There is abundant evidence that in postnatal life, hypoxia elicits multiple cellular and
systemic physiological responses, including angiogenesis, erythropoiesis and glycolysis, to
help an organism survive and develop under hypoxic stress. Hypoxia also plays a
pathophysiological role in many human disorders, including heart attack, stroke, and
cancer. The hypothalamic-pituitary-adrenal axis controls many stress responses mediated
by aldosterone and cortisol (produced in the adrenal gland). High levels of these stress
hormones are associated with hypertension and may lead to heart, brain, and kidney
damage in humans. Hypoxia reduces aldosterone and cortisol production in vivo and in
vitro and may affect steroidogenesis in the adrenal gland. However, the molecular
mechanisms by which hypoxia impairs steroid hormone production are poorly understood.
The hypoxia-inducible factors (HIFs) − HIF-1, HIF-2 and HIF-3 - modulate a variety of
genes involved in various physiological and pathophysiological processes under hypoxia.
MicroRNAs are post-transcriptional regulators that control numerous biological processes,
including cell proliferation, migration, differentiation and metabolism. Little is known
about the possible role(s) of HIFs and miRNAs in the regulation of aldosterone and cortisol
production under hypoxia. Moreover, numerous intracellular signalling pathways and
several signalling molecules regulate steroidogenic gene transcription in the adrenal cortex.
However, the roles of signalling pathway-related genes in regulating aldosterone and
cortisol synthesis under hypoxia have yet to be determined.
Using H295R cells as an in vitro model, I tested the hypothesis that HIFs play an
important role in controlling aldosterone and cortisol biosynthesis through direct or indirect (via miRNAs and signal transduction pathway genes) regulation of certain steroidogenic
enzyme genes of the adrenal cortex. The H295R cell line possesses the characteristics of
undifferentiated human foetal adrenal cells, is capable of synthesising all the enzymes and
hormones involved in steroidogenesis, and has been employed extensively to study the
molecular responses to and regulation of steroidogenesis. The effects of hypoxia were
determined by analysing the expression of (1) HIF-1α, -2α and -3α by qRT-PCR and
Western blot analyses; (2) three steroidogenic enzyme genes (CYP21A2, CYP11B1, and
CYP11B2) by qRT-PCR; (3) five transcription factor genes [NR5A1 (SF-1), NROB1 (DAX-1), NR4A1 (NURR-77), NR4A3 (NOR-1) and CITED2], which modulate stress steroid
production, by q-RT-PCR; and (4) two stress steroid hormones (aldosterone and cortisol)
by ELISA in H295R cells.
Overexpression and knockdown of the human HIF-1α, -2α and -3α proteins in H295R
cells were carried out using a lentiviral delivery system (Invitrogen). The effects of these
three HIF-α proteins on specific steroidogenic genes, regulatory transcription factors and
stress hormone levels were monitored by the techniques described above. In addition,
miRNA expression profiling and signal transduction pathway gene expression profiling
were performed in normoxic, hypoxic, HIFα-overexpressing and HIFα-knockdown H295R
cells. Computational analysis revealed several distinct groups of miRNAs (upregulated and
downregulated) in response to hypoxia or overexpression/knockdown of HIF-α proteins.
Based on findings from bioinformatic analyses, the effects of overexpression and
knockdown of miR-10b on specific steroidogenic enzyme genes and stress hormone levels
were investigated in H295R cells. Further, the functions of miR-10b binding sites in specific steroidogenic enzyme genes, namely CYP11B1 and CYP11B2, were characterised
by luciferase reporter assay.
Among the three HIF isoforms analysed, HIF-2α was expressed at a higher level than
HIF-1α, whereas HIF-3α was undetectable under both normoxic and hypoxic conditions in
H295R cells. Hypoxia induced the expression of CYP21A2, CYP11B2, NUR-77, NOR-1
and CITED2 but reduced SF-1 and DAX-1 expression. Importantly, aldosterone and
cortisol were significantly reduced in hypoxic H295R cells. CITED2 and DAX-1 were the
only two genes that showed reverse expression patterns under HIF-1α overexpression and
HIF-1α knockdown. A similar expression pattern was observed for NURR-77 and NOR-1
in HIF-2α overexpressing and knockdown H295R cells. The three steroidogenic enzyme
genes and the stress steroid hormones showed the same expression patterns under both
HIF-1α and HIF-2α overexpression and knockdown. Interestingly, HIF-3α overexpression
downregulated all three steroidogenic enzyme genes and all five regulatory transcription
factor genes. Moreover, computational analysis of the 5'-flanking regions of CYP21A2 and
CYP11B2 genes revealed a few putative hypoxia responsive elements (HREs).
The miRNA expression profiling experiments showed that the expression of 29% of
the miRNAs detected under normoxia (379) was altered under hypoxia. Comparative
analyses of the miRNA expression profiles in hypoxic, HIFα-overexpressing, and HIFα-
knockdown H295R cells revealed that the highest number of miRNAs were induced and
downregulated, respectively, under HIF2α overexpression and knockdown, including miR-
210. In addition, computational analyses using three miRNA target prediction algorithms
uncovered several miRNAs - miR-10b, miR-31, miR-329, and miR-504 - that putatively
targeted adrenal steroidogenic enzyme genes, especially CYP11B1 and CYP11B2. miR-10b overexpression and knockdown showed that miR-10b significantly downregulated the
mRNA expression of CYP11B1 and CYP11B2 and the levels of the steroid hormones
aldosterone and cortisol. The 3'-UTR analyses of CYP11B1 and CYP11B2 revealed that
both genes contained three putative sequences in their respective 3'-UTRs that
complemented the miR-10b "seed" sequence. Further functional characterisation of the
miR-10b binding sites in the respective 3'-UTRs of CYP11B1 and CYP11B2 by luciferase
reporter assay demonstrated only 0.80- to 0.75-fold reduction in luciferase activity,
suggesting other miRNAs along with miR-10b have roles in the regulation of aldosterone
and cortisol biosynthesis under hypoxia.
The human signal transduction pathway-related gene expression profiling revealed
that hypoxia modulated a number of human signal transduction pathways, for example, the
mitogenic, Wnt, Hedgehog, calcium and protein kinase C, phospholipase C and insulin
pathways in H295R cells. HIF1α had a positive regulatory effect on the Hedgehog and
insulin pathways by targeting several genes, such as WNT1, WNT2, HK2, and LEP. Several
genes of the mitogenic and insulin signal transduction pathways, such as EGR1, FOS, JUN,
and LEP, modulate adrenal steroidogenic enzyme genes, including StAR, CYP11B1, and
CYP11B2. Further investigations are needed to better understand the relationships among
the specific genes of signal transduction pathways and HIFs and their roles in regulating
adrenal steroidogenic enzyme genes under hypoxia.
Overall, the findings in this study suggest that HIFs potentially induce several
miRNAs and specific genes of signal transduction pathways under hypoxia, thereby
modulating the major steroidogenic enzyme genes and the final outcome of stress steroid
production in H295R cells.
| Date of Award | 15 Jul 2013 |
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| Original language | English |
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| Awarding Institution | - City University of Hong Kong
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| Supervisor | Yuen Chong Richard KONG (Supervisor) |
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