Control of antioxidative response by the tumor suppressor protein PML through regulating Nrf2 activity

Oxidative stress is a consequence of an imbalance between reactive oxygen species (ROS) production and the ability of the cytoprotective system to detoxify the reactive intermediates. The tumor suppressor promyelocytic leukemia protein (PML) functions as a stress sensor. Loss of PML results in impaired mitochondrial complex II activity, increased ROS, and subsequent activation of nuclear factor erythroid 2–related factor 2 (Nrf2) antioxidative pathway. We also demonstrate that sulforaphane (SFN), an antioxidant, regulates Nrf2 activity by controlling abundance and subcellular distribution of PML and that PML is essential for SFN-mediated ROS increase, Nrf2 activation, antiproliferation, antimigration, and antiangiogenesis. Taking the results together, we have uncovered a novel antioxidative mechanism by which PML regulates cellular oxidant homeostasis by controlling complex II integrity and Nrf2 activity and identified PML as an indispensable mediator of SFN activity. INTRODUCTION Oxidative stress is a consequence of an imbalance between reactive oxygen species (ROS) production and the ability of the cytoprotective system to detoxify the reactive intermediates. Basal levels of reactive intermediates are critical for relaying signal transduction and the maintenance of cellular function (Sena and Chandel, 2012). However, excessive production of ROS leads to oxidative stress and is correlated with the onset and progression of many diseases, such as atherosclerosis, diabetes, neurodegeneration, and cancer (Andersen, 2004; Nishikawa and Araki, 2007; Heistad et al., 2009; Trachootham et al., 2009). Nuclear factor erythroid 2–related factor 2 (Nrf2) is a member of the cap’n’collar-related basic region-leucine zipper transcription factors and plays a pivotal role in the cellular defense system against oxidative stress (Moi et al., 1994; Itoh et al., 1997). In resting cells, Nrf2 resides in the cytosol through association with Kelch-like ECH-associated protein 1 (Keap1). Keap1 complexes with cullin 3 to form a ubiquitin E3 ligase that acts as a substrate adaptor that binds Nrf2 and promotes its ubiquitination-dependent proteasomal degradation (Itoh et al., 1999). Upon ROS exposure, several reactive cysteine residues in Keap1 are covalently modified, resulting in Keap1 inactivation and subsequent stabilization and nuclear translocation of Nrf2 (Zhang and Hannink, 2003). Nuclear Nrf2 heterodimerizes with a small Maf protein and binds to antioxidant response elements (AREs) in the promoter regions of a subset of antioxidative genes that includes NAD(P)H dehydrogenase [quinone] 1 (NQO1; Venugopal and Jaiswal, 1996; Tong et al., 2006; Yamamoto et al., 2008). Induction of Nrf2 by ROS leads to a marked increase in the expression of target genes that protect the cells from oxidative stress–mediated cytotoxicity (Itoh et al., 1997). In addition to antioxidative enzymes, Nrf2 target genes also include cytoprotective genes involved in several protective systems, such as conjugating/detoxification enzymes, molecular chaperones, transporters, and anti-inflammatory signaling molecules (Kensler et al., 2007). Sulforaphane (SFN) belongs to the isothiocyanate family and is widely used as an antioxidant supplement and applied in cancer chemoprevention. The precursor of SFN, glucoraphanin, is abundant in cruciferous vegetables, with the highest concentration found in broccoli (Zhang et al., 1992). Among known SFN targets, Keap1, Monitoring Editor A. Gregory Matera University of North Carolina Received: Nov 20, 2013 Revised: May 1, 2014 Accepted: Jun 10, 2014 This article was published online ahead of print in MBoC in Press (http://www .molbiolcell.org/cgi/doi/10.1091/mbc.E13-11-0692) on June 18, 2014. Address correspondence to: Hung-Ying Kao (hxk43@cwru.edu) © 2014 Guo et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0). “ASCB®,” “The American Society for Cell Biology®,” and “Molecular Biology of the Cell®” are registered trademarks of The American Society of Cell Biology. Abbreviations used: APL, acute promyelocytic leukemia; ARE, antioxidant response element; ChIP, chromatin immunoprecipitation; CHX, cycloheximide; DAPI, 4′,6-diamidino-2-phenylindole; EC, endothelial cell; HUVEC, human umbilical vein endothelial cell; Keap1, Kelch-like ECH-associated protein 1; MEF, mouse embryonic fibroblast; NAC, N-acetyl cysteine; NB, nuclear body; NQO1, NAD(P)H dehydrogenase [quinone] 1; Nrf2, nuclear factor erythroid 2–related factor 2; PML, promyelocytic leukemia protein; ROS, reactive oxygen species; SFN, sulforaphane.