title: Luteolin inhibition of V-ATPase a3-d2 interaction decreases osteoclast resorptive activity.
abstract: V-ATPase-mediated acid secretion is required for osteoclast bone resorption. Osteoclasts are enriched in V-ATPase a3 and d2 subunit isoforms, and disruption of either of their genes impairs bone resorption. Using purified fusion proteins of a3 N-terminal domain (NTa3) and full-length d subunits we determined in a solid-phase binding assay that half-maximal binding of d1 or d2 to immobilized NTa3 occurs at 3.1 ± 0.4 or 3.6 ± 0.6 nM, respectively, suggesting equally high-affinity interactions. A high-throughput modification of this assay was then used to screen chemical libraries for a3-d2 interaction inhibitors, and luteolin, a naturally occurring flavonoid, was identified, with half-maximal inhibition at 2.4 ± 0.9 µM. Luteolin did not significantly affect NIH/3T3 or RAW 264.7 cell viability, nor did it affect cytokine-induced osteoclastogenesis of RAW 264.7 cells or bone marrow mononuclear cells at concentrations ≤ 40 µM. Luteolin inhibited osteoclast bone resorption with an EC(50) of approximately 2.5 µM, without affecting osteoclast actin ring formation. Luteolin-treated osteoclasts produced deeper resorption pits, but with decreased surface area, resulting in overall decreased pit volume. Luteolin did not affect transcription, or protein levels, of V-ATPase subunits a3, d2, and E, or V(1) V(0) assembly. Previous work has shown that luteolin can be effective in reducing bone resorption, and our studies suggest that this effect of luteolin may be through disruption of osteoclast V-ATPase a3-d2 interaction. We conclude that the V-ATPase a3-d2 interaction is a viable target for novel anti-resorptive therapeutics that potentially preserve osteoclast-osteoblast signaling important for bone remodeling.
authors.affiliation: Dental Research Institute, University of Toronto, Toronto, Ontario, Canada M5G 1G6.
authors.forename: Gazelle J
authors.initials: GJ
authors.lastname: Crasto
authors.forename: Norbert
authors.initials: N
authors.lastname: Kartner
authors.forename: Yeqi
authors.initials: Y
authors.lastname: Yao
authors.forename: Keying
authors.initials: K
authors.lastname: Li
authors.forename: Liv
authors.initials: L
authors.lastname: Bullock
authors.forename: Alessandro
authors.initials: A
authors.lastname: Datti
authors.forename: Morris F
authors.initials: MF
authors.lastname: Manolson
mesh_terms: D000199:Actins / Q000378:metabolism; D000818:Animals; D001854:Bone Marrow Cells / Q000187:drug effects / Q000378:metabolism; D001862:Bone Resorption / Q000473:pathology*; D002454:Cell Differentiation / Q000187:drug effects; D045744:Cell Line, Tumor; D048429:Cell Size / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D057166:High-Throughput Screening Assays; D007527:Isoenzymes / Q000378:metabolism; D047311:Luteolin / Q000494:pharmacology*; D051379:Mice; D046912:Multiprotein Complexes / Q000037:antagonists & inhibitors*; D041681:NIH 3T3 Cells; D010010:Osteoclasts / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D010012:Osteogenesis; D025941:Protein Interaction Mapping; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D025262:Vacuolar Proton-Translocating ATPases / Q000378:metabolism*
chemical_list: D000199:Actins; C484226:Atp6ap1 protein, mouse; D007527:Isoenzymes; D046912:Multiprotein Complexes; D011993:Recombinant Fusion Proteins; C542312:Atp6v0d2 protein, mouse; D025262:Vacuolar Proton-Translocating ATPases; D047311:Luteolin