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

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 þ -ATPase (V-ATPase), resulting in replacement of a highly conserved amino acid (R740S). The þ/R740S mice have normal appearance, size, and weight but exhibit high bone density. Osteoblast parameters are unaffected in bones of þ/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 þ/R740S osteoclasts. However, V-ATPases from þ/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 V 0 complex has proton-pumping-independent functions in mammalian cells. ß

The R740S mutation in the V-ATPase a3 subunit increases lysosomal pH, impairs NFATc1 translocation, and decreases in vitro osteoclastogenesis

“…Heterozygous mice with a point mutation in the a3 subunit of V-ATPase (þ/R740S) display mild osteopetrosis, even though the number of osteoclasts in bones of þ/R740S mice is increased. (10) This is similar to Tcirg1 À/À (a3 knockout) (7) and to oc/oc (a3 truncation) (11) mouse models, all of which display reduced proton translocation at the plasma membrane into the resorption lacunae. This increase in osteoclast numbers in vivo is most likely a compensatory response to the increased bone density or decreased resorption capacity of the osteoclasts, and because of the higher expression of Rankl and Csf1 (M-CSF) in þ/R740S bones, (10) consistent with what is seen in oc/oc mice.…”

“…(10) This is similar to Tcirg1 À/À (a3 knockout) (7) and to oc/oc (a3 truncation) (11) mouse models, all of which display reduced proton translocation at the plasma membrane into the resorption lacunae. This increase in osteoclast numbers in vivo is most likely a compensatory response to the increased bone density or decreased resorption capacity of the osteoclasts, and because of the higher expression of Rankl and Csf1 (M-CSF) in þ/R740S bones, (10) consistent with what is seen in oc/oc mice. (27) In contrast, we show that in vitro osteoclastogenesis is impaired, resulting in smaller and fewer osteoclasts in þ/R740S cells compared with þ/þ cells, a phenomenon not observed in the other murine models.…”

“…(9) We previously demonstrated that heterozygous mice with a point mutation (R740S) in the a3 subunit of V-ATPase (þ/R740S) have mild osteopetrosis and impaired bone resorption, (10) even though the number of osteoclasts in þ/R740S bones is increased, a finding consistent with that seen in Tcirg1 À/À (a3 knockout) (7) or oc/oc (a3 truncation) mouse models. (11) We also showed that proton translocation was inhibited by almost 90% in membranes from þ/R740S osteoclasts.…”

“…Heterozygous mice carrying the R740S mutation bred onto the C3H/HeJ and FVB backgrounds were generated as described previously (10) and are referred to as þ/R740S mice. All experimental procedures received approval from the local Animal Care Committees and were conducted in accordance with the guidelines of the Canadian Council on Animal Care.…”

Inhibitors of V-ATPase Proton Transport Reveal Uncoupling Functions of Tether Linking Cytosolic and Membrane Domains of V0 Subunit a (Vph1p)

“…V-ATPases carefully control the acidic pH essential for endocytic and exocytic vesicular transport, zymogen activation, and protein sorting and degradation. Cells specialized for active proton secretion also express V-ATPases at the plasma membrane, where the efflux of cytosolic protons sustains the acidic luminal pH necessary for sperm maturation (4), urinary acidification (5), and bone resorption (6).…”