title: Ofloxacin induces apoptosis via β1 integrin-EGFR-Rac1-Nox2 pathway in microencapsulated chondrocytes.
abstract: Quinolones (QNs)-induced arthropathy is an important toxic side-effect in immature animals leading to the restriction of their therapeutic use in pediatrics. Ofloxacin, a typical QN, was found to induce the chondrocytes apoptosis in the early phase (12-48 h) of arthropathy in our previous study. However, the exact mechanism(s) is unclear. Microencapsulated juvenile rabbit joint chondrocytes, a three-dimensional culture system, is utilized to perform the present study. Ofloxacin, at a therapeutically relevant concentration (10 μg/ml), disturbs the interaction between β1 integrin and activated intracellular signaling proteins at 12 h, which is inhibited when supplementing Mg(2+). Intracellular reactive oxygen species (ROS) significantly increases in a time-dependent manner after exposure to ofloxacin for 12-48 h. Furthermore, ofloxacin markedly enhances the level of activated Rac1 and epidermal growth factor receptor (EGFR) phosphorylation, and its inhibition in turn reduces the ROS production, apoptosis and Rac1 activation. Silencing Nox2, Rac1 or supplementing Mg(2+) inhibits ROS accumulation, apoptosis occurrence and EGFR phosphorylation induced by ofloxacin. However, depletion of Nox2, Rac1 and inhibition of EGFR do not affect ofloxacin-mediated loss of interaction between β1 integrin and activated intracellular signaling proteins. In addition, ofloxacin also induces Vav2 phosphorylation, which is markedly suppressed after inactivating EGFR or supplementing Mg(2+). These results suggest that ofloxacin causes Nox2-mediated intracellular ROS production by disrupting the β1 integrin function and then activating the EGFR-Vav2-Rac1 pathway, finally resulting in apoptosis within 12-48 h exposure. The present study provides a novel insight regarding the potential role of Nox-driven ROS in QNs-induced arthropathy.
authors.affiliation: State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Science, Chinese Academy of Sciences, 18 Shuangqing Road, Beijing 100085, PR China.
authors.forename: Zhi-Guo
authors.initials: ZG
authors.lastname: Sheng
authors.forename: Wei
authors.initials: W
authors.lastname: Huang
authors.forename: Yu-Xiang
authors.initials: YX
authors.lastname: Liu
authors.forename: Ye
authors.initials: Y
authors.lastname: Yuan
authors.forename: Ben-Zhan
authors.initials: BZ
authors.lastname: Zhu
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology; D051017:Apoptosis Regulatory Proteins / Q000502:physiology*; D002478:Cells, Cultured; D019902:Chondrocytes / Q000187:drug effects / Q000502:physiology*; D004339:Drug Compounding / Q000379:methods; D066246:ErbB Receptors / Q000502:physiology*; D019012:Integrin beta1 / Q000502:physiology*; D008562:Membrane Glycoproteins / Q000502:physiology*; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases / Q000502:physiology*; D015242:Ofloxacin / Q000633:toxicity*; D011817:Rabbits; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D020830:rac1 GTP-Binding Protein / Q000502:physiology*
chemical_list: D051017:Apoptosis Regulatory Proteins; D019012:Integrin beta1; D008562:Membrane Glycoproteins; D015242:Ofloxacin; C056558:CYBB protein, human; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases; D066246:ErbB Receptors; D020830:rac1 GTP-Binding Protein