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.
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.
Organisations
- Queen Mary University of London (Lead Research Organisation)
- University College Hospital (Collaboration)
- National Institute of Health and Medical Research (INSERM) (Collaboration)
- Technical University of Dresden (Collaboration)
- BCH Charities (Collaboration)
- UNIVERSITY OF BIRMINGHAM (Collaboration)
- University of Leuven (Collaboration)
People |
ORCID iD |
Louise Metherell (Principal Investigator) |
Publications
Smith C
(2022)
181 Sphingosine 1-phosphate lyase insufficiency induced ichthyosis is due to sphingolipid imbalance, increased differentiation, and aberrant intercellular junctions
in Journal of Investigative Dermatology
Smith C
(2023)
769 Reduced expression of PPOX, the variegate porphyria gene, causes intrinsic pathological changes to keratinocytes
in Journal of Investigative Dermatology
Chatterjee Sumana
(2019)
GHR transcript heterogeneity may explain the phenotypic variability in patients with homozygous
GHR pseudoexon (6?) mutation
in HORMONE RESEARCH IN PAEDIATRICS
Howard Sasha R.
(2016)
IGSF10 mutations dysregulate gonadotropin-releasing hormone neuronal migration resulting in delayed puberty
in EMBO MOLECULAR MEDICINE
Prasad R
(2013)
A mutation in thioredoxin reductase 2 (TXNRD2) is associated with a predominantly adrenal phenotype in humans
in Endocrine Abstracts
Poukoulidou T
(2014)
A novel homozygous mutation of the IGFALS gene in a female adolescent: indirect evidence for a contributing role of the circulating IGF-I pool in the pubertal growth spurt.
in Hormone research in paediatrics
Da Costa, Alexandra Rodrigues
(2018)
A Novel Stem Cell Model for the Triple a Syndrome
in Hormone Research in Paediatrics
Smith C
(2022)
A rapid genetic diagnosis for >80% individuals with non-CAH Primary Adrenal Insufficiency is achievable by candidate gene sequencing combined with WES
in Endocrine Abstracts
Cottrell E
(2022)
A rare heterozygous IGFI variant impairing IGF-I cleavage and causing postnatal growth failure: a novel disease mechanism offering insights into IGF-I physiology
in Endocrine Abstracts
Maharaj A
(2022)
A retrospective analysis of endocrine disease in sphingosine-1-phosphate lyase insufficiency: case series and literature review.
in Endocrine connections
Maharaj A
(2022)
A retrospective analysis of endocrine disease in sphingosine-1-phosphate lyase insufficiency: case series and literature review.
in Endocrine connections
Maharaj A
(2020)
A Sphingosine-1-Phosphate Lyase Mutation Associated With Congenital Nephrotic Syndrome and Multiple Endocrinopathy.
in Frontiers in pediatrics
Meimaridou E
(2013)
ACTH resistance: genes and mechanisms.
in Endocrine development
Novoselova TV
(2019)
ACTH signalling and adrenal development: lessons from mouse models.
in Endocrine connections
Corkery-Hayward M
(2023)
Adrenal Dysfunction in Mitochondrial Diseases
in International Journal of Molecular Sciences
Smith C
(2023)
Adrenal Insufficiency Associated with Biallelic Mutations in Porphyria Genes
in Endocrine Abstracts
Smith C
(2023)
Adrenal insufficiency can be associated with biallelic mutations in porphyria genes
in Endocrine Abstracts
Yates R
(2013)
Adrenocortical development, maintenance, and disease.
in Current topics in developmental biology
Chortis V
(2017)
Antioxidant pathway targeting as a therapeutic approach in adrenocortical carcinoma
in Endocrine Abstracts
Ali N
(2022)
Can Digenic, Tri-Allelic Inheritance of Variants in STAR and CYP11A1 Give Rise to Primary Adrenal Insufficiency? A Case Report.
in Frontiers in endocrinology
Costa A
(2018)
Can novel stem cell models help unpick the pathogenesis of the Triple A syndrome?
in Endocrine Abstracts
Cottrell E
(2022)
Characterisation of the first heterozygous missense HMGA2 variant helps delineate the crucial functional roles of a novel growth gene
in Endocrine Abstracts
Kidd E
(2023)
Choice of gDNA isolation method has a significant impact on average murine Telomere Length estimates
in Preparative Biochemistry & Biotechnology
Musa Salwa A.
(2022)
Clinical and Genetic characterization of primary adrenal insufficiency in Sudanese children
in HORMONE RESEARCH IN PAEDIATRICS
Zhou J
(2021)
CTNNB1-Mutant Aldosterone-Producing Adenomas With Somatic Mutations of GNA11/GNAQ Have Distinct Phenotype and Genotype
in Journal of the Endocrine Society
Kallali Wafa
(2019)
CYP11A1 (side-chain cleavage enzyme) defect in three brothers causing glucocorticoid and mineralocorticoid deficiency and development of testicular adrenal rest testicular tumour
in HORMONE RESEARCH IN PAEDIATRICS
Maharaj A
(2016)
Cytochrome p450 side chain cleavage enzyme (CYP11A1) mutations: Phenotypic variability and identification of p.E314K as a recurrent, pathogenic variant
in Endocrine Abstracts
Prasad R
(2013)
Deficiency of ALADIN impairs redox homeostasis in human adrenal cells and inhibits steroidogenesis.
in Endocrinology
Klammt J
(2018)
Dominant-negative STAT5B mutations cause growth hormone insensitivity with short stature and mild immune dysregulation.
in Nature communications
Williams J
(2022)
Elevated SGPL1 expression is associated with increased metabolic rate in cells and reduced survival in individuals with adrenocortical carcinoma
in Endocrine Abstracts
Williams JL
(2023)
Elevated sphingosine-1-phosphate lyase leads to increased metabolism and reduced survival in adrenocortical carcinoma.
in European journal of endocrinology
Williams JL
(2023)
Elevated sphingosine-1-phosphate lyase leads to increased metabolism and reduced survival in adrenocortical carcinoma.
in European journal of endocrinology
Habeb AM
(2013)
Familial glucocorticoid deficiency: a diagnostic challenge during acute illness.
in European journal of pediatrics
Meimaridou E
(2013)
Familial glucocorticoid deficiency: New genes and mechanisms.
in Molecular and cellular endocrinology
Smith C
(2023)
Genetic Aetiology of Primary Adrenal Insufficiency in Sudan
in Endocrine Abstracts
Smith C
(2023)
Genetic aetiology of primary adrenal insufficiency in Sudan
in Endocrine Abstracts
Buonocore Federica
(2021)
Genetic analysis of paediatric primary adrenal insufficiency of unknown aetiology over 25 years
in HORMONE RESEARCH IN PAEDIATRICS
Buonocore F
(2021)
Genetic Analysis of Pediatric Primary Adrenal Insufficiency of Unknown Etiology: 25 Years' Experience in the UK
in Journal of the Endocrine Society
Storr HL
(2015)
Genetic characterisation of a cohort of children clinically labelled as GH or IGF1 insensitive: diagnostic value of serum IGF1 and height at presentation.
in European journal of endocrinology
Smith C. J.
(2019)
Genetics of Familial Glucocorticoid Deficiency over the Decades: Phenotypic Variability and Associated Features
in HORMONE RESEARCH IN PAEDIATRICS
Meimaridou E
(2013)
Genome sequencing reveals mitochondrial thiol systems are essential for antioxidant defence in human adrenal glands
in Free Radical Biology and Medicine
Storr HL
(2015)
Heterogeneity of the growth phenotype and birth size in acid-labile subunit (ALS) deficiency.
in Journal of endocrinological investigation
Howard SR
(2018)
HS6ST1 Insufficiency Causes Self-Limited Delayed Puberty in Contrast With Other GnRH Deficiency Genes.
in The Journal of clinical endocrinology and metabolism
Smith C. J.
(2021)
Ichthyosis associated with sphingosine 1-phosphate lyase deficiency is due to aberrant calcium and sphingolipid regulation
in JOURNAL OF INVESTIGATIVE DERMATOLOGY
Smith CJ
(2023)
Ichthyosis linked to sphingosine 1-phosphate lyase insufficiency is due to aberrant sphingolipid and calcium regulation.
in Journal of lipid research
Smith CJ
(2023)
Ichthyosis linked to sphingosine 1-phosphate lyase insufficiency is due to aberrant sphingolipid and calcium regulation.
in Journal of lipid research
Högler W
(2014)
IGFALS gene dosage effects on serum IGF-I and glucose metabolism, body composition, bone growth in length and width, and the pharmacokinetics of recombinant human IGF-I administration.
in The Journal of clinical endocrinology and metabolism
Description | clinical practice |
Geographic Reach | Multiple continents/international |
Policy Influence Type | Influenced training of practitioners or researchers |
Description | Identifying disease-causing variants in endocrine diseases using machine learning approaches in bioinformatics |
Amount | € 46,563 (EUR) |
Organisation | European Commission |
Sector | Public |
Country | European Union (EU) |
Start | 10/2017 |
End | 12/2018 |
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 |
Description | Role of the sphingolipid pathway in primary adrenal insufficiency |
Amount | £318,860 (GBP) |
Organisation | Barts Charity |
Sector | Charity/Non Profit |
Country | United Kingdom |
Start | 04/2017 |
End | 03/2021 |
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 | Mouse substrain RNAseq in adrenals |
Description | RNAseq data from adrenals of three mouse models with varying NNT levels; Nnt null (C57BL/6J), Nnt wild-type (C57BL/6N and Nnt overexpressor (C57BL/6J-BAC) |
Type Of Material | Database/Collection of data |
Year Produced | 2018 |
Provided To Others? | Yes |
Impact | Recognition that oxidative stress impacts steroidogenesis |
Title | Mouse substrain RNAseq in hearts |
Description | RNAseq data from C57BL/6N, C57BL/6J and C57BL/6J-BAC mouse hearts |
Type Of Material | Database/Collection of data |
Year Produced | 2020 |
Provided To Others? | Yes |
Impact | Recognition by International Mouse Phenotyping Consortium that the genotype of C57BL/6N mice, having a mutation in Mylk3, may contribute to this strains susceptibility to cardiomyopathy/cardiac phenotypes |
URL | http://www.ncbi.nlm.nih.gov/bioproject/531833 |
Description | Adrenal genetics |
Organisation | University College 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 | 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 |
Description | School Careers Fair |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Schools |
Results and Impact | I attended a School Career Fair to talk about my research and inspire students to follow a research career |
Year(s) Of Engagement Activity | 2015,2016,2017,2018,2019 |
Description | Work experience for school students |
Form Of Engagement Activity | Participation in an open day or visit at my research institution |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Schools |
Results and Impact | Work experience for students in school years 12 and 13, those just about to make decisions about applying to universities. Purpose was to see if the students would enjoy a career in biological sciences and, in particular, whether research is something they would consider as a career. Some students have returned to do projects/further work experience with us. |
Year(s) Of Engagement Activity | 2016,2017,2018,2019,2020 |