Interleukin-1-induced Nuclear Factor-
B-I
B
Autoregulatory
Feedback Loop in Hepatocytes
A ROLE FOR PROTEIN KINASE C
IN POST-TRANSCRIPTIONAL
REGULATION OF I
B
RESYNTHESIS*
Youqi
Han
,
Tao
Meng
,
Nicole R.
Murray§,
Alan P.
Fields§, and
Allan R.
Brasier
¶
From the
Department of Internal Medicine,
§ Sealy Center for Oncology and Hematology, and the
¶ Sealy Center for Molecular Sciences, University of Texas Medical
Branch, Galveston, Texas 77555-1060
 |
ABSTRACT |
The I
B inhibitors regulate the activity of the
potent transcription factor nuclear factor-
B (NF-
B). Following
signal-induced I
B proteolysis, NF-
B translocates into the nucleus
to activate transcription of target genes, including I
B
itself,
initiating the "NF-
B-I
B
autoregulatory feedback loop."
Upon I
B
resynthesis, NF-
B is subsequently inactivated and
redistributed back into the cytoplasm. We have previously reported a
robust NF-
B-I
B
autoregulatory feedback loop in HepG2
hepatocytes. Sixty minutes after tumor necrosis factor (TNF-
)
stimulation, I
B
is resynthesized to ~2-fold greater level than
in control cells and completely inhibits NF-
B binding. Here we
investigate the mechanism for I
B
resynthesis comparing the effect
of stimulation of TNF-
with that of interleukin-1 (IL-1
).
Although either TNF-
or IL-1
stimulation of protein kinase C
(PKC)-down-regulated cells equivalently induces NF-
B translocation,
the kinetics of I
B
resynthesis is slowed. Moreover, pretreatment
with selective calcium-dependent PKC inhibitors selectively
slowed the kinetics of the IL-1
-induced overshoot without affecting
that produced by TNF-
. Down-regulation of PKC
by antisense
phosphorothioate oligonucleotides and expression vectors selectively
blocked the IL-1
-induced I
B
overshoot. In the absence of
PKC
, although IL-1
induced similar amounts of I
B
transcription and changes in steady-state mRNA, a greater component
of I
B
mRNA was retained in the nucleus. These data indicate a
selective role for PKC
in IL-1
-induced I
B
resynthesis, which is mediated, at least in part, by post-transcriptional control of
mRNA export.
 |
INTRODUCTION |
Multicellular organisms have evolved hormone-inducible signal
transduction pathways for expression of homeostatic genes under conditions of stress. One prominent example, which is highly conserved in vertebrates, is the hepatic acute-phase response
(APR),1 where infectious
processes activate mononuclear cells to secrete the cytokines
interleukin-1
(IL-1
) or tumor necrosis factor-
(1, 2). After
these cytokines enter the circulation, they bind to high affinity
receptors on the hepatocyte plasma membrane, activating expression of
genes required for blood pressure regulation (angiotensinogen (3)) and
immune activation (interleukins-6 and -8 (1, 4)). A prominent mechanism
for inducible gene expression during the APR is at the level of
transcriptional initiation.
The transcription factor nuclear factor-
B (NF-
B) is an important
signal transduction mediator of the APR. NF-
B is a family of related
proteins that includes potent transactivator subunits (Rel A and c-Rel)
and DNA-binding subunits (NF-
B1). In unstimulated hepatocytes,
NF-
B is inactivated in the cytoplasm through reversible association
with the inhibitory proteins, I
B
, -
, and -
(5). Following
cytokine stimulation, I
B
and -
are inducibly phosphorylated by
a ubiquitous multisubunit I
B kinase and selectively degraded (Refs.
5 and 6 and references therein). The release of previously inactivated
cytoplasmic NF-
B allows it to enter the nucleus where it binds to
high affinity sites in the promoters of inducible genes and initiates
transcription (3, 7, 8). NF-
B target genes include acute-phase
responsive genes and, curiously, one of the NF-
B inhibitors,
I
B
. I
B
resynthesis is directly activated by NF-
B through
a mechanism that, at least in part, involves increased transcription
(9-11).
Following its nuclear translocation, NF-
B remains only transiently
in the nuclear compartment, redistributing back into the cytoplasm in
its inactivated (non-DNA binding) form (5, 7). In contrast to its
initial translocation, NF-
B inactivation requires protein synthesis
(5, 11), indicating termination of NF-
B is an active event. Studies
from our laboratory have shown that the timing of NF-
B inactivation
coincides temporally with I
B
resynthesis (5, 7). In unstimulated
cells cytoplasmic NF-
B is complexed to many I
B isoforms. In
contrast, immediately following TNF-
stimulation, NF-
B is
primarily associated only with I
B
(5, 7). Because I
B
can
dissociate DNA-bound NF-
B, and capture it from the nucleus, I
B
apparently plays a role as an initial terminator of NF-
B activity
(12). The role of I
B
in terminating nuclear NF-
B activity has
been underscored in studies on i
b
-deficient mice.
Following TNF-
stimulation, NF-
B binding is sustained
indefinitely in i
b
/
fibroblasts (13).
Together, these data implicate the NF-
B-I
B
autoregulatory
feedback loop as one mechanism for terminating NF-
B activity.
In our previous studies on the mechanism for TNF-
-induced NF-
B
activation in HepG2 hepatocytes, we observed the presence of a robust
NF-
B-I
B
autoregulatory feedback loop (5). Sixty minutes
following TNF-
administration, nuclear NF-
B binding activity is
completely but only transiently terminated due to I
B
resynthesis
and reassociation with Rel A. Termination of NF-
B binding is
dependent on new protein synthesis and occurs temporally when I
B
levels peak at 2-fold greater than that observed in control cells.
Moreover, termination occurs concomitantly with reassociation of
I
B
with Rel A, before detectable resynthesis of any other I
B
isoform has occurred (5). Because the resynthesized I
B
continues
to be proteolyzed, NF-
B reappears in the nucleus (at a lower level)
and persists until other I
B isoforms are resynthesized (14).
In this report, we focus on understanding the detailed mechanism for
the NF-
B-I
B
feedback loop because this inhibitory pathway
could potentially be modulated by anti-inflammatory therapeutics. We
observe that the exaggerated NF-
B-I
B
autoregulatory feedback loop also occurs with the cytokine IL-1
and PKC activator, phorbol 12-myristate 13-acetate (PMA). Surprisingly, we found that
down-regulation of DAG-sensitive PKC isoforms blocked the rapid
kinetics of IL-1
-induced I
B
overshoot resynthesis, implicating
a requirement for the phorbol ester-sensitive PKC isoforms PKC
,
-
II, or -
. We further demonstrate that IL-1
requires
PKC
to induce I
B
overshoot resynthesis as follows: 1) IL-1
induces translocation and degradation of PKC
; 2) the specific
inhibitor of calcium-activated PKC (cPKC) isoforms, Gö 6976, blocks IL-1
-induced but not TNF-
-induced overshoot; and finally,
3) selective inhibition of PKC
expression by antisense
oligonucleotide treatment blocks IL-1
-induced but not
TNF-
-induced overshoot. The role of PKC
is independent of I
B
transcription, as IL-1 induces equivalent I
B
mRNA
levels in the presence of PKC inhibitors. These observations
indicate a role for activated PKC
in modulating IL-1
-induced
NF-
B-I
B autoregulatory feedback loop and identifies PKC
as a target for manipulating the inflammatory response in hepatocytes.
 |
EXPERIMENTAL PROCEDURES |
Cell Culture and Treatment--
The human hepatoblastoma cell
line HepG2 was obtained from ATCC (Rockville, MD) and grown in
Dulbecco's modified Eagle's medium (Life Technologies, Inc.)
supplemented with 10% (v/v) heat-inactivated fetal bovine serum, 2 mM L-glutamine, 0.1 mM nonessential
amino acids, 1 mM sodium pyruvate, and antibiotics
(penicillin/streptomycin/fungizone) in a humidified atmosphere of 5%
CO2. Recombinant human TNF-
(30 ng/ml), IL-1
(4 ng/ml), or PMA (1 µM) were added in serum-free culture
medium, and cells were incubated for the indicated periods at 37 °C.
For pretreatments, 3 µM bisindolylmaleimide I (Gö
6850), 3 µM Gö 6976, or 0.2 µM
calphostin C were added in medium for 30 min, 30 min, or 6 h,
respectively, prior to stimulation. The calcium-activated PKC (cPKC)
and novel PKC (nPKC) isoforms in HepG2 cells were down-regulated by
treatment with 0.5 µM PMA for 20 h at 37 °C,
followed by a standard recovery period of 3 h in the normal growth
medium for TNF receptor expression (15, 16). All above chemicals were
obtained commercially (Calbiochem).
Plasmid Construction--
346/+ 18 base pairs of the human
I
B
promoter driving luciferase reporter was constructed.
I
B
-specific primers,
5'-TCAGGATCCGACGACCCCAATTCAAATCG-3' and
5'-CGGAAGCTTGTGGGCTCTGCAGCGCCG-3' were used in the
polymerase chain reaction with HepG2 genomic DNA as a template under
standard conditions (17). Following purification of the 364-base pair product, the fragment was restricted with unique BamHI and
HindIII sites (underlined) and ligated into the same sites
in the promoterless luciferase reporter pOLUC (18). The sequence was
confirmed by automated sequencing and purified using ion exchange
chromatography prior to transfection. Rel A expression plasmid was
constructed by ligating the BamHI/HindIII
fragment of the human Rel A cDNA into the expression plasmid
pEGFP-C1. Expression vectors for the full-length cDNAs encoding
human PKC
and -
II were cloned into the appropriate episomal
expression vectors in the antisense orientation (19). Antisense PKC
was generated by excision of the full-length PKC
cDNA from pGEM4
with KpnI (5') and HindIII (3') and subsequent directional cloning into the KpnI and HindIII
sites of pREP10. Antisense PKC
II was generated by excision of the
full-length PKC
II cDNA in pBlueBac with BamHI and
KpnI and cloned into the BamHI and
KpnI sites of pMEP4 in the antisense orientation.
Transfections--
HepG2 cells were transiently transfected
using Lipofectin (Life Technologies, Inc.) into triplicate 60-mm plates
with 3 µg of I
B
/Luc reporter, 1 µg of SV40 driven alkaline
phosphatase internal control, and 4 µg of either antisense PKC
plasmid or pREP4 vector according to manufacturer's recommended
protocol. After 48 h, cells were stimulated with ligand for 3 h and harvested for the measurement of luciferase and alkaline
phosphatase activities. Luciferase activity was determined by
subtracting machine background and normalizing each plate to alkaline
phosphatase activity. Fold induction was calculated by dividing
treatment values by control. For the transient expression of Rel A,
antisense PKC
or -
II in HepG2 cells, a mixture of 2.5 µg of DNA
and 20 µl of Lipofectin was added to each 60-mm plate. Cells were
harvested at 48 h for immunoblot analysis. Antisense PKC
II
stable transfection was performed using 25 µg of DNA and 30 µl of
LipofectAMINE for each 100-mm plate. After 42 h, cells were
cultured in 200 µg/ml hygromycin-containing growth medium for 2 months with medium change twice each week. The pMEP4 vector was taken
as control. For the antisense down-regulation of PKC
,
phosphorothioate-modified PKC
antisense oligodeoxynucleotide (ODN,
sequence 5'-GTTCTCGCTGGTGAGTTTCA -3') and scrambled version (5'-GGTTTTACCATCGGTTCTGG-3' obtained from Genemed Biotechnologies, South San Francisco, CA) were introduced into HepG2 cells as described (20). Where indicated, transiently transfected cells were isolated following cotransfection with 5 µg of plasmid CMV.IL2R encoding the
IL-2 receptor. Transient transfectants in 100-mm plates
(108 cells) were purified by adding anti-human CD25
(Caltag) and captured on magnetic beads conjugated to rabbit anti-mouse
IgG (Dynabeads, Dynal Inc.) as described (21, 22).
Preparation of Subcellular Extracts--
For cytosol and
particulate fractionation, HepG2 cells were incubated in hypotonic
buffer (20 mM HEPES, pH 7.5, 10 mM potassium acetate, 1.5 mM magnesium acetate) for 5 min on ice. Lysis
was completed in a Dounce homogenizer and verified by microscopic examination. Nuclei and unbroken cells were removed by low speed centrifugation, and supernatants were centrifuged at 100,000 × g for 1 h at 4 °C in a Beckman SW 55Ti rotor. The
resultant supernatants were taken as cytosolic extract, and pellets
were resuspended with the equal volume of 1% Nonidet P-40-containing
hypotonic buffer as particulate extract. All extracts were normalized
for protein amounts determined by Coomassie G-250 staining using bovine serum albumin as a standard (Bio-Rad).
Sucrose Density-purified Nuclear Extracts--
For the
purification of nuclei, HepG2 cells were resuspended in Buffer A (50 mM HEPES, pH 7.4, 10 mM KCl, 1 mM
EDTA, 1 mM EGTA, 1 mM dithiothreitol, 0.1 µg/ml phenylmethylsulfonyl fluoride, 1 µg/ml pepstatin A, 1 µg/ml
leupeptin, 10 µg/ml soybean trypsin inhibitor, 10 µg/ml aprotinin,
and 0.5% Nonidet P-40). After 10 min on ice, the lysates were
centrifuged at 4,000 × g for 4 min at 4 °C. After
discarding the supernatant, the nuclear pellet was resuspended in
Buffer B (Buffer A with 1.7 M sucrose) and centrifuged at
15,000 × g for 30 min at 4 °C (5). The purified nuclear pellet was then incubated in Buffer C (10% glycerol, 50 mM HEPES, pH 7.4, 400 mM KCl, 1 mM
EDTA, 1 mM EGTA, 1 mM dithiothreitol, 0.1 µg/ml phenylmethylsulfonyl fluoride, 1 µg/ml pepstatin A, 1 µg/ml
leupeptin, 10 µg/ml soybean trypsin inhibitor, and 10 µg/ml
aprotinin) with frequent vortexing for 30 min at 4 °C. After centrifugation at 15,000 × g for 5 min at 4 °C, the
supernatant is saved for nuclear extract. Both of cytoplasmic and
nuclear extracts were normalized for protein amounts determined by
Coomassie G-250 staining.
Electrophoretic Mobility Shift Assays (EMSAs)--
EMSAs were
performed as described previously with minor modifications (5). Nuclear
extracts (10 µg) were incubated with 40,000 cpm of
32P-labeled APRE WT duplex oligonucleotide probe and 2 µg
of poly(dA-dT) in a buffer containing 8% glycerol, 100 mM
NaCl, 5 mM MgCl2, 5 mM
dithiothreitol, and 0.1 µg/ml phenylmethylsulfonyl fluoride in a
final volume of 20 µl, for 15 min at room temperature. The complexes
were fractionated on 6% native polyacrylamide gels run in 1× TBE
Buffer (89 mM Tris, 89 mM boric acid, and 2.0 mM EDTA), dried, and exposed to Kodak X-AR film at
70 °C. Competition was performed by the addition of 100-fold molar
excess nonradioactive double-stranded oligonucleotide competitor at the
time of addition of radioactive probe. The sequences of the APRE
double-stranded oligonucleotides are shown in Oligonucleotide APRE WT,
M2, and M6.
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Western Immunoblots--
For immunoblot analysis, a constant
amount of indicated cellular extracts (200-300 µg as indicated) were
boiled in Laemmli Buffer, separated on 10% SDS-PAGE, and transferred
to polyvinylidene difluoride membranes (Millipore, Bedford, MA).
Membranes were blocked in 8% milk and immunoblotted with the affinity
purified rabbit polyclonal antibodies (Santa Cruz Biotechnology) for
either I
B
(reactive with amino acids 297-317), Rel A (reactive
with amino acids 3-19), NF-
B1 (reactive with amino acids 350-363), PKC
(reactive with amino acids 651-672), PKC
II (reactive with amino acids 657-673), PKC
(reactive with amino acids 554-673), PKC
(reactive with amino acids 723-737), or PKC
(reactive with amino acids 557-592). Anti-PKC
and -
antibodies were purchased from Upstate Biotechnology Inc., and all others were from Santa Cruz
Biotechnology. Immune complexes were detected by binding donkey
anti-rabbit IgG conjugated to horseradish peroxidase (Pierce) followed
by reaction in the enhanced chemiluminescence assay (ECL, Amersham
Pharmacia Biotech) according to the manufacturer's recommendations.
Northern Blots--
50 µg of total cellular RNA was extracted
from HepG2 cells using acid guanidinium/phenol extraction (RNazol B,
Tel-Test Inc., Friendswood, TX), fractionated by electrophoresis on a
1% agarose-formaldehyde gel, capillary-transferred to a nylon membrane
(Hybond, Amersham Pharmacia Biotech, United Kingdom), and prehybridized
for 4 h in hybridization buffer (0.5 M sodium
phosphate, pH 7.2, 7% SDS, 1 mM EDTA, 1% bovine serum
albumin) at 65 °C. The membrane was hybridized with 1 × 106 cpm 32P-I
B
cDNA probe per ml
overnight in the same buffer, washed three times (20 min each) with 1%
SDS-containing phosphate buffer at 65 °C, and exposed to X-AR film
for 24-48 h. For the 18 S RNA hybridization, the same membrane was
reprobed with 32P-labeled human 18 S cDNA and briefly
exposed. Bands in the autoradiogram were quantitated by exposure to
Molecular Dynamics PhosphorImager cassette.
 |
RESULTS |
Ligand Independence of the I
B
Overshoot Resynthesis--
A
time course of nuclear NF-
B binding activity following TNF-
(30 ng/ml) stimulation in HepG2 cells is displayed in Fig. 1A to demonstrate salient
features of the NF-
B-I
B
autoregulatory feedback loop. Three
major complexes that bind to the angiotensinogen APRE are seen by EMSA.
Of relevance here, the TNF-
-inducible C2 complex is rapidly induced
within 15 min of stimulation; we previously reported that C2 binds with
NF-
B binding specificity and is supershifted by the addition of Rel
A or NF-
B1 antibodies, indicating C2 is a Rel A·NF-
B1
heterodimer (5). Thirty minutes after TNF-
stimulation, C2 binding
begins to diminish, and by 60 min, it is completely abolished (at
subsequent times C2 binding reappears at a lower level as a result of
ongoing I
B
proteolysis before the resynthesis of I
B
). Also
in Fig. 1A (bottom), cytoplasmic I
B
abundance is determined by Western immunoblot under conditions where we
have shown that the I
B
signal is linear to input protein (23).
I
B
, present in unstimulated cell cytoplasm, completely disappears
15 min following TNF-
administration. At 30 min following TNF-
stimulation, I
B
begins to be resynthesized, and resynthesis peaks
after 60 min to levels ~2-fold greater than that observed in control
cells. In previous studies, at 60 min, all of the resynthesized I
B
is associated with Rel A as determined by nondenaturing
coimmunoprecipitation assay, and inhibition of its resynthesis blocks
the 60 min termination of C2 binding (5).

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Fig. 1.
A, time course of
TNF- -induced NF- B binding and I B overshoot resynthesis.
Top, autoradiogram of EMSA using 10 µg of nuclear protein
prepared from cultured HepG2 cells stimulated for the various times
(minutes) with 30 ng/ml TNF- binding to the radiolabeled
angiotensinogen NF- B-binding site (APRE WT, see "Experimental
Procedures"). The bound complexes are shown. C2, the Rel
A·NF- B1 complex (5), is rapidly induced with biphasic kinetics,
the first peak at 15 and 30 min, and by 60-min nadirs. Relative to
control values, C2 binding increases 5.1- (15 min), 4.9- (30 min), 0.6- (60 min), 4- (120 min), and 3-fold (360 min). Bottom,
Western immunoblot of 37-kDa cytoplasmic I B from the same samples
(I B is not detectable in the nuclear fraction (5)). I B is
rapidly degraded at 15 min and subsequently is resynthesized, peaking
at 60 min (corresponding to the nadir in NF-kB binding). Relative to
control values, I B is 0.008- (15 min), 0.7- (30 min), 2.2- (60 min), 1.6- (120 min), and 1-fold (360 min). B, time course
of IL-1 -induced NF- B binding and I B overshoot resynthesis.
Cells were stimulated with 4 ng/ml IL-1 for indicated times.
Top, autoradiogram of EMSA (as described in A).
Relative to control values, C2 binding increases 42- (15 min), 38- (30 min), 1.3- (60 min), 28- (120 min), and 20-fold (360 min).
Bottom, Western immunoblot of cytoplasmic I B from same
samples. I B is rapidly degraded at 15 min and resynthesized
thereafter, concomitantly with the appearance of a phosphorylated
isoform migrating ~3 kDa slower seen at the 30- and 60-min time
points. Relative to control values, I B is 0.6- (30 min), 1.7- (60 min), 0.9- (120 min), and 1.2-fold (360 min). C, time course
of PMA-induced NF- B binding and I B overshoot resynthesis.
Cells were stimulated with 1 µM PMA for indicated times.
Top, autoradiogram of EMSA (A). Relative to
control values, C2 binding increases 12- (15 min), 12- (30 min), 9- (120 min), and 9-fold (360 min). The 60-min point is undetectable.
Bottom, Western immunoblot of cytoplasmic I B from same
samples. Relative to control values, I B is 0.2- (15 min), 0.3- (30 min), 1.2- (60 min), 0.6- (120 min), and 0.8-fold (360 min).
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In comparison to that produced by TNF-
, we next determined whether
the phenomenon of I
B
overshoot resynthesis occurs following treatment with IL-1
(4 ng/ml, Fig. 1B). IL-1
is a more
potent inducer of C2 complex; however, the 60-min nadir in the
Rel A·NF-
B1 binding is produced concomitantly with I
B
overshoot resynthesis. The same phenomenon occurs with the PKC
activator, PMA (1.0 µM, Fig. 1C), where a
nadir in Rel A·NF-
B1 binding is also seen concomitantly with the
I
B
overshoot resynthesis at 60 min. Together, these data indicate
that the overshoot in I
B
resynthesis is ligand-independent.
The mechanism underlying the overshoot in I
B
resynthesis was
investigated by measurement of relative changes in steady-state I
B
mRNA abundance by Northern blot analysis (Fig.
2). In control cells, I
B
mRNA
is detectable as a 1.8-kilobase pair transcript. For each treatment
condition, TNF-
(Fig. 2A) and IL-1
(Fig. 2B), or PMA (Fig. 2C), I
B
mRNA was
detectable in control cells and rapidly depleted at 15 min, perhaps
indicating the presence of translation-coupled degradation (24).
However, I
B
mRNA was robustly increased at 60 min, a time
point when the peak in I
B
resynthesis occurs, and gradually
decays over the next 5 h. These data indicate that the I
B
overshoot resynthesis is dependent on the enhanced expression of
I
B
mRNA.

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Fig. 2.
Mechanism for I B overshoot
resynthesis. A, Northern blot analysis of 50 µg
of total cellular RNA isolated from HepG2 cells stimulated for the
indicated times with 30 ng/ml TNF- . Top, autoradiogram
following hybridization for I B . The 1.8-kilobase pair mRNA is
shown. Bottom, autoradiogram following rehybridization of
the same membrane for 18 S ribosomal RNA (18 S). For each time point,
the I B /18 S hybridization ratio was determined and expressed
relative to control values. I B /18 S increases 1.2- (15 min),
2.9- (30 min), 3-(60 min), 1.5- (120 min), and 2.3-fold (360 min).
B, Northern blot analysis following stimulation with 4 ng/ml
IL-1 . Experiment shown is as described in A. Relative to
control values, I B /18 S increases 0.2- (15 min), 0.9- (30 min),
3.4- (60 min), 1.8- (120 min), and 0.8-fold (360 min). C,
Northern blot analysis following stimulation with 1 µM
PMA. Experiment shown is as described in A. Relative to
control values, I B /18 S increases 0.4- (15 min), 0.4- (30 min),
1.7- (60 min), 0.8- (120 min), and 0.9-fold (360 min). D,
Rel A activates I B expression. HepG2 cells were transfected with
IL-2 receptor expression plasmid in the absence ( ) or presence of Rel A expression plasmid (Rel A). Transient
transfectants were subsequently enriched using anti-IL-2 receptor
antibody captured on magnetic beads (see "Experimental
Procedures"). Shown is a Western immunoblot for I B and as
control NF- B1. Relative to empty expression plasmid, Rel A activates
I B protein abundance 8.5-fold.
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Other studies have shown that cytokines are potent activators of
I
B
expression through a mechanism dependent on NF-
B
translocation (9, 11, 25). To confirm this effect in HepG2 cells, the effect of transactivator subunit (Rel A) expression on I
B
abundance was determined in transient overexpression assays. We
observed that Rel A expression strongly induced I
B
protein
abundance (Fig. 2D), indicating that NF-
B is sufficient
for activation of the autoregulatory feedback loop.
Phorbol Ester-sensitive PKC Isoforms Are Involved in the Kinetics
of I
B
Overshoot Resynthesis--
Because both TNF-
and
IL-1
cytokines have been shown to activate PKC activity (16), we
explored the PKC dependence for I
B
overshoot resynthesis. For
this, DAG-sensitive PKC isoforms were down-regulated by chronic
exposure to PMA prior to cytokine stimulation (Fig.
3). Following 15 min stimulation with
either TNF-
or IL-1
, Rel A·NF-
B1 binding was strongly
induced and I
B
proteolyzed, indicating signals necessary for
initial Rel A·NF-
B1 translocation are PKC-independent (16).
Surprisingly, however, we observed the 60-min nadir in Rel A·NF-
B1
was eliminated by PKC down-regulation for either cytokine. Moreover, in
PKC down-regulated cells, although I
B
resynthesized, its
reaccumulation was gradual, without exceeding control levels at the
60-min time point. As expected, acute exposure of PKC down-regulated
cells to PMA produced no induction of Rel A·NF-
B1 binding activity
or I
B
proteolysis (Fig. 3C). These data indicate a
requirement for PMA-sensitive PKC isoforms for the rapid kinetics of
TNF-
and IL-1
-induced I
B
resynthesis.

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Fig. 3.
Phorbol ester-sensitive PKC isoforms
are required for cytokine-induced I B overshoot resynthesis.
DAG-sensitive PKC isoforms were down-regulated by chronic agonist
exposure prior to stimulation. A, time course of TNF-
stimulation. Top, autoradiogram of EMSA following 30 ng/ml
TNF- (described in Fig. 1A). C2, the Rel A·NF- B1
complex (5), is rapidly induced and declines with a monophasic profile,
peaking at 15 and 30 min and declining thereafter without a detectable
nadir. Relative to control values, C2 binding increases 7.2- (15 min),
6.6- (30 min), 4.4- (60 min), 3.5- (120 min), and 3-fold (360 min). The
nadir in C2 binding, previously seen at 60 min, is absent.
Bottom, Western immunoblot of cytoplasmic I B from the
same samples. The overshoot resynthesis of I B , previously seen at
60 min, is absent. Relative to controls, I B abundance decreases
0.01- (15 min), 0.8- (30 min), 1.3- (60 min), 1.4- (120 min), and
1.5-fold (360 min). B, time course of IL-1 stimulation.
Top, autoradiogram of EMSA following 4 ng/ml IL-1
(described in Fig. 1A). Rel A-NF- B1 binding (C2)
similarly follows a monophasic profile, peaking at 15 min and declining
thereafter without a detectable nadir. Relative to control values, C2
binding increases 6- (15 min), 5.3- (30 min), 4- (60 min), 1.5- (120 min), and 1.7-fold (360 min). The 60-min nadir in C2 binding is
similarly absent. Bottom, Western immunoblot of cytoplasmic
I B from the same samples. The overshoot resynthesis of I B
is absent. Relative to controls, I B abundance decreases 0.01- (15 min), 0.07- (30 min), 0.8- (60 min), 1- (120 min), and 1.3-fold (360 min). C, time course of PMA stimulation. Top,
autoradiogram of EMSA following 1 µM PMA (as described in
Fig. 1A). Bottom, Western immunoblot of
cytoplasmic I B from the same samples. No significant induction of
C2 binding or change in I B abundance was detected, indicating
that DAG-activated PKC isoforms were completely hydrolyzed and required
for NF- B activation.
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Effect of TNF-
and IL-1
on Particulate-Cytosol Redistribution
of PKC Isoforms--
Following PKC activation, cytosol-particulate
(membrane) redistribution and degradation occurs. To tentatively
identify PKC isoforms involved in I
B
resynthesis, the effect of
cytokines on subcellular distribution of various PKC isoforms was
determined. At various times following cytokine stimulation, HepG2
cells were fractionated into cytosolic and particulate fractions by
ultracentrifugation (see "Experimental Procedures"), and PKC
isoform abundance was determined by Western immunoblot (Fig.
4A). Unstimulated HepG2 cells
express cPKC isoforms, PKC
and PKC
II, the nPKC isoforms, PKC
and PKC
, and the atypical PKC (aPKC) isoform PKC
(Fig. 4A) (26). All of these isoforms were distributed in both the cytosolic and particulate fractions, except PKC
II (which was primarily distributed in the particulate fraction).

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Fig. 4.
PKC isoforms activated by cytokines and
PMA. A, Western immunoblot for PKC isoforms
distribution in subcellular fractions. HepG2 cells were stimulated with
TNF- or IL-1 and fractionated into cytosolic (Cytosol)
or particulate (Particulate) fractions at various times
(indicated at top). Samples (200 µg) were fractionated,
and changes in PKC isoform abundance were detected by immunoblot using
indicated primary antibody (at left). In unstimulated cells,
78-kDa PKC II ( ) is primarily detected in the particulate
fraction. Distributed in both cytosol and particulate fractions are
80-kDa PKC ( ), 70-kDa PKC ( ), 72-kDa PKC ( ), and
76-kDa PKC ( ). IL-1 induced PKC degradation in the cytosol
fraction (relative to control, PKC declined to 0.6 (5 min), 0.3 (15 min), and 0.3 (45 min)). Both TNF- and IL-1 induced a rapid
PKC translocation to the particulate fraction at 5 min of 6- and
13-fold (respectively). IL-1 also induced a later degradation of
PKC in the particulate fraction where PKC abundance declined to
9.5-fold (15 min) and 5.4-fold (45 min). Because IL-1 induces
initial PKC cytosol-particulate redistribution and degradation in
both compartments, these data indicate IL-1 activates PKC in
HepG2 cells. B, effect of agonist exposure on PKC isoform
abundance. Western immunoblot of cytosol and particulate fractions (200 µg) from control and PMA down-regulated HepG2 cells. PMA
down-regulates cytosolic and particulate PKC , particulate PKC II,
and cytosolic PKC . No significant effects were seen on the PKC or
PKC .
|
|
Cytokine-induced changes were observed for PKC isoforms
,
, and
. Although TNF-
had no consistent effect on cytosolic PKC
,
IL-1
induced rapid cytosolic PKC
degradation, initially detectable after 15 min stimulation. Treatment with either TNF-
or
IL-1
induced translocation of PKC
to the particulate fraction within 5 min after stimulation. In contrast to TNF-
, IL-1
also induced particulate PKC
degradation after 15 min of stimulation. Taken together, these data indirectly indicate that IL-1
is a potent
activator of PKC
. No significant effect by either cytokine was seen
on particulate PKC
II. In contrast, following either TNF-
or
IL-1
stimulation, PKC
also was degraded primarily in the
cytosolic fraction and weakly accumulated in the particulate fraction.
In addition, following IL-1
treatment, PKC
was degraded only in
the particulate fraction.
A similar analysis was performed to determine the effect of PKC
isoforms hydrolyzed by chronic PMA exposure (Fig. 4B).
Chronic exposure to PMA significantly hydrolyzed PKC
(both in
cytosolic and particulate fractions), PKC
II, and PKC
(in the
cytosolic fraction). Together, these data indicate PKC
, -
II, and
-
as candidate isoforms mediating the cytokine-induced I
B
overshoot resynthesis.
cPKC Inhibitors Block IL-1
-induced I
B
Overshoot--
As
additional evidence for the role of PKC isoforms, the potent and
selective PKC inhibitors bisindolylmaleimide I (Gö 6850) and
Gö 6976 were tested for their effects on I
B
overshoot
resynthesis at concentrations that inhibit cPKC isoforms (27). These
agents are highly selective inhibitors for PKC by competitive
inhibition of the ATP-binding site, without effect on tyrosine,
cAMP-dependent protein, myosin, or phosphorylase kinases
(27). Gö 6850 inhibits cPKC and, at higher concentrations, nPKC
isoforms (27). In these experiments, Gö 6850 was used in
concentrations that selectively inhibit the cPKC isoforms. Gö
6850 had no effect on TNF-
-induced I
B
overshoot but completely
blocked the effects of IL-1
(Fig. 5A). As a control for its
inhibitory effect, Gö 6850 blocked the ability of PMA to induce
C2 binding and I
B
proteolysis (Fig. 5A). Gö 6976 is a selective inhibitor of cPKC isoforms (28). Similarly, Gö
6976 interfered with the IL-1
-induced I
B
overshoot but not
that produced by TNF-
(Fig. 5B). Calphostin C, an
inhibitor of the aPKC isoforms by binding to the unique regulatory
domain (without effect on cPKC isoforms (29)) was next tested to
determine whether aPKC plays a role in the autoregulatory feedback
loop. Calphostin C had no effect on either NF-
B activation or
I
B
overshoot resynthesis. Together, these data indicate that cPKC isoforms (PKC
or
II) influence the kinetics of IL-1
-induced I
B
resynthesis (and transient NF-
B termination) in
hepatocytes.

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Fig. 5.
Effect of selective PKC
inhibitors on cytokine-induced NF- B binding and I B overshoot
resynthesis. Top, autoradiogram of EMSA.
Bottom, Western immunoblot of I B from same samples.
A, effect of cPKC and nPKC inhibitor, bisindolylmaleimide I
(Gö 6850). Cells were preincubated with 3 µM
Gö 6850 prior to stimulation for various times with indicated
ligands (30 ng/ml TNF- , 4 ng/ml IL-1 , and 1 µM
PMA). C2 binding (relative to control) following TNF- stimulation is
increased 2.4- (15 min) and 1.2-fold (60 min); following IL-1
stimulation is increased 2.6- (15 min) and 2.0-fold (60 min); following
PMA stimulation is increased 0.6- (15 min) and 0.6-fold (60 min).
I B abundance (at 60 min relative to control) following TNF-
stimulation is increased 1.6-fold, following IL-1 stimulation is
1-fold, and following PMA is increased 0.8-fold. Gö 6850 blocks
IL-1 -induced I B overshoot resynthesis but not that induced by
TNF- . B, effect of cPKC inhibitor, Gö 6976. Cells
were preincubated with 3 µM Gö 6976 prior to
stimulation for various times with indicated ligands (30 ng/ml TNF- ,
4 ng/ml IL-1 , and 1 µM PMA). C2 binding
(relative to control) following TNF- stimulation is 4.6- (15 min)
and 1.5-fold (60 min), following IL-1 stimulation is 5.2- (15 min)
and 5-fold (60 min), and following PMA stimulation is 3.2- (15 min) and
3.4-fold (60 min). I B abundance (at 60 min relative to control)
following TNF- stimulation is 1.1-fold, following IL-1
stimulation is 0.3-fold, and following PMA is 0.7-fold. Gö 6976 partially blocks PMA-induced NF- B activation and I B
proteolysis. In addition, Gö 6976 blocks IL-1 -induced I B
overshoot resynthesis but not that induced by TNF- . C,
effect of aPKC inhibitor, calphostin C. Cells were preincubated with
0.2 µM calphostin C prior to stimulation for various
times with indicated ligands (30 ng/ml TNF- , 4 ng/ml IL-1 , and 1 µM PMA). Calphostin C has no effect on NF- B
activation, I B proteolysis, or I B overshoot resynthesis by
any ligand, making a contribution of atypical PKC isoforms
unlikely.
|
|
Effect of PKC
Down-regulation on IL-1
-induced I
B
Overshoot Resynthesis--
To isolate selectively the contribution of
PKC
in IL-1
signaling, we initially sought to make stable HepG2
cells with a down-regulated PKC
isoform by constitutive expression
of a PKC
antisense expression plasmid. However, we were unable to
isolate PKC
-deficient cells in numerous attempts, even though we
were successful in down-regulating the PKC
II isoform, perhaps
indicating an important role for PKC
in permitting cell
proliferation. As an alternative approach, we transiently
down-regulated PKC
by introducing phosphorothioate-modified
antisense oligodeoxynucleotides (ODN) using liposome-mediated
transfection (20). In preliminary experiments, a time course for
optimal PKC
down-regulation following introduction of the antisense
PKC
ODN was performed. Peak inhibition of PKC
was observed at
24 h (Fig. 6A). At this
time point, the antisense ODN inhibited expression of PKC
by 81%,
whereas the control scrambled ODN affected PKC
abundance by less
than 11%. Following PKC
down-regulation, the effect of TNF-
and
IL-1
on the I
B
overshoot resynthesis was determined (Fig.
6B). PKC
was required for IL-1
- induced but not the
TNF-
-induced I
B
overshoot. To exclude an additional role of
PKC
II, HepG2 cells deficient in PKC
II were generated (Fig.
6C). Down-regulation of PKC
II had no effect on either the
TNF-
- or IL-1
-induced overshoot (Fig. 6D). Together,
these data strongly implicate a requirement for PKC
in mediating
I
B
overshoot resynthesis selectively for IL-1
.

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Fig. 6.
Effects of down-regulating PKC
isoforms on cytokine-induced I B overshoot resynthesis.
A, changes in steady-state PKC abundance following
antisense phosphorothioate ODN treatment. HepG2 cells were treated with
antisense or scrambled phosphorothioate ODN (see "Experimental
Procedures"). Whole cell lysates were prepared and analyzed by
Western immunoblot for PKC . PKC abundance was 0.2-fold
(antisense) and 0.9-fold (scrambled) that seen in control HepG2 cells.
B, effect of PKC down-regulation on cytokine-induced
I B overshoot resynthesis. HepG2 cells were treated with antisense
or scrambled ODN (as in A) and stimulated with indicated
cytokine for various times (top). Western immunoblot is
shown. Top, I B antibody. Bottom, internal
control Rel B. For antisense-treated cells stimulated with TNF- ,
I B abundance increased 3-fold at 60 min, whereas following
IL-1 treatment, I B resynthesis was blocked (0.3-fold at 60 min). In the scrambled ODN-treated cells, I B increased to 4.7- and 5.9-fold at 60 min for both TNF- and IL-1 , respectively.
These data indicate PKC is required for IL-1 -induced I B
overshoot resynthesis. C, down-regulation of PKC II. HepG2
cells were transfected with PKC II antisense expression plasmid
(AS PKC II) or empty expression plasmid (pMEP4)
and stably selected with hygromycin. Western immunoblot of total
cytoplasmic lysate using primary anti-PKC II antibody
(top) or internal control Rel B (bottom).
D, effect of PKC II down-regulation on cytokine-induced
I B overshoot resynthesis. Stably transfected HepG2 cells (shown
in C) were stimulated with TNF- (top) or
IL-1 (bottom) for various times (minutes, indicated at
top) prior to extraction of cytoplasmic protein. Shown is a
Western immunoblot for I B . Down-regulation of PKC II does not
interfere with TNF- - or IL-1 -induced I B overshoot
resynthesis.
|
|
TNF-
and IL-1
Activate I
B
Transcription in a
PKC
-independent Manner--
The effect of PKC
in blocking the
I
B
overshoot could be the result of inhibition of I
B
gene
expression. As shown earlier (Fig. 2D), I
B
expression
is dependent on Rel A translocation. To exclude the trivial possibility
that IL-1
-induced Rel A translocation is dependent on PKC
, a
Western immunoblot for changes in the Rel A isoform was determined
under conditions where the signal is proportional to input protein (5).
Following addition of IL-1
in the absence or presence of PKC
inhibitors (Gö 6976 or Gö 6850) or following PKC
down-regulation, equivalent amounts of Rel A translocated into the
nucleus (data not shown). To demonstrate additionally that TNF-
or
IL-1
affect I
B
promoter expression independent of PKC
activity, transient transfections of the I
B
promoter linked to
luciferase reporter gene were performed in the presence of PKC
antisense expression plasmid. TNF-
induced a 3.8-fold and IL-1
induced a 7.5-fold increase in I
B
promoter activity (Fig.
7A). The level of stimulation
was not affected by transient PKC
down-regulation. Demonstration
that PKC
was down-regulated is presented in Fig. 7B.

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Fig. 7.
IL-1 activates I B transcription
independently of PKC . A, PKC -independent
activation of the I B promoter. HepG2 cells were transfected with
I B promoter/luciferase reporter in the absence (pREP4)
or presence of antisense coding sequences for PKC (AS
PKC ) and stimulated with nothing (con), 30 ng/ml
TNF- , 4 ng/ml IL-1 , or 1 µM PMA as indicated. Cells
were harvested after 6 h, and reporter activity was normalized to
control values. All three agonists are inducers of I B
transcription independently of PKC . B, transient
down-regulation of PKC by antisense expression vector. HepG2 cells
were transiently transfected with IL-2 receptor expression plasmid and
empty expression vector (pREP4), antisense PKC (AS
PKC ), or antisense PKC II (AS PKC II). Transient
transfectants were isolated by affinity purification using anti-IL-2
receptor antibody (see "Experimental Procedures"). Shown is a
Western immunoblot for steady-state PKC (top) or internal
control Rel B (bottom). C, cytokines induce
equivalent changes in I B mRNA in the presence of PKC
inhibitors. HepG2 cells were stimulated with TNF- or IL-1
pretreated in the absence ( ) or presence of chronic agonist
stimulation (PMA-20 h) or the PKC inhibitors Gö 6850 or Gö 6976 as indicated at top. Shown is autoradiogram of Northern blot hybridization of I B mRNA
(top) or 18 S ribosomal RNA (bottom). Relative
to control values, TNF- increased I B mRNA levels by 2.4- (no inhibitor), 2.1- (PMA-20 h), 2.2- (Gö
6850), and 3.4-fold (Gö 6976). IL-1 increased
I B mRNA levels by 5.9- (no inhibitor), 6.5- (PMA-20
h), 6.2- (Gö 6850), and 6.0-fold (Gö
6976). Stimulation in the presence of PKC inhibitor has no effect
on the magnitude of I B mRNA induction.
|
|
Similarly, Northern blot analysis was done to determine the effect of
PKC inhibition on cytokine-induced steady-state I
B
mRNA
expression. Following TNF-
administration, I
B
mRNA
increased by 2.5-fold (Fig. 7C). The induction by TNF-
was not altered by presence of PKC inhibitors (Gö 6976 or
Gö 6850) or following PKC down-regulation. Similarly, IL-1
induced a 6-fold increase in steady-state I
B
mRNA that was
not affected by PKC inhibitors or down-regulation. Together, these data
indicate IL-1
produced similar levels of Rel A translocation and
I
B
gene expression in the absence or presence of PKC
.
Post-transcriptional Role of PKC Is, in Part, through Facilitation
of I
B
mRNA Nuclear Export--
Because equivalent amounts of
steady-state I
B
mRNA were produced following IL-1
stimulation in the absence or presence of PKC
, we concluded that
PKC
apparently plays a role in post-transcriptional control of
I
B
resynthesis. One potential mechanism, previously described to
control I
B
resynthesis in monocytes, was at the level of mRNA
decay in the nucleus (24). To determine the relative distribution of
I
B
RNA, RNA from cytoplasmic and nuclear fractions from control
and IL-1
-stimulated HepG2 cells were analyzed for changes in
I
B
abundance by Northern blot (Fig.
8). Following IL-1
stimulation,
I
B
mRNA primarily increased in the cytoplasmic fraction with
15% of the total signal detected in the nuclear compartment. However,
IL-1
stimulation in cells following PKC down-regulation resulted in
detectable accumulation of nuclear I
B
RNA. In this case, 32% of
the total signal was nuclear. These data indicate a potential role for
DAG-sensitive PKC isoforms in controlling I
B
RNA export.

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Fig. 8.
Post-transcriptional mechanism for I B
overshoot resynthesis. Effect on I B mRNA
nuclear-cytoplasmic distribution. HepG2 cells were stimulated with
IL-1 (lanes 3-6) pretreated in the absence (lanes
3 and 4) or presence of chronic agonist stimulation
(lanes 5 and 6). Lanes 1 and
2 are control cells. Cells were fractionated into nuclear
(lanes 2, 4, and 6) and cytoplasmic (lanes
1, 3, and 5) fractions. Equivalent cell numbers were
analyzed for I B mRNA abundance by Northern blot
hybridization. Shown is autoradiogram following hybridization to
I B mRNA probe (top) or 18 S ribosomal RNA probe
(bottom). For each time point, nuclear 18 S RNA signal is
~50% that of the cytoplasmic signal. Following IL-1 treatment, a
strong 6-fold increase in cytoplasmic I B abundance is seen.
Following normalization to 18 S signal, nuclear I B RNA abundance
increases 7.2-fold in IL-1 treated cells and 17.7-fold in IL-1
treated/PKC down-regulated cells.
|
|
 |
DISCUSSION |
IL-1
is a proinflammatory cytokine that plays an important role
in initiating the cytokine cascade in sepsis and inflammation. This
peptide exerts its biological effects by binding a single transmembrane
spanning receptor on diverse cell types (30). Following activation of
the IL-1 receptor, cytoplasmic proteins associate with the cytoplasmic
tail, generating second messengers and activating intracellular kinases
(31-33). An important intracellular target for NF-
B-activating
agents is the I
B signalsome, a multisubunit complex of proteins
responsible for the phosphorylation of I
B. IL-1
- and
TNF-
-induced signals converge on the NF-
B-inducing kinase, a
protein complexed with the I
B kinase IKK
(Refs. 6 and 31 and
references therein). Following I
B phosphorylation the inhibitor is
targeted for proteolytic degradation. In hepatocytes, we have shown
that IL-1
and TNF-
activate the NF-
B transcription factor in a
process involving its nuclear translocation coincidentally with I
B
proteolysis (5, 34, 35). Once translocated into the nucleus,
NF-
B-responsive (APR and cytokine) genes are inducibly transcribed.
Following NF-
B translocation, the i
b
gene is also
expressed, resulting in its rapid resynthesis. Resynthesized I
B
captures nuclear NF-
B protein and redistributes it back into the
cytoplasm to restore cellular homeostasis, resulting in what is called
the NF-
B-I
B
autoregulatory pathway. Studies by others (9-11)
have shown that an important component of this autoregulatory pathway depends on transcriptional activation of the I
B
promoter. The marked induction of I
B
promoter activity following cytokine stimulation dependent on NF-
B-binding sites and enhanced I
B
expression following Rel A overexpression are in support of this transcriptional component (10, 11). However, post-transcriptional control mechanisms of I
B
expression are also important. Here we
report evidence for a multistep mechanism for NF-
B-I
B
autoregulatory pathway induced by IL-1
in hepatocytes. We
demonstrate the unanticipated findings that IL-1
-induced I
B
resynthesis also requires the participation of activated PKC
.
IL-1
stimulation following selective antagonists of PKC
results
in nuclear retention of a fraction of I
B
mRNA.
PKC are a family of multifunctional serine-threonine
lipid-dependent kinases grouped broadly into three families
based on structural and functional criteria. The
calcium-dependent or classic cPKC isoforms (
,
,
),
novel PKC isoforms (
,
,
,
), and atypical PKC isoforms
(
,
,
) fulfill distinct, nonredundant functions in a cell
involved in intracellular signaling and cell proliferation (19). We
have shown that HepG2 cells express the cPKC (
,
II), nPKC (
,
), and aPKC (
) isoforms, confirming observations of others (26).
Our data extend these observations to show that several of these PKC
isoforms are responsive to cytokine stimulation. PKC
is apparently
activated by IL-1
because IL-1
induces cytosol-to-particulate
translocation of PKC
and, later, its proteolysis. PKC
is also
apparently activated by either TNF-
or IL-1
, as the protein is
rapidly degraded in the cytoplasm.
Here we report that selective down-regulation of PKC
affects
intracellular signaling produced by IL-1
but not TNF-
. However, the I
B overshoot produced by TNF-
is influenced by PKC
down-regulation, probably indicating that a different PMA-sensitive PKC
isoform, perhaps PKC
, subserves the role that PKC
plays in IL-1
signaling. The role of PKC
in TNF-
signaling will require
additional investigation. In a variety of cell types, IL-1
induces
generation of a variety of lipid-derived second messenger including
ceramide, DAG, and arachidonic acid (33). In IL-1
-stimulated
T-lymphocytes, DAG is rapidly produced as a consequence of
phosphatidylcholine hydrolysis, peaking 1 min after addition of ligand
(33). Of the known second messengers that activate PKC
, DAG is
likely to be an important mediator because IL-1
does not induce
measurable changes in bulk intracellular calcium concentrations (33).
The second messenger(s) responsible for IL-1
activation of PKC
in
hepatocytes will require further study.
In other studies, nuclear processing of I
B
mRNA was proposed
to be important in regulating I
B
protein levels following monocyte adherence (24). In these cells, I
B
mRNA is
predominantly nuclear. This contrasts with our observations in
hepatocytes where in both unstimulated and cytokine-stimulated
hepatocytes, I
B
mRNA is predominantly cytoplasmic. However,
in PKC down-regulated cells, retention of newly synthesized nuclear
I
B
mRNA is observed. By its indistinguishable size with
cytoplasmic mRNA, the nuclear I
B
RNA is apparently spliced
and polyadenylated, but this will require direct demonstration.
Together these observations indicate that I
B
nuclear processing
may be a general mechanism for control of its cytoplasmic abundance.
The role for PKC
in IL-1
-induced I
B
resynthesis is
apparently at a post-transcriptional level, mediated in part through
facilitating rapid kinetics of mRNA export. Whether other potential
mechanisms play a role in I
B
resynthesis, such as efficiency of
translational initiation, have not been directly tested in these
studies, and their additional contribution(s) cannot be excluded. In
eukaryotic cells, nuclear export of mRNA is dependent on the
association with RNA-binding proteins. Apparently there are
requirements for helicases (to disassociate the nascent RNA from
spliceosome complex) and "zipcode" RNA-binding proteins (to form
suitable ribonucleoprotein complexes) for mRNA to exit the nuclear
pore (36). Presumably some component of this pathway is influenced by
PKC
activation. Both functional (16) and immunoblot data (presented
here, see Fig. 5) indicate that, following cytokine stimulation, PKC
translocates to the particulate (membrane) fraction. In this
compartment, membrane PKC activity may regulate the proteins or motors
controlling nuclear mRNA export.
Although our study focuses on I
B
expression as a model for
understanding intracellular mechanisms in IL-1
signaling, other pathways are required for complete termination of NF-
B activity. Our
data indicate that activation of the I
B
autoregulatory feedback loop only transiently terminates NF-
B signaling. Following its overshoot resynthesis, I
B
undergoes a second wave of proteolysis allowing liberation of NF-
B and its subsequent re-entry into the
nucleus after 2 h, probably indicating that the inducible proteolytic pathway is still active at this time. However, we note that
NF-
B binding activity observed following I
B
resynthesis is
significantly reduced from that observed immediately after I
B
proteolysis (cf. Fig. 1, A-C) indicating that
resynthesized I
B
sequesters and removes a significant component
of NF-
B from the nuclear compartment. This is in apparent
contradiction to the absolute requirement of I
B
in termination of
NF-
B activity in i
b
/
fibroblasts
(13) and may be a consequence of differences in cell type as the
spectrum and regulation of the I
B inhibitors is cell
type-dependent (14, 23). Additional studies will be required to determine the later mechanism for NF-
B termination in hepatocytes.
NF-
B is a signaling molecule important in mediating cytokine and
acute phase reactant gene expression in a variety of inflammatory pathophysiological conditions. Understanding the mechanisms involved in
termination of NF-
B activity may identify novel target for anti-inflammatory therapeutics. Here we describe a
ligand-dependent role for PKC
in controlling subcellular
distribution of I
B
mRNA during the NF-
B-I
B
autoregulatory feedback loop. We hypothesize that novel
anti-inflammatory therapeutics could be designed that modulate the
I
B
mRNA nuclear export pathway.
 |
ACKNOWLEDGEMENTS |
Plasmid CMV.IL2R was a generous gift of B. Howard and R. Padmanabhan, NICHD, National Institutes of Health.
 |
FOOTNOTES |
*
This work was supported by Grant 1R01 55630-01A2 from NHLBI,
National Institutes of Health (to A. R. B.), Grant 4017 from the
Council for Tobacco Research (to A. R. B), and Grant P30 ES06676 from
NIEHS, National Institutes of Health (to R. S. Lloyd, University of
Texas Medical Branch).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.
Established Investigator of the American Heart Association. To
whom correspondence should be addressed: University of Texas Medical
Branch, 301 University Blvd., Galveston, TX 77555-1060. Tel.:
409-772-2824; Fax: 409-772-8709; E-mail: arbrasie{at}utmb.edu.
The abbreviations used are:
APR, acute-phase
response; aPKC, atypical PKC isoform; APRE, acute-phase response
element; cPKC, calcium-regulated PKC isoform; DAG, diacylglycerol; EMSA, electrophoretic mobility shift assay; I
B, inhibitor of
NF-
B; IL-1
, interleukin-1
; NF-
B1, nuclear factor-
B
50-kDa subunit; nPKC, novel PKC isoform; PKC, protein kinase C; Rel A, NF-
B 65-kDa subunit; TNF-
, tumor necrosis factor
; PMA, phorbol 12-myristate 13-acetate; WT, wild type; ODN, oligodeoxynucleotides.
 |
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