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What processes underlie chronic tissue damage due to aging, inflammation and disease?

Dr. John Wilkinson

I believe that oxidant stress is a key player in this area. The goal of my research is to elucidate the relationship between iron biology, oxidant stress, and chronic disease. The key role that iron may play in oxidant stress development is illustrated in the diagram below:

Oxident Stress

Figure One: Iron Is a Central Mediator of Oxidant Stress

This diagram depicts the Fenton equation wherein hydrogen peroxide and divalent iron react to form trivalent iron, hydroxide, and the hydroxyl radical. This hydroxyl radical can chemically damage the constituents of cells including DNA, lipids, and proteins. Note that the cell has many mechanisms that strive to regulate the abundance of the hydrogen peroxide and divalent iron, presumably to restrict the random occurrence of this reaction. During my second post-doc, working with Dr. Frank Torti and Dr. Suzy Tortiat the Wake Forest University School of Medicine, I developed a novel transgenic model system. In this model, ferritin, the key iron storage protein in mammals, is regulated in a tissue– and temporal- specific manner. This is accomplished in mice using the tetracycline-based (TET) genetic model developed by Prof. Dr. Hermann Bujard for transgene control. In our model, ferritin can be highly over expressed, leading to strong changes in proteins that regulate iron homeostasis. Note how ferritin is positioned to store free iron in the diagram above. Our hypothesis is that this induction of ferritin, which yields changes in free or available iron, will influence the development of oxidant stress in vivo by shifting the equilibrium of the above equation. If this is the case, this model will prove an invaluable resource for unraveling the role of oxidant stress in the development of chronic disease.

Our Transgenic Ferritin H Model

In the TET system, there are two components – the gene to be regulated, cloned into a tet-responsive promoter, containing the tet responsive element (TRE), and the transgene which acts as a transcription factor for the tet responsive promoter, called the tet-transactivator (tTA or rtTA). In our model the TREis complex, directing expression in two directions, permitting two genes to be simultaneously regulated by the tTA or rtTA transcription factors. The genes we selected to be so regulated were ferritin H, encoding the heavy chain of the iron storage protein, and the enhanced green fluorescent protein (EGFP).

Figure Two: A Bi-Cistronic Transgenic Vector

Our evidence, published in the Biochemical Journal is that induction of transgenic ferritin in vivoleads to an increase in transferrin receptor expression, as well as an increase in iron regulatory protein (IRP) activity. These changes are reflective of the relationship between iron responsive proteins and the labile iron pool, depicted in the diagram below. Note that the transgenic ferritin is mutated such that its iron regulatory element (IRE) is no longer responsive to normal regulatory feedback via the IRP.

Ferritin H/EGFP Transgene Expression

Figure Three: Ferritin H/EGFP Transgene Expression in a Renal Model

Our evidence, published in the Biochemical Journal is that induction of transgenic ferritin in vivoleads to an increase in transferrin receptor expression, as well as an increase in iron regulatory protein (IRP) activity. These changes are reflective of the relationship between iron responsive proteins and the labile iron pool, depicted in the diagram below. Note that the transgenic ferritin is mutated such that its iron regulatory element (IRE) is no longer responsive to normal regulatory feedback via the IRP.

Figure Four: Transgenic Ferritin H Drives Changes in Cellular Iron and Iron Responsive Proteins

Our Central Hypothesis is that these changes in iron driven by ferritin H over-expression will lead to changes in oxidant stress. This is because iron is a central mediator of oxidant stress, depicted in Figure One.

We are currently studying the impact of ferritin expression on oxidant stress related damage in vivo in a number of systems in the following project areas:

Nutritionally Oriented Projects Underway In Our Laboratory

Atherosclerosis. For this project I am collaborating with Dr. Nalini Santanam from the department of Pharmacology and Physiology here at MUSOM. To study the impact of iron on oxidant stress and resultant atherosclerosis in blood vessels we are using a partial ferritin knockout model developed by the Torti’s and Khristy Thompson in Dr. Jim Connors Lab. This model has lower expression of ferritin in most tissues we have tested. We will be feeding partial (heterozygous) ferritin knockout mice an atherogenic diet and comparing their resultant phenotype with wild type littermate controls.

Curcumin and Iron. Recently I completed a collaboration with Yan Jiao and the Torti’s which was aimed at determining the impact of curcumin, a widely studied nutritional chemopreventive compound, on iron metabolism. We found that curcumin, a component of curry powders, which is also used as a food coloring agent throughout the world, acts as an iron chelator.

Ethanol and Mammary Cancer. Ethanol is a substantial risk factor for mammary cancer, and iron is a known co-factor for ethanol in liver cancer. My COBRE project centers on adapting a well known transgenic mammary carcinogenesis model, C3(1)TAG mice, developed in the laboratory of Dr. Jeffrey E. Green, M.D. for the purposes of ethanol research. Dietary iron will also be manipulated to determine the impact of this variable on mammary tumorigenesis.

Transsulfuration, Oxidant Stress and Epigenetics. I have chosen to focus on how oxidant stress might cause changes in epigenetic mechanisms, as I think this is a likely route for how a chronic stress might impact cells. The transsulfuration pathway is a biochemical link between the methionine cycle, which regenerates the cells principal methyl-donor, S-Adenosyl-Methionine (SAMe), and the biosynthetic pathway for glutathione, the cells principal antioxidant. My hypothesis is that oxidant stress could lead to depletion of glutathione, which in turn would draw sulfur down the pathway from homocysteine, depleting the methionine cycle of its intermediates, leading to an inhibition of processes involving methyl donation. These processes would potentially include DNA methylation or histone base methylation, prominent epigenetic reactions. Work by Lertratanangkoon, L. et al (Cancer Lett. 1997 Dec 9;120(2):149-56.) demonstrated that this can take place in liver cells.

Transsulfuration and Liver Cancer. I have an R21 application submitted that is pending as a collaborative effort with Dr. Vincent Sollars, to examine the impact of ethanol and iron on epigenetic mechanisms of importance to liver cancer development that may be influenced by transsulfuration.

The world of iron research can be fun.

In May 2005, I presented my model at the International BioIron Society’s Meeting in Prague. The whole meeting was being run and hosted by Dr. Prem Ponka. I was fortunate in that a Torti-lab post-doc and friend of mine, Dr. Joan Buss was a former post-doc in Prem’s laboratory, and knew both Prem and his students well. Thus, I got to tag along with one of the leaders in the field of Iron Biology – who happened to know where the best beer, sausage, and knedlicky (dumplings) were to be found in the city! Our first day, Suzy Torti hiked into the downtown with us and took time to pose with Joan in front of the hotel Prem’s Students were staying at. Note: the stand in front of the Bran Mere had the best sausage in the city.

Hotel Prem

We went walking for a time with Prem. Here is the whole group (Natasha, Billy, Shaan, Prem and Joan). We are in a plaza which has the famous clock (?).

Famous Clock in Prague

This clock is probably the most famous attractionin Prague. Here is its picture.

Prague Clock   

For dinner that day, Prem took us walking through town (largely uphill, I remember) to a secretive restaurant named U Slatne. It had a magnificent view of the rooftops of Prague. Later that evening, he took us to an old haunt of his – a bar where he and his friends had once engaged in mischief while graduate students themselves. Here is a shot of Joan and Prem with some of his lab members.

U Slatne

Of course, it wasn’t all fun and games. After attending two of their meetings, I have to say that BioIron is the best scientific meeting I have been at. There are not so many simultaneous sessions (yet) that you can usually see all the talks and posters that are of interest. And the talks are generally of very high quality.

Here is a picture of Billy (a student of Kostas Pantopoulos) at his poster.

Billy (a student of Kostas Pantopoulos) at his poster

Altogether this was the best meeting I have been too. Great science, great food, and a city that is breathtaking.

 

 

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