Statins modulate transcriptional activity of heme-oxygenase-1 promoter in NIH 3T3 Cells.

Statins, inhibitors of HMG CoA reductase, have pleiotropic effects independent of their capacity to lower cholesterol. Heme-oxygenase-1(HO-1) plays an important role as an anti-oxidant and anti-inflammatory enzyme. In the present study, we used NIH 3T3 cells which express HO-1 to investigate the molecular mechanisms of HO-1 induction by statins. Simvastatin or fluvastatin induced a significant increase in HO-1 protein expression and mRNA levels. Both statins stimulated activity of a mouse HO-1 promoter (-1,287 to +73 bp)/luciferase reporter gene, 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. This effect was more pronounced in the short proximal promoter than the full promoter of HO-1. Gel retardation experiments for C/EBP and upstream stimulatory factor (USF) DNA-binding activities using simvastatin- or fluvastatin-treated cells showed significant nuclear protein-DNA complexes which were supershifted with antibodies specific for C/EBP β and δ or USF-1 and USF-2. Point mutations of the proximal HO-1 promoter (-149 to +73 bp) for the myc/max which binds USF or the C/EBP binding sequences showed a reduction in statin-induced reporter activity whereas no role of the distal C/EBP binding elements located at -4 kb was observed. Moreover, overexpression of mutated C/EBP β and USF factor or the siRNA for both factors supported a role of these transcription factors in statin-dependent induction of HO-1, with a clearer effect for C/EBP.