Parallel induction of heme oxygenase-1 and chemoprotective phase 2 enzymes by electrophiles and antioxidants: regulation by upstream antioxidant-responsive elements (ARE).
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?


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.
Prestera
T
T
Department of pharmacology and molecular sciences, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
Talalay
P
P
Alam
J
J
Ahn
Y I
YI
Lee
P J
PJ
Choi
A M
AM
D000975:Antioxidants / Q000494:pharmacology; D001483:Base Sequence; D002478:Cells, Cultured; D004790:Enzyme Induction; D004798:Enzymes / Q000096:biosynthesis / Q000235:genetics*; D015971:Gene Expression Regulation, Enzymologic; D014453:Glucuronosyltransferase / Q000096:biosynthesis / Q000235:genetics; D005982:Glutathione Transferase / Q000096:biosynthesis / Q000235:genetics; D006419:Heme Oxygenase (Decyclizing) / Q000096:biosynthesis / Q000235:genetics; D008969:Molecular Sequence Data; D016660:NAD(P)H Dehydrogenase (Quinone) / Q000096:biosynthesis / Q000235:genetics; D010084:Oxidation-Reduction; D018384:Oxidative Stress / Q000235:genetics*; D012045:Regulatory Sequences, Nucleic Acid
D000975:Antioxidants; D004798:Enzymes; D006419:Heme Oxygenase (Decyclizing); D016660:NAD(P)H Dehydrogenase (Quinone); D014453:Glucuronosyltransferase; D005982:Glutathione Transferase