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Modeled microgravity and hindlimb unloading sensitize osteoclast precursors to RANKL-mediated osteoclastogenesis

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Abstract

Mechanical forces are essential to maintain skeletal integrity, and microgravity exposure leads to bone loss. The underlying molecular mechanisms leading to the changes in osteoblasts and osteoclast differentiation and function remain to be fully elucidated. Because of the infrequency of spaceflights and payload constraints, establishing in vitro and in vivo systems that mimic microgravity conditions becomes necessary. We have established a simulated microgravity (modeled microgravity, MMG) system to study the changes induced in osteoclast precursors. We observed that MMG, on its own, was unable to induce osteoclastogenesis of osteoclast precursors; however, 24 h of MMG activates osteoclastogenesis-related signaling molecules ERK, p38, PLCγ2, and NFATc1. Receptor activator of NFkB ligand (RANKL) (with or without M-CSF) stimulation for 3–4 days in gravity of cells that had been exposed to MMG for 24 h enhanced the formation of very large tartrate-resistant acid phosphatase (TRAP)-positive multinucleated (>30 nuclei) osteoclasts accompanied by an upregulation of the osteoclast marker genes TRAP and cathepsin K. To validate the in vitro system, we studied the hindlimb unloading (HLU) system using BALB/c mice and observed a decrease in BMD of femurs and a loss of 3D microstructure of both cortical and trabecular bone as determined by micro-CT. There was a marked stimulation of osteoclastogenesis as determined by the total number of TRAP-positive multinucleated osteoclasts formed and also an increase in RANKL-stimulated osteoclastogenesis from precursors removed from the tibias of mice after 28 days of HLU. In contrast to earlier reported findings, we did not observe any histomorphometric changes in the bone formation parameters. Thus, the foregoing observations indicate that microgravity sensitizes osteoclast precursors for increased differentiation. The in vitro model system described here is potentially a valid system for testing drugs for preventing microgravity-induced bone loss by targeting the molecular events occurring in microgravity-induced enhanced osteoclastogenesis.

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Acknowledgments

This work was supported by National Institute of Health Grants R01 AR050235 and P30 AR046031 and a NASA grant, NNJ04HB27G (NAG 9-1562).

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Authors and Affiliations

  1. Department of Cancer Biology and Pharmacology, University of Illinois College of Medicine at Peoria, Peoria, Illinois, 61605, USA

    Ritu Saxena

  2. Department of Pediatrics, Emory University Medical School, Atlanta, GA, 30329, USA

    George Pan

  3. Animal Resources Program, The University of Alabama at Birmingham, Birmingham, AL, 35294-2800, USA

    Erik D. Dohm

  4. Departments of Pathology and Cell Biology, The University of Alabama at Birmingham, LHRB 509, 701 South 19th Street, Birmingham, AL, 35294-0007, USA

    Jay M. McDonald

  5. Veterans Administration Medical Center, Birmingham, AL, 35233, USA

    Jay M. McDonald

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  1. Ritu Saxena
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Correspondence to Jay M. McDonald.

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774_2010_201_MOESM1_ESM.tif

Supplementary figure: RAW264.7 cells were incubated overnight in low-adhesion plates. Cells attached around the beads which were then incubated in gravity (G) and modeled microgravity (MMG) for 24h. Figure shows that the cells remain attached around the beads after incubation (10X magnification) (TIFF 518 kb)

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Saxena, R., Pan, G., Dohm, E.D. et al. Modeled microgravity and hindlimb unloading sensitize osteoclast precursors to RANKL-mediated osteoclastogenesis. J Bone Miner Metab 29, 111–122 (2011). https://doi.org/10.1007/s00774-010-0201-4

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  • DOI: https://doi.org/10.1007/s00774-010-0201-4

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