Duloxetine suppresses BMP-4-induced release of osteoprotegerin via inhibition of the SMAD signaling pathway in osteoblasts.

Duloxetine, a selective serotonin-norepinephrine reuptake inhibitor, is currently recommended for the treatment of chronic painful disorders such as fibromyalgia, chronic musculoskeletal pain, and diabetic peripheral neuropathy. We previously demonstrated that bone morphogenetic protein-4 (BMP-4) stimulates osteoprotegerin (OPG) production in osteoblast-like MC3T3-E1 cells, and that p70 S6 kinase positively regulates OPG synthesis. The present study aimed to investigate the effect of duloxetine on BMP-4-stimulated OPG synthesis in these cells. Duloxetine dose-dependently suppressed OPG release stimulated by BMP-4. Fluvoxamine, a selective serotonin reuptake inhibitor (SSRI), reduced BMP-4-stimulated OPG release, whereas a selective and specific norepinephrine reuptake inhibitor, reboxetine, failed to affect OPG release. In addition, another SSRI sertraline also inhibited BMP-4-stimulated OPG release. On the other hand, siRNA of SMAD1 reduced the OPG release stimulated by BMP-4, indicating the involvement of the SMAD1/5/8 pathway in OPG release. Rapamycin inhibited BMP-4-stimulated p70 S6 kinase phosphorylation, and compound C suppressed the SMAD1/5/8 phosphorylation stimulated by BMP-4. Duloxetine did not affect BMP-4-induced phosphorylation of p70 S6 kinase but suppressed SMAD1/5/8 phosphorylation. Both fluvoxamine and sertraline also inhibited BMP-4-elicited phosphorylation of SMAD1/5/8. These results strongly suggest that duloxetine suppresses BMP-4-stimulated OPG release via inhibition of the Smad1/5/8 signaling pathway in osteoblasts.

TNF Receptor: Fc Fusion Protein Downregulates RANKL/OPG Ratio by Inhibiting CXCL16/CXCR6 in Active Ankylosing Spondylitis.

BACKGROUND: Clinical studies indicate that recombinant tumor necrosis factor receptor:Fc fusion protein (rhTNFR:Fc) quickly alleviates symptoms and physical signs of active Ankylosing Spondylitis (AS), improving the manifestation of spinal inflammation on radiological imaging. However, the regulatory mechanism of rhTNFR:Fc in the chemokine pathway is unclear. Thus we study the mechanism of phlogogenic activity of CXCL16/CXCR6 in AS and the related mechanism of rhTNFR: Fc treatment. METHODS: Thirty-two cases of active AS were treated with rhTNFR:Fc for 3 consecutive months. Clinical response was evaluated at baseline and after treatment. CXCL16/CXCR6 expression as well as Receptor Activator Of Nuclear Factor-Κb Ligand (RANKL)/Osteoprotegerin (OPG), essential molecules for osteoclast differentiation, were studied in AS before and after treatment. Further, the proliferation of lymphocytes and the RANKL level stimulated by recombinant human CXCL16 (rhCXCL16) were measured in vitro. RESULTS: Thirty cases responded to rhTNFR:Fc treatment. The RANKL level, RANKL/OPG ratio, CXCLl6 level in serum, and CXCLl6 and CXCR6 mRNA levels in active AS were higher than those in controls and treated patients (P<0.001). rhCXCL16 treatment increased lymphocyte proliferation and RANKL level in active AS (P<0.001), but not in controls or treated patients (P>0.05). A positive linear correlation was noted between CXCL16 serum levels and RANKL/OPG ratio and between CXCL16 levels and C-reactive protein results (P<0.001). CONCLUSIONS: Our findings suggest that rhTNFR:Fc suppresses inflammation and bone destruction of AS by reducing the RANKL/OPG ratio through inhibition of the CXCL16/CXCR6 pathway.

Rosmarinic acid-induced apoptosis and cell cycle arrest in triple-negative breast cancer cells.

Rosmarinic acid (RA) is a polyphenolic compound with various pharmacological properties, including, anti-inflammatory, immunomodulatory, and neuroprotective, as well as having antioxidant and anticancer activities. This study evaluated the effects and mechanisms of RA in two racially different triple-negative breast cancer (TNBC) cell lines. Results obtained show that RA significantly caused cytotoxic and antiproliferative effects in both cell lines in a dose- and time-dependent manner. Remarkably, RA induced cell cycle arrest-related apoptosis and altered the expression of many apoptosis-involved genes differently. In MDA-MB-231 cells, RA arrested the cells in the G0/G1 phase. In contrast, the data suggest that RA causes S-phase arrest in MDA-MB-468 cells, leading to a 2-fold increase in the apoptotic effect compared to MDA-MB-231 cells. Further, in MDA-MB-231 cells, RA significantly upregulated the mRNA expression of three genes: harakiri (HRK), tumor necrosis factor receptor superfamily 25 (TNFRSF25), and BCL-2 interacting protein 3 (BNIP3). In contrast, in the MDA-MB-468 cell line, the compound induced a significant transcription activation in three genes, including TNF, growth arrest and DNA damage-inducible 45 alpha (GADD45A), and BNIP3. Furthermore, RA repressed the expression of TNF receptor superfamily 11B (TNFRSF11B) in MDA-MB-231 cells in comparison to the ligand TNF superfamily member 10 (TNFSF10) and baculoviral IAP repeat-containing 5 (BIRC5) in MDA-MB-468 cells. In conclusion, the data suggest that the polyphenol RA may have a potential role in TNBC therapies, particularly in MDA-MB-468 cells.

Long-Term Exposure to Low-Dose Lead Induced Deterioration in Bone Microstructure of Male Mice.

The aim of this study was to investigate the long-term effects of low-dose lead exposure on bone microstructure in mice. Ten SPF 12-week-old male C57BL/6J mice were randomly divided into two groups: control (deionized water) and lead exposure (150 ppm of lead acetate in drinking water). After 24 weeks treatment, mice were weighed and the left femurs were collected and stored at - 80 °C. The right femurs of the mice were scanned by Micro-CT for three-dimensional reconstruction, and bone mineral density, bone volume fraction, trabeculae thickness, trabeculae number, and trabeculae separation were measured. The right tibia was collected to investigate histopathological changes in H&E-stained sections. The gene expression of osteoprotegerin (OPG), RANKL, and runt-related transcription factor 2 (Runx2) was determined using real-time PCR. The bone density of femoral cancellous bone and the number of cancellous bone trabeculae in the lead exposure group were both significantly decreased compared with the control group. Bone marrow stromal cell numbers were decreased following lead administration, and lipid droplet vacuoles were observed in the lead group. Levels of OPG were significantly decreased in the lead group, and lead also inhibited the expression of Runx2 compared with the control group. Long-term exposure to low doses of lead can cause bone damage without inducing other obvious symptoms through decreasing bone density and the number of cancellous bone trabeculae, further suppressing bone formation. It suggests that lead may exacerbate bone loss and osteoporosis, especially in the elderly.

Cepharanthine ameliorates titanium particle-induced osteolysis by inhibiting osteoclastogenesis and modulating OPG/RANKL ratio in a murine model.

Periprosthetic asepteic loosening, caused by wear debris, is one of the most severe complications, generally resulting in implant failure. Extensive osteoclast formation and activation are considered as the cause for periprosthetic osteolysis. However, few approaches have been approved to be used for preventing early-stage periprosthetic osteolysis. In this study, we investigated the preventive effects of CEP on titanium particles-induced osteolysis in a murine calvaria model. This inhibitory effect was confirmed to be realized by attenuating osteoclastogenesis in vivo. In addition, CEP markedly reduced wear particles-induced elevation of receptor activator of nuclear factor kappa B ligand (RANKL)/Osteoprotegerin (OPG) ratio in vivo. In conclusion, these data concluded that CEP demonstrated a preventive effect of CEP on titanium particles induced osteolysis, suggesting that CEP might be a novel therapeutic for periprosthesis loosening.

Endocrine modulation of Brucella abortus-infected osteocytes function and osteoclastogenesis via modulation of RANKL/OPG.

Osteoarticular brucellosis is the most frequent complication of active disease. A large amount of cells in bone are osteocytes. Since bone remodeling process is regulated by hormones we sought to study the effect of cortisol and DHEA in Brucella abortus-infected osteocytes. Cortisol treatment inhibited the expression of IL-6, TNF-α, MMP-2 and RANKL in B. abortus-infected osteocytes. DHEA could reverse the inhibitory effect of cortisol on MMP-2 production. B. abortus infection inhibited connexin 43 (Cx43) expression in osteocytes. This expression was increased when cortisol was incorporated during the infection and DHEA treatment partially reversed the effect of cortisol. Osteocytes-infected with B. abortus induced osteoclast's differentiation. Yet, the presence of cortisol, but not DHEA, during osteocyte infection inhibited osteoclastogenesis. Glucocorticoid receptor (GR) is implicated in the signaling of cortisol. Infection with B. abortus was able to increase GRα/ß ratio. Levels of intracellular cortisol are not only dependent on GR expression but also a result of the activity of the isoenzymes 11ß-hydroxysteroid dehydrogenase (11ß-HSD)-1 (cortisone to cortisol conversion), 11ß-HSD2 (cortisol to cortisone conversion). B. abortus infection increased 11ß-HSD 1/2 ratio and cortisone mimicked the effect of cortisol. Our results indicated that cortisol and DHEA could modulate osteocyte responses during B. abortus infection.

Jagged1 inhibits osteoprotegerin expression by human periodontal ligament cells.

BACKGROUND AND OBJECTIVE: Notch signaling regulates bone homeostasis. The present study investigated the effect of Jagged1 on osteoprotegerin (OPG) and receptor activator of nuclear factor kappa-B ligand (RANKL) expression in human periodontal ligament stromal (hPDL) cells. MATERIAL AND METHODS: hPDL cells were seeded on to indirect immobilized Jagged1 surfaces. OPG expression was determined using real-time polymerase chain reaction and enzyme-linked immunosorbent assay. Lentiviral small hairpin RNA particles against NOTCH2 were employed to inhibit NOTCH2 expression. Osteoclast formation was evaluated using RAW264.7 cells. An influence of exogenous OPG on osteogenic differentiation was determined by real-time polymerase chain reaction and Alizarin Red S staining. RESULTS: Jagged1 significantly enhanced HES1 and HEY1mRNA expression in a dose-dependent manner. Furthermore, OPG mRNA and protein levels dramatically decreased upon exposing hPDL cells to Jagged1. However, RANKL mRNA levels were not significantly different. There was also no difference in M-CSF and MCP-1mRNA expression. A γ-secretase inhibitor and cycloheximide treatment rescued Jagged1-attenuated OPG expression. Furthermore, shNOTCH2 overexpressing hPDL cells did not exhibit a decrease in OPG expression upon exposure to Jagged1, implying the involvement of NOTCH2 in the regulatory mechanism. Culturing RAW264.7 cells with conditioned medium from Jagged1-treated hPDL cells enhanced osteoclast formation compared with those cultured with conditioned medium of the control group. Lastly, OPG treatment did not influence osteogenic differentiation by hPDL cells. CONCLUSION: These results suggest that Jagged1 activates Notch signaling in hPDL cells, leading to decreased OPG expression. This may imply an indirect role of Jagged1 on the regulation of osteoclast differentiation via hPDL cells.

Inhibitory effects of autologous γ-irradiated cell conditioned medium on osteoblasts in vitro.

Skeletal complications from radiation therapy have been reported in patients with breast, brain and pelvic cancer, and types of blood cancer. However, it remains to be elucidated whether localized radiotherapy may result in systemic adverse effects on the unirradiated skeleton through an abscopal mechanism. The present study investigated the abscopal effect of radiation on osteoblasts mediated by autologous γ-irradiated cell conditioned medium. Osteoblasts obtained from calvarial bones were incubated with irradiated cell conditioned medium (ICCM) and changes in cell viability, alkaline phosphatase (ALP) activity, mineralization ability, cell apoptosis and the gene expression levels of ALP, osteocalcin (BGP), osteoprotegerin (OPG), receptor activator of nuclear factor-κB ligand (RANKL) and caspase 3 were observed. Notably, ICCM regulated osteoblast function, inhibiting viability and differentiation, resulting in apoptosis or cell death. ICCM at 10 or 20%, from osteoblasts irradiated with 10 Gy γ-rays, significantly inhibited the proliferation of osteoblastic cells (P<0.001). In addition, an increase in apoptosis was noted in the osteoblasts incubated with ICCM at 40% with increasing doses of radiation, accompanied by an upregulation in the mRNA expression of caspase 3. In addition, ICCM at 20% inhibited the ALP activity in the 5 and 10 Gy groups and osteoblast mineralization, particularly at 10 Gy ICCM. Additionally, the mRNA expression levels of ALP, BGP, OPG and RANKL of the cells treated with ICCM at 20% were downregulated significantly compared with those treated with medium from unirradiated cells. The present study provided novel evidence to elucidate radiation-therapy-associated side effects on the skeleton.

The comparative effect of pioglitazone and metformin on serum osteoprotegerin, adiponectin and intercellular adhesion molecule concentrations in patients with newly diagnosed type 2 diabetes: a randomized clinical trial.

AIM: The etiologic role of inflammatory pathways in the development of diabetic complications, especially cardiovascular events, has been established. The anti-inflammatory role of metformin and pioglitazone has been described; however, no study to date has compared the efficacy of these common oral agents in this regard. In this study, the authors aimed to compare the anti-inflammatory properties of pioglitazone and metformin, with respect to their effect on serum concentrations of highly sensitive C-reactive protein (hsCRP), osteoprotegerin (OPG), intercellular adhesion molecule-1 (ICAM-1) and adiponectin. METHODS: In an open-label randomized clinical trial, 117 patients with newly diagnosed type 2 diabetes mellitus were visited; 84 fulfilled the inclusion criteria, and were randomly allocated to 2 arms receiving either 1,000 mg/d metformin or 30 mg/d pioglitazone, respectively. Biochemical assessments were made at baseline and the end of the 3 months trial. RESULTS: Significant reduction in FPG, insulin and HbA1c in women and men of both arms were observed. Log-hsCRP values significantly decreased in both arms. A decreasing, but non-significant trend in log-OPG levels was observed in women of the metformin arm (p=0.063). A greater reduction in log-ICAM levels was identifiable in men receiving pioglitazone compared to the other arm (p=0.008); in addition, the same trend was observed in log-OPG values (p=0.029). Nonetheless, reduction in log-ICAM and log-OPG levels was comparable between the 2 arms. A significant increase in adiponectin was observed in both men and women in the pioglitazone arm (p<0.001), whereas changes were non-significant in the metformin arm. CONCLUSION: Remarkably, patients receiving pioglitazone revealed more significant reduction in inflammatory markers.

Manipulation of receptor oligomerization as a strategy to inhibit signaling by TNF superfamily members.

Signaling by receptor activator of nuclear factor κB (RANK) in response to its ligand RANKL, which is a member of the tumor necrosis factor (TNF) superfamily of cytokines, stimulates osteoclast formation and bone resorption. Thus, this ligand-receptor pair is a therapeutic target for various disorders, such as osteoporosis and metastasis of cancer to bone. RANKL exists as a physiological homotrimer, with each monomer recognizing a single molecule of RANK or the decoy receptor osteoprotegerin (OPG), which inhibits osteoclastogenesis. We engineered a RANKL protein in which all three monomers of RANKL were encoded as a single polypeptide chain, which enabled us to independently control receptor binding at each binding interface. To generate an effective RANK inhibitor, we used an unbiased forward genetic approach to identify mutations in RANKL that had a 500-fold increased affinity for RANK but had decreased affinity for the decoy receptor OPG. Incorporating mutations that blocked receptor binding into this high-affinity RANKL variant generated a mutant RANKL that completely inhibited wild-type RANKL-induced osteoclastogenesis in vitro and bone resorption in mice. Our approach may be generalized to enable the inhibition of other TNF receptor signaling systems, which are implicated in a wide range of pathological conditions.

Betulinic acid, a bioactive pentacyclic triterpenoid, inhibits skeletal-related events induced by breast cancer bone metastases and treatment.

Many breast cancer patients experience bone metastases and suffer skeletal complications. The present study provides evidence on the protective and therapeutic potential of betulinic acid on cancer-associated bone diseases. Betulinic acid is a naturally occurring triterpenoid with the beneficial activity to limit the progression and severity of cancer, diabetes, cardiovascular diseases, atherosclerosis, and obesity. We first investigated its effect on breast cancer cells, osteoblastic cells, and osteoclasts in the vicious cycle of osteolytic bone metastasis. Betulinic acid reduced cell viability and the production of parathyroid hormone-related protein (PTHrP), a major osteolytic factor, in MDA-MB-231 human metastatic breast cancer cells stimulated with or without tumor growth factor-ß. Betulinic acid blocked an increase in the receptor activator of nuclear factor-kappa B ligand (RANKL)/osteoprotegerin ratio by downregulating RANKL protein expression in PTHrP-treated human osteoblastic cells. In addition, betulinic acid inhibited RANKL-induced osteoclastogenesis in murine bone marrow macrophages and decreased the production of resorbed area in plates with a bone biomimetic synthetic surface by suppressing the secretion of matrix metalloproteinase (MMP)-2, MMP-9, and cathepsin K in RANKL-induced osteoclasts. Furthermore, oral administration of betulinic acid inhibited bone loss in mice intra-tibially inoculated with breast cancer cells and in ovariectomized mice causing estrogen deprivation, as supported by the restored bone morphometric parameters and serum bone turnover markers. Taken together, these findings suggest that betulinic acid may have the potential to prevent bone loss in patients with bone metastases and cancer treatment-induced estrogen deficiency.

Protective effect of metformin on periapical lesions in rats by decreasing the ratio of receptor activator of nuclear factor kappa B ligand/osteoprotegerin.

INTRODUCTION: Metformin, one of the antihyperglycemic agents commonly used for the treatment of type 2 diabetes, was shown to inhibit osteoclast formation. The current study aimed to investigate the effects of systemically administered metformin on alveolar bone resorption and on the ratio of receptor activator of nuclear factor kappa B ligand/osteoprotegerin (RANKL/OPG) in rats subjected to experimental periapical lesions. METHODS: Forty adult male Wistar rats were divided equally into control and experimental groups, and the pulp chambers of their mandibular first molars were exposed to the oral environment to induce periapical lesions. The experimental group received daily intramuscular injections of metformin at 40 mg/kg doses, whereas the control group received only the saline vehicle. The injections were initiated 1 day before the periapical lesion induction and then were continued daily throughout the entire experimental period. Two or 4 weeks after pulp exposure, the rats were killed, and the mandibles were prepared for histologic analysis, enzyme histochemistry, immunohistochemistry, and immunofluorescence. RESULTS: The number of RANKL-positive and tartrate-resistant acid phosphatase (TRAP)-positive cells in the metformin-treated groups decreased on day 14, whereas the number of OPG-positive cells increased on day 28. The periapical bone loss area in the metformin-treated group significantly decreased on day 28 compared with the control group. CONCLUSIONS: Metformin inhibits the periapical lesions possibly by lowering the RANKL/OPG ratio, subsequently reducing the number of osteoclasts and bone resorption areas.

OPG/RANKL/RANK pathway as a therapeutic target in cancer.

Bone metastases play an important role in the morbidity and mortality of patients with malignant disease. Despite therapeutic advances in the treatment of solid organ malignancy such as lung cancer, less development on metastasis interventions has been forthcoming. More recent research has focused on molecular pathway manipulation in the prevention and treatment of metastatic bone disease and associated complications such as bone pain and hypercalcemia. The osteoprotegerin/receptor activator of nuclear factor-Ðºß ligand/receptor activator of nuclear factor-Ðºß pathway, which is physiologically involved in bone turnover, has been of considerable interest, and recent promising data have been revealed. In this study, we describe this molecular pathway in terms of its natural physiological function, manipulation for therapeutic benefit, and recent clinical trial results.

[The role of RANK/RANKL/osteoprotegerin (OPG) triad in cancer-induced bone diseases: physiopathology and clinical implications]. / Rôle de la triade RANK/RANKL/ostéoprotégérine (OPG) dans les cancers primitifs et secondaires de l'os: aspects physiopathologiques et implications cliniques.

Bone homeostasis is maintained by the remodelling of bone which depends on a balance between osteoclast-mediated bone resorption and osteoblast-mediated bone formation. Malignant bone lesions are very common in patients with cancer; whether they result from a tumor in bone (giant cell tumour of bone, osteosarcoma, multiple myeloma...) or they are bony metastases from advanced cancers of which the most osteotropic are breast and prostate cancer. Malignant cells within the bone disrupt the normal bone remodelling process, leading to increased bone destruction and occurence of pathological fractures. Receptor activator of NF-kB (RANK) and its ligand (RANKL) play a pivotal role in the regulation of bone remodelling; by binding to RANK, RANKL stimulates osteoclastogenesis and bone resorption, whereas its cognate decoy receptor osteoprotegerin (OPG) blocks this process by interacting with RANKL. Tumour cells produce different factors that manipulate the RANK/RANKL/OPG pathway in order to stimulate bone destruction. Furthermore, pending on the tumour type, RANKL plays a role in the migration, invasion and proliferation of malignant cells within the bone, while OPG increases survival of tumour cells. Inhibition of RANK/RANKL system may therefore offer new therapeutic perspectives for the treatment of primitive and secondary bone cancers.

Osteoblast connexin43 modulates skeletal architecture by regulating both arms of bone remodeling.

Connexin43 (Cx43) has an important role in skeletal homeostasis, and Cx43 gene (Gja1) mutations have been linked to oculodentodigital dysplasia (ODDD), a human disorder characterized by prominent skeletal abnormalities. To determine the function of Cx43 at early steps of osteogenesis and its role in the ODDD skeletal phenotype, we have used the Dermo1 promoter to drive Gja1 ablation or induce an ODDD mutation in the chondro-osteogenic linage. Both Gja1 null and ODDD mutant mice develop age-related osteopenia, primarily due to a progressive enlargement of the medullary cavity and cortical thinning. This phenotype is the consequence of a high bone turnover state, with increased endocortical osteoclast-mediated bone resorption and increased periosteal bone apposition. Increased bone resorption is a noncell autonomous defect, caused by exuberant stimulation of osteoclastogenesis by Cx43-deficient bone marrow stromal cells, via decreased Opg production. The latter is part of a broad defect in osteoblast differentiation and function, which also results in abnormal structural and material properties of bone leading to decreased resistance to mechanical load. Thus Cx43 in osteogenic cells is a critical regulator of both arms of the bone remodeling cycle, its absence causing structural changes remindful of aged or disused bone.

Differential expression of determinants of glucocorticoid sensitivity in androgen-dependent and androgen-independent human prostate cancer cell lines.

Glucocorticoids (GCs) are widely used for the treatment of hormone refractory prostate cancer. However, few data are available on the expression and regulation of glucocorticoid and mineralocorticoid receptors (GR and MR) and 11beta-hydroxysteroid dehydrogenase (11beta-HSD) 1 and -2 activities in prostate cancer cells. Here we show that GR is expressed in both the androgen-independent PC-3 cell line and, at very low levels, in the androgen-dependent LNCaP cells, and MR is expressed in both cell lines. IL-1beta increased GR expression in both cell lines. In LNCaP cells IL-1beta also increased MR expression. Significant 11beta-HSD oxidase activity and 11beta-HSD2 protein were found in LNCaP cells, but not in PC3 cells, and no ketoreductase activity was detected in either cell lines. GR function was assessed by measuring the inhibitory effect of dexamethasone on constitutive and IL-1beta-inducible IL-6 and osteoprotegerin (OPG) production. In PC-3 cells, IL-1beta stimulated IL-6 and OPG release, and dexamethasone dose-dependently inhibited IL-1beta-inducible IL-6 release, and constitutive and IL-1beta-inducible OPG release. In LNCaP cells, IL-1beta stimulated only OPG release. While dexamethasone was ineffective, cortisol dose-dependently inhibited IL-1beta-inducible OPG release. Eplerenone (Epl), a selective mineralocorticoid antagonist, reverted this effect. We conclude that different patterns of expression of receptors and 11beta-HSD activity were associated with different responsiveness to GCs in terms of regulated gene expression. GR and MR expression may vary as a function not only of the malignant phenotype, but also of local conditions such as the degree of inflammation. Inhibition of IL-6 and OPG release by GCs may contribute to the antitumor efficacy in prostate cancer.

Thrombin induces osteoprotegerin synthesis via phosphatidylinositol 3'-kinase/mammalian target of rapamycin pathway in human periodontal ligament cells.

BACKGROUND AND OBJECTIVE: Thrombin influences the biological behavior of periodontal ligament cells and plays multiple roles in the early stages of bone healing. Osteoprotegerin (OPG) is one of the key molecules that regulate bone homeostasis and prevent osteoclastogenesis. The purpose of this study was to evaluate the biological effects of thrombin on OPG synthesis in human periodontal ligament (HPDL) cells in vitro. MATERIAL AND METHODS: Cells were treated with various concentrations (0.001, 0.01 and 0.1 U/mL) of thrombin. The mRNA expression and protein synthesis of OPG, as well as of receptor activator of nuclear factor kappaB ligand (RANKL), were determined by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot analysis, respectively. The influence of thrombin on OPG synthesis and its signaling pathway were investigated using inhibitors. RESULTS: Thrombin profoundly induces protein synthesis of OPG at 0.1 U/mL. The inductive effect was inhibited by cycloheximide, but not by indomethacin. The phosphatidylinositol 3'-kinase (PI3K) inhibitor, LY294002, and the mammalian target of rapamycin (mTOR) inhibitor, rapamycin, exerted an inhibitory effect on the thrombin-induced OPG synthesis. In addition, the effect was inhibited by protease-activated receptor (PAR)-1 antagonist. Activation of phospho-Akt (p-Akt) was observed and the effect was abolished by LY294002. CONCLUSION: Thrombin induces OPG synthesis in HPDL cells post-transcriptionally, possibly through PAR-1. The regulation was through the PI3K/Akt and mTOR pathway. This finding suggests that thrombin may play a significant role in alveolar bone repair and homeostasis of periodontal tissue, partly through the OPG/RANKL system.

Heparin enhances osteoclastic bone resorption by inhibiting osteoprotegerin activity.

Heparin is a highly sulfated glycosaminoglycan and has been shown to activate osteoclastic bone resorption though how is not yet clear. Here we investigate the molecule involved in heparin-induced activation of osteoclasts using an in vitro osteoclast culture assay. The formation and activation of osteoclasts are induced by receptor activator of NFkappaB ligand (RANKL) on osteoblasts, and inhibited by osteoprotegerin (OPG), a decoy receptor of RANKL, which is secreted from osteoblasts. In a coculture of mouse bone marrow cells and osteoblasts treated with 1,25-dihydroxyvitamin D(3) and prostaglandin E(2) on dentin slices, the bone marrow cells differentiate into osteoclasts, and resorption pits are formed on the dentin slices. Addition of heparin, various glycosaminoglycans, and chemically modified heparins to the coculture reveals that heparin enhances the pit-forming activity of osteoclasts, and this effect of heparin on the activation of osteoclasts is dependent on its sugar chain structure. By contrast, mRNA expression levels of RANKL, RANK, and OPG in the coculture are not altered by heparin treatment. Furthermore, neither RANK nor RANKL binds to heparin, suggesting that heparin does not directly interact with these proteins. Instead, heparin specifically binds to OPG and prevents OPG-mediated inhibition of osteoclastic bone resorption in the coculture. Heparin treatment does not enhance osteoclastic bone resorption in a monoculture of osteoclasts derived from bone marrow cells, and in the coculture using osteoblasts from OPG-deficient mice. A (125)I-OPG binding assay showed that OPG binds to osteoblasts and that this binding is inhibited by the addition of heparin, suggesting that OPG binds to RANKL on the osteoblast membrane and that heparin blocks this interaction. These results demonstrate that heparin enhances osteoclastic bone resorption by inhibiting OPG activity.

Prostate-specific antigen stimulates osteoprotegerin production and inhibits receptor activator of nuclear factor-kappaB ligand expression by human osteoblasts.

BACKGROUND: Prostate cancer cells produce a large amount of prostate-specific antigen (PSA), which is widely used as a marker for this cancer. Even though it is widely used in the diagnosis of prostate cancer, many aspects of the pathophysiologic role of PSA in bone metastasis remain obscure. The receptor activator of nuclear factor-kappaB ligand (RANKL) is essential for the activation of osteoclasts, while osteoprotegerin (OPG) neutralizes the action of RANKL. Various substances that act on bone have been shown to modulate the production of RANKL and OPG by osteoblasts. METHODS: In this study, we investigated the effect of PSA on the expression of OPG and RANKL mRNA and on protein production in human osteoblast-like cells. RESULTS: After addition of PSA and culture for 72 h, OPG mRNA expression and protein secretion by MG-63 and SaOS-2 cells showed a concentration-dependent increase. When osteoblasts were incubated with PSA (100 ng/ml), OPG mRNA expression and protein secretion increased with the passage of time. alpha1 -antichymotrypsin (ACT), which inactivates the serine protease activity of PSA, inhibited the increase of OPG mRNA expression and protein production in response to PSA, and this effect of PSA was also inhibited by anti-transforming growth factor-beta antibody. CONCLUSIONS: Based on our findings, PSA acts on human osteoblast-like cells via its own serine protease activity and promotes osteoblast differentiation. In addition, PSA stimulates OPG production and inhibits RANKL expression of osteoblasts, and inhibits bone resorption by osteoclasts, suggesting that it contributes to the characteristic osteoblastic features of bone metastases of prostate cancer.