title: Lysosomal pH Plays a Key Role in Regulation of mTOR Activity in Osteoclasts.
abstract: Mammalian target of rapamycin (mTOR) is a serine/threonine kinase involved in the regulation of cell growth. It has been shown to play an important role in osteoclast differentiation, particularly at the earlier stages of osteoclastogenesis. mTOR activation and function, as part of mTORC1 complex, is dependent on lysosomal localization and the vacuolar H(+) -ATPase (V-ATPase) activity; however, the precise mechanism is still not well understood. Using primary mouse osteoclasts that are known to have higher lysosomal pH due to R740S mutation in the V-ATPase a3 subunit, we investigated the role of lysosomal pH in mTORC1 signaling. Our results demonstrated that +/R740S cells had increased basal mTOR protein levels and mTORC1 activity compared to +/+ osteoclasts, while mTOR gene expression was decreased. Treatment with lysosomal inhibitors chloroquine and ammonium chloride, compounds known to raise lysosomal pH, significantly increased mTOR protein levels in +/+ cells, confirming the importance of lysosomal pH in mTOR signaling. These results also suggested that mTOR could be degraded in the lysosome. To test this hypothesis, we cultured osteoclasts with chloroquine or proteasomal inhibitor MG132. Both chloroquine and MG132 increased mTOR and p-mTOR protein levels in +/+ osteoclasts, suggesting that mTOR undergoes both lysosomal and proteasomal degradation. Treatment with cycloheximide, an inhibitor of new protein synthesis, confirmed that mTOR is constitutively expressed and degraded. These results show that, in osteoclasts, the lysosome plays a key role not only in mTOR activation but also in its deactivation through protein degradation, representing a novel molecular mechanism of mTOR regulation.
authors.affiliation: Faculty of Dentistry, University of Toronto, Toronto, ON, Canada.|Institute of Dental Medicine, Qilu Hospital, Shandong University, Jinan, China.
authors.forename: Yingwei
authors.initials: Y
authors.lastname: Hu
authors.affiliation: Faculty of Dentistry, University of Toronto, Toronto, ON, Canada.
authors.forename: Luciene R
authors.initials: LR
authors.lastname: Carraro-Lacroix
authors.affiliation: Faculty of Dentistry, University of Toronto, Toronto, ON, Canada.
authors.forename: Andrew
authors.initials: A
authors.lastname: Wang
authors.affiliation: Centre for Modeling Human Disease, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, ON, Canada.
authors.forename: Celeste
authors.initials: C
authors.lastname: Owen
authors.affiliation: Faculty of Dentistry, University of Toronto, Toronto, ON, Canada.
authors.forename: Elena
authors.initials: E
authors.lastname: Bajenova
authors.affiliation: Dalla Lana School of Public Health, University of Toronto, Toronto, ON, Canada.
authors.forename: Paul N
authors.initials: PN
authors.lastname: Corey
authors.affiliation: Cell Biology Program, The Hospital for Sick Children, Toronto, ON, Canada.|Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada.|Institute of Medical Science, University of Toronto, Toronto, ON, Canada.
authors.forename: John H
authors.initials: JH
authors.lastname: Brumell
authors.affiliation: Faculty of Dentistry, University of Toronto, Toronto, ON, Canada.
authors.forename: Irina
authors.initials: I
authors.lastname: Voronov
mesh_terms: D000818:Animals; D001343:Autophagy; D002478:Cells, Cultured; D004789:Enzyme Activation; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D008247:Lysosomes / Q000378:metabolism*; D008297:Male; D000076222:Mechanistic Target of Rapamycin Complex 1; D008809:Mice, Inbred C3H; D008822:Mice, Transgenic; D046912:Multiprotein Complexes / Q000378:metabolism; D010010:Osteoclasts / Q000201:enzymology*; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D021381:Protein Transport; D059748:Proteolysis; D058570:TOR Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*
chemical_list: D046912:Multiprotein Complexes; C546843:mTOR protein, mouse; D000076222:Mechanistic Target of Rapamycin Complex 1; D058570:TOR Serine-Threonine Kinases; D046988:Proteasome Endopeptidase Complex
keywords: AUTOPHAGY; LYSOSOME; OSTEOCLAST; V-ATPase; mTOR; pH