Stimulation Of Osteoclastogenesis Research Articles

Increased bone resorption by long-term cigarette smoke exposure in animal model

IntroductionClinical and experimental studies have been attesting the deleterious effects of smoking mainly due to the stimulation of osteoclastogenesis and inhibition of osteoblastogenesis. However the physiological mechanisms that can explain these changes are not fully understood. AimsTo evaluate the trabecular bone resorption effect caused by long-term exposure to cigarette smoke and the action of cytokines and reactive oxygen species involved in this process. MethodsSixty young adult C57BL/6 mice were allocated to two groups: control, 30 animals exposed to filtered air for 1, 3 and 6 months; and smoke, 30 animals exposed to cigarette smoke for 1, 3 and 6 months. Femoral and tibial extraction was performed to evaluate the bone mineral matrix, bone cytokines (Receptor activator of nuclear factor-kappa B ligand - RANKL and Osteoprotegerin - OPG) and oxidative stress markers (Thiobarbituric acid reactive substances - Tbars). ResultsExposure to cigarette smoke (CS) generated changes in bone structural parameters in the 6th month of follow-up, demonstrating an evident bone loss; reduction in OPG/RANKL ratio from the 3rd month on and increase in Tbars in the first month, both closely related to the increase in osteoclastogenic activity and bone resorption. ConclusionThese findings reinforce the importance of CS-induced oxidative stress in bone compromising the bone cellular activities with a consequent impairment in bone turn over and changes in bone structure.

Performance of Nano-Hydroxyapatite/Beta-Tricalcium Phosphate and Xenogenic Hydroxyapatite on Bone Regeneration in Rat Calvarial Defects: Histomorphometric, Immunohistochemical and Ultrastructural Analysis.

BackgroundSynthetic biomaterials have played an increasingly prominent role in the substitution of naturally derived biomaterials in current surgery practice. In vitro and in vivo characterization studies of new synthetic biomaterials are essential to analyze their physicochemical properties and the underlying mechanisms associated with the modulation of the inflammatory process and bone healing.PurposeThis study compares the in vivo tissue behavior of a synthetic biomaterial nano-hydroxyapatite/beta-tricalcium phosphate (nano-HA/ß-TCP mixture) and deproteinized bovine bone mineral (DBBM) in a rat calvarial defect model. The innovation of this work is in the comparative analysis of the effect of new synthetic and commercially xenogenic biomaterials on the inflammatory response, bone matrix gain, and stimulation of osteoclastogenesis and osteoblastogenesis.MethodsBoth biomaterials were inserted in rat defects. The animals were divided into three groups, in which calvarial defects were filled with xenogenic biomaterials (group 1) and synthetic biomaterials (group 2), or left unfilled (group 3, controls). Sixty days after calvarial bone defects filled with biomaterials, periodic acid Schiff (PAS) and Masson’s trichrome staining, immunohistochemistry tumor necrosis factor-alpha (TNF-α), matrix metalloproteinase-9 (MMP-9), and electron microscopy analyses were conducted.ResultsHistomorphometric analysis revealed powerful effects such as a higher amount of proteinaceous matrix and higher levels of TNF-α and MMP-9 in bone defects treated with alloplastic nano-HA/ß-TCP mixture than xenogenicxenogic biomaterial, as well as collagen-proteinaceous material in association with hydroxyapatite crystalloids.ConclusionThese data indicate that the synthetic nano-HA/ß-TCP mixture enhanced bone formation/remodeling in rat calvarial bone defects. The nano-HA/ß-TCP did not present risks of cross-infection/disease transmission. The synthetic nano-hydroxyapatite/beta-tricalcium phosphate mixture presented adequate properties for guided bone regeneration and guided tissue regeneration for dental surgical procedures.

Increased Bone Resorption by Long-Term Cigarette Smoke Exposure in Animal Model

Introduction: Clinical and experimental studies have been attesting the deleterious effects of smoking mainly due to the stimulation of osteoclastogenesis and inhibition of osteoblastogenesis. However the physiological mechanisms that can explain these changes are not fully understood. Aims: To evaluate the bone resorption effect caused by long-term exposure to cigarette smoke and the action of cytokines and reactive oxygen species involved in this process. Methods: Sixty young adult C57BL/6 mice were allocated to two groups: control, 30 animals exposed to filtered air for 1, 3 and 6 months; and smoke, 30 animals exposed to cigarette smoke for 1, 3 and 6 months. Femoral and tibial extraction was performed to evaluate the bone mineral matrix, bone cytokines (Receptor activator of nuclear factor-kappa B ligand - RANKL and Osteoprotegerin - OPG) and oxidative stress markers (Thiobarbituric acid reactive substances - Tbars). Results: Exposure to cigarette smoke (CS) generated changes in bone structural parameters in the 6th month of follow-up, demonstrating an evident bone loss; reduction in OPG/RANKL ratio from the 3rd month on and increase in Tbars in the first month, both closely related to the increase in osteoclastogenic activity and bone resorption. Conclusion: These findings reinforce the importance of CS-induced oxidative stress in bone compromising the bone cellular activities with a consequent impairment in bone turn over and changes in bone structure. Funding: None to declare. Declaration of Interests: None to declare. Ethics Approval Statement: This study was approved by the Review Board for human and animal studies from the University of Sao Paulo School of Medicine - Project Number 937/17

S100A4 released from highly bone-metastatic breast cancer cells plays a critical role in osteolysis

Bone destruction induced by breast cancer metastasis causes severe complications, including death, in breast cancer patients. Communication between cancer cells and skeletal cells in metastatic bone microenvironments is a principal element that drives tumor progression and osteolysis. Tumor-derived factors play fundamental roles in this form of communication. To identify soluble factors released from cancer cells in bone metastasis, we established a highly bone-metastatic subline of MDA-MB-231 breast cancer cells. This subline (mtMDA) showed a markedly elevated ability to secrete S100A4 protein, which directly stimulated osteoclast formation via surface receptor RAGE. Recombinant S100A4 stimulated osteoclastogenesis in vitro and bone loss in vivo. Conditioned medium from mtMDA cells in which S100A4 was knocked down had a reduced ability to stimulate osteoclasts. Furthermore, the S100A4 knockdown cells elicited less bone destruction in mice than the control knockdown cells. In addition, administration of an anti-S100A4 monoclonal antibody (mAb) that we developed attenuated the stimulation of osteoclastogenesis and bone loss by mtMDA in mice. Taken together, our results suggest that S100A4 released from breast cancer cells is an important player in the osteolysis caused by breast cancer bone metastasis.

A CTGF-RUNX2-RANKL Axis in Breast and Prostate Cancer Cells Promotes Tumor Progression in Bone.

Metastasis to bone is a frequent occurrence in patients with breast and prostate cancers and inevitably threatens the patient's quality of life and survival. Identification of cancer-derived mediators of bone metastasis and osteolysis may lead to novel therapeutic strategies. In this study, we established highly bone-metastatic PC3 prostate and MDA-MB-231 (MDA) breast cancer cell sublines by in vivo selection in mice. In bone-metastatic cancer cells, the expression and secretion of connective tissue growth factor (CTGF) were highly upregulated. CTGF knockdown in bone-metastatic cells decreased invasion activity and MMP expression. RUNX2 overexpression in the CTGF knockdown cells restored the invasion activity and MMP expression. In addition, CTGF increased RUNX2 protein stability by inducing its acetylation via p300 acetyl transferase. The integrin αvβ3 receptor mediated these effects of CTGF. Furthermore, CTGF promoted RUNX2 recruitment to the RANKL promoter, resulting in increased RANKL production from the tumor cells and subsequent stimulation of osteoclastogenesis from precursor cells. In addition, animal model with injection of CTGF knocked-down prostate cancer cells into 6-week old BALB/c male mice showed reduced osteolytic lesions. More importantly, the expression levels of CTGF and RANKL showed a strong positive correlation in human primary breast tumor tissues and were higher in bone metastases than in other site metastases. These findings indicate that CTGF plays crucial roles for osteolytic bone metastasis both by enhancing invasiveness of tumor cells and by producing RANKL for osteoclastogenesis. Targeting CTGF may lead to the development of effective preventive and therapeutic strategies for osteolytic metastasis. © 2019 American Society for Bone and Mineral Research.

A Unique Triculture Model to Study Osteoblasts, Osteoclasts, and Endothelial Cells.

In this article, we first developed a new medium to culture together primary human osteoblastic, osteoclastic, and endothelial cells (ECs) chosen to represent the three major bone cell tissues. Indeed, no study has been conducted on primary human cells and on the phenotype/activity retention of these three primary human cell types. Thus, we established an original triculture model with osteoblastic, osteoclastic, and ECs, where not only both cell phenotype and cell activity were maintained but also cell culture homeostasis. These promising results will permit further investigations to create in vitro conditions to mimic the bone microenvironment and analyze cell interactions in ex vivo studies.

Abstract 486: MicroRNA-30 family members inhibit breast cancer invasion, osteomimicry, and bone destruction by directly targeting multiple bone metastasis-associated genes

Abstract MicroRNAs (miRNAs), which are master regulators of gene expression, have emerged as key players in cancer metastasis. Here, we established miR-30 family members (miR-30a, miR-30b, miR-30c, miR-30d and miR-30e) as breast cancer bone metastasis suppressor genes. Low expression levels of miR-30s in primary tumors of breast cancer patients were associated with poor relapse-free survival. Additionally, miR-30s levels were significantly lower in estrogen receptor (ER)-negative/progesterone receptor (PR)-negative tumors than in their hormone-responsive counterparts. Overexpression of miR-30s in ER/PR-negative breast cancer cells resulted in the reduction of bone metastasis burden in vivo. MiR-30s overexpression also reduced tumor outgrowth, when tumor cells were implanted subcutaneously in animals. In vitro, miR-30s did not affect tumor cell proliferation, but inhibited tumor cell invasion. Furthermore, miR-30s restored bone homeostasis by reversing the in vitro effects of the tumor cell conditioned medium on stimulation of osteoclastogenesis and inhibition of osteoblastogenesis. We identified a number of genes associated with osteoclastogenesis stimulation (IL-8, IL-11), osteoblastogenesis inhibition (DKK-1), tumor cell osteomimicry (RUNX2, CDH11) and invasiveness (CTGF, ITGA5, ITGB3) that were direct and/or indirect targets for repression by miR-30s. Among these genes, integrin ITGA5 was a previously unknown miR-30 target. By silencing ITGA5 in ER-/PR-negative breast cancer cells, colonization of the bone marrow by tumor cells was drastically reduced in vivo. Overall, our data indicate that miR-30s employ multiple mechanisms to impede breast cancer bone metastasis. These findings may pave the way to a new field of therapeutic interventions in breast cancer patients with bone metastasis. Citation Format: Martine Croset, Francesco Pantano, Casina Kan, Edith Bonnelye, Francoise Descotes, Catherine Alix-Panabières, Charles Lecellier, Richard Bachelier, Nathalie Allioli, Saw See Hong, Kai Bartkowiak, Klaus Pantel, Philippe Clézardin. MicroRNA-30 family members inhibit breast cancer invasion, osteomimicry, and bone destruction by directly targeting multiple bone metastasis-associated genes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 486.

Effect of Alendronate Treatment on Salivary Levels of Osteoprotegrin and TNF-α in Postmenopausal Woman with Osteoporosis and Periodontal Diseases

Background: All diseases concerning bone destruction such as osteoporosis and periodontal diseases share common pattern in which the osteoclast cells are absolutely responsible for bone resorption that occurred when osteoclast activity exceeds osteoblast activity. Osteoprotegrin (OPG) considered as novel soluble decoy receptor known as “bone protector” since it prevents extreme bone resorption through inhibition of differentiation and activity of osteoclast by competing for binding site. It binds to receptor activator of nuclear factor kappa-B ligand (RANKL) and prevent its interaction with receptor activator of nuclear factor kappa-B (RANK), thus inhibits osteoclast formation. TNF-α is a pro-inflammatory cytokines having a broad range of important roles in regulation of immune system and bone resorption through the stimulation of osteoclastogenesis. Alendronate (ALN) diminishes the expression of osteoclast activating factors and cytokines such as RANKL and enhances the production of decoy receptor osteoprotegerin in osteoblast cells. Moreover, it decreases the production of proinflammatory cytokines such as TNF-α by macrophage, stimulates apoptosis of monocyte-macrophage cell lines derivative and reduces inflammatory response. Aims of the Study: 1. To assess the effect of alendronate treatment on salivary levels of osteoprotegrin and TNF-α in postmenopausal women with osteoporosis and periodontal disease 2. To find any possible correlation between salivary levels of osteoprotegrin and TNF-α in control and study groups. Materials and Methods: Total sample of 90 female subjects (55-65 years) were divided into 3 groups, (30 subjects in each group): first control group involved systemically healthy subjects with healthy periodontium, second group involved postmenopausal women with osteoporosis under alendronate treatment for(3-6)months (alendronate group), third group involved postmenopausal women with osteoporosis without alendronate treatment(osteoporosis group). The last two groups were sub- divided in- to two sub –groups (15 subjects in each sub-group) of gingivitis and periodontitis subjects respectively. Salivary samples were collected from all subjects and salivary levels of osteoprotegrin and TNF- α were determined by enzyme –linked immune sorbent assay (ELISA). Results: Highest median value of salivary (OPG) was found in alendronate group followed by control group while the lowest value was found in osteoporosis group. Highest median value of TNF- α was found in osteoporosis group followed by control group and alendronate group respectively with highly significant differences between them. Spearman correlation between salivary levels of TNF-α and OPG showed non- significant correlation at all subgroups. Conclusion: Subjects with osteoporosis in this study had greater levels of TNF-α and decrease in the level of OPG comparing with patients under alendronate treatment. Alendronate treatment for women with osteoporosis and periodontal disease may have beneficial outcome.

Effect of Alendronate Treatment on Salivary Levels of Osteoprotegrin and TNF-α in Postmenopausal Woman with Osteoporosis and Periodontal Diseases

Background: All diseases concerning bone destruction such as osteoporosis and periodontal diseases share common pattern in which the osteoclast cells are absolutely responsible for bone resorption that occurred when osteoclast activity exceeds osteoblast activity. Osteoprotegrin (OPG) considered as novel soluble decoy receptor known as “bone protector” since it prevents extreme bone resorption through inhibition of differentiation and activity of osteoclast by competing for binding site. It binds to receptor activator of nuclear factor kappa-B ligand (RANKL) and prevent its interaction with receptor activator of nuclear factor kappa-B (RANK), thus inhibits osteoclast formation. TNF-α is a pro-inflammatory cytokines having a broad range of important roles in regulation of immune system and bone resorption through the stimulation of osteoclastogenesis. Alendronate (ALN) diminishes the expression of osteoclast activating factors and cytokines such as RANKL and enhances the production of decoy receptor osteoprotegerin in osteoblast cells. Moreover, it decreases the production of proinflammatory cytokines such as TNF-α by macrophage, stimulates apoptosis of monocyte-macrophage cell lines derivative and reduces inflammatory response. Aims of the Study: 1. To assess the effect of alendronate treatment on salivary levels of osteoprotegrin and TNF-α in postmenopausal women with osteoporosis and periodontal disease 2. To find any possible correlation between salivary levels of osteoprotegrin and TNF-α in control and study groups. Materials and Methods: Total sample of 90 female subjects (55-65 years) were divided into 3 groups, (30 subjects in each group): first control group involved systemically healthy subjects with healthy periodontium, second group involved postmenopausal women with osteoporosis under alendronate treatment for(3-6)months (alendronate group), third group involved postmenopausal women with osteoporosis without alendronate treatment(osteoporosis group). The last two groups were sub- divided in- to two sub –groups (15 subjects in each sub-group) of gingivitis and periodontitis subjects respectively. Salivary samples were collected from all subjects and salivary levels of osteoprotegrin and TNF- α were determined by enzyme –linked immune sorbent assay (ELISA). Results: Highest median value of salivary (OPG) was found in alendronate group followed by control group while the lowest value was found in osteoporosis group. Highest median value of TNF- α was found in osteoporosis group followed by control group and alendronate group respectively with highly significant differences between them. Spearman correlation between salivary levels of TNF-α and OPG showed non- significant correlation at all subgroups. Conclusion: Subjects with osteoporosis in this study had greater levels of TNF-α and decrease in the level of OPG comparing with patients under alendronate treatment. Alendronate treatment for women with osteoporosis and periodontal disease may have beneficial outcome.

Evidences for a New Role of miR-214 in Chondrogenesis

miR-214 is known to play a role in mammalian skeletal development through inhibition of osteogenesis and stimulation of osteoclastogenesis, but data regarding other vertebrates, as well as a possible role in chondrogenesis, remain unknown. Here, we show that miR-214 expression is detected in bone and cartilage of zebrafish skeleton, and is downregulated during murine ATDC5 chondrocyte differentiation. Additionally, we observed a conservation of the transcriptional regulation of miR-214 primary transcript Dnm3os in vertebrates, being regulated by Ets1 in ATDC5 chondrogenic cells. Moreover, overexpression of miR-214 in vitro and in vivo mitigated chondrocyte differentiation probably by targeting activating transcription factor 4 (Atf4). Indeed, miR-214 overexpression in vivo hampered cranial cartilage formation of zebrafish and coincided with downregulation of atf4 and of the key chondrogenic players sox9 and col2a1. We show that miR-214 overexpression exerts a negative role in chondrogenesis by impacting on chondrocyte differentiation possibly through conserved mechanisms.

Differential response of bone and kidney to ACEI in db/db mice: A potential effect of captopril on accelerating bone loss

The components of renin-angiotensin system (RAS) are expressed in the kidney and bone. Kidney disease and bone injury are common complications associated with diabetes. This study aimed to investigate the effects of an angiotensin-converting enzyme inhibitor, captopril, on the kidney and bone of db/db mice. The db/db mice were orally administered by gavage with captopril for 8weeks with db/+ mice as the non-diabetic control. Serum and urine biochemistries were determined by standard colorimetric methods or ELISA. Histological measurements were performed on the kidney by periodic acid-schiff staining and on the tibial proximal metaphysis by safranin O and masson-trichrome staining. Trabecular bone mass and bone quality were analyzed by microcomputed tomography. Quantitative polymerase chain reaction and immunoblotting were applied for molecular analysis on mRNA and protein expression. Captopril significantly improved albuminuria and glomerulosclerosis in db/db mice, and these effects might be attributed to the down-regulation of angiotensin II expression and the expression of its down-stream profibrotic factors in the kidney, like connective tissue growth factor and vascular endothelial growth factor. Urinary excretion of calcium and phosphorus markedly increased in db/db mice in response to captopril. Treatment with captopril induced a decrease in bone mineral density and deterioration of trabecular bone at proximal metaphysis of tibia in db/db mice, as shown in the histological and reconstructed 3-dimensional images. Even though captopril effectively reversed the diabetes-induced changes in calcium-binding protein 28-k and vitamin D receptor expression in the kidney as well as the expression of RAS components and bradykinin receptor-2 in bone tissue, treatment with captopril increased the osteoclast-covered bone surface, reduced the osteoblast-covered bone surface, down-regulated the expression of type 1 collagen and transcription factor runt-related transcription factor 2 (markers for osteoblastic functions), and up-regulated the expression of carbonic anhydrase II (marker for bone resorption). Captopril exerted therapeutic effects on renal injuries associated with type 2 diabetes but worsened the deteriorations of trabecular bone in db/db mice; the latter of which was at least in part due to the stimulation of osteoclastogenesis and the suppression of osteogenesis by captopril.

Activation of mTORC1 in B Lymphocytes Promotes Osteoclast Formation via Regulation of β-Catenin and RANKL/OPG.

The cytokine receptor activator of nuclear factor-κB ligand (RANKL) induces osteoclast formation from monocyte/macrophage lineage cells. However, the mechanisms by which RANKL expression is controlled in cells that support osteoclast differentiation are still unclear. We show that deletion of TSC1 (tuberous sclerosis complex 1) in murine B cells causes constitutive activation of mechanistic target of rapamycin complex 1 (mTORC1) and stimulates RANKL but represses osteoprotegerin (OPG) expression and subsequently promotes osteoclast formation and causes osteoporosis in mice. Furthermore, the regulation of RANKL/OPG and stimulation of osteoclastogenesis by mTORC1 was confirmed in a variety of RANKL-expressing cells and in vivo. Mechanistically, mTORC1 controls RANKL/OPG expression through negative feedback inactivation of Akt, destabilization of β-catenin mRNA, and downregulation of β-catenin. Our findings demonstrate that mTORC1 activation-stimulated RANKL expression in B cells is sufficient to induce bone loss and osteoporosis. The study also established a link between mTORC1 and the RANKL/OPG axis via negative regulation of β-catenin. © 2016 American Society for Bone and Mineral Research.

Gentian violet inhibits MDA-MB-231 human breast cancer cell proliferation, and reverses the stimulation of osteoclastogenesis and suppression of osteoblast activity induced by cancer cells.

Gentian violet (GV) is a cationic triphenylmethane dye, with potent antifungal and antibacterial activity. We recently reported that in vitro GV suppresses the differentiation of bone resorbing osteoclasts while stimulating the differentiation and activity of bone forming osteoblasts. Breast cancer is highly metastatic to bone and drives bone turnover that further promotes cancer engraftment and expansion, the so-called vicious cycle. In humans, breast cancer metastases cause osteolytic lesions and skeletal damage that leads to bone fractures, an additional source of patient morbidity. The MDA-MB-231 human breast cancer cell line is a commonly used model of human breast cancer that when injected into mice metastasizes to bone causing osteolytic lesions by promoting osteoclastic bone resorption and/or suppressing osteoblastic bone formation. In the present study, we investigated the direct action of GV on MDA-MB-231 proliferation, and the capacity of GV to reverse the negative impact of MDA-MB-231 cells on osteoclast and osteoblast differentiation. Our data reveal for the first time that GV suppresses proliferation, and induces apoptosis, of MDA-MB-231 cells. We further demonstrated the capacity of GV to reverse the pro-osteoclastogenic and anti-osteoblastic activities of MDA-MB-231 cells in vitro. These data suggest that GV has important applications in the treatment of breast cancer through multiple actions including direct suppression of cancer cell proliferation, breaking the vicious cycle between cancer and bone, and alleviating the skeletal defects induced by 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.

DDR2 (discoidin domain receptor 2) suppresses osteoclastogenesis and is a potential therapeutic target in osteoporosis.

Osteoporosis is a disease characterized by abnormal skeletal fragility due to an imbalance in bone homeostasis. Finding therapeutic targets that promote bone formation is valuable for treating osteoporosis. Discoidin domain receptor 2 (DDR2) is a receptor tyrosine kinase that promotes the differentiation of bone-depositing cells, osteoblasts, and bone formation. We investigated the role of DDR2 in osteoclastogenesis, the formation of cells responsible for bone resorption. We found that, although differentiated osteoclasts had catalytically active DDR2, its abundance was decreased. Overexpression of DDR2 inhibited the expression of osteoclastic markers, osteoclast maturation, and osteoclast-mediated bone resorption in a culture model of bone matrix. Conversely, knocking down Ddr2 through RNA interference accelerated osteoclast differentiation and bone resorption in mice. Further, we identified the co-receptor Neuropilin-1 (Nrp1) as a DDR2-interacting protein. DDR2 facilitated the binding of Nrp1 and the co-receptor PlexinA1 by forming a DDR2-Nrp1-PlexinA1 complex, which blocked PlexinA1-mediated stimulation of osteoclastogenesis. DDR2 prevented PlexinA1 from interacting with the receptor TREM2 and the adaptor DAP12. Moreover, knocking down Nrp1 rescued the expression of osteoclastic markers observed in DDR2-overexpressing cells, whereas ectopic expression of Nrp1 in DDR2-silenced cells inhibited the induction of osteoclastogenesis. Nrp1 enhanced the function of DDR2 in promoting osteoblast differentiation and bone formation in mice. In an ovariectomized mouse model of osteoporosis, adenovirus-mediated delivery of DDR2 to the femur bone alleviated osteopenic phenotypes. Our results revealed that DDR2 functions as an inhibitor of osteoclastogenesis, as well as a promoter of osteoblastogenesis, suggesting that enhancing DDR2 may be therapeutic for patients with osteoporosis.

Crosstalk among IL-23 and DNAX activating protein of 12 kDa-dependent pathways promotes osteoclastogenesis.

IL-23 has been well studied in the context of T cell differentiation; however, its role in the differentiation of myeloid progenitors is less clear. In this paper, we describe a novel role of IL-23 in myeloid cell differentiation. Specifically, we have identified that in human PBMCs, IL-23 induces the expression of MDL-1, a PU.1 transcriptional target during myeloid differentiation, which orchestrates osteoclast differentiation through activation of DNAX activating protein of 12 kDa and its ITAMs. The molecular events that lead to the differentiation of human macrophages to terminally differentiated osteoclasts are dependent on spleen tyrosine kinase and phospholipase Cγ2 phosphorylation for the induction of intracellular calcium flux and the subsequent activation of master regulator osteoclast transcription factor NFATc1. IL-23-elicited osteoclastogenesis is independent of the receptor activator of NF-κB ligand pathway and uses a unique myeloid DNAX activating protein of 12 kDa-associated lectin-1(+)/DNAX activating protein of 12 kDa(+) cell subset. Our data define a novel pathway that is used by IL-23 in myeloid cells and identify a major mechanism for the stimulation of osteoclastogenesis in inflammatory arthritis.

In vitro and in silico analysis of an inhibitory mechanism of osteoclastogenesis by salubrinal and guanabenz

Inactivating bone-resorbing osteoclasts is a prime therapeutic strategy for the prevention of bone loss in patients with osteopenia and osteoporosis. Synthetic agents such as salubrinal and guanabenz, which attenuate stress to the endoplasmic reticulum, are reported to inhibit development of osteoclasts. However, the mechanism of their inhibitory action on osteoclasts is largely unknown. Using genome-wide expression profiles, we predicted key transcription factors that downregulated nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1), a master transcription factor for osteoclastogenesis. Principal component analysis (PCA) predicted a list of transcription factors that were potentially responsible for reversing receptor activator of nuclear factor kappa-B ligand (RANKL)-driven stimulation of osteoclastogenesis. A partial silencing of NFATc1 allowed a selection of transcription factors that were likely to be located upstream of NFATc1. We validated the predicted transcription factors by focusing on two AP-1 transcription factors (c-Fos and JunB) using RAW264.7 pre-osteoclasts as well as primary bone marrow cells. As predicted, their mRNA and protein levels were elevated by RANKL, and the elevation was suppressed by salubrinal and guanabenz. A partial silencing of c-Fos or JunB by RNA interference decreased NFATc1 as well as tartrate-resistant acid phosphatase (TRAP) mRNA. Collectively, a systems-biology approach allows the prediction of a RANKL-salubrinal/guanabenz-NFATc1 regulatory axis, and in vitro assays validate an involvement of AP-1 transcription factors in suppression of osteoclastogenesis.

Effects of angiotensin-converting enzyme inhibitor, captopril, on bone of mice with streptozotocin-induced type 1 diabetes

There are contradictory results about the effect of angiotensin-converting enzyme inhibitors (ACEIs) on bone. This study was performed to address the skeletal renin-angiotensin system (RAS) activity and the effects of the ACEI, captopril, on the bone of streptozotocin-induced type 1 diabetic mice. Histochemical assessment on bone paraffin sections was conducted by Safranin O staining and tartrate-resistant acid phosphatase staining. Micro-computed tomography was performed to analyze bone biological parameters. Gene and protein expression were determined by real-time polymerase chain reaction and immunoblotting, respectively. Type 1 diabetic mice displayed osteopenia phenotype and captopril treatment showed no osteoprotective effects in diabetic mice as shown by the reduction of bone mineral density, trabecular thickness and bone volume/total volume. The mRNA expression of ACE and renin receptor, and the protein expression of renin and angiotensin II were markedly up-regulated in the bone of vehicle-treated diabetic mice compared to those of non-diabetic mice, and these molecular changes of skeletal RAS components were effectively inhibited by treatment with captopril. However, treatment with captopril significantly elevated serum tartrate-resistant acid phosphatase 5b levels, reduced the ratio of osteoprotegerin/receptor activator of nuclear factor-κB ligand expression, increased carbonic anhydrase II mRNA expression and the number of matured osteoclasts and decreased transforming growth factor-β and osteocalcin mRNA expression in the tibia compared to those of diabetic mice. The present study demonstrated that the use of the ACEI, captopril, has no beneficial effect on the skeletal biological properties of diabetic mice. However, this could be attributed, at least partially, to its suppression of osteogenesis and stimulation of osteoclastogenesis, even though it could effectively inhibit high activity of local RAS in the bone of diabetic mice.

Osteoblasts stimulate osteoclastogenesis via RANKL expression more strongly than periodontal ligament cells do in response to PGE2

ObjectivePeriodontal ligament cells (PDLs) produce prostaglandin E2 (PGE2) in response to orthodontic force. PGE2 is a potent osteoclast-inducing factor that induces the receptor activator of nuclear factor-κB ligand (RANKL). Some studies reported that PDLs express RANKL in response to mechanical stress, whereas another study reported that they do not. Based on an immunohistochemical study, RANKL expression is localized around the alveolar bone surface 3 days after tooth movement. However, ankylosed teeth cannot be moved by therapeutic mechanical stress, suggesting that PDLs play a major role in alveolar bone resorption. In this study, we compared the functional difference in osteoclastogenesis between human PDLs (HPDLs) and normal human osteoblasts (HOBs) as a direct effect of PGE2 exposure. DesignWe examined the expression of RANKL, osteoprotegerin, and macrophage colony-stimulating factor after 48-h culture with or without PGE2 (10−11 to 10−5M) in HPDLs and HOBs. Then to confirm whether RANKL produced by PGE2 treatment induces osteoclastogenesis or not, RAW264.7 cells were co-cultured on HPDLs or HOBs pretreated with 10−6M of PGE2. ResultPGE2 exposure increased significantly RANKL expression in HOBs compared with HPDLs. PGE2 exposure significantly decreased osteoprotegerin expression in HPDLs compared with HOBs. The number of tartrate-resistant acid phosphatase staining osteoclast-like cells from RAW264.7 cells increased significantly by PGE2 pretreatment in HOBs and was reduced by small interfering RNA knockdown of RANKL. ConclusionThese results suggest that osteoblasts strongly influence the stimulation of osteoclastogenesis via RANKL, induced by PGE2 in periodontal tissues, compared with PDLs.

Increased Sclerostin Secretion in Multiple Myeloma Results in Stimulation of Osteoclastogenesis and Inhibition of Osteoblastogenesis

Abstract 1819 Objectives:Osteoblasts (OB) and osteoclasts (OC), are an integral part of the bone microenvironment, and play a crucial role in myeloma growth and survival. Their imbalance results in osteolytic disease and elucidating the mechanisms underlying osteolytic lesions is important not only for the improvement of osteolytic bone disease but also for the treatment of multiple myeloma (MM).The osteocyte-secreted protein sclerostin, encoded by the SOST gene, is a potent inhibitor of osteoblastogenesis. It is regarded as a good target for osteoporosis treatment, but its role in MM remains to be determined. Our objective was to study the role of sclerostin in MM bone disease and determine if sclerostin directed strategies were a reasonable approach in MM. Methods and Results:Sclerostin concentration in patients’ blood plasma and MM cell line supernatant stimulated by IL-6, FGF-2, TNFalpha, BMP7 and TGFbeta was detected by ELISA (ALPCO immunoassays). Increased level of sclerostin was detected in MM patient plasma (n=20, median: 4.73 ng/mL, range: 1.5–19.5 ng/mL). Plasma concentrations were significantly higher (p<0.01) when compared to sclerostin concentration in the plasma of leukemia patients (n=3), gastric cancer patients (n=40) and healthy volunteers (n=4). High sclerostin levels were not associated with extent of bone disease but rather correlated with tumor burden (High B2M, creatinine and LDH, and low Hb) suggesting an autocrine loop for sclerostin production. Because sclerostin is derived from mature OB or orteocytes, we measured levels during OB differentiation but we were unable to detect increased levels. We then measured sclerostin levels in RPMI-8226 MM cell line supernatant either alone or stimulated by cytokines D Systems). Conclusions:These data demonstrate that increased sclerostin levels in MM patients inhibit osteoblastogenesis and stimulate osteoclastogenesis. Taken together, sclerostin may be good target to inhibit myeloma bone disease and help restore normal bone homeostasis. Disclosures:Raje:Celgene: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees; Astra Zeneca: Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees; Acetylon: Research Funding.