Abstract 1374
Neonatal Disease Oriented Research: Steroids & Oxygen: Perinatal Effects Poster Symposium, Sunday, 5/2
Metalloporphyrins (Mp) are known inhibitors of heme oxygenase (HO), the rate limiting enzyme in bilirubin production, and are considered for clinical use. However, some of these agents can induce HO-1 mRNA and this may interfere with their therapeutic benefit. We investigated the induction of heme oxygenase-1 (HO-1) by zinc protoporphyrin (ZnPP; 10 micromolar) in cultured hamster fibroblasts as compared to other Mp at a similar concentration. We determined HO-1 mRNA and protein by Northern and Western analysis respectively. Transcriptional factor binding activity was evaluated by electrophoretic mobility shift assay (EMSA). Several markers of oxidative stress (protein oxidation, lipid peroxidation, glutathione depletion) were also assessed to verify whether oxidative stress accounted for any induction of HO-1, as is commonly the case. When compared to chromium (CrPP) or tin protoporphyrin (SnPP), incubation with ZnPP resulted in a much higher degree of HO-1 induction at 24 hours. The induction of HO-1 by ZnPP or other Mp was not related to increased oxidative stress. Analysis by EMSA indicated that there was no enhanced activator protein-1 or metal response element binding activity with any Mp. In contrast, binding activity of the zinc finger protein SP-1 was increased in SnPP and CrPP but not ZnPP whereas increased binding of a competitive zinc finger protein, early growth response-1 (Egr-1) was observed with all Mp. Furthermore, pre-incubation with an Egr-1 antisense oligomer to block Egr-1 synthesis was associated with a 50% decrease in HO-1 mRNA and protein levels after incubation with ZnPP corroborating activation via Egr-1. Addition of cycloheximide to prevent protein synthesis prior to ZnPP or SnPP did not completely inhibit HO-1 mRNA induction nor did pre-incubation with two mitogen-activated protein (MAP) kinase inhibitors, PD98059 and SB203580 (10-20 % decrease with both cycloheximide and MAP kinase inhibitors). Visualization of intracellular ZnPP and SnPP with laser confocal microscopy revealed nuclear translocation of ZnPP only, further suggesting a direct effect of ZnPP on HO-1 transcription. Lastly, incubation with ZnPP but not with CrPP or SnPP was associated with increased p53 and annexin V protein, suggesting increased apoptosis with ZnPP only. These observations suggest that Mp mediate HO-1 induction in part by Egr-1 binding but that there may be a direct effect of ZnPP on HO-1 gene regulation. In addition, since increased Egr-1 binding is associated with apoptosis and that markers of apoptosis were increased with ZnPP, we speculate that ZnPP may mediate apoptotic signaling via Egr-1 binding. This apoptotic signaling may not have been observed with other Mp since competition for Egr-1 binding sites via increased Sp-1 likely obviated Egr-1 effects.
