Unique effects of zinc protoporphyrin on HO-1 induction and apoptosis.
Zinc protoporphyrin (ZnPP), a naturally occurring molecule, is increased in iron deficiency and lead intoxication. ZnPP can also induce heme oxygenase (HO-1), the enzyme it competitively inhibits. In cultured cells (HA-1), ZnPP was the strongest HO-1 inducer of any metalloporphyrin (MP) tested. This was not due to increased oxidative stress, enhanced binding at metal response element, nor increased binding at activator protein-1 (AP-1) or SP-1 sites on HO-1. Only ZnPP, however, increased binding of nuclear proteins to early growth response-1 (Egr-1) protein consensus sequence. Pretreatment of HA-1 with cycloheximide inhibited ZnPP-induced HO-1 messenger RNA (mRNA) by 55%. Incubation with antisense Egr-1 oligomers decreased ZnPP-induced HO-1 expression by 47%. Furthermore, the level of HO-1 mRNA induction by ZnPP was 2-fold less in Egr-1-deficient fibroblasts than in wild-type cells. Because no Egr-1 binding site was previously identified on the HO-1 promoter, HA-1 cells were transfected with HO-1 CAT constructs containing segments of a 12.5-kb enhancer region of HO-1. A 196-bp fragment (RH) located approximately 9.5 kb upstream of the transcription start site mediated HO-1 induction by ZnPP alone. DNase I footprinting analysis further revealed that nuclear proteins bound to a 50-bp sequence in the RH. Within this sequence, a novel 9-bp region with 78% homology to the Egr-1 consensus sequence was identified further suggesting that Egr-1 partially mediates HO-1 induction by ZnPP. Lastly, increased apoptosis and nuclear localization were only seen with ZnPP, suggesting that increased ZnPP in disease states may serve as a cellular signaling mechanism.
Department of Pediatrics, Stanford University School of Medicine, Stanford, CA 94305 USA.
G
G
Yang
X
X
Nguyen
J
J
Ou
P
P
Rekulapelli
D K
DK
Stevenson
P A
PA
Dennery
D021581:Active Transport, Cell Nucleus; D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D001665:Binding Sites; D018929:Cell Culture Techniques; D002453:Cell Cycle / Q000187:drug effects; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D051766:Early Growth Response Protein 1; D005347:Fibroblasts; D006419:Heme Oxygenase (Decyclizing) / Q000187:drug effects* / Q000235:genetics; D051547:Heme Oxygenase-1; D017874:Immediate-Early Proteins; D008565:Membrane Proteins; D008665:Metalloporphyrins / Q000378:metabolism / Q000494:pharmacology; D051379:Mice; D008817:Mice, Mutant Strains; D011485:Protein Binding; D011524:Protoporphyrins / Q000378:metabolism / Q000494:pharmacology*; D012333:RNA, Messenger / Q000187:drug effects; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology
D004268:DNA-Binding Proteins; D051766:Early Growth Response Protein 1; C486109:Egr1 protein, mouse; D017874:Immediate-Early Proteins; D008565:Membrane Proteins; D008665:Metalloporphyrins; D011524:Protoporphyrins; D012333:RNA, Messenger; D014157:Transcription Factors; C017803:zinc protoporphyrin; D006419:Heme Oxygenase (Decyclizing); D051547:Heme Oxygenase-1; C496542:Hmox1 protein, mouse