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INTRODUCTION |
Insulin-like growth factor I
(IGF-I),1 a 70-residue
single-chain growth-promoting polypeptide, is produced in many
organs and tissues and plays a major role in somatic growth, cell
survival, tissue differentiation, and intermediary metabolism (1-3).
Although various tissue-dependent factors as well as
endocrine hormones seem to regulate the IGF-I gene expression, their
mechanisms, except for those involved in prostaglandin
E2 or cAMP (4-6), are poorly understood.
The protein kinase-C (PKC) pathway is among the few that have been
suggested to be involved in IGF-I gene regulation. The result of a
nuclear run-on assay indicated that treatment of human macrophage-like
cells with 12-O-tetradecanoylphorbol 13-acetate (TPA)
increased the transcription rate of the IGF-I gene 4- to 5-fold (7),
suggesting that the human IGF-I gene regulatory sequences contain
something that responds to PKC. Support for this also comes from our
recent observations with the chicken IGF-I gene, i.e. that
the gene promoter can be activated by TPA through an AP-1 binding site
located in it (8). However, it is unknown whether the mammalian IGF-I
genes are activated by PKC, and if they are, how this occurs.
In contrast to protein kinase A, which seems to be involved in
parathyroid hormone or prostaglandin E2-induced IGF-I gene activation, PKC has been often discussed in correlation with
tumorigenesis. Indeed, the best-known activator of PKC, TPA, is
a strong tumor promoter (9, 10). In vitro overexpression
studies have suggested that individual PKC isozymes control cell
proliferation and malignant transformation. For example, when PKC
I
was overexpressed in rat fibroblasts, the cells were partially
transformed and could form tumors in nude mice (11). Overexpression of
PKC
also occasionally leads to transformation of fibroblasts (12).
Because the IGF system is known to play an essential role in inducing
transformation (13) or maintaining the tumor phenotype in some cells,
such as a human neuroblastoma cell line SK-N-MC (14), it is likely that
PKC-dependent activation of the IGF-I gene, if it occurs in
mammals, may be partially involved in the tumorigenesis.
As a step toward elucidating the molecular basis of IGF-I gene
regulation, we examined whether the human IGF-I gene promoter is a
target of PKC regulation and sought to elucidate the physiological roles of the PKC pathway in the gene expression. Here we report that
the major promoter of the gene, which is located within the 5'-flanking
region and untranslated region of exon 1, is indeed a target of TPA
stimulation and a CCAAT/enhancer binding protein (C/EBP) site within
the promoter is responsible for the phenomenon. C/EBP, which is
activated by PKC both at the level of transcription and of
posttranslation, binds to the C/EBP site and thereby mediates the
phenomenon. Interestingly, the posttranslational activation of C/EBP
occurs primarily through activation of p90 ribosomal S6 kinase (RSK).
Moreover, as support for the pathophysiological significance of these
findings, we found that the constitutive IGF-I gene expression in the
human neuroblastoma-derived SK-N-MC cells depends on the intrinsically
activated PKC.
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EXPERIMENTAL PROCEDURES |
Antibodies--
Antibodies to C/EBP (14AA), C/EBP (C-19),
C/EBP
(C-22), HNF-1 (C-19), and to c-Myb (M-19) were purchased
from Santa Cruz Biotechnology, Inc (Santa Cruz, CA). Antibodies to
c-Fos and to c-Jun were purchased from Oncogene Science (Uniondale,
NY). Antibody to HA tag was purchased from Upstate Biotechnology, Inc.
(Lake Placid, NY).
Cell Culture--
HepG2 cells (Riken Cell Bank, Tsukuba, Japan,
catalog no. RCB459) were maintained as previously described (15).
SK-N-MC cells (ATCC catalog no. HTB10) were maintained in
Earle's modified Eagle's medium supplemented with 10%
heat-inactivated fetal bovine serum, non-essential amino acids,
penicillin, and streptomycin (basal condition medium). 293T cells were
maintained as previously described (16).
Plasmids--
Human IGF-I promoter-1-luciferase fusion
genes were constructed as follows. A phage clone containing the
5'-flanking region and exon 1 of the human IGF-I gene was isolated from
a human genomic library and used as a template to make the reporter
gene plasmids. A series of PCR was performed using the phage DNA as a
template to amplify promoter-1 DNA fragments, which were
comprised of either 1600 bp or 300 bp of the human IGF-I gene
5'-flanking region and the 197 bp of the exon 1 untranslated region.
The PCR primers used were 5'-GCGGTACCGCCTCTCAATGACACAATCTG-3'(for the
1600-bp fragment), 5'-GCGGTACCGAGTTTGCTGGAGAGGGTCT-3'(for the
300-bp fragment) and 5'-GGCAAGCTTGCGCAGGCTCTATCTGCT-3' (for
both). To make the plasmid pIGFI-1600 (Fig. 1), the PCR-amplified
1600-bp fragment was made blunt-ended using the DNA Blunting Kit
(Takara, Kyoto, Japan), digested with HindIII, and ligated
into SmaI/HindIII-digested pA3Luc (a kind gift
from I. H. Maxwell, University of Colorado Health Science
Center, Denver, CO) (17). The plasmid pIGFI-600 (Fig. 1) was
constructed by digesting the 1600-bp fragment with KpnI and
HindIII and subcloning the resulting 600-bp fragment into
the KpnI/HindIII-digested pA3Luc. The 300-bp
fragment was digested with KpnI and HindIII and
ligated into KpnI/HindIII-digested pA3Luc to
construct the plasmid pIGFI-300 (Fig. 1). Site-directed mutagenesis was
performed as described previously (8).
C/EBP expression vector pcDNA3C/EBP has been described
previously (5). Wild type and dominant negative (DN) type RSK expression vectors were gifts from Dr. J. Blenis (Boston, MA). The
plasmid pMSV -gal is an expression plasmid of the -galactosidase gene driven by the murine sarcoma virus long terminal repeat (18). The
plasmid pRL-CMV was purchased from Promega Corp (Madison, WI).
Gene Transfer Experiments--
Transfection studies using HepG2
cells were performed as follows. One microgram of IGF-I
promoter-1-lucferase fusion genes were cotransfected with 500 ng of
pMSV -gal to normalize for transfection efficiency. Cultures at 50%
confluent density were rinsed in serum-free medium and exposed to
plasmids in the presence of LipofectAMINETM for 5 h.
After washing the plates two times with PBS, the solution was then
replaced with serum-free medium (15), and the cells were incubated for
24 h. Next, the cells were treated for 24 h with vehicle
(Me2SO) or 10
7 M TPA. After
incubation, the medium was aspirated, the cultures were rinsed with PBS
twice and lysed in cell lysis buffer (Promega), and luciferase activity
was measured as described previously (15).
Transfection studies using SK-N-MC cells were performed as follows. One
microgram of IGF-I promoter-1-lucferase fusion genes were cotransfected
with 5 ng of pRL-CMV to normalize for transfection efficiency. Cultures
at 50% confluent density were rinsed in serum-free medium and exposed
to plasmids in the presence of LipofectAMINETM for 5 h. After washing the plates two times with PBS, the solution was
replaced with culture medium containing 10% fetal bovine serum, and
the cells were incubated for 24 h. Next, the cells were treated for 24 h with vehicle (Me2SO) or 107
M GF109203X. After incubation, the medium was aspirated,
the cultures were rinsed with PBS twice and lysed in cell lysis buffer, and dual-luciferase assay was performed according to the
manufacturer's instructions (Promega).
Transfection studies using 293T cells were performed basically in the
same way as the SK-N-MC cells described above. One microgram of IGF-I
promoter-1-luciferase fusion genes were cotransfected with the
indicated amount of C/EBP expression vector, 1 µg of wild type or
dominant negative type RSK expression vector, when required, and 5 ng
of pRL-CMV. After transfection, the cells were incubated for 24 h
and dual-luciferase assay (Promega) was performed following the
manufacturer's directions.
Gal4 Fusion Protein Reporter Gene Analyses--
The Gal4 fusion
constructs (Gal4C/EBP118 and Gal4C/EBP166) were generated by
isolating (by PCR) and introducing appropriate DNA fragments of
C/EBP into the EcoRI-BglII site of pFACMV
plasmid (Stratagene), which contained the DNA-binding domain (positions 1-147) of Gal4. Site-directed mutagenesis was performed with the QuikChange site-directed mutagenesis kit (Stratagene) using two synthetic complementary oligonucleotides,
5'-CCGAGCAAGAAGCCGGCCGACTACGGTTACG-3' and
5'-CGTAACCGTAGTCGGCCGGCTTCTTGCTCGG-3' (mutated sequence is underlined), to generate Gal4C/EBP118mutAla105 and
Gal4C/EBP166mut105Ala. The Gal4-responsive reporter plasmid pFR-Luc
plasmid containing five copies of Gal4-binding element upstream of the
basic promoter element (TATA box) linked to luciferase structural gene
was purchased from Stratagene. By lipofection, 1 µg of each Gal4
fusion plasmid was cotransfected into the host cell with 1 µg of
pFR-Luc and 5 ng of pRL-CMV. The cells were then incubated for 48 h, followed by a dual-luciferase assay (Promega) performed according to
the manufacturer's directions.
Nuclear Protein Extracts--
HepG2 and SK-N-MC cell nuclear
extracts were prepared by the method of Lee et al. (19) with
minor modifications. Cells were harvested with a cell scraper and
gently pelleted, and the pellets were washed with phosphate-buffered
saline. The cells were then lysed in hypotonic buffer (10 mM HEPES, pH 7.4, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM dithiothreitol). Nuclei were
pelleted and resuspended in hypertonic buffer containing 20 mM HEPES, pH 7.9, 0.42 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 25%
glycerol, 0.5 mM dithiothreitol, and 0.5 mM
phenylmethylsulfonyl fluoride. Soluble proteins released by a 30-min
incubation at 4 °C were collected by centrifugation at 12,000 × g for 20 min, and the supernatant was collected. The protein concentration was measured using a modified Bradford assay (Bio-Rad).
DNA-Protein Binding Studies--
Gel mobility shift experiments
followed previously published methods (4). Radiolabeled double-stranded
DNA probes were synthesized by annealing complementary end-labeled
oligonucleotides. Nuclear protein extracts (5 µg) were preincubated
for 20 min on ice with 2 mg of poly(dI-dC) with or without unlabeled
specific or nonspecific DNA competitor or antibodies in 25 mM HEPES, pH 7.6, 60 mM KCl, 7.5% glycerol,
0.1 mM EDTA, 5 mM dithiothreitol, and 0.025%
bovine serum albumin. After the addition of 5 × 104
cpm of DNA probe for 30 min on ice, the samples were applied to 12%
nondenaturing polyacrylamide gel that had been pre-electrophoresed for
30 min at 12.5 V/cm at 25 °C in 45 mM Tris, 45 mM boric acid, and 1 mM EDTA. Electrophoresis
was conducted for 2.5 h under identical conditions. The dried gels
were exposed to x-ray film at 
80 °C with an intensifying screen.
Deoxynuclease I (DNase) footprinting was performed as described
elsewhere (6). End-labeled double-stranded DNA probes flanking the
C/EBP site in human IGF-I promoter-1, which corresponds to 46 to +96
bp (relative to transcriptional start site) of the human IGF-I gene,
were generated by polymerase chain reaction using one end-labeled
oligonucleotide primer (5'-ATGCTCTGTCTCTAGTT-3') and one unlabeled
primer (5'-ACTGTAGACAGGAAACAGCT-3'). Nuclear protein (10 µg) was
preincubated for 15 min with poly(dI-dC) in 25 mM HEPES, pH
7.6, 60 mM KCl, 7.5% glycerol, 0.1 mM EDTA, 5 mM dithiothreitol, and 0.05% bovine serum albumin,
followed by the addition of labeled probe (5.0 × 105
cpm/sample) and incubation for 60 min on ice. The reaction mixture was
then treated with DNase I (final concentration 1.15 mg/ml, Worthington
Biochemical Corp., Freehold, NJ) in 2.5 mM
MgCl2 and 2.5 mM CaCl2 for 1 min at
25 C. Nuclease treatment was terminated by addition of 20 mM EDTA, 200 mM NaCl, 1% sodium dodecyl
sulfate, and 10 mg of yeast transfer RNA followed by phenol-chloroform extraction and ethanol precipitation. Samples were analyzed after electrophoresis on 8% polyacrylamide, 8 M urea gel, and
autoradiography for 16 h at 80 °C with an intensifying screen.
RNA Isolation and Analyses--
Total RNA was extracted
from HepG2 cells or SK-N-MC cells by homogenization in guanidine
thiocyanate. Northern blots followed standard procedures using 10 µg
of total RNA, and the buffer conditions were as described. The
hybridization probes were 7 × 106 cpm of
32P-labeled rat C/EBP cDNA probe and human IGF-I
cDNA probe. Reverse transcription-PCR were performed using primers
5'-ATCAGCGTCTTCCAACCCAATTA-3' and 5'-TGGCGCTGGGCACGGACAGA-3' (for human
IGF-I), and 5'-AAGGCCGGCTTCGCGGCGA-3' and 5'-CCGGCCAGCCAGGTCCAGAC-3'
(for -actin).
Western Blot Analysis--
293T cell nuclear proteins were
separated by SDS-PAGE and transferred to nitrocellulose membranes.
After the membranes were blocked with 5% nonfat dry milk and 2% fetal
bovine serum in 20 mM Tris-Cl, pH 7.6, and 137 mM NaCl for 1 h at 25 °C, they were incubated with
an antibody to HA for 1 h at 25 °C. Subsequent steps were
performed as described elsewhere (5).
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RESULTS |
Human IGF-I Gene Promoter-1 Is Activated by Phorbol
Ester--
First we examined whether the major promoter (promoter-1)
of the human IGF-I gene is a target of PKC activation. A series of gene
transfer studies were performed with human hepatocellular carcinoma-derived HepG2 cells. Under the basal condition, HepG2 cells
barely express the IGF-I gene according to reverse transcription-PCR results, but the IGF-I mRNA derived from the promoter 1 was induced when the cells were treated with 107 M TPA
for 4 h (data not shown). Each reporter gene plasmid contained various lengths of IGF-I gene 5'-flanking sequences and 197 bp of the
exon 1 untranslated region linked to the firefly luciferase reporter
because this portion of exon 1 untranslated region appeared to be
important for basal promoter activity of promoter-1 in SK-N-MC cells
(20).
As shown in Fig. 1, despite differences
in the basal promoter activities, the promoter activities of the 1600 bp (pIGFI-1600), 600 bp (pIGFI-600), and 300 bp (pIGFI-300) were
activated to a similar extent after treatment with TPA, about 2.5-fold.
This result showed that the human IGF-I gene promoter-1 could be
activated by TPA treatment of the cells and that the major portion of
the cis-active elements mediating this phenomenon is located within the
300 bp of the 5'-flanking sequence and/or the 197 bp of the untranslated region of exon 1.
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Fig. 1.
Putative TPA-responsive site is located in
the 5'-untranslated region of exon 1 of human IGF-I gene. Left
panel, diagrammatic representation of IGF-I promoter-1-luciferase
reporter plasmids. The nucleotide sequence is shown for the region
containing AP-1-like sequence (bold) and C/EBP binding site
(underlined). pIGFI-300M has a point mutation, and the
altered base is in outline lettering. Right
panel, IGF-I promoter-1-luciferase reporter plasmids were
cotransfected with a pMSV--galactosidase control vector into HepG2
cell using LipofectAMINE. After a 24-h incubation, the cultures were
exposed to control medium (containing vehicle) or TPA (100 nM) for 24 h. Cytoplasmic extracts were prepared, and
luciferase activity was determined. Data were normalized for
transfection efficiency (-galactosidase expression) and presented as
means ± S.D. of at least three independent experiments performed
in duplicate.
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Identification of a Putative TPA-responsive Element--
There was
no region that perfectly matched the consensus AP-1 motif (T(G/T)AGTCA)
within the region of the IGF-I gene which revealed the TPA
responsiveness (300 to ~+197). However, a region of high similarity
to the AP-1 consensus was seen within the exon 1 untranslated region
(+23 to ~+29; TTACTCA); indeed, the same sequence in the JE-1 gene
was shown to be a target for TPA-responsive activation in MC3T3-E1
cells (21).
To find whether this portion is involved in the TPA responsiveness, we
performed a mutation analysis. Because this portion was located within
a region where multiple transcription initiation sites are clustered,
it seemed possible that a mutation in this region could cause
unpredictable, nonspecific damage to the promoter activity. To avoid
this, we changed one sequence of the possible TPA-responsive region
(A (+29) to G) so that the sequence became the same as the
homologous regions of the chicken and rat IGF-I genes (22, 23).
As shown in Fig. 1, when one nucleotide mutation was introduced into
the portion (pIGFI-300M), TPA-induced promoter activation was
completely abolished, showing that this portion does play an essential
role in mediating TPA effects on the IGF-I gene promoter. The mutated
promoter also caused a decrease in the basal promoter activity (Fig.
1).
C/EBP Binds to the Putative TPA-responsive
Region--
Interestingly, the putative TPA-responsive region in the
human IGF-I gene also contains the consensus for the C/EBP binding motif, CTTACTCAA. Indeed, Nolten et al. previously
demonstrated in vitro that C/EBP and C/EBP, when
overexpressed, can bind to this region (24).
To characterize the factors involved in the TPA activation of the human
IGF-I gene, we performed gel-mobility shift analyses. As shown in Fig.
2, TPA enhanced specific protein bindings
to the putative TPA-responsive region (lanes 1 and
5). The unlabeled wild type competitor, but not the mutated
competitor to which the same point mutation was introduced as in the
reporter gene construct (A (+29) to G), inhibited the
DNA-protein bindings (lanes 6-11). Also, when the mutation
was introduced to the labeled probe, no binding was observed at all
(lane 12). Thus, a certain factor or factors bind to the
putative TPA-responsive region in the human IGF-I gene promoter in a
TPA-responsive manner, and this mediates the TPA responsiveness of the
gene transcription.
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Fig. 2.
TPA induced specific nuclear protein binding
to the putative TPA responsive site. Gel mobility shift
experiments were performed as described under "Experimental
Procedures" with nuclear protein extracts isolated from HepG2 cell
after incubation with vehicle or 100 nM TPA for 4 h.
Lanes 1-11 show protein binding to the
32P-labeled wild type probe, and lane 12 shows
protein binding to the 32P-labeled mutated type probe. The
sense strand sequence of probes are as follows: wild type probe,
5'-AAGTCCTTACTCAATAACTT and mutated type probe,
5'-AAGTCCTTACTCGATAACTT (mutated nucleotide is
underlined). Unlabeled 20- to 200-fold molar excess
competitor DNAs were added to the binding reaction as indicated.
Similar results were obtained in three independent experiments.
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As mentioned above, the putative TPA-responsive region reveals
similarity to the AP-1 motif but also is a potential C/EBP binding
site. To identify the factor that mediates the TPA responsiveness by
binding to the region, we next performed a gel mobility supershift assay using specific antibodies against C/EBP, C/EBP, C/EBP, c-Fos, c-Jun, HNF-1, and c-Myc, respectively. Each antibody was added to a tube prior to the binding reaction. As shown in Fig. 3, a supershifted band was observed
together with a reduction of the gel-shift complex only when the
C/EBP antibody was added (lane 9). Thus, the results
clearly indicated that the TPA-responsive binding protein includes
C/EBP, and the TPA-responsive region works as a C/EBP binding
site.
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Fig. 3.
The TPA-induced-binding protein is
C/EBP and the TPA responsive site works as a
C/EBP site. Gel mobility shift experiments were performed as
described under "Experimental Procedures" with nuclear protein
extracts isolated from HepG2 cell after incubation with vehicle
(lanes 1,2), 100 nM TPA for 2 h
(lanes 3,4) or for 4 h (lanes 5-14).
32P-labeled wild type probe whose sequence is shown in Fig.
2 was used in all lanes. Unlabeled 200-fold molar excess competitor
DNAs were added to the binding reaction as indicated (lanes
2,4,6). Specific antibodies (1 µg) to C/EBP (lane
8), C/EBP (lane 9), C/EBP (lane 10),
c-Fos (lane 11), c-Jun (lane 12), HNF-1
(lane 13), or c-Myc (lane 14) were added to the
binding reaction as described in "Experimental Procedures". An
arrow shows the supershift band which appeared only when specific
antibody to C/EBP was added. Similar results were obtained in three
independent experiments.
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Next, we performed a DNase footprinting assay with end-labeled
double-stranded DNA probes derived from human IGF-I promoter-1 and
nuclear extracts from HepG2 cell or C/EBP overexpressing 293T cells
(Fig. 4). The results indicated that
overexpressed C/EBP protected the C/EBP site from nuclease digestion
(lanes 4 and 5). With the nuclear extract of
HepG2 cell, the same site was protected (lane 2), and this
protection was enhanced by 4 h of incubation with TPA (lane
3), confirming that C/EBP binds to the C/EBP site in a
TPA-dependent manner in HepG2 cells. Thus, C/EBP binds
to the C/EBP site in IGF-I gene promoter-1 and mediates the TPA effects
on the IGF-I gene promoter.
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Fig. 4.
DNase footprinting assays confirm that the
TPA inducible-binding protein is C/EBP and
that the C/EBP site is protected in SK-N-MC cell nuclear extract.
DNase footprinting assays were performed as described under
"Experimental Procedures" without (lane 1), with nuclear
protein extracts isolated from HepG2 cell after incubation with vehicle
(lane 2), with 100 nM TPA for 4 h
(lane 3), with nuclear protein from 293T cells transfected
with control vector (pcDNA3, lane 4), with C/EBP
expression vector (pcDNA3C/EBP, lane 5), or with
nuclear protein from SK-N-MC cells (lane 6). Similar results
were obtained in three independent experiments.
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C/EBP Transactivates IGF-I Gene
Promoter-1--
To investigate whether C/EBP that binds to the
C/EBP site can activate the IGF-I gene transcription, we overexpressed
C/EBP in 293T cells and evaluated the effects on the IGF-I gene
promoter activity. 293T cells were chosen because they lack intrinsic
expression of C/EBP (data not shown). As shown in Fig.
5, overexpression of C/EBP
transactivates the IGF-I promoter-1 in a dose-dependent manner. When a point mutation was introduced to the C/EBP site, the
transactivation effect of C/EBP disappeared, suggesting that C/EBP transactivates human IGF-I promoter-1 through the C/EBP site.
Taken together, these results indicate that PKC activation induces
human IGF-I gene transcription through enhancement of the C/EBP
binding to promoter 1.
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Fig. 5.
Overexpressed C/EBP
transactivates the human IGF-I promoter-1 through the C/EBP site
in 293T cells. Wild type IGF-I promoter-1-luciferase reporter
plasmid (pIGFI-300) or mutated type IGF-I promoter-1-luciferase
reporter plasmid (pIGFI-300M) was cotransfected with 1 µg of control
expression vector (indicated as ) or indicated amount of C/EBP
expression vector (pcDNA3C/EBP) with a pMSV--galactosidase
control vector into 293T cell using LipofectAMINE. After 48 h of
incubation, cytoplasmic extracts were prepared, and luciferase activity
was measured. Data were normalized for transfection efficiency
(-galactosidase expression) and presented as means ± S.D. of
at least three independent experiments performed in duplicate.
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PKC Transcriptionally Activates C/EBP in HepG2
Cells--
Next we investigated the mechanism that underlies the
PKC-dependent activation of C/EBP. First, the effects of
TPA on C/EBP mRNA were evaluated in HepG2 cells. The results of
Northern blot analysis (Fig. 6) revealed
that TPA stimulation increases the C/EBP mRNA amount by
~3-fold, suggesting that PKC can stimulate C/EBP gene
transcription, and this may in part explain the PKC activation of the
human IGF gene promoter.
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Fig. 6.
TPA stimulates C/EBP
gene transcription in HepG2 cells. Northern blotting
analyses were performed with 10 µg of total RNA isolated from HepG2
cells after incubation with 100 nM of TPA for the times
indicated. The probe was 32P-labeled rat C/EBP probe.
Similar results were obtained in three independent experiments.
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PKC Posttranslationally Activates C/EBP via Activation of
RSK--
Recently, RSK was shown to stimulate C/EBP activity, and
this facilitates TGF-induced hepatocyte proliferation (25). Because RSK is known to be a downstream target of PKC, we investigated the
possibility of RSK also being involved in the PKC-dependent activation of C/EBP and IGF-I gene activation. For this purpose, we
used a DN mutant of RSK.
As shown in Fig. 7, the DN RSK mutant,
when co-overexpressed in the 293T cells with C/EBP, significantly
suppressed the transactivation potential of C/EBP in terms of the
activation of the human IGF-I gene promoter. This effect was not
observed when C/EBP was absent (Fig. 7). These results suggest that
the transactivation potential of C/EBP depends on the RSK activity
in 293T cells.
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Fig. 7.
Overexpressed dominant negative type RSK
inhibits C/EBP transactivation of IGF-I
promoter-1. Upper panel, IGF-I promoter-1-luciferase
reporter plasmids (pIGFI-300) were cotransfected with 1 µg of control
expression vector (pcDNA3, lanes 1-3) or 1 µg of
C/EBP expression vector (pcDNA3C/EBP, lanes 4-6),
and 1 mg of another control expression vector (pKH3. lanes
1, 4) or 1 µg of HA tagged wild type RSK expression
vector (WT RSK, lanes 2, 5) or HA tagged dominant
negative type RSK expression vector (DN RSK, lanes 3,
6), and Renilla luciferase (pRLCMV) control
vector into 293T cell using LipofectAMINE. After 48 h of
incubation, cytoplasmic extracts were prepared, and luciferase activity
was determined. Data were normalized for transfection efficiency
(Renilla luciferase expression) and presented as means ± S.D. of at least three independent experiments performed in
duplicate. Lower panel, Western blotting with cytoplasmic
extracts using anti-HA antibody was performed.
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To further clarify the mechanism underlying this
RSK-dependent activation of C/EBP, we employed the
Saccharomyces cerevisiae GAL4 fusion protein reporter system
(Fig 8). It is known that the N-terminal
region of C/EBP contains a transcription activation domain and a
transrepression domain (Fig. 8b, (26)). It also includes a
Ser residue at 105, which was previously shown to be critical for the
C/EBP activation by TPA (27) and was also identified recently as the
phosphorylated site by RSK (25).
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Fig. 8.
TPA-induced activation function of
C/EBP depends on RSK. The bar
graph (a) depicts the transactivation potential of
GAL4-C/EBP chimeras. The fusion proteins were obtained by fusing the
DNA-binding domain of GAL4 (aa 1-147) to the 118-aa (Gal4C/EBP118)
or 166-aa (Gal4C/EBP166) N-terminal region of rat C/EBP
(b). A single aa substitution (Ser-105 to Ala) was
introduced to Gal4C/EBP166 plasmid to produce
Gal4C/EBP166mut105Ala plasmid. One microgram of each GAL4-C/EBP
fusion plasmid was cotransfected into HepG2 cells with 1 µg of
Gal4-responsive reporter plasmid (pFR-Luc) and 5 ng of an internal
control, pRL-CMV. Where indicated, 1 µg of wild type or dominant
negative type RSK expression plasmid was also cotransfected. After
transfection, cells were incubated for 24 h, and then
107 M TPA or vehicle was added. After another
24-h incubation, dual-luciferase assays were performed. The firefly
luciferase results were normalized with respect to transfection
efficiency assessed by Renilla luciferase results. The data
were presented as means of at least three independent experiments
performed in duplicate. AD, activation domain.
RD, repression domain.
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Accordingly, we prepared GAL4 fusion constructs containing either 118 or 166 amino acids (aa) of N-terminal region of C/EBP fused to the
heterologous DNA-binding domain of the GAL4 transcription factor (Fig.
8b). These chimeric GAL4-C/EBP fusion proteins were expressed in HepG2 cells, and effects on the GAL4 reporter were evaluated. HepG2 cells were used for this experiment because they show
a very good response to TPA and have intrinsic C/EBP.
As shown in Fig. 8a (lane 3), the Gal4C/EBP118
construct transactivated the GAL4 reporter in serum-free medium. On the
other hand, the Gal4C/EBP166 construct did not activate the GAL4
reporter in the basal state (lane 5). This observation is
consistent with former report by Williams et al. (26) and
provides further support for the idea that the transrepression domain
was potent enough to almost totally suppress the transactivation
potential. Unlike the Gal4C/EBP118 construct revealing no further
activation by TPA (Fig. 8a, lanes 3 and
4), the Gal4C/EBP166 construct was converted to a
transcriptional activator by the TPA treatment (lanes 5 and
6). This clearly indicated that TPA activates C/EBP by
inhibiting the activity of transrepression domain rather than activating the transactivating domain.
In support of the implication of RSK, overexpression of DN RSK mutant
abolished the TPA-responsive activation of Gal4C/EBP166 (Fig.
8a, lanes 9 and 10). Moreover, when
the previously identified phosphorylation site for RSK within C/EBP
(Ser-105; Ref. 25) was disrupted (substituted by Ala) in the GAL4
fusion protein (Gal4C/EBP166mut105Ala), it could not transactivate
the GAL4 reporter in response to the TPA stimulation (lanes
11 and 12). These observations thus suggest that TPA
stimulates activation function of C/EBP via RSK activation followed
by the phosphorylation of Ser-105 of C/EBP.
We next investigated the implication of the RSK-C/EBP axis in the
IGF-I gene regulation. As shown in Fig.
9, the coexpression of DN RSK mutant in
TPA-stimulated HepG2 cells suppressed the promoter activity of the
IGF-I gene in the cells. This agrees with the idea that RSK plays a
primary role in mediating the TPA-dependent activation of
C/EBP and, subsequently, the activation of the IGF-I gene
transcription. Taken together, these data demonstrated that PKC
activates C/EBP both at the level of its transcription (Fig. 6) and
of its posttranslation, which is mediated by the activation of RSK
(Figs. 7-9).
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Fig. 9.
Overexpressed dominant negative type RSK
inhibits TPA stimulation of IGF-I promoter activity. IGF-I
promoter-1-luciferase reporter plasmids (pIGFI-300) were cotransfected
with 1 mg of control expression vector or 1 mg of dominant negative
type RSK expression vector as indicated and Renilla
luciferase (pRLCMV) control plasmid into 293T cell using LipofectAMINE.
After 24 h of incubation, cultures were exposed to control medium
(containing vehicle) or TPA (100 nM) for 24 h.
Cytoplasmic extracts were prepared, and luciferase activity was
determined. Data were normalized for transfection efficiency assessed
by Renilla luciferase results and presented as means ± S.D. of at least three independent experiments performed in
duplicate.
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IGF-I Gene Expression in SK-N-MC Cells Depends on
PKC-dependent, Transcriptional, and Posttranslational
Regulation of C/EBP--
SK-N-MC is a human neuroblastoma cell line
that displays constitutive expression of the IGF-I gene (22). It was
previously shown that IGF-I mRNAs expressed in SK-N-MC cells
contained exon 1 sequences but lacked exon 2 sequences and that
promoter-1, but not promoter-2, was active in the cells (28). As
support for physiological implication of the PKC-dependent
pathway in the IGF-I gene regulation, we found that the IGF-I mRNA
amount was significantly decreased in SK-N-MC cells when the cells were
incubated for 24 h in the presence of the PKC inhibitor
staurosporine (Fig. 10).
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Fig. 10.
PKC inhibitor decreases IGF-I mRNA in
SK-N-MC cells. Northern blotting analyses were performed with 10 µg of total RNA isolated from SK-N-MC cells after incubation with
vehicle (lanes 1, 2) or 100 nM of
staurosporine (lanes 3, 4) for 24 h. The
probe was 32P-labeled human IGF-I cDNA probe. Similar
results were obtained in three independent experiments performed in
duplicate.
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To clarify the factors involved in the intrinsic IGF-I gene expression
in SK-N-MC cells, we investigated whether a certain nuclear factor
binds to the C/EBP site of the human IGF-I gene in SK-N-MC cells.
According to results of gel mobility shift assays (Fig.
11), there was a nuclear protein that
specifically binds to the C/EBP site. This agreed with the results of
the DNase footprinting assay shown in Fig. 4 (lane 6). A
group of specific antibodies were added to identify the binding
protein, and the results clearly demonstrated that only C/EBP
occupies the C/EBP site in SK-N-MC cells (Fig. 11; lanes
8-14). Because the intensity of the retarded band (C/EBP) was
weakened when a PKC inhibitor staurosporine was added to the cells
(lanes 4-6), we concluded that PKC, which seems readily
activated even in the basal state (without any stimulation) in SK-N-MC
cells, is involved in the activation of the nuclear protein binding to
the C/EBP site in these cells.
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Fig. 11.
C/EBP binds to the
C/EBP site of IGF-I promoter-1 in SK-N-MC cells in a
PKC-dependent manner. Gel mobility shift experiments
were performed as described under "Experimental Procedures" with
nuclear protein extracts isolated from SK-N-MC cell after incubation
with vehicle (lanes 1-3, 7-14) or 100 nM TPA for 4 h. 32P-labeled wild type
probe, whose sequence is shown in Fig. 2, was used in all lanes.
Unlabeled 200-fold molar excess wild type competitor (lanes
2, 5) or mutated type competitor (lanes 3,
6) was added to the binding. Specific antibodies (1 µg) to
C/EBP (lane 8), C/EBP (lane 9), C/EBP
(lane 10), c-Fos (lane 11), c-Jun (lane
12), HNF-1 (lane 13), or c-Myc (lane 14)
was added to binding reaction as described under "Experimental
Procedures". The arrow shows the supershift band that
appeared only when a specific antibody to C/EBP was added. Similar
results were obtained in three independent experiments.
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We next sought to determine whether this PKC-dependent
binding of C/EBP to the C/EBP site is required for the human IGF-I gene expression in SK-N-MC cells. Results of reporter gene analyses revealed that incubation of SK-N-MC cells with a specific PKC inhibitor
GF109203X decreased the basal promoter activity of 1600-bp (pIGFI-1600), 600-bp (pIGFI-600), and 300-bp (pIGFI-300) human IGF-I
promoter-1 by more than 50% (Fig. 12).
This suppressive effect of the PKC inhibitor was no longer observed
with pIGFI-300M in which the C/EBP site was disrupted (Fig. 12),
suggesting that intrinsically activated PKC enhances the
C/EBP-binding to the C/EBP site and thus is responsible for the
human IGF-I gene transcription.
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Fig. 12.
PKC inhibitor decreases basal promoter
activity of human IGF-I promoter-1 in SK-N-MC cells. Left
panel, diagrammatic representation of IGF-I promoter-1-luciferase
reporter plasmids. The nucleotide sequence is shown for the region
containing the C/EBP site (underlined). pIGFI-300M has a
point mutation, and the altered base is in bold. Right
panel, IGF-I promoter-1-luciferase reporter plasmids were
cotransfected with a Renilla luciferase control plasmid into
SK-N-MC cell using LipofectAMINE. After 24-h of incubation, cultures
were exposed to control medium (containing vehicle) or GF109203X (1 µM) for 24 h. Cytoplasmic extracts were prepared,
and luciferase activity was determined. Data were normalized for
transfection efficiency assessed by Renilla luciferase
results and presented as means ± S.D. of at least three
independent experiments performed in duplicate.
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To investigate whether both transcriptional and posttranscriptional
regulation of C/EBP by PKC is involved in IGF-I gene expression in
SK-N-MC cells, we performed Northern blot analyses and GAL4 fusion
protein reporter analyses. As shown in Fig.
13A, the amount of C/EBP
mRNA was decreased in SK-N-MC cells after a 24-h incubation with
staurosporine, suggesting that the transcriptional control of C/EBP
by intrinsically activated PKC can in part be involved in the
regulation of IGF-I gene expression in SK-N-MC cells.
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Fig. 13.
Dual control of C/EBP
activity by intrinsically activated PKC in SK-N-MC cells.
A, PKC inhibitor decreases C/EBP mRNA in
SK-N-MC cells. Northern blotting analyses were performed using
32P-labeled rat C/EBP cDNA as a probe. Ten
micrograms of total RNA isolated from SK-N-MC cells after incubation
with vehicle (lanes 1, 2) or 100 nM
of staurosporine (lanes 3, 4) for 24 h were
loaded. Similar results were obtained in three independent experiments
performed in duplicate. B, intrinsically activated PKC
stimulates the activation function of C/EBP through RSK activation.
The bar graph depicts the transactivation potential of
GAL4-C/EBP chimeras. The fusion protein (Gal4C/EBP166) was
obtained by fusing the DNA-binding domain (DBD) of GAL4 (aa
1-147) to the 166-aa N-terminal region of rat C/EBP. A single aa
substitution (Ser-105 to Ala) was introduced to Gal4C/EBP166 plasmid
to produce Gal4C/EBP166mut105Ala plasmid. One microgram of each
GAL4-C/EBP fusion plasmid was cotransfected into SK-N-MC cells with
1 µg of Gal4-responsive reporter plasmid (pFR-Luc) and 5 ng of an
internal control, pRL-CMV. Where indicated, 1 µg of wild type
(RSK-WT) or dominant negative type RSK (RSK-DN)
expression plasmid was also cotransfected. After transfection, cells
were incubated in the basal condition medium for 24 h, and then 1 µM GF109203X or vehicle was added. After another 24-h
incubation, dual-luciferase assays were performed. The firefly
luciferase results were normalized with respect to transfection
efficiency assessed by Renilla luciferase results. The data
were presented as means of at least three independent experiments
performed in duplicate.
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On the other hand, the GAL4 fusion protein reporter assays in SK-N-MC
cells revealed that the Gal4C/EBP166 construct transactivated the
GAL4 reporter in SK-N-MC cells kept under the basal condition (Fig.
13B, lane 2). This contrasted with the
observation obtained with HepG2 cells, in which TPA was required for
the transactivation potential activated (Fig. 8, lanes 5 and
6). The transactivation potential was dramatically
suppressed by addition of PKC inhibitor GF10923X (Fig. 13B,
lane 3), suggesting that intrinsically activated PKC readily
activated C/EBP in SK-N-MC cells even without any stimuli.
Substitution of Ser-105 by Ala (Gal4C/EBP166mut105Ala) or
overexpression of the DN RSK mutant also suppressed the
activation function of C/EBP (lane 4-6). Thus
it was demonstrated that the IGF-I gene expression in SK-N-MC cells
depends on the intrinsically activated PKC, which is involved in the
activation of C/EBP probably through two different mechanisms: the
increment of its transcription and the RSK-mediated posttranslational
activation of its transactivation potential.
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DISCUSSION |
In the present study, we showed that the major promoter of the
human IGF-I gene is a target of TPA stimulation (Fig. 1) and that a
C/EBP site within the promoter is responsible for the phenomenon (Figs.
2-5). C/EBP, which is activated by PKC both at the level of
transcription (Figs. 6 and 13a) and of posttranslation
(Figs. 8 and 13b), binds to the C/EBP site and thereby
mediates the phenomenon. In non-C/EBP-expressing 293T cells,
transiently expressed ectopic C/EBP transactivated the human IGF-I
gene promoter (Figs. 5 and 7) and indeed induced intrinsic IGF-I gene
expression in the cells,2
providing support for the physiological implication of C/EBP in the
IGF-I gene expression.
The C/EBP family proteins comprise a diverse group of
transcriptional regulators active in tissue development and
regeneration, inflammation, and intermediary metabolism (29, 30). They
are members of the basic leucine zipper family of transcription factors (29-31) and also reveal strong amino acid similarity in their COOH domains, which have been shown to be responsible for protein
dimerization and DNA binding (29, 30).
Nolten et al. (24) previously reported that overexpressed
C/EBP and C/EBP can bind to the region that we found to be the putative TPA-responsive region and can stimulate the promoter activity
of human IGF-I promoter-1. Our data showed that, at least in the case
of HepG2 and SK-N-MC cells, C/EBP is the major binding factor for
the C/EBP site and that no other C/EBP family proteins such as C/EBP
and C/EBP are included in the binding complexes formed with the
C/EBP site of the human IGF-I gene promoter. Whereas the C/EBP family
proteins share a common binding preference, this may be because the
C/EBP family proteins other than C/EBP are not expressed in those
cells or because they are inactive even when expressed.
The human neuroblastoma cell line SK-N-MC reveals
relatively high expression of IGF-I under the basal condition; the
amount of IGF-I mRNA was even more than that expressed in the human
liver (22). The fact that the addition of neutralized antibody to IGF-IR prevents the cell from proliferating (14) suggests that the
IGF-I autocrine loop is essential for the tumor phenotype of SK-N-MC
cells. As a step toward elucidating the mechanism underlying the
constitutive expression of the IGF-I gene in SK-N-MC cells, we sought
to clarify the physiological role of the C/EBP site in the IGF-I gene
expression. The results of the DNase footprinting assay (Fig. 4) and
supershift assay (Fig. 11) clearly showed that C/EBP binds to the
C/EBP site in SK-N-MC cells under basal condition, and reporter gene
analysis (Fig. 12) showed that the C/EBP binding to the C/EBP site
is crucial for strong activity of the promoter-1 in SK-N-MC cells.
Support for this comes from the observation by Mittanck et
al. (19) showing that the initial 50 base pairs of the exon 1 untranslated region are essential for the promoter-1 of human IGF-I
gene being active in the SK-N-MC cell line; although, for some unknown
reason, they failed to detect any protein binding with a probe
containing the C/EBP site.
The fact that the mutated human C/EBP site we used in this study, whose
sequence completely matches the sequence of the corresponding region of
rat IGF-I promoter-1, could not be bound by C/EBP clearly showed
that this C/EBP site is not conserved in the rat or mouse IGF-I gene.
However, we previously identified another high affinity C/EBP site,
termed the HS3D site, in rat IGF-I promoter-1 located 5'-untranslated
region of exon 1 (4-6). As support for the extensive implication of
C/EBP family proteins in IGF-I gene regulation, another C/EBP family
protein, C/EBP, was shown to mediate cAMP responsiveness of rat
IGF-I promoter-1 through this site (5, 6). Considering the conserved
binding preference for the C/EBP family proteins, it is possible that
C/EBP family proteins are involved in various regulation of the IGF-I
gene through the same C/EBP binding site with their activity being
posttranslationally modulated by various kinases.
A serine/threonine kinase RSK has been shown to function as a
downstream target of PKC (32, 33). Tan et al. (33)
showed that RSK mediates the PKC-dependent prevention of
Bad-mediated apoptosis by directly phosphorylating Bad protein. In
terms of the substrates of RSK, only a few have been identified to date including transcription factors cAMP-response element-binding protein
and Fos (34, 35). Recently, C/EBP was added to the list of direct
substrates of RSK; activated RSK phosphorylates Ser-105 of C/EBP,
and this activation is critical for hepatocyte proliferation (25).
These reports prompted us to consider that RSK may mediate the present
phenomenon; using a dominant negative mutant of RSK, we were able to
show that RSK plays a key role in the PKC-dependent,
C/EBP-mediated activation of the IGF-I gene promoter (Figs. 7-9).
Moreover, the data obtained with S. cerevisiae GAL4 fusion protein reporter system revealed that the TPA-responsive activation of the GAL4-C/EBP chimera depends on a Ser residue at
position 105 (Ser-105) of C/EBP (Fig. 8). Because Ser-105 is known
as a direct phosphorylation site by RSK (25), it is likely that the
TPA-induced transactivation of C/EBP is also mediated by direct
phosphorylation/posttranslational activation by RSK.
Although PKC seems to act as an essential upstream factor for the
RSK-dependent, C/EBP-mediated IGF-I gene activation in SK-N-MC cells, this may not be the case for all cell types. For example, despite the implication of RSK activation in the
C/EBP-mediated IGF-I gene activation in 293T cells (Fig. 7), there
has been no evidence reported to date that supports the constitutive
PKC activation in the cells. Rather, according to a report by Arould
et al. (36), they directly measured PKC activity in 293T
cells but detected no significant PKC activity, thus disputing the
implication of PKC in the RSK activation in 293T cells. Although
speculative, mitogen-activated protein kinases (MAPK), instead of PKC,
may play a key role in the RSK activation in 293T cells. It was shown previously that MAPK can directly activate RSK (37). Also, the 293T
cell was transformed with SV40 large T antigen and E1A, both of which
are well known activators for the MAPK cascade (38). Indeed, 10% serum
stimulation was shown to cause a partial nuclear translocation of MEK
in 293T cells (39). Because our 293T cells were always maintained in
serum-containing (10%) medium, it is possible that the MAPK cascade
was kept activated in the cells and thereby involved in the RSK
activation. Thus, the RSK-C/EBP-IGF-I axis, which is probably
activated not only by PKC but also by some growth factors that can
activate MAPK, may operate in various cell types and contribute to the
expression of the IGF-I gene in those cells.
Apart from this RSK-mediated effect on the activation
function of C/EBP, our present study failed to clarify whether the TPA treatment enhances the DNA-binding affinity of C/EBP. Although we found that TPA stimulation enhanced the C/EBP binding to its target DNA in HepG2 cells (Figs. 2 and 3), this does not necessarily mean that the DNA-binding affinity was increased in response to TPA as
the increment of the C/EBP gene expression can by itself explain the
phenomenon (Fig. 6). Indeed, Trautwein et al. (27) previously reported that TPA stimulates transactivation potential of
C/EBP without changing its DNA-binding affinity in HepG2 cells. Thus
we assume that the RSK-mediated regulation of C/EBP activity takes
place probably independent of controlling its DNA-binding affinity.
IGF-I is known to function as an autocrine or paracrine growth factor
in a variety of mesenchymal and epithelial tumors (40). Recent studies
have demonstrated that expression of the type-I insulin-like growth
factor receptor (IGF-IR), which mediates most of the IGF-I action, is
required for the establishment and maintenance of the transformed
phenotype in some cell lines (13). A gene-targeting study revealed that
cells derived from IGF-IR(/) mouse embryos cannot be transformed by
the SV40 large T antigen or by an activated and overexpressed
Ha-Ras, while stable transformation of cells with human IGF-IR
expression plasmid restored the ability to be transformed (13). These
findings suggest that the IGF system (including IGF-I and -II and
IGF-IR) plays an essential role in, at least, some cases of
transformation or tumorigenesis. On the other hand, it is well known
that the PKC activator TPA is a strong carcinogen. Because the IGF-I
gene expressed in SK-N-MC cells was shown to play a major role in the
proliferation of SK-N-MC, it is likely that the constitutive active PKC
subtypes contribute toward keeping the IGF system active and thereby
maintaining the tumor phenotype of the cells.
In conclusion, our present study revealed that PKC can activate the
IGF-I gene expression through the RSK and C/EBP-mediated pathway.
Whereas the IGF-I system is essential for proliferation of SK-N-MC
cells (14), constitutively active PKC may contribute to determining
tumor phenotype in some cancer cells by keeping the IGF-I gene
expression active.
-mediated Expression of Insulin-like Growth Factor I
Gene*
,
TOP
ABSTRACT
INTRODUCTION
To whom correspondence should be addressed. Tel.:
81-6-6879-3633; Fax: 81-6-6879-3639; E-mail:
kajimoto@medone.med.osaka-u.ac.jp.
