From the
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-
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-
NF-
Approximately 5
Sequencing was performed by the dideoxy chain termination
method using Sequenase (U. S. Biochemical Corp.).
Deletions of the -5.6 ferritin H/growth hormone
reporter construct, which contains
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 Na
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.
RNA probe labeled with [
Bands were excised from dried gels and counted in a
scintillation counter or, in some experiments, scanned by densitometry.
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
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)
Oligonucleotides used in
EMSA were as follows: F/f(NF-
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
To verify that DNA sequence requirements for binding of factors to
the region of the ferritin promoter defined by the F/f(NF-
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-
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
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-
In addition to the canonical
NF-
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
The NF-
We are grateful to Yoshiaki Tsuji for careful reading
of the manuscript and numerous helpful suggestions.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(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.
(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.
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, I
B. 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.
Nuclear Run-off Transcription Assay
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 H
O, 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
Deletion Constructs
5.6 kb of murine ferritin H
5`-flanking region ligated to hGH(16) , were made as follows.
-5.6
An internal EcoRI fragment
from nt -4477 to -941 was removed from -5.6 ferritin
H/growth hormone by EcoRI digestion and religation.
3.5
-5.3
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).
3.5
-5.1
Construction was similar to
that of -5.33.5
3.5, but PCR synthesis was primed from
approximately -5.1 kb utilizing the 5`-primer
5`-GGCTTGGCACGAAGCACCT-3`.
-4.8
-5.63.5
3.5 was digested
with XbaI to remove the region between -5.6 and
-4.8 kb.
-4.8
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.
3.5TT
-4.7
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.
3.5
-4.6
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.
3.5
-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
HPO
, 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
RNase Protection Assay
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
Nuclear Extraction
, 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)
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.
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.
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
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 4.8 kb 5` of the Transcriptional Start Site
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 -5.6
3.5
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 -5.6
3.5 as well as the deletion mutants
-5.3
3.5, -5.1
3.5, and -4.8
3.5. 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 -4.7
3.5, -4.6
3.5, 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.
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-
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-binding Site
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.
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-
To determine whether the
NF-B Site in the Ferritin H Gene
Reduces TNF Responsiveness in Vivo
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 -4.8
3.5, 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
-4.8
3.5TT was unable to respond to TNF to the same extent as
-4.8
3.5. When the results of three independent experiments
were quantitated, construct -4.8
3.5TT 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 4.8
3.5
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,
-4.8
3.5TT. A, shown is a schematic diagram of the
-4.8
3.5 construct and its mutant, -4.8
3.5TT. 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 -4.8
3.5 or -4.8
3.5TT. 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-
As shown in Fig. 6, the GG-to-TT mutations
at nucleotides -4749 and -4750 of construct
-4.8B
Site between nt -4755 and -4736 Are Required for Full
Response to TNF
3.5TT 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.
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 (-4.776
3.5), p7 (-4.751
3.5), and p6
(-4.739
3.5), 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 (-4.751
3.5), and p8
(-4.776
3.5) 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.
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.
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).
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.
(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.
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.
; IL, interleukin; kb, kilobase(s); hGH, human growth
hormone; nt, nucleotide(s); PCR, polymerase chain reaction; EMSA,
electrophoretic mobility shift assay.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.