Elsevier

Atherosclerosis

Volume 195, Issue 2, December 2007, Pages 313-321
Atherosclerosis

Macrophage paraoxonase 2 (PON2) expression is up-regulated by pomegranate juice phenolic anti-oxidants via PPARγ and AP-1 pathway activation

https://doi.org/10.1016/j.atherosclerosis.2007.01.007Get rights and content

Abstract

Paraoxonase 2 (PON2), a member of the paraoxonase gene family, was shown to protect macrophages against oxidative stress. Pomegranate juice (PJ), which contains potent polyphenol anti-oxidants, exhibits similar effects. We questioned possible association between PJ polyphenolics, macrophage oxidative stress, and cellular PON2 expression, in relation to the activation of specific PON2 transcription factors. Incubation of J774A.1 macrophages with PJ (0–50 μM of total polyphenols) dose-dependently increased expression (mRNA, protein) and activity and reduced macrophage oxidative status. These effects could be attributed to the PJ unique polyphenols, punicalagin and gallic acid. PJ polyphenol-induced up-regulation of PON2 was inhibited by 40% upon using the PPARγ inhibitor GW9662 (50 μM). Accordingly, the PPARγ ligand, rosiglitazone, dose-dependently stimulated macrophage PON2 expression, by up to 80%. Inhibition of AP-1 activation with SP600125, attenuated PJ-induced up-regulation of PON2 by 40%. Similarly, incubation of macrophages with PJ polyphenols in the presence of GW9662 or SP600125, significantly reduced their capacity to protect the cells against oxidative stress. We conclude that the anti-oxidative characteristics of PJ unique phenolics punicalagin and gallic acid could be related, at least in part, to their stimulatory effect on macrophage PON2 expression, a phenomenon which was shown to be associated with activation of the transcription factors PAPRγ and AP-1.

Introduction

Atherosclerotic lesion formation is characterized by accelerated oxidative stress [1], [2]. Pomegranate juice (PJ) has anti-atherogenic and anti-oxidative properties, due to its tannins and anthocyanins polyphenolics and phenolics [3], [4]. PJ or PJ byproduct were shown to reduce oxidative stress in serum and in macrophages in atherosclerotic mice [5], as well as in diabetic patients [6], and to reduce cellular cholesterol accumulation and foam cell formation [7]. Moreover, we found that the PJ sugar fraction decreases macrophage oxidative state [8]. PJ was found to be also involved in gene regulation. It was shown to reduce the activation of redox-sensitive genes (ELK-1) and to increase eNOS expression in cultured endothelial cells and in atherosclerosis-prone areas of hypercholesterolemic mice [9]. Anthocyanins, which are major components of PJ, were found to activate PPARγ [10], and pomegranate flower extract was shown to act as a dual activator of PPARα and PPARγ [11].

Paraoxonase 2 (PON2) is a member of the multigene family of paraoxonases (PONs), which is expressed in various tissues and cells [12], including macrophages [13]. PON2 expression increases in monocytes during their differentiation into macrophages, and this effect was shown to be mediated via the transcription factor AP-1 [14]. Cellular PON2, like humoral PON1, was shown to possess anti-oxidative properties [12], [13].

Cellular oxidative stress and PON2 activity show a biphasic U-shape relationship [15]. However, the mechanisms responsible for macrophage PON2 regulation under reduced oxidative stress are not known. Since PJ is known as a potent anti-oxidant, we questioned whether PJ and its unique phenolics reduce the oxidative stress in macrophages via modulation of PON2 expression, in relation to the activation of specific transcription factors.

Section snippets

Reagents

SP600125, ellagic acid and gallic acid were from Sigma (St. Louis, MO, USA). Rosiglitazone and Wy14643 (a generous gift from Prof. B. Staels from Pasteur Institute, France). GW9662 from Cayman Chemicals. Perlargonidin-3-o-β-glucopyranoside was from polyphenols laboratories AS (Norway).

Pomegranate processing

Pomegranate fruits were crushed and squeezed to yield pomegranate juice that was filtered, pasteurized, concentrated and stored at −18 °C.

Total polyphenols measurement

Total polyphenol concentrations of PJ, punicalagin, GA, EA and Pg were

Pomegranate juice polyphenols up-regulate PON2 expression in J774A.1 macrophages

Incubation of the macrophage cell line J774A.1 with pomegranate juice (PJ) (0–50 μM of total polyphenols) resulted in a dose-dependent increase in PON2 expression (Fig. 1). PJ induced an increase in cellular lactonase activity, by up to 5.4-fold (Fig. 1A). In mouse macrophages, lactonase activity is shared by PON2 and PON3 and thus, in order to eliminate the possibility that the increase in lactonase activity is due to an increase in PON3 expression, we measured statinase activity, which is

Discussion

In the present study we demonstrate, for the first time, that macrophage PON2 expression (mRNA and protein) and activity are inversely related to cellular oxidative stress and are up-regulated dose-dependently by pomegranate juice (PJ) and PJ-derived phenolics (punicalagin and gallic acid). The transcription factors PPARγ and AP-1 were shown to play a role in PJ-mediated PON2 up-regulation. Furthermore, this study shows, for the first time, that PON2 protein is present all over the macrophage

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This study was supported by a Grant from the Israeli Ministry of Health, No. 5313, 2002.

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