title: Effects of Bafilomycin A1: an inhibitor of vacuolar H (+)-ATPases on endocytosis and apoptosis in RAW cells and RAW cell-derived osteoclasts.
abstract: Bafilomycin A1, a specific inhibitor of V-ATPases, is a potent inhibitor of bone resorption, but the underlying mechanisms of its action remain unclear. In this study, we investigated the effect of Bafilomycin A1 on endocytosis and apoptosis in RAW cells and RAW cell-derived osteoclasts. Quantitative analysis by flow cytometry showed that Bafilomycin A1 increased total transferrin levels when RAW cells were exposed to labeled transferrin and decreased the total uptake of Dextran-rhodamine B, both in a dose- and time-dependent fashion, indicating that Bafilomycin influences receptor-mediated and fluid phase endocytosis in these cells. Furthermore, Bafilomycin A1 induced apoptosis of RAW cells in a dose dependent manner as evidenced by Annexin V flow cytometry. The action of Bafilomycin A1 on endocytotic events appeared to be more sensitive and occurred earlier than on its apoptosis inducing effects, suggesting that interrupting of endocytosis might be an early sign of Bafilomycin-mediated osteoclast inhibition. Semi-quantitative RT-PCR analysis showed that the gene transcripts of putative Bafilomycin A1 binding subunit, V-ATPase-subunit a3, were expressed in the preosteoclastic RAW cell line, and up-regulated during RANKL-induced osteoclastogenesis. Osteoclasts treated with Bafilomycin A1 exhibited apoptosis as well as altered cellular localization of Transferrin Alexa 647. Given that endocytosis and apoptosis are important processes during osteoclastic bone resorption, the potent effect of Bafilomycin A1 on endocytosis and apoptosis of osteoclasts and their precursor cells may in part account for Bafilomycin A1 inhibited bone resorption.
authors.affiliation: Department of Surgery (Orthopaedics), University of Western Australia, QEII Medical Center, Nedlands WA. jiakexu@cyllene.uwa.edu.au
authors.forename: Jiake
authors.initials: J
authors.lastname: Xu
authors.forename: Hao Ting
authors.initials: HT
authors.lastname: Feng
authors.forename: Cathy
authors.initials: C
authors.lastname: Wang
authors.forename: Kirk H M
authors.initials: KH
authors.lastname: Yip
authors.forename: Nathan
authors.initials: N
authors.lastname: Pavlos
authors.forename: John M
authors.initials: JM
authors.lastname: Papadimitriou
authors.forename: David
authors.initials: D
authors.lastname: Wood
authors.forename: Ming H
authors.initials: MH
authors.lastname: Zheng
mesh_terms: D017209:Apoptosis / Q000187:drug effects; D001862:Bone Resorption / Q000517:prevention & control; D002460:Cell Line / Q000187:drug effects; D002478:Cells, Cultured; D003911:Dextrans / Q000378:metabolism; D004705:Endocytosis / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015870:Gene Expression; D006801:Humans; D018942:Macrolides / Q000494:pharmacology*; D010010:Osteoclasts / Q000187:drug effects* / Q000502:physiology; D017422:Sequence Analysis, DNA; D014168:Transferrin / Q000378:metabolism; D025262:Vacuolar Proton-Translocating ATPases / Q000037:antagonists & inhibitors* / Q000737:chemistry
chemical_list: D003911:Dextrans; D004791:Enzyme Inhibitors; D018942:Macrolides; D014168:Transferrin; C040929:bafilomycin A1; D025262:Vacuolar Proton-Translocating ATPases