(Received for publication, March 8, 1995; and in revised form, June 5, 1995)
From the
Replenishment of ascorbate in cultured cells, which are almost uniformly vitamin-deficient, increases ferritin mRNA translation in response to iron by 20-fold (Toth, I., Rogers, J. T., McPhee, J. A., Elliott, S. M., Abramson, S. L., and Bridges, K. R.(1995) J. Biol. Chem. 270, 2846-2852). We now demonstrate that ascorbate increases cytosolic aconitase activity. The iron-responsive element-binding protein (IRP-1) exists in three states: bound to mRNA without aconitase activity, free in the cytosol without aconitase activity, and free in the cytosol with aconitase activity. Ascorbate converts free IRP-1 to the enzymatically active form. Enhanced ferritin synthesis with subsequent iron stimulation is due to the altered equilibrium of the free IRP-1. The cellular biology of iron is closely intertwined with that of ascorbate.
Ascorbic acid, or vitamin C, is a dietary requirement for primates, guinea pigs, and a few other species that fail to synthesize the compound (Szent-Györgyi, 1928; Burns, 1957; Nishikimi and Yagi, 1991). Additionally, most cells in culture are ascorbate-deficient since the vitamin generally is not added to culture media and is extremely labile in solution (Moser and Bendich, 1991). We have shown previously that ascorbic acid retards lysosomal autophagy of cytosolic ferritin (Bridges and Hoffman, 1986; Bridges, 1987; Hoffman et al., 1991).
More recent work in our laboratory
demonstrated that ascorbic acid greatly increases the ferritin content
of cells subsequently stimulated with iron (Toth et al.,
1995). Base-line ferritin synthesis in ascorbate replete or deficient
cells is identical. Ferritin message translation in response to iron
loading was greater in cells grown in media containing physiological
concentrations of ascorbate relative to those grown under standard
culture conditions. Ascorbate's role as a facilitator of ferritin
synthesis in cells is mediated through the 5`-untranslated region
region of the ferritin message containing the iron-responsive element
(IRE) ()(Rouault et al., 1988; Hentze et
al., 1988; Harrell et al., 1991; Kühn
and Hentze, 1992). We have extended our investigations and examined
ascorbate's effects on IRP-1, a dual function protein that binds
the IRE sequence and has aconitase activity (Constable et al.,
1992; Philipot etal., 1993; Hirling et al., 1994).
Our data indicate that ascorbate transduces enzymatically inactive free
IRP-1 to an active form.
A 2-h incubation with ascorbate increased cytosolic aconitase activity by 2.5-fold (Table 2, 1 and 2a), again, not blocked by cycloheximide. Activity remained well above base-line levels even after a 16-h incubation (Table 2, 3a). When cells were incubated with ascorbate for 16 h and subsequently loaded with iron for 2 h, aconitase activity returned to the peak level (Table 2, 3b). The effect of ascorbate on cytosolic aconitase activity was less pronounced in cells preincubated with iron for 16 h (Table 2, 2a versus 2c). Dehydroascorbic acid, the oxidized form of the vitamin, also modulated cytosolic aconitase activity (Table 2, rows 4 and 5), indicating that the oxidation state of the vitamin in the medium was not crucial to the potentiation of aconitase activity.
We further investigated the effect of antioxidant agents on
cytosolic aconitase activity by incubating cells with oxidized (GSSG)
or reduced (GSH) glutathione (Meister, 1994). Uptake of
[C]GSH by the cells was linear. Both GSH and
GSSG increased cytosolic aconitase activity at 2 h, although less than
ascorbic acid (Table 2, 8a and 10a). Loading cells with iron for
2 h after a 16-h incubation with GSH or GSSG raised aconitase activity
only modestly (Table 2, 9b and 11b), which also differed from the
marked increase with ascorbate (Table 2, 3b). GSH and GSSG
mimicked ascorbate when added to cells for 2 h after a 16-h
preincubation with iron (Table 2, 2c, 8c, and 10c). The
experiments with uric acid and glutathione indicate that, while
alterations in the internal cellular oxidative environment can modify
aconitase activity, ascorbic acid has additional actions.
Figure 1:
The effect of iron, ascorbic acid,
and desferrioxamine on de novo ferritin biosynthesis by K562
human erythroleukemia cells. A total of 2 10
cells
were incubated in RPMI 1640 medium with various supplements. Half of
the cells were used to measure cytosolic aconitase activity. Those
remaining were metabolically labeled with
[
S]methionine, washed with PBS buffer three
times, and solubilized in a buffer containing 1% Triton X-100, 0.15 M NaCl, 0.02 M Tris-HCl, pH 7.5, and 0.2 mM phenylmethylsulfonyl fluoride. The supernatant from a high speed
spin was resolved on a 15% SDS -phosphate-urea gel. Each treatment was
repeated several times. A representative result is shown. Lane1,
C-labeled marker proteins. Lane2, control cells. Cells incubated in medium supplemented
with 10 µg/ml ferric ammonium citrate for 2 h (lane3) or 16 h (lane4). The cells in lane5 were treated with ferric ammonium citrate for
16 h, washed in PBS, and incubated for 2 h in medium supplemented with
150 µM ascorbate (AA). Cells incubated in medium
supplemented with 150 µM ascorbate for 2 h (lane6) or 16 h (lane7). In lane8, the cells were treated with ascorbate for 16 h,
washed, and incubated for 2 h in medium supplemented with 10 µg/ml
ferric ammonium citrate. Lane9, cells incubated for
16 h in medium supplemented with 100 µM desferrioxamine (Des).
Two hours of 150 µM ascorbate did not change ferritin synthesis but increased cytosolic aconitase activity by 2.5-fold. Cytosolic aconitase activity declined at 16 h of ascorbate incubation, but still exceeded control by 1.5-fold with no alteration in ferritin synthesis. Preincubation of cells with ascorbic acid for 16 h followed by FAC for 2 h raised the level of ferritin translation (Fig. 1, lanes3 and 8). The obverse experiment, addition of ascorbate to cells preincubated with iron, increased both ferritin synthesis and aconitase activity (Fig. 1, lane5). Ascorbate demonstrated clearly the independent modification of cytosolic aconitase activity and ferritin synthesis.
Figure 2: The effect of iron, uric acid, and desferrioxamine on de novo ferritin synthesis in K562 cells. Following the appropriate incubations, half the cells were metabolically labeled and the cytosolic supernatant was electrophoretically separated as in Fig. 1. Lane1, control. Lane2, 100 µM desferrioxamine for 16 h. Cells incubated with 10 µg/ml ferric ammonium citrate for 2 h (lane3) or 16 h (lane4). The cells in lane5 were incubated for 16 h in medium containing ferric ammonium citrate, after which they were washed and treated for an additional 2 h in medium supplemented with 500 µM uric acid (UA). Cells incubated in medium containing uric acid for 2 h (lane6) or 16 h (lane7). The cells in lane 8 were incubated for 16 h in medium containing uric acid, washed, and treated for another 2 h in medium containing ferric ammonium citrate. Half of each batch of cells was used to assay cytosolic aconitase activity.
Incubation with 500 µM GSSG changed neither ferritin synthesis nor cytosolic aconitase activity (Fig. 3, lanes7 and 8). Sequential treatment with iron and GSSG increased cytosolic aconitase activity by 50% without changing ferritin synthesis (Fig. 3, lanes4 and 5).
Figure 3: The effect of iron, oxidized (GSSG), and reduced (GSH) glutathione on de novo ferritin synthesis in K562 cells. The experimental protocol was similar to that detailed in Fig. 2except that uric acid was replaced with 500 µM reduced or oxidized glutathione in 100 mM HEPES buffer, pH 7.2.
GSH produced a different spectrum of changes, increasing ferritin synthesis but not aconitase activity (Fig. 3, lanes10 and 11). These data are the obverse of those obtained with ascorbate alone, which did not alter ferritin synthesis, but raised aconitase activity by severalfold. In aggregate, the data indicate that ferritin translation and cytosolic aconitase activity can be modified independently, suggesting a more complicated picture than one in which IRP-1 is either bound to message and enzymatically inactive or free in the cytosol with enzyme activity. The experiments with the antioxidants indicate that the effect of ascorbate involves more than its redox activity.
Figure 4:
Detection of IRP-1 in K562 cells by
Western blot analysis. After various treatments, cells were washed with
PBS twice and lysed as described in Fig. 1. A 50-µg sample
of mitochondrial-free cell lysate (100,000 g supernatant) from each treatment was separated by 10% SDS-PAGE,
immobilized on a nitrocellulose membrane, and hybridized with rabbit
antibody raised against the human IRP-1 (diluted 1:500). The
antibody-antigen complex was visualized with goat antibody against
rabbit IgG with coupled horseradish peroxidase (Bio-Rad) according to
the supplier's manual. The antibody hybridized to a single
protein in the 90-kDa range. Immunoblot analysis was performed three
times, and one representative result is shown. Lane1, control. Lane2, 10
cells treated overnight with 100 µM desferrioxamine.
Cells incubated in medium containing 10 µg/ml ferric ammonium
citrate for 2 h (lane3) or 16 h (lane4). In lane5, cells were treated for
16 h in medium supplemented with iron, followed after washing by a 2 h
treatment with 150 µM ascorbic acid. In another set of
experiments cells were treated with 150 µM ascorbate for 2
h (lane6) or for 16 h (lane7). In lane8, cells incubated with ascorbate were loaded
with iron for 2 h.
Ascorbate, therefore, activates enzymatically dormant aconitase protein. Ascorbate added in vitro to cytosolic extracts from control cells did not alter aconitase activity, indicating that the vitamin does not directly modify the aconitase protein nor act as an allosteric regulator. The ascorbate effect on cytosolic aconitase activity requires cell metabolism, but not protein synthesis.
Gel retardation experiments assessed the effect of
ascorbic acid on the interaction of IRP-1 and the IRE. Cytosolic
extracts from control cells and cells subjected to various treatments
(see Fig. 5and legend) were incubated with a P-labeled IRE probe and then resolved electrophoretically,
revealing the free IRE probe as well as the single band of the
IRE
IRP-1 complex.
Figure 5:
The effect of desferrioxamine, iron, and
ascorbic acid on the capacity of the IRP-1 to bind the IRE. IRE binding
analysis was performed with control cells (lane1),
cells incubated with 100 µM desferrioxamine 16 h (lane2), iron for 2 h (lane3) or 16 h (lane4), iron for 16 h followed by ascorbate for 2 h (lane5), ascorbate for 2 h (lane6) or 16 h (lane7), or ascorbate for
16 h followed by 2 h of iron (lane8). In each case,
2 µg of freshly isolated detergent extract was analyzed for IRE
binding capacity with excess amounts of P-labeled probe
(according to Leibold and Munro(1988) and Hirling et
al.(1992)). The experiment was repeated several times, and one
representative result is shown.
The extract from cells treated with
desferrioxamine or iron behaved as expected, binding more or less of
the IRE probe, respectively. Aconitase activity increased by 50% in
cells loaded with iron for 16 h and subsequently incubated with
ascorbate for 2 h (Fig. 1, lanes4 and 5). Electrophoretic mobility assay of IREIRP-1 complex
from comparably treated cells showed similar patterns in either
circumstance, indicating that the increase in aconitase activity
produced by ascorbate did not reflect a change in affinity between
IRP-1 and the IRE (Fig. 5, lanes4 and 5).
Cells incubated with iron for 2 h after a 16-h exposure to ascorbate produced a more prominent signal by electrophoretic mobility shift assay than did cells merely incubated with ascorbate for 16 h, implying a greater affinity of IRP-1 for the IRE (Fig. 5B, lanes7 and 8). However, aconitase activity increased substantially under these conditions (Fig. 1, lanes7 and 8). Since the quantity of IRP in the cells was constant (Fig. 4, lane8), these data are most consistent with ascorbate activation of latent cytosolic aconitase.
Comparison of the electrophoretic mobility assay data and ferritin synthetic data further emphasizes the dissociation produced by ascorbate between IRE/IRP-1 affinity and translation of ferritin mRNA. The electrophoretic mobility patterns were virtually identical for cells treated with iron for 16 h and those where the treatment was followed by a 2-h incubation with ascorbate (Fig. 5, lanes4 and 5), while ferritin synthesis increased dramatically in the latter case (Fig. 1, lanes4 and 5). A similar dissociation between ferritin synthesis and IRE/IRP-1 affinity is seen with cells treated with ascorbate for 16 h and those where the treatment was followed by iron for 2 h (Fig. 5, lanes7 and 8versusFig. 1, lanes7 and 8). Fig. 5shows, if anything, a slight increase in the affinity between the IRE and IRP-1 in cells treated briefly with iron after ascorbate incubation, although this combination increased ferritin synthesis dramatically (Fig. 1).
Ascorbic acid is crucial to numerous cellular reactions,
ranging from hydroxylation of lysyl -amino groups in collagen to
the conversion of norepinephrine to epinephrine (Levine, 1988). Most
cells in culture are ascorbate-deficient due to the lability of the
vitamin in solution, disturbing a number of biochemical pathways,
including lysosomal degradation of cytosolic ferritin (Englard and
Seifter, 1986; Lipschitz et al., 1971). Our recent
demonstration that ascorbate repletion greatly enhances ferritin
synthesis in cells stimulated with iron indicated that a completely
different arm of cellular metabolism is influenced by this vitamin
(Toth et al., 1995).
We now show that ascorbate induces cytosolic aconitase activity in K562 cells, which are known to be ascorbate-deficient (Bridges and Hoffman, 1986). IRP-1 has been shown previously to acquire aconitase activity when released from the IRE in the 5`-untranslated region of the ferritin message (Haile et al., 1992). Ascorbate, in contrast, activates aconitase without changing ferritin synthesis, meaning that the vitamin does not dissociate IRP-1 from the ferritin message. The most reasonable conclusion is that the vitamin activates a cytoplasmic pool of enzymatically dormant aconitase protein.
Cells contain more than one IRP. The 90-kDa protein, designated IRP-1, is an aconitase when freed from the message (Constable et al., 1992; Müllner,et al., 1989; Goosen, et al., 1990). A second IRE-binding protein, of 110 kDa, termed IRP-2, lacks aconitase activity (Philpott et al., 1994; Henderson et al., 1993; Samaniego et al., 1994). One explanation of our data is that ascorbate induces aconitase activity in IRP-2. The data in Fig. 1argue against this possibility, however. If ascorbate triggered aconitase activity in IRP-2, then the 2.5-fold increase in activity with a 2-h incubation with the vitamin would reflect activation of IRP-2. The low rate of ferritin is consistent with IRP-1 remaining attached to the message. When cells preincubated with iron for 16 h are pulsed with ascorbate for 2 h, ferritin synthesis increases and aconitase activity rises by only 1.5-fold. If ascorbate imparted aconitase activity to IRP-2, enzymatic activity again should have increased by at least 2.5-fold over base-line levels. Furthermore, the increase in ferritin synthesis indicates that IRP-1 has detached from ferritin mRNA, which should have increased cytosolic aconitase activity even further.
We believe that the data are most consistent with ascorbate activation of latent IRP-1 in the cytosol (Fig. 6). Iron releases IRP-1 from the message. A second step, facilitated by ascorbate, converts this enzymatically inactive protein into an aconitase. Ascorbate stimulates aconitase activity in unbound IRP-1 but does not influence IRP-1 that is attached to the message. Eventually, the free IRP-1 reaches a new steady-state between active and inactive aconitase (Fig. 1, 16 h AA). When iron is added to these cells for 2 h, it releases IRP-1 into a smaller pool of free, inactive IRP-1. A larger quantity of IRP-1 is released before steady state is reached, so that both aconitase activity and ferritin synthesis increase (Fig. 1, 16 h AA + 2 h Fe). The three-state model presented in Fig. 6would allow independent regulation of the two activities of IRP-1, permitting greater flexibility in the regulation of the metabolic environment of the cell than does a model in which the IRP-1 either is bound to mRNA with no aconitase activity or free in the cytosol with aconitase activity.
Figure 6: Schematic representation of the role of ascorbate in ferritin synthesis. IRP-1 is released from the message by iron (step1). The cytosolic IRP-1 initially lacks aconitase activity. The enzymatic activation of IRP-1 is facilitated by ascorbate (step2). Superoxide inactivates the aconitase capacity of IRP-1.
Further biochemical studies are required to determine the molecular steps by which ascorbate switches IRP-1 into the aconitase mode. The recent demonstration that cytosolic aconitase is inactivated by superoxide (Gardner and Fridovich, 1992; Gardner and Fridovich, 1993) suggests that ascorbate could modulate the activity of this enzyme, at least in part, by buffering cytoplasmic superoxide. Nitric oxide has been posited to be a physiological regulator of cytosolic aconitase activity (Weiss et al., 1993). The recent data indicating that superoxide and other free radicals, rather than nitric oxide, mediate cytosolic aconitase activity highlight the importance of identifying the physiologically relevant biochemical constituents in the cell (Hausladen and Fridovich, 1994). The cellular physiology of iron and the plethora of proteins that depend on it cannot be understood without this information.