Effect of statins on heme‐oxyegnase‐1 in macrophages and NIH 3T3cells

Abstract

Heme‐oxygenase‐1 (HO‐1) is an antioxidant and anti‐inflammatory enzyme responsible for the degradation of heme into biliverdin, carbon monoxide and iron. Statins have pleiotropic effects in addition to lowering cholesterol. We have inverstigated the effect of statins on HO‐1 in Raw 267.4 cells and primary cells. Induction of HO‐1 was obtained with 25 μM simvastatin or 10 μM fluvastatin in both Raw 267.4 and peritoneal macrophages. Statins alone increased NO formation and iNOS expression in Raw 264.7 cells and pretreatment with inhibitors of NO synthase, l‐NMMA and 1400W blocked this effect. This induction was NO independent in primary eMPM cells. Both statins increased the promoter activity of the mouse proximal and distal promoter in Raw 264.7 cells, effect inhibited by mevalonate and 1400W. Gel retardation experiments were performed and both statins induced nuclear protein‐DNA complexes compared to untreated cells after 12 or 24‐hour incubation with probes for C/EBP, which were supershifted with antibodies specific for C/EBP β, or AP‐1 but not USF. In NIH 3T3 fibroblast cells, statins induced a significant increase in HO‐1 protein and mRNA levels and stimulated activity of mouse HO‐1 promoter (−1287 bp to +73 bp)/luciferase reporter gene by 3.25 ± 0.23 (Mean ± S.E.M., n= 15, p<0.001, t‐test) and 3.13 ± 0.33 (Mean ± S.E.M., n= 6, p<0.001, t‐test), respectively. Gel retardation experiments for C/EBP and USF on statin‐treated cells showed significant nuclear protein‐DNA complexes which were supershifted with antibodies specific for C/EBP or USF. In summary, statins induce HO‐1 and involve signaling molecules and mechanisms that vary among different cell types.