Inorganic phosphate stimulates apoptosis in murine MO6-G3 odontoblast-like cells.

OBJECTIVE: Dental pathologies such as caries are the most prevalent disease worldwide with infectious and social complications. During the process of caries formation, the tooth is degraded and demineralization of enamel and dentine leads to the release of large amounts of inorganic phosphate (Pi) within dental tubuli. As Pi has been shown to induce apoptosis in skeletal cells, including osteoblasts and chondrocytes, we questioned whether high concentrations of Pi could affect odontoblast viability, proliferation and apoptosis. DESIGN: Using the odontoblast-like MO6-G3 cell line as a model, we used cell counting and MTS-based colorimetric assays to measure cell viability and proliferation. Apoptosis was assessed using Hoechst nuclei staining and detection of the early apoptotic markers annexin V and Apo2.7. RESULTS: We show for the first time that a high Pi concentration (7 mM) induced a decrease in odontoblast viability and proliferation together with a large increase in apoptosis. These effects were blunted in calcium-free medium, possibly due to the formation of calcium-phosphate crystals in the presence of high Pi concentrations. CONCLUSION: This study contributes to clarifying the effect of Pi on odontoblast viability and apoptosis, which may improve our understanding of the role of Pi during caries formation.

Colony-stimulating factor-1 (CSF-1) directly inhibits receptor activator of nuclear factor-{kappa}B ligand (RANKL) expression by osteoblasts.

Colony-stimulating factor-1 (CSF-1), released by osteoblasts, stimulates the proliferation of osteoclast progenitors via the c-fms receptor (CSF-1R) and, in combination with receptor activator of nuclear factor-kappaB ligand (RANKL), leads to the formation of mature osteoclasts. Whether the CSF-1R is expressed by osteoblasts and mediates specific biological effects in osteoblasts has not been explored. Wild-type primary calvaria osteoblasts (OB) were analyzed for CSF-1R expression (RT-PCR and Western blot) and functionality (immunocomplex kinase assay). OB were serum starved for 24 h, and the effect of CSF-1 (0-100 ng/ml) on OB biological activities was determined at 48 h. In wild-type mouse bone marrow cultures, CSF-1 was tested for its effect on RANKL mRNA and osteoclast formation. Because ROS influence osteoblast RANKL expression, studies analyzed the effect of CSF-1 on reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity and Nox1 and Nox4 proteins. Results indicate that OB express CSF-1R mRNA and protein and that CSF-1R could be phosphorylated in the presence of CSF-1. In osteoblasts, CSF-1 decreased RANKL mRNA in a dose- and time-dependent manner. Incubation of bone marrow cultures with CSF-1 resulted in a significant decline in tartrate-resistant acid phosphatase (TRACP) activity and CTR expression. RANKL-decreased expression by CSF-1 was correlated with a decrease of NADPH oxidase activity as well as Nox1 and Nox4 protein levels. These findings provide the first evidence that osteoblasts express CSF-1R and are a target for CSF-1 ligand. CSF-1-mediated inhibition of RANKL expression on osteoblasts may provide an important mechanism for coupling bone formation/resorption and preventing excessive osteoclastogenesis during normal skeletal growth.

Inorganic phosphate regulates Glvr-1 and -2 expression: role of calcium and ERK1/2.

Sodium-dependent phosphate cotransporters are key regulators of phosphate homeostasis and play a major role in mineralized tissues remodelling. However, factors influencing their expression remain under consideration. In our study, modulation of type III sodium-dependent phosphate cotransporters expression by inorganic phosphate (Pi) was investigated in the murine odontoblast-like cell line MO6-G3. Experiments were designed to determine the effects of phosphate release on dental cells during tooth decay. By real-time RT-PCR we demonstrated that Glvr-1 and -2 expressions are up-regulated by Pi. The increase in Glvr-1 and -2 expressions was correlated with ERK1/2 phosphorylation and calcium/phosphate crystals formation in cultured wells. Using calcium-free culture conditions or the specific inhibitor of ERK phosphorylation (UO126), we demonstrated that Pi effects on Glvr-1 and -2 up-regulation require the presence of calcium and involve ERK signalling pathways. This study contributes to give new insights in the control of Pi transport during carious diseases.

High d(+)glucose concentration inhibits RANKL-induced osteoclastogenesis.

Diabetes is a chronic disease associated with hyperglycemia and altered bone metabolism that may lead to complications including osteopenia, increased risk of fracture and osteoporosis. Hyperglycemia has been implicated in the pathogenesis of diabetic bone disease; however, the biologic effect of glucose on osteoclastogenesis is unclear. In the present study, we examined the effect of high d(+)glucose (d-Glc) and l(-)glucose (l-Glc; osmotic control) on RANKL-induced osteoclastogenesis using RAW264.7 cells and Bone Marrow Macrophages (BMM) as models. Cells were exposed to sustained high glucose levels to mimic diabetic conditions. Osteoclast formation was analyzed using tartrate resistant acid phosphatase (TRACP) assay, expression of calcitonin receptor (CTR) and cathepsin K mRNAs, and cultures were examined for reactive oxygen species (ROS) using dichlorodihydrofluorescein diacetate (DCF-DA) fluorescence, caspase-3 and Nuclear Factor kappaB (NF-kappaB) activity. Cellular function was assessed using a migration assay. Results show, for the first time, that high d-Glc inhibits osteoclast formation, ROS production, caspase-3 activity and migration in response to RANKL through a metabolic pathway. Our findings also suggest that high d-Glc may alter RANKL-induced osteoclast formation by inhibiting redox-sensitive NF-kappaB activity through an anti-oxidative mechanism. This study increases our understanding of the role of glucose in diabetes-associated bone disease. Our data suggest that high glucose levels may alter bone turnover by decreasing osteoclast differentiation and function in diabetes and provide new insight into the biologic effects of glucose on osteoclastogenesis.

PDGF up-regulates CSF-1 gene transcription in ameloblast-like cells.

Macrophage colony-stimulating factor (CSF-1) is a key regulatory cytokine for amelogenesis, and ameloblasts synthesize CSF-1. We hypothesized that PDGF stimulates DNA synthesis and regulates CSF-1 in these cells. We determined the effect of PDGF on CSF-1 expression using MEOE-3M ameloblasts as a model. By RT-PCR, MEOE-3M expressed PDGFRs and PDGF A- and B-chain mRNAs. PDGF-BB increased DNA synthesis and up-regulated CSF-1 mRNA and protein in MEOE-3M. Cells transfected with CSF-1 promoter deletion constructs were analyzed. A PDGF-responsive region between -1.7 and -0.795 kb, containing a consensus Pea3 binding motif, was identified. Electrophoretic mobility shift assay (EMSA) showed that PDGF-BB stimulated protein binding to this motif that was inhibited in the presence of anti-Pea3 antibody. Analysis of these data provides the first evidence that PDGF-BB is a mitogen for MEOE-3M and increases CSF-1 protein levels, predominantly by transcription. Elucidation of the cellular pathways that control CSF-1 expression may provide novel strategies for the regulation of enamel matrix formation.

Targeted expression of csCSF-1 in op/op mice ameliorates tooth defects.

OBJECTIVE: The aim of this study was to characterize the tooth phenotype of CSF-1-deficient op/op mice and determine whether expression of csCSF-1 in these mice has a role in primary tooth matrix formation. DESIGN: Ameloblasts and odontoblasts, isolated from wt/wt frozen sections using laser capture microdissection, were analysed for csCSF-1, sCSF-1 and CSF-1R mRNA by RT-PCR. Mandibles, excised from 8 days op/op and wt/wt littermates, were examined for tooth morphology as well as amelogenin and DMP1 expression using in situ hybridisation. op/opCS transgenic mice, expressing csCSF-1 in teeth and bone using the osteocalcin promoter, were generated. Skeletal X-rays and histomorphometry were performed; teeth were analysed for morphology and matrix proteins. RESULTS: Normal dental cells in vivo express both CSF-1 isoforms and CSF-1R. Compared to wt/wt, op/op teeth prior to eruption showed altered dental cell morphology and dramatic reduction in DMP1 transcripts. op/opCS mice showed marked resolution of osteopetrosis, tooth eruption and teeth that resembled amelogenesis imperfecta-like phenotype. At 3 weeks, op/op teeth showed severe enamel and dentin defects and barely detectable amelogenin and DMP1. In op/opCS mice, DMP1 in odontoblasts increased to near normal and dentin morphology was restored; amelogenin also increased. Enamel integrity improved in op/opCS, although it was thinner than wt enamel. CONCLUSIONS: Results demonstrate that ameloblasts and odontoblasts are a source and potential target of CSF-1 isoforms in vivo. Expression of csCSF-1 within the tooth microenvironment is essential for normal tooth morphogenesis and may provide a mechanism for coordinating the process of tooth eruption with endogenous matrix formation.

RANKL/RANK/OPG: new therapeutic targets in bone tumours and associated osteolysis.

The emergence of the molecular triad osteoprotegerin (OPG)/Receptor Activator of NF-kB (RANK)/RANK Ligand (RANKL) has helped elucidate a key signalling pathway between stromal cells and osteoclasts. The interaction between RANK and RANKL plays a critical role in promoting osteoclast differentiation and activation leading to bone resorption. OPG is a soluble decoy receptor for RANKL that blocks osteoclast formation by inhibiting RANKL binding to RANK. The OPG/RANK/RANKL system has been shown to be abnormally regulated in several malignant osteolytic pathologies such as multiple myeloma [MM, where enhanced RANKL expression (directly by tumour cells or indirectly by stromal bone cells or T-lymphocytes)] plays an important role in associated bone destruction. By contrast, production of its endogenous counteracting decoy receptor OPG is either inhibited or too low to compensate for the increase in RANKL production. Therefore, targeting the OPG/RANK/RANKL axis may offer a novel therapeutic approach to malignant osteolytic pathologies. In animal models, OPG or soluble RANK was shown both to control hypercalcaemia of malignancy and the establishment and progression of osteolytic metastases caused by various malignant tumours. To this day, only one phase I study has been performed using a recombinant OPG construct that suppressed bone resorption in patients with multiple myeloma or breast carcinoma with radiologically confirmed bone lesions. RANK-Fc also exhibits promising therapeutic effects, as revealed in animal models of prostate cancer and multiple myeloma. If the animal results translate to similar clinical benefits in humans, using RANK-Fc or OPG may yield novel and potent strategies for treating patients with established or imminent malignant bone diseases and where standard therapeutic regimens have failed.

Relevance of an in vitro osteoclastogenesis system to study receptor activator of NF-kB ligand and osteoprotegerin biological activities.

Receptor activator of NF-kB Ligand (RANKL) is an essential requirement for osteoclastogenesis and its activity is neutralized by binding to the soluble decoy receptor osteoprotegerin (OPG). The purpose of this work was to study the effects of RANKL and OPG during osteoclastogenesis using the murine monocytic cell line RAW 264.7 that can differentiate into osteoclasts in vitro. RAW 264.7 cells plated at 10(4) cells/cm(2) and cultured for 4 days in the presence of RANKL represent the optimal culture conditions for osteoclast differentiation, with an up-regulation of all parameters related to bone resorption: tartrate resistant acid phosphatase (TRAP), calcitonin receptor (CTR), RANK, cathepsin K, matrix metalloproteinase (MMP)-9 mRNA expressions. RANKL and OPG biological effects vary according to the differentiation state of the cells: in undifferentiated RAW 264.7 cells, TRAP expression was decreased by OPG and RANKL, RANK expression was inhibited by OPG, while MMP-9 and cathepsin K mRNA expressions were not modulated. In differentiated RAW 264.7 cells, RANKL and OPG both exert an overall inhibitory effect on the expression of all the parameters studied. In these experimental conditions, OPG-induced MMP-9 inhibition was abrogated in the presence of a blocking anti-RANKL antibody, suggesting that part of OPG effects are RANKL-dependent.

Cellular activity and signaling induced by osteoprotegerin in osteoclasts: involvement of receptor activator of nuclear factor kappaB ligand and MAPK.

Osteoprotegerin (OPG) is a decoy receptor for receptor activator of nuclear factor kappaB ligand (RANKL), an inducer of osteoclastogenesis via its receptor RANK. We recently demonstrated that OPG also exerts a direct effect in osteoclasts by regulating protease expression. Herein, we showed that OPG-induced pro-matrix metalloproteinase-9 activity was abolished by ras/MAPK inhibitors in purified osteoclasts. OPG induced the phosphorylation of p38 and ERK1/2 in RAW264.7 cells. Only p38 activation was totally abolished by a blocking anti-RANKL antibody or an excess of RANKL. Surface plasmon resonance experiments revealed that RANK, RANKL and OPG are able to form a tertiary complex. These results suggested a potential formation of a tertiary complex RANK-RANKL-OPG on osteoclasts. Thus, OPG is not only a soluble decoy receptor for RANKL but must be also considered as a direct effector of osteoclast functions.

Regulation of osteoclast protease expression by RANKL.

Receptor activator of NF-kappaB ligand (RANKL) is essential for osteoclast (OC) differentiation/activation and functions through its receptor RANK at the surface of the osteoclastic cells. This study investigated for the first time the direct effects of hRANKL on protease/protease inhibitor expressions and protease activities in purified rabbit osteoclast cultures, using semi-quantitative RT-PCR, gelatin zymography, and enzymatic assays. RANKL was shown to exert in vitro pro-resorptive effects by increasing osteoclast marker expressions (Tartrate resistant acid phosphatase (TRAP) and cathepsin K), MMP-9 expression, and pro-MMP-9 activity and by diminishing TIMP-1 expression, leading to an up-regulation of the MMP-9/TIMP-1 ratio.

Osteoprotegerin differentially regulates protease expression in osteoclast cultures.

Cysteine proteases and matrix metalloproteinases (MMPs) are important factors in the degradation of organic matrix components of bone. Osteoprotegerin (OPG) is an osteoblast-secreted decoy receptor that inhibits osteoclast differentiation and activation. This study investigated the direct effects of human OPG on cathepsin K, MMP-9, MMP-2, and tissue inhibitors of metalloproteinases (TIMP1 and TIMP2) expressed by purified rabbit osteoclasts. The expression of two osteoclast markers, namely tartrate-resistant acid phosphatase (TRAP) and cathepsin K, was inhibited by 100 ng/mL hOPG, whereas MMP-9 expression was enhanced. Gelatinase activities were measured using a zymographic assay, and hOPG was shown to enhance both pro-MMP-9 and MMP-2 activities. Concomitantly, TIMP1 expression was greatly stimulated by hOPG, whereas TIMP2 mRNA levels were not modulated. Overall, these results show that hOPG regulates the proteases produced by purified osteoclasts differentially, producing a marked inhibitory effect on the expression of cathepsin K, the main enzyme involved in bone resorption.