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Calcitonin and alendronate activate Ca2+-permeable cation conductances with distinct transient receptor potential properties in murine osteoclasts
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Abstracts / Bone 46 (2010) S9–S83
138. Expression of a store-operated conductance and mRNA for canonical transient receptor potential and Orai in murine osteoclasts Brenda Lau, Anthony Albert, Timothy Chambers, Kevin Lawrence Basic Medical Science, St. George's, University of London, London, United Kingdom Cytosolic Ca2 level (Ca2+//0) is important in regulating osteoclast activity and therefore has a pivotal role in bone resorptive processes. Stimulation of Ca2+-sensing receptors induce a rise in Ca2+//0 through different mechanisms, including release of Ca2 from internal Ca2 stores that causes opening of store-operated channels (SOCs) and a direct influx of Ca2 ions. Importantly, SOC activity is proposed to be essential for maintaining osteoclast survival and bone resorption. It is suggested that the canonical transient receptor potential (TRPC) subfamily of Ca2+-permeable non-selective cation channels are excellent candidates for mediating SOCs in many cell types. Moreover, the recently discovered Orai proteins mediate calcium-release-activated channels (Icrac) in non-excitable cells. However there is no information on SOCs, TRPCs or Orai in osteoclasts. The aim of the present work was to investigate the presence of SOCs in mature osteoclasts using single channel patch clamp recording, and expression of mRNA for TRPC and Orai channel proteins that are reported to compose SOCs using semi-quantitative RT-PCR. To determine whether store depletion activates SOCs in osteoclasts, we investigated the effect of cyclopiazonic acid (CPA, 20 uM) and BAPTA-AM (50 uM) on quiescent cell-attached patches. CPA depletes internal Ca2 stores by inhibiting Ca2+-ATPases whilst BAPTA-AM, a cell-permeable Ca2 cheletor, passively depletes internal Ca2 stores. Bath application of CPA or BAPTA-AM activated single cation channel currents in cell-attached patches which had unitary conductances between 3-4 pS. In addition, RT-PCR showed that mRNA for TRPC1, C2, C4 and C6 but not C3, C5 and C7 were expressed in mature osteoclasts. Moreover mRNA for Orai1 and the Ca2 sensor STIM1 but not Orai2 or 3 were also expressed in mature osteoclasts. These novel findings show that osteoclasts contain functional SOCs. Moreover our results also shows that osteoclasts express several TRPC isoforms and Orai1/STIM1 which are likely candidates for constituting SOCs. Thus the present work identifies several new potential targets for treatment of bone disorders. This work was funded by the Arthritis Research Campaign. doi:10.1016/j.bone.2010.01.141
139. Calcitonin and alendronate activate Ca2+-permeable cation conductances with distinct transient receptor potential properties in murine osteoclasts Anthony Albert, Timothy Chambers, Kevin Lawrence Basic Medical Science, St. George's, University of London, London, United Kingdom Cytosolic Ca2 (Ca2+//0) regulates osteoclast activity and controls bone resorption. Calcitonin (CT) is a potent anti-resorptive molecule, acting at G-protein-coupled receptors, which induces Ca2 influx. The transient receptor potential family of Ca2+-permeable non-selective cation channels (TRPC, TRPM and TRPV) are excellent candidates for mediating receptor-operated Ca2+-permeable channels (ROCs) and Ca2 influx pathways in many cell types. However there is no information on the molecular composition or properties of ROCs in osteoclasts. The aim of the present work was to investigate ROCs activated by calcitonin using single channel patch clamp techniques, and expression of mRNA for TRPC using semi-quantitative RT-PCR.
Bath application of calcitonin activated two distinct cation channel currents in a concentration-dependent manner in cell-attached patches. At low concentrations (10-20pM), calcitonin induced channel currents which had brief openings to multiple subconductance states between 10-50pS and displayed a characteristic ‘nois’ appearance. In contrast, high concentrations of calcitonin (1 nM), evoked channel currents with much longer openings to a single unitary conductance of about 25pS. Moreover, bath application of the diacylglycerol analogue, OAG (20uM), in the presence of the protein kinase C inhibitor chelerythrine (3uM), and also application of alendronate (100uM) both activated channel activity with multiple sub-conductances between 10-50pS. RT-PCR experiments showed that mRNA for TRPC6, but not C3 or C7, and TRPM4, but not M5, were expressed in mature osteoclasts. TRPC6 mRNA was expressed at a greater level in the early developmental stages of osteoclasts, whereas TRPM4 was expressed throughout development.These findings show that agents that prevent bone resorption activate two distinct types of ROCs, 1) a TRPC6-like ion channel with conductances between 10-50 pS, which is activated by DAG via a PKC-independent pathway, and 2) a TRPM4like conductance with a conductance of 25 pS which is likely to be activated by a rise in Ca2+//0. Thus the present work identifies several new potential targets for treatment of bone disorders. This work was funded by the Arthritis Research Campaign. doi:10.1016/j.bone.2010.01.142
140. GM-CSF released by osteoblasts in response to TNFalpha inhibits osteoclastogenesis in vitro Elvis Atanga1, Silvia Dolder2, Willy Hofstetter3 1 Group for Bone Biology & Orthopaedic Research, Department Clinical Research, University of Bern, Bern, Switzerland 2 Group for Bone Biology & Orthopaedic Research, Department Clinical Research, University of Bern, Bern, Switzerland 3 Group for Bone Biology & Orthopaedic Research, Department Clinical Research, University of Bern, Bern, Switzerland Introduction. Osteoclasts are multinucleated giant cells resorbing bone. Colony-Stimulating Factor-1 (CSF1) and Receptor Activator of NF-kB Ligand (RANKL) are essential for the development of mature osteoclast from precursors (OCP). Inflammatory cytokines, which are potent modulators of bone resorption, are frequently associated with impairment of bone metabolism. Previously, we found TNFalpha to induce the release of Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF), an inhibitor of osteoclatogenesis. Within this study, we investigated at what stage, and by which mechanisms, GM-CSF affects osteoclastogenesis. Methods. The differentiation of OCP in the presence of GM-CSF was followed in co-cultures of osteoblasts and bone marrow cells (BMC) from TNF receptor deficient mice and in cultures of BMC. c-fms and RANK were assessed at transcript levels. Results. Osteoclast numbers were decreased by ∼ 75% and by ∼50% when CM from wt and GM-CSF-/- osteoblasts, respectively, were added during the early phase (0-3 days) but no inhibition was observed when these CM were added during the late phase (3-5 days). To investigate the inhibitory potential of CM on osteoclast formation, 10% CM (wt and GM-CSF-/- osteoblasts treated with TNFalpha/1,25(OH)2D3 for 0h-24 h or 24h-48 h and 48h-72 h of the osteoblast culture period) was added to BMC cultures supplemented with CSF1 and RANKL in the presence of anti-mouse GM-CSF antibodies or control IgG. Osteoclast formation was decreased by ∼80% with rat IgG and ∼50% in the presence antiGM-CSF with CM from wt and by 50% with CM from GM-CSF-/- osteoblasts. To investigate the effect of GM-CSF or CM on RANK and c-fms
Abstracts / Bone 46 (2010) S9–S83
138. Expression of a store-operated conductance and mRNA for canonical transient receptor potential and Orai in murine osteoclasts Brenda Lau, Anthony Albert, Timothy Chambers, Kevin Lawrence Basic Medical Science, St. George's, University of London, London, United Kingdom Cytosolic Ca2 level (Ca2+//0) is important in regulating osteoclast activity and therefore has a pivotal role in bone resorptive processes. Stimulation of Ca2+-sensing receptors induce a rise in Ca2+//0 through different mechanisms, including release of Ca2 from internal Ca2 stores that causes opening of store-operated channels (SOCs) and a direct influx of Ca2 ions. Importantly, SOC activity is proposed to be essential for maintaining osteoclast survival and bone resorption. It is suggested that the canonical transient receptor potential (TRPC) subfamily of Ca2+-permeable non-selective cation channels are excellent candidates for mediating SOCs in many cell types. Moreover, the recently discovered Orai proteins mediate calcium-release-activated channels (Icrac) in non-excitable cells. However there is no information on SOCs, TRPCs or Orai in osteoclasts. The aim of the present work was to investigate the presence of SOCs in mature osteoclasts using single channel patch clamp recording, and expression of mRNA for TRPC and Orai channel proteins that are reported to compose SOCs using semi-quantitative RT-PCR. To determine whether store depletion activates SOCs in osteoclasts, we investigated the effect of cyclopiazonic acid (CPA, 20 uM) and BAPTA-AM (50 uM) on quiescent cell-attached patches. CPA depletes internal Ca2 stores by inhibiting Ca2+-ATPases whilst BAPTA-AM, a cell-permeable Ca2 cheletor, passively depletes internal Ca2 stores. Bath application of CPA or BAPTA-AM activated single cation channel currents in cell-attached patches which had unitary conductances between 3-4 pS. In addition, RT-PCR showed that mRNA for TRPC1, C2, C4 and C6 but not C3, C5 and C7 were expressed in mature osteoclasts. Moreover mRNA for Orai1 and the Ca2 sensor STIM1 but not Orai2 or 3 were also expressed in mature osteoclasts. These novel findings show that osteoclasts contain functional SOCs. Moreover our results also shows that osteoclasts express several TRPC isoforms and Orai1/STIM1 which are likely candidates for constituting SOCs. Thus the present work identifies several new potential targets for treatment of bone disorders. This work was funded by the Arthritis Research Campaign. doi:10.1016/j.bone.2010.01.141
139. Calcitonin and alendronate activate Ca2+-permeable cation conductances with distinct transient receptor potential properties in murine osteoclasts Anthony Albert, Timothy Chambers, Kevin Lawrence Basic Medical Science, St. George's, University of London, London, United Kingdom Cytosolic Ca2 (Ca2+//0) regulates osteoclast activity and controls bone resorption. Calcitonin (CT) is a potent anti-resorptive molecule, acting at G-protein-coupled receptors, which induces Ca2 influx. The transient receptor potential family of Ca2+-permeable non-selective cation channels (TRPC, TRPM and TRPV) are excellent candidates for mediating receptor-operated Ca2+-permeable channels (ROCs) and Ca2 influx pathways in many cell types. However there is no information on the molecular composition or properties of ROCs in osteoclasts. The aim of the present work was to investigate ROCs activated by calcitonin using single channel patch clamp techniques, and expression of mRNA for TRPC using semi-quantitative RT-PCR.
Bath application of calcitonin activated two distinct cation channel currents in a concentration-dependent manner in cell-attached patches. At low concentrations (10-20pM), calcitonin induced channel currents which had brief openings to multiple subconductance states between 10-50pS and displayed a characteristic ‘nois’ appearance. In contrast, high concentrations of calcitonin (1 nM), evoked channel currents with much longer openings to a single unitary conductance of about 25pS. Moreover, bath application of the diacylglycerol analogue, OAG (20uM), in the presence of the protein kinase C inhibitor chelerythrine (3uM), and also application of alendronate (100uM) both activated channel activity with multiple sub-conductances between 10-50pS. RT-PCR experiments showed that mRNA for TRPC6, but not C3 or C7, and TRPM4, but not M5, were expressed in mature osteoclasts. TRPC6 mRNA was expressed at a greater level in the early developmental stages of osteoclasts, whereas TRPM4 was expressed throughout development.These findings show that agents that prevent bone resorption activate two distinct types of ROCs, 1) a TRPC6-like ion channel with conductances between 10-50 pS, which is activated by DAG via a PKC-independent pathway, and 2) a TRPM4like conductance with a conductance of 25 pS which is likely to be activated by a rise in Ca2+//0. Thus the present work identifies several new potential targets for treatment of bone disorders. This work was funded by the Arthritis Research Campaign. doi:10.1016/j.bone.2010.01.142
140. GM-CSF released by osteoblasts in response to TNFalpha inhibits osteoclastogenesis in vitro Elvis Atanga1, Silvia Dolder2, Willy Hofstetter3 1 Group for Bone Biology & Orthopaedic Research, Department Clinical Research, University of Bern, Bern, Switzerland 2 Group for Bone Biology & Orthopaedic Research, Department Clinical Research, University of Bern, Bern, Switzerland 3 Group for Bone Biology & Orthopaedic Research, Department Clinical Research, University of Bern, Bern, Switzerland Introduction. Osteoclasts are multinucleated giant cells resorbing bone. Colony-Stimulating Factor-1 (CSF1) and Receptor Activator of NF-kB Ligand (RANKL) are essential for the development of mature osteoclast from precursors (OCP). Inflammatory cytokines, which are potent modulators of bone resorption, are frequently associated with impairment of bone metabolism. Previously, we found TNFalpha to induce the release of Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF), an inhibitor of osteoclatogenesis. Within this study, we investigated at what stage, and by which mechanisms, GM-CSF affects osteoclastogenesis. Methods. The differentiation of OCP in the presence of GM-CSF was followed in co-cultures of osteoblasts and bone marrow cells (BMC) from TNF receptor deficient mice and in cultures of BMC. c-fms and RANK were assessed at transcript levels. Results. Osteoclast numbers were decreased by ∼ 75% and by ∼50% when CM from wt and GM-CSF-/- osteoblasts, respectively, were added during the early phase (0-3 days) but no inhibition was observed when these CM were added during the late phase (3-5 days). To investigate the inhibitory potential of CM on osteoclast formation, 10% CM (wt and GM-CSF-/- osteoblasts treated with TNFalpha/1,25(OH)2D3 for 0h-24 h or 24h-48 h and 48h-72 h of the osteoblast culture period) was added to BMC cultures supplemented with CSF1 and RANKL in the presence of anti-mouse GM-CSF antibodies or control IgG. Osteoclast formation was decreased by ∼80% with rat IgG and ∼50% in the presence antiGM-CSF with CM from wt and by 50% with CM from GM-CSF-/- osteoblasts. To investigate the effect of GM-CSF or CM on RANK and c-fms