(Received for publication, January 25, 1996)
From the
The cytoplasmic iron regulatory protein (IRP) modulates iron
homeostasis by binding to iron-responsive elements (IREs) in the
transferrin receptor and ferritin mRNAs to coordinately regulate
transferrin receptor mRNA stability and ferritin mRNA translational
efficiency, respectively. These studies demonstrate that thyroid
hormone (T) can modulate the binding activity of the IRP to
an IRE in vitro and in vivo. T
augmented
an iron-induced reduction in IRP binding activity to a ferritin IRE in
RNA electrophoretic mobility shift assays using cytoplasmic extracts
from human liver hepatoma (HepG2) cells. Hepatic IRP binding to the
ferritin IRE also diminished after in vivo administration of
T
with iron to rats. In transient transfection studies
using HepG2 cells and a human ferritin IRE-chloramphenicol
acetyltransferase (H-IRE-CAT) construct, T
augmented an
iron-induced increase in CAT activity by
45%. RNase protection
analysis showed that this increase in CAT activity was not due to a
change in the steady state level of CAT mRNA. Nuclear
T
-receptors may be necessary for this T
-induced
response, because the effect could not be reproduced by the addition of
T
directly to cytoplasmic extracts and was absent in CV-1
cells which lack T
-receptors. We conclude that T
can functionally regulate the IRE binding activity of the IRP.
These observations provide evidence of a novel mechanism for
T
to up-regulate hepatic ferritin expression, which may in
part contribute to the elevated serum ferritin levels seen in
hyperthyroidism.
The iron regulatory protein (IRP, ()previously known
as the iron-responsive element-binding protein, IRE-BP, and iron
responsive factor, IRF) is a trans-acting RNA-binding protein
which binds with high affinity to conserved stem-loop structures,
iron-responsive elements (IREs), present in the ferritin, transferrin
receptor (TfR), and erythroid 5-aminolevulinate synthase
mRNAs(1, 2, 3) . The IRP serves a central
role in the regulation of iron (Fe) homeostasis(1) . In the
absence of iron, the IRP binds to the IRE in the 5`-untranslated region
(5`-UTR) of ferritin and erythroid 5-aminolevulinate synthase mRNAs and
represses translation(4, 5, 6) . Binding of
the IRP to IREs in the 3`-untranslated region (3`-UTR) of TfR mRNA
stabilizes the mRNA and prevents its
degradation(7, 8, 9) . In iron-replete
states, the reverse holds, which results in increased ferritin
translation and decreased TfR mRNA stability. This reciprocal
regulation is achieved at the post-translational level and is
independent of new protein synthesis (10) .
Two IRPs have
been defined in various human and rat
tissues(3, 11, 12) . The most widely
expressed and abundant IRP in human tissues is
IRP1(1, 3) . A second human IRP (IRP2) has been
described recently. IRP2 is 57% identical with IRP1 at the amino acid
level and 2-10 times less abundant than IRP1 in most tissues,
except in the brain(3) . In contrast to IRP1, cellular
concentrations of IRP2 are inversely regulated by iron levels due to
iron-dependent regulation of the half-life of IRP2 protein(3) .
The relative contribution of each of these species to iron homeostasis
remains to be elucidated. The two rat IRPs have been designated BP1 and
BP2 (12) (also known as IRF and IRF, respectively) (11) and may represent rodent counterparts for IRP1 and IRP2.
Significant functional differences exist between BP1 and BP2. In
particular, BP2 does not have functional aconitase activity and, in
contrast to BP1, levels of BP2 protein are regulated by
iron(12) . In addition, IRF
(and presumably BP2) is
expressed most abundantly in intestine, brain, and kidney (11) .
Thyroid hormone (T) plays a central role
in differentiation, development, and maintenance of body
homeostasis(13) . The actions of T
, like the
steroid hormones, are mediated through intracellular
T
-receptor proteins (TRs)(14, 15) which
act predominantly to modulate transcription by binding to specific
T
-response elements in target genes(16) . Recent
studies have demonstrated, however, that T
also has
important effects at the post-transcriptional level to regulate the
expression of several genes, including the
-subunit of thyrotropin
(TSH
)(17) , the thyrotropin releasing hormone
receptor(18) , and the retinoid-X receptor(19) . To
date, there is little understanding of the molecular mechanisms
underlying these T
-induced changes in mRNA stability.
Several groups have documented an association between T levels and ferritin expression. In earlier reports,
hypothyroidism produced by thyroidectomy was associated with increased
rat hepatic ferritin content, which was found to be due to
post-transcriptional changes in the ferritin synthetic
rate(20, 21) . More recently, however, and in
contrast, hyperthyroid rats with elevated T
and T
levels were found to have an increased liver ferritin protein
synthesis rate (38% above control) (22) . Part of this increase
may be due to elevated IRE-mediated ferritin translation, although
T
has also been shown to increase the transcription rate of
H-ferritin mRNA in rat C6 glioma cells(23) , raising the
possibility of a transcriptional effect in the liver. Of particular
interest, are reports from several groups in which T
was
shown to positively regulate serum ferritin measurements in
humans(24, 25, 26, 27, 28) ,
similar to the changes reported in the rat(22) . Elevated serum
ferritin levels were observed in hyperthyroid individuals, and levels
decreased significantly after antithyroid treatment with normalization
of T
levels(24, 25, 26, 28) .
Furthermore, administration of T
to hypothyroid individuals
produced a significant increase in the serum ferritin level (26, 27) . Although the cause of the
T
-induced increase in the serum ferritin level in humans is
unknown, increased synthesis of ferritin in the liver may well be an
important contributor. These links between T
and the
regulation of ferritin expression suggest that a positive correlation
exists between the levels of T
/T
and ferritin
in the serum. However, the molecular mechanisms involved in the hepatic
regulation of ferritin expression by T
remain to be
determined.
We reasoned that a component of the effect of T on ferritin expression in the liver was due to
T
-induced modulation of IRP binding to the ferritin IRE.
Therefore, we used 1) the RNA electrophoretic mobility shift assay
(REMSA) and 2) transient transfection assays to investigate the
regulation of IRE-dependent gene expression by T
, in
vivo and in vitro. Our results demonstrate that T
can functionally regulate the binding activity of the human and
rat IRP to a ferritin IRE. These data provide evidence for a role of
T
in the post-transcriptional regulation of iron-responsive
genes and new insights into the action of T
in the
modulation of iron homeostasis. Furthermore, these data may, in part,
explain the positive association between serum levels of T
and ferritin.
Figure 1: Plasmids for generation of RNA probes and transfection. pgem-IRE is a 118-base sense transcript prepared with SP6 polymerase from a SmaI-digested rat L-subunit ferritin pseudogene (pGL-66). It contains the first 65 bases of the 5`-UTR (large hatching), including a conserved IRE stem-loop (IRE), 33 bases of the 5`-flanking sequence (small hatching), and 20 bases from pGEM2. pgem-vec is pgem-IRE linearized with SmaI but transcribed with T7 polymerase to produce a 45-base transcript containing vector sequence only. The H-ferritin genomic clone, pUC-HFER, contains a 458-base SstI fragment from the H-ferritin gene cloned into pUC12. The insert comprises 164 bases of the H-ferritin gene sequence upstream of the 5` cap site and 294 bases of the first exon including the 5`-UTR. The 302-base SstI-StyI fragment from pUC-HFER was ligated into the unique SstI and HincII sites in the polylinker of pUC12CAT to produce H-IRE-CAT. pUC12CAT contains the CAT gene inserted into the HindIII site of pUC12. H-IRE-CAT has the H-ferritin promoter and 5`-UTR in front of the CAT open reading frame, and the transcript has a correctly positioned IRE. 182 bases of the H-ferritin promoter is present in H-IRE-CAT, and it transcribes the first 142 bases of H-ferritin mRNA as part of a hybrid H-ferritin/CAT transcript.
Figure 2:
The IRPIRE complex is species- and
tissue-specific. A, REMSA was performed using
P-labeled pgem-IRE and 5 µg of different cytoplasmic
extracts: HepG2 cells (lanes 1 and 2) and rat liver (lanes 3-6). The binding reactions were incubated for 30
min at room temperature prior to sequential addition of RNase T1 and
heparin, as described under ``Experimental Procedures.'' The
binding mixtures were analyzed by electrophoresis on a 4% nondenaturing
polyacrylamide gel (REMSA). In lanes 2 and 4, 2-ME
(2%) was added to the reaction mixture at the beginning of the
incubation. A 100-fold excess of specific (unlabeled pgem-IRE, lane
5) or nonspecific (pgem-vec, lane 6) competitor RNA was
incubated with the extract for 10 min at 22 °C prior to addition of
labeled probe. B, UV cross-linking analysis of rat liver
IRE
IRP complexes. Arrows at BP1 and BP2 denote rat RPCs
containing IRPs and IRE(11, 12) . Following incubation
with RNase T1 and heparin,
P-labeled RPCs were
UV-cross-linked as described under ``Experimental
Procedures.'' The complexes were treated with RNase A at 37 °C
for 15 min. After addition of SDS-sample buffer and boiling, the
cross-linked products were separated by 7% SDS-PAGE and analyzed by
autoradiography. RNase A, incubation with RNase A after
UV-cross-linking; the positions of the molecular weight markers are
indicated.
We then confirmed that the cytoplasmic regulation of IRP binding to the ferritin IRE was preserved in these cells. The IRE binding activity of the IRP is typically increased in vitro by reducing agents, such as 2-mercaptoethanol (2-ME)(37) . As predicted, 2-ME (2%) increased the binding of the IRP to IRE RNA with both HepG2 and rat liver cytoplasmic extracts (Fig. 2A, lanes 2 and 4). Interestingly, the BP2 RPC in rat liver was consistently abolished in the presence of 2-ME (Fig. 2A, lane 4), and the intensity of the BP1 RPC was increased. A 100-fold excess of an unlabeled specific competitor RNA (pgem-IRE) competed efficiently for binding to the IRP and abolished BP1 and BP2 RPC formation (lane 5). However, addition of excess unlabeled nonspecific competitor RNA, either growth hormone (data not shown) or pgem-vec (lane 6), did not affect either RPC. The two bands at the bottom of each lane are nonspecific.
To confirm the size of the two rat RPCs, BP1 and BP2, a portion of
the reaction mixture from Fig. 2A, lane 3, was
analyzed after UV cross-linking and digestion with RNase A (100
µg/ml) for 30 min at 37 °C. Two RPCs were identified which
migrated at the positions predicted for the BP1 and BP2 proteins
(90-95 and
105 kDa, respectively; Fig. 2B)(11, 12) . We concluded that
these cells contained IRPs, BP1 and BP2, which were of the predicted
size and which displayed appropriate regulation with reducing agents.
Figure 3:
T modifies IRP binding to a
ferritin IRE in iron-loaded rat liver cells in vivo. REMSA (4%
PAGE) was performed using 5 µg of rat liver cell cytoplasmic
extract and
P-labeled pgem-IRE. Male Spraque-Dawley rats (n = 14) were given a single intraperitoneal injection
with either vehicle alone (lane 1, n = 3),
T
(20 µg/100 g body weight, lane 2, n = 3), FAC (2.3 mg/100 g body weight, lane 3, n = 4), or both (lane 4, n = 4). The rats
were killed 8 h later, and liver cytoplasmic extracts were prepared and
used in REMSA with labeled pgem-IRE as described in Fig. 2. Each
lane, with the exception of lane 5, represents an analysis of
liver extract from a different rat. A portion of the reaction mixture
from lane 4 was incubated separately with 2-ME (2%) prior to
PAGE analysis (lane 5). Arrows at BP1 and BP2 denote
rat liver cell RPCs containing IRPs and IRE (compare with Fig. 2).
Figure 4:
T modifies IRP binding to a
ferritin IRE in iron-replete human liver HepG2 cells. REMSA (4% PAGE)
was performed using HepG2 cell cytoplasmic extracts and
P-labeled pgem-IRE. The cells were cultured in hypothyroid
medium for 36 h, before FAC was added to the medium 16 h prior to
harvesting (110 µM, lanes 2-5). T
(100 nM) was also added at various times (16, 4, and 2
h) prior to harvesting. REMSA was performed using 5 µg of
cytoplasmic extract from each of these different cells and analyzed as
described in Fig. 2. The intensity of the IRP
IRE RPC was
quantitated by Betagen scanning, and the results are presented at the top of each lane. This REMSA is representative of other (n = 4) experiments.
Similar experiments were conducted using HepG2 cells after the
addition of Df (100 mg/ml) in the presence and absence of
T. However, we were unable to detect any effect of T
on the IRP
IRE complex in iron-depleted cells using REMSA
(data not shown).
HepG2 cells, cultured in hypothyroid
medium, were transiently transfected with H-IRE-CAT and then incubated
in the presence or absence of various combinations of FAC,
T, and Df. After 24 h, the cells were harvested and the
level of [
H]acetylchloramphenicol, a measure of
the CAT activity, was measured. The results shown in Fig. 5are
from a representative experiment, although similar results have been
observed in all other HepG2 experiments (n = 3) and in
H-IRE-CAT transfectants of rat GH
pituitary cells. (
)In the absence of iron loading, no increase in CAT
activity was seen after the addition of 100 nM T
alone (lane 2). As expected, incubation with 110
µM FAC alone increased CAT activity (lane 3).
However, co-culture of 100 nM T
with iron further
increased the CAT activity (lane 4) approximately
35-45%. Even co-culture of 1 nM T
with iron
increased CAT activity (lane 5), consistent with a
dose-response relationship. In contrast, incubation of the cells with
Df (100 mg/ml) reduced the CAT activity by
50% from basal (lane 6), reflecting high affinity binding of the IRP to the
IRE and reduced translational efficiency. Addition of 100 nM T
did not further decrease the CAT activity
significantly (lane 7). The 3-fold overall difference in
translational efficiency of H-IRE-CAT after stimulation with FAC and Df
is consistent with similar findings in other recent
reports(5, 6, 11) .
Figure 5:
Effect of T on the
translational efficiency of hybrid CAT mRNA in HepG2 cell H-IRE-CAT
transfectants. Hybrid CAT mRNAs were transcribed from the 182 base
promoter in the H-ferritin gene and contain 142 bases of H-ferritin
mRNA as part of a hybrid H-ferritin/CAT transcript (H-IRE-CAT). HepG2
cells, cultured in hypothyroid medium, were transiently co-transfected
with 10 µg/well of H-IRE-CAT and 0.8 µg/well of Rous sarcoma
virus-
-galactosidase (
-gal) control plasmid. Cells were grown
for an additional 24 h in the absence or presence of combinations of
iron (FAC, 110 µM), Df (100 mg/ml), and T
(either 1 or 100 nM), prior to cell lysis and CAT assay.
-gal activity was determined for each extract, and the
[
H]acetylchloramphenicol (cpm) was normalized
against
-gal activity. The data are a representative experiment (n = 3), performed in quadruplicate. Error bars indicate S.D.
To ensure that the
increased CAT activity induced by T was not a consequence
of increased transcription of H-IRE-CAT, CAT mRNA was quantitated by
slot blotting and RNase protection. CAT mRNA levels were measured in
RNA extracted from the same H-IRE-CAT HepG2 transfectants by slot
blotting. The slot blot was performed in duplicate for two separate
loadings of 4 and 20 µg of RNA. Compared to control (hypothyroid
medium alone), no significant increase in CAT mRNA was detected after
the addition of T
to the cultured cells (Fig. 6A). The radioactivity in each of the bands was
quantitated, and no increase in counts was detectable in the
T
-treated samples (data not shown). These results are
consistent with the data in Fig. 5, lane 2, in which
incubation with T
alone did not increase CAT activity.
Figure 6:
The effect of iron and T on
the levels of CAT mRNA in H-IRE-CAT transfectants. A,
slot/blot analysis of CAT mRNA. RNA was prepared from the transfectants
of lanes 1 and 2 in Fig. 5. The relative
levels of CAT mRNA in 4 and 20 µg of each RNA population were
quantitated by slot blotting and hybridization to a labeled CAT gene
probe. This is a representative experiment (n = 2). Control, hypothyroid medium alone; T
, incubation
with 100 nM T
. B, RNase protection
analysis. Antisense RNA was used to detect the expression of the
transfected transcript by RNase protection in a second set of
experiments. The arrow indicates the size of the input
antisense cRNA (261 nucleotides for CAT cRNA). The RNase T1-protected
cRNA is smaller than the input cRNA, migrating slightly faster during
electrophoresis (254 nucleotides for protected CAT cRNA). The lanes
represent RNase protections using the following transfected or
untransfected RNA sources: lane 1, H-IRE-CAT; lane 2,
H-IRE-CAT + iron (110 µM); lane 3, H-IRE-CAT
+ T
(100 nM); lane 4, H-IRE-CAT
+ iron (110 µM) + T
(100
nM); lane 5, tRNA; lane 6, input CAT cRNA.
This is a representative experiment (n = 3) performed
in duplicate.
RNase protection analysis confirmed that the levels of protected CAT
mRNA in H-IRE-CAT-transfected HepG2 cells do not change in response to
iron, T, or a combination of iron and T
(Fig. 6B, lanes 2-4,
respectively). This is consistent with previous data confirming the
post-translational nature of the regulation of IRP binding activity by
iron. Furthermore, these results indicated that the effect of T
was not due to transcriptional up-regulation of H-IRE-CAT. Taken
together, these results indicate that T
augments Fe-induced
displacement of the IRP from the ferritin IRE independent of changes in
H-IRE-CAT transcription. Moreover, these functional data complement our
findings in the REMSA studies and support the notion that, in
iron-replete cells, T
acts to augment the effect of iron,
by modifying the binding activity of the IRP to the IRE in ferritin
mRNA.
Figure 7:
Lack of effect of T on IRP
binding to a ferritin IRE in iron-replete CV-1 cells. REMSA (4% PAGE)
was performed using 5 µg of CV-1 cell (monkey kidney cell that
lacks TRs) extract and
P-labeled pgem-IRE. The cells were
cultured in hypothyroid medium for 36 h, before iron (FAC (110
µM), lanes 1-4) was added to the medium 16
h prior to harvesting. T
(1 nM, lane 2;
100 nM, lane 3) was added to the reaction mixture at
the beginning of the incubation. REMSA was performed and analyzed as
described in Fig. 2. This is a representative experiment (n = 2).
To
determine if a component of this T effect was a direct
cytoplasmic effect, and independent of nuclear TRs, we incubated
various concentrations of T
(10
-10
M)
with HepG2 cell cytoplasmic extracts containing labeled pgem-IRE probe,
at the commencement of the reaction. As can be seen in Fig. 8,
addition of T
directly to the cytoplasmic reaction mixture (lanes 2-4), did not modify IRP binding activity. These
data support our results in CV-1 cells and are consistent with the
notion that in liver cells T
acts via nuclear TRs to
facilitate displacement of the IRP from the ferritin IRE.
Figure 8:
Lack of effect of direct addition of
T on IRP binding to a ferritin IRE in iron-replete HepG2
cells. REMSA (4% PAGE) was performed using 5 µg of the HepG2 cell
extract and
P-labeled pgem-IRE. The cytoplasmic extract
used was the same as that used in lane 2, Fig. 3, in
which the cells were cultured in hypothyroid medium for 36 h, before
iron (FAC (110 µM), lanes 1-4) was added to
the medium 16 h prior to harvesting. T
(1 nM, lane 2; 100 nM, lane 3; 10 µM, lane 4) was added to the reaction mixture at the beginning of
the incubation. REMSA was performed and analyzed as described in Fig. 2. This is a representative experiment (n =
2).
The molecular mechanisms governing the regulation of ferritin
gene expression by iron are based on regulated changes in the IRE
binding activity of the IRPs. Here we provide the first evidence that
T, a hormone critical for maintaining body homeostasis, can
modulate the binding activity of the IRP to a ferritin IRE both in
vitro and in vivo. Our REMSA results indicate that
T
acts post-translationally to augment the iron-induced
displacement of the rat and human IRP from an IRE present in the 5`-UTR
of ferritin mRNA. Furthermore, this T
-induced effect was
associated with a similar sized functional increase in IRE-dependent
gene expression (
40-50%), as demonstrated in transfection
studies using a human ferritin IRE-CAT construct in human hepatoma
cells. Our results suggest that T
can, possibly in a
TR-dependent manner, functionally regulate the IRE binding activity of
the IRP.
Our data provide further evidence that the binding activity
of the IRP can be modified by agents other than iron and the redox
state. Recent data indicate a direct association with the nitric
oxide/nitric oxide synthase pathway, in which increases in NO activates
IRP binding to IREs in ferritin and TfR mRNAs(39) . The
reactive oxygen intermediate hydrogen peroxide
(HO
) has recently been shown to increase
binding of the IRP, resulting in reduced ferritin synthesis and
increased transferrin receptor expression(41) . In contrast to
activation of IRP by iron depletion which is okadaic acid-insensitive,
induction of IRP by H
O
is okadaic-sensitive
suggesting the involvement of stress-induced kinase/phosphatase
pathways(41) . Changes in the phosphorylation status of the IRP
mediated by protein kinase C may provide another level of
regulation(42) . T
, however, is the first endocrine
hormone that has been shown to modulate the IRP
IRE interaction
and ferritin translation, and our studies suggest that nuclear TRs are
possibly involved, as evidenced by the absence of effect in CV-1 cells
which lack endogenous nuclear TRs. Moreover, T
was unable,
even at high concentrations (10
M), to
reproduce the effect when added directly to the cytoplasmic extracts.
This IRE-dependent action of T
on ferritin translation
differs from other agents, such as interleukin 1, which modify ferritin
translation through IRE-independent mechanisms(43) . It is not
known which TR isoform(s) (40) is involved in mediating this
T
-induced effect, and this question requires further
investigation.
A significant body of evidence exists showing a
positive correlation between the serum levels of T/T
and ferritin (24, 25, 26, 27, 28) in
individuals with thyroid abnormalities. All of these studies documented
elevated serum ferritin levels in patients with hyperthyroidism which
normalized when the T
/T
levels returned to
normal. Interestingly, the positive relationship between serum ferritin
and T
/T
levels holds in hypothyroidism as well.
A similar positive relationship between serum ferritin and
T
/T
levels has been observed in rats rendered
hypo- and hyperthyroid(22) . Furthermore, the hepatic ferritin
synthesis rate increased significantly in hyperthyroid
rats(22) , consistent with the increased serum ferritin levels.
These observations are consistent with our own data. To date, there are
no human data to suggest that serum ferritin levels rise in
hypothyroidism. Thus, differences exist between the recent data and the
results presented herein compared to the earlier reports documenting
increased rat hepatic ferritin synthesis in
hypothyroidism(20, 21) . The reasons for this are
unclear, but may relate, in part, to the nature of thyroid dysfunction
utilized in each study (e.g. thyroidectomy versus T
supplementation). Interestingly, however, the
earlier work did document that the changes induced by T
were at the post-transcriptional level which would be consistent
with our results(21) .
The IRP is one of two trans-acting RNA-binding proteins whose binding activity is
modified by T. The other is an 80-85-kDa pituitary
protein that recognizes a specific region within the 3`-UTR of TSH
mRNA, an anterior pituitary hormone(33) . Remarkably, the RNA
binding site for this T
-regulated pituitary trans-acting factor within TSH
3`-UTR mRNA contains
features of a ferritin IRE. The TSH
3`-UTR sequence similarity
with a ferritin IRE extends over 12 nucleotides (9 of 12 nucleotides,
which includes the loop and a portion of the IRE stem, Fig. 9).
To investigate whether this sequence could compete with the IRE for IRP
binding, REMSA studies were performed. These showed that excess
unlabeled TSH
3`-UTR mRNA could compete efficiently with labeled
pgem-IRE for IRP binding in pituitary cells (33) . Further,
pgem-IRE competed efficiently with TSH
3`-UTR mRNA for binding of
this T
-regulated trans-acting factor(33) .
In summary, TSH
mRNA has an IRE-like element which can interact
with pituitary factors, including the IRP, in a T
-dependent
manner. The cellular consequences of interactions between the IRP with
other mRNAs sharing sequence similarity with the IRE is under further
investigation.
Figure 9:
Nucleotide sequence alignment of the rat
ferritin IRE with rat TSH 3`-UTR region. The stem-loop structure
depicts the rat ferritin IRE contained within the pgem-IRE
plasmid(30) . The six-membered loop contains five nucleotides
that are almost invariant (boxed nucleotides)(44) .
The 12-nucleotide region of sequence similarity comprises the sequence
between the two lines. Nucleotide differences between the IRE and the
consensus region sequence are indicated with arrows.
Our results are consistent with a model of iron
homeostasis in which IRE-dependent ferritin gene expression is
positively regulated by T. T
alone did not
alter expression at the transcriptional or post-transcriptional level.
However, in iron-replete cells, there was a significant T
effect to up-regulate ferritin gene expression, through
modulation of the IRP binding activity and enhanced IRE-dependent
translation. These results provide further insight into a rapidly
emerging model for the regulation of iron homeostasis, by providing
evidence that T
acts post-translationally to augment
displacement of the IRP from a ferritin IRE. Other endocrine hormones
may also modify IRP binding activity and have profound metabolic
effects (e.g. retinoic acid, glucocorticoids etc.). Further
experiments are in progress to investigate this possibility. Given the
important role that both T
and iron play in the maintenance
of body homeostasis, further elucidation of the control mechanisms
governing the interactions between T
and IRE-dependent gene
expression will be an important goal of future studies.