RNA in aggregates of multinucleated osteoclast-like cells present in the bone marrow cavity, a unique pathological feature for these rats. Migrated osteoclast progenitors are thought to be efficiently differentiated into osteoclasts in response to RANKL expressed by the aggregates of osteoclast-like cells under the influence of the MIP-1α. Such positive-feedback regulation of osteoclastogenesis could result in the highest recruitment of active osteoclasts in the area of marked bone destruction.

Inhibition of RANKL-induced osteoclast formation in mouse bone marrow cells by IL-12: involvement of IFN-γ possibly induced from non-T cell population

IL-12 was shown to have the potential to inhibit osteoclast formation in mouse bone marrow cells treated with macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL). When bone marrow macrophages (BMM) were used as osteoclast precursors, IL-12 failed to inhibit M-CSF/RANKL-induced osteoclast formation from BMM. In coculture experiments using transwells, IL-12 did inhibit osteoclast formation from BMM cocultured with whole bone marrow cells. These results indicated that IL-12 indirectly affected M-CSF/RANKL-induced osteoclastogenesis in bone marrow cells and that the inhibition of IL-12 on osteoclast formation was caused by a humoral factor from bone marrow cells treated with IL-12. Experiments with anti-interferon (IFN)-γ antibody and bone marrow cells from IFN-γ receptor knockout mice revealed that IFN-γ might be involved in the inhibition of osteoclast formation in this system. The expression of osteoprotegerin mRNA in bone marrow cells was not affected by treatment with IL-12. The inhibitory effect of IL-12 on osteoclast formation was also seen in the T cell-depleted bone marrow cells of normal mice and the whole bone marrow cells of athymic nude mice, while the inhibitory effect of IL-12 was partially suppressed in the B cell-depleted bone marrow cells. The inhibitory effect of IL-12 on M-CSF/RANKL-induced osteoclastogenesis was not accompanied with cell death, in contrast with our previous finding that the inhibitory effect of IL-12 on M-CSF/TNF-α-induced osteoclastogenesis is attributable to Fas and FasL-mediated apoptosis.