Hypoxic and normoxic activation of NF-kappaB and HIF: investigating their crosstalk in a coordinated cellular response

Hypoxia (low levels of oxygen) is involved in a variety of pathological and physiological conditions such as cancer, ischemia (stroke and cardiac arrest), acute renal failure and intense muscle contraction during exercise. It also constitutes a great challenge to current cancer therapies. To respond to low oxygen levels, the cell activates a series of proteins that control genes. These proteins, called transcription factors, are responsible for increasing or decreasing the levels of other proteins within the cell. Two such important proteins that are also activated by low oxygen levels are called NF-kappaB (standing Nuclear Factor kappaB) and HIF-1 (standing for Hypoxia Inducible Factor-1). Incorrect function of these proteins leads to many human diseases and as such these proteins have been the focus of a lot of interest by pharmaceutical companies with the goal of developing drugs that would inhibit them. However, at present it is not known what would happen if one of these proteins was inhibited independently of each other. I plan to investigate the role of these proteins in determining how cells respond to low levels of oxygen and consequently what happens in tumours, stroke or cardiac arrest. I will determine if these proteins work together or oppose each other in the cell. This study will help determine if it is better to inhibit only one of the proteins or both at the same time to improve the therapeutic benefit of the current inhibitors. Also new proteins will be identified that could represent new targets for already established therapies or entirely new therapies to be used when low oxygen levels are present.

NF-kappaB and HIF-1 are two very important transcription factors involved in a multitude of both physiological and pathological processes, including stroke, diabetes, and cancer. In addition, these transcription factors are often activated by the same stimulus such as hypoxia or the cytokine TNF-alpha. To achieve a coordinated response the cell must integrate all the pathways activated by any given stimulus. As such, in situations where HIF and NF-kappaB are activated simultaneously, I hypothesize that these pathways functionally interact to achieve a coordinated cellular response.
Given the importance of HIF-1 and NF-kappaB in the context of human disease, these proteins have been the focus of much research by not only by academic laboratories but also by the pharmaceutical industry, with the ultimate aim of developing inhibitors. Despite this fact, it is presently unclear what the implications for the HIF and NF-kappaB pathways are when one of them is inhibited independently. The aim of this proposal is to determine how NF-kappaB and HIF-1 modulate each other?s function to mount a cellular response to hypoxic stress. With the use of biochemical, cell biology and molecular biology techniques, this project will investigate how HIF-1 and NF-kappaB activities change following hypoxia or HIF-1 stabilising agents. The direct effects of each of the NF-kappaB subunits on HIF-1 will be investigated. In addition, using a quantitative mass spectrometry technique, SILAC, this research will determine novel NF-kappaB and HIF-1 interacting proteins. These novel interacting proteins will be investigated in terms of functional effects on NF-kappaB and HIF-1. The SILAC analysis will provide valuable insights into how these important transcription factors are regulated and possibly identify novel targets for therapeutic intervention.
A better understanding of the integration of these pathways would help the design of novel treatment regimes with the currently available inhibitors or even develop novel and more targeted inhibitors.