Vacuolar H+-ATPase activity and expression in mouse bone marrow cultures.
We examined vacuolar H+-ATPase (V-ATPase) structure, enzymatic properties, and protein and mRNA expression from mouse marrow cultured in the presence or absence of 1,25-dihydroxyvitamin D3 (1, 25(OH)2D3), which stimulates formation of bone-resorptive osteoclasts. V-ATPases from osteoclast-containing cultures were similar in ion and inhibitor sensitivities to the enzyme from kidney-derived sources. Immunopurified V-ATPase from 1,25(OH)2D3-stimulated cultures exhibited 20-fold greater ATPase activity than the enzyme from unstimulated cultures, which do not contain osteoclasts. In contrast, 1,25(OH)2D3-treated cultures contained only 2-fold more assembled V-ATPase, as determined by immunoprecipitation. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) and immunoblot analysis similarly showed approximately 2-fold increases of V-ATPase mRNA and protein levels in 1,25(OH)2D3-treated cultures. The bulk of the relative difference in V-ATPase activity between the two cultures was due to a 10-fold difference in enzyme specific activity. Quantitative RT-PCR also revealed that expression levels of V-ATPase mRNAs reflected the stoichiometry of enzyme subunits in the assembled complex. These data indicate that in mouse bone marrow cultures, V-ATPase expression is controlled at the level of mRNA, and that increases in subunit expression and assembly cannot account for the 20-fold difference in enzyme activity in osteoclast-containing cultures. Therefore, osteoclast V-ATPase activity may be regulated by subtle alterations in enzyme structure or associated factors.
Lee
B S
BS
Department of Medicine/Renal Division, Washington University School of Medicine, St. Louis, Missouri, USA.
Holliday
L S
LS
Krits
I
I
Gluck
S L
SL
D000818:Animals; D001854:Bone Marrow Cells / Q000201:enzymology*; D002117:Calcitriol / Q000494:pharmacology; D002478:Cells, Cultured; D015971:Gene Expression Regulation, Enzymologic; D017403:In Situ Hybridization; D051379:Mice; D010010:Osteoclasts / Q000378:metabolism; D011233:Precipitin Tests; D006180:Proton-Translocating ATPases / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D025262:Vacuolar Proton-Translocating ATPases
D012333:RNA, Messenger; D025262:Vacuolar Proton-Translocating ATPases; D006180:Proton-Translocating ATPases; D002117:Calcitriol