An increase in intracellular Ca2+ is one of the initiating events in T-cell activation. A calcium-mediated signalling cascade in T-cells involves activation of calcineurin and the dephosphorylation and translocation of NFAT (nuclear factor of activated T-cells), resulting in the transcriptional activation of target genes such as IL-2 (interleukin-2). In the present study, we found that increased intracellular calcium leads to induction of the antioxidant protein ferritin H. We previously reported that the ferritin H gene is transcriptionally activated under oxidative stress conditions through an ARE (antioxidant-responsive element). The facts that the ferritin H ARE contains a composite AP-1 (activator protein 1) site and that NFAT collaborates with AP-1 transcription factors led us to test whether calcium-activated NFAT is involved in the ferritin H induction through the ARE. Treatment of Jurkat T-cells with the calcium ionophore, ionomycin, increased ferritin H mRNA and protein expression. Although NFAT translocated to the nucleus and bound a consensus NFAT sequence located in the IL-2 promoter after ionomycin treatment, it did not activate ferritin H transcription despite the presence of a putative NFAT-binding sequence in the ferritin H ARE. In addition, the calcineurin inhibitor cyclosporin A treatment blocked ionomycin-mediated NFAT nuclear translocation but failed to abrogate the increase in ferritin H mRNA. Analysis of mRNA stability after actinomycin D treatment revealed that ionomycin prolongs ferritin H mRNA half-life. Taken together, these results suggest that ionomycin-mediated induction of ferritin H may occur in an NFAT-independent manner but through post-transcriptional stabilization of the ferritin H mRNA.
- antioxidant-responsive element (ARE)
- nuclear factor of activated T-cells (NFAT)
An elevation in intracellular calcium incites a signalling cascade, leading to the activation of T-cells in the immune response . Increased calcium is responsible for the activation of calcineurin , a calcium-calmodulin-dependent phosphatase that is involved in transcriptional regulation of cytokine genes . Studies have shown that calcineurin is integrally involved in diverse signalling pathways, which correlate with those controlled by calcium concentration, especially pathways that dictate lymphocyte function . The major substrate of calcineurin is NFAT (nuclear factor of activated T-cells), which was originally described for its role in T-cell activation . Classically, NFAT binds a highly conserved promoter sequence originally found in the upstream region of the IL-2 (interleukin-2) gene; however, previously, it has been shown to bind many other promoter targets, including a number of other ILs, GM-CSF (granulocyte/macrophage colony-stimulating factor), interferon-γ, TNFα (tumour necrosis factor α) and E-selectin , through interaction with AP-1 (activator protein 1) family transcription factors [5,6]. Thus various NFAT–AP-1 composite binding sites have been identified in those NFAT-regulated genes .
During T-cell activation, the population of T-cells increases and ROS (reactive oxygen species) are produced; however, following these events, a large proportion of the population dies via an apoptotic pathway . Calcium may lead directly to activation of the apoptotic pathway . Several lines of evidence have shown that calcium may be released from the ER (endoplasmic reticulum) or taken up through channels during cellular stress response [9–11]. It is believed that this calcium increase in the cell may be the compulsory signal for the cell to die . It has also been proposed that ROS produced during T-cell activation may sensitize T-cells to undergo apoptosis .
ROS are potentially damaging to the cells, but because they are ubiquitous, cells have evolved antioxidant systems to convert them into more benign molecules. In response to exposure to reactive metabolites and oxidative stress, Phase II detoxification enzymes are induced and activated. At the transcriptional level, such Phase II genes as GST (glutathione transferase) , NADPH:quinone oxidoreductase-1 [15,16] and haem oxygenase-1 , are induced via a conserved cis-element in their promoter regions, aptly named ARE (antioxidant-responsive element) . Recently, our studies revealed an ARE in the 5′-region of the human ferritin H promoter  that is responsible for activation of transcription in response to oxidative stress-inducing agents such as H2O2, t-BHQ (butylhydroquinone) and haemin [18–20]. The ferritin H ARE is composed of two bidirectional AP-1 motifs, to which AP-1 and Maf/Nrf2 (nuclear factor-erythroid 2 p45 subunit-related factor 1) family members bind [18–21].
Ferritin H serves a cytoprotective role against iron-catalysed formation of ROS under conditions of oxidative stress, where labile Fe(II) participates in the production of the hydroxyl radical, which causes the most deleterious effect in the cells . Ferritin is an iron storage protein that sequesters free intracellular iron before it may become toxic to the cells. In vertebrates, there are two subunits of ferritin, heavy and light, which assemble in 24 subunit collections to create a channel that encloses the iron . The H (heavy) subunit has ferroxidase activity, and thereby oxidizes Fe(II) to Fe(III) and aggregates the iron inside the core [22,23]. Ferritin H functions to protect cells against iron-mediated oxidative stress [24–28]. We demonstrated that the mouse and human ferritin H genes are transcriptionally activated through the conserved ARE sequences [18,21] and the ARE activation is mediated by AP-1 family member transcription factors, including JunD [18,20]. Ferritin translation by iron, in contrast, is controlled by a well-known translational mechanism involving the binding of an IRP (iron-regulatory protein) to an IRE (iron-responsive element) in the 5′-UTR (5′-untranslated region) of ferritin mRNA, in which binding of IRP to IRE blocks translation, and occurs under conditions of iron deficiency [29,30].
Very little is known about the responses that allow cells to survive the concurrent intracellular calcium concentration and ROS production during T-cell activation, which may prevent apoptosis in selected cells in the population. In the present study, we tested our hypothesis that elevated calcium levels may induce the antioxidant protein, ferritin H, and it may be regulated by calcium-activated NFAT through a composite NFAT–AP-1 site in the ferritin H ARE. Indeed, our results showed that ferritin H was up-regulated in response to elevated intracellular calcium. However, the enhanced ferritin H expression was independent of NFAT activity and occurred through a transcription-independent pathway. Instead, increased calcium levels conferred ferritin H mRNA stabilization.
MATERIALS AND METHODS
NIH 3T3 and Jurkat E6-1 cells were purchased from the A.T.C.C. (Manassas, VA, U.S.A.) and cultured in DMEM (Dulbecco's modified Eagle's medium) with 10% (v/v) bovine calf serum (Hyclone) and RPMI 1640 with 10% (v/v) fetal bovine serum (Mediatech), 0.45% glucose, 1 mM sodium pyruvate and penicillin/streptomycin respectively. Cells were maintained in a humidified, 5% CO2 incubator at 37 °C. Ionomycin (free acid) and actinomycin D (Calbiochem), t-BHQ (Sigma–Aldrich) and PMA (‘TPA’; Sigma–Aldrich) were dissolved in DMSO. Cyclosporin A (Calbiochem) was dissolved in ethanol.
Promoter reporter constructs and DNA transfection
pBluescript SK(–) human ferritin H ARE-luciferase reporter plasmids were as described previously . pGL3-mouse ferritin H ARE-Luc and −0.22 kb mouse-ferritin H-Luc were constructed by SmaI digestion of pGL3 −4.8 kb mouse ferritin H followed by either self-ligation or insertion of double-stranded ARE oligonucleotide: 5′-TACCCCCTCCATGACAAAGCACTTTTGGAGCCCAACCCCTCCAAAGGAGCAGAATGCTGAGTCACGGTGGAACAA-3′ . Jurkat cells were transiently transfected via electroporation (Bio-Rad Gene Pulser X-Cell) by utilizing a manufacturer presetting condition. As an internal control for transfection efficiency, 0.1 μg of pRL-EF (elongation factor promoter–Renilla luciferase reporter) was simultaneously co-transfected. Following electroporation of luciferase promoter–reporter constructs into 1×107 Jurkat cells, they were seeded at an initial density of 4–5×105 cells per 35 mm dish containing 2 ml of culture medium. After incubation for 48 h, the cells were treated with the indicated reagents for 24 h. Preparation of cell extracts and luciferase assays were performed using Dual Luciferase Assay Reagents (Promega). Firefly luciferase expression driven by the ARE of the ferritin H gene was normalized to the Renilla luciferase activity.
Western blots were performed using either whole cell lysates prepared using Reporter Lysis Buffer (Promega) or Lysis Buffer A Solution containing 10 mM Na2HPO4, 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS and 0.2% sodium azide (pH 7.4). Cytosolic and nuclear extracts were obtained using a Nuclear Extract kit (Active Motif, Carlsbad, CA, U.S.A.). Unless otherwise noted, 50 μg of total protein was subjected to SDS/PAGE, and all primary antibodies were incubated overnight at 4 °C. NFAT family antibody (k-18X; Santa Cruz Biotechnology) and NFATc1-specific antibody (7a6X; Santa Cruz Biotechnology) were used with a working dilution of 1:500 in TBS (Tris-buffered saline) containing 0.1% Tween 20 and 5% (w/v) skim milk. Ferritin H-specific antibody (H-53; Santa Cruz Biotechnology) was diluted 1:3000. Secondary antibodies (anti-rabbit IgG and anti-mouse IgG from Alpha Diagnostic and anti-goat IgG from Calbiochem) were used at 1:5000 dilutions. Signals were visualized via ECL® or ECL® Advance Western Blotting Detection Reagents (Amersham–GE Healthcare). Rainbow molecular mass marker (Amersham–GE Healthcare) or Prosieve prestained protein marker (Cambrex) was used for protein size markers in SDS/PAGE.
NIH 3T3 or Jurkat cells were treated for 24 h with 0, 1 and 5 μM ionomycin or an equimolar amount of DMSO vehicle. In some experiments, cells were pretreated with 1 μg/ml cyclosporin A for 1 h. Total RNA was isolated using TRIzol® (Invitrogen). Total RNA (5–15 μg) was applied to a 1.1% agarose formaldehyde-containing gel. The separated RNA was blotted on to a 0.45 μm nitrocellulose Protran BA85 membrane (Whatman Biosystems; Schleicher and Schuell) and ferritin H mRNA was detected by using an [α-32P]dCTP-labelled 0.9 kb fragment of ferritin H human cDNA as a probe.
Gel retardation assay
Nuclear and cytosolic extracts were prepared using the nuclear extraction kit from Active Motif. Binding reaction and separation of retarded bands by PAGE was as described previously . NFAT antibody, along with NFAT consensus oligonucleotide, was obtained from Santa Cruz Biotechnology.
35S translabelling/ferritin immunoprecipitation
Jurkat cells were treated with either 2.5 μM ionomycin for 0–24 h or 1–5 μM ionomycin or 10 μM t-BHQ for 24 h. After treatment, cells were pelleted at 1000 rev./min for 5 min, and normal growth medium was replaced with DMEM/methionine/cysteine-deficient medium. Simultaneously, 10 μCi/ml of [35S]methionine/cysteine (>1000 Ci/mmol of PRO-MIX 35S-Cell Labelling Mix; ICN) was added to each cell suspension and incubated for 1 h. Total cell lysates were prepared with Lysis Buffer A Solution, and cleared using non-immunized rabbit serum (Cappel) and Protein A–agarose (Calbiochem) overnight at 4 °C. 35S incorporation was measured by precipitation with trichloroacetic acid (TCA) and lquid-scintillation counting of radioactivity. The amount of input for each immunoprecipitation was determined by adding equal radioactive counts to each immunoprecipitation reaction. A 6 μl portion of anti-ferritin antibody (A133; Dako) and 20 μl of Protein A–agarose were used for overnight immunoprecipitation at 4 °C. Finally, the resulting immunoprecipitates were subjected to SDS/PAGE as described above, and the dried gel was exposed to a film at −86 °C for 2–5 days.
Calcium ionophore increases ferritin H expression in Jurkat cells
We previously demonstrated that ferritin H was induced by various oxidative stress-inducing agents [18–21]. Because T-cells are exposed to high levels of ROS during activation and also may subsequently undergo apoptosis [32,33], we investigated whether an increase in intracellular calcium levels, which is the initiating signal for T-cell activation, may induce ferritin H. Jurkat acute leukaemic T-cells were treated with the calcium ionophore, ionomycin, PMA, or PMA and ionomycin together. PMA is a tumour promoter that activates PKC (protein kinase C) signalling . Co-treatment of PMA and ionomycin is a prototypical model for T-cell activation . Treatment of Jurkat cells with 1 and 2.5 μM ionomycin resulted in a dose-dependent increase in ferritin H protein (Figure 1A). The highest dose of ionomycin treatment still resulted in a modest increase in ferritin H protein synthesis (Figures 1A and 1B, bottom). This may be due to some toxicity observed at higher concentrations of ionomycin. Neither PMA nor PMA/ionomycin induced ferritin H protein; rather ferritin H protein expression appeared to be slightly decreased compared with the control. Expression of GSTP (GST Pi), another Phase II antioxidant enzyme, was unaffected by treatment with ionomycin, PMA or ionomycin plus PMA (Figure 1A), suggesting that ferritin H induction by ionomycin was not the result of the general antioxidative response of cells.
Ferritin is regulated by iron at the translational level via IRPs [29,30]. We previously reported that H2O2 transiently represses ferritin H protein synthesis for 2 h after H2O2 treatment through activation of the IRPs . To examine whether the regulation of ferritin protein synthesis by ionomycin is similar to that by H2O2, we performed [35S]methionine translabelling and ferritin immunoprecipitation. Treatment of Jurkat cells for 1, 2, 4, 8 and 24 h resulted in increased ferritin protein synthesis without transient inhibition between 1 and 2 h (Figure 1B, top), which was different from that previously observed after H2O2 treatment . We also observed that ferritin H protein appears to be more up-regulated in comparison with ferritin L (Figure 1B, top). In addition, treatment with increasing concentrations of ionomycin resulted in a dose-dependent increase in ferritin H protein synthesis (Figure 1B, bottom). The amount of newly synthesized ferritin H following ionomycin treatment was similar to that of t-BHQ (Figure 1B, bottom), a prototypical Phase II gene activator .
Ferritin H mRNA induction following ionomycin treatment is independent of NFAT-mediated transcriptional activation via the ARE
In addition to ferritin translational regulation, the ferritin H gene can also be regulated at the transcriptional level . To address whether or not ionomycin treatment may regulate the expression of ferritin H mRNA, we exposed both Jurkat and NIH 3T3 cells to either 1 or 5 μM ionomycin or vehicle control for 24 h and assessed ferritin H mRNA by Northern blotting. The results in Figure 2 show that ferritin H mRNA was increased by ionomycin treatment in both Jurkat (Figure 2A) and NIH 3T3 (Figure 2B) cells.
Since (i) NFAT is one of the major transcription factors activated by increased intracellular calcium during T-cell activation [3,4,37], (ii) NFAT associates with AP-1 and binds a composite NFAT–AP-1 site [3,5,6], and (iii) the ferritin H ARE contains a putative NFAT–AP-1 composite site (Figure 4), we hypothesized that the increase in ferritin H mRNA by ionomycin may be due to NFAT-mediated transcriptional activation of the ferritin H gene via the ARE. First, we tried to confirm that NFAT was activated by ionomycin treatment in Jurkat cells. Nuclear and cytoplasmic fractions of Jurkat cells treated with ionomycin, PMA or PMA plus ionomycin were subjected to Western blotting to detect the translocation of NFAT to the nucleus. As shown in Figure 3, both ionomycin and, to a greater extent, PMA plus ionomycin treatment resulted in an increase in the amount of NFAT detected in the nuclear fraction, suggesting that NFAT was activated and available to bind to DNA. To reveal the ability of translocated NFAT to bind its target sequence, the same cellular fractions were subjected to the gel retardation assay using an NFAT consensus sequence (derived from the IL-2 promoter). A shifted band was detected in the ionomycin- and PMA plus ionomycin-treated nuclear fractions (Figure 3B), suggesting that the translocated NFAT was capable of binding to specific DNA target elements.
Then, we tested our hypothesis that NFAT may target the ferritin H promoter for activation following ionomycin treatment. Upon examination of the human ferritin H promoter sequence, we identified two putative NFAT-binding sites adjacent to the AP-1/NFE2 sites in the ferritin H ARE (Figure 4A). Although the putative NFAT-binding site was not an exact match to the consensus NFAT-binding sequence, the presence of a contiguous AP-1-binding site increases the likelihood of NFAT binding because NFAT co-operatively binds and functions with AP-1 family transcription factors. Therefore we examined whether or not NFAT binds to the ARE sequence and is increased following ionomycin treatment. A probe containing the putative NFAT site and AP-1/NFE2 site of the ARE was used for the gel retardation assay with nuclear extracts of Jurkat cells treated with ionomycin. We observed no increase in the amount of protein binding to the putative NFAT–AP-1/NFE2 site after ionomycin treatment compared with the untreated control (Figure 4B). PMA and PMA plus ionomycin stimulation both resulted in a strong increase in the amount of protein bound to the putative NFAT–AP-1/NFE2 site in the ferritin H ARE (Figure 4B). This is probably due to the stimulation of AP-1 transcription factor binding by PMA. These results suggest that NFAT activated by ionomycin treatment may not be involved in the ferritin H mRNA induction via the ARE activation.
Next, we asked whether or not the ferritin H enhancer/promoter is activated by ionomycin. We employed 5.2 kb of the upstream region of the human ferritin H promoter containing the ARE fused to a luciferase reporter for transient transfection . The results in Figure 4(C) show that ionomycin treatment failed to activate the ferritin H promoter in Jurkat cells, while it strikingly induced an NFAT-luciferase promoter reporter construct (Figure 4C). The antioxidant t-BHQ, a positive control of the ARE activation used in the present study, induced expression of luciferase driven by the ferritin H promoter (Figure 4C). We also employed a wild-type ARE consensus oligonucleotide inserted into a TATA-luciferase reporter for the transient transfection assay. Similarly, the ARE was also not activated by ionomycin treatment, although it was induced by t-BHQ (results not shown).
To assess further that the ferritin H mRNA induction following ionomycin treatment is NFAT-independent, we employed cyclosporin A. Cyclosporin A is an immunosuppressant that blocks the activation of calcineurin and also prevents downstream calcium-mediated signalling events . To confirm that cyclosporin A abrogates ionomycin-mediated activation of NFAT, we examined the amount of NFAT present in the nuclear fraction following treatment. NFAT was increased in the nuclear fraction after ionomycin treatment alone, but the addition of cyclosporin A completely blocked NFAT translocation (Figure 5A). When ferritin H mRNA levels were examined, cyclosporin A treatment rather slightly enhanced basal expression, and it did not abrogate ionomycin-mediated increase in ferritin H mRNA (Figure 5B). The results also show that ionomycin or cyclosporine A had a marginal effect on ferritin L mRNA (Figure 5B). Taken together, we concluded that induction of ferritin H mRNA by ionomycin treatment was not involved in the transcriptional activation of the ferritin H gene by the activated NFAT.
Ionomycin-mediated induction of ferritin H occurs via a post-transcriptional mechanism involving mRNA stabilization
Our results suggested that ferritin H was not transcriptionally activated by ionomycin and indicated that ferritin H induction was independent of NFAT activation. We then asked whether or not the increase in ferritin H mRNA following ionomycin treatment may be due to a post-transcriptional mechanism. Enhanced mRNA stability is one of the major post-transcriptional mechanisms that can increase mRNA levels. One of the main mechanisms of mRNA stabilization is the binding of RNA-binding proteins to target sequences in the mRNA, which allows them to modulate the stability of the transcript . To test whether or not ferritin H mRNA half-life (t½) is enhanced by ionomycin treatment, Jurkat cells were treated with either vehicle or ionomycin, and then treated for up to 8 h with the transcription inhibitor, actinomycin D. Total RNA was isolated from these Jurkat cells for Northern blotting to measure the t½ of ferritin H mRNA. The results in Figure 6 show that ionomycin treatment appeared to induce a delay in the onset of ferritin H mRNA decay, ultimately resulted in prolonged ferritin H mRNA t½ from 5 to 8 h and conferred mRNA stabilization.
T-cells are subjected to elevated intracellular calcium and increased ROS during activation [37,40]. Our previous studies have demonstrated that ferritin H, an iron-sequestering protein, is transcriptionally activated by a number of oxidative stress-inducing compounds through an ARE in the far upstream region of the promoter [18,20,21]. In the present study, we tested our hypothesis that elevated intracellular calcium may induce ferritin H in the mechanism of ARE activation similar to that evoked under oxidative stress conditions. We observed that, indeed, ferritin H mRNA and protein were increased following treatment with the calcium ionophore, ionomycin (Figures 1 and 2); however, the potent T-cell-activating stimulus of co-treatment with PMA and ionomycin failed to induce ferritin H (Figure 1). This may be a result of PMA-induced activation of proteins that bind the AP-1/NFE2 site in the ferritin H ARE and occupy the ARE (Figure 4). The increased binding of proteins to the AP-1/NFE2 site may compete for binding to the ARE with major ARE-binding proteins such as Nrf2/Maf transcription factors, thereby resulting in the decreased expression of ferritin H caused by PMA alone or ionomycin plus PMA treatment (Figure 1).
NFAT is a critical transcription factor involved in the activation of cytokine genes during T-cell activation [4,37]. We found a putative NFAT-binding site adjacent to the AP-1/NFE2-binding site in the ferritin H ARE (Figure 4). The presence of a composite AP-1-binding site appeared to increase the potential for NFAT binding and transactivation because Fos and Jun family AP-1 transcription factors were shown to associate with NFAT and enhance NFAT-mediated transcriptional activation in cytokine genes [5,6]. Although we confirmed the translocation and transactivating potential of NFAT on a consensus binding site derived from its classical target, the IL-2 promoter, following ionomycin treatment, neither the 5.2 kb upstream region of the ferritin H promoter (Figure 4) nor the wild-type ARE inserted into a TATA-luciferase promoter reporter (results not shown) was activated in our transient transfection. Furthermore, cyclosporin A blocked NFAT activation by ionomycin, but it failed to block induction of ferritin H mRNA by ionomycin (Figure 5). Rather cyclosporin A slightly increased ferritin H mRNA levels (Figure 5), in which it is likely that the inhibition of the phosphatase, calcineurin, may prevent the dephosphorylation of some transcription factors that positively regulate ferritin H transcription. Cyclosporin A may also affect the stability of ferritin H mRNA by a similar mechanism to the increase in parathyroid hormone mRNA stability, in which cyclosporine A inhibits calcineurin leading to modifications of phosphorylation status of the AU-rich element-binding factor, AUF1 . Indeed, we found that sequences of the 3′-UTR of the human ferritin H mRNA contain AU-rich elements (see below). Calcineurin was reported to be inhibited by ROS , suggesting that inhibition of phosphatase activity is a pan-activating action that allows for the activation or maintenance of activity of many DNA- and RNA-binding proteins that are important to a stress response. In the case of NFAT, it is positively regulated by dephosphorylation by calcineurin [2,43]. Our results indicate that the ionomycin-mediated induction of ferritin H mRNA appears to occur through a pathway distinct from the calcineurin–NFAT-mediated transcriptional activation mechanism.
Since ferritin H was not transcriptionally induced by NFAT that was activated by ionomycin treatment, we investigated whether or not it is post-transcriptionally regulated. When we examined the stability of ferritin H mRNA following ionomycin exposure and actinomycin D treatment, the overall degradation of ferritin H mRNA was delayed and the ultimate t½ of ferritin H mRNA was extended from 5 to 8 h by ionomycin treatment (Figure 6). The relatively long t½ of the human ferritin H mRNA observed in the present study appears to be consistent with the previous report that PMA induced ferritin H mRNA in human monocytic THP-1 cells via stabilization of mRNA from 7 to 12 h . The mechanism of the delay in the onset of ferritin H mRNA decay following ionomycin treatment is not clear; however, we found that sequences of the 3′-UTR of the human ferritin H mRNA contain two contiguous AU-rich elements (5′-UAUUUGUAUUUAUUA-3′), suggesting that RNA-binding proteins may target ferritin H mRNA for post-transcriptional regulation. AU-rich elements are localized in the UTRs of mRNA of many short-lived transcripts, including those of immediate early genes and cytokines [45,46]. Of the RNA-binding proteins that are involved in mRNA stability and that target AU-rich elements, most are involved in the destabilization of mRNA . It was demonstrated that IL-2 mRNA is stabilized by ionomycin treatment . Recent studies have indicated that mRNA stabilization may be one event in calcium signalling through an AU-rich element [48,49]. RNA-binding proteins HuR and AUF1 have been implicated to play a role in stabilizing mRNAs through the binding to the AU-rich elements [41,50]. In addition, the p38 MAPK (mitogen-activated protein kinase) pathway was shown to be involved in the stabilization of many AU-rich transcripts following lipopolysaccharide treatment in the human monocytic THP-1 cells .
We observed that ionomycin also increased ferritin L protein synthesis, to a lesser extent than ferritin H, in a dose-dependent manner (Figure 1B) in the absence of its mRNA induction (Figure 5B). The lack of ferritin L mRNA induction by ionomycin treatment may be explained by the absence of the AUUUA sequence in the 3′-UTR of the ferritin L mRNA, although it also contains high AU sequences such as 5′-AAAUAAAGCUUUUUGAU-3′. Ferritin L also contains an ARE ; however, many pro-oxidants preferentially induce ferritin H [53–55]. Furthermore, ferritin H, but not ferritin L, confers cytoprotection against oxidative stress . Up-regulation of ferritin H by calcium ionophore may be part of a cytoprotective response to the concomitant ROS production or subsequent apoptotic signalling. Indeed, we observed that knocking down ferritin H by siRNA (small interfering RNA) enhanced accumulation of ROS and susceptibility to apoptosis that were induced by the mitochondrial complex I inhibitor rotenone .
The mRNA stability of the transferrin receptor, another major protein involved in iron transport, is increased at low iron concentrations via the enhanced interaction between IRPs and IREs in the 3′-UTR of the transferrin receptor mRNA, leading to more transferrin receptor expression and iron uptake . Under the same conditions, the increased IRP–IRE interactions in the 5′-UTR of ferritin H and L mRNAs inhibit ferritin translation initiation, resulting in lower capacity for iron storage in the cells that allows incorporated iron to be readily available . The mechanism by which ferritin H transcripts are stabilized following ionomycin treatment is unclear; however, the present study suggests a new post-transcriptional mechanism of ferritin H mRNA regulation by calcium. The poly-A tail does provide a degree of protection from the degradation machinery, and deadenylation-dependent and -independent pathways have been proposed to be involved in the destabilization and degradation of mRNA . Further studies that elucidate the responsible RNA-binding protein and mechanism of enhanced ferritin H mRNA stability will provide more insight into this unique pathway of post-transcriptional regulation of the ferritin H gene in response to increased intracellular calcium.
This work was supported by NIH (National Institutes of Health) grant number DK-60007 (to Y. T.).
Abbreviations: AP-1, activator protein 1; ARE, antioxidant-responsive element; BHQ, butylhydroquinone; GST, glutathione transferase; GSTP, GST Pi; IL, interleukin; IRE, iron-responsive element; IRP, iron-regulatory protein; NFAT, nuclear factor of activated T-cells; Nrf2, nuclear factor-erythroid 2 p45 subunit-related factor 1; ROS, reactive oxygen species; UTR, untranslated region
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