Department of Medicine, Biochemistry, and Biophysics, University of North Carolina, Chapel Hill, North Carolina 27599
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ABSTRACT |
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Nuclear
factor-B (NF-
B) prevents hepatocytes from undergoing
apoptosis during development and liver regeneration. Mice with inactivated glycogen synthase kinase (GSK)-3
die from hepatocyte apoptosis during development due to a defect in NF-
B
activation (Hoeflich KP, Luo J, Rubie EA, Tsao MS, Jin O, and Woodgett
JR. Nature 406: 86-90, 2000). In this study, we
determined the role of GSK-3 in TNF-
-induced NF-
B activation and
cell death in primary hepatocytes. LiCl, an established inhibitor of
GSK-3, sensitized primary rat hepatocytes toward TNF-
-mediated
apoptosis resulting in 90% cell death after 24 h. This
was accompanied by increased caspase 8-like and 3-like activities,
nuclear fragmentation and DNA laddering. LiCl treatment had no effect
on I
B-
degradation, I
B kinase (IKK) activity, NF-
B binding
activity, and p65 nuclear import and export, but decreased
transcription of the NF-
B-dependent inducible nitric oxide synthase
gene and a NF-
B-driven reporter gene. The p65 sequence revealed four
potential GSK-3 phosphorylation sites within its COOH-terminal
transactivation domains and recombinant GSK-3
phosphorylated
glutathione S-transferase (GST)-p65(354-551), but not
GST-p65(1-305) in vitro. These results indicate that GSK-3 protects hepatocytes from TNF-
-induced apoptosis through p65 phosphorylation and upregulation of NF-
B transactivation.
p65 phosphorylation; p65 transactivation; IB kinase; tumor
necrosis factor-
; nuclear factor-
B
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INTRODUCTION |
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TUMOR NECROSIS
FACTOR- (TNF-
) is a pleiotropic cytokine that may induce
proliferation, apoptosis, or inflammatory reactions in target
cells. These different cellular reactions toward TNF-
result from
the activation of distinct cellular signaling pathways that interact
with each other in a complex signaling network and allow a wide range
of cellular responses (1, 6). Although TNF-
is a known
activator of the apoptotic signaling cascade, TNF-
commonly does
not induce apoptosis in target cells due to the parallel
activation of protective signaling pathways that interfere with the
onset of apoptosis. The nuclear factor-
B (NF-
B) pathway
is strongly activated by TNF-
and prevents hepatocytes from
undergoing TNF-
-induced apoptosis during development, liver regeneration, and other conditions associated with high levels of
TNF-
(4, 20). Under normal circumstances, NF-
B is
bound to its inhibitor I
B and resides in an inactive state within
the cytoplasm. When released from its inhibitor, NF-
B translocates to the nucleus where it activates the transcription of genes with
B
sites. TNF-
controls this crucial step in the regulation of NF-
B
activity through a kinase cascade that results in the phosphorylation of I
B by the I
B kinase (IKK) complex and its subsequent
ubiquitination and degradation (23). Like many other
transcription factors, the activity of NF-
B is believed to be
additionally regulated by phosphorylation of its subunits containing
transactivation domains (36, 37). This is supported by
studies demonstrating that the NF-
B subunit p65 becomes
phosphorylated on TNF-
or interleukin-1
(IL-1
) stimulation
(30, 34, 45) and that the transactivation potential of p65
is hereby increased (28, 45). Several kinases including
IKK-
and -
, casein kinase II, and calmodulin-dependent kinase 4 (22, 29, 34, 46, 47) have been demonstrated to
phosphorylate p65. Recently, it was reported that mice with inactivated
glycogen synthase kinase (GSK)-3
die during development due to
massive hepatocyte apoptosis, because of a defect in NF-
B
activation independent of DNA binding activity, suggesting a role for
GSK-3
in the regulation of p65 transactivation (19).
In this study, we assess the role of GSK-3 in the regulation of
TNF--induced NF-
B activation in hepatocytes. We demonstrate that
1) LiCl, a pharmacological inhibitor of GSK-3, abolishes TNF-
-induced, NF-
B-dependent gene transcription in hepatocytes independent of NF-
B DNA binding activity and that 2)
GSK-3
phosphorylates the COOH terminus of the p65 unit of NF-
B in
vitro. These results suggest a role for GSK-3 in the transactivation of
NF-
B through p65 phosphorylation.
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MATERIALS AND METHODS |
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Hepatocyte isolation and culture.
Hepatocytes were isolated from male Sprague-Dawley rats (200-225
g) by collagenase perfusion as previously described (18). Hepatocytes were plated onto collagen-coated cell culture dishes or
collagen-coated coverslips in Waymouth medium containing 10% fetal
bovine serum for 2 h. Cells were washed with PBS, and the medium
was subsequently changed to hormonally defined medium as previously
described (38). Before treatment with recombinant mouse
TNF- (R&D, Minneapolis, MN), hepatocytes were incubated with either
LiCl (30 mM) or NaCl (30 mM) for 1 h in hormonally defined medium
without insulin, if not indicated otherwise.
Western blot analysis.
Whole cell extracts were prepared as previously described
(38). For the analysis of IB-
degradation, 25 µg
of whole cell extract were loaded onto a 10% SDS acrylamide gel and
analyzed as previously described (38). For the detection
of
-catenin, 25 µg of whole cell extracts from LiCl- and
NaCl-treated hepatocytes were loaded onto an 8% SDS acrylamide gel and
transferred onto nitrocellulose membranes (Schleicher and Schull,
Keene, NH). After blocking in 5% milk powder in Tris-buffered saline
containing 0.5% Triton X-100, the membrane was incubated with
antibodies to
-catenin (Santa Cruz, Santa Cruz, CA) at a dilution of
1:500 for 6 h at room temperature. After extensive washing, the
membrane was incubated with horseradish-peroxidase conjugated anti-goat secondary antibody at a dilution of 1:1,000 for 1 h at room
temperature. After extensive washing, the bands were visualized by
enhanced chemoluminescene (Amersham, Arlington Heights, IL) and
exposure to Biomax film (Kodak, Rochester, NY). To demonstrate equal
protein loading, membranes were incubated with
-tubulin antibody
(Oncogene Science, Beverly, MA) for 1 h at a dilution of 1:1,000
and a horseradish-peroxidase conjugated anti-mouse secondary antibody
(Santa Cruz) at a dilution of 1:1,000 for 1 h.
NF-B reporter gene assay.
Eight hours after isolation, 0.5 ×106 hepatocytes were
infected with an adenovirus containing firefly luciferase linked to three
B sites at a multiplicity of infection (MOI) of 10 for 2 h (35). Cells were cultured for 12 h and then treated
with TNF-
at a concentration of 30 ng/ml. After 6 h of TNF-
treatment, extracts were prepared and analyzed by luminometry.
Luciferase activity was normalized to the protein content of the extracts.
RNA isolation and RT-PCR.
Hepatocytes were pretreated with LiCl (30 mM) or NaCl (30 mM) for
1 h. After treatment with TNF- (30 ng/ml) for 4 h, RNA was
isolated by the TRIzol method. RT-PCR for inducible nitric oxide
synthase (iNOS) or
-actin was performed as previously described (38).
Electrophoretic mobility shift assay.
Nuclear extracts were prepared as previously described
(38). Five micrograms of nuclear extract were incubated
with 1 ng of 32P-labeled probe containing the NF-B
consensus site and incubated at room temperature for 15 min as
previously described. NF-
B DNA binding was determined by running the
samples on a 5% acrylamide gel in 0.25× Tris-Borate-EDTA buffer and
exposure to Biomax film.
Detection of apoptosis.
After pretreatment with 30 mM LiCl or NaCl, cells were treated with
TNF- for the indicated times. For the detection of nuclear fragmentation, cells were fixed in 30% methanol/70% acetic acid and
stained with propidium iodide and visualized under a
fluorescent mircroscope (Olympus Melville, NY) using Spot digital
imaging software (Diagnostic Instruments, Sterling Heights, MI). For
the detection of caspase-like activity, cells were lysed in caspase lysis buffer and incubated in the presence of either
IETD-7-amino-4-trifluoromethyl coumarin (AFC) or DEVD-AFC substrate as
previously described (38). For the detection of
DNA fragmentation, DNA was extracted as previously described
(38) and analyzed on an 1.8% agarose gel.
In vitro kinase assays.
IKK activity was determined by immunocomplex kinase assay as previously
described (38). To exclude a direct inhibitory effect of
LiCl on IKK, which might be lost in the standard immunocomplex assay
due to a decrease in LiCl concentrations after washing, we additionally
added LiCl to the kinase reaction after washing. The GSK-3 kinase
assay was performed by incubating 100 units of recombinant GSK-3
(New England Biolabs, Beverly, MA) in 25 µl kinase buffer containing
20 mM Tris (pH 7.5), 10 mM MgCl2, 5 mM DTT, and 50 µM
ATP, and 0.5 µCi [32P]ATP with either glutathione
S-transferase (GST)-p65 (1-305) or GST-p65
(354-551) as substrate for 30 min at 30°C.
Immunofluorescent staining.
Hepatocytes (500,000) were plated on a glass coverslip and
pretreated with LiCl (30 mM) or NaCl (30 mM) for 1 h. Cells were then treated with TNF- for the indicated time and fixed with methanol for 5 min. Immunofluorescent staining of p65 was performed as
previously described (38).
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RESULTS |
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Inhibition of GSK-3 by LiCl sensitizes hepatocytes toward
TNF--mediated apoptosis.
LiCl is an established inhibitor of GSK-3 (19, 24, 40). To
demonstrate the inhibitory effects of LiCl on GSK-3
activity in
hepatocytes, we analyzed the levels of
-catenin, a target of
GSK-3
, which is degraded more rapidly after phosphorylation by
GSK-3
(40). Two hours of LiCl treatment significantly
enhanced
-catenin expression, demonstrating an effective inhibition
of GSK-3 by LiCl in hepatocytes (Fig.
1A). To investigate the
potential role of GSK-3 in the TNF-
-induced apoptosis
(19), primary hepatocytes were preincubated with LiCl
followed by treatment with TNF-
. LiCl-pretreated hepatocytes
displayed >90% cell death after 24 h of TNF-
treatment
compared with only 9% cell death in hepatocytes pretreated with NaCl
(Fig. 1B) and displayed typical morphological features of
apoptosis including extensive nuclear fragmentation (Fig.
1C). Additionally, LiCl-pretreated hepatocytes displayed a
DNA fragmentation pattern characteristic for apoptosis. (Fig. 1E). To further confirm that the occurring cell death was
apoptotic, we measured caspase 8-like and caspase 3-like activity
after TNF-
. Compared with NaCl-pretreated hepatocytes,
LiCl-pretreated hepatocytes displayed a 16-fold induction of caspase
8-like and a 27-fold induction of caspase 3-like activity after TNF-
(Fig. 1D).
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LiCl inhibits NF-kB dependent gene transcription.
NF-B is a critical factor in protecting hepatocytes from
apoptosis. Previous studies (19), as well as our
findings that LiCl sensitizes hepatocytes to TNF-
-mediated
apoptosis, suggest that GSK-3 is involved in the regulation of
NF-
B activation. To test this hypothesis, we evaluated the effect of
GSK-3 inhibition on the NF-
B-dependent iNOS gene that is one of
several NF-
B-dependent factors protecting hepatocytes from
apoptosis (17). After 4 h of TNF-
treatment, the mRNA coding for iNOS was significantly induced in NaCl
pretreated hepatocytes (Fig.
2A). In contrast, no iNOS mRNA
was detectable after 4 h of TNF-
treatment in LiCl-pretreated hepatocytes. There was no difference in
-actin expression
between LiCl and NaCl treated hepatocytes (Fig. 2A). To
demonstrate that the effects of LiCl on gene transcription were
solely NF-
B-dependent, we transduced hepatocytes with a reporter
gene in which luciferase transcription is driven by three NF-
B
consensus sites. After 6 h of TNF-
, NF-
B-dependent
transcriptional activity was highly upregulated in NaCl-pretreated
hepatocytes (Fig. 2B). LiCl decreased the TNF-
-induced
reporter gene transcription by 85% confirming that the effects of LiCl
on gene transcription are mediated by its effect on the NF-
B
pathway.
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LiCl does not inhibit IKK activity, IB degradation, and p65
nuclear import or export.
To determine at what level GSK-3 influences NF-
B activation, we
assessed the effects of LiCl at different levels of the NF-
B signaling cascade after TNF-
stimulation. Activation of the IKK complex has been demonstrated to be critical for NF-
B activation and
protection from apoptosis after TNF-
as apparent by massive hepatocyte apoptosis of IKK-
/
mice during
development and sensitization of hepatocytes transduced with dominant
negative IKK-
(25, 26, 38, 41). Therefore, we performed
an immunocomplex kinase assay to assess the potential effect of LiCl on
IKK activation. LiCl-treated hepatocytes displayed a robust activation
of IKK after 10 min of TNF-
treatment that did not differ from
NaCl-treated hepatocytes (Fig.
3A, lanes 2 and
4, respectively). Because LiCl acts as a competitive
inhibitor of GSK-3 (33) and its inhibitory effects may be
lost during the immunoprecipitation and washing, we added LiCl directly
to the kinase reaction. We did not detect a significant direct
influence of LiCl on IKK activity (Fig.
4A, lanes
5-8). After the phosphorylation of I
B-
by the IKK
complex, I
B-
is degraded and releases NF-
B, which translocates
to the nucleus. Therefore, we next analyzed the effects of LiCl on
I
B-
degradation by Western blot analysis. In NaCl-pretreated
hepatocytes, I
B-
was degraded 15 min after TNF-
and was
resynthesized after 60 min (Fig. 3B). In LiCl-treated
hepatocytes, I
B-
degradation occurred in a similar manner, but
the resynthesis of I
B-
was strongly inhibited. Because the
resynthesis of I
B-
is a NF-
B-dependent event due to the
B
site in the I
B-
promoter, the inhibition of I
B-
resynthesis
confirms our initial finding that LiCl inhibits NF-
B activation.
Electrophoretic mobility shift assay revealed a significant increase in
NF-
B DNA binding activity in LiCl-pretreated hepatocytes after
TNF-
with no significant difference compared with NaCl-pretreated
hepatocytes (Fig. 3C). It has been demonstrated that GSK-3
influences nuclear factor of activated T cells (NFAT) and cyclin D1 by
upregulating their nuclear export and degradation (2, 11).
To assess whether nuclear NF-
B import and export differed between
LiCl- and NaCl-pretreated hepatocytes, we performed immunofluorescent
staining for p65 at different times after TNF-
stimulation. In both
LiCl- and NaCl-pretreated hepatocytes, p65 was imported into the
nucleus within the first 30 min after TNF-
(Fig. 3D). One
hour after TNF-
, p65 was still predominantly located in the nucleus
and redistributed to the cytoplasm over the next 2 h in both LiCl-
and NaCl-pretreated hepatocytes.
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GSK-3 phosphorylates p65.
Because LiCl exerted its inhibitory effects on NF-
B activation
independent of IKK activation, I
B-
degradation and NF-
B DNA
binding activity, we determined whether GSK-3
was able to phosphorylate p65. Phosphorylation of p65 has been proposed as the
critical modulator of NF-
B transactivation and may enhance the
binding of transcription factor IIB, TATA-binding protein and
cofactors (37, 45). Analysis of the p65 sequence revealed four GSK-3 sites within the COOH-terminal transactivation domain of p65
(Fig. 4A). We analyzed the capacity of GSK-3 to
phosphorylate p65 by incubating recombinant GSK-3
with different p65
substrates in an in vitro kinase assay. We did not observe
phosphorylation of a GST-p65 (1-305) substrate in
this kinase reaction, but the GST-p65 (354-551)
substrate was strongly phosphorylated by GSK-3
(Fig. 4B,
right). The presence of the GST-p65 substrates was confirmed by Comassie blue staining (Fig. 4B, left).
GSK-3
activity was demonstrated by a significant amount of
autophosphorylation in each kinase reaction (Fig. 4B,
right). The lack of phosphorylation of GST-p65
(1-305) showed that the phosphorylation of the
GST-p65 (354-551) substrate did not occur within the
GST sequence. Importantly, phosphorylation occurred only in the
substrate containing the COOH-terminal domain with the two
transactivation domains. The presence of several bands in the gel
indicates multiple phosphorylation of the GST-p65
(354-551) substrate. Pairwise organization of the GSK
sites (Fig. 4A) may facilitate the observed multiple
phosphorylation, because GSK-3 is believed to act as its own priming
kinase in adjacent sites (16). Similar to some of the
GSK-3 substrates including
-catenin and axin (21),
GST-p65 did not require priming of the substrate by another kinase to
be phosphorylated by GSK-3
.
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DISCUSSION |
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In this study, we demonstrate that 1) inhibition of
GSK-3 downregulates NF-B-dependent gene transcription induced by
TNF-
and sensitizes hepatocytes to TNF-
-mediated
apoptosis, and 2) GSK-3
phosphorylates the COOH
terminus of p65. A recent study has demonstrated that GSK-3
/
mice die of massive hepatocyte apoptosis
during development (19) similar to animals with defects in
the NF-
B pathway (4, 25, 26, 32), suggesting a role for
GSK-3 in regulating NF-
B activity. Although GSK3
/
mouse embryonic fibroblasts (MEFs) had no defect in NF-
B DNA binding activity, they displayed a profound reduction in the transcription of a
NF-
B-dependent reporter gene, suggesting that hepatocytes in these
animals underwent apoptosis during development due to a similar
defect in NF-
B activation (19). Embryological lethality of the GSK3
/
mice prevents studying the effect of
GSK3-
in hepatocytes of these animals. Therefore, we chose to
inhibit GSK-3 pharmacologically by LiCl, an established inhibitor of
GSK-3, which downregulated GSK-3 activity in hepatocytes as
demonstrated by an upregulation of its target
-catenin. Our results
corroborate the data obtained by Hoeflich et al. (19) in
GSK-3
/
MEFs in that NF-
B activity was
impaired only at the transcriptional level in LiCl treated
hepatocytes. Other steps in the NF-
B pathways including the
activation of IKK, I
B degradation, and NF-
B DNA binding activity
remained intact in LiCl-treated hepatocytes. The mechanism by which
GSK-3
might have such a profound influence on NF-
B-dependent
gene transcription was not addressed in previous studies. In this
study, we tried to determine how GSK-3
might affect
NF-
B-dependent transcription in hepatocytes. It has been shown that
besides its role in the Wnt pathway and the regulation of
glycogen homeostasis, GSK-3
phosphorylates several transcription factors including c-Jun, NFAT, CCAAT/enhancer binding proteins (C/EBP-
), and the NF-
B family member p105 (2, 5, 10, 31), as well as the cell cycle regulator cyclin D1
(11). GSK-3
-mediated phosphorylation strikingly alters
the function of these transcription factors as seen by the enhanced
transactivation of C/EBP-
(31), the decreased
transactivation of c-Jun (5) and the increased nuclear
export of NFAT (2). GSK-3
-mediated phosphorylation of
cyclin D1 decreases its stability and enhances its redistribution to
the cytoplasm and consequent degradation (11). To assess the potential effect of GSK-3 inhibition on the subcellular
localization of NF-
B and its stability, we stained hepatocytes for
p65 after various lengths of TNF-
stimulation. Previous studies
(38) have shown that the p50-p65 heterodimer is the main
component of NF-
B binding activity after TNF-
in hepatocytes and
that p65 staining accurately reflects the localization of
the majority of biologically relevant NF-
B. LiCl did
not influence TNF-
-induced p65 translocation or cytoplasmic
redistribution and did not decrease the intensity of p65 staining
compared with NaCl. Accordingly, a recent study has demonstrated that
acetylation is a key mechanism to regulate the duration of the
NF-
B response and the cellular localization of NF-
B
(7).
Because the effects of GSK-3 inhibition by LiCl seemed to be
independent of NF-B localization and DNA binding activity, we tested
whether NF-
B might be a substrate of GSK-3
. Phosphorylation of
the COOH-terminal transactivation domains of p65 is believed to enhance
its transcriptional activity, possibly through enhanced binding of
basal transcription factors, such as transcription factor IIB,
TATA-binding protein and cofactors (37). After TNF-
, IL-1
, or CD40 stimulation (34, 38, 39, 45), p65
is phosphorylated, and this phosphorylation upregulates p65
transcriptional activity (28, 45). Our study demonstrates
that GSK-3
phosphorylates a COOH-terminal p65 peptide but not an
NH2-terminal p65 peptide. GSK-3
-mediated phosphorylation
often requires the priming by another kinase, such as by casein kinase
II or cAMP-dependent protein kinase (8, 12, 13). After
initial priming by other kinases, GSK-3 may act as its own priming
kinase and thus phosphorylate multiple adjacent GSK-3 sites with high
efficiency leading to hyperphosphorylation of certain substrates
(16). GSK-3-mediated phosphorylation may also occur
independently of priming as demonstrated for substrates, such as axin
and
-catenin (21). Recently, distinct residues have
been described to be critical for the phosphorylation of primed and
nonprimed GSK-3 substrates (14) indicating that the
phosphorylation of primed and nonprimed substrates may rely on
two different molecular mechanisms. In some substrates, certain residues are phosphorylated weakly in the absence of a priming kinase
but are phosphorylated much more efficiently after priming (15). Our data demonstrate that phosphorylation of p65 by
GSK-3
does not require priming in vitro. However, the four GSK-3
sites within the transactivation domains of p65 are organized in pairs so that phosphorylation of the second site by GSK-3 may be facilitated through a priming effect after phosphorylation of the first site. This
phenomenon might explain the multiple phosphorylation we observed with
the GST-p65 (354-55) substrate. Combined with our findings that inhibition of GSK-3 downregulates NF-
B-dependent gene
transcription it is very likely that phosphorylation of p65 by GSK-3
enhances its transactivation. GSK-3
might be a highly suitable
kinase for this purpose, because multiple phosphorylation of p65
appears to be advantageous for its transactivation (37). Future studies are needed to further define the functional importance of GSK-3-mediated p65 phosphorylation for NF-
B transactivation.
Our study did not assess whether GSK-3 inhibition by LiCl affects other
GSK-3 targets that might explain the observed sensitization toward
TNF-. c-Jun is a target whose function is downregulated by GSK-3.
Observations from our laboratory (R. F. Schwabe, unpublished observations) and a study from another group (27) show
that the c-Jun NH2-terminal kinase (JNK)/c-Jun pathway
strongly enhances TNF-
-mediated apoptosis in rat hepatocytes.
However, proapoptotic effects of JNK/c-Jun require the inhibition
of NF-
B and make it unlikely that c-Jun is the main target of GSK-3
in our experimental model (9, 27, 42). We also cannot
completely exclude that LiCl affects other systems besides GSK-3 in
hepatocytes through which it may possibly enhance the sensitivity of
hepatocytes toward TNF-
. One such target of LiCl is inositol
monophosphatase 1, but to the best of our knowledge, there is no link
between inositol monophosphatase 1 activity and apoptosis.
Considering the critical role of NF-
B in the protection from
TNF-
-mediated apoptosis (3, 43, 44) and the
necessity of GSK-3
for the protection from hepatocyte
apoptosis during development (19), it is
most likely that the LiCl inhibits GSK-3-dependent NF-
B activation and thereby, sensitizes hepatocytes to TNF-
-mediated apoptosis.
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ACKNOWLEDGEMENTS |
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The authors thank Dr. Hiroaki Sakurai (Tanabe Seiyaku, Osaka,
Japan) for providing GST-p65 plasmids, Dr. John Engelhard (University of Iowa, Iowa City, IA) for providing nuclear factor-B-luciferase adenovirus, and the laboratory of Dr. John Lemasters (University of
North Carolina, Chapel Hill, NC) for providing primary rat hepatocytes.
We thank Carrie Purbeck for excellent technical assistance.
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FOOTNOTES |
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This study was supported, in part, by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-34987 and National Institute of General Medical Sciences Grant GM-41804.
Address for reprint requests and other correspondence: D. A. Brenner, Univ. of North Carolina, Dept. of Medicine, CB #7038, Chapel Hill, NC 27599 (E-mail: dab{at}med.unc.edu).
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.
First published February 20, 2002;10.1152/ajpgi.00016.2002
Received 14 January 2002; accepted in final form 7 February 2002.
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