Effect of pitavastatin on transactivation of human serum paraoxonase 1 gene

Abstract

Hepatic hydroxymethyl glutary coenzyme A HMG-CoA reductase inhibitors (statins) have various anti atherosclerosis pleiotropic effects that are independent of cholesterol reduction. Human serum paraoxonase 1 (PON1) is associated with high-density lipoprotein (HDL) and inhibits the oxidative modification of low-density lipoprotein (LDL). We investigated the effects of statins on PON1 gene transcription using a reporter gene assay. Promoter activity of the PON1 gene was estimated by measuring luciferase activity of plasmids with a PON1 promoter region transfected into human hepatoma HepG2 cells and human embryonic kidney (HEK) 293 cells. Pitavastatin, simvastatin, and atorvastatin each significantly increased PON1 promoter activity, and the transactivation by pitavastatin was abrogated by mevalonic acid and farnesyl pyrophosphate (FPP), however, not by geranylgeranyl pyrophosphate. Further, PON1 promoter activity was enhanced by farnesyl transferase inhibitor (FTI), but not by geranylgeranyl transferase inhibitor (GGTI). PON1 gene transcription has been reported to be dependent on Sp1 and the transactivation by pitavastatin was completely abrogated by mithramycin, an inhibitor of Sp1. Our results suggest that pitavastatin activates transcription of the PON1 gene through the FPP pathway, which may play an important role in the anti atherosclerotic effects of statins.

Introduction

Human serum paraoxonase 1 (PON1) is an esterase that hydrolyses aromatic carboxylic acid esters, organophosphate, and carbamates [1], and is associated with high-density lipoprotein (HDL) [2]. Although the natural substrate of PON1 in vivo is unknown, PON1 inhibits the oxidation of not only low-density lipoprotein (LDL) but also that of HDL [3], [4], [5], and plays an important role in the suppression of development or progression of atherosclerosis [6]. Recently, it was reported that PON1-knockout mice were not protected from the progression of atherosclerosis when consuming a high-fat and high-cholesterol diet [7]. However, atherosclerotic lesion formation was decreased in PON1 transgenic mice [8].

This protein has common polymorphic sites, involving Leu-Met (L/M) at position 55 of the amino acid sequence and Gln-Arg (Q/R) at position 192 [9]. Some studies have shown that these genetic polymorphisms are involved in the development of coronary heart disease (CHD) [10], [11], [12], however, others did not find such a relationship [13], [14]. Recently, we and others found a polymorphism, cytosine-thymine (C/T) at position –108 from the ATG start codon in the upstream region of the PON1 gene, which may be associated with PON1 transcriptional activity and serum concentration [15], [16], [17]. This polymorphism is present in a GC box in the PON1 gene promoter region, where a binding site of Sp1 is thought to exist [15].

We and others have also reported that PON1 activity is related to not only macroangiopathy but also microangiopathy, such as retinopathy or nephropathy, in diabetic patients [10], [18], [19]. Based on these results, we speculated that PON1 may have protective effects on various types of oxidation in vivo other than a lipoprotein oxidation.

In a subanalysis of the findings of West of Scotland Coronary Prevention Study (WOSCOP), which conducted a large clinical trial using pravastatin, statins were estimated to have pleiotropic effects [20]. Further, many basic and clinical studies have shown that statins have antiatherosclerosis pleiotropic effects in addition to a cholesterol-lowering action. One of these pleiotropic effects is thought to be antioxidization [21], as it has been reported that simvastatin inhibited macrophage-dependent oxidization of LDL [22] and atorvastatin inhibited Cu-derived oxidization of LDL [23]. In addition, a clinical study reported that simvastatin normalized low levels of PON1 enzyme activity in patients with familial hypercholesterolemia [24], although the mechanism was not elucidated. This background led us to investigate whether PON1 was involved in the anti-oxidative effects of statins, especially transcription of the PON1 gene. We studied the effects of pitavastatin on promoter activity of the PON1 gene using a reporter gene assay method with human hepatoma HepG2 cells and human embryonic kidney (HEK) 293 cells. Our results showed that statins enhanced the promoter activity of the PON1 gene, which may have occurred primarily through a mevalonic acid-derived farnesyl pyrophosphate pathway.