Regulation of adrenal corticosteroidogenesis: the role of microRNAs in the control of aldosterone synthase and 11β-hydroxylase expression

Wood, Stacy (2012) Regulation of adrenal corticosteroidogenesis: the role of microRNAs in the control of aldosterone synthase and 11β-hydroxylase expression. PhD thesis, University of Glasgow.

Abstract

Hypertension is a major risk factor for all cardiovascular disease, which is the
largest known cause of global mortality. Essential hypertension-that is
hypertension of unknown cause-is thought to have genetic and environmental
risk factors. The best studied genetic system is that concerning corticosteroid
biosynthesis. In humans, the principal glucocorticoid is cortisol, the main
function of which is the control of intermediary metabolism; the major
mineralocorticoid is aldosterone, which affects electrolyte and acid-base
homeostasis. These steroid hormones are produced in the adrenal cortex through
a series of biosynthetic reactions and under the influence of multiple regulatory
factors. The final step in cortisol and aldosterone production involves,
respectively, the cytochrome p450 enzymes, 11β-hydroxylase and aldosterone
synthase. These are encoded by the CYP11B1 and CYP11B2 genes which have a
similar sequence and are highly polymorphic and lie, in tandem, on human
chromosome 8.
Regulation of CYP11B1 and CYP11B2 mRNA abundance and of aldosterone and
cortisol production have been extensively investigated. These studies have
identified that there are several polymorphisms located across the locus which
are associated with an increased aldosterone to renin ratio (ARR; used as an
indicator of aldosterone regulation), inefficient 11β-hydroxylation and essential
hypertension. However, to date, no underlying mechanism for these associations
has been established. Regulation of expression by transcription factors has been
widely studied but, in this thesis, it is the role of a novel regulator, microRNA
(miRNA) that is central.
miRNAs are short, non-coding RNAs which negatively regulate mRNA abundance
They are transcribed from endogenous loci, then undergo a series of enzymatic
maturation reactions that result in the production of a single-stranded molecule
of approximately 20 nucleotides. They function by associating with a group of
proteins known as the RNA-induced silencing complex (RISC) and targeting the 3’
untranslated region (3’UTR) of specific target mRNAs which they bind with
imperfect complementarity. There are approximately 1100 human miRNAs,
which have been implicated in the regulation of a range of target mRNAs and in
several pathologies including cancer and cardiovascular disease. The aim of this
project was to investigate what role, if any, miRNAs have in the regulation of
CYP11B1 and CYP11B2 expression and in corticosteroid production.
The studies in Chapter 3 investigated miRNA regulation of corticosteroidogenesis
in the adrenal cell line H295R. miRNA levels were universally reduced by
targeting Dicer mRNA, a key component of the miRNA synthetic pathway, with
short interfering RNA (siRNA). This study identified all of the CYP450 enzymes of
the corticosteroidogenic pathway (CYP11A1, CYP17A1, CYP21A1, CYP11B1 and
CYP11B2) as likely candidates for miR-mediated regulation based on mRNA and
steroid analysis. The study also suggested that StAR, 3βHSDII and 11βHSDII are
not modulated by miRNAs. To determine whether apparent miRNA regulation of
CYP11B1 and CYP11B2 expression occurs by direct action at their 3’UTRs,
reporter constructs were generated and tested. Under both basal and stimulated
(AngII) conditions, these studies support a regulatory mechanism involving the
3’UTR of CYP11B1 and CYP11B2. This chapter therefore provides evidence for
miRNA-mediated regulation of corticosteroidogenesis.
In Chapter 4, putative miRNA target sites in the CYP11B1 and CYP11B2 3’UTR
were identified using bioinformatic prediction algorithms and the miRNA
expression profile of the normal human adrenal, as determined by microarray
analysis. Based on miRNA target site prediction and analyses of the 3’UTR
sequences (including such parameters as relative length, predicted sequence
conservation and RNA secondary structure), in silico methods indicated the
possibility that miRNAs can target CYP11B1 and CYP11B2 mRNA. Furthermore,
the expression of 107 miRNAs in the normal adrenal gland was confirmed.
Cross-referencing of microarray expression and bioinformatic data identified 16
adrenal miRNAs predicted to bind putative sites in CYP11B1 and 16 predicted to
bind CYP11B2; 12 of these miRNAs were common to both genes.
These formed the basis of the miRNA target validation studies in Chapter 5.
Sixteen adrenal miRNAs identified by bioinformatic analysis were tested
individually in vitro. This was achieved by measuring mRNA expression, steroid
production and 3’UTR reporter construct activity following artificially induced
increases or reductions in the levels of specific miRNAs. These studies identified
some miRNAs as being false positive predictions, while certain others were
validated. The miRNA that gave the most striking and consistent results, for
targeting both CYP11B1 and CYP11B2, was miR-24, which significantly decreased
mRNA levels and steroid production. Analysis of adrenal miRNAs predicted only
to target the CYP11B2 3’UTR confirmed miR-125a-5p and miR-125b as novel
regulators, although effects on steroid secretion remain to be assessed. The
studies in this chapter are the first to report of miRNA-mediated regulation of
CYP11B1 and CYP11B2 expression.
Finally, in Chapter 6, the miRNA expression profiles of four aldosterone producing
adenoma (APA) samples were generated and compared to those of
normal adrenal gland. Analysis identified 67 miRNAs expressed within the APAs;
54 were also present in the normal tissue. The levels of several miRNAs,
including miR-24 and miR-125a-5p, were shown to be differentially expressed
between the tissue types. This chapter also describes polymorphisms within the
3’UTR of the CYP11B1 gene, generated from 26 normotensive patients. No novel
SNPs were identified, but three are located in putative miRNA-binding sites.
Previously, sequence analysis of the CYP11B2 3’UTR had been used to map
miRNA binding sites, this identified two miRNA-binding sites which mapped to a
known SNP. Taken together, the studies in this chapter provide a foundation for
exploring altered miRNA function and/or expression within the adrenal gland.
In summary, the results presented in this thesis support a role for miRNA mediated
regulation of corticosteroidogenesis through actions on CYP11B1 and
CYP11B2 expression. It demonstrates that miRNA are present in the adrenal
gland, that miRNA-binding sites are present on the 3’UTR of relevant mRNAs,
and that miRNAs are capable of post-transcriptional regulation that significantly
alters mRNA abundance and steroid production. My findings describe a novel
regulatory mechanism of corticosteroidogenesis. Whether this mechanism is
altered in diseases such as essential hypertension remains to be elucidated. If
so, miRNAs could, in the longer term, be used as targets for novel therapies or
as biomarkers to classify more precisely specific pathologies.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Keywords: Aldosterone, Cortisol, adrenal, APA, CYP11B1, CYP11B2, microRNA, H295R, 3'UTR
• Subjects: R Medicine > R Medicine (General); Q Science > Q Science (General)
• Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Cardiovascular & Metabolic Health
• Supervisor's Name: Davies, Prof. Eleanor and MacKenzie, Dr. Scott
• Date of Award: 2012
• Depositing User: Stacy Wood
• Unique ID: glathesis:2012-3252
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 15 Mar 2012
• Last Modified: 12 Mar 2015 10:01
• URI: https://theses.gla.ac.uk/id/eprint/3252