Pon3, a natural antioxidant protein: its role in development and disease.

Oxygen plays a critical role in maintaining life but because it is highly reactive, cells have multiple defences to protect themselves from oxidative stress and damage. The detrimental effects of oxidative stress are increased with age and oxidative stress is a well-recognised feature of several common major conditions including diabetes, metabolic syndrome, neurodegenerative diseases and age-related macular degeneration. At birth the fetus is exposed to a rapid and substantial increase in oxygen as it begins to breathe and many defence systems are up-regulated at this time. We therefore searched for previously unrecognised or novel endogenous antioxidants which are up-regulated in late gestation in the fetus. Such molecules may be useful therapeutically in the pre-term infant but may also be important components of the adult anti-oxidant defence system. They may also be perturbed in some of the pathologies characterised by oxidative stress.

We carried out this search and identified paraoxonase 3 (Pon3). This protein is strongly induced in the lung and gut prior to birth in rats, mice, sheep and humans and at least in the sheep, this is controlled by glucocorticoid hormones. We conclude that this may be a systemic preparative process for birth.

The paraoxonase family consists of 3 closely related enzymes which break down a wide range of molecules from several different biologically important families. These include hormone-like molecules, lipid peroxides, the oxidation products of arachidonic acid and estrogen esters. Thus, the Pons have the potential to modulate the signalling pathways mediated by such molecules. These enzymes also effectively break down molecules that bacteria use to regulate their growth in situations where many bacteria are present (as may be the case in the intestine for example). They also break down toxic pesticides.

In blood, PON1 is associated with lipid-containing particles and it can reduce the harmful inflammatory action of oxidized lipid. Artificially increasing PON3 levels in mice inhibited atherosclerotic lesion formation and body fat and Pons in general are antioxidants and anti-inflammatory. In humans mutations in the PON1 gene alter the risk of heart attack and stroke. Thus, in addition to their presumed role as potent circulating antioxidants, these enzymes have anti-atherogenic and anti-inflammatory properties; are capable of metabolising derivatives of important regulatory hormones; can degrade pesticides and have the potential to alter host/microbe interaction. As such, they are implicated in diverse and important biological processes and have attracted considerable attention. Despite this, the physiological function of this family of well-conserved enzymes remains unclear.

To understand the normal role of Pon3 we have used a genetically engineered mice in which the Pon3 gene has been knocked-out. When we bred heterozygous mice together there were significantly fewer Pon3-/- animals than expected (25% expected but only 9% found). The mutant mice die early in pregnancy so we conclude that Pon3 has an essential and non-redundant biological role in early embryonic development.

We predict that loss of or a reduction in Pon3 will lead to increased oxidative stress hence embryonic lethality or reduced embryonic growth. We will investigate this using detailed histological, biochemical and molecular methods in normal and Pon3 knockout mice. Furthermore, this reduction of Pon3 may exacerbate oxidative stress and disease sensitivity in adult life. We will engineer conditional knockout mice and study how loss of Pon3 effects mice as they age and are fed a high fat diet.

These studies will determine whether Pon3 plays a role in protecting mice from the detrimental effects of age and a poor diet. The biochemical studies will identify pathways that are likely to be similar in humans allowing us in future studies to determine whether Pon3 may be important in human disease.

Oxidative stress increases with age and is a well-recognised feature of several common major diseases.
The paraoxonase (Pon) family, consists of 3 closely related esterases which hydrolyze lactones in biologically important molecules, including lipid peroxides. Pon3 are anti-inflammatory antioxidants and transgenic expression of PON3 inhibited atherosclerotic lesion formation and adiposity. In addition to their presumed role as potent circulating antioxidants, these enzymes are capable of metabolising precursors or derivatives of regulatory hormones (arachidonic acid and estrogen esters); are anti-atherogenic; anti-inflammatory; can degrade xenobiotics and have the potential to alter host/microbe interaction. As such, they are implicated in diverse biological processes. Despite this, their physiological function remains unclear.

We have identified a novel aspect of PON3 biology, showing it is markedly up-regulated in late gestation in the rat, sheep and human. This may be a systemic preparative process for birth which is (at least in the sheep) controlled by glucocorticoids.

We have commenced studies on a Pon3 knock out. After het x het mating there were significantly fewer Pon3-/- animals than expected. We conclude that Pon3 has an essential non-redundant biological role in embryonic development. Preimplantation embryos are sensitive to oxidative stress and we hypothesise that loss of Pon3 leads to increased oxidative stress and embryonic lethality.
We will determine when and where Pon3 is expressed in the developing mouse embryo and the cellular consequences of its ablation. Early lineage assignment will be followed. "Floxed" Pon3 ES cells are available from the EUCOMM consortium and we will use these to generate conditional animals. We will investigate whether ablated adult animals show increased sensitivity to age and high-fat diet induced oxidative stress and using metabolomics, determine which biochemical pathways Pon3 acts in.

This work will have academic, economic and societal impact. Specifically the work will enhance the knowledge economy by the generation of new knowledge, this will cross disciplines (reproductive biology, animal physiology, developmental biology, biochemistry (metabolomics) and medicine). As basic scientific research it will be widely disseminated and applicable internationally and it's generation will enhance the status of UK science. As the work is cross disciplinary it will contribute to the broad training of a skilled Post-doc and maintain the health of different academic disciplines.

In addition, this work will enhance health and contribute to wealth generation. The University of Cambridge has already filed a patent based on our previous work in this area for the use of Pon3 as a replacement therapy in pre-term infants. The basic mechanistic biology that is addressed in this proposal aims to deepen our understanding of how this enzyme functions. Addressing these questions will aid the exploitation of Pon3 as a possible therapeutic agent. In addition, the enzyme's possible role in aging and disease sensitivity will inform our understanding of cardiovascular disease. This may lead to new therapeutic strategies and it may generate new IP. Furthermore, identification of the endogenous substrate(s) may have relevance to both normal physiological function, disease processes and drug metabolism. This may also generate new IP, for example new biomarkers. In each of these areas there is considerable scope for wealth generation and improvement of health.