Critical promoter region for statin-induced human endothelial nitric oxide synthase (eNOS) transcription in EA.hy926 cells

Abstract

Aim: Statins have many anti-atherogenic effects apart from reducing the serum level of low density lipoprotein cholesterol (LDL-C). For instance, statins can enhance the expression of endothelial nitric oxide synthase (eNOS), at least partly by upregulating its transcription. Although it has been reported that -786 T/C polymorphism of the promoter region has an important influence on statininduced transcription of the human eNOS gene, much remains unclear about statin-induced eNOS transcription. We tried to identify other statin-responsive promoter regions. Methods: A human endothelial cell line (EA.hy926 cells) was treated with pitavastatin, atorvastatin, or fluvastatin, after which eNOS mRNA levels were assessed by quantitative real-time RT-PCR. EA.hy926 cells were also transiently transfected with luciferase reporter genes driven by various lengths of the human eNOS promoter and were treated with statins before luciferase activity was measured. Results: Statin treatment increased eNOS mRNA levels in EA.hy926 cells. In addition, cells transfected with the reporter gene driven by the eNOS promoter fragment starting from position -740 exhibited a pitavastatin-induced increase of luciferase activity, which was not observed in cells transfected with the reporter gene driven by the fragment starting from -727. Similar results were also obtained with atorvastatin and fluvastatin. Conclusions: Statins enhanced eNOS expression in EA.hy926 cells, at least partly by inducing its transcription. Although a statin-responsive sequence that could function even in a heterologous promoter was not precisely identified, the region of the human eNOS promoter around position -730 seems to be critical for statin-induced transcriptional activation.