Regulation of heme oxygenase and metallothionein gene expression by the heme analogs, cobalt-, and tin-protoporphyrin

Abstract Two heme analogs, cobalt- and tin-protoporphyrin (CoPP and SnPP, respectively) have been used to probe the heme-hemopexin interaction, hemopexin receptor binding, and the mechanism of regulation of heme oxygenase (HO) and metallothionein-1 (MT-1) gene expression by hemopexin. Both CoPP and SnPP are HO inhibitors and hemopexin binds SnPP (Morgan, W. T., Alam, J., Deaciuc, V., Muster, P., Tatum, F. M., and Smith, A. (1988) J. Biol. Chem. 263, 8226-8231) and CoPP. The association of CoPP with hemopexin produces characteristic changes in the absorbance spectrum of CoPP and quenches the intrinsic fluorescence of hemopexin. Binding of CoPP is tight (Kd ca. 3 x 10(-7) M) although of lower affinity than heme itself (Kd < pM); and CoPP binding, like heme, produces conformational changes in hemopexin shown by an increase in the molar ellipticity at 233 nm and affords protection from proteolysis of the hinge region between the two structural domains of hemopexin. The coordination of the central cobalt atom is predicted to be similar to that of heme and to involve His56 and His127 of rabbit hemopexin. Furthermore, CoPP-hemopexin, like SnPP-hemopexin, binds to the hemopexin receptor as shown by competitive inhibition studies with radioactive heme-hemopexin. The effect of free heme analogs and their hemopexin complexes on HO and MT gene regulation was investigated and compared with the extent of induction by heme and heme-hemopexin. Free CoPP is a more effective inducer of HO steady state mRNA levels than free heme and produces a 5-fold increase within 1 h compared to only a 2-fold increase with heme, but free SnPP (up to 10 microM) produces no detectable increase in HO mRNA. In contrast, by 3 h heme-hemopexin and SnPP-hemopexin increase HO mRNA levels 11- and 6-fold, respectively; but the CoPP-hemopexin complex causes no detectable change in HO mRNA levels. The complexes of hemopexin with heme or either of the two heme analogs are effective inducers of metallothionein (MT) mRNA. Induction of MT mRNA by heme-hemopexin is rapid, increasing 4-fold within 1 h and 14-fold by 3-4 h. Strikingly, an even more rapid and slightly more extensive induction of MT mRNA is seen in response to either CoPP- or SnPP-hemopexin complexes, with MT mRNA rising 8-fold within 1 h. In contrast, free heme and the free analogs are far less effective inducers, increasing MT and mRNA levels and in vitro transcription rates only 3-4-fold and declining after 2-3 h.(ABSTRACT TRUNCATED AT 400 WORDS)