MECHANISM OF METALLOTHIONEIN GENE-REGULATION BY HEME-HEMOPEXIN - ROLES OF PROTEIN-KINASE-C, REACTIVE OXYGEN SPECIES, AND CIS-ACTING ELEMENTS

Heme-hemopexin or cobalt protoporphyrin (CoPP)-hemopexin (a model liga nd for hemopexin receptor occupancy) is shown to increase transcriptio n of the metallothionein-1 (MT-1) gene by activation of a signaling pa thway. Promoter deletion analysis followed by transient transfection a ssays show that 110 base pairs (-153 to -43) of 5'-flanking region of the murine MT-1 promoter are sufficient for increasing transcription i n response to heme-hemopexin or to CoPP-hemopexin in mouse hepatoma ce lls. The protein kinase C inhibitor, 1-(5-isoquinolinesulfonyl)-2-meth ylpiperazine dihydrochloride (H7), prevented the increase in MT-1 tran scription by heme-hemopexin, CoPP-hemopexin, or phorbol la-myristate 1 3-acetate, but the protein kinase A inhibitor, HA1004, was without eff ect. N-Acetylcysteine (NAC) and glutathione, as well as superoxide dis mutase and catalase, inhibited both the increase in endogenous MT-1 mR NA and the activation of reporter gene activity by heme-hemopexin, CoP P-hemopexin, and phorbol 12-myristate 13-acetate. In sum, these data s uggest that reactive oxygen intermediates are generated by heme-hemope xin via events associated with receptor binding, including protein kin ase C activation. Induction of heme oxygenase-1 expression, in contras t to MT-1, is significantly less sensitive to NAC. Deletion and mutati on analyses of the MT-1 proximal promoter revealed that the sequence 5 '-GTGACTATGC-3' (from -98 to -89 base pairs) is, in part, responsible for the hemopexin-mediated regulation of MT-1 which is inhibited by H7 . Regulation via this element is also induced by H2O2 showing that it is an antioxidant response element. Heme itself acts via more distal e lements on the MT-1 promoter. In contrast to NAC and glutathione, diet hyl dithiocarbamate and pyrrolidine dithiocarbamate, which inactivate reactive oxygen intermediates and chelate Zn(II), synergistically augm ent the induction of MT-1 mRNA levels and reporter gene activity in re sponse to heme-hemopexin via the antioxidant response element by both metal-responsive element-dependent and -independent mechanisms.