טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentShiner Maayan
SubjectRegulation of Macrophage Paraoxonase 2 (PON2) Expression
DepartmentDepartment of Medicine
Supervisors Professor Emeritus Michael Aviram
Ms. Bianca Furman
Full Thesis textFull thesis text - English Version


Abstract

Paraoxonase 2 (PON2) is a member of the paraoxonases gene family. PON2 is expressed in macrophages, which plays a significant role in atherosclerotic lesion formation. Macrophage PON2 can protect against cellular oxidative stress, and its expression increases under excessive oxidative stress. The aim of the present study was to analyze mechanisms involved in the regulation of PON2 expression under atherogenic conditions, which are characterized by excessive cellular oxidative stress. PON2 expression and activity were analyzed using a novel model for macrophage maturation in which mouse peritoneal macrophages (MPM) were harvested at increasing time intervals after intraperitoneal thioglycollate injection. PON2 expression and activity gradually increased during macrophage maturation, in parallel to a gradual increase in superoxide anions production. Supplementation of vitamin E to Balb/C mice inhibited the NADPH oxidase-dependent increase in cellular superoxide anion production, and down-regulated PON2 expression and activity. Furthermore, PON2 expression was significantly lower in MPM isolated from P47phox-/-, who lack NADPH oxidase activity, in comparison to MPM isolated from control mice.

We also questioned possible association between pomegranate juice (PJ), which contains potent polyphenol antioxidants, and cellular PON2 expression. PJ polyphenols, punicalagin and gallic acid, dose-dependently reduced macrophage oxidative status, and increased PON2 expression and activity. PON2 expression was regulated by the transcription factor AP-1, as suggested by decreased JDP2 protein expression in the nucleus, and by decreased PON2 expression in presence of a JNK inhibitor (SP600125). Similarly, PPARg ligand, rosiglitazone, dose-dependently stimulated macrophage PON2 expression. Incubation of macrophages with PJ-polyphenols in the presence of PPARg inhibitor (GW9662) or SP600125, significantly reduced PON2 expression and the cells capacity to protect against oxidative stress, suggesting that PJ reduces oxidative stress in macrophages through upregulation of PON2 expression via activation of AP-1 and PPARg.

We also analyzed the relationship between the extent of cellular oxidative stress and PON2 lactonase activity under various levels of oxidation, which were obtained by cell incubation with either antioxidants or oxidants in J774A.1 macrophage. Overall, PON2 activity and cellular oxidative stress exhibited a U-shape response curve, which could be related to PON2 inactivation under oxidative stress induction at its low range, while at high range of oxidative stress, a macrophage antioxidant compensatory mechanism up-regulates PON2 in order to cope with oxidation burden. If similar processes occur in the arterial wall during early atherogenesis, an increase in macrophage PON2 activity can contribute to the reduction in oxidative stress, which may attenuate macrophage foam cell formation.