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

Lead Research Organisation: University of Dundee
Department Name: Unlisted

Abstract

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.

Technical Summary

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.

Publications

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Culver C (2010) Mechanism of hypoxia-induced NF-kappaB. in Molecular and cellular biology

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Kenneth NS (2008) Regulation of gene expression by hypoxia. in The Biochemical journal

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Kenneth NS (2009) SWI/SNF regulates the cellular response to hypoxia. in The Journal of biological chemistry

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Melvin A (2011) Further insights into the mechanism of hypoxia-induced NF?B. [corrected]. in Cell cycle (Georgetown, Tex.)

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Van Uden P (2008) Regulation of hypoxia-inducible factor-1alpha by NF-kappaB. in The Biochemical journal

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Van Uden P (2011) Evolutionary conserved regulation of HIF-1ß by NF-?B. in PLoS genetics

 
Description CRUK Senior Fellowship
Amount £1,100,000 (GBP)
Organisation Cancer Research UK 
Sector Charity/Non Profit
Country United Kingdom
Start 08/2011 
End 07/2017
 
Description Tenovus Scotland
Amount £10,000 (GBP)
Organisation Tenovus Cancer Care 
Department Tenovus Scotland
Sector Charity/Non Profit
Country United Kingdom
Start 01/2009 
End 12/2011
 
Title reporter cell lines 
Description Luciferase reporter cell lines for NF-kB and HIF-1 
Type Of Material Cell line 
Year Produced 2013 
Provided To Others? Yes  
Impact These cells can be used to screen several platforms of chemical compounds and siRNA libraries 
 
Title stable cell lines 
Description In the project we have created a great number of stable cell lines with shRNA knockdown for different important genes 
Type Of Material Cell line 
Provided To Others? No  
Impact These cells will be crucial in determining the contribution of these genes to a number of cellular responses 
 
Description APC and HIF crosstalk 
Organisation University of Dundee
Department College of Life Sciences
Country United Kingdom 
Sector Academic/University 
PI Contribution We have identified the mechanism by which APC and HIF crosstalk
Collaborator Contribution Expertise in the field of APC biology
Impact This collaboration has resulted in a publication in Mol. Biol. Cell this year.
Start Year 2008
 
Description Cafe Science 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Gave a presentation without audiovisual help to a broad range of people at a local cafe

Received a great number of question from the audience and a great deal of interest in research
Year(s) Of Engagement Activity 2009
 
Description poster presentation 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Participants in your research and patient groups
Results and Impact poster presentation in a focused international meeting. Well received

received a lot of good feedback
Year(s) Of Engagement Activity 2008
 
Description seminar 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Participants in your research and patient groups
Results and Impact 45 minute seminar to a broad scientific audience. Well received.

I was invited again for another visit
Year(s) Of Engagement Activity 2008