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. S ince bilirubin is a potent antioxidant and heme a pro-oxidant, HO may protect cells against oxidative damage. HO-1 is highly inducible by di verse chemical agents, resembling those evoking induction of phase 2 e nzymes (i.e., Michael reaction accepters, heavy metals, trivalent arse nicals, and sulfhydryl reagents). Phase 2 enzymes (glutathione transfe rases; NAD(P)H:quinone reductase; glucuronosyltransferases) are regula ted by antioxidant-responsive elements (ARE), and their induction prot ects against chemical carcinogenesis. Is HO-1 regulated by chemical ag ents and enhancer elements similar to those controlling phase 2 enzyme s? 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 (TGA(C)/(G) (C)/( A)A) which overlap ARE consensus sequences (TGACNNNGC). Therefore, mut ations of the SX2 element designed to distinguish ARE from TRE were in serted 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 in ducers of phase 2 enzymes. Results: All compounds raised HO-1 mRNA and CAT expression constructs containing wild-type SX2. When the SX2 regi on was mutated to alter TRE consensus sequences without destroying the AXE consensus, full inducibility was preserved. Conversely, when the ARE consensus was disturbed, inducibility was abolished. Conclusion: I nduction of heme oxygenase-l is regulated by several chemically distin ct classes of inducers (mostly electrophiles), which also induce phase 2 enzymes, and these inductions are mediated by similar AREs. These f indings support the importance of HO-1 as a protector against oxidativ e damage and suggest that HO-1 induction is pari of a more generalized protective cellular response that involves phase 2 enzymes.