From the Laboratory of Human Carcinogenesis, NCI,
National Institutes of Health, Bethesda, Maryland 20892 and the
§ Department of Biotechnology, The University of Tokyo,
Tokyo, Japan
Received for publication, February 8, 2001, and in revised form, March 29, 2001
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The leucine-rich nuclear export signal (NES) is
used to shuttle large cellular proteins from the nucleus to the
cytoplasm. The nuclear export receptor Crm1 is essential in this
process by recognizing the NES motif. Here, we show that the oncogenic hepatitis B virus (HBV) X protein (HBx) contains a functional NES
motif. We found that the predominant cytoplasmic localization of HBx is
sensitive to the drug leptomycin B (LMB), which specifically inactivates Crm1. Mutations at the two conserved leucine residues to
alanine at the NES motif (L98A,L100A) resulted in a nuclear redistribution of HBx. A recombinant HBx protein binds to Crm1 in
vitro. In addition, ectopic expression of HBx sequesters Crm1 in
the cytoplasm. Furthermore, HBx activates NF Hepatocellular carcinoma
(HCC)1 is one of the most
prevalent malignant diseases worldwide and hepatitis B virus (HBV) is
the major etiologic factor for HCC (1-3). HBV is a DNA tumor virus, which encodes four open reading frames, S/preS, C/preC, P, and HBx (4).
The oncogenicity of HBV is largely the result of HBx, the
smallest gene encoding a 17-kDa protein (3, 5-8). One major cellular
function of HBx is its promiscuous transcriptional activation activity,
a property that is believed to contribute to its oncogenicity (9). A
wide range of cellular genes can be up-regulated or down-regulated by
HBx (9). However, HBx is localized in the cytoplasm mostly and does not
bind to double-stranded DNA. A "universal" effect of HBx, on
otherwise totally different types of promoters with no obvious
consensus sequence, has led to the hypothesis that HBx may regulate
gene expression by interacting directly with host general transcription
factors (10-12), or indirectly via the activation of protein kinase C
and RAS-RAF mitogen-activated protein kinase (MAPK) signaling pathways
(13-15). Although HBx can induce neoplastic transformation, presumably
by preventing p53-mediated apoptosis (16-18), it also can induce
apoptosis in a p53-dependent or -independent manner (19,
20),2 or sensitize cells to
tumor necrosis factor Close inspection of the HBx sequence revealed a short, hydrophobic,
leucine-rich nuclear export signal motif (NES) (Fig. 1A). An
NES is located in the center region of HBx (residues 89-100). The
center region of HBx is retained in HCC frequently and is essential for
its transactivation (22-24). This region also is conserved among HBx
from different subtypes (Fig. 1A). Several viral proteins
including HIV-1 Rev, HTLV-1 Rex, and adenovirus E4 34-kDa proteins
contain functional NESs (Fig. 1A). Similar to HBx, Rev and
Rex also are potent viral and cellular transactivators with no apparent
DNA binding property (25, 26). In addition, NESs also have been
identified in cellular proteins, many of which are involved in
transcription, cell signaling cascade, oncogenic transformation, and
cell cycle regulators. Examples include protein kinase
inhibitor, MAP kinase kinase (MAPKK), TFIIIA, Mdm2, p53, I In this study, we have investigated the hypothesis that the pleiotropic
effects associated with HBx may be contributed by the presence of a NES
motif, and HBx may activate cellular gene expression and induce
oncogenicity through the modulation of Crm1-mediated functions. We have
identified a functional NES motif in HBx. This motif is necessary for
HBx-induced cytoplasmic sequestration of Crm1, and subsequently, the
nuclear translocation and activation of NF Plasmid Construction--
The plasmid pcDNA3-HBxadr-Hatag
was constructed by the insertion of a C-terminal hemagglutinin (HA)
epitope-tagged full-length HBx into the BamHI and
HindIII sites of a pcDNA3 vector (Invitrogen). GFP-HBx
and GFP-HBx-NESM were constructed as follows: a 529-base pair fragment
released from the digestion of pcDNA3-HBxadr-Hatag with
HindIII/ApaI and inserted into pEGFP-C2
(CLONTECH) at the HindIII/ApaI sites. The resulting GFP-HBx is an
N-terminal fusion of HBx with GFP and a C-terminal fusion with HA tag.
GFP-HBx-NESM was made by a PCR-based site-directed mutagenesis protocol
using GFP-HBx as a template. To generate the HBx-NES mutant
(L98A,G99A,L100A), 2 HBx fragments were amplified by PCR using primer
pairs
(5'-GCTGCTGCATCAGCAATGTCAACAACCGAC-3'/5'-ATGCTCTAGAGGCAGAGGTGAAAAAGTTGCATGG-3' and
5'-TGCAGCAGCAGTCCTCTTATGTAAGAGCTT-3'/5'-TATAAAGCTTGGTACCGAGCTCGGATCTGATGGC-3'). The 2 PCR DNA fragments were denatured and reannealed. Then the HBx-NES
mutant was generated by PCR with primers
(5'-TATAAAGCTTGGTACCGAGCTCGGATCTGATGGC-3' and
5'-ATGCTCTAGAGGCAGAGGTGAAAAAGTTGCATGG-3') and was subcloned into the
pEGFP-C2 vector. Mutations were verified by DNA sequencing. The
GFP-NLS-NES and GFP-NLS-NESM constructs were made by the strategy described previously (34). Briefly, the SV40 NLS motif (residues 125-133) was fused to GFP by the cloning of oligos TNLS1
(5'-TCGAGATCCCCCCAAGAAGAAGCGCAAGGTGGAGCA-3'/TNLS2 5'-AGCTTGCTCCACCTTGCGCTTCTTCTTGGGGGGATC-3') into plasmid pGFPF-C1 (CLONTECH) to generate GFP-NLS. GFP-NES and
GFP-NESM were constructed by cloning oligos XNESW1
(5'-AATTCTCAGGTCTTGCCCAAGCTCTTACATAAGAGGACTCTTGGACTCTCAGCAATG-3')/XNESW2 (5'-GATCCATTGCTGAGAGTCCAAGAGTCCTCTTATGTAAGAGCTTGGGCAAGACCTGAG-3') or
XNESM3
(5'-AATTCTCAGGTCTTGCCCAAGCTCTTACATAAGAGGACTGCTGGAGCCTCAGCAATG-3')- /XNESM4
(5'-GATCCATTGCTGAGGCTCCAGCAGTCCTCTTATGTAAGAGCTTGGGCAAGACCTGAG-3'), respectively, into GFP-NLS, resulting in a GFP-NLS-NES or GFP-NLS-NESM fusion protein. The NES motif corresponds to the HBx residues 87-103,
while NESM is the HBx-NES motif with mutations at the conserved leucine
residues (L98A and L100A). pSVCM contains a GFP-CRM1 fusion gene under
the control of a CMV promoter (28). pNF Cell Culture and Media--
Normal primary human fibroblasts
were obtained from Coriell Institute for Medical Research (Camden, NJ).
These cells were grown in Ham's F-10 medium supplemented with 15%
fetal bovine serum and were used before passage 12. A
telomerase-immortalized human fibroblast line (NHF-hTERT), a gift of
Dr. Judy Campisi, was grown in Dulbecco's modified Eagle's media
supplemented with 10% fetal bovine serum. Normal primary human
hepatocytes were purchased from Clonetics (BioWittaker), grown in LCM
(BioWittaker), and used only at passage 1. Hep3B cells were grown in
Eagle's minimum essential medium containing 10% fetal bovine serum.
Microinjection, Transfection, Luciferase, and Indirect
Immunofluorescence Assays--
Microinjection of NHF cells was done
essentially as described previously (35). Transfection of NHF-hTERT and
Hep3B cells was carried out by the LipofectAMINE Plus reagent based on
the recommended protocol described by the manufacturer (Life
Technologies, Inc., Gaithersburg, MD). For the luciferase reporter
assay, cells were seeded into 6-well dishes (Costar) at 50%
confluence, co-transfected with 0.2 µg of pNFkB-Luc or pNFAT-Luc,
along with 2 µg of each of various HBx constructs, in the presence or
absence of TNF In Vitro Protein-binding Assay--
In vitro binding
between HBx and hCrm1 was determined with the in vitro
translated HBx and hCrm1 (16). The HA-tagged HBx and the hCrm1 proteins
were made by the one-step TnT in vitro transcription and
translation system (Promega), as described previously (16), from
pcDNA-HA-HBx and hCrm1Blue744 vectors (gift of Dr. Gerard
Grosveld), respectively. Aliquots of HBx (25 µl) and hCrm1 (45 µl)
were mixed together in CBBL buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 5 mM MgCl2, 1 mM EDTA, 1 mM dithiothreitol, and 0.1% Nonidet
P-40) in the presence of 0.2 mM GTP Immunohistochemistry Analysis--
A total of 12 samples were
included in this part of the study. Histopathologically, they included
5 normal liver samples, 7 viral hepatitis cases showing grades 1 to 2 activity according to the criteria of Scheuer (36). In addition to H & E light microscopy, tissue sections were cut from frozen samples with a
thickness of 4 µm and mounted on electrically charged glass slides.
The sections were fixed in absolute alcohol and then washed with 2 changes of phosphate-buffered saline for 5 min each. They were then
quenched with 3% hydrogen peroxide to block the endogenous peroxidase
activity for 30 min. Following incubation with 10% horse serum to
block the nonspecific binding, the sections were incubated overnight at
4 °C with anti-CRM1 antibodies. The sections were prepared in
duplicate and two anti-CRM1 antibodies were used, including a goat
anti-Crm1 antibody from Santa Cruz at a 1:10 dilution (Santa Cruz) and
a rabbit anti-Crm1 polyclonal antibody (Gift from Gerard Grosveld) at a
1:100 dilution. A rabbit anti-NF HBx Has a Functional Leucine-rich Nuclear Export Signal--
To
test if HBx-NES functions in nuclear protein export, we first
investigated whether the subcellular localization of HBx is sensitive
to LMB, an antitumor agent that selectively binds to and blocks
Crm1-mediated nuclear export (37, 38). Consistent with previous
findings (18), HBx was localized in the cytoplasm predominately with a
punctate staining pattern in appearance when expressed transiently in
normal human fibroblasts (NHF) (Fig. 1)
and in a HCC-derived cell line (Hep3B) (data not shown). However, LMB
treatment causes an increase in nuclear accumulation of HBx (Fig. 1,
B and C). Next, we used a GFP protein export
reporter assay described previously (34) to further test if the HBx-NES oligopeptide functions in nuclear protein export (34). This reporter
uses a nuclear localization signal (NLS) fused to a GFP, thereby
inducing nuclear localization of the fusion protein. Consistently, the
GFP-NLS reporter was localized in the nucleus of NHF exclusively. However, when an HBx-NES was fused to this reporter, the resulting GFP-NLS-XNES fusion protein can be found in the cytoplasm with a
diffused staining pattern in appearance, although no nuclear exclusion
of this reporter protein could be observed (Fig. 1D). In
contrast, GFP-NLS-XNESM that contains a mutated HBx-NES oligopeptide (L98A,L100A) does not localize in the cytoplasm (Fig. 1D).
To further examine if cytoplasmic localization of a full-length HBx is
dependent on the presence of an NES, a GFP-HBx fusion gene (HBx) or an
NES-mutated GFP-HBx fusion gene (HBxNESM) was constructed. Again,
GFP-HBx was localized in the cytoplasm predominantly with a punctate
pattern in appearance, while HBxNESM was found mainly in the nucleus
with a diffused pattern (Fig. 1E). Taken together, these
data demonstrate that HBx-NES is functional in Crm1-mediated nuclear
protein export.
Interaction between Crm1 and HBx--
Nuclear export receptor Crm1
is known to interact with proteins containing NESs and to mediate
nuclear protein export. The majority of Crm1 protein is found in the
nucleus of normal cells (39). Consistently, every NHF cell displays an
endogenous nuclear-diffused Crm1 distribution (Fig.
2). When HBx was expressed transiently in
NHF, Crm1 was often found in the cytoplasm with a punctate staining
pattern. Approximately 39% ± 2 of the HBx-expressing NHF cells
displayed co-localization of Crm1, and over 99% of cytoplasmic punctate Crm1 signals were co-localized with HBx (Fig. 2A).
The punctate staining patterns containing both Crm1 and HBx signals were not always the same in appearance (Fig. 2A, panels
c-l). These results suggest that the cytoplasmic HBx and Crm1 are
not necessarily associated with a single subcellular component. In contrast, no cytoplasmic co-localization was found in NHF cells expressing HBxNESM. These data indicate that HBx was able to partially retain Crm1 in the cytoplasm and suggest that HBx binds to Crm1 in vivo. Consistently, in vitro translated HBx
can physically interact with in vitro translated Crm1 (Fig.
2B).
HBx-mediated Activation of NF Cytoplasmic Localization of Crm1 Associated with HBV-infected Liver
Samples--
To investigate if Crm1 is sequestrated cytoplasmically in
liver samples from chronic active hepatitis patients with HBV
infection, we performed immunohistochemistry analysis of Crm1 in frozen
tissue sections or in paraffin-embedded sections by anti-Crm1
polyclonal antibody. A total of 12 liver samples with samples from 5 normal individuals were included in this study. The presence of HBV DNA including Core, preS, and HBx genes in these samples was verified by
PCR (data not shown). Among 7 HBV positive cases, liver samples show
chronic active hepatitis with grades 1 to 2 activity (Table I). While 5/5 normal samples revealed
nuclear Crm1 staining exclusively, 6/7 HBV samples showed cytoplasmic
Crm1 staining (Fig. 4 and Table I) in
hepatocytes. An anti-NF We have demonstrated that the oncogenic HBx contains a functional
NES, and that this NES is required for the HBx-mediated activation of
the NF The studies described in this report may offer a common mechanism to
explain the pleiotropic effects of HBx. First, Crm1 transports and
controls many cellular proteins including NF An alternative mechanism for HBx to activate NF HBx is thought to be small enough to diffuse passively through the
nuclear pore complex. The fact that this oncogenic viral protein has
acquired NES activity, is preferentially localized in the cytoplasm,
and modified nuclear export implies that nuclear export may be an
important target for viral-mediated oncogenesis. Interestingly,
cellular oncoproteins such as c-Abl and Mdm2 are known to be involved
in a Crm1-dependent nuclear export. Analogous to viral
hepatitis oncoprotein, the cellular oncoproteins, in principle, may
induce neoplastic transformation also through the disruption of the
Crm1-mediated mechanism. It is known that TPR and CAN are nuclear pore
complex-associated proteins that act as the docking sites and are
essential for Crm1-mediated nuclear export (27). Earlier findings
demonstrated that the TPR and CAN genes are
mutated through translocation in thyroid carcinoma, gastric carcinoma,
and acute myeloid leukemia (46-52). We also found that cytoplasmic
sequestration of Crm1 is associated frequently with HCC (data not
shown). In addition, Crm1 and Ran may play a role in mitosis initiation
(29-31). It is possible that the inactivation of Crm1 and Ran may
induce genomic instability, a predisposing factor for cancer
development. Thus, we postulate that the inactivation of the
Crm1-mediated nuclear export is a common event during viral and
cellular oncogenesis.
B by inducing its
nuclear translocation in a NES-dependent manner. Abnormal cytoplasmic sequestration of Crm1, accompanied by a nuclear
localization of NF
B, was also observed in hepatocytes from
HBV-positive liver samples with chronic active hepatitis. We suggest
that Crm1 may play a role in HBx-mediated liver carcinogenesis.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(TNF
)-induced apoptosis (21). Therefore,
the precise mechanism related to its effector remains unknown and none
of these studies satisfactorily explain the pleiotropic effects
associated with HBx.
B
, NF-AT, cyclin B1, c-Abl, and 14-3-3 (reviewed in Ref.
27). The activities of these proteins are tightly regulated by their NESs. The nuclear export receptor Crm1 and its cofactor Ran GTPase are
essential in this process by recognizing NESs and mediating nuclear
protein export (27, 28). In addition, previous results indicate that
Crm1 may be involved in maintaining chromosomal integrity (29) and Ran
may play a key role in regulating mitosis initiation by stimulating
spindle formation (30, 31). Mutation of the hydrophobic leucine
residues to alanines have been shown to disrupt NES function in a
number of proteins, including HIV-rev, E4 34-kDa, p53, Mdm2, and cyclin
B1 (25, 26, 32, 33).
B. Cytoplasmic retention
of Crm1 also is found in liver samples with chronic active hepatitis
infected with HBV, a condition that is predisposing individuals to the
development of HCC. We suggest that the inactivation of the
Crm1-mediated pathway may be an early step during viral
hepatitis-mediated liver carcinogenesis.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B-Luc, provided by John Brady
(NCI), contains a luciferase gene under the promoter containing 4 NF
B responsive elements.
(10 ng/ml). A total of 0.5 µg of pRL-null or 0.003 µg of pRL-CMV was added into each transfection as an internal
control. pEGFPC1 was used to keep the total amount of plasmid DNA
constant. Cells were then incubated for 24 h prior to harvesting.
The luciferase activity was carried out by the Dual Luciferase Reporter
Assay System (Promega) according to the manufacturer's instructions and were measured in a Monolight 2010 luminometer (Analytical Luminescence Laboratory). The luciferase activity was expressed as fold
activation against the untreated reporter by itself, which was
normalized by Renilla luciferase activity for the transfection efficiency. The reported results represent at least three independent transfections. For immunocytochemistry analysis, cells were fixed in
4% paraformaldehyde in phosphate-buffered saline for 10 min, followed
by methanol for 20 min at 24 h postmicroinjection, and washed in
phosphate-buffered saline-plus (0.15% glycine, 0.5% bovine serum
albumin in phosphate-buffered saline). The GFP fusion proteins can be
visualized directly in a fluorescence microscope equipped with an
fluorescein isothiocyanate filter with or without fixation. The HBx
expression also was verified by an anti-HBx monoclonal antibody (data
not shown). NF
B was detected using an anti-NF
B polyclonal
antibody (1:100) (Santa Cruz) and Crm1 was detected using an anti-Crm1
polyclonal antibody (1:100). Both antibodies were incubated for 1 h at 37 °C. Anti-rabbit IgG conjugated to fluorescein or Texas Red
(Vector Laboratories) was used (1:300) for 1 h at room
temperature. Nuclei were stained with 4,6-diamidino-2-phenylindole. The
following criteria were used to define the subcellular localization, preferentially cytoplasmic localization: protein signals mostly intense
in the cytoplasm; preferentially nuclear localization: protein signals
mostly intense in the nucleus. Some of the slides were analyzed with a
blind fashion to avoid any bias. For GFP-NLS reporter expression, cells
on the same coverslip were microinjected with GFP-NLS, GFP-NLS-XNES, or
GFP-NLS-XNESM construct and incubated for 6 h. Cells were
monitored under a Zeiss Axioskop fluorescence microscope and
representative images were taken using a high-performance CCD imaging
system (IP Lab Spectrum) with the same exposure time. For
co-localization of Crm1 and HBx analysis, cells were co-stained with
anti-HBx and anti-Crm1 antibodies with the corresponding secondary
antibodies conjugated with Texas Red or fluorescein, and analyzed by a
Bio-Rad MRC 1024 confocal system. Sequential excitation at 488 and 568 nm was provided by a krypton-argon gas laser. Emission filters of
598/40 and 522/32 were used for sequentially collecting red and green
fluorescence in channels 1 and 2, respectively. Z-sections were
collected at 0.5-µm intervals for each cell using LaserSharp
software. Images were analyzed by Confocal Assistant software.
S and 80 µl of
anti-HA affinity matrix (Roche Molecular Biochemicals, Indianapolis, IN). The mixture was incubated at 37 °C for 60 min. After
centrifugation, beads were washed four times with CBBL buffer. The
bound proteins were separated on SDS-polyacrylamide gel electrophoresis
and Crm1 was detected by Western blotting using anti-Crm1 antibody and followed by the Amersham ECL system. A 10% of in vitro
translated hCrm1 was loaded in parallel as the input.
B antibody (anti-p65) (Santa Cruz)
was used at a 1:100 dilution for staining NF
B. Biotinylated
secondary antibodies and streptavidin peroxidase complex (LSAB) were
used. Chromogenic development was obtained by the immersion of sections
in 3,3'-diaminobenzidine solution (0.25 mg/ml with 3% hydrogen
peroxide). The slides were counterstained with Harris' hematoxylin and
re-hydrated with alcohol and xylene. Slides were evaluated in a blind fashion.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (19K):
[in a new window]
Fig. 1.
The hepatitis B virus HBx protein contains a
functional NES. A, sequence comparison of the
leucine-rich NESs between 3 subtypes of HBx and other proteins with
known NES function. Dots indicate the critical residues, in
which mutations are known to inactivate the activity of the NESs.
B, effect of LMB on subcellular localization of HBx in
primary human fibroblasts. HBx was expressed transiently in normal
human fibroblasts via microinjection. Following 24 h incubation,
cells were treated with (right panels) or without
(left panels) 4 nM LMB for 2 h. HBx protein
was detected by indirect immunofluorescence staining with monoclonal
anti-HBx antibody and followed by fluorescein isothiocyanate-conjugated
anti-mouse IgG antibody (top panels). Nuclei of the same
cells were stained with 4,6-diamidino-2-phenylindole (DAPI)
(bottom panels). Representative images are shown
(magnification is × 600). C, percent of cells with
cytoplasmic localization of HBx from B. D,
HBx-NES is functional in nuclear export. Several GFP fusion constructs
were expressed transiently in normal primary human fibroblasts. Living
cells were monitored under a fluorescence microscope. A nuclear
localization signal from SV40 T antigen directs GFP to the nucleus
(left panel) while the HBx-NES redistributes it to the
cytoplasm (middle panel). Mutations of leucine to alanine in
NES abolish this activity (right panel). E, the
HBx-NES mutant is localized preferentially in the nucleus. Normal human
fibroblasts were microinjected with the GFP-HBx or GFP-HBx-NESM
expression vector. Following a 24-h incubation, cells were fixed and
stained with 4,6-diamidino-2-phenylindole. The percent of cells in
which the GFP signal was found preferentially in the cytoplasm
(left panel), in the nucleus (middle panel), or
both (evenly distributed; right panel) was
determined. Examples of these images are shown. Data are an average of
three independent experiments.
View larger version (27K):
[in a new window]
Fig. 2.
Interaction between the nuclear export
receptor Crm1 and HBx. A, co-localization of HBx and
Crm1 in the cytoplasm. Normal human primary fibroblasts were
immunostained with anti-Crm1 and detected by an fluorescein
isothiocyanate-conjugated secondary antibody (a, c, g, i,
and k). Cells without (a and b) and
with transient expression of HBx (c-l) were immunostained
with anti-HBx and detected by a Texas Red-conjugated secondary antibody
(d, h, j, and l). The following images are from
the same filed to visualize the co-localization of HBx and Crm1:
panels c-f; panels e and f;
panels g and h; panels i and
j; panels k and l. B, HBx
is able to bind to Crm1 in vitro. In vitro translated hCrm1
was incubated without (lane 2) or with in vitro
translated HA-tagged HBx (lane 3). Following
immunoprecipitation with anti-HA antibody, Crm1 was detected by Western
blot analysis with anti-Crm1 polyclonal antibody. One-tenth of the
in vitro translated hCrm1 protein was loaded in parallel as
input (lane 1).
B Is Dependent on the Presence of
NES--
HBx can activate NF
B and other cellular transcription
factors including NF-AT and MAP kinase cascade, although the mechanism of activation is unknown (14, 40, 41). Because NF
B is inactivated normally through its binding to I
B
which is sequestrated in the
cytoplasm by Crm1-mediated nuclear export (42), we examined whether
HBx-NES is responsible for the activation of NF
B. Over 97% of
normal dividing NHF cells display a predominant localization of NF
B
in the cytoplasm (Fig. 3A). In
contrast, ~55 ± 7% of HBx-expressing NHF cells show a
preferentially nuclear distribution of NF
B, while an NES mutated HBx
(HBxNESM) has lost this activity (Fig. 3, A and
B). Next, we used a luciferase reporter that contains NF
B
responsive elements (pNFkB-Luc) to investigate if the HBx-induced nuclear localization of NF
B correlates with its activity.
pNF
B-Luc was co-transfected with HBx or HBxNESM into an
hTERT-immortalized NHF (NHF-hTERT) or a hepatocellular carcinoma cell
line (Hep3B) in the absence or presence of TNF
, an agent known to
sensitize HBx-mediated transactivation (9). Consistently, HBx alone
induced the luciferase activity weakly which could be further enhanced by the treatment of TNF
in both NHF-hTERT and Hep3B cells. In contrast, HBxNESM is devoid of its ability to increase luciferase activity even in the presence of TNF
(Fig. 3C).
View larger version (17K):
[in a new window]
Fig. 3.
HBx-mediated activation of
NF B is dependent on a functional nuclear
export motif. A, normal primary human fibroblasts were
expressed transiently without (a-c), or with HBx
(d-f) or HBxNESM (g-i) for 24 h. Cells were
fixed and stained with anti-NF
B. Texas Red-conjugated secondary
antibody was used to visualize NF
B (a, d, and
g). The HBx signal was detected directly with an fluorescein
isothiocyanate filter (e and h). Percentages of
cells with nuclear staining of NF
B that are positive for HBx are
shown B. C, a luciferase reporter containing 4 NF
B responsive elements was transfected with HBx or an HBx mutant
(HBxNESM, L98A, G99A, L100A) into a telomerase-immortalized human
fibroblast line (NHF-hTERT) or a hepatocellular carcinoma cell line
(Hep3B). Cells were then treated with or without 3 ng/ml TNF
for
24 h. The luciferase activity was measured and data were
normalized by an internal Renilla luciferase vector.
B antibody was also used to determine the
status of NF
B in these liver samples. Again, normal liver samples
revealed mainly a cytoplasmic localization of NF
B while HBV positive
liver samples showed a nuclear distribution of NF
B. The above
findings provide an in vivo physiological significance for
the presence of a cytoplasmic sequestration of Crm1 accompanied by an
activation of NF
B associated with HBV infection.
Cytoplasmic sequestration of Crm1 and nuclear localization of NFB
associated with HBV-infected liver samples
View larger version (159K):
[in a new window]
Fig. 4.
Cytoplasmic sequestration of Crm1 and
nuclear localization of NF B associated with
HBV-infected liver samples. Frozen sections of a normal (A,
C, and E) and an HBV positive (B, D, and
F) liver samples were analyzed by H & E staining
(A and B), anti-Crm1 antibody staining
(C and D), or anti-NF
B antibody staining
(E and F). Representative images are shown.
Arrows indicate representative cells that are positively
stained by anti-Crm1 or anti-NF
B antibodies. Primary magnifications:
H & E images, ×200; IHC images, ×400.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B signaling cascade. We also have shown that HBx is able to
bind to and sequester Crm1 in the cytoplasm, which correlates with the
nuclear localization and activation of NF
B. Furthermore, liver
samples from HBV-infected patients with chronic active hepatitis, a
condition predisposing individuals for the development of liver cancer,
also display cytoplasmic sequestration of Crm1 and nuclear localization
of NF
B. It is possible that HBx may use a Crm1-dependent
mechanism to modulate cellular gene transcription. Our data provide a
plausible model whereby HBx may induce oncogenicity through the
modulation of the Crm1-mediated pathway.
B, NF-AT, MAPKK, and
p53, whose activities are known to be regulated by HBx (14, 17, 40, 41,
43). Second, Crm1 also regulates cytoplasmic mRNA transport.
Inactivation of Crm1 by HBx, in principle, would alter the stability of
mRNA thereby influencing gene expression. Consistent with this
hypothesis is our findings that HBx is able to sequester Crm1 in the
cytoplasm. The HBx-NES is essential for sequestering Crm1 in the
cytoplasm and for the activation of NF
B. Inactivation of
Crm1-mediated nuclear protein export is known to activate this target
(44). Therefore, the promiscuous transactivation-mediated by HBx may be
a secondary effect resulting from alteration of Crm1 function.
B is through the
degradation of I
B
in the cytoplasm by inducing its
phosphorylation and ubiquitination. However, we did not observe any
detectable degradation of I
B
in cells expressing HBx by Western
blot analysis with anti-I
B
antibody (data not shown). This also
correlates with our results that HBx alone only displays a weak
activation of NF
B that can be enhanced significantly by TNF
(Fig.
3C). It is possible that a small degree of
dissociation of the NF
B-I
B
complex, followed by the
degradation of I
B
induced by HBx in the cytoplasm, may contribute
to the nuclear localization of NF
B. A recent study by Weil and
colleagues (45) indicate that HBx may induce the nuclear import of
NF
B/I
B
by binding to I
B
. Interestingly, the region of
I
B
that is critical for HBx-induced nuclear import of I
B
contains a functional NES (45). These findings are consistent with our
data that HBx-induced nuclear localization of NF
B/I
B
is
dependent on the I
B
-NES.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. Gerard Grosveld for generous
gifts of anti-Crm1 antibody and the Crm1 cDNA, Dr. Barbara Wolff
for the leptomycin B, Dr. John Brady for the pNFB-Luc plasmid, Dr.
Judy Campisi for the NHF-hTERT cell line, and Dr. Rodney Markin for the
human liver samples. We are grateful to Dr. Curtis Harris and members of his group for scientific support, Susan Garfield for assistance on
the confocal imaging analysis, and Dorothea Dudek for excellent editorial assistance.
![]() |
FOOTNOTES |
---|
* This work was supported by the Intramural Research Program of the National Cancer Institute.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom reprint requests should be addressed: LHC, NCI, National Institutes of Health, 37 Convent Dr., MSC 4255, Bldg. 37, Rm. 2C07, Bethesda, MD 20892-4255. E-mail: xin_wei_wang@nih.gov.
Published, JBC Papers in Press, April 3, 2001, DOI 10.1074/jbc.M101259200
2 M. Forgues, A. J. Marrogi, E. A. Spillare, C-G. Wu, Q. Yang, M. Yoshida, and X. W. Wang, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
HCC, hepatocellular
carcinoma;
NES, nuclear export signal;
NLS, nuclear localization
signal;
HBx, hepatitis B viral X protein;
HBV, hepatitis B virus;
NHF, normal human fibroblasts;
LMB, leptomycin B;
MAPK, mitogen-activated
protein kinase;
TNF, tumor necrosis factor-
;
HA, hemagglutinin;
PCR, polymerase chain reaction;
GFP, green fluorescent protein;
GTP
S, guanosine 5'-3-O-(thio)triphosphate.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Di Bisceglie, A. M., Rustgi, V. K., Hoofnagle, J. H., Dusheiko, G. M., and Lotze, M. T. (1988) Ann. Intern. Med. 108, 390-401[Medline] [Order article via Infotrieve] |
2. |
Montesano, R.,
Hainaut, P.,
and Wild, C. P.
(1997)
J. Natl. Cancer Inst.
89,
1844-1851 |
3. | Brechot, C., Gozuacik, D., Murakami, Y., and Paterlini-Brechot, P. (2000) Semin. Cancer Biol. 10, 211-231[CrossRef][Medline] [Order article via Infotrieve] |
4. | Ganem, D., and Varmus, H. E. (1987) Annu. Rev. Biochem. 56, 651-693[CrossRef][Medline] [Order article via Infotrieve] |
5. | Unsal, H., Yakicier, C., Marcais, C., Kew, M., Volkmann, M., Zentgraf, H., Isselbacher, K. J., and Ozturk, M. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 822-826[Abstract] |
6. | Paterlini, P., Poussin, K., Kew, M., Franco, D., and Brechot, C. (1995) Hepatology 21, 313-321[Medline] [Order article via Infotrieve] |
7. | Robinson, W. S. (1999) in Microbes and Malignancy: Infection as a Cause of Human Cancers (Parsonnet, J., ed) , pp. 232-266, Oxford University Press, New York |
8. | Kim, Y. C., Song, K. S., Yoon, G., Nam, M. J., and Ryu, W. S. (2001) Oncogene 20, 16-23[CrossRef][Medline] [Order article via Infotrieve] |
9. | Andrisani, O. M., and Barnabas, S. (1999) Int. J. Oncol. 15, 373-379[Medline] [Order article via Infotrieve] |
10. | Cheong, J. H., Yi, M., Lin, Y., and Murakami, S. (1995) EMBO J. 14, 143-150[Abstract] |
11. | Qadri, I., Maguire, H. F., and Siddiqui, A. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 1003-1007[Abstract] |
12. |
Qadri, I.,
Conaway, J. W.,
Conaway, R. C.,
Schaack, J.,
and Siddiqui, A.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
10578-10583 |
13. | Kekulë, A. S., Lauer, U., Weiss, L., Luber, B., and Hofschneider, P. H. (1993) Nature 361, 742-745[CrossRef][Medline] [Order article via Infotrieve] |
14. |
Benn, J.,
and Schneider, R. J.
(1994)
Proc. Natl. Acad. Sci. U. S. A.
91,
10350-10354 |
15. | Wang, H. D., Trivedi, A., and Johnson, D. L. (1997) Mol. Cell. Biol. 17, 6838-6846[Abstract] |
16. | Wang, X. W., Forrester, K., Yeh, H., Feitelson, M. A., Gu, J. R., and Harris, C. C. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 2230-2234[Abstract] |
17. | Wang, X. W., Gibson, M. K., Vermeulen, W., Yeh, H., Forrester, K., Sturzbecher, H. W., Hoeijmakers, J. H. J., and Harris, C. C. (1995) Cancer Res. 55, 6012-6016[Abstract] |
18. |
Elmore, L. W.,
Hancock, A. R.,
Chang, S. F.,
Wang, X. W.,
Chang, S.,
Callahan, C. P.,
Geller, D. A.,
Will, H.,
and Harris, C. C.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
14707-14712 |
19. |
Chirillo, P.,
Pagano, S.,
Natoli, G.,
Puri, P. L.,
Burgio, V. L.,
Balsano, C.,
and Levrero, M.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
8162-8167 |
20. |
Kim, H.,
Lee, H.,
and Yun, Y.
(1998)
J. Biol. Chem.
273,
381-385 |
21. |
Su, F.,
and Schneider, R. J.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
8744-8749 |
22. |
Kumar, V.,
Jayasuryan, N.,
and Kumar, R.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
5647-5652 |
23. | Ritter, S. E., Whitten, T. M., Quets, A. T., and Schloemer, R. H. (1991) Virology 182, 841-845[Medline] [Order article via Infotrieve] |
24. | Takada, S., and Koike, K. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 5628-5632[Abstract] |
25. | Fischer, U., Huber, J., Boelens, W. C., Mattaj, I. W., and Luhrmann, R. (1995) Cell 82, 475-483[Medline] [Order article via Infotrieve] |
26. |
Dobbelstein, M.,
Roth, J.,
Kimberly, W. T.,
Levine, A. J.,
and Shenk, T.
(1997)
EMBO J.
16,
4276-4284 |
27. | Ullman, K. S., Powers, M. A., and Forbes, D. J. (1997) Cell 90, 967-970[Medline] [Order article via Infotrieve] |
28. | Fornerod, M., Ohno, M., Yoshida, M., and Mattaj, I. W. (1997) Cell 90, 1051-1060[Medline] [Order article via Infotrieve] |
29. | Adachi, Y., and Yanagida, M. (1989) J. Cell Biol. 108, 1195-1207[Abstract] |
30. |
Wilde, A.,
and Zheng, Y.
(1999)
Science
284,
1359-1362 |
31. |
Ohba, T.,
Nakamura, M.,
Nishitani, H.,
and Nishimoto, T.
(1999)
Science
284,
1356-1358 |
32. |
Roth, J.,
Dobbelstein, M.,
Freedman, D. A.,
Shenk, T.,
and Levine, A. J.
(1998)
EMBO J.
17,
554-564 |
33. |
Toyoshima, F.,
Moriguchi, T.,
Wada, A.,
Fukuda, M.,
and Nishida, E.
(1998)
EMBO J.
17,
2728-2735 |
34. | Stade, K., Ford, C. S., Guthrie, C., and Weis, K. (1997) Cell 90, 1041-1050[Medline] [Order article via Infotrieve] |
35. | Wang, X. W., Vermeulen, W., Coursen, J. D., Gibson, M., Lupold, S. E., Forrester, K., Xu, G., Elmore, L., Yeh, H., Hoeijmakers, J. H. J., and Harris, C. C. (1996) Genes Dev. 10, 1219-1232[Abstract] |
36. | Scheuer, P. J. (1991) J. Hepatol. 13, 372-374[Medline] [Order article via Infotrieve] |
37. | Wolff, B., Sanglier, J. J., and Wang, Y. (1997) Chem. Biol. 4, 139-147[Medline] [Order article via Infotrieve] |
38. | Kuerbitz, S. J., Plunkett, B. S., Walsh, W. V., and Kastan, M. B. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 7491-7495[Abstract] |
39. |
Kudo, N.,
Khochbin, S.,
Nishi, K.,
Kitano, K.,
Yanagida, M.,
Yoshida, M.,
and Horinouchi, S.
(1997)
J. Biol. Chem.
272,
29742-29751 |
40. | Meyer, M., Caselmann, W. H., Schluter, V., Schreck, R., Hofschneider, P. H., and Baeuerle, P. A. (1992) EMBO J. 11, 2991-3001[Abstract] |
41. |
Cross, J. C.,
Wen, P.,
and Rutter, W. J.
(1993)
Proc. Natl. Acad. Sci. U. S. A.
90,
8078-8082 |
42. |
Tam, W. F.,
Lee, L. H.,
Davis, L.,
and Sen, R.
(2000)
Mol. Cell. Biol.
20,
2269-2284 |
43. |
Lara-Pezzi, E.,
Armesilla, A. L.,
Majano, P. L.,
Redondo, J. M.,
and Lopez-Cabrera, M.
(1998)
EMBO J.
17,
7066-7077 |
44. |
Rodriguez, M. S.,
Thompson, J.,
Hay, R. T.,
and Dargemont, C.
(1999)
J. Biol. Chem.
274,
9108-9115 |
45. |
Weil, R.,
Sirma, H.,
Giannini, C.,
Kremsdorf, D.,
Bessia, C.,
Dargemont, C.,
Brechot, C.,
and Israel, A.
(1999)
Mol. Cell. Biol.
19,
6345-6354 |
46. | von Lindern, M., van Baal, S., Wiegant, J., Raap, A., Hagemeijer, A., and Grosveld, G. (1992) Mol. Cell. Biol. 12, 3346-3355[Abstract] |
47. | von Lindern, M., Fornerod, M., van Baal, S., Jaegle, M., de Wit, T., Buijs, A., and Grosveld, G. (1992) Mol. Cell. Biol. 12, 1687-1697[Abstract] |
48. | Greco, A., Pierotti, M. A., Bongarzone, I., Pagliardini, S., Lanzi, C., and Della Porta, G. (1992) Oncogene 7, 237-242[Medline] [Order article via Infotrieve] |
49. | Park, M., Dean, M., Cooper, C. S., Schmidt, M., O'Brien, S. J., Blair, D. G., and Vande Woude, G. F. (1986) Cell 45, 895-904[Medline] [Order article via Infotrieve] |
50. | Soman, N. R., Correa, P., Ruiz, B. A., and Wogan, G. N. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 4892-4896[Abstract] |
51. |
Fornerod, M.,
van Deursen, J.,
van Baal, S.,
Reynolds, A.,
Davis, D.,
Murti, K. G.,
Fransen, J.,
and Grosveld, G.
(1997)
EMBO J.
16,
807-816 |
52. |
Bangs, P.,
Burke, B.,
Powers, C.,
Craig, R.,
Purohit, A.,
and Doxsey, S.
(1998)
J. Cell Biol.
143,
1801-1812 |