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.

Redox-based regulation of signal transduction: Principles, pitfalls, and promises

“…Various studies have suggested that MMPs and ADAMs can be activated by oxidants or by NO/SNO, which is suggested to involve oxidation and/or S-nitrosylation of the prodomain cysteine residue [137][138][139], although this is generally supported by indirect evidence. Biochemical analysis of oxidative activation of purified MMPs has shown that NO or H 2 O 2 are ineffective at directly activating these proteases, but they can be activated by more potent oxidants such as HOCl or ONOO − [140,141].…”

NADPH oxidases in lung biology and pathology: Host defense enzymes, and more

“…Critical to the activation of these signaling pathways is the proteolytic cleavage of membrane-associated EGFR ligands by cell surface proteases, such as TNFα-converting enzyme (TACE; ADAM17), a member of the a disintegrin and metalloproteinase (ADAM) family of metzincin metalloproteinases (105,108,121), and matrix metalloproteinases (MMPs) such as gelatinase B (MMP-9), which play critical roles in epithelial host defense and in epithelial repair in response to injury (104,122,123). An important common structural and functional feature shared by the ADAM/MMP proteases is the presence of an invariant cysteine-containing pro-domain, which maintains latency by ligating the active site Zn 2+ , and is susceptible to oxidative disruption (124)(125)(126)(127). Hence, the attractive hypothesis has been put forth that DUOX-derived H 2 O 2 causes direct activation of cell surface MMP/ ADAMs and thereby initiates EGFR-mediated cell signaling (87,105,108) (Fig.…”

Tumor necrosis factor α-converting enzyme mediates MUC5AC mucin expression in cultured human airway epithelial cells

“…TACE is produced in a latent form (11,12) and is activated by agents such as phorbol 12-myristate 13-acetate (PMA) (13) and reactive oxygen species (12,14), resulting in substrate cleavage. We hypothesized that TACE activation induces MUC5AC mucin production.…”