title: Activation of the ubiquitin-proteasome system against arsenic trioxide cardiotoxicity involves ubiquitin ligase Parkin for mitochondrial homeostasis.
abstract: Parkin is an E3 ubiquitin ligase involved in the elimination of damaged mitochondria. Ubiquitination of mitochondrial substrates by Parkin results in proteasomal as well as lysosomal degradation of mitochondria, the latter of which is executed by the autophagy machinery and is called as mitophagy (mitochondrial autophagy). The aim of this study is to examine the possible role of Parkin against cardiotoxicity elicited by arsenic trioxide (ATO) exposure in HL-1 mouse atrial cardiomyocytes. HL-1 cells were administered 1-10μM ATO for up to 24h, and the involvements of apoptosis, and the ubiquitin-proteasome and autophagy-lysosome systems (UPS and ALS) were examined. ATO dose-dependently reduced mitochondrial membrane potentials (ΔΨm) in HL-1 cells, indicating that ATO works as a mitochondrial toxin in these cells. Apoptosis was evident in cells exposed to more than 6μM ATO for 24h. Levels of Parkin in mitochondria-rich fractions were increased, suggesting the recruitment of Parkin to mitochondria. Ubiquitination of the voltage-dependent anion channel1 (VDAC1), a substrate of Parkin, was also proved by immunoprecipitation. Accumulation of ubiquitinated proteins including both K48- and K63-lineages was observed in HL-1 cells after ATO exposure, implying an increased demand for proteasomal as well as lysosomal degradation of cellular proteins. Although UPS was activated by ATO as proved by increased proteasomal activity, only slight activation of the ALS marker LC3 was observed, suggesting differential reactions of UPS and ALS to ATO toxicity. The abrogation of UPS by the proteasome inhibitor bortezomib significantly sensitized HL-1 cells to ATO toxicity, showing the contribution of UPS to the maintenance of cellular homeostasis during ATO exposure. Taken together, our results reveal the activation of Parkin as well as UPS during ATO exposure in HL-1 cardiomyocytes, which contributes to the maintenance of mitochondrial as well as cellular homeostasis.
authors.affiliation: Section of Forensic Medicine, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan.
authors.forename: Mayumi
authors.initials: M
authors.lastname: Watanabe
authors.affiliation: Section of Forensic Medicine, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Funakoshi
authors.affiliation: Section of Forensic Medicine, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan.
authors.forename: Kana
authors.initials: K
authors.lastname: Unuma
authors.affiliation: Section of Forensic Medicine, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan. Electronic address: aki.legm@tmd.ac.jp.
authors.forename: Toshihiko
authors.initials: T
authors.lastname: Aki
authors.affiliation: Section of Forensic Medicine, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan.
authors.forename: Koichi
authors.initials: K
authors.lastname: Uemura
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D000077237:Arsenic Trioxide; D001152:Arsenicals; D001343:Autophagy / Q000187:drug effects; D015153:Blotting, Western; D001897:Boronic Acids / Q000494:pharmacology; D000069286:Bortezomib; D006331:Heart Diseases / Q000139:chemically induced* / Q000517:prevention & control*; D006706:Homeostasis / Q000187:drug effects*; D047468:Immunoprecipitation; D008247:Lysosomes / Q000187:drug effects; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D051379:Mice; D008856:Microscopy, Fluorescence; D008929:Mitochondria, Heart / Q000187:drug effects*; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000378:metabolism; D010087:Oxides / Q000633:toxicity*; D046988:Proteasome Endopeptidase Complex / Q000187:drug effects*; D011719:Pyrazines / Q000494:pharmacology; D025801:Ubiquitin / Q000187:drug effects*; D044767:Ubiquitin-Protein Ligases / Q000502:physiology*; D050995:Voltage-Dependent Anion Channel 1 / Q000096:biosynthesis / Q000235:genetics
chemical_list: D000970:Antineoplastic Agents; D001152:Arsenicals; D001897:Boronic Acids; D010087:Oxides; D011719:Pyrazines; D025801:Ubiquitin; C494763:Vdac1 protein, mouse; D000069286:Bortezomib; D050995:Voltage-Dependent Anion Channel 1; D044767:Ubiquitin-Protein Ligases; C111567:parkin protein; D046988:Proteasome Endopeptidase Complex; D000077237:Arsenic Trioxide
keywords: Arsenic trioxide; Cardiomyocyte; HL-1; Mitophagy; Parkin; Ubiquitin–proteasome system