The V–ATPase a3 subunit mutation R740S is dominant negative and results in osteopetrosis in mice

Authors

  • Noelle Ochotny,

    1. Faculty of Dentistry, University of Toronto, Toronto, Ontario, Canada
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  • Ann M Flenniken,

    1. Centre for Modeling Human Disease, Samuel Lunenfeld Research Institute, Mt Sinai Hospital, Toronto, Ontario, Canada
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  • Celeste Owen,

    1. Centre for Modeling Human Disease, Samuel Lunenfeld Research Institute, Mt Sinai Hospital, Toronto, Ontario, Canada
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  • Irina Voronov,

    1. Faculty of Dentistry, University of Toronto, Toronto, Ontario, Canada
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  • Ralph A Zirngibl,

    1. Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada
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  • Lucy R Osborne,

    1. Centre for Modeling Human Disease, Samuel Lunenfeld Research Institute, Mt Sinai Hospital, Toronto, Ontario, Canada
    2. Department of Medicine, University of Toronto, Toronto, Ontario, Canada
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  • Janet E Henderson,

    1. Faculty of Medicine, McGill University, Montreal, Quebec, Canada
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  • S Lee Adamson,

    1. Centre for Modeling Human Disease, Samuel Lunenfeld Research Institute, Mt Sinai Hospital, Toronto, Ontario, Canada
    2. Department of Obstetrics and Gynecology, Mount Sinai Hospital, Department of Physiology, University of Toronto, Toronto, Ontario, Canada
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  • Janet Rossant,

    1. Centre for Modeling Human Disease, Samuel Lunenfeld Research Institute, Mt Sinai Hospital, Toronto, Ontario, Canada
    2. Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada
    3. Program in Developmental and Stem Cell Biology, Hospital for Sick Children Research Institute, Toronto, Ontario, Canada
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  • Morris F Manolson,

    1. Faculty of Dentistry, University of Toronto, Toronto, Ontario, Canada
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  • Jane E Aubin

    Corresponding author
    1. Faculty of Dentistry, University of Toronto, Toronto, Ontario, Canada
    2. Centre for Modeling Human Disease, Samuel Lunenfeld Research Institute, Mt Sinai Hospital, Toronto, Ontario, Canada
    3. Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada
    • Department of Molecular Genetics, Faculty of Medicine, University of Toronto, Room 4245 Medical Sciences Building, 1 King's College Circle, Toronto, Ontario M5S 1A8, Canada.
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Abstract

A mouse founder with high bone mineral density and an osteopetrotic phenotype was identified in an N-ethyl-N-nitrosourea (ENU) screen. It was found to carry a dominant missense mutation in the Tcirg1 gene that encodes the a3 subunit of the vacuolar type H+-ATPase (V–ATPase), resulting in replacement of a highly conserved amino acid (R740S). The +/R740S mice have normal appearance, size, and weight but exhibit high bone density. Osteoblast parameters are unaffected in bones of +/R740S mice, whereas osteoclast number and marker expression are increased, concomitant with a decrease in the number of apoptotic osteoclasts. Consistent with reduced osteoclast apoptosis, expression of Rankl and Bcl2 is elevated, whereas Casp3 is reduced. Transmission electron microscopy revealed that unlike other known mutations in the a3 subunit of V–ATPase, polarization and ruffled border formation appear normal in +/R740S osteoclasts. However, V–ATPases from +/R740S osteoclast membranes have severely reduced proton transport, whereas ATP hydrolysis is not significantly affected. We show for the first time that a point mutation within the a3 subunit, R740S, which is dominant negative for proton pumping and bone resorption, also uncouples proton pumping from ATP hydrolysis but has no effect on ruffled border formation or polarization of osteoclasts. These results suggest that the V0 complex has proton-pumping-independent functions in mammalian cells. © 2011 American Society for Bone and Mineral Research.

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