©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Role for NF-B in the Regulation of Ferritin H by Tumor Necrosis Factor- (*)

Eunice L. Kwak (4), Denis A. Larochelle (1)(§), Carole Beaumont (5), Suzy V. Torti (2)(¶), Frank M. Torti (1) (3)

From the (1)Departments of Cancer Biology, (2)Biochemistry, and (3)Medicine, Bowman Gray School of Medicine and the Comprehensive Cancer Center, Wake Forest University, Winston-Salem, North Carolina 27157, the (4)Department of Medicine, Stanford University Medical School, Stanford, California 94305, and (5)Genetique et Pathologie Moleculaires de l'Hematopoiese, INSERM Unite 409, Faculte Xavier Bichat, 75018 Paris, France

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Ferritin is a ubiquitously distributed iron-binding protein that plays a key role in cellular iron homeostasis. It is composed of two subunits, termed H (heavy or heart) and L (light or liver). In fibroblasts and other cells, the cytokine tumor necrosis factor- (TNF) specifically induces synthesis of the ferritin H subunit. Using nuclear run-off assays, we demonstrate that this TNF-dependent increase in ferritin H is mediated by a selective increase in ferritin H transcription. Transfection of murine fibroblasts with chimeric genes containing the 5`-flanking region of murine ferritin H fused to the human growth hormone reporter gene reveals that the cis-acting element that mediates this response is located 4.8 kilobases distal to the start site of transcription. Deletion analyses delimit the TNF-responsive region to a 40-nucleotide sequence located between nucleotides -4776 and -4736, which we term FER-2. Electrophoretic mobility shift assays and site-specific mutations indicate that this region contains two independent elements: one contains a sequence that binds a member of the NF-B family of transcription factors, and a second contains a novel sequence that partially conforms to the NF-B consensus sequence and may bind a different member of the NF-B/Rel transcription factor family. Thus, effects of an inflammatory cytokine on ferritin are mediated by a family of transcription factors responsive to oxidative stress.


INTRODUCTION

Ferritin is a 24-subunit protein whose major role is to mediate iron storage and homeostasis. The subunits may be of several types, generally H (heavy or heart) or L (light or liver). Although there is a significant degree of conservation between these two subunits at the level of amino acid sequence, ferritin H and L subunits are encoded by different genes (1) and subsume distinct roles within the intact ferritin protein. The H subunit contains a ferroxidase activity that participates in iron oxidation(2) , whereas the L subunit confers stability to the apoprotein and appears to play a role in the long-term storage of iron(3) . Ferritin proteins vary widely in H/L ratios in different tissues and are altered in malignancy and inflammation(4, 5) .

Tumor necrosis factor- (TNF)()is a cytokine derived primarily from macrophages and T cells whose synthesis is triggered by a variety of immunological insults. TNF plays an important role in numerous host responses, including septic shock, cachexia, vascular leakage syndrome, and other systemic manifestations of disease (see Refs. 6 and 7 for review). Previous experiments have demonstrated that the cytokines TNF and interleukin-1 selectively increase ferritin H mRNA and protein in a variety of mesenchymal cell lines(8, 9, 10) . Given the pivotal role of these cytokines in the response to infection, inflammation, and malignancy, this observation suggested the possibility that cytokines might also impinge on some of the changes in iron metabolism that characterize these responses. Here we report that TNF acts at a transcriptional level to induce ferritin H. Using chimeric genes containing the murine ferritin promoter fused to a reporter gene, we demonstrate that TNF targets a specific region in the ferritin H promoter 4.8 kilobases 5` to the ferritin H transcriptional start site. These experiments also identify member(s) of the NF-B/Rel transcription factor family as key regulators of TNF-dependent ferritin H transcription.

NF-B is a transcription factor that regulates genes involved in the cellular response to stress. NF-B was originally described as a factor binding to the sequence 5`-GGGACTTTCC-3` of the immunoglobulin light chain enhancer in B cells(11) . Since then, numerous studies have shown that p50 (NF-B1) and p65 (RelA), the proteins that compose the NF-B dimer, belong to the larger NF-B/Rel family of proteins. Members of this family have been found to be capable of forming multiple homo- and heterodimers with differing DNA sequence-binding specificities and transcriptional activating properties (for review, see Ref. 12). In most cell types, NF-B exists in the cytoplasm bound to its inhibitor, IB. Translocation of NF-B to the nucleus with subsequent activation of transcription occurs in response to a variety of stimuli, including TNF (for review, see Ref. 13). In this report, we describe the identification of the TNF-responsive region in the murine ferritin H 5`-flanking region, which we term FER-2. This region contains two closely spaced sequences between nucleotides -4776 and -4736 that are required for maximal induction of transcription in response to TNF. Both of these sequences were found to possess nuclear protein-binding properties characteristic of NF-B.


EXPERIMENTAL PROCEDURES

Nuclear Run-off Transcription Assay

Approximately 5 10 NIH3T3 cells (American Type Culture Collection) were treated with 1000 units/ml TNF (Perkin-Elmer) for 7 h. Nuclei were collected from TNF-treated and control cells essentially as described(14) . Following the addition of 40 units of RNasin (Promega) to the nuclei, nuclear transcription activity was measured as the incorporation of [P]UTP into RNA transcripts elongated in vitro(14) . After ethanol precipitation, nuclear RNA was resuspended in HO, and an aliquot was subjected to trichloroacetic acid precipitation and scintillation counting. Equal counts of control and TNF-treated labeled RNAs were added to hybridization buffer (5 Denhardt's solution, 4 SSC, 50% formamide) and used to hybridize filters containing immobilized cDNAs. cDNAs used were -actin(15) , pGEM3Zf (Promega), murine ferritin H(8) , murine interleukin-6 (gift of F. Lee, DNAX), and human ferritin L (gift of J. Drysdale, Tufts University). 20 µg of each plasmid was linearized, denatured, and slot-blotted onto two nitrocellulose filters. The filters were baked at 80 °C and prehybridized at 37 °C overnight. Hybridization was carried out for 2-3 days at 37 °C, followed by washing of filters in 0.1% SDS, 2 SSC at 55 °C. If necessary, filters were subsequently washed as described above with the addition of 5 µg/ml RNase A at 37 °C.

DNA Sequencing

Sequencing was performed by the dideoxy chain termination method using Sequenase (U. S. Biochemical Corp.).

Deletion Constructs

Deletions of the -5.6 ferritin H/growth hormone reporter construct, which contains 5.6 kb of murine ferritin H 5`-flanking region ligated to hGH(16) , were made as follows.

-5.63.5

An internal EcoRI fragment from nt -4477 to -941 was removed from -5.6 ferritin H/growth hormone by EcoRI digestion and religation.

-5.33.5

A fragment containing the region from nt -5300 to the EcoRI site at nt -4477 was synthesized by polymerase chain reaction (PCR). The 5`-primer was 5`-GGTTCCCAGAGGCGCAGA-3`, and the 3`-primer was 5`-CCTTGGAGTGAAGTTGATGC-3`. Following the addition of EcoRI linkers and digestion with EcoRI, the fragment was ligated into the EcoRI site of a construct containing 941 nt of ferritin H 5`-flanking sequence (construct -0.9; see below for details).

-5.13.5

Construction was similar to that of -5.33.5, but PCR synthesis was primed from approximately -5.1 kb utilizing the 5`-primer 5`-GGCTTGGCACGAAGCACCT-3`.

-4.83.5

-5.63.5 was digested with XbaI to remove the region between -5.6 and -4.8 kb.

-4.83.5TT

A 2-nt mutation was introduced at nt -4749 and -4750 (GGGAATCCC to GTTAATCCC) by using oligonucleotides containing the desired mutations to prime synthesis of PCR fragments upstream and downstream from the site of mutation. The two resulting fragments (nt -4819 to -4736 and nt -4755 to -4471) were then melted, annealed, and extended by PCR. This resulted in a fragment from -4.8 to -4.5 kb that was cloned into the -0.9 construct.

-4.73.5

A fragment representing sequence from nt -4701 to -4471 was synthesized by PCR. The 5`-primer used was 5`-CCCATTTTCACCCAGCCTTC-3`. EcoRI linkers were added, and the fragment was digested with EcoRI prior to ligation into the EcoRI site of the -0.9 construct.

-4.63.5

A fragment representing sequence from nt -4591 to -4471 was synthesized by PCR. The 5`-primer used was 5`-CGCACTGTCTGGCACGCACC-3`. Following the addition of EcoRI linkers and digestion with EcoRI, the fragment was ligated into the EcoRI site of the -0.9 construct.

-4.8

The EcoRI fragment between nt -4477 and -941 was isolated by restriction digestion and gel purification and cloned into -4.83.5.

-4.5

The EcoRI fragment from nt -4477 to -941 was isolated as described above and cloned into the -0.9 construct.

-0.9

-5.63.5 was digested with EcoRI, followed by the addition of XbaI linkers to blunted EcoRI ends, subsequent XbaI digestion, and religation.

p6, p7, and p8

Fragments representing sequence between nt -4471 and -4739 (p6), nt -4471 and -4751 (p7), and nt -4776 and -4471 were synthesized by PCR, digested with EcoRI, and ligated into the EcoRI site of the -0.9 construct. The respective 5`-primers used were 5`-TATAGAATTCTTTTGCAACACTGGCTGTTT-3`, 5`-TATAGAATTCGGGAATCCCATCCTTTTGCA-3`, and 5`-TATAGAATTCATCAGAATTTCCAGCACACT-3`.

Cell Culture and Transfection

NIH3T3 fibroblasts were grown in 150-mm culture dishes in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 10% calf serum (Hyclone Laboratories). For transfection, cells were washed in phosphate-buffered saline, trypsinized, and washed again in phosphate-buffered saline. After cells were counted and centrifuged for collection, they were resuspended in electroporation buffer (20 mM HEPES, pH 7.05, 137 mM NaCl, 5 mM KCl, 0.7 mM NaHPO, 6 mM dextrose) such that 1 10 cells were present in 0.6 ml of electroporation buffer. 1 10 cells were transferred to each electroporation cuvette (Bio-Rad), and 40 µg of CsCl-purified plasmid DNA was mixed into each cuvette. Cells were electroporated at 300-330 mV and 960 microfarads on a Bio-Rad Gene Pulser. The contents of each cuvette were then plated into two to three 100-mm tissue culture dishes. Following attachment, cells were refed with Dulbecco's modified Eagle's medium, 10% calf serum.

TNF Treatment and RNA Isolation

When transfected cells approached confluence, they were treated with TNF (1000 units/ml) for 12 or 24 h. At the end of the incubation period, cells were washed in phosphate-buffered saline and lysed in guanidium isothiocyanate. Total RNA was collected by ultracentrifugation through 5.7 M CsCl.

RNase Protection Assay

RNA probe labeled with [P]CTP (>400 Ci/mmol; Amersham Corp.) was synthesized using T7 polymerase (Promega), and the protection assay was carried out essentially as described(17) . The probe used spanned the junction between ferritin H and hGH and included the region of ferritin H from nt -225 to +86 and the HindIII-DraIII fragment of hGH (see Fig. 2). Hybridization of 10 µg of total RNA to 1 10 cpm of RNA probe was carried out in 80% formamide at 56 °C for 14-18 h. Samples were then treated with 10 µg/ml RNase A and 2 µg/ml RNase T1 (both from Sigma) for 30 min at 30 °C, and the products were separated on a 7% denaturing urea-acrylamide gel.


Figure 2: Effect of TNF on the expression of ferritin H/hGH fusion genes. A, schematic diagrams of ferritin H/growth hormone deletion constructs used to transfect NIH3T3 cells. B, representative results of RNase protection experiments. Total RNA was isolated from NIH3T3 cells transfected with the indicated deletion constructs and treated with TNF for 12 or 24 h. Digestion with RNase following hybridization with probe (region indicted by asterisks) as described under ``Experimental Procedures'' yielded two protected fragments: an 190-nt band representing transcripts produced from the transfected constructs and an 86-nt band representing endogenous ferritin H transcripts. C, quantitation of results of multiple transfections of the type illustrated in B. In each experiment, -fold induction was calculated separately for endogenous and transfected ferritin H expression by comparing radioactive counts/minute from protected fragments generated in the presence and absence of TNF. Radioactivity measured in the absence of TNF was defined as 1 (hatchedbar). The number of independent experiments evaluated is in parentheses, and errorbars represent standard errors.



Quantitation of RNase Protection Assays

Bands were excised from dried gels and counted in a scintillation counter or, in some experiments, scanned by densitometry.

Nuclear Extraction

Nuclear extracts were prepared by the method of Dignam et al.(18) . Briefly, nuclei were isolated from NIH3T3 cells treated with or without 1000 units/ml TNF for 4 h. Cells were scraped into phosphate-buffered saline and pelleted by centrifugation. The cell pellet was resuspended in hypotonic buffer (10 mM HEPES, pH 7.9, 1.5 mM MgCl, 10 mM KCl, 0.2 mM phenylmethylsulfonyl fluoride, 0.5 mM dithiothreitol) and broken by Dounce homogenization. Nuclei were collected by centrifugation and resuspended in low-salt buffer (20 mM HEPES, pH 7.9, 25% glycerol, 1.5 mM MgCl, 0.02 M KCl, 0.2 mM EDTA, 0.2 mM phenylmethylsulfonyl fluoride, 0.5 mM dithiothreitol), followed by the addition of high-salt buffer (same as low-salt buffer but with 1.2 M KCl) in dropwise fashion. The nuclear extract was collected and dialyzed against 20 mM HEPES, pH 7.9, 20% glycerol, 100 mM KCl, 0.2 mM EDTA, 0.2 mM phenylmethylsulfonyl fluoride, 0.5 mM dithiothreitol. Protein concentrations were ascertained using Bio-Rad reagent, and extracts were frozen at -80 °C.

Electrophoretic Mobility Shift Assay (EMSA)

The protocol described by Sen and Baltimore (11) was followed with modifications. Briefly, nuclear extracts (15 µg) from control and TNF-treated cells were incubated with 20,000-50,000 cpm of radiolabeled oligonucleotides for 15 min at room temperature in the presence of 2 µg of poly(dI-dC)poly(dI-dC), 10 µg of bovine serum albumin, and 7-8 µl of the buffer against which the nuclear extracts had been dialyzed (see above). In some cases, the following double-stranded unlabeled competitor oligonucleotides were added to the mixture: NF-B (5`-GATCGAGGGGACTTTCCCTAGC-3`; Stratagene) or glucocorticoid-responsive element (5`-GATCAGAACACAGTGTTCTCTA-3`; Stratagene). In addition, some samples received antibody to the p65 NF-B protein (Santa Cruz Biotechnology) or anti-ferritin antibody (negative control; Dako Corp.). Incubated samples were electrophoresed on a 5% native acrylamide gel and fixed in 10% isopropyl alcohol, 10% acetic acid; and the gel was vacuum-dried before exposure.

Oligonucleotides used in EMSA were as follows: F/f(NF-B), 5`-CTCGGGGAATCCCATCCTTT-3` and 3`-CCCTTAGGGTAGGAAA-5`; F/f(NF-B)TT, 5`-CTCGGTTAATCCCATCCTTT-3` and 3`-CAATTAGGGTAGGAAA-5`; and E/e, 5`-TACACTTGCAAATATCAGAATTTCCAGCACACTTCTCG-3` and 3`-GAACGTTTATAGTCTTAAAGGTCGT-GTGAAGAGC-5`. After heating and annealing overnight, the double-stranded oligonucleotides were P-labeled (ICN) with the appropriate nucleotides by fill-in reaction.


RESULTS

TNF Increases Transcription of the Ferritin H Gene

Treatment of a variety of mesenchymal cell lines with the cytokine TNF increases levels of ferritin H mRNA in the absence of any effect on ferritin L(8, 9) . To determine whether this results from increased transcription of the ferritin H gene, we measured transcription of ferritin H in the presence and absence of TNF using a nuclear run-off assay(14) . As shown in Fig. 1, NIH3T3 cells treated for 7 h with TNF demonstrated increased transcription of the ferritin H gene. Interleukin-6, a gene previously reported to be transcriptionally activated by TNF (19) and which was used as a positive control, was activated by TNF in these experiments as well; in contrast, transcription of -actin and ferritin L, genes whose steady-state mRNA levels are not altered by TNF treatment(8, 9) , was not affected by TNF. Quantitation of results from several experiments demonstrated an 3-fold increase in ferritin H transcription in response to TNF. Thus, TNF-mediated increases in ferritin H mRNA result from TNF-dependent increases in ferritin H transcription.


Figure 1: Effect of TNF on ferritin H transcription. NIH3T3 cells were either untreated (control) or treated with 1000 units/ml TNF for 7 h. Nuclei were then harvested for use in a nuclear run-off transcription assay as described under ``Experimental Procedures.'' 40 µg of each cDNA was linearized, and 20 µg was applied to each slot as follows: human -actin, pGEM3Zf, murine ferritin H (F), murine IL-6, and human ferritin L (F).



The TNF-responsive Region of Ferritin H Is Located 4.8 kb 5` of the Transcriptional Start Site

To determine which regions of the ferritin H gene are responsible for TNF-dependent increases in transcription, chimeric genes that fused the 5`-flanking region of the murine ferritin H gene to the hGH reporter gene were constructed. These fusion genes were transfected into NIH3T3 cells, and their response to TNF was assessed using an RNase protection assay that enabled the simultaneous detection of the transfected gene and endogenous ferritin H(16) . Preliminary experiments indicated that a reporter gene containing 5.6 kb of ferritin H 5`-regulatory sequence fused to hGH could be regulated by TNF (Fig. 2A) (data not shown). Deletion of a large internal EcoRI fragment from this construct, which removed a 3.5-kb region from nt -4477 to -941, did not diminish TNF responsiveness (Fig. 2A). Using this -5.63.5 construct as a backbone, several additional deletion mutants that removed progressively larger regions from the 5`-end of the ferritin H/hGH gene were constructed and tested for their ability to be regulated by TNF. Fig. 2B shows representative RNase protection assays from these experiments; results of multiple experiments are quantitated in Fig. 2C. These experiments demonstrated that TNF induced transcription of the parental construct -5.63.5 as well as the deletion mutants -5.33.5, -5.13.5, and -4.83.5. These mutants, which deleted 352, 553, and 855 nt from the 5`-end of the parental construct, respectively, exhibited a 4-7-fold induction in expression of the transfected gene, a level remarkably similar to the 4.5-6-fold induction observed with the endogenous gene. In contrast, the -4.73.5, -4.63.5, and -0.9 deletion mutants exhibited a marked reduction in ability to respond to TNF, although measurement of endogenous ferritin H performed in the same samples indicated that TNF responsiveness of the endogenous gene was completely retained. These deletion studies indicate that the TNF-responsive region resides within an 100-nt region located between 4.8 and 4.7 kb upstream of the transcriptional start site.

To verify that these deletion mutants, which contained a large internal deletion not present in the endogenous gene, accurately recapitulated regulatory responses exhibited by the intact ferritin H promoter, chimeric ferritin genes that preserved the entire 5`-flanking region, including the internal 3.5-kb region from nt -4477 to -941, were also constructed. These were transfected into NIH3T3 cells, and their response to TNF treatment was compared with that of the endogenous gene. Quantitation of multiple experiments is shown in Fig. 3. These experiments demonstrated that a transfected gene containing 4.8 kb of 5`-flanking sequence (-4.8) responded to 12 h (Fig. 3) or 24 h (data not shown) of TNF treatment to the same extent as the endogenous gene. Consistent with the results presented above, deletion of an additional 300 nt of 5`-flanking sequence (-4.5) resulted in the loss of TNF responsiveness. Thus, the region of the ferritin promoter necessary for responsiveness to TNF lies 4.8 kb distal to the start site of ferritin H transcription.


Figure 3: RNase protection analysis of ferritin H/growth hormone constructs -4.8 and -4.5 transfected into NIH3T3 cells. The -4.8 or -4.5 ferritin H/growth hormone construct was transfected into NIH3T3 cells, and the transfected cells were treated with 1000 units/ml TNF for 12 h. Results of RNase protection assays were quantitated as described in the legend of Fig. 2C. The number of experiments evaluated is in parentheses, and errorbars represent standard errors. , transfected; , endogenous; , no TNF.



The TNF-responsive Region of Ferritin H Contains a Functional NF-B-binding Site

To probe further into the nature of regulatory elements present in the TNF-responsive region of the ferritin H promoter, the 118-nt region between -4.8 and -4.7 kb was sequenced. As shown in Fig. 4A, sequence analysis demonstrated the presence of the sequence 5`-GGGGAATCCC-3` between nt -4752 and -4743. This conforms very closely to a consensus sequence that binds the NF-B family of transcription factors (GGGRNNYYCC, where R = purine nucleotide, Y = pyrimidine nucleotide, and N = nucleotide)(13) . No other sequences with obvious similarity to binding sites for known transcription factors were identified in this region. To test whether this region of the ferritin promoter could in fact serve as a functional binding site for NF-B transcription factor(s), an EMSA was performed. As shown in Fig. 4B, EMSA revealed that the region of the ferritin H promoter defined by the F/f(NF-B) oligonucleotide (nt -4755 to -4736) is able to bind nuclear factors and that this binding increases dramatically in TNF-treated extracts. Furthermore, binding is specifically competed by unlabeled oligonucleotide containing the mouse immunoglobulin light chain NF-B motif (5` . . . GGGACTTTCC . . . 3`), but not by DNA of an irrelevant sequence (the glucocorticoid-responsive element). These results indicate that the region of the ferritin H promoter defined by the F/f(NF-B) oligonucleotide can bind an NF-B-like factor and that the presence or activity of this factor is augmented upon exposure to TNF.


Figure 4: EMSA using oligonucleotide F/f(NF-B). A, shown is the sequence of one strand of the double-stranded oligonucleotide used in EMSA. The asterisks above GG indicate bases at positions -4749 and -4750 that were mutated in F/f(NF-B) to produce F/f(NF-B)TT (see Fig. 5). B, nuclear extracts from untreated cells and cells treated with 1000 units/ml TNF for 4 h were incubated with radiolabeled F/f(NF-B). 25 ng of unlabeled competitor oligonucleotides was also added to control samples in lanes2 and 3 and to extracts prepared from TNF-treated cells in lanes5 and 6. In addition, nuclear extracts and radiolabeled F/f(NF-B) were incubated with a polyclonal antibody to the p65 subunit of NF-B (lanes7 and 8) or with an antibody to ferritin (negative control (ctrl); lane9). GRE, glucocorticoid-responsive element.



To further assess the relationship between factor(s) binding to this region of the ferritin promoter and NF-B, their immunological cross-reactivity was assessed. Radiolabeled F/f(NF-B) was incubated with nuclear extracts from TNF-treated cells in the presence of a polyclonal antibody to the p65 subunit of NF-B. As shown in Fig. 4B, the inclusion of this antibody inhibited the formation of specific protein-DNA complexes and in addition resulted in the appearance of higher mobility complexes. When a negative control antibody was used, no change in the EMSA banding pattern or signal intensity was observed. These findings indicate that a protein recognized by anti-NF-B antibody can bind to a sequence located between nt -4755 and -4736 of the ferritin H promoter.

To verify that DNA sequence requirements for binding of factors to the region of the ferritin promoter defined by the F/f(NF-B) oligonucleotide were similar to those exhibited by members of the NF-B family, a double-stranded oligonucleotide that incorporated a 2-base pair mutation in the F/f(NF-B) oligonucleotide was synthesized: 5`-CTCGGGGAATCCCATCCTTT-3` was changed to 5`-CTCGGTTAATCCCATCCTTT-3`. This GG-to-TT mutation abolishes binding of an NF-B-like factor to the major histocompatibility class II-associated invariant chain gene(20) . When this mutant oligonucleotide, F/f(NF-B)TT, was radiolabeled and used in electrophoretic mobility shift assays, it failed to bind to nuclear proteins as compared with the wild-type F/f(NF-B) sequence (Fig. 5). Thus, a mutation known to inhibit the binding of NF-B inhibits the ability of nuclear extracts to bind to a target sequence in the ferritin H promoter.


Figure 5: EMSA using F/f(NF-B) or its mutant counterpart, F/f(NF-B)TT. Either radiolabeled F/f(NF-B) or F/f(NF-B)TT double-stranded oligonucleotides were incubated with nuclear extracts from control or TNF-treated cells (4 h, 1000 units/ml). The mutant oligonucleotide contained a 2-base pair change (GG to TT) as illustrated in Fig. 4A.



Mutation of the NF-B Site in the Ferritin H Gene Reduces TNF Responsiveness in Vivo

To determine whether the NF-B site defined by EMSA affects transcription of ferritin H in vivo, a mutated chimeric gene was constructed that contained a specific mutation in this NF-B site. Starting with the TNF-responsive construct -4.83.5, two nucleotide changes that substituted TT for GG at nucleotides -4749 and -4750 were introduced. These mutations were chosen based on their ability to render F/f(NF-B)TT nonfunctional in vitro (Fig. 5). This mutated gene was then introduced into cells by transfection, and its response to TNF was compared with a gene containing the wild-type sequence. As shown in Fig. 6B, the mutated construct -4.83.5TT was unable to respond to TNF to the same extent as -4.83.5. When the results of three independent experiments were quantitated, construct -4.83.5TT demonstrated an 2-fold reduction in response to TNF, as shown in Fig. 6C. Similar results were observed when GG-to-TT mutations were introduced at nt -4749 and -4750 of a construct containing the entire 4.8 kb of ferritin 5`-flanking sequence (data not shown). Thus, the intact promoter, as well as the 4.83.5 ferritin H promoter, requires a functional NF-B sequence for maximal response to TNF.


Figure 6: RNase protection assay of experiments using -4.83.5 and its mutant counterpart, -4.83.5TT. A, shown is a schematic diagram of the -4.83.5 construct and its mutant, -4.83.5TT. The two deletion constructs were identical with the exception of a 2-base pair mutation at nt -4749 and -4750 in the ferritin H upstream portion of the ferritin H/growth hormone reporter construct. This mutation altered a putative NF-B consensus sequence from GG-GAATCCC to GTTAATCCC. B, NIH3T3 cells were transfected with either -4.83.5 or -4.83.5TT. Transfected cells were either untreated or exposed to 1000 units/ml TNF for 12 or 24 h, and transcripts were analyzed by RNase protection. C, autoradiographs of protected bands arising from transfected constructs were quantitated by scanning densitometry. Bars indicate -fold induction of transfected gene expression in control cells (hatchedbar) or TNF-treated cells (solidbars). Results of three experiments at 12 h of TNF treatment are presented; errorbars represent standard errors.



Other cis-Acting Elements in Addition to the NF-B Site between nt -4755 and -4736 Are Required for Full Response to TNF

As shown in Fig. 6, the GG-to-TT mutations at nucleotides -4749 and -4750 of construct -4.83.5TT did not fully inhibit the ability of the transfected construct to respond to TNF. Quantitation of these results (Fig. 6C) suggested that regulatory elements in addition to the NF-B recognition site at nt -4751 to -4742 might be present.

To explore which additional sequences within the TNF-responsive region of ferritin H might be required for full responsiveness to TNF, additional gel shift assays were performed using a series of overlapping oligonucleotides spanning the 118-nt TNF-responsive region. Of these, only one oligonucleotide demonstrated differential binding to nuclear factors when extracts from control and TNF-treated cells were compared (data not shown). The sequence of this oligonucleotide, termed E/e, is shown in Fig. 7A. It contains a reverse-orientation sequence (5`-GGAAATTC-3`) with considerable similarity to a NF-B recognition motif. The E/e oligonucleotide overlaps F/f(NF-B) by 4 nt, but does not contain the NF-B consensus sequence present in the F/f(NF-B) oligonucleotide. As shown in Fig. 7B, treatment of cells with TNF (lanes 6-10) induced the ability of proteins present in nuclear extracts to bind the E/e oligonucleotide. These proteins could not be detected in the absence of TNF treatment (Fig. 7B, lanes 1-5). Furthermore, binding of labeled E/e oligonucleotide could be competed by unlabeled DNA containing the mouse immunoglobulin light chain NF-B motif, suggesting that although it does not contain a canonical NF-B consensus sequence, it can nevertheless bind a member of the NF-B family. This conclusion was supported by the observation that the appearance of the shifted band was specifically blocked by the addition of antibody to the p65 subunit of NF-B (Fig. 7B, lane9). Therefore, the results of EMSA are consistent with a role for two NF-B transcriptional elements in the regulation of ferritin H expression by TNF.


Figure 7: TNF-induced changes in EMSA using oligonucleotide E/e. A, shown is the sequence of one strand of the double-stranded oligonucleotide used in EMSA. B, nuclear extracts from untreated and TNF-treated NIH3T3 cells (4 h, 1000 units/ml) were incubated with the radiolabeled double-stranded oligonucleotide E/e. 25 ng of unlabeled competitor oligonucleotides (NF-B or glucocorticoid-responsive element (GRE)) was included in samples in lanes2 and 3 and lanes7 and 8. Samples in lanes4 and 9 received 2 µg of antibody to the p65 subunit of NF-B, while samples in lanes5 and 10 received anti-ferritin antibody as a negative control (ctrl).



To further define the contribution of these elements of the ferritin H promoter to the TNF-dependent regulation of ferritin H in vivo, three additional deletions corresponding to critical regions defined by the E/e and F/f(NF-B) oligonucleotides were constructed and fused to the hGH reporter gene. As shown in Fig. 8, these constructs contained both, one, or none of the putative NF-B elements within this 118-nt region; they were termed p8 (-4.7763.5), p7 (-4.7513.5), and p6 (-4.7393.5), respectively. These deletion mutants were transfected into NIH3T3 cells, and their responsiveness to TNF was tested using an RNase protection assay. As seen in Fig. 8(B and C), construct p6 did not significantly respond to TNF treatment, consistent with previous experiments indicating the importance of the NF-B site between nt -4755 and -4736. Inclusion of this NF-B site in construct p7 resulted in a 2.5-fold increase in expression in response to TNF. However, the presence of the additional 25 nt present in construct p8 was required for maximal (4.7-fold) response to TNF. Quantitation of three independent experiments showed that TNF induction was reproducibly submaximal using the p7 mutant and maximal using p8 (Fig. 8C). The striking and consistent augmentation of TNF induction with the sequences in p8 strongly argues that two elements are critical for maximal induction of ferritin H by TNF.


Figure 8: RNase protection analysis of ferritin H/growth hormone constructs p6, p7, and p8. A, a schematic of 5`-deletion mutants p6, p7, and p8 is shown with oligonucleotides used in EMSA. Sequences of the 5`-ends of ferritin H/growth hormone constructs p6 (-4.7393.5), p7 (-4.7513.5), and p8 (-4.7763.5) are shown. The relative locations of oligonucleotides F/f(NF-B) and E/e and the FER-2 region are also indicated. B, p6, p7, or p8 was transfected into NIH3T3 cells, and the transfected cells were treated with 1000 units/ml TNF for 12 h, followed by an RNase protection assay. C, results of multiple RNase protection assays were quantitated by densitometry. -Fold increases in expression from the transfected constructs p6, p7, and p8 are indicated with standard errors.




DISCUSSION

The most well studied regulatory influence on ferritin is iron, which increases both ferritin H and L subunits, primarily by increasing the translation of pre-existing ferritin mRNA (reviewed in Refs. 21 and 22). However, perhaps because cellular environmental conditions that require an adjustment in iron homeostasis may occur without a change in levels of exogenous iron, ferritin responds to regulatory influences in addition to iron. Cytokines, a hallmark of the stress response, represent a physiologically important example of such a regulatory influence. Recent evidence further suggests that multiple pathways for the cytokine regulation of ferritin have evolved, which depend in part on the cell type and cytokine. Thus, in hepatic cells, IL-1 induces both ferritins H and L via a post-transcriptional mechanism(23, 24) . However, in muscle cells, adipocytes, and fibroblasts, the induction of ferritin H by TNF occurs in an iron-independent fashion, specifically targeting ferritin H without affecting ferritin L(8, 9) . This evolution of multiple independent regulatory pathways that impinge on ferritin suggests that modulation of ferritin composition and content is an important component of the cellular and organismal response to cytokines, and hence to the underlying pathophysiological state responsible for cytokine induction.

To clarify mechanisms of the TNF-dependent regulation of ferritin in cells of the mesenchymal lineage, we measured the effect of TNF on ferritin transcription in fibroblasts. We observed that TNF increases the transcription of ferritin H in the absence of any effect on ferritin L. Transcriptional induction of ferritin H by TNF was a relatively rapid response, evident after 4 h (data not shown) or 7 h (Fig. 1) of treatment. Steady-state levels of ferritin H mRNA remained elevated for at least 24 h in these experiments and up to 72 h in others (Refs. 8 and 9; data not shown).

Using a transfection assay, we identified the region of the ferritin H gene mediating TNF responsiveness. This region, FER-2, was found to consist of two closely spaced elements, which together span the region between nt -4776 and -4736.

Using an electrophoretic mobility shift assay, we identified transcription factors that bound to FER-2 as members of the NF-B family. Furthermore, results from EMSA suggested that two different NF-B family members may bind to separate domains within FER-2 (Figs. 4 and 7). The NF-B/Rel family of transcription factors includes at least five members that associate as both hetero- and homodimers(13) . The subunits exhibit different properties, including efficiency of transactivation (greater in the p65 subunit) and DNA binding (greater in the p50 subunit)(12) . In addition, specific members of the NF-B family exhibit preferential affinities for different DNA sequences, which is thought to be important in imparting selectivity to this regulatory pathway. The region of FER-2 between nt -4755 and -4736 contained a canonical NF-B sequence and bound a factor recognized by an anti-p65 antibody (Fig. 4), suggesting that the member of the NF-B family that binds to this region of the ferritin promoter is likely to contain a p65 subunit. Experiments showing that the consecutive G nucleotides at nt -4749 and -4750 are required for binding of nuclear proteins in a gel shift assay (Fig. 5) and the importance of these nucleotides in the binding of p50 (12) further suggest that the p50 subunit may be involved in binding to the region between nucleotides -4755 and -4736, perhaps as a p50/p65 heterodimer. This was also suggested by the similar abundance and mobility of complexes that bind to the F/f(NF-B) sequence as compared with those that bind to the NF-B sequence from the immunoglobulin light chain gene (which binds p50/p65) (data not shown).

In addition to the canonical NF-B sequence between nt -4755 and -4736, deletion analysis and EMSA revealed the existence of a second NF-B site in FER-2, located between nt -4776 and -4752 on the ferritin H promoter. Interestingly, although this region contained a sequence with considerable similarity to a NF-B consensus recognition motif (GGAAATTC), it lacked the 5`-terminal G and 3`-terminal C present in the GGGRNNYYCC consensus sequence and therefore may represent a new motif that binds to NF-B. Other genes with different departures from the GGGRNNYYCC consensus site have been reported, including IL-8(31) , E-selectin(32) , granulocyte colony-stimulating factor, and TNF-1(33) . However, departures from the consensus sequence seen in these binding sites are less dramatic than those seen in ferritin H, which differs from the consensus sequence at both the 5`- and 3`-ends. It is of note that the TNF-induced binding of factors to this site in the ferritin H promoter appeared to be much weaker or the factors less abundant than those bound to the canonical NF-B site in FER-2. Since Rel exhibits relatively poorer binding to DNA as well as a more relaxed binding site specificity when compared with either p50 or p65(34) , it is possible that Rel may be contained in the complex that binds to this upstream component of FER-2. In addition, we observed that antibody to the p65 subunit of NF-B interfered with binding of nuclear factors to this region (Fig. 7), suggesting that the p65 subunit may form a component of the NF-B family member that binds to this region of the ferritin H promoter.

The localization of FER-2 >4 kb 5` to the start site of transcription contrasts with TNF-responsive regions of other genes thus far reported, such as IL-8(25) , E-selectin(26) , H-2K (27), and IL-6(28) , which have tended to be localized relatively proximate to the transcriptional start site. Interestingly, several NF-B consensus motifs can be found within the first 941 nt of the ferritin H 5`-flanking region.()However, based on the data presented here, it is clear that FER-2, containing the two sites at nt -4776 and -4755, constitutes the major regulatory TNF-responsive element of ferritin H. Although the significance of the position of these elements is unclear, we note that other regulatory elements of the ferritin H gene have also been localized in the far upstream 5`-flanking region(16) . This contrasts with the position of elements responsible for basal transcription of ferritin H, which have been localized to the first few hundred nucleotides of the 5`-flanking region of the human (29, 30) and murine (16) ferritin H genes. How these basal elements and their associated transcription factors interact with regulatory elements of the ferritin promoter located >4 kb upstream remains to be explored.

The NF-B family is the final common target of multiple signaling pathways initiated by such diverse stimuli as cytokines, viruses, cAMP, UV light, and calcium ionophores(13) . Based on the observation that the induction of NF-B is an early response to oxidative stress, it has recently been suggested that the generation of reactive oxygen intermediates may represent a convergence point for many of these pathways, including those induced by exposure to TNF (36, 37). TNF is known to induce oxidative stress in at least some cell types, and we have speculated that protection from oxidative stress may be a component of the biological rationale underlying the induction of ferritin by cytokines(38) . Thus, given the role of iron in oxygen free radical formation, the induction of the iron-binding protein ferritin may be viewed as a cytoprotective response, designed to limit the availability of iron for participation in free radical generation under conditions of oxidative stress. Indeed, others have shown that ferritin induction can protect both endothelial (39) and tumor (40) cells from oxidative injury. In this context, NF-B may be viewed not only as a ``sensor'' of oxygen free radicals, but also as a transcriptional activator that, by inducing ferritin, serves to limit damage from those radicals.


FOOTNOTES

*
This work was supported by Grant DK-42412 from the National Institutes of Health. Oligonucleotide synthesis was performed in the DNA Synthesis Core Laboratory of the Comprehensive Cancer Center of Wake Forest University supported in part by National Institutes of Health Grant CA-12197. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Dept. of Cell Biology, P. O. Box 3709, Duke University Medical Center, Durham, NC 27710.

To whom correspondence should be addressed: Dept. of Biochemistry, Bowman Gray School of Medicine, Comprehensive Cancer Center, Wake Forest University, Medical Center Blvd., Winston-Salem, NC 27157. Tel.: 910-716-9357; Fax: 910-716-7671.

The abbreviations used are: TNF, tumor necrosis factor-; IL, interleukin; kb, kilobase(s); hGH, human growth hormone; nt, nucleotide(s); PCR, polymerase chain reaction; EMSA, electrophoretic mobility shift assay.

E. L. Kwak, S. V. Torti, and F. M. Torti, unpublished observations.


ACKNOWLEDGEMENTS

We are grateful to Yoshiaki Tsuji for careful reading of the manuscript and numerous helpful suggestions.


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