title: Enoxacin directly inhibits osteoclastogenesis without inducing apoptosis.
abstract: Enoxacin has been identified as a small molecule inhibitor of binding between the B2-subunit of vacuolar H+-ATPase (V-ATPase) and microfilaments. It inhibits bone resorption by calcitriol-stimulated mouse marrow cultures. We hypothesized that enoxacin acts directly and specifically on osteoclasts by disrupting the interaction between plasma membrane-directed V-ATPases, which contain the osteoclast-selective a3-subunit of V-ATPase, and microfilaments. Consistent with this hypothesis, enoxacin dose-dependently reduced the number of multinuclear cells expressing tartrate-resistant acid phosphatase (TRAP) activity produced by RANK-L-stimulated osteoclast precursors. Enoxacin (50 μM) did not induce apoptosis as measured by TUNEL and caspase-3 assays. V-ATPases containing the a3-subunit, but not the "housekeeping" a1-subunit, were isolated bound to actin. Treatment with enoxacin reduced the association of V-ATPase subunits with the detergent-insoluble cytoskeleton. Quantitative PCR revealed that enoxacin triggered significant reductions in several osteoclast-selective mRNAs, but levels of various osteoclast proteins were not reduced, as determined by quantitative immunoblots, even when their mRNA levels were reduced. Immunoblots demonstrated that proteolytic processing of TRAP5b and the cytoskeletal protein L-plastin was altered in cells treated with 50 μM enoxacin. Flow cytometry revealed that enoxacin treatment favored the expression of high levels of DC-STAMP on the surface of osteoclasts. Our data show that enoxacin directly inhibits osteoclast formation without affecting cell viability by a novel mechanism that involves changes in posttranslational processing and trafficking of several proteins with known roles in osteoclast function. We propose that these effects are downstream to blocking the binding interaction between a3-containing V-ATPases and microfilaments.
authors.affiliation: Department of Orthodontics, University of Florida College of Dentistry, Gainesville, Florida 32610.
authors.forename: Edgardo J
authors.initials: EJ
authors.lastname: Toro
authors.affiliation: Department of Orthodontics, University of Florida College of Dentistry, Gainesville, Florida 32610.
authors.forename: Jian
authors.initials: J
authors.lastname: Zuo
authors.affiliation: Department of Pathology, Immunology, and Laboratory Medicine, University of Florida College of Medicine, Gainesville, Florida 32610.
authors.forename: David A
authors.initials: DA
authors.lastname: Ostrov
authors.affiliation: Department of Oral Biology, University of Florida College of Dentistry, Gainesville, Florida 32610.
authors.forename: Dana
authors.initials: D
authors.lastname: Catalfamo
authors.affiliation: Laboratory of Oral Biology, Department of Dental Materials, School of Dentistry, University of São Paulo, 05508-900 São Paulo SP, Brazil.
authors.forename: Vivian
authors.initials: V
authors.lastname: Bradaschia-Correa
authors.affiliation: Laboratory of Oral Biology, Department of Dental Materials, School of Dentistry, University of São Paulo, 05508-900 São Paulo SP, Brazil.
authors.forename: Victor
authors.initials: V
authors.lastname: Arana-Chavez
authors.affiliation: Department of Orthodontics, University of Florida College of Dentistry, Gainesville, Florida 32610.
authors.forename: Aliana R
authors.initials: AR
authors.lastname: Caridad
authors.affiliation: Department of Orthodontics, University of Florida College of Dentistry, Gainesville, Florida 32610.
authors.forename: John K
authors.initials: JK
authors.lastname: Neubert
authors.affiliation: Department of Physiological Sciences, University of Florida, Gainesville, Florida 32610.
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Wronski
authors.affiliation: Department of Oral Biology, University of Florida College of Dentistry, Gainesville, Florida 32610.
authors.forename: Shannon M
authors.initials: SM
authors.lastname: Wallet
authors.affiliation: Department of Orthodontics, University of Florida College of Dentistry, Gainesville, Florida 32610; Department of Anatomy and Cell Biology, University of Florida College of Medicine, Gainesville, Florida 32610. Electronic address: sholliday@dental.ufl.edu.
authors.forename: L Shannon
authors.initials: LS
authors.lastname: Holliday
mesh_terms: D000199:Actins / Q000378:metabolism; D000818:Animals; D017209:Apoptosis; D003598:Cytoskeletal Proteins; D003599:Cytoskeleton / Q000378:metabolism; D015365:Enoxacin / Q000494:pharmacology*; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology; D008565:Membrane Proteins / Q000096:biosynthesis; D051379:Mice; D008840:Microfilament Proteins; D009419:Nerve Tissue Proteins / Q000096:biosynthesis; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology*; D010010:Osteoclasts / Q000166:cytology / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism; D059748:Proteolysis; D012333:RNA, Messenger / Q000378:metabolism; D025262:Vacuolar Proton-Translocating ATPases / Q000037:antagonists & inhibitors* / Q000378:metabolism*
chemical_list: D000199:Actins; D003598:Cytoskeletal Proteins; C499636:DC-STAMP protein, mouse; D008565:Membrane Proteins; D008840:Microfilament Proteins; D009419:Nerve Tissue Proteins; D019384:Nucleic Acid Synthesis Inhibitors; D010750:Phosphoproteins; D012333:RNA, Messenger; C066559:Lcp1 protein, mouse; D015365:Enoxacin; D025262:Vacuolar Proton-Translocating ATPases