title: Reactive oxygen species mediate tumor necrosis factor alpha-converting, enzyme-dependent ectodomain shedding induced by phorbol myristate acetate.
abstract: Ectodomain shedding of cell surface membrane-anchoring proteins is an important process in a wide variety of physiological events(1, 2). Tumor necrosis factor alpha (TNF-alpha) converting enzyme (TACE) is the first discovered mammalian sheddase responsible for cleavage of several important surface proteins, including TNF-alpha, TNF p75 receptor, L-selectin, and transforming growth factor-a. Phorbol myristate acetate (PMA) has long been known as a potent agent to enhance ectodomain shedding. However, it is not fully understood how PMA activates TACE and induces ectodomain shedding. Here, we demonstrate that PMA induces both reactive oxygen species (ROS) generation and TNF p75 receptor shedding in Mono Mac 6 cells, a human monocytic cell line, and l-selectin shedding in Jurkat T-cells. ROS scavengers significantly attenuated PMA-induced TNF p75 receptor shedding. Exogenous H2O2 mimicked PMA-induced enhancement of ectodomain shedding, and H2O2-induced shedding was blocked by TAPI, a TACE inhibitor. Furthermore, both PMA and H2O2 failed to cause ectodomain shedding in a cell line that lacks TACE activity. By use of an in vitro TACE cleavage assay, H2O2 activated TACE that had been rendered inactive by the addition of the TACE inhibitory pro-domain sequence. We presume that the mechanism of TACE activation by H2O2 is due to an oxidative attack of the pro-domain thiol group and disruption of its inhibitory coordination with the Zn++ in the catalytic domain of TACE. These results demonstrate that ROS production is involved in PMA-induced ectodomain shedding and implicate a role for ROS in other shedding processes.
authors.affiliation: LSUMC Gene Therapy Program, Department of Pediatrics, Louisiana State University School of Medicine, New Orleans, Louisiana 70112, USA.
authors.forename: Z
authors.initials: Z
authors.lastname: Zhang
authors.forename: P
authors.initials: P
authors.lastname: Oliver
authors.forename: J R
authors.initials: JR
authors.lastname: Lancaster
authors.forename: P O
authors.initials: PO
authors.lastname: Schwarzenberger
authors.forename: M S
authors.initials: MS
authors.lastname: Joshi
authors.forename: J
authors.initials: J
authors.lastname: Cork
authors.forename: J K
authors.initials: JK
authors.lastname: Kolls
mesh_terms: D051722:ADAM Proteins; D000072198:ADAM17 Protein; D000111:Acetylcysteine / Q000494:pharmacology; D015703:Antigens, CD / Q000187:drug effects / Q000502:physiology*; D000975:Antioxidants / Q000494:pharmacology; D002460:Cell Line; D003513:Cycloheximide / Q000494:pharmacology; D004151:Dipeptides / Q000494:pharmacology; D004789:Enzyme Activation; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D006877:Hydroxamic Acids / Q000494:pharmacology; D019169:Jurkat Cells; D008666:Metalloendopeptidases / Q000378:metabolism*; D008954:Models, Biological; D009000:Monocytes / Q000187:drug effects / Q000502:physiology*; D011480:Protease Inhibitors / Q000494:pharmacology; D017382:Reactive Oxygen Species; D018124:Receptors, Tumor Necrosis Factor / Q000187:drug effects / Q000502:physiology*; D047889:Receptors, Tumor Necrosis Factor, Type II; D013482:Superoxide Dismutase / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D014409:Tumor Necrosis Factor-alpha / Q000502:physiology
chemical_list: D015703:Antigens, CD; D000975:Antioxidants; D004151:Dipeptides; D006877:Hydroxamic Acids; C092152:N-((2-(hydroxyaminocarbonyl)methyl)-4-methylpentanoyl)-3-(2'-naphthyl)alanylalanine, 2-aminoethylamide; D011480:Protease Inhibitors; D017382:Reactive Oxygen Species; D018124:Receptors, Tumor Necrosis Factor; D047889:Receptors, Tumor Necrosis Factor, Type II; D014409:Tumor Necrosis Factor-alpha; D003513:Cycloheximide; D006861:Hydrogen Peroxide; D013482:Superoxide Dismutase; D051722:ADAM Proteins; D008666:Metalloendopeptidases; D000072198:ADAM17 Protein; C000606462:ADAM17 protein, human; D013755:Tetradecanoylphorbol Acetate; D000111:Acetylcysteine