(Received for publication, December 5, 1996, and in revised form, January 31, 1997)
From the Department of Internal Medicine and Sealy Center for Molecular Science, University of Texas Medical Branch, Galveston, Texas 77555-1060
The proinflammatory cytokine, tumor necrosis
factor (TNF
), is a potent activator of angiotensinogen gene
transcription in hepatocytes by activation of latent nuclear
factor-
B (NF-
B) DNA binding activity. In this study, we examine
the kinetics of TNF
-activated translocation of the 65-kDa (Rel A)
and 50-kDa (NF-
B1) NF-
B subunits mediated by inhibitor (I
B)
proteolysis in HepG2 hepatoblastoma cells. HepG2 cells express the
I
B members I
B
, I
B
, and I
B
. In response to TNF
,
Rel A·NF-
B1 translocation and DNA binding activity follows a
biphasic profile, with an "early" induction (15-30 min), followed
by a nadir to control levels at 60 min, and a "late" induction
(>120 min). The early phase of Rel A·NF-
B1 translocation depends
on simultaneous proteolysis of both I
B
and I
B
isoforms;
I
B
is inert to TNF
treatment. The 60-min nadir is due to a
rapid I
B
resynthesis that reassociates with Rel A and completely
inhibits its DNA binding activity; the 60-min nadir is not observed
when I
B
resynthesis is prevented by cycloheximide treatment. By
contrast, selective inhibition of I
B
proteolysis by pretreatment
of HepG2 cells with the peptide aldehyde
N-acetyl-Leu-Leu-norleucinal completely blocks the late phase of Rel A·NF-
B1 translocation. These studies indicate the presence of inducible and constitutive cytoplasmic NF-
B pools in
hepatocytes. TNF
induces a coordinated proteolysis and resynthesis of I
B isoforms to produce dynamic changes in NF-
B nuclear
abundance.
Multicellular organisms have evolved mechanisms for the coordinate expression of inducible genes through ligand-dependent receptors. Ligand binding to high affinity receptors located on the plasma membrane generate second messenger signals that can influence the activity or abundance of transcription factors through post-translational modifications including signal-induced phosphorylation and/or proteolysis. Hormone-activated gene transcription plays an important role in many homeostatic processes, including the cytokine cascade for lymphocyte expansion (1), and the change in expression of liver genes in response to systemic inflammation known as the hepatic acute-phase response (APR1; reviewed in Refs. 2-4).
The APR is the consequence of inducible transcriptional activation of
hepatic genes required for blood pressure regulation, such as
angiotensinogen (2, 5), and those involved in macrophage opsonization
and wound repair (6) through the effects of macrophage-derived interleukins-1, interleukin-6, and tumor necrosis factor (TNF
) (6). Hepatocyte-specific transactivators modified during the APR
include AP-1 (7), signal transducers and activators (8), nuclear
factor-interleukin 6 (9), and nuclear-factor-
B (NF-
B) (10). The
angiotensinogen gene is transcriptionally activated during the APR by
the effect of a single regulatory element, the acute-phase response
element (APRE) (11, 12). The APRE is a target for intracellular
signaling initiated by the liganded TNF
type I receptor that
activates latent DNA binding activity of the potent NF-
B
transcription factor family (3, 12, 13).
NF-B is a family of homo- and heterodimeric proteins related by an
NH2-terminal ~300 amino acid Rel homology domain
including the proteolytic processed NF-
B1 and NF-
B2 subunits, as
well as the Rel A (p65), c-Rel, and Rel B subunits (reviewed in Ref. 10). Dimerization of various NF-
B subunits produce complexes with
various intrinsic DNA-binding specificities (14), transactivation potentials (10, 15-18), and subcellular localization (19). For
example, NF-
B1 homodimers are constitutively nuclear and bind DNA
avidly, but lack significant transcriptional activity; by contrast, Rel
A·NF-
B1, Rel A·c-Rel, and Rel A·NF-
B2 heterodimers are
cytoplasmic and exhibit various degrees of transcriptional activator
properties (reviewed in Refs. 10, 20). UV cross-linking (11), gel
mobility shift assays with subunit-specific NF-
B antibodies (12),
and transient overexpression assays (12, 13) indicate that Rel
A·NF-
B1 heterodimers are the major species of hormone-inducible
NF-
B subunits that bind the APRE in hepatocytes.
The Rel A·NF-B1 complex is sequestered in a latent
cytoplasmic form by association with various inhibitor (I
B)
proteins, including I
B
(pp40/MAD-3) (21-23), I
B
(24),
I
B
(the COOH-terminal product encoded by translation of the
alternative splicing of the p105 NF-
B1 mRNA precursor (25)), and
p105 itself (26, 27) that associate with Rel A through a protein
interactive domain homologous to erythrocyte ankyrin. Dissociation of
Rel A from I
B is prerequisite for Rel A nuclear translocation (23, 28-30); current evidence favors a two-step dissociation that first requires inducible NH2-terminal phosphorylation (I
B
is phosphorylated at serine residues 32 and 36) followed by proteolysis
through the 26 S proteasome (30-32).
The observations that distinct IB family members are expressed, and
perhaps regulated, in a tissue-restricted fashion (24, 31) prompted us
to investigate the kinetics of latent Rel A:NF-
B1 activation in
hepatocytes. We report the unanticipated findings that TNF
produces
a biphasic Rel A·NF-
B1 nuclear translocation, with an "early"
peak at 15-30 min, return to control (1 h), and a later peak (>2 h
induction). In hepatocytes, the I
B family members
,
, and
,
but not the NF-
B1 p105 precursor are expressed. Early Rel
A·NF-
B1 translocation is due to simultaneous proteolysis of both
I
B
and I
B
. By 1 h, I
B
is rapidly synthesized and reassociates with Rel A; this reassociation (due to "overshoot" synthesis) results in complete inhibition of Rel A·NF-
B1 binding even in the continued absence of I
B
. As I
B
levels fall
after 1 h, Rel A·NF-
B1 binding is again detectable in the
nucleus. Inhibition of I
B
proteolysis by the peptide aldehyde
N-acetyl-Leu-Leu-norleucinal completely prevents the second
phase of Rel A·NF-
B1 binding, demonstrating the requirement of
I
B
for prolonged Rel A·NF-
B1 nuclear action.
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., Grand Island, NY) 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 (rTNF
, Genentech) was
added to a final concentration of 30 ng/ml in culture medium and cells were incubated for the indicated time periods at 37 °C. For
pretreatments, calpain inhibitor I (200 µM, CalBiochem,
San Diego, CA) or cycloheximide (50 µg/ml, Sigma) were added in
medium 1 h or 30 min prior to TNF
stimulation,
respectively.
Preparation of Subcellular Extracts
Cytoplasmic ExtractsHepG2 cell pellets were washed two times with phosphate-buffered saline and then resuspended in Buffer A (50 mM HEPES (pH 7.4), 10 mM KCl, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 0.1 mg/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, the supernatant constitutes the cytoplasmic extract.
Sucrose Density-purified Nuclear ExtractsFor the purification of nuclei (33, 34), nuclear pellets were resuspended in Buffer B (1.7 M sucrose, 50 mM HEPES (pH 7.4), 10 mM KCl, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 0.1 mg/ml phenylmethylsulfonyl fluoride, 1 µg/ml pepstatin A, 1 µg/ml leupeptin, 10 µg/ml soybean trypsin inhibitor, and 10 µg/ml aprotinin), and centrifuged at 15,000 × g for 30 min at 4 °C. The resultant nuclear pellets were 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 mg/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 the Bradford assay using bovine serum albumin as a standard (Bio-Rad).
Electrophoretic Mobility Shift Assays (EMSAs)
EMSAs were performed as described previously with minor
modifications (12, 13). 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 mg/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 (25 mM Tris, 25 mM
boric acid, and 0.5 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 as shown below.
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Antibody supershift assays were performed by adding to the
binding reaction 1 µl of affinity-purified polyclonal antibodies and
incubating for 1 h on ice. All of the antibodies used in these assays were obtained commercially (Santa Cruz Biotech, Santa Cruz, CA).
For the NF-B-DNA binding inhibition assays, the nuclear extracts
were mixed with the indicated amounts of bacterially-expressed full-length I
B
protein in binding reaction. Inactivated I
B
was prepared by boiling the recombinant I
B
protein for 30 min in
phosphate-buffered saline.
Expression of Polyhistidine-tagged IB
The full-length cDNA encoding human IB
was subcloned
as an EcoRI fragment into the pRSETB expression plasmid
under the control of the T7 promoter (InVitrogen, San Diego, CA). The
plasmid was transformed into Escherichia coli BL21(DE3)pLysS
and the 47-kDa protein was induced with the addition of 1 mM isopropyl-1-thio-
-D-galactopyranoside (final concentration) during logarithmic growth and purified under native conditions on a nickel-agarose column (35). The protein was
>90% pure as judged by SDS-PAGE and Coomassie Blue staining.
Western Blotting and Coimmunoprecipitation
For Western immunoblot, a constant amount of cytoplasmic or
nuclear extracts (200-300 µg as indicated) from the above
preparation 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 Biotech)
for IB
(reactive with amino acids 297-317), I
B
(reactive
with amino acids 339-358), I
B
(reactive with amino acids
471-490), Rel A (reactive with amino acids 3-19), or NF-
B1 (reactive with amino acids 350-363). 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) according to the manufacturer's recommendations. For
the coimmunoprecipitation assays, cytoplasmic extracts (1 mg) from
either untreated or TNF
-stimulated HepG2 cells were incubated with
Rel A antiserum in a total volume of 1 ml of TST buffer (50 mM Tris-Cl, 5 mM EDTA, 150 mM NaCl,
and 0.05% Triton X-100) for 2 h at 4 °C and collected on
Protein A-agarose beads (Life Technologies, Inc.). After washing 5 times with TST buffer, the precipitates were boiled in Laemmli buffer
and subjected to immunoblotting with anti-I
B
antibody following
the above procedure. For the neutralization experiments, anti-Rel A and
anti-I
B
antibodies were neutralized with a 20-fold excess of the
relevant polypeptide overnight at 4 °C.
Exposure of cultured HepG2 human hepatocytes to 20 ng/ml
rTNF for 6 h results in an induction of NF-
B DNA binding
activity and transcriptional activity (12, 13). To resolve the
induction kinetics of various NF-
B family members in the rTNF
response, we examined a 6-h time course of APRE DNA binding activity
using extracts of sucrose cushion-purified nuclei using EMSAs. In EMSAs performed under conditions that resolve the individual heterodimeric NF-
B species binding to the radiolabeled APRE, four nucleoprotein complexes could be resolved (C1-C4, Fig. 1A).
The C1 complex was weakly and variable inducible at 15 min. By
contrast, the C2 complex was strongly inducible in a biphasic manner
with the first peak occurring at 15 min (a 16-fold induction relative
to control), declining by 30 min, and was undetectable at 60 min (the
early binding phase). At 120, the C2 complex reappeared (4.1-fold
relative to control) and persisted as long as 360 min (the "late"
binding phase). Binding specificity of the complexes was demonstrated using site-specific competitors of the APRE in the EMSA (Fig. 1B). Complexes C1 and C2 both competed with homologous APRE
WT but not APRE M2 or APRE M6 oligonucleotides, indicating
sequence-specific recognition of the NF-
B contact points on the APRE
(5).
A, rTNF induces a biphasic pattern of
Rel A·NF-
B1 binding in HepG2 nuclei. Autoradiogram of EMSA using
10 µg of nuclear protein prepared from cultured HepG2 hepatoblastoma
cells stimulated for the indicated times (in min) with 30 ng/ml rTNF
binding to radiolabeled APRE WT DNA. Migration of various complexes
(C1-C4) is shown at the left. Complexes C3 and C4
are constitutive. Complex C2 exhibits a biphasic induction pattern with
a return to control values at 60 min. C1 is weakly and variably
inducible. After quantitation, the C1 complex increases (values given
in fold increase relative to controls): 5.7 (15 min), 1.8 (30 min), 1.1 (60 min), 1.6 (120 min), and 1.1 (360 min); for the C2 complex: 16.5 (15 min), 6.1 (30 min), 0.3 (60 min), 4.1 (120 min), and 4.9 (360 min).
B, C1 and C2 bind with NF-
B binding specificity.
Autoradiogram of competition EMSA using 10 µg of 15-min stimulated
HepG2 nuclear extract binding to radiolabeled APRE WT in the absence
(
) or presence of 100-fold molar excess of APRE site mutations
("Experimental Procedures"). Location of complexes is located at
left. Complexes C1 and C2 compete with wild type APRE, but
not mutants APRE M2 or APRE M6 (indicating NF-
B binding specificity).
Complex C3 competes with APRE WT and APRE M6 indicating NF-interleukin
6/C/EBP binding specificity (5). Complex C4 competes in a nonspecific
binding pattern. Complex C2 contains both Rel A·NF-
B1 subunits.
Gel mobility supershift assay after addition of subunit-specific
NF-
B antibodies (indicated at top). Location of complexes
is indicated at the left. Top panel, light exposure.
Bottom panel, longer exposure of supershifted bands.
Addition of anti-Rel A antibody reduces intensity of C1 and C2
complexes, producing a supershifted band (small asterisk).
NF-
B1 antibody reduces C2 complex producing supershifted band
(large asterisk). Anti-c-Rel antibody reduces C1 complex
producing supershifted band (small arrow, bottom
panel).
Gel mobility supershift assays using subunit-specific NF-B
antibodies was used to demonstrate the composition of the strongly inducible APRE-binding C2 complex (Fig. 1C). Addition of Rel
A, but not preimmune antibody resulted in the selective diminution of
the C2 complex with the simultaneous appearance of a supershifted band.
Similarly, addition of NF-
B1 antibody also diminished the intensity
of the C2 nucleoprotein complex. Taken together, these data indicate
that the DNA binding activity of the C2 is biphasic upon rTNF
treatment, C2 binds to the APRE with NF-
B binding specificity and is
composed of the Rel A·NF-
B1 heterodimer.
HepG2 cells fractionated
into cytoplasmic and highly purified nuclear extracts (by sucrose
cushion centrifugation) were assayed in Western immunoblots for changes
in relative abundance of Rel A and NF-B1. The Rel A antibody
recognized a single ~65-kDa antigen (Fig. 2,
arrow) that could be specifically blocked by preadsorbtion using recombinant Rel A protein (not shown). In unstimulated cells, the
majority of Rel A was located in the cytoplasmic fraction. By 15 min of
rTNF
treatment, a slight depletion in the cytoplasmic fraction was
noted with a concomitant 3.7-fold increase in nuclear Rel A. At 60 min,
nuclear Rel A has diminished to levels approximating control followed
by a second increase in Rel A abundance at 120 and 360 min. Changes in
nuclear NF-
B1 abundance, detected by a specific polyclonal antibody
as a 50-kDa band, paralleled those observed for nuclear Rel A (Fig. 2),
and for DNA binding of the C2 complex (Fig. 1). These data indicate
that rTNF
controls cytoplasmic:nuclear positioning of Rel A and
NF-
B1 in hepatocytes in a biphasic pattern.
Dynamic Expression and Differential Regulation of I
Cytoplasmic extracts of control and
rTNF-treated HepG2 cells were assayed for the expression and
relative changes in I
B abundance using antibodies that recognized
specific epitopes of I
B
, I
B
, and I
B
as determined by
the appropriate molecular weight and ability of peptide preadsorption
to compete for the immunostaining (Fig. 3A).
In control cells, 37-kDa I
B
was abundantly detected, as was
46-kDa I
B
and 70-kDa I
B
(Fig. 3B). With rTNF
treatment, both I
B
and I
B
, but not I
B
, disappeared
within 15 min of treatment. Abundance of I
B
returned to a 2-fold
greater than control levels at 60 min producing an "overshoot" in
its synthesis; by 120 min, I
B
returned to control levels. In
contrast, although 46-kDa I
B
disappeared simultaneously with
I
B
after TNF
treatment, no resynthesis of I
B
was
observed. These data indicate the abundance of I
B
and
is
regulated by rTNF
treatment, whereas the abundance of I
B
is
not. Moreover the robust I
B
resynthesis at 1 h corresponds
to the "nadir" of Rel A·NF-
B1 DNA binding activity and nuclear
abundance (cf. Figs. 1A and 2).
I
We noted that the
disappearance of nuclear Rel A occurred simultaneously with enhanced
IB
abundance, indicating that enhanced I
B
synthesis
(overshoot) and reassociation with Rel A may underlie the phenomenon of
the 60-min nadir. To directly measure association of Rel A with
I
B
, we performed a two-step immunoprecipitation/Western immunoblot using cytoplasmic extracts from HepG2 hepatocytes. In this
assay, Rel A complexes are captured and washed under nondenaturing conditions on protein A-agarose beads. After the immune complexes are
eluted in denaturing SDS-PAGE loading buffer, I
B
association is
measured by Western immunoblot with I
B
antibodies (Fig.
4A; Rel A antibody is used as a control for
recovery from the immunoprecipitate). Controls demonstrating
specificity of the Rel A·I
B
association include: 1) use of
preadsorbed Rel A antibody as the primary antibody. As shown in Fig.
4A (lane 2), preadsorbed Rel A antibody does not
bring down Rel A nor I
B
; and 2) use of preadsorbed I
B
antibody in the Western immunoblot to confirm that the 37-kDa antigen
is I
B
(Fig. 4A, lane 1).
In control cytoplasmic extracts, Rel A is associated with IB
(Fig. 4A, lane 3), but this association is lost upon 15 min of rTNF
treatment (a time coincident with its proteolysis, Fig. 3B). At 30 min (and later times), I
B
is reassociated
with Rel A; we note at 60 min that more I
B
is associated with Rel
A than at other time points, indicating that there is strong
association of Rel A with I
B
(during the nadir in Rel
A·NF-
B1 binding).
The late phase of Rel A induction occurs in the continued presence of
IB
. To exclude the possibility that Rel A is modified in a way
that prevents inhibition of its DNA binding activity upon I
B
binding, purified recombinant human I
B
(rhI
B
) was added to
nuclear extracts prepared from either the early or the late phases of
NF-
B activation (Fig. 4B). Heat-inactivated I
B
was
used as a control for nonspecific salt or detergent effects in the
EMSA. Addition of 200 ng of rhI
B
completely inhibited DNA binding
activity of the C1 and C2 complexes, the latter representing the Rel
A:NF-
B1 heterodimer, whereas the nonspecific C4 complex was
unaffected. These data demonstrate that Rel A·NF-
B1 complex associated with I
B
is unable to bind DNA at any phase of its induction.
Our data indicated that enhanced IB
synthesis results in
inhibition of Rel A·NF-
B1 binding at 60 min of rTNF
stimulation. To confirm this model, EMSA was used to determine the
pattern of Rel A·NF-
B1 binding after I
B
resynthesis was
inhibited using the protein synthesis inhibitor cycloheximide. In the
presence of 50 µg/ml cycloheximide, the biphasic Rel A·NF-
B1
binding pattern was abolished and converted into a single monotonic
profile as shown in EMSA (Fig. 5). In this same
experiment, cytoplasmic I
B
disappeared at 15 min and was
undetectable at 1 h of rTNF
treatment assayed by Western
immunoblot (data not shown), indicating requirement of new protein
synthesis for the nadir in Rel A·NF-
B1 binding. These data
indicate: 1) I
B
reassociated with the Rel A·NF-
B1 complex at
60 min; 2) I
B
is capable of inhibiting DNA-binding of Rel
A·NF-
B1; and 3) I
B
appearance at 60 min requires new protein
synthesis. We conclude that the enhanced I
B
resynthesis at 60 min
(I
B
overshoot) underlies the biphasic pattern of Rel A·NF-
B1
translocation, by producing the nadir in C2 binding.
I
Previous work has indicated that
chymotrypsin-like enzyme(s) mediate IB proteolysis (36),
prompting us to examine whether inhibitors could selectively interfere
with I
B
proteolysis so that we could determine its role in
biphasic NF-
B activation in hepatocytes. The effect of pretreatment
with the peptide aldehyde N-acetyl-Leu-Leu-norleucinal
(calpain inhibitor I) was determined using Western immunoblot assays of
cytoplasmic extracts (Fig. 6). In response to rTNF
,
I
B
proteolysis was clearly evident at 15 and 30 min. By contrast,
I
B
abundance was similar, or exceeded, control values from 15 to
360 min (compare with Fig. 3B). I
B
was unaffected
throughout the time course of the experiment. We conclude that calpain
inhibitor I preferentially blocks I
B
, but not I
B
proteolysis in hepatocellular cells.
Under conditions where IB
proteolysis was preferentially
inhibited, we next determined the kinetics of Rel A·NF-
B1
translocation. The effect of calpain inhibitor I pretreatment was to
block completely the second late phase of Rel A·NF-
B1 induction
(Fig. 7A). The early appearance of C2 at 15 and 30 min was attenuated, but not abolished, indicating the
contribution of I
B
proteolysis to the early phase of
translocation. Western immunoblots for nuclear Rel A and NF-
B1
abundance confirmed that Rel A and NF-
B1 were induced corresponding
to the early but not the late phase of nuclear translocation (compare
Fig. 7B (360 min) with Fig. 2 (360 min)). These data
indicate calpain inhibitor I markedly affects the biphasic pattern of
Rel A·NF-
B1 induction, converting is induction to a monophasic
pattern. These data provide direct evidence for the dynamic effect of
I
B
and I
B
proteolysis and resynthesis during the biphasic
Rel A·NF-
B1 induction.
An important mechanism in the transcriptional activation of the
angiotensinogen gene during the APR is the TNF-mediated activation of latent NF-
B subunits that bind to the APRE (3, 12, 13). In this
study, we have identified mechanistically distinct phases of Rel
A·NF-
B1 nuclear translocation that depend on dynamic changes in
individual I
B subunit abundance. In this report, we describe the
unanticipated and novel observation that rTNF
produces a biphasic
pattern of Rel A·NF-
B1 binding. Previous studies have only
identified a monophasic induction pattern using 70Z/3 pre-B lymphocytes
(24), Jurkat T-cells (38), and U937 macrophages (36) of varying
duration. In hepatocytes, the biphasic pattern consists of an early
peak (15-30 min), return to control levels (60 min nadir), and a late
peak (>120 min). The early peak is the consequence of simultaneous
proteolysis of both the I
B
and I
B
subunits (without
alterations in I
B
abundance). Although I
B
resynthesis is
not detectable during the 360-min study, I
B
resynthesis, by
contrast, is rapid. As a result of the overshoot of I
B
synthesis,
I
B
appears at greater than control levels at 60 min and is
apparently responsible for the nadir in Rel A·NF-
B1 binding. The
use of calpain inhibitor I, a protease inhibitor that selectively
blocks I
B
proteolysis, allows us to demonstrate that the second
phase of RelA·NF-
B1 translocation is solely dependent on
signal-induced hydrolysis of I
B
.
In non-B lymphocytes,
NF-B proteins are sequestered in an inactivated form in the
cytoplasm by binding various members of the inhibitory I
B protein
family. I
B
, -
, -
and NF-
B1 p105 are all candidate
regulators of Rel A translocation because other studies have shown
direct association with Rel A·NF-
B1 in cellulo and
their ability to inhibit RelA·NF-
B1 DNA binding activity in
vitro (23-25). The pattern of I
B expression and modification by signal transduction systems has not been systematically studied in
liver cells. Relative levels of mRNA encoding the I
B isoforms have been shown to be expressed in a tissue-restricted fashion. For
example, I
B
mRNA is expressed highly in testes (where no I
B
mRNA is detectable) and I
B
is expressed more
abundantly in spleen and lung than I
B
(24). Although we have not
measured transcript levels, we observe that I
B
protein appears to
be more abundant than I
B
in hepatocytes. I
B
is a 70-kDa
translation product of a unique mRNA that represents either an
alternative splice product or a cryptic promoter from the
nf-kb1 gene (25), and is reported to have the most highly
restricted pattern of expression previously detected only in mouse
lymphoid cell lines (25). Although we have not assayed for expression
of the unique I
B
transcript, we can identify I
B
expression
on the basis of antibody specificity and its appropriate 70,000 molecular weight in hepatocellular cells. In marked contrast to the
behavior of I
B
and I
B
, the steady state abundance of
I
B
appears not to be regulated by rTNF
treatment. The presence
of constitutive I
B
may account for the presence of residual Rel A
in the cytoplasm of rTNF
-treated cells at times (15 min) when nearly
complete I
B
and I
B
proteolysis have occurred, and
underscores the concept that there may be pools of Rel A in complex
with I
B proteins whose abundance are themselves differentially
responsive to distinct second messenger pathways. Finally, it is
important to emphasize that our observations are made on a cell
population and hence represent a statistical average of individual
cellular responses to rTNF
. Based on this experimental design, we
therefore cannot differentiate between the following interpretations:
1) two subsets of cells are present in the HepG2 culture, one group
expresses I
B's
and
and a second group expresses I
B
only, with only the former population being rTNF
-responsive;
versus 2) a homogenous cell population is present in the
HepG2 culture that expresses all three I
B isoforms. Although this
cell population is rTNF
-responsive, the abundance of individual
I
B isoforms are controlled by different intracellular signaling
pathways. Additional studies, at individual cell resolution, will be
required to differentiate between these possibilities.
TNF activates Rel A translocation by signal-induced
modifications of the I
B inhibitor. TNF
-activated intracellular
signals induces phosphorylation of I
B
at NH2-terminal
serine residues (amino acids 32 and 36 (28, 30)), by a
ubiquitination-dependent kinase, a process that
subsequently targets I
B
for proteolysis (31, 32).
Phospho-I
B
migrates at a distinct position on SDS-PAGE gels (~3
kDa larger), allowing for its identification (23, 28, 30, 32). Although
we have not directly demonstrated inducible phosphorylation of I
B in
HepG2 cells, indirect evidence for I
B
phosphorylation is seen in
Western immunoblots of cytoplasmic extracts from rTNF
-treated cells
where the slower phospho-I
B
migrating species is seen with longer
exposures (data not shown). Other studies have shown that
phospho-I
B
is itself rapidly polyubiquitinated (Ubn)
at lysine residues 21 and 22 (31) and subsequently proteolyzed through
the 26 S proteasome pathway (29, 32). The 26 S proteasome, a 700-kDa
protease complex, is known to degrade Ubn-conjugated proteins in an ATP-dependent fashion, but the protease(s)
involved and their specificity are incompletely characterized (37).
In epithelial cells and lymphocytes, serine protease inhibitors
(L-1-tosylamido-2-phenylethyl chloromethyl ketone) and the peptide aldehyde (calpain inhibitor I) are effective in blocking IB
proteolysis at concentrations that interfere with proteasome activity (31, 37). Based on this inhibitor sensitivity profile, the
enzyme(s) in the 26 S proteasome complex mediating I
B
hydrolysis has been characterized as chymotrypsin-like. In this regard, we are
surprised to find in hepatocytes, that calpain inhibitor I blocks
I
B
proteolysis relatively selectively. These data indicate a
potential involvement of cell-type specific factors in the inducible proteolysis of I
B
.
Upon Rel A
translocation into the nucleus, the synthesis of IB
is activated
(38, 39). Newly resynthesized I
B
reassociates with Rel A,
inactivating its nuclear location and transcriptional effect resulting
in an autoregulatory feedback pathway. The important role of I
B
in terminating Rel A·NF-
B1 activation is illustrated in the
persistent NF-
B activation upon rTNF
stimulation in fibroblasts cultured from mice with homozygous deletion of i
b
(40). The surprising observation in our studies is that, although
I
B
is involved in terminating NF-
B action, it does so only
transiently by overshoot resynthesis. That I
B
resynthesis results
in a transient inhibition of Rel A·NF-
B1 DNA binding activity is
supported by the following observations: 1) increased I
B
abundance by Western immunoblot (Fig. 3) is coincident with inhibition
of Rel A·NF-
B1 binding at 60 min (Fig. 1A). 2)
Increased I
B
is associated with Rel A by coimmunoprecipitation
assays (Fig. 4A). 3) Rel A·NF-
B1 DNA binding activity
from both early and late phases of induction is completely inhibited by
rhI
B
(Fig. 4B). 4) Rel A·NF-
B1 inhibition at 60 min is dependent on new protein synthesis (Fig. 5). At present, we are
unable to explain why the increased I
B
levels at 60 min return to
control levels at 120 min; this phenomenon is probably the result
of accelerated turnover of I
B
in the presence of TNF
(as
for LPS activation in 70/Z3 cells (23)).
In Jurkat T-lymphocytes (24) and in mouse
embryo fibroblasts (4), rTNF produces only I
B
, but not
I
B
proteolysis, associated with a transient monotonic induction
of Rel A·NF-
B1 binding. In hepatocytes, by contrast, rTNF
produces a biphasic, prolonged induction of Rel A·NF-
B1. A
requirement for I
B
proteolysis in producing the late phase of Rel
A·NF-
B1 translocation is seen under conditions where I
B
proteolysis is abolished. Earlier reports in other cell lines have
shown that i
b
is not subject to the Rel A-induced
autoregulatory pathway, and once proteolyzed, is not resynthesized
during the time course of these experiments (24); our data are
consistent with this phenomenon. I
B
proteolysis apparently allows
for a prolonged nuclear response to the actions of TNF
in a cell
restricted pattern. Based on their similar domain organizations and
similar rapid proteolytic response to TNF
, it might be expected that
I
B
and I
B
would be proteolyzed through similar pathways.
However, several features in HepG2 cells indicate that the mechanism
for signal-induced proteolysis of I
B
is distinct from that used
for I
B
. Phosphorylated I
B
isoforms migrate more slowly that
nonphosphorylated forms (28, 30), allowing their identification by
Western immunoblot. In calpain inhibitor I-treated cells,
rTNF
-inducible slower migrating species of I
B
corresponding to
phosphorylated forms can be detected upon longer exposure. No such
forms are seen with I
B
(Fig. 6, middle panel), even
under conditions when complete inhibition of proteolysis is observed.
Second, and more importantly, proteolysis of I
B
is insensitive to
200 µM calpain inhibitor I whereas proteolysis of
I
B
is completely blocked by this concentration. These data probably indicate that rTNF
activates I
B proteolysis through two
distinct pathways.
In summary, our studies have focused on the mechanisms for the novel
observation of a biphasic induction of Rel A·NF-B1 binding in
hepatocytes. Rel A·NF-
B1 is associated with three I
B isoforms that are differentially regulated by rTNF
, producing pools of constitutive and inducible NF-
B complexes. We provide evidence for
how dynamic changes in I
B
abundance influences the two distinct phases of Rel A·NF-
B1 translocation. Moreover, we report that separate proteolytic pathways are involved in I
B
and I
B
degradation in hepatocytes producing unanticipated complexity in
signal-induced regulation of I
B abundance.
We thank John Morris for the gift of the MAD
3 cDNA, Rebecca Soliz for secretarial assistance, Weili Duan for
technical assistance, and Junyi Li for polyhistidine IB
expression.