- Email: [email protected]
Mice lacking AMP-activated kinase (AMPK) subunits β1 or β2 have low bone mass, while AICAR acts AMPK-independently to increase osteoclast formation
S136
Abstracts / Bone 44 (2009) S131–S141
b
Civil and Environmental Engineering, University of Melbourne, Melbourne, VIC, Australia c Biomedical Engineering, University of Sydney, Sydney, NSW, Australia d St Vincent's Institute, University of Melbourne, Melbourne, VIC, Australia e Center for Bone Biology, Vanderbilt University, Nashville, TN, United States The purpose of this study is to investigate possible therapeutic drug interventions applied to catabolic bone diseases. Among the many catabolic bone diseases we restrict our focus to those related to the RANK-RANKL-OPG system. In order to test various therapeutic strategies we employ a previously proposed bone cell dynamics model which uses cell numbers and bone volume as output functions. Clinically, the latter two quantities can be linked to the action of drugs on bone volume (or bone mass) and bone turnover. The bone model contains the RANK-RANKL-OPG signaling system between osteoblasts and osteoclasts together with the action of TGF-be on bone cells. We use the entire parameter space of the model as possible therapeutic interventions— these include differentiation and apoptosis rates of osteoblasts and osteoclasts together with components of the RANK-RANKL-OPG system (such as for example RANKL production). Based on the numerical simulations we can identify three individual patterns of bone turnover for every successful therapy, i.e., those which are able to store bone volume back to normal. The first “pro-anabolic” pattern is characterized by high bone turnover and is associated with changes of osteoblast differentiation and apoptosis rate. The second “anti-catabolic” pattern is characterized by rather normal bone turnover caused by changes of differentiation and apoptosis rates of osteoclasts together with parameters of the RANK-RANKL-OPG system. Finally, the third pattern shows mixed properties, i.e., have normal active osteoblast and osteoclast cell numbers while having rather low osteoblast precursor numbers associated with changes in the differentiation rate of osteoblast precursor cells. doi:10.1016/j.bone.2009.01.298
385 Mice lacking AMP-activated kinase (AMPK) subunits β1 or β2 have low bone mass, while AICAR acts AMPK-independently to increase osteoclast formation J.M.W. Quinna,b,c, S. Tamb,c, N.A. Simsb,c, H. Saleha,b, N.E. McGregorc, I.J. Poultonc, E.C. Walkerc, J. Scottc, B.E. Kempc,d, M.T. Gillespiea,b, B.J.W. Van Denderenc a Prince Henry's Institute, Clayton, VIC, Australia b Dept. of Medicine, University of Melbourne, Fitzroy, VIC, Australia c St. Vincent's Institute, Fitzroy, VIC, Australia d Molecular and Health Technologies, CSIRO, Parkville, VIC, Australia AMPK is a ubiquitously expressed energy sensing enzyme that regulates whole body and cellular energy homeostasis and protects cells from stresses that deplete the energy charge (i.e. increase in the AMP:ATP ratio), such as exercise, hypoxia and heat shock. However the role of AMPK in the bone has not been investigated. AMPK is a heterotrimeric α, β, γ enzyme with two β-subunit isoforms. We developed a β1 knockout (β1−/−) and a β2 knockout (β2−/−) mouse line. Both types of knockout mice displayed reduced functional AMPK in many cell types, and displayed abnormally low trabecular volume and trabecular number compared to matched wild type littermate controls. β1−/− × β2−/− crosses yielded no β1−/−/β2−/− offspring (which would lack AMPK), indicating embryonic lethality.
Since these observations suggested that AMPK significantly influence bone metabolism, we investigated effects of a direct AMPK activating compound, AICAR. This compound is converted in cells to the AMP analogue ZMP (through the intracellular action of adenosine kinase) which mimics the effect of cellular metabolic stress. AICAR administration caused 10 week old male mice to rapidly lose trabecular and cortical bone mass (determined by pQCT and histomorphometry) with greatly elevated osteoclast numbers in an adenosine-kinase dependent manner. AICAR also stimulated osteoclast formation (but not resorptive activity) in several models of in vitro osteoclast differentiation, suggesting a direct pro-osteolytic effect of this compound on osteoclast progenitors. However, β2 AMPK subunit was not detected in macrophage/osteoclast lineage cells in wild type or β1−/− mice, and indeed we determined that in the latter, macrophage lineage cells were AMPK null. Consistent with our evidence that AICAR acts in an AMPK independent manner to increase osteoclast formation, AICAR greatly stimulated osteoclast formation from β1−/− mouse bone marrow. Our data suggest that while AMPK significantly influences bone mass, it plays no cell-autonomous role in physiological osteoclast formation or activity. This study also showed that AICAR, a compound proposed as a useful treatment for insulin resistance, has powerful pro-osteolytic actions through effects on macrophage/osteoclast lineage cells which are, nonetheless, independent of AMPK. doi:10.1016/j.bone.2009.01.299
386 Osteomacs: Osteoclast precursors during inflammatory bone disease but regulators of physiologic bone remodeling L.J. Raggatta, M.K. Changa, K.A. Alexandera, E.R. Maylina, N.C. Walshb, E.M. Gravalleseb, D.A. Humec, A.R. Pettita a Centre for Clinical Research, University of Queenland, Herston, QLD, Australia b University of Massachusetts Medical School, Worcester, MA, United States c The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Roslin, Midlothian, United Kingdom We have identified a population of resident tissue macrophages (OsteoMacs), defined by expression of F4/80 and anatomical location that are present within osteal tissues. OsteoMacs regulate in vitro mineralization by osteoblasts and are required for the maintenance of mature osteoblasts in vivo. The close lineage relationship of macrophages and osteoclasts leads us to investigate whether OsteoMacs function as osteoclast precursors. Primary OsteoMacs isolated from endosteal bone surfaces formed TRAP+ osteoclasts in the presence of RANKL (40 ng/ml) and CSF-1, confirming that like many myeloid cell populations, they can be driven to form osteoclasts in vitro. Interestingly, similar treatment of calvarial digest cultures, that contain both osteoblasts and OsteoMacs, produced two distinct populations: F4/80+/TRAP-OsteoMacs and mononuclear/binuclear F4/80-/TRAP+ cells. Therefore, in this more physiologic mixed culture, OsteoMacs are not osteoclast precursors. Immunohistochemical analysis of the metaphyseal region in bone sections confirmed that F4/80+ OsteoMacs do not express TRAP and mononuclear TRAP+ cells do not express F4/80, indicating that during physiologic bone turnover OsteoMacs are not osteoclast precursors. Comparison of Mac-3 and F4/80 distribution within the metaphyseal zone identified that a Mac-3+/F4/80myeloid population predominates in the primary spongiosa, a region rich in osteoclasts, confirming that other potential osteoclast precursor populations are present in the appropriate anatomical
Abstracts / Bone 44 (2009) S131–S141
b
Civil and Environmental Engineering, University of Melbourne, Melbourne, VIC, Australia c Biomedical Engineering, University of Sydney, Sydney, NSW, Australia d St Vincent's Institute, University of Melbourne, Melbourne, VIC, Australia e Center for Bone Biology, Vanderbilt University, Nashville, TN, United States The purpose of this study is to investigate possible therapeutic drug interventions applied to catabolic bone diseases. Among the many catabolic bone diseases we restrict our focus to those related to the RANK-RANKL-OPG system. In order to test various therapeutic strategies we employ a previously proposed bone cell dynamics model which uses cell numbers and bone volume as output functions. Clinically, the latter two quantities can be linked to the action of drugs on bone volume (or bone mass) and bone turnover. The bone model contains the RANK-RANKL-OPG signaling system between osteoblasts and osteoclasts together with the action of TGF-be on bone cells. We use the entire parameter space of the model as possible therapeutic interventions— these include differentiation and apoptosis rates of osteoblasts and osteoclasts together with components of the RANK-RANKL-OPG system (such as for example RANKL production). Based on the numerical simulations we can identify three individual patterns of bone turnover for every successful therapy, i.e., those which are able to store bone volume back to normal. The first “pro-anabolic” pattern is characterized by high bone turnover and is associated with changes of osteoblast differentiation and apoptosis rate. The second “anti-catabolic” pattern is characterized by rather normal bone turnover caused by changes of differentiation and apoptosis rates of osteoclasts together with parameters of the RANK-RANKL-OPG system. Finally, the third pattern shows mixed properties, i.e., have normal active osteoblast and osteoclast cell numbers while having rather low osteoblast precursor numbers associated with changes in the differentiation rate of osteoblast precursor cells. doi:10.1016/j.bone.2009.01.298
385 Mice lacking AMP-activated kinase (AMPK) subunits β1 or β2 have low bone mass, while AICAR acts AMPK-independently to increase osteoclast formation J.M.W. Quinna,b,c, S. Tamb,c, N.A. Simsb,c, H. Saleha,b, N.E. McGregorc, I.J. Poultonc, E.C. Walkerc, J. Scottc, B.E. Kempc,d, M.T. Gillespiea,b, B.J.W. Van Denderenc a Prince Henry's Institute, Clayton, VIC, Australia b Dept. of Medicine, University of Melbourne, Fitzroy, VIC, Australia c St. Vincent's Institute, Fitzroy, VIC, Australia d Molecular and Health Technologies, CSIRO, Parkville, VIC, Australia AMPK is a ubiquitously expressed energy sensing enzyme that regulates whole body and cellular energy homeostasis and protects cells from stresses that deplete the energy charge (i.e. increase in the AMP:ATP ratio), such as exercise, hypoxia and heat shock. However the role of AMPK in the bone has not been investigated. AMPK is a heterotrimeric α, β, γ enzyme with two β-subunit isoforms. We developed a β1 knockout (β1−/−) and a β2 knockout (β2−/−) mouse line. Both types of knockout mice displayed reduced functional AMPK in many cell types, and displayed abnormally low trabecular volume and trabecular number compared to matched wild type littermate controls. β1−/− × β2−/− crosses yielded no β1−/−/β2−/− offspring (which would lack AMPK), indicating embryonic lethality.
Since these observations suggested that AMPK significantly influence bone metabolism, we investigated effects of a direct AMPK activating compound, AICAR. This compound is converted in cells to the AMP analogue ZMP (through the intracellular action of adenosine kinase) which mimics the effect of cellular metabolic stress. AICAR administration caused 10 week old male mice to rapidly lose trabecular and cortical bone mass (determined by pQCT and histomorphometry) with greatly elevated osteoclast numbers in an adenosine-kinase dependent manner. AICAR also stimulated osteoclast formation (but not resorptive activity) in several models of in vitro osteoclast differentiation, suggesting a direct pro-osteolytic effect of this compound on osteoclast progenitors. However, β2 AMPK subunit was not detected in macrophage/osteoclast lineage cells in wild type or β1−/− mice, and indeed we determined that in the latter, macrophage lineage cells were AMPK null. Consistent with our evidence that AICAR acts in an AMPK independent manner to increase osteoclast formation, AICAR greatly stimulated osteoclast formation from β1−/− mouse bone marrow. Our data suggest that while AMPK significantly influences bone mass, it plays no cell-autonomous role in physiological osteoclast formation or activity. This study also showed that AICAR, a compound proposed as a useful treatment for insulin resistance, has powerful pro-osteolytic actions through effects on macrophage/osteoclast lineage cells which are, nonetheless, independent of AMPK. doi:10.1016/j.bone.2009.01.299
386 Osteomacs: Osteoclast precursors during inflammatory bone disease but regulators of physiologic bone remodeling L.J. Raggatta, M.K. Changa, K.A. Alexandera, E.R. Maylina, N.C. Walshb, E.M. Gravalleseb, D.A. Humec, A.R. Pettita a Centre for Clinical Research, University of Queenland, Herston, QLD, Australia b University of Massachusetts Medical School, Worcester, MA, United States c The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Roslin, Midlothian, United Kingdom We have identified a population of resident tissue macrophages (OsteoMacs), defined by expression of F4/80 and anatomical location that are present within osteal tissues. OsteoMacs regulate in vitro mineralization by osteoblasts and are required for the maintenance of mature osteoblasts in vivo. The close lineage relationship of macrophages and osteoclasts leads us to investigate whether OsteoMacs function as osteoclast precursors. Primary OsteoMacs isolated from endosteal bone surfaces formed TRAP+ osteoclasts in the presence of RANKL (40 ng/ml) and CSF-1, confirming that like many myeloid cell populations, they can be driven to form osteoclasts in vitro. Interestingly, similar treatment of calvarial digest cultures, that contain both osteoblasts and OsteoMacs, produced two distinct populations: F4/80+/TRAP-OsteoMacs and mononuclear/binuclear F4/80-/TRAP+ cells. Therefore, in this more physiologic mixed culture, OsteoMacs are not osteoclast precursors. Immunohistochemical analysis of the metaphyseal region in bone sections confirmed that F4/80+ OsteoMacs do not express TRAP and mononuclear TRAP+ cells do not express F4/80, indicating that during physiologic bone turnover OsteoMacs are not osteoclast precursors. Comparison of Mac-3 and F4/80 distribution within the metaphyseal zone identified that a Mac-3+/F4/80myeloid population predominates in the primary spongiosa, a region rich in osteoclasts, confirming that other potential osteoclast precursor populations are present in the appropriate anatomical