Abstract
Background
Heme oxygenase (HO; EC 1.14.99.3) catalyzes the conversion of heme to biliverdin, which is reduced enzymatically to bilirubin. Since bilirubin is a potent antioxidant and heme a pro-oxidant, HO may protect cells against oxidative damage. HO-1 is highly inducible by diverse chemical agents, resembling those evoking induction of phase 2 enzymes (i.e., Michael reaction acceptors, heavy metals, trivalent arsenicals, and sulfhydryl reagents). Phase 2 enzymes (glutathione transferases; NAD(P)H:quinone reductase; glucuronosyltransferases) are regulated by antioxidant-responsive elements (ARE), and their induction protects against chemical carcinogenesis. Is HO-1 regulated by chemical agents and enhancer elements similar to those controlling phase 2 enzymes?
Materials and Methods
Induction of HO-1 by phorbol ester and heavy metals is transcriptionally controlled through a 268-bp SX2 fragment, containing two phorbol ester-responsive (TRE) sites (TGAC/GT C/AA) which overlap ARE consensus sequences (TGACNNNGC). Therefore, mutations of the SX2 element designed to distinguish ARE from TRE were inserted into chloramphenicol acetyltransferase (CAT) reporter plasmids, and the response of the CAT activity of murine hepatoma cells stably transfected with these constructs was examined with a wide range of inducers of phase 2 enzymes.
Results
All compounds raised HO-1 mRNA and CAT expression constructs containing wild-type SX2. When the SX2 region was mutated to alter TRE consensus sequences without destroying the ARE consensus, full inducibility was preserved. Conversely, when the ARE consensus was disturbed, inducibility was abolished.
Conclusions
Induction of heme oxygenase-1 is regulated by several chemically distinct classes of inducers (mostly electrophiles), which also induce phase 2 enzymes, and these inductions are mediated by similar AREs. These findings support the importance of HO-1 as a protector against oxidative damage and suggest that HO-1 induction is part of a more generalized protective cellular response that involves phase 2 enzymes.
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Notes
The electrophile-responsive element (EpRE) was so named because it was shown to be activated by dimethyl fumarate, trans-4-phenylbut-3-en-2-one, and tert-butylhydroquinone (4). These compounds all have (or can easily acquire) Michael reaction acceptor groups, which are reactive electrophiles. In addition to its electrophilic potential, tert-butylhydroquinone is a well-known antioxidant and can engage in redox cycling reactions. The antioxidant responsive element (ARE) (5) was so named because it was responsive to redox-active quinone precursors (catechol, hydroquinone) or 1,4-benzoquinone itself, but failed to respond to a redox-inactive diphenol (resorcinol). Redox-active quinones, however, share a common property with most other monofunctional phase 2 enzyme inducers, in that they are also Michael reaction acceptors and thus are powerful electrophiles (6). Consequently, we prefer the term electrophile-responsive element (EpRE), as we have previously shown (6–8) that the common chemical characteristic of nearly all monofunctional phase 2 enzyme inducers is that they are electrophiles. Nevertheless, we respect the originally proposed nomenclature and have elsewhere referred to the enhancer element as ARE/EpRE (9). In this paper, it is less cumbersome to designate this element as ARE.
Two classes of enzymes metabolize xenobiotics: (i) phase 1 enzymes (mostly cytochromes P450), which functionalize molecules by introducing hydroxyl or epoxide groups; and (ii) phase 2 enzymes (10), which detoxify by either conjugating these functionalized molecules with endogenous ligands (e.g., glutathione), thus facilitating their excretion, or by destroying their reactive centers (e.g., hydrolysis of epoxides by epoxide hydrolase, or reduction of quinones by QR). Reasons for considering QR a phase 2 enzyme are presented elsewhere (11–13).
Inducers of enzymes of xenobiotic metabolism belong to two families (14): (i) Bifunctional inducers, which bind to the Aryl hydrocarbon (Ah) receptor and induce both certain phase 1 enzymes and phase 2 enzymes; and (ii) Monofunctional inducers, which induce phase 2 enzymes independently of the Ah receptor.
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Acknowledgments
The studies by TP and PT were supported by a grant from the National Cancer Institute, Department of Health and Human Services (PO1 CA44530). AMKC was supported by a Physician Scientist award (NIH/NIA K11 AG00516) and a research grant from the American Lung Association. JA was supported by a National Institutes of Health grant (RO1 DK43135). TP was supported by a fellowship from the National Cancer Institute (T32 CA09243), and PJL was supported by a Multidisciplinary Training Grant (NHLBI 07534). We thank Gale Doremus for preparing the manuscript and illustrations.
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Contributed by Paul Talalay on August 25, 1995.
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Prestera, T., Talalay, P., Alam, J. et al. Parallel Induction of Heme Oxygenase-1 and Chemoprotective Phase 2 Enzymes by Electrophiles and Antioxidants: Regulation by Upstream Antioxidant-Responsive Elements (ARE). Mol Med 1, 827–837 (1995). https://doi.org/10.1007/BF03401897
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DOI: https://doi.org/10.1007/BF03401897