Antioxidant defence in adrenocortical cells

Lead Research Organisation: Queen Mary, University of London
Department Name: William Harvey Research Institute

Abstract

Oxidative stress (OS) is involved in many human disease states including neurodegenerative diseases, cancer, stroke, diabetes and heart disease. We study patients with a rare disease called Familial Glucocorticoid Deficiency (FGD) in which the body fails to produce a glucocorticoid called cortisol. Patients with this disease do not have a mechanism to cope with stress. If their body becomes stressed, for example by illness, their blood sugar levels drop, they become liable to infections and they may die if untreated. ACTH, acting through its receptor, is the hormone that is produced in response to stress and it causes the cells of the adrenal gland to produce cortisol. We have previously found defects in three genes in this pathway that cause FGD. More recently we have discovered that defects in four genes usually associated with a cells defence against oxidative stress, can also cause the disease. Exactly how OS prevents cortisol secretion is not fully understood and we believe that other genes may also be involved. This project aims to discover the mechanism by which OS affects the cells of the adrenal to prevent them making cortisol. We also hope to find other genes in this pathway by studying the genetic make-up of FGD patients. If we can discover how the OS causes its effect then this might give us clues to the mechanism in other diseases like those listed above and it may then be possible to design drugs to reduce it.

Technical Summary

Decades of research support the idea that oxidative stress (OS) initiates and promotes cardiovascular, neurologic, and immune-related conditions. OS may arise from an imbalance between the production of reactive oxygen species (ROS) and the detoxification of these species by antioxidant defence systems such as the glutathione and thioredoxin systems. At present, relatively little is known about the contribution of individual enzymes to the redox metabolism in different cell types. We have discovered mutations in four antioxidant (AO) genes in patients with Familial Glucocorticoid Deficiency (FGD) a disease of adrenal resistance to ACTH and loss of cortisol secretion. This suggests that the adrenal cortex may be exquisitely sensitive to ROS and AO defence of primary importance to this cell type. Failure of AO defence leads to increased ROS, decreased glutathione ratios, steroidogenic acute regulatory protein reduction and hence reduced steroidogenesis.
Our aims are;
A Delineation of a larger spectrum of anti-oxidant genes involved in the pathogenesis of FGD by whole exome sequencing of 100 affected individuals to identify novel mono- or (possibly) oligogenic variants.
B Assessment of the effect of knockdown of antioxidant genes in human adrenocortical cell lines (H295R and HAC15) on;
i) ROS levels, glutathione ratio, STAR mRNA and protein levels
ii) steroidogenic profiles
iii) altered AO gene expression
C To attempt to rescue the phenotype in knockdown cell lines
i) by pharmacological intervention
ii) by overexpression of another antioxidant system
Understanding the mechanisms by which OS causes adrenal cell dysfunction could have a much wider indirect impact. Such knowledge may be instructive in the wide range of more prevalent human disorders associated with OS (listed above). Utilising knockdown adrenal cell lines as models for OS and AO treatment as proposed here could give us insights into more effective treatments for these conditions.

Planned Impact

Decades of research support the idea that oxidative stress (OS) initiates and promotes cardiovascular, neurologic, and immune-related conditions. OS may arise from an imbalance between the production of reactive oxygen species (ROS) and the detoxification of these species by antioxidant defence systems such as the glutathione and thioredoxin systems. At present, relatively little is known about the contribution of individual enzymes to the redox metabolism in different cell types. We have identified mutations in four different antioxidant genes in patients with Familial Glucocorticoid Deficiency (OMIM 202200) causing isolated adrenal dysfunction. Our data suggest that, in humans, the glutathione and thioredoxin systems are crucial for ROS detoxification in adrenal cells. There are many publications on antioxidant defence by these systems in tissues and organs especially liver, heart and pancreas, but most of these studies have been performed in mice and rats. There has been no study of this system in the human adrenal. Characterising the antioxidant genes that cause FGD may lead to a better understanding of the mechanisms by which OS causes cell dysfunction in the adrenal and could have a much wider indirect impact. Such knowledge may be instructive in the wide range of more prevalent human disorders associated with oxidative stress including neurodegenerative diseases, cancer, stroke, diabetes and cardiac dysfunction. Utilising knockdown adrenal cell lines as models for OS and antioxidant treatment as proposed here could give us insights into more effective treatments for these conditions.

Publications

10 25 50
 
Description clinical practice
Geographic Reach Multiple continents/international 
Policy Influence Type Influenced training of practitioners or researchers
 
Description Large Project Grant
Amount £318,860 (GBP)
Organisation Barts Charity 
Sector Charity/Non Profit
Country United Kingdom
Start 04/2017 
End 03/2021
 
Description PhD fellowship
Amount £125,000 (GBP)
Organisation Ministry of Health, Trinidad and Tobago 
Sector Public
Country Trinidad and Tobago
Start 02/2017 
End 01/2020
 
Title H295RGPX1KD 
Description H295R human adrenocortical cell line with stable knockdown of glutathione peroxidase 1 (GPX1) gene, achieved by lentiviral delivery of shRNA targeting the gene.C 
Type Of Material Cell line 
Provided To Others? No  
Impact Creation of this cell line has allowed us to assess the effect of knockdown of GPX1. 
 
Title H295RNNTKD 
Description H295R human adrenocortical cell line with stable knockdown of nicotinamide nucleotide transhydrogenase (NNT) gene, achieved by lentiviral delivery of shRNA targeting the gene. 
Type Of Material Cell line 
Year Produced 2013 
Provided To Others? Yes  
Impact Creation of this cell line has allowed us to study the effect of knockdown of NNT 
 
Title H295RPRDX3 
Description H295R cell line with stable shRNA knockdown of PRDX3 
Type Of Material Cell line 
Provided To Others? No  
Impact PhD thesis 
 
Title H295RdoubleKO 
Description H295R cell line with stable knockdown of GPX1 and PRDX3 
Type Of Material Cell line 
Provided To Others? No  
Impact PhD thesis 
 
Title Nnt adrenal RNAseq 
Description RNAseq data from adrenals of three mouse models with varying NNT levels; Nnt null, Nnt wild-type and Nnt overexpressor 
Type Of Material Database/Collection of data 
Year Produced 2016 
Provided To Others? No  
Impact Recognition that oxidative stress impacts steroidogenesis 
 
Description Adrenal genetics 
Organisation University College London Hospital
Department University College London Hospitals Charity (UCLH)
Country United Kingdom 
Sector Charity/Non Profit 
PI Contribution Collaboration to improve the diagnosis of primary adrenal insufficiency through exchange of ideas and protocols
Collaborator Contribution Collaboration to improve the diagnosis of primary adrenal insufficiency through exchange of ideas and protocols
Impact publications
Start Year 2013
 
Description FGD consortium 
Organisation National Institute of Health and Medical Research (INSERM)
Department INSERM U870 (Metabolic regulation, Nutrition and Diabetes)
Country France 
Sector Public 
PI Contribution Discovery of novel genes responsible for cases of familial glucocorticoid deficiency
Collaborator Contribution Contribution of patient samplescontribution of patient samples
Impact Outputs are many publications describing disease causing mutations in patients with familial glucocorticoid deficiency. This collaboration is not multi-disciplinary.
 
Description FGD consortium 
Organisation Technical University of Dresden
Department Children's Hospital
Country Germany 
Sector Hospitals 
PI Contribution Discovery of novel genes responsible for cases of familial glucocorticoid deficiency
Collaborator Contribution Contribution of patient samplescontribution of patient samples
Impact Outputs are many publications describing disease causing mutations in patients with familial glucocorticoid deficiency. This collaboration is not multi-disciplinary.
 
Description NNT cancer 
Organisation BCH Charities
Department Birmingham Children's Hospital Research Foundation
Country United Kingdom 
Sector Charity/Non Profit 
PI Contribution intellectual exchange
Collaborator Contribution intellectual exchange
Impact none so far
Start Year 2013
 
Description NNt zebrafish 
Organisation University of Birmingham
Country United Kingdom 
Sector Academic/University 
PI Contribution intellectual exchange
Collaborator Contribution intellectual exchange
Impact no outcomes yet
Start Year 2014
 
Description Roger Cox MRC Harwell 
Organisation MRC Harwell
Country United Kingdom 
Sector Academic/University 
PI Contribution Adrenal histology in NNT mutant mice
Collaborator Contribution Supply of wild-type, mutant and BAC rescued NNT mice
Impact Nature Genetics paper
Start Year 2011
 
Description SGPL1 mice 
Organisation University of Leuven
Country Belgium 
Sector Academic/University 
PI Contribution Discovery that mutations in SGPL1 cause NPHS14, a syndrome of renal, adrenal, skin and neurological defects. The knockout mouse was previously described to have alung phenotype.
Collaborator Contribution The University of Leuven have generated the SGPL1 knockout mouse that we are now using in our studies to elucidate the human phenotype
Impact Publication describing the human phenotype = Prasad et al. Sphingosine-1-phosphate lyase mutations cause primary adrenal insufficiency and steroid-resistant nephrotic syndrome. J Clin Invest. (2017) 127(3):942-953.
Start Year 2015