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Vol. 20, Issue 24, 5236-5249, December 15, 2009

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Mitochondrial Reactive Oxygen Species Mediate GPCR–induced TACE/ADAM17-dependent Transforming Growth Factor- Shedding

Timothy J. Myers^*,, Leann H. Brennaman^*, Mary Stevenson, Shigeki Higashiyama, William E. Russell, David C. Lee^*,,||, and Susan Wohler Sunnarborg^*,

*Department of Biochemistry and Biophysics and Lineberger Comprehensive Cancer Center, University of North Carolina School of Medicine, Chapel Hill, NC 27599; Departments of Pediatrics and Cell Biology, Vanderbilt University, Nashville, TN 37232; and Department of Biochemistry and Molecular Genetics, Ehime University Graduate School of Medicine, Shitsukawa, Toon, Ehime 791-0295, Japan

Submitted January 2, 2009; Revised September 28, 2009; Accepted October 14, 2009
Monitoring Editor: J. Silvio Gutkind

Epidermal growth factor receptor (EGFR) activation by GPCRs regulates many important biological processes. ADAM metalloprotease activity has been implicated as a key step in transactivation, yet the regulatory mechanisms are not fully understood. Here, we investigate the regulation of transforming growth factor- (TGF-) shedding by reactive oxygen species (ROS) through the ATP-dependent activation of the P2Y family of GPCRs. We report that ATP stimulates TGF- proteolysis with concomitant EGFR activation and that this process requires TACE/ADAM17 activity in both murine fibroblasts and CHO cells. ATP-induced TGF- shedding required calcium and was independent of Src family kinases and PKC and MAPK signaling. Moreover, ATP-induced TGF- shedding was completely inhibited by scavengers of ROS, whereas calcium-stimulated shedding was partially inhibited by ROS scavenging. Hydrogen peroxide restored TGF- shedding after calcium chelation. Importantly, we also found that ATP-induced shedding was independent of the cytoplasmic NADPH oxidase complex. Instead, mitochondrial ROS production increased in response to ATP and mitochondrial oxidative complex activity was required to activate TACE-dependent shedding. These results reveal an essential role for mitochondrial ROS in regulating GPCR-induced growth factor shedding.

^||Present address: The Office of the Vice President for Research, University of Georgia, Athens, GA 30602.

Address correspondence to: Susan Wohler Sunnarborg (susan_sunnarborg{at}med.unc.edu).

Abbreviations used: ADAM, a disintegrin and metalloprotease; AR, amphiregulin; BIM-I, bisindolylmaleimide I; DPI, diphenyleneiodonium; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; GPCR, G-protein–coupled receptor; HB-EGF, heparin-binding growth factor; MAPK, mitogen-activated protein kinase; NAC, N-acetyl-L-cysteine; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; RIA, radioimmunoassay; ROS, reactive oxygen species; TACE, tumor necrosis factor-–converting enzyme; TGF-, transforming growth factor-.