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

• Published in: Journal of bone and mineral research Vol. 26; no. 7; pp. 1484 - 1493
• Main Authors: Ochotny, Noelle, Flenniken, Ann M, Owen, Celeste, Voronov, Irina, Zirngibl, Ralph A, Osborne, Lucy R, Henderson, Janet E, Adamson, S Lee, Rossant, Janet, Manolson, Morris F, Aubin, Jane E
• Format: Journal Article
• Language: English
• Published: Hoboken Wiley Subscription Services, Inc., A Wiley Company 01-07-2011; Wiley; Wiley Subscription Services, Inc
• Subjects: Adenosine Triphosphate - metabolism; Amino Acid Substitution - genetics; Animal Model; Animals; Apoptosis; Base Sequence; Biological and medical sciences; Biological Transport; Bone; Cell Count; Cell Shape; DNA Mutational Analysis; Female; Femur - diagnostic imaging; Femur - pathology; Fundamental and applied biological sciences. Psychology; Genes, Dominant - genetics; Heterozygote; Hydrolysis; Male; Mice; Mice, Inbred C57BL; Molecular Sequence Data; Mutation, Missense - genetics; Osteoblasts - metabolism; Osteoblasts - pathology; Osteoclast; Osteoclasts - metabolism; Osteoclasts - pathology; Osteopetrosis; Osteopetrosis - genetics; Osteopetrosis - pathology; Protein Subunits - genetics; Protons; Rodent; Skeleton and joints; Vacuolar Proton-Translocating ATPases - genetics; Vertebrates: osteoarticular system, musculoskeletal system; X-Ray Microtomography; Animal model; RODENT; Diseases of the osteoarticular system; Rodentia; Osteoclast; Subunit; Genetic disease; Osteoarticular system; Vertebrata; Mammalia; Osteopetrosis; Mouse; Mutation; Bone; Osteochondrodysplasia
• ISSN: 0884-0431; 1523-4681
• DOI: 10.1002/jbmr.355

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.