From the Lineberger Comprehensive Cancer Center,
Department of Biology, and § Curriculum in Genetics
and Molecular Biology, University of North Carolina, Chapel Hill, North
Carolina 27599-7295 and the ¶ Department of Pharmacology,
Biochemistry, and Molecular Biology, Merck Frosst Centre for
Therapeutic Research,
Point Claire-Dorval, Quebec H9R 4P8, Canada
Received for publication, September 30, 2002, and in revised form, October 29, 2002
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ABSTRACT |
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The transcription factor NF- The transcription factor NF- Recently, more complex aspects of NF- The vertebrate mitochondrial genome is circular with ~17 kb of DNA
(11) (for a review, see Ref. 12). The mitochondrial genome encodes two
ribosomal RNAs, 22 tRNAs, and at least 13 peptides, which contribute to
complex I, complex III, complex IV, and complex V of the electron
transport system (11, 12). A regulatory region associated with the
origin of replication also serves as a promoter region for two large
mitochondrial RNA transcripts that are processed into individual RNAs
for the structural RNAs and mRNAs (see Ref. 13). For example, the
mRNAs encoding cytochrome c oxidase I, II, and III
(CoxI, -II, and -III, respectively) are derived from a common precursor
RNA (12).
We report here that the NF- Cell Culture and Treatment with Cytokines and Protease
Inhibitors--
Cell lines were obtained from the Lineberger
Comprehensive Cancer Center Tissue Culture Core Facility at the
University of North Carolina (Chapel Hill, NC). U937 cells were grown
in Dulbecco's modified Eagle's medium H supplemented with 10% fetal
bovine serum and antibiotics. HT1080 V and HT1080 I lines were
previously described (15). Cells were treated with 10 ng/ml human
recombinant TNF Electron Microscopy and Immunocytochemical Labeling--
U937
cells were suspended in serum-free medium and were mixed with a
fixative containing 0.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.4. The cells were cooled to 10 °C on ice, followed by microwave irradiation using a 750-watt microwave oven until
of a final temperature of 40 °C was obtained (16). The fixed cells
were pelleted, rinsed in 0.1 M sodium cacodylate buffer, and postfixed with 1% osmium tetroxide in 0.1 M sodium
cacodylate buffer for 30 min. Following postfixation, the cell pellet
was dehydrated through a graded series of ethanol washes and embedded in L. R. White resin (available from Ted Pella, Inc., Redding, CA). 70-nm ultrathin sections were taken of the embedded pellet, mounted on nickel grids, and stained using indirect immunocytochemical methods.
Immunocytochemical labeling, incubations, and washing steps were
carried out at ambient temperature. The incubation and wash buffer for
the primary antibody consisted of 0.05 M Tris-buffered saline with 0.1% fish skin gelatin and 0.01% Tween 20 (TBS/FGT) at pH
7.6. Grid-mounted sections were blocked with 0.2 M glycine in Hanks' balanced salts for 10 min, followed by a secondary blocking step with 5% goat serum in TBS/FGT for 10 min. The grids were incubated for 1 h with primary antibody (p50/NF- Preparation of Mitochondria--
Mitochondria were isolated from
cell lines as described previously (17). Digitonin was added to the
mitochondrial buffer A at 0.05% to aid in the disruption of the cells.
Further purification was performed by ultracentrifugation at 30,000 rpm
in gradient buffer (250 mM mannitol, 1 mM EGTA,
25 mM Hepes, 0.1% bovine serum albumin), pH 7.4, supplemented with 30% Percol (Sigma). The uppermost band was removed
and washed two or three times with mitochondrial buffer A. Mitochondria
that were analyzed by Western blot analysis were boiled in SDS sample
buffer and loaded directly onto SDS-polyacrylamide gels. When
electrophoretic mobility shift assays (EMSAs) were performed, purified
mitochondria were incubated in nuclear extract buffer for 10 min in
ice. The sample was then centrifuged for 10 min at 12,000 rpm at
4 °C. The supernatants were used directly in the EMSA reaction (see
below). Isolation of mitochondria from rat liver was as previously
described (18).
Western Blot Analysis--
For Western blotting analysis of
cultured cells, equal amounts of protein were separated on a 12%
SDS-polyacrylamide gel and transferred to nitrocellulose. Blots were
blocked in 5% milk in 1× TBST (Tris-buffered saline, 0.5% Tween 20)
and probed with either p65 (Rockland, Gilbertsville, PA), p50, IKK EMSAs--
EMSAs were performed as described previously (20). An
oligonucleotide corresponding to an NF- I Northern Blot Analysis--
RNAs were isolated using Trizol as
recommended by the manufacturer (Invitrogen). Northern blot
analysis utilized 10-20 µg of total cellular RNA separated on 1.5%
formaldehyde-agarose gels according to standard procedures. RNA samples
were transferred overnight to nylon filter, UV cross-linked
(Stratagene, La Jolla, CA), and probed with randomly labeled probe
(Amersham Biosciences) corresponding to cytochrome c oxidase
II or III or cytochrome B. Hybridization and wash conditions were
obtained using Expresshyb (Stratagene) solution as described by
the manufacturer. Blots were normalized for equal loading using a
cDNA probe for glyceraldehyde-3-phosphate dehydrogenase.
Mitochondrial Localization of NF- TNF TNF Phosphorylation of Mitochondrial I
When an antibody specific for the phosphorylated form of I
To address whether the proteasome may not be relevant in mitochondrial
I
NF- Evidence for Mitochondrial IKK Activity--
In order to
understand the mechanism of the induced phosphorylation of I NF- The results presented here demonstrate the localization of certain
NF- Although great efforts were made to ensure the purity of the
mitochondrial preparations, we cannot rule out the possibility of small
amounts of contamination from other cellular organelles. We performed
electron microscopy on our mitochondrial preparations and found that
they were ~95% pure mitochondria. The other 5% of the preparation
consisted of membranous debris, which may include Golgi, plasma
membrane, and endoplasmic reticulum. Western blots were also performed
on these preparations using various organelle markers, confirming that
the mitochondrial preparations were highly purified. We did see some
staining for endoplasmic reticulum in our mitochondrial fractions;
however, the percentage of nonmitochondrial membrane in our
preparations was low enough that we feel it did not contribute
significantly to the protein content of the preparations. In addition,
lack of endoplasmic reticulum staining of p65 or I One obvious question is the potential mechanism whereby NF- Another interesting question is how mitochondrial I The localization of NF- NF-B has been shown
to be predominantly cytoplasmically localized in the absence of an
inductive signal. Stimulation of cells with inflammatory cytokines such as tumor necrosis factor
or interleukin-1 induces the degradation of I
B, the inhibitor of NF-
B, allowing nuclear accumulation of
NF-
B and regulation of specific gene expression. The degradation of
I
B is controlled initially by phosphorylation induced by the I
B
kinase, which leads to ubiquitination and subsequent proteolysis of the
inhibitor by the proteasome. We report here that NF-
B and I
B
(but not I
B
) are also localized in the mitochondria. Stimulation
of cells with tumor necrosis factor
leads to the phosphorylation of mitochondrial I
B
and its
subsequent degradation by a nonproteasome-dependent pathway.
Interestingly, expression of the mitochondrially encoded cytochrome
c oxidase III and cytochrome b
mRNAs were reduced by cytokine treatment of cells. Inhibition of
activation of mitochondrial NF-
B by expression of the superrepressor form of I
B
inhibited the loss of expression of both cytochrome c oxidase III and cytochrome b mRNA. These
data indicate that the NF-
B regulatory pathway exists in
mitochondria and that NF-
B can negatively regulate mitochondrial
mRNA expression.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
B has been studied extensively due
to its interesting regulation and to the range of biological processes
that it controls. Five members of the immediate NF-
B/Rel family have
been identified: p50/NF-
B1, p65/RelA, p52/NF-
B2, RelB, and c-Rel.
The classic form of NF-
B is the heterodimer of the p50 and p65
subunits (reviewed in Ref. 1). In most cells, NF-
B is complexed with
members of the I
B family, I
B
, I
B
, and I
B
, which
typically function to inhibit the action of this group of transcription
factors. Stimulation of cells with stimuli such as
TNF
1 leads to activation
of the I
B kinase (IKK), which phosphorylates I
B
or I
B
on
N-terminal serines (1, 2). Phosphorylated I
B then is targeted for
ubiquitination and subsequent degradation by the proteasome, allowing
NF-
B to accumulate in the nucleus (3). In the nucleus, NF-
B is a
positive regulator of gene expression through its ability to bind to
target sequences in the regulatory regions of genes encoding cytokines,
cytokine receptors, antiapoptotic proteins, and cell cycle regulators
(1, 4). Additionally, NF-
B has been shown to negatively regulate
MyoD mRNA through a post-transcriptional mechanism that appears to require the transcriptional activity of NF-
B (5). Presumably through
its ability to regulate gene expression, NF-
B dysregulation contributes to a variety of diseases, including oncogenesis, arthritis, and cancer cachexia (4, 6).
B regulation have been
proposed. For example, studies indicate that NF-
B shuttles into and
out of the nucleus in unstimulated cells (7-9). Thus, it was found
that leptomycin B, an inhibitor of nuclear export, leads to the
accumulation of NF-
B and I
B in the nucleus without an external
stimulus. Whether NF-
B and I
B shuttle in a complex has been
questioned (7). Additionally, it has been proposed that I
B can be
degraded by a nonproteasomal mechanism following stimulation of cells
with cytokines (10). In that study, the use of inhibitors with distinct
specificities indicated that phosphorylated I
B
can be degraded by
calpain in addition to the more characterized proteasome pathway.
B subunits p50 and p65 along with
I
B
, but not I
B
, are found in the mitochondria as well as in
the cytoplasm of proliferating cells. Quiescent liver cells exhibit
largely p50 and I
B
in the mitochondria with little detectable p65. Electron microscopy and biochemical approaches confirm the localization of these proteins to mitochondria. Recently, others have
found I
B
and p65 in the mitochondria of Jurkat T cells (14).
Interestingly, our data indicate that mitochondrial I
B
is
phosphorylated on N-terminal serines in response to cellular TNF
stimulation, followed by nonproteasomal degradation. TNF
treatment
leads to a significant reduction in CoxIII and cytochrome b
mRNA. Inhibition of NF-
B by mitochondrially localized
superrepressor I
B
blocked the loss of both CoxIII and Cyt B
mRNA. These data show that NF-
B is found in mitochondria, where
it regulates mitochondrial specific gene expression.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
diluted in phosphate-buffered saline. Calpeptin,
lactacystin, and MG132 (Calbiochem) were all resuspended in
Me2SO and used at a final concentration of 40 ng/ml.
B1; Upstate
Biotechnology, Inc., Lake Placid, NY). After rinsing with TBS/FGT, the
grids were incubated for 1 h in secondary antibody (goat
anti-rabbit IgG 10 nm colloidal gold (BBI International, Ted Pella,
Redding, CA), diluted 1:50 in 0.1 M TBS/FGT, pH 8.2. Negative controls were performed concurrently, consisting of incubation
in normal rabbit IgG at the same repetitive dilutions and conditions.
The immunogold-stained sections were poststained with uranyl acetate followed by lead citrate, and the grids were observed and photographed using a LEO EM-910 transmission electron microscope (LEO Electron Microscopy, Inc., Thornwood, NY) at 80 kV.
,
IKK
, IKK
(Upstate Biotechnology), I
B
(c-21), I
B
,
Skp-1 (Santa Cruz Biotechnology, Santa Cruz, CA), Lmp-2, (Affiniti
Research Products, Exeter, UK), cytochrome c (PharMingen,
San Jose, CA), or anti-phosphoserine-32 I
B
(New England Biolabs,
Beverly, MA). Blots were probed with a secondary antibody conjugated to
horseradish peroxidase (Promega Corp.) at a 1:15,000 dilution in 1×
TBST. Protein bands were visualized with an enhanced chemiluminescence
detection system (Amersham Life Sciences). Western blotting of rat
liver mitochondria was performed as described (19).
B site in the
H-2Kb gene was radiolabeled using
[
-32P]dCTP and the Klenow fragment of DNA polymerase I
(Roche Molecular Biochemicals). For antibody supershift analysis,
extracts were preincubated 15 min at room temperature with 1 µg of
antiserum before the addition of the radiolabeled gel shift probe.
Antibodies used in supershift analysis were identical to those utilized
for Western blotting analysis.
B Kinase Assay--
Cells were either treated or not treated
with TNF
for specified times and harvested, and mitochondrial and
cytoplasmic extracts were isolated in phosphate-buffered saline
containing phosphatase inhibitors. Equal amounts of protein (500 µg)
were immunoprecipitated using antibodies against the
subunit of IKK
(a gift of Dr. F. Mercurio, Signal Pharmaceuticals, San Diego, CA).
Kinase activity was determined by incubating the immunoprecipitates
with 4 µg of glutathione S-transferase-I
B
(amino
acids 1-54) wild type substrate or a mutated form of I
B
(S32T,S36T) in the presence of [
-32P]ATP, as described
(21). The immunoprecipitates were subjected to SDS-PAGE, dried, and
visualized by autoradiography.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
B--
Studies were initiated
in order to analyze the subcellular localization of NF-
B and I
B
subunits. Electron microscopy of sections of U937 cells in association
with immunocytochemistry indicated the presence of both the
p50/NF-
B1 and p65 subunits localized in the inner matrix of the
mitochondria as well as in the cytoplasm (see Fig.
1). The control (see "Materials and
Methods") showed no detectable labeling. Additionally, I
B
was
detectable in the inner matrix of
mitochondria.2 After
observing the presence of the NF-
B p50 and p65 subunits in the
mitochondria using electron microscopy, we were interested in
confirming the results biochemically. Mitochondria were first isolated
from rat liver and subjected to increasing concentrations of digitonin,
which functions to elute proteins in a manner dependent on the
localization within the mitochondria. Supernatants and mitochondrial
pellets at each concentration were analyzed by Western blotting using
antibodies to I
B
, p50, or p65. The results show that both
I
B
and p50 are present in the rat mitochondria (Fig. 2). With the addition of low
concentrations of digitonin, I
B
protein was not detected in the
supernatant. The addition of 0.4% digitonin (lane
5) leads to release of I
B
from mitochondria so that it
is detected in the supernatant. However, the p50 protein was not
visible in the supernatant until 0.5-0.6% digitonin was added
(lanes 6 and 7). When these results
are plotted against known mitochondrial components, release of p50 from
the mitochondria following digitonin treatment corresponds to the
release of fumarase,2 a marker for the inner matrix of the
mitochondria, whereas I
B
release from the mitochondria occurs
earlier, indicating that I
B
localization is closer to the
mitochondrial surface and presumably also in the inner matrix. p65 was
not detected in the mitochondria of quiescent rat liver (data not
shown, but see below regarding cells in culture).
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Fig. 1.
Visualization of
p50/NF- B1 and p65 in the mitochondria by
electron microscopy. Sections of U937 cells were stained with a
p50 or p65 antibody and visualized with electron microscopy (see
"Materials and Methods"). Bar, 0.25 µm. Pictures were
taken at ×60,000 magnification.
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Fig. 2.
Localization of p50 and
I B
in the
mitochondria of rat liver cells. Mitochondria were isolated from
rat liver and incubated with increasing amounts of digitonin. Pellet
(P) and supernatant (S) from either untreated
sample (lane 1) or samples treated with
0.1-0.8% digitonin (lanes 2-9) were separated
on 10% SDS-PAGE and transferred to nitrocellulose. The membrane was
probed with either an anti-p50 or anti-I
B
antibody.
Treatment of U937 Cells Causes a Loss of I
B
in the
Mitochondria--
To further analyze the localization of I
B
and
NF-
B in the mitochondria and to determine potential responses to
cytokine treatment, U937 cells were studied by Western blotting of
mitochondrial and cytoplasmic extracts (Fig.
3). Cells were stimulated with TNF
over a 1-h time course. After separation of cytoplasmic and mitochondrial components, the mitochondrial pellet was resuspended in a
volume of buffer equal to one-tenth volume of the cytosolic fraction,
consistent with published estimates that mitochondria represent
one-tenth the total volume of a human cultured cell (22). Protein
assays confirmed that concentrations of the two fractions were
approximately equivalent. Assuming that overall protein concentrations
are similar in the mitochondria and cytoplasm of living cells, we
loaded equal amounts of protein from each fraction in order to observe
physiologically comparable levels of each subunit. First, the data
support the EM data indicating that p50 and I
B
were localized in
the mitochondria. Second, the data indicate that the p65 subunit of
NF-
B is located in the mitochondria of U937 cells. Additionally, the
data indicate that the mitochondrial p65 exhibits slightly faster
migration in SDS gels (e.g. compare the mobility of the
mitochondrial p65 with cytoplasmic p65) (Fig. 3), possibly due to a
protein processing event associated with mitochondrial import (see Ref.
12). The results also show that following TNF
treatment, there is
degradation of I
B
by 30 min and resynthesis at 60 min in both the
cytoplasm and mitochondria of U937 cells (Fig. 3). The overall kinetics of loss of mitochondrial I
B
is slightly different from what we
observe in the cytoplasm. It is noted that a slower migrating form of
mitochondrial I
B
is detected following TNF
treatment, suggestive of induced phosphorylation (see below). There appears to be
very little change in p65 in either the mitochondria or cytoplasm of
these cells following TNF
treatment; however, p50 shows a reduction
in the mitochondria following TNF
treatment of cells. To confirm the
absence of mitochondrial contamination of the cytoplasmic fractions,
the Western blot was probed with a cytochrome c antibody.
Other experiments show that there is little or no cytoplasmic
contamination of mitochondria and that I
B
is not found in the
mitochondria (see Fig. 5, B and C). Additionally, c-Rel was not detected at appreciable levels in the mitochondria of
U937 cells. Identical results were found when using the HT1080 cell
line, showing that the localization is not cell type-specific.
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Fig. 3.
Changes in p50 and
I B
in the
mitochondria of U937 cells upon TNF
stimulation. U937 cells were treated with 10 ng/ml TNF
over a 1-h time course, and mitochondria (M) and cytoplasmic
(C) fractions were prepared and analyzed by Western blot
analysis. Proteins were fractioned on SDS-polyacrylamide gels,
transferred to nitrocellulose, and incubated with antibodies specific
for p65, p50, I
B
, and cytochrome c.
Stimulation Results in Increased p50 DNA Binding Activity in
the Mitochondria--
To determine whether the NF-
B components in
the mitochondria were able to bind DNA, mitochondrial extracts were
prepared, and mitochondrial proteins were eluted in nuclear extract
buffer. The results from an EMSA show two binding complexes (complexes I and II). The NF-
B DNA binding activity is low in the mitochondria in untreated cells and increases with TNF
treatment, with the strongest binding occurring at the 1-h time point (Fig.
4). To identify which components of
NF-
B contribute to this binding activity, supershift analysis was
performed on the mitochondrial extract that was treated with TNF
for
1 h. The use of a p50-specific antibody led to a supershifted band
from complex II (Fig. 4). However, when a p65-specific antibody was
added to the binding reaction, there was a reduction in the DNA binding
in complex II without the appearance of a supershifted band. Complex I
could not be supershifted with either antibody. These results indicate that p50 is the major NF-
B subunit found in binding complex II, possibly existing as a homodimer. It remains possible that additional NF-
B components may be part of the DNA binding activity. The data
also suggest that the presence of p50 and p65 in the mitochondria is
presumably not as a heterodimer, possibly suggesting distinct mitochondrial sublocalization for these two NF-
B subunits.
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Fig. 4.
TNF stimulation
increases DNA binding activity of p50 in the mitochondria.
Mitochondria isolated from U937 cells that were treated with TNF
were incubated in nuclear extract buffer, and the resulting protein was
analyzed by EMSA. Supershift analysis was performed on extract from the
TNF
1-h time point. Extracts were preincubated for 15 min with
either a p65 or p50 antibody prior to the addition of the probe.
B
in Response to TNF
Occurs on N-terminal Serines, and Degradation of I
B
in the
Mitochondria following TNF
Stimulation Is
Proteasome-independent--
The fact that TNF
induced the
degradation of I
B
in the mitochondria led us to explore whether
I
B
is phosphorylated and degraded in a manner similar to that of
cytoplasmic I
B
. First, we asked whether I
B
loss was
dependent on a proteasome-mediated mechanism. Cells were pretreated
with proteasome and calpain inhibitors and either left untreated or
stimulated with TNF
. Mitochondria were purified, and proteins were
eluted and analyzed by Western blotting. The results (Fig.
5A) show that upon treatment
of cells with TNF
for 15 min, there is a significant loss of
I
B
in both the mitochondrial and the cytoplasmic compartments.
This loss in the cytoplasm can be blocked following pretreatment with
either lactacystin, a proteasome-specific inhibitor, or with MG132, an inhibitor of both calpain and the proteasome (10) (Fig. 5A). When cells were pretreated with calpeptin, an inhibitor specific for
calpain (10), there was a modest inhibition of the degradation of
cytoplasmic I
B
but considerably less inhibition than with the
other inhibitors. However, in the mitochondrial fraction, lactacystin
had very little effect in blocking the degradation of I
B
.
Additionally, cells pretreated with calpeptin showed more inhibition of
mitochondrial I
B
degradation than lactacystin, unlike the
cytoplasm, which showed the opposite effect. However, it is noted that
calpeptin was able to inhibit the degradation of I
B
in the
mitochondria only partially.
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Fig. 5.
I B
degradation
upon TNF
stimulation is blocked by
proteasome-specific inhibitors in the cytoplasm but not in the
mitochondria. A, U937 cells were pretreated with either
Me2SO (DMSO; lanes 1 and
2), lactacystin (lanes 3 and
4), calpeptin (lanes 5 and
6), or MG132 (lanes 7 and
8) for 15 min followed by stimulation with 10 ng/ml of
TNF
for 15 min (lanes 2, 4,
6, and 8). Mitochondrial (M) and
cytoplasmic (C) proteins were isolated and analyzed on 12%
SDS-polyacrylamide gels. Blots were first probed with a
phospho-I
B
(pI
B
). Blots were stripped
and reprobed with an I
B
antibody. B, proteins isolated
from untreated U937 cells were analyzed on 12% SDS-polyacrylamide
gels. Blots were probed with Lmp-1, Skp-1, and cytochrome c
antibodies. C, mitochondrial (M) and cytoplasmic
(C) extracts isolated from U937 cells were analyzed on 12%
SDS-polyacrylamide gels. Blots were probed with either I
B
,
I
B
, or cytochrome c antibodies.
B
(anti-phosphoserine 32) was used to probe the same blot, phosphorylated I
B
was visualized when cells were pretreated with all of the inhibitors in the cytoplasm but only in cells pretreated with calpeptin
or MG132 in the mitochondria (Fig. 5A). Again, these results
point to a lack of proteasome effect in the mitochondria. It is not
clear whether the degradation of I
B
in the mitochondria is a
calpain-specific pathway, since calpeptin does not completely block the
degradation. This may be a function of the inhibitor, or there may be
other factors involved in this process. Additionally, we cannot rule
out that the lack of effect seen in the mitochondria when the cells are
pretreated with lactacystin was due to the lack of permeability of the
mitochondria to lactacystin.
B
degradation, cytoplasmic and mitochondrial extracts were
prepared from U937 cells, and Western blotting was performed using
antibodies specific for Lmp-2, a functional subunit of the proteasome
(23) and Skp-1, a component of the ubiquitin ligase associated with
I
B
ubiquitination (3). The results show that both proteins can
only be found in the cytoplasm of U937 cells (Fig. 5B).
Blots were also probed for cytochrome c as a mitochondrial marker. These results further support the possibility that the mitochondria lacks proteasome function. They also confirm that there is
no cytoplasmic contamination of the mitochondria preparations.
B is regulated by multiple forms of I
B, including I
B
and
I
B
(1). Mitochondria blots from U937 cells were probed with
I
B
and I
B
antibodies (Fig. 5C). The results
indicate that whereas I
B
is found in both the mitochondria and
the cytoplasm, I
B
is only found in the cytoplasm. Again,
cytochrome c was used as a mitochondrial marker.
B
in
the mitochondria in response to TNF
treatment, we explored whether
IKK family members were present in or potentially associated with the
mitochondria. Western blots of fractions from U937 cells enriched for
mitochondria were probed with IKK
-, IKK
-, and IKK
-specific
antibodies. The results show that all three IKK family members are
present in the mitochondrial fraction (Fig. 6A). Blots were also probed
with a cytochrome c antibody as a marker for mitochondria.
Based on the phosphorylation on I
B
in the mitochondria and the
presence of the IKK family members, we wanted to determine whether
functional kinase activity could be detected in the mitochondria
fraction. Large scale mitochondria and cytoplasmic extracts were
prepared from U937 cells either left untreated or treated with TNF
.
The results show that both in the cytoplasm and mitochondrial extracts,
low levels of IKK kinase activity were seen in untreated cells (Fig.
6B). Following stimulation with TNF
, the I
B kinase
activity in both the cytoplasm and mitochondria was greatly increased.
These results indicate that the IKK family members are associated with
the mitochondrial fraction and are potentially responsible for the
phosphorylation of I
B
seen there.
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Fig. 6.
IKK ,
IKK
, and IKK
are all
present and functional in the mitochondria. A,
mitochondria (M) and cytoplasmic (C) extracts
isolated from U937 cells were run on 12% SDS-polyacrylamide gels and
transferred to nitrocellulose. Blots were probed with either IKK
,
IKK
, IKK
, or cytochrome c. B, U937 cells
were either treated or not treated with TNF
, and mitochondrial and
cytoplasmic extracts were prepared. 500 µg of protein was
immunoprecipated with an anti-IKK
antibody. Immunoprecipates were
incubated with wild-type glutathione S-transferase-I
B
fusion protein in the presence of [
-32P]ATP. The
immunoprecipitates were run on a 10% SDS-PAGE gel, dried, and exposed
to film. The data represent three independent experiments.
B Negatively Regulates Expression of CoxIII and Cytochrome b
mRNAs--
In order to address a possible function of NF-
B
components in the regulation of mitochondrial gene expression, we
utilized HT1080 cells either containing an empty vector or expressing
the modified form of I
B
known as I
B
SR (15). Interestingly,
I
B
SR was shown to be in the mitochondria as well as the cytoplasm
through its ability to be recognized by a FLAG tag
antibody.2 Importantly, the superrepressor form of I
B
was shown to be resistant to degradation in mitochondria of HT1080
cells in response to TNF
stimulation. HT1080 cells stably
transfected with either a vector control or the I
B
SR were treated
with TNF
, and RNA was isolated at several time points. The results
show that HT1080 vector control cells exhibit a loss of CoxIII mRNA
beginning at 1 h following TNF
treatment and continuing through
2 h (Fig. 7). The mRNA returns
to near normal level following 4 h of TNF
stimulation. However,
when HT1080 cells containing the I
B
SR were treated with TNF
,
only a modest change in the CoxIII mRNA was observed. Since
cytochrome c oxidase III plays an integral role in complex
IV of the electron transport machinery (12), it is expected that the
loss of CoxIII would impact changes in ATP and reactive oxygen species.
Efforts to address such changes in response to TNF signaling have been
hampered by significant differences in ATP and ROI levels between
I
B
-expressing cells and vector control cells in the absence of
TNF treatment (see "Discussion"). We have explored whether another
mRNA, encoded in a region downstream of CoxIII, is also regulated
differentially in the HT1080 V and HT1080 I cells. As with CoxIII, we
find that CytB mRNA is down-regulated by TNF
in the HT1080
vector cells but not affected in the I
B
-expressing cells.
Overall, these results indicate a role for NF-
B in the regulation of
specific mitochondrial gene expression.
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Fig. 7.
CoxIII mRNA is maintained following
TNF stimulation in the absence of
NF
B. HT1080 cells transfected with either
a vector control or I
B
-SR were treated with 10 ng/ml TNF
for
the specified times. RNA was isolated, and 10 µg was electrophoresed
on 1.0% formaldehyde gels. RNAs were transferred to nylon membrane and
probed with 32P-labeled cDNAs corresponding to CoxIII
and cytochrome b. The blots were stripped and reprobed with
glyceraldehyde-3-phosphate dehydrogenase as a loading control.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
B subunits and I
B
to the mitochondria and are consistent with a recent publication showing that I
B
and p65 are found in
the mitochondria (14). Interestingly, the major components of the
NF-
B regulatory pathway are also apparently localized with the
mitochondrial fraction, including the I
B kinase subunits IKK
and
IKK
as well as IKK
. Consistent with this, TNF
treatment of
U937 cells leads to the phosphorylation of degradation of the mitochondrially associated I
B
. In contrast with the degradation that occurs in the cytoplasm, the mitochondrial degradation of I
B
appears to be proteasome-independent, since a specific inhibitor of the
proteasome (lactacystin) does not block degradation and because key
components of the proteasome and ubiquitin ligase associated with
I
B
ubiquitination are not found in the mitochondria. The use of
inhibitors suggests that calpain may be partly involved with I
B
degradation in the mitochondria. The DNA binding assay indicates that
an NF-
B complex containing p50, possibly as a homodimer, is released
from I
B
and is capable of binding to DNA following TNF
stimulation. This response is correlated with the loss of CoxIII and
CytB mRNAs following TNF
treatment. The involvement of NF-
B
in this process is suggested by the use of the superrepressor form of
I
B
, which inhibits the loss of CoxIII mRNA. The data raise
many questions about the functions of NF-
B in the mitochondria.
B
in the
electron micrographs further supports the conclusion that NF-
B
staining in our fractions is mitochondrial.
B
regulates CoxIII and CytB mRNA levels. One can envision at least four mechanisms. One mechanism may be that NF-
B, possibly as a p50
homodimer, inhibits the processing of the RNA precursor at a level
inhibiting CoxIII and CytB mRNA release. In this model, the release
of mitochondrial p50 from I
B
may lead to an interaction with the
precursor RNA and an inhibition of processing. Another mechanism could
be that binding of NF-
B to mitochondrial DNA inhibits a
transcriptional mechanism, possibly elongation, again somehow affecting
CoxIII and CytB mRNA production. Another possibility is that p50 in
association with I
B
serves as a positive mechanism in regulating
CoxIII and CytB mRNA but that dissociation of the p50 and I
B
complex following TNF treatment leads to a loss of this control.
Finally, we cannot rule out that a nuclear gene or genes regulated by
NF-
B encode proteins that regulate specific mitochondrial mRNA
accumulation. Obviously, more complex experimentation will be required
to determine how NF-
B may regulate mitochondrial gene expression and
whether other transcripts may be positively or negatively controlled by
NF-
B. Along these lines, it is presently unclear whether p50 and
I
B
are complexed in the mitochondria and whether the detection of
p65 in the mitochondria is due to an interaction with p50 or I
B
.
The recent paper from Bottero et al. (14) indicates that p65
and I
B
are associated in mitochondrial extracts. Western blotting
did not reveal significant levels of other NF-
B subunits in the
mitochondria of U937 cells (data not shown).
B
becomes
phosphorylated and how mitochondrial or mitochondria-associated IKK
becomes activated in response to TNF signaling (Fig. 6B). For cytoplasmic IKK, one current model is that IKK is recruited to
factors associated with cytokine receptors, such as the TNF receptor
(24), and becomes activated to phosphorylate I
B
or I
B
.
Presumably, rapid activation of mitochondrially associated IKK must
occur by a distinct mechanism that is somehow dependent on TNF binding
to its receptor on the membrane. The nature of the signal linking cell
surface TNF binding to its receptor and the activation of the
mitochondrial or mitochondria-associated IKK is unclear. We cannot rule
out the possibility that the mitochondrial I
B
becomes
phosphorylated by a kinase distinct from IKK or becomes phosphorylated
in the cytoplasm and is transported into the mitochondria.
B to the mitochondria raises interesting
questions concerning a potential role in regulating apoptosis. NF-
B activation in response to TNF signaling has been shown to inhibit apoptosis. Thus, it has been shown that NF-
B activates several gene products (namely Bcl-xL, A1/Bfl-2, IAP proteins, TRAF
proteins, etc.) to inhibit the caspase cascade and to block cytochrome
c release from mitochondria (4, 6). It is interesting to
speculate that mitochondrial NF-
B may play a role in the suppression of apoptosis. As described above, it is predicted that modulation of
CoxIII mRNA may ultimately impact ATP production, which is required
for apoptosis induced by TNF. Along these lines, Bottero et
al. (14) provide evidence that mitochondrial I
B
interacts with ANT, the adenine nucleotide transporter, which has been speculated to be involved with apoptosis through its ability to regulate the
mitochondrial permeability transition (see Ref. 14). Additionally, we
have observed that expression of superrepressor I
B
in cells alters basal levels of ATP (data not shown). Future studies will address mechanisms whereby mitochondrial NF-
B may control cell death mechanisms.
B is not the first nuclear encoded transcription factor to be
localized to the mitochondria. Interestingly, the glucocorticoid receptor has been found in the mitochondria (25). Potentially important
are observations that glucocorticoid receptor can physically interact
with NF-
B subunits (26); thus, NF-
B/glucocorticoid receptor
interactions in the mitochondria may function to regulate key processes
involved in cell growth and apoptsosis. Experiments addressing these
issues may reveal novel regulatory mechanisms associated with
mitochondrial function.
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ACKNOWLEDGEMENT |
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We gratefully acknowledge Vicky Madden for expert technical assistance with the electron microscopy.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants AI35098, CA75080, and CA73756 (to A. S. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
** To whom correspondence should be addressed: Lineberger Comprehensive Cancer Center, CB #7295, University of North Carolina, Chapel Hill, NC 27599. Tel.: 919-966-3652; Fax: 919-966-0444; E-mail: jhall@med.unc.edu.
Published, JBC Papers in Press, November 13, 2002, DOI 10.1074/jbc.M209995200
2 P. Cogswell and A. Baldwin, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are:
TNF, tumor necrosis
factor;
IKK, IB kinase;
CoxI, -II, and -III, cytochrome c
oxidase I, II, and III, respectively;
EMSA, electrophoretic mobility
shift assay.
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REFERENCES |
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---|
1. | Ghosh, S., May, M., and Kopp, E. (1998) Annu. Rev. Immunol. 16, 225-260[CrossRef][Medline] [Order article via Infotrieve] |
2. |
Zandi, E.,
and Karin, M.
(1999)
Mol. Cell. Biol.
19,
4547-4551 |
3. |
Maniatis, T.
(1999)
Genes Dev.
13,
505-510 |
4. |
Baldwin, A.
(2001)
J. Clin. Inv.
107,
3-6 |
5. |
Guttridge, D.,
Mayo, M.,
Madrid, L.,
Wang, C.-Y.,
and Baldwin, A.
(2000)
Science
289,
2363-2366 |
6. |
Baldwin, A.
(2001)
J. Clin. Inv.
107,
241-246 |
7. |
Carlotti, F.,
Dower, S.,
and Qwarnstrom, E.
(2000)
J. Biol. Chem.
275,
41028-41034 |
8. |
Huang, T.,
Kudo, N.,
Yoshida, M.,
and Miyamoto, S.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
1014-1019 |
9. |
Tam, W.,
Lee, L.,
Davis, L.,
and Sen, R.
(2000)
Mol. Cell. Biol.
20,
2269-2284 |
10. |
Yan, Y.,
Weinman, S.,
Boldogh, I.,
Walker, R.,
and Brasier, A.
(1999)
J. Biol. Chem.
274,
787-794 |
11. | Anderson, S., Bankier, A., Barrell, B., de Bruijn, M., Coulson, A., Drouin, J., Eperon, I., Nierlich, D., Roe, B., Sanger, F., Schreier, P., Smith, A., Staden, R., and Young, I. (1981) Nature 290, 457-465[Medline] [Order article via Infotrieve] |
12. | Scheffler, I. (1999) Mitochondria , pp. 367-384, Wiley-Liss, Inc., New York |
13. |
Puranami, R.,
and Attardi, G.
(2001)
Mol. Cell. Biol.
21,
548-561 |
14. |
Bottero, V.,
Rossi, F.,
Samson, M.,
Mari, M.,
Hofman, P.,
and Peyron, J.-F.
(2001)
J. Biol. Chem.
276,
21317-21324 |
15. |
Wang, C.-Y.,
Mayo, M.,
and Baldwin, A.
(1996)
Science
274,
784-787 |
16. | Madden, V. J. (1998) Microsc. Microanal. 4 Suppl. 2, 854-855 |
17. |
Yang, J.,
Liu, X.,
Bhalla, K.,
Kim, C.,
Ibrado, A.,
Cai, J.,
Peng, T.-I.,
Jones, D.,
and Wang, X.
(1997)
Science
275,
1129-1132 |
18. |
Mancini, M.,
Nicholson, D.,
Roy, S.,
Thornberry, N.,
Peterson, E.,
Casciola-Rosen, L.,
and Rosen, A.
(1998)
J. Cell Biol.
140,
1485-1495 |
19. |
Gervais, F.,
Thornberry, N.,
Ruffolo, S.,
Nicholson, D.,
and Roy, S.
(1998)
J. Biol. Chem.
273,
17102-17108 |
20. |
Mayo, M.,
Wang, C.-Y.,
Cogswell, P.,
Rogers-Graham, K.,
Lowe, S.,
Der, C.,
and Baldwin, A.
(1997)
Science
278,
1812-1815 |
21. |
Keifer, J. A.,
Guttridge, D.,
Ashburner, B.,
and Baldwin, A.
(2001)
J. Biol. Chem.
276,
22382-22387 |
22. |
Posakony, J. W.,
England, J. M.,
and Attardi, G.
(1977)
J. Cell Biol.
74,
468-491 |
23. |
Kingsbury, D. J.,
Griffin, T.,
and Colbert, R. A.
(2000)
J. Biol. Chem.
275,
24156-24162 |
24. |
Devin, A.,
Lin, Y.,
Yamaoka, S., Li, Z.,
Karin, M.,
and Liu, Z.
(2000)
Mol. Cell. Biol.
21,
3986-3994 |
25. | Scheller, K., Sekeris, C., Krohne, G., Hock, R., Hansen, I., and Scherer, U. (2000) Eur. J. Cell Biol. 79, 299-307[Medline] [Order article via Infotrieve] |
26. |
Tao, Y.,
Williams-Skipp, C.,
and Scheinman, R.
(2001)
J. Biol. Chem.
276,
2329-2332 |