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NF1 tumor suppressor deficient osteoclast mouse model
Abstracts / Bone 48 (2011) S124–S137
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PP108-T In vitro effects of polyunsaturated fatty acids on osteoclastogenesis and bone resorption in raw 264.7 murine macrophages J.C.A. Boeyens a, W.-H. Chua b, M.C. Kruger b, A.M. Joubert a, M. Coetzee a,⁎ a Department of Physiology, University of Pretoria, Pretoria, South Africa b Institute of Food, Nutrition and Human Health, Massey University, Palmerston North, New Zealand
osteoblast-nerve interactions determines the type of autonomous system innervating a particular bone envelope. This article is part of a Special Issue entitled ECTS 2011. Disclosure of interest: None declared.
Abstract: Overactivity of osteoclasts can lead to increased bone resorption in many pathological conditions. Clinical and animal studies have shown that polyunsaturated fatty acids (PUFAs) can have a bone protective effect. The purpose of this study was to determine whether selected PUFAs modulate osteoclastogenesis in RAW 264.7 cells, thereby affecting the potential number of resorbing osteoclasts. Murine RAW 264.7 macrophages were seeded at 1.5 × 104 cells/well in 24-well plates in the presence of 15 ng/ml RANKL. Ethanol (vehicle control), the n-6 PUFA arachidonic acid (AA) and the n-3 PUFA docosahexaenoic acid (DHA) were added to the cultures at 5–20 μg/ ml. Fresh media and all factors were changed on day 3. After 5 days, osteoclastogenesis was evaluated using tartrate resistant acid phosphatase (TRAP)-enzyme staining and subsequently counting the number of TRAP-positive cells with 5 or more nuclei. Each experiment was conducted in triplicate. RAW 264.7 cells were also seeded onto bone analogue plates coated with an inorganic synthetic bone surface. After 7 days incubation, cells were washed off the surface and bone resorption observed with a microscope. Cells were furthermore seeded onto bone slices in the presence of RANKL and fatty acids, and Ca2+ released into the media quantified by colorimetric assay. In order to determine if the PUFAs enhanced apoptosis, thereby affecting osteoclast number, mature osteoclasts were exposed to the test agents for 24 h, and Annexin V and propidium iodide flow cytometry measurement conducted thereafter. Results showed that both AA and DHA dose-dependently inhibited osteoclastogenesis and bone resorption, with DHA having the greatest effect. DHA almost completely inhibited fusion of pre-osteoclasts, resulting in the absence of multinucleated osteoclasts at the end of exposure. Though AA inhibited osteoclastogenesis, a number of multinucleated osteoclasts could still be observed. Resorption surfaces of the bone analogue plates were smaller where cells were exposed to PUFAs, compared to the vehicle control, with DHA showing the greatest antiresorptive effect. Quantification of Ca2+ released from bone slices confirmed this observation. None of the PUFAs had a pro-apoptotic effect on mature osteoclasts at the end of exposure. The results support the notion that PUFAs inhibit the formation of mature functioning osteoclasts and that it may have a protective effect on bone. Further research is required to clarify the mechanisms involved. This article is part of a Special Issue entitled ECTS 2011. Disclosure of interest: J. Boeyens: None Declared, W.-H. Chua: None Declared, M. Kruger: None Declared, A. Joubert: None Declared, M. Coetzee Grant/Research Support from South African Medical Research Council.
PP110-M NF1 tumor suppressor deficient osteoclast mouse model M.H. Alanne a,⁎, S. Peltonen b, K. Väänänen a, J. Määttä a, J. Peltonen a a Cell Biology and Anatomy, University of Turku, Turku, Finland b Department of Dermatology, University of Turku, Turku, Finland
doi:10.1016/j.bone.2011.03.269
Abstract: Neurofibromatosis type 1 (NF1) syndrome is a neurocutaneous-skeletal disorder which results from mutations in the NF1 gene. The manifestations of NF1 include café-au-lait spots of the skin, multiple cutaneous neurofibromas, and learning disabilities. NF1-related skeletal manifestations include e.g. osteoporosis/osteopenia, dystrophic scoliosis, and pseudarthrosis of long bones. Approximately 50% of NF1 patients have some form of a detectable skeletal manifestation. The NF1 protein, neurofibromin, is needed for bone formation and dynamics. More specifically, neurofibromin is expressed in osteoclasts, osteoblasts and chondrocytes. However, skeletal pathophysiology of NF1 disease is far from being understood. Our aim was to create an NF1-deficient osteoclast mouse model as a tool to clarify the role of the osteoclasts in NF1-pathophysiology. Mice harboring tartrate resistant acidic phosphatase (TRACP) promoter-driven Cre recombinase were mated with the NF1flox/flox mice. The result yielded transgenic mice with Nf1+/− or Nf1−/− osteoclasts in otherwise Nf1+/+ background. Osteoclasts and chondrocytes were microdissected separately for PCR analysis. Inactivation of NF1 gene by recombination was confirmed in osteoclasts only. Increased Ras activation was detected in Nf1 deficient osteoclasts. The tibial bone marrow cavity and the perimeter of cortical bone were smaller in Nf1 Ocl −/− mice compared to control mice as revealed by micro computer tomography (μCT). Correspondingly, trabecular tissue volume and perimeter were smaller. However, osteoporotic phenotype was not observed. NF1-deficient mice displayed a narrowed growth plate in the proximal tibia. The size of spleen was two times larger, and histomorphometric analysis showed splenic megakaryocytosis in Nf1 Ocl −/− mice compared to control mice. In conclusion, our Nf1-deficient osteoclast mouse model displayed mild bone phenotype although the original hypothesis was that inactivation of Nf1 gene in osteoclasts would have lead to osteoporotic bone phenotype. We speculate that splenomegaly and high number of splenic MK might participate to bone homeostasis by inhibiting osteoclastogenesis and bone resorption. This article is part of a Special Issue entitled ECTS 2011. Disclosure of interest: M. Alanne Grant/Research Support from The National Graduate School of Clinical Investigation, S. Peltonen: None Declared, K. Väänänen: None Declared, J. Määttä: None Declared, J. Peltonen Grant/Research Support from Academy of Finland.
doi:10.1016/j.bone.2011.03.268 doi:10.1016/j.bone.2011.03.270
PP109-S Different sympathetic pathways control bone metabolism of distinct bone envelopes M. Cherruau a,⁎, C. Bataille a, E. Hay b, P. Marie b, J.-L. Saffar a a EA2496, Montrouge, France b INSERM U606, Paris, France Abstract: Bone remodeling, the mechanism that modulates bone mass adaptation is controlled by the sympathetic nervous system through the catecholaminergic pathway interacting with osteoblasts expressing ß2 adrenergic receptors. However, resorption in the mandible periosteum envelope is associated with cholinergic VIP positive nerve fibers, suggesting that different sympathetic pathways may control distinct bone envelopes. In this study, we assessed the role of distinct sympathetic pathways on rat femur and mandible envelopes. To this goal, adult male Wistar rats were chemically sympathectomized or treated with agonists/antagonists of the catecholaminergic and cholinergic pathways, and femora and mandibles were sampled. The bones were embedded in methyl methacrylate and processed for enzymo- and immunohistochemistry. Morphometric analysis showed that sympathectomy decreased the number of preosteoclasts and RANKL-expressing osteoblasts and increased OPG mRNA in periosteum mandible but had no effect on femur trabecular bone. Pharmacological stimulation or repression of the catecholaminergic system impacted femur trabecular bone and mandible retromolar endosteal envelope, but not the mandible periosteum and alveolar wall. VIP treatment of sympathectomized rats rescued the disturbances of the mandible periosteum and alveolar wall; VIP10–28 (a VIP receptor antagonist) decreased alveolar wall preosteoclasts. In contrast, the cholinergic pathway had no effect on the catecholaminergic-dependent envelopes. We also found that VIP receptor-1 was weakly expressed in periosteal osteoblasts in the mandible and was increased by VIP treatment, whereas osteoblasts of the retromolar envelope constitutively expressed beta-2 adrenergic receptors. These data highlight the complexity of the sympathetic control of bone metabolism. Envelopes of endochondral origin (femur and retromolar zone) are catecholaminergic-dependent whereas envelopes of membranous origin are cholinergic dependent. We suggest that an origin-dependent imprint of bone cells through
PP112-S Influence of melatonin on murine osteoclast formation in culture — Automated detection and quantification by a novel image analysis system M. Schepelmann a,⁎, A. Heindl a,b, A. Seewald b, A. Nussbaumer a,c, G. Bises a, R. Rogojanu a,d, P. Pietschmann a, I. Ellinger a, T. Thalhammer a a Department of Pathophysiology and Allergy Research, Medical University of Vienna, Vienna, Austria b Seewald Solutions, Vienna, Austria c Institute of Specific Prophylaxis and Tropical Medicine, Medical University of Vienna, Vienna, Austria d TissueGnostics GmbH, Vienna, Austria Abstract: Multinucleated osteoclasts (OC) develop from hemopoietic precursor cells of the monocyte-macrophage lineage and they play an important role in the pathogenesis of diseases like osteoporosis. Therefore, factors influencing OC formation are of great interest. The impact of these factors is usually evaluated in cultures of in vitro differentiated OC. Commonly, cultured OC are identified by the OC-specific histochemical tartrate-resistant alkaline phosphatase (TRAP)staining and the number of their nuclei (>3). However, manual counting of the TRAP+ multinucleated OC often produces highly variable results and does not allow determination of parameters other than OC number. In consequence, we have developed an automated immunofluorescence-based quantification system for evaluation of OC parameters such as cell number, area, number of nuclei and expression levels of associated immunofluorescence-tagged proteins. OC were cultured from mouse bone marrow in a medium containing RANKL (receptor activator of nuclear factor κB) and M-CSF (macrophage colony stimulating factor). After fixation, the cells were probed with antibodies against the calcitonin receptor and cytoskeletal proteins to enable automated total cell area identification. Staining of the precursor cell specific marker F4/80
S133
PP108-T In vitro effects of polyunsaturated fatty acids on osteoclastogenesis and bone resorption in raw 264.7 murine macrophages J.C.A. Boeyens a, W.-H. Chua b, M.C. Kruger b, A.M. Joubert a, M. Coetzee a,⁎ a Department of Physiology, University of Pretoria, Pretoria, South Africa b Institute of Food, Nutrition and Human Health, Massey University, Palmerston North, New Zealand
osteoblast-nerve interactions determines the type of autonomous system innervating a particular bone envelope. This article is part of a Special Issue entitled ECTS 2011. Disclosure of interest: None declared.
Abstract: Overactivity of osteoclasts can lead to increased bone resorption in many pathological conditions. Clinical and animal studies have shown that polyunsaturated fatty acids (PUFAs) can have a bone protective effect. The purpose of this study was to determine whether selected PUFAs modulate osteoclastogenesis in RAW 264.7 cells, thereby affecting the potential number of resorbing osteoclasts. Murine RAW 264.7 macrophages were seeded at 1.5 × 104 cells/well in 24-well plates in the presence of 15 ng/ml RANKL. Ethanol (vehicle control), the n-6 PUFA arachidonic acid (AA) and the n-3 PUFA docosahexaenoic acid (DHA) were added to the cultures at 5–20 μg/ ml. Fresh media and all factors were changed on day 3. After 5 days, osteoclastogenesis was evaluated using tartrate resistant acid phosphatase (TRAP)-enzyme staining and subsequently counting the number of TRAP-positive cells with 5 or more nuclei. Each experiment was conducted in triplicate. RAW 264.7 cells were also seeded onto bone analogue plates coated with an inorganic synthetic bone surface. After 7 days incubation, cells were washed off the surface and bone resorption observed with a microscope. Cells were furthermore seeded onto bone slices in the presence of RANKL and fatty acids, and Ca2+ released into the media quantified by colorimetric assay. In order to determine if the PUFAs enhanced apoptosis, thereby affecting osteoclast number, mature osteoclasts were exposed to the test agents for 24 h, and Annexin V and propidium iodide flow cytometry measurement conducted thereafter. Results showed that both AA and DHA dose-dependently inhibited osteoclastogenesis and bone resorption, with DHA having the greatest effect. DHA almost completely inhibited fusion of pre-osteoclasts, resulting in the absence of multinucleated osteoclasts at the end of exposure. Though AA inhibited osteoclastogenesis, a number of multinucleated osteoclasts could still be observed. Resorption surfaces of the bone analogue plates were smaller where cells were exposed to PUFAs, compared to the vehicle control, with DHA showing the greatest antiresorptive effect. Quantification of Ca2+ released from bone slices confirmed this observation. None of the PUFAs had a pro-apoptotic effect on mature osteoclasts at the end of exposure. The results support the notion that PUFAs inhibit the formation of mature functioning osteoclasts and that it may have a protective effect on bone. Further research is required to clarify the mechanisms involved. This article is part of a Special Issue entitled ECTS 2011. Disclosure of interest: J. Boeyens: None Declared, W.-H. Chua: None Declared, M. Kruger: None Declared, A. Joubert: None Declared, M. Coetzee Grant/Research Support from South African Medical Research Council.
PP110-M NF1 tumor suppressor deficient osteoclast mouse model M.H. Alanne a,⁎, S. Peltonen b, K. Väänänen a, J. Määttä a, J. Peltonen a a Cell Biology and Anatomy, University of Turku, Turku, Finland b Department of Dermatology, University of Turku, Turku, Finland
doi:10.1016/j.bone.2011.03.269
Abstract: Neurofibromatosis type 1 (NF1) syndrome is a neurocutaneous-skeletal disorder which results from mutations in the NF1 gene. The manifestations of NF1 include café-au-lait spots of the skin, multiple cutaneous neurofibromas, and learning disabilities. NF1-related skeletal manifestations include e.g. osteoporosis/osteopenia, dystrophic scoliosis, and pseudarthrosis of long bones. Approximately 50% of NF1 patients have some form of a detectable skeletal manifestation. The NF1 protein, neurofibromin, is needed for bone formation and dynamics. More specifically, neurofibromin is expressed in osteoclasts, osteoblasts and chondrocytes. However, skeletal pathophysiology of NF1 disease is far from being understood. Our aim was to create an NF1-deficient osteoclast mouse model as a tool to clarify the role of the osteoclasts in NF1-pathophysiology. Mice harboring tartrate resistant acidic phosphatase (TRACP) promoter-driven Cre recombinase were mated with the NF1flox/flox mice. The result yielded transgenic mice with Nf1+/− or Nf1−/− osteoclasts in otherwise Nf1+/+ background. Osteoclasts and chondrocytes were microdissected separately for PCR analysis. Inactivation of NF1 gene by recombination was confirmed in osteoclasts only. Increased Ras activation was detected in Nf1 deficient osteoclasts. The tibial bone marrow cavity and the perimeter of cortical bone were smaller in Nf1 Ocl −/− mice compared to control mice as revealed by micro computer tomography (μCT). Correspondingly, trabecular tissue volume and perimeter were smaller. However, osteoporotic phenotype was not observed. NF1-deficient mice displayed a narrowed growth plate in the proximal tibia. The size of spleen was two times larger, and histomorphometric analysis showed splenic megakaryocytosis in Nf1 Ocl −/− mice compared to control mice. In conclusion, our Nf1-deficient osteoclast mouse model displayed mild bone phenotype although the original hypothesis was that inactivation of Nf1 gene in osteoclasts would have lead to osteoporotic bone phenotype. We speculate that splenomegaly and high number of splenic MK might participate to bone homeostasis by inhibiting osteoclastogenesis and bone resorption. This article is part of a Special Issue entitled ECTS 2011. Disclosure of interest: M. Alanne Grant/Research Support from The National Graduate School of Clinical Investigation, S. Peltonen: None Declared, K. Väänänen: None Declared, J. Määttä: None Declared, J. Peltonen Grant/Research Support from Academy of Finland.
doi:10.1016/j.bone.2011.03.268 doi:10.1016/j.bone.2011.03.270
PP109-S Different sympathetic pathways control bone metabolism of distinct bone envelopes M. Cherruau a,⁎, C. Bataille a, E. Hay b, P. Marie b, J.-L. Saffar a a EA2496, Montrouge, France b INSERM U606, Paris, France Abstract: Bone remodeling, the mechanism that modulates bone mass adaptation is controlled by the sympathetic nervous system through the catecholaminergic pathway interacting with osteoblasts expressing ß2 adrenergic receptors. However, resorption in the mandible periosteum envelope is associated with cholinergic VIP positive nerve fibers, suggesting that different sympathetic pathways may control distinct bone envelopes. In this study, we assessed the role of distinct sympathetic pathways on rat femur and mandible envelopes. To this goal, adult male Wistar rats were chemically sympathectomized or treated with agonists/antagonists of the catecholaminergic and cholinergic pathways, and femora and mandibles were sampled. The bones were embedded in methyl methacrylate and processed for enzymo- and immunohistochemistry. Morphometric analysis showed that sympathectomy decreased the number of preosteoclasts and RANKL-expressing osteoblasts and increased OPG mRNA in periosteum mandible but had no effect on femur trabecular bone. Pharmacological stimulation or repression of the catecholaminergic system impacted femur trabecular bone and mandible retromolar endosteal envelope, but not the mandible periosteum and alveolar wall. VIP treatment of sympathectomized rats rescued the disturbances of the mandible periosteum and alveolar wall; VIP10–28 (a VIP receptor antagonist) decreased alveolar wall preosteoclasts. In contrast, the cholinergic pathway had no effect on the catecholaminergic-dependent envelopes. We also found that VIP receptor-1 was weakly expressed in periosteal osteoblasts in the mandible and was increased by VIP treatment, whereas osteoblasts of the retromolar envelope constitutively expressed beta-2 adrenergic receptors. These data highlight the complexity of the sympathetic control of bone metabolism. Envelopes of endochondral origin (femur and retromolar zone) are catecholaminergic-dependent whereas envelopes of membranous origin are cholinergic dependent. We suggest that an origin-dependent imprint of bone cells through
PP112-S Influence of melatonin on murine osteoclast formation in culture — Automated detection and quantification by a novel image analysis system M. Schepelmann a,⁎, A. Heindl a,b, A. Seewald b, A. Nussbaumer a,c, G. Bises a, R. Rogojanu a,d, P. Pietschmann a, I. Ellinger a, T. Thalhammer a a Department of Pathophysiology and Allergy Research, Medical University of Vienna, Vienna, Austria b Seewald Solutions, Vienna, Austria c Institute of Specific Prophylaxis and Tropical Medicine, Medical University of Vienna, Vienna, Austria d TissueGnostics GmbH, Vienna, Austria Abstract: Multinucleated osteoclasts (OC) develop from hemopoietic precursor cells of the monocyte-macrophage lineage and they play an important role in the pathogenesis of diseases like osteoporosis. Therefore, factors influencing OC formation are of great interest. The impact of these factors is usually evaluated in cultures of in vitro differentiated OC. Commonly, cultured OC are identified by the OC-specific histochemical tartrate-resistant alkaline phosphatase (TRAP)staining and the number of their nuclei (>3). However, manual counting of the TRAP+ multinucleated OC often produces highly variable results and does not allow determination of parameters other than OC number. In consequence, we have developed an automated immunofluorescence-based quantification system for evaluation of OC parameters such as cell number, area, number of nuclei and expression levels of associated immunofluorescence-tagged proteins. OC were cultured from mouse bone marrow in a medium containing RANKL (receptor activator of nuclear factor κB) and M-CSF (macrophage colony stimulating factor). After fixation, the cells were probed with antibodies against the calcitonin receptor and cytoskeletal proteins to enable automated total cell area identification. Staining of the precursor cell specific marker F4/80