From the Cell and Cancer Biology Branch,
Center for Cancer Research, NCI, National Institutes of
Health, Rockville, Maryland 20850 and the § Medical
Oncology Clinical Research Unit, CCR, NCI, National Institutes of
Health, Bethesda, Maryland 20892
Received for publication, September 24, 2002, and in revised form, December 11, 2002
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ABSTRACT |
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Hypoxia-inducible factor (HIF)-1 Hypoxia-inducible factor
(HIF-1)1 is a heterodimeric
transcription factor composed of the basis helix-loop-helix-PAS -domain containing proteins HIF-1 One of the major components of the cytoskeleton is the microtubule
network. Because of the dynamic instability of tubulin dimers,
microtubules are subject to constant remodeling (13). MDAs are potent
anti-tumor agents that associate with microtubules and disrupt the
microtubular system, thereby blocking cell division (14-16). The
action of MDAs is thought to loosely mimic a wide range of cellular
responses involving cytoskeletal rearrangement, such as wound healing,
tumor cell metastasis, and invasion (17, 18). Microtubule
reorganization has been also shown to correlate with changes in gene
expression (19-21). For instance, it has been reported that
microtubule disruption by MDAs modulates gene expression and activity
of protein kinases and transcription factors such as NF NF The pathways involved in the nonhypoxic stabilization of HIF-1 Reagents--
The MDAs vinblastine, colchicine, and nocodazole,
and the microtubule stabilizing agent paclitaxel were purchased from
Sigma. The colchicine deriviative Cells and Transient Transfection--
A549 cells (human lung
cancer cell line obtained from American Type Culture Collection) were
cultured in F12-K (Kaighn's modification) medium (Invitrogen).
Colchicine-resistant CHO (10193) and wild type CHO (10001) (a gift from
Dr. M. M. Gottesman, National Institutes of Health) were grown in
Western Blotting--
Cells were lysed and nuclear and cytosolic
extracts prepared as described (42). Cell lysates were
electrophoretically separated using either 4-20 or 7.5% SDS-PAGE gels
(BioRad). Proteins were transferred to nitrocellulose membrane
(Protran, Schleicher & Schuell) and imunoblottted with either
monoclonal anti-HIF-1 Quantitative RT-PCR Analysis for HIF-1 MDAs Induce HIF-1
To determine whether the effect of MDAs on HIF-1 MDAs Induce Both NF MDA-dependent HIF-1 Induction Is Dependent upon New
Transcription and NF
We next examined whether the NF
In addition to the NF MDA-induced HIF-1
We reasoned that the ability of MDAs to increase the population of
transcriptionally active HIF-1 would result in the up-regulation of
HIF-1 target proteins. Therefore, we examined whether the
MDA-dependent increase in HIF-1 reporter activity
correlated with an increase in endogenous iNOS protein, which is known
to be a transcriptional target of HIF-1 Microtubule Disruption Is Required for MDA-mediated HIF-1
Induction--
While the dependence for NF MDAs Up-regulate HIF-1
One of the main regulators of HIF-1 In this report, MDAs were used to simulate cellular responses
activated by microtubule change. We demonstrate that cytoskeletal alteration mediated by a variety of microtubule-depolymerizing agents
elevate protein levels of transcriptionally active HIF-1 Our data conclusively demonstrate that transcription, likely
mediated by NF Several reports demonstrate that drugs capable of microtubule
disruption elevate NF Our data demonstrate that the MDA-mediated pathway for HIF-1 Tumor cell invasion and metastasis, hallmarks of the tumorigenic
process, involve microtubule reorganization. We demonstrate that
MDA-mediated activation of NF levels are
elevated in normoxic cells undergoing physiological processes involving
large scale microtubule reorganization, such as embryonic development, wound healing, and tumor cell metastasis. Although alterations in
microtubules affect numerous cellular responses, no data have yet
implicated microtubule dynamics in HIF-1
regulation. To
investigate the effect of microtubule change upon HIF-1
regulation, we treated cells with the microtubule-depolymerizing agents
(MDAs) colchicine, vinblastine or nocodazole. We demonstrate that these
agents are able to induce transcriptionally active HIF-1. MDA-mediated
induction of HIF-1
required microtubule depolymerization, since
HIF-1
levels were unchanged in cells treated with either the
microtubule-stabilizing agent paclitaxel, or an inactive form of
colchicine, or in colchicine-resistant cells. HIF-1 induction was
dependent upon cellular transcription, as transcription inhibitors
abrogated HIF-1
protein up-regulation. The ability of
transcriptional inhibitors to interfere with HIF-1
accumulation was
specific to the MDA-initiated pathway, as they were ineffective in
preventing hypoxia-mediated HIF-1 induction, which occurs by a distinct
post-translational pathway. Moreover, we provide evidence implicating a
requirement for NF
B transcription in the HIF-1 induction mediated by
MDAs. The ability of MDAs to induce HIF-1
is dependent upon
activation of NF
B, since inhibition of NF
B either
pharmacologically or by transfection of an NF
B super-repressor
plasmid abrogated this induction. Collectively, these data
support a model in which NF
B is a focal point for the convergence of
MDA-mediated signaling events leading to HIF-1 induction, thus
revealing a novel aspect of HIF-1
regulation and function.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
and aryl hydrocarbon receptor nuclear translocator (ARNT, HIF-1
) (1). HIF-1
and HIF-1
mRNAs are constantly expressed under normoxic and hypoxic conditions (2). However, HIF-1
protein is significantly increased by hypoxia, whereas the HIF-1
protein remains constant regardless of oxygen tension (3). Under normoxia, HIF-1
protein is remarkably unstable and its degradation by the proteasome is orchestrated by the ubiquitin protein ligase VHL (3-6). Under normoxia, VHL recognizes HIF-1
as a
substrate due to the enzymatic modification of HIF-1
by prolyl
hydroxylases, whose function is inhibited during hypoxia (7, 8).
Hypoxic stabilization of HIF-1
is accompanied by its nuclear
translocation, heterodimerization with HIF-1
, and transcription of
genes encoding proteins that function to increase O2
delivery, allow metabolic adaptation, and promote cell survival (9).
HIF-transactivated genes such as iNOS, IGF, and VEGF play an important
role in tumor metastasis and invasion (10, 11) and HIF-1
protein is
overexpressed in a majority of nonhypoxic metastasic tumors and cell
lines (12).
B
(22-28).
B is an ubiquitous transcription factor known to be activated by a
wide variety of stimuli including infection, inflammation, oxidative
stress, and the aforementioned microtubule disruption (29). NF
B
transactivates a number of proinflammatory, apoptotic and oncogenic
genes that collectively function to foster cellular adaptation to
stress (29, 30). Although the mechanism of activation depends on the
stimulus, most stimuli initiate various intracellular signaling
cascades that result in the phosphorylation of inhibitory protein
B
(I
B) by I
B kinases (IKKs) (31). NF
B is normally associated
with I
B in the cytoplasm, where it is kept in an inactive state
(32). Stimulus-mediated phosphorylation and subsequent proteolytic
degradation of I
B (33, 34) allows the release and nuclear
translocation of NF
B, where it transactivates a number of target genes.
remain unclear but are thought to be regulated by growth factor signaling cascades such as PI 3-kinase/AKT (35, 36). Interestingly, HIF-1
has been reported to be highly expressed in cells during physiological processes that entail massive microtubule reorganization (12, 37-39). However, there are no reports demonstrating a direct relationship between changes in microtubule dynamics and HIF-1
protein regulation. Therefore, in this study, we specifically investigated this connection. We show that reagents interfering with
tubulin polymerization are able to induce NF
B transcription and we
further show that this activation is necessary for the subsequent
increase in HIF-1
protein expression. These results demonstrate a
novel aspect of HIF-1
regulation and suggest that HIF-1
may play
a broader role in sensing cytoskeletal change.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-lumicolchicine and
pyrrolidinedithiocarbamate and the transcriptional inhibitors
actinomycin D (AcD) and 5,6-dichlorobenzimidazole riboside were also
purchased from Sigma. Cobalt chloride and the iron chelator
phenanthroline were obtained from the same supplier. The protein
synthesis inhibitor cycloheximide (CHX) was from Sigma.
-modified minimal essential medium (Sigma). Jurkat cells were
cultured in RPMI 1640 medium (Biofluids). Unless specified, all other
cell lines were grown in Dulbecco's modified Eagle's medium (DMEM,
Biofluids). Media were supplemented with 10% fetal bovine serum,
glutamine (for DMEM), Hepes, and penicillin/streptomycin. For transient
transfections of NF
B super-repressor plasmid (40) or HIF-1
plasmids (41), cells were plated in 6-cm dishes and transfected with
NF
B super-repressor plasmid (5 ug) or HIF-1
plasmids (3 ug) in
the presence of FuGENE 6 (Roche Molecular Biochemicals). After 24 h, cells were subjected to the indicated drug treatments, lysates were
harvested, and HIF-1
levels determined by Western blotting. HIF-1
protein stability was determined by treatment of cells with 200 µM CHX, followed by immunoblot and densitometric analysis. For transient transfection of reporter plasmids, cells were
plated in 12-well plates and the following day cells were cotransfected
with luciferase reporter plasmids containing either 3× NF
B binding
sites (0.4 µg, a gift from Dr. M. Birrer, NCI) or 3× hypoxia
response element (0.4 µg, a gift from Dr. G. Melillo, NCI), in
combination with the internal control CMV Renilla luciferase plasmid (1:100 the amount of reporter plasmid, Promega). Luciferase activities of reporter plasmids were measured using the Dual-Luciferase Reporter Assay System (Promega). Transfection efficiency was evaluated using green fluorescence protein expression plasmid and determined to
be 35-45% under these experimental conditions. Cell viability was
determined by the trypan blue exclusion method. Cell viability was
unchanged in each experimental condition.
, polyclonal anti-iNOS antibodies (1:300 and
1:500, respectively, Transduction Laboratories), monoclonal anti-HA
antibody (1:1000, Covance) or monoclonal anti-
-tubulin antibody
(1:2000, Santa Cruz Biotechnology). HIF-1
in human cell lines was
detected in 20 µg of nuclear extracts and HIF-1
in non-human cell
lines was detected in 30-40 µg of nuclear extracts using monoclonal
HIF-1
antibody (1:750, Novus). iNOS protein was detected in 40 µg
of cytosolic extracts. All blots were developed with SuperSignal chemiluminescence substrate (Pierce) using anti-mouse horseradish peroxidase IgG (Amersham Biosciences).
Expression--
Cells
were treated with MDAs for 3 h and lysed and total mRNA was
extracted using Rneasy Mini Kit (Qiagen). The real-time quantification
of HIF-1
mRNA was carried out using SYBR Green I dye (Applied
Biosystems) with the following primer pairs: human HIF-1
forward,
5'-TCCAGTTACGTTCCTTCGATCA-3'; human HIF-1
reverse, 5'-TTTGAGGACTTGCGCTTTCA-3'. SYBR Green I, double-stranded DNA binding
dye, was detected using the laser-based ABI Prism 7700 Sequence
Detection System (Applied Biosystems). PCR amplification was performed
using an optical 96-well reaction plate and caps. The final reaction
mixture of 25 µl consisted of 200 nM each primer, 1×
SYBR Green PCR Master Mix (Applied Biosystems) containing a reference
dye, and cDNA at the following conditions: 50 °C for 2 min,
95 °C for 10 min, followed by 40 cycles at 95 °C for 15 s
and 60 °C for 1 min. The cDNAs were prepared from each RNA
sample using a TaqMan Reverse Transcription kit (Applied Biosystems).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
in Various Cell Lines under
Normoxia--
Overexpression of HIF-1
and large scale changes in
microtubule organization are common events associated with embryonic
development, wound healing, and tumor cell invasion and metastasis. We
therefore sought to investigate whether a correlation existed between
microtubule disruption and HIF-1
expression. To examine this
question, A549 cells were treated with the MDAs vinblastine,
colchicine, or nocodazole, or with the microtubule-stabilizing agent
paclitaxel, and HIF-1
protein expression was monitored. The
concentrations used represent those required for maximal microtubule
disruption (43). As shown in Fig.
1A, HIF-1
protein levels
were similarly induced by all of the MDAs tested, while HIF-1
protein remained unchanged following treatment with paclitaxel,
indicating that increased HIF-1
protein expression correlated with
microtubule depolymerization, and not with stabilization. In Fig.
1B, the kinetics of vinblastine-mediated HIF-1
induction
were investigated. The data indicate that the increase in HIF-1 levels
is somewhat transient, with maximal induction occurring between 4-5 h
and rapidly declining by 7 h.
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Fig. 1.
MDAs induce HIF-1 in
various cell lines under normoxia. A, A549 cells were
treated with vinblastine (vin, 0.1 µM),
colchicine (col, 1 µM), nocodazole
(noco, 10 µM), or paclitaxel
(taxol, 1 µM) for 4 h. Following these
treatments, nuclear extracts (20 µg) were prepared, and Western
blotting was performed as described under "Materials and Methods."
B, A549 cells were treated with vinblastine for the
indicated times and HIF-1 levels were assessed in nuclear extracts.
Topoisomerase II was used as an internal loading control. C,
MCF-7, Jurkat, or NIH 3T3 cells were treated with vinblastine (0.1 µM; 1 µM for NIH 3T3 cells) or colchicine
(1 µM; 5 µM for NIH-3T3 cells). Following
these treatments, nuclear extracts were prepared, and Western blotting
was performed as in A.
expression was a
general phenomenon, we assessed the ability of MDAs to induce HIF-1
levels in a variety of cell lines. As shown in Fig. 1C, MDAs
induced HIF-1
protein in cells derived from multiple lineages,
irrespective of tumorigenicity or cell adherence, thereby demonstrating
that this is a general signaling pathway shared by many, if not all,
cell types.
B-dependent Transcription and
Up-regulation of HIF-1
Protein--
We previously found that the
inflammatory cytokines TNF-
and IL-1
induce HIF-1
protein
expression via NF
B
activation.2 Coincidentally,
MDAs are reported to activate NF
B gene transcription (24).
Therefore, we wished to determine whether the HIF-1 induction following
MDA treatment was potentially mediated by an
NF
B-dependent pathway. First, we tested whether MDAs
were capable of inducing NF
B activation in A549 cells, as assessed
with transiently transfected NF
B-responsive luciferase constructs.
As shown in Fig. 2A, the MDAs,
but not paclitaxel, which promotes microtubule polymerization and
stabilization, induced NF
B-responsive luciferase activity. Interestingly, MDA-induced NF
B activity correlated with the ability of these agents to induce HIF-1
protein levels (Fig. 1). We
therefore explored the apparent correlation between MDA-induced NF
B
activation and HIF-1
protein induction. To investigate this
association, transiently transfected A549 cells were treated with
increasing concentrations of either vinblastine or colchicine and
NF
B activity was measured in parallel with HIF-1
protein
expression. As shown in Fig. 2B, treatment of A549 cells
with these agents resulted in a maximal level of NF
B activity,
followed by a decline in activity at higher concentrations. When
HIF-1
protein levels were examined from identically treated cells,
the MDA-dependent increase in NF
B activity mirrored the
increase in HIF-1
levels and maximal HIF-1
expression correlated
with maximal NF
B activity. Similarly, at higher doses, HIF-1
protein levels declined in parallel with decreasing NF
B
activity.
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Fig. 2.
MDAs induce both NFkB-dependent
transcription and HIF-1 up-regulation.
A, A549 cells were cotransfected with
NF
B-dependent luciferase plasmid (0.4 ug) and CMV
Renilla luciferase plasmid for 6 h. Subsequently, cells
were treated with vinblastine (0.1 µM), colchicine (1 µM), nocodazole (10 µM), and paclitaxel (1 µM). Reporter activities were measured 6 h later and
normalized to CMV Renilla luciferase activity. Data
represent the mean of three separate experiments. B, a 549 cells were cotransfected as in A, and reporter activities
were similarly measured at the indicated concentrations of vinblastine
or colchicine. For detection of HIF-1
protein, cells were treated
with varying concentrations of vinblastine or colchicine, as indicated,
and nuclear extracts were immunoblotted for HIF-1
as in Fig 1.
B Activation--
We sought to determine
whether the NF
B activation induced by MDAs was responsible for
mediating the increase in HIF-1
protein. First, we examined the
requirement for general transcription in the MDA pathway. Cells were
treated with MDAs in the presence of the transcription inhibitors AcD
or 5,6-dichlorobenzimidazole riboside (DCB) and HIF-1
levels were
examined. As shown in Fig. 3A
(upper panel), HIF-1 induction by MDAs was completely
inhibited by either of these agents. To confirm that the effect of
transcriptional inhibitors upon HIF-1
levels was specific for the
MDA-mediated pathway, these agents were added in combination with
hypoxia mimetics. Cobalt chloride and phenanthroline (an iron chelator)
are known to stabilize HIF-1
by preventing prolyl hydroxylases from
modifying the protein, thereby rescuing HIF-1
from the destabilizing
effects of VHL. As shown in Fig. 3A (lower
panel), the transcriptional inhibitors did not interfere with the
ability of hypoxia mimetics to induce HIF-1
protein. This result
demonstrates a requirement for cellular transcription in MDA-mediated
HIF-1 induction, thus defining this pathway as distinct from the
hypoxia-mediated pathway.
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Fig. 3.
MDA-dependent HIF-1 induction is
dependent upon new transcription and NFkB activation.
A, upper panel, A549 cells were pretreated for 20 min with the transcription inhibitors actinomycin D (AcD,10
µM) or dichlorobenzimidazole riboside (DCB, 70 µM) before a 4-h treatment with MDAs, as indicated, and
HIF-1 expression was monitored in nuclear extracts. Lower
panel, A549 cells were pretreated with the same transcription
inhibitor before treatment with either cobalt chloride (100 µM) or phenanthroline (phe, 200 µM) and HIF-1
levels were similarly detected.
B, upper panel, A549 cells were transfected with
NF
B super-repressor plasmid (NFRP, 5 ug) for 24 h
followed by treatment with either vinblastine or colchicine and
HIF-1
levels were detected. Lower panel, A549 cells were
pretreated for 30 min with the NF
B inhibitor pyrrolidine
dithiocarbamate (PDTC, 100 µM) followed by
treatment with the hypoxia mimetic phenanthroline, or vinblastine or
colchicine, as indicated. Cells were lysed 4 h later and HIF-1
immunoblots were performed using nuclear extract. C, A549
cells were treated with vinblastine for the indicated times. For the
combination treatments of vinblastine and actinomycin D, cells were
first pretreated with vinblastine for 3 h, followed by treatment
with actinomycin D for either 1 or 2 additional hours (in the continued
presence of vinblastine). D, wild type MCF-7 cells and MCF-7
cells stably transfected with dominant negative c-Jun construct
(MCF-7/dn c-Jun) were treated with the indicated MDAs for 4 h and
HIF-1
expression was examined.
B pathway was specifically implicated
in the MDA-mediated induction of HIF-1
. To test this, A549 cells
were transiently transfected with an NF
B super-repressor plasmid
(expressing I
B mutated to resist proteasome-mediated degradation)
that effectively inhibits NF
B transcription (40), and these cells
were subsequently subjected to treatment with MDAs. As shown in Fig.
3B (upper panel) MDA-mediated HIF-1 induction is
severely impaired in the presence of NF
B repressor, thereby demonstrating a requirement for NF
B transcription in this pathway. To ensure that this result was not a nonspecific effect related to the
transfections, the NF
B-inhibiting drug pyrrolidine dithiocarbamate (PDTC) was used to confirm these observations. As shown in Fig. 3B (lower panel), similar to the NF
B repressor
effects, PDTC abrogated MDA-mediated HIF-1 induction. However, it had
no effect upon the ability of hypoxia mimetics to induce HIF-1
,
thereby validating the specificity of this pathway and emphasizing the crucial role of NF
B. To examine the effect of transcriptional inhibition upon MDA-stabilized HIF-1
protein, A549 cells were pretreated with vinblastine for 3 h, followed by either a 1 or 2 h treatment of actinomycin D (in the continued presence of
vinblastine). As shown in Fig 3C, transcription inhibition
was able to moderately reduce, but not eliminate, already stabilized
HIF-1 protein. These data demonstrate that while constitutive
transcription is needed for MDA-mediated up-regulation of HIF-1
,
persistence of the stabilized protein is not dependent on transcription.
B pathway, it has been reported that MDAs can
also activate the transcription factor AP-1 in a
c-Jun-dependent manner (27). Therefore, we tested the
potential contribution of AP-1 in the MDA-mediated HIF-1 induction
using MCF-7 cells stably transfected with a dominant negative c-Jun
construct (dn c-Jun) that inhibits c-Jun-dependent AP-1
activity (44). As shown in Fig. 3D, cells expressing dn
c-Jun induced HIF-1
in response to MDAs to a degree comparable with
wild type cells, thereby discounting an involvement of
c-Jun-dependent AP-1 activity in this process.
Protein Is Transcriptionally Active--
It
was of interest to determine whether the HIF-1
protein induced by
MDAs was transcriptionally active. To test this, A549 cells were
transfected with a HIF-1 responsive reporter plasmid that contains 3 hypoxia response elements of the iNOS gene (45). As shown in
Fig. 4A, consistent with our
Western results (Fig. 1), HIF-1-dependent luciferase
activity was induced by treatment with MDAs. To confirm that HIF-1
reporter activity was induced in a HIF-1-specific manner, this
experiment was repeated in hepa1c1c7 cells that contain wild type Arnt,
and in matched hepa1c4 cells that are unable to transactivate
HIF-1-dependent genes due to a genetic defect in Arnt (46,
47). First, we determined that MDAs induce over a 2-fold induction of
HIF-1
protein in hepa1c1c7 (Fig. 4B). Furthermore, this
increase in HIF-1
protein correlated with over a 2-fold increase in
HIF-1 reporter activity. However, in Arnt-deficient hepa1c4 cells, MDA
treatment failed to increase HIF-1-dependent luciferase
activity, thereby demonstrating that MDA-mediated HIF-1 activation is
dependent upon and accurately represents transcriptionally active
HIF-1.
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Fig. 4.
MDAs induce HIF-dependent
luciferase activity and upregulate iNOS protein in a
HIF-1-dependent manner. A, A549 cells were
cotransfected with a HIF-1-dependent iNOS luciferase
reporter gene (0.4 µg) and CMV Renilla luciferase plasmid
(4 ng) for 6 h. Subsequently, cells were treated with either
vinblastine (0.1 µM), colchicine (1µM), or
nocodazole (10µM). Reporter activities were measured
10 h later and were normalized to CMV Renilla
luciferase activity. The data represent the mean of three separate
experiments. B, upper panel, Hepa1c1c7 cells were
treated for 4 h with either vinblastine (0.5 µM) or
colchicine (5 µM), cells were lysed and HIF-1 was
immunodetected. Lower panel, both hepa1c1c7 and hepa1c4
cells were cotransfected with a HIF-1-dependent iNOS
luciferase reporter construct as in A prior to treatment
with vinblastine (0.5 µM) or colchicine (5 µM). Reporter activities were measured 10 h later
and were normalized to CMV Renilla luciferase activity. The
data represent the mean of three separate experiments. C,
both hepa1c1c7 and hepa1c1c4 cells were treated with either vinblastine
(0.5 µM), or colchicine (5 µM) for 6 h. Following this treatment, cells were lysed and iNOS protein and
-tubulin (loading control) were immunoblotted using cytosolic
extracts (40 µg).
(45). As shown in Fig.
4C, HIF-1
levels in hepa1c1c7 cells were induced
following a 5-h treatment with the MDAs vinblastine or colchicine.
However, these same agents were unable to elicit any iNOS induction in
the Arnt-defective hepa1c4 cells. Therefore, the ability of MDAs to
induce HIF-1 reporter activity in these cell lines reflects their
ability to induce endogenous iNOS protein and is consistent with
up-regulation of transcriptionally active HIF-1.
B in MDA-mediated HIF-1
induction was definitive, it remained to be determined whether
microtubule disruption itself was required for HIF-1 induction. To
examine this issue, MDA-mediated HIF-1 induction was assessed in both wild type and colchicine-resistant Chinese Hamster Ovary (CHO) cells.
The colchicine-resistant cells contain a mutation in tubulin that
alters the association of MDAs with microtubules (48, 49). As shown in
Fig. 5A, while the wild type
CHO cells exhibited a marked increase in HIF-1 protein following MDA
treatment, the colchicine-resistant cells failed to respond to this
treatment. To further verify that microtubule disruption is a component
of the signaling pathway of MDAs, A549 cells were treated with
-lumicolchicine, a structurally similar, but catalytically inactive
analog of colchicine. As shown in Fig. 5B, this analog
failed to induce HIF-1
protein, thereby demonstrating that MDA
interaction with microtubules is required for MDAs to induce
HIF-1
.
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Fig. 5.
Microtubule disruption is required for
MDA-mediated HIF-1 induction. A, wild type and
colchicine-resistant CHO cells were treated for 4 h with either
colchicine (5 µM) or CoCl2 (100 µM), and HIF-1 expression was detected using nuclear
extracts. B, A549 cells were treated for 4 h with
colchicine (1 µM) or with
-lumicolchicine
(
-lumicol, 1 µM), an inactive form of colchicine, and
Western blotting was performed using nuclear extracts.
Protein at the Post-transcriptional
Level--
Our data suggested that NF
B up-regulated HIF-1
at the
transcriptional level, and we therefore examined whether HIF-1
mRNA levels were induced by MDAs. As shown in Fig.
6A (upper panel) RT-PCR analysis reveals that HIF-1
mRNA levels in A549 cells remain unchanged in response to a 3-h treatment of the indicated MDAs.
The lower panel shows the RT-PCR of
-actin, which was
used as an internal control. This result indicates that MDAs
up-regulate HIF-1
at the post-transcriptional level.
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Fig. 6.
MDAs upregulate HIF-1
at the posttranscriptional level and stabilize the protein in a
VHL-dependent manner. A, A549 cells were
treated with vinblastine (0.1 µM) or colchicine (1 µM) for 3 h. Total cellular mRNA was extracted
and subjected to RT-PCR, as described.
-actin mRNA levels were
examined as an internal control. B, renal carcinoma cells
that are deficient for VHL function (UMRC2), or a clonally selected
line with VHL stably expressed (UMRC2/VHL), were treated for 4 h
with vinblastine (vin, 0.1 µM), and HIF-1
protein was detected in nuclear extracts. C, A549 cells were
transfected with either HA-tagged wild type HIF-1
or HA-tagged
pmHIF-1
(HIF-1
proline-mutated at residues 402 and 564), a form
resistant to VHL-dependent degradation. Following
transfection, cells were treated as in B, and HIF-1
was
immunodetected with an anti-HA antibody. D, A549 cells were
either left untreated or were pretreated with vinblastine for 4 h,
followed by addition of cycloheximide for the indicated times.
Endogenous levels of HIF-1 were visualized from nuclear extracts (30 µg for vinblastine-treated cells and 80 µg for untreated cells).
Blots were reprobed for ARNT expression as a control for equivalent
loading within each group. Densitometric analysis of HIF expression was
used to plot the decay rate of the protein.
protein under normoxia is VHL.
Therefore, we examined whether VHL played a role in the MDA-mediated
induction of HIF-1
. The effect of MDAs upon HIF-1
expression was
examined in a matched pair of renal carcinoma lines either lacking VHL
function, or with VHL replaced by stable transfection. As shown in Fig.
6B, vinblastine up-regulated HIF-1
protein in the cell
line containing functional VHL protein (UMRC2/VHL). However, HIF-1
was not further up-regulated in the VHL-mutated parental line
containing stable HIF-1
protein. To confirm these results, transfections were performed in the well-characterized A549 cell line.
As shown in Fig. 6C, vinblastine up-regulated the levels of
transfected wild type HIF-1
expressed in A549 cells, while this
agent failed to upregulate a mutated form of HIF-1
that is
VHL-resistant. These results suggested that MDA treatment stabilized HIF-1
protein to the effects of VHL. To test this hypothesis, the
stability of endogenous HIF-1
protein in A549 cells was determined either in the presence or absence of vinblastine. As shown in Fig.
6D, while endogenous HIF-1
was extremely labile in
normoxic cells, with a half-life of less than 4 min, vinblastine
treatment significantly stabilized the protein and extended its
half-life by more than 5-fold to 20 min.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
in a
pathway dependent upon NF
B activation in a variety of cell lines,
suggesting that this is a basic mode of signaling universally employed
by most, if not all, cell types. While MDA-mediated HIF-1 induction is
not as pronounced as that elicited by hypoxia, it is significant enough
to result in more than a 2-fold increase in HIF-1-regulated iNOS
reporter activity and in a severalfold up-regulation of endogenous iNOS
protein expression. By comparison, hypoxic stimulation of this same
reporter was on the order of 3-4-fold (data not shown).
B activation, is a requirement for the ability of MDAs
to induce HIF-1
. First, we show that transcription inhibitors interfere with the ability of MDAs to induce HIF-1
protein. Second, we demonstrate that MDAs induce NF
B activity, which correlates with
the ability of these agents to induce HIF-1
levels. Third, and most
compelling, treatment of cells with either a drug that inhibits NF
B
or transfection with the NF
B super-repressor plasmid both abrogated
the ability of MDAs to up-regulate HIF-1
protein. Finally, we
demonstrate that the MDA signaling pathway for HIF-1 induction is
distinct from the hypoxia-mediated pathway, in that NF
B inhibitors
had no effect on reducing HIF-1
levels induced by a hypoxia mimetic,
further emphasizing the unique transcriptional dependence of this mechanism.
B activity (24, 50-51) and in agreement with
these reports, we demonstrate that MDAs induced NF
B activity in A549
cells. However, the precise mechanism of MDA-induced NF
B activation
remains unclear. In one model, MDAs are proposed to stimulate I
B
degradation, resulting in the nuclear accumulation and subsequent
activation of NF
B (24, 52). In accordance with this model, we
observed a slight increase in nuclear NF
B upon MDA treatment (data
not shown). In a second model, mechanisms potentially independent of
nuclear NF
B protein levels play a role. These mechanisms include
posttranslational modification of the NF
B protein, such as
phosphorylation or acetylation of p65 (53). Interestingly, although
high doses of either vinblastine or colchicine resulted in decreased
NF
B activity, no such corresponding decrease in nuclear protein
levels was observed (data not shown), consistent with other reports
(52). Therefore, although our data do not preclude the possibility that
MDA-mediated nuclear translocation of NF
B is an important initial
event, the down-regulation of NF
B activity without corresponding
changes in nuclear p65 suggests that MDAs also initiate additional
signaling events culminating in the post-translational modification of
NF
B. Although a definitive signaling pathway remains elusive, it is
notable that MDAs modulate the activities of a variety of kinases, such
as protein kinases A and C, PI 3-kinase, and focal adhesion kinase
(FAK), that regulate NF
B activity by phosphorylation of the p65
subunit (18, 54-55). Consistent with this hypothesis, MDAs at low, but
not high concentrations, increase tyrosine phosphorylation of focal
adhesion proteins such as FAK and paxillin (56). Finally, it has been
demonstrated that microtubule-stabilizing agents such as paclitaxel are
unable to promote activation of these same kinases (51, 57),
correlating with their inability to activate NF
B and upregulate
HIF-1
.
induction is distinct from the hypoxia-mediated stabilization of
HIF-1
. This is most clearly illustrated by data showing the dependence upon NFkB activation for the former, but not the latter pathway. However, similar to the hypoxia-mediated pathway, we find that
MDAs induce HIF-1
protein at the posttranscriptional level. While we
cannot absolutely rule out the possibility of increased translation of
HIF-1
as an explanation of this phenomenon, in a manner similar to
the effect of various growth factors (35, 58), we suggest that MDAs act
by partially protecting HIF-1
protein from VHL-dependent
degradation. MDA-mediated stabilization does not render the protein
completely resistant to VHL, but rather appears to engender a less
efficient degradation, resulting in a 5-fold increase in half-life.
Compelling evidence for this notion is provided by our finding that
HIF-1
levels in a cell line lacking VHL function remained unchanged
upon exposure to vinblastine. However, in a matched line with stably
expressed VHL, HIF-1
accumulated in response to vinblastine,
suggesting that the MDA-stabilizing effect is dependent upon VHL
expression. Similarly, vinblastine elevated the level of transiently
expressed wild type HIF-1
protein in A549 cells, while these agents
had no effect upon a transiently expressed proline-mutated,
VHL-resistant HIF-1
protein. Finally, the ability of MDAs to
activate NFkB was independent of VHL status (data not shown),
supporting our hypothesis that NFkB activation by MDAs occurs prior to
HIF-1 accumulation. Although the complete mechanism of MDA-induced
HIF-1
accumulation remains unclear, the transcriptional dependence
of this pathway suggests that the mediator(s) involved may be labile.
Evidence for a labile mediator is further provided by our data (Fig.
3C) demonstrating that transcriptional inhibition of
MDA-stabilized HIF-1 results in a moderate decrease in protein levels
within the first hour. This labile mediator(s) may modify HIF-1
, or
another protein involved VHL-HIF association, so as to render HIF-1
less susceptible to VHL-mediated degradation.
B and subsequent induction of HIF-1
is initiated by and depends upon microtubule depolymerization. While
the specific role NF
B may play in invasion and metastasis is
unclear, several reports document overexpression and/or hyperactivity of NF
B in cancer lines (59) and tissues (60). Inhibition of NF
B
correlates with suppression of metastasis and invasion (61, 62),
down-regulation of VEGF mRNA (63), and suppression of angiogenesis
(62), effects, which may be mediated through regulation of HIF-1
.
Given that HIF-1
is overexpressed in a majority of tumors (12), the
data in this study suggest that HIF-1
is among the pro-oncogenic
factors induced by NF
B.
![]() |
FOOTNOTES |
---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed. Tel.: 301-402-3128, ext. 318; Fax: 301-402-4422; E-mail: len@helix.nih.gov.
Published, JBC Papers in Press, December 17, 2002, DOI 10.1074/jbc.M209804200
2 Y.-J. Jung, J. S. Isaacs, S. Lee, J. Trepel, Z.-G. Liu, and L. Neckers, submitted manuscript.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
HIF-1, hypoxia
inducible factor-1;
MDA, microtubule-depolymerizing agent;
vin, vinblastine;
col, colchicine;
noco, nocodazole;
taxol, paclitaxel;
AcD, actinomycin-D;
DCB, dichlorobenzimidazole riboside;
phe, phenanthroline;
-lumicol,
-lumicolchicine;
NF
B, nuclear
factor
B;
NFRP, NF
B super-repressor plasmid;
iNOS, inducible
nitric-oxide synthetase;
ARNT, aryl hydrocarbon nuclear translocator;
HA, hemagglutinin;
PDTC, pyrrolidinedithiocarbamate;
CHX, cycloheximide;
PI, phosphatidylinositol;
CMV, cytomegalovirus.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Wang, G. L., Jiang, B. H., Rue, E. A., and Semenza, G. L. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 5510-5514[Abstract] |
2. | Wiener, C. M., Booth, G., and Semenza, G. L. (1996) Biochem. Biophys. Res. Commun. 225, 485-488[CrossRef][Medline] [Order article via Infotrieve] |
3. |
Salceda, S.,
and Caro, J.
(1997)
J. Biol. Chem.
272,
22642-22647 |
4. |
Huang, L. E., Gu, J.,
Schau, M.,
and Bunn, H. F.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
7987-7992 |
5. |
Cockman, M. E.,
Masson, N.,
Mole, D. R.,
Jaakkola, P.,
Chang, G. W.,
Clifford, S. C.,
Maher, E. R.,
Pugh, C. W.,
Ratcliffe, P. J.,
and Maxwell, P. H.
(2000)
J. Biol. Chem.
275,
25733-25741 |
6. |
Tanimoto, K.,
Makino, Y.,
Pereira, T.,
and Poellinger, L.
(2000)
EMBO J.
19,
4298-4309 |
7. |
Jaakkola, P.,
Mole, D. R.,
Tian, Y. M.,
Wilson, M. I.,
Gielbert, J.,
Gaskell, S. J.,
Kriegsheim, A.,
Hebestreit, H. F.,
Mukherji, M.,
Schofield, C. J.,
Maxwell, P. H.,
Pugh, C. W.,
and Ratcliffe, P. J.
(2001)
Science
292,
468-472 |
8. |
Ivan, M.,
Kondo, K.,
Yang, H.,
Kim, W.,
Valiando, J.,
Ohh, M.,
Salic, A.,
Asara, J. M.,
Lane, W. S.,
and Kaelin, W. G., Jr.
(2001)
Science
292,
464-468 |
9. | Semenza, G. L. (1998) Curr. Opin. Genet. Dev. 8, 588-594[CrossRef][Medline] [Order article via Infotrieve] |
10. | Feng, C., Wang, L., Jiao, L., Liu, B., Zheng, S., and Xie, X. (2002) BMC Cancer 2, 8[CrossRef][Medline] [Order article via Infotrieve] |
11. | Long, L., Rubin, R., and Brodt, P. (1998) Exp. Cell Res. 238, 116-121[CrossRef][Medline] [Order article via Infotrieve] |
12. |
Zhong, H., De,
Marzo, A. M.,
Laughner, E.,
Lim, M.,
Hilton, D. A.,
Zagzag, D.,
Buechler, P.,
Isaacs, W. B.,
Semenza, G. L.,
and Simons, J. W.
(1999)
Cancer Res.
59,
5830-5835 |
13. | Cassimeris, L. U., Walker, R. A., Pryer, N. K., and Salmon, E. D. (1987) Bioessays 7, 149-154[Medline] [Order article via Infotrieve] |
14. | Correia, J. J., and Lobert, S. (2001) Curr. Pharm. Des. 7, 1213-1228[Medline] [Order article via Infotrieve] |
15. | Rowinsky, E. K., and Donehower, R. C. (1991) Pharmacol. Ther. 52, 35-84[CrossRef][Medline] [Order article via Infotrieve] |
16. | Jordan, M. A., and Wilson, L. (1998) Curr. Opin. Cell Biol. 10, 123-130[CrossRef][Medline] [Order article via Infotrieve] |
17. | Sammak, P. J., and Borisy, G. G. (1988) Nature 332, 724-726[CrossRef][Medline] [Order article via Infotrieve] |
18. | Gundersen, G. G., and Cook, T. A. (1999) Curr. Opin. Cell Biol. 11, 81-94[CrossRef][Medline] [Order article via Infotrieve] |
19. |
Janmey, P. A.
(1998)
Physiol. Rev.
78,
763-781 |
20. |
Botteri, F. M.,
Ballmer-Hofer, K.,
Rajput, B.,
and Nagamine, Y.
(1990)
J. Biol. Chem.
265,
13327-13334 |
21. | Elbaum, M., Chausovsky, A., Levy, E. T., Shtutman, M., and Bershadsky, A. D. (1999) Biochem. Soc. Symp. 65, 147-172[Medline] [Order article via Infotrieve] |
22. | Ferrua, B., Manie, S., Doglio, A., Shaw, A., Sonthonnax, S., Limouse, M., and Schaffar, L. (1990) Cell. Immunol. 131, 391-397[Medline] [Order article via Infotrieve] |
23. |
Das, K. C.,
Guo, X. L.,
and White, C. W.
(1998)
J. Biol. Chem.
273,
34639-34645 |
24. | Rosette, C., and Karin, M. (1995) J. Cell Biol. 128, 1111-1119[Abstract] |
25. |
Schmid-Alliana, A.,
Menou, L.,
Manie, S.,
Schmid-Antomarchi, H.,
Millet, M. A.,
Giuriato, S.,
Ferrua, B.,
and Rossi, B.
(1998)
J. Biol. Chem.
273,
3394-3400 |
26. |
Subbaramaiah, K.,
Hart, J. C.,
Norton, L.,
and Dannenberg, A. J.
(2000)
J. Biol. Chem.
275,
14838-14845 |
27. |
Wang, T. H.,
Wang, H. S.,
Ichijo, H.,
Giannakakou, P.,
Foster, J. S.,
Fojo, T.,
and Wimalasena, J.
(1998)
J. Biol. Chem.
273,
4928-4936 |
28. | Giannakakou, P., Sackett, D. L., Ward, Y., Webster, K. R., Blagosklonny, M. V., and Fojo, T. (2000) Nat. Cell Biol. 2, 709-717[CrossRef][Medline] [Order article via Infotrieve] |
29. | Pahl, H. L. (1999) Oncogene 18, 6853-6866[CrossRef][Medline] [Order article via Infotrieve] |
30. | Sun, Z., and Andersson, R. (2002) Shock 18, 99-106[CrossRef][Medline] [Order article via Infotrieve] |
31. | Mercurio, F., and Manning, A. M. (1999) Curr Opin. Cell Biol. 11, 226-232[CrossRef][Medline] [Order article via Infotrieve] |
32. | Baeuerle, P. A., and Baltimore, D. (1988) Science 242, 540-546[Medline] [Order article via Infotrieve] |
33. | Henkel, T., Machleidt, T., Alkalay, I., Kronke, M., Ben-Neriah, Y., and Baeuerle, P. A. (1993) Nature 365, 182-185[CrossRef][Medline] [Order article via Infotrieve] |
34. | Traenckner, E. B., Pahl, H. L., Henkel, T., Schmidt, K. N., Wilk, S., and Baeuerle, P. A. (1995) EMBO J. 14, 2876-2883[Abstract] |
35. |
Zhong, H.,
Chiles, K.,
Feldser, D.,
Laughner, E.,
Hanrahan, C.,
Georgescu, M. M.,
Simons, J. W.,
and Semenza, G. L.
(2000)
Cancer Res.
60,
1541-1545 |
36. |
Treins, C.,
Giorgetti-Peraldi, S.,
Murdaca, J.,
Semenza, G. L.,
and Van Obberghen, E.
(2002)
J. Biol. Chem.
277,
27975-27981 |
37. |
Elson, D. A.,
Ryan, H. E.,
Snow, J. W.,
Johnson, R.,
and Arbeit, J. M.
(2000)
Cancer Res.
60,
6189-6195 |
38. | Minet, E., Michel, G., Remacle, J., and Michiels, C. (2000) Int. J. Mol. Med. 5, 253-259[Medline] [Order article via Infotrieve] |
39. | Zagzag, D., Zhong, H., Scalzitti, J. M., Laughner, E., Simons, J. W., and Semenza, G. L. (2000) Cancer 88, 2606-2618[CrossRef][Medline] [Order article via Infotrieve] |
40. | Liu, Z. G., Hsu, H., Goeddel, D. V., and Karin, M. (1996) Cell 87, 565-576[Medline] [Order article via Infotrieve] |
41. |
Isaacs, J. S.,
Jung, Y. J.,
Mimnaugh, E. G.,
Martinez, A.,
Cuttitta, F.,
and Neckers, L. M.
(2002)
J. Biol. Chem.
277,
29936-29944 |
42. | Andrews, N. C., and Faller, D. V. (1991) Nucleic Acids Res. 19, 2499[Medline] [Order article via Infotrieve] |
43. | Remy-Kristensen, A., Duportail, G., Coupin, G., and Kuhry, J. G. (2000) Mol. Membr. Biol. 17, 95-100[CrossRef][Medline] [Order article via Infotrieve] |
44. | Brown, P. H., Chen, T. K., and Birrer, M. J. (1994) Oncogene 9, 791-799[Medline] [Order article via Infotrieve] |
45. |
Melillo, G.,
Taylor, L. S.,
Brooks, A.,
Musso, T.,
Cox, G. W.,
and Varesio, L.
(1997)
J. Biol. Chem.
272,
12236-12243 |
46. |
Li, H.,
Dong, L.,
and Whitlock, J. P., Jr.
(1994)
J. Biol. Chem.
269,
28098-28105 |
47. |
Li, H., Ko, H. P.,
and Whitlock, J. P.
(1996)
J. Biol. Chem.
271,
21262-21267 |
48. | Whitfield, C., Abraham, I., Ascherman, D., and Gottesman, M. M. (1986) Mol. Cell. Biol. 6, 1422-1429[Medline] [Order article via Infotrieve] |
49. | Cabral, F., Sobel, M. E., and Gottesman, M. M. (1980) Cell 20, 29-36[CrossRef][Medline] [Order article via Infotrieve] |
50. |
Bourgarel-Rey, V.,
Vallee, S.,
Rimet, O.,
Champion, S.,
Braguer, D.,
Desobry, A.,
Briand, C.,
and Barra, Y.
(2001)
Mol. Pharmacol.
59,
1165-1170 |
51. | Spencer, W., Kwon, H., Crepieux, P., Leclerc, N., Lin, R., and Hiscott, J. (1999) Oncogene 18, 495-505[CrossRef][Medline] [Order article via Infotrieve] |
52. |
Das, K. C.,
and White, C. W.
(1997)
J. Biol. Chem.
272,
14914-14920 |
53. | Boone, D. L., Lee, E. G., Libby, S., Gibson, P. J., Chien, M., Chan, F., Madonia, M., Burkett, P. R., and Ma, A. (2002) Inflamm. Bowel Dis. 8, 201-212[Medline] [Order article via Infotrieve] |
54. | Garg, A., and Aggarwal, B. B. (2002) Leukemia 16, 1053-1068[CrossRef][Medline] [Order article via Infotrieve] |
55. | Wang, L. G., Liu, X. M., Kreis, W., and Budman, D. R. (1999) Cancer Chemother. Pharmacol. 44, 355-361[CrossRef][Medline] [Order article via Infotrieve] |
56. | Haier, J., and Nicolson, G. L. (1999) Clin. Exp. Metastasis 17, 713-721[CrossRef][Medline] [Order article via Infotrieve] |
57. | Kadi, A., Berthet, V., Pichard, V., Abadie, B., Rognoni, J. B., Marvaldi, J., and Luis, J. (2002) Bull. Cancer 89, 227-233[Medline] [Order article via Infotrieve] |
58. |
Fukuda, R.,
Hirota, K.,
Fan, F.,
Jung, Y. D.,
Ellis, L. M.,
and Semenza, G. L.
(2002)
J. Biol. Chem.
277,
38205-38211 |
59. | Patel, N. M., Nozaki, S., Shortle, N. H., Bhat-Nakshatri, P., Newton, T. R., Rice, S., Gelfanov, V., Boswell, S. H., Goulet, R. J., Jr., Sledge, G. W., Jr., and Nakshatri, H. (2000) Oncogene 19, 4159-4169[CrossRef][Medline] [Order article via Infotrieve] |
60. |
Sasaki, N.,
Morisaki, T.,
Hashizume, K.,
Yao, T.,
Tsuneyoshi, M.,
Noshiro, H.,
Nakamura, K.,
Yamanaka, T.,
Uchiyama, A.,
Tanaka, M.,
and Katano, M.
(2001)
Clin Cancer Res.
7,
4136-4142 |
61. | Sliva, D., English, D., Lyons, D., and Lloyd, F. P., Jr. (2002) Biochem. Biophys. Res. Commun. 290, 552-557[CrossRef][Medline] [Order article via Infotrieve] |
62. | Huang, S., Pettaway, C. A., Uehara, H., Bucana, C. D., and Fidler, I. J. (2001) Oncogene 20, 4188-4197[CrossRef][Medline] [Order article via Infotrieve] |
63. | Shibata, A., Nagaya, T., Imai, T., Funahashi, H., Nakao, A., and Seo, H. (2002) Breast Cancer Res. Treat. 73, 237-243[CrossRef][Medline] [Order article via Infotrieve] |