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

Abstract 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. J. Cell. Biochem. 113: 3466–3475, 2012. © 2012 Wiley Periodicals, Inc.