Up-regulation of Vascular Endothelial Growth Factor C in
Breast Cancer Cells by Heregulin-
1
A CRITICAL ROLE OF p38/NUCLEAR FACTOR-
B SIGNALING
PATHWAY*
Pei-Wen
Tsai
,
Shine-Gwo
Shiah§,
Ming-Tsan
Lin¶,
Cheng-Wen
Wu§, and
Min-Liang
Kuo
From the
Laboratory of Molecular and Cellular
Toxicology, Institute of Toxicology, College of Medicine, National
Taiwan University, Taipei 110, Taiwan, the § President's
Laboratory, National Health Research Institute, Taipei 115, Taiwan, and
the ¶ Department of Surgery, National Taiwan University Hospital,
Taipei 110, Taiwan
Received for publication, May 17, 2002, and in revised form, December 2, 2002
 |
ABSTRACT |
Vascular endothelial growth
factor C (VEGF-C) is a critical activator of tumor lymphangiogenesis
that recently has been strongly implicated in the tumor metastasis
process. In this study, we identified that HRG-
1 stimulated
up-regulation of VEGF-C mRNA and protein of human breast cancer
cells in a dosage- and time-dependent manner and that this
up-regulation was de novo RNA
synthesis-dependent. The HRG-
1-induced increase in
VEGF-C expression was effectively reduced by treatment with Herceptin,
an antibody specifically against HER2. Also, when HER2 was
overexpressed in MCF-7 cells that resulted in an evident increase in
the VEGF-C level, suggesting an essential role of HER2 in mediating
VEGF-C up-regulation by HRG-
1. NF-
B has been shown to be
probably involved in interleukin-1
- or tumor necrosis
factor-
-induced VEGF-C mRNA expression in human fibroblasts.
Here we found that HRG-
1 could stimulate NF-
B nuclear translocation and DNA-binding activity via the I
B
phosphorylation-degradation mechanism. Blockage of the NF-
B
activation cascade caused a complete inhibition of the
HRG-
1-induced elevation of VEGF-C. In promoter-reporter assay, the
luciferase activities of the reporter constructs, including the
putative NF-
B site deleted and mutated form were significantly reduced after HRG-
1 treatment as compared with the 1.5-kb VEGF-C promoter. Although investigating the upstream kinase pathway(s) involved in HRG-
1-elicited NF-
B activation and VEGF-C
up-regulation, we found that HRG-
1 could activate extracellular
signal-regulated protein kinase 1/2, phosphatidylinositol 3'-kinase,
and p38 mitogen-activated protein kinase (MAPK) in MCF-7. However, only
SB203580 (a specific inhibitor of p38 MAPK), not PD98059 nor LY294002,
blocked the up-regulation of VEGF-C by HRG-
1. A similar inhibition
in VEGF-C expression was obtained by cell transfection with
dominant-negative p38 (p38AF). Interestingly, the HRG-
1-induced
NF-
B activation cascade was also effectively blocked by SB203580
treatment or p38AF transfection. Our data thus suggests that
HRG-
1 stimulated a NF-
B-dependent up-regulation of
VEGF-C through the p38 MAPK signaling pathway in human breast cancer cells.
 |
INTRODUCTION |
Tumor progression and metastasis rely mainly on both vascular and
lymphatic systems by which cancer cells can spread widely into regional
or distant tissues. The high tumor metastasis is strongly associated
with short disease-free survival periods and poor prognosis in cancer
patients. In the past decades, many subsets of molecules have been
reported to be critically involved in regulating the blood microvessel
formation in tumor development (1-3). Little is known about how cancer
cells can migrate to regional lymph nodes or promote the proliferation
of lymphatic vessel. Recent evidence showed that
VEGF1-C and VEGF-D, two
members of the VEGF family, are the ligands for VEGF receptor(R)-3,
which can stimulate the lymphatic vessel growth (lymphangiogenesis) and
also enhance lymphatic metastasis in animal model (4-8).
VEGF-C displays a high degree of similarity to VEGF-A, including
conservation of the eight cysteine residues involved in intra- and
intermolecular disulfide binding. The cysteine-rich COOH-terminal region increases the half-life of the VEGF-C protein relative to the
other members of the family (9, 10). VEGF-C mRNA is first
translated into a 58-kDa precursor from which the 29/31-kDa mature
ligand is formed by a general proteolytic process independent of the
cell type (11, 12). The secreted 29/31-kDa VEGF-C polypeptide specifically binds to the VEGF receptor(R)-3, which is predominantly expressed on lymphatic endothelium in adults, and triggers survival and
proliferative activity in the lymphatic endothelium via p42/p44 MAPK
and phosphatidylinositol 3'-kinase (PI3K)/Akt signaling pathways (13-16). Supportively, VEGF-C expression has been detected in most of
the important human cancers analyzed (17), and its expression level
correlated with lymph node metastases in especially thyroid, gastric,
colorectal, lung, prostatic, and breast cancers (18-23). Clinical and
experimental findings strongly suggest a potential role for
VEGF-C-mediated lymphangiogenesis in human cancer metastasis. However,
little is known about the regulation of VEGF-C in tumor cells to date.
HRG-
1, a member of the epidermal growth factor-like growth factor
family, is secreted from mesenchymal cells and acts as a combinatorial
ligand for the HER3 and HER4 receptors in breast cancer cells (24-26).
The binding of HRG-
1 to its receptors activates a diversified
signaling pathway by which many biological functions, including
proliferation (27, 28), differentiation (27, 29), apoptosis (30, 31),
and migration (32, 33), occur. These biological alterations induced by
HRG-
1 result in the invasive and metastasis-related properties in
human breast cancer (25, 34, 35). HRG-
1 has been shown to
participate in the regulation of many important gene expressions such
as, metalloproteinases (36), urokinase plasminogen activator (37),
paxillin (38, 39), and VEGF-A (40). The signaling pathways including
PI3K, ERK1/2, and p38 MAPK have been shown to be responsible for those gene expressions in a complicated manner (36, 39). However, these
identified genes seem not to fully account for the role of HRG-
1 in
the progression of breast cancer. It is important to identify new
downstream effector gene(s) in breast cancer cells in response to
HRG-
1.
This study was undertaken to investigate whether HRG-
1 would
regulate the expression of VEGF-C in human breast cancer cells. Here we
demonstrate that HRG-
1, but not other growth factors, is a potent
agent to induce VEGF-C up-regulation. Using NF-
B decoy or
transfection with dominant-negative (DN)-I
B
to specifically abrogate the HRG-
1-mediated NF-
B activation including nuclear translocation and DNA-binding activity that resulted in an inhibition of VEGF-C up-regulation. Searching the upstream kinase regulator of
NF-
B signaling, we found that SB203580, a p38 MAPK inhibitor, but
not PD98059 or LY294002, strongly abolished HRG-
1-stimulated NF-
B
activation as well as the subsequent VEGF-C up-regulation. A similar
result was obtained using transfection with DN-p38 (p38AF). Our
current data demonstrates for the first time that HRG-
1 potently up-regulates the VEGF-C gene through the activation of the
p38 MAPK/NF-
B signaling pathway.
 |
EXPERIMENTAL PROCEDURES |
Cell Cultures--
Human breast cancer cell lines (MCF-7, SKBr3,
and T47D), MCF-7 HER2/neu stable cell line expressing Her-2/neu (a kind
gift from Dr. Ruey-Long Hung, Department of Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan), and HBL-100 immortalized human breast cell line were maintained in Dulbecco's modified Eagle's
medium supplemented 10% fetal bovine serum with 2 mM
L-glutamine (Invitrogen), 100 µg/ml streptomycin,
and 100 units/ml penicillin in a humidified 5% CO2 atmosphere.
Transient Cell Transfections--
p38AF dominant-negative mutant
(generously provided by Dr. Ching-Chow Chen, Department of
Pharmacology, College of Medicine, National Taiwan University, Taipei,
Taiwan), dominant-negative 32/36A mutated form of I
B
(kindly
provided by Dr. Shuang-En Chuang), and vector were transfected into
MCF-7 cells using transfection reagent FuGENE-6 (from Roche Molecular
Biochemicals) per manufacturer's protocol. Twenty-four hours after
transfection, cells were serum-starved for a further 24 h and
stimulated with HRG-
1, and then cells were lysed for analysis. Each
experiment was repeated with three independent transfections, and
transfection efficiency varied between 20 and 30%.
Reagents--
Recombinant human heregulin-
1, human
interleukin-1
, human interleukin-6, human interleukin-8, human tumor
necrosis factor-
, and human polyclonal VEGF-C antibody were
purchased from R&D Systems (Minneapolis, MN). The kinase inhibitors
LY294002, PD098059, and SB203580 were obtained from Sigma. The
epidermal growth factor receptor inhibitor PD153035 and PMA
(phorbol-12-myristate-13-acetate) were purchased from
Calbiochem. Antibodies to human Her-2/neu, phospho-p38, p38,
phospho-ERK1/2, ERK1/2, NF-
B (p65), NF-
B (p50), proliferating cell nuclear antigen,
-tubulin, and secondary
horseradish peroxidase-conjugated antibodies were obtained from Santa
Cruz Biotechnology. Antibodies to phospho-AKT and AKT were purchased from the Upstate Biotechnology. [
-32P]dCTP was
obtained from Amersham Biosciences. Anti-Her-2 monoclonal antibody,
Herceptin® (Trastuzumab), was provided by Dr. Chih-Hsin
Yang (Department of Oncology, National Taiwan University Hospital,
Taipei, Taiwan).
RNA Isolation and Reverse Transcriptase-Polymerase Chain
Reaction--
A total of 8 breast carcinoma patients' tissue RNA were
obtained from the Department of Pathology, National Taiwan University Hospital, Taipei, Taiwan. Total RNA was isolated using
RNA-BeeTM reagent (Tel-Test, Inc.) as recommended by the
manufacturer's instructions. Total RNA (1 µg) was
reverse-transcribed into single-stranded cDNA with Moloney murine
leukemia virus Reverse Transcriptase and random hexamers
(Promega, Madison, WI). Amplification of growth factor cDNAs and
-actin cDNA as an internal control in each reaction was carried
out by polymerase chain reaction with the primers as described as
follows. VEGF-C cDNA PCR are 5'-CAGTTACGGTCTGTGTCCAGTGTAG-3' (forward) and 5'-GGACACACATGGAGGTTTAAAGAAG-3' (reverse). The primer sequences for VEGF-D cDNA PCR are 5'-TGGGTCATCTTCTCGCGGTT-3'
(forward) and 5'-GATGATGATATCGCCGCGCT-3' (reverse). The primer
sequences were as follows: VEGF-A cDNA PCR are
5'-AGCTACTGCCATCCAATCGC-3' (forward) and 5'-GGGCGAATCCAATTCCAAGAG-3'
(reverse). Primers designed to amplify a fragment of HRG cDNA
(corresponding to position in HRG-
1 mRNA,
GenBankTM M94166), spanning the variable EGF-like domain
(forward primer: 5'-GTGTGAATGGAGGGGAGTGC-3', reverse primer:
5'-CTTGGCGTGTGGAAATCTAC-3'). Primer sequences for HER2 cDNA PCR are
5'-AGGGAAACCTGGAACTCACC-3' (forward) and 5'-TGGATCAAGACCCCTCCTT-3'
(reverse). PCR was carried out using specific primers for
-actin and
GAPDH (
-actin: forward, 5'-GATGATGATATCGCCGCGCT-3', reverse,
5'-TGGGTCATCTTCTCGCGGTT-3'; GAPDH: forward,
5'-CCACCCATGGCAAATTCCATGGCA-3', reverse,
5'-TCTAGACGGCAGGTCAGGTCCACC-3'). Primers were used at a final
concentration of 0.5 µM. Reaction mixture was first
denatured at 95 °C for 10 min. The PCR condition was 95 °C for 1 min, 55 °C for 1 min, and 72 °C for 1 min, for 30 cycles,
followed by 72 °C for 10 min. Polymerase chain reaction products were visualized by ethidium bromide staining after agarose gel
electrophoresis. Mammary tumors from HER2/neu transgenic mice were
generously provided by Dr. Ricci SM (Ovarian and Breast Cancer, The University of Texas M.D. Anderson Cancer Center, Houston, TX). For
the quantitative real-time PCR analysis of human VEGF-C and GAPDH
mRNA levels, a Light Cycler system and reagents (Roche Molecular
Diagnostics) were used with a double-stranded DNA binding dye,
SYBR Green 1, according to the procedure provided by the manufacturer.
The real-time PCR program for VEGF-C consisted of 30 cycles with
denaturation at 95 °C for 15 s, annealing at 56 °C for
5 s, and extension at 72 °C for 10 s. For GAPDH, it
consisted of 30 cycles with denaturation at 95 °C for 15 s,
annealing at 66 °C for 5 s, and extension at 72 °C for
20 s.
Western Blot Analysis--
Cells were starved in serum-free
medium overnight then incubated with HRG-
1 for different times or
for various concentrations. Cells were washed two times with ice cold
phosphate-buffered saline and lysed in buffer (50 mM
Tris-HCl (pH 7.5), 120 mM NaCl, 0.5% Nonidet P-40, 100 mM NaF, 200 mM Na3VO4,
1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin,
and 10 µg/ml aprotinin) for 15 min on ice. The lysates were
centrifuged in an Eppendorf at 4 °C for 15 min. The equal
amounts of protein from the cell lysates were resuspended in gel sample
buffer, resolved by 10% SDS-polyacrylamide gel electrophoresis, and
transferred to nitrocellulose membranes (Millipore). Membranes were
blocked for 1 h in 5% (w/v) dry milk, phosphate-buffered saline,
and 0.1% Tween 20. Primary antibodies as indicated were incubated with
membranes for 2 h, and the membranes were washed in
phosphate-buffered saline with 0.1% Tween 20. Subsequently, horseradish peroxidase-conjugated secondary antibodies were added for
1 h, and the membranes were washed in phosphate-buffered saline with 0.1% Tween 20. Renaissance® (NENTM Life Science
Products) enhanced chemiluminescence reagent was used to detect
membrane-bound protein by luminography. This light is captured on Kodak
X-OMAT Blue Autoradiography film.
NF-
B/Rel-specific Decoy
Oligodeoxynucleotides--
We used a phosphorothioate double-stranded
decoy oligodeoxynucleotide carrying the NF-
B/Rel-consensus sequence
5'-CCTTGAAGGGATTTCCCTCC-3'. The mutated (scrambled) form
5'-CCTTGTACCATTGTTAGCC-3' was used as a control (41). The 1 µM oligodeoxynucleotide was mixed with 5 µg/ml of
TransFastTM (Promega) for 15 min at room temperature, then
the mixture was added to MCF-7 cells in serum-free medium. After 3 h of incubation, the cells were treated with HRG-
1 for further
appropriate times.
Electrophoretic Mobility Shift Assay--
Confluent MCF-7 cells
cultures were serum-starved overnight followed by stimulation with or
without HRG-
1 (50 ng/ml) for various time points. Nuclear extracts
were prepared by using a nonionic detergent method as described
previously (42). In brief, nuclear extracts were prepared from breast
cancer cells in extraction buffer (420 mM KCl, 20 mM HEPES (pH 7.9), 1.5 mM MgCl2,
0.2 mM EDTA, and 20% glycerol) plus protease inhibitors
(0.5 mM dithiothreitol, 0.5 mM
phenylmethylsulfonyl fluoride, and 10 µg/ml leupeptin). After
centrifugation at 14,000 rpm in a microcentrifuge for 1 min, the
supernatant was cytosol protein extracted and then placed into
extraction buffer (the same concentration of the other reagents as in
the extraction buffer except 500 mM KCl and 10%
glycerol). After centrifugation at 14,000 rpm for 5 min, the
supernatant fraction was harvested as the nuclear protein extract and
stored at
70 °C. Detection of NF-
B was performed with a
[
-32P]dCTP-labeled oligo probe containing the NF-
B
recognition site. The probe was synthesized to span the region of the
human VEGF-C promoter comprised between the +395 and +416:
5'-GGGGCCCAGGGGGGGTCGCCGGGAGGGGGG-3' (random sequences added to the wild type sequence are shown in italics), in which the underlined region indicates the core-binding element. Protruding 5' ends were filled in with (exo
) Klenow fragment
(from MBI Fermentas) and [
-32P]dCTP. Nuclear protein
(5 µg) was added to 20 µl of DNA-binding buffer (2 µg of
poly(dI-dC), 1 µg of bovine serum albumin, 5 mM dithiothreitol, 20 mM HEPES (pH 8.4), 60 mM
KCl, and 10% glycerol), which contained 5 × 104 cpm
[
-32P]-labeled oligonucleotide probe. The reaction
mixture was incubation at room temperature for 20 min. For competition
experiments, the cold oligonucleotide probe was added 15 min before
addition of the probe. Super-shift assays were performed with 1 µg of
antibodies against p65 or p50 incubated 30 min at 4 °C after
addition of the probe. The reaction products were analyzed via 5%
non-denaturing polyacrylamide gel electrophoresis using 12.5 mM Tris, 12.5 mM boric acid, and 0.25 mM EDTA (pH 8.3), for 4-5 h at 280-300 V/10-12 mA. The
gels were dried and exposed to AmershamTM film (Amersham
Biosciences) at
70 °C using an intensifying screen.
Preparation of VEGF-C Promoter-Luciferase Fusion
Constructs--
The 2.5-kb VEGF-C promoter fragment was screened and
obtained from a human placenta genomic library (in Lambda FIX II
vector) using full-length human VEGF-C cDNA, which was provided by
Dr. R.-Y. Yu as a probe. The 2.5-kb fragment was restricted with
XbaI (
1059) and RsrII (+493), and the resulting
1552-bp fragment was cloned into the pGL3-basic vector to generate
pGL3-1.5-kb VEGF-C. To delete the NF-
B site (+401 to +410), we used
PCR with following primers: forward (from
1040):
5'-AACTCGAGATTGGGAGTGAAAG; reverse (from +345):
5'-CCAAGCTTGCTCCCGCCTTCCC. The amplified PCR products were restricted
with XhoI and HindIII and cloned into the
corresponding sites of pGL3-basic vector. The construct was designated
as pGL3-1.4-kb/
NF-
B VEGF-C promoter-reporter. To construct a
plasmid containing the NF-
B site mutations in 1.5-kb VEGF-C
promoter, we used 1.5-kb VEGF-C promoter as a template and
designed a mutated NF-
B site (GGGGGTCGCCGG to
GGGATTCTCCGG) by using the QuikChange
Site-Directed Mutagenesis® kit (Stratagene). The mutated
construct was designated as pGL3-1.5-kb/mutNF-
B VEGF-C promoter-reporter.
Promoter Activity Assay--
For cell transfections, MCF-7 cells
were seeded in 12-well plates in triplicate. When reaching about 70%
confluence, the cells were transfected with pGL3-basic vector,
pNF-
B-Luc (BD Bioscience, Clontech),
pGL3-1.5-kb VEGF-C, pGL3-1.4-kb/
NF-
B, and
pGL3-1.5-kb/mutNF-
B VEGF-C using SuperFect®
Transfection Reagent (Qiagen, Valencia, CA). After transfection, the
medium was replaced by fresh normal growth medium, and the cells were
incubated for 24 h. After starvation in serum-free medium for
16 h, the cells were incubated in the presence or absence of
HRG-
1 (50 ng/ml) for another 1 or 6 h. The cells were harvested with passive buffer, and the luciferase activities were determined with
the use of a Dual-Luciferase® Reporter Assay system
(Promega) according to protocols provided by the manufacturer.
 |
RESULTS |
HRG-
1 Up-regulates VEGF-C in Human Breast Cancer
Cells--
Initially, we used the semi-quantitative RT-PCR technique
to analyze VEGF-C and -D mRNA expression in human breast cancer MCF-7 cells treated with various cytokines and growth factors, whose
altered levels have been shown to be associated with invasive or
metastasis behavior in breast cancer cells. Fig.
1A shows that under the same
circumstance, HRG-
1 (50 ng/ml) was the most potent agent to induce
VEGF-C, but not VEGF-D, mRNA expression in MCF-7 cells when
compared with other agents. Among the agents tested, basic fibroblast
growth factor and PMA also had significant inductive effects on
the level of VEGF-C mRNA at the higher concentrations (> 50 ng/ml). As shown in Fig. 1B, upper panel, the
VEGF-C mRNA level was elevated with a peak in the 3-9 h time
period after 50 ng/ml HRG-
1 treatment. Western blot analysis
revealed that the 58-kDa VEGF-C precursor protein was significantly
increased in the MCF-7 cells at 9 h and was sustained for up to
24 h post-HRG-
1 treatment (Fig. 1B, lower
panel). Fig. 1C reveals that the HRG-
1-induced increase in VEGF-C mRNA (upper panel) and protein level
(middle panel) appeared to be dosage-dependent.
An initial increase of VEGF-C mRNA and protein was observed at a
dose of 0.1 ng/ml HRG-
1, and the maximal induction was found at a
dose of 50 ng/ml. Fig. 1C, lower panel, shows
that not only the 58-kDa VEGF-C precursor but also the 29/31-kDa mature
VEGF-C protein could be increased by HRG-
1 treatment. It is strongly
suggested that VEGF-C protein induction by HRG-
1 is functionally
active in breast cancer cells. We next examined whether VEGF-C mRNA
up-regulation by HRG-
1 is due to increased transcription or
increased RNA stability. To this end, we pretreated MCF-7 cells
with 4 µg/ml actinomycin D, a RNA synthesis inhibitor, or 30 µg/ml
cyclohexamide, a protein synthesis inhibitor, for 1 h before
HRG-
1 was added to the culture medium. Fig. 1D shows that
actinomycin D completely abolished HRG-
1-induced VEGF-C
up-regulation, whereas cyclohexamide had no effect on VEGF-C induction
by HRG-
1. This finding suggests that HRG-
1-induced VEGF-C
up-regulation requires de novo RNA synthesis but not new
protein synthesis.

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Fig. 1.
HRG- 1 up-regulates
VEGF-C expressions through transcription regulation in MCF-7 breast
cancer cells. Total RNA and protein were collected from
serum-starved MCF-7 breast cancer cells treated with various dosages of
HRG- 1 or other cytokines and growth factors for various times as
indicated. RT-PCR and Western blot analysis was performed as
described under "Experimental Procedures." A,
HRG- 1-induced VEGF-C mRNA expression in MCF-7 cells. RT- PCR analysis of the VEGF-C and VEGF-D mRNAs in MCF-7 cells
treated with different growth factors for 6 h. B and
C, HRG- 1-mediated up-regulation of VEGF-C mRNA and
protein expression in MCF-7 cells is time- and
concentration-dependent. MCF-7 cells were treated with 50 ng/ml HRG- 1 for 0, 3, 6, 9, 12, or 24 h or with 0, 0.1, 1, 10, 50, or 100 ng/ml HRG- 1 for 6 h (RT-PCR) and 9 h (Western
blot analysis). The 58-kDa VEGF-C precursor and the 29/31-kDa mature
VEGF-C protein could be increased by HRG- (50 ng/ml, 9 h)
treatment. D, RT-PCR and Western blot analysis of VEGF-C
mRNA and protein in MCF-7 cells pretreated with either 4 µg/ml
actinomycin D or 30 µg/ml cyclohexamide for 1 h and then
incubated with 50 ng/ml HRG- 1 for 6 or 9 h.
|
|
The effect of HRG-
1 on VEGF-C mRNA expression in different
breast cancer cell lines was further investigated. As shown in Fig.
2A, HRG-
1 had a broad
capacity to induce a VEGF-C mRNA increase (around 3-4-fold) in
various human breast cancer cell lines, including SKBr3, T47D, and
MCF-7. In support of a previous study (33), we found that VEGF-A
mRNA was also markedly elevated in breast cancer cell lines treated
with HRG-
1. Interestingly, among these cell lines, HBL-100 cells had
a more abundant HRG-
1 level and displayed higher levels of VEGF-C
and -A mRNA, suggesting that endogenous HRG-
1 is capable of
inducing the VEGF-C mRNA expression. As previously described (26),
HRG-
1 preferentially binds to HER3 and HER4 receptors and then
transactivates HER2 by forming a HER3/HER2 or HER4/HER2
heterodimer, which in turn elicits the signaling pathways for certain
malignant properties. Here we asked whether the HER2 receptor is
required for HRG-
1-induced VEGF-C expression. Fig. 2B,
left panel, shows that HER2-overexpressed MCF-7 cells
displayed much more abundant levels of VEGF-C mRNA and protein as
compared with the vector control cells. In addition, HRG-
1-induced
up-regulation of the VEGF-C mRNA or protein was completely blocked
by treatment with Herceptin (an antibody against HER2 function) or
PD153035 (an inhibitor of HER2 kinase) (Fig. 2B, right
panel). This evidence strongly suggests that the activity of the
HER2 receptor is critical for HRG-
1-mediated VEGF-C gene expression.
To demonstrate the relationship between HER2 and VEGF-C in breast
carcinoma, eight breast tumor RNA samples were detected with HER2 and
VEGF-C mRNAs by using RT-PCR. Fig. 2C,
upper panel, reveals that in HER2/neu overexpressing tumors,
the VEGF-C is also overexpressed. Because human tumors are quite
hetergeneous, we obtained mammary tumors from HER2/neu transgenic mice
and isolated RNA for examining VEGF-C mRNA by using real-time
RT-PCR. As expected, we found that the level of VEGF-C mRNA is
significantly elevated in HER2-positive tumors compared with normal
tissues (Fig. 2C, lower panel). This result
suggests that HER2/neu overexpression correlates with increased
expression of VEGF-C in human breast cancers.

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Fig. 2.
HRG- 1 treatment and
HER2 overexpression up-regulate VEGF-C mRNA or protein expression
in human breast cancer cell lines. A, up-regulation of
VEGF-C in multiple breast cancer cell lines treated with 100 ng/ml
HRG- 1 for 6 h. B, left panel, HER2/neu
stable transfectants displayed much more abundant levels of VEGF-C
mRNA and protein. B, right panel, MCF-7 cells were
pretreated with Herceptin (500 ng/ml) and PD153035 (2.7 µM) for 1 h then treated with HRG- 1 (50 ng/ml)
for 6 or 9 h. VEGF-C mRNA or protein were detected by using
RT-PCR and Western blotting in MCF-7 cells. C, upper
panel, RT-PCR analysis of the expression of HER2 and VEGF-C
mRNAs in human breast cancers. C, lower
panel, real-time RT-PCR for examining VEGF-C mRNA in HER2/neu
overexpressed mammary tumors (2 tumors) and normal tissue. The amount
of VEGF-C mRNA in each sample was normalized against the amount of
GAPDH mRNA in the same sample and subsequently expressed as the
mRNA copy number of VEGF-C per 1000 copies of GAPDH mRNA. The
data were the average of three independent experiments. Bar,
standard error.
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|
Activation of NF-
B Is Required for VEGF-C
Expression--
Transcription factor NF-
B is one of the important
factors activated by HRG-
1 in human breast cancer cells (43, 44). A
putative NF-
B-binding site was found to be located within the VEGF-C
gene promoter region. This site may be implicated in the induction of
VEGF-C mRNA by interleukin-1
and tumor necrosis factor-
(45,
46). We thus tested if NF-
B activation would be involved in
HRG-
1-induced VEGF-C mRNA expression. To this end, MCF-7 cells
were exposed to 50 ng/ml HRG-
1, nuclear extracts were prepared, and
NF-
B protein translocation as well as NF-
B-binding activities
were assessed respectively, by Western blotting and EMSA using an
oligonucleotide corresponding to the putative NF-
B-binding site
within the VEGF-C promoter. As shown in Fig.
3A, the p65 and p50 NF-
B
subunits appeared in nuclear fractions at 30-120 min after HRG-
1
treatment. The nuclear proliferating cell nuclear antigen protein level
is an internal control to ensure equal amounts of total nuclear
protein. Consistently, the DNA-binding activity of NF-
B increased
significantly at 30-120 min (Fig. 3B, upper panel, lanes 2-4) after HRG-
1 treatment, and the
induction of DNA-binding activity was completely attenuated by
specifically competing with the non-radiolabeled probe (Fig.
3B, upper panel, lanes 5 and 6). To quantify the NF-
B activity, we transiently transfected a NF-
B luciferase reporter into MCF-7 cells. The data of
luciferase activity (Fig. 3B, lower panel) was
correlated with the DNA-binding activity of NF-
B in gel shift assay.
Super-shift assays were done to confirm the presence of p50 and p65
binding to the NF-
B-binding site, showing that the specific
protein-DNA-binding activity was super-shifted by the addition of
anti-p65 or anti-p50 antibodies (data not shown). We next employed a
NF-
B decoy oligonucleotide, which sequenced as transcription factor
decoys to inhibit NF-
B binding to the native DNA sites, to
specifically block HRG-
1-induced activation of the NF-
B pathway.
Fig. 3C reveals that the NF-
B decoy effectively blocked
p65 subunit nuclear translocation (upper panel) as well as
abolished the DNA-binding activity of NF-
B (lower panel)
induced by HRG-
1. In contrast, the scrambled NF-
B oligonucleotide
had no effect on the nuclear translocation and DNA-binding activity of
NF-
B by HRG-
1 treatment (Fig. 3C). As shown in Fig.
3D, treatment with decoy NF-
B completely inhibited HRG-
1-mediated VEGF-C mRNA up-regulation, suggesting that
NF-
B activation is important for VEGF-C gene up-regulation by
HRG-
1. To identify whether the NF-
B site is actually involved in
transcriptional regulation of VEGF-C by HRG-
1, we transiently
transfected the 1.5-kb-VEGF-C promoter-reporter containing NF-
B
site, a NF-
B-deleted promoter plasmid, 1.4-kb/
NF-
B VEGF-C, and
a NF-
B site mutated promoter plasmid, 1.5-kb/mutNF-
B
VEGF-C (see "Experimental Procedures") into MCF-7 cells and then
examined their luciferase activities after HRG-
1 treatment.
Fig. 3E shows a 4.2-fold induction of a 1.5-kb VEGF-C
promoter activity by treatment with 50 ng/ml HRG-
1. However,
HRG-
1 had no effect on the luciferase activity of control pGL3-basic
vector. When the NF-
B-deleted and -mutated promoter-reporter, pGL3-1.4-kb/
NF-
B VEGF-C and pGL3-1.5-kb/mutNF-
B
VEGF-C, were transfected into MCF-7 cells, their luciferase activities
were decreased around 60% in response to HRG-
1 when compared with pGL3-1.5-kb VEGF-C. These experiments demonstrate that the NF-
B site is indeed important for HRG-
1-mediated VEGF-C gene
up-regulation.

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Fig. 3.
Activation of NF- B
is required for VEGF-C expression. Nuclear extracts were prepared
from serum-starved MCF-7 cells treated with 50 ng/ml HRG- 1 for 0, 30, 60, or 120 min and 100 ng/ml PMA for 60 min as positive control.
A, nuclear extracts were subjected to Western blot analysis
by using indicated antibodies. B, upper panel,
induction of NF- B DNA-binding activity by HRG- 1 in MCF-7 cells.
EMSA was performed as described under "Experimental Procedures." To
test for specificity of binding, samples were incubated with 2 or 5×
excess unlabeled wild-type (URE). B, lower
panel, NF- B transcriptional activity was measured by luciferase
assay using an optimal NF- B-binding site in tandem followed by
luciferase. The data were the representative of three independent
experiments. C, MCF-7 breast carcinoma cultures were
pretreated with phosphorothioate modified oligodeoxynucleotides (1 µM). The sequence of NF- B oligodeoxynucleotides is
shown under "Experimental Procedures." Western blot and EMSA
performed on nuclear extracts after 60 min treated with 50 ng/ml
HRG- 1. D, MCF-7 cells were pretreated with 1 µM decoy NF- B then treated with 50 ng/ml HRG- 1 for
6 h. VEGF-C mRNA was detected by using RT-PCR. E,
identification of NF- B in VEGF-C promoter. MCF-7 were transfected
with 1.5-kb, 1.4-kb/ NF- B, and 1.5-kb/mutNF- B
promoter-driven plasmids. After being incubated with 50 ng/ml HRG- 1
for 6 h, the luciferase activity from transfectants were measured.
The data were the average of three independent experiments.
Bar, standard error.
|
|
The NF-
B activation is intimately associated with the I
B
phosphorylation and degradation. We thus tested whether HRG-
1 could
induce endogenous I
B
phosphorylation and degradation. Western
blot analysis shows that an evident I
B
protein phosphorylation and a subsequent degradation occurred in the MCF-7 cells after HRG-
1
treatment (Fig. 4A). A
DN-I
B
vector, which is resistant to phosphorylation and
degradation, was transfected and overexpressed in the MCF-7 cells.
Overexpression of DN-I
B
significantly repressed the
NF-
B-DNA-binding activity (Fig. 4B) and NF-
B nuclear
translocation in MCF-7 cells after exposure to HRG-
1 (data not
shown). Under the same experimental conditions, we further examined
whether DN-I
B expression would modulate the HRG-
1-induced
VEGF-C gene expression. The data shown in Fig. 4C
reveals that the HRG-
1-mediated increase in VEGF-C mRNA
(upper panel) and protein (lower panel) was
strongly abolished in DN-I
B
overexpressed cells but not in the
control vector-transfected cells. These results suggest that I
B
degradation and subsequent NF-
B activation were required for
VEGF-C gene expression induced by HRG-
1.

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Fig. 4.
Effect of HRG- 1 on
I B phosphorylation
and degradation. A, cytosol extracts were prepared from
serum-starved MCF-7 cells treated with 50 ng/ml HRG- 1 for 0, 30, 60, or 120 min then subjected to Western blotting with indicated
antibodies. Phosphospecific antibody recognizes I B phosphorylated
at serine 32. B, MCF-7 cells were transiently transfected
with dominant-negative I B . The transfectants were treated with 50 ng/ml HRG- 1 for 60 min. Active NF- B was determined by its
[ -32P]DNA-binding activity by EMSA analysis.
C, the level of VEGF-C mRNA and protein in
DN-I B -expressing MCF-7 cells and vector control transfected cells
after treatment with 50 ng/ml HRG- 1 for 6 or 9 h, as measured
by RT-PCR and Western blot analysis.
|
|
p38 MAPK Is Involved in HRG-
1-induced VEGF-C Gene
Up-regulation--
Many of the kinase signaling pathways have been
shown to be activated and involved in HRG-
1-induced diversified
cellular functions (44, 47). Therefore, we examined which of the kinase signaling pathways is/are required for HRG-
1-induced up-regulation of the VEGF-C gene. To address this issue, MCF-7 cells were
pre-treated with SB203580 (p38 MAPK inhibitor), PD98059 (MEK
inhibitor), or LY294002 (PI3K inhibitor) for 1 h and followed by
treatment with HRG-
1 for 6 or 9 h, and total RNA and protein
were used to determine VEGF-C expression. Fig.
5A reveals that 20 µM of SB203580 completely reduced the increased level of
VEGF-C mRNA (upper panel) and protein (lower
panel) induced by HRG-
1, whereas two other kinase inhibitors, PD98059 and LY294002, had no effect on the up-regulation of VEGF-C. As
shown in Fig. 5B, HRG-
1 indeed activated these three
kinase pathways, p38 MAPK, ERK1/2, and PI3K, in MCF-7 cells as
evidenced by the elevation of their phosphorylated form using their
specific antibodies. Our data also demonstrated that these three
signaling pathways were effectively blocked in MCF-7 cells when
treatment with pharmacological inhibitors at the dose indicated (Fig.
5B). It is suggested that those pharmacological inhibitors
were functionally active in inhibiting the specific signaling pathways.
Because SB203580 was demonstrated to significantly abolish
VEGF-C gene up-regulation by HRG-
1, we thus used another
approach to test the importance of p38 MAPK by establishing a MCF-7
cell line expressing dominant-negative p38 (p38AF) and examined the
expression level of VEGF-C mRNA and protein. Fig. 5C,
upper panel, we demonstrated that HRG-
1-stimulation of
the phosphorylation of p38 MAPK was totally reduced in p38AF expressed
cells but not in the control vector expressed cells. Blocking the p38
MAPK pathway by p38AF also significantly diminished the
HRG-
1-induced up-regulation of VEGF-C mRNA (Fig. 5C,
middle panel) and protein (Fig. 5C,
lower panel). In brief, these results suggest the
possible involvement of p38 MAPK, but not ERK1/2 or PI3K, in the
regulation of VEGF-C gene expression in breast cancer cells
induced by HRG-
1.

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Fig. 5.
The p38 MAPK kinase pathway is required for
the induction of VEGF-C by HRG- 1.
A, MCF-7 cells were incubated with or without 75 µM PD98059, 37.5 µM LY294002, or 20 µM SB203580. HRG- 1 (50 ng/ml) was added to the media,
cell lysates were collected after 9 h for Western blot analysis of
VEGF-C expression, and total RNA was prepared from cells for RT-PCR
analysis of VEGF-C mRNA after 6 h of HRG- 1 treatment.
B, to test the efficacy of inhibitors, MCF-7 cells were
starved overnight, pretreated with the inhibitors at the concentrations
as in Fig. 5A for 1 h, and stimulated by 50 ng/ml
HRG- 1 for 5 min. Cell lysates were prepared and subjected to Western
blot analysis using antibodies against p-p38, p-ERK1/2, and p-AKT.
C, MCF-7 cells were transfected with vector or p38AF
and serum-starved for 24 h and treated with HRG- 1 (50 ng/ml)
for 6 or 9 h, analyzed on Western blotting and RT-PCR.
|
|
p38 MAPK has recently been shown to regulate NF-
B activity in
certain cell systems (48, 49), and we therefore explored the possible
link between p38 MAPK and NF-
B in HRG-
1-mediated VEGF-C gene
expression. To this end, we determined the NF-
B DNA-binding activity
and NF-
B transcriptional activity in cells treated with different
pharmacological inhibitors. Fig.
6A shows that the
HRG-
1-stimulated increase in NF-
B DNA-binding activity and
NF-
B reporter activity were nearly abolished by SB203580 but not
affected by PD98059 and LY294002. Consistently, HRG-
1 treatment
neither induced the nuclear translocation of NF-
B p65 (Fig.
6B, upper panel) nor elevated the NF-
B
DNA-binding activity (Fig. 6B, lower panel) in
p38AF-expressed cells as compared with control vector cells.

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Fig. 6.
p38 MAPK inhibitor and dominant-negative p38
reduced HRG- 1-mediated
NF- B activation. A,
upper panel, the effect of inhibitors on HRG- 1-induced NF- B activation in
MCF-7 cells. MCF-7 cells were pretreated with the p38 MAPK inhibitor
SB203580 (20 µM), ERK/MAPK inhibitor PD098059 (75 µM), or PI3K inhibitor LY294002 (37.5 µM)
for 1 h. Subsequently, cells were incubated with HRG- 1 (50 ng/ml) for 60 min, and nuclear extracts were subjected to EMSA with
NF- B probe. A, lower panel, after transfected
with p-NF- B-Luc, MCF-7 cells were treated with the same experimental
conditions as mentioned in the upper panel, then the
luciferase activity was measured. The data were the
representative of three independent experiments. B, MCF-7
cells were transiently transfected with p38AF then treated with 50 ng/ml HRG- 1 for 60 min. Active NF- B was determined by its nuclear
translocation by Western blotting (upper panel) and
[ -32P]DNA-binding activity by EMSA (lower
panel). C, I B phosphorylation-degradation level
in these cytosol extracts was measured by Western blotting.
|
|
Our results suggest that blockage of p38 MAPK could prevent NF-
B
nuclear translocation, NF-
B DNA-binding activity, and NF-
B reporter activity by HRG-
1. We further examined whether the level of
phosphorylated I
B
in cells expressing p38AF or treated with SB203580 would be changed. As expected, Western blot analysis showed
that HRG-
1-induced the elevation of phosphorylated I
B
and its
degradation was nearly completely abolished in cells treated with
SB203580 (Fig. 6C, upper panel) or in cells
expressing p38AF (Fig. 6C, lower panel).
Together, the data obtained here strongly suggests that p38 MAPK may
act as an up-stream kinase to activate the
NF-
B-dependent up-regulation of VEGF-C in response to
HRG-
1.
 |
DISCUSSION |
In this study we demonstrated that activation of a novel signaling
pathway from p38 MAPK to subsequent NF-
B is a potential requirement
for HRG-
1-mediated up-regulation of the lymphangiogenic factor
VEGF-C in human breast cancer cells. Although VEGF-C has been shown to
be highly expressed in a vast array of malignant tissues, its
up-regulation in cancer cells by which factor(s) is completely unknown.
Few, if any, investigations have found that pro-inflammatory cytokines
such as interleukin-1
and tumor necrosis factor-
could
strongly induce VEGF-C gene expression in human fibroblasts
(46). Here we show that neither IL-1
nor TNF-
were capable of
up-regulating VEGF-C in human breast cancer cells (as can be seen in
Fig. 1A). Importantly, this is the first time it has been
demonstrated that HRG-
1 is a strong up-regulator of the
VEGF-C gene in human breast cancer cells. In addition, our
current data and that from others (15) suggest that the regulatory mechanism for the VEGF-C gene varies in different
cellular contexts or in different pathological processes.
Accumulating evidence showed that overexpression of HRG-
1 in human
breast cancer cells resulted in the more aggressive phenotypes, i.e. enhancement in cell migration and invasion abilities
(32, 50). Many of the downstream effector genes, such as urokinase plasminogen activator (37, 51), metalloproteinases (36), paxillin (38),
and autocrine motility factor (52), have been identified and may
partially account for the role of HRG-
1 in the progression of human
breast cancer. However, the detailed mechanism underlying how HRG-
1
induces these downstream effector genes to coordinate with one another
and arrange the aggressive phenotype is largely unknown. Here we
provide evidence that shows the lymphangiogenic factor, VEGF-C, is
induced in human breast cancer cells by HRG-
1. The induction of
VEGF-C does not require protein synthesis, suggesting that it may be
mediated by activation of certain existing transcription factor(s) by
post-translational modification or relocation. Consistent with this
notion, we detected that the transcription factor NF-
B is activated
and critically required for HRG-
1-induced VEGF-C up-regulation as
evidenced by NF-
B decoy treatment (Fig. 3C) and
DN-I
B
transfection (Fig. 4C). Our data further points
out that HRG-
1-stimulation of NF-
B DNA-binding activity is mainly
through a mechanism involving phosphorylation-degradation of I
B
and the subsequent translocation of NF-
B into the nucleus. Supportively, the VEGF-C promoter-reporter assay clearly demonstrated that the putative NF-
B site in the VEGF-C promoter region is critical for HRG-
1-induced VEGF-C up-regulation (Fig.
3E). Although the sequential activation of NF-
B is
commonly observed, this is the first time it has been shown that
HRG-
1 is capable of activating this mechanism in human breast cancer
cells. NF-
B activation plays a key role in drug resistance and
metastasis in human breast cancer (53-55). Here we report that the
lymphangiogenic factor, VEGF-C, acts as one of the downstream effector
genes of NF-
B signaling. This provides a new aspect of NF-
B in
the pathogenesis of human breast cancer.
Many protein kinases including PI3K/Akt, ERK1/2, and p38 MAPK have been
found to integrate into the activation process of NF-
B in different
cell systems in a distinct way (56-58). Our data demonstrates that in
MCF-7 cells these three signaling pathways are all activated by
HRG-
1 (Fig. 5B). Using a pharmacological and genetic
inhibition approach, we found that only p38 MAPK signaling is
specifically and dominantly involved in HRG-
1-stimulated
NF-
B-dependent VEGF-C up-regulation, although a possible
cross-talk may occur between the p38 signaling and NF-
B activation
process. Currently, the mechanism for the interplay between these two
pathways is not yet understood and may vary in different cell models. A
recent study (57) revealed that the p38 pathway did not affect NF-
B binding and transcriptional activity directly. Instead, it acts by enhancing the NF-
B recruitment into a subset of gene promoters in
response to inflammatory stimuli (57). Distinct from that, in our cell
model HRG-
1-stimulated p38 signaling could promote I
B
phosphorylation and degradation. Phosphorylation of I
B
is carried
out by the multisubunit I
B kinase (IKK), which is in turn activated
by the NF-
B-inducing kinase (NIK) or by the mitogen-activated
protein kinase (MEKK1) (59, 60). How could p38 kinase signaling connect
with the NF-
B activation process? One possibility could be through
phosphorylation of the IKK molecule. Several reports showed that not
only NIK and MEKK1 but also Akt could phosphorylate IKK (61, 62),
suggesting that IKK could be as a key molecule to receive various
kinase signaling and transmit into NF-
B activation cascade.
Supporting this hypothesis, we found that p38 kinase is closely
associated with IKK in response to HRG-
1 as demonstrated by
co-immunoprecipitation using the anti-p38 antibody (data not shown).
Alternatively, it is also possible that there may be a critically
intermediate kinase connecting p38 signaling and IKK.
Ectopic overexpression of VEGF-C in different human breast cancer cells
including MCF-7 and MDA-MB-435, all potently increased intratumoral
lymphangiogenesis, resulting in significantly enhanced metastasis in
the regional lymph nodes (5, 7, 63). The 29/31-kDa mature form of
VEGF-C, which specifically activates VEGFR-3, was obviously detected in
both cell lines. Using a soluble form VEGFR-3 fusion protein inhibited
the tumor growth and tumor-associated lymphangiogenesis by blocking the
interaction between the mature form of VEGF-C and its receptor. Under
the same MCF-7 cell system, we demonstrated that HRG-
1-treated MCF-7
cells indeed produced a large amount of mature VEGF-C either in the
total cell lysate (Fig. 1C) or in conditioned media (data
not shown). This strongly suggests that the enhancement of
lymphangiogenesis may partially account for HRG-
1-mediating a more
aggressive phenotype of human breast cancers. Our current data for cell
lines, clinical specimens, and transgenic mice also show that HER2
receptor overexpression caused an increased expression of VEGF-C, thus
suggesting that the level of HER2 receptor is another switch for
promoting lymphangiogenesis in breast cancer. In agreement with our
data, Yang et al. (64) have demonstrated that the
expression of VEGF-A, -C, and -D was positively correlated with
HER2/neu expression in human breast carcinomas (64).
In summary, our findings identified for the first time that HRG-
1
potently induces the up-regulation of VEGF-C mRNA and protein in
human breast cancer MCF-7 cells through a novel signaling pathway from
HER2 receptor, p38 MAPK, to the subsequent activation of NF-
B
cascade (Fig. 7). Our study also provides
a therapeutic rationale for the inhibition of breast tumor
lymphangiogenesis using pharmacological inhibitors to block this
signaling cascade or using Herceptin to inhibit HER2
activity.

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Fig. 7.
The signaling pathway of up-regulation of
VEGF-C in breast cancer cells by HRG- 1.
HRG- 1 potently induces the up-regulation of VEGF-C mRNA and
protein in human breast cancer MCF-7 cells through a novel signaling
pathway from HER2 receptor, p38 MAPK, to the subsequent activation of
NF- B cascade.
|
|
 |
ACKNOWLEDGEMENTS |
We sincerely thank Dr. R.-Y. Yu at the Cancer
Research Center of Veteran Hospital, Taipei, Taiwan, for the VEGF-C
promoter-reporter constructs and Dr. Shuang-En Chuang at the Division
of Cancer Research, National Health Research Institute, Taipei, Taiwan, for DN-I
B
plasmid and technical suggestions.
 |
FOOTNOTES |
*
This research was supported by the National Science Council,
NSC-91-2320-B-002-209, Taipei, Taiwan.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: Laboratory of
Molecular & Cellular Toxicology, Institute of Toxicology, College of
Medicine, National Taiwan University, No. 1, Sec. 1, Jen-Ai Rd., Taipei
110, Taiwan. Fax: 886-2-2341-0217; E-mail:
toxkml@ha.mc.ntu.edu.tw.
Published, JBC Papers in Press, December 5, 2002, DOI 10.1074/jbc.M204863200
 |
ABBREVIATIONS |
The abbreviations used are:
VEGF, vascular
endothelial growth factor;
EGF, epidermal growth factor;
HRG-
1, heregulin-
1;
HER, human epidermal growth factor receptor;
NF-
B, nuclear factor-
B;
I
B, inhibitor of
B;
ERK, extracellular signal-regulated protein kinase;
PI3K, phosphatidylinositol 3'-kinase;
DN, dominant-negative;
MAPK, mitogen-activated protein kinase;
MEK, mitogen-activated protein
kinase/extracellular signal-regulated kinase kinase;
IKK, I
B kinase;
NIK, NF-
B-inducing kinase;
GAPDH, glyceraldehyde-3-phosphate
dehydrogenase;
RT, reverse transcriptase;
EMSA, electrophoretic
mobility shift assay.
 |
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