(Received for publication, July 11, 1994; and in revised form, September 22, 1994)
From the
Ferritin, by regulating the ``free'' intracellular
iron pool, controls iron-catalyzed generation of reactive oxygen
species, but its role in oxidative damage is still unclear. We show
that ferritin synthesis is significantly stimulated in the liver of
rats subjected to oxidative stress by treatment with phorone, a
glutathione-depleting drug. RNA-bandshift assays document reduced
activity of iron regulatory factor, in particular of IRF,
the cytoplasmic protein that post-transcriptionally controls ferritin
mRNA translation. Furthermore, Northern blot analysis shows increased
accumulation of H and L subunit mRNAs, and nuclear run-on experiments
provide evidence of transcriptional activation. Direct measurements of
intracellular free iron levels by EPR indicate that the increased
ferritin synthesis can be mediated by an expansion of the free iron
pool. An early drop of ferritin content after phorone treatment
indicates that part of the iron that fuels the free pool might derive
from ferritin degradation. Present data seem to suggest that, under
conditions of oxidative stress, liver ferritin can represent either a
pro- or an anti-oxidant in a time-dependent manner. In fact, its early
degradation contributes to expand the intracellular free iron pool
that, later on, activates multiple molecular mechanisms to reconstitute
ferritin content, thus limiting the prooxidant challenge of iron.
Ferritin is a multimeric protein composed of 24 subunits of two
types (H and L), which surround a cavity in which iron can be stored in
a readily available but non-toxic form(1, 2) . Changes
in iron availability regulate ferritin gene expression at several
levels, with the translational control being quantitatively more
relevant(3, 4, 5) . Ferritin mRNA translation
is controlled by a cytosolic protein, iron regulatory factor (IRF), ()whose regulated binding to an iron-responsive element
(IRE) in the 5`-untranslated region of the mRNA finely tunes ferritin
mRNA translation to intracellular iron levels (see (6, 7, 8, 9) for review). A number
of factors in addition to iron stimulate ferritin
synthesis(10) , which, in iron-independent pathophysiological
situations, is predominantly regulated at pretranslational
level(11) .
Reactive oxygen species (ROS) are generated in small amounts in the normal metabolism of the cells and in increased amounts under many conditions of altered cell physiology; they are responsible for many kinds of cell injuries (12) and have been recently shown to induce a significant reprogramming of gene expression(13) . Since intracellular iron catalyzes the generation of ROS(14, 15) , ferritin, with its iron-segregating capacity, plays an important role in modulating cellular sensitivity to oxidant insults. While earlier studies pointed to ferritin as a source of catalytically active iron(16) , more recent work showed that ferritin synthesis increases in different types of cultured cells subjected to oxidative stress conferring resistance to a subsequent insult(17, 18, 19, 20, 21) . However, the intracellular triggers of this activation have not been as yet clearly identified. In fact, although the involvement of an increased free iron pool was hypothesized, direct measurements have not been provided. Moreover, it has not been defined whether other sources in addition to heme degradation by heme oxygenase (20, 21) contribute to the increased metal availability. Furthermore, the molecular mechanisms leading to enhanced ferritin synthesis under conditions of oxidative stress have not been studied in detail.
We studied the regulation of ferritin synthesis in an in vivo model in which a condition of oxidative stress is established by administration of phorone, a glutathione-depleting drug(22) , which, by altering the balance between pro-oxidant and anti-oxidant molecules, amplifies the effects of ROS produced by cellular metabolic activity (23) . We describe here that after phorone treatment, an early increase in the free iron pool, caused both by heme destruction and by ferritin degradation, stimulates ferritin synthesis transcriptionally and posttranscriptionally.
Careful calibration procedures were carried out; free iron
concentration was calculated on a calibration plot obtained by adding
to a control homogenate incremental volumes of a stock solution of
FeSO, whose Fe
concentration was measured
using the o-phenanthroline assay. The homogenate was then
treated as described above, and the calibrating curve between amplitude
of EPR signal and concentration of added iron ions was plotted.
Figure 1:
Ferritin synthesis by liver slices.
Equal amounts of labeled proteins synthesized by rat liver slices
incubated in the presence of [S]methionine were
immunoprecipitated with a specific anti-rat liver ferritin antibody and
separated on a 15% SDS-polyacrylamide gel. Ferritin H and L subunits
were visualized by fluorography. The autoradiogram shown is
representative of three separate experiments. C, control; P
and P
, 3
and 6 h after phorone treatment.
Figure 2:
Bandshift assay of IRF activity. Cytosolic
extracts were incubated with an excess of P-labeled IRE
probe in the absence or presence of 2%
-mercaptoethanol, which is
known to activate IRF binding activity. RNA-protein complexes were
separated on nondenaturing 6% polyacrylamide gels and revealed by
autoradiography. The exposure time of autoradiograms was twice longer
for the samples without
-mercaptoethanol. The results shown are
representative of four separate experiments. C, control; P
and P
, 3
and 6 h after phorone treatment.
Figure 3:
Northern blot analysis of ferritin mRNAs
levels. A filter with equal amounts of total liver RNA was hybridized
with probes for H and L ferritin subunits, heme oxygenase (HO), and -actin as indicated under ``Materials and
Methods.'' The autoradiograms shown are representative of three
independent experiments. C, control; P
and P
, 3 and 6 h after phorone
treatment.
Figure 4:
Run-on transcription assay. Equal amounts
of P-labeled nuclear RNA synthesized in vitro by
isolated liver nuclei were hybridized to panels of the indicated DNA
probes immobilized on nitrocellulose filters. The autoradiogram shown
is typical of three separate experiments. C, control; P
and P
, 3
and 6 h after phorone treatment; HO, heme
oxygenase.
Figure 5:
Liver ferritin content. Cytoplasmic
extracts were enriched in ferritin by heating as described under
``Materials and Methods,'' and equal amounts of proteins were
electrophoresed on non-denaturing 7.5% polyacrylamide gels. Ferritin
was revealed by silver staining. The gel shown is representative of
four independent experiments. C, control; P and P
, 3 and 6 h after phorone
treatment.
The susceptibility of the cells to oxidative stress is dramatically influenced by the availability of free intracellular iron (44) . The iron storage protein ferritin, which can rapidly take up and release iron, has been alternatively seen as a potentially harmful iron donor (16, 43) or as an effective anti-oxidant defense(17, 18, 19, 20, 21) . The data of the present paper seem to suggest that, under conditions of in vivo oxidative stress, ferritin can perform both functions in a time-dependent manner. In fact, early after phorone treatment, we found higher levels of heme oxygenase mRNA and a decrease of ferritin content. The increase of heme oxygenase mRNA suggests that heme oxygenase-mediated heme degradation is a source of iron, as demonstrated to occur in UVA-irradiated fibroblasts(20, 21) . However, iron originating from ferritin breakdown might also contribute to increase the pool of loosely bound redox-active iron. Since the estimated half-life of ferritin shells in rat liver is approximately 24 h(43, 45) , the finding of decreased ferritin accumulation as early as 3 h after phorone treatment suggests that oxidative stress specifically induces ferritin degradation. The non-lysosomal calcium-dependent proteases activated in hepatocytes exposed to oxidative injury (46) might be implicated in this process. These findings are in agreement with a previous report indicating autophagic degradation of ferritin as a likely source of iron involved in oxidative injury of cultured rat hepatocytes(43) . As a consequence of these events, an expansion of the free iron pool is likely to occur. Indeed, by EPR analysis, we directly show that phorone treatment increases free iron levels. Present data obtained in vivo extend therefore previous observations of ferritin induction by oxidative stress in cell cultures(20) , in which an increase in the free iron pool was postulated, but no direct measurements were provided.
At the same time, the increased availability of iron stimulates ferritin synthesis. The preferential induction of the H subunit, where the ferroxidase activity of ferritin shells is located(47) , will result in the accumulation of more acidic isoferritins better suitable for rapid uptake and sequestering of iron; this is consistent with the idea that ferritin has an anti-oxidant role.
The increase of ferritin
synthesis seems to be regulated at both transcriptional and
post-transcriptional levels. Indeed, run-on analysis directly shows a
long-lasting increase of ferritin gene transcription that enhances the
steady state levels of ferritin mRNAs; both H and L genes are
activated, but transcription of the H gene seems to be preferentially
stimulated. The increase in transcription might well be a consequence
of the increased iron pool only; however, the marked stimulation of H
subunit gene transcription, which is not affected by iron
administration(4) , suggests that ROS could be directly
involved in the transcriptional activation of this gene. Oxidative
stress further increases ferritin synthesis post-transcriptionally
through inhibition of IRF binding activity. As recently shown to occur
during liver regeneration(30) , regulation is specific for the
IRF complex. Down-regulation of IRF
might be a
consequence of increased free iron levels, but preliminary results
showing inhibition of IRF activity by oxidative stress in a cell-free
system suggest that ROS could directly affect IRF activity also in
vivo.
Taken together, our data suggest that oxidative stress causes in rat liver the following cascade of events. At first, both heme cleavage by heme oxygenase and ferritin degradation cooperate to increase intracellular free iron levels. This expansion of the iron pool induces ferritin gene transcription and, by decreasing IRF binding activity, allows translation of pre-existing ferritin mRNA molecules to proceed; as a result, ferritin synthesis doubles. IRF activity remains low for a few hours and permits efficient translation of the higher amount of mRNAs that results from the previous activation of ferritin gene transcription. This causes a 6-fold increase of ferritin synthetic rate that allows reconstitution of ferritin content in the attempt to limit iron bioavailability.