Simvastatin modulates expression of the PON1 gene and increases serum paraoxonase: a role for sterol regulatory element-binding protein-2.

BACKGROUND: The HDL-associated enzyme paraoxonase protects LDLs from oxidative stress. 3-Hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) appear to favorably influence the atherosclerotic process by different mechanisms. The present study examined the influence of simvastatin on paraoxonase expression and serum paraoxonase levels. METHODS AND RESULTS: Simvastatin upregulated in a dose-dependent manner the activity of the promoter of the paraoxonase gene in expression cassettes transfected into HepG2 cells. Upregulation could be blocked by mevalonate and other intermediates of the cholesterol biosynthetic pathway. Simvastatin increased nuclear factors, notably sterol regulatory element-binding protein-2, capable of binding to the paraoxonase promoter; this was also blocked by mevalonate. Sterol regulatory element-binding protein-2 upregulated promoter activity in vitro. Patients treated with statin showed a significant increase in serum concentrations and activities of paraoxonase. CONCLUSIONS: The data indicate that simvastatin can modulate expression in vitro of the antioxidant enzyme paraoxonase and is associated with increased serum paraoxonase concentration and activity. It is consistent with effects of simvastatin treatment, which have the potential to influence beneficially antiatherogenic mechanisms at the HDL level. The study provides evidence for 1 molecular mechanism by which paraoxonase gene expression could be regulated.

Paraoxonase-1 promoter haplotypes and serum paraoxonase: a predominant role for polymorphic position - 107, implicating the Sp1 transcription factor.

Accumulating data suggest that paraoxonase-1 (PON1) is a primary determinant of the antioxidant and anti-inflammatory capacities of high-density lipoproteins (HDLs). Variations in HDLs and PON1 have been shown to influence the functions of both. There is a wide spectrum of serum PON1 mass in humans, to which promoter polymorphisms make an important contribution. The present studies attempted to define: (i) the relevance in vivo of promoter polymorphisms by analysing haplotype structure; and (ii) molecular mechanisms implicated in promoter activity. Highly significant differences (P <0.0001) in serum mass and activity were observed as a function of haplotype sequence. Of three promoter polymorphisms (-107, -824 and -907), the -107 site was shown to be of predominant importance to serum PON1. Significant increases in serum PON1 mass and activities between haplotype subgroups could be explained by unit increases in the number of high-expresser variants of the -107 site (-107C) alone. No significant contribution was observed for the -824 and -907 sites. The coding-region Leu(55)-->Met (L55M) polymorphism made an independent contribution to serum PON1 mass, which may account for variations in serum PON1 mass and activity within haplotype subgroups defined by the -107 site. A molecular basis for the effect of the -107 polymorphism on serum PON1 was indicated by the greater affinity of the high-expresser variant (-107C) for hepatocyte nuclear extracts, indicating higher affinity for transcription factors. Competition studies with oligonucleotides representing the consensus (and mutated) sequence for Sp1, and the use of Sp1 antibodies, confirmed formation of complexes between the transcription factor and the PON1 promoter during incubation with nuclear extracts. The data underline the importance of the region containing the C(-107)T polymorphism for gene expression in vivo. Differences in the affinity of the -107C and -107T polymorphic fragments for nuclear extracts have been demonstrated, and coincide with their impact on gene expression. A potential role for the transcription factor Sp1 has been demonstrated, which is consistent with the disruption of an Sp1 recognition sequence by the -107 polymorphism.

High expressor paraoxonase PON1 gene promoter polymorphisms are associated with reduced risk of vascular disease in younger coronary patients.

Human paraoxonase-1 is hypothesised to protect serum lipoproteins from oxidative stress. Decreased serum activity of paraoxonase-1 in animal models is associated with an increased risk of vascular disease and has been linked to the anti-oxidant capacity of the enzyme. Promoter polymorphisms of the human paraoxonase-1 gene strongly influence serum concentrations of the enzyme. The present study examined the hypothesis that promoter polymorphisms may be genetic risk factors for vascular disease in man. Genotypes arising from the promoter C(-907)G polymorphism were analysed in the ECTIM2 population. The global odds ratio for myocardial infarction, comparing the high expressor GG genotype to other genotypes, was 0.77 (0.61-0.97) (P=0.024). The association with the promoter genotype was more pronounced in the youngest age group (odds ratio 0.52 (0.31-0.87), P=0.012) and was progressively lost with age (respectively 50 years to <60 years, P=0.26; >60 years, P=0.45). There was no association between the promoter genotypes and serum lipids. The data are consistent with the high expressor promoter genotype being linked to reduced risk of myocardial infarction. The influence of the genotype may be compromised in older patients.

Paraoxonase-1 L55M polymorphism is associated with an abnormal oral glucose tolerance test and differentiates high risk coronary disease families.

Polymorphisms of the gene for the antioxidant enzyme, paraoxonase-1 (PON1), have been identified as risk factors for coronary disease (CHD), notably in diabetic patients. The polymorphisms have also been linked with other diabetic complications. The present study analyzed glucose metabolism as a function of PON1 polymorphisms in young healthy nondiabetic men from families with premature CHD and matched controls. The L55M PON1 polymorphism was independently associated with the glucose response to an oral glucose tolerance test. LL homozygotes had significantly impaired glucose disposal (P = 0.0007) compared with (LM+MM) genotypes. It was particularly marked for subjects from high CHD risk families and differentiated them from matched controls (P = 0.049). The area under the glucose curve (P = 0.0036) and the time to peak glucose value (P = 0.026) were significantly higher in the LL carriers, whereas the insulin response was slower (P = 0.013). Insulin resistance did not differ between L55M genotypes. There was a trend for reduced pancreatic beta-cell function as measured by glucose-induced insulin secretion (LL vs. LM vs. MM, 20.26 vs. 23.74 vs. 25.60; P = 0.077). The frequency of the L55 allele decreased significantly (P = 0.028) across regions defining a north-south European axis. No significant differences for the glucose response or case-control populations were observed as a function of the PON1 Q192R polymorphism. The study demonstrates an association between PON1 gene polymorphisms and glucose metabolism. The L55M-glucose interaction differentiated offspring of high CHD risk families, suggesting that it may be of particular relevance for vascular disease and possibly other diabetic complications.

Enzymatically active paraoxonase-1 is located at the external membrane of producing cells and released by a high affinity, saturable, desorption mechanism.

Paraoxonase-1 (PON1) is a high density lipoprotein (HDL)-associated serum enzyme that protects low density lipoproteins from oxidative modifications. There is a relative lack of information on mechanisms implicated in PON1 release from cells. The present study focused on a model derived from stable transfection of CHO cells, to avoid co-secretion of apolipoprotein (apo) A-I and lipids, which could lead to formation of HDL-like complexes. Our results indicate that, in the absence of an appropriate acceptor, little PON1 is released. The results designate HDL as the predominant, physiological acceptor, whose efficiency is influenced by size and composition. Neither lipid-poor apoA-I or apoA-II nor low density lipoproteins could substitute for HDL. Protein-free phospholipid complexes promoted PON1 release. However, the presence of both apolipoprotein and phospholipid were necessary to promote release and stabilize the enzyme. Immunofluorescence studies demonstrated that PON1 was inserted into the external membrane of CHO cells, where it was enzymatically active. Accumulation of PON1 in the cell membrane was not influenced by the ability of the cell to co-secrete of apoA-I. Release appeared to involve desorption by HDL; human and reconstituted HDL promoted PON1 release in a saturable, high affinity manner (apparent affinity 1.59 +/- 0.3 microg of HDL protein/ml). Studies with PON1-transfected hepatocytes (HuH-7) revealed comparable structural features with the peptide located in a punctate pattern at the external membrane and enzymatically active. We hypothesize that release of PON1 involves a docking process whereby HDL transiently associate with the cell membrane and remove the peptide from the external membrane. The secretory process may be of importance for assuring the correct lipoprotein destination of PON1 and thus its functional efficiency.